Handbook of Plant and Crop Physiology Third Edition

THIRD EDITION
Handbook of
PLANT AND CROP
PHYSIOLOGY
Edited by
MOHAMMAD PESSARAKLI
THIRD EDITION
Handbook of
PLANT AND CROP
PHYSIOLOGY
BOOKS IN SOILS, PLANTS, AND THE ENVIRONMENT
Editorial Board
Agricultural Engineering Robert M. Peart, University of Florida, Gainesville
Crops Mohammad Pessarakli, University of Arizona, Tucson
Environment Kenneth G. Cassman, University of Nebraska, Lincoln
Irrigation and Hydrology Donald R. Nielsen, University of California, Davis
Microbiology Jan Dirk van Elsas, Research Institute for Plant Protection,
Wageningen, the Netherlands
Plants L. David Kuykendall, U.S. Department of Agriculture,

Beltsville, Maryland
Kenneth B. Marcum, Arizona State University, Tempe
Soils Jean-Marc Bollag, Pennsylvania State University,

University Park
Tsuyoshi Miyazaki, University of Tokyo, Japan
Soil Biochemistry, Volume 1, edited by A. D. McLaren and G. H. Peterson

Soil Biochemistry, Volume 2, edited by A. D. McLaren and J. Skujins
Soil Biochemistry, Volume 3, edited by E. A. Paul and A. D. McLaren
Soil Biochemistry, Volume 4, edited by E. A. Paul and A. D. McLaren
Soil Biochemistry, Volume 5, edited by E. A. Paul and J. N. Ladd
Soil Biochemistry, Volume 6, edited by Jean-Marc Bollag and G. Stotzky
Soil Biochemistry, Volume 7, edited by G. Stotzky and Jean-Marc Bollag
Soil Biochemistry, Volume 8, edited by Jean-Marc Bollag and G. Stotzky
Soil Biochemistry, Volume 9, edited by G. Stotzky and Jean-Marc Bollag
Organic Chemicals in the Soil Environment, Volumes 1 and 2, edited by C. A. I. Goring
and J. W. Hamaker
Humic Substances in the Environment, M. Schnitzer and S. U. Khan
Microbial Life in the Soil: An Introduction, T. Hattori
Principles of Soil Chemistry, Kim H. Tan
Soil Analysis: Instrumental Techniques and Related Procedures, edited by
Keith A. Smith
Soil Reclamation Processes: Microbiological Analyses and Applications, edited by
Robert L. Tate III and Donald A. Klein
Symbiotic Nitrogen Fixation Technology, edited by Gerald H. Elkan
Soil–Water Interactions: Mechanisms and Applications, Shingo Iwata
and Toshio Tabuchi with Benno P. Warkentin
Soil Analysis: Modern Instrumental Techniques, Second Edition, edited by Keith A.
Smith
Soil Analysis: Physical Methods, edited by Keith A. Smith and Chris E. Mullins
Growth and Mineral Nutrition of Field Crops, N. K. Fageria, V. C. Baligar,
and Charles Allan Jones
Semiarid Lands and Deserts: Soil Resource and Reclamation, edited by J. Skujins
Plant Roots: The Hidden Half, edited by Yoav Waisel, Amram Eshel, and Uzi Kafkafi
Plant Biochemical Regulators, edited by Harold W. Gausman
Maximizing Crop Yields, N. K. Fageria
Transgenic Plants: Fundamentals and Applications, edited by Andrew Hiatt
Soil Microbial Ecology: Applications in Agricultural and Environmental Management,
edited by F. Blaine Metting, Jr.
Principles of Soil Chemistry, Second Edition, Kim H. Tan
Water Flow in Soils, edited by Tsuyoshi Miyazaki
Handbook of Plant and Crop Stress, edited by Mohammad Pessarakli
Genetic Improvement of Field Crops, edited by Gustavo A. Slafer
Agricultural Field Experiments: Design and Analysis, Roger G. Petersen
Mechanisms of Plant Growth and Improved Productivity: Modern Approaches,
edited by Amarjit S. Basra
Selenium in the Environment, edited by W. T. Frankenberger, Jr. and Sally Benson
Plant–Environment Interactions, edited by Robert E. Wilkinson
Handbook of Plant and Crop Physiology, edited by Mohammad Pessarakli
Handbook of Phytoalexin Metabolism and Action, edited by M. Daniel
and R. P. Purkayastha
Soil–Water Interactions: Mechanisms and Applications, Second Edition, Revised
and Expanded, Shingo Iwata, Toshio Tabuchi, and Benno P. Warkentin
Stored-Grain Ecosystems, edited by Digvir S. Jayas, Noel D. G. White,
and William E. Muir
Agrochemicals from Natural Products, edited by C. R. A. Godfrey
Seed Development and Germination, edited by Jaime Kigel and Gad Galili
Nitrogen Fertilization in the Environment, edited by Peter Edward Bacon
Phytohormones in Soils: Microbial Production and Function,
William T. Frankenberger, Jr. and Muhammad Arshad
Handbook of Weed Management Systems, edited by Albert E. Smith
Soil Sampling, Preparation, and Analysis, Kim H. Tan
Soil Erosion, Conservation, and Rehabilitation, edited by Menachem Agassi
Plant Roots: The Hidden Half, Second Edition, Revised and Expanded, edited by
Yoav Waisel, Amram Eshel, and Uzi Kafkafi
Photoassimilate Distribution in Plants and Crops: Source–Sink Relationships, edited by
Eli Zamski and Arthur A. Schaffer
Mass Spectrometry of Soils, edited by Thomas W. Boutton and Shinichi Yamasaki
Handbook of Photosynthesis, edited by Mohammad Pessarakli
Chemical and Isotopic Groundwater Hydrology: The Applied Approach, Second Edition,
Revised and Expanded, Emanuel Mazor
Fauna in Soil Ecosystems: Recycling Processes, Nutrient Fluxes, and Agricultural
Production, edited by Gero Benckiser
Soil and Plant Analysis in Sustainable Agriculture and Environment, edited by
Teresa Hood and J. Benton Jones, Jr.
Seeds Handbook: Biology, Production, Processing, and Storage, B. B. Desai,
P. M. Kotecha, and D. K. Salunkhe
Modern Soil Microbiology, edited by J. D. van Elsas, J. T. Trevors,
and E. M. H. Wellington
Growth and Mineral Nutrition of Field Crops, Second Edition, N. K. Fageria,
V. C. Baligar, and Charles Allan Jones
Fungal Pathogenesis in Plants and Crops: Molecular Biology and Host Defense
Mechanisms, P. Vidhyasekaran
Plant Pathogen Detection and Disease Diagnosis, P. Narayanasamy
Agricultural Systems Modeling and Simulation, edited by Robert M. Peart
and R. Bruce Curry
Agricultural Biotechnology, edited by Arie Altman
Plant–Microbe Interactions and Biological Control, edited by Greg J. Boland
and L. David Kuykendall
Handbook of Soil Conditioners: Substances That Enhance the Physical Properties
of Soil, edited by Arthur Wallace and Richard E. Terry
Environmental Chemistry of Selenium, edited by William T. Frankenberger, Jr.,
and Richard A. Engberg
Principles of Soil Chemistry, Third Edition, Revised and Expanded, Kim H. Tan
Sulfur in the Environment, edited by Douglas G. Maynard
Soil–Machine Interactions: A Finite Element Perspective, edited by Jie Shen
and Radhey Lal Kushwaha
Mycotoxins in Agriculture and Food Safety, edited by Kaushal K. Sinha
and Deepak Bhatnagar
Plant Amino Acids: Biochemistry and Biotechnology, edited by Bijay K. Singh
Handbook of Functional Plant Ecology, edited by Francisco I. Pugnaire
and Fernando Valladares
Handbook of Plant and Crop Stress, Second Edition, Revised and Expanded, edited by
Mohammad Pessarakli
Plant Responses to Environmental Stresses: From Phytohormones to Genome
Reorganization, edited by H. R. Lerner
Handbook of Pest Management, edited by John R. Ruberson
Microbial Endophytes, edited by Charles W. Bacon and James F. White, Jr.
Plant–Environment Interactions, Second Edition, edited by Robert E. Wilkinson
Microbial Pest Control, Sushil K. Khetan
Soil and Environmental Analysis: Physical Methods, Second Edition, Revised
and Expanded, edited by Keith A. Smith and Chris E. Mullins
The Rhizosphere: Biochemistry and Organic Substances at the Soil–Plant Interface,
Roberto Pinton, Zeno Varanini, and Paolo Nannipieri
Woody Plants and Woody Plant Management: Ecology, Safety, and Environmental
Impact, Rodney W. Bovey
Metals in the Environment, M. N. V. Prasad
Plant Pathogen Detection and Disease Diagnosis, Second Edition, Revised
and Expanded, P. Narayanasamy
Handbook of Plant and Crop Physiology, Second Edition, Revised and Expanded,
edited by Mohammad Pessarakli
Environmental Chemistry of Arsenic, edited by William T. Frankenberger, Jr.
Enzymes in the Environment: Activity, Ecology, and Applications, edited by
Richard G. Burns and Richard P. Dick
Plant Roots: The Hidden Half, Third Edition, Revised and Expanded, edited by
Yoav Waisel, Amram Eshel, and Uzi Kafkafi
Handbook of Plant Growth: pH as the Master Variable, edited by Zdenko Rengel
Biological Control of Major Crop Plant Diseases edited by Samuel S. Gnanamanickam
Pesticides in Agriculture and the Environment, edited by Willis B. Wheeler
Mathematical Models of Crop Growth and Yield, Allen R. Overman
and Richard Scholtz
Plant Biotechnology and Transgenic Plants, edited by Kirsi-Marja Oksman Caldentey
and Wolfgang Barz
Handbook of Postharvest Technology: Cereals, Fruits, Vegetables, Tea, and Spices,
edited by Amalendu Chakraverty, Arun S. Mujumdar, G. S. Vijaya Raghavan,
and Hosahalli S. Ramaswamy
Handbook of Soil Acidity, edited by Zdenko Rengel
Humic Matter in Soil and the Environment: Principles and Controversies, edited by
Kim H. Tan
Molecular Host Plant Resistance to Pests, edited by S. Sadasivam
and B. Thayumanayan
Soil and Environmental Analysis: Modern Instrumental Techniques, Third Edition,
edited by Keith A. Smith and Malcolm S. Cresser
Chemical and Isotopic Groundwater Hydrology, Third Edition, edited by Emanuel Mazor
Agricultural Systems Management: Optimizing Efficiency and Performance, edited by
Robert M. Peart and W. David Shoup
Physiology and Biotechnology Integration for Plant Breeding, edited by
Henry T. Nguyen and Abraham Blum
Global Water Dynamics: Shallow and Deep Groundwater, Petroleum Hydrology,
Hydrothermal Fluids, and Landscaping, , edited by Emanuel Mazor
Principles of Soil Physics, edited by Rattan Lal
Seeds Handbook: Biology, Production, Processing, and Storage, Second Edition,
Babasaheb B. Desai
Field Sampling: Principles and Practices in Environmental Analysis, edited by
Alfred R. Conklin
Sustainable Agriculture and the International Rice–Wheat System, edited by Rattan Lal,
Peter R. Hobbs, Norman Uphoff, and David O. Hansen
Plant Toxicology, Fourth Edition, edited by Bertold Hock and Erich F. Elstner
Drought and Water Crises: Science, Technology, and Management Issues, edited by
Donald A. Wilhite
Soil Sampling, Preparation, and Analysis, Second Edition, Kim H. Tan
Climate Change and Global Food Security, edited by Rattan Lal, Norman Uphoff,
B. A. Stewart, and David O. Hansen
Handbook of Photosynthesis, Second Edition, edited by Mohammad Pessarakli
Environmental Soil-Landscape Modeling: Geographic Information Technologies and
Pedometrics, edited by Sabine Grunwald
Water Flow in Soils, Second Edition, Tsuyoshi Miyazaki
Biological Approaches to Sustainable Soil Systems, edited by Norman Uphoff,
Andrew S. Ball, Erick Fernandes, Hans Herren, Olivier Husson, Mark Laing,
Cheryl Palm, Jules Pretty, Pedro Sanchez, Nteranya Sanginga, and Janice Thies
Plant–Environment Interactions, Third Edition, edited by Bingru Huang
Biodiversity in Agricultural Production Systems, edited by Gero Benckiser
and Sylvia Schnell
Organic Production and Use of Alternative Crops, Franc Bavec and Martina Bavec
Handbook of Plant Nutrition, edited by Allen V. Barker and David J. Pilbeam
Modern Soil Microbiology, Second Edition, edited by Jan Dirk van Elsas,
Janet K. Jansson, and Jack T. Trevors
Functional Plant Ecology, Second Edition, edited by Francisco I. Pugnaire
and Fernando Valladares
Fungal Pathogenesis in Plants and Crops: Molecular Biology and Host Defense
Mechanisms,Second Edition, P. Vidhyasekaran
Handbook of Turfgrass Management and Physiology, edited by Mohammad Pessarakli
Soils in the Humid Tropics and Monsoon Region of Indonesia, Kim H. Tan
Handbook of Agricultural Geophysics, edited by Barry J. Allred, Jeffrey J. Daniels,
and M. Reza Ehsani
Environmental Soil Science, Third Edition, Kim H. Tan
Principles of Soil Chemistry, Fourth Edition, Kim H. Tan
Handbook of Plant and Crop Stress, Second Edition, edited by Mohammad Pessarakli
Handbook of Plant and Crop Physiology, Third Edition, edited by Mohammad Pessarakli
Humic Matter in Soil and the Environment: Principles and Controversies, Second Edition,
Kim H. Tan
THIRD EDITION
Handbook of
PLANT AND CROP
PHYSIOLOGY
Edited by
MOHAMMAD PESSARAKLI
Research Professor
The School of Plant Sciences
and Adjunct Faculty
Honors College
University of Arizona
Boca Raton London New York
CRC Press is an imprint of the

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not live to see this work and my other works, which in no small part resulted
from the unconditional love that they showered on me for many years.
Contents
Preface............................................................................................................................................xvii
Acknowledgments............................................................................................................................xix
Editor...............................................................................................................................................xxi
Contributors.................................................................................................................................. xxiii
Part I Physiology of Plant/Crop Growth

and Development Stages
Chapter 1 Cell Cycle Regulation and Plant Development: A Crop Production Perspective.........3
Paolo A. Sabelli
Chapter 2 Seed Dormancy, Germination, and Seedling Recruitment in Weedy Setaria............ 33

Jack Dekker
Chapter 3 Alterations in Structural Organization Affect the Functional Ability

of Photosynthetic Apparatus..................................................................................... 103
Emilia L. Apostolova and A.N. Misra
Chapter 4 Photoperiodic Control of Flowering in Plants........................................................... 121

Faqiang Wu and Yoshie Hanzawa
Chapter 5 Role of Alternative Respiratory Pathway in Plants: Some Metabolic

and Physiological Aspects......................................................................................... 139
Elena V. Garmash
Chapter 6 Growth Orientation of Underground Shoots: Stolons and Rhizomes

and Aboveground Creeping Shoots in Perennial Herbaceous Plants....................... 157
Alexander M. Markarov and Tamara K. Golovko
Chapter 7 Structure and Metabolism of Underground Shoots in Perennial

Rhizome-Forming Plants.......................................................................................... 167
Svetlana P. Maslova
Chapter 8 Evaluating and Managing Crops Water Requirement............................................... 179

Zohrab Samani
xi
xii Contents
Part II Cellular and Molecular Aspects of Plant/Crop

Physiology
Chapter 9 Biochemistry and Physiology of Carbon Partitioning in Crop Plants...................... 193

Claudia V. Piattoni, Carlos M. Figueroa, Valeria E. Perotti,
Florencio E. Podestá, and Alberto A. Iglesias
Chapter 10 Role of Nitric Oxide in Plant Development.............................................................. 217

Dagmar Procházková and Nad’a Wilhelmová
Chapter 11 Mitochondria in Plant Physiology............................................................................. 227

Farhad Ghavami, Ali Soltani, Penny M.A. Kianian, and Shahryar F. Kianian
Chapter 12 Signaling Molecules Involved in the Postharvest Stress Response of Plants:

Quality Changes and Synthesis of Secondary Metabolites...................................... 259
Luis Cisneros-Zevallos, Daniel A. Jacobo-Velázquez, Jean-Claude Pech,
and Hisashi Koiwa
Part III Plant/Crop Physiology and Physiological Aspects

of Plant/Crop Production Processes
Chapter 13 Quantifying Immediate Carbon Export from Leaves Predicts Source Strength...... 279
Evangelos Demosthenes Leonardos and Bernard Grodzinski
Chapter 14 Physiology of Grain Development in Cereals........................................................... 301

Muhammad Farooq, Abdul Wahid, and Kadambot H.M. Siddique
Chapter 15 C-Repeat Transcription Factors as Targets for the Maintenance of Crop Yield
under Suboptimal Growth Conditions...................................................................... 313
Keshav Dahal, Khalil Kane, Fathey Sarhan, Leonid V. Savitch, Jas Singh,
Bernard Grodzinski, and Norman P.A. Hüner
Chapter 16 Physiology of Crop Productivity in Cold Climate.................................................... 333

Galina N. Tabalenkova and Tamara K. Golovko
Chapter 17 Rates of Processes of Essential Plant Nutrients........................................................ 343

Thomas W. Crawford, Jr.
Chapter 18 Some Interactions of Mineral Nutrients and Organic Substances in Plant Nutrition.......355
Contents xiii
Part IV Physiological Responses of Plants/Crops under

Stressful (Salt, Drought, Heat, Nutrient Deficiency,
and Other Environmental Stresses) Conditions
Chapter 19 Role of Polyamines in Plant Abiotic Stress Responses............................................. 369

Bhaskar Gupta, Kamala Gupta, and Bingru Huang
Chapter 20 Physiological and Biochemical Mechanisms of Plant Tolerance to Heat Stress....... 389
David Jespersen and Bingru Huang
Chapter 21 Drought Resistance in Small Grain Cereal Crops....................................................405

Moustafa Eldakak, Mukhtar Ahmed, Muhammad Asif, Sanaa I.M. Milad,
Ali I. Nawar, Zohra Aslam, Aakash Goyal, and Jai S. Rohila
Chapter 22 Drought Physiology of Forage Crops........................................................................ 427

M. Anowarul Islam and Augustine K. Obour
Chapter 23 Effect of Drought/Water Stress and Adaptation to Unintended Consequences

of Wheat Growth and Development in Pakistan....................................................... 441
Ijaz Rasool Noorka
Chapter 24 Physiological Mechanisms of Nitrogen Absorption and Assimilation in Plants

under Stressful Conditions........................................................................................ 453
Rama Shanker Dubey, Rajneesh Kumar Srivastava, and Mohammad Pessarakli
Chapter 25 Effects of Hyperosmotic Salinity on Protein Patterns and Enzyme Activities......... 487
Hans-Werner Koyro, B. Huchzermeyer, and Christian Zörb
Chapter 26 Reactive Oxygen Species Generation, Hazards, and Defense Mechanisms

in Plants under Environmental (Abiotic and Biotic) Stress Conditions....................509
Pallavi Sharma, Ambuj Bhushan Jha, Rama Shanker Dubey,
and Mohammad Pessarakli
Chapter 27 Implications of Oxidative Stress for Crop Growth and Productivity........................ 549
Abdul Wahid, Muhammad Farooq, and Kadambot H.M. Siddique
Chapter 28 Physiological and Biophysical Responses of Plants under Low and Ultralow
Temperatures............................................................................................................. 557
Jiří Zámečník and Miloš Faltus
xiv Contents
Chapter 29 Stress Tolerance in Some European Resurrection Plants

(Haberlea rhodopensis and Ramonda spp.).............................................................. 585
Iliya Denev, Detelin Stefanov, Maria Gevezova, Ina Kirilova,
Katya Georgieva, Maya Kurteva, and Galina Panayotova
Chapter 30 Salinity and Amenity Horticulture...........................................................................605

Atif Riaz and Peter M. Martin
Chapter 31 Physiological Responses of Cotton (Gossypium hirsutum L.) to Salt Stress............ 635
Mohammad Pessarakli
Chapter 32 Isoprenoid Biosynthesis in Higher Plants and Green Algae under Normal
and Light Stress Conditions...................................................................................... 655
Parisa Heydarizadeh, Justine Marchand, Mohammad Reza Sabzalian,
Martine Bertrand, and Benoît Schoefs
Part V Physiological Responses of Plants/Crops to Heavy

Metal Concentration and Agrichemicals
Chapter 33 Metal Nanoparticles in Plants: Formation and Action.............................................. 683

Elena Masarovičová, Katarína Král’ová, and Smita Sachin Zinjarde
Chapter 34 Arsenic Toxicity and Tolerance Mechanisms in Crop Plants................................... 733

Pallavi Sharma, Ambuj Bhushan Jha, and Rama Shanker Dubey
Part VI Physiology of Plant/Crop Genetics and Development
Chapter 35 Small RNAs in Crop Response to Temperature Stress Noncoding RNAs

in Plants.................................................................................................................. 785
Galina Yahubyan, Elena Apostolova, Ivan Minkov, and Vesselin Baev
Part VII Bioinformatics and Using Computer Modeling

in Plant Physiology
Chapter 36 Comparative Genomics of Grass Genomes Using CoGe.......................................... 797

Eric Lyons, Matthew D. Bomhoff, Shannon L. Oliver, and Andrew J. Lenards
Contents xv
Part VIII Plants/Crops Growth Responses to Environmental

Factors and Climatic Changes
Chapter 37 Carbon Dioxide, Climate Change, and Crops in the Twenty-First Century:
The Dawn of a New World........................................................................................ 819
Glen M. MacDonald
Part IX Future Promises: Plants and Crops Adaptation and

Biotechnological Aspects of Plants/Crops Improvement
Chapter 38 CAM Plants as Crops: Adaptable, Metabolically Flexible, and Highly
Productive Cultivars.................................................................................................. 831
Karina E.J. Trípodi, Florencio E. Podestá, Carlos M. Figueroa,
Claudia V. Piattoni, Alberto A. Iglesias, and Valeria E. Perotti
Chapter 39 Advances in Improving Adaptation of Common Bean and Brachiaria Forage

Grasses to Abiotic Stresses in the Tropics................................................................ 847
Idupulapati Madhusudana Rao
Chapter 40 Improving Maize Production under Drought Stress: Traits, Screening Methods,
and Environments...................................................................................................... 891
Gerald N. De La Fuente, Ivan Barrero, Seth C. Murray, Tom Isakeit,
and Michael V. Kolomiets
Chapter 41 New Approaches to Turfgrass Nutrition: Humic Substances

and Mycorrhizal Inoculation..................................................................................... 917
Ali Nikbakht and Mohammad Pessarakli
Chapter 42 Use of Sewage in Agriculture and Related Activities............................................... 931

Manal El-Zohri, Awatief F. Hifney, Taha Ramadan, and Refat Abdel-Basset
Chapter 43 Water and Crops: Molecular Biologists’, Physiologists’, and Plant Breeders’
Approach in the Context of Evergreen Revolution.................................................... 967
Ijaz Rasool Noorka and J.S. Heslop-Harrison
Preface
Like any other area in science, both the scope and depth of our knowledge on plant and crop physi-
ology are rapidly expanding. Plant/crop physiologists are continuously making new discoveries.
This phenomenon has resulted in the compilation of a large volume of information since the second
edition of the Handbook of Plant and Crop Physiology was published. The abundance of new data
has necessitated an updated edition, which includes, as much as possible, the latest discoveries in
the field. Like the first and second editions, this edition is a unique, comprehensive, and complete
collection of topics in the field of plant/crop physiology.
Over 90% of the material in this edition is entirely new, and these are included in this volume
under new titles. The remaining 10% have been updated and modified substantially. Therefore,
overall, the material in this book is as good as new.
The Handbook of Plant and Crop Physiology is needed to fill the gap in the literature. It has long
been recognized that physiological processes control plant growth and crop yields. This handbook
will, therefore, serve as an up-to-date resource, covering the relevant information in the field.
Several decisions need to be made when compiling a handbook, such as the extent of content
to include, the information to exclude, the depth to which the topics should be covered, and the
organization of the selected content. In this volume, I have chosen to include information that will
be beneficial to students, instructors, researchers, field specialists, and any others interested in the
areas of plant and crop physiology. In order to plan, implement, and evaluate comprehensive and
specific strategies for dealing with plant and crop physiology problems and issues, strategies must
be based on a firm understanding of facts and principles.
The topics selected for discussion are those that I believe are relevant and in which physiology
plays the dominant role. The concepts have been presented in such a manner as to give both begin-
ning students and specialists an opportunity to expand and refine their knowledge. Certain conclu-
sions and solutions provided throughout the text are related to the more significant and multifaceted
problems of plant and crop physiology. They are presented to provide a concise guide to help stu-
dents and specialists achieve their goals.
This practical and comprehensive guide has been prepared by 105 contributors from 17 countries,
among which are some of the most competent and knowledgeable scientists, specialists, and researchers
in agriculture, particularly in plant sciences and plant physiology. It is intended to serve as a resource
for both university courses and for research. Biologists, physiologists, scientists, agriculture research-
ers, agriculture practitioners, and educators and students will benefit from this unique, comprehensive
guide, which covers plant physiological processes from cellular aspects to whole plants.
As with other fields, accessibility of knowledge is one of the most critical factors involved in crop
physiological processes and problems. Without due consideration of all the elements contributing
to a specific crop physiological process and problem, it is unlikely that a permanent solution will be
achieved. Therefore, this handbook includes several physiological factors. To further facilitate the
accessibility of the desired information on plant/crop physiological processes covered in this col-
lection, the volume has been divided into nine parts: Part I—Physiology of Plant/Crop Growth and
Development Stages; Part II—Cellular and Molecular Aspects of Plant/Crop Physiology; Part III—
Plant/Crop Physiology and Physiological Aspects of Plant/Crop Production Processes; Part IV—
Physiological Responses of Plants/Crops under Stressful (Salt, Drought, Heat, Nutrient Deficiency,
and Other Environmental Stresses) Conditions; Part V—Physiological Responses of Plants/Crops
to Heavy Metal Concentration and Agrichemicals; Part VI—Physiology of Plant/Crop Genetics and
Development; Part VII—Bioinformatics and Using Computer Modeling in Plant Physiology;
Part VIII—Plant/Crop Growth Responses to Environmental Factors and Climatic Changes; and
xvii
xviii Preface
Part IX—Future Promises: Plant and Crop Adaptation and Biotechnological Aspects of Plant/Crop
Improvement. Although the parts are interrelated, each serves independently to facilitate the under-
standing of the material presented therein. Each part also enables the reader to acquire confidence
in his or her learning and use of the information offered. Each of these parts consists of one or more
chapters to discuss, independently, as many aspects of plant/crop physiology as possible.
Part I consists of eight chapters and addresses various physiological processes of plant and crop
growth and development.
Part II contains four chapters and addresses the cellular and molecular aspects of plant/crop
physiology, presenting the most recent information on each of these subjects.
Part III contains six chapters and presents detailed information on the physiology of production
processes in plants/crops and discusses the physiology under different growth conditions.
Since plants and crops, like other living things, at one time or another during their life cycle,
encounter biotic or abiotic stressful conditions, Parts IV and V are devoted to the physiological
responses of plants and crops to stress. Several examples of empirical investigations of specific
plants and crops grown under stressful conditions are presented in these parts. Part IV includes
14 chapters. Each of these chapters presents in-depth information on the topics covered.
Part V consists of two chapters, which discuss the interactions between heavy metals and agrichemi-
cals and plant/crop physiological processes and the potential problems caused by the accumulation of
heavy metals in soils and plant growth media and the application of agrichemicals to plants/crops.
Part VI consists of only one chapter, which presents recent findings on small RNAs in crop
response to temperature stress noncoding RNAs in plants.
Part VII also consists of only one chapter, which presents information on large-scale computa-
tions and the use of bioinformatics in plant/crop physiology.
Due to recent climatic changes and increase in CO2 levels that have had a major impact on plant/
crop physiological processes, the resistance of plants to these changes must be considered for cultiva-
tion under these conditions. Part VIII, consisting of a single chapter, presents the most recent infor-
mation on this subject. It deals with rising CO2 levels and climate change (global warming) and their
impacts on plant/crop growth behavior, development, and production in the twenty-first century.
Part IX presents evidence and guidance on plants and crops that can be successfully cultivated
under more stressful conditions that are likely in the future. It consists of six chapters, which address
alleviation of future food security problems.
Numerous tables, figures, and illustrations are included in this technical guide to facilitate com-
prehension of the presented materials. Thousands of words are also included in the index to further
increase accessibility to the desired information.
It is hoped that an individual seeking a solution in the area of plant/crop physiology will turn to this
practical and professional reference book and be able to promptly acquire the necessary assistance.
Like other fields, the area of plant/crop physiology has been growing so rapidly that all plant/crop
physiologists are faced with the problem of constantly updating their knowledge. To grow in their
profession, they need to extend their interests and skills. In this regard, even a casual reading of the
material in this handbook will help them move ahead in the right direction.
Mohammad Pessarakli, PhD

Research Professor and Teaching Faculty
University of Arizona
Tucson, Arizona
Acknowledgments
I would like to express my appreciation for the secretarial and the administrative assistance that
I received from the staff of the School of Plant Sciences, College of Agriculture and Life Sciences,
the University of Arizona. I greatly appreciate the encouragement and support that I receive from
the director of the school, Dr. Karen S. Schumaker and my mentor Dr. Dennis T. Ray for my edito-
rial work, which have been a major driving force for the successful completion of this project.
I would like to acknowledge Randy Brehm (senior editor, Taylor & Francis Group, CRC Press)
whose professionalism, patience, hard work, and proactive methods helped in the completion of this
project as well as my previous book projects. This job would not have been completed as smoothly
and rapidly without her valuable support and efforts.
I am indebted to Jill Jurgensen (senior project coordinator, Taylor & Francis Group, CRC Press)
for her professional and careful handling of this volume as well as my previous publications. I would
also like to acknowledge the eye for detail, sincere efforts, and the hard work put in by the copy
editor and the project editor.
The collective efforts and invaluable contributions of several experts in the field of plant/crop
physiology made it possible to produce this unique source, which presents comprehensive informa-
tion on the subject. Each and every one of these contributors and their contributions are greatly
appreciated.
Last, but not least, I thank my wife, Vinca, a high school science teacher, and my son, Dr. Mahdi
Pessarakli, MD, who supported me during the course of this work.
xix
Editor
Dr. Mohammad Pessarakli, is a professor in the School of Plant Sciences, College of Agriculture
and Life Sciences, at the University of Arizona, Tucson, Arizona. His work at the university
includes research and extension services as well as teaching courses in turfgrass science, manage-
ment, and stress physiology. He is the editor of the Handbook of Plant and Crop Stress and the
Handbook of Plant and Crop Physiology (both titles published by Marcel Dekker, Inc., which has
been acquired by Taylor & Francis Group, CRC Press) as well as the Handbook of Photosynthesis
and the Handbook of Turfgrass Management and Physiology. He has written 18 book chapters, is
an editorial board member of the Journal of Plant Nutrition and Communications in Soil Science
and Plant Analysis and the Journal of Agricultural Technology, is a member of the Book Review
Committee of the Crop Science Society of America, and is a reviewer of Crop Science, Agronomy
Journal, Soil Science Society of America Journal, and HortScience. He is the author or coauthor
of 135 journal articles and 55 trade magazine articles. Dr. Pessarakli is an active member of
the Agronomy Society of America, Crop Science Society of America, and Soil Science Society
of America, among others. He is an executive board member of the American Association of the
University Professors (AAUP), Arizona Chapter. He is also a well-known, internationally recog-
nized scientist and scholar and an esteemed member (invited) of Sterling Who’s Who, Marqueis
Who’s Who, Strathmore’s Who’s Who, Madison Who’s Who, and Continental Who’s Who as well
as numerous honor societies (i.e., Phi Kappa Phi, Gamma Sigma Delta, Pi Lambda Theta, Alpha
Alpha Chapter). He is a certified professional agronomist and certified professional soil scientist
(CPAg/SS), designated by the American Registry of the Certified Professionals in Agronomy, Crop
Science, and Soil Science. Dr. Pessarakli is a United Nations Consultant in Agriculture for under-
developed countries. He received his BS (1977) in environmental resources in agriculture and his
MS (1978) in soil management and crop production from Arizona State University, Tempe, and his
PhD (1981) in soil and water science from the University of Arizona, Tucson. Dr. Pessarakli’s envi-
ronmental stress research work and expertise on plants and crops are internationally recognized.
For more information about Dr. Pessarakli, please visit http://ag.arizona.edu/pls/faculty/pessarakli.htm
and http://cals.arizona.edu/spls/people/faculty
xxi
Contributors
Refat Abdel-Basset Martine Bertrand
Faculty of Science Microorganisms, Metals and Toxicity
Department of Botany and Microbiology Cnam SITT
Assiut University Cherbourg, France
Assiut, Egypt
Matthew D. Bomhoff
Mukhtar Ahmed School of Plant Sciences
Department of Agronomy BIO5 Institute
Pir Mehr Ali Shah-Arid Agriculture University The University of Arizona
Rawalpindi, Pakistan Tucson, Arizona
Elena Apostolova
Luis Cisneros-Zevallos
Department of Plant Physiology
Department of Horticultural Sciences
and Molecular Biology
Texas A&M University
University of Plovdiv
College Station, Texas
Plovdiv, Bulgaria
Emilia L. Apostolova Thomas W. Crawford, Jr.

Institute of Biophysics and Biomedical Bio Huma Netics, Inc.
Engineering Gilbert, Arizona
Bulgarian Academy of Science
Sofia, Bulgaria Keshav Dahal
Department of Biological Sciences
Muhammad Asif University of Toronto
Department of Agricultural, Food and at Scarborough
Nutritional Science Toronto, Ontario, Canada
University of Alberta
Edmonton, Alberta, Canada
Jack Dekker
Weed Biology Laboratory
Zohra Aslam
Department of Agronomy
Iowa State University
Pir Mehr Ali Shah-Arid Agriculture University
Ames, Iowa
Rawalpindi, Pakistan
Vesselin Baev Gerald N. De La Fuente

Department of Plant Physiology Department of Soil and Crop Science
and Molecular Biology Texas A&M University
University of Plovdiv College Station, Texas
Plovdiv, Bulgaria
Iliya Denev
Ivan Barrero Department of Plant Physiology and Plant
Department of Soil and Crop Science Molecular Biology
Texas A&M University University of Plovdiv
College Station, Texas Plovdiv, Bulgaria
xxiii
xxiv Contributors
Rama Shanker Dubey Elena V. Garmash

Department of Biochemistry Institute of Biology
Faculty of Science Komi Scientific Centre Ural Branch
Banaras Hindu University Russian Academy of Sciences
Varanasi, India Syktyvkar, Russia
Moustafa Eldakak Katya Georgieva

Department of Biology and Microbiology Institute of Plant Physiology and Genetics
South Dakota State University Bulgarian Academy of Sciences
Brookings, South Dakota Sofia, Bulgaria
and Maria Gevezova

Faculty of Agriculture Department of Plant Physiology and Plant
Department of Genetics Molecular Biology
Alexandria University University of Plovdiv
Alexandria, Egypt Plovdiv, Bulgaria
Farhad Ghavami
Manal El-Zohri
Department of Plant Pathology
Faculty of Science
University of Minnesota
Department of Botany and Microbiology
Saint Paul, Minnesota
Assiut University
Assiut, Egypt
Tamara K. Golovko
Institute of Biology
Miloš Faltus Komi Scientific Centre Ural Branch
Plant Physiology and Cryobiology Laboratory Russian Academy of Sciences
Department of Molecular Biology Syktyvkar, Russia
Crop Research Institute
Prague, the Czech Republic Aakash Goyal
Bayer Crop Science
Muhammad Farooq Saskatoon, Saskatchewan, Canada
University of Agriculture Bernard Grodzinski
Faisalabad, Pakistan Department of Plant Agriculture
and University of Guelph
Guelph, Ontario, Canada
The UWA Institute of Agriculture
The University of Western Australia Bhaskar Gupta
Crawley, Western Australia, Australia Department of Biological Sciences
(Section Biotechnology)
Carlos M. Figueroa Presidency University
Instituto de Agrobiotecnología del Litoral Kolkata, India
Consejo Nacional de Investigaciones
Científicas y Técnicas Kamala Gupta
and Plant Molecular Biology Laboratory
Facultad de Bioquímica y Ciencias Department of Biological Sciences
Biológicas (Section Botany)
Universidad Nacional de Litoral Presidency University
Santa Fe, Argentina Kolkata, India
Contributors xxv
Yoshie Hanzawa Alberto A. Iglesias

Department of Crop Sciences Instituto de Agrobiotecnología del Litoral
University of Illinois at Consejo Nacional de Investigaciones
Urbana-Champaign Científicas y Técnicas
Urbana, Illinois and
Facultad de Bioquímica y Ciencias
Biológicas
J.S. Heslop-Harrison
Universidad Nacional de Litoral
Department of Biology
Santa Fe, Argentina
University of Leicester
Leicester, United Kingdom Tom Isakeit
Department of Plant Pathology and
Parisa Heydarizadeh Microbiology
MicroMar Texas A&M University
Mer Molécules Santé College Station, Texas
University of Le Mans
M. Anowarul Islam
Le Mans, France
Department of Plant Sciences
and University of Wyoming
Laramie, Wyoming
Department of Agronomy and Plant
Breeding Daniel A. Jacobo-Velázquez
College of Agriculture Department of Biotechnology and Food
Isfahan University of Technology Engineering
Isfahan, Iran School of Biotechnology and Food
FEMSA-Biotechnology Center
Awatief F. Hifney Monterrey Institute of Technology and Higher
Faculty of Science Education
Department of Botany and Microbiology Monterrey, Mexico
Assiut University
David Jespersen
Assiut, Egypt
Department of Plant Biology and Pathology
Rutgers University
Bingru Huang New Brunswick, New Jersey
Department of Plant Biology and Pathology
Rutgers University Ambuj Bhushan Jha
New Brunswick, New Jersey Department of Plant Sciences
Crop Development Centre
College of Agriculture and Bioresources
B. Huchzermeyer University of Saskatchewan
Institute of Botany Saskatoon, Saskatchewan, Canada
Leibniz Universitaet Hannover
Hannover, Germany Khalil Kane
Department of Biological Sciences
Norman P.A. Hüner Université du Québec à Montréal
Department of Biology Montreal, Quebec, Canada
and
The Biotron Experimental Climate Change Penny M.A. Kianian
Research Centre Department of Plant Sciences
The University of Western Ontario North Dakota State University
London, Ontario, Canada Fargo, North Dakota
xxvi Contributors
Shahryar F. Kianian Eric Lyons

Cereal Disease Laboratory School of Plant Sciences
Agricultural Research Service BIO5 Institute
United States Department of Agriculture iPlant Collaborative
University of Minnesota The University of Arizona
Saint Paul, Minnesota Tucson, Arizona
Glen M. MacDonald
Ina Kirilova
Department of Geography
Department of Plant Physiology and Plant
Institute of the Environment and Sustainability
Molecular Biology
The University of California, Los Angeles
University of Plovdiv
Los Angeles, California
Plovdiv, Bulgaria
Justine Marchand
Hisashi Koiwa MicroMar
Department of Horticultural Sciences Mer Molécules Santé
Texas A&M University University of Le Mans
College Station, Texas Le Mans, France
Michael V. Kolomiets Alexander M. Markarov (deceased)

Department of Plant Pathology and Komi State Pedagogical Institute
Microbiology Syktyvkar, Russia
Texas A&M University
College Station, Texas Peter M. Martin
Amenity Horticulture Research Unit
Hans-Werner Koyro Plant Breeding Institute
Institute of Plant Ecology University of Sydney
Justus-Liebig-University Giessen Sydney, New South Wales, Australia
Giessen, Germany
Elena Masarovičová
Katarína Král’ová Faculty of Natural Sciences
Faculty of Natural Sciences Department of Soil Science
Institute of Chemistry Comenius University
Comenius University Bratislava, Slovak Republic
Bratislava, Slovak Republic
Svetlana P. Maslova
Maya Kurteva Institute of Biology
Institute of Biodiversity and Ecosystem Russian Academy of Sciences
Research Syktyvkar, Russia
Bulgarian Academy of Sciences
Sofia, Bulgaria Sanaa I.M. Milad
Faculty of Agriculture
Andrew J. Lenards Biotechnology Lab
BIO5 Institute Department of Crop Science
iPlant Collaborative Alexandria University
The University of Arizona Alexandria, Egypt
Tucson, Arizona
Ivan Minkov
Evangelos Demosthenes Leonardos Department of Plant Physiology and
Department of Plant Agriculture Molecular Biology
University of Guelph University of Plovdiv
Guelph, Ontario, Canada Plovdiv, Bulgaria
Contributors xxvii
A.N. Misra Valeria E. Perotti

Centre for Life Sciences Facultad de Ciencias Bioquímicas y
Central University of Jharkhand Farmacéuticas
Ranchi, India Universidad Nacional de Rosario
and
Seth C. Murray Consejo Nacional de Investigaciones
Department of Soil and Crop Science Científicas y Técnicas
Texas A&M University Centro de Estudios Fotosintéticos y
College Station, Texas Bioquímicos
Ali I. Nawar Rosario, Argentina
Faculty of Agriculture
Biotechnology Lab Mohammad Pessarakli
Department of Crop Science School of Plant Sciences
Alexandria University College of Agriculture and Life
Alexandria, Egypt Sciences
The University of Arizona
Ali Nikbakht Tucson, Arizona
Department of Horticulture
College of Agriculture
Claudia V. Piattoni
Isfahan University of Technology
Instituto de Agrobiotecnología del
Isfahan, Iran
Litoral
Ijaz Rasool Noorka Consejo Nacional de Investigaciones
Department of Plant Breeding and Genetics Científicas y Técnicas
University College of Agriculture and
University of Sargodha Facultad de Bioquímica y Ciencias
Sargodha, Pakistan Biológicas
Universidad Nacional de Litoral
and Santa Fe, Argentina
Department of Biology
University of Leicester Florencio E. Podestá
Leicester, United Kingdom Facultad de Ciencias Bioquímicas y
Farmacéuticas
Augustine K. Obour
Universidad Nacional de Rosario
Department of Plant Sciences
and
University of Wyoming
Laramie, Wyoming
Científicas y Técnicas
Shannon L. Oliver Centro de Estudios Fotosintéticos y
BIO5 Institute Bioquímicos
iPlant Collaborative Rosario, Argentina
The University of Arizona
Tucson, Arizona Dagmar Procházková
Institute of Experimental Botany
Galina Panayotova
Academy of Sciences of the Czech Republic
Institute of Agriculture and Seed Science
Prague, Czech Republic
“Obraztsov chiflik”
Rousse, Bulgaria
Taha Ramadan
Jean-Claude Pech Faculty of Science
Genomique et Biotechnologie des Fruits Department of Botany and Microbiology
University of Toulouse Assiut University
Castanet-Tolosan, France Assiut, Egypt
xxviii Contributors
Idupulapati Madhusudana Rao Kadambot H.M. Siddique

Bean Program and Tropical Forages Program The UWA Institute of Agriculture
Agrobiodiversity Research International The University of Western Australia
Center for Tropical Agriculture Crawley, Western Australia, Australia
Cali, Colombia
Atif Riaz Jas Singh

Institute of Horticultural Sciences Eastern Cereal and Oilseed Research Centre
University of Agriculture Agriculture and Agri-Food Canada
Faisalabad, Pakistan Ottawa, Ontario, Canada
Jai S. Rohila
Department of Biology and Microbiology Ali Soltani
South Dakota State University Department of Plant Sciences
Brookings, South Dakota North Dakota State University
Fargo, North Dakota
Paolo A. Sabelli
School of Plant Sciences
The University of Arizona Rajneesh Kumar Srivastava
Tucson, Arizona Department of Biochemistry
Faculty of Science
Mohammad Reza Sabzalian
Banaras Hindu University
Department of Agronomy and Plant
Varanasi, India
Breeding
College of Agriculture
Isfahan University of Technology Detelin Stefanov
Isfahan, Iran Department Biophysics and Radiobiology
Zohrab Samani “St. Kliment Ohridski” Sofia University
Civil Engineering Department Sofia, Bulgaria
New Mexico State University
Las Cruces, New Mexico Galina N. Tabalenkova
Fathey Sarhan Institute of Biology
Department of Biological Sciences Komi Scientific Centre Ural Branch
Université du Québec à Montréal Russian Academy of Sciences
Montreal, Quebec, Canada Syktyvkar, Russia
Leonid V. Savitch
Eastern Cereal and Oilseed Research Centre Karina E.J. Trípodi
Agriculture and Agri-Food Canada Instituto de Biología Molecular y Celular de
Ottawa, Ontario, Canada Rosario
Benoît Schoefs Científicas y Técnicas
MicroMar and
Mer Molécules Santé Facultad de Ciencias Bioquímicas y
University of Le Mans Farmacéuticas
Le Mans, France Universidad Nacional de Rosario
Pallavi Sharma
Department of Plant Sciences Abdul Wahid
College of Agriculture and Bioresources Department of Botany
University of Saskatchewan University of Agriculture
Saskatoon, Saskatchewan, Canada Faisalabad, Pakistan
Contributors xxix
Nad’a Wilhelmová Jiří Zámečník

Institute of Experimental Botany Plant Physiology and Cryobiology Laboratory
Academy of Sciences of the Czech Republic Department of Molecular Biology
Prague, The Czech Republic Crop Research Institute
Prague, The Czech Republic
Faqiang Wu
Smita Sachin Zinjarde
Department of Crop Sciences
Institute of Bioinformatics and Biotechnology
University of Illinois at Urbana-Champaign
University of Pune
Urbana, Illinois
Pune, India
Galina Yahubyan Christian Zörb

Department of Plant Physiology and Molecular Institute of Biology
Biology Department of Botany
University of Plovdiv University Leipzig
Plovdiv, Bulgaria Leipzig, Germany
Part I
Physiology of Plant/Crop Growth
and Development Stages
1 Cell Cycle Regulation and
Plant Development
A Crop Production Perspective
Paolo A. Sabelli
CONTENTS
1.1 Introduction—Crop Yield: A Cell Cycle Perspective...............................................................3
1.2 Core Molecular Control of the Cell Cycle: An Overview.........................................................4
1.2.1 Mitotic Cell Cycle.......................................................................................................... 5
1.2.1.1 Regulation of the G1/S-Phase Transition........................................................5
1.2.1.2 Regulation of M-Phase...................................................................................7
1.2.1.3 Cytokinesis.....................................................................................................7
1.2.2 Alternative Cell Cycles: Asymmetric Cell Division and Endoreduplication................ 8
1.2.2.1 Asymmetric Cell Division..............................................................................8
1.2.2.2 Endoreduplication Cell Cycle......................................................................... 8
1.3 Endoreduplication as a Potential Yield Determinant.............................................................. 11
1.3.1 Endoreduplication and Tomato Fruit Development..................................................... 11
1.3.2 Endoreduplication and Cereal Endosperm Development............................................ 12
1.3.3 Endoreduplication and Biotrophic Interactions........................................................... 14
1.3.3.1 Endoreduplication and Symbiotic Interactions............................................. 17
1.3.3.2 Endoreduplication and Parasitic/Pathogenic Interactions............................ 17
1.4 Regulation of Plant Biomass and Architecture....................................................................... 18
1.4.1 Reproductive Development.......................................................................................... 18
1.4.1.1 Gametogenesis.............................................................................................. 18
1.4.1.2 Seed Development........................................................................................ 19
1.4.2 Shoot Branching..........................................................................................................20
1.4.3 Leaf Development........................................................................................................ 21
1.4.4 Root Development and Architecture........................................................................... 22
1.5 Plant Tissue Culture................................................................................................................. 22
1.6 Contribution of Cell Cycle Manipulation to Crop Evolution and Breeding............................ 23
1.7 Does the Cell Cycle Determine Yield? Concluding Remarks.................................................25
References.........................................................................................................................................25
1.1 INTRODUCTION—CROP YIELD: A CELL CYCLE PERSPECTIVE

Cell number and expansion are two key parameters controlling the size of tissues, organs, and the
whole plant. The cell division cycle is directly responsible for cell production (i.e., cell number),
but it also influences plant shape, architecture, and morphogenesis through spatial regulation of
cell wall deposition at cytokinesis, and at least in some notable cases of agricultural relevance also
through cell expansion. Thus, it is intuitive that detailed understanding of the cell cycle, coupled
with the ability to manipulate it, has the potential to significantly contribute to maximizing crop
3
4 Handbook of Plant and Crop Physiology
yield and sustaining agricultural output in the face of future depletion of resources and increased
demand. Over the last 20 years or so, remarkable progress has been made in understanding how
the plant cell cycle is regulated in biochemical, genetic, and physiological terms, particularly in the
model species Arabidopsis thaliana. Key molecular players have been identified that govern the
workings of the cell cycle that are highly conserved among plants and that, in some cases, have
critically helped advance the understanding of the cell cycle in animals as well. However, we are
currently faced with an apparent paradox where despite the advances in model plant systems, it is
difficult to translate this knowledge into suitable applications for the improvement of crops, agricul-
ture, and civilization as a whole. This is partly due, on the one hand, to the higher level of biological
complexity of crop species compared to simpler model systems and, on the other hand, to the pau-
city of cell cycle research efforts in agriculturally important plants. As a result, our knowledge of
cell cycle regulation in crops is rudimentary and rather stagnant, thereby undermining or delaying
attempts aimed at transferring basic research findings into agriculture practice.
In this chapter, the regulation of the plant cell cycle is reviewed with an emphasis on key aspects
and factors that may impact crop production and yield. Because of space limitations, however,
several relevant topics are not discussed in detail here, including the regulation of the cell cycle
by phytohormones (Del Pozo et al. 2005; John 2007; Dudits et al. 2011) and the role of the plant
cell cycle in integrating abiotic signals with developmental programs (Granier et al. 2007; Skirycz
et al. 2011; Komaki and Sugimoto 2012), which could contribute to mitigating yield losses due to
environmental stresses.
For simplicity, the concept of crop yield is narrowed down to the level of the individual tissue,
organ, or plant, and is not viewed in terms of a community of plants in the field. Although the lat-
ter is clearly more representative of agricultural practice, many interacting factors (e.g., the number
of seeds per unit of land area, the plant’s ability to intercept and harvest solar radiation, water/
nutrients availability, temperature, source/sink relationships, and the ability of the plant to adapt to
growing seasons of variable duration, to mention a few) contribute to determining the yield in the
field (Evans 1993), which makes the evaluation of the potential of cell cycle regulation for impacting
yield in agricultural settings very complicated. Notwithstanding these caveats, however, it is pos-
sible to provide an initial assessment of the role played by cell cycle regulation in crop production.
Because the cell cycle is paramount for cell production, it is perhaps most obvious to consider its
role in terms of biomass production, though, as described in the following text, the cell cycle can
impact plants in various ways that may not be directly related to biomass, including the regulation
of their architecture or their interaction with symbiotic or parasitic organisms. For example, an
acceleration of growth may not modify final plant size or morphology, yet it may benefit crops in
environment characterized by short growing seasons (Busov et al. 2008). In addition, several sys-
tems relevant to crop production are impacted by a cell cycle variant, known as endoreduplication,
which not only does not involve cell proliferation, but also is typically mutually exclusive to it. The
main goal of this chapter is to provide an overview of how cell cycle regulation can affect plant
development under the perspective of crop production.
1.2 CORE MOLECULAR CONTROL OF THE CELL CYCLE: AN OVERVIEW

Cell cycle regulation in plants has been the subject of several excellent reviews (De Jager et al. 2005;
Gutierrez 2005; Inze and De Veylder 2006; Francis 2007; Inagaki and Umeda 2011), and therefore
only an overview of some key aspects is provided here. In plants, like in other eukaryotes, cell cycle
progression is controlled by the timely activation of complexes between a cyclin-dependent kinase
(CDK) and a regulatory subunit termed cyclin. In eukaryotes, the expression of the kinase compo-
nent is not generally cell cycle regulated whereas that of the cyclin subunit is, and it is from these
noticeable fluctuations in protein accumulation during the cell cycle that cyclin polypeptides and
genes derive their name. The activity of CDK/cyclin complexes is regulated at several different lev-
els. First, as aforementioned, the availability of cyclin subunit is paramount (Nieuwland et al. 2007).
Cell Cycle Regulation and Plant Development 5
This, in turn, is controlled at the level of gene transcription, but critically also by specific degrada-
tion of cyclin proteins by the 26S proteasome, which can be rather abrupt and is thus well suited for
driving cells through unidirectional cell cycle transitions. Second, the catalytic activity of the CDK
kinase moiety is subject to some exquisite protein conformation regulation, which depends in large
part on the presence/absence of phosphate groups on certain amino acid residues. Thus, regulation
of CDK phosphorylation/dephosphorylation is a crucial aspect of cell cycle control (Morgan 1997;
Inze and De Veylder 2006; Inagaki and Umeda 2011). Third, the activity of CDK/cyclin complexes
can be inhibited by the binding of specific inhibitors generally known as CKIs (Wang et al. 2008).
Each of these three primary mechanisms regulating CDK activity is controlled at different levels by
other factors and pathways and sometimes by CDK/CYC complexes themselves through feedback
regulation, which not surprisingly results in an intricate web of protein-to-protein interactions (Van
Leene et al. 2010).
1.2.1 Mitotic Cell Cycle

The standard mitotic cell cycle consists of the four canonical phases Gap1 (G1), DNA synthesis
(S-phase), Gap2 (G2), and mitosis (M-phase) (Figure 1.1). Upon the orderly completion of these
phases, the chromosomes are replicated as sister chromatids, which are segregated into two geneti-
cally identical daughter nuclei, within the mother cell. Subsequently, deposition of new cell wall
partitions in an intervening region between the nuclei during cell division (cytokinesis) generates
two daughter cells. The gap phases derive their names from early microscopic observations that
could only reveal gross structural changes, which suggested that they were “resting” periods of
relative cellular inactivity. We now know that a great deal of regulation occurs in G1 and G2 at the
molecular level in order for subsequent S-phase or M-phase to take place.
Several classes of CDKs have been identified in plants (at least eight in Arabidopsis) based on
amino acid sequence similarities, but it appears only four, CDKA, CDKB, CDKD, and CDKF
are involved in direct cell cycle regulation (Inze and De Veylder 2006; Doonan and Kitsios 2009;
Inagaki and Umeda 2011). CDKA is coded for by a single gene in Arabidopsis but by at least three
genes in maize (Colasanti et al. 1991; Dante et al. 2013) and is believed to control the onset of
the G1/S-phase transition, DNA synthesis, as well as M-phase entry and execution. B-type CDKs
are unique to plants, are characterized by cell cycle–regulated pattern of accumulation from late
S-phase to M-phase, and are thought to specifically regulate the G2/M-phase transition. CDKD
and CDKF function upstream and regulate CDKA and CDKB through phosphorylation of specific
residues and therefore are also known as CDK-activating kinases (CAKs) (Figure 1.1).
Cyclins are encoded by a larger family of genes in plants than in animals—about 50 in Arabidopsis
and rice grouped into several different classes (Wang et al. 2004a; La et al. 2006; Nieuwland et al.
2007)—probably to facilitate the fine-tuning of cell proliferation in sessile organisms in response
to changing environmental conditions (Menges et al. 2005). While for some cyclins the function
is unknown, most cyclin types have been assigned at least a putative cell cycle role. A great deal is
known about individual cyclins, and they display a dazzling array of cell cycle–regulated expres-
sion and protein-to-protein interaction patterns (Menges et al. 2005; Nieuwland et al. 2007; Van
Leene et al. 2010). Although the following is a gross oversimplification and there are notable excep-
tions, CYCDs are generally involved in relying external proliferation stimuli along a pathway that
leads to the onset of DNA synthesis, CYCAs are believed to regulate progression through S- and
M-phases, and CYCBs are primarily required for the G2/M-phase transition (Inze and De Veylder
2006; Nieuwland et al. 2007; Inagaki and Umeda 2011) (Figure 1.1).
1.2.1.1 Regulation of the G1/S-Phase Transition

During G1, exogenous and endogenous cell proliferation stimuli, which may include sucrose as well
as phytohormones such as auxin, cytokinin, and brassinosteroids (Del Pozo et al. 2005; John 2007;
Dudits et al. 2011), are sensed and relayed to the core cell cycle–regulatory machinery resulting
CAK P
P RBR
CDKA
CYCD
KRP
E2F DP S-phase gene E2F

RBR expression
E2F DP X
APC/C
CDKB
P CYCA
P
CDK activity
threshold S-phase KRP
for M-phase
CAK
CDK activity WEE1
G1
threshold
for S-phase
KRP
G2 P P
CDKA/B
Cytokinesis
CYCA/B/D
P
M-phase MYB M-phase gene
expression
PPB
M-phase
cyclins Level of CDK activity
APC/C
FIGURE 1.1 Schematic diagram illustrating the canonical phases during the plant cell division cycle (G1,
S-phase, G2, M-phase, and cytokinesis) and, in a highly simplified fashion, the key molecular mechanisms that
regulate major transitions. Fluctuations in CDK activity are paramount for cell cycle progression (solid line).
CDK activity must exceed an S-phase threshold (inner broken line) for cells to transition from G1- into S-phase
and replicate their DNA, while another increase during G2 above an M-phase threshold (outer broken line) drives
cells into mitosis. Exit from M-phase and acquiring the competence of chromosomal replication origins for DNA
synthesis (origin licensing) require a drop in CDK activity at the end of mitosis and for most of G1. At the G1/S-
phase transition, a wide-ranging gene expression program dependent on heterodimeric E2F/DP transcription
factors is derepressed as CDKA/CYCD complexes inactivate (through phosphorylation) RBR inhibitors. Further
upstream, the activity of CDKA/CYCD is positively and negatively regulated by CAK and KRP activities,
respectively. At the G2/M-phase transition, a spike in CDK activity results in the phosphorylation and activation
of MYB3R transcription factors that drive M-phase-specific gene expression. This CDK activity involves specific
mitotic cyclins and also is stimulated by CAK-dependent phosphorylation and is generally inhibited by WEE1-
dependent phosphorylation and binding by KRP inhibitors. B-type CDKs appear to be required specifically for
the execution of M-phase, in part by stimulating CDK activity through phosphorylation of KRP and targeting it
for disruption by the APC/C-dependent proteasome. Degradation of mitotic cyclins by the proteasome is also key
for the abrupt lowering of CDK activity that is required for M-phase exit. The cell cycle window during which the
plant-specific preprophase band (PPB) is transiently formed is indicated together with postmitotic cytokinesis.
in the formation of active CDKA/CYCD complexes above a threshold required for S-phase entry
(Figure 1.1). These complexes can be inhibited by specific CKIs of the ICK/KRP family (hereaf-
ter termed KRP), which, in turn, can be induced or downregulated in response to hormones such
as ABA or auxin, respectively. Active CDK/CYC complexes go on to phosphorylate many sub-
strates, which include homologs of the retinoblastoma tumor suppressor family of proteins (termed
RBR, for retinoblastoma related), and proteins involved in licensing origins of DNA replication
(Figure 1.1). A large number of genes need to be timely expressed at the G1/S-phase boundary and
throughout S-phase in order to initiate and support chromatin replication. The expression of many
of these genes requires the activity of adenovirus E2 promoter binding factor (E2F) transcription
factors primarily acting in complexes with their protein dimerization partner (DP). However, regu-
lation of E2F-dependent gene expression is complex, and the E2F protein family includes members
that have primarily a repressive function and do not dimerize with DP. In G1, RBR inhibits E2F/
DP-dependent gene expression by several mechanisms, which involve direct binding to the transac-
tivation domain of E2F and the recruitment of chromatin remodeling factors at E2F-bound promot-
ers with ensuing silencing of specific chromatin domains (Sabelli and Larkins 2009a). This block on
the expression of genes required for S-phase and cell cycle progression is relieved at the G1/S-phase
transition by conformational changes of RBR brought about by phosphorylation by CDK.
1.2.1.2 Regulation of M-Phase
As cells prepare to enter M-phase late in G2, several CDK/CYC complexes are formed and
become activated above another critical threshold (Figure 1.1). These complexes include both
CDKA and CDKB proteins, as well as A-, B-, and, likely, D-type cyclins (Inze and De Veylder
2006). A large number of substrates become phosphorylated as a result, and cells progress into
M-phase. Several molecular mechanisms contribute to the timely activation of these complexes:
gene transcription (critically regulating the availability of CDKBs and cyclins); inhibitory phos-
phorylation of the CDK moiety by WEE1 kinase (however, although functionally conserved in
eukaryotes, there is no evidence for this mechanism in Arabidopsis); activating dephosphoryla-
tion that counters WEE1 (the phosphatase responsible for this reaction has not been unambigu-
ously identified in plants, but a CDC25-like activity seems a likely candidate); and, similarly for
the G1/S-phase transition, phosphorylation by CAKs. The execution of the M-phase transcrip-
tional program seems to depend to a large extent on three-repeat MYB (MYB3R) transcription
factors, which are converted from a primarily repressor to an activator type by CDK-dependent
phosphorylation (Araki et al. 2004). Importantly, the 26S proteasome-dependent degradation of
mitotic cyclins, such as CYCB, through polyubiquitination by the E3 ubiquitin ligase complex
known as anaphase-promoting complex or cyclosome (APC/C) is key to lowering CDK activity
at the end of mitosis below a critical threshold, which is required for the assembly of prereplica-
tion protein complexes at DNA replication origins (Cebolla et al. 1999; Bryant and Aves 2011;
Sanchez et al. 2012). This is a key step for resetting cell cycle regulation upon mitosis completion
and for ensuring the unidirectional progress of the cell cycle (Figure 1.1).
1.2.1.3 Cytokinesis
Cell division in plants initiates in late-G2 before the onset of mitosis with the formation of the
so-called preprophase band (PPB), which is a transient structure consisting of microtubules and
actin filaments (Figure 1.1). PPB recruits many proteins cortically in a ring-like zone and it sort of
imprints the site of deposition of the future cell wall at cytokinesis (i.e., the plane of cell division),
before being disassembled in late prophase/early metaphase. A second plant-specific cytoskel-
etal structure, the phragmoplast, forms after mitosis is completed perpendicular to the future cell
division plane from the microtubule and actin microfilament remnants of the mitotic spindle and
expands bidimensionally toward the so-called cortical division site (CDS) at the cell periphery
through depolymerization/polymerization of microtubules and microfilaments under the appar-
ent guidance of cortical proteins previously recruited by the PPB, which act as a beacon for the
growing phragmoplast. There are a plethora of genes involved in different aspects of cytokinesis,
and it is noteworthy that CDKA localizes at cell division sites (Colasanti et al. 1993), and its
activity may be important for microtubule depolymerization (Rasmussen et al. 2011). The phrag-
moplast functions as a cytoskeletal scaffold for the deposition of golgi-derived vesicles and their
fusion at its midline, which leads to the formation of the cell plate that eventually fuses with the
cell wall at the CDS, effectively partitioning the mother cell into two daughter cells. The term
“cytokinesis” encompasses a vastly complex set of processes that include regulation of cytoskeletal
structures proper, cytoskeleton-associated structural and motor proteins, vesicular trafficking,

and membrane dynamics. These processes and the many genes involved have been reviewed in
detail elsewhere (Otegui and Staehelin 2000; Hong and Verma 2008; Rasmussen et al. 2011, 2013;
McMichael and Bednarek 2013).
1.2.2 Alternative Cell Cycles: Asymmetric Cell Division and Endoreduplication

1.2.2.1 Asymmetric Cell Division
Generally somatic cells (typically, meristematic cells) divide symmetrically to generate similar
daughter cells. However, stem cell niches, cell differentiation, and tissue patterning are often associ-
ated with the formation of unequal cells through asymmetric cell division (De Smet and Beeckman
2011; Rasmussen et al. 2011). As daughter cells remain anchored to each other by sharing a cel-
lulosic cell wall and virtually never move relative to one another, regulation of asymmetric cell
division plays a very important role in cell differentiation and plant morphogenesis. According to
classical views, differential cell fates of progeny cells are specified as a result of intrinsic factors
(such as in the case of asymmetric placement of the cell plate along a polarity gradient to partition
cell fate determinants unevenly) or the action of positional cues (such as in the case of apparently
identical daughter cells that go on to take distinct fates under the influence of specific signals from
different neighboring cells). Examples of asymmetric cell divisions include (1) the first division of
the zygote, (2) procambial and hypophyseal divisions in the embryo, (3) root stem cells and initials,
(4) vascular initials in the procambium, (5) stomatal development, and (6) formation of tricho-
blasts during the development of type-2 root hairs (Datta et al. 2011; De Smet and Beeckman 2011;
Rasmussen et al. 2011; Zhang et al. 2012a). While the mechanisms characterizing these instances
of asymmetric cell division have not been elucidated in sufficient detail to reveal any underlying
potentially common themes, core cell cycle genes are known to impact at least some of them. For
example, regulation of asymmetric cell division of the root initials that generate cortex and endoder-
mis in Arabidopsis involves a pathway in which SHORT ROOT (SHR) and SCARECROW (SCR)
transcription factors control the expression, directly or indirectly, of CYCD6;1 as well as B-type
CDKs, which is necessary for proper tissue patterning (De Smet et al. 2008; Sozzani et al. 2010;
Cruz-Ramírez et al. 2012). RBR1, in its hypophosphorylated and active forms, appears to bind and
inhibit SCR, while CYCD6;1-dependent phosphorylation and consequent inactivation of RBR1 pro-
vides a positive feedforward loop potentially generating a bistable circuit. Interestingly, CYCD6;1
expression is stimulated by auxin, which reaches a relatively high concentrations specifically in the
cortex/endodermis initial cell file, thus providing an explanation for the single formative division
that characterizes them. Thus, precise integration of longitudinal auxin gradients and radial distri-
bution of transcription factors, coupled with a narrow, proteasome-dependent temporal window of
activation of the CDK-RBR1 pathway, seem to ensure proper coordination of cell cycle regulation
with cell differentiation and root patterning (Cruz-Ramírez et al. 2012). In both shoot and root cells
of Arabidopsis, the decision to divide symmetrically or asymmetrically depends on the level of
CDKA;1 activity (high CDKA;1 activity promotes asymmetric division while medium levels stimu-
late symmetric divisions) and is mediated by a transcriptional pathways controlled by RBR1, which
appears to be independent from E2F. Thus, RBR1 is emerging as a central player in integrating
distinct pathways and processes and gating them at the cellular level to fine-tune cell cycle activity
and to coordinate it with cell differentiation and tissue/organ growth and development (Sabelli and
Larkins 2009a; Gutzat et al. 2012; Weimer et al. 2012; Sabelli et al. 2013).
1.2.2.2 Endoreduplication Cell Cycle

Endoreduplication (also known as endoreplication or endocycle) is a specialized cell cycle in which
iterated rounds of nuclear DNA replication occur in the absence of chromatin condensation, nuclear
membrane breakdown, mitotic spindle formation, sister chromatids segregation, and cytokinesis,
S-phase
CDKA/CYCD RBR E2F
program
DNA
replication
licensing
G S-phase
CDK
CCS52A APC/C
M-phase
program
CYCA/B
CDK
CDKB
CYCA
CYCB
KRP, SRM, and WEE1
CDK inhibitors
Transcriptional
down-regulation
FIGURE 1.2 Schematic diagram of the regulation of the endoreduplication cell cycle, which consists of
reiterated G- and S-phases in the absence of chromatin condensation, mitosis, and cytokinesis. As a result,
endoreduplicated cells contain multiple genome copies (polyploidy), generally within large nuclei. During
endoreduplication, a variety of mechanisms cause a downregulation of CDK activity, otherwise required for
the execution of M-phase in proliferating cells. These may involve transcriptional downregulation of CDK and
cyclin components, inactivation by specific inhibitors such as KRP, SRM, and WEE1, and enhanced cyclin
degradation through an upregulation of APC/C by CCS52A. Decreased CDK activity may also stimulate
licensing of DNA replication origins. Additionally, upregulation of the CDK–RBR–E2F pathway controlling
the S-phase program may also occur during endoreduplication.
which results in endopolyploidy and usually large cells (Larkins et al. 2001; Sabelli and Larkins
2008; De Veylder et al. 2011) (Figure 1.2). Plant cells are frequently endoreduplicated, and the
occurrence of this particular type of cell cycle is often associated with cell differentiation and tis-
sue growth. Indeed, endoreduplication is the type of cell cycle that has been best characterized
in agriculturally important systems such as the cereal seed endosperm (Sabelli 2012b), the sym-
biotic nitrogen-fixing nodules of legumes (Kondorosi and Kondorosi 2004), and the tomato fruit
(Chevalier et al. 2011) (see later). Among other tissues that undergo endoreduplication, at least in
certain species, are the embryo, suspensor, and cotyledons within the seed, the antipodal and syn-
ergid cells in the female gametophyte, the anther tapetum, the leaf trichomes, the epidermis of the
stem and leaf, the hypocotyl, and the sites of certain biotrophic interactions (Larkins et al. 2001;
Sabelli and Larkins 2008; De Veylder et al. 2011).
1.2.2.2.1 Specific Regulation of Endoreduplication

The mitotic cell cycle is characterized by exquisite coupling of chromosome segregation (M-phase)
to successful execution of DNA synthesis (S-phase). Generally, a G2 DNA replication checkpoint
sensing DNA integrity prevents M-phase entry until S-phase is completed. Equally important,
DNA replication cannot initiate unless M-phase is completed and CDK activity drops below the
threshold that prevents licensing of DNA replication origins. Together, the coordinated regulation
of these two broad mechanisms is crucial for the orderly sequences of events characterizing mitotic
cells, and to a large extent, it appears to depend on the timely activation and inactivation of specific
CDK/CYC complexes. Because endoreduplication consists of repeated rounds of DNA synthesis

and Gap phases without intervening mitosis, it is conceivable that the regulatory programs that are
responsible for mitosis are downregulated or even absent in endoreduplicating cells, while programs
for S-phase are sustained or even upregulated (Figure 1.2). Indeed, the transition to the endocycle is
typically characterized by the downregulation, through several distinct but likely entwined mecha-
nisms, of CDK activity that is normally associated with and required for premitosis and M-phase in
proliferating cells (Kondorosi and Kondorosi 2004; Sabelli and Larkins 2008; Chevalier et al. 2011;
De Veylder et al. 2011). Generally, this involves transcriptional downregulation of B1-type CDKs
and B-type and A2-type cyclins. This might result, in part, from failure to produce phosphorylated,
and active, MYB3R transcription factors that control the expression of B-type cyclins (Araki et al.
2004) (Figures 1.1 and 1.2). Additionally, the levels of certain A-type and B-type cyclins, which are
tightly regulated in mitotic cells by APC/C-mediated proteolysis, can be diminished in endoredu-
plicating cells below a critical threshold normally required for M-phase by increased proteasome
activity. Indeed, the CELL CYCLE SWITCH 52A (CCS52A) protein, which is a CDH1-type activa-
tor of the APC/C involved in the degradation of mitotic B1-, A2-, and A3-type cyclins, is required
for endoreduplication in a variety of plant systems and stimulates the endocycle if overexpressed
(presumably by locking the APC/C in a constitutively active mode) (Cebolla et al. 1999; Vinardell
et al. 2003; Larson-Rabin et al. 2009). Another mechanism for tuning down CDK activity involves
the SIAMESE-RELATED (SMR) family of CKIs first identified in Arabidopsis but also found in rice
(Peres et al. 2007). WEE1 is another well-established inhibitor of CDK activity at the G2/M-phase
transition (Figure 1.2). However, it may also play important roles in the regulation of endoreduplica-
tion because its expression is associated with the endocycle in both tomato fruit and maize endo-
sperm, and its downregulation in tomato results in lower ploidy levels and diminished fruit size (Sun
et al. 1999a; Gonzalez et al. 2007). The sustained expression of WEE1 during endoreduplication in
these systems, and its requirement for this type of cell cycle may be puzzling considering that, in
Arabidopsis, WEE1 is involved in DNA damage checkpoint rather than the G2/M-phase transition.
However, these proposed roles may not need to be mutually exclusive since active monitoring of
DNA integrity might be integral to the regulation of the endocycle (Chevalier et al. 2011). Low CDK
activity in early G1, upon completion of M-phase, is instrumental in mitotic cells for the assembly
of prereplication complexes and the licensing of replication origins for DNA synthesis. Although
the exact mechanisms for origin licensing during the endocycle are not clear, it is likely that they
involve the general post-S-phase decrease in CDK activity that prevents M-phase. In fact, several
proteins required for licensing DNA replication origin also undergo proteasome-dependent degrada-
tion through a CDK-phosphorylation-dependent mechanism (Castellano et al. 2004). Thus, low CDK
activity could result in increased levels of factors required for licensing S-phase in the endocycle.
Upregulation of S-phase in endoreduplicating cells has been documented in plants, and it
revolves largely around the (up)regulation of the CDK–RBR1–E2F pathway (Figure 1.2). This path-
way controls the expression of many genes involved in DNA replication initiation, DNA synthesis,
and S-phase. Consistent with the idea that upregulation of E2F activity leads to increased ploidy
levels, endoreduplication has been stimulated by the downregulation of RBR1 in differentiating
Arabidopsis, tobacco, and maize tissues (Park et al. 2005; Desvoyes et al. 2006; Sabelli et al. 2013),
or directly by the upregulation of E2F/DP transcription factors (De Veylder et al. 2002; Kosugi and
Ohashi 2003). In Arabidopsis, overexpression of genes involved in replication origin activation such
as CDC6 and CDT1 stimulates endoreduplication (Castellano et al. 2001, 2004). Further upstream
in the pathway, solid evidence implicates CDKA activity in inhibiting RBR-dependent suppression
of S-phase (Nowack et al. 2012). In maize endosperm, downregulation of CDKA;1 inhibits endo-
reduplication through an RBR1-dependent pathway, though expression of specific RBR1-repressed
genes is not affected (Leiva-Neto et al. 2004; Sabelli et al. 2013). Downregulation of the M-phase-
specific CDK, CDKB1;1, stimulates endoreduplication (Boudolf et al. 2009), apparently because the
activity of CDKB1;1/CYCA2;3 complex is needed to phosphorylate and activate MYB3R transcrip-
tion factors, which are required for G2/M-phase-specific transcription (Araki et al. 2004).
In addition to the earlier mechanisms, several additional factors have been found to be
important for endoreduplication in Arabidopsis, including atypical E2F-like proteins and DNA
topoisomerase VI, and they have been reviewed elsewhere (Sabelli and Larkins 2008; De Veylder
et al. 2011; Inagaki and Umeda 2011).
1.3 ENDOREDUPLICATION AS A POTENTIAL YIELD DETERMINANT

It is interesting that some of the best-characterized systems in crop species, in terms of the relation-
ship between cell cycle regulation and development, concern the endoreduplication cell cycle. The
main reason for this is that endoreduplication is often associated with cell/tissue growth and may
play a key role in the development of agriculturally important organs and structures. Examples of
this are the tomato fruit, the endosperm of cereal caryopsis, and the sites of interaction between
plants and several symbiotic and parasitic/pathogenic organisms.
1.3.1 Endoreduplication and Tomato Fruit Development

The fleshy fruit of tomato contains two tissues, the mesocarp and the jelly-like locular tissue, in
which cells undergo endoreduplication and can reach extremely large sizes. In general, tomato fruit
development is characterized by a phase of intense cell proliferation (from about 2 to 10–12 days
post anthesis, dpa), which is dependent on hormones released by the embryo, coinciding with the
development of the ovary into a fleshy pericarp and of the placenta into a locular tissue with a
gel-like appearance enveloping the seeds. This is followed by exit from the mitotic cell cycle and
a transition into the endoreduplication cell cycle, which coincides with the growth of the fruit pri-
marily by cell expansion. The phase of endoreduplication and growth by cell expansion continues
essentially until the onset of fruit maturation and can result in ploidy levels in excess of 256C (C = the
haploid DNA content of a given species) (Chevalier et al. 2011). Cell proliferation following fertil-
ization generates most of the cells comprising the fruit and therefore plays a key role in controlling
fruit size, but it has been little characterized. However, it is the endoreduplication phase of fruit
development that has attracted considerable interest because of its potential for impacting fruit
weight. Indeed, convincing evidence indicates that endoreduplication, cell size, and fruit weight or
size are positively correlated (Cheniclet et al. 2005; Nafati et al. 2011).
Investigation has focused on understanding how cell cycle genes control the switch from mitotic
cell proliferation to endoreduplication, and to what extent endoreduplication affects tomato fruit
development and growth. The transition from mitotic cell cycle to endoreduplication and the ensuing
cell expansion phase of fruit development are characterized by downregulation of CDKA expression
and associated kinase activity (Joubès et al. 1999). In addition, the CDK-specific inhibitory kinase
WEE1 is upregulated during endoreduplication. Constitutive downregulation of WEE1 correlates
with increased CDK activity, decreased ploidy levels, and smaller cell and fruit sizes, suggesting
that endogenous WEE1 is required for endoreduplication-dependent cell expansion through inhibi-
tion of CDKA activity (Gonzalez et al. 2004, 2007).
Four CDK inhibitors belonging to the KPR family (KRP1–4) have been identified in tomato.
The expression of KPR2 and KRP4 transcripts is more closely associated with early tomato fruit
development, which is characterized by cell division, whereas KRP1 and KRP3 mRNAs are prefer-
entially expressed during the subsequent endoreduplication phase, suggesting specific roles for the
four inhibitors in the mitotic and endoreduplication cell cycles. Overexpression of KRP1 under the
control of a promoter (PhosphoEnolPyruvate Carboxylase 2) that is highly active in the mesocarp
during the cell expansion phase of fruit development strongly inhibited endoreduplication between
10 and 20 dpa, and had little effect thereafter (Nafati et al. 2011). Interestingly, however, cell area
and fruit size were not altered (but nuclear size was reduced), effectively uncoupling cell size from
endoreduplication in this KRP1-overexpressing mutant and resulting in fruits with altered karyo-
plasmic ratios.
Modulation of APC/C activity also controls endoreduplication and cell/fruit size in tomato fruit
tissue. The APC/C activator CCS52A is normally upregulated in endoreduplicating tomato fruit
pericarp. Downregulation of CCS52A by RNAi had virtually no effect on cell division, but resulted
in low ploidy levels and smaller cells and fruits, apparently through enhanced stability of CYCA3;1.
In CCS52A-overexpressing lines, however, the growth of the fruit was also transiently impaired by
mid-development, whereas it fully recovered at maturity, suggesting that high levels of CCS52A
protein probably interfered with other cell cycle- and tissue patterning–controlling pathways in
a developmental fashion. Recently, it was shown that, in individual pericarp cells, transcription
of 5.8S rRNA, the large subunit of RNA polymerase II, as well as other genes associated with
endoreduplication, such as WEE1 and CCS52A (but not genes associated with M-phase, such as
CDKB2), increases according to ploidy levels. This indicates that endoreduplication may be a means
to selectively upregulate gene expression (Bourdon et al. 2012). Interestingly, the topology of the
nuclear membrane appears to undergo important changes in endoreduplicated cells. Large (i.e.,
endoreduplicated) nuclei have an invaginated and expanded surface, which contributes to maintain-
ing the capacity of the nucleus to exchange molecules with the surrounding cytoplasm roughly con-
stant (which would otherwise decline with increased nuclear volume if the surface were smooth).
The nuclear surface invaginations are populated by relatively large numbers of active mitochondria.
Together, these results support the idea that endoreduplication in tomato fruit entails larger nuclear
and cell sizes, thus supporting the karyoplasmic ratio theory, and leads to specific increases in gene
expression levels. Endoreduplicated nuclei are deeply grooved, thus with a relatively larger surface
area available for molecular trafficking between nucleus and cytoplasm, which would be an asset for
supporting increased levels of gene expression. The presence of large numbers of active mitochon-
dria in the nuclear grooves is in agreement with the long-hypothesized role for endoreduplication in
supporting high metabolic rates (Bourdon et al. 2012).
1.3.2 Endoreduplication and Cereal Endosperm Development

The starchy endosperm of cereal seeds is a key source of dietary calories and raw materials for
myriad manufactured goods worldwide. The development of the endosperm has been best charac-
terized in maize, which recapitulates well the main events also occurring in other cereals (Sabelli
and Larkins 2009b,c) (Figure 1.3). Following double fertilization, endosperm development begins
with a series of acytokinetic divisions of the triploid primary endosperm nucleus to give raise to a
syncytium, which becomes cellularized a few days after pollination (DAP). A phase of mitotic cell
division then ensues, which generates the vast majority of endosperm cells. Subsequently, in cere-
als, cells gradually transition to an endoreduplication phase that coincides with rapid cell expan-
sion, the accumulation of storage compounds, and the massive growth of the endosperm and the
caryopsis (Kowles and Phillips 1985; Larkins et al. 2001; Sabelli and Larkins 2009b; Sabelli 2012b).
Endosperm endoreduplication, which in maize can generate ploidies in excess of 192C, appears to
be ubiquitous among cereal crops but it is absent in dicots (Sabelli 2012a). Starchy maize endosperm
displays a heterogeneous population of endopolyploid cells as endoreduplication begins in the cen-
ter of the endosperm and spreads toward the periphery of the tissue so that inner cells are typically
more highly endoreduplicated, and larger, than peripheral cells. The chromosomes in endoredupli-
cated cells have a loose polythenic structure and appear to be fully replicated and tightly associated
at centromeric and knob regions (Kowles and Phillips 1985; Bauer and Birchler 2006). Analyses of
interploidy crosses and manipulation of key cell cycle genes suggest that maize endosperm endo-
reduplication involves extensive reorganization of chromatin domains (Bauer and Birchler 2006;
Sabelli et al. 2013).
Endoreduplication in maize endosperm appears to entail a downregulation of the M-phase pro-
gram and an upregulation of the S-phase program. Several observations support this view. For exam-
ple, endoreduplicated endosperm (at 16 DAP) displays a peak in S-phase-associated CDK activity,
whereas mitotic endosperm (10 DAP) has a peak in M-phase-associated CDK activity (Grafi and
Cellularization
Syncytium
Antipodals
Central
Embryo sac vacuole
Primary
endosperm Zygote
nucleus nucleus
3n, 3c Embryo
2n, 2c
0 1 2 3 DAP
(a)
Al
SAI
CSEn
6C 12C
3C 3C Pe
Nu 6C 3C 24C
6C Em
Pe 48C
En 12C
Em 96C
TC
G1 Pl
M Mitosis G
G2 S
Endoreduplication
S
PCD
Mitotic index
Average DNA content
Nuclei number
Fresh weight
Nuclei number
Mitotic index
Average DNA content
Fresh weight
4 6 8 10 12 15 20 DAP
(b)
FIGURE 1.3 (See color insert.) Cell cycle regulation during maize endosperm development. (a) Following
double fertilization, early endosperm development involves acytokinetic mitoses starting with the triploid
primary endosperm nucleus, which results in a syncytium surrounding the central vacuole within the embryo
sac. At around 3 days after pollination (DAP), the endosperm becomes cellularized through an open-ended
alveolation process toward the central vacuole until cellularization is complete. (b) After cellularization from
about 4 DAP, the endosperm develops through a phase of mitotic cell proliferation, followed (from around
by 8 to 10 DAP) by endoreduplication (as shown by flow-cytometric profiles), and by programmed cell death
(PCD) (starting around 16 DAP). The endoreduplication phase and the last part of the cell division phase coin-
cide with a dramatic growth of the endosperm and the synthesis and accumulation of storage compounds. The
graph at the bottom illustrates trends in endosperm fresh weight, nuclei number, mitotic index, and mean DNA
content (C value). Al, Aleurone; CSEn, central starchy endosperm; Em, embryo; En, endosperm; Nu, nucel-
lus; Pe, pericarp; Pl, placentochalaza; SAl, subaleurone layer; TC, transfer cells. (Reproduced in part from
Larkins, B.A. et al., J. Exp. Bot., 52, 183, 2001; Sabelli, P.A. et al., Maydica, 40, 485, 2005b. With permission
from Oxford University Press and Maydica.)
Larkins 1995). Downregulation of CDKA;1 activity, through ectopic expression of a dominant-negative

CDKA;1 allele, resulted in an approximately 50% decrease in ploidy levels, indicating that this CDK
is necessary for S-phase in endoreduplicating endosperm cells (Leiva-Neto et al. 2004) (Table 1.1).
Further analyses of a double CDKA;1/RBR1 mutant revealed that CDKA;1 controls endoreduplication
through an RBR1-dependent pathway, most likely by targeting RBR1 for inhibitory phosphorylation
at the G/S-phase transition (Sabelli et al. 2013). The pattern of CYCB1;3 RNA accumulation shows a
dramatic decline at the mitosis–endoreduplication transition during endosperm development, which
suggested a specific involvement for this cyclin in M-phase and supported the idea that the M-phase
program is downregulated in endoreduplicating endosperm (Sun et al. 1999b). Consistent with this
interpretation, the expression of the M-phase CDK-specific inhibitor WEE1 peaks in endoreduplicated
cells (Sun et al. 1999a). CYCA1;3-associated kinase activity was present in 9-DAP endosperm, and
it phosphorylates RBR substrates in vitro (Sabelli et al. 2005a), which suggests that CYCA1;3 could
be a component of S-phase CDK activity at least in mitotic endosperm. Two CDK inhibitors belong-
ing to the KRP family have been characterized during endosperm development, KRP1 and KRP2,
and although they appear to have similar activities toward CDK complexes containing CYCA1;3,
CYCD5;1, and CYCB1;3, they have been shown to have different patterns of protein accumulation.
Whereas KRP1 protein is expressed at a roughly constant level, KRP2 declines substantially in endo-
reduplicated endosperm cells, which may indicate preferential roles for these two CKIs in regulating
the oscillation of CDK activity in the endocycle and mitotic cell cycle (Coelho et al. 2005). Another
key factor controlling S-phase entry is RBR1. In maize, the RBR family of genes comprise at least four
members, RBR1–4, organized into two closely related groups: RBR1-type (comprising also RBR2)
and RBR3-type (comprising also RBR4) (Sabelli et al. 2005a, 2013; Sabelli and Larkins 2006, 2009a).
Within each group, RBR genes have similar expression patterns, but while RBR3-type genes are pref-
erentially expressed during the mitotic phase of endosperm development, the expression of RBR1-type
genes is sustained during the endoreduplication phase. RBR1 represses the expression of RBR3 in
endoreduplicated endosperm just like it does other E2F targets required for DNA replication. CDKA;1
and RBR1 are epistatic with regard to endoreduplication, but it is intriguing that they are uncoupled
in the control of downstream gene expression. This is in contrast with the situation in Arabidopsis
where CDKA;1 and RBR1 are tightly coupled (Nowack et al. 2012). Both CDKA and RBR activities
are encoded by multiple genes in maize compared to a single gene in Arabidopsis. Thus, CDKA- and
RBR-dependent pathways in maize appear to be considerably more complex than their counterparts in
Arabidopsis. Importantly, downregulation of RBR1 activity in maize endosperm stimulates S-phase
gene expression and endoreduplication, but the increase in nuclear DNA content does not result in
proportionally more active chromatin or increases in nuclear size, cell size, or mature kernel weight
(Sabelli et al. 2013). Collectively, these studies suggest that RBR1 controls important aspects concern-
ing epigenetic mechanisms, chromatin structure and organization, and the coupling of cell size to
DNA content. In maize, the role of the APC/C in endoreduplication and endosperm development has
not been investigated, but recent work in rice suggests that the APC/C activator CCS52A homolog
plays an important positive role in these processes (Su’udi et al. 2012), consistent with other systems.
In spite of a large of body of correlative evidence linking endoreduplication to the expansion
of endosperm cells, the accumulation of storage compounds, and the growth of the whole tissue,
definite proof that it controls seed size and yield remains elusive (Sabelli 2012b). This is believed to
be partly due to the difficulty to disentangling cell cycle regulation from cell differentiation during
seed development.
1.3.3 Endoreduplication and Biotrophic Interactions

Interactions between plants and other organisms are often agriculturally very important. In cer-
tain cases, both symbiotic and parasitic interactions involve and require endoreduplication of the
affected plant tissue, probably as part of a metabolic strategy enabling and supporting the relation-
ship (Kondorosi and Kondorosi 2004; Wildermuth 2010).
TABLE 1.1
List of Genes and QTLs Controlling the Cell Cycle That May Impact Crop Yield
Phenotype (Gene Up-
Crop Gene/QTL Encoded Product (Putative) Function or Downregulation)a References
Wheat Ph1 CDK2-like kinase Prevents Down: Homeologous Griffiths et al.
homeologous pairing and stability of (2006)
chromosome pairing polyploidy genome
Barrel CCS52A CCS52A, a Positive regulator of Down: Decreased Vinardell et al.
medic CDH1-type mitotic cycle/ endoreduplication, (2003)
activator of the endocycle transition cell size and
APC/C development of root
nodules. Increased
lethality
CDC16 Component of Positive regulator of Down: Decreased Kuppusamy et al.
APC/C APC/C-dependent sensitivity to auxin, (2009)
degradation of reduced root
mitotic cyclins apparatus, and
increased number of
root nodules
Tomato FW2.2 Transmembrane Repression of cell Down: Increased fruit Frary et al. (2000);
protein proliferation during size/fresh weight Cong and
fruit development Tanksley (2006)
SlCCS52A CDH1-type Positive regulator of Down: Decreased Mathieu-Rivet
activator of the mitotic cycle/ endoreduplication, et al. (2010)
APC/C. Ortholog endocycle transition cell size, and fruit size
of Medicago
CCS52A
Maize tb1 Class II TCP Negative regulator of Up: Reduced Studer et al.
transcription cell cycle–dependent branching/Increased (2011)
factor gene expression grain yield
CNR1 and Related proteins Negative regulator of Up or down: Negatively Guo et al. (2010b)
CNR2 to FW2.2 from cell proliferation correlated with organ
tomato size
CDKA;1 CDKA;1 cell Regulator of Down: Decreased Leiva-Neto et al.
cycle–controlling G1/S-phase and endosperm (2004)
kinase G2/M-phase endoreduplication.
transitions during No effect on kernel size
mitotic cycle
RBR1 RBR1 protein Negative regulator of Down: Increased Sabelli et al.
E2F-dependent gene endosperm (2013)
expression and cell endoreduplication and
cycle progression cell number. No effect
on kernel size/weight
Rice KRP1 CDK/CYC Regulator of mitotic Up: Decreased Barrôco et al.
specific inhibitor cycle/endocycle endosperm (2006)
transition in endoreduplication,
endosperm seed-filling rate and
weight
CYCB1;1 B-type cyclin Positive regulator of Down: Abortive Guo et al. (2010a)
mitotic cell cycle in endosperm due to
syncytial endosperm abnormal cellularization
(continued)
TABLE 1.1 (continued)

List of Genes and QTLs Controlling the Cell Cycle That May Impact Crop Yield
Phenotype (Gene Up-
Crop Gene/QTL Encoded Product (Putative) Function or Downregulation)a References
CYCB2;2 B-type cyclin Positive regulator of Up: Increased root cell Lee et al. (2003)
M-phase number and growth
TE/TAD1/ CDH1-type Positive regulator of Down: Increased Lin et al. (2012);
OsCCS52A activator of the APC/C-dependent axillary meristem Su’udi et al.
APC/C. Ortholog degradation of activation and (2012); Xu et al.
of Medicago MOC1 and mitotic tillering. Decreased (2012)
CCS52A cycle/endocycle endosperm
transition endoreduplication,
cell size, and seed size
(width) and fertility
GW2 RING-type E3 Negative regulator of Down: Increased grain Song et al. (2007)
ubiquitin ligase cell proliferation in width/weight
spikelet hull
GS3 Transmembrane Negative regulator of Down: Increased grain Fan et al. (2006);
protein ovule development/ length/size Takano-Kai et al.
grain length (2009)
GS5 Serine Positive regulator of Up: Increased seed size Li et al. (2011)
carboxypeptidase cell proliferation in (width) and weight
palea/lemma
qSW5/GW5 Nuclear protein Negative regulator of Up: Increased seed size Shomura et al.
interacting with cell number in outer (2008); Weng
polyubiquitin glume et al. (2008)
GW8 OsSPL16 Positive regulator of Up: Increased grain Wang et al. (2012)
transcription CDKA1, CYCD3 and width, filling, and
factor E2F2 expression, and yield
cell proliferation in
spikelet hull
TGW6 IAA-glucose Positive regulator of Down: Delayed Ishimaru et al.
hydrolase CYCB2;2 and E2F1 endosperm (2013)
expression in cellularization;
syncytial endosperm Increased endosperm
cell number, grain
length, and weight
qGL3 OsPPKL1 type 2A Negative regulator of Down: Increased grain Zhang et al.
phosphatase cell proliferation in length, weight, and (2012b)
spikelet hull yield
SG1 Novel protein Negative regulator of Up: small seed size Nakagawa et al.
cell number (length) (2012)
HGW Novel ubiquitin- Positive regulator of Down: Decreased seed Li et al. (2012)
associated cell number in size (width)
(UBA) domain spikelet hull
protein
RSS1 Novel interactor of Positive regulator of Down: Decreased SAM Ogawa et al.
protein meristem cell size, dwarfism, and (2011)
phosphatase 1, and proliferation under short root under salt
substrate of APC/C salt stress stress
a Gene up- or downregulation may refer to mutant loss of function, RNAi downregulation, overexpression, or gain of function.
1.3.3.1 Endoreduplication and Symbiotic Interactions

One of the best characterized symbiotic interactions occurs between legumes, such as Medicago
truncatula or sativa with endosymbiotic, nitrogen-fixing bacteria, such as Sinorhizobium meliloti,
which results in the formation of specialized lateral root organs known as root nodules that provide
nitrogen to the plant. These nodules, which are characterized by indeterminate development, are
induced in the root cortex by infective threads and typically consist of an apical meristematic zone,
an infection zone, a symbiotic zone, and a senescent zone sequentially distributed according to the
longitudinal axis of the growing nodule. Root nodule cells exit the mitotic cell cycle and undergo
endoreduplication below the meristematic zone, particularly in the infection zone and the symbiotic
zone, the latter becoming populated with N-fixing bacteroids (Kondorosi and Kondorosi 2004).
Endoreduplication is a requirement for the development of functional root nodules and is achieved
primarily by enhanced APC/C-dependent proteolysis of mitotic cyclins. Integral to this process is
the upregulation of the activator of APC/C, CCS52A (Cebolla et al. 1999; Vinardell et al. 2003). As
a result, the mitotic program is skipped (Figure 1.2), and cells continue to replicate their DNA (up
to 64C) without cell division, dramatically enlarging in the process.
Endoreduplication is also involved in the establishment of arbuscular mycorrhizal symbiosis
(AMS), where the fungus provides the plant with the majority of its phosphorus requirements. Here,
too, there is evidence that not only chromatin decondensation and nuclear enlargement of host root
cells, typical of endoreduplication, are associated with AMS, but also their onset precedes and may
be required for fungal infection (Genre et al. 2008; Wildermuth 2010).
1.3.3.2 Endoreduplication and Parasitic/Pathogenic Interactions

Powdery mildews (PMs) are obligate biotrophic ascomycetes that cause widespread disease to thou-
sand of angiosperms, including important crops such as wheat, barley, and grape (Glawe 2008). PMs
establish feeding sinks in the plant host by infecting cells with haustorial complexes, which acquire
nutrient resources from the plant. In the case of Arabidopsis leaves infected with Golovinomyces
orontii, the haustorial complexes infect epidermal cells, and this is associated with endoreduplica-
tion (resulting in a mean ploidy of 32C) in the underlying mesophyll cells, with their concomitant
increases in nuclear and cell volumes. Transcript and genetic analyses identified one member of
the MYB3R family of transcription factors associated with M-phase (MYB3R4) as a key factor
for PM-induced host cell endoreduplication (Chandran et al. 2010). Because MYB3R transcription
factors may inhibit transcription when hypophosphorylated and appear to be activated by CDK-
dependent phosphorylation (Figure 1.1), a variety of mechanisms impinging on CDK activity could
contribute to regulating MYB3R4-dependent gene expression in this system. Additionally, three
other Arabidopsis genes (PUX2, PMR5, and PMR6), which are also suspected to be involved in
the regulation of M-phase, have recently been involved in control of PM-associated mesophyll cell
endoreduplication. The evidence obtained so far indicates that endoreduplication in specific meso-
phyll cells underlying infected epidermal cells coincides with a shift in carbohydrate metabolism
toward fermentation, which could favor the metabolic requirements of PM. Endoreduplication of
host cells associated with infection sites has recently been established as a determinant of suscepti-
bility to PM infection (Chandran et al. 2013).
Nematode infections in the root apparatus cause large losses in many crops, such as tomato, soy-
bean, and potato. Two main infection types are relevant to the present discussion; those caused by
cyst nematodes, which include the genera Heterodera and Globodera, and those caused by root-knot
nematodes (genus Meloidogyne) (Williamson and Hussey 1996). These obligate sedentary parasites
typically penetrate the root and migrate toward the vascular tissue where they establish so-called
nematode feeding sites (NFS), causing distinct responses in the affected cells of the procambium.
In the case of cyst nematodes, a procambium cell develops into a multinucleate, metabolically active
feeding site by incorporating adjoining cells through cell wall dissolution and protoplast fusion.
In root-knot nematode infections, instead, feeding sites consist of small groups of giant cells with
multiple (up to 100) enlarged nuclei, resulting from acytokinetic mitoses and endoreduplication.
Eventually, reactivation of cell division in the cell layers surrounding the feeding sites results in
tissue enlargement and the formation of galls (Williamson and Hussey 1996; Wildermuth 2010; de
Almeida Engler et al. 2012).
Thus, stimulation of root cells associated with the vascular tissue to reenter the cell cycle as well
as activation of the endoreduplication program are common themes in both types of nematode infec-
tions (De Almeida Engler and Gheysen 2013). Recently, functional genetic analyses of NFS utiliz-
ing Arabidopsis lines with altered expression of several genes known to impact the G2/M-phase
transition and endoreduplication (i.e., CCS52A, CCS52B, DEL1, and RHL1) have suggested that
coordination of mitotic with endoreduplication cell cycle is important for the establishment and the
expansion of functional feeding sites both for root-knot and cyst nematodes (De Almeida Engler
et al. 2012).
Thus, the alteration of plant cell cycle regulation by other organisms appears to be key both in
the case of symbiotic (i.e., N-fixing root nodules, AMS) and pathogenic/parasitic (i.e., PM/nematode
infections) relationships. Endoreduplication, in particular, may be a strategy to increase the metabo-
lism of plant cells to support symbiotic or parasitic organisms alike (Wildermuth 2010). Thus, targeted
regulation of the cell cycle may be a valuable strategy to control these biotrophic associations in agri-
cultural settings.
1.4 REGULATION OF PLANT BIOMASS AND ARCHITECTURE

1.4.1 Reproductive Development
1.4.1.1 Gametogenesis
A notable involvement of core cell cycle genes in cell fate- and cell differentiation-related deci-
sions occurs during male gametogenesis in Arabidopsis (Iwakawa et al. 2006; Borg et al. 2009).
Following asymmetric division of the microspore at pollen mitosis I into one large vegetative and
one small generative cell, proper differentiation of sperm cells depends on selective proliferation of
the generative cell, while the vegetative cell exits the cell cycle. RBR1 is essential in restricting cell
cycle activity in both cell types. Correct regulation of cell proliferation of the generative cell requires
CDKA;1 and is specifically sustained through FBL17-dependent degradation of KRP inhibitors by
the proteasome. Interestingly, FBL17 is a direct transcriptional target of RBR1 and E2F, which
closes the negative KRP ┤ CDKA;1 ┤ RBR1 ┤ E2F → FBL17 ┤ KRP feedback loop and essen-
tially provides a bistable switch predicted to be heavily dependent on the levels of KRP activity.
This module has been proposed as a general controlling mechanism for the G1/S-phase transition in
Arabidopsis and elements of it have been shown also to control the first two divisions during female
gametophyte development (Zhao et al. 2012). Moreover, recent results in maize endosperm where
downregulation of RBR1 results in increased CDKA;1 activity (Sabelli et al. 2013) suggest that this
pathway may regulate S-phase entry also in monocots. In addition to the module described earlier,
high levels of expression of CYCB1;1 appear to contribute to maintaining CDKA;1 activity high,
which is necessary for the formation of two sperm cells (Brownfield et al. 2009). Additional factors
are important for pollen microspore cell divisions, such as SAMBA, a component of the APC/C,
which is involved in the proteolysis of CYCA2;3 and in maintaining the ratio of M-phase-related
transcripts versus those of CDK inhibitors relatively high (Eloy et al. 2012).
Besides the regulation of cell division during male gametogenesis, cell cycle genes are also
important for other steps in reproductive development. Examples of this are CYCA1;2, which is
required for the correct execution of metaphase at meiosis during female gametophyte develop-
ment (Wang et al. 2004b); MCM7, which is required for cell divisions in the female gametophyte
(Springer et al. 2000); and RBR1, which is required also for female gametophyte development
(Ebel et al. 2004) and for the control of key epigenetic mechanisms during sexual reproduc-
tion (Sabelli and Larkins 2009c). Additionally, R2R3 MYB transcription factors, known to
control M-phase-related cell cycle genes (Figure 1.1), are important for embryo sac development
(Makkena et al. 2012). A number of cell cycle gametophytic mutants have been reviewed else-
where (Liu and Qu 2008).
1.4.1.2 Seed Development
The production of vast amount of grain is one of the most outstanding outcomes of modern agricul-
ture. One monocot family (Poaceae) and one dicot family (Leguminosae or Fabaceae) contribute the
species that are responsible for the vast majority of grain production worldwide. At the individual
seed level, yield is given by two major components: the seed growth rate (SGR) and the seed-filling
duration (SFD), which have been thoroughly discussed by Egli (1998, 2006). Seed development gen-
erally comprises three phases: A first formative phase (phase I), which begins at fertilization and is
characterized by rapid cell division; a middle phase (phase II), during which economically and nutri-
tionally valuable compounds are accumulated and stored; and a third maturation phase (phase III),
which begins with a decrease in the accumulation of storage metabolites, includes physiological
maturity (i.e., maximum seed dry weight), and finally involves dehydration (Sabelli and Larkins
2009b,c). During phase I, rapid cell division is responsible for creating most of all the cells that will
make up the storage structures of the seed or fruit. In the Poaceae, these are primarily the endosperm
but also, in some cases, the embryo, whereas in the Fabaceae, it is (mostly) the cotyledons (Sabelli
2012a). During this phase, the domains of the seed that are important for establishing transfer cells,
as a specialized link with the mother plant’s vascular tissue for the uptake of metabolic precursors
by the seed, are also specified. This phase is characterized by sucrose uptake that is quickly metabo-
lized to glucose and fructose by cell wall–bound invertase to support intense cell division activity.
Although this phase is critical for seed development and grain yield, the resulting cells generated are
very small, and overall the direct contribution of this phase to seed biomass is rather minor. Phase II
is characterized by the deposition of storage reserves according to a linear pattern in which the accu-
mulation of dry matter is constant with time. This phase is characterized by cell expansion resulting,
in part, from an initial increase in seed water content, so that seed size throughout phase II and at
maturity reflects almost entirely cell volume. This phase is characterized in cereals by endoredupli-
cation, discussed earlier, which is associated with the sizes of the cell and the storage compartment
(Sabelli and Larkins 2009b,c) (Figure 1.3). During phase III, the seeds reaches its physiological
maturity, which represents the end of the seed-filling period. At this stage, the seed becomes func-
tionally severed from the vascular system of the mother plant and begins to lose moisture at a rate
that is species-specific for a given environment. Seed size is closely associated not only with SGR
(r = 0.81) but also, to a lesser extent, with SFD (r = 0.5) (Egli 1998; Sabelli and Larkins 2009c). SGR
is determined largely by genetic control, and it is possibly influenced by the flux of assimilates from
the mother plant as well as by intrinsic mechanisms to the seed. A number of studies indicate that
there is a positive correlation between SGR and the number of cells of the storage tissues within the
seed: the cotyledons or the endosperm. Regulation of the cell cycle contributes to seed development
in several important ways: In both monocots and dicots, the first zygotic division is asymmetric, gen-
erating a cytoplasmic-dense apical cell projected toward the chalaza and a large vacuolated basal cell
toward the micropylar end of the embryo sac. This asymmetric division leads to the establishment
of embryo bipolarity and patterning. Subsequent intense cell proliferation coupled to cell differentia-
tion essentially produces all embryo structures including the cotyledon(s) and the suspensor (Sabelli
2012a). In Arabidopsis, embryo development and seed size are negatively affected by SAMBA, a
negative regulator of the APC/C (Eloy et al. 2012). Additionally, a recent comprehensive investiga-
tion on D-type cyclins highlighted the importance of proper spatiotemporal regulation of CYCD
expression for embryo cell division, patterning, and seed development (Collins et al. 2012). DEL1
is known to inhibit the endocycle, and loss of its function results in a small but significant increase
in Arabidopsis seed size, though the underlying mechanism is not known (Van Daele et al. 2012).
Moreover, there is evidence that the coordination of cell proliferation with tissue patterning dur-
ing Arabidopsis seed development depends on a signaling pathway that involves the peptide ligand
CLAVATA3/EMBRYO SURROUNDING REGION-RELATED8 (CLE8) and the transcription fac-

tor WUSCHEL-LIKE HOME-OBOX8 (WOX8) (Fiume and Fletcher 2012).
The endosperm develops most often through the so-called nuclear type pattern of develop-
ment, which involves early acytokinetic divisions of the primary endosperm nucleus that gener-
ate a syncytium. Subsequent cellularization of nuclear domains results in a cellular endosperm
that goes on to proliferate by standard mitosis coupled to cell divisions. However, while the
endosperm is absorbed by the growing embryo in nonendospermic species such as Arabidopsis,
in cereals, endosperm cells exit the mitotic cell cycle and engage in reiterated rounds of genome
replication, known as endoreduplication, which is associated with cell expansion and accumula-
tion of storage compounds (see Section 1.3.2). While endoreduplication correlates with cell and
endosperm sizes in cereals, the rate and potential of grain filling correlate with the number of
starch granules, which, in turn, depends on cell number and therefore the extent of the cell pro-
liferation phase (Brocklehurst 1977; Reddy and Daynard 1983; Chojecki et al. 1986a,b; Jones
et al. 1996; Sabelli and Larkins 2009b). A number of endosperm mutants in Arabidopsis result-
ing in small or aborted seeds display cell cycle defects during the syncytium and cellularization
phases of endosperm development, indicating that correct regulation of early nuclear proliferation
and the timing of the transition to cellularized endosperm are important for the attainment of
proper seed size (Sabelli and Larkins 2009c). In rice, TGW6, encoding an IAA-glucose hydrolase,
appears to stimulate the expression of CYCB2;2 and E2F1 during the first 3 days after fertiliza-
tion and to hasten early endosperm nuclear proliferation leading to premature cellularization,
which reduces endosperm cell number, length, and grain weight significantly (Ishimaru et al.
2013). This indicates that regulation of the duration of the syncytium period is also important for
the development and the yield of cereal grain. Interestingly, the same investigation suggested that
downregulation of TGW6 may play an important role in mitigating yield losses due to climate
warming. Supporting the view that proper syncytium development and endosperm cellularization
are crucial, conserved aspects of seed development, knockdown of OsCYCB1;1 interfered with
cellularization, caused aborted endosperm, enlargement of the embryo, and overall abnormal
seed development (Guo et al. 2010a). Also, the role of KRP3 may be key in specifically inhibiting
CDKA activity during syncytial endosperm proliferation in rice (Mizutani et al. 2010).
Besides playing an important role in controlling the number of cells in the main seed storage
tissue, cell proliferation must be coordinated between endosperm and seed coat for proper seed
development (Ingouff et al. 2006; Li and Berger 2012). In addition, there is increasing evidence
indicating that factors controlling the cell cycle and regulation of epigenetic mechanisms during
seed development are intertwined and that the RBR pathway may be central to their integration
(Sabelli and Larkins 2009c).
1.4.2 Shoot Branching
Following embryonic development, where the apical–basal body axis is established with the formation
of the two apical meristems, the architecture of the aboveground plant structures, which is extremely
diverse, is largely due to the regulation of shoot branching, which depends on the development of sec-
ondary growth axes. These axes ultimately derive from the shoot apical meristem, through the iter-
ated formation of axillary meristems, which may develop into buds, stems, leaves, and reproductive
structures (Müller and Leyser 2011). Shoot branching (known as tillering in grasses) may negatively
affect grain yield, because often branches and the reproductive organs they bear compete for limited
resources. Thus, reduction in tillering has been one of the key traits that has been selected during the
domestication of several cereal crop species, such as maize and millet (Studer et al. 2011; Kebrom
et al. 2013). However, increased shoot branching may be a desirable trait for some cereal crops, such
as rice. Axillary meristem development and activity are controlled by phytohormones as part of a
complex regulatory web of genetic information and external stimuli. Generally, it is repressed by
auxin and strigolactones and stimulated by cytokinin.
The cell cycle plays an important role in branching. Bud activation and outgrowth in pea cor-
relate with cell cycle entry as revealed by increased expression of several cell cycle genes, such
as PCNA, which is involved in the regulation of DNA synthesis, CYCD3, and CYCB1;2 (Shimizu
and Mori 1998). Expression of cell cycle genes and bud outgrowth are inhibited by the TCP (for
TEOSINTE BRANCHED, CYCLOIDEA, PROLIFERATING CELL FACTORS 1 and 2) family of
transcription factors (Müller and Leyser 2011). TCP transcription factors are stimulated further
upstream by strigolactone and inhibited by cytokinin. Although this pathway linking transactiva-
tion of cell cycle genes, cell proliferation, and bud outgrowth with phytohormones, centered on TCP
transcription factors, is conserved in monocots and dicots, there may exist important differences
between the two phyla in its fine-tuning (Kebrom et al. 2013). With regard to the role of cell cycle
regulation in controlling shoot branching, there is evidence to suggest that while increased expres-
sion of certain cell cycle genes (such as D-type cyclins) can enhance bud outgrowth, it is not suf-
ficient to activate buds or stimulate shoot branching. Conversely, upregulation of cytokinin results
in increased bud activation and branching, while it has little effect on bud growth rate. In summary,
the cytokinin-TCP cell cycle pathway appears to play an important role in plant branching and
architecture, though several important aspects concerning its regulation remain to be understood in
more detail (Müller and Leyser 2011). Recent evidence also implicates CCS52A in the regulation of
tillering in rice through proteolysis of MONOCULM1 (Lin et al. 2012; Xu et al. 2012).
1.4.3 Leaf Development
Development and growth of leaves depend on several cell-autonomous and cell-nonautonomous
pathways impinging on cell division and expansion (Fleming 2007; Gonzalez et al. 2012;
Nelissen et al. 2012; Powell and Lenhard 2012). In both monocots and dicots, intense cell prolif-
eration characterizes the growth of the leaf primordium, which is followed by a transition from
the mitotic into the endoreduplication cell cycle that coincides with the onset of cell expansion
and differentiation. Leaf size in Arabidopsis is determined to a large extent by cell number,
which appears to be a common denominator in several mutants seemingly affecting different
pathways (Gonzalez et al. 2010). The RBR1–E2F pathway plays a central role in linking the
cell cycle to leaf development, but its influence is mostly context dependent. Upregulation of
E2F activity results in increased cell proliferation or endoreduplication, depending on whether
cells are undifferentiated within immature leaves or differentiated (Kosugi and Ohashi 2003).
Accordingly, RBR1 is involved in restricting progression of the mitotic or endoreduplication
cycle based on context (Desvoyes et al. 2006). Upregulation of the E2F pathway, with prolon-
gation of the cell division phase and hyperproliferation of leaves, is also obtained by overex-
pression of CYCD3;1 (Dewitte et al. 2003). However, the situation is probably more complex,
and it has recently been proposed that the effect on leaf shape upon downregulation of RBR1
is primarily due to smaller cell size rather than increased cell division rates (Kuwabara et al.
2011). Although changes in the duration of the proliferation phase strongly correlate with final
leaf size (Mizukami and Fischer 2000; Li et al. 2008; Dhondt et al. 2010; Nelissen et al. 2012),
not necessarily increased cell division through the modification of core cell cycle genes results
in larger leaves, often because of compensatory effects on cell size that offset cell proliferation
changes (Powell and Lenhard 2012). Indeed, leaf development is commonly regarded as one of
the best examples of the dependence of (cell-autonomous) cell division activity on higher-order
control at the tissue or organ levels (see later). However, reduced cell proliferation and leaf size
in scr and shr mutants have been linked to E2F-dependent cell cycle defects (Dhondt et al. 2010),
suggesting that the SCR–RBR1 pathway works similarly in leaves and roots, although only in the
latter system, there is an obvious stem cell population, where these two genes are known to play
key roles. Additionally, there is evidence that other genes such as APC10, CDC27a, and SAMBA,
which control different components of the APC/C, play important roles in controlling mitotic
activity, cell number, and leaf growth (Rojas et al. 2009; Eloy et al. 2011, 2012). Development of
the leaf in cereals involves transitioning between a basal division zone, an expansion zone, and
an apical zone along the longitudinal leaf axis. The boundary between the division and the
expansion zones is characterized by a downregulation of genes involved in M-phase, such as
CYCB and CDKB;1, and a switch to the endoreduplication cycle. Although there is a paucity
of functional studies on the role of the cell cycle in cereal leaf growth and development, recent
results in maize indicate that the positioning of the boundary between the division zone and
expansion zone largely depends on a peak of bioactive gibberellins and that this affects cell
proliferation patterns, cell expansion, and the size of the leaf (Nelissen et al. 2012).
1.4.4 Root Development and Architecture

The root system plays fundamental roles in crop production, including the physical anchoring and
support of the plant, the uptake of water and nutrients from the soil, the storage of metabolites, and
the interaction with a range of biotic and abiotic factors. Root growth is controlled in part by the
availability of some of these factors but also by endogenous plant genes. For example, overexpres-
sion of CYCB2;2 in rice resulted in increased root cell number and growth, probably by stimulating
CDKB2 activity (Lee et al. 2003).
Manipulation of root growth and architecture is increasingly recognized as an important strategy
to potentially maximize the utilization of decreasing resources and mitigate potential yield losses.
The architecture and organization of the root apparatus is markedly different in Arabidopsis and
cereal crops, the latter being characterized by a higher complexity, a more fibrous appearance, and
comprising, in addition to lateral roots, crown roots and, often, seminal roots as well. In both mono-
cots and dicots, activation of the cell cycle at the pericycle (and in cereals also at the endodermis)
near the vascular system is a conserved hallmark of the initial stages of lateral root development
(Casero et al. 1995; Dubrovsky et al. 2001; Smith and De Smet 2012). Several core cell cycle genes
play important roles in lateral root formation. In Arabidopsis, KRP2 is downregulated during cell
cycle reentry in the pericycle, and there is genetic evidence that it antagonizes lateral root initiation
and density by inhibiting CDKA/CYCD2;1 (Sanz et al. 2011). Additionally, E2Fa has been shown
to be rate limiting for the initiation of lateral root primordia (Berckmans et al. 2011). However, it
is currently not clear whether KRP2 and E2Fa control later root formation through the same or
distinct pathways. Although cell cycle regulation is important for later root primordium formation,
it does not appear to be sufficient, and a paramount role is played by auxin-dependent signaling
pathways that specify lateral root cell founder fate (Vanneste et al. 2005; Dubrovsky et al. 2008; De
Smet et al. 2010). The understanding of root architecture control in cereals is very limited at present,
due primarily to a lack of suitable mutants.
Manipulation of root hair density could conceivably enhance the ability of roots to uptake water
and nutrients from their surroundings and to impact a range of plant/soil interactions. In many
plants, regulation of asymmetric, formative cell division of specific epidermal cells in the root dif-
ferentiation zone is critical for specifying root hair cell fate. CDT1, a DNA replication factor, has
been implicated in root hair cell differentiation in Arabidopsis through a pathway that appears to
coordinate cell division with modification of histone chromatin marks and cell fate decisions (Caro
et al. 2007). Although this highlights the role of certain cell cycle genes in coordinating cell divi-
sion with differentiation, it is not clear whether the role of CDT1 is conserved in other plants, given
that root hair specification in angiosperms involves distinct cell division patterns, and this process
is clearly different in monocots compared to Arabidopsis (Datta et al. 2011).
1.5 PLANT TISSUE CULTURE

The culturing of cells and tissue in vitro is integral to many processes in plant biotechnology,
such as micropropagation, transgenic plant production, and culturing plant and algal cells in bio-
reactors for the production of many different compounds including pharmaceuticals and biofuels.
Space limitations prevent a detailed description of cell cycle control in plant tissue culture in this
chapter. However, it is worth mentioning that tissue culture has been deployed extensively as a
tool to unravel important aspects of cell cycle regulation in plants, while, at the same time, the
modification of cell cycle parameters has been pursued to improve plant tissue culture technology.
The importance of an appropriate hormonal input (primarily auxin and cytokinin) for plant tissue
culture is well known (Del Pozo et al. 2005; John 2007; Dudits et al. 2011), and there is substantial
evidence to indicate that modulation of cell cycle regulation mediates the action of hormones. For
example, overexpression of E2Fb/DPa transcription factors stimulate proliferation of tobacco BY-2
cells in the absence of auxin, which is otherwise necessary, and supplementing the medium with
auxin increases the levels of E2Fb protein by enhancing its stability (Magyar et al. 2005). Thus,
auxin apparently stimulates the cell cycle by upregulating the E2F pathway. In addition, during the
culturing of Arabidopsis explants, cytokinin stimulates the cell cycle and induces callus forma-
tion by upregulating the expression of D3-type cyclins (Riou-Khamlichi et al. 1999). Likewise,
sucrose signaling stimulates cell cycle progression by upregulating both CYCD2 and CYCD3 (Riou-
Khamlichi et al. 2000). In maize, a key role has been established for the RBR pathway in controlling
callus growth and plant regeneration. RBR1 inhibits these processes, and its inactivation by ectopic
expression of RepA from wheat dwarf virus (an RBR-binding protein) stimulates callus formation
and regeneration of transgenic plants (Gordon-Kamm et al. 2002). Interestingly, expression of RBR3
is also repressed by RBR1 and appears to be required for the transcription of several E2F target
genes and for S-phase, although it is not sufficient for upregulation of cell proliferation (Sabelli
et al. 2009). Thus, appropriate control of the RBR–E2F pathway is required in cell and tissue cul-
ture, and the upregulation of this pathway generally is associated with or required for increased cell
cycle activity in vitro. This is consistent with the observation that the RBR-homolog MAT3 gene is
important for cell cycle progression in the unicellular alga Chlamydomonas reinhardtii (Umen and
Goodenough 2001).
1.6 CONTRIBUTION OF CELL CYCLE MANIPULATION

TO CROP EVOLUTION AND BREEDING
A central question concerning the role of cell cycle regulation in plant development is whether and
to what extent it has been a factor in the evolution and breeding of crops and whether its manipula-
tion has potential for future crop improvement. This is a topic under constant development as infor-
mation gained from model plants such as Arabidopsis is slowly but gradually being applied to crop
plants. A number of genes or quantitative trait loci (QTLs) impacting the cell cycle and yield param-
eters have already been identified in important crops and characterized to some extent (Table 1.1).
Many of the underlying pathways remain to be characterized, and it is possible that these genes may
control the cell cycle in rather indirect ways. However, they may reveal key aspects of how the cell
cycle regulatory engine is linked to plant growth and development.
Several important crop species that sustain human civilization are allopolyploid, such as
wheat, oats, canola, cotton, and sugarcane. Their genomes contain sets of related homeologous
chromosomes in addition to the homologous chromosomes that are also present in diploid species.
The fertility and genetic stability of allopolyploids generally depend on preventing the exchange
of genetic material between homeologous chromosomes at meiosis during gametogenesis. As a
result, allopolyploids behave as diploids at meiosis, with chromatin exchanges limited to true
homologous chromosomes. Although allopolyploid crops are highly productive, they suffer from
the problem that their breeding is generally rather inefficient because the block on homeologous
recombination in a polyploidy hybrid essentially precludes introgression of desirable alleles from
other species such as wild relatives. Homeologous chromosome pairing has been investigated in
durum (Triticum turgidum) and bread (Triticum aestivum) wheats, which are tetraploid and hexa-
ploid, respectively. A major repressor of homeologous chromosome pairing in these species is the
Ph1 locus, which probably arose after the polyploidization events that generated these species.
The Ph1 locus contains a defective cluster of seven genes related to mammalian CDK2 on chro-
mosome 5B (Griffiths et al. 2006). Although this locus additionally presented evidence of inser-
tion of etherochromatic DNA sequences, recent analyses indicated that the CDK2-like genes on
chromosome 5B are the most likely candidates for Ph1 locus activity (Yousafzai et al. 2010a,b).
Although these CDK2-like genes are divergent from the CDKA and CDKB genes (which are more
closely related to mammalian CDK1) commonly believed to control the cell cycle in plants, they
appear to share several properties with CDK2 concerning the control of premeiotic DNA replica-
tion, chromatin condensation at meiosis, the phosphorylation pattern of histone H1, and chromo-
some pairing. Deletion of the Ph1 locus allows homeologous pairing, which is associated with
upregulated expression of related CDK2-like genes at the Ph1 homeologous loci on chromosomes
5A and 5D. Thus, it would appear that low CDK2-like activity prevents homeologous pairing
whereas increased activity allows it (Greer et al. 2012). While the precise mechanism of action of
CDK2-like genes at the Ph1 loci remain to be unraveled, this insight illustrates the importance of
CDK genes in shaping and maintaining the allopolyploid genome of wheat and offers an oppor-
tunity to improve, through strategies targeted at relieving the inhibition on homeologous pairing,
the breeding of allopolyploid crops and increase their genetic diversity.
Maize teosinte branched 1 (tb1) is a classic example of a gene with a paramount role in the
domestication of a major crop species, such as maize from its wild ancestor (teosinte, Zea mays
ssp. parviglumis) (Studer et al. 2011). The TB1 protein belongs to the class II of TCP family of
transcription factors and inhibits axillary bud outgrowth, thereby contributing to apical domi-
nance. tb1 is bud specific and is expressed at higher levels in modern maize than teosinte, result-
ing in reduced shoot branching, reduced development of the branches compared to the main
stalk, reduced number of ears, and increased grain yield. tb1 is conserved in both monocots and
dicots, with a similar negative function on branching. It appears to inhibit axillary bud outgrowth
by negatively regulating cell proliferation. Although the exact role of tb1 in cell cycle regula-
tion remains to be clarified, possible TB1 targets are PCNA, CYCB1;1 and PROLIFERATION
FACTOR 1 and 2 (PCF1 and PCF2) (Müller and Leyser 2011). Thus, specific inhibition of the
cell cycle during axillary bud activation appears to have been a key event in the domestication of
maize and contributing to its high grain yield.
An additional example of the importance of modulating cell proliferation during domestication
of a major crop comes from tomato. The fw2.2 QTL is responsible for up to 30% of the difference
in fresh fruit weight between domesticated tomato and its smaller wild relatives (Frary et al. 2000).
It encodes a putative transmembrane protein believed to be involved together with CKII kinase
in a signaling pathway that represses the mitotic cell cycle early in fruit development (Cong and
Tanksley 2006). In domesticated tomato fruit, FW2.2 is expressed at lower levels than its wild rela-
tives, and this correlates with more cells and a larger fruit size. In addition, FW2.2 appears to be
conserved in plants, with related genes in maize (CNR1 and CNR2) (Guo et al. 2010b) and avocado
(PaFW2.2) (Dahan et al. 2010) that display similar roles in restricting cell proliferation and organ
size. A related gene in soybean (GmFWL1) also controls nodule number and nuclear size, poten-
tially also impacting chromatin structure (Libault et al. 2010).
Regulation of ubiquitination-dependent proteolysis, such as by the APC/C, appears to be a cen-
tral theme in the developmental control of the cell cycle. Factors that control APC/C activity, such
as the activator CCS52A, play important roles in the developmental regulation of the endocycle
and other processes, thereby affecting yield parameters, in different crop systems such as barrel
medic (Medicago truncatula) (Vinardell et al. 2003; Kuppusamy et al. 2009), tomato (Mathieu-
Rivet et al. 2010; Chevalier et al. 2011), and rice (Lin et al. 2012; Su’udi et al. 2012; Xu et al. 2012).
Additionally, other agriculturally important genes and QTLs involved in ubiquitination- and pro-
teasome-dependent proteolysis have been identified in rice (Song et al. 2007; Shomura et al. 2008;
Weng et al. 2008; Ogawa et al. 2011; Li et al. 2012).
As mentioned earlier, regulation of the cell cycle during syncytial proliferation and endosperm
cellularization is another important theme with potential implications for grain yield in cereal crops,
which has been shown to be impacted by KRP1, CYCB1;1, and CCS52A in rice (Barrôco et al. 2006;
Guo et al. 2010a; Su’udi et al. 2012; Ishimaru et al. 2013). However, cereal seeds appear to be more
resilient to alteration of core cell cycle gene activity occurring later in development (Leiva-Neto
et al. 2004; Sabelli et al. 2013).
Regulation of cell proliferation in maternal reproductive structures, such as the palea/lemma
during early spikelet hull development, is emerging as an important grain size and yield determi-
nant in rice, and several key genes have been identified: GW2 (Song et al. 2007), GS3 (Fan et al.
2006; Takano-Kai et al. 2009), GS5 (Li et al. 2011), qSW5/GW5 (Shomura et al. 2008; Weng et al.
2008), GW8 (Wang et al. 2012), qGL3 (Zhang et al. 2012b), and HGW (Li et al. 2012). Intriguingly,
evidence is emerging that HGW, GW2, qSW5/GW5, and GS3 may function in the same ubiquitina-
tion pathways to regulate grain size and weight (Li et al. 2012).
1.7 DOES THE CELL CYCLE DETERMINE YIELD? CONCLUDING REMARKS

The cell cycle is directly responsible for the number of cells, which together with cell expansion
determines overall tissue/organ size. There are documented cases in which stimulation of cell divi-
sion results in increased tissue growth (Lee et al. 2003; Li et al. 2008; Guo et al. 2010b) (Table 1.1).
However, it has long been recognized that even a severe defect in cell proliferation may not impair
plant development in a dramatic fashion (Haber 1962). This is because cell division frequently
appears to be inversely correlated with cell expansion. A larger number of cells resulting from
increased cell division activity may be compensated for by a smaller cell size, leaving the tissue/
organ as a whole largely unaffected. One of the most convincing examples of compensatory regu-
lation between cell division activity and cell size is represented by the development of the leaf in
Arabidopsis (Inze and De Veylder 2006; John and Qi 2008; Massonnet et al. 2011; Powell and
Lenhard 2012). This kind of compensatory effect brings into question the basic role of cell cycle
regulation in driving plant growth and raises concerns about designing strategies for plant improve-
ment through the manipulation of cell cycle–controlling genes (Inze and De Veylder 2006; John
and Qi 2008; Sabelli et al. 2013). If cell-autonomous effects, based on the alteration of cell cycle
regulation, can be dampened or even entirely swamped by counteracting tissue/organ-wide changes
in cell size, should we expect to modify yield parameters, such as those impacting biomass produc-
tion, by manipulating genes controlling the cell cycle? The debate between the so-called cellular
theory (cell-autonomous role of the cell cycle in driving growth) and the organismal theory (cell
proliferation follows a developmental plan) is still largely unresolved, and it seems sensible that
this view represents too a polarized way of framing the central question concerning the role of the
cell cycle in growth and development (Beemster et al. 2003). In this chapter, several examples have
been provided that illustrate how manipulation of the cell division cycle in crop plants can impact
yield components, such as seed and fruit development, plant architecture and size, and the interac-
tion with symbiotic and parasitic organisms. Regulation of the cell cycle is fundamental for many
developmental programs, and there is the growing realization that, far from strictly representing the
mechanism for producing cells, it is embedded in a highly complex web of context-specific path-
ways that spans metabolism, physiology, and genetic and epigenetic processes, to mention just a few.
Thus, it would be sensible to assess the impact of the cell cycle on crop production on a case-by-case
basis, relying on detailed knowledge of the specific system under consideration.
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2 Seed Dormancy, Germination,
and Seedling Recruitment
in Weedy Setaria
Jack Dekker
CONTENTS
2.1 Nature of Weed Seeds and Seedlings......................................................................................34
2.1.1 Weed Complexity........................................................................................................34
2.1.1.1 Complexity.................................................................................................... 35
2.1.1.2 Self-Similarity, Self-Affinity........................................................................ 35
2.1.1.3 Self-Organization, Self-Assembly................................................................ 35
2.1.2 Biological Communication.......................................................................................... 36
2.1.3 General Nature of Weeds, the Specific Nature of Weedy Setaria.............................. 36
2.2 Nature of Weedy Setaria Seed–Seedling Life History........................................................... 37
2.2.1 Seed–Seedling Life History........................................................................................ 39
2.2.1.1 Threshold Events........................................................................................... 39
2.2.1.2 Germination and Seedling Emergence......................................................... 39
2.2.2 Nature of Setaria: Spatial Structure and Temporal Behavior.....................................40
2.2.3 Setaria Model System of Exemplar Species................................................................40
2.3 Setaria Spatial Structure......................................................................................................... 41
2.3.1 Plant Morphological Structure.................................................................................... 42
2.3.1.1 Seed Morphology.......................................................................................... 42
2.3.1.2 Plant Morphology......................................................................................... 45
2.3.2 Plant Genetic Structure................................................................................................ 47
2.3.2.1 Genotype Structure....................................................................................... 47
2.3.2.2 Population Genetic Structure........................................................................ 48
2.4 Setaria Seed–Seedling Life History Behavior........................................................................ 51
2.4.1 Life History Behaviors: Functions, Traits, and Information....................................... 51
2.4.2 Seed Formation and Dormancy Induction.................................................................. 51
2.4.2.1 Inflorescence and Flowering......................................................................... 52
2.4.2.2 Mating and Fertilization............................................................................... 55
2.4.2.3 Seed Formation and Embryogenesis............................................................. 56
2.4.2.4 Seed Fecundity and Plasticity....................................................................... 62
2.4.3 Seed Rain Dispersal.................................................................................................... 63
2.4.3.1 Seed Germinability–Dormancy Heteroblasty: Dispersal in Time............... 63
2.4.3.2 Soil Seed Pool Formation: Dispersal in Space.............................................66
2.4.3.3 Dispersal and Local Adaptation: Evolutionary History of Setaria
Invasion......................................................................................................... 67
2.4.4 Seed Behavior in the Soil............................................................................................ 68
2.4.4.1 Control of Seed Germinability..................................................................... 69
2.4.4.2 Seed Behavior............................................................................................... 75
33
2.4.5 Seedling Recruitment.................................................................................................. 77

2.4.5.1 Seedling Emergence and Community Assembly......................................... 77
2.4.5.2 Patterns of Seedling Emergence................................................................... 77
2.4.5.3 Seed Germinability–Dormancy Heteroblasty Blueprints Seed
Behavior in the Soil...................................................................................... 79
2.5 Setaria Life History as a Complex Adaptive Soil–Seed Communication System..................84
2.5.1 Forecasting Setaria Seed Behavior: FoxPatch............................................................84
2.5.1.1 Seed State and Process Model...................................................................... 85
2.5.1.2 Soil Signal Controlling Seed Behavior: Oxy-Hydro-Thermal Time............ 85
2.5.1.3 Schema of Intrinsic Setaria sp. Seed Traits.................................................. 87
2.5.1.4 Germinability–Dormancy Induction............................................................ 88
2.5.1.5 Rules for Individual Weedy Setaria sp. Seed Behavior................................ 88
2.5.2 Setaria Soil–Seed Communication System.................................................................90
2.5.2.1 Shannon Communication System for Weedy Setaria Seed–Seedling
Life History...................................................................................................90
2.5.2.2 Setaria Soil–Seed Communication Transmission Algorithm......................92
2.5.3 Seed Memory and Adaptive Evolution........................................................................92
2.6 Summary................................................................................................................................. 93
References.........................................................................................................................................94
2.1 NATURE OF WEED SEEDS AND SEEDLINGS

Present knowledge of the behavior of invading species, both successful and unsuccessful, suggests that
it is in phases of germination and seedling establishment that their success or failure is most critically
determined and it would seem reasonable therefore, to look particularly closely at seeds and seedlings
of invading species. The considerations set out in this paper lead one to expect that it is in properties
such as seed number, seed size, seed polymorphisms, and precise germination requirements that the
most sensitive reactions of a species to an alien environment are likely to occur. (Harper, 1964)
The nature of weeds is a complex adaptive system (CAS). Weed life history is an adaptable, change-
able system in which complex behaviors emerge as a consequence of structural self-similarity and
behavioral self-organization.
The nature of weeds is an environment–biology communication system. Biology is informa-
tion. Information comes via evolution, an ongoing exchange between organism and environment.
Information is physical. Biology is physical information with quantifiable complexity.
2.1.1 Weed Complexity
The plant population that is found growing at a point in space and time is the consequence of a catena
of past events. The climate and the substrate provide the scenery and the stage for the cast of plant and
animal players that come and go. The cast is large and many members play no part, remaining dormant.
The remainder act out a tragedy dominated by hazard, struggle and death in which there are few survi-
vors. The appearance of the stage at any moment can only be understood in relation to previous scenes
and acts, though it can be described and, like a photograph of a point in the performance of a play, can
be compared with points in other plays. Such comparisons are dominated by the scenery, the relative
unchanging backcloth of the action. It is not possible to make much sense of the plot or the action as it is
seen at such a point in time. Most of our knowledge of the structure and diversity of plant communities
comes from describing areas of vegetation at points in time and imposing for the purpose a human value
of scale on a system to which this may be irrelevant. (Harper, 1977)
A CAS is a dynamic network of many interacting adaptive agents acting in parallel (Axelrod and
Cohen, 1999). The overall system behavior is a result of a huge number of decisions, made every
moment, by many individual agents acting and reacting in competition and cooperation to what
Seed Dormancy, Germination, and Seedling Recruitment in Weedy Setaria 35
other agents are doing in which control is highly dispersed and decentralized. A CAS is a complex,
self-similar collectivity of interacting adaptive agents with emergent and macroscopic properties.
CASs are open, and defining system boundaries is difficult or impossible. CASs operate far from
equilibrium conditions, and a constant flow of energy is needed to maintain the organization of the
system. CASs are living, adaptable, changeable systems in which complexity, self-similarity, and
self-organization occur.
2.1.1.1 Complexity
Complexity arises in a system composed of many elements in an intricate arrangement. The numer-
ous elements interact with each other by which numerous relationships are formed among the ele-
ments. The relationship among system parts is differentiated from other parts outside of the system.
The first key principle of CAS behavior and evolution is that the basic system units are agents
(e.g., phenotypes, strategic traits, and cells) that scan/sense/perceive their environment (opportunity
space–time, Dekker, 2011b) and develop schema/plans/traits representing interpretive and action
rules (e.g., seed heteroblasty). These schema/plans/traits are subject to change and evolution. Agents
are a collection of properties, strategies, and capabilities for interacting with local opportunity and
other agents. Agents and the complex system are adaptable, with a high degree of adaptive capac-
ity providing resilience to disturbance. The number of elements in a CAS is large such that a con-
ventional description is impractical and does not assist in understanding the system (e.g., Colbach
et al., 2001a,b). The second key principle of CAS behavior and evolution is that system order is
emergent, not predetermined. Emergence is the way complex systems and patterns arise out of a
multiplicity of relatively simple interactions. Interactions are nonlinear: small causes can have large
effects (e.g., pleiotropy in triazine-resistant plants; Dekker, 1992, 1993). Interaction is physical or
involves exchange of information. Systems emerge from the moment-to-moment decisions made by
many players choosing among very many options at each time (Waldrop, 1994). Emergence is the
arising of novel (even radical) and coherent structures, patterns, and properties during the process
of self-organization in complex systems. The third key principle of CAS behavior and evolution is
that system history is irreversible; the system future is unpredictable. All CASs have a history, they
evolve, and their past is coresponsible for their present behavior, a Markov process.
2.1.1.2 Self-Similarity, Self-Affinity
CASs exhibit self-similarity when a component or an object of the system is exactly or approximately
similar to a part of itself. In complex adaptive weed systems, self-similarity is revealed in several ways.
Self-similarity is expressed in phenotypic plasticity within an individual plant in response to local
opportunity. Plastic responses to local opportunity include variation in the numbers and architecture
of the plant body. Similar modular plant units are repeated at several levels of spatial organization:
modular shoot branching or tillering; leaves and photosynthesizing cells; and modular root branches,
roots, root hairs, and absorptive cells. Self-similar phenotypes are expressed in the temporal popula-
tion structure of a species in a local deme as well as in the continuous interacting local-to-global popu-
lations of the species’ metapopulations. Phenotypic traits in the individual are self-similar in variants
produced by parent plants to achieve slightly different strategic roles in local adaptation (e.g., seed
heteroblasty). These strategic traits in turn are self-similar as preadaptations, exaptations, when those
phenotypic traits confront new evolutionary situations in which function can change.
2.1.1.3 Self-Organization, Self-Assembly
“… (there exists a) distinction between planned architecture and self-assembly.” “The key point is that
there is no choreographer and no leader. Order, organization, structure – these all emerge as by-products
of rules which are obeyed locally and many times over, not globally. And that is how embryology works.
It is done by local rules, at various levels but especially the level of the single cell. No choreographer. No
conductor of the orchestra. No central planning. No architect. In the field of development, or manufacture,
the equivalent of this kind of programming is self-assembly.” (Dawkins, 2009)
Self-organization in CASs is the process wherein a structure or global pattern emerges in a system
solely from numerous local parallel interactions among lower-level components. Self-organization
is achieved by elements distributed throughout the system, without planning imposed by a central
authority or external coordinator. The rules specifying interactions among components are executed
using only local information (without reference to global patterns). It relies on multiple interactions
exhibiting strong dynamical nonlinearity; it may involve positive and negative feedback, and a bal-
ance of exploitation and exploration. Self-organization is observed in coherence or correlation of
the integrated wholes within the system that maintain themselves over some period of time. There
exists a global or macro property of “wholeness” (Corning, 2002).
2.1.2 Biological Communication
What lies at the heart of every living thing is not a fire, not a warm breath, not a “spark of life.” It is
information, words, instructions… If you want to understand life, don’t think about vibrant, throbbing
gels and oozes, think about information technology. (Dawkins, 1986)
The nature of weeds is an environment–biology communication system. Biology is information.

Evolution itself embodies an ongoing exchange of information between organism and environment.
For biology, information comes via evolution; what evolves is information in all its forms and trans-
forms. Information is physical. Biology is physical information with quantifiable (Kolmogorov)
complexity. The gene is not the information-carrying molecule, the gene is the information.
Information in biological systems can be studied. An important problem in science is to discover
another language of biology, the language of information in biological systems. “The information
circle becomes the unit of life. It connotes a cosmic principle of organization and order, and it pro-
vides an exact measure of that” (Loewenstein, 1999). This information circle for weedy plants is the
predictable developmental events of the annual life history.
Information theory was developed by Claude E. Shannon (Shannon and Weaver, 1949) to find
fundamental limits on signal-processing operations such as compressing data and on reliably stor-
ing and communicating data. Since its inception, it has broadened to find applications in statistical
inference, networks (e.g., evolution and function of molecular codes, and model selection in ecology),
and other forms of data analysis. A communication system of any type (e.g., language, music, arts,
human behavior, and machine) must contain the following five elements (E) (Figure 2.1):
A Shannon communication system includes these elements, as well as the concepts of message
and signal. A message is the object of communication; being a vessel that provides information,
it can also be this information; its meaning is dependent upon the context in which it is used. A
signal is a function that conveys information about the behavior or attributes of some phenomena
in communication systems; any physical quantity exhibiting variation in time or variation in space
is potentially a signal if it provides information from the source to the destination on the status of a
physical system or conveys a message between observers, among other possibilities.
2.1.3 General Nature of Weeds, the Specific Nature of Weedy Setaria

The nature of communities is revealed with a complete phenotype life history description of each plant
species in a local community. Local plant community structure and behavior is an emergent property
of its component species. Community dynamics emerges from the interacting life histories of these
self-similar, self-organizing specific components. Understanding an individual weed species in detail
can provide the basis of comparison among and within weed species of a local plant community. The
challenge is to discover the qualities of each member of the weed–crop community, the nature and
variation of species traits used to exploit local opportunity. These insights provide a deeper, specific
understanding of biodiversity responsible for community assembly, structure, and (in)stability.
It is in the nature of weedy Setaria to process ambient environmental information as a communi-
cation system in the process of life history seizure and exploitation of local opportunity. The setting
Channel
Information
source Transmitter Receiver Destination
Message Signal Received Message

signal
Noise
source
Communication Element Description

1. Information source Entity, person, or machine generating the message (characters, math function)
2. Transmitter Operates on the message in some ways: it converts (encodes) the message to produce
a suitable signal
3. Channel The medium used to transmit the signal
4. Receiver Inverts the transmitter operation: decodes message or reconstructs it from the signal
5. Destination The person or thing at the other end
FIGURE 2.1 Schematic diagram of Shannon information communication system. (From Shannon, C.E. and
Weaver, W., The Mathematical Theory of Communication, University of Illinois Press, Urbana, IL, p. 31, 1949.)
of weed evolution, the stage upon which diversifying evolution takes place, is a local population of
variable phenotypes of a weed species in a particular locality. The nature of a particular locality is
defined by the opportunity space–time available to the weed population to seize and exploit, and to
survive and reproduce. Opportunity space–time for a population is the locally habitable space for
an organism at a particular time. It is defined by its available resources (e.g., light, water, nutrients,
and gases), pervasive conditions (e.g., heat and terroir), disturbance history (e.g., tillage, herbicides,
and frozen winter soil), and neighboring organisms (e.g., crops and other weed species) with which
it interacts (Dekker, 2011b). It is the local niche, the niche hypervolume.
The weedy Setaria species-group provides an exemplar of widely distributed species whose
complex life history behavior arises from multiple interacting traits. The specific nature of Setaria
seed–seedling biology provides strong inferences of the nature of all weeds, elucidating the range of
adaptations used by individual species to seize and exploit opportunity in agrocommunities.
2.2 NATURE OF WEEDY SETARIA SEED–SEEDLING LIFE HISTORY

The nature of weedy Setaria seed–seedling life history can be described as a complex adaptive
soil–seed communication system arising from its component functional traits. Complex seed struc-
tures and behaviors emerge as a consequence of self-organization of self-similar parts forming this
adaptive soil–seed communication system. Functional traits controlling seed–seedling behavior are
physical information that have evolved in ongoing communication between organism and environ-
ment leading to local adaptation.
The nature of weedy Setaria life history emerges when self-similar plant components self-
organize into functional traits possessing biological information about spatial structure and
Self-Similar Self-Organization: Biological Emergent

Components Functional Traits Information Behavior
Spatial Structure
Plant Morphological Embryo Somatic Environment– Induction of
structure Seed Aleurone polymorphism plant–seed differential
envelopes layer communication dormancy–
Caryopsis system germinability
coat capacity in
Hull individual seeds
Glumes of inflorescence
Seed Somatic polymorphism Light capture
Inflorescence Spikelet Phenotypic plasticity architecture
Fascicle
Panicle
Tiller 1°
2°
3°
Individual plant
Genetic Local population (deme) Self-pollination Gene flow Control of genetic
Intraspecific variants mating system communication novelty
Species associations Polyploidization system Trait dispersal for
speciation local adaptation
Metapopulations Trait reservoir
Temporal Structure
Life Seed Time of embryogenesis of Seed germination Environment– Heterogeneous
history formation individual seeds on heteroblasty: plant–seed seed
behavior inflorescence differential communication germinability
Time of tillering development: hull, system capacity
inflorescence branching placental pore, TACL
membrane; Oxygen
scavenger protein
Seed dispersal Time of abscission of Invasion and Seed dispersal in
individual seeds colonization space
Seed pool Individual seeds from Hull topography: Soil–seed Seed dispersal in
behavior in several inflorescences, water film oxygen time
soil plants, years oxygenation communication
Placental pore: water system Annual dormancy–
film channel germination
TACL membrane: O2 cycling in soil
transport
Seedling Oxygen scavenger: Locally adapted
emergence embryo O2 regulation emergence
patterns
FIGURE 2.2 Plant structure (spatial, temporal) and emergent behavior examples of self-similar components,
self-organization functional traits, and communication-information in the life history of weedy Setaria.
temporal behavior. Spatial plant structure extends through the morphological (embryo to plant) and
genetic (plant to metapopulation). Temporal life history behavior begins in anthesis/fertilization
and embryogenesis; development continues with inflorescence tillering, seed dispersal in space
and time, and resumption of embryo growth with seedling emergence. The interaction of self-
similar plant components leads to functionally adapted traits, self-organization. These heritable
functional traits are the physical reservoirs of information guiding life history development, emer-
gent behavior. Information contained in structural and behavioral traits is communicated directly
between seed and soil environment during development. The specific nature of Setaria is eluci-
dated in Figure 2.2.
2.2.1 Seed –Seedling Life History

2.2.1.1 Threshold Events
Several discrete threshold events characterize Setaria summer annual life history. These threshold
events provide time points allowing individual comparison in the elucidation of development. The
threshold life history events begin and end with successful fertilization, and continue with seed
abscission, germination, and seedling emergence when the new vegetative plant develops to fertil-
ization of new progeny.
2.2.1.2 Germination and Seedling Emergence

One of the most important events in a plant’s life history is the time of seed germination and
seedling emergence, the resumption of embryo growth and plant development. Emergence timing
is crucial; it is when the individual plant assembles in the local community and begins its struggle
for existence with neighbors. Resumption of growth at the right time in the community allows
the plant to seize and exploit local opportunity at the expense of neighbors, allowing development
to reproduction and replenishment of the local soil seed pool at abscission. Soil seed pools are
the source of all future local annual weed infestations and the source of enduring occupation of
a locality.
Community assembly of crops and weeds in agroecosystems, and its consequences, is a complex
set of phenomena (Dekker et al., 2003). Accurate predictions of the time of weed interference, weed
control tactic timing, crop yield losses due to weeds, and replenishment of weed seed to the soil
seed pool require information about how agricultural communities assemble and interact. Despite
attempts at description (e.g., Booth and Swanton, 2002), little is known about the rules of commu-
nity assembly. Their elucidation may remain an empirically intractable problem.
Despite this, there exist two opportunities to understand agroecosystem community assem-
bly during the recruitment phase (e.g., seedling emergence) that predicate future interactions
with other plants. The first advantage derives from the annual disturbance regime in agricultural
fields that eliminates aboveground vegetation (e.g., winter kill, tillage including seedbed prepara-
tion, and early-season herbicide use). Understanding community assembly is most tractable when
starting each growing year with a field barren of aboveground vegetation and possessing only
dormant underground propagules (e.g., soil seed and bud pools), a typical situation in much of
world agriculture.
The second advantage derives from the observation that the time of emergence of a particular
plant from the soil relative to its neighbors (i.e., crops, other weeds) is the single most impor-
tant determinate of subsequent weed control tactic use, competition, crop yield losses, and weed
seed fecundity. Seedling recruitment is the first assembly step in these disturbed agricultural
communities and is therefore the foundation upon which all that follows is based. Information
predicting recruitment therefore may be the single most important life history behavior in weed
management.
2.2.2 Nature of Setaria: Spatial Structure and Temporal Behavior

Any complete description of an organism includes the concept of phenotypic function, which con-
sists of two universes: the physical (the quantitative and formal structure) and the phenomenal
(qualities that constitute a “world”) (Sacks, 1998). The nature of Setaria weed seeds and seedlings
is presented in this review as the story of plant spatial structure (genetic and morphological) and
temporal life history behaviors, instigated by functional traits, and resulting in local adaptation and
biogeography. The weedy phenotype can be described in terms of its morphological structure, the
embryo and specialized structures forming the seed, and the self-similar shoot tiller architecture
on which reproductive inflorescences arise. The individual self-similar seeds form local popula-
tions (the deme) that aggregate, self-organize, into the global metapopulation. The genetic and mor-
phological structure provides an enduring foundation for the evolution of life history behavioral
adaptation: plant functions, functional traits, and regulation of function. The behavioral regulation
of the Setaria phenotype is an emergent property arising from the interaction of several morpho-
physiological seed compartments (embryo, caryopsis, hull, spikelet, fascicle, and inflorescence til-
ler). The emergent property of these interacting, self-organizing, self-similar components is seed
heteroblasty: the abscission of individual seeds from a panicle, each with different inherent dor-
mancy–germination capacities. Seed heteroblasty is the physical information forming memory of
successful past seedling emergence times appropriate to a locality. It is the hedge-bet for seizing and
exploiting future opportunity space–time.
2.2.3 Setaria Model System of Exemplar Species

One major biological question is how different species become unique organisms. To understand
the origins of adaptation… it is particularly useful to investigate multiple species, especially when
they have independently evolved an ability to prosper under similar environmental conditions. With
the sequencing of the Setaria genome, evolutionary geneticists now have an annual, temperate, C4,
drought- and cold-tolerant grass that they can comprehensively compare to other plants that have or
have not evolved these adaptations… particular traits were targeted… for biotechnical improvement,
namely drought tolerance, photosynthetic efficiency and flowering control. With a completed genome
sequence, the door is now open for further development of Setaria as a model plant. This model can be
applied to understanding such phenomena as cell wall composition, growth rate, plant architecture and
input demand that are pertinent to the development of biofuel crops. In addition to its use as a panicoid
model for switchgrass, pearl millet, maize and Miscanthus, Setaria has the model characteristics that
will encourage its development as a study system for any biological process, with pertinence to the
entire plant kingdom and beyond. (Bennetzen et al., 2012)
The weedy Setaria species-group (green foxtail, S. viridis; bristly foxtail, S. verticillata; giant fox-
tail, S. faberi; yellow foxtail, S. pumila; and knotroot foxtail, S. geniculata) (Rominger, 1962) is
presented herein as a weed exemplar of a complex adaptive soil–seed communication system. The
nature of the weedy Setaria species-group as a complex communication system is an exemplar in
the sense of Kuhn (1962), “… concrete problem-solutions.…”
Setaria provides a model system for seed germination, plant architecture, genome evolution,
photosynthesis, and bioenergy grasses and crops. It is used “… as an experimental crop to inves-
tigate many aspects of plant architecture, genome evolution, and physiology in the bioenergy
grasses… whose genome is being sequenced by the Joint Genome Institute (JGI) of the Department
of Energy.” “… it is closely related to the bioenergy grasses switchgrass (Panicum virgatum), napier-
grass (Pennisetum purpureum), and pearl millet (Pennisetum glaucum), yet is a more tractable
experimental model because of its small diploid genome… and inbreeding nature” (Doust et al., 2009).
Setaria provides a “… potentially powerful model system for dissecting C4 photosynthesis…” (Brutnell
et al., 2010) and “… provide novel opportunities to study abiotic stress tolerance and as models for
bioenergy feedstocks” (Li and Brutnell, 2011). A high-quality reference genome sequence has been
generated for S. italica and S. viridis (Bennetzen et al., 2012).
The weedy Setaria species-group (Darmency and Dekker, 2011; Dekker, 2003, 2004a; Dekker
et al., 2012a,b), S. glauca and S. verticillata (Steel et al., 1983), and S. viridis (Douglas et al., 1985)
has been reviewed.
The author has collected a very large (more than 3000 accessions) Setaria spp.-group
germplasm collection stored in the Weed Biology Laboratory, Agronomy Department,
Iowa State University, Ames. It includes Japanese salt-tolerant germplasm (e.g., Dekker and
Gilbert, 2008), herbicide-resistant biotypes (e.g., Thornhill and Dekker, 1993), systematic
pretransgenic crop era US (notably Iowa) collections, and north temperate world populations
(e.g., Wang et al., 1995a,b).
2.3 SETARIA SPATIAL STRUCTURE

The weedy Setaria phenotype can be described in terms of the spatial structure of its seed and
plant morphology, and by its genotypes and population genetic structure (Figure 2.3). This spatial
structure extends from the cells and tissues of the embryo axes, the surrounding seed envelopes, the
individual seed and then plant, the local deme, and ending with the aggregation of local populations
forming the global metapopulation.
Phenotypic traits in the individual are self-similar in variants produced by parent plants to achieve
slightly different strategic roles in local adaptation. These strategic traits in turn are self-similar as
preadaptations, exaptations, when those phenotypic traits confront new evolutionary situations in
which function can change. Setaria self-organization arises from the interaction of self-similar
plant parts (e.g., seed polymorphism and plant architecture) forming functional traits (e.g., seed het-
eroblasty and seed tillering inflorescences) by means of environment–plant–seed communication
Spatial Structure
Self-
Organization: Biological Emergent
Self-Similar Components Functional Traits Information Behavior
Plant Morphological Caryopsis Embryo Somatic Environment– Induction of
structure endosperm polymorphism plant–seed differential
Placental pore communication dormancy–
Seed Aleurone layer system germinability
envelopes Caryopsis coat capacity in
Hull individual seeds
Glumes of inflorescence
Individual seed Light capture
Inflorescence Spikelet Somatic architecture
Fascicle polymorphism
Panicle
Tiller Primary Phenotypic
2° plasticity
3°
Individual plant
Genetic Local population (deme) Self-pollination Gene flow Control of genetic
mating system communication novelty
Intraspecific variants system
Species associations Polyploidization Trait dispersal for
speciation local adaptation
Metapopulations Trait reservoir
FIGURE 2.3 Plant spatial structure and emergent behavior examples of self-similar components, self-
organization functional traits, and communication-information in the life history of weedy Setaria.
(e.g., plastic tiller shoot development and fascicle-spikelet seed number plasticity). These emergent
properties include tiller shoot development and growth of shoots forming plant architecture for
capturing light for photosynthesis and for photoperiod–seed signal communication influencing dif-
ferential dormancy induction in tiller inflorescences. Emergent morphological properties in Setaria
also include dispersal of heteroblastic seed with differential germinability capacities for fine-scale
timing of germination and seedling emergence allowing Setaria to seize and exploit local opportu-
nity space–time at the expense of their neighbors.
2.3.1 Plant Morphological Structure

Plant morphological structure is the emergent property of Setaria seed and plant self-organization
by means of functional traits for plant phenotypic plasticity (e.g., tillering inflorescence branching
and fertile flower number per spikelet) and seed somatic polymorphism (e.g., seed heteroblasty).
The Setaria seed consists of self-similar structural components nested as integrated, self-orga-
nized compartments: hull > caryopsis > embryo (Dekker, 2003; Dekker et al., 1996). The Setaria
plant consists of self-similar structural parts integrated in a hierarchy forming the architecture of
the plant body: shoot branches (tillering panicle inflorescences) > modular leaves and photosynthe-
sizing cells > root branches with modular roots, root hairs, and absorptive cells. Plant morphology
provides the physical and physiological means for local adaptation of Setaria phenotypes.
2.3.1.1 Seed Morphology
The life cycle of the Setaria seed begins with fertilization and embryogenesis: development of the
embryo, endosperm, and living tissues within the enveloping caryopsis; development of the placen-
tal pore (PP) channel connecting the exterior seed and environment to the living interior; and devel-
opment of the enveloping hull and glumes of the seed exterior (Dekker, 2000). Self-similar parental
tissues surround, protect, and modulate the behavior of living interior zygotic tissues. Three sepa-
rate nuclear genomes interact and produce the tissues that compose the Setaria seed. Parental tis-
sues (2N) include the seed glumes, hull (palea, lemma), many of the crushed layers forming the
caryopsis coat (CC), and vascular remnants and residual tracheary elements at the basal abscission
area, the PP (Rost, 1973, 1975). The zygotic tissues include those of the endosperm (3N; aleurone
and aleurone transfer cells) and the embryo (2N).
2.3.1.1.1 Caryopsis
The caryopsis of Setaria seed is enveloped by the hull and consists of dead and living, parental and
zygotic, tissues. The caryopsis surrounds the living components of the seed: endosperm, aleurone
layer, transfer aleurone cell layer (TACL) membrane, and the embryo (Dekker, 2000; Haar et al.,
2012). Within the living caryopsis interior is also a putative oxygen-scavenging, heme-containing
protein that sequesters oxygen to buffer the embryo against premature germination (Dekker and
Hargrove, 2002; Sareini, 2002).
2.3.1.1.1.1 Embryo Within the caryopsis is the embryo: scutellum, coleoptile (CPT), and coleo-
rhiza (CRZ) (Dekker, 2000; Haar et al., 2012). The exterior PP and the interior TACL are located in
close proximity to the scutellum and coleorhizal tissues of the embryo at the basal end of the seed.
A cementing substance causes the outer epidermis of the CRZ and other embryo parts to adhere
to the aleurone layer (Rost and Lersten, 1970, 1973). This intimate contact provides a continuous
route for the uptake of gas-saturated water from the exterior. The embryo is approximately one half the
caryopsis length and is found near the surface of the caryopsis covered only by the CC (Haar et al., 2012).
The embryo scutellum surrounds the embryonic axis below and along the margins, forming a cup-
like structure in which the axis lies (Rost, 1973). Before water imbibition, the caryopsis is dry, the
CC wrinkled, and the embryo sunken within the endosperm.
2.3.1.1.1.2 Endosperm The first zygotic tissue that gas-saturated water contacts is the endo-
sperm, which is entirely sealed from the outside by the CC, except in the PP region (Dekker, 2000).
2.3.1.1.1.3 Aleurone Layer, Transfer Aleurone Cell Layer Membrane The outermost layer of
the endosperm is the aleurone layer, which is continuous around the caryopsis (Dekker, 2000). It
consists of thick tabular cells, each cell is flattened, somewhat rectangular in surface view, and
25–50 μm in length (Rost, 1971a,b). A thick primary cellulose wall encloses each cell. The matrix of
the aleurone cells appears as a gray network intermeshed between storage materials. This cell layer
has an abundance of protein bodies of various types, lipid bodies (oil droplets or spherosomes), as
well as plastids, mitochondria, and other membrane structures, but little starch (Rost and Lersten,
1973). This outermost layer of endosperm is known to produce enzymes and is the site where ger-
mination is first initiated. The aleurone layer is continuous around entire caryopsis, but adjacent to
the placental pad, the aleurone cells are strikingly different in appearance: the transfer aleurone
cells. Transfer aleurone cells occur near the base of the caryopsis, adjacent to the end of the CRZ
where the seed attaches to the parent plant inflorescence. The transfer aleurone endosperm cells are
enlarged, approximately columnar, and somewhat elongate perpendicular to the fruit coat (Rost and
Lersten, 1970). The thickened portion of the cell wall appears heterogeneous, with that part clos-
est to the middle lamella having a fibrous or porous appearance. These specialized aleurone cells
have thick walls bearing ingrowths on the outer radial and outer tangential walls, which extend into
the cell protoplasm. These ingrowths form a labyrinth, the plasmalemma follows the contours of the
ingrowths, thereby significantly increasing the membrane surface area of each cell. Internally, the
wall has a porous, sponge-like appearance. The wall ingrowths sometimes are very deeply lobed
and convoluted. The inner radial and inner tangential parts of the wall lack these ingrowths and
have a middle lamella and typical appearing primary wall. These inner radial and tangential cell
walls have little or no ingrowths, indicating that they are not receptive to outside solute transport.
These aleurone transfer cell wall ingrowths appear similar to those of certain of the transfer cells
described by Pate and Gunning (Gunning, 1977; Gunning and Pate, 1969; O’Brien, 1976; Pate and
Gunning, 1972). These specialized cells have already been described as playing a role in mature
seed of other species (Zee, 1972; Zee and O’Brien, 1971).
2.3.1.1.1.4 Caryopsis Coat Immediately beneath the tracheary elements of the placental pad is a
thick layer of dark, dense, suberized cells, the CC. The CC surrounds the embryo and endosperm. It
appears as a filmy cuticle layer, shiny, oily to the touch, and gray with dark spots and 3–10 μm thick.
Seen in section, it is a gossamer-like film, analogous to the cuticle. The coat’s speckled appearance
derives from the degradation contents in epidermis pericarp cells. The structure of the coat is a
complex of many layers of parental origin formed from crushed cells in various stages of degra-
dation (nucellus, integuments, and pericarp). The expansion of the developing caryopsis causes
these cells to become crushed, thereby forming the complex CC. At maturity, the CC is water- and
gas-impermeable, except at the proximal (basal) end of the caryopsis (the placental pad and pore
region). Entry of water and dissolved gases into the caryopsis occurs only through the PP and TACL
membrane. Entry of water is never restricted through this region of the caryopsis.
2.3.1.1.2 Placental Pad and Pore

At the basal end of the foxtail seed is the site where water and dissolved gases enter the PP. This
hull structure is rounded and hard with a soft center consisting of remnants of the parental vas-
cular connection into seed interior, degraded strands of former xylem and phloem tissues (Rost,
1971b). During the ontogeny of the caryopsis, a single placental vascular bundle from the par-
ent panicle supplies nutrients and water to the developing ovule. The vascular bundle enters the
ovule in a proximal position on the bottom surface of the developing caryopsis. The nonliving
portion of the PP includes residual vascular elements and tracheary elements left from the pedicel
connection of the seed to the parental panicle (Rost, 1972). The morphology of the PP is identi-
cal in dormant and nondormant caryopsis structures. At the base of the caryopsis is a thickened
region called the placental pad. The dark necrotic contents of the placental pad layers make the
structure appear as a dark oval-shaped area (about 0.2 mm) when seen from outside (Dekker
et al., 1996). The CC in the placental pad and PP region is different from that around the rest of
caryopsis. The transfer aleurone cells rest immediately adjacent to the last layer of pad cells. The
placental pad may serve as a second spongy filter, yet allow free entry of gas-saturated water.
At caryopsis maturity, a thick, dark oval pad remains in the position occupied by the placental
bundle. In longitudinal section, this appears as an elongated multilayered placental pad. The
placental pad of the caryopsis shows evidence of reddish coloration, a morphological indicator of
physiological maturity at abscission of the seed (Dekker et al., 1996). The flared tissue beneath
the placental pad is the region where the caryopsis is connected to the palea. This region is the
only water–gas entrance to seed (unless physically damaged) and may serve as the first spongy
filter of debris, fungal spore, and bacteria entry into the seed. The loose arrangement of the pad
cells and the presence of a large number of pits allow for a relatively unimpeded flow of water and
solutes into the transfer aleurone cells.
2.3.1.1.3 Hull
The seed hull surrounds the caryopsis and consists of the concave lemma and the palea (Haar et al.,
2012). Both these nonliving structures have ridges on their surface; in some species they are trans-
verse, and in others they follow the longitudinal axis. These ridges appear like the drainboard of
a sink and may provide drainage channels for liquids, gases, or solid particles in the soil adjacent
to the seed. Glume and hull surface ridges may function together to both to mix water and air at,
and funnel gas-laden water into, the PP (Donnelly et al., 2012). The lemma and palea surround the
caryopsis and together form a hard protective covering commonly referred to as the hull (Gould,
1968; Rost, 1975). A door or lid-like structure known as the germination lid is found at the proximal
end of the lemma. It is through this structure that the CRZ exits the hull during germination. The
germination lid is attached, hinged to the lemma on one side. The three unhinged sides lack any
physical connection to the adjacent lemma. The hinge provides the only resistance to the opening
of the germination lid.
2.3.1.1.4 Glumes
The outmost envelopes of the foxtail seed are the papery glumes that partially surround the seed
hull (Dekker, 2000). These absorbent structures protect the seed as well as wick and funnel water to
the PP region, their point of attachment to the seed. Ridges on the glumes are often at right angles
to hull (lemma, palea) surface ridges. The fragile glumes detach from the seed sometime after entry
into the soil.
2.3.1.1.5 Seed
The dispersal unit for Setaria is referred to as a seed. The perfect or fertile floret above is often
referred to as the fruit, grain, or seed. Each seed consists of a single floret subtended by a sterile
lemma and two glumes. The fertile floret (seed) of Setaria consists of a hull (tightly joined lemma
and palea) that encloses the caryopsis (embryo and endosperm surrounded by the CC) (Dekker
et al., 1996; Haar et al., 2012; Narayanaswami, 1956; Rost, 1973, 1975). The seed is composed of
an indurate, usually transversely wrinkled, lemma and palea (hull) of similar marking and texture,
which tightly enclose the caryopsis within at maturity. The degree of rugosity of the lemma is a
valuable taxonomic diagnostic character. Rugosity varies from smooth and shiny in foxtail mil-
let (S. viridis, subspecies viridis), to finely ridged in S. viridis, to very coarse rugose seed in other
Setaria species (Rominger, 1962). This rugosity may play a role in soil–seed contact (water and gas
exchange) and seed germination. Seed production is an emergent property of Setaria reproductive
morphology. Seed numbers produced by a Setaria are a function of differences in plant architecture:
shoot tillering–panicle formation, panicle–fascicle branching, and fascicle spikelet–floret develop-

ment. Inherent plastic differences in these reproductive structures among Setaria species determine
the reproductive responses of species and populations to available opportunity in its immediate
environment (Haar et al., 2012).
2.3.1.2 Plant Morphology

The branching architecture of Setaria plants consists of hierarchically organized, nested, structural
sets: tiller culm, panicle, fascicle, spikelet, and floret, as in most grasses. As such, Setaria plant
architecture is the emergent property of self-organized self-similar plant parts. Setaria plant archi-
tecture is plastic and includes the ability to form one or more tillering shoots whose stature and
number are precisely sized to local conditions (Dekker, 2003).
2.3.1.2.1 Panicle Inflorescence
Setaria panicle inflorescences develop at the terminal ends of shoot tillers (Haar et al., 2012). The
inflorescence in the subgenus Setaria is usually narrow, terminal on the culm, very dense, cylindri-
cal, and spicate, with very short branches (fascicles) only a few mm long (Narayanaswami, 1956;
Rominger, 1962; Willweber-Kishimoto, 1962). The fascicles are spirally arranged around the main
axis, each bearing a number of branchlets. Setaria spp. panicles are composed of fascicles, which
consist of spikelet (with florets) and bristle (seta only) shoots (Narayanaswami, 1956; Figure 2.4).
Flowering branches (fascicles) are spirally arranged along the cylindrical inflorescence of the
panicle (Dekker et al., 1996). Most fascicles are composed of several branchlets, with fertile and
nonfertile spikelets subtended by bristles (setae). In the mature inflorescence, the number of fasci-
cles per unit length of panicle axis decreased from the distal to the basal end (Figure 2.5). Flowering
branch density along the axis ranged from 16 fascicles cm−1 at the distal end to 5 fascicles cm−1 at
the basal end of the panicle. This arrangement of fascicles could allow greater light penetration to
those flowers at the basal end of the panicle.
The fascicle–spikelet structure differs among Setaria species. In S. faberi or S. viridis, the num-
ber of fascicles within each panicle varies, a plastic response to a plant’s immediate environment
(Clark and Pohl, 1996). Longer, earlier developing S. faberi or S. viridis panicles often have the most
F1
1°
F2
2° F3
2°
F4
3° 3° F5
BS SS BS
SS
BS
SS SS
Setaria viridis
Setaria pumila Setaria faberi
FIGURE 2.4 Schematic diagram of weedy Setaria species-group reproductive shoot architecture and pani-
cle structure; tiller and panicle types (1°, primary; 2°, secondary; 3°, tertiary): left; fascicle branching (F1–5) on
panicle axis: top, right; fascicle structure and arrangement of bristle (seta) shoots (BS) and spikelet shoots (SS)
along rachilla axis: S. pumila (bottom, middle); S. viridis and S. faberi (bottom, right).
Panicle
16 fascicles cm–1 10
20
30
12 fascicles cm–1
Proportion of panicle length (%)

40
50
60
70
9 fascicles cm–1
80
90
5 fascicles cm–1
100
Leaf collar
Culm
FIGURE 2.5 Number of giant foxtail flowering branches (fascicles) per length (cm) of main axis expressed
as a proportion (%) of the total panicle length.
extensive fascicle branching, as well as more spikelets per fascicle (typically—four to six or more per
fascicle). The plastic response of S. faberi or S. viridis to its immediate environment is also revealed
in the number of spikelets–florets that fully mature, which determines the seed number per panicle
length (seed density). Under favorable conditions, more spikelets are able to develop into seeds, while
under unfavorable conditions, spikelets may abort. S. pumila panicle morphology is different from
that of S. faberi or S. viridis. Only a single, terminal spikelet–floret is found in each S. pumila fascicle
(Clark and Pohl, 1996). This stable, fixed morphology limits the ability of S. pumila to respond in a
plastic way to its environment in terms of seed number relative to that of S. faberi or S. viridis.
2.3.1.2.1.1 Spikelet The basic unit of the Setaria inflorescence is a dorsally compressed, two-
flowered spikelet disarticulating below the glumes and subtended by one to several bristle-like setae
(Hitchcock, 1971; Rominger, 1962). The spikelet consists of the rachilla, a sterile or staminate floret
below, a perfect floret above, and three empty glumes (Li et al., 1935; Willweber- Kishimoto, 1962).
For S. viridis, S. faberi, and S. verticillata, the lower floret is entirely degenerated. In S. glauca, the
lower floret has no pistil; instead, it has three well-developed anthers that open 3–7 days after the
upper floret (Willweber-Kishimoto, 1962). Setaria spikelets are subtended by one to several setae
(stalks of abortive spikelets) that persist after the spikelets disperse (Chase, 1937; Hofmeister, 1868;
Prasada Rao et al., 1987). It is these distinctive setae that give the inflorescence of Setaria spp. its
characteristic appearance (foxtails).
S. faberi has longer panicles than S. viridis. S. viridis panicles have a greater number of seed and
higher seed density than S. pumila. Earlier-developing panicle types were always greater than or
similar to the later-developing panicle type for each of these parameters (Haar et al., 2012).
2.3.1.2.1.2 Fascicle The fascicle, the panicle branch, consists of one to six or more spikelets
amid a cluster of setae (bristles) in a complicated system of branching (Narayanaswami, 1956;
Rominger, 1962). Spikelets are clustered on the rachis that diverges from the main panicle axis. The
number of fertile spikelets per fascicle within each S. faberi or S. viridis panicle is variable (e.g., one
to three) and plastic in response to environmental conditions (Clark and Pohl, 1996). S. glauca fas-
cicles, unlike that of S. viridis or S. faberi, bear only a single spikelet. In the mature inflorescence,
the number of fascicles per unit length of panicle axis decreases from the distal to the basal end
(Dekker et al., 1996). This arrangement of fascicles is a second mechanism allowing greater light
penetration to basal flowers of the panicle.
2.3.1.2.2 Tiller
Panicles that develop at the end of the main shoot are referred to as primary panicles (1°; Figure 2.1)
(Haar et al., 2012). Secondary panicles (2°) arise at the nodes of the primary tiller, and tertiary
panicles (3°) are those that branch laterally from secondary tillers. Developmentally, primary panicles
flower first on a plant, followed by secondary, and then tertiary.
2.3.2 Plant Genetic Structure

Setaria genetic structure includes the genotype structure of individuals within each species as well
as the population genetic structure of those individuals aggregated into local populations, species
associations, and the global metapopulation. The plant genetic structure is an emergent property
of Setaria plant self-organization by means of the functional trait for a self-pollination mating sys-
tem controlling gene flow communication between local populations. Self-similar individual plants
share genes from the local population to the global metapopulation to express the genetic spatial
structure of Setaria. Soil–seed pools of local populations form a physical memory of past successful
genotypes whose functional traits can be shared by gene flow communication between continuous
local populations of the larger global metapopulation. Rare (auto- and allo-) polyploidization events
have resulting in new, reproductive, species (e.g., S. faberi) from which self-organization is respon-
sible for wild-crop–weed, species-group and polyploid cluster species associations.
2.3.2.1 Genotype Structure
Setaria genotype structure includes individual plants and variants of each species.
2.3.2.1.1 Individual Plant
Worldwide there are 125 Setaria species divided among several subgenera, 74 of these species from
Africa (Hubbard, 1915; Rominger, 1962). The taxonomy of the genus is very complex, and an accu-
rate classification has been confounded by the high degree of overlapping morphological characters
both within and between species, and the diverse polyploidy levels within the genus. The genus
Setaria belongs to the tribe Paniceae, subfamily Panicoideae, and family Poaceae (Pohl, 1978).
S. faberi, S. pumila, S. verticillata, and S. viridis (both subspp. italica and viridis) are Eurasian
adventives. The origins of S. geniculata are of particular note. S. geniculata, a perennial, is the only
weedy Setaria native to the New World and very closely resembles S. pumila, an annual species of
Eurasian origin (Rominger, 1962; Wang et al., 1995b). S. geniculata and S. pumila are frequently
misidentified. It has been suggested that the cause of this enigma was an ancient, pre-Columbian
dispersal event westward from the Old to New World (Rominger, 1962). Foxtail millet (S. viridis
subsp. italica) and weedy S. viridis subsp. viridis are subspecies of S. viridis and have continuous
and overlapping genetic variation, evidence of the weedy origins of the crop (Prasada Rao et al.,
1987; Wang et al., 1995a). Relative genetic diversity within each of the four weedy foxtail species is
low to exceedingly low (S. geniculata > S. viridis > S. pumila > S. faberi, monomorphic) in com-
parison to an “average” plant species (Hamrick and Godt, 1990) (Wang et al., 1995a,b). The genus
Setaria is cosmopolitan and can be found on every landmass in the world except the polar regions
(Prasada Rao et al., 1987; Rominger, 1962). In the western hemisphere, Setaria has its center of dis-
tribution in Brazil and radiates poleward north and south (Rominger, 1962). The weedy Setaria spp.
are primarily temperate species but are widely distributed between 45° S and 55° N latitudes (Holm
et al., 1977, 1997; Wang et al., 1995a,b). They are found in every state in the continental United
States and every province in Canada (Lorenzi and Jeffery, 1987). The Eurasian Setaria are rare in
the southwest United States, Mexico, and, more southerly, tropical regions.
2.3.2.1.2 Intraspecific Variation
A number of morphological variants of S. viridis have been named (e.g., S. viridis var. major (Gaud.)
Posp., giant green; S. viridis var. robusta-alba Schreiber, robust white; S. viridis var. robusta-
purpurea Schreiber, robust purple; S. viridis var. pachystachys (Franch et Savat.) Makino et Nemoto;
S. viridis var. vivipara (Bertol.) Parl. (Dore and McNeill, 1980; Hubbard, 1915; Kawano and Miyaki,
1983; Schreiber and Oliver, 1971)), although their taxonomic validity has been questioned
(Dekker, 2003; Wang et al., 1995a). Often the most striking morphological differences among
Setaria arise from biotypes with similar allelic variation (e.g., compare S. viridis subsp. viridis var.
pachystachys and S. viridis subsp. italica race maxima), while genotypic variation can be wide
within nearly identical morphologies (e.g., compare Old World S. pumila and New World S. geniculata).
Many of the morphological variants described as biotypes are in fact extreme examples of continu-
ous characters (e.g., leaf coloration in S. viridis var. robusta-purpurea; Schreiber and Oliver, 1971).
One of the most striking observations of Setaria behavior is the occurrence of phenotypic heteroge-
neity, rather than genetic diversity, among the individuals of a population as a means of exploiting a
locality (Scheiner, 1993; Wang et al., 1995a,b). This phenotypic heterogeneity takes the form of phe-
notypic plasticity and somatic polymorphism in many of its most important traits, especially during
reproduction. Most of the phenotypic variation of Setaria has not been characterized. Only the most
obvious behaviors and morphologies have been described, those characters most apparent in crop
management situations. An increase in herbicide-resistant Setaria variants has been observed. The
resistance mechanisms include those that exclude, as well as metabolically degrade, the herbicides
(e.g., Thornhill and Dekker, 1993; Wang and Dekker, 1995). Physiological variation in dormancy
and germinability exists (Dekker et al., 1996; Norris and Schoner, 1980; Tranel and Dekker, 2002).
Variation in drought tolerance among Setaria has been observed (Blackshaw et al., 1981; Manthey
and Nalewaja, 1982, 1987; Taylorson, 1986). Potentially salt-tolerant genotypes of S. viridis and
S. faberi have been observed along the seacoasts of Japan (Chapman, 1992; Kawano and Miyaki,
1983; author’s personal observation, 1992 and 2000, data not reported). There may exist an intimate
physiological and morphological relationship between drought tolerance, salt tolerance, and seed
dormancy (Dekker and Gilbert, 2008). This is most apparent in extreme habitats where foxtail mil-
let remains an important crop and cultivated cereal (e.g., Central Asia including Afghanistan, India,
sub-Saharan Africa).
2.3.2.2 Population Genetic Structure

The emergent property of interacting local Setaria populations and species associations is the global
metapopulation. Self-similar individual seeds and plants aggregate into local soil–seed pools and
communities across the global landscape to form a Setaria metapopulation. This self-organization
occurs by means of a gene flow communication system within the metapopulation. Low-diversity
species are associated in habitats to exploit overlapping niches, opportunity space–time. Individual
demes are highly differentiated among themselves over the landscape and world metapopulation,
populations finely adapted to local opportunity. Appropriate levels of Setaria biodiversity are
maintained by a conservative, self-pollinating mating system with carefully limited outcrossing for
the introduction of novel traits anticipating changing habitats.
2.3.2.2.1 Local Population (Deme)

The pattern of genetic diversity within an individual weedy Setaria sp. is characterized by unusu-
ally low intrapopulation genetic diversity and unusually high genetic diversity between populations,
compared to an “average” plant (Wang et al., 1995a,b). These two patterns of population genetic
structure appear to typify introduced self-pollinated weeds that are able to rapidly adapt to local
conditions after invasion and colonization. Although relative genetic diversity within each of the
several Setaria species is very low, differences between homogeneous populations are high, indi-
cating a strong tendency for local adaptation by a single genotype. Nearly all populations analyzed
in America consisted of a single multilocus genotype, while more diversity could be found within
European and Chinese populations.
2.3.2.2.1.1 Setaria viridis Genetic bottlenecks associated with founder events may have strongly
contributed to that genetic structure. The founder effect has been observed in S. viridis to a certain
degree. S. viridis accessions from North America have reduced allelic richness compared to those of
Eurasia. Genetic drift probably has occurred in S. viridis, as indicated by the many fixed alleles in
North American accessions. Multiple introductions of S. viridis, in the absence of local adaptation,
should have produced a random mosaic pattern of geographic distribution among North American
accessions. Instead, a strong intracontinental differentiation is observed in S. viridis populations,
both in Eurasia and North America (Jusef and Pernes, 1985; Wang et al., 1995a). S. viridis popula-
tions in North America are genetically differentiated into northern and southern groups separated
on either side of a line at about 43.5° N latitude. The northern type is less variable than southern
type. This regional divergence suggests that natural selection has partitioned S. viridis along a
north–south gradient. These observations imply that the present patterning among S. viridis popula-
tions in North America is the consequence of multiple introductions into the New World followed
by local adaptation and regional differentiation.
2.3.2.2.1.2 Setaria pumila–S. geniculata S. pumila populations are genetically clustered into
overlapping Asian, European, and North American groups (Wang et al., 1995b). S. pumila popu-
lations from the native range (Eurasia) contain greater genetic diversity and a higher number of
unique alleles than those from the introduced range (North America). Within Eurasia, Asian popu-
lations have greater genetic diversity than those from Europe, indicating S. pumila originated in
Asia, not Europe. These observations indicate that there have been numerous introductions of S.
pumila from Eurasia to North America, the majority from Asia. This pattern may also explain
the enigma of the origins of S. pumila and S. geniculata. The pattern of S. pumila genetic vari-
ability is North American was unexpected: nearly the entire diversity of this species appears to be
encompassed by accessions from Iowa, whereas populations collected from other North American
locations were nearly monomorphic for the same multilocus genotype (Wang et al., 1995b). In this
respect, it is significant or coincidental that this pattern was repeated in the diversity data for S.
viridis, also a native of Eurasia (Wang et al., 1995a). Iowa possesses a surprising Setaria genetic
diversity: all five weedy Setaria species are present. Typically two or more Setaria species occur
in the same field at the same time. Iowa is the center of the north–south agroecological gradient in
North America, perhaps leading to greater environmental heterogeneity. Despite originating on dif-
ferent continents, the genetic diversity patterns for S. geniculata parallel those for S. pumila and S.
viridis: Greater genetic diversity occurs in accessions from the New World compared to those from the
introduced range (Eurasia) (Wang et al., 1995b). This most likely reflects genetic bottlenecks associ-
ated with sampling a limited number of founding propagules and the history of multiple introduc-
tions from the Americas to Eurasia. The population genetic structure of S. geniculata consists of
three nearly distinct clusters, groups from Eurasia, northern United States, and southern United
States. Accessions from Eurasia and North America are approximately equally diverse genetically.
Within North America, S. geniculata accessions were strongly differentiated into southern and
northern groups at about the Kansas–Oklahoma border (37° N latitude); indicating greater genetic
differentiation within North American populations than between North American and Eurasian
populations.
2.3.2.2.1.3 Setaria faberi S. faberi contains virtually no allozyme variation. Of the 51 acces-

sions surveyed by Wang et al. (1995b), 50 were fixed for the same multilocus genotype.
2.3.2.2.1.4 General and Specialized Genotypes The population genetic structure of many

widely distributed, introduced, self-pollinating weed species clearly indicates that a high level of
genetic variation is not a prerequisite for successful colonization and evolutionary success (Allard,
1965; Barrett and Shore, 1989). Two contrasting adaptive strategies are hypothesized to explain
weedy adaptation and the success of colonizers: genetic polymorphism with the development of
locally adapted genotypes (“specialists”) and phenotypic plasticity for the development of “general
purpose” genotypes (generalists) adaptable to a wide range of environmental conditions (Baker,
1965, 1974; Bradshaw, 1965; Barrett and Richardson, 1986). Weedy Setaria population genetic
structure allows some insight into the dichotomy of generalism vs. specialization. Setaria possess
both generalists and specific strategy types. A key observation is that although a single multilo-
cus genotype predominates or is fixed in all populations, not all multilocus genotypes are equally
prevalent within individual weedy Setaria species (Wang et al., 1995a,b). The most striking exam-
ple of this is in S. pumila, where the most common multilocus genotype was found in 53 (of 94
evaluated) accessions surveyed by Wang et al. (1995b) from widely separated geographic areas in
Europe, Asia, and North America. Overlaying this pattern of homogeneity were other less abundant
S. pumila genotypes, each with a more narrow geographical and ecological distribution. The popu-
lation genetic structure of S. viridis suggests that this species also possesses both generally adapted
and specially adapted genotypes. Many S. viridis populations are genetically strongly differentiated
(e.g., northern and southern North America), while other populations remain identical. The most
widely distributed S. viridis genotype (fixed multilocus genotype) occurred in 25 (of 168 evaluated)
accessions from six countries, from both the Old World and the New World (Wang et al., 1995a,b).
Interestingly, this common genotype has not yet been found in Iowa, despite the diversity of
S. viridis populations found in this state. These observations reveal a complex hedge-betting strat-
egy by individual weedy Setaria species that balances general adaptation with the additional niche
opportunities available with specialization. The ratio of general to special genotypes in locally
adapted populations in a species for invasion is quite different within S. pumila, S. geniculata, and
S. viridis (Wang et al., 1995a,b).
2.3.2.2.2 Species Associations

The several self-similar Setaria species have self-assembled into several genetic and ecologically
adapted associations (Figure 2.6). Setaria is phylogenetically divided into two clades: S. viridis
and S. pumila (Doust and Kellogg, 2002). The diploid parental S. viridis precedes the tetraploid
S. verticillata and S. faberi forming a polyploid species cluster (Darmency and Dekker, 2011;
Dekker, 2011b). The genus Setaria also contains the important world crop foxtail millet (S. viridis,
subspecies italica) whose geographic distribution and evolutionary history are intimately con-
nected with the weedy foxtails. Gene flow communication between millet crop and weeds in
agroecosystems forms the stable, global wild-crop–weed complex (De Wet, 1966) in both clades
(Dekker, 2003, 2004a). The several weedy Setaria frequently exploit the same range, or the same
field, the emergent property of which is the Setaria species-group (a group of closely related spe-
cies, usually with partially overlapping ranges; Lincoln et al., 1998). Frequently more than one
Setaria species coexist together in a locality, possibly allowing a more complete exploitation of
resources left available by human disturbance and management. Several weedy foxtail species
Crop Wild-Weed Weed
Clade Diploid (2N) Diploid (2N) Tetraploid (4N)

S. viridis S. viridis, subsp. italica S. viridis, subsp. viridis
S. verticillata
S. faberi
S. pumila S. pumila
S. geniculata
Clade Species-group
Polyploid cluster
FIGURE 2.6 Setaria species associations: Clade, wild-crop–weed complex, polyploid species cluster (diploid,
tetraploid) and species-group.
often coexist in a single field (most commonly with S. viridis), each exploiting a slightly different
“opportunity space” or niche.
2.3.2.2.3 Metapopulations
Gene flow and seed dispersal linkages form a metapopulation communication system providing
continuity and stability to local demes, conservation of adaptive traits (physical knowledge), and a
long-term hedge-bet for the enduring success of the species-group. The limited diversity contained
within weedy foxtails is partitioned across the landscape, continent, and world among populations
(metapopulation structure) as alternative homozygous genotypes; heterozygosity was rarely detected
(Wang et al., 1995a,b). This structure suggests strong inbreeding in nearly all populations of the four
species. A common multilocus genotype of both S. viridis and S. pumila occurred in many accessions
from widely separated geographic areas and is an indication of general adaptation. Therefore, the
geographic distance (from global to local) separating foxtail populations does not indicate the genetic
distance separating them. Metapopulation structure provides a means of conserving and preserving
useful traits, available as conditions change in local demes. Self-pollination is a conservative mating
system, the means by which Setaria generates appropriate amounts of variability. Natural selection
and stochastic forces both act to keep species genetic diversity low. Each local population is an island
of local adaptation, sharing with neighbors at very low rates of gene flow. Rapid and numerous dis-
persal via human mediation ensures functional traits move between disturbed agrohabitats to ensure
fine-scale adaptation in seizing and exploiting the changing nature of local opportunity space–time.
2.4 SETARIA SEED–SEEDLING LIFE HISTORY BEHAVIOR

2.4.1 Life History Behaviors: Functions, Traits, and Information
Setaria plant spatial structure is the foundation for emergent life history behavior: self-similar
timing of life history processes regulated by functional traits expressed via environment–plant com-
munication (Figure 2.7).
Setaria life history behavior is a Markov chain of irreversible (dormancy induction; seed disper-
sal, germination, seedling emergence, and neighbor interactions) and reversible (seed after-ripening
[AR] and dormancy reinduction) processes of seed–plant state changes (flowering plants, dormant
seed [DORM], seed germination candidate [CAN], germinated seed, and seedling) regulated by
morpho-physiological traits acting through environment–plant communication systems (environment–
plant–seed and soil–seed) (Figure 2.5).
2.4.2 Seed Formation and Dormancy Induction

Individual panicles on a single parent plant produce a diverse array of seeds, each with potentially
different AR requirements for germination (Dekker and Hargrove, 2002). The production of seeds
Temporal Structure
Self-Similar Self-Organization: Biological Emergent
Life History Processes Components Functional Traits Information Behavior
Life Seed Time of Seed germination Environment–plant– Heterogeneous
history formation embryogenesis heteroblasty: seed communication seed
behavior of individual differential system germinability
seeds on an development: hull, capacity
inflorescence placental pore, TACL
membrane
Time of tillering Oxygen scavenger
inflorescence protein
branching
Seed Time of Seed size, number; Seed dispersal
dispersal abscission of location in space
individual
seeds
Seed pool Time of state Hull topography: Soil–seed oxygen Seed dispersal
behavior changes in water film communication in time
in soil individual oxygenation system Annual
seeds from Placental pore: water dormancy–
several film channel germination
inflorescences, TACL membrane: O2 cycling in soil
Seedling plants, years transport Locally adapted
emergence Oxygen scavenger: emergence
embryo O2 regulation patterns
FIGURE 2.7 Plant temporal structure and emergent behavior examples of self-similar components, self-
organization functional traits, and communication-information in the life history of weedy Setaria.
with different levels of dormancy (experimentally revealed by the AR dose (e.g., time at 4°C,
moist, and dark) required for germination) is a function of plant architecture. Earlier-fertilized
seeds (both intra- and interpanicles) are relatively more dormant than later-developing seeds. The
first seeds on an individual panicle were shown to possess relatively greater dormancy (greater
AR requirement) than the last seeds maturing on the same panicle (Haar, 1998). Additionally,
primary (1°) panicles produce seeds with relatively greater dormancy than those produced on
secondary (2°), and again on tertiary (3°), panicles of the same parent plant. Significant hetero-
geneity in dormancy states among seeds shed by a single plant allows these species to emerge at
appropriate times within a cropping season and in different years (Dekker et al., 1996; Forcella
et al., 1997). Soil seed banks consisting of diverse foxtail species and genotypes, each contribut-
ing to a heterogeneous collection of dormancy phenotypes, reveal a hedge-betting strategy for
adaptation to changing conditions within agroecosystems (e.g., Cohen, 1966; Philippi and Seger,
1989) (Figure 2.8).
2.4.2.1 Inflorescence and Flowering
The branching architecture of Setaria plants is plastic, an ability to form one or more tillering shoots
whose stature and number are precisely sized to local conditions. A complex pattern of branching,
from plant to spikelet, provides diverse microenvironments within which different levels of dormancy
are induced in individual seeds of the panicle and among panicles on a common plant (Dekker, 2003).
The pattern of flowering and seed fertilization is a complex process occurring on several different
spatial and temporal scales: within and among individual panicles on the same plant, during the annual
growing season, and during a daily or diurnal period (Darmency and Dekker, 2011; Dekker, 2003).
Neighbor interactions
Flowering plants [G] Seedlings
Seed [1] [5]
dormancy
induction
[A]
Germination Seed lings

growth emergence
Seed Seed [E] Germinated [F]
dispersal germination seed
[B] candidate [3] [4]
Dormancy
After-ripening
renduction
[C]
[D]
Dormant
seed
[2]
Death Death
FIGURE 2.8 Schematic diagram of weedy Setaria sp. seed–seedling life history behavior in soil pools:
plant/seed state pools (1–5) and processes (A–G; C–D are reversible).
2.4.2.1.1 Flowering Pattern on Panicle Main Axis

The first externally visible event in the reproductive phase is the emergence of the immature panicle
from the subtending leaf sheath of its tiller. Once flowering has proceeded basipetally to the bot-
tom of the panicle, the culm elongates and extends out of the leaf collar. Two patterns of flowering
along the inflorescence were observed (Dekker et al., 1996). A consistent pattern of flowering of the
first spikelet in each fascicle along the axis was observed. The flowering of these first spikelets of
the fascicle was first apparent in the distal 30%–40% section of the panicle axis (Figure 2.9). The
second group of spikelets then flowered in two directions at the same time, in one direction toward
the proximal end and in another direction toward the distal end of the axis. Flowering of these first
spikelets reached completion at the distal end first, followed by those at the basal end. A second pat-
tern of flowering was observed among the other fertile spikelets within individual fascicles along
the axis. This second pattern was very complex, and no consistent observations of its nature along the
axis were made in this study.
2.4.2.1.2 Elongation Pattern of Panicle and Culm Axis
A consistent pattern of panicle and culm elongation was observed, namely, elongation ceased distal
to the appearance of the first flower in a section of the axis (Figure 2.9) (Dekker et al., 1996). Axis
elongation then occurred basipetally to that flower toward the culm and leaf collar. As new flowers
appeared along the axis toward the basal end, elongation distal to these flowers also ceased. The
culm supporting the panicle did not elongate until flowering had proceeded to the basal end of the
inflorescence. At that time, the culm elongated and extended out from the leaf collar. The time from
the appearance of the first flower on the axis until the time when the first spikelet of the most basip-
etal fascicle flowered was about 9 days. In S. pumila, the lower floret has no pistil; instead, it has
Panicle
4 10
3 20
Proportion of panicle length (%)

30
1
40
2 50
4 60
5 70
6 80
7 90
8 100
Leaf collar
Culm
FIGURE 2.9 Pattern of S. faberi flowering spikelets on the panicle main axis over time with development
expressed as a proportion (%) of the total panicle length; circled numbers indicate axis area of temporal pro-
gression of flowering: 1, first axis zone of flowering; 8, last area of axis to flower.
three well-developed anthers that open 3–7 days after the upper floret (Willweber-Kishimoto, 1962).
Elongation of the inflorescence and culm could also allow greater light penetration into the panicle
as it rises above its nearby competitors (intraplant, intraspecies, and interspecies). Both fascicle
spacing and axis elongation could serve as mechanisms differentially modifying the light microen-
vironment of individual spikelets along the axis during flowering and embryogenesis. This differen-
tial light reception may be a mechanism by which different seed germinability phenotypes are shed
from a panicle. Differential induction of germinability in S. faberi seed as a response to variable
field light conditions has been observed previously in giant foxtail (Schreiber, 1965a) (Figure 2.10).
2.4.2.1.3 Seasonal Flowering Pattern

Panicles usually emerge after the summer solstice, and flowering commences and continues after that
time into the autumn. There is considerable variation in the patterns of panicle tiller emergence, flower-
ing, embryogenesis, and seed abscission among and within weedy Setaria spp. inflorescences. Time of
seedling emergence, as well as the photoperiod and temperature coinciding with an individual plant’s
seasonal growth period, is the major determinant of these flowering phenomena (Haar, 1998; Stevens,
1960). The time from seedling emergence to the appearance of the first S. faberi and S. viridis panicle is
highly variable and is dependent on the diurnal photoperiod (Dekker et al., 1996; Nieto- Hatem, 1963;
Schreiber, 1965a; Schreiber and Oliver, 1971; Stevens, 1960). Setaria appear to initiate flowering and fer-
tilization in response to the shortening photoperiod (lengthening dark period) after the summer solstice.
Long daylengths appear to prolong these reproductive responses, while continuous light markedly delays
panicle production and inhibits flowering, in all the weedy and crop Setaria spp. (Fabian, 1938; King, 1952;
Peters and Yokum, 1961; Peters et al., 1963; Santelman et al., 1963; Schreiber, 1965a; Vanden Born, 1971).
Triazine-resistant biotypes of S. viridis flower earlier than susceptible variants (Ricroch et al., 1987).
Blooming in wild-type biotypes is negatively correlated with temperature and positively with relative
humidity (Li et al., 1935). The response of flowering to photoperiod in weedy Setaria spp. may be has-
tened by higher temperatures (Ricroch et al., 1987; Schreiber and Oliver, 1971).
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 8 Day 9

0 30
5 A 25
Zone of elongation B
10 20
Zone of elongation
C
Axis length (cm)
Zone of elongation
BS LC
Zone of elongation
Zone of elongation
E
15 15
Zone of elongation
Culm
F
Zone of elongation
BS LC
BS LC
Zone of elongation
BS LC BS
20 10
LC
LC
25 5
LC
LC 0
30
FIGURE 2.10 Pattern of S. faberi panicle and culm axis elongation growth (cm) over time (9 days) with devel-
opment; A–G represent the location of the first flowering spikelet to appear on the individual day indicated,
with the zone of elongation indicated below it; BS, base of inflorescence defined by most basal spikelet; LC, leaf
collar area subtending the panicle; the inflorescence peduncle (culm) is that distance between BS and LC.
2.4.2.1.4 Diurnal Flowering Pattern

Setaria flowering usually occurs during specific time periods of the day, depending on local envi-
ronmental conditions, and is an endogenously regulated rhythm with two daily maxima peaks
(i.e., 04:00–08:00 h and 19:00–00:00 h) in the dark (Dekker et al., 1996; Li et al., 1935; Rangaswami
Ayyangar et al., 1933; Willweber-Kishimoto, 1962). The average time between flower opening and
closing is 70 min. (Kishimoto, 1938).
2.4.2.2 Mating and Fertilization

2.4.2.2.1 Hybridization
The weedy Setaria are primarily a self-pollinated species (Mulligan and Findlay, 1970; Pohl, 1951).
Wind pollination (anemophily) is the mode in those rare circumstances of outcrossing (Nguyen Van
and Pernes, 1985; Pohl, 1951). Pollen and gene flow in the weedy Setaria can be intraspecific (autog-
amy and self-fertilization; allogamy and outcrossing) or interspecific hybridization (introgression
between different Setaria species) (Dekker, 2003). Although the anthers are visible at flowering and
then feathery stigmas also exert from the glumes, the fertilization principally occurs within the flower.
Stigmate maturity is generally synchronous with anther dehiscence, which results in a high probabil-
ity of self-pollination. Some rare cases of protogyny, or in contrast of protandry, can be observed
according to the genotype and the environmental conditions, but in both cases, the more proximate
pollen belongs to the other spikelets of the same panicle, thus leading again to self-pollination. Wind
pollination (anemophily) is the mode in those rare circumstances of outcrossing (Pohl, 1951), with
pollen probably moving at most a few dozen meters (Darmency and Dekker, 2011; Wang et al.,
1997). Natural outcrossing between Setaria plants of the same species, intraspecific hybridization,
does occur and is an important source of new variants. In S. viridis, spontaneous outcrossing rates
among plants in the field have been observed to be between 0% and 7.6% of the autogamous rates
(Darmency et al., 1987a,b; Li et al., 1934; Li et al., 1935; Macvicar and Parnell, 1941; Prasado Rao
et al., 1987; Till-Bottraud et al., 1992; Takashi and Hoshino, 1934). Till-Bottraud et al. (1992) found
0.74% outcrossing for S. viridis plants spaced every 0.25 m. Similarly, a selfing rate exceeding 99%
was reported in Jasieniuk et al. (1994) for S. viridis. Using a dominant herbicide resistance marker,
Volenberg et al. (2002) found an outcrossing rate ranging from 0% to 2.4% for S. faberi planted at
0.36 m interval. Introgression of pollen between weedy species, interspecific hybridization, is a rare
event. When it does occur, it can have very important consequences, although the progeny is almost
always sterile (Clayton, 1980; Li et al., 1942; Osada, 1989; Poirier-Hamon and Pernes, 1986; Stace,
1975; Till-Bottraud et al., 1992; Willweber-Kishimoto, 1962).
2.4.2.2.2 Asexual Reproduction
Asexual (meiotic) reproduction is not a common mode of reproduction in weedy Setaria and is prob-
ably limited to S. geniculata, a perennial species with short, branched, knotty rhizomes (Rominger,
1962). Agamospermy (seed formation without fertilization) in S. viridis has been noted (Mulligan
and Findlay, 1970) and has also been suggested for S. glauca and S. verticillata (Steel et al., 1983).
Apomixis has also been reported in other Setaria species within the Setaria subgenus (S. leucopila,
S. macrostachya, and S. texana; Chapman, 1992; Emery, 1957). S. faberi and S. pumila tillers
readily root in soil when cut, separated from the plant, and buried in moist soil, an important
trait allowing weedy Setaria to reestablish themselves after cultivation and mowing (Barrau, 1958;
Santlemann et al., 1963; Schreiber, 1965b).
2.4.2.2.3 Speciation and Reproductive Barriers between Setaria Species

New Setaria species can be formed by several processes (e.g., mutation, hybridization, and poly-
ploidization), but such events are very rare. Partial reproductive barriers exist between the Setaria
species, a genetic condition favoring introgression and gene flow at very low levels within the species-
group and wild-crop–weed complex (Darmency et al., 1987a,b; Harlan, 1965; Harlan et al., 1973;
de Wet, 1966). Panicle height differences and times of fertilization (pollen shed and stigma recep-
tivity), both prevent hybridization events from occurring between many populations (Willweber-
Kishimoto, 1962). Reproductive barriers between the Setaria species do not occur at the level of
pollen germination and pollen tube growth in the stigma in any combination of pollen and stigma
among S. italica, S. viridis, S. faberi, S. pumila, and S. pallide-fusca (Willweber-Kishimoto, 1962).
Polyploidization (either alloploidy or autoploidy) has played an active role in speciation and delimit-
ing taxa in Setaria (Khosla and Sharma, 1973). Polyploidization of the more diverse and ancient
S. viridis may have been the genesis of the specialized and less diverse Setaria spp. set (S. faberi,
S. pumila, S. verticillata, and S. geniculata). Allotetraploid forms of S. faberi and S. pumila have
been explained as ancient crosses of S. viridis with an unknown diploid species (Kholsa and Singh,
1971; Li et al., 1942; Till-Bottraud et al., 1992). This polyploidization in S. faberi may be a relatively
recent evolutionary event (Wang et al., 1995b). S. verticillata (n = 18) may be the product of chro-
mosome doubling of S. viridis, autotetraploidy (Till-Bottraud et al., 1992). Geographical barriers to
interspecies hybridization in current times are much less important now than in the past.
2.4.2.3 Seed Formation and Embryogenesis

The life cycle of foxtail seed begins with seed development, the development of outer seed enve-
lopes, the inner endosperm, and the embryo (Dekker, 2003, 2004a). Embryogenesis in Setaria
begins with anthesis and fertilization of the new embryo and ends when the embryo, enveloped with
parental tissues (i.e., lemma, palea, and CC), becomes physiologically separated from the parent
plant (abscission). Three separate nuclear genomes interact and produce the tissues that compose
the foxtail seed. The parent contributes sporophytic tissues to the seed that nurture and protect
the developing embryo as well as provide significant contributions to dormancy and control of
postabscission germination timing (e.g., seed envelopes such as the CC) and physical protection in
the soil. The duration of seed development and embryogenesis of an individual S. faberi seed grown
in controlled environmental conditions is 8–15 days (Dekker et al., 1996; Haar, 1998).
The morphology of foxtail seeds also provides important clues about what environmental fac-
tors limit germination and maintain dormancy (Dekker and Hargrove, 2002). Morphologically, the
foxtail seed caryopsis (embryo, endosperm, and aleurone layer) is surrounded by several enveloping
layers that control its behavior (Dekker et al., 1996). The CC is filmy, oily to the touch, water- and
gastight, and continuous except at the PP opening on the basal end of the seed. Although the mature
foxtail seed is capable of freely imbibing water and dissolved gases, entry is restricted and regu-
lated by the PP tissues, where there is membrane control by the TACL. Gases entering the moist
seed interior must be dissolved in the imbibed water passing through the narrow PP and TACL.
The importance of gases entering dry seed is unknown. The morphology of foxtail seeds strongly
suggests that seed germination is restricted by water availability in the soil and by the amount of
oxygen dissolved in water reaching the inside of the seed to fuel metabolism. Oxygen solubility in
the water entering the seed interior is an inverse function of the diurnally, seasonally, and annually
changing soil temperature. The control by this oxygen-limited gastight morphology is supported by
observations of increased germination when foxtail seed envelopes, including the CC, are punctured
(Dekker et al., 1996; Stanway, 1971). Based on these observations, biogeographic distribution of
weedy Setaria, as well as individual seed behavior, is regulated by the amount of oxygen dissolved
in water taken into the seed over time. When adequate amounts of water and oxygen reach the
embryo, sufficient energetic equivalents are generated to support germination metabolism. When
inadequate amounts of oxygen reach the embryo, dormancy is maintained or secondary dormancy
is induced.
An inherent problem with seed germination only being regulated by a restriction of O2–H2O
entry (TACL regulation) (Dekker, 2000) is the observation that foxtail seed germination in natural
soil habitats occurs several weeks after both favorable temperatures and O2–H2O are available in the
spring (Figure 2.1; Forcella et al., 1992, 1997). What is the basis of delayed weed seed germination
timing in the soil after adequate temperature and resource conditions are obtained? Hiltner (1910)
suggested that inside dormant grass seeds, there exists an oxygen-absorbing substance that would
prevent O2 from reaching the embryo. A robust seed regulatory system could result from the inter-
action of the TACL, which restricts O2–H2O entry into the seed, and an oxygen-scavenging system
that delays the transduction of O2 in the symplast. These restrictions in uptake and O2 scavenging
could act together to prolong the time before O2 stimulates the first metabolic events of seed germi-
nation. Heterogeneous foxtail seeds with differing levels and combinations of PP (TACL) size and
O2 absorption could also provide the means by which the seedling emergence of different individu-
als from the same parent plant would occur over the course of an individual growing season and
also over many years. Heterogeneous collections of foxtail seeds in the soil would provide a hedge-
betting strategy well suited to the disturbances typical of agroecosystems.
2.4.2.3.1 Hull Development

The size and shape of most of the panicle tissues of parental origin do not change appreciably
after emergence from the culm and before seed fertilization commences (culm length and the CC
are notable exceptions). Several qualitative changes occur in the hull and the caryopsis with the
development from anthesis to after abscission (Dekker et al., 1996). The seed hull structures are
present at anthesis, but change during seed ontogeny from open soft tissues to a hard enclosing
structure at anthesis (Dekker et al., 1996). The seed hull color and hardness change with devel-
opment as well as the shape of the palea and its position in relationship to the enclosing lemma.
Caryopsis size and shape change with development as did the endosperm, embryo, CC, and the
placental pad. The size of the hull (parental tissue) changed relatively little compared to the
caryopsis. The length of the hull remained relatively constant with development, ranging from
2.4 to 2.8 mm. The width of the hull ranged from 1.4 to 1.7 mm. The color of the hull changed
with development from light green to dark green and dark brown. The hull began development
as a soft and rubbery structure, progressing to hardness. The relative position of the palea to the
enclosing lemma changed with development. At anthesis, the palea was sunken below (inward
toward the seed interior) the edge, or lip of the lemma, and had a concave surface (curved slightly
inward to the cavity formed by the two hull structures). This relation between lemma and palea
changed until the palea was flat, even with lemma lip edge. Later, the palea shape ranged from
flat to convex. The palea became approximately even with the lemma lip edge with time and then
protruded beyond the lemma lip.
2.4.2.3.2 Placental Pore and Pad Development

During the ontogeny of the caryopsis, a single placental vascular bundle from the parent panicle
supplies nutrients and water to the developing ovule. As the caryopsis matures, a thick, dark oval
pad remains in the position occupied by the placental bundle. The point of attachment of the cary-
opsis (zygotic) with the palea (parental) is the placental pad. The placental pad was first visible
as a dark circle at the base of the caryopsis on the side appressing the palea. At excision of the
caryopsis from the hull, this area exhibited a distinctive color that changed with age. The color of
this structure was white from 2 to 10 days after anthesis (DAA). From 9 DAA and thereafter, the
placental pad began to change color. The first color was a very pale pink (9–10 DAA) followed by a
deepening color from pale pink to rust-red. The necrotic contents of the layers of the placental pad
(Rost, 1973) and the red coloration in this area of the caryopsis (Narayanaswami, 1956) have been
noted previously. It is suggested that this red coloration is a morphological marker in S. faberi
indicating physiological maturity (Dekker et al., 1996). The formation of this abscission layer or
zone is directly analogous to the black layer formation in Zea mays L., an indicator of physiological
maturity (Daynard and Duncan, 1969; Sexton and Roberts, 1982).
2.4.2.3.3 Caryopsis Development

The size and the shape of the caryopsis (COP) and its component structures were tubular early
in development, and the internal cavity formed by the lemma and palea was only partially filled
(Dekker et al., 1996). The caryopsis matures and expands, filling the hull width by day 6 and the
length by abscission. During this early time, the remnant style and anther stalks were also present
in the cavity. The CC becomes fully developed at approximately day 4 and appears shiny, oily,
and gray with dark spots. Sometime after day 4, the CC becomes gas impermeable, except at the
proximal (basal) end of the caryopsis (the placental pad and pore region). Entry of water into the
caryopsis occurs only through the PP after day 4. Entry of water is never restricted through this
region of the seed from day 4 until germination occurs (Rost, 1971b, 1973, 1975). With develop-
ment the COP became ovoid and later plump, filling the hull cavity. The endosperm developed
during this time, beginning with only a few cells, then becoming more visible as a clear liquid.
Thereafter the endosperm became firmer, lost moisture, and changed from liquid, liquid dough,
soft dough, crumbly or granular dough, and finally to a firm and hard dough. The COP (mostly
zygotic tissues) grew, progressively filling the hull cavity with development. At anthesis, it was
0.75 mm and increased to 2.2 mm just after abscission. After that time, the COP length remained
constant or decreased slightly, possibly as a consequence of desiccation. The COP width increased
from 0.35 mm just after anthesis, to a maximum of 1.5 mm just prior to abscission. It remained
relatively constant after that time. The smallest difference between the hull and caryopsis length
(0.35–0.5 mm) occurred between 11 and 15 DAA, then increased slightly after that time due to
COP desiccation. The difference between the hull and caryopsis widths did not change (0.1–0.25
mm) after 6–7 DAA, apparently filling the hull cavity in that dimension at that time. Although the
caryopsis appears to fill the cavity formed by the hull completely, arguments implicating the hull in
physical restraint of embryo germination are incomplete (Simpson, 1990). Although germination
must occur through the germination lid of the lemma (Rost, 1973), some space is apparent inside
the seed cavity: the embryo and endosperm both shrink somewhat within the cavity after 16 DAA
(Figure 2.8). Several seeds dissected in the course of these experiments (Dekker et al., 1996) had
partially germinated CPTs and CRZs (never both in the same seed), indicating arrested viviparous
(precocious) germination.
2.4.2.3.4 Embryo Development

The embryo first became visible at 2 DAA as a faint darkish spot and ring at the basal end on the
lemma side of the COP. The remainder of the COP at this time was clear. Later the coleorhizal end
of the embryo axis became prominent and began to protrude from the globular-shaped caryopsis.
This coleorhizal protrusion progressed from a rounded shape to a boxy, fully formed structure that
filled the germination lid cavity on the basal end of the lemma. The embryo shrank below the endo-
sperm surface (14 DAA and thereafter) as the seed became dehydrated.
2.4.2.3.5 Dormancy Induction

Variable germinability–dormancy capacity is induced in developing seeds during embryogenesis
(Dekker, 2003, 2004). S. faberi embryos become capable of independent germination at about day 6
of embryogenesis (Dekker et al., 1996). At that time, embryo germination is very high (i.e., 95%),
and occurs in both axes (CPT, CRZ). By day 8, embryo germination decreases, becomes more
variable, and axis-specific germination first appears (germination of only one axis). This embryo
dormancy induction period occurs from day 8 through anthesis, when embryo germination is very
low (i.e., 10%). The variability in embryo and caryopsis germination among individual seeds shed
by a panicle increases from when dormancy is induced until after abscission. Four qualitatively
different types of embryo germinability were observed (Dekker et al., 1996). The four germina-
tion types were mutually exclusive; only one type appeared in any individual seed. These different
germination types were correlated with the age of the embryo. Immature embryos exhibited three
apparently different types of germinative growth.
The first type was observed in caryopses 0–5 DAA and appeared as disorganized, undifferenti-
ated callus growth at the basal, coleorhizal end of the caryopsis. Germinability was evidenced as a
pointed protrusion of cellular growth overlying the site of the excised pedicel, immediately basipetal
to the placental pad. Also, a slight swelling of the basal, coleorhizal end of the caryopsis was often
associated with this type of germination. It was unclear whether this type of germination arose from
vascular (parental) or coleorhizal (zygotic) tissue. Although this type of germination was observed in
many very young COPs, it may be an artifact associated with the excision of the vascular bundle to the
pedicel. A similar type of germinability, disorganized proliferation of the radicle (callus), was noted
previously in wheat (Triticum aestivum) in which the pericarp was removed (Symons et al., 1983).
The second type of germination of immature embryos was observed from 5 to 7 DAA, the time
when the embryo was first large enough for excision from the caryopsis. At that time, almost all
embryo CPTs and CRZs appeared to be differentiating and growing. This germination took the
form of shortening and thickening of those two axes. The growth never progressed very far, but each
shortened and thickened axis appeared as rounded “balls” of tissue within the underlying scutellar
depression (the scutellar “cup”).
The third type of germination involved germinative growth of the immature (7–11 DAA) scutel-
lum (modified cotyledonary structure of the embryo, intercalated into the endosperm in the caryopsis).
Infrequently cellular growth and swelling would occur in areas of the scutellum appressed to the
endosperm, usually at the CPT end of the embryo. Other types of germinative growth did not occur
when this scutellar germination was observed.
More typically, germination and growth of the CPT and CRZ occurred in embryos of 7 DAA
and older. Several different combinations of this type of axis-specific germinability of S. faberi
embryos were observed. Germination of only the CPT axis of the embryo took several different
forms. This type of germination first took the form of swelling of the basal area immediately
adjacent to the CPT itself. When the embryo was part of the caryopsis, the first indication of this
type of germination was a cracking of the overlying CC. While some embryos did not continue
to germinate after this, some CPTs continued to grow. The next step observed was the growth
of the CPT itself causing it to increase in length and for the axis to curve and lift away from the
scutellar “cup” it was enclosed by before germination. Some portions of the embryos observed
continued to germinate, as evidenced by continued elongation and extension of the axis followed
by the emergence of the first true leaf. Axis-specific germinability also was observed in which
only the CRZ grew. The first indication of this type of germination was a swelling and protru-
sion of the CRZ leading to its extension beyond the end of the caryopsis. In some embryos, this
would appear as a pointed protrusion from the axis end, and in others as the entire blunt, rounded
CRZ end swelling and growing. Growth in some of these embryos would not proceed beyond this
stage. Still in others, continued germination was evidenced by the appearance and extension of
the trichomes. If germination proceeded, the radicle would extend from the end of the CRZ. All
of these different axis-specific germinability states were observed in S. faberi embryos, as well
as germination in which both axes germinated. Axis-specific germinability in wheat has been
reported previously (Mitchell, 1980; Symons et al., 1983).
Germination of embryos differed not only with the stage of development, but also as a function
of the tissues surrounding them: hull plus nonembryo caryopsis, caryopsis, CRZ trichomes (endo-
sperm and CC), and the isolated embryo. Quantitative and axis-specific differences in germinative
capacity among embryos were also observed.
2.4.2.3.5.1 Seed Germinability Seeds were completely nongerminable from fertilization to

abscission and shortly thereafter. No seed germinated in these environmental conditions until AR
had occurred.
2.4.2.3.5.2 Caryopsis Germinability Three different axis-specific types of caryopsis germinabil-

ity were observed: CPT only, CRZ only, and CPT plus CRZ. Caryopses first became germinable at 8
DAA. This was primarily due to CPT germinability from 8 to 16 DAA. Germinability of caryopses, as
well as variability among similar-aged COPs, increased during the second half of embryogenesis, pri-
marily as axis-specific CPT germination. Apparently, other COP tissues are positively influencing the
embryo relative to its behavior in isolation, delaying the onset of full developmental arrest. Coleorhizal,
as well as CPT plus CRZ, germinability was very low from 8 to 16 DAA. After-ripened caryopses were
highly germinable, primarily due to CPT plus CRZ germinability, although some germinability was
restricted to single axes at that time. Variability of all types of caryopsis germinability was greatest
just after abscission (12–16 DAA). Germinability of caryopses was very high after AR had occurred.
2.4.2.3.5.3 Embryo Germinability The same three types of axis-specific germinability

observed in caryopses were also observed in isolated embryos. Embryos first become germinable
at 6 DAA, most of which was CPT plus CRZ “ball”-type germination. Germinability among
embryos was very high at that time. More normal axis germinability was observed at 2–8 DAA.
After 6 DAA, embryo germinability decreased until after abscission (12 DAA). During this time,
both CPT plus CRZ and CRZ-only germinability decreased, while CPT germinability increased,
then decreased. CRZ-only germination remained very low at all ages. This decrease in embryo
germinability in the second half of embryogenesis has been reported for other species (e.g., sun-
flower: Le Page-Degivry et al., 1990). At 12 DAA, just after abscission, embryo germinability of
all types was low. After that time, embryo CPT plus CRZ germinability and variability increased,
while CPT-only and CRZ-only germination remained low. Variability in embryo germinabil-
ity increased with development from 6 DAA until 16 DAA. After-ripened embryos were highly
germinable, primarily due to CPT plus CRZ germinability, but some low levels of single-axis
germinability were observed.
2.4.2.3.5.4 Multiple Germinability Phenotypes There is no single, qualitative state of germina-

bility into which all seeds must enter, remain, and exit (Simpson, 1990). Instead, there are multiple
states of germinability arising from the wide array of environmental conditions that these plants face,
changing sensitivities of seed organs from anthesis through AR to these conditions, different roles
played by the inflorescence, fascicle, spikelet, and seed components, and a wide variation in genetic
constitutions. Although seed germination is a singular (threshold) event in the foxtail seed life cycle,
there are many different developmental paths by which it can be attained. The foxtail seed is a plas-
tic dynamic biological entity situated at all times in a complex changing environment. These selec-
tion pressures over evolutionary time have led to multiple germinability phenotypes within a single
genotype. A single foxtail plant shedding seed with different germination capacities has the ability
to ensure its enduring occupation of a locality with changing environmental conditions. Giant foxtail
seed rain consisting of multiple germinability phenotypes is a consequence of influences exerted at the
level of the inflorescence, the fascicle, the spikelet, and compartments within the seed. Germinability
in S. faberi embryos is achieved quickly after anthesis. This germinability has four different germina-
tion forms, but the importance of some of them is not apparent. Embryo germination can take place in
either or both axes at any time from the middle of embryogenesis through AR. The germinability of
isolated embryos is not necessarily an indication of their germinability when enclosed in the caryop-
sis or hull. The embryo can respond differently in each seed compartment (hull, caryopsis, embryo),
and each compartment probably exerts a regulatory influence independent (as well as dependent) of
the others. This compartment-specific regulation changes with development. For example, at 8 DAA,
the surrounding caryopsis tissues inhibited the enclosed embryo relative to the high germination of
isolated embryos of that age. Conversely, the surrounding caryopsis tissues enhanced the germination
of the enclosed embryo at 12 DAA, just after abscission, relative to the low germination of the naked
embryo at that time. Seed germination does not occur until each compartment independently has
achieved a significant amount of embryo germinability in both axes, especially in the CRZ. The lack
of seed germination may be a direct consequence of asynchrony among these three seed compart-
ments, with seed germination occurring when these three seed compartments act synchronously. The
times of seed abscission, dissemination from the parent panicle, and entry into the soil seed bank may
have an important evolutionary and ecological significance to the species. At these times, seed leav-
ing the parent plant are nongerminable, and isolated embryos are in their least germinable state. At
abscission, both caryopses and isolated embryos possess their maximum variation in germinability as
a population, either in terms of percent germinability or axis-specific germinability. The population of
S. faberi seed leaving a panicle after abscission expresses a large, possibly maximal, number of ger-
minability phenotypes in its life cycle. This may be an important adaptive strategy of foxtail to ensure
enduring survival as it enters the hostile soil seed bank, as the chances of survival of the population
is increased by disseminating the maximum number of germination options for an uncertain future.
2.4.2.3.5.5 Light and Dormancy Induction Atchison (2001) observed that seeds maturing
early in the growing season in partial shade are considerably more dormant than those on nearby
plants in full sun. Axis elongation and fascicle spacing are two mechanisms differentially modify-
ing the light microenvironment of individual spikelets (Dekker et al., 1996). This differential light
reception may be a mechanism by which different levels of dormancy are induced in individual
florets of a panicle: relatively greater light levels are associated with relatively reduced dormancy
in later-maturing seeds on a panicle. Greater light penetration is correlated with relatively less dor-
mancy in the more widely spaced basal florets, while the greater height and light above the soil
surface (and competitors) are correlated with less dormant florets maturing later on an individual
panicle (Atchison, 2001; Dekker et al., 1996; Haar, 1998).
2.4.2.3.5.6 Morpho-Physiological Dormancy Traits The germination of giant foxtail embryos

is modified by the presence of several seed envelopes, notably the hull (glumes, lemma, and palea)
and the CC (Rost, 1973, 1975). Induction of dormancy in caryopses and seeds coincides with the
sealing of the caryopsis by the CC and the maturation of the hull. The variability in germination
among individual caryopses and seeds increases with time after dormancy induction. These events
may describe a “phenocritical” period in seed genesis when dormancy is induced (Christianson and
Warnick, 1984). Compartment germination differences provide evidence of multiple seed tissue
control of germination.
2.4.2.3.5.7 Compartmentalization of Seed Germinability The germinability state of an indi-

vidual seed at any one instance in its life cycle is a function of the germinability of its component
compartments, the embryo, and the enclosing and associated structures of the seed (hull and caryop-
sis) (Dekker et al., 2012). Several different germinability states were observed in S. faberii embryos
as they developed. The hull was observed to exist in two states, depending on the resistance of the
germination lid (Rost, 1973) to internal pressure and opening by the CRZ. The embryo, whether
isolated or in the caryopsis, exists in four different states: both axes nongerminable, both axes
germinable, CPT-only germinable, or CRZ-only germinable. The germinability states of S. faberi
seed can be modeled based on the independent, and at other times dependent, relationship among
the three structural compartments. From the observed germinability states, 32 hypothetical quali-
tative states can be envisioned. Of the 32 states, 28 were observed at some time during develop-
ment. The common feature of these four missing states is that they all have open hulls (H +), and
the isolated embryo has nongerminable CPTs and germinable CRZs. Either these states do not
exist or they were not revealed in the times during the life cycle we studied. We propose that dor-
mancy in S. faberi seed can be defined by the asynchronous germinability of the embryo within
each of the three compartments of the seed (embryo, caryopsis, and hull), and germination can be
defined by the synchronous germinability of the embryo within each of the three compartments.
From these studies, we hypothesize a dynamic developmental basis of germinability regulation
resulting in multiple germinability phenotypes at all stages of development, particularity when
seed is shed from the parent plant and the soil seed bank is replenished.
2.4.2.3.6 Seed Abscission

Weedy Setaria spp. seeds become physiologically independent when the abscission layer in the
pedicel forms, indicated by the red coloration of the placental pad on the caryopsis (Dekker
et al., 1996). Abscission is a singular threshold event in the life history of a new Setaria plant,
but it is only a partial separation of the new and parental generations: parental tissues surround
the embryo, affecting its ability to germinate. The basic unit of the Setaria inflorescence is
a dorsally compressed, two-flowered spikelet, which disarticulates below the glumes, and is
subtended by one to several bristle-like setae (Hitchcock, 1971; Rominger, 1962). S. faberi
seeds abscise from the parent plant almost entirely dormant, a threshold life history event
(Dekker, 2003, 2004).
2.4.2.4 Seed Fecundity and Plasticity

Seed production is an emergent property of Setaria reproductive morphology. Seed numbers
produced by a Setaria are a function of differences in plant architecture: shoot tillering–panicle
formation, panicle–fascicle branching, and fascicle spikelet–floret development. Inherent plastic
differences in these reproductive structures among Setaria species determine the reproductive
responses of species and populations to available opportunity in its immediate environment.
Setaria panicle inflorescences develop at the terminal ends of shoot tillers. Developmentally,
primary panicles flower first on a plant, followed by secondary, then tertiary. Setaria spp. panicles
are composed of fascicles, which consist of spikelet (with florets) and bristle (seta only) shoots.
The fascicle–spikelet structure differs among Setaria species. In S. faberi or S. viridis, the number of
fascicles within each panicle varies, a plastic response to a plant’s immediate environment (Clark and
Pohl, 1996). Longer, earlier developing S. faberi or S. viridis panicles often have the most extensive
fascicle branching, as well as more spikelets per fascicle (typically four to six or more per fascicle).
The plastic response of S. faberi or S. viridis to its immediate environment is also revealed in the
number of spikelets–florets that fully mature, which determines the seed number per panicle length
(seed density). Under favorable conditions, more spikelets are able to develop into seeds, while under
unfavorable conditions, spikelets may abort. S. pumila panicle morphology is different from that of
S. faberi or S. viridis. Only a single, terminal spikelet–floret is found in each S. pumila fascicle (Clark
and Pohl, 1996). This stable fixed morphology limits the ability of S. pumila to respond in a plastic
way to its environment in terms of seed number relative to that of S. faberi or S. viridis.
Setaria seed rain exhibited some stable, and many more plastic, responses. S. faberi panicles were
consistently longer than those of S. viridis. S. viridis parameters were greater than S. pumila. Earlier
panicles were greater than, or similar to, later ones for all parameters. More typically, tillers and pan-
icles responded to local conditions in a plastic way, confounding the formulation of seed production
generalizations. In S. faberi and S. viridis, no consistent relationship between seed number and panicle
length was observed among different tiller types. A more consistent relationship between parameters
was observed for S. pumila compared to the others, making prediction possible for this species. The
stability and plasticity of these relationships is partially due to the differences in S. faberi and S. viridis
panicle, fascicle, and spikelet morphology compared to S. pumila. These stable and plastic responses
provide fine-scale adjustment to a locality, maximizing exploitation of local opportunity.
The numbers and yield of seed produced by a weedy Setaria plant are highly plastic and strongly
dependent on biomass accumulation, plant tillering, and fascicle architecture (Haar, 1998; Haar and
Dekker, 2011; Kawano and Miyake, 1983). Setaria plant biomass is strongly dependent on available
resources, competition from neighbors, and the time of seedling emergence (Dekker, 2004b). In gen-
eral, plant size and seed number are greater for plants emerging earlier in the growing season than
later (Schreiber, 1965b). Reports of seed productivity therefore vary from 1 to 12,000 seeds/plant
(Peters et al., 1963; Rominger, 1962; Slife, 1954; Steel et al., 1983; Vanden Born, 1971). Seed number
per panicle, panicle length, and seed number per unit panicle length (seed density) were found to
vary among Setaria species (S. faberi, S. viridis, and S. pumila), panicle types (1°, 2°, and 3°), and
field locations (Haar, 1998). For example, Haar (1998) found that seed number per panicle varied by
Setaria species, location, and panicle type: S. faberi, 165–2127; S. viridis subsp. viridis, 144–725; and
S. pumila, 54–213. Because the weedy Setaria spp. seed rain is continuous over a weeks-to-months
period, productivity can be underestimated by infrequent collection methods and inefficient capture
techniques. Attempts to provide experimental seed productivity estimates (e.g., relationship between
panicle length and seed number per unit length measurements) have produced inconclusive results
(Barbour and Forcella, 1993; Defelice et al., 1989; Fausey et al., 1997; Haar, 1998).
2.4.3 Seed Rain Dispersal

Setaria seed is dispersed in both time (seed germinability–dormancy heteroblasty) and space (inva-
sion and colonization and formation of soil seed pools).
2.4.3.1 Seed Germinability–Dormancy Heteroblasty: Dispersal in Time

The discovery of clines in seed polymorphism … suggests that seed polymorphism may be a most
sensitive indicator of evolution in weedy species. (Harper, 1964)
Seed polymorphisms seem particularly likely to be sensitive indicators of evolutionary change in alien
invaders. (Harper, 1964)
Weedy Setaria seed is shed from the parent plant primarily in a nongerminable, but viable, state of
developmental arrest. Studies have concluded that all seed shed from the parent plant is dormant
(S. pumila (Rominger, 1962): Peters and Yokum, 1961; Povalitis, 1956; Rost, 1975; S. verticillata:
Lee, 1979), “almost completely” dormant (S. pumila: Kollman, 1970; Taylorson, 1966; S. faberi:
Stanway, 1971), or shed with variable germinability (S. pumila: Norris and Schoner, 1980; S. viridis:
Martin, 1943). Morphological variants of S. viridis (robust white, robust purple, and giant green)
have been reported indicating that they do not possess seed dormancy (Schreiber, 1977). Triazine-
resistant S. faberii seeds were reported to be inherently more dormant than susceptible biotypes
(Tranel and Dekker, 1996, 2002), consistent with observations in other species (Mapplebeck et al.,
1982). Viviparity (premature and precocious germination) has been observed in S. viridis plants
(Dore and McNeill, 1980; Haar, 1998; Hubbard, 1915).
Individual Setaria panicles on a single parent plant produce a diverse array of seeds, each with
potentially different dormancy states (seed heteroblasty) (Atchison, 2001; Dekker et al., 1996; Haar,
1998; Kollman, 1970; Martin, 1943; Moore and Fletchall, 1963; Nieto-Hatem, 1963; Schreiber, 1965a;
Stanway, 1971; Taylorson, 1986; Vanden Born, 1971). Heterogeneity of dormancy among seeds from a
single parent plant, seed heteroblasty, is one of several types of heteroblastic development of repeating,
self-similar plant units (metamers) during plant ontogeny (Gutterman, 1996; Jones, 1999). This phe-
nomenon has been characterized in other plant species (e.g., Xanthium pensylvanicum, Weaver and
Lechowicz, 1983; Chenopodium album, Williams and Harper, 1965). Systematic quantitative charac-
terization of heteroblasty among individuals within a locally adapted population, between local popu-
lations, and their relationship to subsequent life history behaviors has not been reported previously.
Evidence has been provided for a complex model of germinability regulation based on the inde-
pendent asynchronous actions of the embryo, caryopsis, and hull compartments, as well as on their
dependent synchronous action (Dekker et al., 1996). These studies provide evidence for a dynamic
developmental model of S. faberi germinability regulation resulting in phenotypes with a wide range
of germinability shed from an individual panicle. These diverse germinability phenotypes are found at
all stages of development, but particularly when the seed is shed and the soil seed pool is replenished.
An individual weedy Setaria spp. plant synflorescence produces individual seeds whose germina-
tion requirements vary. This seed heteroblasty occurs in any parental environment, even those within
controlled constant conditions for the duration of its life history (Dekker et al., 1996; Harr, 1998).
Seed heteroblasty is therefore a constitutively expressed genetic (or epigenetic) trait. The dormancy
capacity of an individual S. faberi seed refers to its dormancy state at abscission, as well as the quan-
tity of environmental signals (oxy-hydro-thermal time; Dekker et al., 2003) required to stimulate a
change in state (e.g., AR duration) (Dekker and Hargrove, 2002; Dekker et al., 1996; Sareini, 2002).
Germination capacity is an inherent quality of a Setaria seed, which is retained for its entire life.
Changes in germination were correlated with tiller development and relative time of seed matu-
rity within a panicle (Haar et al. 2012). Seeds produced on tillers that developed earlier (panicle age
[PA] and time of the first flower of panicle) were more likely to be dormant than seeds from later-
developing tillers (Figure 2.11). Seeds that matured later on a panicle (panicle position [PP], PDP)
were more likely to germinate than seeds that matured earlier on the same panicle. A consistent
trend toward later-maturing seed (calendar date [CD]) having less dormancy was found for seed
grown under different environments, which implies an inherent or parental source for variation in
giant foxtail seed dormancy. The variation in percentage germination at abscission and following
stratification AR treatments (4°C, dark, moist) indicates that the S. faberi seed rain consists of indi-
vidual seeds, possibly each with a different degree of dormancy.
Studies were conducted to determine the relationship between weedy S. faberi seed dormancy
and subsequent behaviors in the soil culminating in the timing of seedling recruitment (Atchison,
2001; Dekker et al., 2012b; Jovaag, 2006; Jovaag et al., 2011, 2012a,b). A robust characterization of
seed heteroblasty at the time of dispersal for 39 locally adapted S. faberi populations, as influenced
by parental genotype (time of embryogenesis) and environment (year and location) has been pro-
vided. The heteroblastic structure of each population was revealed by the germination response to
increasing durations of AR (in “ideal” conditions). The production of seeds with different germina-
tion requirements (germinability–dormancy) is a function of genotype, plant architecture, and the
environmental conditions during ca. 12 day embryogenic period. Hence, individual Setaria plant
populations are most accurately defined as a unique combination of species, individual parent plant
location, and time of abscission (calendar Julian week [JW] and year).
For Setaria, considerable heterogeneity in germination requirements existed among and within
all populations tested. Year, seasonal time of abscission, and location all proved to be important
factors influencing the germination heterogeneity structure of Setaria populations as revealed by
Panicle
position 1
cohort
POS
1
POS
2 POS
1 CD2
POS POS
1° 3 2
POS
4
1° POS
3
Calendar date cohorts

POS
1
POS
4 CD5
POS2
Time
2° POS3 POS1
POS4
POS2 CD8
POS1 POS3
POS2
2° POS4
3° POS
3 POS1
POS
4 POS
2
CD12
POS
3
3° POS4
Panicle age 1
Panicle age 3
Panicle age 5
Panicle age 2
Panicle age 4
Panicle age 6
All panicle cohort:

Age of first flower
FIGURE 2.11 Schematic description of Setaria faberi seed cohorts from primary (1°), secondary (2°), and
tertiary (3°) panicle types: calendar date (time) of seed abscission (CD); seed position on panicle at time of
abscission (PP, PDP); cohort of seed from entire panicle based on age (PA, time of first flower of panicle).
AR. These observed heteroblastic differences within and among populations revealed a fine-scale
adaptation to local space and time conditions. Earlier-harvested seed was consistently less germi-
nable than later-harvested seed. Earlier-fertilized seeds produced on Setaria 1° synflorescences
(typically August in the Midwest United States) are relatively more dormant than those from 2°
panicles (ca. September), which are in turn more dormant than seed on 3° panicles (ca. October)
and subsequent panicles. This is consistent with earlier studies (Dekker, 2004; Dekker et al.,
1996) suggesting the importance of photoperiod, which is longest while the early (JW32, August)
seeds are developing, then decreases through the middle (JW36, September) and late (JW40,
October) periods of the seed rain.
A B1
Germination
C
B2
After-ripening duration
FIGURE 2.12 Schematic diagram of the hedge-bet structure within the heteroblasty phenotype space. A: High
initial germinability, rapid germination response to AR. B1: Low initial germinability, increasing germinability
with increasing AR duration until a plateau of high germinability is reached. B2: Increasing germinability with
increasing AR, but less overall germination than that in B1. C: Little or no early germinability, but some increase
with long AR durations. D: “Perfect” crop response—all seeds immediately germinable.
Germination responses of seeds to AR were used to visualize the general patterns phenotype
space. A schematic diagram of this generalized germination reaction norm is given in Figure 2.10.
The majority of the populations were differentiated from each other; this variation indicated a
fine-scale adaptation to different local environments. Taken together, the 39 responses represented
Setaria’s “seed dormancy phenotype space” and revealed three different generalized dormancy
patterns. The first pattern, low-dormancy populations, had high initial germination in response to
low doses of AR. The second, high-dormancy populations, had no or low initial germination with
little additional response to increased AR. Most populations had the third pattern, intermediate to
the others, with low initial germination and increasing germination with increasing AR dose. The
third, most common pattern, occurred within the B1 and B2 regions (Figure 2.10). These regions
defined a pattern of low (<10%) initial germination, then increasing germination with increasing
AR, followed by a plateau at about 40–50 days of AR when the germination response to added AR
was saturated (at 70%–100% germination).
These studies emphasize the crucial role that environment (year and season) at the time of seed
formation and embryogenesis plays in the dormancy phenotype produced by plants in a local-
ity. The location itself (soil properties, fertility, past management history, etc.), the “ terroir,” can
also influence the quality of plant phenotypes produced. The germination patterns observed and
dormancy phenotype space occupied defined the heteroblastic hedge-bet structure at abscission.
The B1/B2 regions were the most commonly occupied phenotype spaces, suggesting a popula-
tion’s “best bet” generally laid within this region. Variation among populations within this region
indicated a fine-scale adaptation to the local environment. Regions A and C were less commonly
occupied, suggesting that they were “long shots” at the edge of local adaptation (Figure 2.12).
Germination responses were also used to rank populations based on their dormancy level to
facilitate later comparisons with emergence behavior. Weedy Setaria seed dormancy capacity het-
erogeneity at abscission (seed heteroblasty) provides a “blueprint” for subsequent behaviors in the
soil culminating in seedling recruitment.
2.4.3.2 Soil Seed Pool Formation: Dispersal in Space

Seed heteroblasty inevitably leads to the formation of long-lived seed pools in the soil. A seed
pool begins with heterogeneous DORM dispersed into the soil from local or distant plants. One
of the most important traits weedy Setaria spp. possess for invasiveness, colonizing ability, and
enduring occupation of a locality is their ability to form long-lived soil seed pools, the source
of all future annual weed infestations (Buhler et al., 1997a; Dekker, 1999). Dormancy inherent
in Setaria seeds inevitably leads to the formation of soil seed pools. Significant heterogeneity in
dormancy states among weedy Setaria seeds results in precisely timed germination over large
temporal scales (hours to decades; Atchison, 2001; Dekker et al., 1996; Forcella et al., 1992,
1997). Soil seed pools consisting of a diverse collection of Setaria species, genotypes, and dor-
mancy phenotypes reveal a hedge-betting strategy for adaptation to changing conditions within
agroecosystems (e.g., Cohen, 1966; Philippi and Seger, 1989). The reasons why Setaria spp. are so
successful in disturbed habitats derive from their biodiversity (Dekker, 1997; Wang and Dekker,
1995; Wang et al., 1995a,b), especially their ability to produce diverse phenotypes from a single
parent plant (e.g., Dekker et al., 1996) and their ability to time germination precisely. This results
in individual seedlings emerging from the seed bank at different times over large temporal scales
(hours to decades; Forcella et al., 1992, 1997).
The life of an individual weedy Setaria parental plant ends with reproduction, seed abscission,
and dispersal replenishing the soil seed pool (Dekker, 2003). Weedy Setaria spp. seeds separate
(shatter) from the parent plant soon after abscission, but the time of dispersal varies based on pedicel
deterioration and wind (Dekker, 2003). Seeds of the crop foxtail millet have been selected to resist
shattering, allowing near-complete harvest by the grower. Setaria seed dispersal occurs by gravity,
animal (e.g., birds), wind, water, and human (e.g., farm machinery) vectors (Bor, 1960; Ridley, 1930;
Steel et al., 1983; Wilson, 1980). The retrorsely barbed setae of S. verticillata stick easily to animals
and humans facilitating their dispersal (Bor, 1960). The Setaria seed rain period in the north tem-
perate regions of the world occurs generally from July through December, depending on seasonal
environmental conditions (notably the time of soil freezing and killing frost at the end of the season)
and harvesting (Atchison, 2001; Haar, 1998; Peters et al., 1963; Stevens, 1960). The time of seed
rain is continuous, with the seeds from 1° panicles occurring first, followed by 2° and subsequent
tillers. On individual panicles, the seed rain progresses in approximately the order in which they
flower (Dekker et al., 1996).
2.4.3.3 Dispersal and Local Adaptation: Evolutionary History of Setaria Invasion
Local adaptation of weedy Setaria spp. today is the evolutionary and ecological consequence of past
dispersal events: invasion, colonization, and enduring occupation. The weedy Setaria are a very
ancient group of plants, and their evolutionary history is characterized by invasion and colonization
followed by selection and local adaptation for enduring occupation of a site (Dekker, 2004). There
appears to be five major phases, or waves, of Setaria invasiveness in earth’s history. The tropical
genus Setaria dispersed to Eurasia from its probable first home in Africa. The first invading species
was probably an S. viridis or viridis-like diploid annual (Rominger, 1962; Stapf and Hubbard, 1930).
In the second phase, an S. viridis weedy progenitor species was spread over Eurasia as a wild
colonizing species. With domestication, it then spread both as a weed (Li et al., 1942, 1945; Wang
et al., 1995a,b) and as a crop (foxtail millet, S. viridis, subspecies italica; Fukunaga et al., 1997,
2002; Wang et al., 1995a,b; de Wet, 1995; de Wet et al., 1979; korali, S. pumila). Foxtail millet
was a crop in China 6000 years B.P. (Cheng, 1973; Gao and Cheng, 1988; Ho, 1975; Kawase and
Sakamoto, 1984; Li and Wu, 1996), while its use as a crop in Europe dates to 3500 B.P. (Dembinska,
1976; Kuuster, 1984; Neuweiler, 1946; de Wet and Harlan, 1975). S. pumila seed were gathered from
wild plants and later cultivated in India (de Wet, 1992). Other weedy foxtail species arose from
S. viridis-like ancestors (Li et al., 1942, 1945; Prasado Rao et al., 1987; Rominger, 1962; Willweber-
Kishimoto, 1962; Williams and Schreiber, 1976). These polyploid specialists probably included
S. pumila and S. verticillata.
In the third wave, the weedy foxtails spread to the New World at two different times: pre-Columbian
(before ca. 1500 A.D.) and post-Columbian invasions (after ca. 1500 A.D.). The pre-Columbian
origins of S. geniculata, the only weedy foxtail native to the New World, suggest an ancient dis-
persal event westward from the Old World (Africa) to New World (Rominger, 1962). A species of
Setaria was the oldest cultivated cereal in the Americas, its origins dating to 8000 B.P. (Callen,
1965, 1967; Smith, 1968). Prehistoric migration of Setaria could date from when the ancestors of
contemporary Native Americans first crossed the Bering Strait land connecting Asia and North
America (Beringia; approximately 10,000 B.P.). The S. pumila invasion of the Americas came pri-
marily from Asia (Wang et al., 1995b). The post-Columbian dispersal of weedy foxtails by Eurasian
human emigration over the last 500 years is most likely the major source of the weedy foxtail floral
introductions we see in North American agroecosystems today (Wang et al., 1995a,b). For example,
S. viridis had invaded Canada by 1821 (Rousseau and Cinq-Mars, 1969). The foxtails have expanded
their range since their introduction to North America in the last several hundred years as a conse-
quence of global trade and human-mediated dispersal (Hafliger and Scholz, 1980).
The fourth wave may have been determined recently by an extremely rare allo (tetra) polyploidiza-
tion event giving rise to a fertile hybridization product in southern China, S. faberi (Pohl, 1951, 1966;
Wang et al., 1995b). S. faberi was first introduced to North America in the 1920s near New York City
(Fairbrothers, 1959). Soon thereafter, it was found in Philadelphia (1931) and Missouri (1932). From
this early colonization, S. faberi spread rapidly in the Eastern and Midwestern states (Fernald, 1950;
Reed, 1970; Rominger, 1962). More recently, S. faberi has spread northward into Ontario in the 1970s
(Dore and McNeill, 1980; Warwick, 1990). The largest increase and spread of S. faberi occurred
across the North American corn- and cereal-growing regions in post-WWII era. The introduction and
adoption of the herbicide 2,4-D to control dicotyledonous weeds created an opportunity space for, or
population shift to, grassy weeds like the foxtails (Pohl, 1951; Slife, 1954). Introduction of other new
selective herbicides, as well as expansion of maize and soybean production in North America, acceler-
ated these trends (Warwick, 1990). In little over a half century, S. faberi has colonized a large part of
the most fertile disturbed areas of North America (Hafliger and Scholz, 1980).
Currently we are experiencing a fifth invasive phase, from the late 1970s through the 2000s,
with the appearance of many different types of herbicide-resistant biotypes of foxtail (e.g., Holt and
LeBaron, 1990; LeBaron and Gressel, 1982; Ritter et al., 1989; Stoltenberg and Wiederholt, 1995;
Thornhill and Dekker, 1993). Concurrent with this is the incremental spread of S. faberi and
S. pumila in the northern prairies of North America.
Geographic invasion can simultaneously be viewed from the perspective of genetic invasion:
Setaria polyploidization. Polyploid foxtails more specifically adapted to niche opportunities in
agroecosystems include those four weedy foxtails with more narrow geographic distributions than
diploid S. viridis: knotroot, yellow, S. verticillata, and more recently S. faberi. The fourth geo-
graphic invasive wave was also the fourth genetic polyploidization, weed speciation event.
2.4.4 Seed Behavior in the Soil

Weedy Setaria behavior in the soil is regulated by the amount of oxygen dissolved in soil water
taken into the seed over time.
There exists a cascade of three morpho-physiological mechanisms that act together to modulate
received oxygen, water, and heat and thereby constrain and control Setaria spp. seed embryo behav-
ior. The seed hull and outer envelopes act to attract and accumulate water, enhance gas solubility
in surface water film by means of surface rugosity, and channel that gas-laden water to the PP. The
TACL is a membrane whose diameter and function regulate diffusion of gas-laden water in and out
of the living seed interior. An oxygen-scavenging heme-containing protein in the living interior acts
to buffer the seed against premature germination by sequestering oxygen. This morphology strongly
suggests that seed germination is restricted by water availability in the soil and by the amount of
oxygen dissolved in water reaching the inside of the seed symplast to fuel metabolism.
Soil water, temperature, and oxygen play a unifying role in regulating both the global biogeo-
graphic distribution of weedy Setaria spp. and the responses of individual seeds in a soil microsite.
Environment and morpho-physiological traits interact to stimulate weedy Setaria seed behav-
iors: induction of embryogenic dormancy, induction of summer dormancy, AR, germination, and
seedling emergence. The signal stimulating all these Setaria behaviors is the quantity of oxygen
within the water content of the living seed caryopsis interior per time unit, oxy-hydro-thermal time:
mass O−12 volume H2O time seed .
−1 −1 −1
Setaria seed behavior in the soil is predicated on dormancy capacity heterogeneity at abscission
and modulated by the seasonal environmental conditions experienced in the field: seed germinability–
dormancy heteroblasty is the blueprint for all postabscission behaviors in the soil.
2.4.4.1 Control of Seed Germinability

Seed germination includes all types of embryo growth under any condition. Germinability refers
to the capacity of an embryo to germinate under some set of conditions. AR refers to the period
after dispersal when the seed cannot germinate, even under favorable conditions, and during which
changes occur allowing it to germinate. Dormancy is avoided except when referring to specific
published reports that use the term (Dekker et al., 1996).
2.4.4.1.1 Regulation of Weedy Setaria Seed Behavior

The wide geographic range of adaptation, heterogeneity in dormancy phenotypes, and genotypic
diversity raise the question of what mechanisms in weedy Setaria spp. seeds drive seed behaviors
(e.g., induction of dormancy, AR, germination, induction of summer dormancy, and seedling emer-
gence) (Dekker, 2003). There is evidence that soil water, temperature, and oxygen play a unifying role
in regulating both the global biogeographic distribution of weedy Setaria spp. and the responses of
individual seeds in a soil microsite (Dekker, 2000; Dekker and Hargrove, 2002; Dekker et al., 2001).
The effects that naturally occurring gases (oxygen, nitrogen, and carbon monoxide) may cause
in dormant giant foxtail (S. faberi) seed germination under favorable temperature and moisture
conditions were investigated (Dekker and Hargrove, 2002). The germination responses to gas
mixtures supported the hypothesis that S. faberi germination behavior is regulated by the amount
of oxygen taken into hydrated seed over time. S. faberi seed germination was markedly affected
by O2 concentration (in N2) above and below that of air (20% O2): the largest increase in ger-
mination (from 37% to 60%) occurred between 20% and 25% O2; between 0% and 10% O2,
germination increased from 0% to 30%; and surprisingly germination at 10% and 20% O2 was
similar. These observations reveal an asymmetrical response to incremental changes in O2 above
and below that typically found in agricultural soils. Carbon monoxide had opposite effects on
S. faberi germination in air depending on concentration, stimulation, and inhibition: germination
increased from 37% to 56% with the addition of 1% CO, but decreased from 37% to 14% with
75% added CO. An explanation may be that there are two separate effects of CO, each occurring
in different physiological systems of DORMs at the same time. At high concentrations (75%) in
air, CO inhibited seed germination, probably by inhibiting mitochondrial respiration. But low CO
concentrations (0.1 or 1%) in air stimulated seed germination. It was not apparent which physi-
ological system(s) CO and O2 affected. It seems unlikely that CO-stimulated germination arises
from effects on the respiratory apparatus, but may be a consequence of CO interactions with an
as yet uncharacterized physiological factor in the seed (Sareini, 2002).
The morphology of weedy Setaria spp. seeds provides clues about which environmental fac-
tors limit germination and maintain dormancy. This morphology strongly suggests that seed ger-
mination is restricted by water availability in the soil and by the amount of oxygen dissolved in
water reaching the inside of the seed interior to fuel metabolism. Oxygen solubility in the water
entering the interior is an inverse function of the diurnally, seasonally, and annually changing
soil temperature. The control by this oxygen-limited gastight morphology is supported by obser-
vations of increased germination when Setaria seed envelopes, including the CC, are punctured
(Dekker et al., 1996; Stanway, 1971), as well as by the stimulatory effects of oxygen and carbon
monoxide (Dekker and Hargrove, 2002).
There exists a cascade of three morpho-physiological mechanisms that act together to modulate
received oxygen, water, and heat and thereby constrain and control Setaria spp. seed embryo behavior.
The first is the seed hull and outer envelopes that act to attract and accumulate water, enhance gas
solubility in those water film by means of its surface rugosity, and channel that gas-laden water to
the PP (the basal opening to the seed interior, the only water entry point in the seed) (Donnelly et al.,
2012). The PP terminates with the TACL, a membrane whose diameter and function regulate the dif-
fusion of gas-laden water in and out of the interior (Rost, 1971a,b, 1972, 1973, 1975; Rost and Lersten,
1970, 1973). The foxtail seed is gas- and watertight except at this pore due to the enveloping caryopsis
coat (CC). Together, the PP, TACL, and CC provide the second controlling mechanism. The third
element is an oxygen-scavenging heme-containing protein (“X,” Figure 2.1) in the interior that acts to
buffer the seed against premature germination by sequestering oxygen (Dekker and Hargrove, 2002;
Sareini, 2002). These controlling mechanisms are represented schematically in Figure 2.13.
The interaction of these three mechanisms in any individual seed defines its unique dormancy
state at abscission, which interacts with its immediate environment to define its subsequent behav-
ior. Environmental signals change the initial dormancy state of an individual seed over the course
of its life history in the soil. The quantity of these signals required to change behavior is an intrin-
sic quality of the individual and is retained for the entire life of the seed. The dormancy state an
individual living seed inherits from its parent plant is never lost or “broken.” The behavior of an
individual seed in the soil during its life history is always a consequence of its current intrinsic state
responding to its current extrinsic conditions, a Markov chain process.
2.4.4.1.2 Movement of Water–Gas between Soil and Seed

There is increasing evidence that specialized properties of the seed, its shape, its size and form, and the
nature of its surface determine the type of contact the seed makes with the soil and on this depends the
ability of the seed to germinate successfully. (Harper, 1964)
Seed morphology therefore seems to be a ripe area for investigation, in particular the morphology
of seed surfaces as the interface, the “antenna,” linking the soil-borne weed with its immediate
microsite environment (Dekker and Luschei, 2009; Donnelly et al., 2012). The seed morphological
structures that water and gases encounter as they enter the seed from the soil environment extend
from the glumes to the embryo: glumes–hull–PP–placental pad–CC–TACL membrane–aleurone
layer–endosperm–embryo. The environmental signal regulating weedy S. faberi behavior is the
amount of oxygen dissolved in water received by the embryo over time and temperatures conducive
to both growth and oxygen solubility (i.e., oxy-hydro-thermal time, Dekker et al., 2003). These con-
trolling mechanisms are represented schematically in Figure 2.14.
Temperature, oxygen, and moisture signals are exchanged between three continuous partitions:
(1) the soil matrix, (2) the surface of the seed (hull) that includes the PP, and (3) the interior of the
seed (Figure 2.13). The soil matrix in contact with the seed surface is the local source of moisture
and gases required by the seed to initiate germination. The resupply rate of this external water/gas
pool varies over time in delayed response to macroscopic hydrological and thermal cycles (weather).
Germination requires conditions favorable for rehydration and sustained metabolism. For a variety
of plant species, lower temperatures, causing increased dissolved oxygen content and lower meta-
bolic demands in seeds, can play an important part in seasonal germination timing (Dekker and
Hargrove, 2002). It is therefore logical that the presence of surface films or excessive moisture as
supplied by the environment can alter the degree to which a given internal moisture content and
temperature are favorable for embryonic germination (Figure 2.15).
It is hypothesized that the change in moisture content of the Setaria spp. seed is driven by the
dynamics of two anatomical structures: the hull and the caryopsis. Water saturates the hull by adhe-
sion and the formation of surface films. In contrast, for caryopses with intact suberized coats, water
enters and hydrates the caryopsis only by passing through the PP. Thus, the anatomy of the seed
hull may function as more than a simple protective structure for the Setaria seed; it may represent a
H2O–O2
Glumes Caryopsis coat Soil

Soil particle
particle H2O–O2
Seed hull
H2O–O2
H2O–O2
Aleurone layer
H2O–O2
H2O–O2 Endosperm
H2O–O2
H2O–O2 TACL
H2O–O2 “X” H2O–O2
H2O–O2
H2O–O2
H2O–O2 O2 H2O–O2
H2O–O2
H2O–O2
H2O–O2
Placental
H2O–O2
Soil pore
H2O–O2
particle H2O–O2 Embryo
H2O–O2
H2O–O2 H2O–O2
H2O–O2
Mitochondria
H2O–O2
Seed Dormancy, Germination, and Seedling Recruitment in Weedy Setaria
FIGURE 2.13 Schematic diagram of the Setaria sp. seed, surrounding soil particles, and oxygen dissolved in water (H2O–O2). The seed interior (aleurone, TACL,
endosperm, O2-scavenging protein (X), and embryo) is surrounded by the nonliving glumes, hull, PP, and the gas- and water-impermeable caryopsis coat.
71
H2O–O2
Placental pore
TACL
Seed hull
Endosperm
O2 Caryopsis coat
Aleurone layer
“X”
Mitochondria
Embryo
FIGURE 2.14 Schematic diagram of the Setaria sp. seed and the morpho-physiological components respon-
sible for seed dormancy and behavior. The interior (aleurone layer, transfer aleurone cell layer [TACL], endo-
sperm, oxygen-scavenging protein (X), and the embryo) is surrounded by two enclosing structures, the hull
(lemma and palea) and the gas- and watertight caryopsis coat. Water and dissolved gas entry into the interior
is restricted to the PP at the basal end of the seed.
Heat
Soil matrix
Soil
water–gas
Gas
Embryo
FIGURE 2.15 Schematic diagram of the transduction of water–gas (e.g., H2O–O2) and temperature (HEAT)
signals from the soil matrix to the Setaria sp. seed symplast. Soil water–gas mixtures can be present in three
continuous partitions (gray shaded areas): soil matrix water–gas (top), seed exterior (hull), and apoplast (bot-
tom left gray), and the seed symplast (bottom right gray). Heat from the environment affects oxygen solubility
in water and metabolic behaviors (e.g., germination).
mechanism by which the seed has tuned its germination timing by indirectly altering the frequency
of its internal moisture and gas availability.
Water saturates the hull by adhesion and the formation of surface films (Dekker and Luschei,
2009). In contrast, water typically enters and hydrates the gas- and watertight caryopsis only by
passing through the PP. By analyzing the rate of water loss during a drying period, it was possible to
determine the initial moisture content of the two anatomical structures and to reveal how the loss of
water from the hull (partition 2) and caryopsis (partition 3) varied with moisture availability and ther-
mocycle conditions in the local environment (partition 1). The drying procedure removed water in
three distinct phases. In the first, water was lost quickly from the hull surface and more slowly from
the caryopsis via the narrow PP. In the second, water continued to be lost from the caryopsis alone.
After some time, the interior water was tightly bound in the seed, the final phase. The moisture incu-
bation experiments demonstrated that S. faberi seeds did not absorb water in the manner expected if
it was a homogeneous material. Hydration of dry S. faberi seeds began with preferential partitioning
of water to the interior seed caryopsis and the embryo. With additional local moisture availability, the
hull and caryopsis compartments absorbed moisture in a similar manner until the moisture content
becomes sufficiently high to saturate the caryopsis. The caryopsis saturated at a lower local water
availability content than did the seed hull. With the saturation of the caryopsis, the hull preferentially
absorbed the excess moisture. Hull water was spread uniformly over the seed surface forming a water
film (partition 2). It is speculated that the water film changes the manner in which the seed interfaces
with the external soil microsite environment (partition 1), the locus of oxygen exchange. The delivery
rate of oxygen to the embryo is a function of its phase, partial pressure gradient, and diffusion rate to
the embryo (partition 3). The thickness of the seed surface water film is therefore a powerful mecha-
nism regulating the pool of dissolved oxygen pool (partition 2) directly available to the seed embryo
(partition 3), hence its crucial role in determining subsequent seed behaviors. Evidence that hull sur-
face water quantity, in the physical form of boundary layer film thickness, controls germination was
provided by the observation that maximum seed germination occurs in conjunction with intermedi-
ate levels of moisture availability in the soil environment. Soil moisture levels above and below this
optimal hydration level resulted in lower germination. This observation provides a strong inference
that hull surface water quantity in the physical form of boundary layer film thickness controls ger-
mination. Consistent with the theory of oxygen–water control of S. faberi germination (e.g., Dekker
and Hargrove, 2002), these observations support the concept that optimal water film thicknesses on
the seed surface are those that maximize the trade-off between hull surface area available for oxygen
diffusion and water availability to support and sustain germination metabolism. Relatively low hull
surface water contents provide thinner water films for soil atmosphere gases to penetrate and there-
fore support greater gas diffusion but provide insufficient moisture for germinative growth. Thicker
surface films provide greater resistance to gas diffusion but provide ample moisture for germination.
Intermediate water availability optimizes the trade-off between water film gas diffusion (the dis-
solved oxygen pool) and moisture for germination.
Alternating diurnal thermocycles stimulated greater seed germination than did constant tem-
peratures for all moisture contents greater than 0.25 mL, given the same heat units in each condi-
tion. These results are consistent with the hypothesis that alternating diurnal temperature conditions
provide additional oxygen to the seed embryo and interior caryopsis due to the degassing effect that
occurs when water temperature is increased. When temperatures increase in a water solution with
oxygen at equilibrium with the local atmosphere, gas solubility decreases and excess oxygen is
forced out of solution as a gas to the adjacent environment as it reaches the new equilibrium. This
pulse of gaseous oxygen is immediately available to the seeds’ metabolic physiology, the acute oxy-
hydro-thermal time signal (Dekker et al., 2001, 2003).
These experiments indicate a robust dynamic system of germination control by the transduction
of external moisture and thermocycle signals from the soil to the seed embryo as modulated by the
characteristics of the seed hull surface and the PP. The seed is unable to control its temperature, but
the morphology of its exterior surface provides a means by which the seed is able to regulate the
other two requisites for germinative growth, moisture, and oxygen.
In a study by Donnelly (Donnelly et al., 2012), evidence was provided that seed shape in the
genus Setaria is a product of the trade-off between the constraints of phylogeny and local adapta-
tion. Although there is a general “foxtail shape” (i.e., Setaria seeds from any of the taxa examined
are recognizably similar), phylogenetic relatedness alone cannot explain the pattern of shape dif-
ferences found in this species-group. Green foxtail is more similar in shape to the taxa that share
its ecological niche (invasive–colonizing–weedy) than to the crops that are conspecific with it.
Evidence was found of selection for size, surface-to-volume ratio (S:V), shape, seed surface rugos-
ity, and seed hull permeability to water and gases. From these results, the null hypothesis was
rejected that phylogeny alone is sufficient to explain the pattern of variation in shape between taxa
in Setaria. Because there was a clear divide between taxa of differing life histories (weeds vs.
crops), the possibility that the differences in shape between taxa are random (neutral drift) was also
rejected. Thus, it is likely that there is a functional explanation to explain the pattern of variation
observed.
The results can be explained by the hypothesis that seed shape in Setaria is adapted to transduce
an environmental signal consisting of oxygen, water, temperature, and time (oxy-hydro-thermal
time) (Dekker, 2003; Dekker and Hargrove, 2002). This signal is used by the seed interior to regu-
late seed behavior, particularly germination. Moisture availability plays a key role in the regulation
of germination in S. faberi (Dekker and Luschei, 2009), with too much or too little water inhibiting
germination (Dekker and Luschei, 2009). With the sensitivity Setaria exhibit to temperature fluc-
tuations, oxygen levels (Dekker and Hargrove, 2002), and volume of water forming a film on the
surface of the seed (Dekker and Luschei, 2009), we conclude that transducing an oxy-hydro-thermal
time signal is a crucial role of the seed exterior. To transduce the signal, the outer surfaces of the
seed must accumulate soil water, passively oxygenate the accumulated water, and channel it to the
seed interior via the PP. In other words, the lemma and palea of a Setaria seed act as an antenna to
receive, modulate, and transduce information about the environment that the seed interior can use
to determine germination.
The efficiency of this antenna is affected by several features of the seed’s exterior, size, S:V,
rugosity, and the permeability of the lemma, palea, and lemma/palea (LP) suture to water. The
differences we found between taxa show how each group has evolved outer seed morphology to
increase the signal reception. S. viridis and S. verticillata have very small seeds, which are more
elongate to increase surface area relative to seed volume. Elongation in these species is likely a
result of the trade-off between size and the S:V. S. faberi, with slightly larger seeds, has also evolved
elongation that has resulted in a higher S:V than S. viridis and S. verticillata. The seeds of S. geniculata
are larger and the most elongate with a moderately high S:V. S. pumila has the highest S:V despite
being the least elongate and having the largest seed size of the weedy taxa. It may be that life history
differences between S. pumila and S. geniculata (S. geniculata was the only perennial species in the
study) explain the differences in elongation between them. As a perennial, S. geniculata has mul-
tiple years for reproduction and the potential to reproduce vegetatively (Rominger, 1962). This could
reduce selection pressure for increased seed volume as found in S. pumila. Despite their differences,
all the weeds show a distinct selective pressure acting on seed shape. The seeds are elongate to at
least a limited extent, and all have relatively flat paleae. Foxtail millets’ (S. viridis, subsp. italic;
S. italica) domestication has resulted in much rounder (less elongate), moderately sized seeds with
an intermediate S:V. The palea is extremely rounded in foxtail millet, likely due to the selection for
maximizing starch content.
Rugosity is likely to amplify the differences seen in S:V in the weedy taxa. S. pumila and
S. geniculata, the taxa with the highest S:V, are also the most visibly rugose. S. faberi has more rugose
seed surfaces than S. viridis or S. verticillata and also a greater S:V. However, S:V in foxtail millet will
likely decrease in relation to the weeds when rugosity is factored in. The seeds of foxtail millet are
relatively smooth in comparison to their weedy relatives (Pohl, 1951, 1978; Rominger, 1962).
Although it is hard to separate the crops out from the weeds by looking at seed size and S:V,
there is a large difference between the taxa in terms of water permeability along the LP suture and
seed surface fragility. Because the LP suture is completely fused in the weedy taxa, water must be
channeled to the seed interior via the PP (abscission point). This is not necessary in foxtail millet
because the LP suture is not completely sealed at the distal end and the lemma and palea of foxtail
millet are thin and fragile (Pohl, 1951; Rominger, 1962).
Another difference between the weeds and crops is the germination behavior. The weeds ger-
minate throughout the growing season, adjusting to local agricultural practices (Atchison, 2001;
Jovaag et al., 2011). Foxtail millet, on the other hand, has been bred to exhibit nearly simultaneous
germination. If seed shape in the weedy foxtails is adapted to transduce the environmental signal
the seed interior uses for germination, it is likely that the differences in seed shape and water per-
meability in foxtail millet are due to the release of the selection pressures acting on seed shape. In
addition, selective breeding for increased starch volumes in foxtail millet is likely the reason foxtail
millets are much more spherical than the weedy taxa (maximizing volume while minimizing sur-
face area).
With the results of this study, the oxy-hydro-thermal time signal regulating seed behavior, sen-
sitivity to water film thickness, and the importance of the outer seed surface as the primary inter-
face between the seed interior and its environment, the null hypothesis that seed shape differences
between the taxa in the genus Setaria can be explained by phylogenetic relatedness is rejected. It is
also likely that Harper’s (1964) supposition that seed shape is sensitive to environmental differences
is correct for this weedy/invasive/colonizing species-group.
2.4.4.2 Seed Behavior

2.4.4.2.1 Annual Seed–Dormancy–Germinability Cycling in the Soil
The fate of heteroblastic Setaria faberi seed entering the soil postabscission was elucidated by
Jovaag (Jovaag et al., 2011). Introduction of four populations of S. faberi seeds with heterogeneous
dormancy capacities into the soil of a no-till Glycine max field resulted in the formation of endur-
ing pools with varying seasonal cycles of dormancy, AR, germination, dormancy reinduction, and
death (Figure 2.14).
The buried seed rain of highly dormant seed after-ripened with time and became highly ger-
minable, awaiting favorable temperature and moisture conditions: the heterogeneous germination
CAN pool. As this pool was depleted in the spring and early summer by seedling emergence and
death, dormancy was reinduced in the living seeds that remained in the soil. The seeds remained
dormant throughout the summer, then resumed AR during late autumn. This dormancy–germinability
cycle exhibited complexity both within and among S. faberi populations. Seed heteroblasty within
S. faberi populations was retained, and germinability responses to the yearly seasonal environment
varied among S. faberi populations. Further, local adaptation was shown by the differential germi-
nability responses among S. faberi populations in common location agricultural nurseries. Seed
mortality patterns also exhibited complexity within and among populations. Within an individual
S. faberi population, mortality patterns changed as seeds aged in the soil. Among S. faberi popula-
tions, differential mortality responses were observed in response to yearly seasonal environments
and common nurseries (Figure 2.16).
These observations clearly indicate that S. faberi seeds have an annual dormancy–germina-
bility cycle similar to that of the summer annual weeds reported by Baskin and Baskin (1985):
high spring germinability, summer dormancy reinduction, and higher autumn germinability.
Unlike that previous report, evidence is now provided that individual S. faberi seed popula-
tions retained heteroblastic qualities throughout this annual cycle, that cyclic responses to the
yearly seasonal environment varied among populations, and that adaptation to local condi-
tions occurred in the germinability of an individual population. The retention of heteroblasty
within a population is evident in the variation of the proportions of CAN seeds within a single
Input:
Variable
dorm state
seed
Seedling
(output)
Soil seed pool
erg g
e
em edlin
enc
Se
Dormancy Seed
Dormant reinduction germination Germination Germinated
seed candidate Growth seed
After-ripening
Death Death
FIGURE 2.16 Schematic diagram of weedy Setaria spp. soil seed pool behavior based on the life history of the
seed (seed states and processes (transitions between states)). Life history states: Dormant seeds, seed germination
candidate, germinated seeds, seedlings, and dead seeds or seedlings. Life history processes: induction of dormancy
and input (dispersal) of dormant seed at abscission (seed rain), after-ripening of dormant seed, dormancy reinduc-
tion of germination candidates, germination and growth of germination candidates, seedling emergence, and death.
population and time, and the heterogeneous decline in germinability, from early spring to early
summer. The Lorentzian function model provided a sensitive method to distinguish this hetero-
blastic variation in the proportion of germination CANs over seasonal time among populations.
Germinability responses to yearly environmental conditions were also shown. Local adapta-
tion was evident in the differential germinability responses among S. faberi populations to the
common nursery environments (i.e., a phenotype by environment interaction). This differential
response occurred in both years. These observations provide evidence that the heterogeneous
dormancy capacity among individual seeds remained, consistent with the hypothesis that dor-
mancy capacity is an inherent quality of the seed, and dormancy capacity heterogeneity of the
population remains over time.
2.4.4.2.2 Seed Longevity in the Soil
The length of time that weedy Setaria spp. seeds are able to survive in the soil is quite variable,
but mortality decreases with the depth of burial (Dekker, 2003). The maximum period of survival
in the soil varies by species: S. pumila: typical, 13 years; maximum, 30 years (Darlington, 1951;
Dawson and Bruns, 1975; Kivilaan and Bandurski, 1973; Toole and Brown, 1946); S. viridis: maxi-
mum 10–39 years (Burnside et al., 1981; Dawson and Bruns, 1975; Thomas et al., 1986; Toole and
Brown, 1946); and S. verticillata: maximum, 39 years (Toole and Brown, 1946). More typically,
the majority of weedy Setaria spp. seed live in the soil for much shorter time periods (Atchison,
2001). In general, S. viridis and S. pumila seed on the surface lose viability sooner than buried
seed (Banting et al., 1973; Thomas et al., 1986). The rate of survival of Setaria seed is longer
under uncultivated conditions (Stoller and Wax, 1974; Waldron, 1904). Burial of weedy Setaria
spp. seed increases the level of dormancy, viability, and longevity, of those seeds, possibly due to
decreased oxygen at greater depths or within soil aggregates (Banting et al., 1973; Dekker and
Hargrove, 2002; James, 1968; Pareja and Staniforth, 1985; Pareja et al., 1985; Stoller and Wax,
1974). Dawson and Bruns (1975) showed that precipitation had no effect on S. viridis seed longev-
ity in the soil.
2.4.5 Seedling Recruitment
Weedy Setaria spp. seeds with heterogeneous dormancy states are recruited from soil pools when suf-
ficient oxygen, water, and heat accumulate over time (Dekker et al., 2003). The variable timing of this
recruitment is the first step in assembly with crops and other weeds in annually disturbed agricultural
communities. The timing of seedling emergence is the single most important factor in determining
subsequent weed control, competition, crop losses, and replenishment of the soil seed pool.
2.4.5.1 Seedling Emergence and Community Assembly

Seedling emergence is the birth of a Setaria plant, its entrance into a local community, and the first
committed step in assembly with neighbors. Soil seed pool spatial structure, seed dormancy, and
environment determine where and when seedlings emerge. Successful assembly of Setaria spp. in
local communities with other species is determined by key traits typifying their annual life history.
The first assembly step in many agricultural fields is the emergence of seedlings or herbaceous foli-
age of perennial species. Successful foxtail seedlings then interact with other species in the locality.
The competitive phase concludes with reproduction and entry of new seed into the soil. Seedling
emergence prediction guides early-season risk management and the use of weed tactics. Time of
seedling emergence is the single most important factor determining subsequent yield losses from,
and competitiveness of, a weed. A weed’s competitive success determines yield losses, seed bank
replenishment, and future weed infestations.
2.4.5.1.1 Seedling Emergence

In general, weedy Setaria spp. seeds are capable of germinating and emerging anytime the soil is
unfrozen (Dekker, 2003). Most Setaria seedlings emerge in the early part of the growing season
of a locality (e.g., April–June in the northern hemisphere) (Atchison, 2001; Buhler et al., 1997b;
Forcella et al., 1992, 1997): S. viridis (Banting et al., 1973; Chepil, 1946; Dawson and Bruns, 1962);
S. pumila (Dawson and Bruns, 1962, 1975; Manthey and Nalewaja, 1987; Martin, 1943; Sells, 1965;
Steel et al., 1983; Stoller and Wax, 1974); and S. verticillata (Martin, 1943). With the knowledge
of dormancy states and environmental O2–H2O signals in a local seed pool, it may be possible to
predict Setaria emergence in a locality with precision (Dekker et al., 2001).
2.4.5.1.2 Seed Germination Depth from the Soil
Weedy Setaria spp. seedlings emerge from relatively shallow depths in the soil, usually between
1 and 5 cm depth (Dekker, 2003). Emergence is greatest at 1.5–2.5 cm and declines with depth to
a maximum of about 7.5–14 cm (Buhler, 1995; Buhler and Mester, 1991; Dawson and Bruns, 1962;
Gregg, 1973; Waldron, 1904). Emergence of weedy Setaria spp. does not usually occur on the soil
surface except in the debris of no-till systems, an indication that light may not play an important
role in seed germination. The soil depths from which the maximum S. faberi emergence occurs
is a function of the tillage system: the greater the depth of tillage, the deeper and more varied the
seed placement and seedling emergence: 0–2 cm (no-till); 1–3 cm (disk); 1–4 cm (chisel plow); and
1–5 cm (moldboard plow) (Mester and Buhler, 1986; Yenish et al., 1992). Atchison (2001) observed
that S. faberi in the first year after dispersal emerged from shallower depths (0–5 vs. 5–10 cm), but
emergence of older, more dormant seed was similar from these two depths. Others have correlated
depth of emergence and seed weight, surmising that greater seed weight indicates more seed reserves
(Dawson and Bruns, 1962; King, 1952). Oxygen content decreases with soil depth, inhibiting AR and
germination at greater depths and enforcing dormancy (Dekker and Hargrove, 2002; James, 1968).
2.4.5.2 Patterns of Seedling Emergence

Seedling recruitment of heteroblastic S. faberi seed entering the soil postabscission was elucidated
(Jovaag et al., 2012). Evidence was provided that weedy Setaria seedling recruitment behavior
is predicated on dormancy state heterogeneity at abscission (seed heteroblasty), as modulated by
environmental signals. Complex oscillating patterns of seedling emergence were observed during
the first half of the growing season in all 503 soil burial cores of the 39 populations studied. These
patterns were attributed to six distinct dormancy phenotype cohorts arising from inherent somatic
polymorphism in seed dormancy states. Early-season cohorts were formalized using a mixture
model consisting of four normal distributions. Two, numerically low, late-season cohorts were also
observed. Variation in emergence patterns among Setaria populations revealed a fine-scale adapta-
tion to local conditions. Seedling recruitment patterns were influenced by both parental-genotypic
(time of embryogenesis) and environmental (year, common nursery location, and seed age in the
soil) parameters. The influence of seed heteroblasty on recruitment behavior was apparent in that
S. faberi populations with higher dormancy at the time of dispersal had lower emergence num-
bers the following spring, and in many instances occurred later, compared to those less dormant.
Heteroblasty was thus the first determinant of behavior, most apparent in recruitment number,
less so in pattern. Environment modulated seedling numbers, but more strongly influenced pat-
tern. The resulting pattern of emergence revealed the actual “hedge-bet” structure for Setaria seed-
ling recruitment investment, its realized niche, and an adaptation to the predictable mortality risks
caused by agricultural production and interactions with neighbors. These complex patterns in seed-
ling recruitment behavior support the conjecture that the inherent dormancy capacities of S. faberi
seeds provide a germinability “memory” of successful historical exploitation of local opportunity,
the inherent starting condition that interacts in both a deterministic and a plastic manner with envi-
ronmental signals to define the consequential heterogeneous life history trajectories.
S. faberi is a very successful invasive agricultural weed because of its ability to form long-lived
soil seed pools of heterogeneous seed (Jovaag et al., 2012a,b) that cycle annually between dormancy
and germination candidacy until environmental conditions allow germination and emergence to
occur. Seedling recruitment is an irreversible committed step in the life history of a plant. It is the
time when the annual S. faberi plant resumes autotrophic growth, assembles in local agrocommuni-
ties, and begins interactions with neighbors. As such, its timing is crucial to subsequent behaviors,
including survival and reproductive success. Seedling emergence timing is a trade-off between the
risks of mortality from disturbance and competition with neighbors and exploitation of opportu-
nity space–time to accumulate biomass and produce seed. S. faberi has evolved in agroecosystems
characterized by annual disturbance and selection by predictable farming practices (e.g., planting,
tillage, herbicides, and harvesting). The variable risks in these agricultural habitats have not led to a
single best time to germinate. Is there a pattern to the periodicity of S. faberi seedling recruitment?
Past research reveals only a qualitative pattern: an early flush of seedling emergence in the spring
(April–June) that decreases as temperatures warm, followed by low numbers during the late season
(e.g., Forcella et al., 1992, 1997). Is it possible to discover a more precise quantitative pattern to
seedling emergence? If so, does it bear a relationship to seed heteroblastic patterns among the same
seeds at abscission? A successful survival strategy for a prolific seed-producing species like Setaria
may be to disperse heteroblastic seeds able to take advantage of as many recruitment opportunities
as are available, in both seasonal time and field space. The resulting pattern of seedling emergence
timing may reveal the “hedge-bet” for individual Setaria fitness. It is hypothesized that there exists
a pattern to Setaria seedling recruitment timing, which is predicated in the first instance on the
dormancy state heterogeneity (seed heteroblasty) at abscission. This pattern in the second instance
is modulated by the environment (year, common nursery location, seed age in the soil). Thus, pat-
terns in Setaria emergence will reflect the historically advantageous recruitment times (opportu-
nity) enabling survival and reproductive success.
2.4.5.2.1 General Seedling Recruitment Pattern

A complex pattern of S. faberi seedling emergence was observed consisting of six discrete cohorts
during the growing season: JW 16–49 (Jovaag et al., 2012). The majority were recruited during four
early-season (JW16–31) emergence periods, and a small minority during two later-season (JW32–49)
emergence flushes. This complex pattern of discrete discontinuous seedling emergence was unexpected.
A priori expectations were of a large continuous flush of emergence in early season followed by low
sporadic numbers during the late season, as reported previously (e.g., Forcella et al., 1992, 1997).
2.4.5.2.2 Early-Season Recruitment

A complex oscillating pattern of S. faberi seedling emergence was observed in the first half of the
growing season (JW16–31) in all 503 cores of the 39 populations studied and formalized using a mix-
ture model consisting of four normal distributions, which consistently fit data from different popula-
tions and years (Figure 2.15). The consistent appearance of these four normal distributions provides
strong evidence of the existence of discrete recruitment cohorts arising from inherent somatic poly-
morphism in seed dormancy states (seed heteroblasty). Changes in the recruitment of early-season
S. faberi cohorts in response to several factors were reflected in their timing, the proportional distri-
bution among the individual seasonal cohorts, and the total number of seedlings that emerged.
2.4.5.2.3 Parental and Environmental Influences on Recruitment

Fine-scale changes in seedling emergence patterns in response to changes in parental qualities (time
of abscission) and environmental conditions (year, common nursery, and seed age in the soil) indicate
that S. faberi has the ability to adapt its behavior on a fine scale to a locality by generating multiple
well-timed and numbered pulses of seedlings over the course of the growing season (Figure 2.17).
2.4.5.2.3.1 Parental Influences Seed heteroblasty is induced during embryogenesis and modu-

lated by the specific seasonal conditions experienced by the parental plant (Dekker, 2003). The
influence of seasonal environment on dormancy induction is apparent at the time of seed abscis-
sion from the parent plant: the earliest maturing (August) seed was more dormant than middle
(September) seed, which in turn was more dormant than late (October) seed (Jovaag et al., 2012a).
The influence of seed heteroblasty was more apparent in recruitment number, less so in pattern.
Total seedling recruitment was often inversely related to the time of seed maturity from August to
October. This inverse relationship between the time of abscission and the number of seeds subse-
quently emerging during the spring and early summer was observed in 1999 and the second half
of 2000. Further, midsummer to autumn emergence during both 1999 and 2000, while low, came
only from middle- and late-maturing populations. During the first half of 2000, there was generally
no difference in the emergence numbers, proportions, or timing among seeds with different abscis-
sion times. This similarity indicated that environmental conditions dominated emergence patterns
in 2000. The greater variation in emergence proportion and number during 1999 indicated that
environmental conditions were less dominant, and parental variability more influential, than that in
2000. Seed heteroblasty had some influence on emergence pattern: less dormant populations some-
times emerged earlier than more dormant. In years producing more germinable seeds, the majority
generally emerged earlier compared to those years producing relatively more dormant seeds.
2.4.5.2.3.2 Year Seasonal conditions (year) had the greatest influence on S. faberi recruitment
relative to other environmental parameters studied.
2.4.5.2.3.3 Seed Age in the Soil There was no consistent relationship of seed age in the soil
with the timing and proportion of emergence, evidence of the inherency of seed dormancy capacity.
Recruitment numbers (as well as the soil seed pool size) declined rapidly during the early season
(JW16–31) as seed age in the soil increased from 1 to 3 years.
2.4.5.3 Seed Germinability–Dormancy Heteroblasty Blueprints Seed Behavior in the Soil

2.4.5.3.1 Relationship of Seed Heteroblasty and Seedling Emergence
Fine-scale differences in seed dormancy capacities (heteroblasty; Jovaag et al., 2012a), dormancy
cycling in the soil (Jovaag et al., 2011), and seedling emergence behavior (Jovaag et al., 2012b) were
observed among S. faberi populations. Further, a clear relationship between these behaviors of an
0.4
Relative frequency
0.3
0.2
0.1
0.0
15 20 25 30
Julian week
Burial year: 1997

Year 1: 1998 Year 3: 2000
Year 2: 1999
0.4
Relative frequency
0.3
0.2
0.1
0.0
15 20 25 30 15 20 25 30 15 20 25 30
Julian week Julian week Julian week
Time of seed abscission

August: JW 32 September: JW 36 October: JW 40
0.4
Relative frequency
0.3
0.2
0.1
0.0
15 20 25 30 15 20 25 30 15 20 25 30
Julian week Julian week Julian week
FIGURE 2.17 S. faberi seedling emergence (proportion of total) with time (Julian week, JW) during the
spring and the early summer of the 1st year after burial for all populations (top panel); year of emergence (year
1, 1998; year 2, 1999; year 3, 2000) of all populations buried in 1997 (middle panel); the 1st year after burial of
populations with a common time of seed abscission (August, JW 32; September, JW 36; and October, JW 40)
(bottom panel). Bars: relative frequency; solid line: mixture model estimate (four normal components with
equal variance); dashed lines: model’s four components weighted by the mixing proportions. (From Jovaag, K.
et al., Int. J. Plant Res., 2(3), 46, 2012a; Jovaag, K. et al., Int. J. Plant Sci. 2(6), 165, 2012b.)
S. faberi population during its seed–seedling life history stages was observed, evidence consistent
with the hypothesis that seedling recruitment is predicated at the outset by seed heteroblasty and
subsequently modulated by the environment.
The life history trajectory of the individual S. faberi seed, from anthesis and embryogenesis to
germination and seedling emergence, was determined in the first instance by the inherent, paren-
tally induced, dormancy capacity of the seed, but was sensitive to the environmental conditions
confronted on different time scales. This, consequential, life history trajectory was first apparent
in dormancy–germinability cycling in the soil (Jovaag et al., 2011). At the widest temporal scale
studied, heteroblastic qualities largely determined the numbers of seeds that are recruited in a single
year and locality. On a finer temporal scale (weekly or daily), the distribution and timing
(pattern) of seedling emergence were strongly influenced by the unfolding seasonal environment.
The life history trajectory is therefore both deterministic and plastic, conditional on starting condi-
tions (parentally induced dormancy capacity) and modulated by environmental conditions confronted
subsequently. The resulting seedling recruitment in a locality is an emergent property of these
complex interacting factors.
A relationship between seed heteroblasty at abscission and its subsequent behavior in the soil
was observed. Individual S. faberi seed populations retained heteroblastic qualities throughout their
annual soil germinability cycle (high spring germinability, summer dormancy reinduction, and
increased autumn germinability). Retention of heteroblasty was evident in the variable proportions
of CAN seeds within a single population in the soil at individual seasonal times. The influence of
heteroblasty was also apparent in the heterogeneous decline in the germinability of seed in the soil
from early spring to early summer. Further, the germinability pattern of the CAN pool in the soil
(high in spring, low in summer, and somewhat increased in autumn) was consistent with the emer-
gence pattern (high in spring and early summer, low in midsummer, none in late summer–early
autumn, and low in mid–late autumn).
A relationship between seed heteroblasty at abscission from the parent plant and its subsequent
seedling recruitment behavior was also observed. This influence, modulated by environment, was
apparent in both seedling recruitment numbers and temporal emergence patterns. Evidence of this
relationship between heteroblasty and emergence numbers was provided by the positive Spearman
correlation between dormancy capacity at abscission and the cumulative number of seeds emerged
during the first year after burial for both the 1998 and 1999 S. faberi populations. Additionally,
more dormant populations had lower emergence numbers during the first year after burial than less
dormant populations. This was more apparent during the 1999 season than the 2000 season. Further
evidence was provided by the inverse relationships between the time of abscission and subsequent
emergence numbers during 1999 and the second half of 2000. Early-maturing seed was the most
dormant and had the least number of seeds emerging. Seed maturing late in the season was the
least dormant and had the greatest number of seeds emerging. During the first half of 2000, envi-
ronmental conditions strongly dominated emergence numbers and patterns such that no relation-
ship with the time of abscission was observed. The 1998 seed dormancy heteroblasty (AR) groups
(Jovaag et al., 2011) also provided evidence of this relationship: the most dormant seed (AR group 3)
had the least emergence, while the least dormant seed at abscission (AR group 1) subsequently had
the greatest number of seeds emerging. Further, the least dormant seed generally emerged earlier:
AR group 1 had the most early spring emergence and the least early summer emergence.
2.4.5.3.2 Seedling Emergence Hedge-Betting for Assembly in Agrocommunities

These studies by Atchison and Jovaag (Jovaag et al., 2011, 2012a,b) provide insights about weedy
hedge-betting strategies of S. faberi for invasion, colonization, and enduring occupation of cen-
tral Iowa agricultural fields. Fitness in S. faberi is conferred by strategic diversification of seedling
recruitment when confronted by risks from agricultural disturbance and heterogeneous, change-
able habitats. Evolutionarily, hedge-betting is a strategy of spreading risks to reduce the variance
in fitness, even though this reduces intrinsic mean fitness. Hedge-betting is favored in unpredictable
environments where the risk of death is high because it allows a species to survive despite recur-
ring, fatal disturbances. Risks can be spread in time or space by either behavior or physiology. Risk
spreading can be conservative (risk avoidance by a single phenotype) or diversified (phenotypic vari-
ation within a single genotype) (Cooper and Kaplan, 1982; Philippi and Seger, 1989).
Hedge-betting of seedling recruitment time and pattern reduces temporal variance in the fitness
of S. faberi in agroecosystems characterized by annual cycles of disturbance and unpredictable
climatic conditions. Earlier emergence allows for greater biomass and potentially explosive seed
production at season’s end (Dekker, 2004b), but also has a high risk of death from agricultural
practices. Later emergence has a lower potential for seed production, but also a lower risk of death.
Thus variation in germination time results in both a decrease in the maximum and an increase in the
minimum potential seed production, reducing fitness variability and ensuring enduring occupation
of the locality. Variation in germination time has the additional benefit of reducing sibling competi-
tion, which enhances individual fitness.
Hedge-betting by S. faberi begins with the birth of seeds and the induction of heteroblastic
differences that are subsequently echoed in seedling emergence behavior. The complex pattern of
emergence observed in this study (Jovaag et al., 2012b) reveals the actual hedge-bet structure for
central Iowa S. faberi seedling recruitment, its realized niche. The relative seedling investment
made by S. faberi at different times during the season is a consequence of the changing balance
between the risk of mortality and the potential fecundity during those periods.
There exists some predictability to the primary sources of mortality and recruitment opportuni-
ties for S. faberi in Iowa agroecosystems over the course of their annual life histories (Figures 2.18
and 2.19). The majority of seedlings are recruited in the spring when the risk of mortality is very
high from crop establishment practices (seedbed preparation, planting, and weed control), and
the rewards are the greatest. Weed seedlings emerging early have the greatest time available for
biomass accumulation and competitive exclusion of later-emerging neighbors. Subsequent fitness
devolves on those individual S. faberi plants that escape these disturbances.
Seedling recruitment in the late spring and early summer is affected by the cessation of all
agricultural disturbances and weed control tactics, a time termed “layby” in the Midwestern United
States. Seeds emerging after layby avoid mortality from cropping disturbances, and the risk of death
shifts to that from interactions with neighbors. Freed from cropping disturbance risk, seedlings
emerging postlayby face increasing competition from crop and weed neighbors and decreasing time
for biomass production. Subsequent fitness devolves on those plants freed from human intervention
that compete effectively with neighbors until harvest, an advantageous opportunity space–time
apparent from the seedling investment observed.
Cohort 1 2 3 4 5 6
Early Late
Season Early Spring Mid Spring Late Spring Early Summer Summer Autumn
Time (JW) 16–18 18–20 22–24 26–28 32–35 45–49
Fecundity potential Very high Very high High Medium Low Very low
Mortality risk Very high Very high High Low Low Low
Source(s) of risk Crop Crop Crop Neighbors Neighbors Crop disturbance
disturbance disturbance disturbance climate
Weed strategy Escape Escape Escape Postlayby Postlayby Postharvest
cropping cropping postlayby opportunity opportunity opportunity
opportunity
Seedling investment 12% 38% 30% 20% 0.2% 0.1%
FIGURE 2.18 S. faberi seedling recruitment cohort (time, Julian week [JW]) exploitation of changing
opportunity space–time in Iowa maize–soybean cropping fields. (From Jovaag, K. et al., Int. J. Plant Sci.,
2(6), 165, 2012b.)
Month APRIL MAY JUNE JULY AUGUST SEPTEMBER OCTOBER NOVEMBER DECEMBER
Julian Week 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Maize Seedbed prep
Planting
Weed control
Layby
Harvest
Autumn tillage
S. faberi Early spring 12
Recruitment Mid-spring 38
Cohorts Late spring 30
Early summer 20
Summer 0.2
Autumn 0.1
Soybeans Seedbed prep
Planting
Weed control
Layby
Harvest
Autumn tillage
Seed Dormancy, Germination, and Seedling Recruitment in Weedy Setaria
FIGURE 2.19 Calendar of historical, seasonal times (Julian week, month) of agricultural field disturbances (seedbed preparation; planting; weed control, including
tillage and herbicides; time after which all cropping operations cease, layby; harvest and autumn tillage), and seedling emergence timing for Central and Southeastern
Iowa, USA, Setaria faberi population cohorts (all S. faberi combined: Time, +/− S.E.; Mean Proportion 3). (From Jovaag, K. et al., Int. J. Plant Sci., 2(6), 165, 2012b.)
83
Very small S. faberi seedling investments are made late in the season when the risks of mor-
tality are much lower. Seedlings recruited late experience a senescing crop light canopy. Those
emerging during or after harvesting experience mortality risks only from the approaching winter
climate. Floral induction in S. faberi is increasingly stimulated by decreasing photoperiod, allow-
ing a seedling with as few as two to three leaves to produce a few seeds very late in the season
(e.g., Oakwood site, 1998; data not reported). This justifies a small hedge-bet investment yield-
ing low numerical returns but ensuring some potential increase in soil seed pool size for future
infestations.
The time of seedling emergence determines what individuals assemble in plant communi-
ties and how they interact with neighbors. Recruitment timing therefore determines how much
biomass successful plants can accumulate, which directly predicates seed productivity (Dekker,
2004b). Fitness devolves on preadapted individual S. faberi plants that anticipate the unfolding
opportunity space–time. The hedge-betting structure of S. faberi revealed in these papers sup-
ports the conjecture that the inherent variation in seed dormancy capacities among seeds dispersed
by a parent plant provides a germinability “memory” of historically successful seedling recruit-
ment responses to local disturbance allowing exploitation of local opportunity in space and time
(Trewavas, 1986).
2.5 S
ETARIA LIFE HISTORY AS A COMPLEX ADAPTIVE
SOIL–SEED COMMUNICATION SYSTEM
The nature of the weedy Setaria is revealed in the physical (morphological and genetic spatial
structures) and the phenomenal (life history behavior instigated by functional traits). The nature of
Setaria is the consequence of structural self-similarity and behavioral self-organization. Structural
self-similarity in morphology was revealed in seed envelope compartmentalization and individual
plant tillering, in genetics by local populations and Setaria species-associations forming the global
metapopulation. Behavioral self-organization was revealed in the self-pollinating mating system
controlling genetic novelty, seed heteroblasty blueprinting seedling recruitment, and phenotypic
plasticity and somatic polymorphism optimizing seed fecundity.
The consequence of structural self-similarity and behavioral self-organization has been the
evolution of a complex adaptive seed–soil communication system. Weedy Setaria life history is
represented in algorithmic form as FoxPatch, a model to forecast seed behavior. The environment–
biological informational system with which weedy Setaria life history unfolds is represented in the
seed–soil communication system.
2.5.1 Forecasting Setaria Seed Behavior: FoxPatch

Weedy Setaria spp. seeds with heterogeneous dormancy states are recruited from soil pools when
sufficient oxygen, water, and heat accumulate over time (Dekker, 2004). The variable timing of this
recruitment is the first step in assembly with crops and other weeds in annually disturbed agricul-
tural communities. The timing of seedling emergence is the single most important factor in deter-
mining subsequent weed control, competition, crop losses, and replenishment of the soil seed pool.
FoxPatch is a computational tool to predict the behavior of individual weedy Setaria spp. seeds,
including AR, dormancy reinduction, germination, and seedling emergence from the soil (Dekker,
2009, 2011a; Dekker et al., 2003). It is based on intrinsic morpho-physiological traits in the seed (the
germinability–dormancy state) at abscission responding to extrinsic oxy-hydro-thermal time signals
it receives from the environment (Dekker et al., 2003). Three rules of behavior define the interaction
of individual seeds with signals from their immediate environment and thus determine patterns of
seed–seedling life history behaviors.
Algorithmically, FoxPatch consists of a schema derived from the morpho-physiological traits
in the seed determining its dormancy state at any time in its life history. This schema responds
to soil signals in predictable ways. These predictable responses are the foundation for three seed
behavior rules that together define the seed–seedling portion of the weedy Setaria sp. life history.
Forecasting Setaria seed behavior relies on three separate sources of information: dormancy state
heterogeneity, environmental signals, and patterns of seedling emergence.
2.5.1.1 Seed State and Process Model

FoxPatch is a predictive tool, a formalization of what we empirically know about the Setaria spp.
seed traits (schema) and the life history behaviors that arise from the interaction of these traits with
the environments in which they thrive (behavior rules). The life history of weedy Setaria sp. seed is
schematically represented in Figures 2.16 and 2.20.
The seed portion of the life history can also be represented by the behavioral processes leading
to transitions between seed states:
AR
æ æ æÆ
DORM AB ¨ æDORM
æ æ æ æ ææ CAN æ GROW
RE-INDUCT
æ æ Æ GERM æ EMERGE
æ æ æÆ SEEDLING

where DORMAB is the dormancy state at abscission.
2.5.1.2 Soil Signal Controlling Seed Behavior: Oxy-Hydro-Thermal Time

Weed seeds like Setaria require only three resources/conditions (signals) from their immediate
soil environment to germinate and emerge as seedlings: temperatures (T) favorable for germina-
tion and seedling emergence (thermal-time), adequate but not excessive soil moisture (hydro-
time), and oxygen dissolved in imbibed water to support germination metabolism (oxy-hydro
Input:
Heteroblastic Flux states = circles Output:
Germ-dorm state Terminal states = boxes seedlings
seed Processes = arrows
[4]
[1]
[A] Soil seed pool

erg g
[E]
e
em edlin
enc
[B]
Se
Afer-ripening Seed
Dormant germination Germination Germinated
seed [1] candidate growth seed
Dormancy [2] [D] [3]
reinduction
[C]
[F1] [F2] [F3] [F4]
Death [5] Death [5]
FIGURE 2.20 Schematic diagram of weedy Setaria sp. soil seed pool behavior based on the life history of the
seed (seed states and transitions between states). Life history states (terminal, boxes; flux, circles): [1] dormant seeds
(DORM), [2] germination candidate seeds (fully after-ripened; CAN), [3] germinated seeds (GERM), [4] seedlings
(SEEDLING), and [5] dead seeds or seedlings (DEAD). Life history processes (arrows), or transitions between
states: [A] induction of dormancy and input (dispersal) of dormant seed at abscission (the seed rain), [B] after-ripening
of dormant seed (AR), [C] reinduction of (2°) dormancy in germination candidates (DORM RE-INDUCT), [D]
germination growth (GROW), [E] seedling emergence (EMERGE), and [F: F1–F4] death (from states 1 to 4).
time). For each of these three resources/conditions, there is a threshold amount an individual seed
needs to germinate. Each of these three thresholds has a minimum and maximum value range
within which seeds germinate. When any of these three signals is insufficient or excessive, a liv-
ing seed in the soil will remain dormant. When all three signals are within their thresholds, ger-
mination will occur. The conditions within all three thresholds can also be conceptualized as the
germination “window” through which a seed must pass to germinate and emerge as a seedling,
germination phenotype space.
FoxPatch can be expressed quantitatively by defining the environmental signal required by an
individual to complete each of the component processes, given the initial dormancy state of indi-
viduals. All viable weedy Setaria spp. seed in the soil obey the following rules:
General seed The behavior of an individual weedy Setaria seed in the soil is regulated by the amount of oxygen dissolved
behavior in water (the oxygen–water signal) that accumulates in the seed symplast, and temperatures favorable
rule: (or not) to germination growth (the germination temperature signal), over some time period
(cumulatively oxy-hydro-thermal time)
(Atchison, 2001; Dekker, 1999, 2000, 2003, 2004a; Dekker and Hargrove, 2002; Dekker et al.,
1996, 2001; Donnelly et al., 2012; Sareini, 2002).
All FoxPatch predictions are based on the assumption that when an individual, dormant foxtail
seed receives the minimum environmental signal required for AR and germination in the soil,
it will emerge (barring mortality). Therefore, the parameters critical to predicting the seedling emer-
gence of an individual seed are (1) the soil signals (T, H2O, O2) required by an individual Setaria
sp. seed to overcome its inherent level of dormancy to after-ripen and germinate (the schema), and
(2) the signals that an individual seed in the soil actually experiences and utilizes (soil signal).
Provided these two critical pieces of information are available, seedling emergence predictions in
FoxPatch are based on the following:
General An individual weedy foxtail seed will change state when the minimum inherently required
prediction oxy-hydro-thermal time signal is received from its realized environment (plus signals not
algorithm: causing an effect due to inefficient transduction or insensitivity).
There are two signals in the soil that interact to control weedy Setaria seed behavior. Both are
based on temperature and moisture. The oxygen signal is OxSIG, oxy-hydro time. The heat signal
is TSIG, germination thermal time. The combined influence of these two signals on foxtail seed
behavior is the oxy-hydro-[thermal] time signal.
2.5.1.2.1 Oxy-Hydro-Time Signal: OxSIG

One of the most striking features of the soil in the temperate regions of the globe is the diur-
nal fluctuation in soil temperatures that occur every day, and the ample moisture in springtime
soils contains the seasonally maximum amount of oxygen immediately after thawing. This diurnal
phenomenon (the “big spring temperature oscillator”) plays a crucial role in AR and germina-
tion (Dekker et al., 2001). During the cool night period, water entering the seed PP brings with
it much oxygen due to its high solubility. During the midday, temperatures rise and O2 solubility
decreases causing a degassing effect. The water is purged, sending a pulse of free oxygen into the
seed symplast because diffusion out of the narrow pore opening is restricted. The contribution of
both chronic O2 availability (based on solubility alone) and acute O2 availability (based on
degassing with temperature change) is the two sources of the oxy-hydro signal to which Setaria spp.
have adapted and synchronized their life history in the soil.
The signal controlling Setaria seed AR, 2° dormancy reinduction and the continuing supply of O2
during the germination processes is OxSIG: the oxygen mass per water volume of seed symplast (cary-
opsis) per time period per seed. An individual Setaria seed experiences two different OxSIGs (chronic
and acute) from the soil environment depending on the diurnal pattern of temperature (constant, alter-
nating, or both). The OxSIG affecting any single seed is the summation over time of the O2 received:
OxSIG Total = [(OxSIG Chronic ) + (OxSIG Acute )]
The chronic O2 signal (SIGChronic) is that amount of dissolved O2 (g L−1) that diffuses into the seed
symplast when the ambient temperature is constant for some time period (g L−1 Time−1):
OxSIG Chronic = (OXYTemp∞C * VH 2OSeed ))* (TIME Temp∞C ) seed -1

where
OXYTemp°C is the O2 solubility (g per L) at a particular temperature
VH2OSeed is the H2O volume (g or mL) of a single seed symplast
TIMETemp°C is the time (h) at a particular temperature
We have experimentally determined the seed symplast water volume of a locally (Ames, IA, USA)
adapted S. faberi population (author lot # 3781; K99-40) to vary from 0.00036 to 0.00050 mL
(360–500 nL) per seed, depending on the immediate microsite moisture availability and tempera-
ture (Atchison and Dekker, unpublished data).
The acute O2 signal (SIGAcute) is the difference in O2 (g) in solution that is degassed (lost from
solution) when the symplast H2O changes from a low to a high temperature, a process that occurs
every day of the year in the soil:
OxSIG AR Acute = (OXYLOW Temp∞C * VH 2OSeed ) - (OXYHIGH Temp∞C * VH 2OSeed )
where OXYLOW Temp°C and OXYHIGH Temp°C are the oxygen solubilities in H2O at the low and high
temperatures, respectively.
2.5.1.2.2 Thermal Signal: TSIG

Temperature (T) plays two different roles as a signal controlling Setaria seed behavior. The ambi-
ent temperature determines O2 solubility (inversely related to T) as well as the germination growth
environment. Temperature’s two roles often have opposite effects on an individual seed: cool pro-
vides more O2, while inhibiting germination; warm provides less O2 but promotes embryo growth.
2.5.1.2.3 Oxy-Hydro-[Thermal]-Time: OxSIG and TSIG
The combined influence of these two signals on foxtail seed behavior is the oxy-hydro-[thermal]
time signal (chronic and acute OxSIG and TSIG). Experimentally, local soil moisture and tempera-
ture are measured at depth in the soil hourly, continuously, for accurate signal quantification.
2.5.1.3 Schema of Intrinsic Setaria sp. Seed Traits
Each individual Setaria sp. seed possesses inherent traits, morphological and physiological, that
determine how it will behave in a variety of environmental conditions it might encounter. These
traits of the seed are inherited from the parent plant at abscission and can be conceptualized as a
plan for future action, a schema. The schema is a blueprint constraining an individual’s unique
set of reactions to local environmental conditions (biotic and abiotic), the realized phenotype.
This schema is biologically complete in the sense it predicates all the behaviors an individual will
perform such as AR and germination. What is the schema that weedy Setaria sp. seed possess? In
its most basic computational form, it is the amount of oxy-hydro-thermal time signal required to
germinate an individual seed. Experimentally, this is determined by germination assays at seed
abscission under optimal controlled conditions (see dormancy induction later).
2.5.1.4 Germinability–Dormancy Induction
The local environment surrounding an individual Setaria panicle during embryogenesis, and the
physiology of dormancy induction traits in developing embryos, conspires to produce a heteroge-
neous collection of seeds, each with potentially different dormancy states at abscission, the time
they first become independent of the parent plant, seed germinability–dormancy heteroblasty.
The amount of dormancy in each seed, and the variability in dormancy quantity among individu-
als, is a direct display of the future timing of recruitment. The consequence of past selection and
local adaptation determines the “germinability–dormancy heterogeneity bandwidth” among a
cohort of seeds produced by a single successful parent plant panicle. The precise mechanisms
and environmental conditions leading to particular dormancy states in individual seeds are
unknown, but the role of light may be important (Atchison, 2001; Dekker, 2003, 2004a; Dekker
et al., 1996). When this process is understood, the definition of a dormancy induction rule during
embryogenesis may be possible. Until that time, the quantity of dormancy induced in an indi-
vidual seed at the time of abscission (DORM AB) can be experimentally determined in optimal
conditions:
Initial individual seed The inherent dormancy state of an individual foxtail seed at abscission is defined
dormancy state: as the minimum oxy-hydro-thermal time signal required to stimulate germination.
The initial dormancy state is experimentally determined by the minimum amount of AR required
for a DORM to become a germination CAN and for that CAN to germinate (GROW), in favorable
(controlled, ideal) environmental conditions (e.g., AR: 4°C, moist, dark; GROW: 15°C–35°C alternat-
ing diurnal, moist, light). The population data are presented in frequency or cumulative distribution
diagrams of AR dose vs. percent germination. The signals required to after-ripen and germinate indi-
vidual foxtail seeds can be expressed in a number of ways, including AR time and oxy-hydro-thermal
time units.
2.5.1.5 Rules for Individual Weedy Setaria sp. Seed Behavior

The rules that control individual weedy Setaria sp. seed behavior are illustrated in its life history
(Figure 2.16). Setaria sp. seed behavior is the consequence of the interaction between extrinsic local
environment influences and intrinsic seed traits (the schema). These rules of behavior are the inevi-
table consequence of past selection and adaptation molding the Setaria sp. phenotypes we see in the
field today. They pertain to living processes and changes in state, as opposed to the more stochastic
and unpredictable factors involved in mortality.
FoxPatch is based on three rules of individual weedy Setaria sp. seed behavior. These rules
are defined by field observations of dormancy state heterogeneity among seeds at abscission from
an individual panicle, as well as by AR, dormancy reinduction, and germination. What particular
behavior occurs is dependent on the initial state of the seed (the schema) and the rate and amount
of signal accumulated. Each of these behavioral transitions is governed by a specific foxtail seed
behavior rule, presented here in their qualitative forms.
2.5.1.5.1 After-Ripening Rule and Its Inverse, the Dormancy Reinduction Rule

From the time a Setaria spp. seed is shed (typically August–November in North America), it is
exposed to moisture–temperature conditions that either after-ripen (AR) the seed (generally cool,
moist) or maintain (or induce) dormancy (generally hot, dry). When sufficient AR has occurred, and
the seed symplast accumulates enough oxygen (dissolved in water) over a period of time, it becomes
a germination CAN.
AR (see Figure 2.16):

DORM AB æ æ
AR
Æ CAN
AR rule: An individual dormant foxtail seed (DORM) will after-ripen when the rate of oxygen dissolved
in water entering the symplast exceeds the capacity to remove that oxygen for some time
period, during which the minimum inherently required amount of oxygen has accumulated in
the symplast (CAN) allowing germination (GERM) to occur at some temperature.
AR prediction An individual dormant foxtail seed (DORM) will after-ripen to a germination CAN when the
algorithm: minimum inherently required oxy-hydro time signal is received from its realized environment
(plus signals not causing an effect due to inefficient transduction or insensitivity) allowing
germination (GERM) to occur at some temperatures.
When conditions in the field are generally hot and dry for some time (e.g., summer), the oxygen and
water levels in the seed decrease, and dormancy is reinduced as the effects of previous AR are lost.
Dormancy reinduction
DORM AB ¨ æDORM æ æ ææ CAN
æ æRE-INDUCT
(see Figure 2.16):
Dormancy reinduction An individual after-ripened foxtail seed (CAN) will reacquire dormancy when the rate of
rule: oxygen dissolved in water entering the symplast is less than the capacity to remove oxygen
for some time period, during which the minimum inherently required amount of oxygen
is no longer present.
Dormancy reinduction An individual foxtail seed germination CAN will have dormancy reinduced when the
algorithm: minimum inherently required oxy-hydro time signal is not received from its realized
environment (plus signals not causing an effect due to inefficient transduction or
insensitivity) preventing germination from occurring at some temperatures.
Experimentally we determine AR and DORM by removing seed from the field at intervals through
the year and determine the number of viable (tetrazolium assay) seeds that immediately germinate
(GERM) or not (DORM) under optimal conditions.
2.5.1.5.2 Germination Candidate Threshold State

Once a seed becomes fully after-ripened, saturated with sufficient dissolved oxygen in the symplast,
it is poised on the threshold of germination, a germination CAN, awaiting only favorable tempera-
ture conditions to germinate. It is from the after-ripened seed pool (CAN) in the soil that seedlings
are recruited. It is the least dormant seed in the soil that after-ripen the earliest in a season, and
therefore they compose the most likely germination CANs for recruitment.
2.5.1.5.3 Seed Germination Rule

When a fully after-ripened weedy Setaria sp. seed, a germination CAN, is exposed to some tem-
peratures (usually higher than that for AR) for some time, it germinates.
Germination
CAN æ GROW
æ æ Æ GERM
(see Figure 2.16):
Seed germination An individual foxtail germination CAN (an after-ripened seed with sufficient free symplastic
rule: oxygen for sustained respiration) will germinate when exposed to minimally favorable
temperatures for some time period.
Seed germination An individual foxtail germination CAN seed will germinate when exposed to minimally
algorithm: favorable temperatures from its realized environment (plus signals not causing an effect
due to inefficient transduction or insensitivity) for some time period.
Minimally favorable temperature often is a narrow temperature range that increases as the oxygen
in the symplast increases (AR depth).
The most complex part of FoxPatch at this time is elucidation of a quantitative function that
predicts germination of a germination CAN based on both oxy-hydro (OxSIG) and thermal (TSIG)
time. Two opposing forces are at work at this time: lower temperatures (e.g., O°C liquid water)
possess high oxygen solubility and inhibit germination; higher temperatures (35°C is more favor-
able than 15°C) stimulate germination but have low oxygen solubility. AR is favored by cool, moist
conditions. Germination is favored by relatively high symplast oxygen saturation and high tempera-
tures. The correct function to predict these opposing forces will be a computational trade-off weigh-
ing their relative influences and interaction. These opposing elements provide a very powerful, yet
sensitive, environment-sensing mechanism allowing Setaria spp. to detect very precisely favorable
conditions leading to recruitment and subsequent success.
Forecasting the seed behavior of weedy Setaria spp. seeds in agricultural soils based on intrinsic
dormancy qualities, environmental oxy-hydro-thermal time signals, and consistent seasonal pat-
terns of seedling emergence could provide us with a robust tool of immense value, an algorithm of
the first assembly step of disturbed agricultural communities.
2.5.2 Setaria Soil–Seed Communication System

2.5.2.1 Shannon Communication System for Weedy Setaria Seed–Seedling Life History
The nature of weedy Setaria seed–seedling life history is a complex adaptive soil–seed commu-
nication system arising from its component functional traits (Table 2.1). Functional traits control-
ling seed–seedling behavior are physical information that has evolved in ongoing communication
between organism and environment leading to local adaptation. A Shannon communication system
of any type must contain the five elements (E) presented in Figure 2.1, as well as message and signal
(Shannon and Weaver, 1949).
Weedy Setaria seed life history behaviors are controlled by environmental information (signals) flow-
ing from the soil to the seed embryo. The Setaria seed is constructed in such a way as to be receptive to
specific signals contained within the entire soil information available to it. The specific signal to which
Setaria is tuned affecting seed behavior in the soil is the amount of oxygen and heat (T, thermal) in soil
water over time, oxy-hydro-thermal time (O2–H2O–T time). The message that directly controls Setaria
seed behavior is oxygen and heat accumulating in the embryo modulating seed respiration.
The Shannon environmental–biological communication system between the soil and the Setaria seed
contains the five elements (E) and components as schematically organized in Figures 2.17 and 2.21.
O2–H2O–T time; the message is O2–H2O–T stimulating embryo respiration.
2.5.2.1.1 Information Source (E1): The Soil Adjacent to the Setaria Seed

The information source selects a desired message out of a set of possible messages. (Shannon and
Weaver, 1949)
The source of information generating the message affecting weedy Setaria seed life history development
is the soil surrounding the individual seed in a particular locality. There are many sources of information
impinging on the seed, including changing cycles of oxygen–water–heat in seasonal climate and environ-
ment, site latitude–longitude–elevation–slope–aspect, soil quality (tilth, structure, texture, compaction,
organic matter, and chemicals), neighboring seeds and plants, microbial and animal activity, and agri-
cultural and other human disturbances. This information flows constantly within the soil. The soil-borne
seed responds to only a limited portion of the entire soil information, the message-controlling behavior.
The message is heat and oxygen dissolved in soil water that the Setaria embryo requires for continued
development, whether continued quiescence or germination. The growth and development behaviors of
the embryo include “remain dormant” or “resume growth and development.” The message was naturally
selected during Setaria evolution from among the larger entire set of soil information as a dependable
Channel:
Soil-seed H2O films
E1: E2: E4:

Information Transmitter: Receiver: E5:
source: Soil-seed H2O Living seed destination:
The soil contact interior Embryo
Message: Signal: Received Message:

O2–H2O–T O2–H2O–T–Time signal O2–H2O–T
Noise
source
FIGURE 2.21 Schematic diagram of Shannon environment–biology information–communication system for

weedy Setaria seed–seedling life history development. Communication elements (E): E1, information source, soil;
E2, transmitter, soil particle contact with seed surface water films; E3, channel, continuous soil particle–seed sur-
face water films; E4, receiver, living seed interior from TACL membrane to aleurone layer to embryo; E5, destina-
tion, embryo. The signal is soil O2–H2O–T–Time; the message is O2–H2O–T stimulating embryo respiration.
signal stimulating–inhibiting seed germination–dormancy behaviors. The specific signal that affects
Setaria behavior is the amount of heat and oxygen dissolved in soil water films connecting soil particles
with the seed with time, oxy-hydro-thermal time (O2–H2O–heat time).
2.5.2.1.2 Transmitter (E2): Water Film Contact between Soil

Particle–Seed Surfaces Modulating Oxygen Content
The transmitter changes this message into the signal which is actually sent over the communication
channel from the transmitter to the receiver. (Shannon and Weaver, 1949)
The transmitter converts/encodes/changes the message to produce a suitable signal. The transmitter
is the water film adhering to soil particles adjacent to, and continuous with, seed exterior surfaces
(hull and PP). The message sent is heat and oxygen dissolved in soil water; information is physical.
The specific signal transmitted is the amount of heat and oxygen dissolved in soil water films con-
necting soil particles with the seed interior–exterior with time (oxy-hydro-thermal time) received by
the embryo–endosperm affecting Setaria behavior. The transmitter modulates the heat and oxygen
content of water films that physically connect soil particles with seed surfaces and that are received
by the receiver, the living tissues of the seed interior. The transmitter changes, encodes, the amount
of oxygen dissolved in soil water by the formation of continuous water films physically connecting
soil particle–seed surfaces. Signal encoding is the change in the water film message by the morphol-
ogy of the seed surface. Setaria seed hull surface morphology and topography accomplish these
changes that encode the signal; information is physical.
2.5.2.1.3 Channel (E3): Soil–Seed Water Films

The Setaria communication channel is the continuous water film connecting soil particles with the
seed exterior (hull and PP) and interior (TACL, endosperm, and embryo). This water film com-
munication channel transmits the oxygen–water–heat signal to the living seed interior receiver and
the embryo destination. The capacity of this water film channel can be described in terms of the
water–oxygen information it transmits per unit time from the adjacent, continuous, soil source.
2.5.2.1.4 Noise Source
Introduction of noise in the communication channel means that the received message exhibits
increased uncertainty. The received signal is selected out of a more varied set than the transmitted
set. There can be several sources of noise between the transmitter and the receiver, everything that
corrupts the water–oxygen signal: water film discontinuities, physical interference from soil biota
and chemicals, soil structural changes, seed damage, localized areas of anomalous gas, and water
content/exchange.
2.5.2.1.5 Receiver (E4): The Seed Interior Beginning with the TACL Membrane
Continuous with the Exterior Water Film Terminating with the Placental Pore
The receiver is a sort of inverse transmitter, changing the transmitted signal back into a message, and
handling this message on to the destination. (Shannon and Weaver, 1949)
The signals transmitted from exterior water films (terminating with the PP) are received by the seed
interior by TACL membrane transport and diffusion equilibrating heat and dissolved gases between
the exterior–interior seed compartments. Water oxygenated by the seed surface water films is the
signal that is decoded by the seed interior receiver in the form of the free oxygen message available
to stimulate respiration and hence embryo germination (the destination).
2.5.2.1.6 Destination (E5): The Embryo, Whose Behavior Is Provoked by

Accumulation of Seed Interior Oxygen Stimulating Respiration
The resumption of life history growth and development by the embryo is stimulated by the accu-
mulation of adequate interior oxygen leading to germination and seedling emergence. Insufficient
oxygen maintains or stimulates dormancy and embryo quiescence. This message is “remembered”
by the weedy Setaria seed in the process of evolutionary adaptation. Successfully surviving and
reproducing plants arising from seed utilizing this communication system pass on these functional
traits to their polymorphic progeny, each of which in turn passes on the ability to generate its own
range of heteromorphic seeds appropriate to continuing local adaptation.
2.5.2.2 Setaria Soil–Seed Communication Transmission Algorithm

The Setaria soil–seed communication system seed behavior described earlier (Figure 2.17) can be
also expressed as operations (processes) computed by seed algorithms (Figure 2.22).
2.5.3 Seed Memory and Adaptive Evolution

Information is physical: memory resides in several locations in the Setaria seed. Setaria seed mem-
ory has both short- and long-term expressions. In the short term, memory is expressed by the amount
of oxygen accumulated in the seed interior. Setaria seed memory is the current germination–
dormancy state of each living seed in a local soil pool. The seed germination–dormancy capacity is
“hardwired” (intrinsic) to seed at birth, embryogenic seed dormancy induction. Information storage
is an important component of communication. The ability of Setaria seeds to accumulate oxygen
in the seed interior provides a message storage capability for a time interval determined by the
seed’s germination–dormancy capacity. Memory is expressed in the long term by responsiveness
to O2–H2O–heat messages as determined by the morpho-physiological soil–seed communication
system (hull, TACL membrane, and scavenger protein). Memory resides in the functional–adaptive
traits that regulate all seed behaviors by transducing/transforming/encoding and decoding inor-
ganic soil O2–H2O–heat signals over time: the three morpho-physiological mechanisms forming the
Soil–Seed Communication System

Algorithmic Operations Computed Life History Developmental Processes
Step 1: Acquire information from soil
water source adjacent to seed (E1)
Step 2: Transmit oxygen–water Step 2A: Assemble transmitter encoder and antenna by adhesion of continuous
signal to seed exterior encoder and water films in contact with soil particles and seed exterior
antenna Step 2B: Encode transmitter signal by amplifying soil–seed water film oxygen
content. Change oxygen content of water films by contact with seed hull
surface (e.g., increased surface area exposed to dissolve soil atmosphere gases)
Step 2C: Seed exterior antenna transmits signal by diffusive equilibration of
oxygen content throughout continuous soil–seed water film communication
channel (hull, placental pore) connected to TACL membrane receiver
Step 3: Transmit oxygen–water Step 3A: Form water films continuous with hull, placental pore, and TACL
signal to seed interior receiver membrane entrance to seed interior
Step 3B: Oxygen–water signal movement across seed surface facilitated by hull
topography channeling to basal placental pore entrance
Step 3C: Oxygen–water signal physically filtered by apoplastic placental pore
tissues
Step 3D: Oxygen–water signal entrance into seed interior receiver is physically
restricted (tuner, resistor) to TACL membrane by continuous surrounding
gas- and watertight caryopsis coat surrounding the embryo and endosperm
compartment
Step 3E: Equilibrate oxygen–water content throughout soil–TACL channel by
water–gas film diffusion
Step 3F: Equilibrate seed exterior–interior oxygen–water content by membrane-
regulated diffusion
Step 4: Receive oxygen–water signal Step 4A: Accumulate oxygen–water message information in
in seed interior embryo–endosperm capacitor
Step 4B: Remove oxygen in embryo–endosperm water by action of interior
oxygen scavenger protein
Step 5: Provoke embryo behavior Step 5A: Stimulate–inhibit respiration and embryo germination with adequate
with oxygen–water message at seed oxygen–water accumulation
interior destination
FIGURE 2.22 Seed algorithms, operations (developmental processes), computed by weedy Setaria seeds
during life history development by means of the shannon soil–seed communication system.
soil–seed communication system. The message is remembered: plants arising from seed utilizing
this communication system pass on these functional traits to their polymorphic progeny, each of
which in turn passes on the ability to generate its own range of heteromorphic seeds appropriate to
continuing successful local adaptation.
2.6 SUMMARY
The nature of weeds is a complex adaptive soil–seed communication system. The nature of weedy
Setaria life history is an adaptable, changeable system in which complex behaviors emerge when
self-similar plant components self-organize into functional traits possessing biological information
about spatial structure and temporal behavior. The nature of the weedy Setaria is revealed in the
physical (morphological and genetic spatial structures) and the phenomenal (life history behavior
instigated by functional traits). Structural self-similarity in morphology is revealed in seed envelope
compartmentalization and individual plant tillering, in genetics by local populations and Setaria
species-associations forming the global metapopulation. Behavioral self-organization is revealed in

the self-pollinating mating system controlling genetic novelty, seed heteroblasty blueprinting seed-
ling recruitment, and phenotypic plasticity and somatic polymorphism optimizing seed fecundity.
The weedy Setaria phenotype can be described in terms of the spatial structure of its seed and plant
morphology, and by its genotypes and population genetic structure. This spatial structure extends
from the cells and tissues of the embryo axes, the surrounding seed envelopes, the individual seed,
and then plant, the local deme, and ending with the aggregation of local populations forming the
global metapopulation. Setaria plant spatial structure is the foundation for emergent life history
behavior: self-similar timing of life history processes regulated by functional traits expressed via
environment–plant communication. Temporal life history behavior begins in anthesis, fertilization,
and embryogenesis; development continues with inflorescence tillering, seed dispersal in space and
time, and resumption of embryo growth with seedling emergence. Setaria life history behavior is
a Markov chain of irreversible (dormancy induction; seed dispersal, germination, seedling emer-
gence, and neighbor interactions) and reversible (seed AR and dormancy reinduction) processes of
seed–plant state changes (flowering plants, DORM, seed germination CAN, germinated seed, and
seedling) regulated by morpho-physiological traits acting through environment–plant communica-
tion systems (environment–plant–seed and soil–seed). Heritable functional traits are the physical
reservoirs of information guiding life history development, emergent behavior. Information con-
tained in structural and behavioral traits is communicated directly between soil environment and
seed during development. Functional traits controlling seed–seedling behavior are physical infor-
mation that has evolved in ongoing communication between organism and environment leading to
local adaptation. The consequence of structural self-similarity and behavioral self-organization has
been the evolution of a complex adaptive seed–soil communication system. Weedy Setaria life his-
tory is represented in algorithmic form as FoxPatch, a model to forecast seed behavior. The environ-
ment–biological informational system with which weedy Setaria life history unfolds is represented
in the soil–seed communication system.
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3 Alterations in Structural
Organization Affect the
Functional Ability of
Photosynthetic Apparatus
Emilia L. Apostolova and A.N. Misra
CONTENTS
3.1 Introduction........................................................................................................................... 103
3.2 Molecular Organization and Composition of Thylakoid Membranes................................... 104
3.2.1 Organization and Function of Photosystem II........................................................... 104
3.2.2 Organization and Function of Photosystem I............................................................ 105
3.2.3 Lipid Composition of Thylakoid Membranes............................................................ 106
3.3 Role of Oligomerization of Pigment–Protein Complexes in Thylakoid Membranes............ 107
3.4 Influence of Modification of Pigment Composition on Function and Organization of
Photosynthetic Apparatus...................................................................................................... 107
3.4.1 Influence of Decreased Chlorophyll Content............................................................ 107
3.4.2 Influence of Decreased Carotenoid Content.............................................................. 109
3.5 Influence of Modification of Lipid Composition on Function of Photosynthetic
Apparatus............................................................................................................................ 110
3.6 Conclusions............................................................................................................................ 111
Acknowledgments........................................................................................................................... 112
References....................................................................................................................................... 112
3.1 INTRODUCTION
Photosynthesis in oxygenic organisms such as higher plants, green algae, and cyanobacteria is
driven by cooperation of two photosystems, photosystem I (PSI) and photosystem II (PSII), which
are embedded in thylakoid membranes and are responsible for converting the light energy into
chemical energy [1–4]. These membranes of higher plants and some green algae consist of two
main domains: (1) the grana lamellae, which are stacked or the appressed regions of thylakoid
membranes, and (2) the stroma (exposed) lamellae, which are unstacked or the unappressed regions
of thylakoid membranes [5–8]. Both these domains are well connected. Mem-brane models, derived
through freeze fracture, immune-localization, and electron micrographs, show a spatial distribution
of PSII and its antenna pigment–protein complexes, the light-harvesting complex of PSII (LHCII),
reside mainly in the appressed regions of the stacked grana thylakoid membranes [9], while
that of PSI reside predominantly in the unstacked and unappressed stroma (exposed) thylakoid
membranes [9,10]. This heterogeneous distribution of PSII, PSI, and the LHCs plays a vital role
in the maintenance of structural integrity and functional ability of the photosynthetic apparatus of
higher plants and algae under varying environmental stress conditions [3,10–16].
103
In contrast, the cyanobacterial photosynthetic membranes do not form grana stacking regions
[7,17,18]. Cyanobacterial strains display differences in intracellular organization, in particular hav-
ing various arrangements of the thylakoid within the cell. In addition, it was found that the distances
between thylakoid membranes are correlated with the size of the phycobilisome (PBS) antenna and
that they change reversibly and rapidly upon illumination [19].
Light harvesting occurs in the pigment bed of the pigment–protein complexes that are orga-
nized as antenna complexes and reaction center complexes of PSII and PSI, with their characteristic
absorption and emission characteristics [1–4,10].
The structure of the thylakoid membrane is not rigid, akin to other biological membranes, it is
fluidic in nature due to the presence of the lipid bilayer and is composed of lipoprotein complexes
dispersed in it. The organization of the thylakoid membranes undergoes dynamic changes as per
the environmental cues [16,20–22]. On the other hand, the structure of the thylakoid membranes
depends on the species of the photosynthetic organisms. The relationship between the organization
of pigment–protein complexes and the function of photosynthetic apparatus as well as the role of
the different oligomeric structure in the photosynthetic apparatus is particularly important. Taking
these facts into consideration, the relationship between the structural organization of the photosynthetic
apparatus and its functional activity is described in this review.
3.2 MOLECULAR ORGANIZATION AND COMPOSITION

OF THYLAKOID MEMBRANES
Thylakoid membranes are highly specialized and vectorially oriented membranes, which provide
an ideal system for studying the relationship between the structure and the function of these mem-
branes. Functional characteristics of these membranes include the light-driven reactions that are
defined by a highly ordered structural arrangement [23].
3.2.1 Organization and Function of Photosystem II

PSII is a multisubunit chlorophyll–protein complex, embedded in the lipid environment of the thy-
lakoid membrane of plants, green algae, and cyanobacteria [10,24,25]. The light-driven reaction
carried out in this complex leads to the photolysis of water, which releases oxygen, electrons, and
protons. The function of PSII is associated with charge separation across the thylakoid membranes
[26]. The charge separation between the excited chlorophyll in the reaction center (P680 •) and
pheophytin (Phe) molecule produces the primary charge-separated state (P680+•Phe−•), which is
followed by rapid charge stabilization by secondary electron transport reactions [27]. The electron
from the reduced Phe is transferred to the quinine acceptors (QA and QB) and subsequently to the
mobile pool of plastoquinone (PQ). P680+• is reduced by the redox-active tyrosine of the D1 protein
(Tyr-Z), which received electrons from the oxygen-evolving complex (OEC) [see Ref. 27]. It can be
assumed that the modification of PSII, which influenced the charge stabilization processes in this
complex, leads to increasing the lifetime of P680+•Phe−• [27].
PSII complex is composed of a PSII core and a light-harvesting antenna complex system. The
LHCII in the thylakoid membrane of the higher plants binds more than 40% of the total chlorophyll
and is the most abundant pigment–protein complex [28,29]. LHCII comprises six polypeptides
(Lhcb 1–6) [30]. Lhcb 1–3 form the major antenna complex, which are present as trimers [31], while
Lhcb 4–6 (CP29, CP26, and CP24) are present as monomers in the thylakoid membranes [32,33].
The amino acid sequence studies of the isoforms of Lhcb 4 led Klimmek et al. [34] to suggest the
extension of LHCII into eight (Lhcb 1–8) groups.
The function of LHCII is to absorb light and transfer the excitation energy to the reaction center of
PSII. As such, this complex plays a vital role in efficient light harvesting or photoprotection. LHCII
in the granal thylakoid membranes forms large chiral-aggregated structures [35,36]. This higher-
order oligomeric structural organization of the major LHCII stabilizes the membrane ultrastructure
Structure and Function Relationship of Thylakoid Membranes 105
and is important for the dynamics and efficient functioning of the photosynthetic apparatus [37–40].
This process is regulated by the changes in lumen acidification and redox regulation of the
protein phosphorylation in the photosystems, leading to the dynamic rearrangement of antenna
pigment–protein complexes in the appressed grana lamellae, contributing exciton transfer to PSII, or
move away to unappressed stroma lamellae donating photon to PSI [41,42]. The antenna system regu-
lates the quantum efficiency of PSII and prepares the photosystems to avoid the deleterious effects
of high light or photoinhibition and other stress conditions. The photochemical efficiency is higher
in nonstressed photosynthetic systems growing under ambient climatic conditions [43]. However,
under stress conditions, the excitation pressure yields 3Chl* and subsequently by intersystem crossing
yields 1O2 [44]. These conditions are overcome by transferring LHCII complexes between PSII and
PSI, thereby balancing the exciton transfer through the change in antenna size [6] or by the process of
nonphotochemical quenching (NPQ) that dissipates excess energy into heat [45]. A recent hypothesis
propounded by Grieco et al. [46] postulates that the LHCII phosphorylation, NPQ, electron transfer
via cyt b6 f, and turnover of PSII in the maintenance of the photosynthetic machinery in the thylakoid
membrane occur in order to prevent oxidative damage of PSI, also.
The role of light-harvesting antenna in cyanobacteria is performed by PBSs, which are attached
to the outer surface of the thylakoid membranes [47,48]. PBSs are large multisubunit assemblies
of phycobiliproteins. The molecular mass of PBS depends on the species and light quality of the
environment [49]. PBS can interact with PSI as well as PSII, and the lipids play a role in controlling
PBS–reaction center interaction [50].
The core complex of PSII also contains LHCs, CP43 and CP47, which transfer excitation to pig-
ments in the reaction center. CP43 and CP47 are located nearer to the D1/D2 heterodimer, and two
chlorophyll molecules from these complexes are probably involved in excitation energy transfer [51].
The precise structure of the PSII core complex from higher plants is not available at present, but
it is supposed to be very similar to that of cyanobacteria. The main difference in the protein com-
position is related to the extrinsic protein(s) involved in the stabilization of the OEC [51]. The PSII
core polypeptides are complemented by three tightly bound polypeptides, PsbO, PsbP, and PsbQ (in
eukaryotes) or PsbO, PsbV, and PsbU (in cyanobacteria) [52]. The catalytic center of OEC is located
in the luminal side of PSII. This complex contains inorganic cofactors, Mn, Ca, and Cl atoms.
The rate of the oxygen evolution, corresponding to an electron flow from the OEC to the PQ pool,
and the kinetics of P680 reduction depend on the Si state transitions [53,54]. It is well known that
the OEC accumulates successively four positive charges in the Mn-binding site. These states are
denoted as S0 –S4 states, which reflect different oxidation states of the Mn atoms. These state transi-
tions take place before the evolution of a molecule of oxygen [55–57]. The operation of two parallel
mechanisms (noncooperative and cooperative) is suggested to be involved in the oxygen evolution
process [58–60]. The oxygen evolution by noncooperative Kok’s mechanism [55] is faster, whereas
the cooperative mechanism is slower because it involves recombination between various oxygen-
evolving centers. Our investigation revealed that organization of the PSII complex (in particular, the
peripheral antenna of this complex) strongly influenced the ratio of the PSII centers evolving oxygen
by cooperative and noncooperative mechanisms [61–63].
3.2.2 Organization and Function of Photosystem I

PSI is a large macromolecular pigment–protein complex embedded in thylakoid membranes, which
catalize the transfer of electrons from plastocyanin to ferredoxin [64,65]. This complex is com-
posed of 15 core subunits, with the associated LHCI consisting of four subunits, Lhca1 to Lhca4
in higher plants [65–68], while in cyanobacteria, PBS acts as peripheral antenna systems under
normal growth conditions. The proteins of LHCI are organized in dimers [69]. The core complex of
PSI binds to about 100 molecules of chlorophyll a and 30 molecules of β-carotene as well to a large
number of cofactors, which act as inner antenna, but not involved in electron transfer reactions [64].
Some of the antenna chlorophyll molecules absorb at wavelengths longer than the reaction center
itself and are often referred as the “red forms” [64]. The main part of antenna chlorophylls in cyano-
bacteria is located in PSI [70]. Cyanobacterial PSI is assembled as a trimer with a molecular weight
of more than 1 million Da [71]. PSI antenna among the cyanobacteria differs in content and spectral
characteristics of long-wavelength chlorophylls. In some species, the amount of these chlorophylls
in monomers and trimers of PSI differs [72 and ref. therein].
The light absorbed by antenna pigments is transferred to the reaction center, P700, which is
located in the heterodimeric protein PsaA/B in the central part of the complex [72]. It has been
shown that P700 is tightly bound to the core antenna chlorophylls, and so the PSI reaction center
cannot be isolated without the antenna chlorophylls [72]. The primary charge separation occurs,
and the electron transfer is initiated by P700 in PSI. Electrons are transferred from P700 to the pri-
mary electron acceptors, A0 (chlorophyll a molecule). The charge separation is stabilized by series
of electron transfer steps involving the acceptor, A1 (phylloquinone molecule) and from there to the
three [4Fe4S] clusters, FX, FA, and FB [71,73]. The electron transport carriers can carry out both
cyclic and linear electron flows. The cyclic electron flow creates a proton gradient across the photo-
synthetic membrane and allows ATP synthesis independent of PSII activity. During linear electron
flow, reduced Fd provides the electron necessary for NADP reduction in a reaction catalyzed by
ferredoxin:NADP-oxidoreductase [74].
3.2.3 Lipid Composition of Thylakoid Membranes

Thylakoid membranes of photosynthetic organisms are composed of monogalactosyldiacylglycerol
(MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phos-
phatidylglycerol (PG) [75]. The lipids are important not only for the formation of the lipid bilayer
but also for the structure and function of the complexes of the photosynthetic apparatus. Duchêne
and Siegenthaler [76] proposed that the current model of thylakoid membrane lipid composition
is one that consists simultaneously of bulk and specific lipids. PG and SQDG possess negatively
charged head groups, whereas MGDG and DGDG are noncharged lipids. Depending on the nature
of its head group, each lipid is expected to have a specific role in the photosynthetic process [75].
Various studies have indicated the influence of lipids in the assembly and function of PSII. Loll
et al. [77] found that 14 different lipids are integrally bound to isolated PSII complex. There are
four DGDG, six MGDG, three SQDG, and one PG associated with PSII complex [77]. The specific
role of different classes of lipids has been studied by biochemical and molecular genetic approaches.
Using mutants of Arabidopsis, it has been concluded that the total content of anionic lipids is limit-
ing for chloroplast structure and function, and is critical for overall photoautotrophic growth and
plant development [78]. It was established in cyanobacteria Synechocystis and Synechococcus that
the anionic lipids SQDG and PG could be functionally complement each other, as their total content
is compensated by the synthesis of each other [79].
PG, which is the only and the indispensable anionic phospholipid component of thylakoid mem-
branes [80], plays an important role both in the structure and in the function of the photosynthetic
apparatus [81–87]. PG is essential for cell growth and is required for the maintenance of chlorophyll–
protein complexes and of normal conformation and activity of both PSII as well as PSI complex [88].
It has been shown that three PG molecules are strongly bound the PSI reaction centers [89] and one
to the PSII reaction center between CP43 and D1 [77]. PG is needed for the binding of CP43 to the
reaction center core [90]. PG is also essential for the PSII dimer formation and for the electron transfer
between the primary and secondary PQ electron acceptors, QA and QB [81,83,84,91,92]. It plays a
pivotal role in the structural stability and function of the donor side of PSII, mainly at the OEC [87].
Besides this, PG is associated with the formation of the oligomeric form of LHCII [93] and with the
binding of LHCII to the reaction center of PSII [94]. In addition, this lipid is involved in the stacking
of thylakoid membranes forming grana [95].
MGDG and DGDG are major components of thylakoid membranes, amounting to about 50%
and 30% of total lipids, respectively [96]. These lipids not only take part in the formation of the lipid
bilayer, but are also very important for the structure and function of the photosynthetic complexes.
Mutant analysis showed that DGDG plays an important role in the structural organization of the
photosynthetic apparatus [97]. Only a small fraction of DGDG and MGDG is essential for a fully
active PSII [75]. In addition, it has been shown that this small fraction of DGDG is very important
for the proper structure and function of PSII, mainly at the donor side [87,98,99]. These studies
revealed that a part of the total violaxanthin pool is located in an MGDG phase surrounding the
LHCII, whereas another part was bound to the LHCII apoproteins.
The lipids of thylakoid membranes are characterized by high levels of unsaturation of their fatty
acids, which determined their physical and biochemical characteristics and play an important role
in the function of the photosynthetic apparatus [100,101]. The level of unsaturation is mediated by
the activity of fatty acid desaturase. The expression of lipid desaturase genes is light regulated and
thereby modulates the thylakoid membrane assembly [102].
3.3 ROLE OF OLIGOMERIZATION OF PIGMENT–PROTEIN

COMPLEXES IN THYLAKOID MEMBRANES
Pigment–protein complexes in thylakoid membranes exist mainly as oligomers that are functionally
active as monomers but more stable due to their ability to dissipate excess energy [72]. Aggregates
of LHCII are the structures characterized by a relatively high rate of excitation quenching, and these
structures defend the photosynthetic apparatus against oxidative damage [103,104]. It is suggested
that dissipation of excess absorbed energy of aggregates of LHCII takes place with a contribution
of peripherally located chlorophylls and carotenoids [72]. The high-light-grown plants show the for-
mation of a dimeric (C2S2) PSII supercomplex [105,106], through the binding of monomeric CP29
and CP26, located near the core of PSII, which mediate a tight or strong (S) binding of trimeric
LHCII antenna complex [107]. This supercomplex, however, becomes enlarged (C2S2M2) in low-
or moderate-light-grown plants, in order to increase the absorption cross section of PSII, by binding
to two more LHCII trimers (M: moderately and L: loosely bound) and one more monomer CP24
[108,109]. The conserved sequence of the antenna polypeptides gives an insight into their specific
role in photosynthetic function during evolution [110–114]. In cyanobacteria and green algae, it has
been reported that several small subunits are involved in the stabilization of the dimeric form of the
PSII complex [115–118].
The PSI complex in the thylakoid membranes of cyanobacteria is organized as a trimer [119–121],
while in the higher plant exists as monomer [67]. Earlier experiments reveal that the PSI trimers
contain the most “red chlorophylls,” and it is suggested that long-wavelength chlorophylls contrib-
ute to the dissipation of the excess energy in PSI complex [72]. The physiological relevance of the
trimers in cyanobacteria has not yet been fully elucidated.
3.4 INFLUENCE OF MODIFICATION OF PIGMENT

COMPOSITION ON FUNCTION AND ORGANIZATION
OF PHOTOSYNTHETIC APPARATUS
3.4.1 Influence of Decreased Chlorophyll Content
In higher plants, chlorophyll b is specifically required for the assembly, stability, and function of
light-harvesting proteins. Chlorophyll-deficient mutants have often been used to investigate the
role of chlorophyll proteins for photosynthetic efficiency and organization of the photosynthetic
apparatus.
The chlorophyll molecules per antenna pigment complexes associated with PSII vary up to 350
chlorophyll a and chlorophyll b molecules, but that in the antenna of PSI vary up to 300 chlorophyll a
molecules only [108,122]. Most of these pigment molecules are organized into LHCs (Lhcb1–6 or 8 in
PSII and Lhca1–4 in PSI). The amount of these LHCs determines the size of the functional antenna
of the photosystems. So the antenna size shows dynamism in every photosynthetic organism vary-
ing substantially as per the genetic, physiological, and environmental conditions thereby regulating
the developmental aspect of these antenna supercomplexes and their stability. Usually, the size of the
antenna matters in the photo-adaptation of plants. Plants growing under low-light intensities show a
large chlorophyll antenna size and that under high light have a smaller antenna size [122]. The antenna
size in response to light intensity is a mechanism of the chloroplasts to prevent overexcitation of the
pigment systems in PSII and PSI, and thereby preventing the possible photooxidative damage [108,122].
Earlier studies in Hordeum vulgare, pea, and soybean mutants revealed that a decrease in the
synthesis of chlorophyll b leads to (1) a smaller functional antenna size for both photosystems,
(2) a higher PSII/PSI ratio, and (3) an enhanced PSIIβ content [61,123–125]. Further studies on
chlorophyll b–less mutants of higher plants reaffirmed that the mutants either lack or have signifi-
cantly lower amounts of LHCII and LHCI in their thylakoid membranes [126–132]. The relative
increase in PSII/PSI ratio is interpreted as a response of the plants to the lowered light-harvesting
capacity of PSII in the photosynthetic apparatus of these mutants [123].
In contrast to higher plants, the chlorophyll b–less mutant of the green algae—Chlamydomonas
reinhardtii—showed a decrease in the functional antenna size of PSII, but that of PSI remained
fairly constant [133,134]. In addition, Polle et al. [134] suggested the presence of the inner subunits
of LHCII and the entire complement of LHCI in these mutants. However, the decreased antenna size
of PSII in C. reinhardtii leads to a decrease in the quantum yield, saturated rate of photosynthesis,
and the photochemical efficiency of PSII [134].
It is well known that PSII complexes exist in two different populations, known as PSIIα and
PSIIβ centers [135]. PSIIα centers display a lower chlorophyll a/b ratio, larger antenna size, and are
located in the appressed region of grana stacks forming a cluster of three to four PSIIα centers. But
PSIIβ centers have smaller chlorophyll antenna size and form isolated units in unappressed stroma-
exposed region of chloroplast thylakoid membranes [135]. Under certain conditions, interconversion
between the two types of PSII centers can be carried out. Accumulation of PSIIβ in the chlorophyll
b–less thylakoid membranes is correlated with the degree of chlorophyll b deficiency [124]. PSIIβ
centers are supposed to be an intermediated state in the development of the mature PSII com-
plex [123,124]. Chlorina f2 chlorophyll b–less mutant thylakoid membranes, in which main LHCII
(Lhcb 4–6) is absent, lack the differentiation of PSII into PSIIα and PSIIβ centres [123].
The photosynthetic oxygen evolution is altered with the modifications in the organization in PSII
complex. The size and degree of the oligomerization of LHCII influence the rate of the oxygen evolu-
tion and on the S0 –S1 state distribution, which suggest the structural changes in the Mn clusters. The
enhanced population of centers in the S0, observed in the membranes with a higher degree of LHCII
oligomerization, indicates a reduction of Mn3+ to Mn2+ [61]. Nugent et al. [136] also have shown that
the oxygen evolution process is particularly sensitive to structural changes, which may alter the redox
potentials of the Si state intermediates, hydrogen bonding, and pKa values of amino acids surround-
ing the complex. Moreover, thylakoid membranes from Chlorina f2 mutant of barley, with strongly
reduced antenna size and lack of the trimeric structure of LHCII [137,138], do not register flash oxygen
yields [61]. The earlier facts clearly show the role of the structural organization of the PSII complex for
oxygen-evolving activity. On the other hand, the decreased light-harvesting efficiency in mutant cells
of Synechocystis sp. PCC6803 with partial and complete elimination of PBS decreases the amount of
the functionally active PSII centers and the rate of the oxygen evolution [63].
The changes in the chlorophyll a/b ratios were observed in plants grown at different light irradia-
tions [139]. Anderson and Aro [140] revealed that the ratio of chlorophyll a/b is a sensitive index of
membrane staking, that is, the relative proportion of stacked versus unstacked membrane domains,
as measured by the cross-sectional area of stacked membranes per chloroplast. Recently we have
shown that changes in the ratio of chlorophyll a/b correlate with the ratio of the oligomeric (het-
erotrimers) to monomeric forms of the LHCII [61]. This is also correlated with the negative surface
charge density of the thylakoid membranes, which influence the energy redistribution between the
two photosystems. Our data suggest that higher degree of oligomerization of LHCII, which
correlates with lower values of the membrane electric moments, causes a decrease in the energy
spillover between PSII and PSI. The degree of LHCII oligomerization (high ratio of the trimeric to
monomeric forms of LHCII) has also a determining role in the energy transfer within the LHCII–
PSII complex when the chlorophyll b is excited, which reveals a significant role of the LHCII
oligomerization in the energy transfer between the molecules of chlorophyll b and chlorophyll a.
Therefore, the protein conformational changes in the LHCII complex, related to the oligomeriza-
tion of the major LHCII and the variation of the electric properties, might regulate the energy
transfer between chlorophyll–protein complexes [61]. The influence of the electric properties of
the membrane on the energy transfer between the pigment–protein complexes was also shown in
the thylakoid membranes from mutants of Synechocystis sp. PCC6803 with the modification of the
pigment–protein complexes of the photosynthetic apparatus [63].
The variation in the chlorophyll b content, which influenced the structure of the PSII complex,
also influenced its functional activity. The decreased content of chlorophyll b leads to a decreased
photochemical activity of both photosystems as well as the electron transport activity of whole-
chain electron transport from water to NADP in the pea thylakoid membranes [141,142].
3.4.2 Influence of Decreased Carotenoid Content

Carotenoids are light-harvesting accessory pigments, and they are very important for the function
and the stability of pigment–protein complexes in the photosynthetic apparatus [143–145]. There
are two main classes of carotenoids: the carotenes that are cyclized or uncyclized hydrocarbons
(e.g., β-carotene), and the xanthophylls that are oxygenated derivatives of carotenes (e.g., lutein,
violaxanthin, antheraxanthin, zeaxanthin, and neoxanthin) [146]. These pigments are located in the
antenna and core complexes of both photosystems and have a special role in the thermal dissipation
of excess light energy [147,148].
The most important function of carotenoids is the photoprotection of the photosynthetic apparatus
by quenching triplet chlorophyll, singlet oxygen, and other reactive oxygen species [149]. β-Carotene
performs the critical role of photoprotection in the reaction centers by quenching triplet chlorophyll
and singlet oxygen [150]. Carotenoids are structural components of PSII and PSI, and they are involved
in the stabilization of the LHCII trimers as well as in the assembly of LHCII monomers and PSII core
complex [151–154]. Ruban et al. [155] found that violaxanthin and zeaxanthin induce disaggregation
and aggregation of the major LHCII, respectively. The monomeric forms of LHCII undergo confor-
mational changes upon lumen acidification [156], preferentially the xanthophyll binding site in L2 sub-
units of these polypeptides, and facilitate the exchange of violaxanthin to zeaxanthin for the enzymatic
de-epoxidation [157] under high-light or photo-inhibitory conditions and is correlated to NPQ [158].
This process is essential for the photoprotection of plants [159,160].
The amount and composition of the carotenoids depend on the environment conditions [161–
166]. Demmis-Adams et al. [167] showed that the ratio of the carotenoid molecules per chlorophyll
molecule is typically greater in the sun compared with shade leaves of higher plants. There is a
strong increase in the fraction of xanthophyll cycle pigment in sun leaves, which play an impor-
tant role in the photoprotection of photosynthetic apparatus under environmental stress [168]. On
the other hand, carotenoids are indispensable constituents of the photosynthetic apparatus, being
essential not only for antioxidative protection but also for an efficient synthesis and accumulation of
photosynthetic proteins, especially that of PSII antenna subunits [169].
Moreover, it has been ascertained that carotenoids can also operate in thylakoid membranes as
a stabilizer of the lipid phase [169]. Recently, it was shown that the fluidity of photosynthetic mem-
branes in cyanobacteria is influenced by the ratio of polar to nonpolar carotenoid pools under dif-
ferent environmental conditions [166]. The elimination of both xanthophylls and/or polyunsaturated
fatty acids caused remarkable alterations of the molecular architecture mainly in the inner side of
lipid bilayer.
Rakhimberdieva et al. [170] proposed that the quenching induced by carotenoids could be a new
regulatory mechanism protecting photosynthetic apparatus of cyanobacteria against photodamage.
The authors presented spectral and kinetic evidence that cyanobacterial carotenoids activated by
absorbing blue light induce reversible quenching of PBS emission.
A decrease in the carotenoid content is accompanied by changes in the amount of chlorophylls
[62,171]. It was suggested that the decrease in the amount of chlorophylls could be the result of a
carotenoid deficiency–induced photooxidation of the chlorophylls. Recently, we have shown the
structural changes in the photosynthetic apparatus and a decrease in functionally active PSII centers
as a result of decreased carotenoid content [62]. In addition, it has been shown that the core antenna
complex of PSII is more damaged under illumination than the peripheral complexes, which is a
result of higher sensitivity of these complexes to high light [62,172]. Carotenoid depletion leads to
the inhibition of the photochemistry of both photosystems, a decrease in the ratio of the functionally
active PSIIα to PSIIβ centers, as well as the rate constant of the oxygen evolution [62]. In addition, it
has been shown that the changes in the carotenoid composition, after treatment with bleaching her-
bicide (fluridone), influence the lipid-to-protein ratio, membrane fluidity, the structural organization
of the chloroplasts and antenna complex of PSII, as well as the stability of the membrane complexes
[173,174]. All these changes influence the function of both photosystems, with relatively more influ-
ence on PSII than on PSI [62]. Also it was shown that these effects have stronger influence on the
structural organization and function of PSI in the grana margins than in the stroma lamellae [62].
3.5 INFLUENCE OF MODIFICATION OF LIPID COMPOSITION

ON FUNCTION OF PHOTOSYNTHETIC APPARATUS
Studies of a Synechocystis PCC6803 mutant, in which a gene encoding PG phosphate synthase
(pgsA) has been disrupted, revealed that long-term deprivation of PG resulted in a decrease in the
activities of both photosystems and in the degradation of PSI trimers and monomerization of PSII
[85,86]. In the PG-depleted thylakoid membranes, there is a strong decrease in the energy transfer
from PSII to PSI antenna pigments as well as the changes in the surface charge density of the thy-
lakoid membranes [175].
The depletion of the second anionic lipid (SQDG) in thylakoid membranes of C. reinhardtii leads
to a decrease in the PSII activity [176]. This observation could be due to impairment in PSII reac-
tion center or a decrease in the efficiency of the energy transfer from LHC to the reaction center.
A diminished PSII activity due to a decreased amount of SQDG is probably brought about either by
a conformational change in PSII complex caused by the lack of specific binding of SQDG of the PSII
complex in the thylakoid membrane or by the changes in the lipophilic surrounding at the QB bind-
ing site of the PSII complex even without the conformational changes in the protein complex [177].
Investigation with the mutants of Arabidopsis thaliana reveals that the total content of
the anionic lipids is limiting for chloroplast structure and is critical for the photoautotrophic
growth and development of plants [78,178]. The DGDG-deficient mutant of A. thaliana showed
a decreased ratio of PSII to PSI, which is a result of a decrease in the amount of PSII and
a concomitant increase in the amount of PSI [179]. This decrease in the amount of PSII is
compensated with an increase in the amount of peripheral (main) LHCII relative to the inner
antenna complex of PSII [179].
The role of polyunsaturated lipids is established by the studies on fatty acid desaturation-
deficient mutants [180,181]. The enzyme brings about different saturation levels of membrane lip-
ids. The modifications in the membrane lipids bring about a change in the energy redistribution
between PSII and PSI, and changes in the surface electric charge distribution as a consequence of
modification of the lipids and/or changes in the organization of the supramolecular complexes in
the thylakoid membranes [182]. On the other hand, the changes in the membrane as a result of a
decrease in lipid unsaturation do not inhibit cation-induced changes in the excitation energy dis-
tribution between chlorophyll–protein complexes of thylakoid membranes as well as their lateral
rearrangement [180,183]. It has been shown also that a decrease in the proportion of the polyunsatu-
rated fatty acids of the thylakoid lipids increases the stability of protein–protein interaction between
and within PSII complex [184]. The replacement of all polyunsaturated fatty acids by monounsatu-
rated fatty acids suppressed the growth of the cyanobacterium Synechocystis PCC 6803 [185] due
to severe impairment of photosynthetic function.
3.6 CONCLUSIONS
In conclusion, the influence of the modification of the structural organization of the photosyn-
thetic apparatus, in particular the pigment and lipid composition, can be summarized as follows
(Figure 3.1):
1. The variation of the chlorophyll and carotenoid content affect the organization of the
LHC and subsequently modify the PSII supercomplex. The earlier alterations influence
the stability of the complex, its light-harvesting ability and photoprotection functions, as
well as the primary photochemistry and oxygen-evolving activity.
2. The changes in the pigment composition also infuence the lipid phase of the membrane.
3. The amount of the PG affects the oligomerization of the thylakoid membrane pigment–
protein complexes and the functional activity of both the photosystems, PSII and PSI.
4. A decrease in the amount of SQDG leads to an impairment of PSII reaction center and/
or a decrease in the efficiency of the energy transfer from light-harvesting complex to the
reaction center.
Chl depletion
Car depletion
PG depletion
Organization of LHCII
Grana stacking
Surface charge density
hν
Energy transfer
NPQ
Functional activity
PSII stronger than PSI
FIGURE 3.1 Effect of decreased amount of carotenoids, chlorophylls, and lipids on the structure and
function of the thylakoid membranes.
The present review illustrates that changes in the pigment and lipid composition strongly
influence the organization and function of the photosynthetic antenna complexes. Extensive studies
on the role of the antenna complex on the structural and functional stability of photosystems (PSII and
PSI) so far emphasize its regulatory role on energy distribution, dissipation, and reorganization of the
pigment–protein complexes in the thylakoid membranes leading to their functional stability. There is
a myriad of information available on the role of these complexes preferably for the stability of PSII.
However, antenna chlorophyll–protein complexes, through their redistribution and mobility in the
thylakoid membranes, also play a vital role in the stability of PSI. With the advent of membrane pro-
teomics, x-ray crystallographic data, and single-cell imaging systems, studies on the role of antenna
pigment protein systems are certainly going to change, in the changing environment of the future.
ACKNOWLEDGMENTS
This chapter is the result of cooperation under Bulgarian-Indian Inter-Governmental Programme of
Cooperation in Science and Technology, project BIn-01/07 of the National Science Fund of Bulgaria and
project Grant No. INT/BULGARIA/B70/06 by Department of Science & Technology, Govt. of India.
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4 Photoperiodic Control
of Flowering in Plants
Faqiang Wu and Yoshie Hanzawa
CONTENTS
4.1 Introduction........................................................................................................................... 121
4.2 Photoperiodic Flowering Research in the Premolecular Era................................................ 122
4.2.1 Photoperiodism in Plants........................................................................................... 122
4.2.2 Photoperiodic Time Measurement............................................................................. 122
4.2.2.1 Plants Measure the Duration of Darkness.................................................. 122
4.2.2.2 External Coincidence Model...................................................................... 123
4.2.3 Florigen...................................................................................................................... 123
4.3 Circadian Clock in Plants...................................................................................................... 124
4.4 Photoreceptors in Photoperiod.............................................................................................. 125
4.4.1 Phytochromes............................................................................................................ 125
4.4.2 Cryptochromes.......................................................................................................... 126
4.4.3 Zeitlupe Family.......................................................................................................... 127
4.5 Molecular Mechanism of Photoperiodic Flowering in Plants............................................... 128
4.5.1 Arabidopsis................................................................................................................ 128
4.5.2 Rice............................................................................................................................ 129
4.5.3 Morning Glory........................................................................................................... 130
4.5.4 Soybean...................................................................................................................... 131
4.5.5 Sugar Beet.................................................................................................................. 132
4.5.6 Poplar......................................................................................................................... 132
4.5.7 Sunflower................................................................................................................... 133
4.6 Conclusions............................................................................................................................ 133
References....................................................................................................................................... 134
4.1 INTRODUCTION
Photoperiod, the daily cycle of day and night, is one of the most fundamental environmental signals
for living organisms. Photoperiods regulate numerous biological events across all life forms, includ-
ing cell division in yeast, diapause of insects, and coat color and sexual behavior of birds and
animals (Classen et al. 1991, Danilevskii 1965, Eppley et al. 1967, Hoffmann 1973). In humans, dis-
ruptions of the photoperiod cycles result in metabolic and psychological disorders. Photoperiodism,
a word derived from the Greek words for “light” and “duration of time,” refers to the developmental
and physiological reactions of organisms to the length of day and night, enabling them to adapt to
seasonal changes in their environment. Photoperiodism thus may imply the ability and the means
of organisms not only to recognize but also to memorize and anticipate seasonal changes in the
length of day and night. Such mechanisms are especially important for organisms in the temperate
zones, where a “clock” mechanism controlling seasonal developmental and physiological alterna-
tives apparently measures the length of day and night (Saunders 2002). The mechanisms of photo-
periodism are studied particularly well in plants. Photoperiodism involves various aspects of plant
121
development, including seedling growth, dormancy, tuberization, leaf fall, and flowering induction.
This chapter introduces the pioneering work and the recent progress in photoperiodism studies in
plants with a specific focus on flowering, plants’ decision to switch to reproductive growth from
vegetative growth in response to seasonal changes in photoperiods.
4.2 PHOTOPERIODIC FLOWERING RESEARCH IN THE PREMOLECULAR ERA

4.2.1 Photoperiodism in Plants
Since Henfrey reported the importance of latitudinal variation in summer daylength in influenc-
ing the distribution of plants in nature (Henfrey 1852), numerous experiments have been per-
formed in order to demonstrate the importance of the daily cycle of day and night in flowering
induction and to characterize the possible mechanisms. Now that photoperiodism is opening up to
molecular and genetic analyses, these classical experiments and the models they propose become
increasingly important in guiding future approaches to clarify the underlying mechanisms of
photoperiodism.
Most of the plants can be classified into three classes according to their flowering response to
photoperiods. The first class encompasses long-day (LD) plants that flower when the length of day
exceeds their critical photoperiod, the daylength in every 24 h duration. Examples include ort, barley,
spring wheat, pea, spinach, and Arabidopsis. The second class contains short-day (SD) plants that
flower when the daylength is less than their critical photoperiod. Examples include maize, rice, sug-
arcane, cotton, hemp, tobacco, and soybean. Finally, the third class is day-neutral plants that do not
respond to photoperiods, including cucumber, rose, and tomato. LD plants and SD plants are often
classified further as obligate (qualitative) and facultative (quantitative), depending on the strictness of
the daylength requirement. Other plants fall into none of the aforementioned three classes. Instead,
some plants can distinguish both shortening and lengthening of days to avoid seasonal ambiguity,
such as the same daylength in spring and fall. Such dual-daylength plants represent two categories:
long-SD plants that flower only after a sequence of LDs followed by SDs, and short-LD plants that
flower only after a sequence of SDs followed by LDs. Another grouping involves plants responding
to intermediate daylength. Intermediate-day plants flower in response to intermediate daylength and
remain vegetative under either excessive or insufficient daylength, while amphophotoperiodic plants
flower under daylength of SD and LD (8 and 18–24 h) and show delayed flowering under intermediate
daylength (12–14 h in case of Media elegans) (Hopkins and Hüner 2008).
The earlier examples of plants responding to specific photoperiods indicate plants’ ability to
measure the duration of light and/or darkness in a 24 h period. To achieve this regulation, plants
must possess both a clock that measures time and a photoreceptor that discriminates light from
darkness. The identity and function of these key factors of photoperiodic mechanisms in plants are
explained in Sections 4.3 and 4.4.
4.2.2 Photoperiodic Time Measurement

4.2.2.1 Plants Measure the Duration of Darkness
Do plants measure the length of light, darkness, or both? Hamner and Bonner (1938) showed that
flowering of the SD plant Xanthium is determined primarily by the absolute duration of darkness;
flowering occurred only under night of sufficient length (>8.5 h), regardless of the daylength. A sim-
ilar result was observed in the SD plant Pharbitis nil (Knapp et al. 1986). Providing further evidence
of the importance of night length, a short light interruption or a night-break of a few minutes to as
long as 30 min was sufficient to prevent flowering in many SD plants, including Perilla, Glycine cv
Biloxi, Kalanchoe, Pharbitis, and Xanthium. Many LD plants flower with long nights when given a
short night-break; therefore, the measurement of night length represents a critical factor for deter-
mining the flowering response of LD flowering plants, as in SD plants. However, photoperiodism of
Photoperiodic Control of Flowering in Plants 123
LD plants appears more complex than SD plants. For example, some LD plants show no response to
night-breaks and remain vegetative under short daylength, although a single LD treatment induced
flowering in Anagallis arvensis (Ballard 1969). Brassica campestris and carnation both show insen-
sitivity to a short night-break as long as 30 min but show the flowering response as the night-break
is lengthened over several hours (Friend 1969, Harris and Scott 1969). Therefore, some species,
especially LD plants, seem to respond differently to a night-break than do SD plants.
4.2.2.2 External Coincidence Model

Over the last century, considerable efforts have been made to clarify possible mechanisms of
night-length measurements. Two major hypotheses emerged: (1) night-length measurement
is accomplished by components of the plants’ circadian system, and (2) such measurement is
accomplished by an hourglass-like timer. Erwin Bünning published the first hypothesis in 1936
(Bünning 1936), which proposed that plants’ endogenous circadian rhythms provide the yard-
stick for day- and night-length measurements, thus suggesting an adaptive function for circa-
dian rhythms. Bünning’s hypothesis supplied the theoretical basis for current photoperiodism
research; however, the hypothesis remained little known until 1959, when it was reintroduced by
Colin Pittendrigh at the Cold Spring Harbor Symposium on Biological Clocks and became widely
known (Saunders 2005). Bünning proposed that light played two roles in photoperiodism: the
entrainment of the circadian system, and the illumination during a particular light-sensitive phase
as photoperiods change with seasons, representing a “coincidence” of a specific external light
phase with the endogenous phase of the circadian rhythm that triggers the initiation of flowering.
According to the hourglass hypothesis, proposed by Tony Lees in 1950s (Lees 1950, 1953a,b), an
hourglass-like timer measures either short or long night-length from dusk. Although Lees’s hour-
glass model resembles the circadian clock model in the measurement of night-length, it provides
no fundamental mechanism but a black box to explain night-length measurement. Further confir-
mation of the external coincidence model and its molecular nature, at least in part, has emerged
in the past decade through the progress in genetic and molecular studies of photoperiodism, as
described later in this chapter.
4.2.3 Florigen
Evidence from a number of approaches indicates that the site of photoperiod perception and signal
generation lies in the leaves, whereas flower production takes place elsewhere in the plants, in many
cases at the shoot apical meristems (SAMs). The first demonstration of photoperiod perception by
the leaves was performed by Knott (1934) for the LD plant spinach. He found that exposing leaves
to LD photoperiods caused the initiation of flower production at the shoot apex. The same phenom-
enon appeared in many other LD and SD plants. In some plants, treatment of only a single leaf is
sufficient to induce flowering (Naylor 1941). Importantly, these studies indicate that in addition to
perceiving photoperiods, leaves must be able to export a stimulus that evokes the captured photope-
riod signals at the receptive site where flower production occurs. This observation led to the idea of
a mobile floral hormone, or florigen, originally proposed by the Russian plant physiologist Mikhail
Chailakhyan, who defined florigen as a floral stimulus generated in the leaves under inductive pho-
toperiods and translocated from the leaves to the shoot apex (Chailakhyan 1936).
Grafting experiments provided further confirmation of the florigen hypothesis. Donor leaves
exposed to flowering-inductive photoperiods caused flowering when grafted to a noninduced stock
plant. Interspecific and intergeneric grafting experiments indicated that the nature of the florigen
might be common to many plants of different photoperiodic classes. For instance, the LD plant
Hyoscyamus flowers when grafted to the SD plant tobacco under SDs but not under LDs (Lang
et al. 1977). Conversely, tobacco flowers when grafted to Hyoscyamus under LDs. It was assumed
that the florigen moves in the phloem with the flow of the phloem sap. For example, a strong flower-
ing response in Perilla induced by a donor leaf moves at about the same velocity as photosynthetic
assimilates (Sachs and Hackett 1976). Efforts to identify biochemical substances that possess the
florigen action had been unsuccessful, and the nature of florigen remained mysterious for many
years, only becoming clearer during the past decade.
4.3 CIRCADIAN CLOCK IN PLANTS

The path between the cause (the durations of light and darkness and light inputs) and the effect
(flowering initiation) in photoperiodic flowering, although multiple models were proposed, had
been a black box that contains a series of cryptic physiological, biochemical, and molecular
events. Growing attention and significant progress in genetic approaches in model systems such as
Arabidopsis, however, led to our current understanding of photoperiodism at the molecular level.
The circadian clock is an endogenous timekeeping mechanism that has evolved in organisms in
order to coordinate biological processes, such as metabolic, developmental, and behavioral activi-
ties, with diurnal and periodic environmental fluctuations, such as photoperiods and temperature.
The presence of such a clock allows the organism not only to time biological events appropriately
but also to anticipate upcoming environmental changes. Given its self-sustainable nature, the cir-
cadian clock shows robustness to environmental changes (Gould et al. 2006, Salomé et al. 2008);
however, it is also entrainable by environmental cues that set its phase. Light perceived by multiple
types of photoreceptors serves as such an environmental cue. The nature of photoreceptors and their
roles in photoperiodism are described in the next section.
Recent progress in genetic and molecular studies of photoperiodism clarified the basic circadian
clock architecture being composed of three major domains. The first domain includes input pathways
that receive and transmit environmental signals, such as light through photoreceptors, to the second
domain, the central clock oscillator consisting of negative feedback loops. The timing signals gener-
ated by the central oscillator are transmitted through the third domain, comprising output pathways to
the various clock-controlled processes such as the regulation of primary metabolism, photosynthesis,
hormone biosynthesis, nutrient uptake, defense responses, development, and flowering transition.
Eukaryotic systems share the basic structure of the central oscillator, in which interconnected
negative feedback loops with species-specific clock components sustain robust rhythms. Numerous
components in the central oscillator have been identified and characterized in the LD model plant
Arabidopsis thaliana (Arabidopsis). Among these components, two MYB domain-containing
transcription factors, CIRCADIANCLOCK-ASSOCIATED1 (CCA1) and LATE ELONGATED
HYPOCOTYL (LHY), and a transcription factor that belongs to the PSEUDE RESPONSE
REGULATOR (PRR) gene family (Gendron et al. 2012, Huang et al. 2012, Pokhilko et al. 2012),
TIMING OF CAB EXPRESSION1 (TOC1), play a pivotal role in the core oscillator loop, in which
CCA1 and LHY repress TOC1 mRNA expression by directly binding to the regulatory promoter
region of TOC1 through the evening element, a cis-regulatory element commonly found in pro-
moters of evening-expressed genes. In turn, TOC1 represses CCA1 and LHY, creating a negative
feedback loop that generates circadian rhythms. In addition to the core feedback loop, two phase-
specific loops that appear in the morning or evening were identified in the central oscillator of
the Arabidopsis circadian clock. The core oscillator loop, the morning loop, and the evening loop
are interconnected by common components. In a morning-specific loop, CCA1 and LHY induce
mRNA expression of PRR5, PRR7 and PRR9, which subsequently repress CCA1 and LHY. In an
evening loop, CCA1 and LHY directly bind to the promoter of the LUX ARRHYTHMO (LUX)
gene and inhibit its expression, while LUX in turn promotes the expression of CCA1 and LHY.
Many additional components also participate in these loops, sustaining robust rhythms in concert.
In addition to transcriptional regulation, multiple layers of regulation at posttranscriptional, post-
translational, and chromatin remodeling levels have emerged as critical mechanisms for normal cir-
cadian rhythmicity (Jones et al. 2012, Nagel and Kay 2012, Wang and Ma 2013, Wang et al. 2012).
Moreover, alternative splicing is known to be part of essential mechanisms for the regulation of
some clock genes (Jones et al. 2012, Wang et al. 2012).
The molecular nature of the link from the central oscillator of the circadian clock to one of the
major output events, flowering transition, has been a target of intense study in recent years. The
current consensus is that the mechanism identified in Arabidopsis is highly conserved among flow-
ering plants. GIGANTEA (GI), a flowering gene and a component of the central oscillator, acts as
a master regulator that transmits the circadian signal to the flowering regulators CONSTANS (CO)
and FLOWERING LOCUS T (FT). The regulatory interaction of these central components of pho-
toperiodic flowering is referred as the CO–FT module or the GI–CO–FT module. The details of the
downstream events of the circadian clock are described in Section 4.5.
4.4 PHOTORECEPTORS IN PHOTOPERIOD
Since the discovery of phytochrome (PHY) in the 1940s, several classes of photoreceptors have
been identified, and their biochemical nature and functions clarified. All photoreceptors contain
an organic nonprotein component known as chromophore that serves as the primary site of photon
absorption (Möglich et al. 2010). Photoreceptors are classified by the chemical nature and pho-
tochemistry of their chromophore. At present, six classes of photoreceptors are known: PHYs,
cryptochromes (CRYs), light–oxygen–voltage (LOV) sensors, blue light sensors utilizing flavin
adenine dinucleotide (FAD), rhodopsins, and xanthopsins (Möglich et al. 2010). Three of these
photoreceptors, the red/far-red light receptor PHYs, the blue light receptor CRYs, and the LOV-
domain containing blue light receptors, the Zeitlupe family, are known to be associated with photo-
periodism by providing the light input and entrainment of the circadian clock.
4.4.1 Phytochromes
Phytochromes (PHYs) are dimeric proteins consisting of two identical apoproteins covalently
linked with the linear tetrapyrrole phytochromobilin that acts as a chromophore and absorbs the
red to far-red light. Flowering transition of SD plants that require sufficiently long nights to flower
was inhibited when a short pulse of the red light interrupted a long night (Parker et al. 1946). The
inhibitory effect of the red light could be canceled by a subsequent pulse of far-red (Borthwick
et al. 1952), which led to the discovery of a pigment, named phytochrome that was activated by red
and inactivated by far-red. PHY is synthesized in the red-absorbing form Pr (absorption maximum
∼660 nm) and undergoes a rapid conformational change to the far-red-absorbing form Pfr (absorp-
tion maximum ∼730 nm) when irradiated with the red light. Irradiation with the far-red light con-
verts Pfr back to Pr. Pfr is the biologically active form in plants and interacts with other proteins
to induce light response. The ratio of Pr to Pfr is dynamically determined primarily by the relative
proportions of red and far-red in ambient light and reflects biological outputs.
Three modes of PHY responses to different radiation energy of light (fluence) are known. In
dark-imbibed seeds or dark-grown seedlings in which all PHY is in the Pr form, extremely low
fluences of light raise the level of Pfr. Responses saturated by the low levels of Pfr under such low
fluences are called very-low-fluence response (VLFR). VLFR is not reversible and is sensitive to
a broad spectrum of light between 300 and 780 nm. Reponses saturated at intermediate fluences
are low-fluence response (LFR) and possess repeated reversibility by red/far-red. LFR is critical
at all stages of plant development, including chloroplast movement, ion fluxes seed germination,
de-etiolation, leaf expansion, and flowering induction. VLFR and LFR require transient exposures
to light for lesser or greater durations, respectively. In contrast, high irradiance responses require
continuous or high-frequency intermittent illumination of red or far-red (Chen et al. 2004).
The PHYs constitute a small gene family in most of plant species. Phylogenetic analysis sug-
gests that the origin of plant PHYs splits into two major gene lineages that have descendants in all
existing seed plants (Mathews 2006). One lineage includes PHYP of gymnosperms and PHYB and
PHYB-related genes of angiosperms. The other lineage includes PHYO and PHYN of gymnosperms
and PHYA and PHYC of angiosperms. As the results of evolution, different plant species contain
different PHY gene family members: five PHY genes in Arabidopsis (PHYA–PHYE) (Mathews and
Sharrock 1997), eight in soybean (PHYA1, PHYA2, PHYA3, PHYA4, PHYB1, PHYB2, PHYE1, and
PHYE2), three in rice (PHYA–PHYC) (Mathews and Sharrock 1996), and three in Ginkgo (PHYP,
PHYN, and PHYO). In Arabidopsis, PHYD is a recent duplicate of PHYB.
Plant PHYs share remarkably conserved protein domain structures. The N-terminal photo-
sensory domain contains four subdomains: P1, P2/PAS, P3/GAF, and P4/PHY. The P1 domain is
important for the stability of PHYA, but not essential for PHYB regulation. P2/PAS and P3/GAF
are tightly linked and form a chromophore-binding module that regulates limited photoconver-
sion from the dark state. Addition of the P4/PHY domain creates the three-domain P2/PAS-P3/
GAF-P4/PHY photosensory core module that displays the full photoconversion properties of full-
length proteins (Rockwell et al. 2006). The C-terminal effector domain contains two additional
PAS domains (PAS-A and PAS-B) and a histidine-kinase-related domain (HKRD). The PAS-A
and PAS-B domains are considered to be responsible for both dimerization and nuclear localiza-
tion of PHYB. However, bacterial PHYs that contain the HKRD domain but no PAS-A and PAS-B
domains can dimerize, suggesting that HKRD may also contribute to the dimerization of PHYs.
In addition, PAS-A, PAS-B, and HKRD are required for proper nuclear speckle formation. Unlike
PHYB, the C-terminal domain of PHYA is responsible for the dimerization but not for nuclear
localization. Instead, PHYA nuclear localization is regulated by PHYA interacting proteins, FAR-
RED ELONGATED HYPOCOTYL 1 (FHY1) and FHY1-LIKE (FHL) (Bae and Choi 2008).
Understanding the function of plant PHYs has been greatly deepened by the recent progress in
the model plants Arabidopsis and rice. Different PHY members show similar but different molecu-
lar and physiological properties and play shared but distinct roles in plant development, including
seed germination, seedling photomorphogenesis, shade avoidance, flowering, and many other adap-
tive responses (Bae and Choi 2008). While PHYA is light liable, all the other PHYs are light stable.
PHYA and PHYB represent the principal mediators of far-red- and red light–mediated develop-
ment, respectively. PHYA is predominant in etiolated seedlings, whereas PHYB and the others
predominate in light-grown plants. In Arabidopsis, PHYA plays a role in photoperiodic perception
(Johnson et al. 1994, Reed et al. 1994) and promotes flowering (Neff and Chory 1998), whereas in
rice, PHYA delays flowering under LD conditions but promotes flowering under SD conditions,
especially in the absence of PHYB (Takano et al. 2005). Another important function of PHYA is the
regulation of de-etiolation in response to FR light in both Arabidopsis and rice (Dehesh et al. 1993,
Takano et al. 2005, Whitelam et al. 1993). PHYB delays flowering and promotes seed germination
and de-etiolation in response to red light. In addition, PHYB inhibits shade avoidance responses
under a high ratio of R:FR light (Takano et al. 2005). In Arabidopsis, the other three PHYs (PHYC–
PHYE) play less important roles than PHYA and PHYB, with overlapping but distinct functions.
4.4.2 Cryptochromes
CRYs are a class of blue light–sensitive flavoproteins widely distributed in higher plants, most ani-
mals, and a handful of other eukaryotes and prokaryotes. Designed as a pun, the name cryptochrome
combines the cryptic nature of the photoreceptor and the cryptogamic organisms on which many
blue light studies were carried out (Gressel 1979). Most of our current knowledge about CRYs is
derived from Arabidopsis. Among the three CRY genes in Arabidopsis (CRY1, CRY2, and CRY3),
CRY1 mediates primarily blue light inhibition of hypocotyl elongation, and CRY2 mediates photo-
periodic control of floral initiation. CRYs also regulate a variety of other light responses, including
circadian rhythms, tropic growth, stomata opening, guard cell development, root development, de-
etiolation, pathogen responses, and abiotic stress responses (Cashmore et al. 1999).
CRYs possess two domains: the N-terminal PHR (photolyase-homologous region) domain that
binds the chromophore FAD and the CRY C-terminal extension domain that appears intrinsically
unstructured but critical to the function and regulation of CRYs (Fankhauser and Ulm 2011). The
PHR domain is closely related to DNA photolyases (Brautigam et al. 2004). CRYs are thought to
have evolved from photolyases and have lost or reduced the DNA repair activity of photolyases but
have gained a novel role in signaling. The PHR domain is required for chromophore binding and
homo-dimerization of CRY1 and CRY2 in Arabidopsis.
Most CRYs accumulate in the nucleus and undergo blue light–dependent phosphorylation or
ubiquitination. It is hypothesized that photons excite electrons of the flavin molecule, resulting in
redox reaction or circular electron shuttle and conformational changes of the photoreceptors. The
photo-excited CRYs are phosphorylated to adopt an open conformation, which interacts with sig-
naling partner proteins to alter gene expression at both transcriptional and posttranslational levels
and consequently the metabolic and developmental programs of plants (Yu et al. 2010). Arabidopsis
CRY1 and CRY2 are soluble proteins and localize in the nucleus. Light-activated and phosphory-
lated CRY2 undergoes degradation via ubiquitination and the subsequent 26S proteasome pathway
in the nucleus. In contrast, CRY1 is considered stable and shows dynamic cellular localization.
Nuclear localized CRY1 is responsible for blue light inhibition of hypocotyl elongation, whereas
cytosolically localized CRY1 mediates blue light stimulation of cotyledon expansion and root elon-
gation (Wu and Spalding 2007). Nuclear-localized CRY1 also mediates blue light–induced mem-
brane depolarization upon light exposure. Autophosphorylation of CRY1 is observed in vitro in a
blue light–dependent or -independent manner; however, its functional importance remains unclear
(Bouly et al. 2003, Özgür and Sancar 2006). Arabidopsis CRY3 belongs to a different class of CRYs
that contains a shorter N-terminal extension of the PHR domain and localizes in chloroplasts and
mitochondria (Kleine et al. 2003). The function of CRY3 in signal transduction is unclear.
Rice possesses two CRY1 genes (OsCRY1a and OsCRY1b) and one CRY2 gene (OsCRY2). CRY and
PHY cooperatively but independently reduce active gibberellin content in rice seedlings under light
irradiation (Hirose et al. 2012). Tomato possesses two CRY1 genes (CRY1a and CRY1b), one CRY2,
and one CRY3 gene. Ectopic expression of CRY2 revealed its role in vegetative development, flowering
time, and fruit antioxidant content (Lopez et al. 2012). In soybean, four CRY1 genes and two CRY2
genes have been identified (Zhang et al. 2008). In pea and rapeseed (B. napus), blue light–mediated
CRY1 controls flowering and photomorphogenesis (Chatterjee et al. 2006, Platten et al. 2005).
4.4.3 Zeitlupe Family
LOV domains utilize flavin nucleotide cofactors to detect blue light. In plants, three families of
photoreceptors utilizing LOV domains have been identified (Möglich et al. 2010). The first fam-
ily consists of phototropins 1 (phot1) and 2 (phot2), which mediate a wide variety of light-induced
responses including phototropism, stomata opening, chloroplast, and leaf movement (Möglich et al.
2010). The second family, the Zeitlupe family, comprises ZERITLUPE (ZTL), FLAVINBINDING,
KELCH REPEAT, F-BOX 1 (FKF1), and LOV KELCH PROTEIN 2 (LKP2) in Arabidopsis and
mediates ubiquitin-dependent protein degradation in a light-controlled manner; this protein degra-
dation ultimately leads to photoperiodic expression and accumulation of key proteins involved in
the entrainment of the circadian clock and flowering onset, which represents relatively slower light
responses (Imaizumi et al. 2005). The third family consists of the Arabidopsis PAS–LOV protein,
a putative photoreceptor whose flavin incorporation and LOV photoreaction have not yet been dem-
onstrated (Nakasone et al. 2006).
Phot1 and phot2 possess an N-terminal photosensory domain composed of two LOV domains and a
C-terminal Ser/Thr kinase domain (Demarsy and Fankhauser 2009). Blue light triggers covalent bind-
ing of the flavin nucleotide chromophore to a cysteine residue within each LOV domain, resulting in a
conformational change and enhanced kinase activity (Briggs 2007, Christie 2007). The Zeitlupe family
possesses an N-terminal LOV domain followed by an F-box and six Kelch repeats. Light activation of
the LOV domain in ZTL family members modulates their interaction with both a master regulator of
flowering, GIGANTEA (GI), and ubiquitin E3 ligase activity (Demarsy and Fankhauser 2009).
Detailed actions of the Zeitlupe family, as well as PHYA, CRY1, and CRY2 in flowering control,
are described in the following section.
4.5 MOLECULAR MECHANISM OF PHOTOPERIODIC

FLOWERING IN PLANTS
In addition to the recognition of the timekeeping circadian clock and the light-perceiving photore-
ceptors described earlier, thorough understanding of photoperiodic flowering requires identification
of the nature of “florigen” and its regulation. Here we summarize accumulated knowledge in the
past decade on the molecular and biochemical basis of “florigen” and “the external coincidence
model” underlying photoperiodic flowering of Arabidopsis and other plant species. While many
of the major flowering genes are conserved among species, they demonstrate notable variation and
surprising complexity in their regulation and evolution (Ballerini and Kramer 2011).
4.5.1 Arabidopsis
Two genes occupy the core of the photoperiodic flowering gene pathway in the LD flowering model
plant Arabidopsis: CONSTANS (CO) and FLOWERING LOCUS T (FT) (Samach et al. 2000). The CO
gene encodes a putative zinc finger transcription factor and activates mRNA expression of the FT gene,
which encodes a small transcription cofactor that is a major component of florigen. This regulatory
interaction, known as the CO–FT module, is highly conserved among distantly related flowering plants.
CO is an output of the circadian clock and is known as a point of “external coincidence” in which
the internal circadian clock and the external day–night cycles integrate (Pittendrigh and Minis
1964, Sawa et al. 2008, Turck et al. 2008). Rhythmic expression of CO mRNA is regulated partly
by both GIGANTEA (GI), a component of the circadian clock and an activator of CO (Fowler et al.
1999, Huq et al. 2000), and CYCLING DOF FACTOR1 (CDF1), a repressor of CO (Song et al.
2010) (Figure 4.1). Under blue light, GI forms a complex with the FLAVINBINDING, KELCH
REPEAT, F-BOX 1 (FKF1) in the late afternoon under flowering-inductive LD. The GI–FKF1
complex degrades CYCLING DOF FACTOR 1 (CDF1) on the regulatory promoter of CO, resulting
in the activation of CO transcription at the end of the day under LD (Sawa et al. 2007). Under SD,
the membrane-bound E3 ligase DAY NEUTRAL FLOWERING (DNF) has been shown to repress
CO expression, maintaining low CO expression under SD (Morris et al. 2010).
The CO mRNA oscillation coincides with specific light quality at a specific time of the day,
which triggers posttranslational regulation of CO protein (Valverde et al. 2004). In the early morn-
ing under LD, the red-light receptor PHYB promotes degradation of CO protein (Srikanth and
Schmid 2011), but the far-red receptor PHYA and the blue light receptors CRY1 and CRY2 stabilize
CO protein toward the end of the day through inhibition of proteasome-dependent CO degradation.
Other factors, including SUPRESSOR OF PHYA 1 (SPA1), SPA3, SPA4, and the E3 ubiquitin ligase
CONSTITUTIVE MORPHOGENIC 1 (COP1), regulate CO protein stability during the night (Jang
et al. 2008, Laubinger et al. 2006). The concerted action of light and the circadian clock control-
ling CO mRNA oscillation and protein stability allows CO protein to accumulate highly only at
CCA1 PHYA CRY2 LFY AP1

LHY COP1
PHYB
TOC1 SPA1 DNF
SOC
FKF1 CO
CDFs FT FT- FD
GI
miR172 AP2
Leaf Phloem Meristem
FIGURE 4.1 Photoperiodic flowering gene network in Arabidopsis.

the end of the day under flowering-inductive LD. This high CO accumulation in turn activates the
floral inducer FT, a mobile florigen signal synthesized in the leaves and transmitted to the SAM to
cause flowering transition (Abe et al. 2005, Corbesier et al. 2007, Kardailsky et al. 1999, Kobayashi
and Weigel 2007, Wigge et al. 2005). In the SAM, FT interacts with the bZIP transcription factor
FLOWERING LOCUS D (FD) and induces the expression of the floral identity gene APETALA1
(AP1). FT also activates the flowering inducer SUPRESSOR OF OVEREXPRESSION OF CO 1
(SOC1) expression, and SOC1 in turn induces another floral identity gene, LEAFY (LFY). LFY and
AP1 initiate the development of floral primordial on the flanks of the SAM (Figure 4.1).
The regulatory mechanisms of CO explain very well the molecular basis for the flowering of
Arabidopsis under LD. However, the molecular basis of photoperiodic flowering in reality may be
more complex. Multiple factors and pathways that regulate flowering in a CO-independent man-
ner have been reported (Castillejo and Pelaz 2008, Jung et al. 2007, Mathieu et al. 2009, Sawa and
Kay. 2011, Yant et al. 2010). For example, GI regulates the processing of the small noncoding RNA
microRNA172 (miR172), which induces FT expression through repression of APETALA2-like
(AP2-like) transcription factors independently of CO (Jung et al. 2007). In addition, GI is shown to
directly bind to the FT promoter and enhances its expression without affecting CO expression (Sawa
and Kay 2011) (Figure 4.1). These observations suggest two important insights in Arabidopsis photo-
periodic flowering. First, plants have evolved multiple genetic pathways that mediate photoperiodic
flowering. Second, there are multiple points of external coincidence of internal factors and external
lights in the flowering gene networks.
4.5.2 Rice
Rice (Oryza sativa) is one of the most well-studied SD flowering plants and provides an insightful
point of comparison with the LD plant Arabidopsis. Many genes involved in Arabidopsis photoperi-
odic flowering are conserved in rice. Nevertheless, rice shows unique variations in their regulations
and novel components involved in photoperiodic flowering.
Three key components of photoperiodic flowering in Arabidopsis, GI, CO, and FT exist in rice and
function in a similar manner to their Arabidopsis orthologous genes. As observed in Arabidopsis, the
rice GI ortholog OsGI activates a CO ortholog Heading date1 (Hd1) under flowering-inductive SD.
Hd1 then activates the expression of FT homologs Hd3a and RFT1, indicating that the CO–FT
module is conserved among distantly related Arabidopsis and rice (Hayama and Coupland 2004,
Yano et al. 2000). A notable difference in CO function in rice is its “dual-functionality”; rice Hd1
acts as an inducer of Hd3a under SD but a repressor under LD. The current model for the opposite
regulation of Hd3a by Hd1 under different daylengths involves the coincidence of the Hd1 mRNA
oscillation and light signaling through PHYs, which suppresses the Hd3a induction by Hd1 (Doi
et al. 2004). Similar to Arabidopsis, PHYs are thought to posttranscriptionally regulate Hd1 to
activate Hd3a.
In addition to the upstream regulatory mechanism, many of the downstream flowering initia-
tors in rice act differently compared to their homologs in Arabidopsis. Unlike Arabidopsis SOC1’s
induction by FT in the meristem, the rice SOC1 homolog OsMADS50 induces the FT homolog
RFT1 under LD in the leaves, leading to eventual flowering under noninductive LD conditions
(Komiya et al. 2009). RICE FLORICAULA/LEAFY (RFL), a rice homolog of the floral identity gene
LFY, is expressed in leaves and promotes flowering through the activation of OsMADS50 and RFT1
expressions, unlike Arabidopsis LFY, which is expressed in the meristem (Figure 4.2). Therefore,
despite the striking similarity of the core components in photoperiodic flowering, key differences
exist in regulatory interactions of the flowering gene networks in rice; these differences play a role
in the opposite flowering responses of Arabidopsis and rice under different daylengths.
Another more significant difference is that rice utilizes unique components in flowering regu-
lation that appear to have no counterparts in Arabidopsis. Early heading date1 (Ehd1) encodes a
B-type response regulator and activates Hd3a expression and flowering under SD (Kim et al. 2007).
PHY
CRY
Ghd7
OsCOL4
OsGI
Ehd2
OsMADS51
Hd1 Ehd1
OsMADS50
Osld1
RFL
OsMADS56
RFT1
Hd3a
FIGURE 4.2 Photoperiodic flowering gene network in rice.
Ehd1 expression is strongly induced when the morning phase set by OsGI-dependent circadian
clocks coincides with blue light (Itoh et al. 2010). Ehd1 is also induced by a unique rice gene,
OsMADS51 (Kim et al. 2007). The Ehd1 expression is suppressed by exposure to red light during
night through another unique flowering gene in rice, Ghd7, which encodes a CCT domain protein.
Additionally, a rice CO homolog, OsCOL4, is reported to repress Ehd1 as well as Hd3a under con-
trol of OsPHYB in a night-break-insensitive pathway to regulate flowering time (Lee et al. 2010).
Contrary to the dual-functional Hd1, Ehd1 acts as an inducer of flowering under both SD and LD
through activation of different FT homologs: Hd3a in SD and RFL1 in LD (Kim et al. 2007); activa-
tion of these homologs ensures eventual flowering under nonoptimal LD for survival and perhaps
adaptation to various light regimens. Similarly, the LD facultative plant Arabidopsis eventually
flowers under SD conditions, possibly through alternative CO-independent pathways.
The flowering gene networks in rice, therefore, are significantly different from those in Arabidopsis
despite conservation of the GI–CO–FT pathway. Nevertheless, as observed in Arabidopsis, external
coincidence of internal factors and light inputs at multiple points in the flowering gene networks
governs photoperiodic flowering of rice.
4.5.3 Morning Glory
Although the photoperiodic flowering response of morning glory (P. nil or Ipomoea nil) has long
been studied, only limited information is available for the molecular mechanisms of its SD flow-
ering habit. Despite the fact that Pharbitis possesses homologs of the major flowering genes GI
(PnGI), CO (PnCO), and FT (PnFT), and that PnCO is capable of rescuing the late-flowering phe-
notype of the co mutant in Arabidopsis (Hayama et al. 2007, Liu et al. 2001), the mode of action
and regulation of the flowering genes in Pharbitis show little similarity to the GI–CO–FT pathway
found in Arabidopsis and, in a variant form, in rice. PnCO is a functional CO protein; however, it
does not influence PnFT1 or PnFT2 expression levels or activity. Instead, PnFT1 is directly regu-
lated by a circadian clock under SD, unlike either Arabidopsis or rice (Hayama et al. 2007). While
a coincidence of peak PnFT1 expression and PnCO expression during SD may indicate some level
of PnCO involvement in the regulation of PnFT1, these peaks do not overlap during increasing day-
lengths. It is thus assumed that two clock mechanisms govern photoperiodic flowering of Pharbitis,
one of which regulates PnFT expression in response to the cessation of light at dusk, while another
clock regulates PnCO in response to light at dawn. So far, no conclusive evidence for the function
of PnCO in flowering in Pharbitis itself has been demonstrated.
Similarly, the mode of PnGI action departs from that of Arabidopsis GI. Although both
Arabidopsis GI and PnGI are circadian clock-regulated and show similar responses to photoperiod
in their mRNA expression, Arabidopsis GI induces flowering mainly through activation of CO,
leading to FT expression, while PnGI suppresses flowering by acting directly on PnFT1, repress-
ing its expression under flowering-inducing SD (Higuchi et al. 2011). This also contrasts with rice,
in which OsGI works through the CO ortholog Hd1 to influence the FT ortholog Hd3a. However,
rice does possess an alternate pathway that connects OsGI to Hd3a through Ehd1, a unique rice
flowering gene. Similarly, Arabidopsis GI regulates FT through miR172 and AP2-like transcription
factors in a CO-independent manner. Pharbitis may also possess alternative cascades between PnGI
and PnFTs other than PnCO; these remain a target for future investigation.
4.5.4 Soybean
In addition to morning glory, the SD flowering plant soybean (G. max) has long been a key model to
study photoperiodic flowering, yet little is known about molecular mechanisms and genetic regula-
tion of floral initiation in soybean. Key flowering gene homologs are present in soybean as in rice
and Pharbitis, including several CO homologs (GmCOs) and FT homologs (GmFTs), as well as at
least two GI homologs (GmGIs). Expression analysis of some of these genes indicates that GmCOs
show circadian expression patterns (Huang et al. 2011, Jiang et al. 2011, JinHua et al. 2009, Liu et al.
2011), although thus far GmCOs’ involvement in photoperiodic flowering has not been established.
One of the GmGIs has been identified as a causal gene for the soybean maturity quantitative trait
locus (QTL) E2 and a flowering QTL, FT2. Plants that carry recessive e2/ft2 alleles show early flow-
ering and elevated expression of a soybean FT homolog, indicating the involvement of GmGIs in the
initiation of flowering (Watanabe et al. 2011). The best-studied flowering gene homologs in soybean
are GmFTs. Of the at least 10 FT homologs found in the soybean genome, GmFT2a and GmFT5a
demonstrate increased expression under SD and decreased expression under LD (Kong et al. 2010,
Thakare et al. 2010). When ectopically expressed in Arabidopsis, both GmFT2a and GmFT5a pro-
duced an early flowering phenotype similar to that of ectopically expressed Arabidopsis FT (Kong
et al. 2010, Thakare et al. 2011). These observations suggest that GmFT2a and GmFT5a are induc-
ers of floral transition in soybean as seen in their Arabidopsis counterparts; however, further infor-
mation is required to clarify the function of GmCOs and the regulatory interaction between GmCOs
and GmFTs.
Including E2/FT2, nine major maturity QTLs controlling flowering and maturity in soybean have
been identified: the maturity loci E1–E8 and J (Bernard 1971, Bonato and Vello 1999, Buzzell 1971,
Buzzell and Voldeng 1980, Cober and Voldeng 2001, Cober et al. 2010, McBlain and Bernard 1987,
Ray et al. 1995), as well as several flowering QTLs (Gai et al. 2007). Of these, E1, E3, E4, and
E7 have shown sensitivity to photoperiod (Watanabe et al. 2012). E3 causes a delay in flowering
under LD with a variety of light conditions (Cober et al. 1996). E1, E4, and E7 induce late flower-
ing under LD with red and far-red light (R:FR) only (Cober and Voldeng 2001, Cober et al. 1996),
with E1 demonstrating the strongest inhibitory effect (Thakare et al. 2010). Recessive e1 alleles lead
to increased expression of both GmFT2a and GmFT5a (Watanabe et al. 2011). The causal gene of
E1 has been isolated to a 17.4 kb region on chromosome 6 (Xia et al. 2012). It encodes a transcription
factor without close homologs in Arabidopsis that acts as a repressor of both flowering and matura-
tion (Xia et al. 2012). E3 and E4 have been identified as homologs of the Arabidopsis photoreceptor
PHYTOCHROME A (PHYA), GmPHYA3 and GmPHYA2, respectively (Liu et al. 2008, Watanabe
et al. 2009). E3 and E4 are reported to affect both GmFT2a and GmFT5a expressions, similar to
PHYA in Arabidopsis (Kong et al. 2010, Liu et al. 2008), indicating that at least part of the external
coincidence mechanism is likely conserved in soybean photoperiodic flowering control. In addition,
GmCRY1a, an ortholog of the Arabidopsis photoreceptor CRY1, promotes floral initiation and exhib-
its a rhythmic expression pattern, highlighting its role in photoperiodic flowering (Zhang et al. 2008).
4.5.5 Sugar Beet
Flowering transition of the biennial sugar beets (Beta vulgaris) is initiated upon both an extended
cold period during winter called vernalization and subsequent exposure to LD. For the annual vari-
eties, exposure to LD without vernalization is sufficient to flower (Owen et al. 1940). While sugar
beet possesses CO and FT homologs, mechanisms of photoperiodic flowering in sugar beet may
have undergone a unique evolutionary history.
Two FT homologs, BvFT1 and BvFT2, have been identified in sugar beets. Modification of their
expression by transgenic approaches suggested that BvFT2 is the functional FT ortholog and an activator
of flowering. In contrast, BvFT1 possesses antagonistic function as a repressor of flowering. This finding
suggested that the beet had evolved a different strategy relative to Arabidopsis and cereals to regulate
flowering (Pin et al. 2010). Furthermore, BvFT1 and BvFT2 are regulated by a unique factor in sugar
beet. The bolting locus B is considered a master switch distinguishing annuals from biennials (Abegg
1936). The causal gene of the B locus was recently revealed to be the pseudo-response regulator gene
BOLTING TIME CONTROL 1 (BvBTC1) (Pin et al. 2012). BvBTC1 regulates both photoperiod and
vernalization signals through the regulation of sugar beet FT homologs. Sugar beets carrying functional
BvBTC1 behave as annuals and flower as a direct response to LD by repressing BvFT1 and activat-
ing BvFT2. During the domestication of beets, a rare partial loss-of-function BvBTC1 allele has been
selected; this loss-of-function allele does not block expression of the floral repressor BvFT1 and thus
results in reduced sensitivity to photoperiod. Vernalization restores the function of the loss-of-function
BvBTC1 to repress BvFT1 and induce BvFT2, thereby promoting floral initiation in response to LD,
which confers bienniality (Andrés et al. 2012, Pin et al. 2012).
Several CO homologs have been identified in sugar beets; however, their function in photoperi-
odic flowering control requires further investigation (Chia et al. 2008).
4.5.6 Poplar
The perennial tree poplar (Populus trichocarpa) requires several years to reach the sexual maturity
to flower. Shoot meristems undergo repeated seasonal transitions between vegetative and reproduc-
tive growth each year, providing a unique model for photoperiodic and seasonal control of flowering
in trees. Exposure to low temperature during winter initiates reproduction and production of floral
buds during spring, whereas vegetative growth occurs by exposure to warm temperature and LD
during summer. The repeated cycles of vegetative and reproductive growth in poplar are coordi-
nated by the functionally diverged poplar FT homologs PtFT1 and PtFT2. PtFT1 does not show
diurnal expression and appears to be regulated strictly by temperature, while PtFT2 is regulated
by photoperiod. Low temperatures during winter promote PtFT1 expression, which in turn triggers
the formation of floral buds. In spring and summer, however, warm temperatures turn off PtFT1
expression, and in turn LD induces PtFT2 expression, leading to vegetative growth (Andrés and
Coupland 2012, Hsu et al. 2011).
Contrasting results were reported on poplar CO homologs. PtCO1 and PtCO2 are the closest CO
homologs in poplar. PtCO2 appears to mediate the seasonal dormancy response. Transgenic poplar
that has reduced expression of PtCO2 shows low expression of PtFT1 and PtFT2, providing some
evidence that the CO–FT module is conserved in popular (Bohlenius et al. 2006, Hsu et al. 2006).
However, a recent study reported that ectopic expression of PtCO1 and PtCO2 in transgenic popu-
lar plants failed to alter the flowering time, raising a possibility that the function of the poplar CO
homologs might not overlap with that of Arabidopsis CO (Hsu et al. 2012).
4.5.7 Sunflower
Sunflowers (Helianthus annuus) flower in response to either LD or SD depending on the acces-
sion. Four sunflower FT homologs (HaFT1–HaFT4) represent another interesting functional evolu-
tion of FT underlying the diverse photoperiodic flowering response of poplar. HaFT1, HaFT2, and
HaFT4 are functional genes that complement the late-flowering phenotype of the Arabidopsis ft
mutant when ectopically expressed, whereas HaFT3 is a putative loss-of-function gene (pseudogene)
carrying several nonsynonymous mutations in the coding sequence with no detectable expression
(Blackman et al. 2010). A striking effect of genetic interaction between HaFT1 and HaFT4 in pho-
toperiodic flowering is observed in domesticated sunflower. A domesticated variety of sunflower
carries a frameshift mutation in HaFT1, leading to a protein with 17 amino acids longer than that of
wild sunflower. The frameshift allele is associated with earlier flowering and maps to a major flower-
ing QTL that affects flowering only under LD. Although the frameshifted HaFT1 is expressed under
both LD and SD, it is suggested that the frameshifted HaFT1 interferes the function of HaFT4 that is
expressed only under LD in a dominant negative manner, resulting in the photoperiod-specific early
flowering effect of the frameshifted HaFT1 in the domesticated sunflower (Blackman et al. 2010).
The precise mechanism of the interference of HaFT4 by HaFT1 is currently unknown.
Two sunflower CO homologs, HaCOL1 and HaCOL2, have been identified (Blackman et al.
2011); however, currently no information exists as to their function in photoperiodic flowering.
4.6 CONCLUSIONS
Over the last century, it was not possible to dissect fully the mechanisms underlying photo-
periodic flowering of plants due to the complex and cryptic physiological and developmental
processes at play. Thanks to the recent technological advances in genetic, molecular, and bio-
chemical approaches in the model species, during recent decades; however, we have experienced
a sudden explosion of novel information and a deeper understanding of molecular mechanisms
of plants’ photoperiodic response. The major landmarks of this achievement are the discoveries
of the nature and action of photoreceptors, the circadian clock and the florigen, as well as the
flowering gene networks linking photoperiod signals and the florigen, and external coincidence
regulating the flowering gene networks. More recently, the advancement of genomic approaches
has allowed increased understanding of the remarkable diversity in the photoperiodic flowering
response among flowering plants. Significant conservation of the genes and mechanisms among
distantly related plant species has been observed, as well as considerable modifications contrib-
uting to species’ diverse responses to seasonal environmental changes. In particular, the activa-
tion of the florigen FT (and its homologs) is a universal feature of the photoperiodic flowering
response highly conserved among plant species, although different plants employ different regu-
latory mechanisms of the florigen, which ensures that diverse environmental factors can achieve
activation of the florigen. Notably, FT can also control other seasonal responses of plants, such as
tuberization, growth, and floral bud dormancy.
Despite such progress, several important questions remain. A major question is how the florigen
FT promotes flowering or other developmental processes. Although FT’s involvement in transcrip-
tional regulation of floral identity genes through interaction with the bZIP transcription factor FD
has strong support in Arabidopsis and rice, the mechanisms controlling FT and FD regulation and
activation require further clarification. In addition, the molecular mechanisms controlling the sea-
sonal regulation of FT remains unclear in many plant species that have evolved diverse flowering
responses, including the difference between annual and perennial species or between LD and SD
species. Moreover, the molecular nature of the interaction between photoperiods and other environ-
mental factors such as temperature and humidity is mostly unanswered. Addressing these questions
further will allow thorough understanding of the photoperiodic flowering response of crop plants
and provide novel strategies and opportunities for agricultural applications.
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5 Role of Alternative Respiratory
Pathway in Plants
Some Metabolic and
Physiological Aspects
Elena V. Garmash
CONTENTS
5.1 Introduction........................................................................................................................... 139
5.2 Respiratory System in Plants................................................................................................. 140
5.2.1 Course of Respiratory Process.................................................................................. 140
5.2.2 Mitochondrial Electron-Transport Chain.................................................................. 140
5.2.3 Respiratory Control................................................................................................... 141
5.3 Characteristics of Alternative Oxidase.................................................................................. 141
5.3.1 AOX Structure........................................................................................................... 141
5.3.2 AOX Genes................................................................................................................ 142
5.3.3 AOX Activation: Transcriptional and Posttranslational Mechanisms of Regulation.......142
5.3.4 AOX Capacity and Activity....................................................................................... 143
5.4 Physiological Role of Alternative Respiration in Plants........................................................ 143
5.4.1 Cyanide Resistance.................................................................................................... 143
5.4.2 Thermogenesis........................................................................................................... 143
5.4.3 Energy Overflow: Balancing the Respiratory Metabolism....................................... 144
5.4.4 Prevention of Oxidative Stress................................................................................... 145
5.4.5 Maintenance of Interaction between Respiration and Other Metabolic Processes..........148
5.4.6 “Antiapoptotic Protein”............................................................................................. 150
5.5 Concluding Remarks............................................................................................................. 150
References....................................................................................................................................... 151
5.1 INTRODUCTION
Respiration is a universal process and a fundamental basis of life of any living cell. Respiration pro-
duces energy (adenosine triphosphate [ATP]), reducing equivalents (NAD(P)H) and carbon skeletons
to sustain synthesis de novo and maintain integrity and functional activity of cellular structures, tissues,
and organs of the whole organism. Respiration is an indicator of functional stability of living objects.
Respiration acts as the mechanism accelerating evolution. It is known that DNA oxidation by reactive
oxygen species (ROS) formed during respiration is the major tool of natural mutagenesis (Skulachev
1999). Therefore, the respiration has captured the interest of scientists for more than two centuries.
In plants, the main substrate for respiration is carbohydrates assimilated during photosynthesis.
Each day, a large fraction of all assimilated carbohydrates (from 30% to 70%) are expended in
respiration (Golovko 1999, Lambers et al. 2005, Van der Werf et al. 1992). Production of energy as
139
ATP proceeds in specialized respiratory organelles—mitochondria in the electron-transport chain

(ETC). The terminal part of the respiratory path consists of, among other components, two respira-
tory pathways for electron transport—the main cytochrome pathway (CP) and the alternative path-
way (AP) through alternative oxidase (AOX). The major characteristic of AP is that it is not coupled
with the synthesis of ATP and therefore considered a wasteful pathway (Moore and Siedow 1991).
The unproductive characteristic of AP has, for a long time, raised questions regarding its regulation
in vivo and the role in plant metabolism. Many important reviews have discussed the hypotheses
concerning its role (Juszczuk and Rychter 2003, Millenaar and Lambers 2003, Moore and Siedow
1991, Shugaev 1999, Vanlerberghe and McIntosh 1997, Vanlerberghe et al 2009, Wagner and Krab
1995). Plants as sessile organisms developed biochemical strategies, and alternative respiratory
pathway is considered to play an important role for plant acclimation.
The progress in AOX research was achieved by biochemical studies, by the identification of AOX
gene families and by using the methodology of reverse genetics to manipulate AOX respiration in
plants. Despite our knowledge about the molecular nature of AOX, its regulation mechanisms, and
functions, the understanding regarding the role of alternative respiration in plant metabolism is still
incomplete. In many respects, it is connected with the fact that respiration does not function in isola-
tion but rather must integrate with other processes, and the AP would likely be engaged in the total
flow of plant responses to different internal and external fluctuations as a highly efficient additional
mechanism for optimizing the energetic metabolism and redox balance.
This chapter briefly reviews the information about the structure and mechanisms of AOX activa-
tion as well as discusses the known functions of the nonenergy-conservating AP in plants. Based
on our own experimental data, the engagement and physiological significance of AP in plants in
dependence on their growth rate, developmental status, as well as stressful conditions are shown.
We underline that the physiological role of AOX is to provide metabolic homeostasis in response to
fluctuating cellular conditions.
5.2 RESPIRATORY SYSTEM IN PLANTS

5.2.1 Course of Respiratory Process
Respiration is the stepwise process proceeding in different cell compartments. The first step in
the production of energy for respiration occurs in cytoplasm, when glucose (or other storage car-
bohydrates) is metabolized in glycolysis or in the oxidative pentose phosphate pathway. The main
end-products of glycolysis—pyruvate and malate—are exported to mitochondria where they are
oxidized in the tricarboxylic acid or the Krebs cycle, functioning in the matrix of the mitochon-
drion. During Krebs cycle operation, the reducing equivalents—pyridine nucleotides (NADH and
FADH2)—are yielded. At the final step of respiration, the reducing equivalents donate their elec-
trons to the ETC localized in the inner membrane of mitochondria where ATP is produced.
5.2.2 Mitochondrial Electron-Transport Chain

In the mitochondrial ETC, there are four major complexes associated with electron transport, and
one (ATP synthase) associated with oxidative phosphorylation (Figure 5.1). In addition, there are
two small redox molecules, ubiquinone and cytochrome c, which play an important role in electron
transfer. The ATP production is driven by the proton gradient across the inner mitochondrial mem-
brane and linked to the flow of electrons in ETC. Transfer of a couple of electrons on the main CP
from NADH to O2 through the four complexes with the subsequent synthesis of three molecules of
ATP is universal for animals and plants.
Respiratory ETC in plants is more complicated and functionally flexible than ETC in animal
mitochondria (pиc. 1). The mitochondrial ETC in higher plants consists of not only the phosphory-
lating CP pathway but also several nonphosphorylating pathways. The additional enzymes include
Role of Alternative Respiratory Pathway in Plants 141
External NADH
dehydrogenases
NAD+ NAD+
4H+ NADH NADH 4H+ 2H+ 3H+
Intermembrane
space
Cyt c
Complex
Inner I Ubiquinone
Complex
membrane pool AOX Complex IV
Complex III
II
Succinate O2
NADH Fumarate H2O
NAD+ NADH O2
Matrix H 2O
NAD+ NAD+
Internal NADH ADP
dehydrogenases ATP
ATP synthase
FIGURE 5.1 Organization of the electron-transport chain in the inner membrane of plant mitochondria.
Discontinuous arrows indicate electron transfer; solid arrows indicate proton translocation. AOX—alterna-
tive oxidase.
type II NAD(P)H dehydrogenases, which reduce ubiquinone bypassing complex I, and the AOX,
which transfers electrons from the ubiquinone pool directly to oxygen and produces water. This
pathway through AOX is called alternative pathway (AP) (Vanlerberghe and McIntosh 1997). The
AP is insensitive to cyanide (complex IV inhibitor); therefore, it is called the cyanide-insensitive
respiratory pathway. Transfer of electrons from ubiquinone to oxygen via AP is not coupled with the
extrusion of protons from matrix to the intermembrane space, and the energy is lost as heat. Hence,
the transfer of electrons from endogenous NADH to oxygen via AP yields only one-third of the
amount of ATP that is produced when CP is used (Moore and Siedow 1991).
5.2.3 Respiratory Control
In isolated mitochondria, the rate of electron transport, and hence the rate of oxygen uptake, is con-
trolled by the so-called respiratory control (Siedow and Day 2000). There are four respiratory states
during the course of mitochondrial respiratory activity. The respiratory control represents the ratio of
oxygen consumption rate of mitochondria in the presence of both substrate and ADP (state 3) to the rate
in the presence of substrate but the absence of ADP (state 4) since all the ADP has phosphorylated to
form ATP. The respiratory control ratio indicates the tightness of the coupling between respiration and
phosphorylation. In intact tissues, the respiratory control also occurs. It is one of the mechanisms to
control plant respiration rate by demand of different processes (growth, transport, nutrient uptake and
assimilation, and maintenance) for energy (ATP) (Farrar 1985, Golovko 1999, Lambers et al. 2005). The
respiration rate is enhanced when the demand for ATP increases. The cytochrome respiration as path-
way coupled with the ATP production is effectively controlled by the availability of ADP and dependent
on ADP/ATP ratio (adenylate control), whereas the AP is not restricted by adenylate demands.
5.3 CHARACTERISTICS OF ALTERNATIVE OXIDASE

5.3.1 AOX Structure
AOX was isolated from Sauromatum guttatum and characterized for the first time almost 30 years
ago (Elthon and McIntosh 1986). The progress in elucidation of the AOX structure was achieved by
the difficult procedure of purifying protein and the subsequent analysis by spectroscopic techniques
(Berthold and Sidow 1993, Berthold et al. 2002, Moore et al. 2008).
AOX (EC. 1.10.3) is an approximately 32 kDa homodimeric interfacial quinol-oxidizing protein

peripherally associated with the matrix side of the inner mitochondrial membrane (Andersson and
Nordlund 1999, Siedow and Umbach 2000). Both terminal domains face the matrix. The N-terminal
domains are responsible for the maintenance of the dimeric AOX as they form the intermolecular
disulfide bond. The C-terminal domains contain a coupled di-iron center on the basis of a conserved
sequence motif consisting of the proposed iron ligands, four glutamate and two histidine amino
acid residues (Berthold et al. 2002). The hydroxo-bridged mixed-valent Fe(II)/Fe(III) binuclear
iron center within a four-helix bundle is the active site responsible for the reduction of oxygen to
water. Some conformational changes in AOX such as “carboxylate shift,” altering ligation of the
iron atoms, may cause movement of the helices thus providing additional room for oxygen access
and resulting in protein activation (Albury et al. 2009).
To explain the structure of quinone-binding site in AOX, a model, in which the hydrophobic
pocket, between two α-helices (II and III), acts as a channel enabling the substrate to gain access
to the active site from the membrane-binding domain, has been proposed (Albury et al. 2009,
McDonald 2009).
5.3.2 AOX Genes
Plant AOXs are encoded by a small nuclear multigene family that is divided into two subfami-
lies, AOX1 and AOX2 in eudicots, whereas only AOX1 members have been found in monocots
(Polidoros et al. 2009). For example, Arabidopsis contains four AOX1 genes (a–d) and one AOX2
(Considine et al. 2002), whereas two AOX1 genes (a, c) in common wheat have been characterized
(Takumi et al. 2002).
AOX genes in both families present a conserved intron–exon structure that in many species con-
sists of four exons interrupted by three introns at highly conserved splice site positions (Considine
et al. 2002).
5.3.3 AOX Activation: Transcriptional and Posttranslational

Mechanisms of Regulation
The AOX is induced in response to a wide range of stresses and adverse environmental condi-
tions as well as respiratory inhibitors (Clifton et al. 2006, Rasmusson et al. 2009, Vanlerberghe
and McIntosh 1997, Vanlerberghe et al. 2009). It was proposed that ROS and H2O2, produced as
a result of different stresses, may be involved in inducing AOX expression (Polidoros et al. 2005,
Vanlerberghe and McIntosh 1996, Wagner 1995). There is also the so-called ROS-independent
pathway that signals the induction of AOX based on the ability of added citrate to induce AOX
transcript and protein without any observed increase in ROS (Grey et al. 2004).
The expression of AOX is triggered by a variety of signals indicating that it is a common
response to many stresses or mitochondrial dysfunction (Clifton et al. 2006, Van Aken et al.
2009, Yu et al. 2001). AOX is considered as a target and regulator of stress responses. It means
that the expression of AOX is regulated by stress-related and developmental anterograde signals,
as well as by retrograde signals induced by perturbed mitochondrial function (Juszczuk et al.
2012, Van Aken et al. 2009).
It is known that the promoter (regulatory region of gene) of AOX1a in Arabidopsis is a 93-bp region
that was defined as essential for induction by antimycin A and monofluoroacetate (Dojcinovic et al.
2005). The promoter region of AOX1a was analyzed, and functional analysis identified 10 cis-acting
regulatory elements, some of which have been previously associated with stress responses (Ho et al.
2008). At least three distinct signaling pathways regulating stress-induced transcript abundance for
genes encoding mitochondrial proteins including AOX1a were proposed (Ho et al. 2009). It means
that AOX expression is a part of a general cellular stress response. Certain signal transduction path-
ways responsible for AOX induction under different conditions need to be further clarified.
Posttranslational regulation of AOX is controlled by two interrelated mechanisms. AOX is

present in angiosperm as a noncovalent dimer (Umbach and Sidow 1993). The first mechanism
is connected with regulation by covalent redox state through oxidation and reduction of an inter-
subunit disulfide bond (Umbach et al. 1994). In its oxidized state, AOX is less active, whereas in
its reduced state, it is more active and can be further activated by some α-keto acids like pyruvate
(Umbach et al. 1994). Pyruvate appears to bind to the same cysteine residue that is used for dimer-
ization (Rhoads et al. 1998, Vanlerberghe et al. 1998). Understanding the biochemical control of the
AOX activity remains incomplete. The partitioning of electrons between AP and CP is very respon-
sive to metabolic changes within the mitochondrion and the cell (Finnegan et al. 2004).
5.3.4 AOX Capacity and Activity

AP capacity (or maximal activity) is defined as the O2 uptake resistant to the CP inhibitor and
sensitive to the AOX inhibitor (Møller et al. 1988). A range of specific inhibitors is used to assess
the respiratory pathways presence. Cyanide (CN) blocks complex III and is used to reveal the
presence of the AP. The inhibitors of AOX are some hydroxamic acids such as salicylhydroxamic
acid (SHAM), and n-propyl gallate. SHAM is thought to act as competitive inhibitor of ubiquinol.
Application of inhibitors allows to determine the maximum activity (often called capacity) of the
AP or the CP of a plant cell or tissue. These inhibitors can have side effects on respiration. To mini-
mize potential problems, the appropriate concentration of inhibitor for that species, organ, age, and
growth condition must be determined by an entire titration curve (Møller et al. 1988).
The inhibitor method for AOX assessment is based on the assumption that AOX respiration only
occurred once the CP was saturated with electrons (Bahr and Bonner 1973). AOX capacity reflects
maximum possible flux of electrons and can give an indication that some level of engagement was
present in a tissue. AOX is currently known to be regulated by α-keto acids and covalent modifica-
tion as well as by additional mechanisms not yet characterized. This activation allows the AP to
compete with unsaturated CP for electrons (Day et al. 1996).
In order to measure the AOX activity in vivo, the oxygen isotope discrimination technique origi-
nally developed by Guy et al. (1989) is used. This method is based on the observation that AOX
and cytochrome oxidase discriminate to different extents against heavy O2 (18O16O) (Henry et al.
1999, Ribas-Carbo et al. 1995). In some cases, the results obtained with the isotope discrimination
method are similar to the results from the inhibitor method (Rachmilevitch et al. 2007).
5.4 PHYSIOLOGICAL ROLE OF ALTERNATIVE RESPIRATION IN PLANTS

The alternative respiration as energetically wasteful pathway has a large impact on the energy
conservation, but ubiquity of AOX has raised vast discussions about its biological function.
5.4.1 Cyanide Resistance
There is an opinion that alternative respiration is arisen during evolution of plants as a way to
maintain respiration process in the presence of cyanide. Many plants (about 2500 species) contain
cyanogenic glycosides from which hydrogen cyanide can be released (Vetter 2000). The biological
role of these secondary metabolites can be protective, directed against phytopathogens or eating by
herbivores. However, it does not explain why all plants have AOX and what its physiological role is.
5.4.2 Thermogenesis
The only well-known function of the AP is related to thermogenesis during anthesis in aroid spadi-
ces and other thermogenic plants (Meeuse 1975). During anthesis, mitochondria use AOX to respire
at very high rates. Free energy is released as heat raises tissue temperatures and volatilizes aromatic
compounds to attract pollinators. Owing to this phenomenon described in the eighteenth century by
Lamarck, the cyanide-insensitive respiration was discovered in the 1930s (Meeuse 1975, Okunuki
1932, 1939, Van Herk 1937). In nonthermogenic tissues, a general role of AOX is still intensively
discussed, and several hypotheses regarding its role have been postulated.
Nevertheless, some facts do not exclude a thermoregulatory role of AOX in nonthermogenic
tissues at low temperatures (Grabelnych et al. 2003, Moynihan et al. 1995, Vojnikov et al. 1984).
Although the heat generated by respiration via AOX did not have a pronounced effect on tissues
heating (Breidenbach et al. 1997), the respiratory heat could be enough to prevent the loss of fluidity
of mitochondrial membranes that is essential for the dynamics and functions of membrane proteins.
5.4.3 Energy Overflow: Balancing the Respiratory Metabolism

The first explanation of physiological role for AP has been suggested by Lambers (1982). According
to his “energy overflow” hypothesis, the AP has a function of the removal of an excess of sugar
that is not needed for growth and maintenance processes. The “energy overflow” hypothesis was
in accordance with the state that AOX is engaged when the CP is at or near saturation (Bahr and
Bonner 1973). After it had been accepted that both pathways compete for electrons (Day et al. 1996),
the “overflow” hypothesis was reconsidered but did not lose its rationality. It is supposed that AOX
functions as a balance regulator between carbon metabolism and electron transport (Simons and
Lambers 1999, Vanlerberghe and McIntosh 1997). The ability of the mitochondrial ETC to divert
electrons to the AOX, when CP is restricted by adenylate control, can be necessary for continua-
tion of the Krebs cycle and for the prevention of the ubiquinone pool overreduction and hence ROS
production (Millenaar and Lambers 2003, Vanlerberghe and McIntosh 1997). Such situation can be
expedient for plants or tissues during their active period of growth, when the availability of ATP
might not be as critical as the supply of carbon skeletons for biosynthesis. A positive correlation
between the activity of the AP and relative growth rate in roots of six wild monocotyledonous grass
species with different relative growth rate was found (Millenaar et al. 2001). Respiration and AP
capacity in fast-growing barley plants were significantly higher than that in slow-growing plants
(Garmash and Golovko 2011). The results suggest that growth rate of plants controls the energeti-
cally wasteful AP engagement.
“Energy overflow” hypothesis considers AP as a control of carbohydrate metabolism (Simons
and Lambers 1999). The relationship between AOX engagement and soluble carbohydrate content
has been shown in some experiments, especially in leaves with different sugar levels caused by
photosynthetic activity depending on diurnal changes or changes of growth light intensities. At the
end of night, respiratory rates in wheat and spinach leaves enhanced by exogenous sucrose or gly-
cine were sensitive to SHAM (Azcon-Bieto et al. 1983a). In spinach and bean primary leaves, the
AOX activity measured by oxygen-isotope fractionation technique was also high early during the
night when carbohydrate concentration was high, while rates were slow later during the night when
carbohydrate status was low (Noguchi et al. 2001). This suggested that AOX is engaged early during
the night, and AOX would consume a large proportion of carbohydrates, when sugar status is high.
A significantly lower AP contribution to the respiration and carbohydrate content was found in
the leaves of winter-green perennial plant Ajuga reptans grown in its natural habitats (mixed-conifer–
aspen forest) under irradiance of 5% maximum illumination compared to plants grown in an experi-
mental plot under full sunlight conditions (Golovko and Pystina 2001). However, the overwintered
leaves were not SHAM-sensitive but were characterized by high carbohydrate content. Also, soluble
carbohydrates were directly related to the AOX activity in Cucumis sativus wild-type (WT) and
MSC16 mutant plants characterized by mitochondrial genome rearrangement (Florez-Sarasa et al.
2009). Plants grown under high light intensity had a higher AOX activity and sugar content than
plants grown under low light intensity. In addition, AOX activity and soluble sugar contents were
higher in MSC16 than in WT regardless of the growing light intensity. However, AOX activity did
not correlate with the amount of AOX protein. In our experiments, there was no correlation among
6
d
O2 uptake (nmol g–1 FW s–1)

5
ac ac
b
4 a c
a
b
3 d
c
2 ec c
e
a
b
1 b
0
0 1 2 4 6 12 24 48
Time (h)
FIGURE 5.2 Capacity changes in alternative pathway (black part of columns) and cytochrome pathway
of respiration (white part of columns) in wheat (Triticum aestivum L., cv. Irgina) leaves during the green-
ing process. Time indicates hours after the start of illumination. Data are presented as mean values ±
SE (n = 4–6) taken during four independent experiments. Significant differences between mean values
(ANOVA, Duncan’s test, P < 0.05) are indicated by different letters. (Redrawn from Garmash, E.V. et al.,
Photosynthetica, 2013.)
Time of greening (h)
0 6 24
100 70 135
AOX
FIGURE 5.3 Purified mitochondria preparation immunoblots of AOX protein in wheat leaves after differ-
ent periods of de-etiolation. Twenty micrograms of mitochondrial protein were loaded per lane, separated by
SDS-PAGE, immunoblotted, and visualized by chemiluminescence. Blots were quantified by densitometry
using Quantity One 4.6.9 software (Bio-Rad Ltd.). Results are expressed relative to control; the amount of
AOX protein (reduced form, M about 32 kDa) in mitochondria isolated from etiolated leaves is set as 100.
respiratory activity, AP capacity (Figure 5.2), and AOX protein level (Figure 5.3) in leaves during
de-etiolation of wheat seedlings. Lack of correlation between carbohydrate status and respiratory
rates as well as AOX activation (Gonzàlez-Meler et al. 2001, Millenaar et al. 2000, Noguchi et al.,
2001a, Ribas-Carbo et al. 2000), and AOX protein level (Florez-Sarasa et al. 2009, Jusczcuk et al.
2007, Ribas-Carbo et al. 2000) would suggest the presence of an additional posttranslational control
of AOX protein. This regulation might be energetically more favored than AOX protein degradation
and resynthesis depending on different metabolism changes (Florez-Sarasa et al. 2009, Rasmusson
and Escobar 2007).
5.4.4 Prevention of Oxidative Stress

Another proposed function for AOX is the prevention of ROS formation. Most of the ROS produced
originates from chloroplasts and peroxisomes, but is also generated in mitochondria (Foyer and
Noctor 2003, Navrot et al. 2007). ROS can act as signaling molecules, especially in response to vari-
ous stresses (Gechev et al. 2006). On the other hand, the surplus of ROS is harmful because of their
detrimental effect on all important macromolecules: proteins, pigments, lipids, and nucleotides, and
the ability to diffuse through the cellular compartments (Sweetlove et al. 2002).
An important hypothesis that AOX may act to alleviate the formation of ROS has been proposed
(Purvis and Shewfelt 1993, Wagner and Krab 1995). When the CP is inhibited or restricted, AOX
as the terminal oxidase can accept electrons from ubiquinone and reduce molecular oxygen to water
preventing overreduction of the ubiquinone pool (Millenaar and Lambers 2003). It was shown that
a high membrane potential correlates with highly reduced ETC components, and this can stimulate
superoxide formation (Møller 2001). Respiration via AOX, as well as other nonphosphorylating
pathways (alternative type II NAD(P)H dehydrogenases, uncoupling mitochondrial proteins), can
result in uncoupled respiration, lowering the membrane potential (Millenaar and Lambers 2003,
Møller 2001, Navrot et al. 2007).
The role of AOX in lowering ROS formation in plant mitochondria was supported by the impor-
tant findings. The inhibition of AOX strongly stimulated the production of H2O2 by plant mitochon-
dria (Popov et al. 1997). Transgenic cultured tobacco cells with suppressed AOX levels accumulated
the higher level of ROS in the presence of ETC inhibitors (Maxwell et al. 1999). Moreover, the
regulation of the AOX protein synthesis in which ROS and especially H2O2 play the crucial role as
second messengers in the signal transduction pathway from the mitochondria to the nucleus was
presented (Polidoros et al. 2005, Vanlerberghe and McIntosh 1996, Wagner 1995).
ROS accumulation causes oxidative stress that is often a result of the impact of unfavorable fac-
tors. AOX stimulation (gene expression, protein level, AP activity, and AP capacity) in response
to various biotic and abiotic stresses has been revealed. AOX is shown to be induced by pathogen
infection (Amirsadeghi et al. 2007), drought (Atkin and Macherel 2009), mineral nutrient defi-
ciency (Lambers et al. 1981, Sieger et al. 2005), anoxia (Skutnik and Rychter 2009), low tempera-
tures (Fiorani et al. 2005, Grabelnych et al. 2003, Ribas-Carbo et al. 2000), high light (Dinakar
et al. 2010, Florez-Sarasa et al. 2009, Kumar et al. 2007, Yoshida et al. 2008), and other factors.
Among stress factors, the heavy metal (HM) pollution belongs to the most dangerous anthropo-
genic stresses. Excess HM of even the necessary microelements is toxic for plants. HM stimulates
formation of ROS, either by direct electron transfer involving metal cations or as a consequence
of metal-mediated inhibition of metabolic reactions (Dietz et al 1999, Sharma et al. 2011). In our
experiments, high cadmium concentrations suppressed the respiration rate of barley plants and
induced engagement of SHAM-sensitive respiration (Garmash and Golovko 2009, Garmash et al.
2011). The contribution of AP increased to 40% of the total respiration in Cd-treated roots as organs
performing barrier function. At the same time, lipid peroxidation is intensified, which is an indica-
tion of increased oxidative stress. AOX induction under Cd stress is evidently required for the pre-
vention of ROS generation and likely for the continuation of the Krebs cycle. Organic acids of the
cycle are able to bind Cd in vacuoles (Sanità di Toppi and Gabrielli 1999, Seregin and Ivanov 2001).
In other experiments, the responses of aquatic macrophyte Elodea (Egeria) densa Planch. Casp.
exposed to two concentrations of Cd were studied. Interestingly, at 100 μM Cd concentration, the strong
protective antioxidant responses have been observed (Maleva et al. 2011), but there were no changes
in the respiratory activity and pathway ratio (Table 5.1). Plants exposed to higher Cd concentration
(300 μM), on the contrary, were characterized by the decreasing antioxidant enzyme activity and by the
increasing AP capacity. The study of the effect of organic phosphorus xenobiotic—methylphosphonic
acid (MPA)—on physiological and biochemical parameters of reed canary grass (Phalaroides arun-
dinacea L. Rausch) revealed the same pattern (Maslova et al. 2010). The treatment of the aboveground
plant organs with 0.01 and 0.1 M MPA caused an oxidative stress in leaves and decreased peroxidase
activity. At the same time, the respiratory rate and the contribution of AP increased (Table 5.1). A devel-
opmental stepwise response of cellular antioxidant systems evidently occurs.
Increases in solar ultraviolet (UV) radiation (B and A spectra), reaching the ground due
to stratospheric ozone depletion layer, have a negative impact on living organisms (Caldwell
et al. 2003, Robson and Aphalo 2012). Plants respond to UV radiation by stimulating protection
mechanisms including antioxidant systems (Galatro et al. 2001). To study the role of alternative
TABLE 5.1
Influence of Heavy Metals and Methylphosphonic Acid (MPA)
on Respiration and Alternative Pathway Capacity (AP), Lipid
Peroxidation (TBARS), and Activity of Some Antioxidant Enzymes
in Leaves of Two Plant Species
Variant
Cd Concentration (μM)
Maleva et al. (2011)
Species
Elodea (Egeria) densa Planch. Casp. Control 100 300
TBARS (nmol g FW)−1 45.9 ± 2.4 37.9 ± 4.2 51.6 ± 1.9
Respiratory activity (nmol g−1 FW s−1) 3.2 ± 0.3 3.5 ± 0.5 4.1 ± 0.3
AP capacity (nmol g−1 FW s−1) 1.1 ± 0.2 1.3 ± 0.2 2.4 ± 0.2*
SOD activity (U mg−1 protein) 4.2 ± 0.1 7.7 ± 0.3* 3.7 ± 1.2
Catalase activity (mM mg−1 protein s−1) 0.17 ± 0.01 0.29 ± 0.03* 0.11 ± 0.01*
MPA Concentration (M)
Maslova et al. (2010)
Phalaroides arundinacea L. Rausch 0.01 0.1
TBARS (nmol g FW)−1 37.2 ± 2.8 44.2 ± 5.2 56.0 ± 2.4*
Respiratory activity (nmol g−1 FW s−1) 2.6 ± 0.2 2.3 ± 0.2 2.8 ± 0.2
AP capacity (nmol g−1 FW s−1) 0.6 ± 0.1 0.5 ± 0.2 1.2 ± 0.1*
Peroxidase activity (mL I2 g−1 FW) 20.1 ± 0.2 6.5 ± 0.3* 13.1 ± 0.8*
Sources: Data from Maleva, M.G. et al., Effect of the exogenous anthocyanins on pro-/
antioxidant reactions in Elodea canadensis and E. densa exposed to cadmium and
manganese, in: Abstracts of the 11th International Conference on the
Biogeochemistry of Trace Elements (ICOBTE 2011), Florence, Italy, 2011
(partially); Maslova, S.P. et al., Agrokhimia (Agrochemistry), 1, 73, 2010.
Note: Data are presented as mean values ± SE (for TBARS content, antioxidant enzymes
activity n = 3–5, for respiration n = 5–8). Significant differences between the control
and treated plants are indicated by * (P ≤ 0.05).
respiratory pathway in plant growth reactions to chronic UV radiation, Arabidopsis thaliana

plants transformed with self-AOX lines under the control of the constitutive CaMV 35S promoter
were used: AtAOX1a overexpressors (XX-2) and AtAOX1a antisense plants (AS-12) (Umbach
et al. 2005). Mutants were compared with the untransformed WT Columbia (Col-0). Three-week-
old Arabidopsis rosettes (growth stage 1.08, 8 rosette leaves; Boyes et al. 2001) were exposed to
supplemental UV radiation. The calculated daily doses of UV-A and UV-B were 38 и 0.5 kJ m−2
respectively. After 10 days, plants reached growth stage 3 (Boyes et al., 2001), when the final
number of leaves (14) had been produced. Ten days of UV acclimation showed a substantially
reduced leaf area that is likely the result of UV-mediated inhibition of cell division and expansion
of adaxial epidermis (Hectors 2010). However, leaf area in the AOX-overexpressing line after
10 days of UV treatment decreased to a lesser extent compared to the WT Col-0 and the antisense
line AS-12 (Figure 5.4a). As a result of the leaf area decrease, the plants of the mutant line XX-2
had a larger seed yield per plant than those of the line AS-12 and the WT (Figure 5.4b). Since
in these transformed plants, AOX protein level affects ROS formation when the CP is restricted
(Umbach et al. 2005), a higher AOX engagement may lead to less ROS production and hence may
maintain more optimal conditions for growth. H2O2-induced AOX expression under enhanced
UV-B radiation was revealed (Zhao et al. 2007).
a a
8
Total leaf blade area (sm2)

7 a
6
5 b
4 bc
c
3
2
1
(a) 0
a
a
40
Seed yield (mg plant–1)
ab
30 d
cd
c
20
10
0
(b) Col-0 XX2 AS-12
FIGURE 5.4 Effect of UV on leaf blade area (a) and seed yield (b) of Arabidopsis AOX transgenic plants
during the vegetative phase. Fifteen to twenty plants of each genotype in each experiment on day 31 (after
10 days of UV treatment) were measured. White bars (–UV) and black bars (+UV) represent the mean ± SE
of two replicated experiments. Significant differences between the mean values (ANOVA, Duncan’s test,
P < 0.05) are indicated by different letters. Arabidopsis mutant seeds were obtained from NASC (UK) and
kindly provided by Dr. V. Tarasenko (Siberian Institute of Plant Physiology and Biochemistry, Irkutsk, Russia)
after checking AOX-transformed plants for the presence of gene construction.
5.4.5 Maintenance of Interaction between Respiration and Other Metabolic Processes

Respiration closely interacts with other processes in a living cell and organism. Respiration rate
is controlled by different ATP-demanding processes via adenylate control. The ability of the plant
mitochondrial respiration to involve nonphosphorylating AP may have an important consequence
for plant energy metabolism. Considering the impact of AOX on the ROS network, this respiratory
pathway is thought to provide a degree of signaling homeostasis from the mitochondrion and to
control cellular response to stress (Vanlerberghe et al. 2009). All these suggest that AOX may par-
ticipate in the regulation of plant life activity.
It is known that respiration of a plant or its organ decreases with age that is connected with
decreasing the part of meristematic active tissues, the metabolic activity, and as a whole—the
growth processes (Amthor 1989, Golovko 1999, James 1953). But how the AP is involved in the
respiratory shift with tissue aging is still debated.
The idea of age-related changes in respiratory pathways was contributed by O.A. Semikhatova.
She noticed that during leaf development, different respiratory systems function in it (Semikhatova
1969). The cytochrome oxidase activity prevails in a young leaf, whereas the cyanide-insensitive
respiratory system, whose nature was unknown at that time, previals in a mature leaf. A decrease in
total respiration during leaf maturation associated with a decline in the CP activity has been shown
while the AP activity remains stable (Azcon-Bieto et al. 1983b, Florez-Sarasa et al. 2007, Priault
et al. 2007). A higher contribution of the CP to growth respiration was suggested. The activity of AP
was mostly involved in the maintenance respiration component, while the AP also plays a role for
providing optimal conditions for growth (Florez-Sarasa et al. 2007, Priault et al. 2007). Changes in
partitioning between the pathways in mature leaves during whole plant ontogenesis are also shown
(Shugaev et al. 1998).
6
2.5
O2 uptake (nmol g–1 FW s–1) 5
2.0
4
1.5
3
1.0
2
1 0.5
0 0
(a) Young Mature (b) Young Mature
FIGURE 5.5 Capacity changes in alternative pathway (black part of columns) and cytochrome pathway of
respiration (gray part of columns) in young and mature leaves of annual spring wheat (Triticum aestivum L.,
cv. Irgina) (a) and winter rye (Secale cereale L. cv. Vjatka 2) (b). White part of columns represents residual
respiration. Data are presented as mean values ± SE (n = 6–8).
The respiratory pathway ratio in mature leaves was observed to depend on the adaptive strategy
of plants. The contribution of the AP to the young leaves of spring wheat and winter rye was
30%–40% of the total O2 consumption (Figure 5.5). The respiration via AOX in winter rye mature
leaves was the same as in young leaves and 25% higher in spring wheat. Fully expanded wheat
leaves did not grow during intensive plant tillering (RGR of whole plant was equal to 0.22 d−1) and
exported assimilates to young leaves. In early autumn, the adaptive strategy of winter rye plants
during the active vegetative phase likely aimed at the maintenance of the maximal energy efficiency
of respiration. When a cold-resistant barley cultivar was grown in a controlled-environment growth
chamber under two temperature regimes (13/8°C and 21/17°C, day/night) limiting the zone of the
cultivar temperature growth optimum, the plant respiration at 13/8°C was not inhibited in the pres-
ence of SHAM—the inhibitor of AP (Garmash and Golovko 2011). In addition, the temperature
coefficient of heat production rate (Q10) of the plants grown at 13/8°C was twice less than Q10 of the
plants grown at 21/17°C (Garmash 2005). As discussed by Priault et al. (2007), plant cells possess
highly efficient control mechanisms to avoid AOX engagement when it is not necessary or deleteri-
ous to energetic metabolism.
At present, there is evidence for the importance of alternative respiration in optimizing photo-
synthesis and protection against photoinhibition (Dinakar et al. 2010, Kumar et al. 2007, Noguchi
and Yoshida 2008). We studied the developmental changes in respiration and photosynthetic rates
and thermal dissipation processes connected with chloroplasts and mitochondria activity in etiolated
wheat (Triticum aestivum L., cv. Irgina) seedlings during the greening process under continuous
light conditions (190 μmol(photon) m−2 s−1) (Garmash et al. 2013). During the greening process,
the respiratory activity of wheat seedlings and AP capacity changed dramatically at the later phase
(Figure 5.2), when most prolamellar bodies converted into thylakoids; however, photosystems were
not completely developed. The positive linear correlation between AP capacity and heat production in
greening t issues indicates the role of the AP as energy-dissipating system. The increase in AP activ-
ity closely correlated with the time course of the change in AOX gene expression during the green-
ing process in soybean cotyledons was observed earlier (Finnegan et al. 1997, Ribas-Carbo et al.
2000). Feng et al. (2007) revealed the increased AOX1c expression during the greening of etiolated
rice seedlings only after 4 h of continuous illumination. When etiolated Arabidopsis aox1a mutant
seedlings were exposed to illumination, the reduction of the photosynthetic ETC and the accumu-
lation of reducing equivalents were greater in the mutant during de-etiolation (Zhang et al. 2010).
All these s uggest that AOX activation in light during greening is associated with the oxidation of
NADH, which could be imported to mitochondria from chloroplasts via malate/oxaloacetate shuttle
(Padmasree and Raghavendra 1999). The increase in mitochondria’s adjacent location to chloroplasts
VXC (%) 0 23 95 100 90 88 83

0.7
d
d
0.6 c
bc
0.5 b
b
qN 0.4 a
0.3
0.2
0.1
0
1 2 4 6 12 24 48
Time (h)
FIGURE 5.6 Nonphotochemical quenching of chlorophyll fluorescence (qN) and relative level of violax-
anthin cycle (VXC) conversion in wheat (Triticum aestivum L., cv. Irgina) leaves during greening. Data are
presented as mean values ± SE (for qN n = 5–12, for VXC pigment content n = 3) taken during three inde-
pendent experiments. “-”—data are absent. Significant differences between mean values (ANOVA, Duncan’s
test, P < 0.05) are indicated by different letters. (Redrawn from Garmash, E.V. et al., Photosynthetica, 2013.)
during the wheat seedlings’ greening (Garmash et al. 2013) indicated possible metabolite exchange
between these organelles. The AP activation was accompanied by the increasing processes of non-
photochemical quenching of chlorophyll fluorescence and the activity of violaxanthin cycle in devel-
oping chloroplasts (Figure 5.6). Our results show that the induction of the energy-dissipating systems
in mitochondria and chloroplasts is regulated by the level of photosynthetic machinery development
and light stress susceptibility of greening leaves, in general.
5.4.6 “Antiapoptotic Protein”
Programmed cell death (PCD) is a genetically regulated process of cell suicide, which accomplishes
the central role in development, homeostasis, and integrity of multicellular (eukaryotic) organisms
(Vianello et al. 2007). Mitochondria are shown to take an active role in PCD pathways of both ani-
mals (Green and Reed 1998) and plants (Lam et al. 2001, Vanlerberghe et al. 2002, Vianello et al.
2007). One of the main hallmarks of PCD is the release of cytochrome c from mitochondria, which
precedes the appearance of the nonreversible PCD changes (blebbing, cell shrinkage, nuclear and
DNA fragmentation, and formation of apoptotic bodies). ROS acts as direct and indirect signals
interacting with other signaling pathways (NO-, salicylic acid-, and ethylene-mediated pathways)
leading to PCD (Overmyer et al. 2003). Regulation of the mitochondrial pathway inducing PCD
involves mechanisms controlling ROS formation and scavenging. There is evidence that the elec-
tron transport via AOX lowers ROS accumulation and sustains mitochondrial respiration when the
CP is restricted by inhibitors or death-inducing compounds, and hence influences PCD progression
(Maréchal and Baldan 2002, Robson and Vanlerberghe 2002, Vanlerberghe et al. 2002, Yip and
Vanlerberghe 2001). The activation of the AOX can be considered as one of the mechanisms con-
trolling homeostasis and preventing self-destruction of the cell (Vianello et al. 2007). Therefore,
AOX is called “antiapoptotic protein” and “survival protein” (Maréchal and Baldan 2002, Robson
and Vanlerberghe 2002).
5.5 CONCLUDING REMARKS
Thus, the nonphosphorylating alternative respiratory pathway can be considered as a mecha-
nism supporting cellular homeostasis and functioning of a plant under varying conditions. AOX
may increase adaptive potential opportunities of a plant organism, provide maintenance of
optimal conditions for growth, and protect against stress. Evidence relating to function for the
AOX in heat generation of specialized thermogenic tissues, the maintenance of redox balance
during mitochondrial electron transport, and avoiding the formation of ROS especially during
period of stress is convincing. The role of AOX in growth rate optimization, maintenance of
relationship between photosynthesis and respiration, protection against photooxidation, and
progression of PCD is intensively investigated. Applying molecular–physiological approach,
the understanding of the details of AOX metabolism, regulation, and signaling is constantly
improved. Analysis of current information shows that AOX engagement varies depending on
the physiological status and metabolism plasticity of a plant, its developmental stage, duration,
and extent of stress impact.
The research of the AP has already left the framework of just a theoretical problem. Changes
in respiration and respiratory pathway ratio may be used for evolutionary investigations and future
projections of plant adaptation to global climate change. It is suggested that mitochondrial energy
coupling and AP-mediated responses of respiration to changes in atmospheric CO2 enhanced sur-
vival of plants at low CO2 levels during the Pleistocene to help overcome a low carbon balance
(Gonzàlez-Meler et al. 2009). Selection experiment with Arabidopsis thaliana revealed that respira-
tion of plants selected at elevated CO2 was less coupled with ATP production than in current and
low-CO2 plants. An important step on the way to counteract human mitochondrial respiratory chain
defects was made by successful engineering of the AOX gene from urochordate Ciona intestinalis
(AOX was also found in some animals but not in arthropods or vertebrates) for expression in mam-
malian mitochondria (Dassa et al. 2009, Rustin and Jacobs 2009). It is established that the expres-
sion of AOX rescues cells with the mitochondrial phosphorylation dysfunction from this stressful
condition.
Intensive obtaining of evidence about the functioning of this unique respiratory pathway in plants
on one hand makes us closer to a solution of the problem of AOX activation in vitro, in vivo, and in
situ, but on the other hand, it sets new questions for researchers. Knowing the laws of respiratory
pathway ratio regulation, it is possible to regulate life activity not only in plants, but also in humans.
There will be a lot of discoveries in this field.
ACKNOWLEDGMENTS
I am grateful to my scientific supervisor Prof. T.K. Golovko, who has inspired me to study the
alternative respiration. This work is supported by the Program of the Ural Branch of the Russian
Academy of Sciences, project No 12-Y-4-1008.
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6 Growth Orientation of
Underground Shoots
Stolons and Rhizomes and
Aboveground Creeping Shoots
in Perennial Herbaceous Plants
Alexander M. Markarov* and Tamara K. Golovko
CONTENTS
6.1 Introduction........................................................................................................................... 157
6.2 Evolution Aspects of Diatropism........................................................................................... 158
6.3 Geodiagravitropism of Perennial Herbaceous Plant Shoots.................................................. 159
6.3.1 Decapitation of Aboveground Shoots and Various Photoperiods
Do Not Change Rhizome and Stolon Growth Orientation........................................ 160
6.3.2 Effect of Light on Rhizome and Stolon Growth Orientation.................................... 161
6.3.3 Sarment Growth Orientation and Development........................................................ 162
6.3.4 Light Control of Diatropic Growth Orientation of Detached Stolons....................... 162
6.4 Potato Stolon Diageotropic Growth Orientation and Morphophysiological Changes
of Stolons at Their Transition into Tuber............................................................................... 162
6.5 Epigeodiagravitropism of Perennial Herbaceous Plant Shoots............................................. 163
6.6 Summary and Conclusion...................................................................................................... 164
References....................................................................................................................................... 165
6.1 INTRODUCTION
Plant organism is an entire system of special organs. The entirety of plant organism is ensured by
interaction among its parts and by the presence of dominating centers located in shoot apex, root
tip, etc. (Polevoi and Salamatova 1991). But within the entire plant organism, plant organs remain
relatively independent and have specific physiological–biochemical processes.
A perfect example for the earlier-said testament is tropisms, that is, movements of the attached
organism or its organs caused by unilateral irritation (Hart 1990). When tropism is positive, organ
moves toward irritation source, and against it when tropism is negative. In general, plant shoots are
negatively gravitropic and positively phototropic. They grow vertically against the force of gravity
toward the light. In case of root, gravi- and phototropisms have a diverse direction as compared to
shoots. There are, however, numerous exceptions to this rule such as strawberry stem, for instance.
If movement is directed at a certain angle to irritation source, this is plagiotropism. Movement at
right angle (90°) is called diatropism. The tropism phenomenon is based on differential growth.
Tropism reactions usually involve growth via elongation, much more rarely via cell division.
* Deceased (August 12, 1935–June 4, 2013).
157
Growth orientations of axial organs compensate for their sessile nature and make it possible for
the plant to optimize its position in space for better usage of environmental resources (Smith 1982,
Esmon et al. 2005).
Shoot tropisms are believed to be related to redistribution of hormones—auxins (Evans 1992,
Lomax et al. 1995, Chen et al. 1999). Shoot apex senses exterior stimulus and redistributes auxins.
Concentration of auxins increases at shady side and so provides for intensive cell growth in subapi-
cal zone. Growth via elongation is typical more of shady side cells and less of daylight side cells.
Consequently, shoot apex bends toward light (phototropism). By the auxin hypothesis, after gravit-
ropic stimulation, higher plants demonstrate auxins to be redistributed to the physically low side. If
shoot has changed its position in space (bent, windfallen, etc.), auxins redistribute to the physically
low side. Subapical zone tries to recover vertical growth (negative gravi- or geotropism).
In nature, diverse herbaceous perennial plants are divided into species with underground hori-
zontally growing (hypogeodiagravitropic) shoots as stolons and rhizomes and into creeping grasses
with aboveground horizontally growing (epigeodiagravitropic) shoots as stolons, sarments, and fla-
gella. Horizontally growing rhizomes and stolons are usually side metamers of basal part at the
leading aboveground orthotropic shoot (Markarov 1996).
This chapter covers some physiological aspects of the growth orientation of the under- and
aboveground diatropic shoots. Evolution aspects of diatropism are also discussed.
6.2 EVOLUTION ASPECTS OF DIATROPISM

Comparing representatives of different phylogenetic lines and taxa by morphostructure shows that
diatropism effect as a cell, organ, or organism property appeared in the ancient times and still exists
(Markarov 1996; Markarov and Golovko 2007). Physiological mechanism of diagravitropism—
growth of plant part transversely to the Earth’s gravitation centerline—is possibly among the oldest
mechanisms of growth orientation. A pronounced horizontal growth orientation of plant part is
observed in the subkingdom of lower plants (thalloid) in case of representatives of the Chlorophyta
order, the Chaetophoraceae family. The Fritschiella tuberosa alga adapted to exist in soil has an
underground thallus (horizontal cell row) with hypogeodiatropic growth (Vinogradova 1978). From
thallus up to the soil surface, the alga forms vertical cell rows. It can be considered a prototype of
land plants with hypogeodiagravitropically functioning organism parts. Important is that diatro-
pism as an oriented growth process can localize either in apical cell (e.g., at Fritschiella tuberose)
or in apical body part (as in the case with Caulerpa prolifera, another alga from the same family).
Higher plants are considered to be descended from rhyniophytes (Takhtadzhian 1964, 1978a).
There are fossil remains of Rhynia major. Gametophyte of this plant was underground, and sporo-
phyte had a clear underground horizontal part (rhizomoid) that branched dichotomically. Telome-
stems rose upward from rhizomoid to soil surface. Rhyniophytes were poorly adapted to exist in air
medium (on dry land) because they had imperfect stele. Transition from water to land could happen
only in case of the development of various adaptation mechanisms.
Morphophysiological body differentiation is an adaptation mechanism providing for growth ori-
entation in the environment. Information about rhyniophytes has allowed for evolutionary origina-
tion succession of plant organs and to prove that among other plant organs stem was first formed
(Takhtadzhian 1964). In case with higher plants, hypogeodiatropism is an evolutionary ancient fea-
ture of plant organ formed in need of adaptation.
Fossil lycopsids had orthotropic aboveground shoots growing from underground horizontal rhi-
zomes (Snigirevskaia 1978). The ancient Lycopsida sample Asteroxylon (Asteroxylaceae family)
that died 400 million years ago possessed stems growing from underground plant part that was
a horizontally prostrate and dichotomically branching rhizomoid. Hypogeodiatropic rhizomoid
grew from apex. Consequently, the apical rhizomoid part of fossil lycopsids had the mechanisms of
underground orientation and diatropic growth.
Growth Orientation of Underground Shoots 159
Horsetail plants also have an expressed underground horizontal rhizome. For plants of the
Equisetum genus, it is typical that formation of specimen from germ is accompanied by the devel-
opment of side shoot from initial shoot’s basal node (Filin 1978). Third and other shoots are formed
at second shoot’s base. Their apical part bends and deepens into soil with transition to horizontal
position and, then, forms rhizome under soil surface. Thus, ancient representatives of higher plants,
that is, horsetails, provide a sample of evident negative photo- and positive geotropism.
There are numerous samples of hypogeodiatropic rhizome orientation in case of grassy pteroid
plants (Takhtadzhian 1978b). The cosmopolite species Pteridium aquilinum (Cyatheaceae family)
annually has two buds formed on apical rhizome part. One of them (parent bud), the so-called
frond, continues horizontal growing under soil surface without leaves, and the other (child bud)
develops a short horizontal rhizome branch. Fronds are formed simultaneously with next branch
development at short side part and appear at a significant distance (more than 3 m) from apical zone
of leading hypogeodiatropic rhizome.
In phylogenetic aspect, it is interesting that grassy forms of angiosperms have hypogeodiatropic
shoots. According to Takhtadzhian (1964), grassy forms of flowering plants appeared independently
in different ways and lines and at different evolutionary levels. Environmental conditions were also
different. According to Khokhriakov (1981), angiosperms have an evolutionary row of living forms
as dense bunch (tussock), short rhizome, and long rhizome. This row of living forms is predeter-
mined by climatic, edaphic, and cenotic conditions.
So in spite of different environmental conditions and various transformation routes of grassy
perennials, numerous plant species have hypogeodiatropic underground axial organ. The concerned
plant group became passively (possibly under certain conditions) hypogeodiatropic at early evo-
lution stages. This passive property became active due to adaptation needs. To exist under soil
surface, shoot needs assimilates. Underground existence is required to form and conserve areas of
meristems under soil surface. In case of hypogeodiatropic growth and sympodial branching, shoot
steadily forms photophilous aboveground sympodia (shoots) whose principal function is synthesis
of assimilates and generative reproduction (in certain conditions).
Along with the phylogenic series of living forms’ transformation “trees–shrubs–long–and–
short-rhizomatous grasses,” there exists another series “trees–shrubs–creeping grasses” among
angiosperms. The epigeodiagravitropicity as a shoot property of the living form creeping grasses
appeared long ago and was characteristic of ancient representatives of the plant kingdom (Markarov
and Golovko 2007). Ancestors of plants with aboveground horizontal (epigeogravitropic) growth of
body axis are lower plants of the Chlorophyta order. The water alga Caulerpa prolifera has a hori-
zontal shoot on the bottom of water body. In the subkingdom of higher plants, the Lycopodiophyta
specimens possess an epigeodiatropic body part (sporophyte).
Thus, the plant kingdom has three independent physiologically different growth orientation types
of shoot organs, which have been formed historically in various phylogenetic lines as (1) orthogravi-
tropic (vertical and parallel to the Earth’s gravitation axis); (2) hypogeodiagravitropic (underground,
horizontal, and perpendicular to gravitation axis); and (3) epigeodiagravitropic (aboveground, horizon-
tal, and perpendicular to gravitation axis). As a rule, horizontally growing shoots are side metamers of
basal part of the leading aboveground orthotropic shoot. At present, physiological mechanism of dia-
gravitropism, which seems to be one of the oldest growth orientation mechanisms, is still understudied.
6.3 GEODIAGRAVITROPISM OF PERENNIAL HERBACEOUS PLANT SHOOTS

The species of perennial herbaceous plants that produce underground diatropic shoots, rhizomes,
and stolons are widely distributed in the plant kingdom. They are actively used for practical pur-
poses (for hayland and grassland rehabilitation, land recultivation, phytoremediation, as sources of
food and medicine, etc.). Rhizomatous and stoloniferous species are characterized by their rela-
tively high productivity and tolerance to environmental stress factors. Rhizomes and stolons of
perennial herbaceous plants are organs of vegetative propagation, dissemination, and expansion.
Underground shoots provide successful wintering and early regrowth of plants in spring.
6.3.1 Decapitation of Aboveground Shoots and Various Photoperiods

Do Not Change Rhizome and Stolon Growth Orientation
Plant organism is built up by a modular principle. Modules are phytomers that are formed by
apical shoot and root meristems. Shoot phytomer includes node, internode, auxiliary bud, and leaf.
Phytomers are not fully independent and are under whole organism control. A bright sample of
systemic development is apical domination, which is observed in developmental inhibition of aux-
iliary bud by apical bud at apical zone of orthotropic shoot (Polevoi and Salamatova 1991). Shoot
apex removal usually results in the rapid growth of lateral buds. Their hormonal status changes
significantly. ABA concentration decreases, and IAA and cytokinin activity increases (Cline 1991).
Phytohormone and assimilate transport to the lateral buds is activated as a result of connection
appearance between the conducting system of the orthotropic shoot and its lateral buds. Finally, new
correlations between the buds and other organs are established. Many morphogenetic and physi-
ological processes in shoots and roots are determined by the shoot apex.
Wareing and Phillips (1981) have found that the apical bud of the aboveground potato shoot con-
trols the growth orientation of lateral shoots. With this in mind, they supposed that apical bud controls
growth orientation in the underground shoots (stolons) as well. Some other authors (Ewing 1985,
Cline 1991) also carried out the experiments with aboveground shoots for studying the apical and
lateral bud interaction mechanisms and developed a concept of the apical bud as an organizing cen-
ter at the whole plant level (Polevoi 1984, Polevoi and Salamatova 1991).
We put forward and experimentally tested a hypothesis that rhizomes and stolons are autono-
mous (i.e., independent of the aboveground orthotropic shoot) in their diatropic growth orienta-
tion (Markarov and Golovko 1995a). For these purposes, the responses of underground shoots to
decapitation of aboveground shoots were studied in some perennial herbaceous plants (milfoil,
meadow vetchling, and couch grass) and three Solanum species (S. demissum, S. cardenazii, and
S. tuberosum). The shoot apex of the aboveground stem was removed (2–5 cm).
The experiments with plant species producing rhizomes and stolons have shown that aboveg-
round shoot apical dominance does not spread to the underground metameric complex (Markarov
1996, Golovko et al. 2004). The growth orientation of the underground shoots of second—fifth basal
metamers does not change after the decapitation of orthotropic shoots or removal of the greater part
of aboveground shoot. Furthermore, in darkness, the linear diatropic growth continues in the sto-
lons cut off from the mother plant.
The day length is the most important factor determining the growth and developmental pattern
in many plant species. It is assumed that photoperiod time length can induce stolon transformation
into aboveground shoot. Our experiments with the photoperiod-sensitive species S. demissum have
not detected any changes in the underground shoot growth orientation in response to variation in
day length (Markarov and Golovko 1995a). Growth orientation remains the same in the decapitated
plants under various photoperiodic regimes. We have shown that the activity of cytokinins and ABA
increases and that of gibberellins decreases in the leaves of photoperiod-sensitive potato species
exposed to a shorter photoperiod. The changes in leaf hormonal balance do not affect stolon growth
orientation, but result in a greater underground biomass due to the tuber formation. The stolon api-
cal part gains the capacity to be transformed into aboveground shoot only after tuber formation. The
tuber apical bud produces aboveground shoot after the dormancy release.
So, the perennial rhizomatous and stoloniferous plants appear to have two metameric com-
plexes: aboveground orthotropic shoots with apical zone as dominant and growth-regulating cen-
ter, and underground shoots with autonomic growth orientation, independent of the aboveground
shoot apex. The removal of apical dominance by decapitation or excision of the greater part of
aboveground orthotropic shoot does not induce transformation of the underground diatropic shoots
into orthotropic shoots. The day length also does not affect growth orientation in the underground
shoots. Therefore, one can conclude that the aboveground part of perennial herbaceous plant does
not control diatropism in the underground shoots.
6.3.2 Effect of Light on Rhizome and Stolon Growth Orientation

Light is critical for plants because they depend on it as a source of energy and information. Besides pho-
tosynthesis, plant morphogenesis is also a light-dependent process. Photomorphogenesis is a process of
light control over organism development and is typical of most plants. Light signals provide information
of crucial ecological value at many developmental stages (Smith 1982). Light signal perception may be
thought of as the means by which the plant determines where it is in space and time (Neff et al. 2000).
The three classes of plant signal-transducing photoreceptors—phytochromes, cryptochromes,
and phototropin—have characterized molecular details and function (Ahmad and Cashomore 1996,
Batschauer 1999, Neff et al. 2000, Christie 2007).
The phytochromes (Phy) are signal-transducing photoreceptors that convert between inactive
and active forms in response to different wavelengths of light. This conversion is used to syn-
chronize plant development to exigencies of the light environment (Smith 1995). Phytochrome-like
genes occur not only in all green plants, including gymnosperms, ferns, mosses, and algae, but also
in cyanobacteria and even in certain other bacteria (Smith 2000).
In addition to phytochromes, which are involved in the perception of red (R) and far-red (FR) light,
two other photoreceptors, cryptochromes and phototropins, have been identified, which are associated
with ultraviolet-A wavelengths (Briggs and Olney 2001, Quail 2002, Christie 2007). Phytochromes
A–E are R- and FR-light photoreceptors that secondarily also absorb blue light. In higher plants, unilat-
eral red light induces weak positive phototropism in the roots of Arabidopsis (Ruppel et al. 2001, Kiss
et al. 2003). Whereas shoots bend toward the direction of incoming blue light, improving the chances
of light-harvesting organs to collect light for photosynthesis, roots bend away from the direction of
incoming blue light stimuli, avoiding the stressful conditions of upper soil layers (Esmon et al. 2005).
Depending on soil type, light can penetrate to a depth from 1 to 3–5 cm (Tester and Morris 1987).
Phytochrome is known to regulate the transcription of numerous genes. The stimulation and
repression of transcription by light can be very rapid, with a lag time of few minutes. Such early
gene expression is likely to be regulated by direct activation of transcription factors by one or more
phytochrome-initiated signal transduction pathways. The activated transcription factors then enter
the nucleus, where they stimulate the transcription of specific genes.
Phytochrome is now known to be a protein kinase. Kinases are enzymes that have the capacity to
transfer phosphate groups from ATP to amino acids such as serine or tyrosine, either on themselves
or on other proteins. Kinases are often found in signal transduction pathways in which the addition
or removal of phosphate groups regulates enzyme activity. Higher plants’ phytochromes are serine/
threonine kinases. It is shown that unilateral blue light enhances phytochrome kinase substrate 1
(PKS1), a plasma membrane–associated protein, expression in the subapical region of Arabidopsis root
(Boccalandro et al. 2008). The negative phototropism and the enhanced PKS1 expression in response
to blue light require Phy A.
Another protein kinase associated with phytochrome is nucleoside diphosphate kinase 2 (NDPK 2).
Phy A has been found to interact with this protein, and its kinase activity is increased about twofold
when Phy A is bound in the PhyR form.
We studied the phytochrome-dependent growth responses of rhizomes and stolons to reveal the
role of phytochrome in controlling underground diatropic growth (Markarov and Golovko 1995b).
For this purpose, phototropism of underground shoots was investigated in response to irradiation
with R (600–680 nm) and FR light (710–780 nm). The apical zone (1–2 cm) of underground shoots
was irradiated with 1 W/m2 of R or FR light during 5 min every night for 2 weeks. Irradiation of
apical zone of Mentha piperita and Achillea millefolium rhizomes with FR did not change its dia-
tropic growth orientation. Red light induced a negative phototropic response that resulted in apex
curvature toward soil. A similar phototropic response was observed in the rhizomes irradiated with
natural (white) light. This response was reversible if R treatment was followed by FR. Phytochrome-
controlled growth responses were also revealed in stolons of Solanum species and Oxalis tuberosa.
Irradiation of the stolon apical zone with FR did not affect its diatropic growth pattern. Light treat-
ment with R after FR induced a negative phototropic response.
So, it was shown that irradiation with R or white light induced a negative phototropic response in
the apical zones of rhizomes and stolons, and FR reversed the R effect. Therefore, one can assume
that PhyR supports plagiotropic growth of rhizomes and stolons under soil surface, and PhyFR pre-
vents the shoot apex appearance.
6.3.3 Sarment Growth Orientation and Development

Many herbaceous perennials produce sarments, shoots that are formed by buds of above- or under-
ground shoots. Unlike rhizomes and stolons, underground shoots if they produce sarments are capa-
ble of changing diatropic (horizontal) growth to orthotropic (vertical) growth without any period of
dormancy (Markarov 1996, Maslova et al. 2006). We have studied morphogenesis of sarment and
effects of light on its growth orientation and development (Markarov and Golovko 1995c). In the
potato seedlings grown from seeds, sarments originate from second-order buds, and stolons develop
from third-order buds in the first–fourth nodes of the basal part of the orthotropic shoot. Unlike
rhizomes and stolons, sarments’ apex differentiation already occurs in current growing season.
Sarments experience diatropic growth under soil surface while their apexes are undifferentiated.
Differentiation results in the formation of leaf primordia that has determined the appearance of the
photophilous response of the apical zone. Sarments lose their negative phototropic reaction from
this time, and their orthotropic growth starts. Aboveground shoots rise from the apical part, and the
formation of juvenile plant occurs within current growth season. The rhizomes and stolons are able
to transform into aboveground shoots after the period of dormancy.
Experiments with the irradiation of the sarment apical zone with R and FR have shown that their
negative phototropic reaction during the period of underground growth is mediated by phytochrome
system. Phytochrome control of diatropic growth orientation is effective till the beginning of the
formation of leaf primordia.
6.3.4 Light Control of Diatropic Growth Orientation of Detached Stolons

The stolons excised from the maternal plants (O. tuberosa, S. cardenasii, and other Solanum species)
are capable of changing apex growth orientation in response to light (Markarov and Golovko 1995d,
Markarov et al. 2001). White and red light induces a negative phototropic curvature of the excised
stolon, like intact ones. In darkness and after interruption of dark period with FR, the excised sto-
lons demonstrate diatropic growth orientation.
The preliminary treatment of the orthotropic shoot of maternal plant by hormones (IAA+GA or
IAA+ABA) has no effect on the detached stolon growth orientation. In contrast to stolons, the lateral buds
on cuttings from the aboveground orthotropic shoots produce new shoots with changed growth orientation
in response to hormonal application (Markarov and Golovko 1995c). No changes in IAA, ABA, and GA
activity in the apex of excided stolons irradiated with R or FR are revealed. It is possible to suggest that
phytochrome control of diatropic growth orientation is not related to changes in hormone activity.
6.4 POTATO STOLON DIAGEOTROPIC GROWTH ORIENTATION

AND MORPHOPHYSIOLOGICAL CHANGES OF
STOLONS AT THEIR TRANSITION INTO TUBER
The structure of mature potato plant is represented by two shoot systems—overground (orthotropic)
and underground (hypogeodiatropic) shoots. Stolons are lateral shoots formed by buds on the basal
nodes of orthotropic shoot under soil surface. The underground shoot-forming buds initiate at the
second stage of organogenesis (Markarov et al. 2001, Markarov and Sverdlova 2007). During bud
initiation, vegetation cone of monocarpic orthotropic shoot has from two to five leaf primordia on
two—five nodes of metamers. Lateral shoot buds of orthotropic shoot overground part are initiated
after the buds form underground lateral shoots—stolons. Many tuber-forming species are able to
form sarments, underground shoots with diatropic–orthotropic growth orientation. Sarments distin-
guish from stolons by morphological properties (Markarov and Golovko 1995c). The apical part of
stolon reduces leaves, giving curved shape to stolon. During stolon development, apical zones stay
undifferentiated. Apical part of a sarment during a certain period of underground diatropic growth
forms photophilous leaf primordia. Most potato cultivars are devoid of sarments, which stop the
development of secondary auxiliary buds.
As genetically determined organ-forming process, tuber formation manifests itself at certain
stage of organogenesis. The volume (radial) growth in subapical zone of a stolon starts at stage VI of
organogenesis (Markarov et al. 2001, Golovko et al. 2004). During this stage, generative sphere of
the main orthotropic shoots undergoes processes of micro- and macrosporogenesis. Linear growth
of potato stolons ceases with transition to radial growth.
Radial expansion of stolon subapical zone is the result of intensive periclinal divisions of cells
belonging to perimedulla zone of the pith, and to pericycle and cambium. On the whole, dividing
cells of perimedullar zone mainly contribute to stolon swelling.
Expansion of tissues in subapical zone of stolon when tuber development begins is a complex
process including hormones. In the North, under natural long-day conditions, treatment with exo-
genic ABA intensifies tuber formation in a quantitative short-day species S. andigenum and weakly
affects tuber formation in neutral species S. cardenasii and S. phureja (Tabalenkova et al. 1998).
Treatment with ABA and cytokinins of qualitative short-day species S. demissum at short-day con-
ditions strengthens tuber formation. At long day, these hormones are unable to induce tuberization.
Consequently, exogenous hormones do not induce tuberization but only strengthen tuber formation
in inducing conditions. Only short day is able to induce tuberization in species being absolute in
their photoperiodic response.
6.5 EPIGEODIAGRAVITROPISM OF PERENNIAL HERBACEOUS PLANT SHOOTS

The Cucurbitaceae representatives (cucumber, watermelon, and cucurbit) have the leading shoot
growing horizontally on soil surface. But if we use any supporting device and put it into vertical posi-
tion, then it will grow orthotropically and would not change its position to horizontal one. In other
words, the leading shoot of the Cucurbitaceae family representatives is characterized by positive pho-
totropism and negative gravitropism. There is a question if shoots of aboveground-creeping grassy
perennials (e.g., Ranunculus repens) have tropisms that usually characterize shoot-like organs.
The living form aboveground-creeping grassy perennial plant is presented in different taxo-
nomic and ecological groups. In phylogenetic aspect, this living form is a chain in the transforma-
tion series from tree to annual grasses and has been formed as a result of ecological–morphological
adaptation changes.
A possible model to study growth reaction of creeping shoot is samples of creeping buttercup
(R. repens), creeping five-finger (Potentilla anserina), alehoof (Glechoma hederacea), and north-
ern twinflower (Linnaea borealis). In case of alehoof and twinflower, the leading shoot axis grows
horizontally to soil surface. As for buttercup and five-finger, creeping shoots are specialized into
aboveground stolons. Aboveground stolons of buttercup grow quicker than those of five-finger.
In experiments with wild plants, reposition of horizontal creeping shoot with support device
at different angle to soil surface (from 10° to 130°) was found not to affect grow orientation of
apex; it retained its horizontal position (Markarov 1996). Apex of creeping shoots continued hor-
izontal growth independently of photoperiod duration change or the complete absence of light.
Consequently, light is not a factor required for creeping shoot to fulfill epigeodiatropism reaction.
Nevertheless, diagravitropism reaction rate increased in light and decreased in darkness.
Within creeping shoot, there are zones with different morphophysiological reaction to light and
gravitation. Apical shoot part does not react to unilateral action of light and gravitation force, that is,
does not show positive phototropism and negative gravitropism signs. Side leaves and shoots are posi-
tively phototropic and negatively gravitropic. Differences in reaction are quite understandable from
the point of strategy of the living form creeping plant, which is vegetative expansion on soil surface.
The constancy of epigeodiagravitropic reaction cases in apical plant zone is evidenced by the
experimental results on replacement of creeping shoots under soil surface. If buried in soil, creep-
ing buttercup apex grows upward to soil surface and finally reaches aboveground position (negative
phototropism). Having left soil, apical plant zone renews epigeodiagravitropic growth. If the leading
orthotropic shoot is repositioned horizontally above soil surface (35–40 cm), it forms side shoots
that grow toward soil surface (positive gravitropism) and at a distance of 10–15 cm from soil surface
started diatropic growth.
Apex of creeping shoot does not react to hormones; treatment with GA and IAA does not change
growth orientation of apical plant part. Side shoots elongate internodes, which is a typical reaction
against GA. Apical zone of creeping shoot seems to possess the correction mechanisms of position
in space to maintain epigeodiagravitropic growth on soil surface. Apex of creeping shoot does not
show phototropism signs, does not have a typical reaction against GA, and steadily maintains hori-
zontal growth above soil surface. It allows for a thesis that the plant kingdom has a specific growth
movement and shoot’s orientation type, the so-called epigeodiagravitropism.
For development and establishment of the living form aboveground-creeping herbaceous plant,
changes that cause formation of neutral reaction of apical zone of creeping shoot to light and gravi-
tation are of decisive importance. Light is not an obvious factor for horizontal orientation of creep-
ing shoot. However, light strengthens the correction process into horizontal position provided that
the apical part is not in this position.
6.6 SUMMARY AND CONCLUSION

Transition of plant industry to intensive technologies, developing methods of growing isolated cells
and tissues in culture, microclonal reproduction, and other biotechnologies is based upon the knowl-
edge on basic principles of plant growth and development. That is why the study of regularities
and mechanisms of growth regulation is of a great theoretical and implied importance. In nature,
diverse herbaceous perennials also include plant species with underground horizontally growing
(hypogeodiagravitropic) shoots as stolons and rhizomes, as well as creeping plants with aboveg-
round horizontally growing (epigeodiagravitropic) shoots as stolons, sarments, and flagella.
Horizontally growing rhizomes and stolons are side metamers of basal part of the leading
aboveground orthotropic shoot. The underground shoot-forming buds initiate at the second stage
of organogenesis. Type and morphophysiology of underground shoot is determined at early plant
ontogenesis stages. Growth orientation of underground shoot does not depend on photoperiod and
photoperiodic reaction of aboveground plant part. Apical part of underground shoots of grassy
perennials reacts to light and shows a reaction of negative phototropism when treated with RL low-
energy irradiation. Negative phototropic reaction to R light is neutralized with FR-light irradiation.
This provides evidence that negative phototropism of underground shoots as rhizomes, stolons, and
sarments is regulated by phytochrome. Phytochrome in PhyR form maintains diagravitropic growth
of underground shoot thereby neutralizing growth reaction of negative gravitropism. Phytochrome
in PhyFR form prevents appearance of apical underground shoot part on soil surface. Regulatory
functions of phytochrome depend on the organ-forming processes of underground shoot apex grow-
ing point. Phytochrome control of underground shoot diatropism removes as soon as the growing
point of underground shoot starts forming primordia of photosynthesizing leaves. In soil, apical
part of horizontal underground shoot fulfills a transition from diagravitropic to orthotropic growth,
whereas photophilous bud becomes aboveground and forms orthotropic aboveground shoot.
Creeping plants have a specific growth movement and shoot orientation type, the so-called epi-
geodiagravitropism, which is characterized by the neutral reaction of creeping shoot apical part to
light and gravitation, positive tropism to soil surface, and horizontal growth on soil surface.
Diagravitropic growth orientation is a principal physiological reaction of rhizomes, stolons, and
aboveground creeping shoots of perennial herbaceous plants. But our information on the shoots’
diagravitropic growth maintaining mechanisms is very limited. In this relation, growth diatro-
pism of above- and underground shoots is to be studied using complex methods and approaches of
structural biology, ecology, physiology, and molecular biology. Identification of physiological and
molecular-genetic mechanisms of diagravitropism in rhizomes, stolons, and aboveground creeping
shoots of herbaceous perennials will form a scientific basis for targeted growth and organ-formation
regulation, improving the application efficiency of plants and development of ecologically favorable
methods of weed controlling.
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7 Structure and Metabolism
of Underground Shoots
in Perennial Rhizome-
Forming Plants
Svetlana P. Maslova
CONTENTS
7.1 Introduction........................................................................................................................... 167
7.2 Growth and Morphogenesis of Underground Shoots in Rhizome-Forming Plants.............. 168
7.3 Metabolic Activity and Energy Balance of Rhizomes.......................................................... 170
7.4 Phytohormone and Trophic Growth Regulation of Underground Shoots............................. 172
7.5 Underground Shoot Complex in Source–Sink System of Rhizome-Forming Plants............ 174
7.6 Summary and Conclusion...................................................................................................... 176
References....................................................................................................................................... 176
7.1 INTRODUCTION
The realization of the plant life strategy is related to the formation of protective and adaptive
mechanisms that are responsible for the functional plasticity and efficient usage of environmental
resources (Bazzaz 1996; Larcher 2003). Taking this into consideration, rhizomatous plant species
are of special interest. These plant species easily grow in different botanic–geographic zones. The
formation of plant rhizomes is an important adaptive sign, which has been formed during the evolu-
tion and is nothing else but the adaptation to the cold and dry climates. Rhizomes provide vegetative
reproduction, dissemination, and overwintering of perennial grasses. The underground shoot com-
plex forms the vegetative meristematic fund of buds and is characterized by its own mechanisms
of growth regulation and growth orientation (Markarov and Golovko 1995; Maslova 2001; Maslova
et al. 2005).
At present, the ontogenetic and ecologic growth and developmental regulation of underground
shoots in rhizome-forming plants are not sufficiently studied (Pearce et al. 1995; Taylor et al. 1995;
Belynskaya et al. 1997; Kondrat’eva 2000). The relationship between the structure and the func-
tional activity of the rhizome during the morphogenesis is not clear. Knowledge on the growth,
metabolic activity, and overwintering regulation of underground shoots is poor. The problem of the
seasonal growth dynamics and metabolism changes of underground shoots is especially important,
as well as the one of the adaptive reactions of rhizomatous plants to different temperatures. These
questions are important for general biology as they promote better insight into the general rules of
the plant adaptive evolution, which simultaneously proceeds in two environmental components, that
is, in the air and soil media. The study results on complex morphophysiological and biochemical
parameters of rhizome-forming plants, which dominate in many phytocenoses, can be applied for
the development of the functional species classification to forecast species behavior in the changing
environmental conditions. Practically, knowledge on the formation mechanisms of underground
167
shoot complex of rhizomatous plants is applicable for pasture and hayfield management, restoration
of disturbed lands, and yield improvement of medicinal and food plants.
In this chapter, we present information on the growth, metabolism, and adaptation mechanisms
of the underground of rhizome-forming perennial plants to the environmental conditions. The
morphological structure, growth, biomass accumulation, and physiological characteristics (heat gen-
eration, respiration, content of soluble carbohydrates and free phytohormones, and distribution of
assimilated carbon) of plant rhizomes in natural populations and in in vitro conditions have been
studied (Maslova et al. 2005, 2007, 2010a,b, 2013; Maslova and Tabalenkova 2010).
7.2 GROWTH AND MORPHOGENESIS OF UNDERGROUND

SHOOTS IN RHIZOME-FORMING PLANTS
Rhizome is an underground shoot, which is responsible for storage function, vegetative reproduc-
tion, and overwintering. Most plant species form rhizome on the basal part of the leading orthotropic
shoot as an auxiliary plagiotropic under- or aboveground shoot. Buds from which underground
shoots grow are formed at the second ontogenesis stage of the aboveground shoot (Markarov
and Golovko 1995). This stage corresponds to the third-leaf stage of monocotyledonous grasses
(Kuperman 1977). At the concerned stage, the shoot apex is differentiated into initial nodes, inter-
nodes, and stem leaves. The longer the second stage is, the more new buds and vegetative shoots
are. It is important to state that buds of aboveground lateral shoots are formed after the buds of
underground shoots such as rhizomes and stolons. This priority is a vegetative reproduction assur-
ance mechanism; it guarantees perennial grasses to survive and to grow successfully in cenosis.
At the initial formation stages of the aboveground shoots, vegetative organs are characterized
by a low growth rate; underground shoots grow in length and generally get two to four metamers
(Maslova et al. 2005, 2013). Such intensive growth of the underground shoot and formation of new
metamers last until aboveground shoots enter the sexual development stage. Thereafter, the under-
ground shoot starts branching and lateral shoots are formed. Plant species with long rhizomes form
many lateral shoots and have numerous underground metamers or nodes, which represent meriste-
matic activity sources. For example, the Phalaroides arundinacea 3-year-old clones have formed
over 50,000 (Maslova et al. 2007) and those of Elytrigia repens over 26,000 underground nodes/1 m2.
Most rhizomatous nodes are formed in the second half of the vegetation period after the end of
vegetative growth of the plants. The reserve of underground nodes provides high efficiency of veg-
etative reproduction and resistance of rhizome-forming plants to different ecological conditions.
The perennial rhizome-forming grasses (Panicum virgatum, Andropogon gerardii, Sorghastrum
nutans, etc.) were shown to constitute the basis of underground meristematic bank in tallgrass prai-
ries of North America (Kansas) (Benson et al. 2004). The underground bank of buds in grasses
varies from 360 to 1800 buds/1 m2 depending on climatic conditions and fire frequency. The authors
point out that the vegetative reproduction by means of underground meristematic bank of buds
largely predetermines the structure and population dynamics of tallgrass prairie plants composed
of perennial grasses (70%) and semishrubs (30%).
Rhizome-forming species are usually highly productive and often dominate in plant communi-
ties. The most productive among them are meadow plant species, especially the grasses with long
rhizomes Bromopsis inermis and P. arundinacea, which compete with each other (Maslova et al.
2010b). On average, the dry weight of these species was 400–450 g m−2. The proportion of rhizomes
in total plant biomass varied from 30% to 75% (Figure 7.1). Fast-growing meadow species were
characterized by a more even distribution of biomass between above- and underground organs. The
biggest part of stress-tolerant forest plant biomass (70%) was in their rhizomes. This is evidently
related to the vegetative propagation of such plants and the deposition of assimilates in their rhi-
zomes in connection with unfavorable conditions in their habitats. Stress-tolerant plants on poor
nutrition diet accumulate 40%–60% of photosynthesis-assimilated carbon in underground shoots
(P’yankov et al. 2000).
Structure and Metabolism of Underground Shoots in Perennial Rhizome-Forming Plants 169
a b
Elytrigia repens
Hypericum maculatum
Phalaroides arundinacea CR
Bromopsis inermis
Mentha arvensis
Achillea millefolium
Pyrola rotundifolia
Vaccinium vitis-idaea
Oxalis acetosella S
Paris quadrifolia
Majanthemum bifolium
0 20 40 60 80 100
Biomass, % of plant biomass
FIGURE 7.1 Biomass distribution in rhizome-forming plants with different adaptive strategies.
(a) Aboveground organs; (b) underground organs (rhizomes and roots). S—species with stress-tolerant
strategy; CR—species with competitive–ruderal strategy. (Redrawn from Maslova, S.P. et al., Rus. J. Plant
Physiol., 57, 631, 2010b, in Russian.)
The biomass accumulation dynamics of rhizome-forming species depends on the life strategy of
plants. The sexual and vegetative reproduction means are of equal significance for long-rhizomatous
grasses P. arundinacea. They form their aboveground biomass in summer and their underground
biomass in autumn (Figure 7.2) (Maslova et al. 2007). It reflects the time separation of the sexual
and vegetative reproduction, necessary for the optimal use of assimilates during the seed ripen-
ing and rhizome growth. When plants, such as Mentha arvensis and Achillea millefolium, have
Phalaroides arundinacea
2000
a
1600
Biomass (g DW m–2)
1200
b
800
400
0
May June July September
30 Achillea millefolium
Biomass (g DW plant–1)
25
20 a
b
15
10
0
May June July September
FIGURE 7.2 Biomass accumulation by perennial rhizome-forming plants. (a) aboveground part and
(b) underground part. (Redrawn from Maslova, S. et al., Rus. J. Plant Physiol., 54, 491, 2007, in Russian;
Maslova, S. et al., Rus. J. Plant Physiol., 60, 2013, in Russian.)
vegetative reproduction as dominating, they develop rhizomes mainly in summer simultaneously

with the aboveground part (Maslova et al. 2013).
7.3 METABOLIC ACTIVITY AND ENERGY BALANCE OF RHIZOMES

Respiration is a basis of plant vital activity; it determines the metabolic activity of plant species. The
measurements of the CO2 evolution rates showed that the respiration rate of heterotrophic tissues in
the underground organs of rhizome-forming plants was 2–3 times lower than that of aboveground
organs measured at 20°C (Maslova et al. 2010b). The respiration rate of rhizomes varied from
0.1 to 2 mg CO2 h−1 g−1 DW depending on the growing conditions and ecological strategy of
plant (Figure 7.3). The respiration rate of rhizomes of competitive–ruderal meadow plant species
was on average 1 mg CO2 h−1 g−1 DW. The respiration rate of rhizomes of stress-tolerant forest spe-
cies growing in unfavorable conditions was less (by three times) than that of meadow species. The
intensive respiration of rhizomes in fast-growing meadow plant species is due to a higher growth
rate value in contrast to that of slowly growing forest species (Poorter et al. 1990; Lambers et al.
1998). The differences in the modes of assimilate translocation could evidently be one of the factors
determining the respiration rate. Light-requiring plants, if they grow in meadow phytocenoses, usu-
ally are apoplastic species (Gamalei 2005). Symplastic species are shade-enduring plants. They can
grow in poor illumination conditions when the competition between plant species is insignificant
(forest cenoses). A transport form of assimilates on apoplastic phloem loading is sucrose. Apoplastic
sucrose loading demands more energy than symplastic loading because transmembrane transport of
1 mol sucrose requires 1 mol ATP (Penning de Vries 1975).
Age changes in the respiration rate fixed at a constant optimal temperature highlight the meta-
bolic activity status of plants and/or plant organs at different growth and development stages. The
seasonal dynamics of underground shoot respiration rate reflects the growth rhythm of rhizome-
forming plants and their ecological strategy (Maslova et al. 2010a). The respiration rate of rhizomes
of the meadow ever-green species A. millefolium was on average 1.5 mg CO2 h−1 g−1 DW in summer,
which decreased by 30% by the end of October. The respiration rate of the perennial grass B. inermis
rhizomes was high at the earing development phase and decreased by half at the seed-ripening
phase (Maslova et al. 2005). The respiration rate of underground shoots of the ever-green forest
species Pyrola rotundifolia increased toward the end of June and remained high till the end of
September (Maslova et al. 2010a).
2.5
CR
CO2 evolution (mg g–1 DW h–1)
S
2
1.5
1 a
b
0.5
0
1 2 3 4 5 6 7 8 9 10 11
FIGURE 7.3 Respiration rate of leaves (a) and rhizomes (b) of plants with stress-tolerant (S) and c ompetitive–
ruderal (CR) types of adaptive strategy. 1—Pyrola rotundifolia, 2—Vaccinium vitis-idaea, 3—Paris quadri-
folia, 4—Maianthemum bifolium, 5—Oxalis acetosella, 6—Gymnocarpium dryopteris, 7—Bromopsis
inermis, 8—Tussilago farfara, 9—Mentha arvensis, 10—Achillea millefolium, 11—Elytrigia repens.
(Redrawn from Maslova, S.P. et al., Rus. J. Plant Physiol., 57, 631, 2010b, in Russian.)
Heat is a final respiration product. Over 90% of cell-generated metabolic heat has been identified
to be produced during O2 regeneration in mitochondria (Hopkin 1991). High heat generation rate
in rhizomatous apexes has been identified for summer period when underground shoots actively
grow and form lateral shoots (Maslova et al. 2013). At the end of vegetation period when next-
year buds are formed at initial stage, heat generation rate in rhizomatous apexes remains relatively
high (Maslova et al. 2013). This may be an aftereffect of active metabolism during morphogenetic
changes in rhizomatous meristems when plants prepare for wintering.
To study the energy balance and the temperature optimum of rhizome growth, we have applied
a simulation model (Hansen et al. 1994) that links growth, respiration, and heat production together
and thus allows to evaluate the growth as heat storing. The growth rate temperature curve of rhizomes
from P. arundinacea and A. millefolium has been estimated using heat and CO2 generation indexes.
It has been shown that in accordance with the temperature, the activity of the energy storing in the
biomass increases in summer and autumn (Maslova et al. 2007, 2013). Underground shoots can grow
in the temperature range from 2°C to 20°C (Figure 7.4). In higher temperatures, the energy storing
rate decreases and attains its lowest value at 30°C. Thus, low and moderate positive temperatures are
optimal for the growth of rhizomes, which successfully overwinter in the cold climate of the North.
Phalaroides arundinacea
16
12
Growth rate (µW mg–1 DW)
4 a
0
2 5 15 25 35
–4 Temperature (°C)
–8
–12
b
–16
Achillea millefolium
30
25
Growth rate (µW mg–1 DW)
20
15
a
10
5 c
0
5 10 15 20 25 30
–5 Temperature (°C)
FIGURE 7.4 Temperature dependence of metabolic activity of the rhizomes. (a) July, (b) November, and
(c) January. Growth rate calculated from heat and respiration rate. (From Hansen, L.D. et al., Planta, 194, 77,
1994; Redrawn from Maslova, S. et al., Rus. J. Plant Physiol., 54, 491, 2007, in Russian; Maslova, S. et al.,
Rus. J. Plant Physiol., 60, 2013, in Russian.)
Results of microcalorimetric studies show that the rhizomes of perennial plants continue growing
at low positive temperatures in late autumn and even in winter when the soil temperature decreases
to 0°C and −3°C (Maslova et al. 2007, 2013). It is important to note that cells of rhizome apexes
contain water up to 90% independent from the vegetation period or soil temperature. In winter, the
water–ice transition temperature in tissues of apical part equals −10°C, which is three times as low
as the temperature in the soil around them. This adaptive mechanism allows perennial rhizome-
forming plants to overwinter and regrowth in early spring.
7.4 PHYTOHORMONE AND TROPHIC GROWTH

REGULATION OF UNDERGROUND SHOOTS
The integration of the plant organism is dependent on the interaction of intercellular regulation
systems as trophic, hormone, and electrophysiological systems. Trophic dependences are visual
between the shoot and the root, leaves, and attracting organs. Carbohydrates are the usual material
for the synthesis of structural and reserve plant biomass; their concentration is influenced by the
plant growth rate, development phase, and environmental factors. Nonstructural sugars are the ini-
tial material for the plastic and energy exchange; they raise the plant resistance to low temperatures
(Travert et al. 1997; Zabotina et al. 1998; Savitch et al. 2000; Deryabin et al. 2004, 2007; Sinkevich
et al. 2008). Carbohydrates may serve as signal molecules and participate in the regulation of physi-
ological and morphogenetic programs (Hänisch and Breteler 1981; Veyres et al. 2008).
The results of our study prove that the content of nonstructural carbohydrates in leaves and
underground shoots of rhizome-forming perennials depends on the ecological strategy and the
growth rate (Maslova et al. 2010b). Stress-tolerant forest species were characterized by the low
content of soluble sugars in rhizomes and the high one in leaves. The accumulation of carbohy-
drates, especially of monosaccharides, in leaves can possibly indicate the low carbon transport and
utilization rates in rhizomes of slowly growing forest species under the conditions of poor illumina-
tion. Stress-tolerant plants differ from other plants by low-rate outflow of photosynthesis products
(P’yankov et al. 2000). Additionally, some assimilates may be used for the formation of strengthen-
ing tissues, cuticle, and for the synthesis of secondary compounds, the concentration of which is
higher in stress-tolerant plant organisms (Lambers and Poorter 1992).
Meadow plant species preferably accumulate soluble sugars in rhizomes, not in leaves. The low
concentration of soluble carbohydrates in leaves of fast-growing meadow plants is the result of inten-
sive utilization of assimilates for growth and respiration in aboveground shoots and of transport of
sugars into rhizomes. Underground shoots of meadow rhizome-forming species grow actively and
form numerous underground vegetative meristems (Maslova et al. 2007, 2010b). The accumulation
of sugars is known to promote cell division and differentiation processes in meristematic root zones
(Hänisch and Breteler 1981).
The concentration and qualitative composition of soluble carbohydrates in underground tis-
sues of rhizome-forming perennials undergo seasonal changes. In summer, the content of soluble
sugars in rhizome dry mass of perennial herbaceous plant A. millefolium was 15%. In autumn, to
the end of ontogenesis of current-year aboveground shoot, the portion of soluble carbohydrates in
rhizomatous tissues increases by 30% (Maslova et al. 2013). In winter, when underground shoots
stop growing, the content of carbohydrates remains at the autumn level. During overwintering, we
registered a considerable accumulation of oligosaccharides, the content of which composed 60%
of the total sum of carbohydrates. The oligosaccharides are considered to be cryoprotectants and
are able to increase low-temperature resistance of the plants in autumn and winter (Zabotina et al.
1998; Ayupova et al. 2001). At this period, rhizomes actively form lateral shoots and buds for next-
year regrowth. Sugars lower the water–ice transition temperature of intracellular solution, resist
to the injury of protein–lipid complex in cell membranes, function as the main material to synthe-
size stress-proteins and lipids, and prevent from free-radical oxidation processes of biologically
important macromolecules under low temperatures (Deryabin et al. 2007).
90
80
Carbohydrate content (mg g–1 DW)

a
70
60
50
40
b
30
20
10 c
0
June 16 October 17 May 17
FIGURE 7.5 The content of carbohydrates in the rhizomes of Phalaroides arundinacea.

(a) Monosaccharides, (b) disaccharides, and (c) oligosaccharides. (Redrawn from Maslova, S. et al., Rus. J.
Plant Physiol., 54, 491, 2007, in Russian).
Rhizomes of the perennial grass P. arundinacea vary in balance between elementary and
compound soluble carbohydrates depending on the year season, but the total concentration of sugars
is always the same and makes up 10% of underground shoots’ dry biomass (Figure 7.5) (Maslova
et al. 2007). In autumn, rhizomes contain less monosaccharides and more oligosaccharides. In
spring, when aboveground shoots regrow, rhizomes contain less oligo- and monosaccharides and
considerably more disaccharides (mainly sucrose), the content of which was 75% of the total amount
of carbohydrates. Spring is typical of a quick inclusion of sugars into metabolic processes and active
transport of dimeric carbohydrates. In this season, rhizomes are the main donor of nutrients and
hormones for actively regrowing aboveground shoots and newly forming rhizomes. The accumula-
tion of sugars is known to promote cell division and differentiation in meristematic zones of roots
(Hänisch and Breteler 1981). As a source material for plastic and energy exchange, sugars can act as
signal molecules and thus can regulate morphogenesis of plants (Veyres et al. 2008).
Phytohormones play a major role in the regulatory systems of plants. They start and regulate dif-
ferent physiological and morphogenetic programs. The intensity and character of metabolism, the
growth and development rhythms of underground shoots, as well as their interaction with other plant
organs are largely influenced by the content and ratio of phytohormones (Belynskaya et al. 1997;
Kondrat’eva 2000; Maslova et al. 2007). We have demonstrated the hormone growth and the resis-
tance regulation of underground shoots in rhizome-forming plant species to be actual (Maslova et al.
2007, 2013; Maslova and Tabalenkova 2010). Considerable seasonal changes in levels of cytokinins,
gibberellins (GA), auxin (IAA), and abscisic acid (ABA) in rhizomatous tissues are shown. The rhi-
zomes of A. millefolium were characterized by the high content of phytohormones, gibberellins
excluded, in summer, particularly in July (Figure 7.6). In autumn, gibberellins significantly increase
in activity against the activity decrease in other hormones. The GA/ABA and cytokinins/ABA ratios
essentially rise up at this period. The high GA activity is required to stimulate the linear growth of
metamers in rhizomes and axial organs of lateral buds in nodes of newly forming metamers in the
underground shoot. Nodes of rhizomatous metamers represent lateral growth-active zones, which
allow the underground diagravitropic and aboveground orthotropic branching of the rhizome.
The increase in relative content of cytokinins in autumn may be affected by the transport
decrease in this hormone group to aging and dying aboveground organs. Rhizomes of the perennial
grass P. arundinacea contain 30 times as many cytokinins in November as compared to summer
(Maslova et al. 2007). Cytokinins in underground buds can stimulate morphogenesis processes
including initial organogenesis stages of lateral buds (cell division and tissue differentiation of lat-
eral meristems). Cytokinins can increase the tissue-attracting activity and thus raise the influx of
IAA 4500 Cytokinins

700
3500
500
2500
300
Phytohormone content (ng g–1 DW)
1500
100 500
0 0
July October May July October May
4500 3000
GA ABA
2500
3500
2000
2500
1500
1500 1000
500
500
0 0
July October May July October May
FIGURE 7.6 The content of free phytohormones in the rhizomes of Achillea millefolium. (Redrawn from
Maslova, S. et al., Rus. J. Plant Physiol., 60, 2013, in Russian.)
nutrients to underground buds (Belynskaya et al. 1997). Cytokinins affect the expression of many
genes (Hare and van Staden 1997; Rashotte et al. 2003; Romanov 2009) and contribute to the bet-
ter cold resistance of plants by the activity regulation of protein-synthesizing apparatus (Hare et al.
1999) and by the activation of antioxidant system enzymes (Wang et al. 2009). This is particularly
important for underground shoots in winter period when the lipid peroxidation level rises because
of low temperatures (Maslova et al. 2013).
The highest ABA content is fixed in July in the period of the active formation of young rhizomes
(Belynskaya et al. 1997; Maslova and Tabalenkova, 2010; Maslova et al. 2013). We refer it to the role
that ABA plays in the maintenance of the regular water balance in underground shoots. The water
exchange regulation mechanism is believed to be dependent on the ABA ability to heighten the gene
expression and phosphorylation levels of aquaporins (Schaffner 1998).
Thus, the carbohydrate and hormone metabolism plays an important role in the morphogenesis
regulation of underground shoots and in the adaptation of rhizome-forming perennials to unfa-
vorable winter conditions. In autumn, the accumulation of growth hormones and cryoprotectant
substances, particularly oligosaccharides, in rhizome tissues was noted. This favors morphogenetic
changes in underground shoots as initiation of buds (initial shoot organogenesis stages, and cell and
tissue differentiation) in the autumn period.
7.5 UNDERGROUND SHOOT COMPLEX IN SOURCE–SINK SYSTEM

OF RHIZOME-FORMING PLANTS
The source–sink system conception of plants is maintained in the interaction of organs that pro-
duce and consume assimilates (Wareing and Jennigs 1980; Mokronosov 1990). Acceptors (sink)
are represented by zones of storing and intensive utilization of assimilates, the so-called meri-
stems; donors (source) are leaves and other green plant parts. Sink–source relationships, the carbon
distribution, and the utilization during the ontogenesis of the plant have been studied mostly for
crops whose economic organs such as ears, tubers, and roots are active acceptors (Crompton et al.
1981; Chikov 1987). For tuber-forming plants, the transition of stolon to the tuber causes a new level
of sink–source relations within the whole plant system where most of the assimilates are transported
underground for the growth of storage organs. The utilization intensity of assimilates during the
formation of tubers, in turn, predetermines the photosynthesis intensity according to the feedback
principle (Mokronosov 1990).
Rhizome-forming plants that deposit carbon in underground shoots, that is, rhizomes, are here of
special interest. We have obtained the data on morphophysiology of the underground shoot complex
and its contribution into the sink–source system of rhizome-forming perennials (Maslova et al. 2005,
2010a,b). The underground shoot complex of perennial grasses is an important link in the regulation of
the source–sink system and characterizes the life form perennial grass. It is a stable formation whereas
aboveground shoots die every year; rhizomes with winter buds survive and overwinter. Rhizomes
take a large part in the whole plant biomass (30%–80%), form numerous vegetative meristems, and
are characterized by a high metabolic activity. Consequently, the underground shoot complex plays a
significant role in the source–sink system of perennial grasses (Maslova et al. 2007, 2010b).
According to the obtained results, manipulations with aboveground part of long-rhizomatous
grasses (plant decapitation or spraying of the aboveground plant part with growth regulators) do not
essentially affect the amount and the biomass values of rhizomes (Maslova et al. 2005). Decapitation
experiments have evidenced a high ability of the perennial grass P. arundinacea to form shoots.
Just 3 weeks later (at the earing phase) after the decapitation, the experimental plants contain more
aboveground shoots than the control plants. In 7 weeks (at the seed-ripening phase), experimen-
tal and control plants have equal quantities of aboveground shoots of about 160 units. After the
removal of aboveground shoots, the respiration rate of rhizomes of experimental plants increased
by 25%–30%. It is connected with high morphogenetic and metabolic activity of rhizomes needed
to form new aboveground shoots.
Treated with chlorocholine chloride retardant, the B. inermis plants are characterized by the
longer metamers and higher concentrations of soluble sugars in rhizomes than the control plants
(Maslova et al. 2005). To the end of the vegetation period, experimental and control plants dif-
fer neither by the morphological structure of rhizomes, nor by the biomass accumulation and the
physiological activity. Consequently, underground shoots of long-rhizomatous grasses are relatively
independent from other plant organs and realize genome morphogenetic program in certain condi-
tions (vegetation period). Such autonomic growth of the underground shoot complex provides a high
competitive capacity and resistance of grassy perennials in plant communities.
The formation of the source–sink system is regulated by the plant genotype and the environ-
mental conditions (Mokronosov 1983). The direction of assimilate translocation depends on the
synthesis intensity in leaves and the activity of attracting organs and tissues (Mokronosov 1983;
P’yankov et al. 2000). Wild plant species have a broad variety of sink–source systems as they are
represented by numerous life forms, pheno-rhythm-types, and adaptive strategies.
Long-rhizomatous plants with different seasonal development rhythms growing in different
ecologic–cenotic conditions significantly vary in the assimilated carbon uptake rate and the distri-
bution (Maslova and Tabalenkova 2010). Leaves of meadow summer-green species (M. arvensis,
A. millefolium, and E. repens) assimilate more 14CO2 compared to forest ever-green species
(P. rotundifolia and Vaccinium vitis-idaea). This fact may be an evidence for a higher photosyn-
thetic activity of summer-green species. For example, leaves of the summer-green plants Betonica
officinalis and Artemisia absinthium photosynthesize at a rate of 10–30 mg CO2 h−1 g−1 DW, which
is more than those of the ever-green species from the Pyrola genus by 5–15 times (P’yankov et al.
2000). Such great differences may be caused by anatomic–morphologic particularities of leaves
from plants with different pheno-rhythm-type. Annually vegetating plant species have a larger frac-
tion of nonphotosynthesizing tissues (cuticle, primary cuticle, and sclerenchyma) in the leaf, and as
a result the CO2 assimilation rate decreases.
Assimilates in the summer-green short-vegetating species M. arvensis are often used to grow
young rhizomes, which annually causes morphologic plant disintegration and contributes to the
isolation and self-existence of specimens (Maslova and Tabalenkova 2010). Fifty percent of under-
ground shoots in the summer-green plant A. millefolium are underground shoots, which form assim-
ilating organs in the current vegetation period (sarments), and so most of the carbon is used to form
the new assimilating surface of sarments where 14C-assimilates mostly sink. The majority of assimi-
lated carbon in the ever-green plant P. rotundifolia is consumed for the formation of new rosettes of
leaves, which function a year round. Leaves of the ever-green undershrub V. vitis-idaea function for
3–4 years; for this reason, 50% of photoassimilates are stored in structural elements of leaves and
stems to be used the next year for the growing process.
Generally, the obtained data demonstrate the interrelations of the seasonal development rhythm
with carbon assimilate distribution in the organism of long-rhizomatous plants. Assimilated carbon
in summer-green fast-growing plants is translocation into the underground part of plants, whereas
ever-green species largely deposit assimilates in perennial aboveground shoots or utilize them to
form leaves that function the year round.

As a result of the long research of the structural–functional organization of the underground shoot
complex of rhizome-forming plants, metabolic activities of rhizomes during their morphogenesis
have been studied; sink–source interrelations in the whole plant system have been demonstrated.
The underground shoot complex plays a major role in the sink–source system of grassy perennials;
it is characterized by intensive metabolism, forms big reserve of vegetative meristems, and is highly
capable of self-restoration and self-regulation. Rhizomes demonstrate certain autonomy and realize
the morphogenetic program of the plant genome under definite conditions of the growth period.
The underground shoots contribute to the adaptation of perennial rhizome-forming plants to low
temperatures in winter. Hormones and carbohydrates play an important role in the growth and resis-
tance regulation of rhizomes and prevent underground meristem from the frost damage. In autumn,
the rhizomes were characterized by the high content of growth hormones, oligosaccharides, and
the respiration rate of rhizomatous apexes. We conclude that the plant adaptive strategy, ecological
conditions, and the seasonal development rhythm are strongly linked to the physiological properties
of rhizome-forming plants. Meadow species with pronounced competitive–ruderal properties are
characterized by a high yield, active metabolism, carbon investing in assimilating regrowing shoots,
and rhizomes. Forest species with stress-tolerant features are characterized by a low physiological
activity and the deposition of assimilated carbon to the underground plant part.
In general, the results of the study contribute to the understanding of the formation mechanisms
of the underground shoot complex of rhizome-forming plants and can be applied for the develop-
ment of functional classification of species that will allow to forecast their behavior in the changing
climatic conditions. The aim of the future work in this field of plant ecological physiology is related
to the genetic plasticity mechanisms and diversity of the life form rhizome-forming perennial, anal-
ysis of molecular basis of rhizomatous plant species evolution, and revealing of mechanisms and
principles of this plant group integration from a molecule to a cenotic level.
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8 Evaluating and Managing
Crops Water Requirement
Zohrab Samani
CONTENTS
8.1 Introduction........................................................................................................................... 179
8.2 Evapotranspiration................................................................................................................. 179
8.2.1 Crop Coefficient (Kc)................................................................................................. 180
8.2.2 Calculating Growing Degree Days............................................................................ 180
8.2.3 Irrigation Interval...................................................................................................... 185
8.2.4 Alternative Method “Calculating ET without GDD”................................................ 187
8.2.5 Perennial Crops......................................................................................................... 189
References....................................................................................................................................... 190
8.1 INTRODUCTION
Crops use water for cooling, nutrient transport to the leaves, and photosynthesis. Most of the water
used by the crop evaporates from the surface of the leaves. Plants function like an evaporative cooler
where large amount of water is used for cooling, which is an integral part of the photosynthesis pro-
cess. The growth of plant is tied to the ability of leaves to produce carbohydrate via photosynthesis.
The carbohydrate is the basis for the accumulation of biomass and growth of the plant. The leaves
capture light over the surface area of the leaves exposed to the sun. Upon exposure to the solar
energy, the leaves open thousands of tiny pores (stomata) to let in carbon dioxide (CO2) and solar
energy to produce carbohydrate. The open stomata and solar energy also result in water evapora-
tion at the same time. The water evaporated through the stomata is referred to as transpiration. The
sum of water loss through transpiration and evaporation outside of stomata such as soil surface is
referred to as evapotranspiration (ET). The ET is a function of climate condition, plant leaf area, and
the stomata behavioral characteristic of the plant. The water that evaporates through ET is supplied
from the stored moisture within the root zone of the plant. Thus the success of the crop growth and
yield production depends on the climate, the plant, and the ability of the producer to provide the soil
reservoir with timely and sufficient quantity of water. Water is the single most important production
parameter in irrigated agriculture. This chapter describes how the crops’ water requirement (ET) can
be estimated and how the soil moisture reservoir can be managed to achieve successful production.
8.2 EVAPOTRANSPIRATION
To estimate the crops’ water requirement, the ET is divided into two distinct parameters: the crop
coefficient (Kc) representing the crop, and reference evapotranspiration (ETr), which is calculated
from the climate parameters. The ET thus can be described as
ET = K c ¥ ETr (8.1)
ETr is calculated using various equations of Hargreaves and Samani (1982, 1985) and
Penman–Monteith (Allen 1986), Allen et al. (1998), and ASCE-EWRI standardized reference
179
evapotranspiration (2005). The ASCE-EWRI (2005) has recommended two equations for calculat-
ing reference evapotranspiration (ETr). Those are standardized Penman–Monteith and Hargreaves
and Samani (1985). The Hargreaves–Samani equation is as follows:
ETr = 0.0023 ¥ RA(Tc + 17.8)(TD)0.5 (8.2)
where
RA is the extraterrestrial radiation representing the amount of solar energy received at any lati-
tude above the atmosphere
Tc is the average of daily maximum and minimum temperatures in degree Celsius
TD is the difference between daily maximum and minimum temperatures in degree Celsius
ETr is the daily ET value in mm
The extraterrestrial values as a function of the latitude of location are shown in Table 8.1. Equation
8.2 uses the temperature difference to account for the loss of solar energy in the atmosphere due to
cloudiness or humidity.
8.2.1 Crop Coefficient (Kc)

The most difficult parameter to be determined in assessing ET and managing water is the Kc. The
Kc value changes not only with crop, but also with time or stage of growth of the crop, crop density,
and general growth conditions. The Kc is also temporally and spatially variable. This means that a
Kc value determined for one climatic condition cannot be used for another climatic condition. One
of the easiest ways to determine the Kc value for a given crop under a given climatic condition and
growth stage is through the use of growing degree days (GDDs) as described in the following text.
The crop growth starts with the start of growing season, where plants develop leaves and start to
actively grow. In annual crops, regardless of when the seed is planted, the crop growth stage starts
with a minimum average temperature called the base temperature and the growth stops once a
minimum temperature occurs. The same is true for the perennial crops where the plants start to
grow when a minimum base temperature is reached and will stop growth when a final minimum
temperature occurs. Table 8.2 shows the base temperature and final (end) temperature for some
common crops.
8.2.2 Calculating Growing Degree Days

The GDD calculation starts when the minimum temperature has reached the base temperature
shown in Table 8.2. The equation to calculate GDD is as follows:
Tmax + Tmin
GDD = - Tbase (8.3)
2
where
GDD is the growing degree day calculated for each day
Tmax is the maximum daily temperature
Tmin is the minimum daily temperature
Tbase is the minimum start-up temperature
In calculating GDD, the Tmax is normally limited to a maximum cutoff daily temperature, which is
normally 86°F. For example, if the Tmax of the day for corn crop is 90°F, Tmax is set equal to 86°F.
Some crops like alfalfa and pecan do not have cutoff temperature.
TABLE 8.1
Extraterrestrial Radiation (RA) (mm/Day)
Northern Hemisphere Southern Hemisphere
Jan Feb Mar Apr May June Jul Aug Sep Oct Nov Dec Lat° Jan Feb Mar Apr May June Jul Aug Sep Oct Nov Dec
3.8 6.1 9.4 12.7 15.8 17.1 16.4 14.1 10.9 7.4 4.5 3.2 50 17.5 14.7 10.9 7.0 4.2 3.1 3.5 5.5 8.9 12.9 16.5 18.2
4.3 6.6 9.8 13.0 15.9 17.2 16.5 14.3 11.2 7.8 5.0 3.7 48 17.6 14.9 11.2 7.5 4.7 3.5 4.0 6.0 9.3 13.2 16.6 18.2
4.9 7.1 10.2 13.3 16.0 17.2 16.6 14.5 11.5 8.3 5.5 4.3 46 17.7 15.1 11.5 7.9 5.2 4.0 4.4 6.5 9.7 13.4 16.7 18.3
5.3 7.6 10.6 13.7 16.1 17.2 16.6 14.7 11.9 8.7 6.0 4.7 44 17.8 15.3 11.9 8.4 5.7 4.4 4.9 6.9 10.2 13.7 16.7 18.3
5.9 8.1 11.0 14.0 16.2 17.3 16.7 15.0 12.2 9.1 6.5 5.2 42 17.8 15.5 12.2 8.8 6.1 4.9 5.4 7.4 10.6 14.0 16.8 18.3
6.4 8.6 11.4 14.3 16.4 17.3 16.7 15.2 12.5 9.6 7.0 5.7 40 17.9 15.7 12.5 9.2 6.6 5.3 5.9 7.9 11.0 14.2 16.9 18.3
6.9 9.0 11.8 14.5 16.4 17.2 16.7 15.3 12.8 10.0 7.5 6.1 38 17.9 15.8 12.8 9.6 7.1 5.8 6.3 8.3 11.4 11.4 17.0 18.3
7.4 9.4 12.1 14.7 16.4 17.2 16.7 15.4 13.1 10.6 8.0 6.6 36 17.9 16.0 13.2 10.1 7.5 6.3 6.8 8.8 11.7 14.6 17.0 18.2
7.9 9.8 12.4 14.8 16.5 17.1 16.8 15.5 13.4 10.8 8.5 7.2 34 17.8 16.1 13.5 10.5 8.0 6.8 7.2 9.2 12.0 14.9 17.1 18.2
8.3 10.2 12.8 15.0 16.5 17.0 16.8 15.6 13.6 11.2 9.0 7.8 32 17.8 16.2 13.8 10.9 8.5 7.3 7.7 9.6 12.4 15.1 17.2 18.1
8.8 10.7 13.1 15.2 16.5 17.0 16.8 15.7 13.9 11.6 9.5 8.3 30 17.8 16.4 14.0 11.3 8.9 7.8 8.1 10.1 12.7 15.3 17.3 18.1
9.3 11.1 13.4 15.3 16.5 16.8 16.7 15.7 14.1 12.0 9.9 8.8 28 17.7 16.4 14.3 11.6 9.3 8.2 8.6 10.4 13.0 15.4 17.2 17.9
Evaluating and Managing Crops Water Requirement
9.8 11.5 13.7 15.3 16.4 16.7 16.6 15.7 14.3 12.3 10.3 9.3 26 17.6 16.4 14.4 12.0 9.7 8.7 9.1 10.9 13.2 15.5 17.2 17.8
10.2 11.9 13.9 15.4 16.4 16.6 16.5 15.8 14.5 12.6 10.7 9.7 24 17.5 16.5 14.6 12.3 10.2 9.1 9.5 11.2 13.4 15.6 17.1 17.7
10.7 12.3 14.2 15.5 16.3 16.4 16.4 15.8 14.6 13.0 11.1 10.2 22 17.4 16.5 14.8 12.6 10.6 9.6 10.0 11.6 13.7 15.7 17.0 17.5
11.2 12.7 14.4 15.6 16.3 16.4 16.3 15.9 14.8 13.3 11.6 10.7 20 17.3 16.5 15.0 13.0 11.0 10.0 10.4 12.0 13.9 15.8 17.0 17.4
11.6 13.0 14.6 15.6 16.1 16.3 16.1 15.8 14.9 13.6 12.0 11.1 18 17.1 16.5 15.1 13.2 11.4 10.4 10.8 12.3 14.1 15.8 16.8 17.1
12.0 13.3 14.7 15.6 16.0 15.9 15.9 15.7 15.0 13.9 12.4 11.6 16 16.9 16.4 15.2 13.5 11.7 10.8 11.2 12.6 14.3 15.8 16.7 16.8
12.4 13.6 14.9 15.7 15.8 15.7 15.7 15.7 15.1 14.1 12.8 12.0 14 16.7 16.4 15.3 13.7 12.1 11.2 11.6 12.9 14.5 15.8 16.5 16.6
12.8 13.9 15.1 15.7 15.7 15.5 15.5 15.6 15.2 14.4 13.3 12.5 12 16.6 16.3 15.4 14.0 12.5 11.6 12.0 13.2 14.7 15.8 16.4 16.5
13.2 14.2 15.3 15.7 15.5 15.3 15.3 15.5 15.3 14.7 13.6 12.9 10 16.4 16.3 15.5 14.2 12.8 12.0 12.4 13.5 14.8 15.9 16.2 16.2
13.6 14.5 15.3 15.6 15.3 15.0 15.1 15.4 15.3 14.8 13.9 13.3 8 16.1 16.1 15.5 14.4 13.1 12.4 12.7 13.7 14.9 15.8 16.0 16.0
13.9 14.8 15.4 15.4 15.1 14.7 14.9 15.2 15.3 15.0 14.2 13.7 6 15.8 16.0 15.6 14.7 13.4 12.8 13.1 14.0 15.0 15.7 15.8 15.7
14.3 15.0 15.5 15.5 14.9 14.4 14.6 15.1 15.3 15.1 14.5 14.1 4 15.5 15.8 15.6 14.9 13.8 13.2 13.4 14.3 15.1 15.6 15.5 15.4
14.7 15.3 15.6 15.3 14.6 14.2 14.3 14.9 15.3 15.3 14.8 14.4 2 15.3 15.7 15.7 15.1 14.1 13.5 13.7 14.5 15.2 15.5 15.3 15.1
15.0 15.5 15.7 15.3 14.4 13.9 14.1 14.8 15.3 15.4 15.1 14.8 0 15.0 15.5 15.7 15.3 14.4 13.9 14.1 14.8 15.3 15.4 15.1 14.8
Lat°, Latitude in degree.

181
TABLE 8.2
Plant Growing Season Start-Up and End Temperature in °F
Crop Earliest Mean Temperature (°F) End Temperature
Perennial crops
Alfalfa 50 28°frost
Cool season grasses 45 45°F
Deciduous orchards 50 45°F
Grapes 55 50°F
Pecan 60 25°frost
Pomegranate and almond 50 28°frost
Annual crops
Dry beans 60 32°frost
Corn 55 32°frost
Cotton 62 32°frost
Spring grain 45 32°frost
Potatoes 60 32°frost
Sorghum, grain 60 32°frost
Sugar beets 28 28°frost
Wheat, winter (Fall season) — 45°
Wheat, Spring season 45° —
Tomato 50 32°frost
TABLE 8.3
Relationship between Kc and GDD for Various Stages of Growth (Corn—Grain)
Corn—Grain Tbase 55°F
Time % to full cover 10 20 30 40 50 60 70 80 90 100
GDD 5.3 23.4 53.5 102 164 222 318 425 541 644
Kc, before full cover 0.345 0.345 0.345 0.345 0.368 0.483 0.633 0.805 0.98 1.093
Days-after-full cover 10 20 30 40 50 60 68
GDD 833 1002 1168 1341 1472 1567 1603
Kc 1.104 1.093 1.081 1.035 0.978 0.909 0.85
Once the daily GDD is calculated, the cumulative (sum-GDD) is calculated starting from day 1
and related to crop coefficient Kc. There is a direct relationship between sum-GDD and Kc of crop
throughout the growing season. Tables 8.3 through 8.7 show the relationship between sum-GDD
and Kc for several crops. Tables 8.3 through 8.7 were created using 8 years of experimental data
presented by Wright (1981).
Example 8.1
Table 8.8 shows the procedure to calculate daily ET and growing season ET of grain corn crop
for Laguna, New Mexico, using GDD. In this example, Table 8.3 for grain corn was used to cal-
culate GDD from temperature data. The calculation starts from day 120 of the year when the
average daily temperature reaches 55°F, and a positive GDD can be calculated. The daily GDD
is then summed up starting from day 120 until the maximum GDD value of 1603°F when the
crop matures. The daily Kc is then calculated based on the cumulative GDD (sum-GDD) value.
Evaluating and Managing Crops Water Requirement 183
TABLE 8.4
Relationship between Kc and GDD for Various Stages of Growth
(Corn—Sweet)
Corn—Sweet Tbase 55°F
GDD 5.3 23.4 53.5 101 164 222 318 423 541 644
Kc, before full cover 0.345 0.345 0.345 0.345 0.368 0.483 0.632 0.805 0.98 1.093
Days after full cover 10 20 30
GDD 833 1002 1168
kc, after full cover 1.07 1.07 1.035
TABLE 8.5
Relationship between Kc and GDD for Various Stages of Growth (Bean)
Bean Tbase 60°F
Time % to full cover 30 40 50 60 70 80 90 100
Sum-GDD 20 35 70.3 106 164 220 292 341
Kc 0.345 0.403 0.518 0.633 0.78 0.92 1.035 1.093
Days after full cover 10 20 30 40 45
Sum-GDD 463 582 698 808 865
kc 1.093 1.035 0.77 0.38 0.173
TABLE 8.6
Relationship between Kc and GDD for Various Stages of Growth (Barley)
Barley Tbase 45°F
GDD 8.23 25.6 67.1 140 204 293 421 531 613
kc, before full cover 0.345 0.368 0.46 0.748 0.978 1.093 1.14 1.15 1.15
Days after 20 30 40 50 55
GDD 1030 1276 1529 1781 1827
kc 1.15 1.035 0.575 0.288 0.173
TABLE 8.7
Relationship between Kc and GDD for Various Stages of Growth (Alfalfa)
Alfalfa 1146 GDD/Cut Tbase 41°F
Time % of total cycle 10 20 30 40 50 60 70 80 90 100
Kc, first cut 0.805 0.94 1.05 1.104 1.15 1.15 1.127 1.104 1.093 1.093
Kc, second + third cuts 0.46 0.575 0.92 1.104 1.13 1.15 1.15 1.13 1.093 1.093
Fourth cut 0.46 0.51 0.69 0.75 0.63 0.58 0.52 0.4 0.35 0.29
TABLE 8.8
ET Calculation for Grain Corn in Laguna, New Mexico
DOY Sum-GDD ETr Kc ET (mm) DOY Sum-GDD ETr Kc ET (mm)
120 0.69 4.8 0.345 1.65 166 354 6.8 0.805 5.49
121 1.11 4.7 0.345 1.62 167 368 6.8 0.805 5.44
122 1.59 4.7 0.345 1.61 168 382 6.8 0.805 5.44
123 2.07 4.8 0.345 1.65 169 397 6.8 0.805 5.51
124 4.27 4.9 0.345 1.7 170 411 7 0.805 5.6
125 7.63 5 0.345 1.74 171 427 6.9 0.805 5.52
126 10.6 5 0.345 1.73 172 442 6.8 0.98 6.64
127 13.4 5 0.345 1.74 173 458 6.8 0.98 6.7
128 16.4 5.1 0.345 1.75 174 473 6.9 0.98 6.8
129 20.3 5.2 0.345 1.78 175 489 7 0.98 6.82
130 23.9 5.2 0.345 1.79 176 504 7 0.98 6.83
131 27.6 5.2 0.345 1.8 177 520 7 0.98 6.86
132 31.4 5.3 0.345 1.82 178 536 6.9 0.98 6.76
133 35.9 5.3 0.345 1.84 179 552 7 1.093 7.62
134 40.7 5.3 0.345 1.84 180 568 7 1.093 7.6
135 46.3 5.4 0.345 1.85 181 585 6.8 1.093 7.46
136 51.8 5.4 0.345 1.87 182 602 6.7 1.093 7.37
137 58 5.5 0.368 2.03 183 618 6.8 1.093 7.45
138 64.4 5.5 0.368 2.03 184 635 6.6 1.093 7.24
139 71.5 5.6 0.368 2.08 185 652 6.6 1.093 7.27
140 79.4 5.7 0.368 2.08 186 669 6.7 1.104 7.35
141 86.1 5.6 0.368 2.05 187 686 6.7 1.104 7.44
142 92.8 5.7 0.368 2.09 188 703 6.7 1.104 7.45
143 99.9 5.7 0.368 2.1 189 720 6.7 1.104 7.45
144 108 5.8 0.368 2.12 190 737 6.7 1.104 7.37
145 116 5.8 0.368 2.12 191 754 6.6 1.104 7.32
146 125 5.7 0.368 2.11 192 772 6.6 1.104 7.32
147 134 5.7 0.368 2.11 193 789 6.6 1.104 7.26
148 142 5.9 0.368 2.15 194 807 6.6 1.104 7.27
149 151 6 0.368 2.21 195 824 6.6 1.104 7.33
150 161 6 0.368 2.22 196 841 6.5 1.104 7.12
151 170 6 0.483 2.91 197 859 6.5 1.093 7.11
152 180 6.1 0.483 2.94 198 876 6.4 1.093 7.01
153 190 6.1 0.483 2.94 199 894 6.4 1.093 7.02
154 201 6.1 0.483 2.96 200 912 6.4 1.093 7.01
155 211 6.1 0.483 2.96 201 929 6.4 1.093 6.97
156 222 6.2 0.483 2.99 202 946 6.3 1.093 6.87
157 234 6.3 0.633 4.01 203 964 6.3 1.093 6.91
158 247 6.4 0.633 4.05 204 981 6.4 1.093 6.97
159 260 6.4 0.633 4.06 205 998 6.3 1.093 6.9
160 273 6.4 0.633 4.07 206 1016 6.3 1.081 6.76
161 285 6.4 0.633 4.08 207 1034 6.2 1.081 6.67
162 299 6.4 0.633 4.06 208 1051 6.2 1.081 6.67
163 312 6.4 0.633 4.03 209 1068 6.2 1.081 6.65
164 326 6.6 0.633 4.17 210 1086 6.1 1.081 6.58
165 340 6.6 0.805 5.34 211 1103 6.2 1.081 6.71

ET Calculation for Grain Corn in Laguna, New Mexico
DOY Sum-GDD ETr Kc ET (mm) DOY Sum-GDD ETr Kc ET (mm)
212 1121 6.1 1.081 6.58 227 1380 5.6 0.978 5.47
213 1139 6.1 1.081 6.64 228 1396 5.6 0.978 5.48
214 1156 6.1 1.081 6.59 229 1413 5.6 0.978 5.48
215 1174 6 1.081 6.47 230 1429 5.6 0.978 5.5
216 1191 5.9 1.035 6.07 231 1446 5.6 0.978 5.48
217 1208 5.8 1.035 6.05 232 1462 5.5 0.978 5.38
218 1226 5.9 1.035 6.1 233 1479 5.4 0.978 5.26
219 1243 5.9 1.035 6.06 234 1495 5.4 0.909 4.88
220 1261 6 1.035 6.17 235 1511 5.4 0.909 4.92
221 1278 5.9 1.035 6.09 236 1527 5.3 0.909 4.83
222 1295 5.8 1.035 5.99 237 1542 5.4 0.909 4.95
223 1312 5.8 1.035 5.99 238 1558 5.4 0.909 4.88
224 1329 5.7 1.035 5.92 239 1574 5.3 0.909 4.86
225 1346 5.7 1.035 5.88 240 1590 5.3 0.85 4.48
226 1363 5.7 0.978 5.53 241 1606 5.2 0.85 4.43
The daily ET values are then calculated by multiplying daily ETr by daily Kc values as shown in
Equation 8.1. Note that even though in Table 8.2 the end temperature is shown as 32°frost, the
actual maturity and end of growing season occur on day 241 of the year when the cumulative
GDD reaches the 1603°F value, which is the required GDD for maturity. In this example, the
length of growing season for grain corn is 122 days (from day 120–241). The same crop will reach
maturity in only 106 days in Las Cruces, New Mexico, due to higher daily temperature. The grow-
ing season will also vary depending on when the crop is planted. If a crop is planted later than day
120, then it will mature at a later day, but most likely at a shorter growing season.
8.2.3 Irrigation Interval
One of the main questions to be answered in an irrigation management is when to irrigate. In drip,
and sprinkler irrigation, it is possible to irrigate every day. However, from practical, agronomic, and
economic points of view, the best time of irrigation is when the readily available water (RAW) is
consumed. One can calculate the RAW by having the knowledge of soil type, root depth, and crop
management allowed depletion known as MAD. To calculate RAW, one needs to know the soil field
capacity (FC) and the crop root depth. The MAD is also a function of crop.
The RAW is calculated as
RAW = MAD ◊(F )(FC)(RD)† (8.4)

where
RAW is the readily available water
MAD is the management allowed depletion from Table 8.9
RD is the root depth
F is the soil factor, which is taken as 0.5 for medium soil, 0.4 for clay soil, and 0.6 for sandy soil
Tables 8.9 and 8.10 show MAD and root depth for various crops. The root depth can vary depending
on the soil type and age of the crop. If not sure, one can always use the lower value of root depth to
calculate RAW and irrigation interval.
TABLE 8.9
Typical Root Depth and MAD Values
for Various Crops
Crop MAD Root Depth (ft) Root Depth (m)
Almond 0.6 3–4 1–1.3
Alfalfa 0.55 3–10 1–3
Banana 0.35 2–3 0.6–0.9
Barley 0.55 3–5 1–1.5
Beans 0.45 1–2 0.3–0.7
Beets 0.5 2–3 0.6–1.0
Cabbage 0.45 1–2 0.3–0.6
Carrots 0.35 1–3 0.3–1.0
Celery 0.20 1–2 0.3–0.5
Citrus 0.5 4–5 1.2–1.5
Clover 0.35 2–3 0.6–0.9
Corn 0.6 3–5 1–1.7
Cotton 0.6 3–5 1–1.7
Cucumber 0.5 2–4 0.7–1.2
Dates 0.5 5–8 1.5–2.5
Flax 0.5 3–5 1.0–1.5
Grains, small 0.55 3–5 1–1.5
Grapes 0.35 3–6 1–1.8
Grass 0.5 2–5 0.6–1.5
Lettuce 0.30 1–2 0.3–0.6
Melons 0.35 3–5 1–1.5
Olives 0.65 4–5 1.2–1.5
Onions 0.25 1–2 0.3–0.6
Peas 0.35 2–3 0.6–1.0
Pepper (Chile) 0.25 2–3 0.6–1.0
Pecan 0.55 3–6 1.0–2.0
Pineapple 0.5 1–2 0.3–0.6
Potatoes 0.25 1–2 0.3–0.6
Pomegranate 0.55 3–6 1–2
Safflower 0.6 3–6 1–2
Sorghum 0.55 3–6 1–2
Soybeans 0.5 2–4 0.6–1.2
Spinach 0.2 1–2 0.3–0.6
Strawberries 0.15 0.5–1.0 0.2–0.6
Sugar beets 0.5 2–4 0.6–1.2
Sugarcane 0.65 4–6 1.2–2
Sunflower 0.45 3–5 1–1.5
Sweet potatoes 0.65 3–5 1–1.5
Tomatoes 0.4 2–5 0.6–1.5
Vegetables 0.2 1–2 0.3–0.6
Wheat 0.55 3–5 1–1.5
TABLE 8.10
Almond Crop Coefficient (Kc) Recommended by UN-FAO
Month Dec/Jan Feb Mar April May June/July/Aug Sep Oct Nov
Kc 0.85 0.85 0.85 0.95 1.05 1.15 1.10 0.90 0.85
with cover crop
Kc 0.00 0.00 0.50 0.70 0.85 0.90 0.80 0.75 0.65
without
cover crop
Example 8.2
Table 8.10 shows the Kc values for mature almond. If the ETr for the month of June is 8 mm/day and
the crop coefficient from Table 8.10 is 0.9, then the ET can be calculated as
ET = kc × ETr = 0.9(8) = 7.2 mm/day
Taking the MAD value of 0.6 and root depth of 1 m from Table 8.9, for a medium soil with an FC
of 0.3 (30%), the RAW can then be calculated as
RAW = 0.6(0.5)(0.3)(1.0) = 0.09 m = 90 mm
That means that the soil can hold 90 mm of water within the root zone of the crop without caus-
ing stress. If the ET is 7.2 mm/day in the month of June, then there is enough water to last 13 days
(90/7.2) before the next irrigation. One factor that can change is the effective rainfall. There are
various ways to calculate the effective rainfall. One of the simplest equations is the USDA/SCS
TR-21 (1970), which is described as
Re = (0.70917 ◊[Rt]0.82416 - 0.11556)◊(100.02426[U ]) (8.5)
where
Re is the effective rainfall
Rt is the monthly rainfall in inches
U is the monthly consumptive use in inches
In this example, if we had 1 in. (25.4) of rainfall during the 13 days, that would be equivalent to
2.3 in./month. Using Equation 8.5, and monthly equivalent of 8.5 in. (216 mm) of ET, the effective
rainfall for the equivalent month is calculated as
Re = 2.08 in. = 52.8 mm
Adjusting the monthly Re for 13 days, the effective rainfall during the 13 days would be 22.9 mm.
This can then prolong the irrigation for another 3 days (22.9/7.2). This results in the total irrigation
interval of 13 + 3 = 16 days.
8.2.4 Alternative Method “Calculating ET without GDD”

In the absence of Kc–GDD relationship shown in Tables 8.3 through 8.7, a simplified approach can
be used to calculate Kc and ET. Allen et al. (1998), in FAO-56, have provided some information
Kc3
Kc4
Kc2
Kc-end
Kc
Kc1
Stages of growth
FIGURE 8.1 Kc versus the stages of growth in a typical crop.
on the stages of growth and the relevant Kc for various climate conditions. Allen et al. (1998) have
divided the stages of growth into four phases of initial, growing stage, midseason, and late season
similar to Figure 8.1. The definition of each stage is as follows:
Stage 1. From planting to 10% ground cover

Stage 2. From 10% ground cover to maximum growth
Stage 3. From maximum growth to the start of maturity
Stage 4. From the start of maturity to maturity or harvest
Allen et al. (1998) have then provided corresponding number of days for each stage and kc values
for initial, midseason, and end date. Table 8.8 shows an example of such data. For growing crops
in a new region or a different planting date, one can construct Kc versus days or GDD similar to
Figure 8.1, by knowing the duration of each growth stage and the corresponding Kc values for Kc1,
Kc3, and Kc-end shown in Table 8.11. However, the information on stages and corresponding kc need
to be reconfigured for a new location or new planting date. Example 8.3 shows how information on
stage and kc can be transferred for a new location or a new planting date.
Example 8.3
Research has shown that tomato requires between 1600 and 1850 GDD with a base tem-
perature of 50°F from planting to maturity depending on the variety. If we intend to plant an
early variety tomato in Las Cruces, New Mexico, the crop can be planted as early as day 83.
TABLE 8.11
Stages of Growth and Kc Values for Typical Crops
Stages (1, 2, 3, 4 in Kc
Crop Days) Total (Days) Kc1 kc3 kc-end
Tomato 35 40 50 30 155 0.6 1.15 0.8
Cantaloupe 30 45 35 10 120 0.5 0.85 0.60
Potato 25 30 45 30 130 0.5 1.15 0.75
Source: Allen, R.G. et al., Crop evapotranspiration: Guidelines for computing

crop water requirements, Food and Agriculture Organization, FAO
Irrigation and Drainage Paper No. 56, Rome, Italy, 1998.
Calculating cumulative GDD, for the area, it shows that the crop will reach maturity on day 176,
a total of 94 days. Looking at Table 8.8, the total growing season is given as 155 days. A simple
way to determine the Kc–days relationship would be to scale the growing season by multiply-
ing the length of each stage by the ratio of (94/155 = 0.605). The new stages of growth for the
tomato will then be calculated as follows:
Stages 1 2 3 4
Days 21 24 31 18
And the corresponding kc for each stage can then be calculated using the Kc values in Table 8.8.
A more accurate relationship can be established by modifying the length of each stage by
observing the duration of the first two stages in the field and calculating the duration of the third
and fourth stages using the remaining days.
8.2.5 Perennial Crops
The crop coefficients for perennial crops such as almond, pecan, or grapes are normally shown
on monthly basis as is shown in Tables 8.10 and 8.12. The growing season in such crops starts
as the minimum beginning temperature is reached and ends when minimum temperature is
reached at the end of season. In perennial crops, the ET values are normally calculated for
growing season, and irrigation is practiced during growing season. However, the perennial crops
never stop using water even during what is called the dormant season. When perennial crops
lose their leaves, their physiological activities slow down but do not cease. During this period,
the Kc reduces to basic Kc, which is typically 0.3–0.40. During this dormant period, the plants
continue to lose water and may need irrigation. In dry climates, such as that of Southwestern
United States, crops like pecans are often irrigated during the dormant season to maintain suf-
ficient moisture in the root zone.
TABLE 8.12
Monthly Kc Values for Grapes Based on Four Climatic
Conditions
Cond. Mar Apr May Jun Jul Aug Sep Oct Nov
Mature grapes grown in areas with killing frost, ground cover 40%–50%
1 — — 0.50 0.65 0.75 0.80 0.75 0.65 —
2 — — 0.50 0.70 0.80 0.85 0.80 0.70 —
3 — — 0.45 0.70 0.85 0.90 0.80 0.70 —
4 — — 0.50 0.75 0.90 0.95 0.90 0.75 —
Mature grapes grown in areas of light frost, ground cover 30%–35%
1 — 0.50 0.55 0.60 0.60 0.60 0.60 0.50 0.40
2 — 0.50 0.55 0.65 0.65 0.65 0.55 0.55 0.40
3 — 0.45 0.60 0.70 0.70 0.60 0.60 0.60 0.35
4 — 0.45 0.65 0.75 0.75 0.75 0.75 0.65 0.35
Mature grapes grown in hot dry areas, ground cover 30%–35%
3 0.25 0.45 0.60 0.70 0.70 0.65 0.55 0.45 0.35
4 0.25 0.45 0.65 0.75 0.75 0.70 0.55 0.45 0.35
Notes: 1—Humid, light to moderate wind; 2—humid, strong wind; 3—dry, light
to moderate wind; 4—dry, strong wind.
REFERENCES
Allen, R.G. (1986). A penman for all seasons. Journal of Irrigation and Drainage Engineering Division ASCE
112(4): 348–368.
Allen, R.G., L.S. Pereira, D. Raes, and M. Smith (1998). Crop evapotranspiration: Guidelines for computing
crop water requirements. Food and Agriculture Organization, FAO Irrigation and Drainage Paper No. 56.
Rome, Italy.
ASCE-EWRI (2005). The ASCE standardized reference evapotranspiration equation. In: Allen, R.G., Walter,
I.A., Elliot, R.L., Howell, T.A., Itenfisu, D., Jensen, M.E., and Snyder, R.L. (eds.), American Society of
Civil Engineers (ASCE) Task Committee on Standardization of Reference Evapotranspiration. Reston,
VA: ASCE, 0-7844-0805-X, 204p.
Hargreaves, G.H. and Z.A. Samani (1982). Estimating potential evapotranspiration. Journal of Irrigation and
Drainage Engineering Division ASCE 108(3): 225–230.
Hargreaves, G.H. and Z.A. Samani (1985). Reference crop evapotranspiration from temperature. Applied
Engineering in Agriculture 1(2): 96–99.
USDA/SCS Engineering Division (April, 1967). Irrigation water requirements. Technical Release No. 21.
Revised September 1970.
Wright, J. (1981). Crop coefficients for estimates of daily crop evapotranspiration. ASAE Irrigation Scheduling
Conference, Chicago, IL.
Part II
Cellular and Molecular Aspects
of Plant/Crop Physiology
9 Biochemistry and Physiology
of Carbon Partitioning
in Crop Plants
Claudia V. Piattoni, Carlos M. Figueroa, Valeria E. Perotti,
Florencio E. Podestá, and Alberto A. Iglesias
CONTENTS
9.1 Carbon Fixation and Photosynthate Partitioning.................................................................. 193
9.1.1 Photosynthetic Carbon Assimilation......................................................................... 194
9.1.1.1 Carboxylation.............................................................................................. 194
9.1.1.2 Reduction.................................................................................................... 196
9.1.1.3 Regeneration............................................................................................... 196
9.1.1.4 Regulation of the Benson–Calvin Cycle..................................................... 196
9.1.1.5 Adaptations of the Carbon Fixation Metabolism........................................ 197
9.1.2 Intra- and Intercellular Photosynthate Partitioning................................................... 198
9.2 Carbon Metabolism in Source Tissues.................................................................................. 199
9.3 Carbon Metabolism in Sink Tissues......................................................................................200
9.4 Crops Producing Different Major Photosynthetic Products.................................................. 201
9.4.1 Starch and Soluble Sugars......................................................................................... 201
9.4.1.1 Starch Metabolism...................................................................................... 201
9.4.1.2 Sucrose Metabolism.................................................................................... 203
9.4.2 Oils and Fatty Acids..................................................................................................205
9.4.3 Sugar Alcohols...........................................................................................................207
9.4.4 Proteins and Amino Acids......................................................................................... 210
9.5 Plant Crops Ahead: Developing Strategies for a Sustainable World..................................... 210
References....................................................................................................................................... 211
9.1 CARBON FIXATION AND PHOTOSYNTHATE PARTITIONING

Carbon fixation is a biological process that converts atmospheric CO2 (and H2O) into organic
carbon compounds (carbohydrates) and O2 due to the photosynthetic capacity of some particular
organisms like plants, algae, and cyanobacteria. For the synthesis of carbohydrates, these organ-
isms have a particular set of enzymes acting in cycle to condensate the CO2 with a five-carbon
acceptor, ribulose-1,5-bisphosphate (Rul-1,5-bisP). This process has high energy demands pro-
vided as ATP and NADPH, energy-containing compounds that are synthesized by harvesting the
solar energy through a specific electron transport chain. The metabolic process generally called
“photosynthesis” (more strictly oxygenic photosynthesis) is used to denote the combined capture of
light and synthesis of reduced organic molecules, mainly carbohydrates, but both steps occurring
by different sets of enzymes (Nelson and Cox 2004). The overall photosynthetic energy balance is
represented in Table 9.1 (Equation 9.1).
193
TABLE 9.1
Equations for Some Steps of Plant Carbon Metabolism
(9.1) 6CO2 + 6H2O + light → C6H12O6 + 6O2 → ∆G0′ = 2870 kJ mol−1
(9.2) 6CO2 + 18ATP + 12NADPH + 12H+ + 12H2O → C6H12O6 + 18Pi + 18ADP + 12NADP+
(9.3) Ga3P + NAD+ + Pi ⇆ 1,3-bisPGA + NADH + H+
(9.4) 1,3-bisPGA + ADP ⇆ 3-PGA + ATP
(9.5) Ga3P + NADP+ + H2O → 3-PGA + NADPH + 2H+
(9.6) ATP + Glc-1P ⇆ ADP-Glc + PPi
(9.7) ADP-Glc + (α-1,4-polyglycan)n → ADP + (α-1,4-polyglycan)n+1
(9.8) (α-1,4-polyglycan)lineal → (α-1,4-polyglycan)branched
(9.9) Glc-1P + UTP ⇆ UDP-Glc + PPi
(9.10) UDP-Glc + Fru-6P ⇆ Suc-6P + UDP
(9.11) Suc-6P → Suc + Pi
(9.12) UDP-Glc + Fru ⇆ Suc + UDP
(9.13) Suc + H2O → Glc + Fru
(9.14) Ga3P + DHAP ⇆ Fru-1,6-bisP
(9.15) Glc-6P + NADPH + H+ ⇆ Gol-6P + NADP+
(9.16) Gol-6P + H2O → Gol + Pi
(9.17) Gol + NAD+ ⇆ Fru + NADH + H+
One particular feature in plants is the occurrence of two different tissue types: photosynthetic
(or autotrophic) and nonphotosynthetic (or heterotrophic). The processes of carbon fixation and
light capture occur only in photosynthetic tissues and specifically inside the chloroplast, an exclu-
sive organelle of such tissues. Because higher plants are also composed of heterotrophic tissues,
photosynthates have to be partitioned not only within the cell, but also between different tissues. In
general, intracellular carbon partitioning occurs between the chloroplast and the cytosol in the form
of triose-phosphate (triose-P), whereas sucrose (Suc) is the main metabolite involved in intercellular
carbon partitioning (Iglesias and Podestá 2005) (Figure 9.1).
9.1.1 Photosynthetic Carbon Assimilation

The reductive pentose phosphate pathway assimilating atmospheric CO2 is generally known as the
Benson–Calvin cycle (BCC, see Figure 9.1a) in honor of Melvin Calvin and Andrew Benson (and
their coworkers), who unraveled this metabolic process more than 60 years ago (Benson and Calvin
1950). This carbon-fixing metabolism, common to all oxygenic photosynthetic organisms, occurs
inside the chloroplast stroma during the day when the photosynthetic electron transport chain is
synthesizing ATP and NAPDH (Leegood 1999b; Martin et al. 2004). Reactions involved in the
BCC can be subdivided into three steps: carboxylation, reduction, and regeneration. The sum of all
steps is represented by the global reaction shown in Table 9.1 (Equation 9.2), clearly indicating the
high amount of energy demanding the synthesis of a molecule of glucose (Glc), provided by light.
9.1.1.1 Carboxylation
In the first step of the BCC, CO2 is combined with the acceptor Rul-1,5-bisP to produce two
molecules of 3-phosphoglycerate (3-PGA) in a reaction catalyzed by the Rul-1,5-bisP carboxylase/
oxygenase (RuBisCO, EC 4.1.1.39) (Quayle et al. 1954). RuBisCO has low carboxylation efficiency
due to its low specific activity and because CO2 atmospheric concentrations are lower than those
required to reach the half of the maximum enzyme activity. This inefficiency is compensated by
plants through the synthesis of high RuBisCO levels, being the most abundant protein (higher than
50% of the total soluble protein) in leaves (Ellis 1979). As many other enzymes, RuBisCO is tightly
regulated. RuBisCO activity works out after a reversible carbamylation reaction occurring between
Biochemistry and Physiology of Carbon Partitioning in Crop Plants 195
Mitochondria
Photosynthetic electron TCA
Light Citrate
transport chain OAA
Malate
+ Pyr
ATP NADPH NAD
Pi ATP
NADH
CO2 3-PGA OAA HCO2
– 2.7.1.40
4.1.1.31 ADP
1.2.1.13 PEP
RuBisCO
BCC 3PGA
Rul-1,5bisP Pi Pi ATP
Ga3P DHAP NADPH
ADP
1,3-bisPGA
TPT NADH
1.2.1.9
Chloroplast 1.2.1.12 NAD++Pi NADP
+
Glc-1P
DHAP Ga3P
Pi Fru-1,6bisP
ADP
Starch 2.7.1.90 2.7.1.11
PPi ATP
Cytosol Fru-6P
Glc–6P
Sucrose
(a)
Phloem
Starch Sucrose
2.4.1.18 3.2.1.26 2.4.1.13
2.4.1.21 GlC Fru UDP UDP-GlC
ADP-Glc ATP PPi
PPi ATP 2.7.7.9
2.7.7.27 2.7.1.1 ADP
UTP
+ ATP Glc-1P
NADP Glc-1P Pi Pi ADP
1.1.1.49 5.4.2.2
NADPH 5.3.1.9
NADP+ Glc-6P HPT Glc-6P Fru-6P Glc-6P
1.1.1.44 Pi 5.3.1.9
Pi Rul-5P
OPPP OPPP
NADPH complete Xul-5P XPT Xul-5P incomplete
Rib-5P
Plastid Triose-P Triose-P
Pyr
PEP PEP
Cytosol
Pyr
Respiration Biosynthesis
Mitochondria
(b)
FIGURE 9.1 Schematic representation of carbon partitioning in plants: (a) carbon metabolism in source
tissues during light periods; (b) carbon metabolism in sink tissues.
a CO2 molecule (a different one from that involved in the carboxylation of Rul-1,5-bisP) and a Lys
residue positioned in the active site of the enzyme (Leegood 1999b; Martin et al. 2004). The activa-
tion reaction is catalyzed by a RuBisCO activase in the presence of Mg2+ (Robinson et al. 1988). In
addition to the carboxylation reaction, RuBisCO can also condensate O2 with the Rul-1,5-bisP accep-
tor molecule producing one molecule of phosphoglycolate and another of 3-PGA. Such oxygenase
activity starts a process called photorespiration, which involves more than 15 enzymes and trans-
porter proteins distributed between chloroplasts, peroxisomes, and mitochondria. Photorespiration
releases one CO2 molecule per two phosphoglycolate synthesized and reduces carbon fixation pro-
ductivity under normal atmospheric conditions (21% O2 and 0.036% CO2) (Douce and Heldt 2004;
Leegood 1999b).
9.1.1.2 Reduction
Following Rul-1,5-bisP carboxylation, the produced 3-PGA is reduced to triose-P (a term used
for both forms, glyceraldehyde-3-phosphate [Ga3P] and dihydroxyacetone phosphate [DHAP]) at
the expense of ATP and NADPH produced by the electron transport chain. First, 3-PGA kinase
(PGKase, EC 2.7.2.3) transfers the γ-phosphate moiety from ATP to the 3-PGA carboxylic group
producing 1,3-bisphosphoglycerate (1,3-bisPGA). In a second step, NADP-dependent Ga3P dehy-
drogenase (Ga3PDHaseA/B, EC 1.2.1.13) reduces 1,3-bisPGA to Ga3P using NADPH and inor-
ganic orthophosphate (Pi). Ga3P and DHAP are interconverted by a triose-P isomerase (TPIase,
EC 4.1.2.13) (Leegood 1999b).
9.1.1.3 Regeneration
In the last step of the BCC, Rul-1,5-bisP is regenerated to restart the pathway. The process needs one
molecule of the previously synthesized Ga3P and ATP. First, aldolase (EC 4.1.2.13) links Ga3P and
DHAP giving fructose-1,6-bisphosphate (Fru-1,6-bisP). The latter is then irreversibly hydrolyzed
by Fru-1,6-bisP phosphatase (FBPase, EC 3.1.3.11) to fructose-6-phosphate (Fru-6P). Thereafter,
transketolase (EC 2.2.1.1) produces erythrose-4-phosphate and xylulose-5-phosphate (Xul-5P) by
transferring two carbons from Fru-1,6-bisP to Ga3P. Then, aldolase acts again to join erythrose-
4-phosphate with DHAP giving sedoheptulose-1,7-bisphosphate, that is irreversibly hydrolyzed to
the respective monosaccharide-7-phosphate by a specific phosphatase (SBPase, EC 3.1.3.37). In
the next reaction, transketolase transfers two carbons from sedoheptulose-7P to DHAP producing
ribose-5-phosphate (Rib-5P) and Xul-5P. Rib-5P is isomerized to ribulose-5-phosphate (Rul-5P)
by a specific isomerase (EC 5.3.1.6), and Xul-5P is converted to Rul-5P by a specific epimerase
(EC 5.1.3.1). Finally, the Rul-1,5-bisP initial molecule is regenerated by irreversible phosphoryla-
tion of Rul-5P in a reaction catalyzed by phosphoribulokinase (PRKase, EC 2.7.1.19) (Iglesias et al.
1997; Leegood 1999b). The global analysis evidences that after the condensation of three CO2 mol-
ecules, one triose-P is generated. This cyclic conversion is autocatalytic, as plants can produce more
acceptor than is consumed until a photosynthetic steady state is reached. In consequence, there is a
net carbon gain allowing the use of triose-P for the synthesis of the final photosynthetic products,
Suc and starch (Iglesias et al. 1997) (Figure 9.1).
9.1.1.4 Regulation of the Benson–Calvin Cycle

The overall photosynthetic metabolism is regulated to coordinate the activity of key enzymes dur-
ing light hours, when photosynthetic organisms can capture the energy from the Sun. Enzyme
regulation controls the metabolic flux through the BCC, thus coupling carbon fixation to ATP and
NADPH production by the electron transport chain. Coordination of metabolism reduces metabolite
fluctuations and unnecessary enzyme activities under changeable environmental conditions (Martin
et al. 2004; Portis 2001). The first signal within the cell indicating the presence of light is reducing
power (NADPH) availability. Part of the NADPH provided by photosynthesis is used by the fer-
redoxin/thioredoxin reductase to reduce a small protein called thioredoxin (TRX) (Buchanan 1980;
Wolosiuk et al. 1993). TRX, ubiquitous to all organisms, pertains to a protein family of disulfide
reductases capable of reducing disulfide bridges (S–S) to the thiol form (–SH) in numerous target
proteins. In such a way, TRX regulates the activity of the target by posttranslational redox mecha-
nisms (Buchanan 1984, 1991; Buchanan et al. 1994). Plants have many TRX isoforms with differ-
ent primary structure, specificity, and subcellular localization. TRX-m, TRX-fa, and TRX-fb are
found inside the chloroplast regulating the photosynthetic process. Many enzymes from the BCC
(Ga3PDHaseA/B, FBPase, SBPase, and PRKase) are known to be regulated by TRXs (Martin et al.
2004; Portis 2001).
The Ga3PDHaseA/B is a heterotetrameric enzyme composed of two A and two B subunits.
The catalytic activity depends on its oligomerization state being modified by redox mechanisms
according to light availability. During light periods, the enzyme in the oligomeric A2B2 (∼150 kDa)
form is highly active, but in the dark, the enzyme aggregates into ∼600 kDa oligomers and loses
enzymatic activity. When light is again available, NADPH is synthesized, TRXs are reduced,
and Ga3PDHaseA/B activated when its quaternary structure A2B2 is restored by TRX reduction
of –S–S– to the –SH from in the ∼600 kDa oligomers. Reduction occurs in the presence of low
1,3-bisPGA concentrations and increases ∼20-fold the enzyme affinity for the substrate 1,3-bisPGA
(Baalmann et al. 1995; Wolosiuk and Buchanan 1976, 1978a). This was the first Benson–Calvin
enzyme where redox regulation of activity according to light/dark transitions was discovered, but is
not the only enzyme in the cycle regulated by this mechanism. The irreversible reactions catalyzed
by FBPase, SBPase, and PRKase are also redox modulated, but in these cases, regulation occurs
on cysteine residues that need to be reduced for catalysis, and this takes place during the day when
reduction equivalents are available.
Regulation of chloroplastic FBPase is carried out by TRX-f in association with increments in
Fru-1,6-bisP concentration. This activation outline is a positive feedback because Fru-1,6-bisP
increases during light periods (Ballicora and Wolosiuk 1994; Ballicora et al. 1998; Chiadmi et al.
1999). As well as FBPase, SBPase is inactive in the oxidized state and its activity is restored by reduc-
tion few minutes after the light cycle begins and TRXs are reduced. It has been shown that TRX-fa, -fb,
and -m can reduce and activate the enzyme in vitro (Cadet and Meunier 1988; Hutcheson and
Buchanan 1983). Also, PRKase is activated in vivo by TRX-dependent reduction during light. The
redox activation process is dependent of the substrate (Rul-5P) presence, and once activated, the
enzyme becomes sensitive to regulation by assorted metabolites (Wolosiuk and Buchanan 1978b).
Besides the fact that redox modulation of enzyme activity is a critical regulatory process, other
signals also indicate the presence of light in the chloroplast stroma. One is pH increment in such
compartment: during dark periods pH is near 7.0, while during the light period it approximates to
8.0. Another change, occurring in consequence, is the increment in Mg2+ concentrations. The men-
tioned changes take place due to proton influx into the thylakoids’ lumen, which is associated with
an efflux of Mg2+ ions (Heldt et al. 1973). It has been reported that the activity of RuBisCO, FBPase,
and SBPase increases in the light, since their optimal pH is near 8.0 or because they use Mg2+ as a
cofactor (Portis and Heldt 1976; Portis et al. 1977; Werdan et al. 1975).
9.1.1.5 Adaptations of the Carbon Fixation Metabolism

The oxygenase activity of RuBisCO is highly costly for the photosynthetic productivity, being the
CO2/O2 relationship inside the chloroplast stroma a critical factor determining the plant carbon fixa-
tion efficiency (Iglesias et al. 1997; Portis 2001). An increase of 30%–40% in photosynthesis has
been estimated if photorespiration could be abolished. Another key issue in plants is to balance the
water loss occurring through the stomata during the CO2 intake (Portis 2001). To increase
the efficiency during carbon fixation, some plants have developed additional systems. According to
the type of photosynthetic carbon fixation process, higher plants are classified into C3, C4, and those
performing crassulacean acid metabolism (CAM). The BCC is common to all types, but additional
routes operate separately in space or time in C4 or CAM plants, respectively.
In order to bypass photorespiration, in C4 plants, the step of atmospheric CO2 fixation occurs
in a different place than the BCC. The C4 metabolism is related to the particular anatomy of C4
plant leaves, characterized by the presence of two photosynthetic cell types: mesophyll and bundle-
sheath. These different cells are disposed semiconcentrically with mesophyll cells in the outer and
bundle-sheath cells in the inner circle, an organization known as kranz anatomy. Assimilation
of atmospheric CO2 occurs in mesophyll cells, while the BCC functions in bundle-sheath cells
(Leegood 1999a). During light periods, C4 plants fix CO2 (under the form of bicarbonate) by com-
bining it with the three-carbon acceptor phosphoenolpyruvate (PEP), producing oxaloacetic acid
(OAA) in a carboxylation reaction catalyzed by PEP carboxylase (PEPCase, EC 4.1.1.31) (Iglesias
et al. 1997). The four-carbon product OAA is then metabolized to malate or an amino-acid,
which is transported to be decarboxylated in bundle-sheath cells, rendering CO2. The additional
carbon-fixing route is completed to constitute a cycle by regenerating PEP by the reaction of pyru-
vate phosphate dikinase present in mesophyll cells. Carbon fixation by PEPCase is more efficient
compared to RuBisCO because PEPCase cannot accept O2 as substrate and photorespiration is
notably diminished. In C4 plants, RuBisCO remains isolated from O2 and saturated with the CO2
released by the decarboxylative step of the C4 metabolism, which operates as a pump driving CO2
from the atmosphere to the chloroplasts of bundle-sheath cells (Iglesias and Podesta 2008).
Concerning CAM, it is a photosynthetic adaptation allowing plants to efficiently assimilate
carbon in arid environments, avoiding water lost during CO2 intake. CAM plants fix CO2 into
four-carbon compounds (through carboxylation of PEP by PEPCase) like C4 plants, although the
process occurs during the night and in a single photosynthetic cell. In this way, CAM operates sepa-
rated in time with the BCC (Ranson and Thomas 1960). By this way, stomata for CO2 intake are
open during the night in CAM plants, when the loss of water by transpiration is practically avoided.
At this period, CO2 is fixed and accumulated as malate into the vacuole. During the day, when ATP
and NADPH are photosynthesized, RuBisCO can condensate the CO2 released through malate decar-
boxylation by the action of the NADP-dependent malic enzyme (NADP-ME, EC 1.1.1.40), the latter
occurring when the stomata are completely closed (Leegood 1999a; Portis 2001).
9.1.2 Intra- and Intercellular Photosynthate Partitioning

In plants, photosynthesis takes place mainly in leaves, and the photosynthetic machinery is exclu-
sively located inside the chloroplast. This is the place of triose-P de novo synthesis, the source for the
major products of the whole autotrophic process (Suc and starch). Inside the chloroplast, the main fate
of triose-P is starch synthesis as a transitory reserve of carbon and energy. Alternatively, triose-P are
exported to the cytosol to serve for Suc synthesis or to supply carbon for energetic cell metabolism
demands (Figure 9.1a). With some exceptions, starch is the principal energy reserve compound accu-
mulated in photosynthetic and heterotrophic tissues. Starch storage in leaves is transitory and con-
centrations are highly variable during the day, whereas long-term storage is restrained to specialized
organs like tubers or seeds (within the amyloplast). Besides being the principal accumulated product,
starch constitutes a stationary carbon reserve and plants need to mobilize the assimilated carbon
among different tissues. This generally arises through the transport of Suc, which constitutes the
mobile carbon reserve (Iglesias and Podestá 2005) (see Figure 9.1). For the synthesis of these major
carbohydrates (starch and Suc), carbon metabolites produced by photosynthesis (photosynthates)
need to be partitioned inside the cell among different compartments and then among different tissues.
Plants, as highly evolved multicellular organisms, have distributed different functions in special-
ized organs. Leaves are responsible for carbon assimilation (source tissues), whereas nonphoto-
synthetic organs specialize in different functions. Thus, roots are dedicated to water and minerals
uptake and other tissues (sink tissues) like seeds or tubers mainly accumulate reserve biomolecules.
In any case, communication of all organs and metabolites interexchanged between source and sink
tissues is critical for the organism. To allow compounds to move among tissues, plants have devel-
oped two kinds of transport conducts: the xylem (with the function of water and mineral transport
from roots to organs like leaves and stem) and the phloem (transporting nutrients like Suc from the
leaves to organs like roots, shoots, reproductive organs, and other heterotrophic tissues) (Lalonde
et al. 2004). Once in the tissue, exchanged metabolites enter the cells by transporter proteins pres-
ent in the cell membrane. Each kind of organ has different transporter proteins to interconnect
not only the cells, but also different cell compartments, which is critical to coordinate the whole
metabolism. Inside the cell, the molecules are metabolized and further partitioned intracellularly to
supply carbon and energy demands. Intracellular metabolite partition is an example of how superior
organisms have evolved to increment the control of metabolic pathways, allowing the simultaneous
operation of metabolic fluxes competing for the same substrates and avoiding the occurrence of
futile cycles (Iglesias and Podestá 2005; Lunn 2007).
9.2 CARBON METABOLISM IN SOURCE TISSUES

During the light periods, when photosynthesis reaches the stationary phase, triose-P not destined
to the acceptor regeneration in the BCC are available to be converted in the final photosynthetic
products. Approximately 30% of the fixed carbon is used for starch synthesis inside the chloroplast,
which is transitorily accumulated to provide carbon and energy resources for the upcoming night
(Iglesias and Podestá 2005; Portis 2001). Starch synthesis within the chloroplast mainly occurs from
Fru-6P, which is converted to Glc-6P by a specific isomerase (EC 5.3.1.9) and to Glc-1P through
phosphoglucomutase (EC 5.4.2.2), generating a hexose-P pool near equilibrium. Glc-1P is used for
starch synthesis by the metabolic pathway involving ADP-Glc pyrophosphorylase (ADP-Glc PPase,
EC 2.7.7.27), starch synthase (EC 2.4.1.21), and branching enzyme (EC 2.4.1.18) (Ballicora et al.
2004; Iglesias and Podestá 2005) (see details of this metabolic pathway under Section 9.4; see also
Figure 9.1 and Table 9.1, Equations 9.6 through 9.8).
Alternatively, triose-P forms photosynthetically synthesized are transported to the cytosol via a
transporter protein in interexchange with Pi. The triose-P transporter (TPT) is located in the inner
chloroplastic membrane and is found mainly in photosynthetic cells (Figure 9.1a). TPT is specific for
triose-P and 3-PGA, and cannot transport PEP, pentose-P, or hexose-P (Tegeder and Weber 2007;
Toyota et al. 2006). Once in the cytosol, triose-P can be further metabolized to produce hexose-P
by gluconeogenesis or used to obtain energy and the building blocks necessary for cell maintenance
through glycolysis. Even more, hexose-P produced by gluconeogenesis could also be partitioned
between the oxidative pentose phosphate pathway (OPPP, discussed later in Section 9.3) and the
pathway synthesizing Suc (gluconeogenesis and Suc synthesis are detailed in point 9.4.1.2). Suc
produced from hexose-P is then exported to the phloem and transported to other tissues (Figure 9.1).
If Suc synthesis exceeds Suc export, the metabolite is accumulated into the vacuole to feed carbon
and energy demands during the night, when photosynthesis is not active.
In photosynthetic tissues, during the day, cytosolic glycolysis starts from triose-P. Ga3P
can be further metabolized to 3-PGA by two different Ga3P dehydrogenases (Givan 1999).
Through the classic glycolytic pathway, the process occurs by two consecutive enzymes (Iglesias
1990; Plaxton 1996). First, the reversible NAD-dependent Ga3P dehydrogenase (Ga3PDHase,
EC 1.2.1.12) oxidizes Ga3P with coupled phosphorolysis to generate NADH and 1,3-bisPGA
(Table 9.1, Equation 9.3). The latter, a high-energy content compound, allows phosphorylation
at the substrate level (Table 9.1, Equation 9.4) catalyzed by a cytosolic PGKase. The alternative
pathway is performed irreversibly in one step by oxidation tied to hydrolysis (rather than phos-
phorolysis) mediated by the nonphosphorylating NADP-dependent Ga3PDHase (np-Ga3PDHase,
EC 1.2.1.9) to generate NADPH (Table 9.1, Equation 9.5) (Figure 9.1a). The parallel occurrence
of both routes sets out an important difference for cell energetics: depending on the relative
activity level of each triose-P dehydrogenase, the oxidation of Ga3P to 3-PGA will provide dif-
ferent amounts of energy (ATP) and/or reducing power (NADPH) in the cytosol (Iglesias 1990;
Plaxton 1996). In fact, it was shown that Ga3P partitioning between Ga3PDHase and np-Ga3P-
DHase would change under different metabolic scenarios, with a different balance in energy
or reducing power synthesis. Posttranslational regulation by redox mechanisms would fine-tune
the fate of Ga3P, where oxidative conditions will toward Ga3P metabolism via np-Ga3PDHase,
which would increase the synthesis of reducing power, favoring antioxidant system’s action to
cope with the oxidative situation (Piattoni et al. 2013).
Metabolism of 3-PGA continues to be converted into 2-PGA by a phosphoglyceromutase (EC
5.4.2.1). Next, enolase (EC 4.2.1.11) dehydrates and redistributes the energy of the 2-PGA molecule
elevating the phosphate group from position 2 to highest energy state producing PEP (Nelson and
Cox 2004). PEP metabolism presents a variety of alternatives in the cytosol of plant cells. Classically,
PEP is transformed to pyruvate (Pyr) by Pyr kinase (PKase, EC 2.7.1.40) in a reaction that pre-
serves the energy as ATP. Alternatively, PEP can be metabolized by PEPCase in a carboxylation
reaction using bicarbonate and producing OAA, which is further metabolized to malate by an
NAD-dependent malate dehydrogenase (MDHase, EC 1.1.1.37). The synthesized malate is then
transported to the mitochondria or plastids, where it is converted to Pyr by the action of the NAD-
(EC 1.1.1.39) or NADP- (EC 1.1.1.40) ME (Plaxton 1996) (Figure 9.1a). The glycolytic bypass
produced by PEPCase/MDHase/ME is of great importance during Pi starvation, when PKase activ-
ity is limited by the low ADP availability (Plaxton 1996). In fact, it was shown that synthesis of
PEPCase is induced in response to stresses like Pi starvation or cold. PEP can also be transported
into the vacuole and can be dephosphorylated by the PEP phosphatase (PEPase, EC 3.1.3.60). The
last alternative already known to PEP metabolism is PEP carboxykinase (PEPCKase, EC 4.1.1.49),
which catalyzes a reversible reaction producing OAA, but probably this enzyme is more related to
gluconeogenesis than glycolysis (Iglesias and Podesta 2008). Glycolysis produces Pyr (that enters
into the mitochondria to feed cell respiration by the tricarboxylic acid cycle), ATP, reducing power
[NAD(P)H], and building blocks for anabolic reactions like amino acid, protein, organic acid, and
lipid syntheses, supporting cell energetics and carbon demands for growth and maintenance.
9.3 CARBON METABOLISM IN SINK TISSUES

In nonphotosynthetic tissues, carbon is supplied by Suc transported from leaves through the phloem
(Figure 9.1b). Once loaded into the tissue, Suc enters the cell by transporter proteins and provides
the carbon needed for respiration and biosynthesis. In heterotrophic tissues, it can be considered
that glycolysis starts with Suc cleavage into the composing monosaccharides or their derivative
products. There are two different alternatives: invertases (EC 3.2.1.26) split Suc into Glc and Fru,
while sucrose synthase (SucSase, EC 2.4.1.13) uses UDP to produce UDP-Glc and Fru (these
metabolic steps are detailed in Section 9.4) (Winter and Huber 2000). Intracellular partitioning of
Suc depends on the type of tissue and its developmental stage. It was reported, for example, that
legume-developing seeds use invertase to split Suc during the cell division phase, when high energy
demands and biosynthesis are associated with elevated mitotic activity. Instead, when cell division
ends, a transition phase triggers the synthesis of reserve compounds (mainly starch and oils), and
Suc metabolism changes to cleavage by SucSase (Weber et al. 2005).
Depending on the enzyme responsible for Suc cleavage, the products are metabolized by differ-
ent ways. Glc and Fru are phosphorylated to produce Glc-6P and Fru-6P by hexokinase (EC 2.7.1.1).
UDP-Glc is converted into Glc-1P and UTP by UDP-Glc pyrophosphorylase (UGP-Glc PPase,
EC 2.7.7.9), and Glc-1P is then converted to Glc-6P by a cytosolic phosphoglucomutase. Once Glc-6P
is produced, it can be partitioned between many pathways; for instance, in the cytosol, Glc-6P can be
further metabolized by glycolysis (described later) or by the OPPP (Figure 9.1b).
Following the glycolytic route, Fru-6P is phosphorylated in carbon 1 to produce Fru-1,6-bisP. In
the cytosol of plants, this reaction is alternatively catalyzed by an ATP- (ATP-PFKase, EC 2.7.1.11)
or a PPi-dependent phosphofructokinase (PPi-PFKase, EC 2.7.1.90) (Plaxton 1996). ATP-PFKase is
the classic glycolytic enzyme and is a key step precisely regulated to control the quantity of Fru-6P
following glycolysis, while PPi-PFKase (present only in plants, other photosynthetic organisms, and
several microorganisms) is regulated in a different way and constitutes a bypass to this critical step
(Iglesias and Podesta 2008) (Figure 9.1b). ATP and AMP are not critical effectors of plant ATP-
PFKase, thus adenine nucleotides (hence energy state) do not play a prominent role in regulating
plant glycolysis at this level, like in other nonplant organisms. Instead, regulation of plant glycolysis
is exerted by PEP, Pi, Fru-2,6-bisP, and tricarboxylic acid cycle intermediates. All ATP-PFKase
studied so far showed potent inhibition by PEP, and this inhibition is released by the activator Pi;
in contrast, Pi is a potent inhibitor of PPi-PFKase in the forward direction (Plaxton 1996). Thus,
the Pi/PEP ratio would be critical to regulate Fru-6P metabolism by these two enzymes. Also,
Fru-2,6-bisP is an important regulator of Fru-6P partition, as it activates PPi-PFKase but have no
effect on plant ATP-PFKase. Synthesis of Fru-2,6-bisP is also regulated by the cytosolic Pi/3-PGA
ratio, being Pi an activator and 3-PGA and PEP inhibitors of 6-phosphofructo-2-kinase (the enzyme
producing Fru-2,6-bisP) (Plaxton 1996).
9.4 CROPS PRODUCING DIFFERENT MAJOR PHOTOSYNTHETIC PRODUCTS

9.4.1 Starch and Soluble Sugars
9.4.1.1 Starch Metabolism
Living organisms have developed the capacity of storing carbon and energy as α-1,4-polyglucans.
Animals, fungi, and bacteria (including cyanobacteria) accumulate glycogen, whereas green algae
and plants utilize starch as Glc storage polymer (Ballicora et al. 2004). As stated before, starch syn-
thesis and degradation in leaves is a dynamic process that occurs within the chloroplasts. During
light hours, triose-P forms produced as a result of the photosynthetic carbon fixation are in part
used for synthesizing starch, which accumulates during the light period in relatively small insoluble
granules. In the dark period, starch is actively degraded and used to provide carbon to the whole
plant (Smith and Stitt 2007). In developmental stages, storage tissues (such as tubers and fruits) also
synthesize starch (within plastids). Then, during the germination process (in tubers) or maturation
(in fruits), starch is degraded, and the resulting metabolites are used as carbon and energy sources.
Thus, the processes of starch biosynthesis and degradation in these tissues are temporarily sepa-
rated (Ballicora et al. 2004).
The starch granules may vary in size, shape, and composition depending on the plant and tissue.
In storage tissues, the amount of starch can be really high: for instance, the starch content in potato
tubers, maize endosperm, and sweet potato roots represents between 65% and 95% of the dry mat-
ter (Ballicora et al. 2004). Two different types of Glc polymers can be distinguished within starch:
amylose and amylopectin, which are distributed within the granule in such a way that their reducing
groups are localized inside, whereas the opposite nonreducing ends are orientated to the outside.
Both molecules are composed of Glc molecules polymerized by α-1,4 bonds. Amylose is essentially
linear with few α-1,6 bonds, while amylopectin is highly branched with α-1,6 bonds, localized in
every five to six Glc moieties (Buleon et al. 1998).
The synthesis of starch in plants occurs by using ADP-Glc as glycosyl donor for the elongation
of a preformed α-1,4 chain. ADP-Glc is synthesized from ATP and Glc-1P in a reaction cata-
lyzed by ADP-Glc pyrophosphorylase (ADP-Glc PPase, EC 2.7.7.27; Table 9.1, Equation 9.6), in
the presence of a divalent cation (Mg2+). This reaction is freely reversible in vitro, but ADP-Glc
consumption for the synthesis of the polyglucan and PPi hydrolysis by an inorganic pyrophos-
phatase turns it almost irreversible in vivo (Ballicora et al. 2004). Starch synthesis is completed
with the reactions catalyzed by starch synthase (EC 2.4.1.21; Table 9.1, Equation 9.7) and branch-
ing enzyme (EC 2.4.1.18; Table 9.1, Equation 9.8). Different isoforms of starch synthase and
branching enzyme have been reported, which might play differential roles for the synthesis of
amylose and amylopectin (Ball and Morell 2003).
ADP-Glc PPase is an allosteric enzyme regulated by key metabolites from the main carbon
assimilatory pathway of the respective organism. These effectors represent signals of high or low
carbon and energy content within the cell. Also, as ATP is one of the substrates of the enzyme, it is
expected that starch accumulation in plants would be maximal when carbon and energy are in excess,
and vice versa (Ballicora et al. 2004). Numerous studies demonstrated the relevance of the ADP-Glc
PPase regulatory mechanisms for carbohydrate metabolism in different organisms. For instance, the
Arabidopsis thaliana TL25 mutant, which is deficient in both ADP-Glc PPase subunits, accumu-
lates only 2% of the starch that is usually found in normal plants (Lin et al. 1988a), indicating that
starch synthesis depends almost exclusively on ADP-Glc synthesis. In addition, the A. thaliana TL46
mutant, deficient in the ADP-Glc PPase regulatory subunit, showed abnormal starch accumulation
and possessed only 7% of the ADP-Glc PPase activity compared to that observed in wild-type plants
(Lin et al. 1988b). More recently, a study realized with the A. thaliana aps1 mutant, which has neither
ADP-Glc PPase activity nor starch, demonstrated that it is possible to overcome these characteristics
by transformation with an inactivated form of the aps1 gene, implying that some regulatory subunits
also have activity in vivo (Ventriglia et al. 2008).
9.4.1.1.1 Allosteric Regulation of ADP-Glc PPase

As previously mentioned, most ADP-Glc PPases characterized so far are allosterically regulated.
Considering their affinity for allosteric effectors and quaternary structure, these enzymes have been
classified into nine different classes (Ballicora et al. 2004). In plants, this enzyme is usually acti-
vated by 3-PGA and inhibited by Pi; however, four patterns of 3-PGA/Pi interaction can be dis-
tinguished. The first one, observed for most enzymes, is that where 3-PGA and Pi affect enzyme
activity separately and where increasing concentrations of 3-PGA might overcome Pi inhibition
(for instance, in the potato tuber enzyme) (Ballicora et al. 1995). Another pattern is the one observed
in ADP-Glc PPases from sink tissues of some cereals, which possess special regulatory properties.
The enzymes from peanut embryos, barley endosperm, pea cotyledons, and wheat endosperm are
relatively insensitive to 3-PGA. However, a complete characterization of the wheat endosperm
enzyme showed that it is regulated by several metabolites. This enzyme is inhibited by Pi, ADP, and
Fru-1,6-bisP, and this inhibition can be reverted by 3-PGA and Fru-6P, which have no effect in the
absence of the inhibitors (Gomez-Casati and Iglesias 2002). Consequently, this enzyme is affected
by the 3-PGA/Pi ratio in a particular way.
A third class of 3-PGA activation and Pi inhibition is that reported for enzymes from leaves of
CAM plants (Singh et al. 1984) and maize endosperm (Plaxton and Preiss 1987). These enzymes
are activated between 10- and 25-fold by 3-PGA, but in the absence of the activator, they are not
sensitive to the inhibitor Pi. The last pattern of interaction among allosteric regulators is observed
in the barley endosperm enzyme, which is poorly activated by 3-PGA or inhibited by Pi. However,
3-PGA lowers the Km for ATP up to threefold, and at low ATP concentrations, the activation by
3-PGA is fourfold and Pi can revert the effect (Kleczkowski et al. 1993). Thus, in this enzyme, the
effects of 3-PGA and Pi could be important for increasing or lowering the apparent affinity for the
substrate ATP.
9.4.1.1.2 Redox Regulation of ADP-Glc PPase

ADP-Glc PPase from potato tuber has an inter-subunit disulfide bridge that links both small sub-
units through the Cys12 residue. It has been demonstrated that the enzyme can be activated by
reducing this bridge. Both reduced TRX-f and -m from spinach leaves activate the enzyme in pres-
ence of micromolar concentrations of 3-PGA (Ballicora et al. 2000). This Cys residue is well con-
served in ADP-Glc PPases from several plant species, and it has been suggested that reduction plays
a key role in the fine-tuning of ADP-Glc PPase activity in photosynthetic tissues. Recently, Hadrich
et al. (2012) proved the importance of Cys81 (analog to Cys12) in the enzyme from A. thaliana for
the accumulation of transitory starch in leaves.
The relevance of TRX regulation in the potato tuber ADP-Glc PPase is less clear. It is feasible
that a TRX isoform could be located in amyloplasts, because these are widely distributed proteins
that are located in different subcellular compartments, including cytosol, mitochondria, chloroplast,
and nucleus (Buchanan and Balmer 2005). The question that arises is which is the reducing power
that drives regulation by TRX in these tissues? Even though the photosynthetic electron transport
chain is not found in amyloplasts, the oxidative pentose-P pathway can generate reducing power in
the form of NADPH in a light-independent process. This NADPH can reduce a ferredoxin isoform
present in the plastids of heterotrophic cells, thus transferring reducing power to other enzymes.
It has not been proved if these systems are operative; however, dithiothreitol (DTT) stimulates
starch accumulation in potato tuber disks (Tiessen et al. 2002), suggesting that the reductive activa-
tion of ADP-Glc PPase observed in vitro has a regulatory role in vivo. Recently, Michalska et al.
(2009) showed that an unusual plastid-localized NADP-TRX reductase C (NTRC) plays a key role
in the regulation of ADP-Glc PPase. In the presence of NADPH, NTRC activated ADP-Glc PPase
in vitro. Moreover, an A. thaliana mutant in the ntrc gene showed decreased levels of both reduced
ADP-Glc PPase and starch in leaves and roots. These results confirmed the importance of redox
regulation for starch synthesis in vivo (Michalska et al. 2009).
In recent years, several studies demonstrated that TRX-mediated ADP-Glc PPase activation
in photosynthetic tissues is strongly related with the levels of the regulatory signal trehalose-6P
(Tre-6P) (Smith and Stitt 2007). A. thaliana plants with altered levels of Tre-6P, alternatively trans-
formed with the genes coding for Tre-6P synthase and Tre-6P phosphatase, showed differences in
the fraction of reduced (more active) small subunit of the ADP-Glc PPase. This fact was directly
correlated with higher or lower levels of starch, respectively, which demonstrates the physiological
relevance of the finding (Kolbe et al. 2005). Even though many efforts have been done to elucidate
the mechanism underlying the regulation of starch accumulation by Tre-6P, it is far from being fully
understood.
9.4.1.2 Sucrose Metabolism
Suc plays key roles in plants: (1) it is the major form by which carbon is transported from source to
sink tissues; (2) it is used for carbon accumulation in many plants; and (3) it acts as a gene expres-
sion regulator. Suc is an appropriate molecule for transporting and storing photosynthetically fixed
carbon due to its high solubility in water and its nonreducing properties (Winter and Huber 2000).
Suc is synthesized from hexose-P by the combined action of three cytosolic enzymes: UDP-Glc
PPase (Table 9.1, Equation 9.9), Suc-6-phosphate (Suc-6P) synthase (EC 2.4.1.14, Equation 9.10), and
Suc-6P phosphatase (EC 3.1.3.24, Equation 9.11). UDP-Glc can be used in other metabolic routes,
like cellulose synthesis; thus, UDP-Glc PPase is not specific for the Suc synthesis pathway. Suc-6P
synthase catalyzes a reversible reaction (Table 9.1, Equation 9.10), and its direction would be deter-
mined by the relative concentrations of the reactants. The activity of Suc-6P phosphatase exerts a
critical influence in the direction of the Suc-6P synthase reaction, as the hydrolytic step is essentially
nonreversible (Table 9.1, Equation 9.11). The phosphatase activity ensures a low Suc-6P concentra-
tion and makes the whole process almost irreversible toward Suc production in vivo, even at low
concentrations of UDP-Glc and Fru-6P. In addition, Suc-6P synthase is precisely regulated, which
increases the complexity of the disaccharide synthesis (Winter and Huber 2000).
Another enzyme capable of catalyzing the synthesis of Suc is SucSase (Table 9.1, Equation 9.12),
which is mainly localized in the cytosol. However, the differential spatial distribution of this and
other enzymes in different plant tissues suggests that Suc-6P synthase is responsible for the syn-
thesis of Suc, whereas SucSase plays a major role in the degradation of the disaccharide. In Suc-
synthesizing tissues, the activity of Suc-6P synthase is higher than that of SucSase. Conversely, the
activity of SucSase is usually higher in nonphotosynthetic tissues (Winter and Huber 2000). Also,
Suc can be degraded by different invertase isoforms (Table 9.1, Equation 9.13), which generate Glc
and Fru. Invertases are classified based on the optimal pH of reaction and the intracellular local-
ization. Alkaline invertases have maximal activity at pH 7.0 and are localized in the cytosol; acid
invertases have an optimal pH near 5.0 and can be found within the vacuole or outside the plasma
membrane (Winter and Huber 2000).
Albeit Suc is mostly synthesized in leaf cells, it can also be produced in other tissues, photosyn-
thetic or not, through different pathways. For example, in germinating castor oil seeds, most stor-
age lipids are converted into PEP through the glyoxylate cycle and associated reactions; afterward,
PEP can be derived to Suc synthesis for exporting carbon from the seed to developing tissues.
In other seeds, starch degradation generates Glc-1P and Glc, which can directly enter the hexose-P
pathway to provide a carbon source for Suc synthesis (Smith 1999). In mature leaves, most triose-P
produced during photosynthesis are converted into Suc and transported through the phloem to
heterotrophic tissues. However, during the light period, the rate of Suc synthesis usually exceeds
that of phloem loading. High Suc concentrations are supported in the cytosol, though it is in part
translocated and stored into the vacuole. During the next dark phase, most of this Suc will disappear
from the source leaf. A part is utilized to sustain biosynthetic reactions and cell respiration, whereas
another fraction is transported to heterotrophic plant tissues. Regardless of the destiny, Suc does
not remain within the leaf. This type of storage acts as a photosynthetic buffer, ensuring the highest
photosynthetic rate when Suc cannot be exported quickly enough outside the cell. In certain plant
species, Suc also accounts for long-term carbon accumulation. For example, sugar cane and sugar
beet store Suc in the vacuole of specialized nonphotosynthetic cells (Smith 1999).
9.4.1.2.1 Allosteric Regulation of Suc Metabolism

Suc is one of the major photosynthetic products in plants. Thus, several mechanisms coordinate
Suc synthesis in the cytosol with photosynthate transport from the chloroplast and demands from
nonphotosynthetic tissues. Plants need to maintain a balance between the reductive pentose-P path-
way (the BCC) and Suc and starch syntheses. Altogether, these processes are regulated in PEP
and such a way that the highest photosynthetic rates are maintained while carbon is processed and
assimilated. Several regulatory mechanisms are involved, including the modulation of the activity
of the cytosolic enzymes FBPase (cFBPase) and Suc-6P synthase.
Triose-P forms exported to the cytosol are used for synthesizing Fru-1,6-bisP in a reaction
catalyzed by aldolase (Table 9.1, Equation 9.14). Then, the activity of cFBPase renders Fru-6P,
which can also occur through the action of PPi-PFKase. However, the activity of the latter would
not be relevant for Suc synthesis because it is more significant in the glycolytic pathway (Plaxton
1996). The activity of cFBPase is controlled by several metabolites, including AMP, Pi, and
Fru-2,6-bisP. Triose-P may also alter the activity of cFBPase, not directly but affecting the con-
centration of the regulatory molecule Fru-2,6-bisP. At nanomolar concentrations, Fru-2,6-bisP
inhibits cFBPase by diminishing its affinity toward Fru-1,6-bisP and increasing its sensitivity
to the inhibitors AMP and Pi (Stitt and Heldt 1985; Stitt et al. 1985). Thus, a high level of
Fru-2,6-bisP will inhibit Suc synthesis. The concentration of this metabolite is tightly modulated
by the relative activities of Fru-6P 2-kinase (EC 2.7.1.105) and Fru-2,6-bisP 2-phosphatase (EC
3.1.3.46), generally present as a bifunctional enzyme. The kinase activity is inhibited by t riose-P
and activated by Pi, whereas the phosphatase activity is subject to product inhibition (Pi and
Fru-6P) (Macdonald et al. 1989).
High cytosolic concentrations of triose-P will have several effects in promoting their usage:
(1) by releasing the inhibition of cFBPase by Fru-2,6-bisP through the inhibition of the Fru-6P
2-kinase; (2) by increasing the substrate of cFBPase because the cytosolic conversion of triose-P
into Fru-1,6-bisP is close to equilibrium in vivo; and (3) an increase in Suc synthesis will drive to
a higher Pi release, thus favoring Fru-2,6-bisP degradation. Conversely, accumulation of hexose-P
in the cytosol will lower Fru-1,6-bisP utilization because Fru-6P activates Fru-6P 2-kinase, which
restricts the metabolic flux in the direction of Suc synthesis (Smith 1999). Studies performed with
mutant plants with altered levels of Glc-6P isomerase have shown that increased Fru-6P levels
agree with a higher Fru-2,6-bisP content and a the partition of carbon towards starch. As a conse-
quence of its activity over cFBPase and because its concentration depends on the level of triose-P
exported from chloroplasts, Fru-2,6-bisP acts as a signal of the amount of carbon available for
Suc synthesis (Smith 1999). Additionally, Suc synthesis is regulated at the Suc-6P synthase level.
This enzyme is allosterically activated by Glc-6P and inhibited by Pi. During the photoperiod, the
level of hexose-P in the cytosol increases, whereas Pi decreases after its transport into the chloro-
plast. The resulting effect is a higher Glc-6P/Pi ratio, which stimulates Suc-6P synthase activity
(Winter and Huber 2000).
9.4.1.2.2 Regulation of Suc Metabolism by Protein Phosphorylation

The activity of Suc-6P synthase can be modulated by phosphorylation. It has been described that
this enzyme is phosphorylated in several Ser residues in vivo, with different physiological implica-
tions. One of them is related to the light/dark regulation of the enzyme. In spinach leaves, Ser158
is involved in the inactivation observed during dark hours. This residue is highly conserved among
the enzyme from different species. Its phosphorylation affects Suc-6P synthase activity by lowering
the affinity toward substrates and effectors, with no alteration of the maximal velocity (Winter and
Huber 2000). A second phosphorylation site in the spinach leaf enzyme is Ser424, which could be
important under osmotic stress conditions. Phosphorylation of this highly conserved site activates
the enzyme, probably by competing with the other phosphorylation site (Ser158) and thus allow-
ing Suc synthesis to occur under situations where it would be restricted (Winter and Huber 2000).
Other studies have shown that 14–3–3 regulatory proteins can associate with Suc-6P synthase from
spinach leaves in the presence of Mg2+. In this case, the proposed interaction site is the phosphory-
lated Ser229. The interaction between these two proteins was evidenced by coimmunoprecipitation
and coelusion in gel filtration chromatography. The resulting effect of 14–3–3 binding to Suc-6P
synthase is a lower activity, even at saturating substrate concentrations (Winter and Huber 2000).
However, the physiological role for this interaction is still unknown.
The activity of Suc synthase is not affected by metabolites; therefore, it was first suggested
that its activity depends only on the relative concentrations of substrates. However, several stud-
ies demonstrated that this enzyme has a particular type of regulation. Suc synthase 1 (SUS1) from
maize leaves is phosphorylated in the highly conserved Ser15 residue by a sucrose nonfermenting 1
(SNF1)-related protein kinase (Hardin et al. 2003). The principal effect of this phosphorylation is to
increase the activity in the Suc degradation direction. In addition, it would be critical for membrane
association (Hardin et al. 2004). The latter would be relevant for the synthesis of cellulose and other
polysaccharides present in the cell wall because it could channel the UDP-Glc synthesized by Suc
synthase (Koch 2004). Also, it has been demonstrated that Ser170 in the enzyme from maize leaves
can be phosphorylated by a calcium-dependent protein kinase (Hardin et al. 2003). Phosphorylation
of this residue is the determinant for the degradation of the enzyme by the 26S proteasome, prob-
ably through a mechanism mediated by ubiquitination. This would regulate the intracellular levels
of this protein (Hardin and Huber 2004).
9.4.2 Oils and Fatty Acids

Many seeds (including agriculturally important species like soybean and sunflower) as well as some
fruits (such as olives and avocado) accumulate oils instead of starch as the main carbon reserve
(Taiz and Zeiger 2006). Lipids store carbon in a more reduced form than carbohydrates, thus releas-
ing more than twice energy during catabolism (Baud and Lepiniec 2010; Theodoulou and Eastmond
2012). Plant seeds accumulate oil as triacylglycerols (TAGs), but they also produce polar glyc-
erolipids to synthesize cellular membranes, waxes, and terpenoids (Taiz and Zeiger 2006). Lipid
metabolism in plants requires the cooperation of different organelles (Figure 9.2). Thus, synthe-
sis of TAG mainly involves plastids (for fatty acid synthesis) and the endoplasmic reticulum (ER;
for TAG assembly) (Figure 9.2a), whereas lipid degradation principally occurs between glyoxy-
some and mitochondria (Figure 9.2b). Here we will focus on TAG synthesis for long-term storage.
Accumulation of TAG occurs in the cytosol, and they are stored (either in cotyledons or endo-
sperm) as oils bodies or oleosomes. Oil bodies are spherical organelles surrounded by a phospho-
lipid monolayer and stabilized by the presence of proteins (oleosins) imbibed in the lipid membrane
(Baud and Lepiniec 2010).
De novo fatty acid synthesis in plants occurs in the plastid, unlike other organisms where the
process takes place in the cytosol (Ohlrogge and Jaworski 1997). As it was stated before, Suc rep-
resents the major form by which photosynthetically assimilated carbon is transported into sink
tissues, thus providing the precursors for lipid synthesis (Figure 9.2a). Two glycolytic intermediates
Sucrose
Starch
+
NADP ADP-Glc
Pi Pi
NADPH Fru-6P
+ +
NADP NADH NAD
OPPP Glc-6P HPT Glc-6P
complete Pi Pi Ga3P
Rul-5P 1.1.1.8
NADPH OPPP DHAP
Xul-5P XPT Xul-5P incomplete
Rib-5P
Gro-3P
PEP PPT PEP
ADP
Plastid ATP Pi Pi
Pyr Pyr
TAGs
CO2 Respiration Biosynthesis oil
bodies
Mitochondria
FAS Acetyl-CoA
Cytosol
C18:1 Fatty acid C20:1

elongation C21:1
(a)
Sucrose
Mitochondria Fumarate
Malate Fru-6P
Succinate
NAD+
Glyoxylate FADH2 FAD NADH Triose-P

NADH
cycle
NAD+
Glyoxylate Acetyl-CoA PEP
Glyoxysome CO2
NAD+ β-oxidation ADP
OAA ATP
NADH Acyl-CoA
CoA OAA
Cytosol Fatty acids TAGs
oil bodies
Lipase
(b)
FIGURE 9.2 Schematic representation of lipid metabolism in plants: (a) lipid synthesis; (b) lipid degradation
by β-oxidation and glyoxylate cycle during seed germination.
are the building blocks for TAG synthesis. Glycerol-3-phosphate (Gro-3P) is produced from DHAP
by the Gro-3P dehydrogenase (EC 1.1.1.8), while Pyr provides the acetyl-CoA by decarboxylation
in a reaction catalyzed by the pyruvate dehydrogenase complex that resides inside the plastid (Baud
and Lepiniec 2010; Schwender et al. 2003).
Fatty acid biosynthesis involves the cyclic condensation of acetyl-CoA by a set of enzymes that
are thought to be forming a complex collectively referred to as fatty acid synthase (see Figure 9.2a),
that is, the strategic control point (Taiz and Zeiger 2006). Pyr, the carbon source that provides
the acetyl-CoA units, derives almost entirely from glycolytic cleavage, responsible for most of the
embryo hexose catabolism, as it was demonstrated in Brassica napus (Schwender et al. 2003).
In plants, one particular feature is the presence of duplicated glycolytic and OPPP pathways, each
occurring in the cytosol and inside the plastid. Considering the complexity and redundancy of plant
metabolic pathways leading to the production of precursors, the proportion of carbon fluxes through
these pathways is hard to address (Baud and Lepiniec 2010). Data gathered to date suggest that
each species has a particular metabolic flux. In Brassicaceae (B. napus or A. thaliana), imported
Suc goes through cytosolic glycolysis until PEP. Then, it is imported into the plastid, converted to
Pyr inside the organelle, and further transformed into acetyl-CoA (Schwender et al. 2003). In non-
green heterotrophic embryos, like sunflower, Suc metabolism goes through hexose-P in the cytosol,
hexose-P are imported into the plastid, and any further metabolism to Pyr seems to take place inside
the plastid (Baud and Lepiniec 2010).
Fatty acid synthesis has a high demand of energy and reducing power, and many pathways con-
tribute to ATP and NADPH production. It has been reported that in green seeds, photosystems are
one of the sources for reducing power and ATP, whereas in nongreen seeds (where cytosolic and
plastidic metabolism are connected at the hexose-P level), the OPPP is the main pathway producing
NADPH and glycolysis the one providing ATP (Baud and Lepiniec 2010; Schwender et al. 2003).
Once fatty acids are synthesized, TAG assembly takes place in the ER from Gro-3P and acyl-CoA
(Baud and Lepiniec 2010). During germination, oil seeds metabolize oil reserves by β-oxidation to
obtain the energy and carbon needed for growth and development until photosynthesis turns active.
In the germinating process, the glyoxylate cycle plays a critical function to effectively mobilize car-
bon skeletons from oils to carbohydrates by gluconeogenesis (Taiz and Zeiger 2006) (Figure 9.2b).
9.4.3 Sugar Alcohols
In addition to starch and Suc, many plants utilize sugar alcohols to transport carbon and energy
from source to sink tissues (Figure 9.3). Glucitol (Gol), also known as sorbitol, is a sugar alcohol
that constitutes a major photosynthetic product in many economically important plant species from
the Rosaceae family, including apple, peach, and pear (Loescher and Everard 2004). It has been
reported that the capacity of sugar alcohols synthesis and degradation largely varies among dif-
ferent plant tissues or within a certain tissue during development. Developing leaves experience a
transition from sink (with high rates of polyol oxidation) to source (with increasing capacity of sugar
alcohol synthesis). In apple and peach leaves, it was shown that this transition is accompanied by an
increase in the biosynthetic and a decrease in the degrading enzymes (Loescher and Everard 2004).
Gol is synthesized in mature leaves from Glc-6P (Figure 9.3a), which is converted into Gol-6P
in a reaction catalyzed by a NADPH-dependent aldose-6P reductase (Ald-6PRase, EC 1.1.1.200;
Table 9.1, Equation 9.15) (Zhou et al. 2003b). Thereafter, the phosphate group is hydrolyzed by
a Gol-6P phosphatase (EC 3.1.3.50; Table 9.1, Equation 9.16) (Zhou et al. 2003a). Both enzymes
are thought to be localized in the cytosol (Loescher and Everard 2004). The NADPH necessary to
sustain the synthesis of the polyol would be produced by np-Ga3PDHase, which has been found in
celery leaves in levels high enough to support the high rate of mannitol production observed in this
species (Gao and Loescher 2000).
The sugar alcohol synthesized in mature leaves is then transported to developing or sink tissues
(Figure 9.3b), like immature leaves or fruits, where it is converted into Fru by an NAD-dependent
Gol dehydrogenase (GolDHase, EC 1.1.1.14; Table 9.1, Equation 9.17) (Oura et al. 2000). It was first
suggested that GolDHase is located in the cytosol and that it is not associated with intracellular
compartments. However, a recent study realized with apple leaves and fruits indicated that the
enzyme is located within plastids and other compartments. Even more, the authors proposed that
starch levels within plastids are directly related to the level of GolDHase in these compartments and
that its activity could provide hexoses for the synthesis of the polyglucan (Wang et al. 2009).
Based on the type of reaction catalyzed and sequence homology, Ald-6PRases were included
within the aldo/keto reductase superfamily (Hyndman et al. 2003). This superfamily is mainly
208
Pi
Suc-6P Sucrose
Photosynthetic electron ST
Light transport chain UDP
Gol UDP
ATP NADPH
2.7.1.11 3.1.3.50 Pi GT Sucrose 2.4.1.13
CO2 3-PGA Starch Plastid
Gol-6P UDP-Glc
UDP-Glc +
1.2.1.13 NADP Gol
RuBisCO PPi 2.7.7.9
BCC 1.1.1.200 +
NAD UTP
Rul-1,5-bisP NADPH Glc-1P NADPH
Pi Pi Fru-6P Glc-6P 1.1.1.14 Glc-1P NADPH
Triose-P 5.3.1.9 Pi Pi + NADP+
5.4.2.2 NADP
NADH
Chloroplast TPT
Fru Glc-6P HPT Glc-6P
Triose-P Ga3P
Starch NADP+ OPPP
1.2.1.12 2.7.1.1 Rul-5P Pi Pi complete
1.2.1.9
Cytosol 2.7.2.3 Fru-6P OPPP Xul-5P Xul-5P
NADPH XPT
incomplete Rib-5P
3PGA
Cytosol Triose-P Triose-P
(a) (b)
FIGURE 9.3 Schematic representation of sugar alcohol metabolism in plants: (a) synthesis of Gol in source tissues; (b) Gol metabolism in sink tissues.
Handbook of Plant and Crop Physiology
composed of proteins that are monomers in their native state. However, some proteins included in the
classes 2, 6, and 7 can be found as multimers, like Ald-6PRase from apple leaves, which is a dimer
(Figueroa and Iglesias 2010) and has been included in class 2 (Hyndman et al. 2003). The kinetic and
regulatory properties of Ald-6PRase were studied in a limited number of works. The enzymes from
apple seedlings (Kanayama and Yamaki 1993) and loquat leaves (Hirai 1981) were the first ones to
be purified from source tissues and kinetically characterized. Then, Zhou et al. (2003b) showed that
Mg2+ reduces the Km for Glc-6P of the enzyme from apple leaves, whereas Pi inhibits the enzyme by
increasing the Km for Glc-6P. In addition, these authors showed that the enzyme is inhibited by Zn2+
and Cu2+, while incubation with NADP+ or NADPH protects the enzyme from Zn2+ inhibition. These
results suggest that there might be thiol groups important for cofactor binding (Zhou et al. 2003b).
More recently, the enzyme from apple leaves was recombinantly expressed in Escherichia coli cells
and kinetically characterized. The authors described the use of a system for obtaining high amounts
of pure enzyme, which represents an important molecular tool for advancing in the analysis of the
kinetic and regulatory properties of this enzyme (Figueroa and Iglesias 2010).
Sequence analysis of different plant GolDHases strongly suggests that these enzymes belong
to the NAD-dependent medium-chain dehydrogenase/reductase family (Persson et al. 2008).
GolDHase was initially identified in mammals and then its presence in plants was reported. The
enzyme from apple callus was the first to be purified and characterized. In this work, the inhibition
by heavy metals and compounds that react with thiol groups (such as iodoacetate) was also reported.
Also, enzyme activity was inhibited by incubation with l-Cys, but it was recovered by the addi-
tion of ZnSO4, which suggests that the enzyme needs Zn2+ for catalyzing the reaction (Negm and
Loescher 1979). The first fruit enzyme to be purified was that from apple (Yamaguchi et al. 1994).
In this work, a complete kinetic characterization was performed, and the results were in good agree-
ment with those reported for the apple callus enzyme. Later, the gene coding for GolDHase from
apple fruits was cloned, which showed that the enzyme is a heterotetramer composed of subunits
of 40 kDa (Yamada et al. 1998). This result was further confirmed during the characterization of
the enzyme purified from Japanese pear fruits, where the authors determined that the enzyme is a
tetramer of 160 kDa (Oura et al. 2000).
In addition to their role as photosynthates, sugar alcohols play a key role as protectors under
certain abiotic stress conditions (Loescher and Everard 2004). Many organisms accumulate low-
molecular-weight compounds, such as nonreducing sugars (Suc and trehalose), cyclic and aliphatic
polyols (mannitol, Gol, and Gro), quaternary ammines (glycine betaine), and amino acids (pro-
line). These molecules, known as compatible solutes, might allow these organisms tolerate some
abiotic stress conditions (like salinity, drought, and cold) by osmotic regulation. Another proposed
mechanism to exert this protection could be by stabilizing the quaternary structure of proteins
(Bohnert and Jensen 1996). In addition, sugar alcohols are molecules that could increase the toler-
ance against oxidative stress (Shen et al. 1997), micronutrients deficiency (Brown et al. 1999; Shen
et al. 1997), and salinity (Gao et al. 2001).
Recent studies have increased our knowledge on the molecular mechanisms that take part
in peach fruit development (Morandi et al. 2008) and in the changes observed after girdling
(Borsani et al. 2009). It was proposed that the amount of Gol transported to peach fruits could
have a key role in the regulation of their development, because the conversion of Gol into Fru is
directly related to the fruit growing rate. An advantage of Gol oxidation over Suc degradation
is a higher NADH production (compare Equations 9.12 and 9.13 with Equation 9.17 in Table 9.1),
which could increase growth efficiency and lower the respiration costs (Borsani et al. 2009). Even
though sugar alcohols are important for carbon partition and abiotic stress tolerance, the stud-
ies realized with enzymes of Gol metabolism in plants are scarce. Thus, it would be desirable to
characterize such enzymes to better understand both the physiological regulation of sugar alcohol
biosynthesis and the stress tolerance mechanisms in plants.
9.4.4 Proteins and Amino Acids

A part of photoassimilated carbon is utilized for amino acid production in plants. Of course, the flux
of carbohydrates derived toward the amino acidic metabolism is interconnected with the nitrogen
assimilation either from organic (urea)/inorganic (nitrate, ammonia) nitrogen forms in nonlegume
or by fixing atmospheric molecular N2 via the symbiosis established by legumes with specific bacte-
ria (Iglesias et al. 2005). The routes for amino acid synthesis are important in plants, as many inter-
mediate metabolites play key roles for the regulation of responses to changes in the environment
affecting the availability of nutrients or light as well as provoking different stress conditions (Galilli
and Höfgen 2002). Plants synthesize all 20 amino acids by metabolic routes described elsewhere
(Taiz and Zeiger 2006). The synthesis of tryptophan, phenylalanine, and tyrosine takes place by
the shikimate pathway, a metabolism of particular relevance for many reasons: (1) the biosynthetic
route might take up to one-third of carbon fixed by photosynthesis; (2) aromatic amino acids are
precursors of pigments, alkaloids, hormones, and other natural products having significance both
for plant functioning and in their use as commercial goods; (3) these aromatic amino acids are
essentials for humans; and (4) because of its absence in animals, the biosynthetic pathway has been
the main target for the design of herbicides and the associated strategy to generate genetically modi-
fied plants with resistance (Maeda and Dudareva 2012).
Concerning polypeptides, different plants accumulate relatively large quantities of proteins (as
protein bodies) in seeds. For example, in mature cereal grains, storage globulins and prolamines might
represent nearly 50% of the total protein (Shewry and Halford 2002). A recent study (Abirached-
Darmency et al. 2012) reports the biogenesis of protein bodies in grain legumes, determining the
occurrence of an ER vacuole trafficking route. The use of the ER to store proteins in huge amounts
and highly condensed formation is characteristic of plants and seems a mechanism to avoid degrada-
tion of the polymeric product (Vitale and Ceriotti 2004). In an analysis of carbohydrate partitioning
into different storage products in developing legume seeds, Golombek et al. (2001) clearly stated that
after reducing the levels of ADP-Glc PPase (via antisense transformation), plants contained less starch
but increased levels of Suc and protein. A little explored field is that of protein quality improvement
in plants, because they are deficient in certain amino acids as is the case for lysine and tryptophan in
cereals or methionine and cysteine in legumes and vegetables.
9.5 PLANT CROPS AHEAD: DEVELOPING STRATEGIES

FOR A SUSTAINABLE WORLD
Plants, as organisms with autotrophy to assimilate inorganic carbon, represent a major source of
natural resources for food and other industries. Consequently, historically, many efforts were dedi-
cated to inbreed species for improvement in their productivity. In the current global panorama
of, with increased demands for food and the necessity to turn different industrial procedures
more ecologically friendly (specially replacing fosil fuels by biofuels and generating biodegradable
chemicals and plastic products), the use of plants for production of critical compounds is of the
utmost importance. Thus, plants currently are key target organisms to apply metabolic engineering
strategies via genetic transformation with different productive purposes, mainly with three main
objectives (Cortassa et al. 2012). One possibility is to incorporate new traits, to enhance resistance
to chemicals as well as to biotic or abiotic stress conditions. In this field, important goals have been
reached with the first generation of transgenic plants. For example, the production of crop with resis-
tance to a number of pests using genes encoding endotoxins from Bacillus thuringiensis (Collins
and Shepherd 1996) or generation of plant with resistance to the broad-spectrum herbicide glypho-
sate (Duke 2010; Herrmann and Weaver 1999). Also for these purposes, it is important to associate
the above detailed on the metabolism of sugars and sugar alcohols, as its accumulation through the
handling of their metabolism could probably improve the tolerance to drought or cold stresses.
A second approach is to genetically manipulate plants to improve their natural primary

p roducts in quantity and/or quality. To have success in this objective, it is critical the meticulous
understanding on the occurrence of different metabolic pathways and their respective r egulation.
To afford only a few examples in this field, we could mention: (1) the project of the “golden
rice” allowing to obtain the major food resource of poor population with healthy quality
because of increased contents of carotenoids (Ye et al. 2000); (2) the modification of plant
fatty acid composition (Collins and Shepherd 1996; Somerville and Bonetta 2001; Thellen and
Ohlrogge 2002) for uses as better food or material for biodiesel production; (3) the manipu-
lation to modify the amounts of starch (Collins and Shepherd 1996; Stark et al. 1992) as a
critical tool for increased p roduction of the quantitatively major aliment of numerous animals
(including man) as well as to better manage the field of biofuels and biodegradable polymers
(Mooney 2009); and (4) the redesign of plant secondary metabolism, with the potential to obtain
a series of phytochemicals of high value in different industries, including pharmaceuticals
(Dudareva and Pichersky 2008; McChesney et al. 2007).
An emergent, highly relevant field is the use of plants as “molecular farming” to produce com-
pounds of non plant origin. A good example is the recombinant expression of heterologous pro-
teins of eukaryotic origin, of value for medicinal, pharmaceutical, and nutraceutical applications
(e.g., antibodies, vaccines, and diagnostic polypeptides) (Drake and Thangaraj 1995; Obembe et al.
2011). In this respect, plants have the advantages of higher safety and less ethical concerns, while
possessing the biological machinery to synthesize fully folded polypeptides with posttranslational
modifications. The alternative to accumulate the recombinant product in some tissues, as seeds, could
be convenient to improve the stability of proteins during storage. As well, plants may be considered
as appropriate bioreactors to produce enzymes used for biofuels production, polymers biosynthesis,
and the whole processes for refinery and fuel biological generation (Collins and Shepherd 1996).
ACKNOWLEDGMENTS
The authors acknowledge support from CONICET (PIP 2519 to FEP and AAI), FONCyT (PICT
2011 01122 to FEP, PICT 2008 1754 to AAI, and PICT 2011 1986 to CVP), and UNL (CAI+D Redes &
Orientado to AAI). All the authors are members of the investigator career from CONICET.
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10 Role of Nitric Oxide
in Plant Development
Dagmar Procházková and Nad’a Wilhelmová
CONTENTS
10.1 Introduction........................................................................................................................... 217
10.2 NO and Related Reactive Nitrogen Species.......................................................................... 217
10.2.1 Physicochemical Properties of NO............................................................................ 217
10.2.2 NO Biosynthesis........................................................................................................ 218
10.2.3 Reactive Nitrogen Species......................................................................................... 218
10.3 NO in Plant Development...................................................................................................... 219
10.3.1 NO in Pollen Grains.................................................................................................. 219
10.3.2 NO during Seed Formation and Germination........................................................... 220
10.3.3 NO in Root Growth................................................................................................... 220
10.3.4 NO in Leaf Growth.................................................................................................... 221
10.3.5 NO in Flowering........................................................................................................ 222
10.3.6 NO in Senescence...................................................................................................... 222
10.3.7 NO in Programmed Cell Death................................................................................. 223
10.4 Conclusions............................................................................................................................ 223
Acknowledgment............................................................................................................................ 223
References....................................................................................................................................... 223
10.1 INTRODUCTION
For more than 50 years, the only gaseous signaling molecule in the living world known to science
was the plant hormone ethylene. The 1998 Nobel Prize for Medicine heralded the establishment of
another, even a smaller one, player of this kind in mammalian cells—nitric oxide (NO) (Wojtaszek
2000). With the finding that NO has many functions in mammalian cells, such as regulation of
vascular tone, neuronal signaling, or immune response to infection (Knowles and Moncada 1994),
various studies have reported its presence in the plant kingdom as well. It has been shown that NO
can regulate many processes related to plant growth and development, which led some researchers
to define NO as a plant growth regulator (Leshem et al. 1998). Indeed, NO fits most of the criteria
required for plant growth regulators: it is synthesized by plants and affects physiological processes
at low concentrations, which excludes its nutritional effect. Additionally, a cross talk among NO,
ethylene, gibberellic acid (GA), auxin, and cytokinins supports the idea that it is involved in hor-
mone signaling and triggers many physiological responses (Kopyra and Gwóźdź 2004).
10.2 NO AND RELATED REACTIVE NITROGEN SPECIES

10.2.1 Physicochemical Properties of NO
Nitric oxide (common name) or nitrogen monoxide (systematic name) is a chemical compound
with the chemical formula NO. It is one of the smallest diatomic molecules with a high diffusivity
217
(in pure water, 2.21 × 10 −5 cm2 s−1 at 25°C and 3.0 × 10 −5 cm2 s−1 at 37°C), exhibiting hydrophobic
properties (Popova and Tuan 2010; Zacharia and Deen 2005).
NO is a gaseous radical with an unpaired electron in its π orbital and with a half-life of 3–5 s in
biological systems (Tuteja et al. 2004). NO may not only easily migrate in the hydrophilic regions of
the cell (due to its high diffusivity in water), such as the cytoplasm, but also freely diffuse through
the lipid phase of membranes (Arasimowicz and Floryszak-Wieczorek 2007). Its short half-life
reflects the highly reactive nature of NO, which reacts directly with transition metals forming com-
plexes and other radicals and indirectly as reactive nitrogen species (RNS) with DNA, proteins, and
lipids (Popova and Tuan 2010; Wink and Mitchell 1998).
10.2.2 NO Biosynthesis
Plants produce NO using different enzymes. Nitrate reductase (NR) is involved in nitrogen assimila-
tion by converting nitrate to nitrite; however, it can also mediate NO production from nitrite (Rockel
et al. 2002) using succinate as electron donor (Meyer and Stöhr 2002). The reduction efficiency is
about 1% of total NR activity, and it is pronounced more in a low-oxygen environment and requires
nitrite levels higher than normal ones. NR has been found both as a cytosolic form and as a plasma
membrane-bound form (Stöhr 1999).
Nitrite:NO reductase (NI-NOR), a root-specific enzyme whose activity markedly differs from
cytosolic NR, has been studied mainly in Nicotiana tabacum. It does not use reduced nicotine
adenine nucleotides; instead reduced cytochrome c can serve as an electron donor in vitro. However,
participation of cytochrome c at the plasma membrane in vivo seems unlikely, and the physiological
electron donor has not been identified so far (Stöhr et al. 2001).
The enzyme responsible for NO generation in animal organisms is nitric oxide synthase (NOS)
catalyzing five-electron oxidation of one of the atoms in l-arginine (N3− to N2+) with the partici-
pation of O2 and NADPH (Arasimowicz and Floryszak-Wieczorek 2007). Many plant biologists
searched intensively for NO-generating enzyme similar to NOS (Corpas et al. 2006). Ten years
ago, a gene of a plant protein capable of generating NO from l-arginine and, therefore, having NOS
activity, was identified in Arabidopsis and named AtNOS1 (Guo et al. 2003). The encoded protein,
AtNOS1, had similarity to one from a snail that was possibly involved in NO synthesis. Importantly,
AtNOS1 was shown to possess the biochemical characteristics of NOS because it reduced arginine
to citrulline (Neill et al. 2008). However, neither gene nor protein with sequences similar to the
large animal NOS genes or proteins has been found even in the sequenced Arabidopsis genome.
Moreau et al. (2008) identified that AtNOS protein belongs to the group of circularly permutated
GTPase, and it was renamed to AtNOA1. These authors further questioned AtNOS/A1 as an authen-
tic NOS as it cannot bind and oxidize arginine but show GTPase activity. These results suggest that
the NOS activity in plants comes from a different type of enzyme (Gas et al. 2009).
Yamasaki and Sakihama (2000) described a nonenzymatic mechanism for the synthesis of
NO from NO2 under acidic conditions, where nitrite is protonated to form nitrous acid (HNO2)
in a freely reversible reaction. In addition, yet another form of oxidative NO formation has been
described, where hydroxylamine and salicylhydroxamate were oxidized to NO (Rümer et al. 2009).
10.2.3 Reactive Nitrogen Species

In plant biology, knowledge of the physiological functions of NO and other RNS, that is, peroxyni-
trite (ONOO −), peroxynitrous acid (HOONO), nitrogen dioxide (NO2), dinitrogen trioxide (N2O3),
S-nitrosothiols (RSNOs), and S-nitrosoglutathione (GSNO), has experienced a significant advance
in recent years.
NO can react with superoxide radicals forming peroxynitrite and hydroxyl radical, eventually
(Kissner et al. 1997). Peroxynitrite is a relatively short-lived RNS at the physiological pH range and
temperature, which may readily migrate through biological membranes, and in animals, it could
Role of Nitric Oxide in Plant Development 219
influence surrounding target cells within one to two cell diameters (∼5–20 μm) (Szabó et al. 2007).
It is a powerful oxidant, which can react with DNA, leading to cellular damage and cytotoxicity
(Szabó et al. 2007), and with thiol groups of proteins and with polyunsaturated radicals of fatty
acid lipids of membrane, causing serious damage to cell structures (Arasimowicz and Floryszak-
Wieczorek 2007).
Peroxynitrite is also known to cause nitration of phenolic rings in a molecule, including tyrosine
residues in proteins (Alamillo and Garcia-Olmedo 2001; Jasid et al. 2008). Tyrosine nitration, simi-
lar to other posttranslational modifications, has been shown to be capable of changing the function
of a protein in several ways: (1) gain of function as well as no effect on function; and (2) inhibition
of function being a much more common consequence of protein tyrosine nitration (Radi 2004). It
has been also reported that nitration of a tyrosine residue may either prevent further phosphorylation
or stimulate phosphorylation (Rayala et al. 2007; Shi et al. 2007).
The formation of RSNOs implies the reaction of nitrosonium with a thiol group present in free
cysteine, peptides, or proteins (Chaki et al. 2009). These compounds perform important biologi-
cal reactions, including NO release, transnitrosation, S-thiolation, as well as direct actions (Hogg
2000; Stamler et al. 2001). For example, it was described in mammalian cells that they inhibit NOS
(Stamler et al. 2001). Under physiological conditions, RSNOs are considered to provide protection
against cellular damage induced by oxidative and nitrosative stress (Valderrama et al. 2007).
GSNO results from the reaction between NO and reduced glutathione or by a process of transni-
trosation from other RSNOs with reduced glutathione. GSNO may function both as an intracellular
NO reservoir and as a transporter for NO throughout the cell (Singh et al. 1996a,b).
10.3 NO IN PLANT DEVELOPMENT

Most of the experiments investigating the participation and effects of NO in plant development
employ pharmacological access, based on the application of gaseous NO or compounds known under
certain conditions to release or scavenge NO in vivo. The widely used NO donors are sodium-nitro-
prusside (SNP), S-nitroso-N-acetyl penicillamine (SNAP), GSNO, and 3-morpholinosydnonimine
(SIN-1); less used is N-tert-butyl-α-phenylnitrone. On the contrary, the most used NO scavengers
are [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] (c-PTIO), methylene
blue, and NG-monomethyl-arginine monoacetate. In some experiments, NG-nitro-l-arginine methyl
ester is employed to inhibit NOS activity.
There is little information on the enzymatic source of endogenous NO during normal develop-
ment of plants, and still less is known on the loci where NO is generated (Corpas et al. 2006).
10.3.1 NO in Pollen Grains

NO generation by pollen is more widespread among angiosperms and could influence the internal
biochemistry of the pollen grain and pollen tube (Wilson et al. 2009).
It is well known that pollen tubes must be precisely oriented inside the anatomically complex
female ovary in order to deliver sperm. Prado et al. (2004) showed that NO could play a role in this
guidance in lily: when external NO reached a critical concentration, the growth axis underwent a
subsequent sharp reorientation, after which normal growth was attained. This response was abro-
gated in the presence of the NO scavenger cPTIO and affected by drugs interfering in the guanosine
3′, 5′-cyclic monophosphate (cGMP) signaling pathway. These experiments indicate that the rate and
orientation of pollen tube growth is regulated by NO levels at the pollen tube tip and suggest that this
NO function is mediated by cGMP. The NO participation in pollen tube orientation was confirmed in
Arabidopsis pollen tubes as well (Prado et al. 2008). NO is also involved in configuration and distribu-
tion of cell wall components in Pinus bungeana pollen tubes by altering extracellular Ca2+ influx and
F-actin organization (Wang et al. 2009) and partly mediate extracellular nucleotide-induced suppres-
sion of pollen germination and pollen tube elongation (Reichler et al. 2009).
10.3.2 NO during Seed Formation and Germination

Barley (Hordeum vulgare) aleurone layers produced NO rapidly when nitrite was added to the
medium in which they were incubated. NO production was accompanied by a loss of nitrite from
the medium. Phenolic compounds in the medium increased the rate of NO production (Bethke
et al. 2004).
Abscisic acid (ABA) is known to prevent the early interruption of seed dormancy. The rapid
NO-induced decrease in ABA in the early stage of Arabidopsis seed imbibition was mediated by a
rapid increase in cyp707a2 (encodes (+)-abscisic acid 8′-hydroxylase, a key enzyme in ABA catabo-
lism) expression and increased amounts of CYP707A2 protein. In this manner, NO produced in the
endosperm layer during imbibitions regulates cyp707a2 expression, preceding the increase in ABA
catabolism required for seed germination (Liu et al. 2009). Indeed, A. thaliana seeds treated with
SNP increased germination to 90%, but when pure NO was mixed with air and passed over dormant
seeds, 30% of the seeds germinated (Libourel et al. 2006). Imbibition of embryos isolated from dor-
mant apple seeds in medium with SNP or SNAP resulted in enhanced germination (Gniazdowska
et al. 2007). Moreover, NO treatment removed morphological abnormalities of seedlings developing
from dormant embryo. The NO scavenger cPTIO removed the effects of enhanced germination and
also morphological abnormalities (Gniazdowska et al. 2007). Similarly, deep dormancy of apple
embryos was removed by short-term NO pretreatment (Gniazdowska et al. 2010).
The prerequisite for seed germination is imbibition. The stimulation of rice seed germination by
treatment with SNP and GSNO was accompanied by the increased expression of several aquaporin
genes (OsPIP1;1, OsPIP1;2, OsPIP1;3 and OsPIP2;8), which likely results in an enhanced seed capac-
ity for water uptake during imbibition and its further development. These results suggest that water
channels play an important role in seed germination, acting, at least partly, in response to the NO sig-
naling pathway (Liu et al. 2007). NO donor effect seems to be dose dependent because high concentra-
tion of SNP contrarily caused the retardation of germination of tobacco seeds (Seregélyes et al. 2003).
It has been shown that NO donors are able to promote seed germination, while NO scaven-
gers inhibit germination and reduce the stimulative effects of NO donors during germination. For
example, SNP and GSNO promoted and cPTIO inhibited rice seed germination (Liu et al. 2007).
Similarly, exogenous NO was shown to break dormancy of lettuce seeds, being even more effective
than GA (Beligni and Lamattina 2000). Interestingly, after the treatment with NO donors (organic
nitrates and SNP), only phytochrome-specific induced germination was affected during light-
induced A. thaliana seed germination, and to a far lesser extent, these substances were able to affect
phytochrome B-specific induced germination (Batak et al. 2002). NO also enhanced the germina-
tion of light-independent lupine seeds in a dose-dependent manner (Kopyra and Gwóźdź 2003).
The relation between reactive oxygen species (ROS) and NO was confirmed by the fact that H2O2
effects during seed germination were abolished by the NO scavenger cPTIO, suggesting that H2O2
control of ABA levels was mediated by NO signaling (Liu et al. 2010).
NO is also able to influence seed physiology. The addition of SNP was able to induce a rapid
increase in β-amylase activity without affecting α-amylase (Zhang et al. 2005).
10.3.3 NO in Root Growth

NO involvement in mediating root development in response to nitrate and in signaling at the
root–environment interface is inferred from the coordinated activity of the root-specific plasma
membrane-bound enzymes, NR and NI-NOR (Stöhr and Stremlau 2006). NOS in root has been
discussed as well: Zhao et al. (2007) suggested that inhibition of root elongation in maize by high
external nitrate is likely to result from a reduction of NOS-dependent endogenous NO levels in
maize root apical cells.
During pea root development, the NOS activity in primary roots reached a maximum in 7-day-
old seedlings and then it decreased. From this time, the number and length of lateral roots increased,
and, therefore, the NO generated by the NOS activity could be involved in the stimulation of this
process. The presence of NO observed in the epidermal cells and root hairs could indicate the
involvement of NO in the cascade of signals during the development and formation of new roots
(Corpas et al. 2006).
NO donors SNAP, SNP, and GSNO reduced primary root cell division in A. thaliana, affecting
the distribution of mitotic cells and meristem size by reducing cell size and number compared with
the same plants treated with cPTIO. In addition, both elevated NO supply and the NO-overproducing
Arabidopsis mutant cue1/nox1 exhibited reduced expression of the auxin reporter markers, and
these effects were accompanied by a reduction in auxin transport in primary roots. The use of
both chemical treatments and mutants with altered NO levels demonstrated that high levels of NO
reduce auxin transport and response by a PIN1-dependent mechanism, and root meristem activity is
reduced concomitantly (Fernández-Marcos et al. 2011).
Different results were obtained by Gouvea et al., who employed the same NO donors plus nitro-
socysteine and described that NO induced cell elongation in maize roots in a similar way to auxin,
and the elongation was inhibited by methylene blue, an NO scavenger (Gouvea et al. 1997). In
tomato seedlings, SNP induced lateral root emergence and elongation as well (Correa-Aragunde
et al. 2004).
Similarly, in cucumber (Cucumis sativus) hypocotyls, treatment with SNP or SNAP plus auxin
indole-3-acetic acid (IAA) resulted in longer roots than those from hypocotyls treated with the NO
donors or IAA alone. Application of cPTIO delayed adventitious root emergency and significantly
reduced the root length and number of the IAA-treated explants. The inhibitory effect of cPTIO was
reversible because adventitious root emergency was triggered by the addition of SNP after cPTIO
treatment. In addition, a direct inactivation of IAA by cPTIO per se may be discarded because a
mixture of IAA plus cPTIO was able to induce epinasty, an IAA-mediated effect, at the same extent
as IAA alone (Pagnussat et al. 2002). In addition, the treatments with SNP and SNAP, applied to
cucumber hypocotyl cuttings, where primary roots were removed, were able to mimic the effect of
the IAA in inducing de novo root organogenesis. Both NO- and IAA-induced roots presented simi-
lar anatomic structure. This NO-mediated effect was prevented when PTIO was added with SNP or
SNAP (Pagnussat et al. 2002).
As cyclic guanosine monophosphate (cGMP) is an important component of NO signaling in ani-
mals (Mc Donald and Murad 1995), the involvement of cGMP in adventitious root development was
analyzed. The results showed that NO operates downstream of IAA, promoting adventitious root
development through the guanylate cyclase–catalyzed synthesis of cGMP (Pagnussat et al. 2003).
Further, NO mediates the IAA induction activation of mitogen-activated protein kinase cascade
involved in adventitious root development in cucumber. It brings about a new mode of NO signaling,
independent of cGMP pathway (Pagnussat et al. 2004).
It is well known that NO plays important role during plant stress. With regard to root growth, it was
demonstrated that exogenous application of NO exacerbated Al-induced root growth inhibition in rice
bean (Zhou et al. 2012). This finding is contrary to a previous report on Cassia tora L. (Wang and Yang
2005). The discrepancy could be due to different plant species, because Al stress resulted in different
effects on endogenous NO concentrations dependent on plant species. Similarly, NO donors counteract
the inhibitory effect of cadmium, lead, and salinity on root growth (Kopyra and Gwóźdź 2003).
10.3.4 NO in Leaf Growth

Corpas et al. (2006) showed that pea leaves are the plant organs with lower NOS activity compared
to roots and stems, and this activity did not undergo significant changes during seedling develop-
ment. This suggests that the NO generated in pea leaves must be sufficient for the optimal develop-
ment of these plant organs.
NO showed to have concentration-dependent effects on pea leaves, inducing leaf expansion at low
concentrations, but exerting a negative effect at high concentrations (Leshem and Haramaty 1996).
For example, NOS inhibitor reduced leaf biomass while the NO donor increased leaf biomass in
maize. The inhibitor also reduced exo- and endo-beta-glucanase activity and leaf biomass, while the
donor increased the enzyme activity and leaf biomass. This suggests that the changes of NOS activity
showed a positive correlation to glucanase activity and leaf biomass (An et al. 2005).
10.3.5 NO in Flowering
NO is synthesized in specific cells and tissues in the floral structure, and its production increases
with floral development until anthesis. In the gynoecium, NO synthesis occurs only in differentiated
stigmatic papillae of the floral bud, and in the stamen, only anthers that are producing pollen grains
synthesize NO. Sepals and petals do not show NO production (Seligman et al. 2008).
NR-deficient mutant Arabidopsis plants showed increased caulinar/rosette leaf number and the
decrease in the number of days to bolting and anthesis, and this phenotype seems to result from the
markedly reduced NO levels in roots and leaves during vegetative growth (Seligman et al. 2008).
The application of SNP resulted in a delay of flowering induction in Arabidopsis. Through
genetic screening of Arabidopsis, mutants with high or low endogenous NO levels were identified.
These mutants delayed flowering or blossomed precociously, respectively, when compared with
wild-type plants. Moreover, high levels of NO suppressed expressions of the gene participating
in floral initiation process in the floral meristem and the gene involved in floral promotion, which
positively affected flowering and, additionally, increased the expression of the floral repressor gene
FLC. These results suggest a role for NO in suppressing floral transition by affecting the expression
of genes involved in the regulation of the flowering process (He et al. 2004).
SNP, SNAP, and SIN-1 induced flowering in duckweed Lemna aequinoctialis and in L. aequi-
noctialis under noninductive conditions, while these effects of NO donors on flowering were sub-
stantially negated by c-PTIO (Khurana et al. 2011).
10.3.6 NO in Senescence
NO was found to have senescence preventing effects. NO may affect ethylene (a senescence-
promoting factor) biosynthesis: it is suggested that both gases act antagonistically. The NO treatment
leads to the inhibition of methionine adenosyltransferase activity and results in the reduction of the
pool of ethylene precursor S-adenosylmethionine (Arasimowicz and Floryszak-Wieczorek 2007).
The mechanism through which NO may counteract plant senescence could also be related to the
ability of this molecule to interrupt the chain reactions leading to lipid peroxidation, the preserva-
tion of photosynthetic pigments by a direct effect on chlorophyll biosynthesis or by the modification
of the activity, or the turnover of proteins by posttranslational modifications (Jasid et al. 2009). NO
can also prevent Fenton reaction and thus avoid the formation of hydroxyl radical (Wink et al. 1995).
The application of exogenous NO modulated the generation of ethylene (Leshem and Haramaty
1996), delayed senescence (Leshem et al. 1998), and attenuated the induced senescence (Mishina
et al. 2007). SNP also delayed petal wilting in cut carnation flowers, maintained water content, the
antioxidative enzyme activity, and malondialdehyde content as well as cell membrane stability, while
methylene blue had the ability to reverse the active effects of NO (Zeng et al. 2011). On the contrary,
the exposure of plants to NO gas increased ethylene levels and the inhibition of NO synthesis did not
affect ethylene production in Arabidopsis (Magalhães et al. 2000). Selcukcan and Cevahir (2008)
described that lower concentrations of exogenous NO delayed senescence while higher one induced it.
Tobacco floral (Leshem et al. 1998) and leaf (Wilhelmová et al. 2006) senescence was associated
with a significant decrease in NO emission. In senescent pea leaves, endogenous NO generation was
clearly reduced in the vascular tissues together with the downregulation of NOS activity (Corpas
et al. 2004). Endogenous NO was detected in soybean cotyledons at day 10 after germination, but
was undetectable after 25 days. Rejuvenated soybean cotyledons, which did not show marks of
senescence, had higher endogenous levels of NO (Jasid et al. 2009). Expression of a NO-degrading
dioxygenase in A. thaliana initiated a senescence-like phenotype, an effect that proved to be more

pronounced in older than younger leaves (Mishina et al. 2007). Moreover, inhibition of NO syn-
thesis by NG-monomethyl-arginine monoacetate resulted in senescence-associated gene SAG 12
expression and mortality (De Michele et al. 2009).
10.3.7 NO in Programmed Cell Death

There are numerous and often contradictory reports concerning NO and programmed cell death
(PCD). For example, infection of suspension-cultured soybean cells with the bacterial pathogen
Pseudomonas syringae caused NO and ROS increase, which resulted in hypersensitive response
and PCD. NO had also been implicated in Cd-induced PCD in Arabidopsis suspension cultures
(DeMichele et al. 2009).
The increase in either NO or ROS separately did not induce cell death in tobacco, whereas the
simultaneous increase in both NO and ROS activated PCD (De Pinto et al. 2002). On the other
hand, elevated levels of NO were sufficient to induce cell death in Arabidopsis cell suspension, inde-
pendently from ROS (Clarke et al. 2000). The interaction between NO and ROS in PCD induction
in soybean cell suspension showed that NO by itself does not induce PCD, but the key factor is the
NO:superoxide ratio (Delledonne et al. 2001).
On the contrary, Beligni et al. (2002) provided data indicating an NO-dependent delay of devel-
opmental PCD induced by GA in barley aleurone layers. Although NO is able to inhibit PCD in
GA-treated cells, it does not have a general effect on cellular metabolism and is only predicted as a
specific endogenous modulator of PCD (Beligni et al. 2002).
10.4 CONCLUSIONS
Most of the current information about the function of NO in plants has come from pharmacologi-
cal approaches using NO donors, NO scavengers, and NOS inhibitors. The results indicate that NO
mediates either negative or positive effects dependent upon the concentrations of NO used. In addi-
tion, results are influenced by tissue types and age, plant species, treatment duration, etc. The major
challenge ahead is to clarify the metabolism of endogenous NO and related RNS.
ACKNOWLEDGMENT
This work was supported by Grant Agency of the Czech Republic Grant No. P501/11/1239.
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11 Mitochondria in
Plant Physiology
Farhad Ghavami, Ali Soltani, Penny M.A. Kianian,
and Shahryar F. Kianian
CONTENTS
11.1 Introduction.......................................................................................................................... 227
11.2 Plant Mitochondria and Respiration.................................................................................... 228
11.3 Plant Mitochondrial Genome............................................................................................... 231
11.4 Plant Mitochondria and Oxidative Stress............................................................................ 232
11.5 Alternative Oxidase Pathway in Plant Physiology............................................................... 234
11.6 Role of Mitochondria in Programmed Cell Death............................................................... 237
11.7 Cross Talk between the Organelles Affects Plant Physiology.............................................240
11.8 Importance of Cytoplasmic Male Sterility in Crop Plants.................................................. 241
11.9 Heteroplasmy and Its Potential Role in Plant Physiology....................................................244
11.10 Alloplasmic Conditions in Plants Alter Their Physiological Responses............................. 247
11.11 Conclusions.......................................................................................................................... 250
References....................................................................................................................................... 250
11.1 INTRODUCTION
Mitochondria are cellular centers of energy metabolism responsible for the production of ATP
needed for daily functions. Coordination of gene expression among the genomes present in the
mitochondria and nucleus is of crucial importance for cells of all eukaryotes. Hundreds of genetic
diseases in humans and thousands of phenotypic variations in plants and other organisms are known
to be the result of alterations affecting this critical communication. Defects in this communication
are known to affect any organ in the human body and at any age resulting in debilitating and often
fatal diseases. It is believed that in the United States alone, over 50 million adults suffer from
diseases that may in part be due to defects in nuclear–mitochondrial (NM) interaction. Due to the
nature of mitochondrial inheritance, most disorders in NM interaction are maternally inherited.
The mitochondrial genome is highly conserved among diverse organisms, and the functional
relationship with the nuclear genome is critical to all major cellular activities. The size of mitochon-
drial genome varies within the plant kingdom from 186 kbp to more than 2400 kbp. However, this
size variation does not alter the presence of many critical genes needed for proper function and is
mainly a reflection of changes in repeat sequences and open reading frames (ORFs) of unknown
function. In plants, the nucleus coordinates biogenesis and the function of mitochondria and chloro-
plast on a developmental basis. Nuclear genes encode a majority of the proteins needed for organelle
function. Most mutations affecting NM interactions lead to cytoplasmic male sterility (CMS). It is
believed that pollen-forming tissues have a lower threshold value for respiratory deficiencies than
other plant tissues. In all cases examined, the CMS phenotype is believed to result from the expres-
sion of novel mitochondrial polypeptides that may interfere with mitochondrial function and pollen
227
development or alterations in mitochondrial gene expression patterns. Several lines of evidence

from primate cell culture and alloplasmic lines of Tigriopus and Drosophila indicate NM coad-
aptation, and disruption of critical enzyme activities lead to impaired or deleterious phenotypes
emphasizing the importance of NM interaction in the development of all eukaryotes.
Recent data indicate that heteroplasmy (presence of a mixture of more than one type of mito-
chondrial genome within a cell or an individual), which is considered prevalent among plants, also
persists among human. The mechanism and possible means by which heteroplasmy is generated and
maintained are discussed. The important role of heteroplasmy in plant development, physiology is
beginning to be realized. As plants cannot escape, the adverse environmental conditions, manipula-
tion of mitochondrial genotypes or mitotypes may provide a mechanism to tolerate stress conditions.
Alloplasmic lines (lines with alien cytoplasm) have been generated for many plant species,
but those of Triticum–Aegilops species represent the largest collection available to date. This col-
lection, unequalled in the plant kingdom, has been established by the substitution backcrossing
method (almost all at backcross 14 and above). Contrasting (alloplasmic vs. the control euplasmic
[true cytoplasm] wheat lines exhibit an array of phenotypic variations in the formation of gametes,
embryos, anthers, and in the overall plant vigor and height that can be exploited to understand the
influence of NM interaction on eukaryotic cell development. In addition, these lines represent an
exciting opportunity to change the paradigm of traditional crop improvement strategies to meet the
dual challenge of rapid environmental change and population growth.
11.2 PLANT MITOCHONDRIA AND RESPIRATION

The mitochondrion is a spherical or rod-shaped double membrane organelle in the cytoplasm of a
eukaryotic cell. Unlike the nucleus of the cell that has only a single membrane layer to separate the
nuclear contents from the cytoplasm, the mitochondrion has a double-layer membrane (Figure 11.1).
The outer member is permeable to 10 kDa size molecules. The second membrane, known as the
inner member, is impermeable and vaginated to maximize the surface area (Buchanan et al. 2002).
The impermeable nature of the inner membrane requires transmembrane proteins within the mem-
brane to facilitate moving molecules from inside to outside the membrane and vice versa (Jacoby
et al. 2012). The transmembrane proteins are important for regulating the movement of protons
from inside the inner membrane to the inter membrane space. The inter membrane space is between
the inner and outer membranes. This region accumulates the proton needed for the electrochemical
proton gradient for energy production in the form of ATP and NADPH (Jones et al. 2012). The inner
membrane surrounds the matrix. The matrix is where respiration takes place to create energy in the
form of ATP.
The mitochondrion is responsible for generating the energy needed by a cell. The double mem-
brane structure allows this organelle to create an electrochemical proton gradient for the produc-
tion of energy through the conversion of carbon molecules into ATP and NAPH. The source of the
carbon molecule determines if the plant is undergoing cellular respiration or photorespiration (Jones
et al. 2012). With cellular respiration, the carbon source is from a stored carbon molecule in the cell
typically in the form of starch or sugar. For photorespiration, carbon molecules created within the
chloroplast are converted and transported into the mitochondria for energy production.
In plants, through photosynthesis, sugar and starch molecules are created and stored. These mol-
ecules serve as the stored energy for use through glycolysis and the tricarboxylic acid (TCA) cycle
(also known as the citric acid cycle or Krebs cycle). These processes, with the addition of oxygen,
break down carbohydrates and convert them into cellular energy (Jones et al. 2012). The process
of glycolysis takes place within the cytoplasm of the cell. The sugar is broken down into four pyru-
vate molecules through the multistep process of glycolysis. A six-carbon sugar, like glucose, with the
addition of two ATP molecules and nine enzymes (hexokinase, phosphoglucose isomerase, phospho-
fructokinase, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate, 3-phosphoglycerate kinase,
Mitochondria in Plant Physiology 229
Pyruvate
Citrate Acetyl-CoA
FADH2
TCA
Outer membrane
GTP NADH
Mitochondrion CO2 Inter membrane
+ AOX e–
2H+ e– 2H+ 2H Inner membrane
UQ C
I III IV
II V
NADH FAD 2H+ 6H+
1/2O2
NAD+FADH2 H2O
ADP ATP
Citrate
ROS
? TPR, PPR, and
mitochondrial proteins
ROS
?
Isocitrate
ROS
?
Chloroplast Mg-Proto IX Nucleus
FIGURE 11.1 Interrelationships within the cell between organelles with emphasis on mitochondrion (Mackenzie
and McIntosh, 1999). Plant mitochondria are the powerhouse of the cell. The electrons gained by FADH2 and
NADH during the TCA cycle are a source for the ETC in the inter membrane including ubiquinone (UQ), com-
plex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (the cytochrome bc1 complex),
and complex IV (cytochrome oxidase; COX). Under environmental stress, changes in chloroplast redox and
reactive oxygen species (ROS), a by-product of the electron transport pathway along with citrate, induce the
nuclear-encoded mitochondrial genes like alternative oxidases (AOXs) to bypass the cytochrome pathway.
phosphoglyceromutase, enolase, and pyruvate kinase), is converted into two pyruvate molecules, four
ATP molecules, and four NADH cytosolic molecules (Jones et al. 2012).
The TCA cycle within the mitochondrial matrix (Figure 11.2) further converts the carbon mol-
ecule into energy by the removal of hydrogen molecules to create the electrochemical proton gradi-
ent in the inner membrane space or to replenish NAD or FAD to NADH or FADH2, respectively
(Jones et al. 2012). To begin the process, the pyruvate molecules in the cytoplasm created during
glycolysis are transported into the mitochondrial matrix through a protein transport channel driven
by pH differences between the inter membrane space and the matrix. The pyruvate molecule, 2 ADP,
8 NAD+, 2 FAD, and phosphate will be converted into 12 CO2 molecules, 4 ATP, 16 mitochon-
drial NADH, and 4 FADH2 molecules through 9 enzymatic reactions of TCA. Pyruvate is con-
verted into acetyl-CoA, a three-carbon molecule, through pyruvate dehydrogenase. The acetyl-CoA
molecule is converted into a variety of four- and five-carbon molecules including citrate, isocitrate,
alpha-ketoglutarate (2-OG), succinyl-CoA, succinate, fumarate, and malate, before converting back
to a three-carbon molecule, oxaloacetate, to begin the TCA cycle again. The TCA cycle uses eight
matrix enzymes (pyruvate dehydrogenase, citrate synthase, aconitase, isocitrate dehydrogenase,
alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, fumarase, and malate dehydrogenase)
to complete the cycle in addition to the inter membrane–bound protein succinate dehydrogenase.
Hexose
Phosphoenolpyruate (PEP)
ADP
ATP NADH NAD
Amino acids Pyruvate Ethanol
Pyruvate
Citrate
te
eta
ac
alo
Tricarboxylic
Ox
acid cycle
NAD(P)H
Mitochondrion
Succinate
e–
e– Complex II
e–
Complex I UQ Complex III c Complex IV
e–
O2 H2O
AOX
O2 H2O
FIGURE 11.2 A schematic illustration of alternative oxidase and cytochrome c oxidase in the plant mito-
chondrial respiratory chain: complex I; complex II; complex III; complex IV; UQ, ubiquinone; AOX, alterna-
tive oxidase; AOX reduces oxygen to water but, in contrast to complexes III and IV, does not generate any
transmembrane potential, wasting the energy as heat. Although the phosphorylation potential from complex I
is retained resulting in some energy production. Excess pyruvate in the cell is controlled by AOX overexpres-
sion and reduction, regulates ethanol production.
The electrons gained by FADH2 and NADH during the TCA cycle are a source of energy for
the electron transport chain (ETC) in the inter membrane (Jones et al. 2012). These electrons aid in
the movement of the hydrogen protons across the inner membrane to the inter membrane space to
create the electrochemical proton gradient. These electrons are then passed through the ETC also
known as the cytochrome pathway (CP). The CP includes complexes I, II, III, and IV (Figure 11.1),
and results in the production of 12 H2O molecules. Complexes of the CP transfer an electron
from NADH and FADH2 created during the TCA cycle through the electron carrier, ubiquinone,
within the inner mitochondrial membrane. NADH transfers an electron to complex I composed
of many polypeptides with several Fe–S sites. This complex moves the electrons from NADH to
ubiquinone generating NAD+, while pumping four hydrogen protons to the inter membrane space.
At complex II, FAD is created through the membrane-bound protein, succinate dehydrogenase,
of the TCA cycle. Complex II is similar to complex I by containing multiple subunits composing
the protein and several Fe–S sites. This complex also transfers electrons to ubiquinone, but the
substrate that electrons are removed from is FADH2 and not NADH like complex I. Complex II
also pumps two hydrogen protons from the matrix to the inter membrane space. Complex III also
known as the cytochrome bc1 complex is the next. This complex is made from several subunits
with active sites containing heme and Fe–S. Complex III accepts electrons from ubiquinone
and passes them to cytochrome c, allowing for the pumping of four protons per two electrons to
the inter membrane space.
Cytochrome c is not a membrane-bound protein and is found on the outer surface of the inner
mitochondrial membrane. Cytochrome transfers the electrons of the CP to the final complex of
the sequence known as complex IV or cytochrome oxidase (COX). It is composed of several poly-
peptide subunits with heme and Cu active sites. The transfer of electrons leads to the conversion
of oxygen and two hydrogens into a water molecule and the pumping of two protons for each two
electrons. The pumping of hydrogen protons during the transfer of electrons through the CP creates
a chemical gradient between inter membrane space and the matrix of the mitochondria (Jones et al.
2012). The hydrogen protons in the inter membrane space are pumped back into the matrix region
by the membrane-bound protein, ATPase, sometimes referred to as complex V, to regenerate ATP.
ATPase is a multisubunit protein creating a channel within the inner membrane. The energy of the
movement of protons through the channel from the inter membrane space of high proton potential to
the matrix of lower proton potential regenerates ADP into ATP through ATPase. The net output in
ATP molecules potentially generated from one glucose molecule through the mitochondria is about
30 ATP molecules. These energy molecules are then further used for the growth and development
of the plant (Jones et al. 2012).
11.3 PLANT MITOCHONDRIAL GENOME

Mitochondrial genome (mt genome) architecture has been observed as supercoiled DNA, one cir-
cular, multicircular, linear multimeric head-to-tail concatamers, and branched structures (Burger
et al. 2003; Kubo and Newton 2008). There are also linear plasmids present inside mitochondrion
that can insert into the main circular mt genome changing its overall organization (Burger et al.
2003). The size of mt genome varies within the plant kingdom (Table 11.1) from 186.6 kbp in
liverworts (Marchantia polymorpha) (Oda et al. 1992), to more than 2400 kbp in muskmelon
TABLE 11.1
Mitochondrial Genome Sizes from Selected Plant
and Animal Species
Species Accession Numbera Size (bp) Genes
Pongo pygmaeus NC 001646 16,389 13
Pan paniscus NC 001644 16,563 13
Homo sapiens NC 001807 16,571 37
Drosophila melanogaster NC 001709 19,517 37
Saccharomyces cerevisiae NC 001224 85,779 43
Marchantia polymorpha NC 001660 186,609 110
Brassica napus NC 008285 221,853 100
Arabidopsis thaliana NC 001284 366,924 135
Beta vulgaris subsp. vulgaris NC 002511 368,801 171
Nicotiana tabacum NC 006581 430,597 193
Triticum aestivum NC 007579 452,528 78
Sorghum bicolor NC 008360 468,628 54
Oryza sativa NC 007886 491,515 96
Zea mays subsp. mays NC 007982 569,630 218
Zea perennis NC 008331 570,354 59
Tripsacum dactyloides NC 008362 704,100 55
Cucumis melo JF412800 2,428,112 78
JF412792
a GenBank identifiers of assembled or partially assembled mt genomes.

(Cucumis melo) (Ward et al. 1981). The range of size variations seen in plants is not present in
any other eukaryotes including animals. Animal mt genomes are nearly 10 times smaller than
the smallest plant, liverworts. In comparison to animals with a compact mt genome with about
37 genes, higher plant mt genomes contain about 70 genes. Like animals, plants contain genes
encoding tRNA, ribosomal genes, genes responsible for the formation of complex I–V for respira-
tion, and for proteins involved in cross membrane transport (Marienfeld et al. 1999; Notsu et al. 2002).
The additional genes present in plant mt genomes are likely a consequence of gene duplication
due to the repetitive sequences in the plant mt genome.
Rearrangement in mt genome through insertion, deletion, and recombination is common due
to the multicopy nature of this genome in the chondriome (Burger et al. 2003). There are differ-
ent kinds of repeated sequences in the plant mt genome that are active in recombination (for more
details, see Section 11.7). The repeat sequences are not only active sites for exchanging segments
between multiple copies of the mt genome but also the locations for integration and excision of small
circles to/from the master circle (Dong et al. 1998). The repeated sequences in rice cover about 26%
of the mt genome (Notsu et al. 2002), while in a larger mt genome, these repeats can exceed more
than 40% of the genome (Rodriguez-Moreno et al. 2011). The presence of repeats allows for recom-
bination within the genome. If during the process of recombination the exchange between repeats
is unequal, it can lead to segmental duplication or loss. The consequence of this leads to changes in
genome size. Recombination can also restore the function of genes lost through point mutation to
maintain the integrity of the DNA (Barr et al. 2005). The results from these changes due to recom-
bination can cause heteroplasmy and stoichiometric changes (see Section 11.7 for more details).
11.4 PLANT MITOCHONDRIA AND OXIDATIVE STRESS

Oxygen (O2) is needed for respiration in the mitochondria for energy production. Reactive oxygen spe-
cies (ROS) are molecules of oxygen that are partially reduced or are reactive oxygen deviations. These
reactive oxygen deviations include singlet oxygen (1O2 instead of O2), superoxide (O2−), hydrogen per-
oxide (H2O2), and hydroxyl radical (HO•). These molecules serve as signals within the cell to indicate
a stable growth environment when at optimum levels. Molecules such as H2O2 can be transported
within the plant and are necessary for normal plant growth (Kwak et al. 2006). Without the H2O2
signal molecule, plant growth is disrupted (Foreman et al. 2003; Kwak et al. 2003), showing stunted
growth and decreased sensitivity to abscisic acid (ABA). In crop plants, a range of 1–5 μmol/FWg is
the expected range of H2O2 in tissue (Cheeseman 2006). However, increases in ROS levels indicate
a response in the plant to a changing environment. These molecules can damage the plant cell when
present at high levels. The connection between ROS and the molecular changes within the plant is
represented as the ROS network. In Arabidopsis, over 150 genes are responsible for the ROS network
recognizing H2O2 as a cellular signal while controlling for damage from these molecules (Mittler et al.
2004). The ROS network includes genes involved in the production, sensing, signaling, regulation, and
conversion of ROS molecules (Mittler et al. 2011).
ROS molecules are made in the chloroplast and mitochondria. The majority of ROS in the
chloroplast are created from the process of photosynthesis (reviewed in Apel and Hirt 2004). In
the chloroplast, increases in ROS are suspected due to the ETC and photosystem II through the
breakdown of water molecules (reviewed in Apel and Hirt 2004). ROS can also be generated in
the mitochondria through its CP. However, plant mitochondria compared to animal mitochondria
produce a lower amount of ROS due to the use of the alternative oxidase (AOX) pathway levels
(for details see Section 11.4). In the mitochondria, ROS molecules are formed during the produc-
tion of water from the CP at complexes I and III as a consequence of normal plant energy production
(Laloi et al. 2004; Moller 2001).
Abiotic and biotic stresses lead to increased ROS production in the plant cell in the chloroplast
and/or the mitochondria. These stresses include drought, temperature, light, wounding, pathogen
attack, pollution, and herbicides (Keunen et al. 2011; Liu et al. 2000, 2009; Lyons and Raison 1970;
Moran et al. 1994; Orozco-Cardenas and Ryan 1999; Pastore et al. 2007; Prasad et al. 1994; Rhoads
et al. 2006; Torres et al. 2005). The additional ROS molecules can cause cell injury and death
through damage to DNA, cell membranes, and/or proteins. The damage caused by the increased
concentration of ROS molecules is called oxidative stress.
With increases of ROS beyond levels necessary for normal plant growth and development, dam-
age to the cell begins from the free radicals. Cell damage includes breakdown of lipid membranes
causing cell leakage, protein modifications reducing enzyme function, and/or damage to DNA causing
mutations (Jones et al. 2012). The free radical oxidation of the nucleotide guanine allows for pairing
with adenine in the double-stranded DNA helix. This pairing is not detected by DNA damage repair
enzymes (Brieba et al. 2004). The inappropriate pairing creates point mutations in the DNA during
replication. Membranes composed of lipids are also susceptible to ROS. The phospholipids making up
membranes can interact with ROS and become unstable through lipid peroxidation due to autooxida-
tion (Verma and Dubey 2003). This disrupts their stability and ability to maintain membrane integrity
causing organelle/cellular leakage. Further breakdown of the membranes results in end product such
as HNE (4-hydroxy-2 nonenal), which can become attached to active sites of proteins making them
inactive. HNE and other free radicals of ROS molecules interact with proteins and disrupt their func-
tion. These radicals can have a range of effects on the CP and other cellular proteins making them to
become inactive due to changing their chemical charges, cross-linking with other proteins, binding to
Fe–S active site, modifying site-specific amino acid, or increasing protein degradation (reviewed in
Sharma et al. 2012). In Arabidopsis, cell cultures treated with H2O2 to stimulate stress condition showed
decreases in protein levels related to energy production including aconitase, E2 subunit of 2-oxoglu-
tarate dehydrogenase complex, fumarase, succinyl-CoA ligase, two complex I subunits, and β-subunit
of ATP synthase complex (Sweetlove et al. 2002; Taylor and Millar 2007; Taylor et al. 2002). Several
of these proteins also appear to be degraded in the cell during the stress event (Sweetlove et al. 2002;
Taylor and Millar 2007; Taylor et al. 2002). When H2O2 is bound to aconitase, it cannot bind citrate
affecting the TCA and energy production of the cell (Moller 2001). These effects on proteins lead to a
decrease in dry weight and oxygen uptake (Sweetlove et al. 2002). Similar effects are seen with metals
and other pollutants. Metals and herbicides can also interact directly with the CP and other mitochon-
drial proteins interfering with their enzyme function (reviewed in Halliwell 2006 and Keunen et al.
2011). These chemical pollutants result in decreased growth and/or death due to lower protein function.
In nonphotosynthetic organisms, mitochondria are the major sites for ROS production; however,
chloroplasts in plants become a major site of ROS production during photosynthesis (Moller 2001).
Photosynthesis involves O2 consumption. About 1% of O2 will be converted to ROS, which will be
scavenged or controlled by different plant pathways in the cell. In the case of high light intensity,
light oversaturates the PSI and PSII in the chloroplast leading to the creation of singlet oxygen,
H2O2, and superoxide ROS molecules (Foyer and Noctor 2005). The response of this stress is com-
municated from the chloroplast to the mitochondria inducing ROS production in this organelle.
Stress induced by limited water due to salinity, drought, or high temperature results in changes to
cell solute concentrations (Jones et al. 2012; Liu et al. 2000). One of the many cellular solutes is the
amino acid proline. Proline acts as a free radical scavenger protecting the cell from damage by free
radicals. As the water concentration changes in the cell, the concentration of proline also changes,
influencing its effectiveness as a scavenger of excess ROS. Low temperatures do not have the same
effect as high temperatures. Lower temperature affects the efficiency of the mitochondrial enzymes
leading to increases in ROS (Lyons and Raison 1970; Prasad et al. 1994). Pathogens also induce
ROS production in the mitochondria. Several pathogens secrete toxins that interact with the mito-
chondria CP disrupting its function resulting in ROS production (Rhoads et al. 2006). In response to
wounding ROS increases, however, unlike pathogen attacks, no toxin is involved. It is suggested that
plant proteins are damaged during wounding leading to damage of the antioxidative proteins within
the cell leading to an increase in ROS (Orozco-Cardenas and Ryan 1999). In the situation where the
plant is unable to control ROS production, the mitochondria initiates programmed cell death (PCD)
to protect the organism from further ROS damage (reviewed in Laloi et al. 2004).
In comparison to animal mitochondria, plants maintain lower ROS levels using three approaches:
(1) avoidance through control of CPC, (2) detoxification of ROS species, and (3) repair of ROS
damage (Moller 2001). The structure of the mitochondria allows for the controlled production of
electrons and electrochemical gradient for cellular energy. However, when ROS levels increase,
the ability of the mitochondria to continue production of energy via the CP is limited. The ROS
molecules interact with the Fe–S active sites. During stress, a plant needs to protect itself from
additional ROS while maintaining its ability to continue energy production in the mitochondria.
The first mechanism plant utilizes is through the activation of AOX, an uncoupling protein (UCP),
and use of an insensitive NAD(P)H dehydrogenase. These three proteins work together to reduce
ROS by removing electrons from a saturated CP (reviewed in Moller 2001), discussed in the next
section. Removal of the electrons from CP slows/stops the production of more ROS during stress.
The second mechanism plant employs to decrease ROS in the cell is through detoxification.
The superoxide (1O2) ROS molecule is a free radical, which is highly reactive compared to H2O2
(Halliwell 2006). The conversion of 1O2 by manganese superoxide dismutase (SOD) in the mito-
chondria to the less reactive ROS, like H2O2 and O2 protects the cell. The reactive free radical 1O2
can interact with lipids, DNA, and protein, damaging these structures beyond repair (Moller 2001).
Levels of H2O2 also need to be decreased to eliminate the oxidative stress in the cell. The detoxifi-
cation of H2O2 as an ROS can be done through multiple pathways. H2O2 can be catalyzed to water
and ½O2 by catalase (CAT) or through a redox cycle catalyzed using ascorbate peroxidase (APX),
monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase (Jones
et al. 2012). Two additional detoxification systems may be at work in plants including the glutathi-
one peroxidase (GPx) system and the thioredoxin system. However, the existence of these cycles
functioning in plants has not been found (Moller 2001; Pubmed search no new data—personal
communication).
The ROS network connects many components of the cell together. This network is necessary
for the signaling of H2O2 in the cell for normal growth and development in coordination with other
hormone and chemical pathways (Laloi et al. 2004; Mittler et al. 2004). The ROS network is vital
for protecting the plant from abiotic and biotic stresses through the sensing of ROS levels. Through
the sensing of ROS levels, the network can regulate the protection against the excess ROS molecules
and regulate the conversion of the free radicals into less harmful molecules. If the cell is not able
to correct for the ROS imbalance in the cell, the ROS network is able to induce PCD to localize the
stress and protect the plant from further damage (reviewed in Laloi et al. 2004). Work continues to
understand how the cell balances the role of H2O2 as a signal for stressed and unstressed environ-
ments with this network.
It is not clear if the H2O2 levels killing the plant cell are direct or through induction of PCD path-
ways (Mittler 2002). But it is known that oxidative stress is the central common factor in all biotic
and abiotic stresses. Therefore, the overlaps between gene expression patterns seen in most biotic
and abiotic stresses rely on this fact that they produce a common stress in the cell (Fujita et al. 2006;
Seki et al. 2002). Although excessive oxidative damage to the mitochondria can induce PCD, even
mild oxidative stress to the cell perturbs the energy supply for normal growth and shifts it to repair
mechanisms (Taylor and Millar 2007). The consequence of this energy shift includes delayed plant
growth, low vigor, and reduced yield. Though the abiotic and biotic stress sources may be differ-
ent, they result in similar damage phenotypes due to the activation of the ROS pathways for plant
protection.
11.5 ALTERNATIVE OXIDASE PATHWAY IN PLANT PHYSIOLOGY

The AOX pathway is an alternative pathway for respiration when the CP of the mitochondria is
inhibited by ROS molecule. AOX is not sensitive to cyanide in contrast to cytochrome c oxidase
of ETC. Although the AOX pathway is tolerant to a variety of toxins like azide, nitric oxide
(NO), and sulfide, it is inhibited by hydroxamic acids like salicylhydroxamic acid (SHAM) and
n-propyl gallate (nPG) (McDonald 2008). AOX functions as a quinol oxidase to transfer elec-
trons and allows respiration to occur in the absence of heme-containing terminal oxidases found
in the CP. As a quinol oxidase, AOX has to be associated with a membrane where the quinols,
such as ubiquinone, are present (Figure 11.2). Therefore, in plants and animals, AOX is associ-
ated with the inner membrane of the mitochondria and catalyzes the four-electron reduction of
oxygen to water (McDonald 2008). The electron transport by this pathway does not contribute to
a transmembrane electrochemical potential except for phosphorylating potential from complex I
resulting in wasted energy. Although the AOX pathway is not very efficient and produces a great
amount of heat in the cell, some energy will be produced through this pathway (Vanlerberghe
and McIntosh 1997).
The first physiological function of the AOX was discovered while studying thermogenesis
(Vanlerberghe and McIntosh 1997). Thermogenesis is a phenomenon in which the temperature
of a particular part of the plant (mostly flowers) increases by endogenous heat production (Onda
et al. 2008). Many plants raise the temperature by using the AOX pathway to attract a pollina-
tor, to release odors, or to tolerate the cold temperatures. The flowers of the sacred lotus can
control its heat production by changing the AOX expression pattern. In this thermogenic plant,
the activity of AOX was stimulated by succinate rather than pyruvate (Zhu et al. 2011). It is
known that increasing pyruvate activates the AOX pathway to draw down the pyruvate level
(Vanlerberghe and McIntosh 1997). Most of the thermogenic plants produce small amount of
heat for a couple of days. In contrast to most plants, skunk cabbage (Symplocarpus foetidus) can
bloom in spring keeping its temperature at 20°C for a week when temperatures are freezing (Ito
et al. 2004). In this unique plant, increasing pyruvate levels will boost the AOX pathway flux,
as most thermogenic plants, but it seems that coexpression of a UCP helps the heat production
(Onda et al. 2008).
In addition to using the AOX to generate heat, plant can also use it to balance carbon metabolism
and electron transport in the mitochondria. Simultaneous monitoring of oxygen consumption and
level of ubiquinone reduction (Q electrode technique) in isolated mitochondria shows that partition-
ing of electrons to the CP linearly relates to the amount of ubiquinone reduction (Vanlerberghe
and McIntosh 1997). However, the AOX pathway will become activated when levels of ubiquinone
reduction pass a threshold. Therefore, AOX acts as an overflow for the CP when too many electrons
enter (Vanlerberghe and McIntosh 1997). If the supply of inorganic phosphate and ADP is low,
the AOX can help the continuation of electron transport while CP flux will be limited (McDonald
2008). The AOX pathway is not always helping the CP and sometimes competes for respiratory
electrons (McDonald 2008). Using the Q electrode technique, it was discovered that the reduced and
activated AOX enzyme has a higher affinity for reduced ubiquinone. The increased affinity makes
the threshold lower to compete with the CP for electrons (Vanlerberghe and McIntosh 1997). The
ability of the substrates, such as pyruvate, available for respiration to reduce and activate the AOX
enzyme determines if the AOX pathway should act as overflow or to compete for respiratory elec-
trons (Vanlerberghe and McIntosh 1997). AOX activation by pyruvate is a sensitive mechanism to
regulate pyruvate accumulation, which has a central role in carbon metabolism. Pyruvate accumula-
tion increases the AOX pathway flux, which results in drawing down the pyruvate level. A decrease
in pyruvate prevents the production of large quantities of ethanol as the side product and toxic to the
cell (Vanlerberghe and McIntosh 1997).
Photosynthesis efficiency has shown to be affected by the CP in mitochondria through dissipat-
ing the reducing equivalents and changing the amount of carbon supplies and ATP pool in the cell.
The mitochondrion electron transport in the light may have some role in oxidizing the excess of
photosynthetic reducing power. The production of hydroxypyruvate and glyoxylate in the photores-
piration process activates AOX similar to pyruvate. This shows the potential for interaction between
AOX and photosynthesis (Vanlerberghe and McIntosh 1997). It is now clear that both AOX and the
CP have essential roles in photosynthesis efficiency. It seems that the CP helps the process of photo-
synthesis by providing more ATP to the cytosol for sucrose synthesis, while the AOX pathway tends
to dissipate the excess reducing equivalents produced by the chlorophyll. If all the flowing electrons
from the ubiquinone enter the AOX rather than CP, it produces ATP no more than one-third of CP
capacity. This emphasizes the reality of the previous statement that AOX acts as an antioxidant
system for photosynthesis (Yoshida et al. 2006).
AOX has a primary role in controlling ROS generated in the mitochondria. Although ROS has
important signaling functions in the cell in small quantities, overproduction of these species can be
of great danger to the cell. AOX can prevent ROS production by controlling the overreduction of
the CP. Being active as a monomer and dimer, AOX has an advantage over other multisubunit com-
plexes. It can be produced rapidly and use the excess electron production in the CP during biotic and
abiotic stress conditions while controlling ROS production (McDonald 2008). Other pathways such
as the glycolate oxidase pathway are also enhanced during abiotic stress similar to AOX. Although
ROS is an unavoidable by-product of respiration and photosynthesis, NADPH oxidases, amine oxi-
dases, and cell wall–bound peroxidases are also involved in ROS production. While overproduction
of ROS is mainly controlled by AOX expression and function (Mittler 2002), ROS scavenging has
been conducted by different mechanisms like that of SOD, APX, and catalyse (CAT).
When AOX1 was suppressed by antisense RNA, ROS formation in tobacco suspension culture
was significantly higher than wild type (Maxwell et al. 1999). In contrast, transgenic cell cul-
tures with AOX1 overexpression had lower amounts of ROS than wild type (Maxwell et al. 1999).
Inhibition of the CP with potassium cyanide in Arabidopsis plants produced a large quantity of
ROS in the wild type and transgenic plants expressing the antisense RNA of AtAOX1. However,
ROS production was minimal while overexpressing the AtAOX1 gene. The microarray analysis on
the antisense transgenic lines showed that genes associated with carbon metabolism and photosyn-
thesis were affected (Umbach et al. 2005). Several other studies also have indicated that AOX plays
an important role in lowering ROS formation in plants as reviewed in detail (Juszczuk and Rychter
2003; McDonald 2008). However, the direct proof of AOX’s role in controlling ROS in intact plants
seems to remain controversial. In a recent study, Tarasenko et al. (2012) indicated that changes in
AOX expression did not affect ROS content or stress sensitivity in the whole plant, while its effect
is seen in suspension cultures.
AOX genes have been found in all organisms except archaebacteria. AOX is a nuclear-encoded
gene in eukaryotes and appears as a small gene family in most plants studied (McDonald 2008). The
AOX1 gene is present in all the angiosperms and is known for its expression under stress conditions,
while AOX2 has mostly been found in dicots and has a constitutive expression as a housekeeping
gene (Juszczuk and Rychter 2003). However, its constitutive presence in some tissues determines its
potential role in normal plant condition (Juszczuk and Rychter 2003). AOX gene expression under
a variety of environmental stresses indicates that this gene belongs to the stress-induced proteins.
The upregulation of AOX expression and the increased activity of the AOX pathway during biotic
and abiotic stresses gives flexibility to the ETC. This flexibility is an advantage for a plant during
environmental stress (McDonald 2008).
It has been shown that cold stress changes the expression of AOX in monocots and dicots, but
the response was different for various members of the AOX family. In Arabidopsis, AtAOX1a is
upregulated by cold stress, while in sugarcane, SsAOX1c expression increases after 6 h of cold stress
and decreases back to its normal expression by 24 h (Borecky et al. 2006). By inhibiting the AOX
pathway in watermelon using 2 mM salicylhydroxamic acid (an AOX pathway inhibitor), the plants
become susceptible to the cold stress treatment (Li et al. 2012). It seems that AtAOX1a and AtAOX1d
(also known as AtAOX3) are upregulated by a wide range of plant stresses in Arabidopsis showing
that the AOX pathway plays an important role in plant response to environmental stress (Van Aken
et al. 2009b).
Induction of AOX has been reported in a number of other abiotic stresses like drought (Bartoli et al.
2005), salinity (Ferreira et al. 2008), high light (Yoshida et al. 2011), and nutrition deficiency (Sieger
et al. 2005). Arabidopsis plants constitutively overexpressing the AtAOX1 gene had lower ROS for-
mation and 30%–40% increased growth than normal plants in salt stress conditions (Smith et al. 2009).
In a study on citrus cell suspension culture exposed to salinity, an increase in the amount of AOX
protein could be detected 24 h after 200 and 400 mM NaCl treatment. However, a moderate salt
stress of 100 mM could not trigger AOX overproduction as other mechanisms of ROS scavenging and
detoxification are more likely to handle this situation (Ferreira et al. 2008). Expression of AOX1 in
the leaves of pea irrigated with 150 mM of NaCl shows upregulation of the gene in the early stage of
salt stress (5 days after irrigation), but a downregulation after 14 days. However, the protein content
was maintained and significantly higher at 14 days, indicating posttranscriptional or posttranslational
regulation of the protein (Marti et al. 2011).
In wheat, the AOX pathway is upregulated by drought stress protecting the photosynthetic appa-
ratus against overreduction of chloroplast electron carriers (Bartoli et al. 2005). The enhanced
capacity of the AOX pathway is seen also in rice during drought stress (Feng et al. 2009).
AOX1 in Arabidopsis is induced not only by cold, salt, and drought stresses but also under other
treatments including UV-B and ozone (Kilian et al. 2007). This shows that triggering AOX is a com-
mon response to environmental stresses. Analysis of the AtAOX1a promoter region revealed bind-
ing motifs for WRKY and Dof (Dojcinovic et al. 2005). Later, Ho et al. (2008) found 10 different
unique cis element motifs in the AOX1a promoter region; some of them related to stress responses.
So, it seems that AOX can be induced by a variety of environmental stresses through different tran-
scription pathways.
AOX not only is stress responsive but also plays an important role in defining stress responses,
although it is not clear whether the H2O2 or AOX levels or even the release of cytochrome c from
mitochondria are responsible for inducing PCD in the cell (see Section 11.5 for more details). But it
seems that the AOX level determines the threshold for the induction of PCD in plants being the major
player for controlling ROS formation. This means that the amount of AOX formation and reduction
has a key role in defining the stress responses in an organism, reviewed by Van Aken et al. (2009a).
AOX has also an important role in fruit development. Transgenic tomato plants overexpressing
LeAOX has the same fruit ripening pattern as the wild type. However, the transgenic RNAi plants
with reduced LeAOX mRNA level showed retarded ripening, reduced carotenoid, decreased respi-
ration, and ethylene production (Xu et al. 2012). In the process of fruit ripening in mango, it was
observed that MnAOX2 declines steadily toward a minimum at the ripening stage, while MnAOX1
increased up 10-fold by that stage (Considine et al. 2001). It seems that enhancement of ethylene
biosynthesis increases the level of HCN in the cell inhibiting the CP while inducing the AOX path-
way. However, the action of CN as a signaling molecule for AOX induction or as a toxin for the CP
inhibition is not yet clear (Xu et al. 2012).
11.6 ROLE OF MITOCHONDRIA IN PROGRAMMED CELL DEATH

PCD is a highly regulated process of cell suicide in many eukaryote organisms including plants
and animals as part of normal growth and development (Vianello et al. 2007). PCD, in many cir-
cumstances, can be considered as a necessary pathway critical for an organism’s normal lifecycle.
Pathogens try to override (overcome) the PCD of a host cell to survive and propagate. For example,
baculovirus produces a viral protein (p35) to inhibit PCD pathway in animal cells (Clem et al.
1996). The ability of p35 protein to inhibit PCD pathway in both animal and plant systems may
indicate a similar pathway in all eukaryote organisms. However, necrotrophic pathogens promote
PCD through cell death and consequent pathogen spread (Govrin and Levine 2000).
In higher plants, PCD has been recognized as a crucial mechanism for normal development of
almost all cells and tissues including megaspore formation, endosperm and aleurone degradation
(in monocots), tracheid, root cap and aerenchyma formation, and leaf senescence (Gray 2004). PCD
also plays an important role in an organism’s response to biotic and abiotic stresses. For instance,
PCD has been documented in plant root cell response to hypoxia (Drew et al. 2000). This study
showed that hypoxia stimulates ethylene hormone synthesis. This hormone via a signaling cascade
triggers PCD and, consequently, aerenchyma formation. PCD stimulation was also detected in plant
cells in response to other abiotic stresses such as temperature extremes (Vacca et al. 2004) and
ozone (Langebartels et al. 2002). Furthermore, it is recognized that plants use PCD in response
to pathogens for defending themselves. This defensive process, which is known as hypersensitive
response (HR), was explained by Hatsugai et al. (2004) in detail.
Animal and plant cells share some similarities in their PCD characteristics such as cell shrink-
age, chromatin condensation, mitochondrial electric potential (m∆Ψ) dissipation, endonuclease,
and cytochrome c release (Vianello et al. 2007). The most obvious difference between animal and
plant PCD pathways is in their regulators (antiapoptotic/proapoptotic proteins). In animals, Bcl-2
gene members are the best characterized PCD regulators. This family contains both PCD sup-
pressors and promoters (Droin and Green 2004). The mechanism of action of Bcl-2 gene was first
explained in B-cell leukemia/lymphoma 2 disease. However, in plants, no Bcl-2 homologous genes
have been detected in genomic surveys. In plants, Bcl-2-associated X protein (BAX) members may
act as the proapoptotic proteins. Lacomme and Santa Cruz (1999) showed that BAX overexpres-
sion in tobacco cells is associated with the occurrence of symptoms similar to HR-induced PCD.
Interestingly, this protein is functional only when targeted to mitochondria, indicating the critical
role of mitochondria in HR response of plants.
In plants, generally, two types of cell death have been identified: necrosis and PCD. In necro-
sis, plant cells are accidentally dying in response to an extrinsic factor such as a phytotoxin (Van
Breusegem and Dat 2006). Necrosis is usually associated with cytosolic membrane loss of function.
However, PCD itself can be categorized in two groups: autophagy and cell death during HR. In
autophagy, vacuole and its membrane (tonoplast) play the key role in the development of cell death
during xylem and aerenchyma formation (Lam 2004). Usually in this form of PCD, vacuole rupture
immediately precedes nuclear DNA fragmentation (van Doorn and Woltering 2005). PCD during
HR leads to nuclear DNA fragmentation before tonoplast rupture highlighting involvement of other
pathway(s) in PCD.
It is well documented that mitochondria is one of the central PCD regulator elements in ani-
mal cells (Desagher and Martinou 2000; Green and Kroemer 2004) and at least in the HR of
plant cells (Lam et al. 2001). In animals, mitochondrial-mediated PCD regulators involve proteins
that can be categorized into two groups: (1) proapoptotic proteins that promote the apoptosis path-
way and (2) antiapoptotic proteins that are integrated in the outer surface of the mitochondrial
outer membrane. These antiapoptotic proteins prevent translocation of proapoptotic proteins in
the mitochondria and consequently preclude PCD (Kaufmann and Hengartner 2001). Although
the mitochondrial-mediated apoptosis pathway is not yet understood, several models have been
proposed. These models all emphasize on the higher permeability of mitochondrial outer or inner
membrane for apoptosis commence (Bernardi et al. 2001; Desagher and Martinou 2000; Kroemer
and Reed 2000). Higher permeability of outer/inner mitochondrial membranes, depending on the
existence of apoptotic proteins, allows more soluble proteins to translocate into the mitochondrial
matrix. Higher concentration of soluble proteins inside the matrix results in m∆Ψ dissipation, more
osmotic swelling, mitochondrial outer-membrane rupture, and consequently the release of mito-
chondrial intermembrane proteins into cytosol (Kroemer and Reed 2000). These proteins contain
several enzymes that activate apoptosis activation. Endonuclease G (Ameisen 2002; van Gurp
et al. 2003) is among them, which upon its release can be transferred to the nucleus and digest
nuclear DNA. Cytochrome c also triggers caspase-dependent apoptosis pathway via sequestration
of apoptosis protein inhibitors (van Gurp et al. 2003). In this model, cytochrome c causes apoptotic
protease activating factor 1 (Apaf1), an adaptor protein, to dimerize. Upon dimerization, another
Apaf protein (procaspase 9) comes in close proximately to Apaf1. Consequently, these proteins
transactivate each other as well as other downstream caspases leading to PCD (Zou et al. 1999).
Support for the cytochrome c model inducing apoptosis has also been detected in differ-
ent plants. The first evidence was observed in carrot cells, where addition of cytochrome c pro-
moted DNA digestion of mouse nuclei suspended in carrot cytoplasmic extracts (Zhao et al. 1999).
Several other studies support the association between mitochondrial cytochrome c release into the
cytoplasm and PCD symptoms in various plant cells. These studies include response of maize to
Agrobacterium infection (Hansen 2000) and Arabidopsis thaliana to mannose (Stein and Hansen
1999). Critical question remains as to how cytochrome c can trigger cell death in plants considering
that there are no caspase homologous genes detected in any plant genomes (Jones 2000). It has been
proposed that the release of mitochondrial cytochrome c induces PCD by interrupting the electron
flow from complex III to complex IV, decreasing ATP synthesis, and elevating the ROS formation.
Elevated ROS concentration serves as a PCD amplification signal (Blackstone and Green 1999).
Also there may be genes in the plant genome with similar function to caspase but enough sequence
divergence that prevent them to be detected based on comparative studies. Besides cytochrome c
release, other PCD-related symptoms were also observed in plant cells including m∆Ψ dissipation
and overexpression of AOX and heat shock proteins (Krause and Durner 2004). In another study,
a mutation in the sunflower mitochondrion was associated with cytochrome c release and PCD in
tapetal cells, resulting in CMS (Balk and Leaver 2001). Similar to animal PCD, it was observed that
endonuclease activity associated with cytochrome c release plays a crucial role in PCD pathway in
response to different stimuli (Balk et al. 2003).
Both outer and inner membranes play critical role in releasing cytochrome c and endonucleases
via several mechanisms. In wheat and potato tubers, mitochondrial permeability transition pore
has been detected on the inner membrane that can allow the entrance of soluble proteins into the
mitochondrion (Fortes et al. 2001; Virolainen et al. 2002). This may cause mitochondria swelling
and rupture of its outer membrane and consequently release of cytochrome c. Another channel has
been detected in mitochondrial inner membrane that potentially can be involved in cytochrome c
release. This is a K+ channel that in mammals can be involved in cytochrome c release even with-
out mitochondrial outer membrane disruption (Gogvadze et al. 2004). In this mechanism, it was
hypothesized that only a subtle change in mitochondrion volume can induce cytochrome c release
into the cell cytosol. However, a mitochondrial volume modification is not a necessity for all mod-
els of cytochrome c release. Voltage-dependent anion channels (VDACs), which are also present
on mitochondrial outer membrane, can be involved in nonswelling mechanisms of cytochrome c
release in plant cells. It was observed that VDAC expression elevated in HR and senescence in
A. thaliana, indicating the potential role of these channels on PCD (Lacomme and Cruz 1999).
ROS such as H2O2, superoxide ion, and nitric oxide (NO) has also been identified as the main
regulators of PCD in animals and plants. Therefore, it seems that PCD pathways can be regulated via
several mechanisms and signals including hormones (Hoeberichts and Woltering 2003). At above-
threshold concentrations, ROS molecules can react with almost any biomolecule and be destructive.
But a well-defined mechanism by which ROS regulate cell death has remained unknown. Indeed, it
was shown that ROS can trigger cell death by several different pathways. In a hormone-dependent
pathway, oversynthesis of specific phytohormones, such as salicylic acid and ethylene, is positively
associated with ROS overproduction and cell death (Overmyer et al. 2005). In a hormone-independent
mechanism, ROS can regulate cell death by directly interacting with proteins. For instance, it was
shown that three zinc finger proteins (LSD1, LOL1, and LOL2) can sense the ROS concentration in
Arabidopsis cells and regulate the downstream PCD-related genes (Epple et al. 2003). Considering
the effect of ROS in PCD regulation, any gene that can regulate the ROS concentrations in a cell can
indirectly control PCD pathway. In fact, it was shown in the Arabidopsis genome that there are more
than 158 genes involved in ROS formation-scavenging network indicating the complex scheme of
ROS regulation (Mittler et al. 2004).
Mitochondrion is highlighted as the main initiation site for PCD pathway in both animals and
plants. This central compartment determines the cells’ fate to die or live. In this model of PCD,
mitochondria are considered as a warehouse of PCD inducer proteins (like cytochrome c), which
can be released in the presence of upstream signals such as anti- or proapoptotic proteins. The
function of AOX in regulating PCD and ROS production (reviewed in previous section) has left
scientists with an uncertainty of the first inducer of this phenomenon especially when the AOX and
cytochrome c pathways are tightly associated.
11.7 CROSS TALK BETWEEN THE ORGANELLES AFFECTS PLANT PHYSIOLOGY

It is hypothesized that mitochondria and chloroplasts descended from free-living bacterial ances-
tors (Dyall et al. 2004). Following their acquisition by eukaryotic cells during endosymbiotic evo-
lution, most of the genes in these organelles were either lost or transferred to the nucleus. Today,
most of the mitochondrial proteins (over 1500, 93%–99%) are encoded in the nucleus, synthesized
in the cytoplasm, and then imported into the organelles (Woodson and Chory 2008). For instance,
human mtDNA contains only 37 genes and codes for 13 polypeptides, 22 tRNAa, and 2 rRNAs
(Ryan and Hoogenraad 2007). Therefore, it is essential for the organelles to cross talk and to
communicate effectively for protein exchange and gene expression regulation with the nucleus.
Hundreds of genetic diseases in humans and thousands of phenotypic variations in plants and other
organisms are known to be the result of alterations affecting NM communication/relationships.
These communication mechanisms include nucleus to organelle (anterograde) and organelle to
nucleus (retrograde) signaling. Anterograde mechanisms coordinate gene expression in organelles
in response to endogenous and environmental stimuli that are perceived by the nucleus. Retrograde
mechanisms transmit signals that originate in the organelles to regulate nuclear gene expression,
which can then alter anterograde control (Woodson and Chory 2008).
In anterograde signaling, nuclear-encoded organelle proteins can control gene expression in
mitochondria and chloroplast at several levels. The first level of organelle gene expression regula-
tion can be transcriptional control. It is documented that NEP (nuclear-encoded RNA polymerase)
and PEP (plastid-encoded RNA polymerase) are involved in chloroplast gene expression and are
encoded by the nucleus and chloroplast, respectively. During plant development, chloroplastic genes
are transcribed dominantly by PEP, rather than NEP (Maliga 1998). In general, PEP has a higher
affinity to transcribe photosynthesis-related genes (Maliga 1998). This example clearly indicates
the role of anterograde signaling on physiological development of chloroplast. However, most of the
gene regulation does not happen at transcriptional level and therefore is not correlated with protein
levels in mitochondria (Giege et al. 2005) or chloroplast (Eberhard et al. 2002). Posttranscriptional
and posttranslational modifications are the main mechanisms of organelle protein regulations medi-
ated by nuclear genes (Leon et al. 1998). Nuclear-encoded regulators are found in three classes of
proteins: pentatricopeptide repeat (PPR) or tetratricopeptide repeat (TPR) proteins that are con-
served in most eukaryotes, or pioneer proteins that are less conserved. The pioneer-type proteins
do not have any identifiable motifs other than those for localization to the organelle. It is believed
that both PPR and pioneer proteins are functional via binding to a specific site of organellar mRNA
and then recruiting RNA metabolism enzymes for further RNA modification. The TPR proteins
are active in protein–protein interactions especially in posttranslational regulations (Woodson and
Chory 2008). Organelles consist of several protein complexes encoded by nuclear and organelle
genes simultaneously. Thus a balanced coregulation of both genomes seems necessary for the intact
function of TPR proteins. An example of anterograde signaling with TPR proteins is in sucrose-
starved Arabidopsis thaliana plants. These plants had lower levels of mitochondrial ribosomes and
respiration protein complexes (Giege et al. 2005). Further investigations showed this lower level of
mitochondrial protein complexes resulted from a lower abundance of transcripts/translation product
of nuclear-encoded genes that form the functional complexes together with mitochondrial-encoded
proteins (Giege et al. 2005).
Retrograde signaling is another form of communication from organelles to nucleus. In this case,
chloroplast and mitochondria modify the expression profile of nuclear genes in response to stress
or developmental factors. Despite consideration of anterograde signals, retrograde signals seem to
be highly diverse in different species and tissues. ROS is identified as the most universal member
of the retrograde signals for both mitochondria and chloroplast (Woodson and Chory 2008). In
stress conditions sensed by mitochondria or chloroplast, a less efficient ETC is usually associated
with increased levels of ROS. Although high levels of ROS has destructive effects to normal cel-
lular functions, it can trigger a retrograde signaling pathway resulting in upregulation of nuclear
target genes such as AOX (Rhoads and Subbaiah 2007). This retrograde signaling is indispens-
able for organism’s survival under stress conditions by inducing cellular protection via the AOX
protein, which reduces the amount of excessive ROS (Rhoads et al. 2006). Chloroplast also can
produce its own retrograde signals. The most well-known chloroplast-specific retrograde signal is
Mg-protoporphyrin (Mg-proto) IX. This was the first unique intermediate of chlorophyll biosyn-
thesis (Woodson and Chory 2008). It was shown that in undeveloped chloroplasts, photooxidative
damage can happen due to lack of enough carotenoids. In this situation, accumulation of Mg-proto
in chloroplast and cytoplasm can act as a retrograde signal for repressing nuclear genes responsible
for photosynthesis-related protein synthesis (Strand et al. 2003). ABA Intensive 4 (ABI4) was char-
acterized as a transcription factor that can be regulated by Mg-proto signals and repress chloroplast
protein-encoding genes (Koussevitzky 2007).
Other crucial cross talks exist between chloroplast and mitochondria in plant cells. Although
several sets of evidence confirm the presence of this type of communication between organelles,
no direct signals have been detected. Several studies suggest that for efficient photosynthesis,
integrity of both mitochondria and chloroplast is necessary (Raghavendra and Padmasree 2003).
It is elucidated that although some TCA cycle reactions are inhibited under light conditions, the
ETC and phosphorylation are conducted supporting the photosynthesis processes in chloroplast
(Padmasree et al. 2002). There are three hypotheses for the crucial role of mitochondria in photo-
synthesis (Raghavendra and Padmasree 2003): (1) mitochondrial oxidative metabolisms can dis-
sipate excessive redox equivalents from chloroplast in both carbon and ammonia assimilation,
(2) photosynthetic carbon assimilation, and (3) protection of chloroplast against photoinhibition.
Close interrelationships between the chloroplast and the mitochondria are also thought to exist
due to evidence based on nucleotides. Comparison of mitochondria and chloroplast tRNA mol-
ecules finds that they can be exchanged between the two organelles (Bennoun and Delosme 1999).
It was also shown that mutations in mitochondrial glycine decarboxylase (Heineke et al. 2001;
Igamberdiev et al. 2001) and mitochondrial NADH dehydrogenase (Sabar et al. 2000) result in
impaired function of the chloroplast. The defective glycine decarboxylase was observed in potato
and barley with impaired photorespiration. However, in mitochondria, an NADH dehydrogenase
mutation had a drastic reduction in photosynthesis. This decrease was particularly observable in
dark-to-light transition or whenever the photorespiration and carbon fixation occurred at the same
time. In a mutant Arabidopsis, which is unable to edit ATP9 transcript, an increase in levels of
chlorophyll degradation was observed (Busi et al. 2011). However, it is not clearly elucidated if the
main cause of this degradation is the nuclear-encoded enzymes or the excessive ROS production
by mitochondria. Vice versa, in the barley mutant albostrains, nonfunctional chloroplast results in
an increase in both mitochondrial gene copy numbers and transcripts (Hedtke et al. 1999). Cross
talk between organelles can also be mediated via the nuclear genome. For example, the maize
nonchromosomal stripe 6 mutation in the mitochondrial genome can alter the expression pattern
of PSI-related genes in nucleus and chloroplast (Jiao et al. 2005). These examples illustrate the
coordination of organellar function through proper communications.
11.8 IMPORTANCE OF CYTOPLASMIC MALE STERILITY IN CROP PLANTS

Mitochondria and chloroplasts are recognized as organelles for energy production and photosyn-
thesis, but they also control several other useful traits including fertility, aging, stress tolerance,
biomass production, and plant growth regulation. Among these organelle-originated traits, CMS
is one of the most important economic traits that has been utilized widely by breeders for hybrid
seed production. By definition, male sterility is a physiological characteristic, which results in the
failure of functional pollen production in specific lines. Since its first observation in early 1900s in
flax (Bateson and Gairdner 1921), the role of cytoplasm was highlighted as the primary cause of
majority of male sterility conditions. Today it is well documented that changes in the mitochondrial
genome is the main reason for this phenotype. After the Bipolaris maydis epidemic on maize lines
A C A C
Stamen
Sepal
Sepal
Sepal
Pistil
Pistil
Pistil
Petal
(a) (b)
FIGURE 11.3 ABC model for floral organ identity. (a) Precise expression of three gene classes is necessary
for correct flower formation in all angiosperms. Expression of A gene class converts meristematic cells to
sepal. Expression of the B and A gene classes resulted in petal formation, B and C in stamen formation, and
finally C class expression is necessary for pistil formation. (b) Loss of the B gene class function results in
pistillody phenotype in which stamen transforms to pistil and petals show sepal characteristics.
having Texas type of CMS (Pring and Lonsdale 1989), attention was directed to the effect of cyto-
plasm on floral development. Three different physiological abnormalities can result in CMS phe-
nomenon: (1) homeotic changes, (2) stamen degeneration, and (3) functionality of pollen. Homeotic
changes are when male reproductive organs (stamens) are converted to petals (petaloid phenotype)
or pistils (pistillody phenotype). Stamen degeneration is when anther or tapetal cells cannot be
developed and consequently pollen will not be produced. The third abnormality is when anther and
tapetal are normal but normal viable pollen cannot be developed itself.
Homeotic CMS can be explained with the ABC model of floral organ identity. In normal flo-
rets, precise spatial expression of three gene classes is necessary for the correct formation of floral
organs (Figure 11.3). These three gene groups belong to the same family of transcription factors,
the MADS-box gene family (Schwarzsommer et al. 1990). Alterations of these gene functions can
be induced by either mutation of nuclear genes or the presence of particular mitochondrial ORFs.
Mitochondrial-induced homeotic CMS indicates that retrograde signaling from mitochondria to
nucleus should regulate the expression level of at least these three gene classes. Loss of class-B
gene function was identified as the pistillody in which stamen transforms to pistil and petals have
sepal characteristics. The loss of class-B gene function in CMS phenotype of several different allo-
plasmic lines including wheat (Triticum aestivum L. cv. Norin 26) containing the cytoplasm of the
wild relative species, A. crassa L., showed the pistillody phenotype (Krizek and Meyerowitz 1996).
Interestingly, this alloplasmic line shows the homeotic CMS phenotype when grown under long-day
conditions, but is fertile in short-day conditions (Murai and Tsunewaki 1993). Reduced expression
of the wheat APETALA3 homologue, which is a class-B gene member, is believed to be the main
reason for this pistillody form of CMS. It was hypothesized that ATP depletion resulting from mito-
chondrial dysfunction can elevate the activity of negative regulators of class-B genes. The activity
of the negative regulators will consequently trigger the carploid phenotype (Teixeira et al. 2005).
Replacement of class C by A in stamen-defined regions results in a petaloid phenotype. Specific
carrot CMS lines have been identified with petaloid phenotypes (Wright et al. 1996).
Stamen degeneration is another reason for CMS in some alloplasmic conditions such as
Helianthus annuus (sunflower) plants, carrying H. petiolaris cytoplasm. Maize plants, with the
Texas type of CMS (CMS-T), show a high degree of tapetal cell necrosis making the plant com-
pletely male sterile. The candidate mitochondrial locus responsible for CMS-T was recognized as
an ORF (urf13) encoding a protein integrated into the mitochondrial inner membrane. In the vegeta-
tive tissues of these maize lines in the presence of Bipolaris maydis toxin, URF13 can form a pore
across the membrane. This pore disrupts the mitochondrion inner membrane leading to sterility
(Braun et al. 1990).
To date, more than 14 mitochondrial ORFs were determined to be associated with CMS (Hanson
and Bentolila 2004). These are usually chimeric ORFs containing normal regulatory elements of
a particular gene fused to a functionally unknown segment. Since there is no homologous region
in the nucleus or in the chloroplast for the unknown segment, the origin of these DNA segments is
usually not clear. It is believed that mitochondrial intra- or intergenomic homologue recombination
results in the emergence and establishment of chimeric ORFs. These chimeric ORFs, in most cases,
contain a promoter and a portion of the ATP synthase subunits 4, 6, 8, or 9. The regulatory element
from the partial mitochondrial genes may allow the chimeric ORFs to be transcribed and integrated
into mitochondrial membrane interrupting the proper function of the mitochondrion (Hanson and
Bentolila 2004).
Several hypotheses have been emerged concerning the role of these ORFs on the obstruction of
pollen production. One hypothesis is based on URF13 in maize CMS-T lines and its possible role
in mitochondrial inner-membrane disruption. Although it is unproven, it is suggested that in tapetal
cells, the presence of an altered gene product can form a similar pore as seen in PCD and triggers
necrosis of tapetal cells. Another hypothesis of how ORFs create sterility is based on a work in
rice (Wang et al. 2006). CMS in rice (Oryza sativa) with Boro II cytoplasm has been shown to
involve a novel mitochondrial ORF (orf79) encoding a cytotoxic peptide. Interestingly, the ORF79
protein accumulated in microspore leads to male sterility (Wang et al. 2006). In other CMS condi-
tions, related ORFs cannot integrate into mitochondrial membrane but can modify the expression
of neighboring cotranscribed normal genes. For instance, in the pol CMS system in Brassica, a
chimeric ORF was identified as a CMS-associated gene located directly on the upstream region
of a normal atp6 gene. This ORF contains 58 codons of atp8 fused to 13 codons of rps3 and an
unknown sequence (Singh and Brown 1991). Expression comparisons of 14 different mitochondrial
genes between Brassica, pol CMS, and fertile lines showed an alteration of atp6 expression in CMS
lines. This altered expression likely resulted from cotranscription of this gene with the mentioned
ORF. Another hypothesis regarding the association of CMS-related ORFs in pollen dysfunction
emerged from CMS sunflower. It is believed that mitochondrial-triggered PCD is the main reason
for this type of CMS (Balk and Leaver 2001). In this case, the characteristic features of PCD such
as cytochrome c release were observed. Another hypothesis regarding the role of mitochondria
in functional pollen production arose from the evidence of a high number of mitochondria in the
tapetal cells. In these cells, the number of mitochondria is 20- to 40-fold higher than vegetative
cells (Warmke and Lee 1978). Higher energy demands of tapetal cells can be the reason for higher
number of mitochondrion in these cells. It is speculated that an incomplete interaction of the mito-
chondria and nucleus causes severe mitochondrial dysfunction within these cells. This results in low
ATP production and premature death of these cells.
In most of CMS cases with mitochondrial dysfunction, the functionality can be restored by the
introduction of one or two nuclear genes into the plant. For example, in CMS-T type of maize,
complete male fertility can be attainable with the presence of dominant alleles in both Rf1 (restorer
of fertility1) and Rf2 (restorer of fertility 2) loci (Wise et al. 1999). Rf genes can be sporophytic or
gametophytic. In the sporophytic system, all pollens produced by a heterozygous genotype (Rf/rf)
are functional. In the gametophytic system, just those pollens with the dominant allele are func-
tional and the pollens with recessive alleles cannot survive (Chase 2007).
In terms of function, rf genes are generally categorized into two different groups: (1) functional
genes which their products can directly ameliorate a defective biochemical pathway or (2) regula-
tory genes that modify the expression pattern of other mitochondrial gene(s). Aldehyde dehydro-
genase, which was previously known as rf2 in CMS-T type of maize, is the best example of a
functional gene. The rf genes indirectly modify the associated biological pathways to recover the
fertility. To date, most cloned rf genes were identified as a member of regulatory genes that can
modify the expression profile of a single or multiple mitochondrial gene(s) at the transcriptional
and/or translational levels (Chase 2007). Almost all regulatory elements are encoded by nuclear
genes and targeted to the mitochondria or chloroplast. Mutations in these genes or the absence of
proper mitochondrial inner-membrane transporter result in improper mitochondrial function caus-
ing male sterility. It seems that restorer alleles can recover the fertility of CMS line at either the
RNA processing or posttranslational levels. Examples of processing at transcriptional level can be

seen in CMS lines of Brassica with nap and pol cytoplasms. Nap and Pol (Polima) are two differ-
ent cytoplasm types that can confer male sterility in B. napus (Lhomme and Brown 1993). In these
lines, the presence of an rfp gene in the nucleus is associated with an enhanced level of processing in
dicistronic orf224/atp6 transcripts. This leads to more abundant atp6 transcripts and fewer orf224
transcripts (Brown 1999; Li et al. 1998). Also in the CMS-S system in maize, rf3 can modify the
transcript pattern of CMS associated with orf355/orf77 (Wen and Chase 1999). However, there
are certain Rf genes that do not change the CMS-associated ORFs at the transcript level. Indeed,
they are altering the proteome profile of these ORFs, indicating posttranscriptional gene control.
For instance, in CMS-T of maize, rf1, although reduces the URF13 abundance at the protein level,
causes no significant decrease in transcript levels (Wise et al. 1999).
Some rf genes belong to PPR proteins that are nuclear-encoded regulators of posttranscriptional
and posttranslational modifications. These PPR proteins are proposed to act as an adaptor that can
bind to specific RNAs and mediated their enzymatic modifications (Lurin et al. 2004; Shikanai
2006). In Arabidopsis, it has been predicted that 54% of all PPR genes are targeted to the mitochon-
dria and 19% to the plastids (Lurin et al. 2004). Several cloned rf genes from various species to date
are characterized as PPR genes that can alter mitochondrial CMS-associated ORFs in either the
transcript or protein levels (Bentolila et al. 2002; Brown et al. 2003; Desloire et al. 2003; Kazama
and Toriyama 2003; Klein et al. 2005; Koizuka et al. 2003; Wang et al. 2006). In another mecha-
nism, fertility of CMS line can be recovered directly via changes in substoichiometric changes of
a particular CMS-associated mitotype. For instance, in common bean, CMS-associated mitochon-
drial ORF (pvs) can be reduced to low substoichiometric levels in the presence of the nuclear Fr
locus (Janska et al. 1998). Since the Fr gene has not been cloned, its precise mechanism of function
is not clear. However, based on similar phenotype observed in the Arabidopsis chloroplast mutator
(CHM), it is predicted that Fr can change the stoichiometric level of mitotypes via the regulation of
mitochondrial genome recombination or replication (Martinezzapater et al. 1992).
In summary, CMS-associated ORFs can cause male sterility via several diverse pathways includ-
ing mitochondrial inner-membrane disruption (CMS-T of maize), cytotoxicity (rice Boro II), tran-
script alteration of other genes (Brassica pol and nap systems), triggering PCD (sunflower) and
altering proper floral gene expression (CMS wheat line). Most of genes cloned and characterized
as restorer of fertility belong to PPR gene family. These genes can change the expression pattern of
mitochondrial genes at the posttranscriptional or posttranslational levels, indicating that the mito-
chondrial transcriptome and proteome alteration is a universal mechanism for fertility recovering
in CMS lines.
11.9 HETEROPLASMY AND ITS POTENTIAL ROLE IN PLANT PHYSIOLOGY

Heteroplasmy is the state when more than one mitochondrial genotype is present in an individual
organism. Usually one type of mitochondria (mitotype) is predominant and the others exist in low
proportion (Kmiec et al. 2006). Heteroplasmy was first observed in pathological (Holt et al. 1990;
Matthews et al. 1995) or nonnatural plant populations (Holt et al. 1990; Matthews et al. 1995;
Yamato and Newton 1999). However, at the beginning of the twenty-first century, heteroplasmy
was observed as a common state of plant mitochondria (Kmiec et al. 2006). Some mitochondrial
rearrangements are found at a very low level in healthy human tissues due to recombination (Kmiec
et al. 2006). In a study on 100 single hair roots from 35 human individuals, 24 different hetero-
plasmic positions were identified in the hypervariable human mitochondrial control region (HV1).
Even highly variable levels of heteroplasmy are observed in a single individual (Grzybowski 2000).
In plants, heteroplasmy is well documented in CMS studies. The first evidence of heteroplasmy
in plants was reported in maize normal (N) and sterile (S and T) cytoplasms (Small et al. 1987).
Prolonged exposures of an atpA probe in DNA hybridization revealed that the mitotypes previously
thought to be unique to male sterile S and T cytoplasms are present in N cytoplasm, but at a low
proportion (Small et al. 1987). Heteroplasmy in natural plant populations has been observed as the
result of biparental inheritance of mitochondrial genome in angiosperms (Mogensen 1996). In the
gynodioecious plant, S. vulgaris, heteroplasmy seems to be a common feature in its natural popula-
tion (Welch et al. 2006). Using quantitative real-time PCR for atp1 and cox1, 61 individuals exhibit
heteroplasmy out of the 408 selected from 22 natural populations (Pearl et al. 2009) indicating
heteroplasmy as a widespread phenomenon in this species.
Three different events can lead to heteroplasmy in the mitochondrial genome of an organism.
The main cause is the recombination between the long and short repeats in the genome resulting
in structural changes (Woloszynska 2010). Point mutations as well as duplications and deletions
are other sources of mitochondrial genome variation in a cell. These mutations are produced by
replication errors, defective repair mechanisms, and also the action of ROS produced in the mito-
chondria (Kmiec et al. 2006). Besides replication and mutations, paternal leakage has been reported
as a factor in mitochondrial heteroplasmy in an organism. Although mitochondrial genomes are
mainly inherited maternally, there are evidences of paternal mtDNA transmission in plants, ani-
mals, and humans (Kmiec et al. 2006). Heteroplasmy seems to be an evolutionary strategy of mater-
nally inherited plant mitochondrial genomes to compensate for lacking of sexual recombination.
Therefore, different mitotypes in the cell accumulate as a reservoir of genetic variability and may
allow accelerated evolution. Occasionally, certain mitotypes are selected and amplified in the pro-
cess called substoichiometric shifting. This substoichiometric shifting allows a minor mitotype to
take over the role of the main genome.
In the past, it was assumed that no recombination occurs in animal mtDNA. The first evidence for
recombination in mtDNA of vertebrates was in the study of tandem repeats in flatfish (Platichthys
flesus). There are two types of repeat arrays in flatfish populations named C and T. However, a mix-
ture of C/T tandem repeats was found in an individual, a direct evidence of recombination in verte-
brates (Hoarau et al. 2002). In contrast to animal, recombination in plants has been known for many
years. One of the first evidence of mitochondrial genome recombination was in the cytoplasmic
hybrid of tobacco through protoplast fusion (Belliard et al. 1979). In plant mitochondria, repeated
sequences are common features that are actively functional in mitochondrial recombination. In
mitochondria, the master circle goes through different recombination events to produce small linear
or circular subgenomes in the cell (Dong et al. 1998). In Brassica, the recombination occurs across a
pair of direct repeats. However, in some crops such as maize, the existence of six direct repeats and
one set of inverted repeat made the prediction of the recombination results complicated (Dong et al.
1998; Fauron and Casper 1994). There are also a dozen short repeats in maize, which produce stable
irreversible recombinations, in contrast to larger repeats that are reversible. Large (or long) repeats
(>1 kb) undergo a high frequency of reciprocal recombination in plant mitochondria. The high fre-
quency of recombination is responsible for dividing the genome into several reversible sublimons
at relatively equal frequencies. The recombination in short (or small) repeats (a few to hundreds
of bp) is in low frequencies and asymmetric. This results in one of the two possible recombinants,
as recently shown (Sloan et al. 2012b). The repeats shorter than 50 bp do not participate in recombi-
nation events, as seen in Arabidopsis, and all asymmetric recombination occurs in the intermediate
repeats ranging from 50 to 556 bp (Davila et al. 2011). Although the reversible recombination is
used temporarily for increasing the copy number of specific DNA region in the mitochondria and/or
changing regulation of gene expression, it is the stable recombination in intermediate repeats that
are the source of heteroplasmy in the mitochondrial genome.
Besides recombination, mutation seems to be the main reason for heteroplasmy in plants. The
occurrence of heteroplasmy in self-pollinating crops is mainly due to mutation. In these plants,
paternal and maternal mtDNA are the same, and paternal leakage is not a factor in heteroplasmy
(Woloszynska 2010). Point mutations and insertions/deletions are considered the main sources of
heteroplasmy in self-pollinated plants such as Arabidopsis (Davila et al. 2011), Triticum–Aegilops
(Tsukamoto et al. 2000), and rice (Bentolila and Stefanov 2012). The mutations responsible for
heteroplasmy are found in coding as well as noncoding regions (Woloszynska 2010). The rate of
synonymous substitution in angiosperm mitochondrial genes has been reported to be three times
lower than chloroplast DNA and at least sixfold lower than nuclear genes (Wolfe et al. 1987). These
rates are not consistent between plant species. Reports of lower (Palmer and Herbon 1988) or higher
(Cho et al. 2004) rates compared to chloroplast and nuclear DNA have been published. The muta-
tion rate is not only variable between different plant lineages but also within each lineage. A 100-
fold difference in the mutation rate has been detected in the genus Silene. There is also variation
within this species for mitochondrial genome size (Sloan et al. 2012a). The high mutation rate is
seen not only in some species (Cho et al. 2004), but also in CMS alloplasmic plants (Darracq et al.
2011). The variable rate of mutation in mtDNA could be related to the efficiency of replication
accuracy and also DNA repair. It is also thought that the production or detoxification of ROS in the
mitochondria could affect the rate of mutation (Cho et al. 2004). Therefore, alloplasmic plants with
modified nuclear–cytoplasmic interaction (NCI) property have a tendency to have higher mutation
rate compared to the normal euplasmic lines with proper NCI established through plant evolution.
Although the inheritance of mitochondria and chloroplast is known to be uniparental (Birky
1995), different mechanisms for cytoplasm inheritance have been observed, which is reviewed by
Mogensen (1996). The paternal leakage of cytoplasm is observed in different studies (Bentley et al.
2010; Pearl et al. 2009; Yang and Griffiths 1993). The inheritance of plastids in a majority of plants
is known to be through the female parent, but there are examples of paternal inheritance or biparen-
tal inheritance for some species. For mitochondria, the inheritance seems to be more often maternal
than paternal except in some conifers (Mogensen 1996). The first evidence of paternal leakage for
mtDNA was observed in triticale, a hybrid crop generated by a cross between wheat and rye. This
study investigated orf25 variants (Laser et al. 1997). The different orf25 variants observed by RFLP
in rye and wheat were all detected in wheat by PCR but in a smaller proportion. This study could not
prove whether the coexistence of orf25 in the hybrid is due to paternal leakage or a stoichiometric
shift of an existing mitotype. In other interesting studies, the paternal leakage has been observed by
quantitative real-time PCR in homoplasmic mothers (Bentley et al. 2010; Pearl et al. 2009). Using
homoplasmic mothers eliminates the chance of having heteroplasmy because of the stoichiometric
level changes of the minor sublimons. Bentley et al. (2010) observed 2.5% of offspring being het-
eroplasmic from crosses between two homoplasmic parents carrying two different mitotypes in
S. vulgaris. They observed 0.5%–100% of paternal contribution in the offspring, showing different
amounts of paternal leakage in this species. Another study showed that the percentage of paternal
mitotypes will increase by further reciprocal backcrossing compared to the F1 in an alloplasmic
line of wheat (Kawaura et al. 2011). The paternal leakage has been proved by additional studies on
S. vulgaris (Bentley et al. 2010), jack pine (Pinus banksiana Lamb.), and lodgepole pine (P. contorta
Dougl.) (Wagner et al. 1991), alloplasmic wheat with A. plasmons (Kawaura et al. 2011; Tsukamoto
et al. 2000), Nicotiana sylvestris (Thyssen et al. 2012), and many other plants (Woloszynska 2010).
Paternal leakage followed by recombination with the maternal mtDNA will yield intermediate
mitotypes different from both parents in their properties (Woloszynska 2010).
The dynamic nature of heteroplasmy results in different composition of mitotypes often referred
to as sublimons. During the analysis of sublimons, they are found to be at a very low copy number
compared to the main genome (Woloszynska 2010). The copy number shifting of pvs-orf239 in
common bean, responsible for male sterility, has been detected at 1000- to 2000-fold difference
in a population of common bean. In some cases, the pvs-orf239 was the predominant mitotype, but
in other cases, it was at substoichiometric levels (Arrieta-Montiel et al. 2001). In triticale, 85% of
the mitotypes were similar to maternal (wheat) parent, and 12% were similar to paternal (rye) parent
mitochondrial genome. However, 0.1% of the orf25 sequences in the wheat chondriome were simi-
lar to the rye version, but in a substoichiometric level. It was suggested that the substoichiometric
shifts of mitotypes may be affected by the alien nuclear genome in triticale (Laser et al. 1997). In an
alloplasmic wheat with A. crassa cytoplasm, the proportion of minor mitotypes similar to wheat’s
mtDNA increased by successive backcrossing in each generation and reached 30% of chondriome
(Kawaura et al. 2011). It is not clear if this shift resulted from paternal leakage or a stoichiometric
shift controlled by the nuclear genome as it becomes more similar to the male genome after each
backcrossing. Therefore, heteroplasmy should not be simply considered as the coexistence of mul-
tiple mitotypes in a cell or an organism but a situation of having variant mitotypes to be selected
preferentially by the nucleus based on different conditions (Woloszynska 2010).
A single mutation in the nucleus can affect the rate of recombination in a particular repeat in the
mtDNA and change the stoichiometric level of a particular mitotype. In N. sylvestris, two different
mitotypes T and U are predominant in regenerated plants from protoplasts of the wild type and
mutant for a nuclear gene “mp4,” respectively. The plants with predominant U mitotype show male
sterility, higher stem height, and late flowering compared to the wild types with T mtDNA (Albert
et al. 2003). Most often, the stoichiometric changes in the chondriome are not controlled by a single
gene. The preferential amplification of different mitotypes has been detected during the dediffer-
entiation and regeneration process in plant tissue culture. The dedifferentiation of the green plant
tissue to callus reduced the main mtDNA to a substoichiometric level of 25%. This changed to 50%
of the total mtDNA by chlorophyll formation in callus and to 86% by regeneration to a green plant
(Kanazawa et al. 1994). Therefore, it seems that mtDNA heteroplasmy and stoichiometric shift are
complicated and may be the reason for a portion of epigenetically occurring somaclonal variations.
It has also been shown that heteroplasmy and the mixture of parental mitochondria in hybrids show
heterosis with regard to oxidation and phosphorylation in maize and wheat (Sarkissi and Srivasta
1967, 1969). It is still not clear whether the stoichiometric shifts in the heteroplasmic condition are
proactive or reactive responses to the new physiological conditions of the plants. However, they can
be potential factors behind many unknown plant physiological behaviors that cannot be addressed
by the nuclear genome itself.
11.10 ALLOPLASMIC CONDITIONS IN PLANTS ALTER

THEIR PHYSIOLOGICAL RESPONSES
Alloplasmic lines can be developed by replacing the nucleus of one species through substitution
backcrossing. In an alloplasmic line, a new combination of nuclear and cytoplasmic genomes is
created that is not observed naturally. The first attempt to obtain these lines was by Michaelis in
1954 (Michaelis 1954). He made extensive investigation of the plasmons of the genus Epilobium.
However, these studies were limited to E. hirsutum and E. luteum due to the strong cross incom-
patibility in Epilobium species. The largest collection of alloplasmic lines is with the Triticum
(wheat genus) and related Aegilops genera (goat grass genus). In these genera, the genetic barriers
to artificial interspecific and intergeneric crossing are less pronounced. All alloplasmic lines can
be derived by using cytoplasmic donors as the female parent and a polyploid wheat as the nuclear
donor parent in a series of backcrosses (Tsunewaki et al. 1996). A large collection of alloplas-
mic wheat lines carrying different plasmons from other Triticum species, Aegilops, Secale, and
Agropyron have been developed by various research groups (Tsunewaki 2009; Tsunewaki et al.
1996). This collection includes 551 alloplasmic lines, between 12 nuclear genotypes of hexaploid
wheat and 46 alien plasmons. The plasmons are derived from all the species of the two related
genera, Triticum (8 species) and Aegilops (24 species), which differed in their nuclear genome
constitutions. Due to these efforts, wheat has the largest array of alloplasmic lines, larger than any
other mammalian, insect, or plant system, making it a model species to study the effect of cyto-
plasm on major physiological traits. The physiological effects of the plasmons in alloplasmic lines
were analyzed for many traits in wheat. Compared with euplasmic lines as controls, 46 plasmons
were classified in 17 distinct groups, based on their effects on 21 characters (Tsunewaki et al.
2002). Plasmon classification based on phenotypic effects was basically in agreement with the
genotyping results through RFLP analysis (Wang et al. 2000), with only two exceptional plasmon
groups. In one, the plasmons show clear differences in their organellar genomes and no obvious
differences in phenotypic effects. In the other group, they have identical organellar genomes, but
showed clear differences in phenotypic effects. From these data, it was concluded that functional
divergence among plasmons was in accordance with their molecular divergence. It was proven
that all studied characters are mostly under direct or indirect control of the plasmons with signifi-
cant NCIs. Among the studied characters, the number of selfed seed production, an indication of
male sterility, was the most sensitive character to alien cytoplasm. Whereas ear length is the least
sensitive to alien cytoplasm substitution. However, female fertility, measured by backcrossed seed
fertility, was not affected by cytoplasm exchange. Other economically important traits, in wheat,
were also measured in the alloplasmic lines including heading time, dry matter weight, plant
height, and number of spikelets per ear. Interestingly, it was shown that the effect of cytoplasm was
significantly different on all studied characters. This indicates the indispensable role of the mito-
chondria and chloroplast on general plant physiology. Furthermore, the significant effect of the
interactions between nuclei and cytoplasms reveals that various nuclear alleles present in distinct
cultivars responded differently to a specific plasmon type.
The cytoplasmic and NCI effects were not limited to common physiological and phenotypic
characteristics of plants. Some novel phenotypes emerged in alloplasmic lines that were important
in studying fertility. The most remarkable ones were pistillody, germless grain formation, premature
sprouting, haploid and twin seedling formation, and variegation under low temperature (Tsunewaki
1993). Germless grains were found more frequently in Triticum aestivum var. erythrospermum and
strain salmon nuclei possessing alien cytoplasms. It seems that germless grains result from par-
thenogenesis of egg cells and the consequent death of these parthenogenetic cells after endosperm
development (Tsunewaki 1993).
A reason for producing alloplasmic lines in wheat is the potential contribution they have for pro-
ducing hybrid wheat. Therefore, CMS and rf genes were analyzed extensively in these alloplasmic
lines. Using extensive cytological and genetic studies, the location and the number of different rf
genes were identified in various wheat varieties (Mukai and Tsunewaki 1979; Muramatsu 1959;
Tahir and Tsunewak 1971; Tsujimoto and Tsunewaki 1984; Tsunewaki 1974, 1982). Most of the
identified rf genes are located on chromosome 1B, but others are located on chromosomes 2B, 6B,
7B, and 1D (Maan et al. 1984). Maan (1985) developed four R-lines with homozygous gene pairs
from single rf genes. However, the rf genes do not overcome hybrid incompatibility, just sterility in
certain situations (Maan 1992a). For example, incompatibility between T. longissimum cytoplasm
and the nuclei of T. turgidum L. var. durum resulted in inviable shriveled seeds. In this case, a key
gene named species cytoplasm specific (scs) can restore the compatibility but not the fertility (Maan
1992b). It seems that this gene exists in all the species belonging to the Triticum–Aegilops tribe. The
scs gene can restore compatibility between each genome and its related plasmon type. The isolation
and characterization of this gene will impact the development of compatible NC combinations. This
will allow for an increase in the number of alloplasmic lines of wheat and other species.
Alloplasmic condition also has proven useful to combat environmental stress conditions. Klimov
et al. (2005) proposed that alloplasmic lines of wheat can serve as a novel type of synthetic plants
beneficial to breeding programs. The alloplasmic lines having the common wheat nucleus and wild
relatives’ cytoplasms have shown their potential for improving plant responses to biotic and abiotic
stresses (Hou et al. 2000; Klimov et al. 2005; Liu et al. 2002). The novel synthetic wheat lines have
shown tolerance to drought and salt stress conditions (Hou et al. 2000; Kholodova et al. 2007).
Some of these lines also performed better than their parents in vigor and yield (Tsunewaki et al.
2002). The advantage of these lines as a source of tolerance over the classic synthetic wheat is that
integrating them will be easy in any adapted cultivars without any adverse effect on the quality or
the genes that are already stabilized in the nucleus (Figure 11.4). This method eliminates the need
for making a big bi/multiparental population and recurrent selection in the breeding programs. This
decreases cost and time of breeding (Figure 11.4). The most difficult part of this breeding strategy is
to establish an array of alloplasmic lines in the same subspecies (e.g., hexaploid wheat or tetraploid
wheat). Establishment of materials is time consuming and requires skills in sexual crossing. After
this initial step, making alloplasmic lines in any other cultivar of that species requires only recur-
rent backcrossing (Wu et al. 1998).
A cultivar A cultivar
A wild relative with
drought tolerance
X X
B A A B
X X
Several rounds of selection in the field and more

backcrossing and selection to have a new cultivar
that is time consuming and costly
(a) (b) An alloplasmic cultivar
FIGURE 11.4 A schematic comparison of using plant improvement through traditional breeding and mak-
ing novel synthetic wheat or alloplasmic cultivars. (a) In traditional breeding, the adapted cultivars (line B) are
crossed to a wild relative (line A) having a few desirable characteristics (such as drought tolerance) but full
of undesirable traits. After mating, it takes several rounds of selection and backcrossing to break the linkages
with undesirable traits in the nuclear genome. (b) In the alternative strategy for making alloplasmic cultivars,
a wide array of alloplasmic lines is needed that contains certain cytoplasmic genomes with desirable effect
such as drought tolerance (line A). Later, the cytoplasm of the interest can be easily replaced through recurrent
backcrossing. This creates a new alloplasmic cultivar with the same nucleus but in a new cytoplasm.
A minimum NM compatibility is required for the survival of an alloplasmic plant. Otherwise,

small amino acid differences in the protein encoded by nuclear and mitochondrial genes could
interrupt their assembly or interactions and lead to the death of the organism. Even with a minimum
compatibility, the enzymes of the inner mitochondrial membrane containing subunits encoded from
mitochondria and nucleus may not be compatible and work properly in the newly formed alloplasmic
line (Nair 1993). The pressure of incompatibility and the increased recombination of the mtDNA
may result in improper function of the nuclear gene(s). Improper function of genes like MSH1
and RECA3 (Shedge et al. 2007), controlling the recombination in the new mitochondrial genome,
would cause a high rate of heteroplasmy and also substoichiometric changes. These changes speed
up evolution and produce mitotypes that survive in the new condition. This accelerated evolution
of the newly formed mitotypes will lead to multiple mitotypes differing from the maternal mito-
chondrial type in organization and genetic information. Therefore, in the alloplasmic lines, a larger
number of newly formed mitotypes exist, and stoichiometric shifts change the major mitotype from
the maternal type to the type fitting the new condition better (Woloszynska 2010). Similar data
from a CMS T. aestivum line describing mtDNA rearrangements were reported (Liu et al. 2011).
This information shows the importance of the alloplasmic situation to accelerate the evolution of
mitochondrial genome and a higher level of heteroplasmy. These changes could help organism use
any mitotype in this pool to cope with the special physiological condition via stoichiometry shifts.
Therefore, the cytoplasm in the newly formed alloplasmic conditions can be different from the
maternal parent. These alloplasmic conditions bring together a new NC-compatible combination
and also engineer new versions of mitochondria by accelerating its evolution.
11.11 CONCLUSIONS
The environmental challenges of warming climate and rapid population growth confront plant
scientists to produce high-yielding crops that perform under stressful conditions. Traditional means
of looking at the nuclear genome variation of wild and related species for crop improvement have
not been effective in terms of yield improvement for crop plants. Therefore, we are faced with
looking outside the current genetic pools for more diversity while maintaining the agronomic quali-
ties that represent current agricultural production system. Nuclear–cytoplasmic incompatibilities
tend to slow progress toward utilizing this diverse gene pool. However, past studies have clearly
illustrated the value of manipulating cytoplasmic genomes (those present in the mitochondria and
chloroplast) to improve crop performance. Thus, it is imperative that we utilize this knowledge and
existing resources to manipulate the mitochondrial genome to increase the potential of crops to
yield more under stressful conditions or in marginal lands.
ACKNOWLEDGMENTS
We acknowledge the partial funding support from the United States Army Research Office grant no.
W911NF-08-1-0319 and the National Research Initiative of the USDA Cooperative State Research,
Education and Extension Service, grant no. 2008-01099.
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12 Signaling Molecules Involved
in the Postharvest Stress
Response of Plants
Quality Changes and Synthesis
of Secondary Metabolites
Luis Cisneros-Zevallos, Daniel A. Jacobo-Velázquez,
Jean-Claude Pech, and Hisashi Koiwa
CONTENTS
12.1 Introduction........................................................................................................................... 259
12.2 Abiotic Stresses during Postharvest: Quality Effects............................................................260
12.2.1 Climacteric and Non-Climacteric Fresh Produce and Quality Changes..................260
12.2.2 Ethylene, Temperature, and Altered Gas Composition Stress and Quality Changes...... 262
12.2.3 Wounding Stress and Quality Changes..................................................................... 263
12.3 Abiotic Stresses during Postharvest: Synthesis of Secondary Metabolites...........................264
12.3.1 Stress-Signaling Molecules Mediating Postharvest Abiotic Stress Responses.........264
12.3.2 Wounding Stress: Primary and Secondary Metabolism...........................................266
12.3.3 Wounding Stress: ATP and Reactive Oxygen Species as Signaling Molecules........ 267
12.3.4 Potential Uses of Postharvest Stresses: Plants as Biofactories of Secondary
Metabolites................................................................................................................ 269
12.3.5 Potential Uses of Stresses: Genetic Modification of Plants
for Hyperaccumulation of Flavonoids....................................................................... 270
12.4 Summary and Conclusions.................................................................................................... 272
References....................................................................................................................................... 273
12.1 INTRODUCTION
It is well known that fruits and vegetables undergo a series of changes once harvested from the fields
until their use by the consumers. In this process, typically fresh produce are harvested, transported to
packing houses, lowered their temperature, conditioned or treated, sorted, packed, stored, transported,
and displayed. The sequence and steps used will depend on the type of produce and market destination;
however, the perishable nature of fresh produce defines their storage life. Through the past few decades,
extensive research has been conducted to define optimal postharvest handling for shelf life extension
of various commercial crops. Many available postharvest techniques developed are based on expos-
ing these fresh produce to a diverse range of abiotic stresses. For example, temperature management,
controlled atmospheres, modified atmospheres, ozone, ethylene treatments, minimal processing, and
irradiation are based on controlling abiotic stresses like temperature, altered gas composition, use of
chemicals/hormones, wounding, and radiation exposure, respectively. Other stresses that may have an
259
impact on fresh produce, but their use on commercial technologies have not been implemented, include
partial dehydration, gravitational exposure, and overall total pressure.
The term abiotic stress has several definitions according to the context of pre- and postharvest
conditions in which the plant is exposed. For example, under preharvest conditions, an abiotic stress
can be defined as a scenario where plants are exposed to unfavorable environments (e.g., water defi-
cit, chilling, oxygen deficiency, air pollution) resulting in some degree of stress and the expression
of only a fraction of the plant’s genetic potential. As a consequence, plants adapt to unfavorable
conditions through genetically stress resistance (Drew, 1997, 1998). Similarly, abiotic stress can
be defined as a scenario when plants are exposed to conditions outside the boundaries of a narrow
optimum span. The stress is an external factor or succession of factors of such magnitude that it
tends to disrupt the normal physiology process of the organism; accordingly, it interrupts, restricts,
or accelerates normal metabolic processes in an adverse or negative manner (Kays, 1991). In the
area of postharvest, an abiotic stress is defined as an external factor that will result in undesirable
changes in quality if the plant or plant parts are exposed to it for sufficient duration or at sufficient
intensity (Kays, 1991). In all cases, abiotic stress has a negative connotation as observed in the
aforementioned definitions. However, as will be presented in the following chapter, abiotic stresses
may also have a positive connotation specially when focusing on secondary metabolite synthesis.
Secondary metabolites with health-promoting properties are usually synthesized under stress condi-
tions (Cantos et al., 2000; Espin and Tomas-Barberan, 2001; Heredia and Cisneros-Zevallos, 2002;
Kang and Saltveit, 2002; Cisneros-Zevallos, 2003, Reyes and Cisneros-Zevallos, 2003; Heredia and
Cisneros-Zevallos, 2009a,b; Jacobo-Velázquez et al., 2011; Becerra-Moreno et al., 2012; Jacobo-
Velázquez and Cisneros-Zevallos, 2012; Surjadinata and Cisneros-Zevallos, 2012). These same
favorable conditions for secondary metabolite synthesis may have opposite effects on quality during
postharvest handling (Cisneros-Zevallos, 2003).
The following chapter is organized in two main topics: first, the discussion of abiotic stresses during
postharvest and associated quality effects including the influence of climacteric and non-climacteric
fruit and vegetables, and second, the discussion of abiotic stresses during postharvest and their effects
on secondary metabolite synthesis including potential stress-signaling molecules, effects on primary
and secondary metabolism, role of ATP and reactive oxygen species (ROS), and the use of plants
as biofactories to produce health-promoting compounds. The discussions will include examples of
potential use in abiotic stress manipulation, genetic transformation, and revisiting a former diagram of
wounding stress effects on postharvest events associated to quality changes and synthesis of polyphe-
nols (Saltveit, 1997) and updating it with the most recent findings on signal molecules.
12.2 ABIOTIC STRESSES DURING POSTHARVEST: QUALITY EFFECTS

In this section, we will discuss how abiotic stresses applied during postharvest handling affect the
quality of fruits and vegetables. The response will be influenced by the type of fresh produce including
climacteric and non-climacteric and the type of stress applied. We will cover three common stresses,
ethylene exposure, temperature, and altered gas composition, that have been used for decades in
research and industry. In addition, wounding stress will be discussed since it is the basis for an emerg-
ing industry of minimally processed or fresh-cut produce that appeared in the 1990s.
12.2.1 Climacteric and Non-Climacteric Fresh Produce and Quality Changes

Fruit and vegetables are classified as climacteric or non-climacteric according to their ripening pattern,
presence of a burst in respiration, and ethylene production (Kays, 1991; Kader, 2002). Accordingly,
climacteric produce show a postharvest ripening pattern characterized typically by an increase in
sugars and decrease in acids, synthesis of volatile compounds, pigment formation or degradation, cell
wall depolymerization, and membrane degradation that are usually associated to changes in quality
parameters including taste, odor/aroma, color, and softening, respectively (Figure 12.1a). This ripening
Signaling Molecules Involved in the Postharvest Stress Response of Plants 261
Aromas
Ripening Odor
volatiles
Climacteric Ethylene
Non-climacteric
other signals
Cell wall
Autocatalytic H — H
C C
Taste H H
H — H
C C
H H Perception
Ethylene
Ethylene biosynthesis
Sugars Aroma
Signal transduction
acids
Color
Nucleus
Vacuole
Respiration Pigments
Chromoplast Transcription factors

Ripening-related DNA
Mitochondria genes
Membrane degradation Genetic program
Cell wall degradation
(a) Softening
Ripening Aromas Odor Wounding

volatiles
Climacteric Ethylene
Non-climacteric
Other
Cell wall signals
Autocatalytic H — H
C C
Taste H H
H — H
C C
H H Perception
Ethylene
Ethylene biosynthesis
Sugars Aroma Signal transduction
acids
Se
co
nd
Color ary
m Nucleus
Vacuole et
ab
ol
Respiration Pigments ism
Chromoplast Transcription factors

Ripening-related DNA
Mitochondria genes
Membrane degradation
Genetic program
Cell wall degradation

(b) Softening
FIGURE 12.1 Physiological and associated quality changes taking place in the fruit cell during the ripening
process in climacteric and non-climacteric fresh produce in the absence of wounding stress (a) and during the
application of wounding stress (b). (Adapted from Bouzayen, M. et al., Mechanism of fruit ripening, in: Pua,
E.C. and Davey, M.R. (eds.), Plant Development Biology—Biotechnological Perspectives, Vol. 1, Springer-
Verlag, Berlin, Germany. pp. 319–339, 2010.)
process is associated with an increase in ethylene production and respiration. In climacteric produce,
it is accepted that ethylene is the primary signal that triggers the climacteric response, through an
ethylene autocatalytic mechanism that allows ethylene biosynthesis. As the hormone ethylene binds
to the receptor and perception takes place, proteins in the signal transduction pathway are activated
including transcription factors that trigger the DNA genetic program and the ripening-related genes
(Bouzayen et al., 2010). In a short period of time, the ripening events take place and the produce under-
goes a process of loss of homeostasis driven by an increase in oxidative stress (Romani, 1987, 1979;
Puerta-Gomez and Cisneros-Zevallos, 2011). When maintenance of homeostasis is lost, the produce
enters the stage of senescence characterized by a loss of quality and commercial value. If climacteric
produce is left on the plant in the field, it will show a similar ripening pattern as described earlier;
however, the process time probably may differ.
On the other hand, non-climacteric produce go through a ripening process mainly during prehar-
vest conditions and small changes during postharvest conditions. If harvested before full ripening,
non-climacteric produce typically undergo small changes or no changes in sugars, acids and volatile
compounds and in cell wall depolymerization. They may however exhibit some changes in pigment
formation or degradation (e.g., green to red peppers) and some senescence-associated membrane
degradation (Figure 12.1a). Thus, changes in quality parameters including taste, odor/aroma, color,
and softening are events that take place before harvest and have small changes during its shelf life.
In non-climacteric produce, there is a lack of burst in ethylene production and respiration. The lack
of autocatalytic ethylene goes along with very small ethylene production in non-climacteric pro-
duce and the respiration pattern may be high or low depending on the type of tissue (e.g., asparagus
meristematic tissue). In non-climacteric produce, ethylene plays a secondary role and signals that
trigger most of the ripening pathways are unknown. However, other signals (e.g., hormones such as
ABA) bind to their respective receptors and perception takes place; proteins in the signal transduc-
tion pathway are activated including transcription factors that trigger the DNA genetic program
and the ripening-related genes (Bouzayen et al., 2010). Despite that non-climacteric produce do not
undergo major biochemical changes after harvest, the produce will eventually undergo a process of
loss of homeostasis due to oxidative stress. Similarly to climacteric produce, once maintenance of
homeostasis is lost, the non-climacteric produce enters the stage of senescence characterized by a
loss of quality and commercial value.
12.2.2 Ethylene, Temperature, and Altered Gas Composition

Stress and Quality Changes
When climacteric and non-climacteric produce are exposed to abiotic stresses, the response will
differ depending on the type of stress applied. For example, exposure to ethylene will initiate the
ripening process in climacteric produce triggering the different events described earlier associ-
ated to quality changes. However, for non-climacteric produce, ripening is not initiated, and
only certain quality changes may take place like color change (e.g., citrus degreening through
chlorophyll degradation), increase in respiration, and synthesis of secondary metabolites such as
phytoalexins (e.g., isocoumarin synthesis in carrots) during the ethylene exposure (Kays, 1991;
Kader, 2002).
Similarly, exposure to increasing temperatures will differentially affect both types of produce.
Increasing temperatures will accelerate the ripening process in climacteric produce due to an
increase in ethylene production, respiration, and rates of activities of key enzymes associated to
quality changes. While in non-climacteric produce, there will be mainly an overall increase in
respiration and rates of activities of key enzymes associated to quality changes. In both types of
produce, exposure to temperature higher than normal may actually alter metabolic pathways due
to key enzymes being affected (e.g., irregular ripening in heat-treated climacteric mango fruit) and
favor synthesis of heat shock proteins (e.g., conditioning of tomatoes to reduce chilling sensitivity)
(Kays, 1991; Saltveit, 2000; Kader, 2002).
Exposure to decreasing temperatures will reduce the overall rate of metabolic process in both
types of produce and thus quality change; however, low-temperature exposure below the optimal
window of storage may trigger physiological disorders known as chilling injury (Kader, 2002;
Karakurt and Huber, 2003). These chilling injury alterations are associated to a series of events
including changes in membrane fluidity and irregular enzyme activities, among others. The symp-
toms are more evident after the chilling exposure and subsequent storage at higher temperatures.
Tropical fruits are mainly affected by these disorders; however, temperate crops may be affected
as well (e.g., tissue discoloration in avocado fruits, sugar accumulation in potatoes, epidermal cell
death in cucumbers).
Exposure to altered gas composition is the basis for technologies of controlled and modified
atmospheres. These gas alterations will certainly affect metabolic processes in both climacteric and
non-climacteric produce by reducing quality changes and deterioration. Typically, altered gas com-
position exposure includes reduced oxygen levels (e.g., 0.5%–5% O2) and increased carbon dioxide
(e.g., 2%–15% CO2) and their combinations. It is accepted that reduced oxygen levels may reduce
respiration, ethylene production, and oxidation of phytochemicals, while increased carbon dioxide
levels may reduce ethylene perception and respiration and alter enzyme activities (e.g., pH changes
associated to carbonic acid due to CO2 solubility). When combined, these altered gases may elicit
additive and synergistic effects in metabolic processes and quality changes delaying ripening and
extending the postharvest shelf life.
However, altered gas composition may also have detrimental effects on quality and trigger physi-
ological disorders. For example, anoxia and hypoxia may induce anaerobic respiration and elicit the
fermentative process with production of off-flavors (e.g., acetaldehydes, ethanol in lettuce tissue) and
even partial lactic acid production (e.g., carrot tissue) (Leshuk and Saltveit, 1991; Ke et al., 1993;
Kato-Noguchi and Watada, 1997). Recently, an oxygen-sensing mechanism has been proposed in
plants (Arabidopsis thaliana) based on the ubiquitin-dependent N-end rule pathway for protein deg-
radation and an associated ethylene response factor (ERF)-transcription factor RAP2.12 dedicated
to an oxygen-dependent sequence of posttranscriptional modifications. Under hypoxia conditions,
RAP2.12 is released from the plasma membrane accumulating in the nucleus and activating gene
expression for hypoxia acclimation (Licausi et al., 2011). Knowledge of this new described system
in plants may open possibilities to avoid low-oxygen detrimental effects in fresh produce.
Similarly, high CO2 exposure may induce anaerobic metabolism, cell death, and tissue discolor-
ation, among other disorders (Kays, 1991; Kader, 2002). Less is known about the effects of exposure
to higher levels of oxygen (hyperoxia) or the combination of high levels of oxygen and carbon diox-
ide on quality changes. However, there have been reports on secondary metabolite synthesis under
hyperoxia in carrots, strawberries, and blueberries (Zheng et al., 2003; Ayala-Zavala et al., 2007;
Jacobo-Velázquez et al., 2011) and under high levels of carbon dioxide in grapes (Becatti et al.,
2010) and tannin polymerization and astringency removal under the latter conditions in persimmons
(Kato, 1990).
12.2.3 Wounding Stress and Quality Changes

During the 1990s, a new industry emerged based on developing products that were ready to eat,
were minimally processed, were metabolically active, and showed convenience to the consumers.
These products derived from whole fruits and vegetables and went through a process of sectioning,
peeling, and cutting. As a result, these tissues were exposed to wounding stress that altered their
physiology and induced an increase in the rate of quality changes and a drastic reduction in post-
harvest shelf life.
As observed in Figure 12.1b, it was thought that wounding stress induced quality changes and
altered the physiology of the tissue through an unknown primary signal that bounded to a receptor,
perception taking place, and proteins in the signal transduction pathway being activated includ-
ing transcription factors that triggered the DNA genetic program and the ripening-related genes.
This wounding stress would alter the sugar and acid content and the synthesis of volatile com-
pounds, induce synthesis of secondary metabolites or accelerate their degradation, oxidation, or
polymerization, and induce cell wall depolymerization and membrane degradation (Reyes et al.,
2007; Tavarini et al., 2010). All these changes would eventually be associated to changes in quality
parameters including taste, odor/aroma, color, and softening.
The wounding stress affects both, climacteric and non-climacteric produce, in differing ways. In
climacteric produce, ripening and quality changes are accelerated due to an increase in wound-induced
ethylene production and an increase in respiration, while in non-climacteric produce, the main effect is
due to an increase in respiration and a transient increase in ethylene. Thus, climacteric produce exposed
to wounding stress have a shorter shelf life compared to non-climacteric. The reduced shelf life of
wounded tissue is an indication that the produce undergoes an accelerated process of loss of homeosta-
sis driven by an accelerated increase in oxidative stress (Puerta-Gomez and Cisneros-Zevallos, 2011).
In general, application of wounding stress on fresh produce goes along with the manipulation of
other stresses to reduce the impact of accelerated deteriorations. This includes low-temperature expo-
sure, the use of altered gas composition (e.g., modified atmosphere packaging), and the exposure to
high-humidity environments to avoid water loss due to the removal of the cuticle/epidermal layers. The
latter is achieved necessarily with the use of semipermeable plastic films. Through the use of these
combined stresses, the shelf life of wounded tissue can be increased depending on the type of produce.
12.3 ABIOTIC STRESSES DURING POSTHARVEST:

SYNTHESIS OF SECONDARY METABOLITES
In this section, we will discuss how abiotic stresses applied during postharvest handling affect the
biosynthesis of secondary metabolites in fruits and vegetables with health-promoting properties.
There is a diverse range of phytochemicals present in fresh produce including phenolic compounds,
triterpenes, glucosinolates, and alkaloids, among others. We will focus on the former group since
more work has been gathered for that category of phytochemicals compared to other compounds.
Polyphenolics are a family of compounds that are produced in both vascular and nonvascular
plants. The functions of polyphenolics include forming physical barriers, biochemical and visual
signals to symbiotic partners and pollinators, protection from UV damage, and regulation of auxin
transport during development (Dixon and Paiva, 1995; Shadle et al., 2003). For animal consump-
tion, polyphenolics are known for health-promoting effects, featuring their antioxidant activity
and prevention of chronic degenerative diseases and obesity (Korkina, 2007; Iriti and Faoro, 2009;
Jacobo-Velázquez and Cisneros-Zevallos, 2009).
We will cover the three potential signal molecule candidates in postharvest abiotic stress responses,
the primary and secondary metabolism involved in phenolic biosynthesis, the possible role of ATP
and ROS as primary and secondary signals in wounding stress, and the use of plants as biofactories to
produce health-promoting compounds. The final discussions will include examples of potential use in
abiotic stress manipulation, genetic transformation, and revisiting a former diagram of abiotic stress
effects on postharvest events associated to quality changes and synthesis of secondary metabolites.
12.3.1 Stress-Signaling Molecules Mediating Postharvest Abiotic Stress Responses

The general cellular process and regulation for activating plant secondary metabolite starts with
a signal, either extracellular or intracellular, and is perceived by a receptor on the surface of the
plasma membrane, which then initiates a signal transduction network that leads to activation or
de novo synthesis of transcription factors to regulate gene expression involved in the plant sec-
ondary metabolism (Figure 12.2; Low and Merida, 1996; Koiwa et al., 1997; Morgan and Drew,
1997; Taiz and Zeiger, 1998; Vranova et al., 2002; Zhao et al., 2005). This signal transduction is
a complex network with multiple sequential reactions to establish an efficient defense mechanism
S-Adenosy1
Methionine
methionine
Abiotic stress
ACC 1–MCP
SOD CAT
C2H4 O2 O2– H2O2 H2O
Plasma NADPH
membrane
Lipase oxidase
Receptor Receptor Receptor
Protein dimer G-proteins NADPH NADP+
α-Linolenic acid Ca2+ H2O2
ETR1 Phenidone LOX
Kinases DPI
13(S)-Hydroperoxylinolenic acid
Regulator
CTR1 Proteins
P-p67-phox
Protein p-p47-phox ROS
Jasmonic acid
ETN2
Gene expression Gene expression Gene expression
Phenylalanine Phenylalanine Phenylalanine

PAL PAL PAL
Phenolics Phenolics Phenolics
FIGURE 12.2 Stress-induced signal transduction pathways in plants for ethylene, jasmonic acid, and ROS
associated to the phenylpropanoid metabolism. (Adapted from Low, P.S. and Merida, J.R., Physiol. Plant.,
96, 533, 1996; Koiwa, H. et al., Trends Plant Sci., 2, 379, 1997; Morgan, P.W. and Drew, M.C., Physiol.
Plant, 100, 620, 1997; Taiz, L. and Zeiger, E., Plant Physiology, 2nd edn., Sinauer Associates, Inc. Publishers,
Sunderland, MA, 1998, 656p.; Vranova, E. et al., The role of active oxygen species in plant signal transduc-
tion, in: Scheel, D. and Wasternack, C. (eds.), Plant Signal Transduction, Oxford University Press, Inc., New
York, pp. 45–73, 2002.)
(Zhao et al., 2005). It has several components that consist of some parallel or cross-linking signaling
pathways that eventually lead to different target responses. An improved knowledge of signal trans-
duction will be beneficial in developing strategies to modify the production of target compounds by
either activation or inhibition of certain metabolic pathways (Zhao et al., 2005).
Remarkable progress has been made in understanding how plants perceive and respond to ethyl-
ene (Morgan and Drew, 1997). A complex network of genes has been identified: ETR1/ETR4 CTR
1 EIN2 EIN3/EIN5 system (Morgan and Drew, 1997; Taiz and Zeiger, 1998). Some of these genes
are negative and some are positive regulators. However, not much is understood about the mecha-
nism of stress perception and although interactions with several other plant hormones have been
suggested, there is no study where the entire series of events can be traced from stress perception to
ethylene response (Morgan and Drew, 1997). It could be assumed, however, that when a plant expe-
rienced a stress, first, the plant must detect and measure the intensity of the stress. After the stress
is perceived, a signal is sent to the receptor and transduced to synthesize the production of ethylene,
which eventually leads to observable symptoms (Morgan and Drew, 1997). Another plant hormone
that has been observed to increase as a result of stress is jasmonic acid (Wasternack and Hause,
2002). This compound is derived from the octadecanoid pathway and originated from α-linolenic
acid. Oxygenation by lipoxygenase (LOX) at carbon atom 13 resulted in (13S)-hydroperoxy linole-
nic acid, which then converts to cis-(+)-12-oxo-phytodienoic acid (OPDA) by the action of allene
oxide synthase (AOS) and allene oxide cyclase (AOC) (Wasternack and Hause, 2002). All of these
processes eventually lead to the formation of (+)-7-iso-jasmonic acid. There are two parts of stress-
induced jasmonic acids formation: (1) early gene activation, mainly local generation of signals in
the vascular bundles that lead to both local and systemic responses, and (2) late gene activation,
preferentially defense-related gene expression in the mesophyll cells upon activation in the systemic
tissue by a systemic signal (Wasternack and Hause, 2002).
As an aerobic organism, plant needs oxygen to produce energy. During the reduction of O2 to
H2O, ROS such as O2−, H2O2, and hydroxyl radical can be formed (Vranova et al., 2002). When
stress occurs, Ca2+ influx, alkalinization of the apoplast, protein phosphorylation, and translocation
of the p-47-phox and p-67-phox subunits lead to the synthesis of NADPH oxidase and trigger the
oxidative burst (Low and Merida, 1996; Vranova et al., 2002). NADPH oxidase is considered to be
the main source of the early and sustained accumulation of ROS (Vranova et al., 2002). NADPH
oxidase is responsible for the reduction of O2 to O2− and with the presence of SOD enzyme, O2−
is then converted to H2O2 (Low and Merida, 1996; Zhao et al., 2005). Both O2− and H2O2 have
been shown to be involved in plant stress-induced defense responses, either locally or systemically.
Expression of genes related to plant defense pathway has been demonstrated as consequences of
pathogen attack, chilling injury, wounding, and excess light (Vranova et al., 2002).
Recently, we have shown that phenolic biosynthesis under several stress conditions including
wounding alone, and in combination with UV light (types A, B, and C) stresses or in combination
with ethylene and jasmonic acid exposure, is an event mainly mediated by ROS as a signal molecule
(Cevallos-Casals, 2006; Heredia, 2006; Surjadinata, 2006; Jacobo-Velazquez, 2010). In these stud-
ies using carrots and seed sprouts as model systems, ROS production was inhibited using DPI as a
specific inhibitor of NADPH oxidase activity, jasmonic acid production was inhibited using phe-
nidone as a specific inhibitor of LOX activity, and ethylene perception was inhibited using 1-MCP
(Figure 12.2). These inhibitors were used alone or combined, and the outcome showed that all
three pathways (ROS, ethylene, and jasmonic acid) were involved in the stress responses, but their
contribution differed depending on the type of stresses applied; however, overall ROS was the main
signal molecule. More recently, Jacobo-Velázquez (2010) has shown a complex cross talk among
these three pathways eliciting the expression of genes from the primary and secondary metabolism.
12.3.2 Wounding Stress: Primary and Secondary Metabolism

When wounding is applied either alone or in combination with other stresses including UV light,
ethylene, or hyperoxia, the biosynthesis of chlorogenic acid derivatives is enhanced drastically in
carrot tissue allowing the accumulation of 3–20 times the original content of a specific polyphe-
nol in nonwounded samples (Table 12.1). The wound-induced accumulation of hydroxycinnamic
acids, flavonoids, lignin, and suberin in plants has to be related with the activation of metabolic
events involved on the supplementation of carbon skeletons to the phenylpropanoid metabolism
(Figure 12.3). Carbohydrates (e.g., starch and sucrose) are the substrates needed for the synthesis
of phenolic compounds. Phenolic biosynthesis, as well as respiration, starts in the cytosol of plant
cells where sucrose is cleaved to produce glucose 6-P and fructose 6-P. Part of the hexose-P pool
is transported to the plastid and converted to erythrose 4-P by the oxidative pentose phosphate
pathway (OPPP). The other fraction of the hexose-P pool is converted to glycerone-P in the cyto-
sol by glycolysis. Glycerone-P is transported to the plastid and converted to phosphoenolpyruvate
(PEP). Erythrose 4-P and PEP are the substrates for the shikimate pathway occurring in the plastid
(Herrmann and Weaver, 1999). The shikimate pathway produces chorismate, which is used by cho-
rismate mutase to synthesize prephenate. Prephenate is then converted to arogenate, which is subse-
quently transformed to l-phenylalanine. l-Phenylalanine is used by phenylalanine ammonia-lyase
(PAL) to initiate the synthesis of phenolic compounds as part of the phenylpropanoid metabolism
that occurs in the endoplasmic reticulum of plant cells (Dixon and Paiva, 1995; Amthor, 2003).
As previously reported, carrots can be used as biofactories of chlorogenic acids when subjected to
extreme conditions of wounding stress (Jacobo-Velazquez et al., 2011). Therefore, it is suggested that
all these metabolic pathways involved in the synthesis of l-phenylalanine are wound induced in the
plant cell to supply the carbon flux needed for phenolic biosynthesis (Figure 12.3). It is well known
that respiration is increased in wounded carrot tissue and that the respiration rate is dependent on the
TABLE 12.1
Effect of Different Abiotic Stresses (Wounding, UV Light, Ethylene, and Hyperoxia)
on the Accumulation of Individual Phenolic Compounds in Carrots
Phenolic Content
Abiotic
(mg/kg FW)
Stress Storage
Compound Applied Conditions Beforea After Increase (%) Reference
Chlorogenic acid W 6 day/15°C 148 628 324 Heredia and Cisneros-
Zevallos (2009b)
W 2 day/20°C 38.2 803.3 2002 Jacobo-Velázquez et al.
(2011)
W + UV-C 4 day/15°C 100 588 488 Surjadinata (2006)
W+E 6 day/15°C 148 926 526 Heredia and Cisneros-
Zevallos (2009b)
W+H 2 day/20°C 38.2 1171.4 2966 Jacobo-Velázquez et al.
(2011)
4,5-Dicaffeoylquinic W 6 day/15°C 32 236 637 Heredia and Cisneros-
acid Zevallos (2009b)
W+E 6 day/15°C 32 281 778 Heredia and Cisneros-
Zevallos (2009b)
3,5-Dicaffeoylquinic W 4 day/15°C nd 49.1 — Surjadinata (2006)
acid
Sources: Jacobo-Velázquez, D.A. and Cisneros-Zevallos, L., Agriculture, 2, 259, 2012.

a
Initial values of individual phenolics are variety dependent; data shown were obtained from independent studies
(Jacobo-Velázquez et al., 2011; Heredia and Cisneros-Zevallos, 2009b; Surjadinata, 2006).
Abbreviations: W, wounding; UV-C, ultraviolet light C; E, ethylene; H, hyperoxia; FW, fresh weight.
degree of wounding (Surjadinata and Cisneros-Zevallos, 2003). Likewise, the upregulation of glyco-
lytic enzymes in wounded tissues such as sugar beet has been previously reported and proposed as
a potential mechanism to increase the flux of carbon compounds to support wound-healing process
(Klotz et al., 2006). The activation of the OPPP by wounding is believed to play an important role
in providing reducing power (NADPH) for lignin biosynthesis (Pryke and Rees, 1977). The wound-
induced upregulation of 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase (DAHP synthase) and
chorismate mutase, key regulatory enzymes of the shikimic acid pathway, has also been reported in
Brassica juncea seedlings, tomato, and potato (Dyer et al., 1989; Sharma et al., 1999).
12.3.3 Wounding Stress: ATP and Reactive Oxygen Species as Signaling Molecules

Recently, Jacobo-Velázquez et al. (2011) proposed a model for wounding stress and phenolic bio-
synthesis and identified ATP as the primary signal and ROS as the secondary signal of the stress
response (Figure 12.4). The proposed model was based on previous work of wounding stress in A.
thaliana in which wounding releases ATP that in turn increases ROS levels in the cells (Song et al.,
2006; Demidchick et al., 2009). In Jacobo-Velázquez et al.’s adapted model, wounding stress releases
ATP from damaged cells that in turn diffuse through intercellular spaces and bind to ATP receptors
on undamaged cells (these receptors have not been identified in plants so far) and elicits the response.
As a consequence, calcium channels increase calcium ion flux into the cytosol activating NADPH
oxidase at the membrane level. Superoxide radical is produced, which is transformed into hydrogen
peroxide by superoxide dismutase. Hydrogen peroxide diffuses into the cytosol and initiates the signal
transduction pathway and activation of transcription factors that trigger PAL gene expression and pro-
tein expression and activity and the corresponding biosynthesis of phenolic compounds. Furthermore,
Wounding
Signaling molecules
JA ET ROS
Primary metabolism Secondary metabolism

Glucose Aromatic amino acids
Glycolysis OPPP
L-Phenylalanine
NADPH
Phenylpropanoid
PAL
PEP E–4P metabolism
t-Cinnamic
Shikimic acid acid
pathway
C4H
Chorismate
p-Coumaric acid
4CL
Aromatic
amino acids p-Coumaroyl CoA
Hydroxycinnamic Flavonoids Lignin

acids
FIGURE 12.3 Wound-induced activation of pathways related with the biosynthesis of phenolic compounds in
plants. Abbreviations: JA, jasmonic acid; ET, ethylene; ROS, reactive oxygen species; OPPP, oxidative pentose
phosphate pathway; PEP, phosphoenolpyruvate; E-4P, erythrose-4 phosphate; PAL, phenylalanine ammonia-
lyase; C4H, coumarate 4-hydroxylase; 4CL, 4-coumarate CoA ligase. (From Jacobo-Velázquez, D.A. and
Cisneros-Zevallos, L., Agriculture, 2, 259, 2012.)
in the proposed model, when a second stress is applied such as hyperoxia (Figure 12.4), there is an
increase in respiration that induces a higher level of the hydrogen peroxide pool that is additionally
favored by an inhibitory effect of hyperoxia on two detoxifying enzymes, catalase and ascorbate per-
oxidase. In this model, hydrogen peroxide would be the primary signal for hyperoxia stress.
A few years ago, Saltveit (1997) proposed a model of wounding effects on physiological pro-
cesses in fresh-cut produce. This model was the first attempt in describing and bringing together
physiological and quality changes in wounded tissue and has been used as a key reference in the
area of fresh-cut research work up to date. In that model, wound elicits an unknown signal that
triggers an increase in respiration with the known consequences of a reduction in carbohydrates,
organic acids, and ascorbic acid generating poor flavor of the wounded tissue. That same unknown
signal elicits ethylene production that accelerates ripening and softening of the tissue. Parallel to
these events, the signal elicits the phenolic metabolism that triggers the synthesis of phenolic com-
pounds through PAL activity while inducing browning through PPO and O2. Finally, this same
unknown signal elicits the synthesis of suberin and wound healing in the tissue. Since the model
was proposed in 1997, there were many attempts to find the actual signal; however, the only known
facts were that the signal was removable by washing and that the signal could be diffused or move
from the actual wounding site as reported in several papers by Saltveit’s group.
Based on the recent model proposed by Jacobo-Velázquez et al. (2011), the primary
ATP and secondary ROS signals proposed in their work fit and describe Saltveit’s model
Wounding
ATP
ROS
NADPH oxidase
ATP O2 O2 –
ATP SOD
receptor
Plasma
H2O2
membrane
Ca2+ PAL
APX
CAT
Detoxification Phenolic
compounds
Mitochondria 3–CQA
FA
3,5–diCQA
Hyperoxia 4,5–diCQA
FIGURE 12.4 Hypothetical model explaining the role of extracellular ATP and ROS on the wound stress-
induced production of phenolic compounds in carrots. The role of hyperoxia as a second stress is described as
well. (From Jacobo-Velázquez, D.A. et al., J. Agric. Food Chem., 59, 6583, 2011.)
appropriately (1997). Accordingly, the missing signal unknown at that time will correspond
to the ATP and ROS proposed by Jacobo-Velázquez et al. By adding these primary and sec-
ondary signal molecules, the model for wounding proposed originally by Saltveit (1997) is
finally completed after 15 years of research and presented as a modified model in this chapter
as Figure 12.5.
Wound
ATP Primary signal
ROS
Secondary signal
Respiration Ethylene Phenolic Wound

metabolism healing
Heat
Suberin
Ripening PAL
Reduced PPO Periderm

carbohydrates
organic acids O2
Phenolic Cell
ascorbic acid compounds
division
Softening
Browing
Poor flavor Lignin
Toughening
FIGURE 12.5 Diagram indicating the wounding stress effects on physiological process and associated qual-
ity changes in plant tissues. This model is adapted from Saltveit (1997) to introduce the role of extracellular
ATP as the primary signal and ROS as the secondary signal as proposed by Jacobo-Velázquez et al. (2011)
(model shown in Figure 12.4).
12.3.4 Potential Uses of Postharvest Stresses: Plants

as Biofactories of Secondary Metabolites
The implication and potential impact of Jacobo-Velázquez et al.’s model (Figure 12.4) in differ-
ent plants is such that opens the possibility to study and explore different stress combinations, to
understand previous published results from the literature that for many years were unable to identify
the signal molecules, and finally to revisit actual processes utilized by the industry to enhance the
content of bioactive compounds in plant tissues. For example, in actuality, the fresh-cut indus-
try washes wounded tissues right after the cutting process, eliminating the ATP primary signal.
However, if washing takes place only before cutting and not after wounding, the ATP primary
signal remains and will elicit the biosynthesis of secondary metabolites with health-promoting
properties. Interestingly, the wounding stress through these signals elicits both quality detrimental
effects and secondary metabolite synthesis in the tissue (Figure 12.5), making it a challenge to find
a delicate balance between maintenance of quality attributes and promoting biosynthesis of health-
promoting compounds.
Thus, when secondary metabolite synthesis has to be favored using wounding stress so as to
convert plants as biofactories of bioactive compounds, quality attribute maintenance will not be
necessarily important in the process. An example of potential use of wounding stress to induce
plants as biofactories can be seen in the recent proposal by Becerra-Moreno et al. (2012) for the pro-
duction of the intermediate compound shikimic acid (Figure 12.6). Shikimic acid is extracted from
star anise (Illicium verum) and is used as the basis to produce the antiviral TAMIFLU. Shikimic
acid is not normally accumulated in plants and is mainly present in very small quantities. Star anise
is an exception and thus its high demand in the pharmaceutical market. Shikimic acid is an inter-
mediate compound in the pathway for the biosynthesis of l-phenylalanine that is mediated by the
activity of 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase. l-Phenylalanine is utilized in the
phenylpropanoid metabolism to synthesize phenolic compounds. In their proposal, Becerra-Moreno
et al. demonstrated that by using wounded carrot tissue and an inhibitor of EPSP, it was possible
to accumulate high amounts of shikimic acid. On the one hand, wounding elicited an increase in
the primary and secondary metabolism at the gene level, enhancing the carbon flux through the
primary metabolism. On the other hand, the use of glyphosate inhibited EPSP activity in a counter-
effect. As a result, there were more shikimic acid accumulation in carrot tissue and biosynthesis of
chlorogenic acid derivatives in large amounts compared to wounding stress alone (Becerra-Moreno
et al., 2012).
12.3.5 Potential Uses of Stresses: Genetic Modification

of Plants for Hyperaccumulation of Flavonoids
In addition to using solely stresses to enhance the accumulation of active compounds as described
earlier, there is the possibility of genetically modifying the plants to favor the accumulation of
active compounds when specific stresses are applied. Most importantly is the identification and
use of transcription factors associated to stresses that can be applied in preharvest or postharvest
conditions. For example, anthocyanin, kaempferol, and quercetin are several major types of com-
pounds that have been most extensively studied in Arabidopsis and other species. Several tran-
scription factors including myb-type transcription factors production of anthocyanin pigment 1
(PAP1) and production of anthocyanin pigment 2 (PAP2) (Kranz et al., 1998; Borevitz et al., 2000;
Stracke et al., 2001; Gao et al., 2008) and homeobox gene anthocyaninless2 (ANL2) (Kubo et al.,
1999) have been identified in the anthocyanin branch of flavonoid pathways.
Ectopic overexpression of PAP1 and other myb transcription factors have successfully
enhanced the biosynthesis of anthocyanins in various plant species (Vom Endt et al., 2002;
Xie et al., 2006; Zhou et al., 2008; Peel et al., 2009; Li et al., 2010; Velten et al., 2010).
Primary
PEP E-4-P
metabolism
DAHP synthase
DAHP
3–DHQA
Quinic acid 3–Dehydroquinic acid
+
Wounding Shikimic acid
+
+ SK
S3P
eATP +PEP EPSP synthase
Primary signal EPSP
+
+ –
L-phenylalanine
ROS PAL Glyphosate

+ +
Secondary signal
t-Cinnamic acid
C4H
Secondary
p-Coumaric acid
metabolism
4CL
p-Coumaroyl CoA
HCT/HQT HCL
p-Coumaroylquinic acid p-Coumaroylshikimic acid
C3΄H
CQAs Chlorogenic acid
FIGURE 12.6 Hypothetical model explaining the mechanism by which wounded carrot tissue treated with
glyphosate produces high levels of shikimic acid and phenolic compounds. Phosphoenolpyruvic acid (PEP);
erythrose 4-phosphate (E-4-P); 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP); 3-dehydroquinic acid
(3-DHQA); shikimate kinase (SK); shikimic acid 3-phosphate (S3P); 5-enolpyruvylshikimate 3-phosphate
(EPSP); phenylalanine ammonia-lyase (PAL); cinnamic acid 4-hydroxylase (C4H); 4-coumarate:CoA ligase
AMP forming (4CL); p-coumarate 3′-hydroxylase (C3′H); hydroxycinnamoyl-CoA shikimate/quinate hydroxy-
cinnamoyl transferase (HCT); hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT); reactive
oxygen species (ROS); and extracellular ATP (eATP). (From Becerra-Moreno, A. et al., J. Agric. Food Chem.,
60(45), 11378, 2012.)
Transcriptomic analysis of activation-tagging mutant of PAP1 in Arabidopsis (pap1-D) revealed

that PAP1 strongly upregulates anthocyanin biosynthesis pathway genes specifically, without
effective interference with the expression of early phenylpropanoid pathway and flavonoid path-
way (Tohge et al., 2005). While constitutively overexpression of PAP1 may cause vegetative
growth retardation, an inducible system could be applied to achieve PAP1 upregulation at a
proper stage of plant development.
Feng et al. (2011) proposed a three-component gene expression system (Figure 12.7) as well
as its application to cold-inducible anthocyanin production. A gene of interest (PAP1) was cloned
downstream of cold-inducible RD29A promoter, and Arabidopsis plants were cotransformed with
RD29A-PAP1 and a feedforward effecter gene of the cold signal (RD29A-CBF3). A mutation in host
repressor CPL1 (Koiwa et al., 2002; Xiong et al., 2002) seemed to be an essential third component
Osmotic
stress
cpl1–2
Osmotic stress signals
DRE CBF3 DRE PAP1

RD29a RD29a
promoter promoter
PAP1
CBF3
CBF3
PAP1
Feedback PAP1
activation PAP1
PAP1
PAP1 PAP1
PAL CHS
FIGURE 12.7 Design of a three-component inducible system. Low temperature and osmotic stress acti-
vates RD29a promoter that drives PAP1 and CBF3 transgenes. RD29a–CBF3 cassette functions as a positive
feedback system and further activates RD29a promoter. Resulting PAP1 overproduction drives expression of
endogenous phenylpropanoid pathway enzymes such as PAL and chalcone synthase (CHS). Low-temperature
and osmotic signal-induced phenylpropanoid production will lead to the overexpression of PAP1 and hyperac-
cumulation of flavonoids. (Adapted from Feng, Y., Biochemical characterization of plant small CTD phospha-
tases and applications of CTD phosphatase mutant in hyper accumulation of flavonoids in Arabidopsis, PhD
thesis, Texas A&M University, College Station, TX, 2010; Vikram, M. et al., Acta Hortic., 841, 615, 2009.)
for the success of this expression system. Cold induction activated expressions of PAP1 and antho-
cyanin biosynthetic genes, which was accompanied with overproduction of anthocyanins. Resulting
phytochemical profile of transgenic plants showed synergism of native and PAP1-induced anthocy-
anin production. The results establish that a three-component system using a native plant promoter
is sufficient to drive high expression of transgene upon induction.
The three-component system consisting of RD29a-PAP1, RD29a-CBF3, and the cpl1 mutation
was shown to effectively enhance specific anthocyanin accumulation. Expression of PAP1 using
inducible three-component system can minimize severe vegetative growth inhibition caused by the
constitutive expression of transgenes. Unlike several inducible systems, such as dexamethasone-
inducible system, the three-component system described here does not require any constitutive
expression of the system components, therefore, expected to be more resistant to gene silencing.
Since cpl1 mutation can enhance expression of other inducible promoters in addition to osmotic
stress pathway genes (Koiwa et al., 2002, 2004; Xiong et al., 2002; Matsuda et al., 2009; Aksoy
et al., 2013), the three-component system with cpl1 is applicable for other inducible promoter–
transcription factor combinations (Feng et al., 2011). This same approach can potentially be used
in other plant species under pre- and postharvest stress; however, more work is needed in this area.
12.4 SUMMARY AND CONCLUSIONS

This chapter covered the topic of abiotic stresses in the context of postharvest applications and
their effects on quality attributes and synthesis of secondary metabolites. There are several stresses
applied in postharvest handling including temperature, altered gas composition, plant hormones,
chemicals, light, radiation, and wounding, among others. One key component to understand how
plants respond to stresses is to identify the signals associated to them. If a common signal triggers
both quality changes and secondary metabolite synthesis, then two scenarios may take place: either
changes may occur in opposite direction like in wounding stress, where detrimental quality effects
are elicited while phenolic compound synthesis is promoted or changes may occur in the same
direction like in the use of temperature to promote quality changes during ripening and synthesis of
secondary metabolites including volatiles and pigments.
More studies are needed to understand how stresses favor secondary metabolite synthesis without
quality changes or vice versa, as well as how the combinations of stresses may favor one respond or
both of them. Since in the past few years much attention has been focused on the nutraceuticals
or health-promoting compounds of fresh produce, understanding how they are synthesized will be
of major interest.
There is also a need to identify the transcription factors that regulate secondary metabolite syn-
thesis under different types of stresses as well as other mechanism of posttranscriptional regulation
including microRNAs. This will allow the design of more efficient genetic transformations and or
design more appropriate strategies for controlling abiotic stresses.
The concept of abiotic stresses and unfavorable effects in postharvest has been associated tra-
ditionally to quality changes. However, the idea that stresses can be applied to tissues to favor sec-
ondary metabolite synthesis without considering quality may derive in defining a new concept for
abiotic stresses associated to secondary metabolites in more positive terms. We attempt in doing so
in this chapter by defining that “plants under extreme external environmental conditions may favor
the genetic potential of plants and trigger the response of secondary metabolite synthesis” either for
the plant’s use as defense or adaptive mechanisms or for human use as biofactories of nutraceuticals
or health-promoting compounds.
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Part III
Plant/Crop Physiology and
Physiological Aspects of Plant/Crop
Production Processes
13 Quantifying Immediate
Carbon Export from Leaves
Predicts Source Strength
Evangelos Demosthenes Leonardos and Bernard Grodzinski
CONTENTS
13.1 Introduction........................................................................................................................... 279
13.2 Methodologies Used to Estimate Immediate Carbon Export................................................ 281
13.2.1 Gas Exchange and Differential Dry Weight Analysis............................................... 281
13.2.2 Isotopes of C.............................................................................................................. 281
13.2.2.1 Mass Isotope 13C......................................................................................... 282
13.2.2.2 Radioisotope 11C......................................................................................... 282
13.2.2.3 Radioisotope 14C......................................................................................... 283
13.3 Steady-State 14CO2 Labeling and Measurement of Immediate C Export Rates during
Photosynthesis.......................................................................................................................284
13.3.1 Open-Flow Gas Analysis System..............................................................................284
13.3.2 14CO2 Feeding and Monitoring of 14C Retention....................................................... 285
13.3.3 GM Detector Counting Efficiency............................................................................. 286
13.3.4 Calculation of Concurrent Export during Steady-State 14CO2 Feeding.................... 286
13.3.5 Estimating Export of 14C Assimilates during a Light or Dark Chase Period.....289
13.3.6 Partitioning of 14C in the Fed Leaf and Plant............................................................ 289
13.4 Case Studies........................................................................................................................... 290
13.4.1 Photosynthesis and Export under Stress.................................................................... 290
13.4.2 Immediate Export in Natural Photosynthetic Variants............................................. 291
13.5 Summary............................................................................................................................... 293
References....................................................................................................................................... 294
13.1 INTRODUCTION
In vascular plants, three elements, carbon (C), hydrogen, and oxygen, comprise 96% of the plant’s
dry weight. Source leaves are the primary sites of C reduction and the main photosynthetic organs
exporting reduced C to growing sinks. It is well known that in almost all species, sugars, starch,
and amino acids accumulate in leaves during the daytime and export of assimilates derived
from these reserves occurs both concurrently with photosynthesis and subsequently during night
periods (Fondy and Geiger, 1982; Gordon, 1986; Kalt-Torres et al., 1987; Wardlaw, 1990; Geiger
and Servaites, 1994; Weise et al., 2011). Our overall knowledge of translocation processes has
been derived from diverse experimental approaches (Canny, 1973; Geiger, 1980; Milburn and
Kallarackal, 1989; Olrich and Komor, 1989; Farrar, 1993a,b; Heldt, 1997; Gamalei, 2002). For
example, imaging techniques that include light, electron, and fluorescent microscopy using dyes
or proteins provide valuable qualitative data on intercellular connections and export (Peterson
279
and Currier, 1969; Gamalei, 1985; Knoblauch and van Bel, 1998; Côté et al., 1992a; Fricker and
Oparka, 1999). Generally, these imaging techniques are destructive. However, procedures using
isotopes of carbon (e.g., mass isotopes, 13C, and radioisotopes, 11C and 14C) to study export can
also be employed and be both quantitative and nondestructive (Geiger, 1980; Minchin, 1986; Jiao
and Grodzinski, 1996; Verscht et al., 1998; Minchin and Thorpe, 2003). Phloem sap exudation
from cut sieve tubes or from aphid stylectomy has provided a practical means of sampling mobile
assimilates (Weibull et al., 1990). Collection of apoplastic fluids (Ntsika and Delrot, 1986; Tetlow
and Farrar, 1993a,b) and measurements of pH and membrane potential (Delrot, 1981) further dem-
onstrate the physiological and biochemical interactions that operate intercellularly as sugars are
loaded or unloaded from the phloem (Gamalei, 2002). More recently, molecular techniques have
led to the characterization of sugar transporters (Kuhn et al., 1999; Bräutigam and Weber, 2011) and
the engineering of transgenic plants that can be designed to provide important information regard-
ing the role of specific export processes in the leaves (Frommer and Sonnewald, 1995; Heldt, 1997).
Most researchers who have studied translocation and tried to determine export rates (Canny,
1973; Zimmermann and Ziegler, 1975; Stitt et al., 1987; Van Bel, 1993; Geiger and Servaites, 1994;
Turgeon, 1996) acknowledge that it is very difficult to actually quantify simultaneously (1) the CO2
assimilation by the leaf, (2) the rates of C recycling within the leaf, and (3) the turnover of transient
pools of key intermediates within the leaf and relate these processes to (4) the immediate C efflux
rate from the organ via the phloem. It becomes even more challenging to relate any of these leaf
processes to daily export patterns that affect relative growth rates (RGRs) at the whole-plant level
(Leonardos, 1999; Leonardos and Grodzinski, 2011).
One of our interests has been to examine the importance of product removal (i.e., export) from
the leaf on processes such as C recycling in the leaf specifically during periods of active photosyn-
thesis and photorespiration (Madore and Grodzinski, 1984; Grodzinski, 1992; Jiao and Grodzinski,
1996; Grodzinski et al., 1998; Leonardos and Grodzinski, 2000). By knowing what is happening
quantitatively to leaf export concurrently with CO2 fixation, we are better able to evaluate concepts
such as feedback inhibition of photosynthesis and quantify intercellular movements of metabolites
that link long-distance export and sink activity.
In leaves of C3 plants, the stroma of the chloroplast is the site of the reductive pentose pathway
(i.e., the Calvin cycle) and CO2 fixation (Bassham and Calvin, 1957). The primary inorganic sub-
strate of ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco) is CO2. The primary organic
substrate for C fixation is ribulose 1,5-bisphosphate (RuBP). Although the Calvin cycle fixes
inorganic substrate, CO2, the cycle must regenerate the CO2 acceptor, RuBP. The regeneration of
RuBP represents a key feedback mechanism for control of the rate of photosynthesis in the chloro-
plast. However, metabolism and recycling of reduced C and N occurs in subcellular compartments
other than the chloroplast and even outside the cell, which may have been the primary site of C
reduction. During photorespiration, for example, the C in the phosphoglycolate molecule that is
generated by the oxygenase reaction is not recycled conservatively within the chloroplast (Artus
et al., 1986; Husic et al., 1987) or in the cell (Grodzinski, 1992). Similarly, the sucrose that is the
major phloem mobile leaf product is not synthesized in the chloroplast but in the cytosol (Stitt et al.,
1987; Huber and Huber, 1996; Heldt, 1997; Weise et al., 2011). The rate of removal of recent photo-
assimilates from the active metabolite pools in the cell that is fixing C is a major factor regulating
the fixation of C via the Calvin cycle. It is noteworthy that phloem mobile sugars and starch need not
be synthesized in the same cells or tissues that initially fixed the CO2 (Weise et al., 2011). It suffices
here to note that some auxiliary phloem sugars such as raffinoses are made in phloem cells distant
from the site of CO2 fixation (Turgeon, 1995) and also that intercellular exchanges of assimilates
preceding export from the leaf are known to be even more complex in leaves of C4 and C3–C4 inter-
mediate types than in those of C3 species since sucrose synthesis and CO2 fixation occur in sepa-
rate cells and tissues (Edwards and Ku, 1987; Ohsugi and Huber, 1987; Huber and Huber, 1996).
Clearly, we are dealing with a complex set of biochemical, compartmentalized processes when we
try to understand how the rate of net photosynthesis, which is measured as substrate utilization
Quantifying Immediate Carbon Export from Leaves Predicts Source Strength 281
(i.e., μmol of CO2 fixed per second per square meter), is affected by sink demand that involves
product removal from the leaf (μmol of CO2 exported per second per square meter). Subcellular,
cellular, and tissue level interactions are all involved (Gamalei, 2002). The literature suggests that
source leaf photosynthesis and sink demand for assimilates are linked mechanistically, in part, by
the operation of specific site exchanges. In the light, the phosphorylation status of the chloroplast
and the removal of reduced C are mediated by the operation of the phosphate translocator at the
chloroplast membrane (Stitt et al., 1987; Frommer and Sonnewald, 1995; Huber and Huber, 1996;
Heldt, 1997). It is known that reserves of sugars and starch buffer sink demand and affect growth
and development (Wardlaw, 1990; Geiger and Servaites, 1994). In order to determine which pri-
mary leaf parameters best reflect the capacity of the source leaf to meet the demand of developing
sinks, we have developed a working hypothesis that necessitates quantifying the immediate export
rates from leaves during photosynthesis (i.e., in the light). Our hypothesis simply stated that in most
cases with vascular plants, the RGR of the plant is tightly correlated with the capacity of the leaves
to export C in the light as the C is being fixed (i.e., immediately). How can one quantify or estimate
the immediate C flux through a complex heterogeneous organ such as a leaf resulting from CO2
fixation and being removed via the translocation stream?
13.2 METHODOLOGIES USED TO ESTIMATE IMMEDIATE CARBON EXPORT

In this chapter, we describe how steady-state 14CO2 labeling of leaf tissue is achieved and why data
obtained when 14C-isotopic equilibrium exists between photosynthesis and export provide useful
estimates of the immediate mass efflux of C from a source leaf. To appreciate the advantages and
disadvantages of steady-state 14CO2 labeling, one needs to consider other methodologies that have
been used to quantify C export (Canny, 1973; Geiger, 1980; Milburn and Kallarackal, 1989; Olrich
and Komor, 1989; Farrar, 1993a,b).
13.2.1 Gas Exchange and Differential Dry Weight Analysis

Perhaps the least costly method to estimate mass rates of export from source leaves is to measure
the net CO2 exchange rate with an infrared analyzer (IRGA) and the changes in the dry weight of
the leaf overtime (Terry and Mortimer, 1972; Ho, 1976; Silvious et al., 1978; Hendrix and Huber,
1986; Kalt-Torres et al., 1987; Grimmer and Komor, 1999). The IRGA quantifies the net amount
of C fixed and thus the total dry weight that should have been retained in the leaf in the absence
of export. Photorespiratory CO2 losses are accounted for because net CO2 exchange is measured.
The difference in dry weight between samples in the light and the theoretical amount of C that
should have been retained can only be due to export. Whereas the IRGA measurements are non-
destructive, the differential dry weight estimations of leaf tissue are destructive, and many small
samples need to be taken from several leaves. Subsampling reduces disturbance of a single leaf
and eliminates the large variability in dry weight within the same leaf and heterogeneity among
different leaves. A disadvantage of this protocol is that it is difficult to evaluate short-term changes
in export. Also, measurable changes in leaf dry weight do not occur rapidly, usually taking several
hours. Nevertheless, destructive sampling does provide the tissue necessary for biochemical char-
acterization of the leaf intermediates and coupled with labeling procedures can provide important
information regarding the specific pools of assimilates contributing to leaf metabolism and export
(Jiao and Grodzinski, 1996).
13.2.2 Isotopes of C
In most instances, 95% of the photoassimilates being transported via the phloem are carbohydrates
such as sucrose. The mass (e.g., 13C) and radioactive (e.g., 11C, 14C) isotopes of C introduced as
labeled CO2 are excellent tools for directly tracing the movement of photoassimilates in plants.
Two of the advantages of using radioactive and mass isotopes as probes of source leaf export are that
information about metabolism can be obtained and a less invasive method of measuring C export
from individual leaves or leaflets can be achieved. However, sufficient time is required to properly
label the pools of the primary phloem mobile assimilates, and care must be exercised in determin-
ing instrument sensitivity and isotope discrimination.
13.2.2.1 Mass Isotope 13C

The mass isotope 13C has been used extensively in studies of isotopic discrimination as a tool
to distinguish photosynthetic pathways in C4, C3, and C3 –C4 intermediate plants (Monson et al.,
1988; von Caemmerer and Hubick, 1989; Farquhar et al., 1989). Mass isotopes such as 13C and
15N are currently not used as extensively as they should be to study export patterns (Schnyder
and de Visser, 1999 and refs. therein). One problem at the moment is the lack of an inexpensive
detection method for the mass isotopes that does not require destructive sampling (Hirano et al.,
1997; Warringa and Marnissen, 1997). In most studies, a destructive sampling step is required to
determine the enrichment levels of the assimilates in samples that are often prepared for analysis
by mass spectrophotometry coupled to gas or liquid chromatography. However, it is now possible
to use mass isotopes to probe assimilate transport (i.e., fluxes) by coupling, with improving tech-
nologies such as flux sensors, proteins that can respond via a conformational change to binding
of a ligand (Ehrhardt and Frommer, 2012; Okumoto et al., 2012). Fluorescence resonance energy
transfer (FRET) can be used to follow the conformational change of the ligand. Mass isotope
analysis can also be linked to nuclear magnetic resonance (NMR) imaging (Lauterbur, 1973;
Verscht et al., 1998; Kalusche et al., 1999). NMR imaging in medicine has revolutionized remote
sensing of tissues (Jelinski, 1988). With steady-state labeling of leaves with mass isotopes such
as 13CO2, we should be able to replace other methods of quantifying export, as well as moni-
tor partitioning and allocation patterns. It is theoretically possible to analyze export of labeled
assimilates from leaves, movement within individual bundles of phloem cells, and sink metabo-
lism noninvasively.
13.2.2.2 Radioisotope 11C
The short-lived radioisotope 11C has been used extensively as a noninvasive probe of transloca-
tion processes (Minchin, 1979, 1986; Thompson et al., 1979; Grodzinski et al., 1984; Pickard and
Minchin, 1990; Côté et al., 1992b; Pickard et al., 1993; Keutgen et al., 1995; Leonardos et al., 1996;
Minchin and Thorpe, 2003; Minchin and Lacointe, 2005; Thorpe et al., 2010, 2011). However,
due to the short half-life of 11C (i.e., 20.4 min), experiments are restricted in time and must
be performed in the proximity of a particle accelerator. The main advantage of 11C is that the
isotope emits β+ particles of much higher energy than the β − particles that are emitted from
14 C, and thus, translocation of the 11C-labeled intermediates is easier to monitor remotely using
Geiger–Müller (GM) detectors. However, heavy shielding of the detectors is required (Fensom
et al., 1977) that limits an analysis of partitioning of label within different tissues in the same
organ (Kalusche et al., 1999). Nevertheless, 11C has been used to study directional movements
and translocation speed in stems (Thompson et al., 1979; Woodrow et al., 1988; Leonardos et al.,
1996). In other studies, transfer function and compartmental analysis have been used to quan-
tify 11C-photoassimilate export from source leaves (Minchin and Troughton, 1980; Young, 1984;
Keutgen et al., 1995; Minchin and Thorpe, 2003). Given the cost, time, and effort of setting up
labeling experiments, the overall value of using short-lived isotopes must be considered carefully.
Although the procedure is noninvasive and the short half-life of 11C and 13N permits repetition of
tests using the same leaf (Fensom et al., 1977; Grodzinski et al., 1984, Côté et al., 1992b), a signifi-
cant disadvantage of these isotopes is their short half-life that makes analysis of labeled assimi-
lates by current biochemical techniques very difficult. Sap samples and tissue extracts must be
purified and analyzed immediately. We have been able to use 13N (half-life 10 min) fed as 13NH3 to
directly probe leaf photorespiration (Grodzinski et al., 1984). In the first hour, 13N-labeled gluta-
mate, alanine, serine, and glycine were detected (unpublished), and export of these photorespira-
tory intermediates (using 14C) was observed (Madore and Grodzinski, 1984, 1985). However, it is
impossible to follow the fate of the intermediates using short-lived isotopes during pulse–chase
experiments that extend into a normal day–night cycle.
13.2.2.3 Radioisotope 14C
The most widely used radioisotope in the study of leaf photosynthesis and phloem translocation
is 14C. The main advantage of 14C is its commercial availability in different forms (e.g., 14CO2,
14 C-sucrose) and its long half-life (5730 years). It can be used in both short- and long-term stud-
ies of export and partitioning. Although 14C emits β − (negatron) of low energy, translocation of
14 C-labeled assimilates can be monitored in a noninvasive manner with GM detectors, and prod-
ucts can be analyzed following sampling (Geiger and Fondy, 1979; Jiao and Grodzinski, 1996).
Unfortunately, GM detectors cannot measure 14C in tissues further than 1 mm from the plant sur-
face, and a correction must be made for each leaf (Geiger and Fondy, 1979; Jiao and Grodzinski,
1996). Interestingly, several studies with plants have used the x-ray detectors to monitor the
Bremsstrahlung radiation that is also emitted from the 14C. Although the efficiency of count-
ing is low, 14C-photoassimilates that were mobile following pulse labeling of a source leaf were
detected within thick plant tissues (sinks) including the roots and fruits (Sowinski et al., 1990,
1998; Black et al., 2012). The manner in which 14CO2 is introduced and export of 14C assimilates is
monitored varies, but all techniques should be assessed carefully when trying to interpret the data
obtained using 14C as the probe of metabolism and fluxes in and among source and sink tissues.
Both pulse–chase and steady-state 14CO2-labeling protocols have been used to obtain useful data
regarding export of photoassimilates from leaves.
Pulse–chase labeling with 14CO2 is the most common procedure used to study translocation of
14C-photoassimilates (Hofstra and Nelson, 1969; Mor and Halevy, 1979; Madore and Grodzinski,
1984; Farrar and Farrar, 1985, 1986; Moing et al., 1992; Jeannette et al., 1995). Typically the label
is fed to a source leaf, as 14CO2, and either its disappearance from the leaf or appearance in sink
tissue is analyzed. Sizes and rates of turnover of sugar pools (e.g., transport and vacuolar sucrose
pools) in the light have been estimated by monitoring the translocatory efflux of 14C from leaves
pulse-labeled with 14CO2 and employing compartmental models (Moorby and Jarman, 1975; Bell
and Incoll, 1982; Farrar and Farrar, 1985, 1986). However, pulse–chase experiments may not pro-
vide precise measurements of the mass transfer rate of C during photosynthesis since the specific
activity of 14C in the pools changes dramatically during chase periods especially in leaves subjected
to different environmental conditions (Geiger and Fondy, 1979; Jiao et al., 1996). Nonetheless, we
have used pulse–chase experiments primarily to follow the export and respiration of transitory car-
bon reserves during the dark period when 14C is no longer incorporated directly into the transport
products via ongoing photosynthesis (Jiao and Grodzinski, 1998; Jiao et al., 1999; Leonardos et al.,
2003, 2006). Steady-state labeling is our method of choice to quantify the mass transfer rate of
immediate export during photosynthesis.
During steady-state labeling, transport pools of sugars can achieve isotopic equilibrium with
the 14CO2 that is supplied continuously at a constant specific activity (Geiger and Fondy, 1979; Jiao
and Grodzinski, 1996). There are different protocols for establishing steady-state labeling with
14CO (Geiger and Fondy, 1979; Geiger, 1980; Shishido et al., 1987; Jiao and Grodzinski, 1996;
2
Leonardos and Grodzinski, 2000). By calculating export fluxes only when isotopic equilibrium has
been achieved, errors associated with determining immediate export rates under non–steady-state
labeling and pulse–chase experiments are significantly reduced (Jiao and Grodzinski, 1996; Jiao
et al., 1996).
13.3 STEADY-STATE 14CO2 LABELING AND MEASUREMENT OF

IMMEDIATE C EXPORT RATES DURING PHOTOSYNTHESIS
13.3.1 Open-Flow Gas Analysis System
Leaf photosynthesis and C export rates were obtained using an open-flow gas analysis and
14CO -labeling system (Figure 13.1) similar to that described previously (Leonardos et al., 1996,
2
Grodzinski et al., 1998, Leonardos and Grodzinski, 2000). During experiments, plants were held
in an illuminated whole-plant growth cabinet (GS20 BDAF LT big foot, Biochambers, Winnipeg,
Canada) under constantly controlled irradiance, temperature, CO2, and humidity levels. Individual
leaves on these plants were placed in specialized leaf chambers. A personal computer (690 Precision,
Dell) using a custom-designed combination of hardware (National Instruments Corporation, Austin,
TX, United States) and software (LabView 2009, National Instruments Corporation) was used to
control and monitor all devices and log data. Control boards allowed the measurement of analogue
signals from sensors and outputted digital and analogue signals to devices that controlled the envi-
ronment of the gas flowing through the leaf chambers and over the leaves of measurement.
The system included four leaf chambers. The middle portion of a leaf was enclosed in a brass leaf
chamber, which had been chrome plated to reduce problems associated with water exchange (Dixon and
Grace, 1982). The leaf chamber consisted of a top part (16 cm2 exposing area through a glass w
indow)
and a bottom part in which was mounted a GM detector (model 7231, LND Inc., Oceanside, New York,
United States). Both upper and lower sections of the leaf chamber were designed as water-circulating
jackets for leaf temperature control. Temperature of the water circulating through the leaf chamber
was controlled by a water bath (RTE10 NESLAB, Thermo Scientific). Leaf and air temperatures
inside the leaf chamber were measured with two thermocouples (Type T model 5TC-TT-T-30-36 0.010
Dia./Ga., Omega Engineering Inc., Stanford, CT, United States) inside the leaf chamber. One ther-
mocouple was placed in contact with the lower leaf surface (leaf temperature), and the other was
under the leaf but not in contact with it (air temperature). Light (400–700 nm, photosynthetic photon
WPC FH
MFC MFC LC/GM SVM FM
P T
CO2
MFC MFC LC/GM FM
P T
O2 WB
PG MFC MC H FM RC/GM FM
SV SV SV P T
Air
MFC LC/GM FM
P T
N2
MC MFC LC/GM FM
T
14CO2
FM
SP
StdG
SV
MFM
MFM
IRGA
OA
FIGURE 13.1 A schematic of the open-flow gas analysis and 14CO2-labeling system used to measure photo-
synthesis and C export rates in source leaves. For simplicity, only major components and the flow of gasses are
shown. The abbreviations used are as follows: FM, flowmeter; FH, fume hood; H, humidifier; IRGA, infrared
gas analyzer; LC/GM, leaf chamber/Geiger–Müller detector; MFC, mass flow controller; MFM, mass flow-
meter; MC, mixing chamber; OA, oxygen analyzer; PGG, purge gas generator; P, pump; RC/MG, reference
chamber/Geiger–Müller detector; SV, solenoid valve; SVM, solenoid valve manifold; StdG, standard gas; SP,
syringe pump; T, trap; WB, water bath; WPC, whole-plant chamber.
flux density [PPFD]) was provided by high-pressure sodium and metal halide lamps (400 Watt,
Sylvania, GTE, Toronto, Ontario, Canada) and was measured with a LI-COR quantum sensor (model
LI-189, LI-COR Inc., Lincoln, NE, United States) positioned at the surface level of the leaf.
Compressed air was used to provide air for all leaf chambers. To avoid large variations in the
CO2 concentration in this compressed air during the day and over seasons, all of the air was passed
through an FT-IR purge gas generator (model 75–52, Parker Balston, Analytical Gas Systems,
Haverhill, MA, United States) to remove all CO2 and H2O from the air. The desired CO2 con-
centration (e.g., 40 Pa) was obtained by mixing the CO2-free air from the purge gas generator
with pure CO2 (Linde, Guelph, Ontario, Canada) using two mass flow controllers (MFCs) (Smart-
Trak, 100 series, Sierra Instruments, Inc., Monterey, CA, United States). A third MFC was used to
achieve different O2 concentrations when desired. In this case, pure N2, O2, and CO2 were mixed
in appropriate ratios using these three gas MFCs. The total volume of gas from the two MFCs
(e.g., 2500 mL min−1) entered a mixing/buffer container. Humidity (dew point) in the gas was
controlled by passing the gas stream through a gas bubbler placed in a temperature-controlled
water bath (RTE17 NESLAB, Thermo Scientific). To avoid water condensation within the stain-
less steel tubing, all the gas lines were traced with a heating cable and maintained at temperatures
well above the dew point. A 14CO2 injection and mixing loop was used during labeling experi-
ments. The main gas line was then split into a reference line and four more lines one for each of
the leaf chambers. The flow rate in each chamber lines (chambers 1–4) was maintained constant
(e.g., 0.25–0.70 L min−1) by MFCs (Smart-Trak, 100 series, Sierra Instruments Inc.). The flow rate
in the reference line was controlled by a variable rate flow meter (model N112-02, Cole-Parmer
Instrument Co., Niles, IL, United States). The reference flowmeter was also used to control the
air pressure in the gas stream before the four chamber MFCs. This pressure was set around 3 psi
needed for the operation of the MFC and was monitored by an in-line pressure sensor (Cole-
Parmer Instrument Co.). Each line passed through a leaf chamber. An electronic flowmeter (Smart-
Trak, Sierra Instruments, Inc., Monterey, CA, United States) precisely measured the flow rate in
each line after the leaf chambers. The flow rate from this electronic flowmeter was compared
to the flow from the MFC before the leaf chamber and thus used to detect potential leaks from
each leaf chamber. A manifold of solenoid valves (model 8320G222/3 Red-Hat, ASCO, Florham
Park, NJ, United States) was used to direct the gas of each line through a CO2/H2O infrared gas
analyzer (IRGA) (model 7000, LI-COR) and an O2 analyzer (model P110, California Analytical
Instruments Inc., Orange, CA, United States), one line at a time. The CO2 concentration entering
each leaf chamber (same as in Reference line) and that exiting each leaf chamber (chamber line)
was measured with the IRGA, thus monitoring the gas exchange of each leaf.
Photosynthesis rates were calculated using the recorded CO2 concentrations, gas flow rates and
the leaf area enclosed in the leaf chamber. Gas sampling cycles/modes in the system’s software
were used for manual or automatic monitoring of the CO2 concentration and humidity in each leaf
chamber and for operating and calibrating the IRGA in a specific mode. In auto mode, each leaf/
line was sampled for 60 s and photosynthesis was monitored every 5 min. All five gas lines were
finally directed to a fume hood. A set of five air pumps after the leaf chambers and the IRGA and O2
analyzer drew the air from each line plus additional air from the room preventing a buildup of back
pressure in the instruments and the leaf chambers. After the air pumps, five variable rate flowmeters
(model N092-04, Cole-Parmer Instrument Co.) set at flow rates at least twice those set in each line
ensured that all gasses were directed to the fume hood where 14CO2 was trapped in KOH.
13.3.2 14CO2 Feeding and Monitoring of 14C Retention

An acclimation period of 15–30 min was usually required before a steady-state rate of photosyn-
thesis was monitored after inserting the leaf in the chamber. Only after this period, the 14CO2
was supplied when conducting a short-term feed (e.g., 30–180 min). However, during longer feeds
(e.g., during the whole photoperiod), 14CO2 was supplied when lights were turned on at the beginning
of the photoperiod. 14CO2 was generated by reacting NaH14CO3 and H2SO4 in a flask and then stored
in a large airtight syringe (500 mL, model S0500, Hamilton Company, Reno, NV, United States).
During steady-state 14CO2 labeling, a small volume of labeled air, taken with a 60 mL syringe,
was injected into the gas stream by using a precision syringe pump (PHD 2000 Infusion, Harvard
Apparatus, Holliston, MA, United States). The specific activity of 14CO2 in the gas steam was moni-
tored by a GM detector in the reference chamber in line after the 14CO2 mixing chamber. A standard
curve (GM cpm vs. specific activity) was obtained by trapping a volume (e.g., 5 mL) of the inlet gas
in ethanolamine/ethylene glycol monomethyl ether (e.g., 1.0 mL; 1:2 v:v) (Fisher Scientific, Toronto,
Ontario, Canada). Samples of the inlet gas were taken from a 14C-sampling port at which point the
temperature of the gas was measured by a thermocouple (Omega Engineering Inc.). During each
feed, the specific activity of the 14CO2 in the inlet gas remained constant. This specific activity
varied among experiments from 50–250 (whole-day feeds) to 300–3000 (2–4 h feeds) Bq μmol−1 C
depending on the leaf photosynthetic rate, the CO2 concentration, and other characteristics of the
experiment. The duration of the feed also depended on the nature of the experiment.
The GM detector positioned under the leaf surface was used to monitor the radioactivity accumu-
lated in the leaf during the feed period in a manner similar to that described by Geiger and Fondy (1979).
The GM detectors were controlled by a custom-made power supply (model 045-001, Lou Champagne
Systems Inc. Oakville, Ontario, Canada), and the output through a rate counter (model NI-PCI-6602,
National Instruments) was recorded continuously during the feed. At the end of the experiment, the
leaf was taken out of the chamber and the maximum recorded counts were corrected for the total
radioactivity recovered in the fed portion of the leaf over the GM detector inside the leaf chamber. The
total radioactivity recovered in the fed portion of the leaf being over the GM detector was determined
destructively by liquid scintillation counting (Winspectral 1414, Wallac, Turku, Finland).
13.3.3 GM Detector Counting Efficiency

Due to the differences in morphological, anatomical, and biochemical leaf characteristics (e.g.,
leaf thickness, venation pattern, partitioning) among species that we have examined, the counting
efficiency (CE) of the GM detectors varied between 0.1% and 1.0%. The counting efficiencies of
the GM detectors for a representative number of different photosynthetic types of Panicum species
(Leonardos, 1999; Leonardos and Grodzinski, 2000) are shown in Figure 13.2. In spite of the low
counting efficiency (i.e., 0.5%–1.0%), for each species, there was a high linear correlation between
the radioactivity determined by destructive analysis and that counted by the GM detector. The
coefficient of determination (r2) varied from 0.79 to 0.98. These data support the view that the GM
detectors can be used to monitor 14C in the leaf nondestructively.
13.3.4 Calculation of Concurrent Export during Steady-State 14CO2 Feeding

The theories behind determining mass fluxes of C during photosynthesis using either the method
of differential weight analysis or 14CO2 steady-state labeling coupled with net gas exchange are
very similar. Figure 13.3 shows the net CO2 assimilation calculated from the photosynthetic rate
obtained from the IRGA for representative C3, C3–C4 intermediate, and C4 species that transport
sucrose (panels A, B, and C, respectively) and a C3 species that translocates auxiliary sugars as well
as sucrose (panel D). The IRGA was used to estimate the rate of assimilation (dashed line) through-
out the experiment. In each case, the photosynthetic rate was constant before 14CO2 was supplied
at a constant specific activity. The retention of 14C was measured nondestructively with the GM
detector and was corrected with measurements of radioactivity made by destructive sampling at the
end of the feeding period (solid line in Figure 13.3a through d). Immediate export of 14C assimilates
(dotted line) was calculated as the difference between fixation and retention rates given by the data
derived from the IRGA and the GM detector during an appropriate period (shaded area in Figure
13.3a through d).
(a) (b)
30,000 C3 C3
20,000
P. laxum P. bisulcatum
10,000
CE = 0.76% ± 0.02% CE = 0.89% ± 0.02%
r 2 = 0.91 r 2 = 0.98
0
(c) (d)
30,000 C3-C4 C4(NAD-ME)
GM reading (cpm)
20,000
P. milioides
10,000 P. capillare
CE = 0.92% ± 0.04% CE = 0.58% ± 0.04%
r 2 = 0.89 r 2 = 0.98
0
(e) (f )
30,000 C4 (NADP-ME) C4 (PEP-CK)
20,000
10,000 P. bulbosum P. maximum

CE = 0.70% ± 0.04% CE = 0.74% ± 0.02%
r 2 = 0.98 r 2 = 0.95
0
0 2 × 106 4 × 106 0 2 × 106 4 × 106
Radioactivity in leaf (dpm)
FIGURE 13.2 CE of the GM detectors in experiments with Panicum species. Radioactivity was measured
nondestructively by monitoring 14C accumulation in the leaf with a GM detector and by scintillation counting
after destructive sampling of the leaf. Data shown are those of the following: two C3 species, P. laxum (a) and
P. bisulcatum (b); a C3–C4 intermediate species, P. milioides (c); a NAD-ME C4 species, P. capillare (d); a
NADP-ME C4 species, P. bulbosum (e); and a PEP-CK C4 species, P. maximum (f). Each point is the mea-
surement of one leaf. The data are from measurements made at different CO2 (35 and 90 Pa) and at the end of
different experimental periods (30, 60, 90, and 120 min and 17 h). Feeding periods were usually 120 min, but
in pulse–chase experiments, we frequently extended the period of noninvasive monitoring of 14C retention to
17 h. Respiration data in these pulse–chase experiments were used to correct for total export (see Leonardos
et al., 1996, Jiao and Grodzinski, 1998, and Jiao et al., 1999). CE is the value of radioactivity obtained by the
GM detector, divided by the radioactivity determined after destructive analysis of the leaf tissue, times 100.
Values for CE are means ±SE. The variation explained by a linear model fitted to the data is indicated by the
coefficient of determination (r2).
In order to determine what was the appropriate period, a series of destructive experiments were
designed to approximate the time required for transport pools to reach isotopic equilibrium. When
the leaf was sampled during a typical 2 h feeding period, the pattern of 14C partitioning in the
transport sugars indicated that isotopic equilibrium between the 14CO2 in the air stream and the major
14C-translocates was generally not achieved in the first hour (Figure 13.3m through p). A period of
Sucrose +Auxiliary sugars
C3 C3–C4 C4 C3
150 (a) (b) (c) (d)

Fixation, retention, and export
(mmol C m–2)
100 Fixation
Retention
50
Export
0
(e) (f) (g) (h)
40
C-partitioning
(mmol C m–2)
Sugars
20
Starch
14
0
(i) (j) (k) (l)
20
(mmol C m–2)
C-sugars
10
14
Suc
Raf
Stac
0
Raf
(mol 14C-sugar mol–1 sugar)
(m) (n) (o) (p)
0.4
Specific activity
Suc
0.2
Stac
0.0
0 30 60 90 120 30 60 90 120 30 60 90 120 30 60 90 120
Time (min)
FIGURE 13.3 Total 14C fixation, 14C retention, 14C export, and 14C partitioning in major intermediates dur-
ing a 2 h 14CO2 feeding of source leaves. Data are those for species that transport sucrose (a C3 species,
P. bisulcatum [panels a, e, i, and m]; a C3–C4 intermediate species, P. decipiens [panels b, f, j, and n]; and a
C4 species, P. miliaceum [panels c, g, k, and o]) and for a species that transports auxiliary sugars as well as
sucrose (a C3 species, C. sativus [panels d, h, l, and p]). Measurements were made under saturating irradiance
(1500, 1600, and 1750 μmol m−2 s−1 for the C3, C3–C4 intermediate, and C4 species, respectively), 35 Pa CO2,
21 kPa O2, and 30°C. Cumulative net C fixation (dashed line in panels a, b, c, and d) was calculated from
IRGA data, whereas 14C retention in the leaf was measured both nondestructively by monitoring 14C with
a GM detector continuously (solid line) and destructively at the end of the feeding. Export (dotted line) was
estimated as the difference between total fixation (dashed line) and 14C retention in the leaf (solid line). Panels
e, f, g, and h show partitioning of total 14C in the total sugar fraction (λ) and in ethanol insolubles (starch) (∆).
Panels i, j, k, and l show partitioning of total 14C into sugars (sucrose and auxiliary sugars), and panels m, n,
o, and p show the specific activity of the transport sugars, sucrose (Suc, ▴), raffinose (Raf, ▪), and stachyose
(Sta, •). Each point is the average of at least four leaves on four different plants, and each error bar represents
the SE of the mean. The shaded areas represent the 90–120 min period during which export was estimated.
60–90 min was usually required before the specific activity of the major sugar (sucrose) reached a
steady level. The sugar pools in the C4 species generally reached isotopic equilibrium earlier than
in the C3 and C3–C4 intermediates species. Normally, the data between 90 and 120 min were used
to calculate values for photosynthesis and the corresponding concurrent export rate (shaded area in
Figure 13.3a through d). During this period, the 14C-sucrose pool was in isotopic equilibrium with the
14CO being assimilated. Similarly, in species such as Cucumis sativus, the 14C-stachyose pool (auxil-
2
iary phloem mobile sugar that was used as a marker of transport) was in isotopic equilibrium. Panels
I, G, and F show the accumulation of sucrose that is the main form of assimilates being exported in
the Panicum species. In C. sativus, auxiliary sugars accumulated as well as sucrose (Figure 13.3l).
However, in some species, there was a large pool of labeled hexoses (e.g., in the C4 species Panicum
miliaceum and in the C3 species C. sativus) (data not shown). In all species, 14C accumulated in sugar
and starch (Figure 13.3e through h) that sustain metabolic requirements within the leaf and export
during subsequent periods of light or darkness (Leonardos et al., 1996; Jiao and Grodzinski, 1998;
Grodzinski et al., 1999; Leonardos, 1999). The fate of these pools and their contribution to export and
respiration could be determined in pulse–chase experiments.
The rates of photosynthesis and immediate export obtained when isotopic equilibrium was first
established (e.g., 90–120 min) were the data sets that we have used to evaluate changes in imme-
diate export rates in leaves challenged with environmental stresses (Jiao and Grodzinski, 1996;
Leonardos et al., 1996; Grodzinski et al., 1998; Leonardos and Grodzinski, 2000), or diseases (Jiao
et al., 1996, 1999). These data also provide comparisons of immediate export capacity among leaves
with naturally different CO2 fixation pathways (i.e., C3, C3–C4 intermediate, and C4) (Grodzinski
et al., 1998; Leonardos, 1999; Leonardos and Grodzinski, 2000) or transgenic with specifically
altered C metabolism (Grodzinski et al., 1999).
13.3.5 Estimating Export of 14C Assimilates during a Light or Dark Chase Period

The rate of C export during a chase period following the feed can also be obtained. The fate of
14C previously fixed during a specific time during the daytime can be determined throughout a
subsequent chase period representing a natural day/night period. These experiments are timed to
approximate the normal periods of light and darkness to which the plants had been acclimatized.
For example, in steady-state labeling experiments, which were usually conducted between 15:00
and 17:00 h, the 2 h feed period was followed by a 5 h light and a 10 h dark chase period, during
which the gas stream did not contain 14CO2. The disappearance of 14C from the source leaf during
the chase is continuously monitored by the GM detector. The respiratory release of 14CO2 is also
determined by trapping of the outlet gas in 40–100 mL of ethanolamine/ethylene glycol mono-
methyl ether (1:2 v:v) and by further liquid scintillation counting (Leonardos et al., 1996). Export
during the chase in dark is then corrected for respiration losses (Leonardos et al., 2003, 2006).
13.3.6 Partitioning of 14C in the Fed Leaf and Plant

The total 14C in the fed leaf area inside the chamber is needed to correct for the efficiency of
the GM detector. The total 14C in the fed area of the leaf is determined at the end of the feed
or the end of the feed–chase period. After rapid extraction of the leaf tissue in boiling ethanol
(80% v:v) (three times), the extract is vacuum dried, rehydrated, and partitioned against chloro-
form (chloroform/water, 2:1 v:v). Ion-exchange chromatography (AG50-X8 and AG1-X8, BioRad
Laboratories, Hercules, CA, United States) can be used to separate the aqueous phase into neutral
(sugars), acidic (organic acids), and basic (amino acids) fractions. Individual sugars can be separated
by high-performance liquid chromatography (HPLC). Each peak is calculated using an integrator
to estimate the total sugar pools (labeled and non-labeled). The radioactivity in each fraction can be
determined by an in-line radioisotope detector, following the HPLC separation of the fractions. Each
fraction can be pooled and its radioactivity is determined by liquid scintillation counting. The radio-
activity remaining in the ethanol-insoluble fraction is counted by liquid scintillation after oxidizing
the oven-dried tissue samples (BIO-OX, RJ Harvey Instrument Co., Hillsdale, NJ, United States).
Additionally, the 14C-starch in the ethanol-insoluble fraction can be determined using a starch diges-
tive assay. The distribution of 14C within the plant can also be determined by dividing the plant
into predetermined parts/organs at the end of the feed/chase period. These plant parts are oven- or
freeze-dried and grinded, and subsamples are oxidized to quantify the amount of 14C they contain.
13.4 CASE STUDIES
13.4.1 Photosynthesis and Export under Stress
As pointed out previously, the primary functions of a source leaf are to fix light energy and to pro-
vide that energy in the form of exported photoassimilates for growth of the vascular plant. A leaf
is not a homogeneous structure. In the light, photosynthesis and photorespiration occur simultane-
ously, while C is being exported from the tissue. Our analyses showed that in healthy source leaves
when photorespiration was suppressed by low O2 and high CO2 levels (i.e., non-photorespiratory
conditions), both photosynthesis and immediate export increased (Jiao and Grodzinski, 1996;
Leonardos et al., 1996). Significantly, using our steady-state 14CO2-labeling procedure, we were
able to show that leaf warming resulted in a reduction in immediate export prior to any inhibition
of the photosystems that would have altered the C fixation processes per se (Jiao and Grodzinski,
1996; Leonardos et al., 1996). Our data show that net CO2 fixation can be maintained while export
of the fixed C is inhibited. These data challenge conventional literature that focuses on the concept
that disruption of thylakoid membranes in chloroplasts and loss of the photosystem activity or the
downregulation of Rubisco are the primary leaf traits limiting source strength during moderate
heat stress of the leaf canopy. One applied outcome of our studies of immediate export at elevated
leaf temperatures (Jiao and Grodzinski, 1996) has been a reexamination of the effect of high tem-
perature on the maintenance of valuable greenhouse crops, such as alstroemeria (Leonardos et al.,
1996) and roses (Jiao and Grodzinski, 1998). In the case of roses, for example, our data provide an
explanation for reduced production of flowers and abortions that accompany high temperatures.
Photosynthesis is maintained but export during photosynthesis is inhibited leading to poorer flower
development and commercial yield. Carbohydrate limitation due to the abiotic stress imposed by
elevated canopy temperature during the daytime does not appear to be compensated for by night-
time export of stored reserves since most of the C in roses is exported in the light even when the
plants have acclimated to high CO2 and more C was stored in the light (Jiao and Grodzinski, 1998).
Similarly, in a series of studies with wheat plants that were acclimated or not acclimated to cold con-
ditions, low temperatures reduced export of photoassimilates to sinks and that C not being exported
in the daytime appeared to be a major factor in reducing sink development (Leonardos et al., 2003).
Our measurement of the diel patterns of 14C export confirmed that under both ambient and enriched
CO2 conditions, the bulk of the C was being exported in the light rather than during subsequent
nighttime chase periods. During development of vascular plants where sink and source metabolisms
combine to regulate overall C allocation patterns and plant development, the optimal temperature
window for leaf export appears to be narrower than the corresponding temperature range for photo-
synthesis (Leonardos and Grodzinski, 2011).
Studies of biotic stress during which leaf export rates were estimated using steady-state labeling
also show that immediate export rates can change before noticeable changes in leaf photosynthe-
sis are noted and that export at night does not relieve the stress on sink development imposed by
reduced daytime export rates. In bean leaves infected with bacterial blight (i.e., Xanthomonas),
immediate export rate was reduced before a drop in photosynthesis was observed (Jiao et al., 1996).
This conclusion was not readily evident from other analyses of export. In a related study with gera-
nium infected with bacterial blight photosynthesis, immediate and subsequent nighttime export
rates were inhibited more at high CO2 than at ambient levels even though bacterial numbers in the
leaves were lower in plants grown at high CO2 (Jiao et al., 1999). The data show that a classical
definition of disease severity (virulency) based on bacteria numbers (e.g., colony-forming units
[CFUs]) is not accurate. In plants that were acclimated to high CO2, but not infected, the immediate
and daily export rates were greater than those in control plants grown at ambient CO2. Collectively,
these results are interesting since they provide some insight into the acclimation processes that
might operate in plants growing in a CO2-enriched world as well as the manner in which plants
compete with pathogens or symbiotes for assimilates. Using 14C pulse–chase analysis, it appears in
geranium that the healthy uninfected control plants (Jiao et al., 1999) like the healthy rose plants
(Jiao and Grodzinski, 1998) export the bulk of the 14C fed during a light period (i.e., during photo-
synthesis). Long-term CO2 enrichment increased the storage of sugars and starch in the leaves, but
proportionally both ambient-grown plants and those that were acclimated to CO2 enrichment had
similar patterns of immediate (daytime) and nighttime export. Nighttime respiration was generally
higher in the CO2-enriched geranium plants.
13.4.2 Immediate Export in Natural Photosynthetic Variants

Differences in photosynthetic capacity are recognized to occur naturally among genetically
similar species and genera. Anatomical, biochemical, and physiological differences among C3,
C3 –C4 intermediate, and C4 photosynthetic pathways in genera such as Panicum and Flaveria
are well documented (Edwards et al., 1982; Edwards and Walker, 1983; Edwards and Ku,
1987; Hatch, 1987). One advantage of examining the leaf photosynthesis and export patterns in
Panicum and Flaveria genera more closely is that these genera represent a range of naturally
occurring variants of photosynthetic types. Both genera contain C3 –C4 intermediate types in
addition to species with well-defined C3 and C4 traits. Although the impact of these differ-
ent photosynthetic pathways on photosynthetic and photorespiration rates has been examined,
studies that provide measurements of export capacity during photosynthesis in leaves with dif-
ferent photosynthetic types are limited and have often employed different techniques to quan-
tify translocation (Hofstra and Nelson, 1969; Gordon, 1986; Grodzinski et al., 1998; Leonardos
and Grodzinski, 2000).
Hofstra and Nelson (1969) showed based on the rate of disappearance of 14C during a 6 h chase
period that C4 species corn and sorghum had higher export rates than did C3 species such as soybean
and tomato. Leaves of six C3 dicots and four C4 monocots from different families were compared.
Their conclusion was substantiated by Gordon (1986) who employed differential weight analysis
and infrared gas analysis to estimate immediate export. Using a steady-state 14CO2-labeling pro-
tocol to measure photosynthesis and immediate export rates, we have examined over 42 monocot
and dicot species from different families and genera including a number from the Panicum and
Flaveria genera (Grodzinski et al., 1998; Leonardos and Grodzinski, 2000). We examined imme-
diate export both as an absolute rate and as a rate relative to the fixation rate (i.e., as a percent of
photosynthesis). Collectively, the data for all species showed that the faster the photosynthesis,
the faster the immediate export (Figure 13.4). At ambient CO2, there was a high correlation coef-
ficient (r = 0.88) between the rate of photosynthesis and the absolute rate of immediate export
(Figure 13.4a). Among all species and within each of the Panicum (#22–35) and Flaveria (#10–16)
genera, photosynthesis and export rates of the C4 species were higher than those of C3 species
(Figure 13.4a(ii)) (Leonardos and Grodzinski, 2000). Previous studies also show that at ambient
CO2, C4 species have higher translocation rates than C3 species (Hofstra and Nelson, 1969; Lush
and Evans, 1974; Gallaher et al., 1975; Lush, 1976; Gordon, 1986). However, the concept that leaves
with a functional C4 metabolic pathway inherently export newly fixed C more readily than those
with C3 metabolism was challenged (Grodzinski et al., 1998). At ambient CO2, the percent of C
exported immediately relative to photosynthesis was high in a number of C3 dicot species (#3, 7, 8,
19, 38; Figure 13.4c(i); Grodzinski et al., 1998), which produce auxiliary transport sugars (Turgeon,
1995). The notable exception was sunflower (#17; Figure 13.4c(i)), which translocated only sucrose,
but also had a relatively high immediate export capacity (Hofstra and Nelson, 1969; Grodzinski
Ambient CO2 Elevated CO2

25
(i) (ii) (i) (ii)
20
Export (µmol C m–2 s–1)
15
(iii) (iv) (iii) (iv)

10
5 r = 0.88 r = 0.51
(a) (b)
0
(i) (ii) (i) (ii)
80
(% of photosynthesis)
Relative export
60
(iii) (iv) (iii) (iv)

40
20 r = 0.42 r = 0.04
(c) (d)
0
0 5 10 15 20 25 30 0 5 10 15 20 25 30 35
Photosynthesis (µmol C m–2 s–1)
FIGURE 13.4 Photosynthesis and immediate export rates of source leaves of 42 species measured at ambient
CO2 (35 Pa, panels a and c) and at short-term exposure to elevated (90 Pa, panels b and d) CO2 levels. Export rates
were expressed in absolute values (panels a and b) and as relative C efflux rate calculated as a percentage of the
photosynthesis rate (panels c and d). Each panel was divided into four areas (quadrants) so that the data among
panels could be compared easily. The species included C3 types (simple numbers), as well as C4 and C3–C4 inter-
mediate types that are indicated by * and **, respectively. The 42 species were as follows: 1, Alstroemeria sp. cv.
Jacqueline; 2*, Amaranthus retroflexus; 3, Apium graveolens; 4, Avena sativa L. cv. Elgin; 5, Capsicum annuum cv.
Cubico; 6, Chrysanthemum morifolium; 7, Coleus blumei; 8, Cucumis sativus cv. Revenue; 9*, Flaveria bidentis;
10**, Flaveria chloraefolia; 11**, Flaveria floridana; 12**, Flaveria linearis; 13, Flaveria pringlei; 14, Flaveria
robusta; 15*, Flaveria trinervia; 16*, Gomphrena globosa; 17, Helianthus annuus; 18, Hordeum vulgare; 19, Nepeta
faassenii; 20, Nicotiana tabacum; 21*, Panicum antidotale; 22, Panicum bisulcatum; 23*, Panicum bulbosum; 24*,
Panicum capillare; 25**, Panicum decipiens; 26*, Panicum dichotomiflorum; 27, Panicum laxum; 28*, Panicum
laevifolium; 29*, Panicum makarikariense; 30*, Panicum maximum; 31*, Panicum miliaceum; 32**, Panicum mili-
oides; 33, Panicum trichanthum; 34*, Panicum virgatum; 35, Phaseolus vulgaris; 36, Pisum sativum cv. Improved
Laxton’s Progress; 37, Rosa hybrida cv. Samantha; 38, Salvia splendens cv. Bonfire; 39, Sandersonia aurantiaca;
40*, Sorghum bicolor cv. Sudan; 41, Triticum aestivum cv. Karat; 42*, Zea mays. Measurements were made under
light saturating conditions and at the growing temperature (25°C except for the Panicum species, which was 30°C).
Each point is an average of at least four leaves on four different plants. The SE of the means is not shown for graphi-
cal clarity. Starting from top to bottom and from left to right, species’ numbers that overlap within each panel were
as follows: panel A (30*, 31), (16*, 26*), (15*, 24*), (9*, 14), (34*, 32**), (28*, 30), (13, 18), (35, 10**, 5), and (22, 37);
panel b (36, 15*), (32**, 6, 34*), and (27, 1); panel C (24*, 26*, 16*), (32**, 8), (18, 13), (29*, 28*, 30), and (10**, 5); and
panel d (19, 16*), (34*, 25**), (32**, 29*), and (31*, 15*, 36).
et al., 1998). Among the Flaveria species, F. robusta (#14; Figure 13.4c(ii)), a C3 which also trans-
located only sucrose, seemed to have a relatively high export flux.
An interesting finding was that the C3–C4 intermediate species can be very different in their
ability to export C immediately. When immediate 14C efflux was examined relative to the rate of
14C assimilation, type I C –C intermediate Panicum species (#25**, 32**) exported newly acquired
3 4
14C as quickly as the C species (#21*, 23*, 24*, 26*, 28*, 29*, 30*, 31*, 34*) did (Figure 13.4c). In
4
contrast to this pattern, among the Flaveria species, type II C3–C4 intermediates (#10**, 11**, 12**)
had the lowest export rates of the three photosynthetic types (Leonardos and Grodzinski, 2000).
Collectively, the data in Figure 13.4a and c show that the C3–C4 intermediate type I and type II
species of the two genera behave differently with respect to immediate export. The reason for this
difference in immediate export capacity remains unclear.
In both type I and type II C3–C4 intermediate species, a special anatomy and biochemistry leads
to reduced rates of photorespiration compared to those of C3 species (Edwards and Ku, 1987; Hylton
et al., 1988; Rawsthorne et al., 1988; Dai et al., 1996). In leaves of type I C3–C4 intermediates, the
mitochondrial enzyme glycine decarboxylase is localized in the bundle sheath cells (Hylton et al.,
1988; Rawsthorne et al., 1988). Photorespired CO2 that is released in the bundle sheath may be refixed
by Rubisco before escaping from the leaf and result in reduced rates of apparent photorespiration at
ambient CO2 (Dai et al., 1996). Anatomical features such as partially developed kranz anatomy and
localization of a higher number of organelles (e.g., mitochondria) in the bundle sheath cells would
further facilitate the refixation of CO2 (Brown et al., 1983a,b; Edwards and Ku, 1987; Hylton et al.,
1988; Brown and Hattersley, 1989; Rawsthorne, 1992). In addition to compartmentation of glycine
decarboxylase in the bundle sheath cells (Hylton et al., 1988; Rawsthorne et al., 1988), some elements
of C4 metabolism are found in type II C3–C4 intermediate species (Ku et al., 1983; Monson et al.,
1986; Edwards and Ku, 1987). Although not as well developed as in C4 Flaveria species, aspects of
kranz-type anatomy are also evident (Edwards and Ku, 1987; Brown and Hattersley, 1989). Clearly,
both anatomical and biochemical characteristics need to be considered to explain why type I C3–C4
intermediate Panicum species export newly fixed 14C as quickly as their C4 cousins, whereas type II
C3–C4 intermediate Flaveria species exported less 14C (Figure 13.4c).
Consistent with the expected suppression of photorespiration and the increased availability of
CO2 for fixation (Ku and Edwards, 1978; Edwards et al., 1982; Ku et al., 1991; Dai et al., 1996),
short-term CO2 enrichment increased photosynthesis in all C3 and type I and type II C3–C4 inter-
mediate species (Figure 13.4b). In most species except for a few C4 species, the absolute export rate
increased at high CO2, but not proportionally with photosynthesis (Figure 13.4b). In all species,
the relative export rates decreased under CO2 enrichment (Figure 13.4d). Collectively, these data
indicate that during CO2 enrichment, all species tended to accumulate excess C in their leaves in
the light. Taken together, plant productivity of C3 and C3–C4 intermediate and C4 species depends
on many factors including the ability of the leaves to export C immediately (Edwards and Ku, 1987;
Wardlaw, 1990; Geiger and Servaites, 1994; Leonardos, 1999).
Interestingly, in studies of a Flaveria linearis line with altered leaf sucrose–starch partitioning,
we noted that in spite of increased starch during diel tests, most of the carbon exported occurred
during the light period underscoring, as discussed previously the importance of being able to quan-
tify the immediate rate of export during photosynthesis (Leonardos et al., 2006).
13.5 SUMMARY
Over the last half of the twentieth century, the availability of radioisotopes of C (e.g., 14C) led to
the elucidation of the major photosynthetic processes in algae and higher plants. For example, the
discovery of the Calvin cycle (Bassham and Calvin, 1957) helped to define the manner in which
net CO2 assimilation occurs in all plants (Hatch, 1987; Heldt, 1997) and predicted the involve-
ment of secondary regeneration cycles such as photorespiration (Artus et al., 1986; Husic et al.,
1987; Grodzinski, 1992). Although the first function of the leaf as the major site of C fixation is
fairly well understood today, the second function of the leaf as a source of reduced C for develop-
ing sinks is not well understood at the whole-plant level (Wardlaw, 1990; Farrar, 1993a,b; Geiger
and Servaites, 1994). Terms such as source strength and sink strength or sink demand define
concepts affecting C partitioning and allocation in plant tissue (Farrar, 1993a,b; Leonardos and
Grodzinski, 2011). However, there are a limited number of methodologies that provide quantita-
tive data regarding fundamental C fluxes and exchanges such as the immediate export rate from
source leaves. The mass (13C) and radioactive (11C, 14C) isotopes of C are valuable probes for
quantifying assimilate movements within the plant. The potential exists to use the mass isotopes
such as 13C and 15N more extensively; however, to date, user-friendly noninvasive techniques for
mature plants have not been devised. Although the radioisotope 11C emits particles of sufficient
energy to be used to study phloem transport in a noninvasive manner, its use has been restricted.
The more stable form of C, 14C, has limitations as a noninvasive probe. However, it remains a
powerful tool in studying export during photosynthesis. The steady-state labeling methodology
outlined in this chapter, which depends on measurements of immediate export being made when
the transport sugar pools are in isotopic equilibrium with the 14CO2 being assimilated, provides
estimates of mass transfer rates of C through export during photosynthesis.
As pointed out in this chapter, plant productivity in natural photosynthetic variants (C3, C3–C4
intermediate, and C4 types) depends on many factors including the ability of the leaves to export C
(Edwards and Ku, 1987; Wardlaw, 1990; Geiger and Servaites, 1994; Leonardos, 1999). During the
last 25 years, researchers have attempted to increase productivity of plants through genetic engineer-
ing by altering the primary metabolic steps involved in the reduction of CO2. It has been proposed
that modifications of properties of key photosynthetic enzymes such as Rubisco (Frommer and
Sonnewald, 1995; Furbank and Taylor, 1995; Whitney et al., 1999) or the transfer of C4 genes into
C3 species will alter leaf photorespiratory and photosynthetic capacity (Mann, 1999; Ashida and
Yokota, 2011). Numerous groups have noted that there are many challenges in the area of improving
photosynthesis and partitioning and thus plant productivity yet there are many opportunities also to
use the large knowledge based on the enzymes of primary carbon pathways to enhance crop pro-
duction. Both fixation and export processes are functions of a source leaf that can be controlled by
new breeding techniques. More data are needed at the whole-plant level that integrate quantitatively
photosynthesis and export processes with the regulation imposed by key enzymes of C metabolism
and transport mechanisms at the cellular level (Okumoto et al., 2012). A better understanding of
specific site mutations and their effects on diurnal patterns of C partitioning including export during
photosynthesis is needed. Nevertheless, the existence of so many naturally occurring plants with
variations in their photosynthesis, partitioning, and export patterns suggests that by altering key
steps in sugar and starch metabolism, major changes in plant growth can be achieved (Serrato et al.,
2009; Ainsworth and Bush, 2011; Brautigam and Weber, 2011; Weise et al., 2011). With the integra-
tion of techniques and the use of metabolic engineering coupled with traditional biochemical and
physiological approaches, there is ample evidence to predict that we will be able to improve crop
production globally by targeting photosynthetic performance and assimilate partitioning and export
in our major vascular crop plants.
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14 Physiology of Grain
Development in Cereals
Muhammad Farooq, Abdul Wahid,
and Kadambot H.M. Siddique
CONTENTS
14.1 Introduction........................................................................................................................... 301
14.2 Flowering Initiation and Development.................................................................................. 301
14.3 Gametophyte Development and Anthesis.............................................................................. 303
14.4 Pollination, Fertilization, and Grain Initiation......................................................................304
14.5 Grain Development................................................................................................................304
14.5.1 Endosperm Development........................................................................................... 305
14.5.2 Starch Synthesis.........................................................................................................306
14.5.3 Synthesis of Grain Storage Proteins..........................................................................307
14.5.4 Seed Coat Development.............................................................................................307
14.6 Conclusions............................................................................................................................307
References.......................................................................................................................................307
14.1 INTRODUCTION
Cereal grains are one of the major sources of human caloric intake as food and animal feed
worldwide. Cereal crops are well adapted at all latitudes (from 60′ N to 50′ S), on variety of soils
(from slightly acid to alkaline) in both arid and well-watered regions (Chopra and Prakash 2002)
throughout the world with total annual grain yields exceeding 2300 million tons (FAO 2012).
The life cycle of cereals follows the phasic pattern with vegetative and reproductive stages as the
two major phases. Grain development takes place during the reproductive phase of crop cycle. The
reproductive phase starts with the transformation of a vegetative meristem into an inflorescence and
a floral primordium, and ends when the grains attain the maximum dry matter—the physiological
maturity. The entire reproductive phase is a sequential process and may be divided into subphases
viz. flowering initiation and development, development of male and female gametophytes, pollina-
tion and fertilization, and grain development (GRDC 2005).
Caryopses, single-seeded fruits of cereals, are formed in a single carpel. Embryo and endosperm
produced during the fertilization are surrounded by adherent seed coat and pericarp tissues for-
merly made of carpel wall (commonly known as grains). In this chapter, physiological processes
through flowering, pollination, fertilization, and grain development are discussed with special refer-
ence to cereals (wheat, rice, and maize).
14.2 FLOWERING INITIATION AND DEVELOPMENT

Cereals are members of monocotyledonous subclass of angiosperms, characterized by their unique
wind-pollinated flowers. The inflorescence consists of spikelets having florets (individual flowers).
With the initiation of reproductive growth, the inflorescence meristem is differentiated into spikelet
meristem, which is then changed into the floret meristem (Shitsukawa et al. 2009). Spikelets are
301
TABLE 14.1
Base Temperatures for Various Reproductive Phases in Wheat,
Maize, and Rice
Stage Wheata Maizeb Riceb
Terminal spikelet 1.8 ± 0.25 15.3 ± 0.25 13.5 ± 3.5
Anthesis 9.7 ± 0.43 07.0 ± 0.25 19.5 ± 2.5
Grain filling 9.6 ± 0.75 08.0 ± 2.00 15.5 ± 3.0
Sources: aAdapted from Farooq, M. et al., Crit. Rev. Plant Sci., 30, 491, 2011. bData
from Ghoshal, D., Modern Agronomic Practice of Maize, National Academy of
Agricultural Research Management, Hyderabad, India, available online at
http://www.slideshare.net/guest2a2f705/maize-2, (accessed on June 6, 2013),
2011; Lee, M.H., Low temperature tolerance in rice: The Korean experience, in
Increased Lowland Rice Production in the Mekong Region, J. Basnayake and
S. Fukai, eds., ACIAR Proceedings 101 (printed version published in 2001),
2001; McDonald, D.J., Aust. J. Exp. Agric., 34, 878, 1994; Moldenhauer, K. and
Slaton, N., Rice Growth and Development, available online at baegrisk.ddns.
uark.edu/test/Books/PDF/chapter1sl3.pdf (accessed on June 7, 2013), 2004;
Shimono, H. et al., Field Crops Res., 73, 67, 2002; Tollenaar, T., Corn Maturity
and Heat Units, available online at http://www.plant.uoguelph.ca/research/
homepages/ttollena/research/cropheatunits.html, (accessed on June 7, 2013),
2013; Warrington, I.J. and Kanemasu, E.T., Agron. J., 75, 749, 1983; Yoshida, S.,
Tropical climate and its influence on rice, IRRI Research Paper Series 20,
International Rice Research Institute, Los Baños, Philippines, 1978.
bract leaves called glumes together with their florets. Glumes may either encompass the florets like
in maize with two florets per spikelet or may enclose multiple florets as in wheat or can be a reduced
structure as in rice (Calder 1966). The “floret” has lemma, palea, lodicules, stamens, and carpels in
its structure (Bell 1991).
Transition of indeterminate leaf and internodes into determinate floral structures indicates the
commencement of reproductive stage (Chimonidou-Pavlidou 2004). Temperature and photoperiod
determine the timing of flowering (Table 14.1; Guardiola 1997; Cockram et al. 2007), along with
vernalization, which influences the timing of flowering through the activity of actively dividing
cells (Chouard 1960). Vernalization can either be an obligate factor—reproductive phase will not
occur without it as found in wheat in temperate zones (Guardiola 1997)—or facultative, where flow-
ering is accelerated by vernalization (Lozano et al. 1998), while in other crops like rice and maize,
this effect has been eliminated (Cockram et al. 2007).
Yield potential of cereal grains largely depends upon the fertile florets formed before anthesis
(Wang et al. 1996), which may differ either due to genotypic difference (Miralles et al. 1998) or
environmental effects (Serrago et al. 2008). The spikelet development is the integration of corre-
lated mechanisms responsible for floret development. Floret primordial development coincides with
the stem elongation phase; stem elongation time, therefore, is critical for the yield determination
(Miralles and Slafer 1999; González et al. 2011).
Abiotic stresses have strong influence on the flowering initiation and development. Abortive
ovaries and infertile pollens are very common effects of drought stress in maize (Boyer and Westgate
2004). Other than drought, temperature extremes also affect the floral development. For example,
incidence of chilling at floral initiation suppressed the development of branch meristem and substan-
tially decreased the tassel branching and pairs of spikelet in maize (Bechoux et al. 2000), whereas
Physiology of Grain Development in Cereals 303
in rice, both drought (Farooq et al. 2009) and chilling during the reproductive stage increased the
spikelet sterility (Gunawardena et al. 2003).
Nutrient deficiency also affects the pollen fertility. For instance, boron deficiency at premei-
otic interphase to late tetrad stages of microsporogenesis is very sensitive in wheat, whereas the
period of mitosis I–II is less sensitive as during this stage starch accumulation takes place in pollen
grains (Huang et al. 2000). In addition, nitrogen also influences the floret developmental from the
third floret primordium onward (Ferrante et al. 2013). Likewise, plant growth regulators also influ-
ence the floret development in cereals (Youssefian et al. 1992; Guo et al. 1995; Wang et al. 1999).
For instance, exogenous application of cytokinin (zeatin) promoted the floret development whereas
exogenous application of abscisic acid, indole acetic acid, and gibberellic acid inhibited the floret
development (Wang et al. 1999).
14.3 GAMETOPHYTE DEVELOPMENT AND ANTHESIS

The male gametophyte, the pollen grain, is developed within the anthers (Borg et al. 2009), whereas
development of female gametophytes, the embryo sacs, takes place in the ovule (Yadegaria and
Drews 2004). The embryo sac contains seven cells of four types viz. the egg cell, two accessory cells
called synergid cells, the central cell, and three cells of unknown function called antipodal cells.
Embryo sac is enclosed within the diploid sporophytic tissues, the integuments, from which seed
coat is developed in the mature seed (Yadegaria and Drews 2004). The pollen grain contains a veg-
etative cell, which performs the metabolic functions of the pollen grain and delivers the gametes to
the embryo sac, and two sperm cells, which fertilizes the egg and central cell to produce the embryo
and endosperm, respectively (Borg et al. 2009).
During early developmental stages of pollen grains, high metabolic activity and active growth
take place in the anther. To support the early development, large quantity of sugars is translocated
to the anthers, which possess the highest sink strength in the flower (Clément et al. 1996; Castro and
Clément 2007). The tapetum functions at its maximum capacity, the cell wall of pollen is deposited,
and the locular fluid is produced to provide the nutrition for developing pollen grains at the young
microspore stage (Clément et al. 1994). Any incidence of environmental stresses during the tapetal
development may result in pollen sterility (Jung et al. 2005).
Pollen produced from the same flower may not be necessarily of the same size or shape show-
ing different nourishing patterns by the microspore tapetum layer (Pacini 2010). Lodicules are
important structures for anthesis. They are not necessarily the parts of the perianth as they develop
from leaf or stem like primordia in some genera. They are functionally important for the opening of
florets at the time of anthesis by swelling via cell expansion causing an increased spread of lemma,
palea, and glumes, allowing the wind to access mature stamens for pollen dispersion (Keijzer et al.
1996). At anthesis, anthers dehisce, flowers open, and become fully functional.
Low temperature induces pollen and ovule infertility, flower abortion, causing failure of fertil-
ization and poor grain filling, and eventually low grain yield (Thakur et al. 2010). Cold stress at
reproductive phase induces flower drop, ovule abortion, pollen infertility, pollen tube rupture of,
and poor fruit setting causing low yield (Staggenborg and Vanderlip 1996; Verheul et al. 1996).
Likewise, heat stress during anthesis increases floret abortion (Wardlaw and Wrigley 1994).
Temperatures above 20°C, between spike initiation and anthesis, may substantially reduce grain
number per spike (Saini and Aspinall 1982). Anthesis is also sensitive to drought, and their failure
directly affects the kernel number, thus causing substantial decrease in grain yield. Incidence of
even a brief episode of drought during meiosis of pollen mother cells disrupts subsequent microspo-
rogenesis resulting in pollen sterility, which can decrease the grain set by ∼50% (Saini and Aspinall
1981; Saini and Westgate 2000). Nevertheless, similar level drought does not affect megasporogen-
esis in wheat (Saini and Aspinall 1981).
TABLE 14.2
Influence of Drought during Grain Pollination and Grain Filling on Grain
Number, Grain Weight, and Grain Yield in Wheat, Maize, and Rice
Decrease over Control (%)
Wheat Maize Rice
Stage of
Drought GN GW GY GN GW GY GN GW GY
Pollination 39.94 16.79 46.68 14.78 12.5 8.85 30.51 38.75 5.68
Grain filling 19.67 1.79 27.41 1.04 15.0 32.74 16.55 14.26 3.09
Sources: Data from Ghooshchi, F. et al., Am.-Eurasian J. Agric. Environ. Sci., 4, 302, 2008; Bakul,
M.R.A. et al., Bangla. Res. J., 3, 934, 2009; Shamsi, K. and Kobraee, S., Ann. Biol. Res., 2,
352, 2011.
Note: GN, number of grains per spike/cob/panicle; GW, 1000-grain weight; GY, grain yield.
14.4 POLLINATION, FERTILIZATION, AND GRAIN INITIATION

Pollination, pollen transfer from anther to the stigma, starts with maturation of pollen grains and
anther dehiscence. Pollen maturation and anther dehiscence depend on the rate of cell division in
gametophytic tissue and development of endothecium. Adhering of the pollen grain with papil-
lary cell initiates signaling continuum between pollen and maternal pistil tissues (Heslop-Harrison
1987). Vegetative cell produces a pollen tube, which grows through the style toward ovary reaching
the ovule, where it enters into the embryo sac. Interaction with the synergid causes the rupture of
pollen tube and release of two sperm cells, one to fertilize the egg and the other to fuse with polar
nuclei to form triploid endosperm (Hiscock and Allen 2008). Embryo and endosperm, produced as
a result of fertilization, are surrounded by adherent seed coat and pericarp tissues formerly made of
carpel wall. The endosperm has an outer aleurone layer and inner layer of starchy endosperm cells.
Any incidence of drought before, during, and after pollination strongly influences the number of
grains, grain weight, and grain yield in wheat, maize, and rice (Table 14.2; Ghooshchi et al. 2008;
Bakul et al. 2009; Shamsi and Kobraee 2011).
14.5 GRAIN DEVELOPMENT

The process of grain development starts with double fertilization (Yang and Zhang 2006). Cereal
grains consist of endosperm and the embryo surrounded by the seed coat (Emes et al. 2003). The
endosperm, the largest organ in the cereal grain, is surrounded by a single layer of cells, the aleurone
layer. In cereals, grain development follows a triphasic pattern in terms of fresh and dry weights
and grain water contents (1) grain expansion; (2) grain filling, and (3) grain maturation (Figure 14.1;
Bewley and Black 1994). Grain expansion involves quick and early division of the zygote and trip-
loid nucleus. Cell division is followed by the water intake, which drives cell extension (Altenbach
et al. 2003; Emes et al. 2003).
Cell division and expansion take place with water uptake, and when this expansion stops, cells
are destined to maturity. The endosperm cell division stops after 2–3 weeks of anthesis with the
net increase in water per grain representing the maximum grain size (Schnyder and Baum 1992).
Grain-filling duration is controlled by kernel water content. In early developmental stages, grains
accumulate more water, which is then reserved as volume attained by grains is more important
than the density in the beginning (Saini and Westgate 2000). This accumulated water is termed as
maximum water content accumulation (Saini and Westgate 2000). Grain filling duration is inversely
proportional to the water loss and biomass deposition (Gambin et al. 2007).
1 2 3
FW
Weight (mg)
DW
WC
Days after anthesis
FIGURE 14.1 Typical triphasic pattern of grain development in cereals in terms of fresh weight (FW), dry
weight (DW), and water contents (WC). (1): Grain expansion, (2): grain filling, and (3): grain maturation.
(Adapted from Bewley, J.D. and Black, M., Seeds: Physiology of Development and Germination, 2nd edn.,
Plenum Press, New York, 1994.)
In cereals, grain filling depends on the assimilate supply from current photosynthesis and
retranslocation of reserves in vegetative tissues (Schnyder 1993). During the later grain-filling stages
and dark period of the diurnal cycle, assimilate supply for the developing grains comes from the
hydrolysis of reserve photosynthates (Schnyder 1993). Contribution of assimilate supply from stem
reserves may exceed up to 40% under periods of heat or deficit moisture conditions (Bidinger et al.
1977; Gebbing and Schnyder 1999). Remobilization of stem reserves to the developing grains is thus
very important to harvest good grain yield if the plants are subjected to abiotic stresses (Asseng and
Herwaarden 2003; Plaut et al. 2004).
Abiotic stresses strongly influence the grain development process. For instance, drought stress
during grain development restricts the grain sink capacity by decreasing the number of endosperm
cells (Ober et al. 1991; Saini and Westgate 2000), thus lowering the grain weight by reducing the
starch accumulation capacity of endosperm by decreasing the duration and rate (Yoshida 1972;
Brocklehurst 1977). Stress-induced decrease in assimilate supply also influences the grain develop-
ment process (Sharkey 2005; Subrahmanyam et al. 2006; Farooq et al. 2009).
14.5.1 Endosperm Development
In cereals, pattern of endosperm development is more or less same (Sabelli and Larkins 2009). After
fertilization, endosperm development begins with a syncytium layer surrounding a central vacuolar
space around the nucleus. The endosperm nuclei then undergo division followed by migration to the
periphery of the central cell (Sabelli and Larkins 2009). The nuclei are arranged around the periph-
ery; with the formation of anticlinal cell walls, the process of cellularization starts for producing the
alveoli (Becraft and Yi 2011). Each alveolus is open centripetally to the common cytoplasm and has
a nucleus. Cellular peripheral and alveolar interior layers are then produced by periclinal divisions
(Becraft and Yi 2011). In the inner alveolar layers, this process is repeated several times until the
cellularization of the endosperm is complete (Olsen 2001; Lohe and Chaudhury 2002; Becraft and
Yi 2011). This is followed by cell expansion due to increase in water content; this water is replaced
by proteins and starch later on (Lopes and Larkins 1993).
In cereals, major nutritional value is derived from the endosperm (Lopes and Larkins 1993).
The mature endosperm is a bag of carbohydrate (principally starch), protein, lipids, and minerals.
The starchy endosperm is surrounded by a specialized layer of highly differentiated tissues of

aleurone, which is in fact a lipid–protein storage tissue and remains viable upon maturity (Becraft
and Yi 2011). Many of the digestive enzymes are secreted by this layer, which can mobilize the
reserves of endosperm (Becraft and Yi 2011).
14.5.2 Starch Synthesis
Starch synthesis is the basic biosynthetic activity of grain development (Dai 2010). Starch is made
up of two glucose polymers, amylose and amylopectin. Amylose is a linear polymer linked through
α-1, 4 linkages. Amylopectin, comprising ∼70% of the starch, contains a backbone of glucose resi-
dues linked through α-1, 4 linkages and nonrandomly placed α-1, 6 branch points comprising ∼5%
of the glycosidic bonds (James et al. 2003). Amylopectin structurally contributes to the crystalline
organization of the starch granule in cereals. Amylopectin is biosynthesized through coordinated
enzymatic reactions involving ADP glucose pyrophosphorylase (AGPase), soluble starch syn-
thase, starch branching enzyme, and starch debranching enzyme, whereas biosynthesis of amylose
requires AGPase and granule-bound starch synthase (Jeon et al. 2010).
The process of starch synthesis is initiated by AGPase, which determines the possible sink
strength of the growing grain (Priess 1991; Jeon et al. 2010). Starch synthesis then starts with the
converting glucose 1-phosphate to glucosyl donor ADP-glucose (ADPG) catalyzed by AGPase
(Figure 14.2; Beckles et al. 2001). The process of ADPG synthesis takes place in the cytosol,
and ADPG is then transported into plastids (Figure 14.2; James et al. 2003). In cereals, ADPG
form of carbon is solely utilized for starch synthesis and regulated by 3-phosphoglycerate and
Starch
Amylopectin amplification
Amylose Amylopectin
Cytosol Amyloplast
DBE SS
BE
GBSS I
DBE SS
Glucan initiation
BE
G-6-P G-6-P G-1-P
AGPase
AGPase
G-1-P
ADPG ADPG
FIGURE 14.2 Starch biosynthesis in developing cereal grain. Starch synthesis starts with the conversion of
glucose 1-phosphate to glucosyl donor ADP-glucose (ADPG) catalyzed by AGPase. Soluble starch synthase
(SS) catalyzes the formation of α-1, 4 linkages during elongation of amylopectin. Starch branching enzyme
(BE) mediates the formation of α-1, 6 linkages in glucose polymer creating the branched amylopectin. Starch
debranching enzyme (DBE) isoamylase removes some of the α-1, 6 linkages and forms the crystalline shape
of amylopectin. Filled circle indicates G-6-P transporter and open circle is ADPG transporter.
orthophosphate (Pi) (Hannah 1997; Smidansky et al. 2002). Starch synthase enzyme has two
subclasses: (1) granule-bound starch synthase and (2) soluble starch synthase, each with distinct
functions (Viriten and Nakamura 2000; Jeon et al. 2010). Soluble starch synthase catalyzes the for-
mation of α-1, 4 linkages during elongation of amylopectin (Leterrier et al. 2008). Starch branch-
ing enzyme mediates the formation of α-1, 6 linkages in glucose polymer creating the branched
amylopectin (Regina et al. 2005). Starch debranching enzyme, isoamylase, removes some of the
α-1, 6 linkages and forms the crystalline shape of amylopectin (Jeon et al. 2010).
14.5.3 Synthesis of Grain Storage Proteins

Cereal grains contain ∼10–12% proteins with prolamins as the principal form of grain storage
proteins with the exception of rice, which has glutelin as the major form of grain storage pro-
teins (Kawakatsu and Takaiwa 2010). Based on the solubility, cereal grain proteins are classified
into prolamin, glutelin, globulin, and albumin (Shewry and Casey 1999). Albumins are soluble in
water, prolamins in diluted alcohol, glutelins in alkaline solutions, and globulins in saline solutions
(Kawakatsu and Takaiwa 2010).
Grain storage proteins are biosynthesized in the rough endoplasmic reticulum as secretory
proteins (Yamagata and Tanaka 1986; Muench et al. 1999). Immediately after the cleavage of
N-terminal peptide, grain storage proteins are transferred from the cytoplasmic side of the rough
endoplasmic reticulum to its lumen (Kawakatsu and Takaiwa 2010). These proteins are accumulated
in the protein bodies derived from protein sorting vacuole (Chrispeels 1985; Shewry and Casey
1999). In some cases, specifically in rice and maize, grain storage proteins are deposited within the
lumen of rough endoplasmic reticulum, which is then converted into protein bodies (Coleman and
Larkins 1999; Muench et al. 1999).
14.5.4 Seed Coat Development

Seed coat controls the embryo development and may regulate the seed dormancy and germination
(Moise et al. 2005). Seed coat is developed from the integument, which surrounds the ovule prior to
fertilization. Cell of integuments are undifferentiated before fertilization; however, after fertiliza-
tion, cell layers are differentiated. During seed coat development, some cell layers accumulate large
quantities of mucilage or pigment, which influence the seed morphology (Moise et al. 2005).
14.6 CONCLUSIONS
Wheat, maize, and rice are the major world cereals providing bulk of global caloric intake. These
crops are well adapted across the continents in a variety of environments. Reproductive phase, in par-
ticular the grain development, is the most important phase in the crop cycle determining the crop yield
potential. Starch occupies the major part in the cereal grains. Starch biosynthesis in cereals, taking
place in cytosol and plastids, is regulated by several enzymes including ADP glucose pyrophospho-
rylase, soluble starch synthase, granule-bound starch synthase, starch branching enzyme, and starch
debranching enzyme; ADP glucose pyrophosphorylase is a rate-limiting enzyme in process. Grain
development is regulated by plant hormones and is under the strong influence of environment. An
understanding into the physiology of grain development can help to improve the grain production level.
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15 C-Repeat Transcription
Factors as Targets for
the Maintenance of Crop
Yield under Suboptimal
Growth Conditions
Keshav Dahal, Khalil Kane, Fathey Sarhan, Leonid V. Savitch,
Jas Singh, Bernard Grodzinski, and Norman P.A. Hüner
CONTENTS
15.1 Introduction........................................................................................................................... 313
15.2 Energy Conversion Efficiency and Crop Productivity........................................................... 315
15.2.1 Photosynthetic Energy Conversion Efficiency vs. Photoprotection.......................... 315
15.3 Photosynthetic Performance at Low Temperature................................................................ 318
15.3.1 Sensing Low Temperature......................................................................................... 318
15.3.2 Cold Acclimation Enhances Photosynthetic Performance........................................ 319
15.3.3 Enhancement of Energy Conversion Efficiency Is Cultivar Dependent.................... 320
15.4 CBF Transcription Factors and Energy Conversion Efficiency............................................. 322
15.5 Photosynthetic Responses to Elevated CO2........................................................................... 323
15.5.1 Short-Term Response of Photosynthesis to Elevated CO2......................................... 323
15.5.2 Long-Term Response of Photosynthesis to Elevated CO2......................................... 323
15.5.3 Long-Term Exposure to Elevated CO2 Suppresses Biotic Stress Genes.................... 324
15.6 Summary, Conclusions, and Future Perspectives.................................................................. 325
References....................................................................................................................................... 326
15.1 INTRODUCTION
The intergovernmental panel on climate change has predicted that the atmospheric CO2 concentration
will double from the present 380 μmol C mol−1 to approx. 700 μmol C mol−1 by the end of the twenty-
first century, which may be coupled to an increase in average global temperature by the end of this
century (IPCC 2007). The predicted increase in atmospheric CO2 and high temperature associated
with global warming may increase the severity of water stress as well as the incidence of biotic stresses
(Hatfield et al. 2011; DeLucia et al. 2012; Vandegeer et al. 2012). Such suboptimal growth conditions
due to global warming and climate change may substantially affect the photosynthetic performance of
plants and, consequently, plant biomass and seed yield. The anticipated increase in global temperature
313
will result in the inhibition of photosynthetic carbon assimilation, which is closely correlated with a
high-temperature-induced decrease in the activation state of Rubisco as well as irreversible photosys-
tem II (PSII) damage and increased photorespiratory rates (Long et al. 2004; Salvucci and Crafts-
Brandner 2004; Ainsworth and Rogers 2007; Kumar et al. 2009; Dahal et al. 2012c). This is associated
with the inhibition of respiration and grain filling (Salvucci and Crafts-Brandner 2004; Ainsworth
and Rogers 2007; Kumar et al. 2009; Dahal et al. 2012c), increased risk of sterility, and complete
crop failure (Teixeira et al. 2013). In addition, the anticipated rise in temperature will increase the
evapotranspiration leading to soil water deficit and agricultural drought (Hatfield et al. 2011) and will
increase the likelihood of crop infestation by insect, pests, diseases, and weeds leading to yield losses
(Ziska et al. 2011, DeLucia et al. 2012). Thus, although the projected rise in the atmospheric CO2 may
increase crop yield potential in certain plant species, the yield deterioration due to high temperature,
water limitations, and biotic stresses associated with global warming induced by elevated CO2 may
exceed the benefit achieved by any increase in CO2. For example, high CO2 is expected to increase
global crop yields by about 1.8% per decade over the next few decades, whereas global warming will
likely decrease the crop yield by a 0%–4% over the same period due to other coincident factors such
as high-temperature stress, water limitations, and biotic stress (Lobell and Gourdji 2012). Recently,
Tonkaz et al. (2010) reported that although a 6°C decrease in growth temperature increases yield by
37%, a 6°C increase in growth temperature decreases winter wheat yield by 30%. In a study of eight
different crops, Hlavinka et al. (2009) revealed that drought was the leading factor causing yield vari-
ability. Similarly, Oliveira et al. (2012) reported a considerable decrease in wheat biomass and grain
yield under drought stress regardless of growth CO2 levels. Furthermore, Sardans and Penuelas (2012)
conclude that the effects of global warming on crop productivity are expected to vary widely depend-
ing on the ecosystem in question, for instance, cold, wet temperate regions vs. hot, dry ecosystems.
To add to the complexity of the effects of climate change on crop productivity, the suboptimal
growth conditions associated with global warming are occurring at a time when the world popula-
tion is likely to increase by about 30% by 2050 (UN report 2011). It has been predicted that the
world food demand will double from its present demand of 12 to about 25 Tetracalories d−1 in 2030
due to increased world population as well as increased per capita food consumption (Murchie et al.
2009). This has triggered an immediate need to substantially enhance seed yield and productivity
of major food crops such as rice, wheat, and maize to meet the nutritional demand of the increas-
ing population over the next 50 years (Murchie et al. 2009). For instance, the world production of
wheat in 2010 was 651 million tons (FAO 2012), which needs to be increased to about 840 million
tons per year by 2025 to meet the required demand of increasing population (Murchie et al. 2009).
The projected increased food demand is occurring in a context of losses of agricultural land due to
urbanization, desertification in most of the developing countries, increasing proportion of grain for
animal nutrition and biofuel generation, and suboptimal conditions to attain maximum crop yield
(Murchie et al. 2009; Zhu et al. 2010).
The anticipated global climate changes will affect plants differentially, depending on their
genetic characteristics as well as habitats (Higgins and Scheiter 2012). For instance, the predicted
increases in CO2 and temperature may favor C4 grasses relative to C3 plants (Morgan et al. 2011). In
addition, the increases in atmospheric CO2 associated with global warming will induce abrupt local
shifts in vegetation characterized by increased plant biomass due to the predominance of woody
plant species (Higgins and Scheiter 2012).
Powell et al. (2012) suggest that the maintenance of crop yield stability through enhanced tolerance
to environmental stresses such as drought, low temperature, and high temperature associated with
global climate change remains a crucial challenge for plant biologists and agronomists to maximize
crop productivity in the future. We summarize experimental evidence that targeting the C-repeat
binding factor (CBF) pathway in major crop species may be a novel approach to improve crop yield
and productivity through enhanced photosynthetic energy conversion efficiency. We suggest that this
approach may provide important insights into potential molecular approaches focused on the main-
tenance of plant productivity under suboptimal growth conditions associated with climate change.
C-Repeat Transcription Factors 315
15.2 ENERGY CONVERSION EFFICIENCY AND CROP PRODUCTIVITY

The increase in the yield of major food crops since the mid-1950s has been achieved mainly through
genetic improvement and increased use of agricultural inputs such as fertilizers, pesticides, and
water (Murchie et al. 2009). Zhu et al. (2010) have suggested that the yield of major food crops
since the last decade is increasing slowly, which may indicate that yield increase due to improved
agricultural practices has reached an upper theoretical limit. Thus, it appears that further enhance-
ment in the crop yield can be achieved only by enhancing genetic yield potential, that is, the seed
yield that a crop can achieve per unit ground area under optimum growth conditions without biotic
and abiotic stresses. The maximum potential biomass and grain yield that a plant can produce are
determined by a number of yield variables (Long et al. 2006; Amthor 2007; Zhu et al. 2008, 2010).
They are (1) the amount of incident solar radiation available over the growing season of a plant,
(2) light interception efficiency, that is, the efficiency of the photosynthetic pigments to intercept
photosynthetically active radiation (PAR), (3) energy conversion efficiency, that is, the ratio of the
biomass energy produced over a given period to the radiative energy intercepted by the canopy over
the same period, (4) translocation of photosynthates to sinks, as determined by sink strength, and
(5) partitioning efficiency, that is, the amount of total biomass energy partitioned into seed produc-
tion, also termed harvest index (Long et al. 2006; Amthor 2007; Zhu et al. 2008, 2010).
The increase in yield potential of major crops over the past 50 years was, by and large, accounted
for by the improved partitioning efficiency and light interception efficiency of crop plants (Long et al.
2006; Murchie et al. 2009; Zhu et al. 2010). Increased partitioning efficiency has chiefly resulted
through the release of dwarf cultivars producing higher numbers of seeds per plant. Increased light
interception efficiency is a consequence of increased leaf area index associated with larger-leafed
cultivars as well as the development of dwarf cultivars with improved lodging resistance against the
adverse weather conditions, such as rain, wind, and hail (Long et al. 2006; Murchie et al. 2009).
Since energy partitioning efficiency and light interception efficiency have approached the theo-
retical upper limit (Amthor 2007; Zhu et al. 2008, 2010), further increase in yield potential can
be achieved only by an increase in the energy conversion efficiency into biomass. Since plant dry
matter consists of about 40% carbon by weight, an increase in total biomass production can be
achieved through enhanced photosynthetic carbon assimilation (Murchie et al. 2009). Although
photosynthesis is the ultimate basis for the conversion of light energy into biomass and seed yield
to date, improving photosynthetic carbon assimilation has played only a minor role in enhancing
energy conversion to biomass and seed yield (Long et al. 2006; Zhu et al. 2010).
15.2.1 Photosynthetic Energy Conversion Efficiency vs. Photoprotection

Figure 15.1 illustrates the photosynthetic conversion of solar incident energy to final biomass for a
C3 plant and processes involved in the losses of energy and, thus, a decrease in energy conversion
efficiency between the sun and final plant biomass. Of the total solar incident energy impinging the
leaf surface, 51.3% is outside of the PAR range of 400–700 nm. Thus, only the remaining 48.7% of
the incident solar energy is intercepted by photosynthetic pigments. However, since photosynthetic
organisms have very weak absorbance in the green region of the visible spectrum, 4.9% of total
solar energy is reflected or transmitted through leaves leaving only 43.8% remaining for absorption
by the leaf. Photosynthetic pigments have highest absorbance in the blue (400 nm) and red (700 nm)
regions of the visible spectrum. Since photochemistry in PSI and PSII reaction centers is driven
with the energy of red photons irrespective of the wavelength initially intercepted, the absorbed
energy of blue photons is rapidly converted to the lower energy level of red photons. This results in
the loss of 6.6% of the absorbed energy as heat and hence a contribution to photosynthetic ineffi-
ciency (Figure 15.1) leaving about 37.2% of the initial energy trapped and stored in the form of ATP
and NADPH. Subsequently, this energy is consumed in the assimilation of CO2 and its conversion
to carbohydrates through reductive biosynthesis at a cost of about 24.6% of the total energy, which
Total solar energy
% Retained Energy losses % Lost
48.7 Non-PAR irradiance 51.3
43.8 Reflected/transmitted 4.9
37.2 Photochemistry 6.6
12.6 Carbohydrate synthesis 24.6
6.5 Photorespiration 6.1
4.6 Respiration 1.9
C3 biomass conversion (4.6%)
FIGURE 15.1 Conversion of solar incident energy to final plant biomass and processes involved in the losses of
energy and, thus, a decrease in energy conversion efficiency between the sun and final plant biomass. (Modified
from Zhu, X.G. et al., Curr. Opin. Biotechnol., 19, 153, 2008; Zhu, X.G. et al., Annu. Rev. Plant Biol., 61, 235, 2010.)
results in the conservation of about 12.6% of the total energy (Figure 15.1). In C3 plants, the oxida-
tive carbon pathways of photorespiration and respiration are antagonistic to the light-dependent
assimilation of CO2 by contributing to the evolution of CO2. Consequently, photorespiration (6.1%)
and respiration (1.9%) expend a total of 8.0% of the total energy. Therefore, it is estimated that
about 4.6% of the initial energy impinging the leaf surface is conserved as fixed carbon and plant
biomass (Figure 15.1). However, this estimate does not take into account the energy consumed in
the reduction of N and S, which should also be considered photosynthetic since they are dependent
upon photosynthetically generated electrons (Foyer and Noctor 2002).
In addition to the inefficiencies due to the normal processes of light absorption and energy trans-
formations associated with photosynthesis, photorespiration, and respiration in C3 plants (Figure 15.1),
plants are frequently exposed to an irradiance that is in excess of which can be used for C, N, and S
reduction and ultimately growth. Such conditions lead to photoinhibition of photosynthesis, that is, light-
dependent inhibition of photosynthesis (Krause 1988; Baker and Ort 1992; Aro et al. 1993; Osmond
1994; Melis 1999; Takahashi and Murata 2008). Excess light is not only necessarily due to an increase
in incident irradiance but also generated with no change in absolute irradiance when coupled with either
low temperature (Krause 1994; Hüner et al. 1998; Farage et al. 2006; Takahashi and Murata 2008)
or other abiotic and biotic environmental stresses (Murata et al. 2007; Takahashi and Murata 2008).
Chronic photoinhibition results in the photodamage of the psbA-encoded 32-kD PSII reaction center
polypeptide, D1, resulting in a decreased efficiency in charge separation that can be monitored as
decreased Fv/Fm (Raven 2011). Chronic photoinhibition is determined by the balance between the rate
of D1 damage and the rate of its repair (Takahashi and Murata 2008). Repair of photodamage to D1
involves the proteolytic removal of the damaged D1 protein and its replacement with newly synthe-
sized protein (Raven 2011). Historically, the consensus was that PSII was the only site of photoinhibi-
tion (Powles 1984). However, recent experimental evidence has indicated that PSI is also sensitive to
photoinhibition although less so than PSII when measured in vivo (Sonoike 2011).
In contrast, dynamic photoinhibition results in the reversible short-term reduction of photosyn-
thetic efficiency with minimum changes in the photosynthetic capacity and is considered to be
a photoprotective mechanism. Photoprotection through dynamic photoinhibition involves mech-
anisms to dissipate excitation energy through heat loss as nonphotochemical quenching (NPQ)
within the light-harvesting complex of PSII (LHCII), hence called antenna quenching (Figure 15.2).
Antenna quenching occurs through the xanthophyll cycle, which is the reversible light-dependent
conversion of the light-harvesting carotenoid, violaxanthin, to the quenching carotenoids, antherax-
anthin, and zeaxanthin (Demmig-Adams and Adams 1996; Horton et al. 1999; Niyogi et al. 2005).
Thus, while the violaxanthin harvests light energy and transfers that excitation energy to PSII reac-
tion centers, antheraxanthin and zeaxanthin play a pivotal role in the photoprotection of PSII by
dissipating excess energy as heat. Recently, Amarnath et al. (2012) reported that NPQ is the result
of the combination of charge transfer quenching in the minor antenna complexes of PSII as well as
quenching due to aggregated LHCII trimers.
In addition to dissipation through antenna quenching, the excess energy can also be lost through
xanthophyll-independent constitutive energy quenching (Hendrickson et al. 2004) (Figure 15.2).
Although the molecular mechanism of constitutive quenching remains equivocal, recent evidence
Fluorescence
Photosystem II
Lhcb1+2+3
S
Lhcb4+5+6 N M
QA L
Fe 2+ Q H X
K B Carbon
Electron transport Biomass
Lhcb1+2+3
D(D2) A(D1) synthesis

J
E Pheo
559
F
qP
Cyt b
Chl Chl
P680 Y
Y D
C(CP43)
B(CP47)
Z
P e– O
2Mn 2Mn
I
s
los
Q R
2H2 O +
at
O2 + 4H
Constitutive
He
quenching
NPQ
FIGURE 15.2 Photosynthetic conversion of solar energy to biomass. The light-harvesting antenna complex
absorbs the light energy and becomes excited. The excitation energy resulting from light absorption is basi-
cally used in the photochemistry initiating linear electron transport, the process known as photochemical
quenching (qP), and finally used in CO2 assimilation and growth. The irradiance, that is in excess of which is
used for CO2 assimilation and growth, is dissipated as heat through nonphotochemical quenching (NPQ) in
the light-harvesting complex and through constitutive energy quenching in PSII reaction centers.
indicates that it is associated with reaction center quenching (Ivanov et al. 2008). This involves
modulation of the redox potential difference between QA, the primary electron acceptor quinone of
PSII and QB, and the secondary electron acceptor quinone of PSII such that the redox potential of
QA mirrors that of QB. In the model cyanobacterium, Synechococcus sp. PCC 7942, the modulation
in the relative redox states of QA and QB is governed by the transient exchange of the D1:1 form
of the PSII reaction center with the D1:2 form (Sane et al. 2002). This increases the probability of
charge recombination between reduced QA and QB on the acceptor side of PSII with the positively
charged S-states on the donor side of PSII resulting in the nonphotochemical release of energy
(Ivanov et al. 2008; Sane et al. 2012). Although eukaryotes also exhibit reaction center quench-
ing (Ivanov et al. 2008), these photoautotrophs have only one form of the gene (psbA) encoding the
D1 reaction center polypeptide. Consequently, the mechanism by which the relative redox states of
QA and QB are modulated in eukaryotes remains unknown.
The discussion provided earlier illustrates that photoautotrophs exhibit several mechanisms
to protect themselves from excess irradiance by decreasing the photochemical efficiency of PSII.
Although this decrease in energy utilization efficiency reduces the efficiency of CO2 assimilation
and decreased biomass production, these photoprotective mechanisms are essential for the sur-
vival of myriad environmental stresses such as low and high temperatures and water stress (Sane
et al. 2012). Even though inhibition of these photoprotective mechanisms would increase energy
conversion efficiency in the very short term, this is simply not an option for long-term survival of
plants exposed to environmental conditions that fluctuate on an hourly, daily, and annual basis.
Photoprotection is essential for plant survival.
15.3 PHOTOSYNTHETIC PERFORMANCE AT LOW TEMPERATURE

15.3.1 Sensing Low Temperature
Molecular, biochemical, and physiological analyses of cold acclimation and freezing tolerance
have been examined in an array of northern temperate plant species such as Arabidopsis thaliana
(Seki et al. 2001; Tamminen et al. 2001; Thomashow 2001, 2010; Stitt and Hurry 2002; Benedict
et al. 2006; Yamaguchi-Shinozaki and Shinozaki 2006), wheat and rye (Sarhan et al. 1997;
Danyluk et al. 1998; Ndong et al. 2002; Griffith and Yaish 2004), alfalfa (Monroy and Dhindsa
1995; Monroy et al. 1998), and Brassica napus (Hurry et al. 1995; Savitch et al. 2005) as well
as the Antarctic plant species Colobanthus quitensis (Kunth) Bartl and Deschampsia antarctica
(Bravo et al. 2001; Bravo and Griffith 2005). Historically, research was focused on changes in
the physical structure of the cell membrane as a primary sensor involved in acclimation to low
temperature (Levitt 1980; Murata and Los 1997; Chinnusammy et al. 2007). Low-temperature-
induced increase in cell membrane viscosity has been shown to activate two-component histidine
kinases, which stimulate the expression of specific fatty acid desaturases involved in the modula-
tion of membrane fluidity in cyanobacteria (Los and Murata 2002; Xin 2002). Modification of the
physical properties of the cell membranes also activates Ca2+ channels, which results in the accu-
mulation of Ca2+ in the cytosol. Such a low-temperature-induced Ca2+ signal occurs rapidly upon
exposure to low-temperature stress (Monroy et al. 1998; Plieth et al. 1999; Orvar et al. 2000).
Such accumulation of Ca2+ in the cytosol activates a protein kinase, which phosphorylates ICE1
(Inducer of CBF Expression1) (Chinnusamy et al. 2007). ICE1 is necessary to induce the expres-
sion of CBFs/DREBs (C-Repeat-Binding Transcription Factors/Dehydration Responsive Element
Binding Factors) that bind to the promoter region of CBF-regulons and initiate the expression
of CBF-regulons necessary to acquire freezing tolerance (Jaglo-Ottosen et al. 1998; Zarka et al.
2003; Gilmour et al. 2004; Benedict et al. 2006; Chinnusamy et al. 2007; Thomashow 2010;
Medina et al. 2011; Lee and Thomashow 2012). CBFs/DREBs are a family of transcriptional
activators (Liu et al. 1998; Kasuga et al. 1999; Gilmour et al. 2000; van Buskirk and Thomashow
2006; Chinnuswamy et al. 2007; Badawi et al. 2008; Lee and Thomashow 2012; Theocharis
et al. 2012) required for the induction of a suite of cold-regulated genes (COR) such as WCS120,
WCOR410, and WCS19 that enhance plant freezing tolerance (Sarhan et al. 1997).
All plants utilize sunlight as their primary source of energy. Consequently, all photoautotrophs
are predisposed to maintain a balance between the energy trapped by temperature-insensitive
photochemical reactions (energy source) and the energy utilized through temperature-dependent
metabolism, growth, and development (energy sinks) (Hüner et al. 1998, 2012a,b; Ensminger et al.
2006). An imbalance between energy trapped through photochemistry vs. energy either utilized
through biochemistry or dissipated through NPQ will occur whenever the rate at which the energy
absorbed through PSII and the rate at which electrons are injected into photosynthetic electron
transport exceed the rates of temperature-dependent nonphotochemical processes and metabolic
electron sink capacity. Such an imbalance is defined as excitation pressure, which is a measure of
the relative redox state of QA, the first stable quinone electron acceptor of PSII reaction centers and
reflects the modulation of the redox state of the plastoquinone pool and components of the inter-
system photosynthetic electron transport chain (Hüner et al. 1998, 2012a,b; Ensminger et al. 2006).
This can be detected in vivo as an accumulation of closed PSII reaction centers and quantified
by in vivo Chl a fluorescence as 1-qP (Dietz et al. 1985; Hüner et al. 1998) or 1-qL (Hendrickson
et al. 2004; Kramer et al., 2004; Baker 2008). Energy imbalance can be created by numerous envi-
ronmental stresses such as high light, low temperature, drought stress, and nutrient stress (Hüner
et al. 1998, 2012a,b; Ensminger et al. 2006). Adjustments in photosynthesis to balance the flow of
energy can either occur through an increase in the rate of energy utilization and storage via sink
processes such as respiration, N-assimilation, and ultimately growth and biomass production or by
increased rates of energy dissipation through photoprotective processes such as NPQ, which can
be considered to be energy “safety valves” (Demmig-Adams and Adams 1992, 1996). Thus, redox
energy imbalances detected through modulation of excitation pressure within the chloroplast and
the photosynthetic apparatus represent an extremely sensitive mechanism for all photoautotrophs
to sense changes in temperature. Thus, there does not appear to be a single low-temperature sensor
used by plants and other photoautrophs to detect and respond to changes in temperature. Rather,
we suggest that photoautrophs integrate the information generated by the primary low-temperature
sensors, which detect temperature-induced changes in membrane viscosity, cellular Ca2+ fluxes, and
chloroplast redox status in order to establish the cold-acclimated (CA) state.
15.3.2 Cold Acclimation Enhances Photosynthetic Performance

Recently, Anderson et al. (2012) discussed the issue of whether plant adaptation can keep pace with
the rate of climate change. In order to survive, plant populations must be able to either adapt to
hotter drier climate under elevated CO2 or shift their latitudinal distributions from warmer tropical
and subtropical areas where water may be limiting to more temperate regions with increased water
availability but cooler temperatures. The success of migrating populations will be, in part, due to
their inherent phenotypic plasticity and their ability to adjust to environmental stress combined
with elevated CO2. Wheat is second only to rice as the leading food source for human and animal
nutrition (FAO 2012). Crop yield stability required to maintain seed production and food security
for the growing human population under increasingly suboptimal climatic conditions will undoubt-
edly require greater dependence on crops grown in cooler temperate regions (Anderson et al. 2012;
Powell et al. 2012). Since cereals are among the most cultivated crops worldwide, the effect of low
temperature and elevated CO2 on their performance and productivity will have a major impact on
human nutrition worldwide.
Previous studies have reported that cold-tolerant cultivars and species such as winter wheat,
winter rye, barley, spinach, Brassica, and A. thaliana exhibit an increase in photosynthetic capacity
through upregulation of carbon metabolism during cold acclimation (Hurry and Hüner 1991; Hurry
et al. 1995, 2000; Hüner et al. 1998; Öquist and Hüner 2003; Dahal et al. 2012a,b). The upregulation
of carbon metabolism of cold-grown, cold-tolerant species appears to be associated with increased
expression and subsequent activities of CO2-fixing enzyme, Rubisco (Hurry et al. 1994; Strand
et al. 1999; Dahal et al. 2012a,b), as well as enhanced activities of the cytosolic sucrose biosynthetic
enzymes, cFBPase and SPS (Hurry et al. 1995; Strand et al. 1999; Savitch et al. 2000; Rapacz et al.
2008; Dahal et al. 2012a,b), in response to low growth temperature. In addition, winter cultivars of
wheat (Savitch et al. 2000; Leonardos et al. 2003) and A. thaliana (Stitt and Hurry 2002) enhance
sink capacity and concomitant sucrose export to the sinks during cold acclimation. In most cereals,
this sucrose is stored as fructans in the crown tissue as well as in the leaf vacuoles during cold accli-
mation (Pollock and Cairns 1991; Savitch et al. 2000). Consequently, cold acclimation of winter
wheat, winter rape (Hurry et al. 1995), and A. thaliana (Stitt and Hurry 2002) results in enhanced Pi
cycling and increased capacity for RuBP regeneration through increased utilization of phosphory-
lated intermediates. Furthermore, cold acclimation results in the suppression of photorespiration,
thus diverting ATP and NADPH from oxygenation to carboxylation in winter wheat (Savitch et al.
2000) and also increases water use efficiency by about twofold in winter wheat, rye and B. napus
(Leonardos et al. 2003; Dahal et al. 2012a,b). Consequently, it was suggested that cold acclimation
of winter wheat and rye leads to a feed-forward stimulation of photosynthetic carbon assimilation
through the global reprogramming of photosynthetic carbon metabolism (Hurry et al. 1994, 1995;
Strand et al. 1999; Stitt and Hurry 2002). This is supported by a detailed, comparative metabolomics
study of the CA vs. nonacclimated (NA) A. thaliana (Gray and Heath 2005). Furthermore, there is
a strong positive correlation between the cold acclimation–induced stimulation in photosynthetic
capacity and the development of freezing tolerance measured as LT50 (Öquist et al. 1993), as well as
an increased resistance to low-temperature-induced photoinhibition in spinach (Krause 1988; Boese
and Hüner 1992), winter rye and winter wheat (Öquist et al. 1993; Gray et al. 1996; Pocock et al.
2001), A. thaliana (Savitch et al. 2001), and B. napus (Savitch et al. 2005).
These adjustments at the physiological, biochemical, and molecular levels are associated with
coordinated changes in leaf anatomy and plant phenotype (Hüner 1985; Gray et al. 1996; Strand et al.
1999; Dahal et al. 2012a,b) and induction of freezing tolerance in cold-tolerant species (Sarhan
et al. 1997; Pocock et al. 2001; Thomashow 2001; Savitch et al. 2005; Chinnuswamy et al. 2007;
Rapacz et al. 2008). With respect to phenotypic plasticity, cold acclimation of winter wheat, winter
rye, Brassica, and spinach as well as A. thaliana generally results in a compact dwarf phenotype,
altered leaf morphology and anatomy with increased leaf thickness and specific leaf weight
relative to their NA counterparts (Hüner et al. 1981; Hüner 1985; Boese and Hüner 1990; Strand
et al. 1999; Savitch et al. 2005; Gorsuch et al. 2010a,b; Dahal et al. 2012a,b). The increased leaf
thickness associated with the CA state can be accounted for by either increases in leaf mesophyll
cell size (Hüner et al. 1981; Gorsuch et al. 2010a) and/or increases in the number of palisade meso-
phyll layers (Boese and Hüner 1990; Dahal et al. 2012b). At the ultrastructural level, CA winter rye
and A. thaliana exhibit an apparent increase in cytoplasmic volume and an apparent decrease in
vacuolar volume (Hüner et al. 1984; Strand et al. 1999). This is accompanied by an increase in the
content of sucrose and other structural carbohydrates (Guy et al. 1992; Hurry et al. 1995; Strand
et al. 1999, 2003; Savitch et al. 2000; Gorsuch et al. 2010b). The dwarf phenotype of winter hardy
plants previously assumed to be regulated by low temperature is, in fact, regulated by excitation
pressure (Gray et al. 1997).
15.3.3 Enhancement of Energy Conversion Efficiency Is Cultivar Dependent

Cold acclimation–induced adjustments at the physiological, morphological, biochemical, and molec-
ular levels of winter wheat and B. napus are associated with enhanced rates of respiration and bio-
mass accumulation at either ambient or elevated CO2 levels compared to NA controls (Table 15.1).
This is translated into increased grain yield in CA vs. NA control plants (Table 15.2). To assess energy
conversion efficiency, one may compare the balance between the efficiency for the utilization of pho-
tosynthetically generated electrons for CO2 assimilation and the efficiency for energy dissipation
through NPQ (Dahal et al. 2012a). The former can be measured as the apparent number of photons
TABLE 15.1
Rates of Dark Respiration (Rdark), Specific Leaf Weight (SLW), and Total Dry Mass
for NA (20°C/16°C; 20) and CA (5°C/5°C; 5) Norstar Winter Wheat and Wild
Type (WT) and BnCBF17-Overexpressing (BnCBF17) Brassica napus Grown at
Either Ambient (380 μmol C mol−1) or Elevated (700 μmol C mol−1) CO2
Rdark SLW Total Dry Mass at Flag
Species/Cultivars (μmol C Evolved m−2 s−1) (g DM m−2 Leaf Area) Leaf Stage (g Plant−1)
Winter wheat
20/380 3.07 ± 0.22a 30 ± 3.6a 8.1 ± 0.9a
20/700 3.41 ± 0.30ab 38 ± 2.5a 12.3 ± 0.4b
5/380 3.88 ± 0.16b 72 ± 4.9b 11.7 ± 0.6b
5/700 3.97 ± 0.27b 77 ± 3.1b 16.6 ± 1.4c
Brassica napus
20/380 (WT) 2.19 ± 0.13a 27 ± 1.7a 4.3 ± 0.3a
20/700 (WT) 2.61 ± 0.20ab 36 ± 1.3b 5.2 ± 0.2b
20/380 (BnCBF17) 2.82 ± 0.10b 50 ± 3.5c 5.4 ± 0.2b
20/700 (BnCBF17) 3.18 ± 0.29b 61 ± 4.0cd 7.7 ± 0.7c
5/380 (WT) 3.02 ± 0.31b 63 ± 2.9d 5.7 ± 0.4b
5/700 (WT) 4.1 ± 0.26c 69 ± 3.2d 7.4 ± 0.3c
Note: Data represent the mean of nine plants from three different pots ± SE. Significant differences due to
growth CO2 or temperature within each species are indicated by the superscribed letters (P ≤ 0.05).
TABLE 15.2
Effects of Elevated CO2 on Grain Yield and Its Components in Winter
and Spring Wheat
Growth CO2 Effective Panicle Grain No. Grain Yield
Cultivars (μmol C mol−1) Tiller No. Plant−1 No. Plant−1 Panicle−1 (g Plant−1)
Norstar 380 15 ± 1.2ab 14 ± 1.3b 30 ± 1.8a 9.2 ± 0.7b
700 21 ± 1.2c 18 ± 0.5c 32 ± 1.1a 14.8 ± 1.2c
Katepwa 380 12 ± 0.6a 8 ± 0.7a 35 ± 1.3ab 6.6 ± 0.2a
700 17 ± 1.7bc 9 ± 1.1a 38 ± 1.9b 9.4 ± 0.5b
Note: The plants were grown at either ambient CO2 (380 μmol C mol−1) or elevated CO2 (700 μmol C
mol−1). Katepwa was grown at 20°C/16°C until grain maturity while Norstar was first grown at
5°C/5°C for 75 days and transferred to 20°C/16°C until grain maturity. Data represent the mean of
nine plants from three different pots ± SE. Significant differences due to growth CO2 within each
cultivar are indicated by the superscribed letters (P ≤ 0.05).
required to close 50% of PSII reaction centers and the latter as the apparent number of photons
required to induce one unit of NPQ (Dahal et al. 2012a). An increased efficiency in the utilization of
reductant to assimilate CO2 will be reflected in an increase in the quantum requirement, that is, an
increase in the number of photons required to close a PSII reaction center. Concomitantly, this should
be associated with an increased number of photons required to induce photoprotection through NPQ,
which reflects a decreased dependence on photoprotection through NPQ. This explains why CA
plants are more resistant to chronic photoinhibition and less dependent on NPQ for photoprotection
(Hüner et al. 1993, 1998).
The apparent quantum requirements to close 50% of the PSII reaction centers and the apparent
quantum requirements to induce one unit of NPQ under ambient CO2 conditions were about 35% and
50% higher in CA Norstar winter wheat relative to its NA counterpart (Dahal 2012; Dahal et al. 2012b).
This is coupled to a 45% increase in dry biomass accumulation of CA vs. NA Norstar winter wheat
(Table 15.1) and a 40% increase in grain yield of CA Norstar winter wheat relative to NA Katepwa
spring wheat at ambient CO2 (Table 15.2). Comparable results for cold acclimation–induced increases
in biomass and concomitant increases in the apparent quantum requirements for PSII closure and NPQ
induction under ambient CO2 conditions were reported for Musketeer winter rye (Dahal 2012, Dahal
et al. 2012b). This means that in the CA state, the winter cereals are able to trap a greater percentage
of the incident radiation through photochemistry and convert this energy into biomass and seed pro-
duction through enhanced rates of CO2 assimilation. Concomitantly, the CA winter cereals dissipate
less energy as heat through NPQ because of the enhanced capacity to utilize and subsequently store
absorbed energy as biomass. Consequently, this results in an increase in energy conversion efficiency.
In contrast to winter cereals, CA spring cereals such as SR4A rye and Katepwa wheat exhibit an
inhibition of light-saturated rates of CO2 assimilation compared to NA controls. This results in a
significant decrease in the apparent quantum requirements for PSII closure as well as NPQ induc-
tion during cold acclimation at ambient CO2 (Dahal 2012, Dahal et al. 2012b). Thus, the inhibition of
CO2 assimilation in CA spring cultivars results in increased sensitivity to the closure of PSII reaction
centers. To compensate, fewer absorbed photons are required in CA than in NA spring cultivars to
activate NPQ, which dissipates the energy that cannot be used in the assimilation of CO2. Thus, spring
cultivars are able to grow and survive under cold acclimation conditions through NPQ-mediated pho-
toprotection at a cost of decreased energy conversion efficiency and lower biomass accumulation.
15.4 CBF TRANSCRIPTION FACTORS AND ENERGY CONVERSION EFFICIENCY

What regulates the increased energy conversion efficiency observed for CA winter cereals?
Compared to NA wild type, the BnCBF17-overexpresser (BnCBF17-OE) grown at 20°C mimicked
CA wild-type B. napus with respect to increased biomass and specific leaf weight (Table 15.1), com-
pact dwarf phenotype, increased light-saturated rates of photosynthesis and photosynthetic electron
transport, improved water use efficiency, and enhanced levels of key Calvin cycle enzymes and
components of photosynthetic electron transport at ambient CO2 (Dahal et al. 2012a,b). BnCBF17-
overexpression as well as cold acclimation of wild-type B. napus increased the dry biomass by
25%–35% relative to NA wild type at ambient CO2 (Table 15.1), which were associated with
increased amount of key regulatory photosynthetic enzymes such as stromal-localized Rubisco
(rbcL) and cytosolic FBPase (cFBPase) important in regulating sucrose biosynthesis (Dahal et al.
2012a). These results for the BnCBF17-OE and CA wild-type B. napus are comparable to the
enhanced photosynthetic performance reported for CA winter wheat and winter rye (Dahal et al.
2012b) and consistent with the report of Savitch et al. (2005), who reported that the BnCBF17-OE
exhibits significant enhancement in the gene expression as well as enzyme activities of Rubisco,
SPS, and cFBPase.
Concomitantly, this was associated with increased quantum requirements to close PSII reaction
centers and to induce energy dissipation by NPQ in CA wild type and the BnCBF17-OE relative to
NA wild type (Dahal et al. 2012a,b). Furthermore, CO2 response curves for excitation pressure and
NPQ indicated that both CA wild type and the BnCBF-OE exhibited significantly lower proportions
of closed PSII reaction centers as well as lower NPQ as a function of CO2 relative to NA wild-type
B. napus (Dahal et al. 2012a). This indicates that compared to NA wild type, BnCBF17-OE as well
as CA wild-type B. napus exhibits an enhanced capacity to utilize absorbed light energy and con-
vert fixed CO2 into biomass with a concomitant decreased reliance on NPQ to dissipate absorbed
energy for photoprotection. Therefore, we suggest that CBFs/DREBs are critically important factors
not only in the regulation of plant freezing tolerance and phenotypic plasticity (elongated vs. dwarf
phenotype) but also in the governance of photosynthetic energy conversion efficiency into biomass.
15.5 PHOTOSYNTHETIC RESPONSES TO ELEVATED CO2

15.5.1 Short-Term Response of Photosynthesis to Elevated CO2
Plants respond directly to increased CO2 concentrations through photosynthesis (Sage et al. 1989;
Long and Drake 1992; Long et al. 2004). In the short term (minutes–hours), the photosynthetic rates
of C3 plants are stimulated following shifts to an elevated CO2 (Cheng et al. 1998; Long et al. 2004;
Ainsworth and Rogers 2007; Dahal et al. 2012c). This photosynthetic stimulation following a short-
term shift to elevated CO2 is attributed to two reasons: (1) Rubisco is CO2 substrate-limited at ambi-
ent CO2 as indicated by the K m (CO2) for Rubisco, which is close to the current atmospheric CO2
concentration of 380 μmol CO2 mol−1 (Long et al. 2004; Tcherkez et al. 2006). Thus, an immediate
increase in carboxylation velocity is expected by increased CO2 substrate availability. (2) Elevated
CO2 competitively inhibits photorespiratory CO2 drain because CO2 is a competitive inhibitor of
the oxygenation of RuBP by Rubisco (Long et al. 2004). Consequently, the assimilatory power,
NADPH and ATP, is used preferentially in the carboxylation reaction to fix CO2 as opposed to the
oxygenation reaction. However, the observed stimulation of photosynthetic rates upon a short-term
shift to elevated CO2 is dependent upon the cultivar and plant growth temperature. Although short-
term exposure to elevated CO2 increased the CO2 assimilation rates of spring and winter cereals by
55%–65% in the NA state, exposure to short-term elevated CO2 had minimal effects on the CO2
assimilation rates of CA spring wheat and spring rye (Dahal et al. 2012c). It was concluded that
although cold acclimation suppresses the short-term CO2 stimulation of photosynthesis in spring
cereals, cold acclimation appears to further increase the CO2 stimulation of photosynthetic rates of
winter wheat and winter rye (Dahal et al. 2012c).
15.5.2 Long-Term Response of Photosynthesis to Elevated CO2

Plant species have developed several acclimatory strategies to survive the consequences of
increased CO2 concentrations. The mechanisms involved in acclimation to elevated CO2 range
from modifications at the molecular and biochemical levels to modifications at the physiological
and morphological levels. Feedback inhibition of photosynthesis induced by long-term growth and
development at elevated CO2 has been reported in a wide range of plant species (Long et al. 2004;
Ainsworth and Rogers 2007; Dahal et al. 2012a; Smith and Dukes 2013; Thilakarathne et al. 2013).
In contrast to the short-term shift, long-term growth and development of plants at elevated CO2
may lead to an inhibition of photosynthetic capacity due to accumulation of nonstructural carbo-
hydrates in the cytosol (Stitt and Quick 1989; Stitt 1991; Long and Drake 1992; Drake et al. 1997).
The increased carbon assimilation resulting from initial stimulation of rates of photosynthesis by
elevated CO2 results in the accumulation of sucrose in the source leaf. This leads to feedback inhi-
bition of photosynthesis due to Pi regeneration limitation in the short term and downregulation of
the expression and activities of key regulatory photosynthetic enzymes such as Rubisco, cFBPase,
and SPS in the long-term (Sharkey and Vanderveer 1989; Stitt and Quick 1989; Arp 1991; Stitt
1991; Drake et al. 1997; Pego et al. 2000). Plants exhibiting feedback-limited photosynthesis may
not benefit in terms of biomass and seed yield from the future increase in atmospheric CO2. It has
been suggested that sensitivity to feedback inhibition of rates of photosynthesis at elevated CO2 is
predominantly determined by sink strength. Any factors that limit sink capacity lead to a greater
rate of feedback inhibition of photosynthesis at elevated CO2.
Our recent study (Dahal et al. 2012a) indicates that although wild-type B. napus grown at 20°C
exhibits feedback inhibition of photosynthesis upon growth at elevated CO2, wild-type B. napus
was rescued from feedback inhibition of photosynthesis by either overexpressing BnCBF17 or by
cold acclimation (Dahal et al. 2012a). In contrast to B. napus, neither winter nor spring wheat and
rye exhibited feedback inhibition of photosynthesis in response to growth at elevated CO2 in either
the NA or CA state (Dahal 2012). We suggest that the elevated CO2-induced differential sensitivity
to feedback-limited photosynthesis may be associated with differential stimulation of sink capacity
as reflected in differences in biomass accumulation in cereals vs. Brassica in response to growth at

elevated CO2 (Table 15.1). For instance, elevated CO2 appears to enhance the dry matter accumula-
tion by 52% in Norstar winter wheat (Table 15.1) and by 75% in rye (Dahal 2012) as opposed to 21%
in wild-type B. napus in the NA state (Table 15.1). This differential sensitivity to feedback-limited
photosynthesis between cereals and B. napus may be accounted for, in part, by the fact that cereals
are natural fructan accumulators and, consequently, are able to partition carbon to fructans in addi-
tion to sucrose and starch (Pollock and Cairns 1991; Cairns et al. 2000; Savitch et al. 2000, Li et al.
2013). This additional carbon sink may contribute to enhanced sink capacity and biomass accu-
mulation in the cereals, which may be translated into a decreased sensitivity to feedback-limited
photosynthesis under elevated CO2 (Cairns et al. 2000; Xue et al. 2013). Thus, we suggest that
the sensitivity to feedback-limited photosynthesis induced by growth at elevated CO2 is species
and cultivar dependent, which is consistent with the recent results of Thilakarathne et al. (2013).
Furthermore, overexpression of BnCBF17 appears to play a crucial role in reducing the sensitivity
of photosynthesis to feedback inhibition during the long-term growth of B. napus at elevated CO2.
BnCBF17-OE as well as CA wild-type B. napus exhibits about 45% higher biomass accumula-
tion compared to NA wild type at elevated CO2 (Table 15.1). Concomitantly, BnCBF17-OE and CA
wild-type B. napus exhibit a lower excitation pressure for a given irradiance and a given CO2 con-
centration, and thus, a greater capacity to keep QA oxidized as compared to NA wild-type B. napus
(Dahal et al. 2012a). This enhanced capacity to utilize absorbed light energy and convert it to
biomass is reflected in decreased reliance on NPQ to dissipate absorbed energy for photoprotec-
tion and an increased quantum requirement to close PSII reaction centers during long-term growth
and development under elevated CO2 conditions (Dahal et al. 2012a). This is coupled to a lower
Ci requirement to keep PSII reaction centers open and a lower propensity to dissipate absorbed
energy through NPQ under CO2-saturated conditions (Dahal et al. 2012a,b). Thus, overexpression
of BnCBF17 enhances photosynthetic energy conversion efficiency into biomass as observed upon
cold acclimation of B. napus not only at ambient CO2 but also at elevated CO2. This is consistent
with the results for CA winter rye and winter wheat (Dahal 2012). In all cases, the increased energy
use efficiency in rye, wheat, and B. napus at either ambient or elevated CO2 was associated with a
change in plant morphology characterized by a dwarf phenotype and increased specific leaf weight
(Table 15.1; Dahal 2012; Dahal et al. 2012a,b). Recently, Thilakarathne et al. (2013) reported that an
increase in leaf mass per unit area was important in determining the grain yield response of wheat
cultivars during growth at elevated CO2.
15.5.3 Long-Term Exposure to Elevated CO2 Suppresses Biotic Stress Genes

Elevated CO2 along with high temperature changes the leaf chemistry as indicated by increases in
C/N and C/P ratios in plants grown at elevated CO2 (Sardans and Penuelas 2012). This may favor
infestation by certain insects and pathogens (Zavala et al. 2008; Oehme et al. 2013). For instance,
elevated CO2-induced stimulation of photosynthetic rates increases the leaf carbohydrate content
promoting an increased growth of Colletotrichum gloeosporioides (Ziska et al. 2011). Since ele-
vated CO2 increases the C/N ratio and the chewing insects are mostly limited by N content in their
diet, they must consume more leaf tissue to fulfill their nutritional demands (DeLucia et al. 2012).
In addition, because elevated CO2 increases the plant biomass at the whole plant level, increasing
the host biomass available for infection by plant diseases would have a negative impact on crop
productivity in a high-CO2 world (Ziska et al. 2011).
Using wheat Affymetrix microarrays, we performed a transcriptome analysis of winter wheat
(cv Norstar) leaves grown and developed at either control, NA conditions (20°C/16°C, NA), or CA
conditions (5°C/5°C, CA) at either ambient or elevated CO2 (Kane 2012). Growth and development
of NA Norstar at elevated CO2 resulted in the downregulation of 121 genes associated with biotic
stress. These downregulated genes were generally associated with pathogen-related (PR) or sys-
temic acquired resistance genes involved with cell wall and antimicrobial activities that help plants
avoid the pathogen attacks. In addition, several down-regulated transcripts encoding protein kinases
and WRKY transcription factors were identified in NA Norstar grown at elevated CO2 (Kane 2012).
In contrast to NA Norstar, CA Norstar exhibited minimal downregulation of the same set of genes
associated with biotic stress (Kane 2012).
CO2-induced downregulation of transcripts associated with these defense-related genes indi-
cates that NA Norstar winter wheat grown at elevated CO2 may be vulnerable to attack by insect,
pest, and diseases compared to their CA counterparts (Kane 2012). The cold acclimation–induced
protection of winter hardy cereals, such as wheat and rye against the biotic stress, in part, through
the low-temperature induction of PR proteins, has been established (Griffith and Yaish 2004) and
is associated with adjustment at the molecular, physiological, and biochemical levels during cold
acclimation (Houde et al. 1992; NDong et al. 2002; Badawi et al. 2007, 2008). Since CBF expression
governs the establishment of the CA state, we suggest that overexpression of CBFs will not only
enhance energy conversion efficiency, biomass accumulation, and crop yield but also concomitantly
enhance plant resistance to biotic stress under conditions of elevated CO2 associated with climate
change. However, this requires experimental verification.
15.6 SUMMARY, CONCLUSIONS, AND FUTURE PERSPECTIVES

Atmospheric CO2 is projected to rise from present 380 μmol C mol−1 to approx. 700 μmol C mol−1,
which may likely be coupled with increases in the average global temperature, the frequency of severe
drought events, as well as the frequency and extent of crop failures due to disease and pest infestations
by the end of this century. These changes in atmospheric CO2 associated with global warming may
have considerable effects on photosynthetic performance of plants and therefore on plant biomass and
grain yield. This has raised global concerns regarding food security, which highlights an urgent need
to double the yield and productivity of major food crops such as rice and wheat to feed the increasing
population over the next 50 years. In order to achieve this rising food demand, we need to identify spe-
cific characteristics to develop and exploit new crop cultivars with increased biomass and seed yield
potential under projected future suboptimal conditions for crop production.
Research on CBF overexpression in plants has been primarily focused on freezing tolerance at the
molecular level (Jaglo-Ottosen et al. 1998; Zhu et al. 2007; Lee and Thomashow 2012; Theocharis
et al. 2012). Although increasing energy conversion efficiency is critical for plant biomass and seed
yield, few studies have attempted to integrate CBF overexpression with photosynthetic performance
and energy conversion efficiency. We demonstrate that CBFs appear to play a key role not only
in enhancing plant resistance to abiotic and biotic stresses and phenotypic plasticity, but also in
increasing photosynthetic energy conversion to biomass and grain yield. Brassica BnCBF17 tran-
scription factor has advanced our understanding of CBF as a central component, which plays a vital
role in producing superior hardy crops that can not only withstand multiple abiotic stresses, but also
exhibit outstanding photosynthetic performance and energy conversion efficiency in a high-CO2
environment. Therefore, targeting the CBF pathway in major crop species may be a novel approach
to improve crop yield and productivity, which may assist in meeting the nutritional demands of
a rising population. We suggest that this approach may provide important insights into potential
molecular approaches focused on the maintenance or even the enhancement of plant productivity
under suboptimal growth conditions associated with elevated CO2, global warming, and climate
changes. In addition, this may aid in the identification of novel crop cultivars that can sustain stress
or even perform better under changing environmental conditions. The results obtained from CA
overwintering plants and BnCBF17-overexpressing Brassica have provided new insights into the
central role of the CBF transcription factor(s) and the identification of novel characteristics in wheat,
rye, and B. napus that offer considerable potential for breeding programs aimed at enhancing or
monitoring crop productivity in response to global warming and climate change.
Although the molecular mechanism(s) by which CBFs regulate freezing tolerance through the
expression of COR genes has been and continues to be an active area of research (Thomashow 2010;
Lee and Thomashow 2012; Theocharis et al. 2012), its broader role in governing phenotypic plastic-
ity, photosynthetic performance, and energy conversion efficiency is still in its infancy. Therefore,
in our opinion, future research focused on elucidation of the mechanism by which CBFs regulate
phenotypic plasticity and photosynthetic energy conversion efficiency should be a priority. For
example, plant CBFs represent a family of transcription factors. Therefore, does each CBF exhibit
a specific role in the regulation of photosynthetic energy conversion efficiency? Since CBFs affect
plant phenotype, this family of transcription factors must interact with sensing/signaling pathways
associated with various plant growth regulators. Overexpression of CBFs induces a CA state and a
dwarf phenotype typically in the absence of exposure to low temperature. Cold acclimation is typi-
cally associated with the developmental process of vernalization (Sung and Amasino 2005; Trevaskis
et al. 2007; Trevaskis 2010). Thus, CBF expression must in some way be connected to the regulation
of vernalization genes. We conclude that CBF transcription factors may be a master regulator that
governs processes involved in plant growth and development, freezing tolerance, photosynthetic per-
formance, energy conversion efficiency, and plant biomass production. Clearly, a systems approach
will be necessary to unravel the interacting network of genes, proteins, metabolic pathways, and plant
growth regulators that govern such a complex biological response to a constantly changing environ-
ment with respect to abiotic and biotic stresses associated with global warming and climate change.
ACKNOWLEDGMENTS
This work was supported, in part, by the Natural Sciences and Engineering Research Council
(NSERC) and industrial and government partners, through the Green Crop Research Network
(GCN). N. P. A. H., F. S., and B. G. also acknowledge research support through their individual
NSERC Discovery grants, Canada Foundation for Innovation (CFI) grants, and Canada Research
Chair (CRC) grants. JS and LVS acknowledge the Agriculture and Agri-Food Canada for their
research grants. Figure 15.2 was provided by Dr. Alexander Ivanov, Biotron Experimental Climate
Change Research Centre, the University of Western Ontario, and modified by author.
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16 Physiology of Crop
Productivity in Cold Climate
Galina N. Tabalenkova and Tamara K. Golovko
CONTENTS
16.1 Introduction........................................................................................................................... 333
16.2 Physiological and Biochemical Basis of Plant Production Process....................................... 333
16.3 Production Process of Some Crops in the Northern Conditions........................................... 335
16.3.1 Potato Crop................................................................................................................ 335
16.3.2 Cereal Crops: Barley and Oat.................................................................................... 337
16.3.3 Annual and Perennial Forage Herbs.......................................................................... 338
16.4 Summary and Conclusion......................................................................................................340
References.......................................................................................................................................340
16.1 INTRODUCTION
Crop productivity depends on genotypic and physiological–biochemical properties of plant species
and varieties and their interaction with the environment. The genetically determined growth and
development processes provide a basis of crop productivity. Substrate and energy provision of plant
growth depend on photosynthesis, respiration, transport, and distribution of assimilates. Especially
important are regulatory interactions between the functions that are realized through the sink–
source system of the whole plant.
The up-to-date principles of highly productive agrocenosis formation are based upon the maxi-
mum efficient usage of environmental resources (light, water, mineral elements, etc.) and realization
of the culture productivity potential. For better understanding, the production process of agricul-
tural plants grown in particular agroclimatic conditions, physiological interrelations between pho-
tosynthetic parameters, metabolism, and biomass accumulation are to be studied.
The next sections will be devoted to consider the physiological–biochemical aspects of produc-
tivity formation and the production process peculiarities of the main agricultural plants grown in
the Northeast of the Europe.
16.2 PHYSIOLOGICAL AND BIOCHEMICAL BASIS

OF PLANT PRODUCTION PROCESS
For the first time, theoretical conception on the photosynthetic plant productivity was formulated in
the mid of the former century (Nichiporovich, 1956, 1988). It was shown that the agrocenosis pho-
tosynthetic activity depends on leaf area, its building-up rate, and time length of leaf function in the
season. The leaf photosynthetic potential (LPHP) is an integral index, which accounts for leaf area
value and duration of leaf photosynthetic activity during vegetation period. An important indicator
of phytocenosis ability to absorb light effectively is a leaf area per phytocenosis area unit, or the
so-called leaf area index (LAI, m2/m2). The LAI value depends on the thickness of sowing. Crops
with the LAI of about 4 m2/m2 are able to assimilate near 90%–95% of photosynthetic active radia-
tion (PAR). The LAI value of highly productive crop cultures equals 5–6 m2/m2 (Dwyer et al., 1991;
333
Bondada, 2001). For most agrocenosis, the optimal LAI is 4–5 m2/m2. If the LAI increases it may
have a negative effect on yield in consequence of the increase in cenotic interaction between plants
and of strong competition for light, moisture, and mineral elements. The costs on plant biomass
maintenance also increase.
To assimilate more light, plants tend to increase a leaf mass per plant mass (LWR, g/g) or a leaf
area per leaf mass (specific leaf area, SLA, m2/g). The SLA value varies within 0.01–0.06 m2/g for
agricultural plants. The SLA is most dependent on light intensity. The SLA is an integral structural
instrument with the help of which plant regulates its architecture in phytocenoses for a better use
of PAR.
Specific leaf mass (SLM) is an index characterizing the efficiency of assimilate use to form a leaf
surface unit. The SLM is the amount of leaf mass per unit leaf area, g/m2. The SLM value consider-
ably changes depending on plant species and growing conditions (Potter and Jones, 1977; Muller
and Moorby, 1990; Lambers and Poorter, 1992; Hunt and Cornelissen, 1997). It varies within the
range of 40–100 g/m2 and correlates with the net photosynthesis (Pn) rate and phytomass accumula-
tion (Bedenko and Kolomeichenko, 2005).
Crop productivity depends on photosynthetic assimilated carbon allocation. Sink–source trophic
relations unify all parts and organs of plant into one system (Mokronosov, 1983). Studying the
regulation mechanisms of sink–source relations is necessary for understanding the crop formation
mode and for finding the ways of the crop management. Geiger (1969), Wareing and Patrick (1973),
Kursanov (1976), and Mokronosov (1982, 1983) contributed much to the development of sink–source
conception. According to this conception, photosynthetic intensity and crop productivity are depen-
dent on epigenetic factors such as growth (de novo formation of structural biomass), assimilate accu-
mulation, and reserve formation processes. Many ecological factors affect photosynthesis through
their impact on plant demand for carbohydrate. In general, processes that increase carbohydrate
demand cause an increase in photosynthetic rate, whereas factors that reduce carbohydrate demand
reduce photosynthesis. The environmental conditions can significantly modify epigenetic regula-
tion of photosynthesis by limiting the growth, transport, and distribution of assimilates. Increase
or decrease in metabolic activity centers (growth or storage compound deposition) leads to changes
in carbon requirement and, consequently, influences the photosynthesis (Mokronosov, 1983, 1988).
Assimilates’ transport processes are normally more sensible to temperature than the photosynthetic
carbon metabolism. Suppression of carbohydrate transport by low temperature is related to the
inhibition of phloem loading in source leaves and to a decrease in utilization rate of photosynthetic
products in sink organs (Gamalei, 2004). Low demand for assimilates can lead to starch accumula-
tion in chloroplasts and inhibition of the photosynthesis by the law of feedback (Mokronosov, 1983;
Kursanov, 1984; Morcuende et al., 1996; Kochar and Pollock, 2003). Plants store carbon in leaves,
stems, roots, or specialized storage organs. Annual crops allocate relatively little of their assimilates
to storage as they are short-lived plants. During seed formation and filling, carbohydrate reserves in
stem of cereal plants are remobilized and transported into seeds. In unfavorable conditions for pho-
tosynthetic up to 50% of wheat grain mass can form by stem reserves reutilization (Kumakov et al.,
1983; Bedenko and Kolomeichenko, 2005). Competitive interrelations between acceptors, which
become apparent in particular plant organs (shoot–roots) or processes (growth–storage), are also
to be considered (Wardlaw, 1990; Aufhammer and Hannapfel, 1991; Ho, 1992; Ganeshaiah et al.,
1995; Munier-Jolain et al., 1998; Murchie et al., 2002; Kiriziy, 2004). In general, sink–source plant
system is built up by the principle of balance between the demand from the sink and supply from the
source. When it is broken, changes in photosynthetic activity and redirection of carbohydrate fluxes
usually maintain the supply of acceptors by assimilates.
Plants exist in the environment where conditions greatly affect the life activities. The sensibility of
growth processes to unfavorable conditions is higher than the process of CO2 assimilation. Cell divi-
sion and elongation inhibition during the leaf growth decrease the area of assimilation apparatus. This,
in turn, lowers the cenosis productivity (Guliaev et al., 1989). The productivity of genotypes resistible
to unfavorable environmental influences is more stable than that of nonresistible ones (Evans, 1993).
Physiology of Crop Productivity in Cold Climate 335
Photoperiod is one of the environmental factors influencing upon the productivity of agricultural
plants. The length of the photoperiod affects the flowering response of long-day and short-day crops,
as well as aspects of vegetative plant development that are not directly related to reproduction.
The numerous agricultural plants become neutral during selection, but their productivity depends
on photoperiod because it determines the daily duration of the positive CO2–gas exchange crop. In
the middle taiga subzone of Northeast of the Europe, plants assimilate on 3–5 h longer than in mod-
erate latitudes in June–July. A long day on the north partly compensates a short period, favorable
for plant vegetation.
Photoperiod affects the ratio of phytohormones in plants; under long-day the gibberellins activity
increases (Rallton and Wareing, 1973; Metrger, 1990; Olsen et al., 1995; Markarov et al., 2001). The
high content of gibberellins enhances the growth of potato top and retards the biomass accumula-
tion in tubers (Tabalenkova et al., 1998; Konstantinova et al., 1999; Markaropv et al., 1993, 2001).
Photoperiod influences the carbohydrate synthesis in leaves and transport to sink organs (Wang and
Ouebedeaux, 1997), and carbon inclusion into plant biomass, and, consequently it influences the
plant productivity (Talanov et al., 2005).
16.3 PRODUCTION PROCESS OF SOME CROPS

IN THE NORTHERN CONDITIONS
Plant cultivation in the taiga zone of European North is limited by short vegetation period, poor
podzolic soils, and cold damp climate with an annual precipitation of 600–800 mm. The mean
monthly temperature in July is +16.8°C. Period with air temperatures above +15°C lasts for 47 days
on average. Frost-free period takes nearly 100 days. Numerous days with rainy and cloudy weather
negatively affect plant development, especially the ripening stage. In general, vegetation period is
characterized by favorable for plant growth light conditions. In June–July, the length of daylight
hours is above 18, and total daylight hours from May till September equal 2518. Bioclimatic condi-
tions of the region are appropriate for cultivating potato, some cereals, and fodder legumes.
16.3.1 Potato Crop
Potato is a crop with high potential productivity and ability to adapt to a wide spectrum of climatic
conditions. Leaf appearance intensity and leaf growth rate determine the pattern of agrocenosis
assimilation surface formation. In the north, the linear growth rate of middle-layer leaves (7–12th
leaves from the shoot base) of the short season varieties of potatoes was on average 2 cm/day
(Markarov et al., 2001). Lifespan of these leaves varies within 60–70 days. Upper-layer leaves
(16–17th leaves) grew slower (0.7–1.0 cm/day) and their areas were less.
In the flowering stage (first half of July), potato plant leaf area amounted to 50% of finite value.
Leaf area reached its highest value (0.8–1.3 m2/plant) 2–3 weeks before harvesting (end August–
early September). The LAI value of potato agrocenosis varied from 3 to 5 m2/m2, depending on
cultivar and weather conditions of a season. Slow growth of leaf surface in the first half of vegeta-
tion (June) is determined by deficiency of heat. Late completion of leaf surface formation of potato
agrocenosis is one of the major factors limiting crop-producing power of this culture in the north.
The contribution of the organs in the whole plant biomass changed during potato plant develop-
ment. Plant biomass increased through the fast growth of the leaves and stems during June–July,
and due to tuber formation in August. Depending on seasonal weather conditions before harvest-
ing, the part of leaves in the total plant biomass amounted to 10%–30%, stems 10%–20%, and
tubers 57%–70%. As compared to early ripening varieties, late-ripening varieties of potato crops
are considered to be more productive because they have higher LPHP value. In cold climate, this
advantage of late-ripening varieties cannot be realized due to short vegetation period. In the north,
the potato productivity is dependent more on the weather conditions of vegetation period than on
the ripening rate of variety.
Potato crop leaves are characterized by high photosynthetic activity. The photosynthetic rate of
middle-layer leaves amounted to 0.4 mkmol CO2/g DW/s. They made the largest contribution to the
tuber yield formation (Markarov et al., 2001).
Potato leaves have a wide temperature optimum for Pn, 15°C–25°C (Winkler, 1971; Ku et al., 1977;
Dwelle, 1975; Dwelle et al., 1981; Shvetcova, 1987). The light saturation of leaves in cenosis occurs
at fluence rates of 10%–15% of full sunlight. At light intensity of 180–270 mkmol/m2 s PAR and leaf
temperature of 18°C–20°C, photosynthetic rate was equal to 9–13 mkmol CO2/m2/s (Shvetcova, 1987).
At low temperature (7°C), it did not exceed 4–6 mkmol CO2/m2/s. At a temperature of 10°C–15°C,
potato cultivars that are adapted to northern conditions can photosynthesize with an average rate of
6–9 mkmol CO2/m2/s and so provide for daily assimilation equal to 26 g CO2/m2 of leaf area.
In the northern conditions, the highest contents of chlorophyll (1.7 mg/g FW) and carotenoids
(0.4 mg/g) were observed during the budding stage (Kurenkova, 1998). Chlorophyll content in
potato leaves depended on a potato variety. Early and middle–early varieties accumulated less
by 25% pigments in comparison to middle–late varieties. Potato agrocenoses are characterized
by comparatively high chlorophyll productivity values, over 200 kg carbon/kg chlorophyll for the
vegetation period.
High plant productivity is attained at an optimal ratio between photosynthesis and respiration of
the whole plant (Golovko, 1985). At 20°C, the maximal respiration rate (0.05 mkmol CO2/g DW/s)
was demonstrated by young leaves of potato plant (Golovko, 1990, 1999). When the leaves have
40%–50% of finite area, they became an exporter of assimilates, and its respiration rate decreased
by two to three times. The respiration rate in mature leaves on average amounted to 0.02 mkmol
CO2/g DW/s. Stem and root respiration rates were equal to 0.01 mkmol CO2/g DW/s. Respiration
rate in tubers was significantly less than that in leaves. Even in very young tubers (diameter near
1 cm), respiration rate was equal to 0.02 mkmol CO2/g DW/s. During tuber growth, their respiration
rate decreased to 0.004 mkmol CO2/g DW/s (Golovko, 1990, 1999).
For a majority of vegetation period, the aboveground organs (especially leaves) dominated in the
respiration of the whole potato plant. Contribution of tubers in CO2 production amounted to 40%
at the end of vegetation, when their part in the whole potato plant dry biomass amounted to 70%
(Golovko, 1999). These data show that growth and maintenance of leaves as the main photosyn-
thetic organ of potato plants have much more respiration cost than that of special organs attracting
assimilates and accumulating them in the form of nonstructural carbohydrates.
Interactions between source and sink organs are a major factor regulating the plant photosyn-
thetic function and are responsible for plant productivity. The relation between leaf photosynthetic
activity and tuber attractive ability was found (Tabalenkova and Golovko, 2010). The leaf effi-
ciency as the source of assimilates for tuber formation depends on leaves’ age and their position in
sink–source plant system. The majority of assimilates (about 70%) needed for tuber formation is
produced by middle-layer leaves (7–12th leaves from the shoot base). They are characterized by a
long life, the largest area of lamina, and highest photosynthetic activity.
For the period of intensive biomass accumulation, tubers become the main acceptors of assimi-
lates. About 60% of plant-assimilated carbon is transported into tubers. The considerable amount
of assimilated carbon (over 30%) remains in a potato aboveground part for the maintenance of
functional activity of shoots.
In the northern conditions, tubers remain immature at harvesting time. This is evidenced by the
high content of soluble sugars in tuber biomass, a relatively high respiration rate in tubers, and their
thin periderm.
Starch content in tubers varies depending on earliness of the potato variety, age, and tuber matu-
rity. The conditions of cultivation (photoperiod, temperature, moisture, mineral nutrition, and etc.)
have appreciable effect on starch accumulation rate. Starch content in tubers depended on leaf
ability to synthesize enough soluble sugars. We found that the correlation coefficient between the
sugar content in leaves and the starch synthesis intensity was about 0.7. The increase in starch-
synthesizing ability is directly coupled with an increase in the activity of ferments responsible for
starch biosynthesis in tubers. We found the enhanced content of the soluble protein in tubers during
maturation (Tabalenkova and Golovko, 2010). Potato varieties with the high starch content were
characterized by the active protein-synthesizing system in tubers.
Potato culture needs high level of the mineral nutrition. Before the flowering phase, potato plants
take up over 70% of total required mineral elements (Yel’kina, 2008). Nitrogen requirement of
potato plants is the highest during vegetative growth when top biomass accumulates. After the ini-
tiation of tuber formation, the demand in potassium and phosphorus increases. Carryover of mineral
elements with the tuber yield varied within the diapason of 220–380 kg/ha. High consumption of
mineral elements by potato culture is mostly related to the growth of the aboveground part than to
tuber crop formation. It often occurs in the years with a wet and relatively warm summer.
A potato plant can be successfully cultivated in a temperate climate condition. In the colder cli-
mate of northern latitudes, a potato planting begins usually at the end of May and in the years with
a late spring in the beginning of June. The period from the planting till the emergence of seedlings
may take nearly one-fourth of the whole vegetation. In the north, potato crops rarely suffer from
deficient moisture. Potato plants consume 60–90 kg of water in order to form 100 kg of tubers. Low
positive temperature with possible frosts and abundant rains in August may shorten the vegetation
of potato crop to 10–15 days; it lasts 75–80 days usually.
Inherently, a native species of potato belongs to plants with short-day reaction tuberization
(Markarov et al., 2001). This property was lost under selection, and the present varieties of potato
react weakly to the length of the photoperiod. But we found that in the north, a long day has an
appreciable effect on the physiological–biochemical characteristics of the middle- to late-ripening
potato variety Ideal. The value of SLM decreased by 10%–15%, and respiration rate in leaves
decreased by 25% when photoperiod was shortened artificially. At a long day, plants accumulated
two times more aboveground biomass than that at a short day. At a short light day, tubers consume
up to 40% of plant-assimilated carbon and only about 8% at a long day.
Temperature affects the tuber growth and the chemical composition of tuber biomass greatly. In
the north, a potato culture ripens at the end of August. A moderate average daily air temperature
(13.9°C) is typical for this month. Cool weather together with high soil moisture has a negative
effect on tuber biomass accumulation and tuber maturation. As a result, the starch-to-sugar ratio
varied significantly, from 10 to 24, but was still lower two to four times than that in the central part
of Russia. In the northern conditions, the potato tuber yield is 20–25 t/ha.
16.3.2 Cereal Crops: Barley and Oat

Barley is especially important among the cereal crops appropriate for cultivation in the north,
because it has a highly adaptive potential. The northern varieties are characterized by intensive
growth and leaf area formation. Depending on growing conditions, the LAI value varies from
1.5 to 3, and the SLM attains 70 g/m2. The positive dependence of barley productivity on LAI value
was found (Golovko et al., 2004).
The light saturation of photosynthetic rate in the under-flag barley leaf was observed at
450 mkmol/m2 s. The rate of CO2 uptake reached up to 20–25 mkmol/m2/s at light saturation.
Under-flag barley leaf continued to assimilate actively till the end of the vegetation period. So, at
grain-ripening period, the current photosynthesis of leaves is also important as well as reutilization
of the stored carbon from the stems.
The photosynthetic pigment accumulation reflects the state of photosynthetic apparatus. The
highest content of chlorophyll (3 mg/g FW) and carotenoids (0.7–1.2 mg/g) was found in barley
leaves at the earing phase of plants. The majority of the whole-plant chlorophyll (62%–67%) was
located in the barley leaf laminas. At grain-ripening phase, the chlorophyll allocation was as fol-
lows: the leaves contained 18%–27%, stems with leaf sheaths 42%–44%, and ears 28%–30%. Stem
and leaf sheath contribution into photosynthetic gas exchange increased with the growth of their
part in the total plant biomass.
The analysis of the assimilate distribution showed that a large carbon part was located in stems
with leaf sheaths in the tillering and during the period of vegetative growth. At seed formation stage
when an ear demand for assimilates is high, reutilization of deposited carbon from stems into ears
was observed. Nonleaf plant organs supply up to 30% of carbon required for grain formation.
Northern barley accumulated dry biomass of about 880 g/m2 of agrocenoses, and grain productivity
amounted to nearly 350 g/m2. Under unfavorable weather conditions at an early vegetation period, bar-
ley plants formed less shoots and so they had less grain productivity. Cool and rainy weather in August
initiated the growth of vegetative shoots and depressed the processes of grain formation and ripening.
The productivity of barley culture in the northern conditions depended on the weight of seeds in
an ear and on the number of caryopses per 1 m2 of agrocenoses. Northern agrocenoses were largely
behind most southern ones by these parameters. Grain yield of the south agrocenoses comprised
about 700 g/m2, that is, twice more as compared to northern agrocenoses (Golovko et al., 2004;
Tabalenkova et al., 2006). In the northern conditions, the barley seed contained more soluble sugars
by 1.6 times (26–28 mg/g DW) and less starch and reduced nitrogen; it indicates incompleteness of
maturing process due to the short vegetative period and the warmth deficiency in August.
In contrast to barley, oats is of a more late-ripening plant culture. The intensive growth of its leaf
area belonged to 10–30th day from the emergence of seedlings. Leaf biomass accumulation con-
tinued for 50–60 days. The highest growth of the stem biomass belonged to 40–60th days from the
emergence of seedlings. The LAI of the oat agrocenoses amounted to 3.5–6.5 m2/m2. The average
daily value of productivity of leaf area unit equaled 4.2–6.7 g DW/m2. The dry biomass of the oat
crop was 117–195 kg/ha.
As in barley, the highest content of green pigments in the oat leaves (4.4 mg/g FW) was observed
during tillering. At the flowering stage, chlorophyll content in leaf laminas decreased for 3.4–3.9
mg/g FW. Carotenoid content varied within 0.3–0.8 mg/g FW and attained its maximum at the ear-
ing stage. At the period of grain maturing, the oat leaves contained 35%–40%, stems with sheaths
about 40%, and heads about 20% of the whole-plant chlorophyll. The oat leaves assimilated daily
from 3.5 to 7.5 mg DW/mg chlorophyll.
The respiration rate in the leaves of the young oat plants equaled 0.03 mkmol CO2/g DW/s.
In the period of grain ripening, the rate of CO2 release in leaves decreased significantly (three to
four times). Stems with leaf shealths made an essential contribution into the total plant respiration
value. The respiration rate in the stems with sheaths of young plants attained 0.05 mkmol/g DW/s
(at 20°C), and during the grain filling, it did not exceed 0.003 mkmol CO2/g DW/s. In the north,
oat seeds do not have enough time to complete the ripening processes, and so, the harvest of grain
is carried out in the phase of milky or milky-wax seed ripening. At this period, the oat heads are
characterized by the high respiration activity (0.03 mkmol CO2/g DW/s at 20°C). The productivity
of the northern oat agrocenoses equals 15–18 t of dry biomass, but grain part is only 20%–22%.
16.3.3 Annual and Perennial Forage Herbs

The number of the annual forage plant varieties, which can be cultivated in the northern conditions,
is not large. Lolium multiflorum (Italian ryegrass) is a fast-growing and cool-season crop. Ryegrass
requires the accumulated temperatures of 680°C–700°C to form vegetative biomass and of 900°C
to form the mature seeds. Leaf area of ryegrass agrocenoses grow rapidly. LAI of agrocenosis
amounted to 1 m 2/m 2 in 2 weeks after seedling emergence, and LAI attained highest value (3 m2/m2)
in the earing stage (in 30–35 days after seedling emergence). The value of LAI depended greatly on
cenosis density (amount of plants per m2). The biomass productivity of ryegrass cenosis correlated
closely (r = 0.76) to LAI value.
In the northern conditions, ryegrass is used as two-mowing culture. Depending on the weather,
the crop productivity for two-mowing procedures amounted to 6–10 t (dry mass)/ha. Ryegrass plants
pass all ontogenetic stages and produce full-value seeds for a short vegetation period (65–70 days).
Ryegrass plants annually formed 500–600 kg/ha of ripe seeds.
The photosynthetic pigment content in ryegrass leaves changed depending on the plant age,
density, and the nitrogen supply of agrocenoses. Chlorophyll content in leaf blades equaled
1.7–2.0 mg/g FW at the earing phase. The leaves contained 64%–75%, stems with leaf sheaths
23%–29%, and ears 1%–8% the chlorophylls of the whole plant. The contribution stems with
leaf shealths and ears in total plant pigment pool increased to 60%–75% at the begining of seed
ripening stage. The highest daily biomass increments per chlorophyll unit (18–30 mg DW/mg
chlorophyll) were observed at the earing stage, when plant growth rate was also high.
The leaves assimilated over 80% of carbon in the tillering phase, and the stems with leaf sheaths
contributed more into carbon assimilation (60%–70%) at the earing phase. Data on the ability of green
nonleaf organs of cereals (stems, heads, and ears) to positive CO2–gas exchange are presented by other
authors (Bedenko and Kolomeichenko, 2005). Positive correlation between CO2 absorption and chlo-
rophyll amount in whole ryegrass plants was shown (Golovko et al., 1992). Daily positive CO2–gas
exchange of ryegrass plants in the northern cenoses lasts 18–20 h in June. CO2 assimilation rate of
plants in the evening hours (7–8 p.m. local time) amounted to 30%–35% of values determined in the
morning sun hours.
During vegetation period, highly productive ryegrass agrocenoses spent for transpiration up to
5000 t water/ha. The transpiration coefficient (water amount evaporated during the synthesis of one
biomass unit) was 430–670 g H2O/g DW.
Fertilization is the most efficient way to increase the productivity of forage crop cultivated on the
poor podzolic soils (Beznosikov, 1997). Productivity of the ryegrass agrocenoses was 15.4 t (green
biomass)/ha, when phosphate-potassium fertilizers were used. Nitrogen fertilizer in the doses of 30, 60,
and 90 kg/ha increased the yield by 1.3–2 times. Nitrogen in the doze of 120–150 kg/ha did not have
positive result on productivity. The nitrogen abundance was accumulated in biomass in the nitrate form.
In the northern region, the fodder production is based upon using perennial plant cultures, which
have more chances to realize their productive potential in contrast to annuals. The regrowing of the
perennial grasses (Bromopsis inermis, Phalaroides arundinacea, Poa pratensis, and Alopecurus
pratensis) is started in the first half of May, immediately after snow cover melting. LAI of the peren-
nial grass agrocenoses may reach considerable values (10–12 m2/m2) at flowering period (early of
July). Yield of the aboveground biomass varied from 0.3–2 kg DW/m2 depending on the varieties
and weather conditions. The most productive were B. inermis and P. arundinacea plants in which
biomass was dominated by stems. The agrocenoses of P. pratensis and A. pratensis plants were
less productive, but the leaf part in their biomass was essentially higher.
Leaves of perennial grasses were characterized by considerable chlorophyll and carotenoid accu-
mulation. The maximum of chlorophyll (2.0–3.0 mg/g FW) and carotenoid (0.5–0.8 mg/g FW) con-
tents were observed at the begining of the earing period, when the grass canopy leaf area amount to
its maximal development. The chlorophyll a/b ratio was 1.4–2.1, which pointed to high fraction of
the antenna chlorophylls. The increase in the light-harvest chlorophyll content was related to plant
competition for light due to the intensive growth of shoots (Kurenkova et al., 2007).
A positive CO2 –gas exchange of grass plants lasts for 18–20 h in June–July. At optimal light
and temperature conditions, the photosynthetic rate of A. pratensis and B. inermis leaves reached
2.5 g CO2/m 2/h (Shvetcova, 1987). The highest leaf assimilation rates were measured for the
earing–early flowering stage in the morning hours, when carbon pools were still empty and light
conditions were favorable.
The leaf photosynthetic activity depended on the leaf age and leaf position on a stem. The
photosynthetic rate in the low-layer leaves of the main shoots was by three times lower as com-
pared to the upper leaves, which were younger and received more light. The photosynthetic rate
in the leaves of middle and upper layers was saturated by light at 540–670 mkmol/m 2 s PAR.
The intensity of the leaf radiation adaptation (Tooming, 1988) was equal to 180–220 mkmol/m2 s
PAR. Daily the perennial grasses assimilated on average 180–250 g CO2/m2 (leaf area). The tem-
perature optimum for leaf photosynthesis was within a zone of 12°C–17°C (Shvetcova, 1987).
However, photosynthetic apparatus can be efficient in the wider temperature range of 5°C–30°C.
The wide temperature amplitude evidences that assimilation apparatus of grasses is adapted for
rapid temperature changes, which normally occurs in cold climate regions.
After flowering, the contribution of stems with leaf sheaths into carbon assimilation increased,
although their photosynthetic rate was significantly lower (by 5–10 times) as compared to the leaf
blade. The cenosis ability of biomass accumulation is determined by carbon balance. Monitoring of
postphotosynthetic 14C dynamics in grass plants has revealed a considerable decrease in 14C amount
in leaves due to the outflow of assimilates toward stems and underground organs. Increase in 14C
amount in other plant organs did not compensate for its decrease in leaves. Respiration plays an
important role in the carbon balance of perennial grasses. At earing–flowering stage, the respiration
losses were comparable with biomass increment in agrocenosis.

Productivity formation of the northern agrocenoses is determined by specific climatic and edaphic
factors. Northern agrocenoses are prominent through slow development of assimilating surface at
initial vegetation stages. Mostly moderate air temperatures, appropriate water provision, and a long
light day activate vegetative mass growth, which is important for the harvest of perennial grasses.
Dominating vegetative growth and leaf surface increase worsen radiation regime in agrocenosis
and require substantial energy-plastic expenditures for the growth and maintenance of the vegeta-
tive plant organs. These factors have a negative effect on the harvest of potato and cereals. Period
of the formation of economically important biomass of cereal and potato crops is often noted by
rains and low temperatures, which decreases the transport and usage of assimilates in biosynthetic
processes and, consequently, results to delay of the physiological ripening of grain and tubers. At
harvest time, the high content of low-molecular nitrogen compounds in the barley grain and soluble
sugars in potato tubers were found. Despite the high adaptation level of modern plant varieties, the
northern climatic conditions considerably impact productivity, biomass chemical composition, and
yield quality of agricultural plants, and they retard the realization of productivity potential.
Thus, the study of the production process as an integral plant function was managed to improve
the understanding of the productivity and resistance formation of modern varieties of potato, barley,
oats, and annual and perennial grasses adapted to be grown in cold climate. Knowledge of general
mechanisms of plant functions provides a basis for production process optimization and allows to
estimate the degree of their metabolism fitness the agroclimatic conditions, to forecast the effect of
global and regional climatic changes on the productivity of different plant cultures.
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17 Rates of Processes of
Essential Plant Nutrients
CONTENTS
17.1 Introduction........................................................................................................................... 343
17.2 Movement of Elements into Whole Plants and Compartments of Multicompartment
Plants.....................................................................................................................................344
17.3 Movement of Essential Nutrient Elements in Plants............................................................. 345
17.3.1 Rates of Transport of Essential Plant Nutrients across Cell Membranes.................. 345
17.3.2 Rates of Transport of Essential Plant Nutrients in the Xylem and Phloem...............348
17.3.3 Acquisition of Elements through Stomata and Cuticle of Leaves............................. 349
References....................................................................................................................................... 350
17.1 INTRODUCTION
The goal of the present chapter is to give the reader a clearer sense of some of the many processes
that involve mineral nutrients and ranges of rates at which those processes occur. Mineral nutri-
tion of plants involves the acquisition of elements from the environment, and the organization and
functioning of essential plant nutrients are a consequence of the interaction of deoxyribonucleic
acid (DNA) of the plant with the environment. Because of the great complexity of plants, processes
involving essential nutrient elements vary from relatively slow to relatively fast. Plants require 17
essential nutrients, and several other elements, such as cobalt, sodium, and silicon, have been found
to stimulate the growth of some plants (Epstein and Bloom, 2005).
Green plants obtain carbon from the air, whereas nonphotosynthetic plants such as fungi obtain
carbon as saprophytes, breaking down organic materials of living or dead organisms. Unicellular
and multicellular, photosynthetic, nonvascular plants such as algae obtain carbon and other essen-
tial elements with little transport from the environment to the site of photosynthesis, and the prod-
ucts of photosynthesis are transported relatively short distances to become new living tissue or to
be expelled from the plant to the environment. Vascular plants, on the other hand, have evolved to
move essential plant nutrients within the xylem and the phloem over distance far greater than the
dimensions of the uncharged atoms, molecules, or ions containing the essential plant nutrients. The
anatomy of vascular plants as well as nonvascular plants can be understood to include sources and
sinks, between which mineral nutrients move. In addition, the plant itself can be viewed as a sink,
which, as a result of the organizing force of DNA, is the recipient of essential nutrients of the plant
that originate from the environment, which can be considered a source.
Several means of measuring rates of transport of mineral nutrients in plants, other than veloc-
ity, include volume transfer (cm3 h−1), mass transfer (g h−1), and specific mass transfer (g cm−2 h−1)
(Canny, 1960).
343
17.2 MOVEMENT OF ELEMENTS INTO WHOLE PLANTS AND

COMPARTMENTS OF MULTICOMPARTMENT PLANTS
Within a range of concentrations from 0.1 to 10,000 mmol m−3, net rates of uptake of Ca 2+, K+,
N, P, S, and Zn2+ were found to range from less than 0.001 to greater than 1.0 μmol g FW root−1 h−1
(Pitman, 1975). In maize, net rates of accumulation and loss of nitrogen in various parts of the
maize shoot during reproductive growth have been estimated by the first derivatives of regres-
sion equations based upon periodic sampling and chemical determination of N content of various
compartments of the multicompartment system. Estimated maximum rates of accumulation of
total N in the grain were estimated to be approximately 80 mg N day−1 plant−1, based upon esti-
mated rates of accumulation of exogenous N and endogenous N in the grain. N fluxes (net flux
resulting from influx and efflux) for stem, leaves below the ear, leaves above the ear, shank, cob,
and husk were also estimated, based on the first derivatives of the regression equations (Crawford
et al., 1982). Net rates of accumulation and loss of nitrogen in the China 17 sorghum (Sorghum
bicolor (L.) Moench) genotype, which is an efficient user of nitrogen, were estimated, per plant,
to have varied among the following ranges: lowest half of the stalk, 0 to −40 g N week−1; third
highest of the four sections of the stalk, +0.04 to −0.04 g N week−1; leaves of the third highest of
the four sections of the stalk, +0.08 to −0.03 g N week−1; fourth highest of the four sections of
the stalk, +0.16 to −0.07 g N week−1, leaves of the fourth highest of the four sections of the stalk,
+0.11 to −0.04 g N week−1; and the grain, 0–0.12 N g week−1. Comparable data for the sorghum
genotype, TX623, show that fluxes differ during the same period of growth for a less efficient
user of nitrogen (Crawford et al., 2009). Flux rates of N, P, K, Cu, Fe, Mn, and Zn varied in the
roots and shoots of cucumber (Cucumis sativus L.) plants, as affected by deficiency, sufficiency,
or toxicity of Mn. During the period of 43–58 days after germination, Mn deficiency caused the
roots to change from sink to source of N and K on days 56 and 53, respectively, and caused the
shoot to change from sink to source of P and Fe on days 57 and 58 respectively. During the same
period, Mn toxicity caused the roots to change from sink to source of N, K, and Cu on days 46,
51, and 46, respectively, and caused the shoot to change from sink to source of Fe on day 55
(Crawford et al., 1990).
Mycorrhizal associations with plant roots have been shown to increase the uptake of phosphate.
Sanders and Tinker (1973) showed that during 14 days, the rate of mycorrhizal phosphate uptake by
the roots of onion plants was 0.17 pmol cm−1 s−1, compared to nonmycorrhizal uptake, which was
0.050 pmol cm−1 s−1. Similarly, during a 10-day experiment, they found that the rate of mycorrhizal
phosphate uptake by the roots of onion plants was 0.13 pmol cm−1 s−1, compared to nonmycorrhizal
uptake, which was 0.042 pmol cm−1 s−1.
Eight-day-old maize (Zea mays L.) seedlings replete with nitrogen, when placed in N-deficient
solutions, increased their rate of absorption of nitrate and ammonium uptake from 200 μM
NH4NO3. Patterns of uptake changed during a 72-h period. During the 72 h of exposure, the rate of
uptake of ammonium increased from about 5 to 9 μmol g−1 h−1, whereas the rate of uptake of nitrate
increased from 2 to 5 μmol g−1 h−1, then declining to about 4 μmol g−1 h−1. More detailed rate data for
shorter periods of time during the 72 h are also included in the report by Jackson and Volk (1992).
Selenate and selenite are absorbed differently in the roots of wheat. Plants absorbed similar
amounts of Se within 1 d when supplied with selenite or selenate. Uptake of selenate and selenite
was enhanced in sulfur-starved and phosphorus-starved plants, respectively. During a 30-min period
when selenite concentration was increased from 0 to 10 μM, Se uptake rate increased from 0 to
about 7 mmol Se g−1 root h−1 when no phosphorus was present, but when 0.1 mM phosphorus was
present, Se uptake rate increases from 0 to only about 2 mmol Se g−1 root h−1; the investigators
surmised that selenite is absorbed by a mechanism similar, if not identical, to that of phosphate
(Li et al., 2008). Selenate and sulfate are oxyanions of selenium and sulfate, respectively, and are
in Group 6A of the periodic table. These two elements both have oxidation states of +4, +6, and −2
and display similar chemical behavior.
Rates of Processes of Essential Plant Nutrients 345
17.3 MOVEMENT OF ESSENTIAL NUTRIENT ELEMENTS IN PLANTS

Three categories of movement of essential elements in vascular plants are (1) movement across
membranes of cells and or organelles such as the plasmalemma, mitochondrion, and vacuoles;
(2) longer-distance transport through the vascular system composed of xylem and phloem; and
(3) movement through stomata and cuticle of leaves.
17.3.1 Rates of Transport of Essential Plant Nutrients across Cell Membranes

Rates and patterns of ion absorption by plant roots from the soil solution are partially influenced by
concentration of the elements outside the plant roots or, in the case of nonvascular plants, outside the
cell membranes that are in contact with the environment. The concept of saturation kinetics was first
observed in 1937 by Dutch plant physiologist T. H. Van den Honert (1937), who studied phosphate
uptake by sugarcane (Saccharum officinarum) and found that as the concentration of phosphate
outside the plant increased, it finally reached a maximum rate of absorption. Subsequently, Emanual
Epstein and C. E. Hagen described the transport of ions across cellular membranes in terms used in
enzymology (Epstein and Hagen, 1952). They observed that as with enzyme systems, saturation kinet-
ics occurred as the concentration of a nutrient ion was increased outside a cell membrane. The rate of
absorption in one direction across a plant cell membrane, v, if there is no counterflow in the opposite
direction can be expressed as the product of capacity and intensity factors: v = (Vmax · [S])/(Km + [S]).
This Michaelis–Menten equation relating the rate of enzymatic catalysis to the concentration of sub-
strate was found by Epstein and others to be equally valid to describe the rate of movement of an ion
across a cell membrane as a function of the concentration of the ion outside the membrane, [S]. In
the case when the rate of absorption of the ion is one-half the maximal rate, v = ½Vmax. So, Vmax/2) =
(Vmax · [S])/(Km + [S]), (Km + [S]) = 2[S], and Km = [S]. So, Km, the “Michaelis constant,” equals the
concentration of the substrate ion resulting in one-half the maximal rate of absorption. The lower the
value of Km for an ion, the higher is the affinity of the carrier sites for the ion (Epstein, 1972).
In subsequent work, Epstein and others demonstrated that during the absorption of potassium,
when increasing the concentration of K+ in the substrate from 0 to 50 mM, by barley roots, the
rate of absorption of K+ increased, plateaued, then increased, plateaued at a higher rate, and so on
until the maximal rate was reached at a concentration of 50 mM. This research pointed to more
than one mechanism controlling the absorption of potassium across barley root cell membranes.
Such experimental results were explained by Epstein and others as being the result of one uptake
mechanism (“Mechanism 1”) prominent at the plasmalemma, or cell membrane, when substrate
concentrations were on the order of 2 × 10 −6 M, whereas a second mechanism or additional mecha-
nisms were operative when absorption of K was from substrates with K+ concentrations between
0.50 and 50 mM. Mechanism 1 obeys simple Michaelis–Menten kinetics, whereas absorption by
Mechanism 2 at higher concentrations is a result of a more complex set of mechanisms. The rate, v,
of uptake of K+ corresponding to the range of substrate concentrations from 0 to 50 mM is approxi-
mately 0–40 μmol g−1 h−1. Welch and Epstein (1968, 1969) concluded that both mechanism 1 and
mechanism 2 exist in parallel across the plasmalemma. A recent thorough review of mechanisms
of potassium uptake in plants (Cuin et al., 2008) indicates that there is a functional overlap between
the high- and low-affinity mechanisms of K+ uptake in plants and that potassium can be absorbed
via a number of different kinds of channels in cell membranes. Following the pioneering work of
Epstein and others, subsequent research has linked such factors as root:shoot ratio and relative
growth rate to nutrient uptake rate (v), maximum uptake rate (vmax), and uptake rate of the whole
plant (vplant) (Gutshick and Pushnik, 2005). For zinc, a table summarizing values of Michaelis–
Menten kinetics variables through membranes shows Vmax in a range from 2.3 to 18,300 nmol
[DW] g−1 h−1 (Broadley et al., 2007). This broad range of maximum rate of movement of zinc
across membranes is collected from many investigators using various crop species under various
conditions to measure zinc flux.
Effects of external concentration of nutrient ions upon rates of uptake by plants have been dem-
onstrated using the enzyme-kinetic hypothesis of ion absorption (Epstein, 1976). When “low-salt”
roots grown in a nutrient-deficient medium are transferred to a solution containing the deficient
ion, a transient high rate of absorption will be achieved. As the concentration of the ion inside the
compartment (e.g., cell, organelle) increases, the concentration of the ion in the compartment is
maintained at an adequate level, and the uptake rate declines to a steady rate (Glass and Siddiqi,
1984). Other changes in the uptake rates of nutrient ions are diurnal, such as the case of net nitrate
uptake in perennial ryegrass following the peak of diurnal CO2 fixation after a lag of 5–6 h (Clement
et al., 1978) or circadian patterns of K+ uptake in Lemna gibba (Kondo, 1982).
The pH of the soil solution has been shown to affect the rate of absorption of potassium. In
studying the effect of pH on the absorption of K+, Jefferies et al. (1969) found that maximal rates of
absorption of K+ by two species of liverworts occurred in the laboratory at pH values close to the
pH of the natural environments from which the samples were collected. K influx into Cephalozia
connivens was maximal, approximately 0.5 pmol cm−2 s−1 at pH 5, but when the pH of the solution
with which the liverworts were in contact was higher or lower than the pH of the environments in
which the liverwort samples had been collected, the rate of K+ uptake was less than the maximum.
This research points to the evolution of the ability of plants to maximize the rate of acquisition of
essential mineral nutrients from the environments influenced by key factors such as pH of the solu-
tion in contact with the organs of the plant that absorb the nutrients directly from the environment.
The absorption of the transition metal cations from concentrations approximately 1 μM
is strongly inhibited by alkali and alkaline earth cations and by other transition metal cations
(Robson and Pitman, 1983). The rate of zinc absorption by wheat roots was shown to have been
decreased from about 300 ng atoms (f FW roots)−1 day−1 with no K+ present to about 100 ng
atoms (g FW roots)−1 day−1 when 750 μM K+ was present with the concentration of Ca++ less than
1000 μM. The rate of K+ absorption was approximately the same, about 30 ng atoms (g FW roots)−1
day−1 when Ca++ was present, regardless of whether K+ was present or not in the solution from
which the zinc was absorbed.
Because absorption of ions by the roots is an energy-consuming process, it is crucial that
supplies of sugars from the leaves be available to provide energy for the uptake of ions by the
roots. The rate of absorption of potassium by the roots of sunflower, Helianthus annuus, was
reduced from 0.140 mequiv h−1 to approximately 0.030 mequiv h−1 by cooling the stem of the
plant from 25°C to 0°C to reduce downward translocation of sugars. The rate of uptake of potas-
sium returned to greater than 0.140 mequiv h−1 once the temperature of the phloem was returned
to its original temperature (Weatherly, 1969). Weatherley et al. interpreted the decrease in the
rate of absorption of K+ when the temperature of the phloem was lowered as a result of less sugar
reaching the roots. It is possible, however, that the cooler phloem reaching the roots may have
depressed the rate of K+ absorption due to the lower temperature within the roots, since the rate
of absorption of mineral nutrients has been shown to be temperature-dependent (Sutcliffe, 1962).
The external concentration of KCl ranging from 0 to 80 mM resulted in rates of influx of Cl−,
which increased from 0 to approximately 10 × 10 −13 mol cm−2 s−1 across the plasmalemma and
from 0 to approximately 1.4 × 10 −13 mol cm−2 s−1 into the vacuole of cells of maize. A similar
pattern was evident for the influx of Cl− across the plasmalemma and into the vacuole of carrot
cells (Cram, 1974).
The rate of uptake of mineral nutrients by a cell or whole plant is affected by temperature, and the
temperature effects can be related to temperature coefficient (Q10) values. The Q10 value represents
the factor by which the rate of a reaction increases for every 10° rise in the temperature. Absorption
of mineral nutrients across cell membranes, as affected by temperature, can be categorized by
absorptive processes with a Q10 of about 1–2, and another group of absorptive processes with a Q10
of 2 or greater. At a temperature of 1°C–2°C, most absorption across cell membranes is thought to
occur mainly by physical processes such as diffusion, mass flow, exchange, and adsorption. This
physical absorption at low temperatures occurs fairly rapidly, whereas metabolic absorption with a
higher Q10 is prolonged. The amount of uptake of K+ by washed carrot tissue slices increased from
0 to 4 μequiv (g fresh wt.)−1 from 2 to 6 h later at 2°C, the rate of uptake having gone to essentially
0 at 2 h. On the other hand, the amount of K+ absorbed continually increased from 0 to 16 μequiv
(g fresh wt.)−1 at 20°C during the same 6-h period. As a function of temperature, from 2°C to 50°C,
the amount of K+ absorbed by washed carrot tissue slices increased during a 30-min period from
about 4 μequiv (g fresh wt.)−1 to a maximum of about 7 μequiv (g fresh wt.)−1 at 40°C, but was about
5 μequiv (g fresh wt.)−1 at 50°C. In contrast, when the temperature was raised more gradually during
a 2-h period from 2°C to 50°C, the amount of K+ absorbed by washed carrot tissue slices was only
about 5 μequiv (g fresh wt.)−1 at 2°C but increased to about 12 μequiv (g fresh wt.)−1 at 40°C, but
the amount of K+ absorbed was only about 3 μequiv (g fresh wt.)−1 at 50°C (Sutcliffe, 1962). These
data showing the influence of temperature on the absorption of a monovalent ion, K+, are indicative
of the paramount importance of temperature in regulating rates of absorption of mineral nutrients
across cell membranes.
Diurnal rhythms of light and dark strongly influence the rate of absorption of ionic nutrients
across cell membranes. Alberda (1948) showed that when the rate of absorption of phosphorus by
maize plants was 0.20 mg P2O5 h−1 in the daylight, it decreased during a 4-h period to almost 0 mg
P2O5 h−1 in darkness. When light was again supplied to the maize plants, the rate of uptake of phos-
phorus increased relatively rapidly during a 2-h period from about 0 to 0.20 mg P2O5 h−1, compared
to the slower rate of decline. This experiment shows the importance of light as regards the uptake of
mineral nutrients by photosynthetic plants and illustrates the diurnal fluctuation of rate of uptake of
phosphorus, as it is affected by diurnal changes in the presence and absence of light. In experiments
with pea, Nobel (1969) showed that absorption of K+ could be almost completely stopped by depriv-
ing pea plant leaf fragments of light. This finding indicates the importance of light for maximal
absorption of fertilizer nutrients applied directly to leaves of crops.
Uptake of inorganic phosphate, Pi, is affected by external Pi concentration. Spirodela plants of a
control treatment that included phosphorus were exposed to 1000 μM Pi and absorbed phosphorus at
a rate of 500 nmol g−1 fresh wt. h−1, whereas the same species of the control treatment when exposed
to 1 μM Pi absorbed phosphorus at a rate of 20 nmol g−1 fresh wt. h−1. Spirodela plants that had been
deprived of phosphorus for 4 days and had a 30% reduced growth rate but no marked symptoms of
P deficiency were exposed to 1000 μM Pi and absorbed Pi at a rate of 1200 nmol g−1 fresh wt. h−1. At
a lower concentration of 1 μM Pi, Spirodela plants that had been deprived of phosphorus for 4 days
and had a 30% reduced growth rate but no marked symptoms of P deficiency absorbed Pi at a rate of
40 nmol g−1 fresh wt. h−1. Temperature has also been shown to affect the uptake of Pi by Spirodela.
When the alga was exposed to 1000 μM Pi, the uptake rate decreased from 460 nmol g−1 fresh
wt. h−1 at 25°C to 80 nmol g−1 fresh wt. h−1 at 5°C (McPharlin, 1981).
Aerobic organisms such as green plants require oxygen to absorb ions (Mengel and Kirkby,
1978). It was shown that the rate of absorption of phosphate increased from 0 to about 3 mol
P × 10 −7 (g root) −1 h−1 as the oxygen tension was increased from 0% to about 2.4% (Hopkins,
1950). This is but one example of the necessity of oxygen for active uptake of a mineral nutrient
across cell membranes.
Research reported more than 100 years ago by Brezeale (1906) indicated that when an inorganic
nutrient was withheld from the roots of hydroponically grown wheat plants for a period of hours,
the absorptive capacity of the element was increased several-fold. Brezeale showed that after 18 h
without each of the following nutrients, uptake rates of four mineral nutrients increased signifi-
cantly: 3.3-fold for NO3-, 2.2-fold for Ca2+, 4-fold for K+, and 2.5-fold for inorganic P. According to
Glass (2005), following the resupply of a withheld nutrient, rapid reduction of the increased capac-
ity to absorb the nutrient at a high rate occurs, and he surmises that if plants are able to rapidly
“upregulate” and “downregulate” uptake rates of mineral nutrients, plants may be able to minimize
fluctuations of availability of mineral nutrients within the plant. Since concentrations of mineral
nutrients in soils may vary across orders of magnitude (Reisenauer, 1966), this flexibility on the part
of plants may have contributed to their survival and evolution.
The mechanism for nitrate ion uptake into barley (Hordeum vulgare) plants was investigated, and
the net uptake rate of nitrate by barley plants that had previously received little nitrogen decreased
from approximately 2 μmol NO3- g -1 min -1 to slightly more than 0 μmol NO3- g -1 min -1 after 14 min
of exposure. During the same period, the accumulation of NO3- increased from 0.4 μmol
NO3- g -1 min -1 to about 2.0 μmol NO3- g -1 min -1 , which was explained by Deane-Drummond
(1984) as being due to loss of nitrate from the cells by “facilitated diffusion.”
Copper is an element that is preferentially absorbed through thylakoid membranes. The inter-
nal volume of thylakoid membranes was determined by Shingles et al. (2004) to be about 3.3 μL
(mg chlorophyll)−1 (Heldt et al., 1973). Assuming a protein to chlorophyll ratio of 4:1 for thylakoid
membranes, the calculated initial rate of Cu+ transport is approximately 70 pmol min−1 mg protein−1.
By comparison, Fe+, Cd+, and Mn2+ were slowly transported across the thylakoid membranes, giving
initial rates of transport of 5.0 and 2.0 pmol min−1 mg protein−1, respectively. Mn2+ transport was
negligible.
17.3.2 Rates of Transport of Essential Plant Nutrients in the Xylem and Phloem

Peel (1974) provides an insightful discussion of the measurement and concepts of velocity and mass
transfer of solutes in the vascular systems of plants. At the outset, he states that volume transfer
(cm3 h−1) = area (cm2) × velocity (cm h−1), and to calculate mass transfer, mass transfer (g h−1) =
volume transfer (cm3 h−1) × concentration (g cm−3), or specific mass transfer (g cm−2 h−1) = velocity
(cm h−1) × concentration (g cm−3). The xylem of vascular plants is the specialized tissue that con-
ducts solutes from the root to shoot; it not only can be considered a continuous system of intercon-
nected tubes with little resistance to the flow of water but also includes living parenchyma cells that
are involved with loading solutes into the xylem (Shabala, 2007). Dead lignified cells called vessels
and tracheids transport solutes and water, and xylem flow rates are about 13 mm s−1 in trees with
large vessels (Taiz and Zeiger, 1991). Flow rates of water and solutes in wheat have been measured
at a mean speed of 0.8 mm s−1 (Passioura, 1988).
The rate of Pi movement in xylem vessels is about 200 cm h−1, and phosphorus redistribution
in vascular plants is very rapid, the transport of Pi in the phloem occurring at a rate of about
50 cm h−1 (Kolek and Kozinka, 1992), and in maize plant xylem, 79%–82% of the total activity of
32P-compounds transported in xylem exudate was inorganic phosphorus, 3%–7% was the fraction
of phosphoric esters of glycides, and 13%–17% was free nucleotides (Michalík and Ivanko, 1971).
Sodium flux rates in the roots have been measured at rates greater than 250 nmol m−2 s−1 (Yeo and
Flowers, 1986).
Root pressure exudation experiments entail cleanly cutting the stem of a root system and mea-
suring the volume and chemical composition of the xylem sap at various points in time. From the
primary experimental data of the rate of xylem exudate production and the concentration of any
ion in the xylem sap, the ion flux through the xylem may be calculated as Js = Jv · Cs where Jv is the
exudation volume flux normalized over unit area of root surface or over unit fresh weight of root
for highly branched root systems, and Cs is the concentration of the ion “s” in the exuded xylem
sap. Js is the ion flux emerging at the cut end of the root (Anderson, 1975). Values of Js reported by
different investigators for K+, Cl−, SO2-4 , NO3 in xylem exudates of Zea mays, Ricinus communis,
-
Sinapis alba, Triticum aestivum, Avena fatua, A. sativa, H. annuus, and Allium cepa are organized
in tabular format by Anderson (1975).
Sieve tubes of the phloem carry both organic compounds synthesized in the leaves and inorganic
ions. Zimmerman measured rates of movement of a mixture of stachyose, raffinose, sucrose, and
d-mannitol in the sieve tubes of American ash to range between 30 and 70 cm h−1 (Zimmermann,
1969). Using radiotracer methods and employing two colonies of aphids to remove honeydew sam-
ples, Peel and Weatherley (1962) found the rate of movement of the phloem to have been between
25 and 33 cm h−1. Using a thermoelectric technique on exposed phloem tissue of a species of
Heracleum, Ziegler and Vieweg (1961) measured movement of solutes in the phloem within a range
of velocity from 37 to 70 cm h−1. Canny (1960) has reported mass transfer rates in the phloem of
various plant species with a range from 0.14 to 4.8 g dry weight cm−2 phloem h−1.
In the phloem, different rates of transport of different substances have been measured. Radioactive
phosphorus, when injected into cotton leaves, was found to be phloem-mobile, with downward
rates of translocation greater than 21 cm h−1 (Biddulph and Markle, 1944). Applying 14CO2, titrated
water (3H2O), and 32P-labeled inorganic phosphate to leaves of red kidney bean plants, Biddulph
and Cory (1957) found that after 15-min migration time, the tracers moved at different rates: 32P at
86.5 cm h−1, 3HHO at 86.5 cm h−1, and 14C at 107 cm h−1. Subsequent research with excised strands
of Heracleum phloem injected with [14C]sucrose and 42K into a single sieve element showed after
2 min that 14C traveled at about 200–500 cm h−1 and 42K traveled at 30–60 cm h−1 (Fensom, 1972).
Plant hormones, as defined by a Committee of the American Society of Plant Physiologists in
1954, “are regulators produced by plants, which in low concentrations regulate plant physiological
processes. Hormones usually move within the plant from a site of production to a site of action”
(American Society of Plant Physiologists, 1954). Plant hormones and inorganic nutrients are both
factors that influence the growth and development of plants, since hormones internally regulate
growth and inorganic nutrients meet the mineral requirements of the plant by maintaining osmotic
potential of cells and tissues and by serving as components of organic compounds and cofactors
in biological reactions (Karmoker, 1985). Auxin is a generic term for plant hormones that induce
elongation in shoot cells, and they resemble indole-3-acetic acid (IAA) in their physiological action
(Marumo, 1986). Auxin moves in a basipetal direction that is away from meristematic tissue. The rate
of movement of IAA in short tissue sections has been measured within a range of 5.7–15 mm h−1 in
tissues such as Phaseolus epicotyl, Zea coleoptile, Zea root, and Avena coleoptile (Goldsmith, 1968).
The rate at which sugar is translocated in the phloem is directly related to the net photosynthesis
rate. Servaites and Geiger (1974) demonstrated a linear relationship between the rate at which 14C was
translocated via the phloem from the leaves of 19 sugar beet plants and the net rate of photosynthesis.
Translocation rates of 14C in the phloem that they measured range from ∼5 to 50 μg C d m−2 min−1
for a range of net photosynthesis rate from 0 to ∼260 μg C dm−2 min−1. This research supports the
hypothesis that the mass transfer rate of translocation of sugars from the leaves under conditions of
sufficient sink demand is limited by the net photosynthesis rate or more specifically by the rate of
synthesis of sucrose, and that this limitation is independent of light intensity per se.
17.3.3 Acquisition of Elements through Stomata and Cuticle of Leaves

Carbon dioxide moves into leaves through stomata, and the uptake of CO2 is regulated, in large part, by
diffusion (Gastra, 1959). Immediately outside the stomata in still air, a typical concentration of CO2 is
4 × 10−4 mg cm−3 (corresponding to a volume content of CO2 of 0.02%), and at the chloroplasts, the con-
centration of CO2 may be considered to be 0 (Meidner and Mansfield, 1968). Fick’s Law, m/t = Dα∆ρ/l
where m/t is the mass of the gas in grams diffusing in 1 s; D is the coefficient of diffusion, α is the cross-
sectional area of the path in cm2; ∆ρ is the difference in density in g cm−3, and l is the length of the path
in cm. The diffusion coefficient is expressed in cm2 s−1. Meidner and Mansfield have estimated rates of
photosynthetic intake of CO2 through stomata at 17 mg CO2 dm−2 h−1, and with a wind speed of 5 km h−1,
they estimate that CO2 would move through stomata at a rate of 60 mg CO2 dm−2 h−1. Wiitjacsono et al.
(1999) measured the assimilation of CO2 by in vitro and ex vitro avocado plantlets under conditions of
ambient CO2 concentration and enriched CO2 concentration. They found that CO2 assimilation through
the stomata was greater (31 ± 7 μmol CO2 m−2 s−1), whereas enrichment with CO2 resulted in a lower
assimilation rate (17 ± 2 μmol CO2 m−2 s−1). The net photosynthetic rate of pea (Pisum sativum L. cv.
‘Meteor’) has been found to be influenced by CO2 levels in the air. As air–CO2 level was increased from
100 to 500 ppm, the net photosynthesis rate increased from 10 to 75 μg dm−2 min−1 (Harvey, 1977).
Organic liquids (Stalfelt, 1916), and particularly oils (Turrell, 1947) with low surface tension,
undoubtedly infiltrate stomata. Aqueous solutions with a surface tension near that of pure water
(∼72 dyne cm−1) do not penetrate (Greene and Bukovac, 1974). In contrast, stomatal penetration
of aqueous solutions has been demonstrated in the laboratory when the surface tension is lowered
sufficiently with surfactants (Dybing and Currier, 1961). Using fluorescent and radioactive tracers
and a precipitation method to investigate foliar penetration of aqueous solutions, Dybing and Currier
(1961) found that stomatal penetration by aqueous solutions occurred rapidly if an efficient surfac-
tant was used at the proper concentration. Surfactants varied in their ability to promote stomatal
entry, and the concentration of surfactant necessary for stomatal penetration varied with the species
being tested. The leaves of Zebrina pendula Schnizl., Pyrus communis L., Prunus armeniaca L., and
Lactuca scariola L. were readily penetrated via the stomatal route. Phaseolus vulgaris L. leaves,
however, required a greater concentration of surfactant for stomatal entry, and cuticular penetra-
tion through areas over the veins took place quite rapidly. Rates of stomatal penetration of Zebrina
leaves by C14-labeled urea during a 5-min period were 234 counts min−1 (cpm) with 0.1% Vatsol
surfactant and 6 cpm with no surfactant with open stomates. With closed stomates, corresponding
rates of penetration were 2 cpm with 0.1% Vatsol surfactant and 4 cpm with no surfactant. Rates of
penetration of Zebrina leaves by H3P32O4 during a 5-min period were 787 cpm with 0.1% Vatsol sur-
factant and 155 cpm with no surfactant with stomates open and 321 cpm with 0.1% Vatsol surfactant
and 175 cpm with no surfactant with stomates closed (Dybing and Currier, 1961).
The absorption of 59Fe3+ was studied with tomato, sorghum, kidney bean, and small white
bean. Total stomatal area per unit leaf area was found to be a major factor in determining the
rate of foliar uptake of iron per unit dry weight or per unit of leaf area. There were distinct differences
in rates of uptake of iron by the four different plant species. Use of a surfactant caused a large increase
in iron uptake in both the sorghum and the red kidney bean leaves during the day. An increase also
occurred due to surfactant for the sorghum during the night. The essentially linear rate of uptake for
the first 30–40 min followed by a sharp decrease in rate is highly suggestive of a mass flow mecha-
nism. The sharp decrease in uptake rate may occur due to the filling of the substomatal chamber
with treatment solution. The poorer agreement between species as submersion time increases can be
explained as an expression of internal leaf characteristics, such as the size of the substomatal chamber
and the arrangement of the mesophyll cells surrounding the chamber (Eddings and Brown, 1967).
Studying cuticles of tomato fruit, with no stomatal pores, and onion leaves, with stomatal pores,
Yamada et al. (1964) found that 45Ca2+, 86Rb+ (an analogue for K+), 36Cl−, and 35 SO2- 4 moved more
rapidly from the outside of the cuticle to the inside of the cuticle, compared to movement of the
ions from inside the cuticle to outside. Moreover, they found that during a 40-h period, the rate of
movement of the four ions was initially rapid, declining with time. They concluded that the perme-
ability of the cuticular membranes to these four ions was greater from outside to inside, compared
to permeability of the cuticles from inside to outside. They observed that these differences were
greater in the tomato fruit cuticle, which lacks stomata, than in the cuticle of the green onion leaf,
which possesses stomata.
Penetration of monovalent cations was investigated using isolated leave cuticles of apricot
(P. armeniaca L.). The penetration rates of the monovalent cations in group IA followed a normal
lyotropic series, that is, Cs+ > Rb+ > K+ > Na+ > Li+. Absorption of 1 mM Rb+ and phosphate by leaves
of bean seedlings occurred during a 24-h period at constant rates. Rates were constant at 0.785 and
6.81 mμmol cm−2 leaf × hour for phosphate and Rb, respectively (McFarlane and Berry, 1974). The
absorption rates were obtained as the slopes of the linear regressions calculated by the least square
method (Jyung and Wittwer, 1964).
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18 Some Interactions of Mineral
Nutrients and Organic
Substances in Plant Nutrition
CONTENTS
18.1 Introduction........................................................................................................................... 355
18.2 Properties of the Essential Plant Nutrients............................................................................ 355
18.3 Properties of Soil Organic Matter......................................................................................... 356
18.4 Interaction of Mineral Nutrients and Organic Substances Near and at the Soil–Root
Interface................................................................................................................................. 358
18.5 Interaction of Mineral Nutrients and Organic Substances at the Leaf..................................360
18.6 Some Aspects of Transport of Mineral Nutrients in the Plant.............................................. 362
References....................................................................................................................................... 363
18.1 INTRODUCTION
The mineral nutrition of plants is dependent upon complex inorganic and organic sources of essen-
tial plant nutrients. Carbon is assimilated from the air by photosynthetic plants, and parasitic plants
absorb some or all of their nutrients from other plant or animal material. Essential plant nutrients
other than carbon are absorbed directly through cells in contact with the environment (e.g., roots,
leaves, and surface of cells of other organs) or from other plants, in the case of parasitic plants such
as Cassytha spp. or Striga spp. The acquisition of mineral nutrients can be directly facilitated by
other organisms, such as mycorrhizae, which can increase the surface area of the root system of a
plant, and indirect action by organisms such as fungi and bacteria can mineralize elements, making
them available for absorption by plants. The purpose of this chapter is not encyclopedic, but rather
to orient the reader regarding various aspects of interaction of mineral nutrients with organic sub-
stances in plant nutrition; further, in-depth information is available via the references.
18.2 PROPERTIES OF THE ESSENTIAL PLANT NUTRIENTS

There are 17 essential plant nutrients, and an element is essential if it fulfills either one or both
of two criteria: (1) it is part of a molecule that is an intrinsic component of the structure or
metabolism of a plant, or (2) plants deprived of this element exhibit abnormalities in growth,
development, or reproduction (Epstein and Bloom, 2005). For the purpose of better understand-
ing of interactions of organic (carbon-containing) substances with mineral nutrients (elements
other than carbon), it is useful to categorize the 17 essential plant nutrients as 9 metals and 8
nonmetals. The metals are calcium, copper, iron, magnesium, manganese, molybdenum, nickel,
potassium, and zinc, and the nonmetals are boron, carbon, chlorine, hydrogen, nitrogen, oxygen,
phosphorus, and sulfur. Of the metals, molybdenum is found principally in anionic form (MoO2- 4 )
355
in well-aerated agricultural soils with pH > 5.0 (Lindsay, 1972). Cationic competition in nutrient
uptake by plants was clearly shown by Martin (1933).
Plants accumulate more base cations (Ca2+, Mg2+, Na+, and K+) than anions from the soil, so
in the plant, the electrical imbalance between cations and anions is eliminated, or balanced, with
organic anions. In terms of equivalents of inorganic cations (Ci), inorganic anions (Ai), and organic
anions (OA), Ci – Ai – OA ≈ 0 in biomass. Inorganic cations (mainly K+ and Ca2+) and organic anions
(anions of weak acids such as malate and citrate) buffer the internal pH of plants in a physiological
range near pH 6.5. One consequence of the Ci – Ai imbalance in biomass, however, is a surplus of
Ai relative to Ci in soil, so for each organic anion created in plant metabolism, one H+ accumulates
in soil. Another cause of acidity produced as a result of biological activity in soils is acidity that
occurs when NH +4 is oxidized, producing H+ and NO3- as the result of microbial processes (Loomis
and Conner, 1992).
18.3 PROPERTIES OF SOIL ORGANIC MATTER

Soil organic matter (SOM) from outside the plant contains and interacts with mineral nutrients
outside the plant. SOM can be divided into nonhumic and humic substances, and SOM can facili-
tate transport and absorption of mineral nutrients. Nonhumic substances such as carbohydrates,
proteins, amino acids, fats, waxes, alkanes, and low-molecular-weight organic acids are readily
attacked by microorganisms and can disappear rapidly (Schnitzer, 1978). Humic substances in
soils comprise most of SOM, and they decompose slowly under natural conditions. The chemical
composition of SOM is approximately 50% C, 39% O, 5% N, 5% H, 0.5% P, and 0.5% S (w/w),
but these values can vary from soil to soil (Barber, 1995). There is a synergistic effect of min-
eral nutrients and humic substances on plant growth. Long-term experimentation since 1852 at
Rothamsted, United Kingdom, has shown that spring barley grown on soil that was amended with
farmyard manure yielded more barley grain than did the same soil that was fertilized only with
mineral (NPK) fertilizers (Johnston et al., 1994). Stimulatory effects of SOM on plant growth can
be considered indirect by improving supply of nutrients, improving soil structure, increasing soil
microbial population, increasing cation exchange capacity (CEC) and buffering capacity of the
soil, supplying defined biochemical compounds to plant roots, and supplying humic substances
that serve as carriers of micronutrients or growth factors. Direct stimulatory effects of humic
substances on plant growth include those that require uptake of organic macromolecules resulting
in various biochemical effects in the cell wall, cell membrane, or cytoplasm (Chen et al., 2004).
Some specific indirect effects of humic substances on mineral nutrition of crop plants include
(Stevenson, 1994)
• Thorough incorporation of N and S into stable structures of humic substances during

mineralization/immobilization
• Chemical transformations of inorganic N forms, namely, stabilization of N through chemi-
cal fixation of NH3 and conversion of NO2- - N to N2 and N2O through nitrosation
• Solubilization of phosphates by complexation of Ca in calcareous soils and Fe and Al in
acid soils
• Alleviation of metal ion toxicities, including Al3+ in acid soils
Direct or indirect effects of organic matter in soil can influence the incidence of pathogenic
organisms, since an abundant supply of organic matter may result in more growth of sapro-
phytic microorganisms relative to pathogenic microorganisms, reducing populations of the latter.
Moreover, biologically active organic compounds in soil, such as antibiotics or certain phenolic
acids, may protect plants from soil-borne pathogens (Stevenson, 1994).
Organic matter is present as solid material in soils and is also present in soil solution. Humus that
can be extracted from mineral soils in alkali can be separated into humic and fulvic acids, which are
Some Interactions of Mineral Nutrients and Organic Substances in Plant Nutrition 357
procedural artifacts existing only in the laboratory. Another way of characterizing humic substances
is to use a mild step-wise fractionation of the components of supramolecules by a “humeomics”
approach using various advanced analytical techniques (Nebbioso and Piccolo, 2011). Particulate
organic matter, often used as an index of SOM, contains humic materials that are heterogeneous in
age and function. SOM can be categorized in pools and related fractions: labile or active SOM with
half-life of days to a few years; slow or intermediate SOM with half-life of a few years to decades;
and recalcitrant, passive, and stable SOM with half-life of decades to centuries (Wander, 2004). The
bulk of organic matter in most soils occurs as stable humus (Stevenson, 1994). A recent conference
in Hangzhou, China, was devoted to analytical and ecological aspects of natural organic matter (Xu
et al., 2012), and a partial bibliography of recent humic literature is available at the website of the
International Humic Substances Society (Olk, 2006).
The importance of CEC in soils is that it serves to store essential plant nutrient cations that can
then be released to the soil solution by cation exchange with other cations, such as hydrogen ion,
becoming available for uptake by plants. SOM, depending upon pH, may be more effective in cat-
ion exchange per unit mass than are clay minerals (Bohn et al., 1979). Metal cations exchange read-
ily with protons of the amino (–NH2), carboxyl (–COOH), and hydroxyl (–OH) groups of SOM.
Representative CEC values for clay range from 30 to 100 meq/100 g, while humus may be as great
as 300 meq/100 g (or cmol (+)/kg). Although humus usually constitutes only 2%–5% of the weight
of agricultural soils, it makes a significant contribution to the CEC (Loomis and Conner, 1992).
CEC, when viewed as the ability of a negatively charged surface to be saturated with hydrogen
ions, can be considered “acidity.” Soil scientists tend to view this property as “CEC,” whereas
many chemists view the same property as “acidity.” Humus is highly colloidal and amorphous,
rather than crystalline, and the absorptive capacities of humus are greater than those of layer
silicate minerals. The surface properties of humic materials are significant. For example, the ion
exchange capacities of well-developed humus can range from 150 to 300 meq/100 g, and surface
areas of well-developed humus may be as high as 800–900 m2/g (Bohn et al., 1979). There are
a variety of functional groups of organic matter in soils, including carboxyl (–COOH), phenolic,
hydroxyl (–OH), quinone, hydroxyquinone, lactone, ether, and alcoholic hydroxyl (–OH). Fulvic
acids have somewhat higher contents of –COOH groups than humic acids. The –COOH content
of humic substances appears to be inversely related to molecular weights, and the proportion of
oxygen that occurs in the form of –COOH is higher for fulvic acids than for humic acids. The
reactivity of humic substances, compared to that of clay minerals, is due in large measure to their
high content of oxygen-containing functional groups, including –COOH, phenolic- and/or enolic
–OH, alcoholic –OH, and the C=O of quinones, hydroxyquinones, and possibly α,β-unsaturated
ketones. Total acidities of fulvic acids (640–1420 cmol (+)/kg) are generally higher than total
acidities of humic acids (560–890 cmol (+)/kg, and the content of oxygen-containing functional
groups in fulvic acids appears to be substantially higher for any other naturally occurring organic
polymer (Stevenson, 1994). In 20 Colorado soils, the organic matter in soil solution was measured
in the range of 2–25 mmol (+)/L, and in 10 New York soils, organic matter in soil solution was
measured in the range of 15–75 μmol (+)/L (Hodgson et al., 1966).
Two types of cation–organic matter interactions are the interactions of mineral nutrient cations
with negatively charged carboxyl groups or hydroxyl groups and more complex interactions where
coordinate linkages with organic ligands are formed (Baldock and Nelson, 2000). Complexation of
inorganic cations by SOM can influence soil properties and processes in the following, among other,
ways: increased availability of insoluble mineral P through complexation of Fe3+ and Al3+ in acid
soil and Ca2+ in calcareous soil, competition for P adsorption sites, and displacement of adsorbed
P (Stevenson, 1994; Cajuste et al., 1996) and release of plant nutrients through weathering of rocks
and soil parent materials by the removal of structural cations from silicate minerals (Robert and
Berthelin, 1986; Tan, 1986). Recent investigation into the interaction of Fe with humic substances
of high molecular weight has shown that cucumber plants can absorb Fe2+ from Fe2+ complexed with
high-molecular-weight humic substances (Colombo et al., 2011).
18.4 INTERACTION OF MINERAL NUTRIENTS AND ORGANIC

SUBSTANCES NEAR AND AT THE SOIL–ROOT INTERFACE
The soil–root interface, or rhizosphere, is that zone of the soil influenced by the plant, as compared
to the “bulk soil,” which is the zone of soil that lies outside the rhizosphere zone and is not directly
influenced by growing roots except by the withdrawal of water and nutrients. Biological aspects of
the rhizosphere include the walls of the outermost root cells that form the inner boundary of the
interface. In relatively young roots, these tissues are the epidermis and root hairs, and other princi-
pal components of the rhizosphere are mucigel, soluble exudates, and the microbiological flora of
the rhizosphere and rhizoplane, the substrates of which are mucigel, soluble exudates, and, in some
cases, cellular contents of living plant tissues (Russell, 1977). The provision of plant nutrients by the
cycling of minerals is one of the most significant activities of microorganisms in soil (Lynch, 1983).
It has been proposed that the macronutrients, N, P, and S, are made available to plants in the soil
by the mineralization of these elements from organic matter through a dichotomous system involv-
ing both biological and biochemical mineralization (McGill and Cole, 1981). Biological mineraliza-
tion is the result of microbial decomposition of organic matter as an energy source, resulting in the
mineralization of N- and C-bonded S. Biochemical mineralization refers to the release of oxidized
forms of P and S—phosphate and sulfate—from the P and S ester pool via enzymatic hydrolysis
outside the cell membrane.
Rhizosphere studies have, in part, focused on the exudation of organic substances from
roots, which can increase the uptake of essential mineral nutrients. Several investigators have
reported that carbon released from roots growing in soil amounted to approximately 20% of
total plant dry matter (Rovira, 1979). Wheat roots have been reported to lose to the soil up to
39% of carbon translocated to the roots (Martin, 1977). In wheat and barley, with a 16 h pho-
toperiod, growth at 15°C constant or 18°C day/14°C night gave a loss of 33%–40% of the total
net fixed carbon (defined as 14C retained in the plant plus 14C lost from the root). Photoperiod
can affect the loss of organic compounds from the roots to the rhizosphere. With a 12 h pho-
toperiod and a temperature regime of 18°C/14°C, carbon loss from the roots was decreased to
17%–25% of the total fixed carbon. The proportion of 14C translocated to the roots that was
released into the soil did not change with temperature, so carbon distribution within the plant
must have changed (Whipps, 1984).
Although some P enters the soil solution through dissolution of precipitates of phosphate, decom-
position of organic matter is a more important source. Decay and mineralization of phytic acid and
polyphosphates occur slowly, but decay of nucleic acids (such as DNA and RNA) and phospholipids
is rapid (Lynch, 1983). Corn roots have been shown to be able to enrich the phosphatase activity
in the rhizosphere between 17% and 40%, which enables increased liberation of phosphorus from
organic sources. The experiment did not allow determination as to what proportion of the increased
phosphatase activity in the rhizosphere was due to exudate from the corn roots and what proportion
was contributed by soil microbes growing on or near the roots. Phosphatase activity, measured in
μmol/g/h was highest at the root surface and decreased by about 5% or less at a distance of 15 mm
from the root surface (Boero and Thien, 1979).
Rhizosphere acidification as a result of exudation of H+ by plant roots in an alkaline soil will
generally be the sole process stimulating the solubilization of alkaline rock phosphates and of native
or residual soil phosphates. Such rhizosphere acidification can result from (1) exudation of H+ due
to (a) excess cation-over-anion uptake, (b) excess cation-over-anion uptake as a consequence of NH +4
nutrition, and (c) excess cation-over-anion uptake as a consequence of symbiotic N2 fixation; or
(2) exudation of organic acids as a reaction to P starvation (Van Diest, 1991).
Biological fixation of gaseous N (N2) in nodules of legumes (plants in the family Fabaceae
[identified by others as Leguminosae]) by nitrogen-fixing bacteria is an organic system that pro-
duces ammonia that is assimilated into the amide group of glutamine. Most often, glutamine is fur-
ther metabolized to asparagine in temperate leguminous plant species and into ureides, allantoin,
and allantoic acid in tropical species. These solutes are the principal forms of N translocated from
the nodules through the xylem (Atkins, 1987). At least six genera of diazotrophic (N2-fixing) bac-
teria have been isolated from the roots of grasses and cereals of agricultural importance (Boddey
and Döbereiner, 1988). In 1984, experimentation conducted with nonleguminous plants, specifi-
cally wheat, has shown that inoculation of wheat roots with Bacillus C-11–25 resulted in 23.9% of
the N absorbed by the crop was derived from the atmosphere (Rennie and Thomas, 1987). Besides
legumes that are nodulated by Rhizobium species, about 200 plant species covering 8 families
and at least 17 genera in the tropics and subtropics are nodulated by N2-fixing actinomycetes of
the genus Frankia (Peoples and Craswell, 1992). Nonsymbiotic nitrogen fixation is another aspect
of the interaction of organic matter and mineral nutrients near the root surface. Clostridium pas-
teurianum, isolated in 1893, Azotobacter choococcum, Azomonas agilis, and other free-living
microorganisms, including some blue–green algae and yeasts, can convert atmospheric nitrogen
to ammonia, which is then available to plants. Bacteria of the genera Beijerinckia, Azotobacter,
and Clostridium comprise the major groups of free-living, nitrogen-fixing bacteria that can make
nitrogen available for uptake by plant roots (Curl and Truelove, 1986). Bacteria of the genus
Azospirillum, also aerobic, can inhabit the cortical layers of root tissue and utilize the root exu-
date energy source as they benefit the plant by fixing substantial quantities of atmospheric nitro-
gen (Atlas and Bartha, 1981).
Plant growth-promoting rhizobacteria (PGPR) are about 2%–5% of rhizobacteria (Kloepper
and Schroth, 1978). While all PGPR are free-living bacteria, some of them invade tissues of liv-
ing plants and cause unapparent and asymptomatic infections (Sturz and Nowak, 2000). PGPR
may induce plant growth promotion by direct or indirect modes of action (Beauchamp, 1993;
Kloepper, 1993; Kapulnik, 1996; Lazarovits and Nowak, 1997). Direct modes of action include
production of stimulatory bacterial volatile compounds and phytohormones, lowering of the eth-
ylene level in the plant, improvement of the plant nutrient status (liberation of phosphates and
micronutrients from insoluble sources, and nonsymbiotic nitrogen fixation), and stimulation of
disease-resistance mechanisms. Indirect effects originate in cases such as when PGPR act like
biocontrol agents reducing diseases and when they stimulate xenobiotics in inhibitory contami-
nated soils (Jacobsen, 1997). PGPR have been classified as biofertilizers (increasing the availabil-
ity of nutrients to plants), phytostimulators (plant growth promoting, usually by the production
of phytohormones), rhizoremediators (degrading organic pollutants), and biopesticides (control-
ling diseases mainly by the production of antibiotics and antifungal metabolites) (Somers et al.,
2004). Among the PGPR, Azospirillum species head the list of bacterial biofertilizer products,
yet except those formulated with Azospirillum, all bacterial biofertilizer products are applied as
biopesticides or as biocontrol agents (Burdman et al., 2000; Lucy et al., 2004). To develop PGPR
biofertilizers, the strain(s), inoculum production, and, in general, development of appropriate
formulations and strategies of field experimentation are fundamental conditions for a successful
application of PGPR species, at least in the case of Azospirillum inoculants (Fuentes-Ramirez and
Caballero-Mellado, 2006).
Root exudates include carbohydrates, amino acids, organic acids, nucleotides, flavonones,
enzymes, and miscellaneous compounds, among which are hydrocyanic acid, glycosides, and sapo-
nins, which are toxic to microorganisms. Root exudates can stimulate or inhibit the growth of soil
microorganisms (Rovira, 1965). Organic acids have a prominent role in cell metabolism, and they
affect soil pH and soil microbial activity. Moreover, organic acids are good metal-chelating com-
pounds, and they play an important role in the absorption and translocation of mineral nutrients
(Curl and Truelove, 1986).
Hunt et al. (1986) have identified five classes of chemical bonds in humic substances (C–C, N–C,
S–C, S–O–C, and P–O–C). Microorganisms oxidizing carbon provide energy to mineralize the
compounds characterized by the first three types of bonds. Compounds with sulfur and phosphorus
atoms present as esters can be mineralized by the action of extracellular hydrolases, according to
the need of the element (McGill and Cole, 1981).
18.5 INTERACTION OF MINERAL NUTRIENTS AND

ORGANIC SUBSTANCES AT THE LEAF
Materials such as urea, antibiotics, and herbicides applied to plant foliage may, depending upon the
plant species and the nature of the chemical compound, be translocated to the roots from which
either the compound or a by-product may be released to the rhizosphere where it can affect microbes
(Curl and Truelove, 1986). The “skin” of plant leaves is known as the cuticle, a continuous extracel-
lular membrane made of highly recalcitrant material that can easily resist decay for millions of years
under favorable conditions (Edwards et al., 1996; Ewbank et al., 1996). The major functions of the
plant cuticle include transpiration control, control of loss and uptake of polar solutes, controlling
the exchange of gases and vapors, transport of lipophilic substances, water and particle repellence,
attenuation of photosynthetically active radiation and UV radiation, mechanical containment, sepa-
rating agent in plant development, and serving as an interface for biotic interactions (Riederer, 2006).
Penetration of the cuticle of leaves by nonpolar compounds and polar compounds, such as inor-
ganic ions and charged organic compounds like organic acids or amino acids, occurs via different
paths in the cuticle (Schreiber, 2006). The lipophilic transport path is composed of lipophilic cutin
and wax domains, and a polar transport path is presumed to be composed of polar aqueous pores
(Schreiber et al., 2001; Schönherr and Schreiber, 2004; Schlegel et al., 2005). While the chemical
nature of polar domains in cuticles is still being determined, neither the size nor the chemical nature
of the postulated paths of transport was known in 2006. Nonetheless, plant cuticles are known to
contain polar functional groups that may account for polar aqueous pores. One hypothesis is that
nonesterified carboxyl and/or hydroxyl groups of cutin monomers or wax molecules could con-
tribute to the formation of polar transport paths. Another hypothesis is that polar carbohydrates
known to be associated with isolated cuticles could form the basis for polar transport paths, possibly
extending through the lipophilic cuticle from the epidermal cells to the outer surface of the cuticle,
forming sites where polar compounds such as inorganic nutrients or organic acids could diffuse
through the cuticle to the cells contained by the epidermal cell membranes (Schreiber, 2006).
One factor in the absorption of mineral nutrients in leaves is the presence of ectodesmata, which
are plasmodesmata in the outer walls of epidermal cells. Ectodesmata are microscopic channels in
the walls of plant cells, allowing movement of solutes between them. Leaves of Plantago major and
Helxine soleirolii were examined in connection with foliar absorption, and leaf structures such as
guard cells, conical hairs, anticlinal walls, and the epidermal cells adjacent to the leaf veins were
shown to consistently contain large numbers of ectodesmata, while in neighboring cells ectodes-
mata were low in number or lacking. The ectodesmata were explained by the investigator to be a
means by which mineral nutrients could enter the leaves from their surface (Franke, 1961).
Cuticular membranes, the first barriers in nutrient uptake by leaves, appear highly permeable
to monovalent and divalent cations and anions. Yamada et al. (1964) in experiments with Ca45Cl2,
Rb86Cl, FeS35O4, or RbCl36 and green onion leaf cuticles and tomato fruit cuticles found that pen-
etration of the inorganic cations and anions studied occurred equally well through cuticles from
plant surfaces with or without stomata. These investigators found that absorption (penetration from
the outside surface inward) occurred more readily than leaching (penetration from the inside sur-
face outward), and they surmised that uptake dominates loss of nutrients through foliar surfaces.
Active uptake of phosphorus, and rubidium, an analogue of potassium, into leaf tissue has been
measured in primary leaves of bean seedlings. Criteria for active uptake included time-course
analysis, temperature, oxygen, energy dependence, sensitivity to metabolic inhibitors, accumulation
against a concentration gradient, irreversibility, and pH dependence. The results suggest that the
overall process of foliar absorption of rubidium and phosphate by bean leaves, beginning with zero
time and extending over a 24 h period, is metabolic. The investigators proposed that carriers play an
important role in uptake and that they are proteinaceous (Jyung and Wittwer, 1964).
Urea, ammonium, and nitrate are common forms used for foliar application of nitrogen to fertil-
ize crops. Norway spruce shoots that included both needles and twigs absorbed ammonium ions,
and there was no evidence that ammonium uptake was accompanied by exchange for base cat-
ions such as calcium (Wilson, 1992). Pathways for the absorption of solutes are known to exist
in the region of radial rays across the twig bark (Klemm, 1989). Neither spruce nor pine shoots
absorbed nitrate (Wilson, 1992). The fact that ammonium was absorbed by the stems of spruce
and pine, but nitrate was not, suggests that the form of nitrogen used for foliar fertilization may
be important in determining the efficiency of foliar nitrogen fertilizers. Uptake of urea by foliar
application can be toxic to plants. Leaf-tip necrosis often observed after foliar fertilization of
soybean plants with urea is usually attributed to ammonia formed through hydrolysis of urea by
plant urease. Research using a urease inhibitor demonstrated that although addition of this urease
inhibitor to foliar-applied urea increased the urea content and decreased the ammonia content
and urease activity of soybean (Glycine max. (L.) Merr.) leaves fertilized with urea, it increased
the leaf-tip necrosis observed after fertilization. The investigators concluded that this necrosis
resulted from accumulation of toxic amounts of urea rather than from formation of toxic amounts
of ammonia (Krogmeier et al., 1989). Ammonium was absorbed more rapidly than was nitrate
by Ricinus through the stomates and cuticle around the stomates, although the rates of uptake of
both were slow (Peuke et al., 1998).
Salicylic acid and zinc, when applied to the leaves of mungbean (Vigna radiata L. Wilczek), were
found to significantly (p ≤ 0.05) increase plant height, number of branches/plant, number of pods/
plant, number of seeds/pod, 1000 seeds weight, seed weight/plant, and seed yield/ha as compared
with control (untreated plants), and the superiority was due to the high salicylic acid concentration
(150 ppm). Significant (p ≤ 0.05) increases in all the earlier-mentioned traits occurred with foliar
application of zinc, as compared to untreated plants. Based on the experimental results, the investi-
gators recommended foliar application of salicylic acid and zinc to mungbeans, using salicylic acid
and zinc at concentrations of 150 and 400 ppm, respectively (Ali and Mahmoud, 2013).
The size of the hydrated ion is a major factor affecting foliar absorption of mineral nutri-
ents. Mandair and Harris (2003) investigating foliar application of N, P, K, Na, and glucose with
Prosopis chilensis found that the most important factor affecting foliar mineral uptake was the
size of the hydrated ion. They note that it is well established that the cuticular penetration rates of
mineral nutrients are inversely related to the radius of the hydrated ion (Swietlik and Faust, 1984;
Kannan, 1986), an observation with which their results concurred. Of the four mineral nutrients
examined, K had the smallest hydrated ionic radius at 1.88 Å and was most readily absorbed.
K uptake was followed in descending order by Ca, NO3-, and PO3- 4 ions, which exhibit succes-
sively larger hydrated ionic radii. As early as 1969, Kannan (1969) reported that for 14C-labeled
molecules, rates of penetration of the cuticlular membranes enzymatically isolated from tomato
fruit were higher for smaller molecules. A negative correlation between molecular weights of the
organic solutes and their rates of penetration was obtained by regression analysis. Higher cor-
relation was obtained for penetration through cuticles from the stomatous surfaces than for those
from the astomatous surfaces.
The form of iron used to supply the element through foliar application is important, since the size
of the compound containing iron affects the rate at which the element moves through the cuticle of
the leaf. Experimentation with 59Fe and intact bean leaves has shown that while specific absorption
of 59FeSO4 and 59FeSO4 + urea (10 mM) was greater than that of 59Fe-EDTA and 59Fe-EDDHA,
the percentages of the two chelated forms of iron were greater than those of the two 59FeSO4 forms
(Wittwer et al., 1965). Kannan (1969) observed that the penetration of Fe through cuticles of tomato
fruit was reduced by chelation with EDDHA, the presence of urea, or both. He speculated that the
chelated form of Fe was less penetrable because of its greater molecular size. In earlier studies
(Wittwer et al., 1965), uptake of Fe from FeEDDHA was also very much less than that from FeSO4.
This poor absorption of Fe from FeEDDHA by leaves of intact bean plants could be related to low
permeability of cuticles to FeEDDHA, as obtained herein with isolated cuticles (Kannan, 1969).
Naturally occurring chelates such as proteins, amino acids, organic acids, flavonoids, purines,
and riboflavin can facilitate the absorption of cationic plant nutrients, and the stability of metal
chelates, or the replacing power of the elements is Fe3+ > Cu2+ > Zn2+ > Fe2+ > Mn2+ > Ca2+ > Mg2+, so
in a solution, the ferric ion would be expected to replace other ions, if all were in equivalent amounts
(by charge) (Hsu, 1986).
18.6 SOME ASPECTS OF TRANSPORT OF MINERAL NUTRIENTS IN THE PLANT

To maintain the concentration of essential metals within physiologically tolerable limits and to min-
imize their detrimental effects, all eukaryotes have evolved a number of mechanisms that control
the uptake, accumulation, trafficking, and detoxification of metals. The main components of metal
homeostasis are transport, chelation, and compartmentation (Shingles et al., 2004).
As living cells constantly exchange energy, matter, and information with their environment,
the plasma membrane surrounding them cannot be impermeable and is, indeed, a selective barrier
though which many solutes can be transported, sometimes against a concentration gradient. This
active transport requires the plasma membrane to be energized by cation-translocating adenosine
triphosphatases (ATPases) and, possibly to a lesser extent, by redox reactions. Cation-translocating
ATPases reside inside the plasma membrane, and they actively pump cations against their concen-
tration gradient. The process is powered by hydrolysis of adenosine triphosphate (ATP) to adenos-
ine diphosphate, and inorganic phosphate (Pi) for transported cations such as H+, Ca2+, Na+/K+,
and H+/K+ (Michelet and Boutry, 1996). In the roots, minerals are absorbed by root hair, epidermal,
and cortical cells (Taiz and Zeiger, 1991), and once the mineral nutrients are in the cell, they are
transported to the stele. It appears that H+-ATPase activity is needed not only in the cortex and
epidermis of the roots for ion absorption, but also in the stele for loading ions into the conductive tis-
sues (Parets-Soler et al., 1990; Samuels et al., 1992; Cowan et al., 1993). As an example of the abil-
ity of ATPases to transfer mineral nutrient ions against concentration gradients, the concentration
of K+ in the soil solution is often below 100 μM, and the intracellular concentration of K+ in cells
within the plant is on the order of 100 mM, 1000-fold greater than outside the plant (Maathuis and
Sanders, 1993). Uptake of K+ occurs across plasma (cell) membranes by two transports systems—
low affinity and high affinity—demonstrated by Epstein et al. (1963).
During early growth of soybean, it has been shown using radioactive 14C tracers that organic
compounds containing nitrogen can be transferred directly from the xylem to the phloem, an indi-
cation that inorganic N compounds may also be transferred directly from the xylem to the phloem
(Da Silva and Shelp, 1989–1990). Organic N compounds produced in root nodules of legumes have
been shown to be mobile in both the xylem and the phloem of soybean (Shelp and Da Silva, 1990).
In pea shoots, some amino acids arriving via the xylem in the mature leaves were translocated from
the mature leaves to the young leaves via the phloem with relatively little metabolic conversion.
Substantial amounts of another amino acid, glutamine, were converted to glutamate, which was
exported (with unchanged amide) to younger leaves with little further conversion (Urquhart and
Joy, 1982).
Zinc is the most common crop micronutrient deficiency, particularly in high-pH soils with low
[Zn]bss(Graham et al., 1992; White and Zasoski, 1999; Cakmak, 2002, 2004; Alloway, 2004). Fifty
percent of cultivated soils in India and Turkey, a third of cultivated soils in China, and most soils in
Western Australia are classed as Zn deficient (Broadley et al., 2007). In plant cells, high Zn-status
leaf epidermal cell vacuoles, cell walls, and cytoplasm can contain, respectively, 74,305, 11,577,
and 3,205 μg Zn/g DW (dry weight); lower Zn-status leaf mesophyll cell vacuoles, cell walls, and cyto-
plasm contain, respectively, 327, 9353, and ≤262 μg Zn/g DW; and root cortical vacuoles, cell walls,
and cytoplasm contain, respectively, ≤262, 589, and ≤262 μg Zn/g DW (Freye et al., 2000). Several
studies have shown no correlation between zinc efficiency and root Zn uptake, Zn translocation to
shoot, or shoot Zn accumulation. While it has not been possible to conclusively link differences in leaf
subcellular Zn compartmentation with zinc efficiency of plants, biochemical Zn utilization, including
the ability to maintain the activity of Zn-requiring enzymes in response to Zn deficiency, may be a key
component of zinc efficiency.
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Part IV
Physiological Responses of Plants/Crops
under Stressful (Salt, Drought, Heat,
Nutrient Deficiency, and Other
Environmental Stresses) Conditions
19 Role of Polyamines in Plant
Abiotic Stress Responses
Bhaskar Gupta, Kamala Gupta, and Bingru Huang
CONTENTS
Abbreviations.................................................................................................................................. 369
19.1 Introduction........................................................................................................................... 370
19.2 PA Biosynthesis and Catabolism in Relation to Abiotic Stress Tolerance............................ 371
19.3 Polyamines Involved in Abiotic Stress Signaling and Responses......................................... 373
19.3.1 Salt Stress................................................................................................................... 374
19.3.2 Osmotic Stress........................................................................................................... 375
19.3.3 Water Stress............................................................................................................... 376
19.3.4 Extreme Temperature Tolerance................................................................................ 378
19.3.5 UV Stress................................................................................................................... 379
19.3.6 Oxidative Stress......................................................................................................... 380
19.3.7 Mineral Deficiency and Heavy Metal Tolerance....................................................... 380
19.3.8 Mechanical Stress...................................................................................................... 381
References....................................................................................................................................... 382
ABBREVIATIONS
ABA Abscisic acid
ABF ABRE-binding factor
ABRE ABA-responsive elements
ACC Oxidase 1-amino-cyclopropane-1-carboxylic- acid oxidase
ACC Synthase 1-amino-cyclopropane-1-carboxylic- acid synthase
ADC Arginine decarboxylase
AdoMet S-adenosyl-l-methionine
AdoMetDC AdoMet decarboxylase
AIH Agmatine iminohydrolase
COR Cold responsive
CPA N-carbamoylputrescine amidohydrolase
CuAO Copper-containing amine oxidases
DAO Diamine oxidases
DAP Diaminopropane
DcAdoMet Decarboxylated S-adenosyl-l-methionine
DHH Deoxyhypusine hydroxylase
DHS Deoxyhypusine synthase
DRE Dehydration-responsive element
GABA γ-Aminobutyric acid
H2O2 Hydrogen peroxide
LAT l-Type amino acid transporter
369
MAP Mitogen-activated protein

MAPK Mitogen-activated protein kinase
MAT Methionine adenosyltransferase
m-RNAs Messenger ribonucleic acids
NO Nitric oxide
ODC Ornithine decarboxylase
PA Polyamine
PAO Polyamine oxidases
PEG Polyethylene glycol
Pro Proline
PT Permeability transition
Put Putrescine
RMV1 Resistant to methyl viologen 1
ROS Reactive oxygen species
SAM S-adenosylmethionine
SAMDC S-adenosylmethionine decarboxylase
SMO Spermine oxidase
Spd Spermidine
SPDS Spermidine synthase
Spm Spermine
SPMS Spermine synthase
TMV Tobacco mosaic virus
uORFs Upstream open reading frames
19.1 INTRODUCTION
Abiotic stresses, especially salinity and drought, are the significant causes of crop loss worldwide.
Maintaining crop yields under adverse environmental stresses is probably the major challenge faced
by modern agriculture where polyamines (PAs) can play an important role (Ahmad et al. 2012).
Plant adaptation to environmental stress is dependent upon the activation of cascades of intricate
molecular networks involved in stress perception, signal transduction, and the molecular expression
of specific stress-related genes and metabolites (Vinocur and Altman 2005). Stress-induced gene
expression leads to the synthesis of enzymes for the synthesis of compatible solutes such as amino
acids, sugars, alcohols, and PAs (Quinet et al. 2010).
PAs, including the diamine putrescine (Put2+), triamine spermidine (Spd3+), and tetramine
spermine (Spm4+), are ubiquitous, low-molecular-weight, straight-chain aliphatic amines, which
are involved in various molecular, biochemical, and physiological processes controlling plant
growth, development, and abiotic stress responses (Alcázar et al. 2010a; Hussain et al. 2011; Gupta
et al. 2013). Because of their polycationic nature at physiological pH, PAs are able to interact with
nucleic acids, acidic domains of proteins, membrane phospholipids, and cell wall constituents,
thereby stabilizing these molecules and elucidating diverse functions (Flink and Pettijohn 1975;
Roy et al. 2005; Gupta et al. 2012a,b; Gupta et al. 2013). The unique feature of PA structure com-
pared to inorganic cations like Mg2+ or Ca2+ is that they have methylene groups harbored between
them and the positive charges are at defined distances. These methylene groups have been shown
to participate in hydrophobic interactions and thus influence PA activity as a whole (Wallace et al.
2003). Recently it was shown that plant and mammalian PA metabolisms share critical features,
giving new insights into plant PA biosynthesis and breakdown (Moschou et al. 2008). The inter-
action between PAs and plant cellular membranes is suggested to be an intermediate in impor-
tant cellular events such as membrane fusion, functioning of tonoplast proton pumps (Schuber
1989; Roy et al. 2005; Janicka-Russak et al. 2010), and transmission of receptor-mediated signals
(Koenig et al. 1983). Ivanov et al. (2010) reported the presence of “upstream” open reading frames
Role of Polyamines in Plant Abiotic Stress Responses 371
(uORFs) in mRNAs of PA biosynthesis, which has been shown to act as sensors for regulatory cir-
cuits and amplify signals. These autoregulatory circuits exemplify the dynamic interface between
components of the proteome and metabolism, regulated by translation.
A large body of literature has revealed the potential roles of PAs in myriad biological processes,
including cell division and differentiation, cell proliferation, homeostasis, gene expression, ion
channel regulation, protein phosphorylation, macromolecular synthesis, control of cell cycle, and
apoptosis (Cohen 1998; Igarashi and Kashiwagi 2000; Childs et al. 2003; Kuehn and Phillips 2005;
Bachrach 2010; Mattoo et al. 2010; Gupta et al. 2012a,b). PA metabolism has been extensively
investigated in plants and other organisms by many research groups (Bagni and Tassoni 2001;
Martin-Tanguy 2001; Hanfrey et al. 2001; Illingworth et al. 2003; Wallace et al. 2003; Bortolotti
et al. 2004; Alcázar et al. 2005, 2011a; Knott et al. 2007; Zahedi et al. 2007; Kakehi et al. 2008;
Kusano et al. 2008; Casero and Pegg 2009; Takahashi et al. 2010; Angelini et al. 2010; Hewezi et al.
2010; Igarashi and Kashiwagi 2010; Quinet et al. 2010; Toumi et al. 2010; Fincato et al. 2011) and
has been elaborately reviewed by Hussain et al. (2011) and Gupta et al. (2013). Therefore, this chap-
ter reviews PA biosynthesis and catabolism in the context of stress responses of plants and provides
a comprehensive review of recent literature on the functions and roles of PAs in the regulation of
plant tolerance to various environmental stresses.
19.2 PA BIOSYNTHESIS AND CATABOLISM IN RELATION

TO ABIOTIC STRESS TOLERANCE
Since the time, many decades ago, an increase in Put levels in plants due to potassium deficiency
was reported; PAs have been extensively studied when plants are exposed to single or combined
stresses (Rowland-Bamford et al. 1989; Tiburcio et al. 1994; Scalet et al. 1995; Nam et al. 1997;
Santa-Cruz et al. 1997; Shen et al. 2000; Mo and Pua, 2002; Urano et al. 2003; Camacho-Cristobal
et al. 2004; Kuthanova et al. 2004; Liu et al. 2006). Bouchereau et al. (1999) showed that stress led
to the accumulation of free or conjugated PAs, pointing to the fact that PA biosynthesis might act
as indispensable component of stress-induced plant responses. In the past few years, genetic and
molecular studies using mutants and transgenic plants with altered activity of enzymes involved
in PA biosynthesis have contributed immensely to a better understanding of the biological func-
tions of PAs in plants (Capell et al. 2004). A number of authors have reported the relationship of
PA metabolism with plant responses to various environmental stimuli, such as drought, salinity,
heavy metal toxicity, chilling, light intensity, etc. Increased PA biosynthesis has been linked
to increased plant resistance against drought and salt stresses (for reviews, see Martin-Tanguy
2001; Wallace et al. 2003; Alcazar et al. 2006b, 2010a; Kusano et al. 2008; Hussain et al. 2011;
Tavladoraki et al. 2012; Gupta et al. 2013).
The biosynthesis of plant PAs has been well documented, encompassing decarboxylation of orni-
thine or arginine, catabolized by ornithine or arginine decarboxylases (ODC and ADC) respectively
to yield diamine Put. Apart from being a precursor of arginine biosynthesis, ornithine also acts as
a precursor of other important metabolic pathways like, PA biosynthesis via ODC and also, gluta-
mate and proline via ornithine δ-aminotransferase activities (Kalamaki et al. 2009). The resulting
intermediate agmatine, synthesized from arginine, is subsequently converted to Put, by agmatine
iminohydrolase (AIH) and N-carbamoylputrescine amidohydrolase (CPA). Spermidine (Spd) and
spermine (Spm) are formed by the sequential addition of aminopropyl groups to Put and Spd respec-
tively from decarboxylated S-adenosylmethionine (dcSAM) by SAM decarboxylase (SAMDC)
(Pang et al. 2007; Kusano et al. 2008; Hussain et al. 2011). Ethylene is produced from SAM via
1-amino-cyclopropane-1-carboxylic-acid (ACC) with the help of ACC synthase and ACC oxidase
(Figure 19.1; Alcazar et al. 2006a,b). It has been extensively studied that the cellular PA content
in plants has been modulated by either overexpression or downregulation of the key genes ADC,
ODC, or SAMDC (Kumar et al. 1997; Walden et al. 1997; Capell et al. 1998; Kumar and Minocha
1998; Malmberg et al. 1998; Rajam et al. 1998; Roy and Wu, 2001; Bhatnagar et al. 2002). PAs are
Arginine Ornithine
Arginine
CO2
decarboxylase
Agmatine CO2
Ornithine
Agmatine Succinate
decarboxylase
iminohydrolase
DAO NAD+ NADH

Putrescine
N-Carbamoylputrescine NH2(CH2)4NH2 D1-pyrroline GABA
N-Carbamoylputrescine amidohydrolase O2+H2O H2O2 PDH
CO2
S-Adenosylmethionine Decarboxylated
(AdoMet) AdoMet
SAMDC
Spd synthase
1,5-Diazabicylononane
1-(3-Aminopropyl)-pyrroline (Propylamino transfer)

O2+
H2O2 O2+ b-Alanine
H2O PAO
H 2O H2O2
Spermine Spermidine
NH2(CH2)3NH(CH2)4NH(CH2)3NH2 1,3-Diaminopropane
NH2(CH2)3NH(CH2)4NH2
Spm PAO
synthase
FIGURE 19.1 Metabolic pathway of polyamines. The anabolic pathway is denoted with dark arrows and
catabolic pathway with lighter arrows respectively.
also synthesized parallelly from the amino acid, methionine. Minois et al. (2011) have illustrated
this metabolic pathway as described here. l-methionine is converted to S-adenosyl-l-methionine
(AdoMet) by methionine adenosyltransferase, which in turn is converted by AdoMet decarboxylase
(AdoMetDC) to decarboxylated S-adenosyl-l-methionine (DcAdoMet). This DcAdoMet now func-
tions as a donor of aminopropyl group, either to putrescine to produce spermidine by spermidine
synthase, or to spermidine to produce Spm by spermine synthase (SPMS).
Apart from biosynthesis, degradation of PAs plays a crucial role in the regulation of cellular PA
titers, which is mainly ascribed to the two amine oxidases, namely, diamine oxidase (DAO) and
polyamine oxidase (PAO) (Liu et al. 2007). Put undergoes oxidation in the presence of DAO to give
pyrroline, which is further metabolized to γ-aminobutyric acid (GABA), which is again metabo-
lized to succinate (Cona et al. 2006). On the other hand, PAO catalyzes the conversion of Spd to
pyrroline and that of Spm to 1-(3-aminopropyl)-pyrroline, along with 1, 3-diaminopropane in plants
(Figure 19.1; Martin- Tanguy 2001; Sebela et al. 2001).
Minois et al. (2011) have extensively reviewed and documented PA catabolism too. They have
observed that the higher-order PAs, like Spm and Spd, can well be converted back to Put. The rate-
limiting enzyme of PA degradation, spermidine/spermine N1-acetyltransferase (SSAT), which is
present in the cell cytosol, is integral for the formation of Put from Spd. The enzyme SSAT acety-
lates Spm and Spd, which then enter into the peroxisomal space, where PAO oxidizes them. H2O2
and acetaminopropanal are by-products of this oxidation. Back-conversion of Spm to Spd can also
be carried out by spermine oxidase (SMO) in the cell cytoplasm. Unlike PAO, the preferred sub-
strate of SMO is Spm and not its acetylated form, acetylspermine. They have also demonstrated
the synthesis of eiF5A from Spd. It is a two-step process, where, first, Spd is converted to eiF5A-
deoxyhypusine by deoxyhypusine synthase (DHS), and then deoxyhypusine hydroxylase converts
eiF5A-deoxyhypusine to eiF5A-hypusine.
Both PA synthesis and breakdown are responsive to abiotic stresses, and different pathways
may be affected by different types of stresses. Figure 19.2 gives a gist of various abiotic stress
Ion channel regulation

Environmental stimuli
Wa Protein phosphorylation
ter
str
ess
Met
al st Macromolecular synthesis
ress
Osmotic
stress
Salinity stress Regulation of gene expression
Increase in
ss polyamine content
ive stre
Oxidat ss
stre ABA biosynthesis
UV ss
tre
e at s ess
H str
al Induction of stomatal closure
a nic
h
ec
M
Antioxidant activity
FIGURE 19.2 Role of polyamines in several metabolic processes owing to different environmental stimuli.
Different forms of abiotic stress cause increase in the PA content. This results in an array of cellular response
leading to physiological as well as metabolic changes within the plant.
factors affecting cellular PA content and the plant biochemical response. Plant adaptation to abi-
otic stress is controlled by cascades of molecular networks. These intricate networks work syn-
ergistically to activate various stress-responsive mechanisms to reestablish cellular homeostasis
and to protect and repair damaged membranes and proteins. The initial environmental signals
(e.g., extremes of temperature, osmotic stress, dehydration) stimulate/activate various stress-
responsive genes via a plethora of osmosensors (second messengers, protein kinases, transcrip-
tion factors, and osmoprotectants) triggering downstream physiological and molecular response
at the transcriptional and translational levels. PAs are reported to be coherent part of this intricate
molecular network, and their ameliorative effect against abiotic stressors is elicited through pro-
tein phosphorylation, ion channel regulation, macromolecular synthesis, gene expression regula-
tion, antioxidant activity, etc. (Figure 19.2). Inadequate responses at one or more steps in the
signaling and gene activation process might ultimately result in irreversible changes in cellular
homeostasis and in the destruction of functional and structural proteins and membranes, leading
to cell death (Vinocur and Altman 2005). For an orderly discussion, PAs’ responses to different
abiotic stresses and their involvement in plant tolerance to individual stress are discussed in the
following text.
19.3 POLYAMINES INVOLVED IN ABIOTIC STRESS

SIGNALING AND RESPONSES
In response to abiotic stresses, such as salinity, high temperature, drought, freezing, heavy metal
toxicity, and other anaerobic stresses, plants display a host of signaling components, including PAs
(Grover et al. 2001). Much attention of recent research has also been devoted to the involvement of
PAs as second messengers in response to different abiotic stresses such as osmotic stress, drought,
heat, chilling, high light intensity, mineral nutrient deficiency, heavy metals, pH variation, UV irra-
diation, and exposure to pollutants (Groppa and Benavides 2008; RoyChoudhury et al. 2008, 2011;
Igarashi and Kashiwagi 2010; Hussain et al. 2011; Gupta et al. 2012a,b; Gupta et al. 2013). PA may be
involved in stress signaling through the interaction with abscisic acid (ABA), which has a pivotal role
as a regulator of abiotic stress signaling (Cutler et al. 2010; Hubbard et al. 2010; Raghavendra
et al. 2010; Umezawa et al. 2010). As shown by Xiong et al. (1999), salt-, cold-, and drought-induced
signaling pathways in plants interact with ABA and further converge at multiple steps. The presence
of stress-responsive, drought-responsive (DRE), low-temperature-responsive, and ABA-responsive
elements (ABRE and/or ABRE-related motifs) has been reported in the promoters of the PA biosyn-
thetic genes. PAs such as Put modulate ABA biosynthesis in response to abiotic stress. This reinforces
the view that in response to drought and salt treatments, the expression of some of the genes involved
in PA biosynthesis is regulated by ABA (Alcázar et al. 2006b). Activation of an SnRK protein in
rice cultivars by NaCl, ABA, and Spd has been reported by Gupta et al. (2012a,b), which exemplifies
the involvement of ABA and PAs in abiotic stress signaling. They hypothesized that salinity stress,
which causes an increase in the level of Spd and ABA in vivo, differentially activates members of
SnRK2 group of protein kinases, which in turn upregulates the expression of many stress-responsive
genes through stimulation of a wide array of transcription factors. One such mechanism is by phos-
phorylation of different trans-acting factors, as evident in this report by the activation of OSBZ8 by
OSPDK. It is interesting to note that three completely different compounds like Spd, ABA, and NaCl,
with variable ionic and structural properties, could give similar phosphorylation signals (Gupta et al.
2012a,b). The over-expression of plant specific SnRK2 gene family members by hyperosmotic stress
and some by abscisic acid is well established. In a recent report, Saha et al. (2013) have analyzed the
evolution of SnRK2 gene family in different plant lineages including green algae, moss, lycophyte,
dicot and monocot. Their results provide some evidences to indicate that the natural selection pres-
sure had considerable influence on cis-regulatory promoter region and coding region of SnRK2 mem-
bers in Arabidopsis and Oryza independently through time. Hydrogen peroxide (H2O2), derived from
PA catabolism, also exerts signaling effects (Moschou et al. 2008) as it is capable of diffusing from
its site of production to neighboring cells and tissues thereby activating a defensive response (Neill
et al. 2002). Similarly, nitric oxide (NO) is also known to trigger several signal transduction pathways
including cyclic nucleotides, Ca2+ ions, and protein kinases in response to stress in plants. Tun et al.
(2006) reported PAs, like Spm and Spd, to be potent inducers of NO-mediated signals. ABA synthe-
sis in roots of wheat plants subjected to drought was much higher in the presence of NO and reactive
oxygen species (ROS), suggesting synergistic action of ROS and NO (Zhao et al. 2001). Involvement
of NO and H2O2 in ABA-induced stomatal closure in Vicia faba (Garia-Mata and Lamattina 2002)
and Arabidopsis (Bright et al. 2006) has thrown light on the intricacy of signaling cascade triggered
by these metabolites. An et al. (2008) reported that PAs are also involved in ABA-induced stomatal
regulation under abiotic stresses. These multifunctional molecules have been shown to be involved in
numerous physiological and biological responses during stress responses forming a complex network
(Neill et al. 2003; Grün et al. 2006; Alcázar et al. 2010a,b). Evidence obtained so far has being com-
piled, showing the involvement and interaction of ABA, H2O2, NO, and PAs during stress responses
by a complex network (Figure 19.3). However, a complete PA-mediated abiotic stress signaling path-
way remains elusive.
19.3.1 Salt Stress
It has been reported from several plant species that PAs play a very important role in salt stress
tolerance (Evans and Malmberg 1989; Durner and Klessig 1999; Alcázar et al. 2006b, 2010a; Liu
et al. 2007). In the majority of the studies concerning salt stress (Wimalasekara et al. 2011), it has
been revealed that PA accumulation occurs. This accumulation of PAs might be a consequence of
PA biosynthesis regulation or catabolism by DAO or PAO (Moschou et al. 2008). Several PAO genes
of Arabidopsis are shown to be induced due to salt stress (Cona et al. 2006). Krishnamurthy and
Bhagwat (1989) studied and compared PA accumulation in nine cultivars of rice with different salt
sensitivity and thereby came to the conclusion that high levels of Spd and Spm were accumulated
by salt-tolerant cultivars, while lower concentrations of Spd and Spm and excessive Put were accu-
mulated by salt-sensitive rice cultivars. This is in accordance with the report of Roy et al. (2005)
Abiotic stress
Catabolism
Polyamines H2O2
ABA No ?
synthesis synthesis
ABRE Ca2+, cyclic

nucleotides,
protein kinases
Stress
response
FIGURE 19.3 Abiotic stress signaling PA, NO, ABA, and H2O2. Abiotic stress activates PA biosynthe-
sis, which in turn increases ABA as well as NO synthesis. ABA and NO trigger several second messengers
and kinases upregulating stress-responsive genes through ABA-dependent pathway via ABRE or ABA-
independent pathway. PAs may directly activate second messengers via an unknown metabolite-activating
stress-responsive gene (depicted by ?). On the other hand, PA catabolism leads to the production of H2O2,
which again triggers a signaling cascade via similar second messengers.
showing the presence of higher amount of plasma membrane–bound Spd and Spm in salt-tolerant
rice cultivars as compared to salt-sensitive cultivars.
Xing et al. (2007) have studied salinity stress in soybean roots. They have found a negative cor-
relation between the levels of PAs and GABA, that is, the degradation of PAs is directly proportional
to elevated GABA levels, but, conversely, during stress recovery process, the GABA concentration
is reduced concomitantly to an increase in the levels of PAs (Figure 19.4). It has been suggested
by Bitrián et al. (2012) that, during salinity stress, PA catabolism has a contribution to increased
concentrations of GABA. Hu et al. (2012) have extensively studied the effects of exogenous applica-
tion of Spd on the metabolism and PA content in tomato plants exposed to a mixed stress, namely,
salinity–alkalinity stress. Roy et al. (2005) reported that exogenous application of Spd could restore
the salinity-induced plasma membrane damage.
19.3.2 Osmotic Stress
The possible involvement of Spm in the acclimation of soybean to osmotic stress was studied
by Radhakrishnan and Lee (2013a) by determining the changes occurring in photosynthetic
pigments, plant hormone levels, and antioxidants, in response to applied Spm. Fujii and Zhu
(2012) reported that SNF1-related protein kinase (SnRK) 2 kinase play crucial roles in osmotic
stress-induced responses. According to their review, osmotic stress activates the SnRK2s, and a
mutant lacking SnRK2s is hypersensitive to osmotic stress. Radhakrishnan and Lee (2013b) in
their studies to determine the ameliorative role of Spm in reproductive phase of soybean dur-
ing polyethylene glycol (PEG)-induced osmotic stress observed that Spm applied exogenously
during osmotic stress was able to recover the reduced fresh weight of pods and seeds as well as
Salinity Drought
ADC1 SPDS1 SPMS

Arginine Putrescine Spermidine Spermine
ADC2 SPDS2
DAO
OSPDK like kinase

SAM
GABA
ABRE/ABF
SAMDC1
SAMDC2
dcSAM
Unknown sensors ?? Transcription factors (OSBZ8, TRAB1.....)
Rab16, Osem, rd29A, SalT....
FIGURE 19.4 Effect of salinity and drought on the metabolism of polyamines, and subsequent activation of
downstream effectors involved in stress tolerance.
their protein contents. Exogenous application of Spm increased the stress effects by the reduc-
ing lipid peroxidation and elevating total polyphenol as well as enzyme activities of catalase
and superoxide dismutase. They showed that the concentration of endogenous ABA was higher
in pods collected from PEG treatment, which decreased upon exposure to Spm. These stud-
ies exemplify the role of exogenous Spm in improving the reproductive health of plant under
osmotic stress conditions.
19.3.3 Water Stress
Stress-specific roles of plant PAs have been confirmed by significant increase in its endogenous
levels in water-stressed plants (Galston and Tiburcio 1997; Groppa and Benavides 2008). It has been
shown in Arabidopsis that overexpression of ADC2 that increases Put levels enhanced drought tol-
erance by the induction of stomatal closure (Alcázar et al. 2010b). It is a known fact that PAs have
involvement in the regulation of stomatal movements by exercising a control over inward K+ chan-
nels in guard cells (Lie et al. 2000).
Crop productivity and plant growth are severely affected by drought stress (Mahajan and
Tuteja 2005). An extensive transcriptional reprogramming occurs in plants in response to
drought stress, which includes both ABA-dependent and ABA-independent metabolic path-
ways (Yamaguchi et al. 2006; Tuteja 2007). As a result of drought stress, plants accumulate
a wide range of osmolytes, including PAs, sugars, proline, etc., to combat dehydration condi-
tions (Bartels and Sunkar 2005; Alcázar et al. 2006b; Urano et al. 2009). Figure 19.5 depicts
the probable molecular mechanism for the upregulation of PA anabolic pathway genes lead-
ing to the stress tolerance response by plants. Quite interestingly, the changes in PA titers
in response to drought conditions can be broadly correlated with the characteristics that are
typical of drought resistance (Bouchereau et al. 1999; Kusano et al. 2008). Yamaguchi et al.
(2007) reported that Spm levels increase in drought-stressed plants, which in turn might affect
Ca 2+ -permeable ion channels and as a result elevate the concentration of Ca 2+ in cytoplasm.
Water
stress
ABA independent ABA dependent IP3
DREB2 Ca2+
CBF4 MYC/MYB bZIP
Upregulation of Strees
CRT/DRE MYB/CRS ABRE ADC2, SPDS1, SPMS tolerance
FIGURE 19.5 Upregulation of polyamine biosynthetic genes during water stress.
As a result, there is inactivation of the K+ inward rectifier at the plasma membrane, which in
turn stimulates the closure of stomata (Willmer and Fricker 1996).
In a recent study by Alcázar et al. (2011a), a Put to Spm metabolic canalization was revealed in
A. thaliana and the resurrection plant Craterostigma plantagineum in response to drought stress.
They analyzed the levels of PAs in response to drought in the mutants of A. thaliana and found
impairment at different stages of the PA biosynthetic pathway. Thus they could monitor the accu-
mulation of PAs during the course of dehydration. The authors revealed that even though there is
no accumulation of Spd or Spm in wild-type A. thaliana under conditions of drought, a canaliza-
tion of Put to Spm is reported, which is not translated to Spd or Spm increases. However, the
drought-tolerant species C. plantagineum showed drastic increase in the levels of Spd and Spm that
were shown to correlate with drought tolerance. The metabolic canalization of Put to Spm is sup-
posed to be a conserved mechanism between species, whereas the ability to accumulate high Spd
and Spm levels (e.g., by oxidative inhibition) may discern between species tolerant and intolerant
to drought.
For the determination of the role of ABA in the transcriptional regulation of the PA biosynthetic
pathway as a result of drought stress, Alcázar et al. (2006a) analyzed the expression of the differ-
ent genes of PA biosynthesis, namely, ADC1, ADC2, AIH, CPA, SPDS1, SPDS2, SPMS, ACL5,
SAMDC1, and SAMDC2 in A. thaliana wild-type plants and mutants impaired in ABA biosynthe-
sis (aba2–3) or signaling (abi1–1) and elaborated the results. According to their experiments, the
genes, ADC2, SPDS1, and SPMS were among the most responsive ones to drought stress. Their
results also revealed that the expression of ADC2 and SPDS1 increased many fold by treatment
under drought, but the expression of their gene paralogs, ADC1 and SPDS2, did not show any
substantial change. In their report, Alcázar et al. (2006a) have demonstrated that ABRE or ABRE-
related motifs are also found in the promoters of ADC2, SPDS1, and SPMS. In response to drought,
the ABRE are highly upregulated. Upon analysis carried out in aba2–3 and abi1–1 mutants, much
more moderate increases in ADC2, SPDS1, and SPMS expressions were seen. Evidences derived
from these experiments confirmed that transcriptional upregulation of the genes ADC2, SDPS1, and
SPMS by drought stress is mediated by ABA. Subsequently, it was established that drought induced
PA biosynthetic pathway with ABA as the upstream regulator (Bitrián et al. 2012). In the experi-
ment conducted by Alcázar et al. (2006a), the contents of Put, Spd, and Spm levels in response to
drought stress were analyzed to study the effect of the transcriptional regulation of PA biosynthetic
genes on PA levels; it was observed that there was a progressive accumulation of Put in response to
drought conditions in wild-type plants, whereas there was complete absence of this accumulation in
aba2–3 and abi1–1. Thus, an effective Put accumulation occurs as a result of the ABA-dependent
upregulation in ADC2 expression under drought (Bitrián et al. 2012).
The role for ADC2 overexpression in conferring tolerance against drought by A. thaliana trans-
formation with the homologous gene, ADC2, under the constitutive CaMV 35s promoter, was stud-
ied and elucidated in a contemporary work by Alcázar et al. (2010b). Contrasting degrees of ADC2
expression and Put accumulation were found upon the analysis of different lines (Alcázar et al.
2005). Based on the transgenic line, the total Put content was between 12- and 2-folds higher than
the wild type (Alcázar et al. 2005, 2010b). A notable observation was that the plants that accumu-
lated higher levels of Put were more drought-resistant varieties, and there was a correlation between
the enhanced drought tolerance with a reduced stomata aperture and transpiration rate (Alcázar
et al. 2010b).
19.3.4 Extreme Temperature Tolerance

Differential accumulation of PAs during low- and high-temperature tolerance has been demon-
strated in a number of plant species (Evans and Malmberg 1989; Groppa and Benivades 2008;
Alcazar et al. 2010a). A very high accumulation of PAO and PA biosynthesizing ADC along with
substantial increase in free, conjugated, and long-chained PAs was observed in heat-tolerant cot-
ton and rice species that underwent heat stress (Evans and Malmberg 1989; Cona et al. 2006). An
increase in the level of activities of ADC and PAO was detected in the callus of heat-tolerant rice
cultivars by Roy and Ghosh (1996).
During recent years, transcriptomic and metabolomic approaches have been instrumental in
identifying the cold-responsive genes and metabolites, some of which are known to have protec-
tive effects against the damaging effects of cold temperatures (Alcázar et al. 2011b; Sanghera
et al. 2011). Chinnusamy et al. (2007) observed that in nature, cold acclimation by exposure
to low temperature counters damage caused due to freezing in most temperate plants. Global
approaches aimed to identify correlations between genes and/or metabolites, which have under-
gone cold treatments, very frequently recognize a crucial role of the PA biosynthetic pathway in
the cold responses (Cook et al. 2004; Vogel et al. 2005; Usadel et al. 2008). Put plays a pivotal
role in the cold stress metabolome that occurs in the plant Arabidopsis (Cook et al. 2004; Usadel
et al. 2008). Cold stress regulates the expression of 4%–20% of genes in the Arabidopsis genome
(Hannah et al. 2005; Lee et al. 2005). Increases in the levels of ADC1 transcript in Arabidopsis
were able to be detected after treatment with cold (Urano et al. 2003). Recently quantitative
expression analyses carried out by Cuevas et al. (2008) indicate that transcription of both ADC1
and ADC2 genes of Arabidopsis is induced as early as 30 min after exposure to cold stress,
where the amplitude and mRNA transcript levels of ADC1 were observed to be higher than that
of ADC2. Sequence analysis of the ADC1 promoter was carried out, and it revealed the presence
of CRT/DRE, which confer cold-mediated responsiveness, desiccation, and salinity and more-
over, could mediate the early and transient ADC1 upregulation under conditions of cold stress
(Yamaguchi-Shinozaki et al. 1994; Hummel et al. 2004; Alcázar et al. 2006a; Figure 19.6).
Recent studies by Todorova et al. (2012) have given some insights into the role of natural and
synthetic PAs as growth regulators in the enhancement of freezing tolerance of winter wheat
(Triticum aestivum L.).
Cold
Unknown sensors ?? Arginine
ADC1 Cold acclimation

Ca2+ influx ADC2
Agmatine
Kinase/phosphatase
Zeaxanthine
CRT/DRE Putrescine
ABA
Spermidine Protective
ABRE/ABF protein and
metabolites
Spermine ABRE/COR
FIGURE 19.6 Signal transduction mechanism involving PA anabolic pathway during cold stress.
19.3.5 UV Stress
Terrestrial plant life was made possible by the formation of an ozone layer in the stratospheric layer
of the atmosphere, which absorbs all of the solar UV-C and part of the UV-B radiation (Rozema et al.
1997). Owing to the advent of industrialization and increased anthropogenic activities, this protec-
tive layer has gradually disintegrated resulting in a greater influx of the lethal UV-B radiations. This
becomes an environmental stress for several ecosystems as demonstrated by many researchers. The
solar UV-B rays have several deleterious effects on plants, such as damaging proteins, DNA, mem-
brane lipids (Teramura 1983; Quaite et al. 1992; Teramura and Sullivan 1994), and an increase in the
levels of ROS (Brosche and Strid 2003). According to Mapelli et al. (2008), UV radiation triggers pro-
tective stress responses in plants, such as changes in antioxidant enzyme activities and content of PAs.
Free PAs are known to show a remarkable decrease in Phaseolus vulgaris in response to UV-B
radiation (Smith et al. 2001). In tobacco cultivars, Lutz et al. (2005) demonstrated an increase in the
levels of total PAs (especially Put in thylakoid membranes) as one of the primary protective mecha-
nisms of the photosynthetic apparatus against the harmful UV-B irradiations. The penetration of
UV radiation varies among different plant species, and this can be well reflected in the sensitivity of
these species (Day et al. 1992; DeLucia et al. 1992). Kondo et al. (2011) showed that UV-C irradiation
increased the PAs in contrast to the decreased ROS thereby suggesting the role of PA as antioxidant.
Scientific investigations have been carried out regarding the content and distribution of PAs
(free, conjugated to low-molecular compounds, or bound to macromolecules) in Mesembryanthemum
crystallinum L., Thellungiella halophila Mey. (halophytes), and Plantago major L., Geum urbanum
L. (glycophytes) when these species are affected by UV-B irradiation (Mapelli et al. 2008). PA bio-
synthesis was significantly upregulated in the red seaweed P. cinnamomea in response to UV-B, with
the greatest proportional increases being in bound soluble Put, Spd and Spm, and bound insoluble
Spm. An exposure to UV-B caused increases in the activities of both ADC and ODC; the increase
in ADC activity was 10-fold greater than that of ODC, suggesting that the ADC pathway was the
principal route by which PA levels increased in response to UV-B (Schweikert et al. 2011).
19.3.6 Oxidative Stress
There are certainly some roles of PAs in oxidative stresses. PAs demonstrate their antioxidant
effects through a combination of their anionic and cationic binding properties in radical scaveng-
ing, inhibiting properties of lipid peroxidation, metal-catalyzed oxidative reaction, and production
of H2O2 by DAO and PAO, and moreover, it has been well established that ozone damage and
ozone-derived oxidative damage can be countered by plants by the modulation of their PA levels
(Groppa and Benivades 2008). Antioxidative defense responses in certain oxidative stress condi-
tions may be promoted by H2O2 produced by plant PA catabolism (Wimalasekara et al. 2011).
The accumulation of potentially toxic molecules in tissues such as O2-, OH·, and H2O2 (which are
collectively known as reactive oxygen species or ROS) can cause oxidative damage to DNA, lip-
ids, proteins, and other molecules (Apel and Hirt 2004). Phenylpropanoid–PA conjugates behave
as antioxidants when they encounter ROS and reactive nitrogen species during stress conditions
(Yamasaki and Cohen 2006). Reliable evidence of free PA acting as ROS scavengers was derived
from experiments performed in vitro in a system generating free radicals (Ha et al. 1998; Kuznetsov
et al. 2007). Radyukinaa et al. 2011 suggested that PA could stimulate proline synthesis via H2O2
formation during oxidative stress.
19.3.7 Mineral Deficiency and Heavy Metal Tolerance

It is a well known fact that PA metabolism is altered in response to mineral deficiencies (Evans and
Malmberg 1989; Groppa and Benivades 2008). Watson and Malmberg (1996) have observed the
accumulation of Put in K+-deficient plants, and they have related it to increased ADC activity. The
deficiencies of minerals, such as boron, ammonium, and Mg2+, induce alterations in endogenous
levels of PAs in several plant species. For example, Evans and Malmberg (1989) had demonstrated
changes occurring in the PA content of grapevine due to Mg and N deficiencies. Qiao et al. (2012)
showed that Pb stress induced a continuous accumulation of perchloric acid–insoluble bound (PIS-
bound) Spm and an initial accumulation of PIS-bound Put and Spd. While the ratio of free (Spd
and Spm)/Put significantly declined at low Pb concentrations, the ratio of total (Spd + Spm)/Put
rose, and PAO activity rose gradually with an increase in Pb concentration. The ODC activity first
increased and then declined.
Groppa and Benavides (2008) have also observed changes in PA metabolism in response to
metal toxicity in plants. It was stated by Wimalasekara et al. (2011) that PA metabolism was dif-
ferentially affected in Cd- and Cu-treated sunflower and wheat and was well related to increased
ODC and ADC activities. Alterations in DAO and PAO activities in response to heavy metal tox-
icity were also observed by Groppa et al. (2003). Hossain et al. (2012) emphasized the capacity
of PAs for scavenging free radicals and ROS, thus exerting a strong antioxidant function during
various types of stress.
Pal Choudhary et al. (2012) extensively studied the involvement of brassinosteroids (BRs) and
PAs in the regulation of copper (Cu) homeostasis in plants exposed to toxic levels of Cu. Their
study has analyzed the effects of exogenously applied BRs and PAs on radish (Raphanus sativus)
plants exposed to toxic Cu concentrations. Enhanced tolerance to Cu stress occurred due to the
interaction of 24-epibrassinolide (EBR, an active BR) and Spd which had considerable effect on the
pattern of gene expression and the physiology of radish plants. It was derived from the results that
the combined application of Spd and EBR modulated the expression of the genes that encode the
PA enzymes and genes impacting the metabolism of indole-3-acetic acid and ABA, which leads to
enhanced Cu stress tolerance. The main effect of EBR and Spd leading to Cu stress alleviation in
radish was the altered expression of genes implicated in Cu homeostasis. Processes, such as comet
assay, ion leakage, in vivo imaging of H2O2, and improved tolerance of Cu-sensitive yeast strains,
have demonstrated the ability of EBR and Spd to improve Cu tolerance remarkably. The exogenous
application of PAs such as Put, Spd, and Spm in Crocus sativus improved plant performance under
Al stress, with respect to control (Chen et al. 2008). Blancheteau (2012) attributed this protective
role of PA to lower Al content in the root tips and subsequent less lipid peroxidation and oxidative
stress. Overexpression of Spd synthase of apple (MdSPDS1) in transgenic European pear (Pyrus
communis) revealed that the performance of transgenic was much better than that of wild type,
indicating that Spd is implicated in elevating Al-stress tolerance (Wen et al. 2009). Roychoudhury
et al. (2012) highlighted the importance of Spm in conferring protection against metal-induced
toxicity in rice.
19.3.8 Mechanical Stress
PAs also respond to mechanical stress besides environmental stress factors as discussed earlier
(Wimalasekara et al. 2011). A significant increase in Put level was observed in mechanically injured
Arabidopsis and oilseed rape (Perez-Amador et al. 2002; Cowley and Walters 2005). The role of
DAO and PAO in wound healing has been demonstrated by Angelini et al. (2010). In wounded
chickpea, an increase in the levels of CuAO/DAO was detected both locally and systemically, and
this induction of CuAO/DAO was shown to be essential for H2O2 production in response to mechan-
ical stress or wounding (Rea et al. 2008). The importance of PAO-mediated H2O2 in wound healing
was well demonstrated in maize (Zea mays), and it was revealed that along the wound area, high
levels of PAO activity accelerated the deposition of lignin and suberin (Angelini et al. 2008). The
intensified lignin and suberin deposition as a consequence of H2O2 release explains the function of
DAO and PAO in wound healing (Wimalasekara et al. 2011).
Plant stress tolerance encompasses complex processes involving numerous metabolites and
metabolic pathways. Analyses of metabolic adjustments that the plants make in response to
different levels and categories of stress tolerance and transgenic approaches provide an insight
into the role of different metabolites in adjusting to extremes of environmental conditions. There
are numerous evidences supporting the fact that PAs are involved in a wide range of plant pro-
cesses, including DNA replication, transcription of genes, cell division, etc., with an emphasis
on abiotic stresses. However, the exact contribution of PAs in different processes is yet to be
ascertained.
Recent developments of PA-deficient mutants and transgenic plants as well as molecular and
genetic investigations should further our understanding of their roles in plant growth and develop-
ment. The use of mutants, reverse genetics, suicide inhibitors, and transgenic plants has made it
easier to manipulate different PA metabolic pathways, which will help in revealing the core compo-
nents of plant PAs. The molecular and biochemical elucidation of PA pathway in plants has made it
easier to make progressions and modulations with the help of advanced biotechnological method-
ologies. The signaling networks in plants induced by abiotic stresses have been extensively studied
by many scientists.
Innovative studies by Green et al. (2011) have provided a correlation between gene fusions and
PA biosynthetic pathways. Gene fusion is crucial to the evolution of proteins and metabolic path-
ways. Being ubiquitous in nature and having short biosynthetic pathways, PAs are ideal for carrying
out investigations regarding the occurrence, function, and evolution of gene fusions in metabolic
pathways. AdoMetDC activity was exhibited by purified recombinant Pelagibacter fusion pro-
tein, which was not stimulated by magnesium ions but was stimulated though weakly by diamines
and spermidine. The aminopropyltransferase activity was highly promiscuous, and it recognized
putrescine, cadaverine, spermidine, and agmatine with similar efficiency as substrates. This sub-
strate promiscuity explains the diversified products detected when the Pelagibacter gene fusion was
expressed in Escherichia coli.
In conclusion, research works on PA and stress tolerance are fast advancing and generating some
interesting results for the scientific community. More vigorous research is required to have a better
knowledge of the diverse functions of these relatively simple, yet mysterious molecules occurring
ubiquitously. Further studies will enhance the chances of exploration and elucidation of the detailed
molecular and biochemical aspects of PAs during stress tolerance in plants and shed light upon
newer stress tolerance strategies in plants.
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20 Physiological and Biochemical
Mechanisms of Plant
Tolerance to Heat Stress
David Jespersen and Bingru Huang
CONTENTS
Abbreviations.................................................................................................................................. 389
20.1 Introduction........................................................................................................................... 389
20.2 Membrane Stability............................................................................................................... 390
20.3 Antioxidant Metabolism........................................................................................................ 391
20.4 Carbon Metabolism............................................................................................................... 392
20.5 Protein Metabolism............................................................................................................... 395
20.6 Hormone Metabolism............................................................................................................ 397
20.7 Conclusion............................................................................................................................. 398
References....................................................................................................................................... 399
ABBREVIATIONS
ABA Abscisic acid
ACC 1-Aminocyclopropane-1-carboxylic acid
APX Ascorbate peroxidase
CAT Catalase
CLP Caseinolytic protease
GR Glutathione reductase
HSF Heat shock factor
HSP Heat shock protein
POX Peroxidase
PSI Photosystem I
PSII Photosystem II
ROS Reactive oxygen species
Rubisco Ribulose-1,6-bisphosphate carboxylase oxygenase
SA Salicylic acid
SOD Superoxide dismutase
20.1 INTRODUCTION
Predictive models forecast that future summers will increasingly see record-breaking high tempera-
tures, with today’s highest temperatures become the future’s norm, as global warming continues to
cause temperatures to rise (Battisti and Naylor, 2009). It is predicted that elevated temperatures will
result in decreased food security worldwide (Lobell and Field, 2007; Battisti and Naylor, 2009),
which further necessitates the need for a better understanding of mechanisms of plant adaptation to
389
heat stress to improve plant growth and productivity in increasing temperature environments. Even
relatively short periods of high temperatures during key developmental phases of a plant’s life cycle
can have significant negative impacts on plant growth and production, as reported in various crop
species. The adverse impact of elevated temperatures on annual crop yields in certain regions in
the past few decades has already been observed, including rice (Oryza sativa) in China (Peng et al.,
2004), maize (Zea mays) in Africa (Lobell et al., 2011), as well as other major grain crops (Lobell
and Field, 2007). High temperatures also can be detrimental to the growth of perennial grasses,
particularly for C3 cool-season species (DiPola and Beard, 1992; Fry and Huang, 2004).
The level of heat damage depends on a number of factors including plant species, developmental
stage, duration, as well as the intensity of heat during the stressful period (Wahid et al., 2007). The dif-
ferences in the levels of heat tolerance also exist within an individual species. Various process changes
occur at physiological, cellular, and molecular levels when plants are exposed to elevated temperatures,
and most frequently, these changes are greatest in cool-season or temperate plant species. It is impor-
tant to understand the underlying effects and responses to supraoptimal temperature. It is through this
understanding that we can begin to develop more heat-tolerant plants and management practices to help
negate the effects of ever-increasing temperatures and maintain plant growth and productivity.
This chapter reviews current research on major physiological and metabolic processes and path-
ways associated with heat responses and heat tolerance in various plant species, including membrane
stability, carbon metabolism, protein metabolism, antioxidant metabolism, and hormone metabo-
lism. These processes can be interrelated and interactively involved in plant response and tolerance
to heat stress. For an orderly discussion, literature regarding each process is presented separately.
20.2 MEMBRANE STABILITY
Membranes are an essential structural component for all cell life and also are of key importance
for a host of cellular functions. Membranes contain many proteins integrated onto their surface or
into their structure. These proteins are responsible for a wide array of metabolic processes, from
signal recognition and transduction, transporter proteins, to proteins in the thylakoid membranes
of chloroplasts responsible for photosynthetic processes and those in the mitochondria responsible
for the breakdown of sugars to generate energy in the form of ATP (Taiz and Zeiger, 2010). It is
because of their importance in function as well as their sensitivity to fluctuations in temperatures
that make them an important component of tolerance to elevated temperatures. Membrane stability
is a commonly used indicator of cellular integrity and viability in plant tolerance to various stresses
(Shanahan et al., 1990; Marcum, 1998; Liu and Huang, 2000; Sung et al., 2003; Camejo et al., 2005).
Membrane stability can be estimated by measuring the leakage of electrolyte from a cell or tissue
and has been shown to have strong positive correlations to plant heat tolerance (Blum et al., 2001).
With increasing temperature, membranes have increased fluidity as the thermal energy disrupts
bonds between adjacent fatty acid molecules, lowering the stability and interrupting membrane inte-
gral processes (Savchenko et al., 2002). One of the main factors believed to affect membrane stabil-
ity is lipid composition of the membrane. Lipids contain unsaturated fatty acid molecules, which
have at least one double bond between carbon molecules that introduce a slight bend in the fatty acid
chain. The more saturated the fatty acid with hydrogen atoms, the lesser the number of double bonds
present, resulting in a straighter fatty acid chain. This presents greater opportunity to get near and
interact to form bonds with the adjacent fatty acid molecules, resulting in a more rigid lipid layer.
Increased levels of saturation have been shown to increase as the temperature rises, and increased
saturation is associated with membrane stability at high temperatures (Thomas et al., 1986; Vigh
et al., 1989; Larkindale and Huang, 2004). Due to the relative ease of isolating chloroplasts, numer-
ous studies have looked at the effects of lipid saturation on thylakoid membranes, demonstrating
that improved membrane stability can have beneficial effects on the maintenance of photosynthetic
capability by maintaining thylakoid integrity as well as the function of thylakoid-bound proteins
such as the pigment–protein complexes needed to harvest light (Raison et al., 1982). Membranes are
Physiological and Biochemical Mechanisms of Plant Tolerance to Heat Stress 391
also sites of temperature stress recognition, leading to signaling cascades that alter gene expression
(Vigh et al., 1998). It is clear that whether through maintaining cell integrity, through signaling
of stressors, or through important metabolic functions like molecule transport between cells or
the generation of energy in the chloroplast through photosynthesis, membrane integrity is impera-
tive for maintaining cellular functions, particularly since their integrity not only is vulnerable to
changes in structure caused by changes in temperature but also is a major site of damage by reactive
oxygen species (ROS) as discussed in the next section.
20.3 ANTIOXIDANT METABOLISM
ROS are a group of oxygen-containing molecules where the oxygen is in a reduced state causing the
molecule to be highly reactive. These reactive radicals can then interact with cellular components
leading to oxidative damage. These ROS can take many forms and commonly include such mol-
ecules as super oxide (O2- ), singlet oxygen (1O2), hydrogen peroxide (H2O2), and hydroxyl radicals
(OH−). ROS production is frequently associated with photosynthesis in the chloroplasts, respiration
in the mitochondria, as well as metabolic activities in peroxisomes. ROS are produced in the per-
oxisomes from the catabolism of fatty acids and other organic compounds (Gill and Tuteja, 2010).
In the chloroplasts, ROS production is induced when there is overexcitation of electron transport
associated with the light-dependent reactions, or CO2 fixation is limited (Suzuki and Mittler, 2006).
This leads to electrons not moving through the normal chain of photosynthesis but instead ener-
gizing oxygen molecules, which leads them to form into radicals and damage other molecules.
Additionally, the photorespiration pathway can lead to the creation of ROS in peroxisomes through
the action of glycolate oxidase (Mittler et al., 2004). In mitochondria, heat stress leads to an uncou-
pling of respiration with ATP generation, which can lead to a depletion of ATP, and eventually to
possible cell death, and similar to ROS production in chloroplast, it causes an overreduction of
electron transport leading to the formation of more of these damaging radicals (Tiwari et al., 2002).
When these highly chemically ROS are produced and the cell cannot detoxify them quickly,
ROS accumulation causes oxidation of cellular components. Lipids, proteins, and DNA molecules
can all be damaged by ROS leading to their destruction and impairment of cell functions (Gill and
Tuteja, 2010). The production of ROS in response to abiotic stresses, including heat stress, is well
documented. Lipid peroxidation is a commonly used method to estimate overall oxidative damage
due to the relative ease in the ability to measure the resulting compounds produced when ROS dam-
age membrane lipids. Heat stress increases oxidative damage associated with ROS in various plant
species (Sairam et al., 2000; Huang et al., 2001; Chaitanya et al., 2002; Larkindale and Knight,
2002). These damages lead to impairment of metabolic functions including photosynthesis, respi-
ration, and protein metabolism. The accumulation of these damages eventually leads to apoptosis,
programmed cell death.
To prevent oxidative damages from ROS, cells have developed antioxidant pathways to scavenging
radicals before they can damage the cell. These pathways include nonenzymatic mechanisms, where
various compounds including flavonoids, carotenoids, alkaloids, and other phenolic compounds are
able to scavenge ROS and react with them to limit the amount of free radicals present to cause dam-
ages. Other metabolites like ascorbic acid and glutathione also have the ability to accept the free
electrons from ROS and are key substrates for enzymatic antioxidant pathways such as ascorbate per-
oxidase (APX) cycle and glutathione reductase (GR) cycle (Alscher et al., 1989; Gechev et al., 2006).
In the roots of maize plants exposed to heat stress, an increase in glutathione was found, which was
associated with increased antioxidant metabolism (Nieto-Sotelo and Ho, 1986).
Antioxidant enzymes consist of a wide range of gene families common among plant species with
an array of functions and expression profiles (Mittler et al., 2004). Superoxide dismutase (SOD)
is one of the first enzymes required for antioxidant pathways as it is responsible for converting
superoxide into hydrogen peroxide, which is then further broken down by other antioxidants. Due
to its unique ability to neutralize superoxide, it is often expressed in the cellular compartments
mentioned like chloroplasts where superoxides are generated, and during heat stress events, expres-
sion and subsequent activity of SOD are increased to tolerate heat-induced ROS damages (Alscher
et al., 2002). Catalase (CAT) is a family of enzymes that are able to convert hydrogen peroxide
directly to water, and increases in activity have also been associated with more active antioxidant
systems that confer heat tolerance (Sairam et al., 2000; Chaitanya et al., 2002). Hydrogen peroxide
is also broken down through two other major pathways, the ascorbate–glutathione cycle and the
glutathione–peroxidase cycle (Apel and Hirt, 2004). During the ascorbate–glutathione cycle, hydro-
gen peroxide is converted to water and ascorbate is converted to monodehydroascorbate and then
dehydroascorbate by their respective reductase enzymes resulting in glutathione disulfide, which
is then reduced to glutathione by the enzyme GR. In the glutathione–peroxidase cycle, hydrogen
peroxide and glutathione are converted to water and glutathione disulfide by glutathione peroxidase,
which is then again converted back to glutathione by GR.
When comparing tolerant and sensitive lines of wheat (Triticum aestivum), tolerant lines were
found to have greater activities of SOD, CAT, APX, GR, and peroxidase (POX), which in turn
were associated with less severe membrane damages and maintaining higher levels of chlorophyll
(Sairam et al., 2000; Almeselmani et al., 2006). The alteration of antioxidant activities is a well-
documented response during exposure to high temperatures and has been noted in various plant
species (Rainwater et al., 1996; Chaitanya et al., 2002; Gulen and Eris, 2004; Almeselmani et al.,
2006). Potatoes (Solanum tuberosum) transgenically modified to increase the expression of SOD and
APX in response to stress had increased heat tolerance, demonstrating the importance of antioxi-
dants for heat stress tolerance (Tang et al., 2006). It is however possible for these pathways to become
overwhelmed, and stress damages accumulate beyond their control. Studies involving arabidopsis
(Arabidopsis thaliana) and creeping bentgrass (Agrostis stolonifera) showed that while antioxidant
activities may increase during moderate stress, as temperature and the level of stress continue to
increase, there is an eventual decrease in the levels of these enzymes and their associated activities
(Panchuk et al., 2002; Xu and Huang, 2004). Decreases in antioxidant activity are then accompanied
by increases in ROS damages (Jiang and Huang, 2001). Additionally, decreases in SOD and CAT
activities have been associated with the onset of leaf senescence (Dhinsda et al., 1981).
While ROS can be injurious to plants, they are also important signal molecules important for the reg-
ulation of cell growth and development, and of abiotic and biotic stress responses, ROS have been found
to influence various types of signal cascades including histidine and mitogen-activated protein kinases
(Apel and Hirt, 2004). Additionally, ROS are believed to interact with a number of transcription factors
to regulate gene expression in response to abiotic stress. Large gene families of transcription factors are
regulated in response to heat stress and in turn affect the transcriptional response to heat stress including
the regulation of genes involved in ROS pathways. WRKY and ZAT proteins are two such transcription
factors, some of which are activated under heat stress and can lead to the production of antioxidants such
as APX (Miller et al., 2008). Heat shock factors (HSFs) are thought to be able to sense ROS and lead to
changes in signal pathways. An arabidopsis mutant was shown to have increased APX activity associ-
ated with increased HSF expression (Panchuk et al., 2002). Plants pretreated with hydrogen peroxide
prior to heat stress had higher antioxidant activity (Uchida et al., 2002), which demonstrate that transient
increases in ROS may activate antioxidant systems, which in part may explain how pretreating plants
with a minor heat stress event may confer higher tolerance levels during more extreme events. In some
situations, however, ROS can signal the production of more ROS, most likely through NADPH oxidases
(Gechev et al., 2006). Excessive ROS accumulation ultimately can cause cell death, particularly when
antioxidant defense is weakened under severe stress conditions.
20.4 CARBON METABOLISM
Photosynthesis is also one of the first processes to be damaged by increased temperatures due to
many of its components being highly thermoliable, as found in various plant species (Al-Khatib and
Paulsen, 1999; Camejo et al., 2005; Ciu et al., 2006). The inhibition of photosynthesis can cause
detrimental shifts in carbon balance resulting in plants without the energy reserves to put toward
heat tolerance mechanisms or recovery during post-stress periods. While not all aspects of photo-
synthesis are equally damaged by heat stress, key components of both the light and dark reactions
are damaged in ways that affect both the capturing of light energy and carbon fixation.
The decline in chlorophyll content or leaf chlorosis is one of the earliest symptoms of heat-
induced leaf senescence. Under prolonged heat stress, chlorophyll levels decline. Along with the
decline in the levels of chlorophyll, the activities of chlorophyll-degrading enzymes such as chlo-
rophyllase and chlorophyll peroxidases (POXs) increase, while chlorophyll biosynthesis enzymes
including 5-aminolevulinic dehydratase, porphobilinogen deaminase Mg-chelatase, S-adenosyl-
L-methionine: Mg-protoporphyrin IX methyltransferase, and Mg-protoporphyrin monoester-
cyclase exhibit reduced activities (Tewari and Tripathy, 1998; Todorov et al., 2003; Yamauchi et al.,
2004). Both heat-accelerated chlorophyll degradation and heat inhibition of chlorophyll synthesis
may occur simultaneously, causing leaf chlorosis. However, the relative contribution of these two
processes to heat-induced leaf chlorosis may vary with the level of stress and phases of leaf develop-
ment (young vs. old leaves). Ultimately, the loss of chlorophyll is due to an unfavorable shift in the
balance between chlorophyll synthesis and degradation, with both aspects playing important roles.
However, recent literature has proposed that the inhibition of protein synthesis may be the more
imperative factor leading to photoinhibition and subsequent loss of chlorophyll during heat-induced
senescence (Murata et al., 2007; Takahashi et al., 2008).
Another early sign of heat stress to photosynthetic machinery is the damage in photosystem II
(PSII) reaction center housed in the thylakoid membranes of the chloroplasts. The PSII complex
is the first protein–pigment complex involved in the light-dependent reactions of photosynthesis
and is responsible for splitting water into hydrogen and oxygen, as well as generating electrons
for transport to other photosynthetic machinery and generating hydrogen ions to create the pro-
ton gradient needed for ATP synthesis (Taiz and Zeiger, 2010). Many studies have found that the
PSII complex is highly thermoliable so even moderate increases in temperature may damage it and
lead to a reduction in photosynthesis (Havaux, 1993; Haldimann and Feller, 2005). Chlorophyll
fluorescence uses the light reemitted from chlorophyll molecules to estimate quantum yield and is
widely used to estimate the photosynthetic health of light-dependent reactions (Yamada et al., 1996;
Maxwell and Johnson, 2000). Quantum yield is a measure of how efficiently the photosynthetic
complex PSII is capturing light energy to use for electron transport. Under severe stress, PSII dam-
age may accumulate to such a level that thylakoids are unable to restore light-dependent functions
to prestress levels (Sharkova, 2001). The loss of PSII activity has been attributed to a number of
functional changes that alter the structure of the protein complex. Damages to chloroplast mem-
branes and increases in membrane fluidity caused by temperature stress may lead to the integral
membrane proteins becoming dislodged from their location in the thylakoid. The idea that loss of
PSII function is due to a dissociation with the thylakoid membrane is supported by studies that have
shown that increased thylakoid stability is associated with better maintenance of photosynthetic
capability (Thomas et al., 1986; Sharkey, 2005; Ristic et al., 2007) Another theory as to the loss
of PSII activity is that certain components within the protein complex become denatured. This
denaturation can be the result of direct damage due to high temperatures or the creation of oxygen
radicals that then damage cellular components by oxidizing them. The oxygen-evolving complex
responsible for the splitting of water molecules into hydrogen and oxygen is a major site of damage
within the PSII complex (Bukhov et al., 1999; Toth et al., 2005). Yamane et al. (1998) have shown
that the manganese-stabilizing protein needed to split water molecules dissociates from the oxygen-
evolving complex under high temperatures. Furthermore, it has been demonstrated that the recovery
of PSII during poststress periods relies on the synthesis of new protein components of the complex,
which may be further hindered by the altered protein metabolism associated with heat stress (Toth
et al., 2005; Murata et al., 2007). Another aspect of the light-dependent reactions altered under high
temperatures is the increased activity of PSI, another protein complex responsible for absorbing light
energy in the chloroplasts (Havaux, 1996). This commonly observed phenomenon has been attributed
to alteration of the thylakoid membrane and conformational changes of the b6f cytochrome protein,
which is a membrane-bound protein essential for electron transport (Thomas et al., 1986). This
conformational change has been associated with the exposure of additional electron acceptor sights,
which would help facilitate the increased electron flow. This increased activity has been associated
with cyclic electron flow, a pathway in the thylakoids, which does not involve PSII but instead cycles
electrons through PSI, as well as plastoquinone and cytochrome b6f. This increase in cyclic electron
flow is believed to act as a protective mechanism that acts as a sink for excess energy as well as a
means to generate additional ATP (Munekage et al., 2004; Sharkey, 2005).
The dark reaction, also known as Calvin cycle of photosynthesis, includes three major processes:
carbon fixation, carbon reduction, and ribulose bisphosphate regeneration, which works coordi-
nately to produce carbohydrates and intermediate metabolites supporting plant growth. Under heat
stress conditions, it has been found that there is a reduction in the level of metabolites associated with
the Calvin cycle, which demonstrates a reduced level of carbon-fixation activities (Law and Crafts-
Brandner, 1999). The rate of ribulose bisphosphate regeneration and carbon reduction processes in
the Calvin cycle are inhibited by heat stress (Wise et al., 2004). High temperatures also reduced
many essential proteins of the Calvin cycle including both large and small subunits of ribulose-1,6-
bisphosphate carboxylase oxygenase (Rubisco) as well as Rubisco-binding proteins and Rubisco
activase (Demirevska-Kepova et al., 2005). Rubisco, which is essential for the photosynthetic pro-
cess, is estimated to be the most abundant protein on earth and is the enzyme responsible for fixing
carbon dioxide to longer chain carbon molecules to create sugars, which act as energy stores for
later use. It has been shown under elevated temperatures that heat inhibition of carbon assimilation
is largely due to a reduction in the activation state of Rubisco (Law and Crafts-Brandner, 1999;
Salvucci and Crafts-Brandner, 2004). This activation state refers to the conformational shape of
the protein, which needs to be open and free of sugar residues or other inhibitors to be able to bind
and enzymatically react with carbon dioxide. When Rubisco is in an inactive state, it will be unable
to assimilate the carbon needed to complete photosynthesis. Rubisco activase is the protein that is
responsible for returning Rubisco from the inactive to the enzymatically functionally active state.
Rubisco activase is believed to be more thermoliable than Rubisco itself (Crafts-Brandner and Law
2000). As temperatures rise, Rubisco activase activity decreases, which in turn leads to a higher
percentage of Rubisco molecules being in the inactive state opposed to being active, which in turns
leads to decreases in carbon fixation and reduces photosynthesis.
Rubisco has the ability to bind both with the carbon dioxide needed to complete the photosyn-
thetic process and with oxygen; it is this binding of oxygen that leads to the photorespiratory pathway.
Photorespiration is a much more serious concern in C3 plants because they lack the carbon dioxide
concentrating mechanisms of C4 plants, which act to reduce the possibility of Rubisco binding with
oxygen. This is due to the fact that at increasing temperatures, the solubility of carbon dioxide and
oxygen shift in a way that favors oxygen binding as well as Rubisco having a decreased specificity
for preferential carbon dioxide binding or during certain abiotic stresses that create stomatal limi-
tations (Jordan and Ogren, 1984). Once oxygen is bound to Rubisco, the cell must expend energy
resources to remove the oxygen molecule. Although photorespiration has widely been viewed as a
wasteful process leading to reduced photosynthesis, there is the belief that it is valuable as a means
to stress protection because it can potentially act as an energy sink to prevent photoinhibition from
excess light energy absorbed, as well as the production of useful metabolites (Wingler et al., 2000).
In contrast to photosynthetic responses to increasing temperature, increases in respiration rates
have been found in a number of plant species (Sato et al., 2000; Liu and Huang, 2001; Rizhsky et al.,
2002, 2004). A study investigating the effects of heat stress on transcription in tobacco (Nicotiana
tabacum) found that most photosynthetic genes were downregulated (except a PSI and cytochrome
gene that may be related to cyclic electron flow), whereas respiration-related genes were largely
upregulated (Rizhsky et al., 2002). The increases in respiration and decreases in photosynthesis
can cause carbohydrate depletion, particularly during prolonged periods of heat stress. Decreases in
starch accumulation as well as activities of starch-related enzymes have been associated with heat
damages (Wilhelm et al., 1999; Chaitanya et al., 2001; Majoul et al., 2003). Additionally, there is
a decreased export of carbon from leaf tissues to roots under high-temperature conditions (Dinar
et al., 1983; Lafta and Lorenzen, 1995). This decrease in carbon mobilization coupled with the fact
that respiration also increases in root tissues, which relies entirely on carbon generated from pho-
tosynthesis, can be detrimental for plant growth under heat stress (Gunn and Farrar, 1999; Lyons
et al., 2007). Maintaining balanced photosynthesis and respiration and carbohydrate accumulation
is critically important for plant survival of long-term heat stress.
20.5 PROTEIN METABOLISM
Decreased levels of protein is a well-documented response to heat stress (Thomas, 1978; Ferguson
et al., 1990, 1994; Gulen and Eris, 2004; Xu and Huang, 2004), which is one of the major char-
acteristics of heat-induced leaf senescence (Thomas and Stoddart, 1980). Heat stress inhibits
protein synthesis but accelerates protein degradation. Duncan and Hershey (1989) found that
decreased protein synthesis is partly due to decreased ribosome activity and increased protein
phosphorylation. Ferguson et al. (1994) found that as temperatures increased, decreased ribosome
activity, mRNA levels, as well as decreases in the uptake of radiolabeled methionine into new
proteins occurred, demonstrating that protein synthesis was inhibited. One of the most common
responses to high temperatures is the increase in free amino acid content, which may be due in
part to increased protein degradation, releasing free amino acids (Guy et al., 2008). Although not
all amino acids are accumulated equally under heat stress, which may demonstrate perturbations
in some metabolic pathways or preferential accumulation of certain amino acids for protective
functions (Mayer et al., 1990).
Proteases are responsible for the catabolism of proteins and polypeptides. During heat stress,
the level of proteases in the cell often increases and is likely responsible, at least in part, for pro-
tein degradation. Two major classes of proteases found to be upregulated during heat stress are
cysteine proteases and serine proteases (Palma et al., 2002; Schaller, 2004). Additionally, pro-
tease inhibitors have been found to reduce the amount of protein degradation during heat stress
and inhibit cell death, confirming the regulatory role of proteases (Tiwari et al., 2002). Ubiquitin-
mediated protein degradation is also another major pathway for protein destruction. Ubiquitins
are small highly conserved proteins that act to target proteins for degradation. Ubiquitins are
ligated to proteins to be degraded, and the subsequent addition of additional ubiquitin units
results in a polyubiquitin chain. The addition of the polyubiquitin chain onto the protein tar-
gets it to a proteasome complex, which degrades the protein to smaller polypeptides (Belknap
and Garbarino, 1996). In wheat roots exposed to heat, protein degradation was associated with
decreases in the pool of free ubiquitin, but an increase in ubiquitin conjugations demonstrated
that ubiquitin-mediated degradation is an important element of protein degradation during heat
stress (Ferguson et al., 1990).
Proteomic profiling by two-dimensional electrophoresis and mass spectrometry is a powerful
tool for the quantification of protein abundance and identification of specific proteins that change
in response to a variety of events, such as heat stress. In response to heat stress, proteins asso-
ciated with protein biosynthesis such as initiation factors and ribosomal proteins were found to
be downregulated, while proteins such as cysteine protease associated with protein degradation
were upregulated (Majoul et al., 2004; Zou et al., 2011). Changes related to carbon metabolism
were also noted, with many glycolysis proteins downregulated, but a few such as glyceraldehyde-3-
phosphaste dehydrogenase were upregulated by heat stress, and many photosynthetic proteins were
downregulated as well, with the exception of Rubisco activase, which as previously mentioned may
play a role in maintaining the activity of the Calvin cycle (Majoul et al., 2004; Zou et al., 2011).
In two turfgrass species, superior heat tolerance was associated with the maintenance of higher
levels of photosynthetic proteins, such as Rubisco and Rubisco activase, and proteins related to car-
bon metabolism, such as glyceraldehyde-3-phosphaste dehydrogenase and malate dehydrogenase
(Xu and Huang, 2010). Proteins in PSII components in photosynthesis declined, while PSI compo-
nents increased in response to heat stress (Ferreria et al., 2006). Alteration of proteins imparting
critical cellular functions is an important mechanism of plant adaptation to heat stress.
When plants are exposed to elevated temperatures, proteins are broken down to be either reformed
as new proteins or transported to other areas of the plant so the nutrients can be reused elsewhere.
Heat stress can directly damage proteins by altering the three-dimensional shape of proteins so
they are no longer in active conformations. These damaged proteins then often aggregate together.
Proteins may be damaged by ROS produced during heat stress. The oxidative damage inflicted by
ROS will also render proteins unable to carry out their metabolic functions. Proteins with altered
conformations due to damage caused directly by high temperatures or through oxidation caused by
ROS are selectively targeted to be degraded. Damaged proteins not only tie up and make unusable
nutrients in the cell but are also potentially damaging by inhibiting metabolic functions and generat-
ing additional ROS.
Another important change in protein metabolism in response to heat stress is the accumulation
of heat shock proteins (HSPs). HSPs can be found in almost all organisms, and although they were
originally discovered as heat-induced proteins, they can be induced by many other stresses, and some
are functional during nonstress steady-state conditions (Feder and Hofmann, 1999). HSPs are often
induced by high temperature and are strongly associated with heat tolerance (Vierling, 1991). HSPs
can range in size from small proteins less than 15 kDa to larger ones over 100 kDa in size. Acting as
chaperone proteins, HSPs assist in the folding or refolding of proteins into their active conformations
(Hendrick and Hartl, 1995). Additionally, some members of this protein family also act as signal mol-
ecules affecting signal transduction and eventual expression of genes (Wang et al., 2004).
Small HSPs are those that are under 40 kDa in size, and while it is a diverse group of proteins,
they all contain a highly conserved α-crystallin domain (Sun et al., 2002). Small HSPs are important
for stabilizing proteins and assist in chaperone functions by binding to denatured proteins to prevent
further denaturation and targeting them to be refolded by other chaperones (Wang et al., 2004). It
is believed that small HSPs bind with the hydrophilic domains of proteins, which become exposed
as a protein denatures but before protein aggregates form (Basha et al., 2012). These proteins are
expressed during heat stress events, as well as in response to many other stressors. Expression pat-
terns may change based on the level and duration of stress, but it is believed that several heat shock
factors (HSFs) may control the expression of small HSPs (Schoffl et al., 1998). Analysis of HSF
structure confirms that they can interact with DNA to influence transcriptional regulation (Nover
et al., 1996). Three classes with over 20 different HSFs have been identified in plants demonstrat-
ing the diversity of this family of proteins responsible for influencing the genetic regulation of HSP
and other heat responses (Kotak et al., 2007). While many details of the pathway still need to be
determined, it is believed that HSFA1 is a major factor responsible for leading to further responses
to high temperatures, while HSFB1, which belongs to a different class of HSF, can be induced by
HSFA1 and is an important coregulator that interacts with other transcriptional regulatory units
(Baniwal et al., 2004).
The HSP 100 protein family is closely related to caseinolytic protease (CLP) family of proteins
and is believed to make up subunits of CLP proteases (Agarwal et al., 2001). Additionally, HSP 100
proteins have been found to play a role in breaking apart aggregated proteins so that other chaperones
can refold or degrade them (Glover and Lindquist, 1998). In maize roots, mutants lacking HSP 101
were shown to have deficient thermotolerance (Nieto-Sotelo et al., 2002). Similar results were found
in arabidopsis mutants lacking HSP 101 expression having reduced heat tolerance; additional mutants
overexpressing this gene had increased tolerance compared to controls (Queitsch et al., 2000).
Other major families of HSPs include HSP 60, 70, and 90. HSP 60, sometimes referred to as
chaperonin, has been shown to prevent the inactivation of proteins under high temperatures (Martin
et al., 1992). HSP 70 has been shown to interact with HSP 100 to refold proteins that have aggregated
(Glover and Lindquist, 1998), but in addition to its chaperone functions, HSP 70 is not only impor-
tant for stress tolerance but has roles in regulating cell development (Su and Li, 2008). In maize
exposed to heat stress, HSP 70 was shown to be involved in the regulation of antioxidant defenses
(Hu et al., 2010). HSP 90 has the chaperone functions of refolding proteins similar to other HSPs;
however, many of its targets are involved in signal transduction, including steroid receptors and pro-
tein kinases, which show that HSP 90 influence the regulation of cell responses to stress (Krishna
and Gloor, 2001). Regardless of the size of HSP, the induction of HSP generally is positively associ-
ated with heat tolerance in plants.
20.6 HORMONE METABOLISM
Plant hormones play key roles in regulating plant growth and development. Hormones are also impor-
tant for inducing responses to stress and the processes of acquired thermotolerance (Larkindale and
Huang, 2004; Kotak et al., 2007). Several hormones that are reported to have major effects on heat
stress responses in plants are discussed here.
Cytokinins regulate cell division and cell differentiation and play roles in plant tolerance to heat
stress (Hare et al., 1997). Cytokinin content decreases under heat stress (Cheikh and Jones, 1994).
Exogenous applications of cytokinins have been found to elevate endogenous cytokinin levels, sup-
pressing leaf senescence and heat-inhibition of photosynthesis, leading to improvement in heat toler-
ance (Tetley and Thimann, 1974; Caers et al., 1985; Veerasamy et al., 2007; Zhang and Ervin, 2008).
Application of cytokinins has been shown to delay heat-induced senescence in creeping bentgrass
(A. stolonifera) leading to improvements in photosynthetic processes, including the maintenance of
chlorophyll content and photochemical efficiency, as well as improved antioxidant function and an
associated reduction in oxidative damage (Liu et al., 2002; Veerasamy et al., 2007; Xu and Huang,
2009). Increases in cytokinin production through genetic transformation have also been reported to
be effective to improve plant tolerance to heat stress. Transformation of tobacco with an isopentenyl-
transferase gene, the enzyme of the rate-limiting step of cytokinin biosynthesis, on a heat shock pro-
moter induced heat tolerance–related genes and suppressed heat-induced senescence (Smart et al.,
1991; Harding and Smigocki, 1994). Studies that used an isopentenyl-transferase gene expressed under
control of a senescence-activated promoter found increased heat tolerance in creeping bentgrass, man-
ifested as maintenance of higher chlorophyll content, as well as improved both shoot and root growth
(Xing et al., 2009; Xu et al., 2009). Investigations of protein changes associated with these transgenic
bentgrasses have found that plants with delayed senescence due to increased cytokinins have altered
accumulations of photosynthetic proteins, HSPs, and antioxidant proteins, which reflect important
physiological pathways of thermotolerance (Xu et al., 2009). Increases of cytokinins in a plant have
been associated with a delay in the expression of senescence-associated genes and an upregulation of
genes associated with photosynthesis (Gan and Amasino, 1996).
Ethylene accumulation has been associated with heat-induced leaf senescence and is an impor-
tant signal regulating the timing of senescence and activation of stress-related genes, as well
as the associated breakdown of cellular components including proteins, membranes, and pig-
ments (Davies et al., 1989; Lim et al., 2007). Periods of high temperatures have been shown to
induce endogenous accumulations of ethylene (Morgan and Drew, 1997; Balota et al., 2004; Hays
et al., 2007). In oat (Avena sativa) leaves, applying an ethylene precursor, 1-aminocyclopropane-
1-carboxylic acid, stimulated senescence while inhibiting ethylene synthesis suppressed senes-
cence (Gepstein and Thimann, 1981). In arabidopsis, similar effects were seen where exogenous
applications of ethylene lead to an induction of senescence-associated genes, while an ethylene-
insensitive mutant had the opposite effect of delayed expression of senescence-associated genes
and the maintenance of photosynthetic genes (Grbic and Bleecker, 1995). In other plant spe-
cies, similar results were found where blocking ethylene production or reception ameliorated
the effects of heat stress (Hays et al., 2007; Xu and Huang, 2009). In creeping bentgrass, the
application of aminoethoxyvinylglycine, a compound that inhibits the synthesis of ethylene, led
to improved heat tolerance through the maintenance of higher chlorophyll content as well as
improved antioxidant functions (Xu and Huang, 2009).
Abscisic acid (ABA) is believed to be responsible for general stress responses and has been
found to increase in response to many stresses, including heat stress (Daie and Campbell, 1981;
Cheikh and Jones, 1994). ABA signaling pathways lead to the induction of transcription fac-
tors that then in turn lead to the expression of genes, which confer tolerance to stress condition
including heat (Kim et al., 2004). ABA was shown to act synergistically with heat to increase
the expression of stress-associated genes, demonstrated by increased expression of genes con-
trolled by a stress-responsive promoter when exposed to both heat and exogenous ABA (Xiong
et al., 1999). Exogenous applications of ABA has been shown to increase heat tolerance, which
has been associated with an increase in antioxidant metabolism resulting in higher activities of
important antioxidant enzymes including APX, CAT, and SOD (Larkindale and Knight, 2002;
Agarwal et al., 2005). Larkindale and Knight (2002) showed that an ABA-insensitive mutant
had a greater sensitivity to heat stress as demonstrated by increased accumulations of oxidative
damage, which further demonstrates ABA’s role in acquired thermotolerance particularly relating
to antioxidant pathways. Additionally, ABA has been further implicated in acquired stress toler-
ance by increased accumulations of HSP proteins in response to exogenous applications of ABA
(Heikkila et al., 1984; Campbell et al., 2001).
Salicylic acid (SA) increases during heat stress and may also play a role in the signaling of gen-
eral stress responses, ultimately leading to increases in heat tolerance (Dat et al., 1998; Horvath
et al., 2007). While exogenous applications of SA result in increases in hydrogen peroxide, possibly
as part of the ROS signaling pathway, it was also shown to increase thermotolerance associated with
increases in antioxidant enzyme activity (Dat et al., 2000). Increases in thermotolerance, as indi-
cated as improved survival, increases in antioxidant function, and maintenance of photosynthetic
health, associated with exogenous applications of SA have been demonstrated in a number of spe-
cies, although frequent application of higher rates leads to declines in thermotolerance showing the
delicate balance needed for appropriate stress signaling (Senaratna et al., 2000; He et al., 2005; Shi
et al., 2006). Furthermore, transgenic arabidopsis with inhibited SA production was shown to have
increased oxidative damage and severely decreased survival during exposure to heat stress, while
conversely mutants with increased SA accumulation were shown to have enhanced thermotolerance
(Clarke et al., 2004; Larkindale and Huang, 2004). SA has been implicated in the genetic regulation
of stress tolerance through experiments that demonstrate that it can induce small nuclear proteins
that then bind to known stress-associated genes, as well as SA leading to increased accumulation
of HSPs (Goldsbrough et al., 1993; Clarke et al., 2004). Although stress-signaling pathways are far
from being fully elucidated, it is clear that plant hormones play roles in the signaling and coordina-
tion of responses to heat stress.
20.7 CONCLUSION
Heat stress causes changes in various metabolic processes at multiple levels from physiology to
molecular biology. Plant adaptation to heat stress involves complex and multiple traits. Those
traits that are strongly correlated with heat tolerance, such as active photosynthesis, membrane
thermostability, induction of HSP, accumulation of senescence-suppressing hormones, and oxida-
tive scavenging, could be used for improving heat tolerance through molecular marker–associated
breeding and/or the candidate genes controlling heat tolerance traits being incorporated through
genetic transformation. Such approaches have been found effective in generating heat-tolerant
germplasm in various plant species. For example, several studies have already shown that the
insertion and regulation of key genes can lead to improved tolerance such as HSPs in carrot
(Daucus carota subsp. Sativus) and arabidopsis (Malik et al., 1999; Queitsch et al., 2000) or
increased cytokinin production in perennial grasses (Xu et al., 2009). However, the efficiency
of heat tolerance modification can be further improved as more insights about heat tolerance
mechanisms are explored and multiple interactive traits may be incorporated due to the nature of
complex multigenic traits of heat tolerance.
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21 Drought Resistance in
Small Grain Cereal Crops
Moustafa Eldakak, Mukhtar Ahmed, Muhammad Asif,
Sanaa I.M. Milad, Ali I. Nawar, Zohra Aslam,
Aakash Goyal, and Jai S. Rohila
CONTENTS
21.1 Introduction...........................................................................................................................405
21.2 Importance and Production of Wheat as a Major Small Grain Cereal Crop........................407
21.3 International Institutes Working on Wheat Drought Resistance...........................................408
21.4 Exploring Genetic Diversity for Drought Resistance............................................................409
21.5 Examples of Physiological Effects of Drought Stress on Crop Plants.................................. 410
21.5.1 Photosynthesis........................................................................................................... 411
21.5.2 Growth Responses..................................................................................................... 411
21.5.3 Development.............................................................................................................. 412
21.5.4 Reproductive Processes and Crop Yields.................................................................. 412
21.6 Molecular Approaches for Obtaining Drought Resistance in Crop Plants........................... 413
21.6.1 sRNAs Can Regulate the Gene Expression during Drought..................................... 413
21.6.2 Transcription Factors Can Regulate the Expression of Many Genes........................ 414
21.6.3 Differential-Regulated Functional Genes and Proteins Provide Tolerance
to Drought Stress....................................................................................................... 415
21.6.4 Overview of Gene Revolution Following Green Revolution..................................... 416
21.7 Understanding Drought Tolerance Mechanisms................................................................... 416
21.7.1 Genetics and Molecular Approaches......................................................................... 417
21.7.2 Genomics-Based Approaches.................................................................................... 418
21.7.3 Biotechnological Approaches.................................................................................... 418
21.7.4 Modeling Approaches................................................................................................ 419
21.7.5 Root Studies............................................................................................................... 420
21.8 Challenges and Future Perspectives...................................................................................... 420
References....................................................................................................................................... 421
21.1 INTRODUCTION
Historical evidences have linked drought with the collapse of the Mayan civilization from 1020
to 1100 A.D. (Kennett et al. 2012). This statement tells the drastic effects of drought in very
plain words and also cautions us to find the ways to tackle this problem, which limits the food
productions, for the benefit of the future generations. Drought tolerance or resistance is defined
as “the potential of a plant to display flower and produce an economic yield under limited supply
of water” (Farooq et al. 2009). The viable components of drought resistance in plants include the
avoidance as well as the tolerance to water-limited conditions. Plant mechanisms that contribute
405
primarily to the drought resistance in wheat are early maturity so that the crop ripens ahead of
periods of drought stress; a vigorous and deep root system to efficiently utilize the available soil
moisture; an active mechanism that senses the water-limited environment and closes the stomata
in response to the drought stress without hindering the photosynthetic output; a waxy layer on the
leaf surface to reduce transpiration loss; and a complex mechanism in which the macromolecules
and several small biological molecules such as proteins, nucleic acids (microRNA, RNA, and
DNA), lipids, carbohydrates, free radicals, hormones, mineral elements, and ions play crucial
roles toward making the plant to cope with the drought stress. Drought is also associated with
various stresses, such as high temperatures, excess salt, acidity, cold, and alkalinity, and a number
of physiological reactions including pathological reactions, growth, senescence, embryogenesis,
development, wound healing, signal transduction, damage by UV-B radiation, flowering, and
many other processes. Hence, in general, drought stress is associated with nearly all features of
biology of a plant (Hong-Bo et al. 2006).
Wheat is the major small grain cereal crop in the drier regions of the temperate zone and is
widely grown in areas subject to frequent drought stress (Poehlman and Sleper 1995). As water
availability for irrigation is likely to decline in the coming decades, the regions dedicated to wheat
productions will be threatened by, or exposed to, increasingly low levels of irrigation. Therefore,
the modification of wheat to tolerate drought stress is necessary to maintain wheat productions
and to maintain socioeconomic balance at global levels (Zhao et al. 2008). Drought-tolerant plants
will provide a practical solution toward alleviating the problem of water scarcity and enabling the
sustainability of agriculture. At present, drought analysis has been one of the major focuses of plant
breeding and plant biology worldwide. Many advances in this important topic and many promising
approaches, including biotechnological breeding and the development of molecular mechanisms to
resist drought, have been developed; however, these methods and practices must be translated to the
field in the near future (Hong-Bo et al. 2006). Osmotic regulators include micromolecules such as
soluble sugars, proline, potassium, and betaine; all these small molecules are useful physiological
indicators of the capacity for drought resistance and the osmotic adjustment ability of wheat geno-
types and species (Hong-Bo et al. 2006). In plants, the production of reactive oxygen species (ROS)
such as superoxide anion radicals, hydrogen peroxide, singlet oxygen, and hydroxyl radicals can
cause significant dehydrating effects. ROS are mainly produced in chloroplasts; as a result, photo-
synthetic activity is compromised during the stress conditions. Drought resistance is unequivocally
related to antioxidant cellular processes. Antioxidative activity can be enzymatic (e.g., peroxidase,
metallothionein, superoxide dismutase, and catalase) and/or nonenzymatic (e.g., vitamins E and C,
flavonoids, glutathione, polyamines, carotenoids, and alkaloids) (Xoconostle-Cazares et al. 2010).
Developing drought and heat tolerance in crop plants is the demand of time, because the popula-
tion increase is already posing a serious threat to food security (Berg et al. 2013); moreover, the
projections coming out of the climate models predict that the global temperature will rise another
2.0°F–11.5°F by the end of this century (Marcott et al. 2013), causing more stress conditions for
major field crops (Teixeira et al. 2013) such as rice, wheat, maize, and soybeans. In this chapter, we
are trying to provide the latest information available in public domain that help in understanding
the importance of water for a plant, especially a cereal crop plant, and the cellular mechanisms that
plant physiologists, molecular biologists, and breeders can utilize to enhance the drought toler-
ance levels of their plant of interest, which may mitigate the impact of climate change through the
improved plant breeding practices and better cultivar development. This information is continually
enriching our current understanding at a much faster pace than we could realize, especially by the
utilization of transcriptomics, proteomics, metabolomics (collectively called systems biology), and
biotechnology approach in the omics era; thus, these technologies hold a great promise in the future
to help the plant breeders significantly (Cominelli et al. 2012; Cramer et al. 2011; Eldakak et al.
2013; Varshney et al. 2011). The new knowledge and the technologies are a boon for modern plant
breeders, as the available tools are helping them to bring the next green revolution, which is now
expected to come from the dry lands (Cominelli et al. 2012; Cooper et al. 2008).
Drought Resistance in Small Grain Cereal Crops 407
21.2 IMPORTANCE AND PRODUCTION OF WHEAT

AS A MAJOR SMALL GRAIN CEREAL CROP
Humankind is dependent on crops and other plant materials for food and feed. At present, crops
utilize almost one-fifth of the Earth’s vegetated surface and constitute the biggest imprint of man
on the planet and its landscapes. Cropping is the principal source of livelihoods and employment
worldwide, with well over one billion plant breeders and small farmers in progressing the global
agriculture forward (Anand et al. 2003; Andersson et al. 2004; Apel and Hirt 2004).
Wheat (Triticum aestivum L.) is the major cereal food crop, after rice, used to meet the global
food demands (Khan and Iqbal 2011). In the twenty-first century, the increased water demands
of urban populations, industry, and agriculture will decrease the water availability for irrigation.
Moreover, it is predicted that in various agricultural systems, the emerging climate change will
increase the variability of rainfall in some places and reduce rainfall in others. Thus, the challenge
for crop scientists is to enhance productivity with reduced rainfall in dryland agriculture and less
available of water for irrigation in many parts of the globe (Hong-Bo et al. 2006). Developing high-
yield wheat cultivars under drought conditions in semiarid and arid regions has now become an
important objective of breeding programs (Leilah and Al-Khateeb 2005).
Of the many abiotic stresses that can affect crops (e.g., salinity, extreme temperature, drought),
drought is considered to be the most limiting factor in wheat production in many countries (Khan
and Iqbal 2011). The production of wheat is adversely influenced by drought in 70% of developed
countries and 50% of developing countries. In many parts of the world, water has driven the agricul-
tural production. The “green revolution,” which resulted in the fruitful increase in crop production
due, in part, to increased irrigation, was noteworthy and successful in reducing hunger (Hong-Bo
et al. 2006), but now with climate change, the drought is creating a scary picture for the future
cereal crop production.
For a long time, wheat has provided sustenance for the global population. Wheat is produced in
many geographic regions and climatic environments, as shown in Table 21.1. Wheat serves a broad
range of purposes, including providing basic food for a majority of poor consumers and farmers
of the world. While developing and developed countries appear to have similar average yields,
the resemblance is deceptive. In developing countries (especially in India and China, which are
large wheat producers), more than 50% of cultivated wheat is irrigated; in developed countries,
approximately 90% of wheat is rain-fed. In addition, there are huge differences in wheat production
between countries deploying similar agronomic practices. Thus, the need to enhance wheat produc-
tivity is considerable in many countries (Ortiz et al. 2008).
Wheat is an important staple food in countries like China, India, the United States, Russia,
France, Canada, Germany, Turkey, Pakistan, Australia, the United Kingdom, Iran, Poland,
Egypt, Italy, Romania, Argentina, Uzbekistan, Argentina, Kazakhstan, and Ukraine (FAO 2007).
Internationally, wheat is the most traded food crop, the most imported crop in developing nations,
and also constitutes a main portion of the crisis food aid program (Ortiz et al. 2008). In the future,
the global population is anticipated to rise steadily to approximately 9 billion in 2050. Based on
stock exchanges and production, the demand for wheat is projected to rise from 621 million t (from
2004 to 2006) to 760 million t (in 2020) and to nearly 813 million t (in 2030) to 900 million t
(in 2050); in other words, the rise in demand will occur at rates of 1.6% growth rates for 2005–2020
and 0.9% for 2005–2050 (FAO 2007).
The global average of wheat production has increased for many decades due to the support of
the International Wheat Improvement Network (IWIN), a consortium of the International Maize
and Wheat Improvement Center (CIMMYT), the National Agricultural Research System (NARS),
the Advanced Research Institutes (ARI), and the International Center for Agricultural Research in
the Dry Areas (ICARDA). In developing countries, IWIN has deployed avant-garde science and
multidisciplinary practical approaches that were used in the germ plasm development; these efforts
have modernized the livelihoods of farmers and have improved the food security (Ortiz et al. 2008).
TABLE 21.1
Production of Wheat in Selected Regions around the Globe
Regions Wheat Production (Million t)
Australia and New Zealand 19
Central Asia and Caucasus 22
Developing countries 308
East Asia 98
Eastern Europe and Russia 69
European Union 27 137
North Africa and Middle East (including Turkey) 61
North America 88
South America 22
South Asia (including Afghanistan) 97
Sub-Saharan Africa world 9
World 621
Nations/land governed by rain-fed production
Canada 27
Kazakhstan 12
United Kingdom 15
Nations/land governed by irrigated production
Egypt 8
India 70
Source: FAOSTAT, http://faostat.fao.org/site/567/default.aspx#ancor, 2012.
21.3 INTERNATIONAL INSTITUTES WORKING

ON WHEAT DROUGHT RESISTANCE
In the developing world, the CIMMYT acts as a leader and catalyst in a wheat and maize worldwide
innovative networks that works with and helps the poor farmers (Ortiz et al. 2008). The value of
CIMMYT resides in its relevant use of crop genetic diversity: CIMMYT studies conserve, share,
and add worth and value to crop genetic diversity, by working with clients worldwide. The main
undertakings of this organization include the following:
• The development of strategic germ plasm through innovative genetic enhancement

• Exploiting the original or untapped significant of crop genetic resources through the
discovery of crucial traits required for future and current target beneficiary generations
• Understanding the genetic diversity of wheat and maize (the two most important staples)
globally
• The safe and long-term preservation of the world heritage of wheat and maize crop
resources for future generations
These undertakings are in line with the ceremonial agreements of the Plant Genetics Resources for
Food and Agriculture International Treaty of 2004 (Ortiz et al. 2008).
The cultivation of wheat is linked to ancient and modern history of human civilization. The grain
is broadly used to prepare bread. Half of the wheat production is carried out under the conditions of
limited water availabilities. Conventional breeding at CIMMYT yielded polyploid plants that pro-
vided lines with drought tolerance and new genetic variability. Conventional breeding worldwide is
performed in both private and public research centers. The International Crops Research Institute for
the Semi-Arid Tropics (ICRISAT) in India and the International Centre for Agricultural Research
in Dry Areas (ICARDA) in Syria are worth mentioning here for their significant contribution in this
field. The specific portions of wheat genomic DNA fragment regulating the production of abscisic
acid (ABA) were discovered on the 5A chromosome. Transgenic bread wheat lines produced by
means of genetic engineering harboring the Arabidopsis thaliana (DREB1A) that showed drought
tolerance were developed at CIMMYT, Mexico, underneath the control of rd29a, a stress-inducible
promoter (Xoconostle-Cazares et al. 2010).
The CIMMYT strategy for improving the efficiency of breeding for drought tolerance is to cer-
tify that drought-tolerant germ plasm manages to respond positively if extra moisture becomes
available in a season. Tolerance to drought and high-yield potential are not mutually exclusive
and can be bred concurrently in alternate generations by selecting segregating populations under
drought stress and favorable conditions.
Drought resistance is a multivariate characteristic that is also affected by root diseases of
plants. Healthier roots use the mixture of available soil more efficiently than the sick roots do.
Luckily, resistance to nematodes and to certain root diseases and pathogens is often inherited,
and molecular markers to employ marker-assisted selection (MAS) are available to assist breed-
ers in cultivar selection. CIMMYT uses these molecular markers to breed drought-resistant
wheat material (Xoconostle-Cazares et al. 2010). While CIMMYT focuses on value-added germ
plasm and higher grain yields, it also plays an “integrative” role in promoting crop management
research, lower production costs, the efficient usage of irrigation water resources and other inputs,
the management of biotic stresses, and the improved resilience and diversity of systems (Ortiz
et al. 2008).
Finally, the CIMMYT must confirm that its products reach end users and that their livelihoods
improve. CIMMYT is the main public, transnational source of wheat seed-embedded technology
to alleviate poverty, reduce vulnerability, and help breeders/agriculturalists to move forward from
subsistence to income-generating production systems (Ortiz et al. 2008).
21.4 EXPLORING GENETIC DIVERSITY FOR DROUGHT RESISTANCE

Enhancing crop productivity is a daunting challenge in drought-prone environments (DPE) due to
the many factors involved and their interactions with the surrounding environment. Wide crossing,
conventional breeding, and trait-based approaches have produced positive results in DPE. To widen
gene pools, three main approaches can be manipulated:
• Wide crosses, which involve intergeneric and interspecific hybridization

• Genetic transformation
• Introgression with appropriate or compatible genomes from germ plasm (Xoconostle-
Cazares et al. 2010)
Landraces to introduce genes for abiotic and biotic stress resistance have been extensively used in
various breeding programs.
MAS for genetically simple traits that are indirectly related to drought and disease resistance has
also routinely been applied. Because drought adaptive traits are multigenic, quantitative trait loci
(QTLs) have become essential tools for the molecular tailoring of crop plants. However, during the
past decade, despite the impressive progress of various “omics” platforms (e.g., proteomics, micro-
arrays, sequence information, and bioinformatics) and MAS, the overall impact on the development
and dissemination of drought-resistant cereal cultivars remains insignificant (Xoconostle-Cazares
et al. 2010).
21.5 EXAMPLES OF PHYSIOLOGICAL EFFECTS

OF DROUGHT STRESS ON CROP PLANTS
The physiological factors relevant to resisting the drought include the ability of plant tissues to
regulate its cellular water content (Jones 2007). In turn, this depends on the relative fluxes of water
throughout the plant and within the soil–plant–atmosphere continuum. Thus, in addition to water
shortages, the water vapor pressure gradient from the air to the leaf and soil can also impose drought
situations on the plant (Cattivelli et al. 2008). Once a decrease in water potential develops, a broad
range of physiological responses are induced in the plant. Some of these physiological responses are
brought about by changes in the signaling hormones associated with water status, while others are
triggered directly by the changing water content of tissues (Chaves et al. 2003).
The physiological crop traits that respond to water inadequacy and that are changed by subopti-
mal water supply (Table 21.2) span a wide variety of basic processes. As a result, it can be strongly
assumed that there is no one distinct response pattern that is highly correlated with yield under
drought environments.
TABLE 21.2
Plant Physiological Traits Relevant for Response to Drought Conditions
Physiological Traits Modulation under Stress Appropriate Effects for Yield References
Utilization of stem By increased remobilization, Lower/Higher reserves remobilization Slafer et al. (2005)
reserve and rectification of reduced for grain filling from stems,
partitioning current leaf photosynthesis affecting kernel weight
Availability of starch Photosynthetic activity Reduction in starch availability leads Boyer and Westgate
during development inhibition, reduces starch to abortion and grain number (2004)
of embryo/ovary availability reduced
Rooting depth Total mass reduced but ratio Lower/higher soil tapping resources Hoad et al. (2001),
of root/shoot increased of water Sharp et al. (2004)
Leaf temperature/ Under stress, stomatal Less/more rapid consumption of Lawlor and Cornic
stomatal conductance hindrance increase water. Temperature of leaf reflects (2002)
evaporation and hence is a function
of stomatal conductance.
Photosynthetic Reduction under stress Modulation of enzymes concentration Lawlor and Cornic
contents of the Calvin cycle and the light (2002)
reaction elements
Stress-relevant Accumulate or assembled Involved in the cellular structure, Ramanjulu and
proteins under stress protein activities, and protection Bartels (2002)
accumulation
Antioxidative defense Defense system acclimation Protection or stability against active Ramachandra Reddy
species of oxygen et al. (2004)
Osmotic adjustment Slow or inactive response to Solutes accumulation: sugars, ions, Serraj and Sinclair
water potential glycine betaine, amino acids, (2002)
poly-sugars
Timing of Wheat advanced flowering Early/late flowering. Growth duration Richards (2006),
phenological phases and maturity, reduced grain number, Slafer et al. (2005)
and synchrony of anthesis and silk
emergence
Membrane Regulation in response to Changes in aquaporin function and Tyerman et al.
composition changes in water potential membrane stability increased (2002)
Single plant leaf area Reduction under stress Related to plant productivity and size Walter and Schurr
(wilting senescence, (2005)
abscission)
21.5.1 Photosynthesis
Keeping a good photosynthesis system till the grains or fruits of a plant are completely filled is a pre-
requisite of the drought-tolerant crop, and this character is genotype dependent (Snider et al. 2012).
Drought stress primarily affects the photosynthesis in three aspects, that is, physiological, biochemi-
cal, and molecular levels (Xu et al. 2013). Drought limits the photosynthesis by several means includ-
ing stomatal regulation, which is primarily by stomatal closure that leads to a decrease in CO2 inflow
to the mesophyll tissue (Chaves et al. 2003; Flexas et al. 2004). The primary metabolic and physi-
ological changes upon the onset of drought stress are through limiting the regeneration efficiency
of ribulose bisphosphate (RuBP) and ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo)
protein, which hampers the carbon fixation cycle directly (Bota et al. 2004), and this is because of
the decreased activity of carbon fixing enzyme RuBisCo (Parry et al. 2002) and damage to the ATP
synthesis machinery in the chloroplast. In general, a decreased stomatal conductance is observed
during the early phases of drought stress, which in turn leads to a deterioration of photosynthesis
machinery. On the other hand, in some plant species during the early stages of drought, a transitory
internal CO2 level gets increased, by the closure of stomata (Briggs et al. 1986). Zhou et al. (2007)
reported that both the diffusive limitations (by stomatal closure) and the non-stomatal limitations
(such as oxidative damage to chloroplasts) are key factors for lowering the photosynthesis efficiency.
21.5.2 Growth Responses
During drought conditions, plant has to make a decision whether it wants to divert its limited energy
resources preferentially toward growth and development or toward keeping a steady rate of physiologi-
cal reactions. Two processes that have a direct link with the plant’s growth are cell division and cell
enlargement. Cell division in meristematic tissues is known to be affected by drought stress in plants,
even before the photosynthesis and respiration processes start affecting. Under conditions of severe
water deficiency, cell elongation can be inhibited from the xylem to surrounding elongating cells due to
the interruption of water flow, especially in tissues that are farthest from the rhizosphere. Under drought
stress, cell elongation, expansion, and the impaired mitosis result in reduced crop growth, leaf length,
and leaf area index (Farooq et al. 2009). Cell growth is also influenced by the cell turgor pressure. Leaf
expansion is among the most sensitive growth processes to drought (Alves and Setter 2004). A poorly
expanded leaf, which is a result of drought, has smaller size of cells and has a fewer number of total
cells produced in leaf meristem tissue (Tardieu et al. 2000). Alves and Setter (2004) showed that both
the cell expansion and number of cells contribute in leaf area index, depending on the developmental
stage of the plant at which the drought stress has occurred. The mature leaves, which are no longer
involved in new cell divisions, have less effect on the average cell expansion rate. On the contrary, in
younger plants, the inhibition of cell division was found prominent and resulted in reduced numbers
of leaves, as a general rule of thumb. Because of this reason, the consequence of mild drought stress
on plants is seen as reduction in total leaf number, expansion rate, and average leaf size. On the other
hand, under severe and extended period of drought stress, the rate of leaf elongation decreases and leaf
growth can come to a total halt. A steady and longer duration of drought stress can accelerate the rate of
senescence in plants (De Souza et al. 1997) and cause death of leaf tissue and leaf abscissions of old and
mature leaves. Besides, the rewatering after short stress period did not revert the effects of drought and
the process of senescence (Brevedan and Egli 2003). Simonneau et al. (1993) explained that the stem
diameter can shrink in response to significant fluctuations occurring for the internal water status of
the plant. The changes in stem diameters were found to be highly correlated with the leaf water poten-
tial under control and the prolonged drought stress conditions (Katerji et al. 1994). Yatapanage and
So (2001) reported the similar results when they measured stem diameter after drought in Sorghum.
During drought stress, the responses of root growth are variable; in case of moderate moisture stress,
root growth is found to be significantly higher because of increased levels of carbohydrates partitioning
toward the roots. Antagonistically, the severe drought results in decreased root growth.
21.5.3 Development
It is not only the growth curve of the plant that is affected by the stress, but the transition period of the
developmental stage can also be affected by the drought stress. Drought, as well as heat stress, alters
the timing of initiation and the duration of developmental phases of the plant. The total time period
from floral initiation to anthesis is decreased as a result of moderate drought in the life of a plant, but it
is increased as the severity of the drought increases. If the onset of drought stress happens at flowering
stage, the success rate of fertilization process is significantly decreased because the pollen develop-
ment and the ovule functions get affected. Further, drought stress also reduces the total duration for
ear development and the grain filling period. Drought stress at advanced stages of flower or panicle
development decreases the total numbers of filled spikelet. Frederick and Camberato (1995) showed
a high correlation between the reduced leaf area index before anthesis and the reduction in number of
kernels per spike, during the drought stress. Although the onset of drought after flowering does not
affect the percentage of seed filled, it shortens the seed filling duration, and this leads to the small-size
seeds and eventually leads to reduced seed yield (Wardlaw and Willenbrink 2000).
21.5.4 Reproductive Processes and Crop Yields

As discussed earlier, drought causes a significant decrease in the photosynthetic rates, especially at
flowering stage, and thus results in the decreased amount of allocated photosynthates to the floral
organs. This is the main reason of lower rates of seed settings in crop plants (Raper and Kramer
1987). ABA is the most prominent plant hormone related to stress, which helps in maintaining the
root growth and increase the root hydraulic activities during drought periods. This leads to the
increased uptakes and hydraulic activities of water. ABA also plays an important role in cellular
signal transduction at whole plant level and to remote plant organs including those of developing
embryos leading to sterility and abortion during drought stress. As a result of dehydration of leaf
cells during drought stress, ABA starts reaching to the shoots through xylem and causes the clo-
sure of stomata, lowering the rates of leaf expansions. The ABA can translocate to the developing
embryos that lead to the poor seed settings. This hypothesis is well tested through experiments
where ABA was applied exogenously to the developing embryos under normal irrigation conditions.
The exogenous application of ABA mimicked the drought stress conditions in flower organs and
caused abortion and sterility (Mambelli and Setter 1998). If the onset of drought occurs after the
fertilization phase, the seed size is affected more than the seed numbers.
The response of the plant’s reproductive processes to drought at the whole plant level may differ
on the basis of their determinate or indeterminate nature of flowering. The indeterminate flowering
crops, such as peanuts, pea, canola, Brassica, and dry bean, undergo floral initiation over a longer
period of time during non-stress or mild stress environments and can compensate in yields, for the
inhibited development loss during the periods of high stress. In other words, increased failures of
embryos and seeds at one stage of development can be compensated with an increased success at
later stages of flowering. On the other hand, the crops that show determinate nature undergo floral
initiation over a short period of time, and if this period, unfortunately, coincides with that of the
drought stress, the reproductive phase will severely be affected and so the yield.
In plants, many yield-determining physiological processes respond to water deficits. These yield-
determining physiological processes are integrated among each other in a complex way. Thus, with
the current knowledge and understanding, it is difficult to elucidate how plants combine, display, and
accumulate the ever-changing and indefinite physiological processes over their life span. Severity
of water stress, timing and the duration of stress, the response of the plant after the removal of the
stress, and the interaction between other factors and stress are highly critical to consider in breeding
for drought tolerance. In many crop species, drought-induced yield losses dependent on the dura-
tion and severity of stress have been reported in crops such as barley, maize, rice, wheat, sunflower,
potato, canola, chick pea, cowpea, pigeon pea, common beans, and soybean (Farooq et al. 2009).
In cereals, grain filling is a process of synthesizing starch from carbohydrates. Four enzymes
play a strategic role in this process: starch branching enzyme, synthase, starch synthase, and ade-
nosine diphosphate glucose pyrophosphorylase (Taiz and Zeiger 2010). A reduction in the grain
growth rate in water-stressed wheat has resulted from a decline in the activity of sucrose synthase,
although growth cessation can also result from adenosine diphosphate glucose pyrophosphorylase
inactivation (Ahmadi and Baker 2001).
21.6 MOLECULAR APPROACHES FOR OBTAINING

DROUGHT RESISTANCE IN CROP PLANTS
In DPE, conventional breeding has been supplemented by adopting exotic germ plasm to amplify
the crop gene pool and by examining physiological mechanisms such as harvest index (HI), water
uptake (WU), and water-use efficiency (WUE) as yield drivers (Reynolds and Tuberosa 2008).
WUE under stress is considered an important component of drought tolerance and yield of a
crop. Rain-fed plant production can be enhanced per unit use of water, according to the “more crop
per drop” mission. As long as the photosynthesis biochemistry is not improved genetically, WUE
and transpiration efficiency (TE), two extremely significant traits for plant production, are driven by
plant traits that minimize crop water-use and transpiration. As the production of biomass is closely
linked to transpiration, breeding for transpiration to obtain the maximal capture of soil moisture
under conditions of drought stress is the most important target for yield enhancement. The effective
use of water (EUW) for transpiration implies the maximal capture of soil moisture; this process
requires the minimal loss of water by soil evaporation and reduced non-stomatal transpiration.
Osmotic adjustment is a main adaptive trait that can increase transpiration and soil moisture capture
to resist the drought. A high HI describes the partitioning of assimilates toward the reproductive
functions of the plant and is expressed as an indicator of successful yield and high levels of plant
production capabilities. By improving the status of plant water, EUW helps sustain reproductive
success and the partition of assimilates; therefore, EUW is a major target for improving yields in
water-limited conditions (Blum 2009).
To obtain new individual cultivars, conventional improvement is based on the incorporation
of better characteristics into the offspring/progeny by selecting desirable genetic traits. For this
purpose, two plants with desirable traits are crossed such that these traits are exchanged and the
progeny acquires new genetic arrangements in the nucleus. This way, the plant breeders are devel-
oping the fittest cell for the plant’s standing in stress environment. To express the appropriate traits
and to maintain their expression in future generations of plants, individual plants are needed to be
tested. In practice, tolerance to drought and plant productivity can both be selected; noncommercial
varieties showing drought tolerance are crossed with exposed/susceptible plants of higher yields
(Xoconostle-Cazares et al. 2010).
21.6.1 sRNAs Can Regulate the Gene Expression during Drought

Genome-wide transcriptomics and proteomics discoveries have highlighted a key fact that drought
stress changes the genes and protein expressions inside a plant cell at significant levels. The next
question arises—how? The pre-transcriptional control regulated by transcription factors is an estab-
lished fact, but posttranscriptional control is not very clear at molecular levels. Recent studies in
different crop plants are establishing the fact that small RNAs (sRNAs) regulate the conserved gene
expressions of various mechanisms, including plant physiology, at posttranscriptional levels during
various stresses (Kantar et al. 2011; Shinozaki and Yamaguchi-Shinozaki 2007; Shukla et al. 2008).
These sRNAs are called microRNAs, when they are produced as distinct sRNA species from MIR
genes (Kruszka et al. 2012), and are known to be involved in various developmental and physiologi-
cal processes that require intercellular signaling (Marín-González and Suárez-López 2012). The
numbering of the miRNAs (e.g., miR397 that is found to increase during dehydration in Arabidopsis
[Sunkar and Zhu 2004]) is sequential from the miRNA database. If a sequence of miRNA from one
species already exists in the database and other researchers identify similar sequence of miRNA in
a different species, then the number remains the same and is attached to the new species. An inter-
esting aspect of miRNA-regulated gene expression is that a same sequence of miRNA could involve
slightly divergent mechanisms in different plants, which suggest that it is a highly adapted mecha-
nism (Kruszka et al. 2012), for example, OsMIR169g is upregulated in response to water stress in
rice and is known to have two cis-acting dehydration-responsive elements (DREs) in the promoter
region of its gene. Unlike rice, the AtMIR169a and AtMIR169c are downregulated in response to
water stress in Arabidopsis. As a result, the target gene (NFYA5) of AtMIR169 is strongly upregu-
lated and contributes toward drought resistance in Arabidopsis (Li et al. 2008).
21.6.2 Transcription Factors Can Regulate the Expression of Many Genes

The transcriptomics approach involves the study of transcripts (mRNA) for drought tolerance in
crop plants and may lead to novel identification of genes encoding transcription factors. These
transcription factors are known to regulate the expression of several genes by interacting with the
promoter region of genes. So far, various studies have shown that drought and other stresses affect
the abundance of certain transcripts, but it is not clear yet whether this pattern is conserved among
certain species of plants or it is specific to each genotype and each environment. The transcription
factors are the molecular switches and regulate the expression of other genes that would provide
tolerance to certain stresses, for example, drought. Two successful examples in the late 1990s were
shown independently (Jaglo-Ottosen et al. 1998; Kasuga et al. 1999) by overexpressing the tran-
scription factor gene, which lead to the overexpression of several other stress-responsive functional
genes. These independent expressions of transgene established the fact that transcription factors
may work as master switch to turn several genes on simultaneously and can be utilized to enhance
the plant tolerance to various stresses. Among the several genes, Myb proteins are one member of
such conserved and ubiquitous transcription factor family. The Myb transcription factor motif is
made up of helix–turn–helix repeats, which are imperfect in nature. Myb-related plant genes make
a large family and are likely to participate in a variety of functions, including drought tolerance
(Meissner et al. 1999). Similarly, Myc-like proteins, another class of transcription factors, contain
the basic helix–loop–helix domain and are comprised of two subdomains: the basic region that is
accountable for DNA binding and the helix–loop–helix region accountable for its dimerization. The
rd22 BP1 gene in Arabidopsis, which encodes a Myc homolog transcription factor, has been shown
to get upregulated by stresses like drought and salt. The upregulation of rd22 BP1 gene is correlated
with the activation of rd22 gene (Abe et al. 1997). Homeodomain–leucine zipper (HD–ZIP) genes,
another class of transcription factors, encode proteins that regulate the development of a plant and
its responses to interactions with several environmental conditions. HD–ZIP proteins have two
unique characteristics in its structure: (1) DNA-binding homeodomain present with (2) a leucine
zipper motif. The specific activity of an HD–ZIP protein lies in the specific DNA-binding property
of its homeodomain and the ability of its leucine zipper to mediate the protein–protein interactions
with other members of HD–ZIP protein family. Three Arabidopsis genes of the HD–ZIP family II
(ATHB-7, ATHB-6, and ATHB-12) are shown to be responsive to drought in an ABA-dependent
signaling pathway (Lee and Chun 1998; Söderman et al. 1996). During a study involving the water
stress and low-temperature-regulated genes in Arabidopsis, C-repeat motif CCGAC, which is a part
of DRE, was discovered. This led to the first report that explained the promoter of water stress and
low-temperature-regulated gene rd29A (Yamaguchi-Shinozaki and Shinozaki 1993). DREB1A and
DREB2A, the two subclones, were later isolated by Liu et al. (1998). These clones were described
as DRE-binding protein, which specifically bind to the DRE region of the rd29A promoter gene.
Overexpression of the DREB IA driven by the constitutive 35S CaMV promoter, or the stress-
inducible rd29A promoter, shows an increased level of tolerance to low temperature, drought, and
salt stresses (Kasuga et al. 1999). Twelve stress-inducible genes were identified by Seki et al. (2001),
which were the potential targets of DREB1A transcription factor. Several basic leucine zipper
(b-ZIP) transcription factors are found to contain a DNA-binding domain, are rich in basic residues,
and have a dimerization domain next to the leucine zipper. These b-Zip transcription factors, which
bind to ABA-responsive elements (ABREs), have been cloned in the past and are considered as
candidates for ABA-responsive transcription factors that induce the gene expression by dehydration
and/or ABA. Uno et al. (2000) isolated two cDNAs encoding b-ZIP-type ABRE-binding proteins
(AREB1 and AREB2) and found that their transcript was upregulated by drought, salt, and ABA.
Another big family of transcription factors, which is plant specific, is known as WRKY. This family
of proteins got its name because proteins of this family contain a conserved domain consisting of
WRKYGQK amino acids. The protein sequences, which come under this family, can be recognized
by a WRKY domain on its -NH2 terminus and a zinc finger motif at its -COOH terminus. The mem-
bers of this family are recently demonstrated to be involved in regulation of drought stress tolerance
genes (Rabara et al. 2013; Rushton et al. 2012; Tripathi et al. 2012). Zinc finger proteins (ZFPs) are
a large family of proteins that works as transcription factors in plants. Rai et al. (2013) and many
other references show that ZAT12, encoding C2H2 ZFP, confers multiple abiotic stress tolerance,
including drought stress tolerance as evidenced by elevated catalase activity, low electrolyte leak-
age, and higher relative water content in leaves.
21.6.3 Differential-Regulated Functional Genes and Proteins

Provide Tolerance to Drought Stress
Late embryogenesis abundant (LEA) proteins are one of the major functional protein families
that accumulate in abundance during late stages of embryogenesis and during drought and other
water-related stresses. Predominantly, they are distributed among monocot and dicot plant species.
The LEA proteins are characterized by a biased amino acid composition and their high hydro-
philic nature. The homology among different LEA proteins and the presence of conserved protein
domains (e.g., LEA3 contains consensus sequence of TAEAAKEKAXXXE) and their expression
during water stress conditions assure their role in drought tolerance. This group of proteins has
been proposed to play key roles, such as membrane and protein stabilizers, in the protection against
cellular collapse after dehydration (Ramanjulu and Bartels 2002; Tunnacliffe and Wise 2007).
Aquaporins are a complex family of channel proteins that facilitate the transport of water across the
transmembrane water potential gradients in the cell and that can increase the hydraulic conductivity
of the membrane and increase the water potential by 10–20-folds (Maurel and Chrispeels 2001).
Earlier, several aquaporin genes were found to be upregulated under drought stress in Arabidopsis
plants (Yamaguchi-Shinozaki and Shinozaki 1993). Recently, different forms of aquaporins have
been reported in rice that are highly regulated under severe drought stress conditions (Mirzaei et al.
2012). During various stresses, including drought, an oxidative stress environment is generated that
is a consequence of the accrual of ROS, which are known to impair the cellular structures (Smirnoff
1993). However, plants are well found with several antioxidant enzymes and metabolites that help
to handle with these ROS. The accrual and the upregulation of enzymes such as ascorbate peroxi-
dases, superoxide dismutases, glutathione S-transferases, and catalases have been reported under
stress environments in different cell compartments (Mittler 2002). The fate of the cell that comes
under drought and whether this cell will continue to function or will face photorespiration depend
upon the capacity of the antioxidative defense system (Foyer et al. 1994). Several nonspecific lipid
transfer proteins (nsLTPs) have been reported to increase in Lycopersicon pennellii in drought con-
ditions (Treviño and O’Connell 1998). This increased level of nsLTP indicates the induction of the
epidermal cell-specific expression that represents an adaptive response to drought stress. Proteolytic
activity is high under stress environments and has a positive correlation with programmed cell
death (PCD) (Fukuda 1997). The expression of cysteine protease inhibitors enhances the drought
tolerance ability of plants (Solomon et al. 1999; Zhang et al. 2008) because the cysteine protease
inhibitors block the PCD pathway in the cell. Habib and Fazili (2007) reported that the increase in
plant protease inhibitors expression is a result of the response to several biotic and abiotic stresses.
The stability of photosynthetic complex is very crucial in determining the drought tolerance of a
plant species. Since the photosynthetic complex is very sensitive to disintegration during drought
stress, it needs to be recovered properly from damage during rehydration and recovery phase after
stress (Godde 1999). Because membrane proteins make a large part of this apparatus, they have a
special significance for the stable functionality of the photosynthetic apparatus during stress and
its recovery phase. In Craterostigma plantagineum, three genes, encoding for chloroplast localized
stress protein (DSP), are expressed preferentially under drought stress (Schneider et al. 1993). The
first two, DSP22 and DSP34, are located in the thylakoid membrane, and the third DSP21 is found
in the stromal compartment of the chloroplast. These three proteins have been proposed to play
crucial roles in the maintenance of chloroplast structures. Further, it was shown that the translation
of DSP22 is dependent on the degree of photo-inhibitory damage (Alamillo and Bartels 2001).
It is not only upregulation, but the downregulation of gene expression may also contribute to the
adaptation of plants to drought stress. For example, ProDH gene expression is downregulated dur-
ing water stress, resulting in the accumulation of proline (Yoshiba et al. 1997). In C. plantagineum
studies, researchers have revealed that the transcripts encoding proteins relevant to photosynthesis
are downregulated and have been estimated to represent 36% of the total number of genes altered
during the dehydration process (Mariaux et al. 1998). The exact function of several downregulated
genes is largely unknown. A logical hypothesis for downregulation of specific genes and proteins
can be made because of the fact that their product might not be suited to the new physiological con-
dition developed by dehydration stress (Chandler and Robertson 1994) in plant.
21.6.4 Overview of Gene Revolution Following Green Revolution

Globally, food shortages, increasing populations, and abiotic stresses on plants (e.g., salinity,
drought, and extreme temperatures) have resulted in focused efforts in the gene revolution. The path
from the green revolution to the gene revolution is referred to as the transgenic approach or genetic
engineering. This avenue offers new opportunities for enhancing the plant tolerance to abiotic
stresses. Drought stress is a major environmental constraint on agricultural productivity worldwide.
In agricultural biotechnology, improving drought tolerance is a long-standing goal. The current
strategies (i.e., genetic engineering/transgenic approach) depend on the transfer of one or several
genes to the target plant. The overexpression of genes in transgenic plants enhances their abilities
to tolerate drought stress and other abiotic stresses. These genes are associated with regulatory/
signaling pathways or encode enzymes that catalyze the synthesis of structural and functional pro-
tectants (Hussain et al. 2011).
Efforts to improve the performance of crops under conditions of drought have lacked successes
for two major reasons: a lack of knowledge of the interactions of different stresses and an incomplete
understanding of the fundamental mechanisms of stress tolerance. In crop plants, the engineering of
drought tolerance has important economic implications. The genetic engineering of drought stress
tolerance has had limited successes due to the limited availability of specific promoters and genes
in the pre-genomic era. Now, on a genome-wide scale, it is possible to simultaneously study the
function and structure of many genes. However, the potential side effects on plant growth and the
multigenic nature of stress tolerance mechanisms make this task difficult (Hussain et al. 2011).
21.7 UNDERSTANDING DROUGHT TOLERANCE MECHANISMS

By initiating various physiological, biochemical, and morphological responses, plants can adapt to
and survive drought stress (Farooq et al. 2009). Drought stress influences the role of water in plants
at the cellular, tissue, and organ levels, inducing specific and undefined adaptation reactions and
damage (Beck et al. 2007). Tolerant plants initiate successful defense mechanisms to cope with
drought stress (Chaves and Oliveira 2004).
The study of the biochemical, physiological, and molecular mechanisms that plants use to respond
to drought stress has provided significant scientific knowledge toward plant breeding. Though
drought resistance is a quantitative trait, key genes can contribute considerably to mitigate the dam-
age produced by water limitation. A number of genetically improved drought-tolerant crop plants
have been developed by conventional breeding, transgenic methods, and marker-assisted breeding.
A combination of the aforementioned techniques will likely be needed to produce new drought-
resistant cultivars for modern agriculture (Xoconostle-Cazares et al. 2010). The mechanisms of
drought resistance, ascertained breeding, molecular biology, genetics, biotechnology, genomics,
modeling, and root studies are presented in the following sections.
21.7.1 Genetics and Molecular Approaches

Conventional plant breeding is time-consuming, slow, expensive, and labor intensive and only
allows access to genes from sexually compatible or closely related species. These problems can be
overcome with molecular breeding and MAS, two techniques that are based on DNA-based marker
development (Vasil 2007).
With genetics and the arrival of molecular biotechnologies, new openings to study the relation-
ship between wheat traits and their genetic control have become available to plant physiologists. The
functional determinations of wheat genes that contribute to stress tolerance are believed to provide
an integrated understanding of the physiological and biochemical basis of responses to drought
stress (Zhao et al. 2008).
In wheat, the drought response is an adaptation strategy, and it is paramount to clarify the cen-
tral control element from the reductionistic, atomistic view. The dissection can be regulated at the
genetic, transcriptional, or translational levels to further manipulate the components of the geno-
typic response. Over the previous three decades, information and genetic tools (i.e., comparative
mapping, bulked segregant analysis, linkage mapping, gene discovery, candidate gene, and func-
tional genomics) have been adopted and developed to understand the basic aspects of the expression
and genetics of different wheat species.
To develop crops with improved drought stress tolerance, a basic understanding of the physi-
ological, biochemical, and regulatory networks of genes is needed.
Several tools of functional genomics have advanced our knowledge of stress signal transduction
and linked molecular regulatory networks. These functional genomics tools regulate the inducible
systems that respond to drought stress and that have many transcription factors and several stress-
inducible genes (Table 21.3).
TABLE 21.3
Examples of Transgenic Wheat Lines Developed for Drought Conditions
Target Gene Effect References
Mannitol mtlD After 30 days in pot experiment, drought transgenic Abebe et al. (2003)
line had greater tiller number, plant height, and dry
weight than the control plants.
Lipid transfer protein TaLTP1 Enhancing by water stresses such as by hormone Jang et al. (2004)
treatments, treatment by NaCl, and PEG
concentration.
DREB DREB1A For 10 days after water was within held, there was Pellegrineschi et al. (2004)
less wilting in transgenic lines than control plant.
Membrane protein- TaVAP Increasing in mild drought stress response in the Singh et al. (2007)
associated protein flag leaf.
LEA proteins HVA1 Higher shoot biomass and root under drought than Sivamani et al. (2000)
control and improved recovery after drought stress.
21.7.2 Genomics-Based Approaches
The genomics-based approaches provide routes to obtaining desirable agronomic traits that affect
responses at QTLs, thus enabling the improvement of crop yields and drought tolerance under the
conditions of water deficit; these methods are generally more effective and speedy than conven-
tional approaches. MAS has already improved the development of drought-related traits and has
helped breeders. The analysis of gene products and sequence data should facilitate the cloning and
identification of genes at target QTLs (Tuberosa and Salvi 2006). Furthermore, bioinformatics,
sequencing information, microarrays, and new genomics platforms have all added new dimensions
and made new methods available for manipulation and deciphering of the genetic basis of drought
resistance (Table 21.4).
The genomics-based approach contributes novel information that can help identify and analyze
candidate genes and elucidate their regulation and function under water-limited conditions. Further
information on the role of the candidate genes at target loci can be obtained, and their effect on the
phenotype can be determined through EcoTILLING, a platform for classifying single-nucleotide
polymorphism (SNP) haplotypes (Tuberosa and Salvi 2006).
To enhance drought tolerance, the successful exploitation of genomics will only be possible
within an interdisciplinary, coherent context that can provide a complete understanding of the limit-
ing factors of crop yield in DPE (Tuberosa and Salvi 2006).
21.7.3 Biotechnological Approaches
Progress has been made in identifying genes and pathways that can be manipulated to improve
drought tolerance using biotechnological models (Deikman et al. 2012).
The impact of the genomics age on crop improvement is just beginning to be realized. Basic
scientific research in the last few decades has been geared toward understanding the ABA recep-
tors, the signal transduction pathways of ABA components, and the ABA biosynthesis. The compo-
nents of the complex ABA signaling pathways are valuable, and new mechanistic understandings of
these pathways should expedite innovations that are aimed at controlling crop responses to drought
(Deikman et al. 2012).
To enhance tolerance to drought stress, biotechnological approaches in plants may involve the
overexpression of genes involved in specific features of cellular homeostasis, such as the production
of chaperone proteins, antioxidants, or osmotic adjustments. Alternatively, the suppression or ectopic
expression of regulatory genes could simultaneously activate multiple mechanisms of stress tolerance.
TABLE 21.4
Web Resources for Drought-Related Information on Crop Plants
Websites/Resources Main Features
http://www.plantstress.com/ Articles on plant environmental stresses and discussions and their impact in agriculture
and mitigation. It includes news, events, congress announcements, and reference
database.
http://www.generationcp.org/ The generation challenge program is focused on discoveries and using genomic tools
in developing countries to enhance drought tolerance in staple crops. It has five
subprograms that span the research spectrum in genomics, germ plasm, molecular
breeding, and bioinformatics for agricultural development.
http://rarge.gsc.riken.jp/ RRGE is a website that provides resource data searching services and presents resource
data of Arabidopsis.
http://rootgenomics.rnet. The plant root genomics website is dedicated to root physiology and genetics. The aim
missouri.edu/prgc/index.html is to develop molecular mechanisms and understanding used by plant roots to acquire
minerals and water from soil under multiple stresses.
TABLE 21.5
Improvement of Drought Resistance in Wheat through Transgenic Technology
Transgenic Discovery
Pathway Gene Gene Family Expression Strategy References
Osmoregulation AhBADH Betaine aldehyde Zm.Ubiquitin Hypothesis Wang et al. (2010)
dehydrogenase
betA Choline Zm.Ubiquitin Hypothesis He et al. (2011)
ABA response GmbZIP1 AREB b-ZIP rd29A (drought); Arabidopsis Gao et al. (2011)
wheat: ubiquitin
Stress response GhDREB AP2/ERF Zm.Ubiquitin and Arabidopsis Gao et al. (2009)
At.rd29 (drought)
TaDREB2 double 35S and Arabidopsis Morran et al. (2011)
TaDREB3 maize RAB17
TaNAC69 NAC HvDhn8s Reverse genetics Xue et al. (2011)
(constitutive) or in wheat for
HvDhn4s (drought stress-induced
inducible) genes
Genes in the family of the ERF/AP2 transcription factor, including NAC transcription factor, ABRE-
binding proteins, and DRE-binding proteins, have all shown promise in developing drought-tolerant
crops; these genes encode proteins involved in signal transduction mechanisms, such as enzymes
involved in protein modification and kinases (Rohila et al. 2009). In addition, breakthroughs in the
identification of microRNAs and their target genes will provide inspiring new sites and targets for
controlling response pathways during drought stress conditions (Deikman et al. 2012).
Many traits that can improve drought tolerance have been identified, but the commercialization
of these genes has been slow. The importance of biotechnology for enhancing crop yields under
stressful environments is becoming evident with the first demonstration of improved drought stress
tolerance in wheat crop in the field (Table 21.5).
21.7.4 Modeling Approaches
An understanding of the impacts of drought stress is being motivated by increases in water demand for
domestic use, climate change, and variability in hydrometeorological parameters/variables. Drought
hydrology has been gaining significant attention. Forecasting drought is a critical component of stress
mitigation, drought preparedness, and drought hydrology, all of which play key roles in risk manage-
ment. Considerable work has been performed on miscellaneous aspects of drought modeling, includ-
ing the prediction and identification of its severity and duration. However, the development of suitable
techniques is a major research challenge for forecasting the initiation and termination of drought. The
inability to accurately predict drought stress situations and conditions years or months in advance is
one of the main obstacles to mitigating the effects of drought (Mishra and Singh 2011).
Over the past three decades, there has been significant improvement in drought modeling.
For long lead-time drought forecasting, hybrid models seem to be the most promising approach,
as they are able to incorporate large-scale climatic indices. Additional research related to cli-
mate change (due to changes in spatiotemporal rainfall variability) is needed to understand the
complexities of drought (spatiotemporal). To achieve a multivariate characterization of drought,
copula-based models seem to be promising. Advanced studies of decision support systems
(DSS) for evaluating risks, issuing warning, and taking precautionary measures are also needed.
Effective ways of managing the flow of information from decision makers to users need to be
developed (Mishra and Singh 2011).
21.7.5 Root Studies
Drought-resistant or drought-adapted plants are characterized by vigorous and deep root systems:
Root length, rooting depth, and resistance to flow of water within the roots may be important char-
acteristics to relate to the previously mentioned environmental conditions. Among plants, the shape
and importance of the root system differ noticeably; even in the drought-adapted plants, both very
deep root systems and superficial root systems can be observed. The attributes of appropriate root
systems also diverge with climatic and edaphic conditions:
• A deep rooting system will be essential if water is deeply stored in the soil profile.
• A sparse root setup that can extract water at slow rates may be advantageous over a pro-
longed period, especially at sites where the crops must survive on water stored in the soil
and where rainfall amounts are uncertain.
• When evaporation uses up a significant amount of the total water at the soil surface, a dense
root setup may be favorable.
Important intraspecific variability can be found in most root attributes (e.g., root/shoot weight, root
length, and root depth) in many crops; genetic variability in root size was found in wheat, rice, oat,
and sorghum (Monneveux and Belhassen 1996).
21.8 CHALLENGES AND FUTURE PERSPECTIVES

There are a number of key predicted trends that are informing agricultural decision makers: In par-
ticular, the increasing global population is resulting in an increased demands for energy, food, and
feed to supply an ever-growing global demand for animal products; the effects of climate change
are also increasing abiotic stress levels in many areas of wheat production, as a result decreasing the
water that should be available for crop irrigation. The application of biotechnologies toward improv-
ing crops and developing wheat genetic resources is needed. Indeed, these technologies offer new
possible ways of increasing yield.
The philosophy underlying wheat improvement programs involves enhancing the science of
wheat improvement while conserving the human touches or art necessary for the paradigm of
research for development. The global population is projected to increase steadily. The fundamen-
tal challenge facing current and future crop researchers is to increase crop yield and agricultural
productivity in a sustainable manner (Deikman et al. 2012). Many achievements have been made
using both traditional breeding and biotechnological and genomic approaches. Currently, sustain-
able development is an important global objective. The use of biological measures to produce crops
with desirable traits is the main way of achieving this goal (Hong-Bo et al. 2006).
The invasion of crop fields by pathogens is increasing because of increasing human mobility,
globalization, climate change, and the evolution and adaptation of pathogens. In 2005, CIMMYT
estimated that two-thirds of the wheat grown in Pakistan and India (which amounts to about 20%
of world production) were very susceptible to wheat stem rust Ug99. In Africa and Asia, 80% of
the varieties of wheat that are grown are potentially exposed to stem rust/fungus. The spores of
this pathogen can be carried by wind across continents and over long distances. Approximately
50 genes that confer tolerance to different races of stem rust (Sr genes) have been identified (Vurro
et al. 2010).
To provide current and continuous information on the potential spread and current status of
the stem rust race of wheat Ug99, CIMMYT has developed the so-called RUSTMAPPER (http://
www.cimmyt.org/gis/rustmapper/) that shows the known status and current sites of Ug99/stem rust
and its derivatives. Summary information on wheat susceptibility to Ug99, areas of major wheat pro-
duction in Asia and Africa, and near-real-time wind trajectories from known sites for each country
are available on this site. Integrating this information for visualization using Google Earth presents
a near-real-time summary of Ug99 (wheat stem rust) status and its derivatives. Certain resistance
genes against Ug99 have been identified; if introduced into cultivated varieties, these genes could
help minimize the impact of the disease (Vurro et al. 2010).
Knowing the enemy is the first step toward controlling the drought losses and minimizing the gap
between attainable and actual yields. Drought stress is a complex trait and is a major limitation to
wheat productivity. The mechanisms of drought resistance are multifaceted and involve a variety of
biochemical and physiological processes that can be activated at different stages of plant develop-
ment and at the entire-plant level. Examples of these mechanisms include the synthesis of osmopro-
tectants, the accumulation of osmolytes, and the increased uptake of water through the development
of deep and large root systems and the increase in stomatal resistance, resulting in reduced water
loss. The expression of aquaporins and stress proteins, the stability of cell membranes, the appro-
priate plant genotypes, the upregulation of plant growth regulators, and other strategies are vital
mechanisms in drought resistance.
The applications of biotechnology, transcriptomics, proteomics, and metabolomics toward under-
standing the molecular basis of plant responses to conditions of drought and improved WUE are
also imperative. Molecular knowledge of the mechanisms involved in drought tolerance can pave
the way for the engineering of plants that can provide better economic yields (Eldakak et al. 2013).
The identification of genomic regions and novel genes associated with the expression of traits
responsible for drought tolerance is needed and will provide the foundation for effective crop man-
agement strategies, leading to the development of improved wheat germ plasm for particular agro-
ecological niches. If any methods of overcoming drought are to be developed, it is paramount to
first understand the adaptation strategies of the plant. Furthermore, to enhance stress tolerance, the
transfer of stress-inducible regulatory genes into wheat plants is a possible method of integrating
information attained from functional genomics into knowledge-based breeding programs.
It is likely that multiple approaches will be needed to develop robust drought tolerance in wheat.
Multiple approaches can also defend against various types of drought stress that may be encoun-
tered across a range of geographical conditions. Drought-resistant crops will eventually be pro-
duced by integrating advanced farming practices and the breeding of genes or traits developed
through biotechnology (Varshney et al. 2011).
ACKNOWLEDGMENTS
The authors acknowledge the funding from USAID/USDA in JSR and SIMM and AIN labs.
JSR also acknowledges financial support from SD Agriculture Experiment Station, SD Wheat
Commission, and SD Soybean Research and Promotion Council.
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22 Drought Physiology
of Forage Crops
M. Anowarul Islam and Augustine K. Obour
CONTENTS
22.1 Introduction........................................................................................................................... 427
22.2 Physiological Impacts of Drought on Forage Crops.............................................................. 429
22.3 Mechanisms of Plant Response to Drought........................................................................... 430
22.3.1 Drought Escape.......................................................................................................... 431
22.3.2 Dehydration Postponement........................................................................................ 433
22.3.2.1 Reduction of Water Loss............................................................................. 433
22.3.2.2 Maintenance or Increase Water Uptake...................................................... 434
22.3.2.3 Osmotic Adjustment................................................................................... 435
22.3.3 Dehydration Tolerance............................................................................................... 435
22.4 Management Considerations in Drought............................................................................... 436
References....................................................................................................................................... 437
22.1 INTRODUCTION
Drought stress is one of the most important constraints on agricultural profitability and sustain-
ability worldwide. Crops with high-use efficiency of limiting resources including water are critical
to producer success. Forage crops can produce high net biomass per unit area with minimum inputs
because of their low requirements of fertilizers, herbicides, and land preparation (Islam et al., 2011).
Forages allow producers to harvest important commodities, like fiber and animal feed, and even
biomass for biofuel, from marginal lands that would otherwise be incapable of high productivity.
Grasslands and native rangelands are predominant land use all over the world. In the United
States, improved pastures and rangeland cover nearly 99 million hectares, representing 27% of
total land use in 2007 (Nickerson et al., 2011). Grasslands do not only support forage production
for livestock but also provide significant ecological benefits. For instance, pasturelands mitigate
drought and flooding, maintain soil quality, prevent nutrient loss to water bodies, maintain bio-
diversity, and serve as habitats for wildlife as well as refuge for endangered plant and animal
species (Bohlen et al., 2009). Though other environmental factors (e.g., temperature, light, and
soil fertility) affect plant growth, variations in precipitation have been identified as an impor-
tant environmental factor affecting forage productivity and ecological distribution of grasslands
(Figure 22.1; Izaurralde et al., 2011).
Variations in soil moisture due to variability in precipitation have greater influence on forage
production. Studies have shown a direct relationship between precipitation and forage dry mat-
ter (DM) production (Rickard and Fitzgerald, 1970; Radcliffe, 1979; Lambert et al., 1983). In the
Northern Great Plains region of North America, there is a significant relationship between available
fall soil moisture and forage yields in the following growing season (Rogler and Haas, 1947). The
current drought in the United States and climate change projections indicate high rainfall variabil-
ity worldwide, with many regions predicted to experience severe decline in annual total rainfall.
427
428
Acres of Non-Federal Grazing Land, 2007
Each dot represents

25,000 acres
Pastureland
Rangeland
Grazed Forest Land
Federal Land
Hawaii
(no data)
There were approximately 583.9 million acres of
Pacific Basin Non-Federal Grazing Land in the conterminous
(no data) United States in 2007. This includes approximately
409.1million acres of rangeland, 118.6 million acres of
Northern Pastureland, and 56.1 million acres of
Marianas grazed forest land.
Note that the dots do not represent
actual feature locations or points.
Guam The dots are distributed randomly
within each area — in this map
state and 8-digit hydrologic unit.
American Samoa
Map ID: 11039
Data Source:
2007 National Resources Inventory Puerto Rico and US Virgin Islands
Alaska US Department of Agriculture, Natural Resources Conservation Service (no data)
(no data) Map Source:
US Department of Agriculture, Natural Resources Conservation Service
Resources Inventory and Assessment Division, Washington, DC, December 2009
FIGURE 22.1 (See color insert.) Distribution of pastureland across the United States in 1997 (1 acre = 0.4047 ha). (Adapted with permission from Izaurralde, R.C.
et al., Agron. J., 103(2), 371, 2011.)
Drought Physiology of Forage Crops 429
This changing precipitation pattern has increased the renewed interest in drought management and
plant strategies to avert the detrimental effects of drought.
Plants including forage crops have developed specialized physiological and morphological char-
acteristics to enable them to adapt to water-stressed environments. Drought adaptations of plants
are changes that make them increase their ability to tolerate drought. Some of these features include
increasing rooting depth and density, early plant vigor, low root hydraulic conductance, osmotic
adjustment, low lethal water status, reduced stomata conductance, leaf movements, leaf reflectance,
low epidermal conductance, and transpiration efficiency. This chapter focuses on physiological
impacts of drought on forage plants and the mechanisms that enable them to withstand long peri-
ods of drought stress. The role of management practices that improve forage productivity during
drought will also be discussed.
22.2 PHYSIOLOGICAL IMPACTS OF DROUGHT ON FORAGE CROPS

As water deficit develops, the plant relative water content and leaf water potential decrease. Since
growth is related to cell turgor (Green, 1968), the first plant process to be affected is the rate of cell
expansion. In forage crops, foliage production is the most important economic yield component.
The direct effect of drought on forages is the decline in leaf expansion and reduction in leaf growth.
This consequently causes a decline in forage DM production, as leaves constitute about 20%–75%
of total DM yield in forage crops (Bittman et al., 1988).
In evaluating the response of shoot growth of native forage grass species to decreasing water
potentials, Majerus (1975) showed that leaf expansion of blue grama [Bouteloua gracilis (Willd. ex
Kunth) Lag. ex Griffiths] ceased when soil water potential reached −8.0 MPa at 5 cm and −0.8 MPa
at 35 cm depths, while corresponding values for little bluestem [Schizachyrium scoparium (Michx.)
Nash] and western wheatgrass (Agropyron smithii Rydb.) were −2.4 and −0.3 MPa and −3.0 and
−1.5 MPa, respectively.
Leaf elongation of perennial ryegrass (Lolium perenne L.) slowed at soil water potential of
−0.15 MPa (Lawlor, 1972). Notwithstanding, when plants are subjected to gradual water stress,
leaf expansion may still persist even after considerable water loss. This may be due to substantial
osmotic adjustment, which helps stressed plants to maintain positive turgor potentials. Increasing
soil moisture deficit showed to have greater effects on tiller number than leaf expansion in Italian
ryegrass (Lolium multiflorum L.) cultivars growing in the field (Norris, 1982). Similarly, leaf expan-
sion of field-grown perennial ryegrass was maintained at one-third the rate of irrigated controls at
leaf water potential of −1.6 MPa (Jones et al., 1980).
One important effect of drought in plants is increased leaf senescence and eventual death of
leaves. Leaf senescence may be an adaptive strategy to prevent water loss through reduction in
transpiration rates from the leaf. Crested wheatgrass (Agropyron cristatum L.) has been reported to
expand the leaf area more rapidly in the spring than smooth bromegrass (Bromus inermis Leyss.) and
Altai wildrye [Leymus angustus (Trin.) Pilg.] and responds to soil water deficits by sharply reduc-
ing its leaf area through senescence of older leaves (Bittman and Simpson, 1987). Bittman et al.
(1988) showed that leaf senescence was greater for floral tillers than vegetative tillers in both crested
wheatgrass and smooth bromegrass in both dryland and irrigated conditions and suggested that leaf
senescence was controlled by hormones. The production of more senescence leaves reduces forage
availability and quality. The decline in forage quality is mainly caused by increased proportion of
stems, loss of leaves by senescence, and increased lignification of both stems and leaves. Crested
wheatgrass forage quality is reduced in the summer dry months due to greater leaf senescence and
high proportion of stems (Bittman et al., 1988).
Drought causes changes in biochemical process in plants that results in accumulation of
chemical compounds like soluble carbohydrates (e.g., sucrose), inorganic ions (such as potas-
sium (K+), calcium (Ca 2+), and sodium (Na+)), and nonprotein amino acids like proline.
These substances tend to help in osmotic adjustment by lowering the osmotic potential of
cells (Ford and Wilson, 1981; Nilsen and Orcutt, 1996). Water-stressed Old World bluestem
(Bothriochloa spp.), tall fescue [Schedonorus arundinaceus (Schreb.) Dumort.], western wheat-
grass, and weeping lovegrass [Eragrostis curvula (Schrad.) Nees] plants produced significantly
greater concentrations of proline than non-stressed plants grown in a growth chamber (Bokhari
and Trent, 1985). Accumulations of proline were significantly greater in the cool-season grasses
tall fescue (7.48 mg g−1) and western wheatgrass (16.25 mg g−1) than the warm-season weeping
lovegrass (2.66 mg g−1).
Drought stress increases sugar concentration in the leaves of forage crops. The type of sugar that
accumulates depends on the length of water deficits. In setaria [Setaria sphacelata (Schumach.)],
short-term water stress caused a reduction in sucrose and starch production, but a long-term gradual
drought stress caused a twofold increase in sucrose and a decrease in starch content (da Silva and
Arrabaca, 2004). The reduction in sucrose accumulation under short-term water stress conditions is
probably due to increased respiration rates. When sugar concentration reaches a threshold level, the
formation of storage polysaccharides commences. In water-stressed conditions, tall fescue tends to
accumulate fructans in the leaves (Shewmaker et al., 2006). Drought stress in the summer triggers
accumulation of water-soluble carbohydrates in leaf bases of orchardgrass (Dactylis glomerata L.)
and sugar accumulation is reported to increase as the drought progresses (Volaire and Lelievre,
1997). The capacity to accumulate high levels of nonstructural carbohydrates (NSC) may facilitate
the ability of plants to grow rapidly recovering after drought stress. Grass species that accumulate
NSC are more persistent under drought stress than species that do not (Boschma et al., 2003).
Hence, the maintenance of higher levels of NSC will increase drought tolerance and the ability of
plants to grow after grazing or clipping.
Drought stress affects various physiological processes involved in photosynthesis. Drought influ-
ences photosynthesis in two ways: either through stomatal closure by decreasing flow of CO2 into
the mesophyll tissue (Chaves et al., 2003; Flexas et al., 2004) or by directly impairing metabolic
activities (Farquhar et al., 1989). During the initial stages of drought stress, inhibitory conductance
through the stomata is the primary cause of decline in photosynthesis. Evidence suggests that in
some plant species, the photosynthetic capacity of the mesophyll tissues does not decline even when
CO2 assimilation was close to zero (Chaves, 1991).
It has been reported that as relative water content drops from 100% to 70%, stomata conduc-
tance decreases, which results in decreasing photosynthetic assimilation of CO2 (Lawlor and
Cornic, 2002). Accumulation of solutes during drought (osmotic adjustment) apparently does
not directly interfere with photosynthesis, although the sugars may be part of a coordinated
regulation between synthesis and translocation, a regulation that may lead ultimately to leaf
senescence (Chaves, 1991). Of course, senescence leaves have less mesophyll and less photo-
synthetic activity.
22.3 MECHANISMS OF PLANT RESPONSE TO DROUGHT

Forage crops have developed varied physiological and morphologic features to respond to drought.
For instance, warm-season grasses such blue grama, buffalograss [Bouteloua dactyloides (Nutt.)
J.T. Columbus], big bluestem (Andropogon gerardii Vitman), and crested wheatgrass are the dom-
inant grass species found in semiarid regions of the North American Great Plains due to their
drought tolerance (Beetle, 1950). The ability of these species to thrive in extreme drought conditions
is primarily due to their profuse stoloniferous morphology and structure, growth habit, and ability
to enter dormancy under stress conditions (Beetle, 1950; Riordan et al., 1993; Islam et al., 2013;
Table 22.1). However, cool-season grass species like tall fescue thrive well in moderately dry envi-
ronments due to limited tolerance to water deficits. Obviously, the response to water deficit differs
among forage crops. As suggested by Turner (1986), plant adaptive strategies to respond to water
deficits can be grouped into three large categories: (1) drought escape, (2) dehydration postpone-
ment, and (3) dehydration tolerance.
TABLE 22.1
Cool- and Warm-Season Grass Dormancy (%) under Irrigated and Rain-Fed Conditions
at the James C. Hageman Sustainable Agriculture Research and Extension Center, Lingle,
Wyoming, during Summer from 2009 to 2011
Species Cultivar July 29, 2009 June 1, 2010 August 5, 2010 June 6, 2011 July 26, 2011
Irrigated
Blue grama Alma 13.8 4.8 17.5 94.2 23.0
Bad River 13.8 8.5 13.8 75.0 17.0
Hachita 3.8 6.0 17.5 97.0 17.5
Buffalograss Bison 4.0 5.8 13.8 95.5 11.3
Bowie 4.0 6.8 10.0 95.0 10.3
Cody 1.8 4.5 22.5 97.0 17.0
Kentucky bluegrass Bandera 15.0 8.3 20.0 22.0 19.5
Common 85/80 9.0 7.0 52.5 21.0 10.5
Midnight 29.0 8.3 10.5 16.3 34.5
Tall fescue Blackwatch 6.3 8.0 50.0 9.5 16.5
Tar Heel II 42.5 8.0 42.5 7.3 20.0
Watchdog 38.8 7.8 38.8 8.3 22.0
Mean 15.2 7.0 25.8 53.2 18.3
SEa 8.3 3.7 10.0 3.7 10.0
Rain-fed
Blue grama Alma 13.8 5.5 53.8 32.5 30.0
Bad River 14.0 8.0 71.0 53.3 49.3
Hachita 10.5 6.3 71.3 81.3 47.5
Buffalograss Bison 4.0 6.8 60.0 100.0 42.5
Bowie 4.0 7.3 50.0 96.5 73.4
Cody 1.8 7.3 62.5 91.3 46.3
Kentucky bluegrass Bandera 10.0 7.0 49.5 5.3 86.3
Common 85/80 4.0 7.5 73.8 29.3 94.5
Midnight 4.0 7.5 93.8 3.5 96.5
Tall fescue Blackwatch 10.0 7.8 81.3 2.8 88.3
Tar Heel II 29.0 8.0 52.5 32.5 61.3
Watchdog 25.0 7.5 68.8 8.0 55.0
Mean 10.8 7.2 65.7 44.7 64.3
SEa 8.3 1.2 22.0 1.2 22.0
Note: Dormancy varies between species, cultivars within species, and between months and also years because of varia-
tions in temperature, moisture, and plant genetics and physiology.
a SE, standard error for mean comparison.
22.3.1 Drought Escape
This mechanism involves the ability of plants to complete their growth cycle before the onset of
water limitation. Annual species can escape droughts by setting seed and then reestablishing quickly
when moisture conditions are favorable. Drought escape through natural reseeding is a successful
drought tolerance strategy in annual legumes. Subterranean clover (Trifolium subterraneum L.)
usually completes its life cycle before summer drought and reseeds itself when moisture returns in
the winter (Rickard and Fitzgerald, 1970). Although efficient in nature, this adaptive strategy is not
very common in perennial pasture species that are expected to provide forage for grazing livestock.
Notwithstanding the obvious limitation, this mechanism has been exploited by breeders to develop
lines that can be grown and established earlier before drought occurs (Rose et al., 1992). This effort
will be particularly important in selecting drought-tolerant annual forage crops.
In extended drought periods, some perennial forage grasses may cease growing with leaves
becoming senescent, but crowns, stolons, and rhizomes may still be alive. This is often referred
to as dormancy, which preserves the vital parts of the plant for regeneration of shoots and roots
when moisture conditions return. By becoming dormant, forage grasses can escape drought stress
by reducing water demand until soil water is replenished through irrigation or precipitation
(Figure 22.2). The length of the dormancy period depends on many factors including soil water
availability, plant growth rate, and the health of the plants at the onset of dormancy. Therefore,
perennial grasses can partially escape droughts by reducing leaf area and relying on dormant buds
and underground organs for rapid regeneration during wet periods.
Regeneration from dormant buds has been reported in perennial ryegrass in Australia (Silsbury,
1961). Also crested wheatgrass is known to lose tissue water under drought and uses drought escape
as an adaptive mechanism by growing rapidly early in the season when soil moisture is available,
undergoing dormancy on the onset of drought in the summer (Bittman et al., 1988). Perhaps this
mechanism has allowed crested wheatgrass to adapt well to the Northern Great Plains where mois-
ture is relatively abundant in the fall and early spring but limited during the summer. Studies in
the Northern Great Plains region of North America have shown a highly significant relationship
between available fall soil moisture and crested wheatgrass forage yields in the following growing
season (Rogler and Haas, 1947).
Tall fescue in June Tall fescue in August
Tall fescue in November

Tall fescue in September
FIGURE 22.2 (See color insert.) Tall fescue grazing plots at the Samuel Roberts Noble Foundation,
Ardmore, Oklahoma, in 2006. Grasses went to complete dormant in August and September during hot and
dry weather and returned to full green growth in November. Through dormancy, forage grasses can escape
drought stress by reducing water need until soil water is replenished through irrigation or precipitation.
(Pictures taken by M. Anowarul Islam.)
22.3.2 Dehydration Postponement
Dehydration postponement involves the development of physiological mechanisms that allows
the plants to maintain favorable internal water content under drought. Some of these mechanisms
include (1) reduction of water loss, (2) maintenance or increase water uptake, and (3) osmotic adjust-
ment (Turner, 1986). Plants exhibiting this mechanism may or may not experience any reduction in
growth because this adaptive strategy allows metabolic processes to continue even though the plant
may be under water stress conditions.
22.3.2.1 Reduction of Water Loss

Leaf architecture and stomatal characteristics play an important role in the ability of forage crops
to limit water loss (Renard and Demessemacker, 1983). Leaf orientation, stomata distribution, leaf
rolling, surface ridging, and leaf senescence all affect water loss. Leaf rolling occurs in many grass
species as a strategy to maintain turgor pressure and to limit water loss via transpiration under
drought conditions. Renard and Demessemacker (1983) reported a 65% reduction in tall fescue
transpiration when leaves were artificially rolled. Similarly, the relatively high productivity of tall
fescue compared to perennial ryegrass and white clover (Trifolium repens L.) under drought is
attributed to the ability to roll its leaves under water stress (Johns, 1978). In this same study, the
relatively high leaf diffusive water conductance of white clover resulted in poorer yields. Also, the
reduction of water loss in orchardgrass was achieved through reduction and cessation of leaf growth
when water potential was −0.2 MPa (Volaire et al., 1998).
Early research efforts have documented the importance of stomatal closure in reducing water loss
through decreasing transpiration rates in plants (Shimshi, 1963). The density, size, and distribution
of stomata on leaf surfaces have important implications to gas exchange and water loss. Anderson
and Briske (1990) showed that a significant positive relationship exists between density and size of
stomata and transpiration among native grass species. Temperate grasses are reported to have more
stomata on the adaxial (top) than on the abaxial (bottom) leaf surface. Obviously, water loss may be
greater from stomata located on the adaxial surface than the abaxial surface. For instance, adaxial
conductance accounted for 80% of the total leaf water conductance in crested wheatgrass under
both irrigated and moisture-deficit situations (Bittman and Simpson, 1989).
The ability to control water loss through stomatal conductance is varied among forage species.
In a greenhouse experiment, Thomas (1986) compared leaf water conductance among orchardgrass,
perennial ryegrass, and Italian ryegrass and concluded that the slow decline in perennial ryegrass
growth was due to lower transpiration rates as a result of decreased leaf conductance compared to
the other grass species. Studies evaluating seasonal leaf conductance in three forage grasses found
greater leaf conductance in Altai wildrye compared to smooth bromegrass and crested wheatgrass
(Bittman and Simpson, 1989). The authors reported a limited relationship between leaf water poten-
tial and stomatal conductance and suggested that other factors like the rate of leaf water loss affect
stomatal response to tissue water loss. Other authors suggested that soil water content, not leaf water
potential, directly regulates stomatal behavior (Turner et al., 1985; Blaikie et al., 1989). This is
thought to be mediated by chemical signals like abscisic acid that is transported from roots induced
by water stress (Davies et al., 1994, 2002; Kim et al., 2010).
Recent studies have demonstrated that abscisic acid may promote and maintain root growth
under water stress (Munns and Sharp, 1993; Sharp, 2002). Plant roots are important sites for the
synthesis of abscisic acid and can be transported to shoots and initiate signals in the guard cells that
alter the membrane transport of ions. This results in the guard cells losing their turgor and closure
of the stomata. The increased concentration of abscisic acid in the leaf has been shown to induce
stomatal closure and reduce leaf expansion during early stages of soil drying in the absence of any
change in leaf water status (Bahrun et al., 2002; Liu et al., 2005). These effects of abscisic acid on
the leaf may significantly decrease the rate of transpiration, which will prevent dehydration of leaf
tissues and enhance the chance for survival under prolonged drought stress.
The role of abscisic acid as metabolic factor regulating drought tolerance in grasses and other
crops has been well studied (King and Evans, 1977; Sharp et al., 1990; Wilkinson and Davis,
2002). Wang et al. (2003) reported that Kentucky bluegrass (Poa pratensis L.) cultivars toler-
ant to drought exhibited slower abscisic acid accumulation rate than drought-sensitive cultivars
during short-term drought stress. This suggests that low accumulation rate of abscisic acid in
leaves would be beneficial for the maintenance of photosynthesis during short-term drought and
allows DM to accumulate to support plant survival during prolonged drought. The importance
of stomatal regulation of water loss has led to several breeding efforts attempting to screen for
higher resistance to water loss in plants, either by screening for low stomatal frequency or for high
stomatal resistance itself.
22.3.2.2 Maintenance or Increase Water Uptake

One of the most important plant responses to drought is continued root growth. The ability of plants
to maintain greater access to available soil moisture is critical to survival during drought. This has
been documented by plants developing greater root–shoot ratio in water-stressed plants compared to
non-stressed controls (Jones et al., 1980) or developing deeper root growth (Caradus and Woodfield,
1998). Differences in root–shoot ratio during drought stress may be due to carbon reallocation from
shoots to roots. This results in the formation of a more extensive and deeper root system. In maize
(Zea mays L.), root growth continues even at soil water potentials that inhibit shoot growth (Sharp
et al., 1990). In a study investigating carbon metabolic responses in depleting soil water content,
both Kentucky bluegrass and tall fescue exhibited decreased respiration rates in shoots and roots in
the upper drying layer but enhanced carbon allocation to roots deeper in soil profile where condi-
tions are wet (Huang and Fu, 2000). Plants in drought conditions with the ability to increase water
uptake will survive long periods.
Rapid water uptake by plants is influenced by root density and distribution. Although majority
of roots (60%–80%) in forage crops occur at the top 30 cm of the soil (Evans, 1978), deeper rooting
can occur in many crops. Tall fescue is an excellent example of a drought avoider by developing
deep root system. It exhibited maximum root extension of 33%–60% greater than buffalograss,
zoysiagrass (Zoysia spp.), or bermudagrass [Cynodon dactylon (L.) Pers.] in a greenhouse study
and extracted over 50% more water at a 90 cm depth than zoysiagrass (Qian et al., 1997). The
ability of canarygrass (Phalaris tuberosa L.) to supply water from the subsoil enables it to sur-
vive long summer droughts (McWilliam and Kramer, 1968). The authors observed the roots of
canarygrass growing more than 200 cm to exploit available soil moisture deeper in the profile. The
deep rooting system of Altai wildrye allowed increase water uptake and maintained higher tissue
water levels under water deficits (Bittman and Simpson, 1987). Volaire et al. (2001) reported that
drought-resistant orchardgrass cultivars had deeper root systems than sensitive cultivars. In an ear-
lier study, it has been reported that under certain conditions and often for prolonged period of time,
plants such as alfalfa (Medicago sativa L.) can be dependent upon subsoil moisture by expanding
roots and extract moisture from a depth down to 900 cm (Kiesselbach et al., 1929).
Accessing water deep in the soil profile through deeper root systems can result in water realloca-
tion and transport of water into drier regions in the upper layers of the soil profile, a phenomenon
referred to as hydraulic lift. Hydraulic lift has been defined as the process of water movement from
relatively moist to dry soil layers using the plant root systems as a conduit (Caldwell et al., 1998).
The deep roots of sagebrush (Artemisia tridentata Nutt.) allow it to extract water in daytime from
deeper layers within the soil profile and release it back to the drier upper soil layers at night. This
water is subsequently reabsorbed during the daytime by the sagebrush and the associated neighbor-
ing forage and grass plants (Caldwell and Richards, 1989).
In buffalograss, water absorbed by roots deep in the soil profile at night helps to support root
growth and function in nutrient uptake in upper soil profile, resulting in delaying drought stress
injury (Huang, 1999). The redistribution of water in the soil profile through hydraulic lift improves
water use efficiency in water-limiting environments in plant exhibiting this mechanism.
22.3.2.3 Osmotic Adjustment
One of the important factors controlling crop tolerance to dehydration is the ability to maintain
adequate turgor pressure during water stress. This is normally done by increasing the concentra-
tion of compatible solutes or osmoregulatants within the plant cells. Osmoregulatants influence the
osmotic potential of a cell by modifying the production of various solutes including inorganic sol-
utes (K+, Ca2+, Na+) and organic solutes such as soluble sugars and acids (Nilsen and Orcutt, 1996).
Accumulation of these solutes in cells under water deficit tends to lower the osmotic potential of the
cell without causing toxic effects. This active accumulation of solutes is known as osmotic adjust-
ment. As suggested by Blum (1988), this is an active accumulation process rather than a simple
accumulation of solutes due to the concentration effects resulting from water loss. Increasing com-
patible solutes in plant cells can lower osmotic potential and thus water potential, which prevents
water loss to intercellular spaces and increase water movement into cells with low water potential.
Osmotic adjustment has been well recognized as an important plant adaptive mechanism to
drought (Martin, 1930; Morgan, 1984), and this has been demonstrated in many plant species.
The ability to lower osmotic potential under water stress by osmotic adjustment helps plants to
maintain positive turgor as water deficits develop enabling them to maintain leaf expansion and
allow the stomata to remain open for photosynthesis (Hsiao et al., 1976; Jones and Rawson, 1979;
Steponkus et al., 1980). Greater root growth at lower soil water potentials has been attributed to
osmotic adjustment (Sharp and Davies, 1979). Continued shoot and root growth of tall fescue under
drought conditions was also attributed to low osmotic potential (Burch and Johns, 1978). In evaluat-
ing turgor maintenance in tall fescue cultivars, White et al. (1992) indicated that osmotic adjustment
and low basal osmotic potential before water stress were responsible for tall fescue survival during
drought. DaCosta and Huang (2006) compared drought tolerance in creeping bentgrass (Agrostis
stolonifera L.) and velvet bentgrass (Agrostis canina L.) and reported that velvet bentgrass showed
a 50%–60% higher magnitude of osmotic adjustment under water deficit that increases visual turf
quality and leaf relative water content under drought stress compared to creeping bentgrass.
Osmotic adjustment is also known to play a significant role in drought recovery. Data by Qian
and Fry (1997) showed a positive correlation between osmotic adjustment and ability of buffalo-
grass, bermudagrass, and tall fescue to recover from drought. Although osmotic adjustment occurs
in response to water deficit in both C4 (warm-season) and C3 (cool-season) grasses, studies indicate
that greater osmotic adjustment occurs in C4 than C3 under water-stressed conditions (Barker et al.,
1993). The greater osmotic adjustment in C4 grasses and associated C4 metabolic pathway make
them well adapted to high-temperature and low-moisture conditions and high water use efficiency
compared to C3 grasses.
22.3.3 Dehydration Tolerance
This adaptation strategy involves the ability of plants to maintain physiological functions even when
tissue water potential is very low. Most plants will die at leaf water potentials lower than −4 MPa,
yet some plants can survive dehydration at leaf water potentials >−100 MPa (Gaff, 1989; Oliver
et al., 2000). Some of the biochemical processes linked to drought tolerance involve increased syn-
thesis of soluble carbohydrates and proteins.
In drought stress, plants are known to exhibit significant changes in protein composition, with
increased synthesis of stress-inducible proteins (Riccardi et al., 1998). Synthesis of stress-induced
proteins such as late embryogenesis abundant (LEA) proteins in response to drought has been associ-
ated with increased adaptive ability and tolerance to drought stress (Riccardi et al., 1998; Bewley and
Oliver, 1992). Stress-induced LEA proteins, known as dehydrins, play a positive role in drought toler-
ance. Dehydrins have been shown to accumulate under drought stress and in response to increased
abscisic acid concentrations in tall fescue (Jiang and Huang, 2002). It has also been reported that
dehydrins are involved in osmotic adjustment regulators thereby enhancing desiccation tolerance
(Nylander et al., 2001).
22.4 MANAGEMENT CONSIDERATIONS IN DROUGHT

The most noticeable effect of drought is reduction in forage productivity in both planted pastures
and rangelands. In forage-based livestock production systems, animal production is based on
continuous availability of forage. Hence, any shortage of forage due to drought interruption may
reduce farm productivity and increase feeding cost if the duration of drought is prolonged. In
environments where droughts are prevalent, growers can plan ahead in anticipation of the drought
occurrence.
Good grazing management is critical during drought. Defoliation through grazing can increase
pasture growth and productivity under wet conditions but the converse is true during drought
(Appadurai and Holmes, 1964). When defoliation occurs during drought, plant recovery and growth
can be severely impacted after the drought. Forage clipping in crested wheatgrass during water
stress resulted in lower tiller formation and regrowth of new leaves (Busso and Richards, 1995).
Proper grazing and defoliation management is essential to ensure persistence and forage produc-
tivity. It is imperative that stocking rates are reduced during drought to a level that will provide
acceptable animal performance. However, defoliation prior to the onset of drought stress has been
reported to increase tiller survival during drought and resulted in faster pasture recovery following
the drought (Barker et al., 1985).
Producers must make best use of the available forage resources during drought. Hence, livestock
should not be allowed to spot graze and waste limited forage. Using rotational grazing will improve
forage utilization efficiency during drought. In rotational grazing, livestock graze a particular area
of the pasture for a limited period of time before moving to a different location. This process pro-
vides forages some resting period to recover and ensures greater utilization of the available forage.
It is also important to know forage species to aid in selecting forage crops that will produce
enough foliage under water stress (Islam et al., 2011). Breeding efforts can identify or develop
forage crops that are specifically suitable to drought-stressed conditions. Cool-season grasses like
tall fescue, orchardgrass, and Kentucky bluegrass are relatively less tolerant to drought and least
productive during drought stress. Conversely, warm-season grasses like bahiagrass (Paspalum
notatum Flüggé), bermudagrass, and blue grama grass are more tolerant to drought. Incorporation
of warm-season grasses in the forage mixture could provide some relief to the producer during
drought. Forage legumes like alfalfa have well-developed and deep rooting systems making them
more drought tolerant. Therefore, having alfalfa or alfalfa-grass mixed pastures can extend the
grazing season in the phase of drought.
Fertilizer applications during drought are not economical especially if the drought is prolonged.
Applying limited amounts of fertilizer to improved pastures can increase forage production when
applied at the beginning of the season to take advantage of any available soil moisture. Proper fer-
tilization will ensure a good recovery following drought. Any fertilization program should be based
on soil test recommendations and fertilizers should be applied before the onset of precipitation.
Forage recovery after regular droughts is very important for perennial pastures. Good regrowth
from an existing desirable forage species will save the producer the cost of establishing new pasture
(Islam et al., 2011). Drought-stressed pasture should be treated as newly established plants until
recovery is complete.

Drought affects morphology and metabolic physiological processes of forage crops, hence affecting
forage and animal productivity. Plants, by nature, develop many external and internal adaptation
mechanisms to tolerate droughts. The most important ones include drought escape, dehydration
postponement, and dehydration tolerance. Selecting appropriate species and cultivars, proper pro-
duction, management and utilization strategies, and finally breeding forage crops tolerant to drought
can improve overall productivity and sustainability of forage crops.
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23 Effect of Drought/Water
Stress and Adaptation to
Unintended Consequences
of Wheat Growth and
Development in Pakistan
Ijaz Rasool Noorka
CONTENTS
23.1 Introduction......................................................................................................................... 441
23.2 Emerging Challenge for Food Security.............................................................................. 442
23.3 Biodiversity Conservation and Interaction between Land and Ecosystem.........................444
23.4 Climate Change Mitigation, Adaptation, and Management of Unintended Consequences...... 444
23.5 Mechanisms Involved in Drought Resistance.....................................................................444
23.6 Methods Used in Breeding for Drought Tolerance............................................................. 445
23.7 Choice of Best Parent by Screening.................................................................................... 445
23.7.1 Emergence Percentage (%)...................................................................................... 445
23.7.2 Emergence Index..................................................................................................... 445
23.7.3 Emergence Rate Index............................................................................................ 445
23.7.4 Energy of Emergence..............................................................................................446
23.7.5 Desiccation Tolerance Index...................................................................................446
23.7.6 Percent Seedling Recovery......................................................................................446
23.8 Effect of Water Stress on Morphological Traits.................................................................446
23.9 Gene Action........................................................................................................................ 447
23.10 Correlation.......................................................................................................................... 447
23.11 Conclusions.........................................................................................................................449
References�� 449
23.1 INTRODUCTION
Pakistan is situated in the north-eastern part of the Indo-Pakistan subcontinent that lies between
23° and 37° north latitude and 62° and 75° east longitude, with a length and width of nearly 1400
and 500 km, respectively. Pakistan spans across a huge area, having a fertile soil, topography, and
a favorable climate, highly suitable for round-the-clock agriculture and good food p roduction.
Pakistan is naturally an agricultural-based economy, which is the main provider of employment
and livelihood. The country produces wheat, maize, rice, cotton, sugarcane, and other cereal in suf-
ficient quantities. Wheat is the predominant food grain in Pakistan (Noorka et al., 2012). Pakistan
underwent a historical green revolution of the 1960s, which paved the way for new, improved early-
maturity and high-yielding dwarf varieties of wheat and rice (Noorka et al., 2013a).
441
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United States of America United Kingdom Saudi Arabia
Russian Federation Pakistan Mexico India
FIGURE 23.1 Wheat production graph of the last 50 years (1961–2009) among the United States, Great Britain,
Saudi Arabia, Russian Federation, Pakistan, Mexico, and India. (From FAO stat 2011, http://faostat.fao.org.)
Our planet has no shortage of water due to an abundance of it in the form of oceans spread
across three-fourths of its surface and some in the form of heavy rainfall throughout the year.
However, aridity is also increasing in most parts of the world due to insufficient irrigation water
and unreliable rainfalls. Under the changing climatic situation and global warming, an uncer-
tainty prevails regarding water provision for agricultural use (Noorka and Haidery, 2011). The
world has experienced long and short spells of drought causing food shortage in general and
its consequences in particular in the form of salinity, crop diseases, reduction in crop yields,
food starvation, and human diseases. Food insecurity is also closely associated with water stress
because it is considered as the major militating factor, affecting food stability in all agricultural-
based economies (FAO, 2011) (Figure 23.1).
Stability in yield has always been a problem in intermittent water stresses at various stages of
crop growth (Shafi et al., 2012). Therefore, genotype x environment interactions are of major con-
cern to plant breeders for developing yield stability in commercial varieties.
23.2 EMERGING CHALLENGE FOR FOOD SECURITY

It is true that food, fiber, and fuel are the basic necessities of life, and food security remains a
venerable issue the world is facing, having to confront a series of challenges including long and
short drought spells, water stress, climate change, land competition for bioenergy production, and
food contamination (Chowdhary et al., 1999; John, 2004; Kelman, 2006; Lenton et al., 2008, 2011;
IPCC, 2011) (Figure 23.2). The world needs an urgent solution that can ensure nonstop food supply,
which can only be possible by supporting small-scale farmers and increasing agricultural efficiency
by adopting research-oriented production, harvesting, and storing options.
1000
–500k
0k
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FIGURE 23.2 Predicted population growth rate of Pakistan, India, and China (1961–2050). (From FAO stat 2011, http://faostat.fao.org.)
2035
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443 Effect of Drought/Water Stress and Adaptation
23.3 BIODIVERSITY CONSERVATION AND INTERACTION

BETWEEN LAND AND ECOSYSTEM
Today we are much concerned with two important facets of research: biodiversity and environmen-
tal effects. Both are deeply rooted in society and nature for its articulated linkages and ecosystem
interaction. Plants are continuously utilizing the potential environmental for their best survival
(Noorka and Khaliq, 2007). However, the land research community has been shaken by land deg-
radation, erosion, and drought particularly in developing countries where land directly affects food
supply and livelihood. Den Biggelaar et al. (2004) estimated that around 2–5 million hectares of
land are lost each year to land degradation, mostly due to soil erosion.
23.4 CLIMATE CHANGE MITIGATION, ADAPTATION, AND

MANAGEMENT OF UNINTENDED CONSEQUENCES
According to the US Department of Agriculture (2011), the number of food-insecure peoples has
increased to 861 million in 77 developing countries. However, the UN has warned that the world
population will definitely cross the 9 billion mark by 2050. The food demand and supply will
disturb the balance of payment of almost all parts of the world (FAO, 2008; Pisante et al., 2009;
Nelson et al., 2010). The immediate implication is to increase farm production for the next 40 years.
The effects of climate change (higher temperatures, unreliable rainfall, shifting of seasons, frequent
and extreme weather conditions predicting unscheduled floods and droughts) and the challenge for
food production will be more daunting than predicted (Min et al., 2011). Climate change and its
modeling resulted in observational evidences that can alter the frequency, strength, and distribution
of extreme events toward the management of food shortage issues (Zheng et al., 1994; Ren et al.,
2000; Noorka et al., 2009).
When this management is scaled up, the mitigation of and adaptation to unintended consequences
can be minimized or avoided to support the climate policies. For instance, the Intergovernmental
Panel on Climate Change (2007, 2011) revealed that some African countries’ crop production may
reduce up to 50% by 2020 if they do not adopt the climate change unintended consequences pre-
cautionary measures.
Drought resistance is defined as the ability of a crop plant to produce an economic product with
minimum loss in a water-deficit environment. Drought resistance is considered as the collabora-
tive outcome of morphological, physiological, and biochemical traits. From a genetics viewpoint,
drought resistance in crop plants is a way to evolve potential genotypes to withstand water stress
environment by conventional or unconventional breeding/biotechnological approaches (Muhammad
and Hussain, 2012).
23.5 MECHANISMS INVOLVED IN DROUGHT RESISTANCE

The mechanisms of drought resistance are categorized in to drought tolerance, drought avoidance,
and drought escape. However, these mechanisms are unconfined and most unstable, involving rapid
phonological improvements. The exploration and characterization of drought resistance–related
plant genetic resources may be an excellent approach toward paradigm shift to conserve desired
traits (Bajji et al., 2000).
Plant genetic engineering can act as transgene to confer drought resistance in desired crops.
Stress proteins like LEA and dehydrin can be amalgamated under severe water stress conditions, and
a relative examination of polypeptides produced as a result of drought, among drought-tolerant and
drought-sensitive genotypes, may be employed to ascertain protein markers to produce transgenic
drought-resistant plants. In this way, by using a multidisciplinary methodology consisting of plant
breeding, physiology, biotechnology, biochemistry, and genetics, crop science can combat this com-
plex and multidimensional drought to plant (Basal et al., 2005).
Effect of Drought/Water Stress and Adaptation 445
23.6 METHODS USED IN BREEDING FOR DROUGHT TOLERANCE

In self-pollinated crops like wheat, usually pedigree and bulk methods are used, while in cross-
pollinated crops, recurrent selection methods are used to fix drought-combating characteristics in
established high yield varieties. However, backcross and biparental mating (half-sib and full sib) are
used for the maintenance of broad genetic to contest genotype of drought resistance.
23.7 CHOICE OF BEST PARENT BY SCREENING

From an agricultural point of view, the breeder is always interested to increase the genetic poten-
tial and to minimize yield loss by genetic manipulation. The best practice is choosing best parent
by screening a large population by creating an artificial water stress environment (Mehmood
and Noorka, 2011; Noorka et al, 2013a). Seedling survivability is a time tested, simple and
an efficient screening method particularly to screen a large germ plasm under an artificially
induced water stress environment. The screening has been used as a selection criterion (Chang
and Loresto, 1986), in cowpea (Singh et al., 1999) and wheat (Winter et al., 1988; Tomar and
Kumar, 2004). Simple and valuable traits may be measured during the screening process accord-
ing to the work of Noorka and Khaliq (2007). The following criteria are mainly used to screen
a large population.
23.7.1 Emergence Percentage (%)

It is the hidden ability and power of the crop seed to emerge from itself. It is considered that
good emergence can give a good crop stand (Allen and Donnelly, 1965; Schonfeld et al., 1988;
Subrahmanyam et al., 2006). Data collection was done immediately after the emergence of the first
seedling in any pot/bag, line, row, etc., onward on a daily basis. The number of visible seedlings was
recorded. Data collection continued until there was no further growth and emergence percentage
was calculated by the formula derived by Smith and Millet (1964):
Total number of seedlings emerged 18 DAP

E% = ¥100
Total number of seedlings grown
DAP = Days after planting
23.7.2 Emergence Index
Emergence Index (EI) is an estimate of the speed of emerging seedlings and can be calculated
according to the method described in the Association of Official Seed Analysis (1983). EI = No. of
seeds emerged at first count + …… + No. of seeds emerged at final count
Days of first count +……………+ days of final count
23.7.3 Emergence Rate Index

Emergence rate index (ERI) is the product of EI and emergence percentage and can be calculated by
the method proposed by Noorka and Khaliq (2007) and Noorka et al. (2013a) as follows:
Emergence index
ERI =
Emergence percentage
23.7.4 Energy of Emergence
It is the power of a seed to emerge from the seed testa and soil. The energy of emergence (EE) is
calculated with the formula given by Ruan et al. (2002). Usually it is considered as the percentage
of emerged seedlings after 3 days of sowing.
Mean emergence time (MET) is the average time taken by the seed to emergence from the soil
and is dependent on genetic potential as shown in the equation of Ellis and Roberts (1981):
SDn
MET =
Sn
where
n is the number of seeds germinated on day D
D is the number of days counted from the beginning of emergence
23.7.5 Desiccation Tolerance Index

To check the potential of the genotype to bear the water stress condition, the plants were watered
until they sprouted two to three leaves; this is considered the best stage to evaluate the genotype
for their water stress tolerance and susceptibility as suggested by ISTA (Anonymous, 1997). To
induce artificial water stress condition, the water provision was withheld; as a result, some of the
water stress–susceptible genotypes will retard their growth and ultimately die. The irrigation was
resumed and survivability was recorded as the regrowth as well as water stress tolerance in each
replication. The numerical data provide information on the live and dead seedlings to check the des-
iccation tolerance parameter (O’Toole et al., 1978; Noorka and Khaliq, 2007). Desiccation tolerance
index was calculated according to Peacock et al. (1990):
Final number of dead seedlings

Desiccation tolerance index =
Final emergence number
23.7.6 Percent Seedling Recovery

After resumption of the irrigation water, drought-tolerant genotypes will recover. The recovery
percentage of the seedlings after desiccation was calculated with the formula (Blum et al., 1980;
Peacock et al., 1990; Noorka et al., 2013c). Hameed et al. (2010) suggest that seedling survivability
(late and early dying) and seedling growth behavior under drought indicated that root/shoot length,
ratio/root fresh and dry weight were the most important traits for screening drought tolerance at
the seedling stage. Selection by combining seedling survivability, growth response, relative water
content (RWC), and leaf water potential may be helpful for rapid evaluation of drought tolerance in
wheat breeding (Kaydan and Yagmur, 2008).
23.8 EFFECT OF WATER STRESS ON MORPHOLOGICAL TRAITS

Water stress is one of the most important abiotic stresses, mainly combined with rise in tem-
perature (Dash and Mohanty, 2001; HongBo et al., 2006; Akhter et al., 2008; Siddiqui et al.,
2008). Breeding programs of bread wheat seeking increased yield have usually attempted to
improve water stress tolerance of plants (McMichael and Quisenberry, 1991; Kumari et al., 2000).
Successful genetic manipulation is the only way to characterize the physiological traits to over-
come water stress tolerance or sensitiveness (Milthorpe, 1950; Bray, 1997). Several m ethods
based on physiological and agronomical traits as selection criteria, such as water use efficiency,
early seedling vigor, shoot and root length, dry root/shoot ratio (Dhanda et al., 2004), and seed
reserve mobilization (Soltani et al., 2006; Noorka, 2013a), have been suggested to screen the germ
plasm for drought tolerance.
Water stress at root formation and tillering stages affect the whole plant life particularly in yield,
contributing traits like flag leaf area, plant height, peduncle length, days to heading, spike length
and number of spikelets per spike, and ultimately decline in harvest index (Chowdhary et al., 1999;
Noorka et al., 2013b). The number of tillers and particularly the fertile tillers, number of grains per
spike, and 1000-grain weight always contribute toward the yield positively. Plant breeders usually
selected these traits in contrasting water provision conditions to raise the crop efficiently in water-
scarce areas. Ultimately, genotypes that have promising values for yield and yield contributing
trait under water stress condition are used in hybridization programs (Chowdhary et al., 1999).
However, Atale and Zope (1991) suggest that number of spikes per square meter, number of days to
heading, spike length, number of grains per spike, grains weight per spike, and 1000-grain weight
are the prime traits to consider for the improvement of yield under irrigated and rain-fed environ-
ments. Amar (1999) reported that the length of vegetative period, plant height, days required to
heading, number of grains per spike, and number of spikes per square meter are the major con-
tributors to grain yield.
23.9 GENE ACTION
Gene action depicts the internal traits and functions of the plant, the characteristics of hereditary
information, traveling from parents to offsprings. Gene action is not exclusively dependent on
genetic conditions but also on environmental conditions (Noorka et al., 2012). Gene action is of
additive and nonadditive nature with dominance, partial dominance, etc. Additive type of gene
action with partial dominance was observed for a number of traits like plant height, days to head-
ing, number of grains per spike, 1000-grain weight, and harvest index. However, the number of
fertile tillers per plant, grain yield per plant, and dry weight per plant were governed by nonad-
ditive and overdominance type of gene action (Sheikh et al., 2000). In the same way, Biljana
and Marija (2007) observed the combining ability and genetic effects of quantitative traits like
plant height, spike length, and number of spikelets per spike in durum wheat and concluded that
nonadditive gene action is more influential than additive type of gene action. Verma et al. (2007)
concluded that under normal fertile and saline–sodic soil environments, predominance of nonad-
ditive gene action for yield contribution and other quality traits was observed, showing desirabil-
ity to offer transgressive segregants in succeeding generations; however, Dhadhal et al. (2008)
and Wu et al. (2010) concluded both additive and nonadditive gene actions for days to heading and
maturity, number of fertile tillers per plant, spike length, number of spikelets per spike, number of
grains per spike, 1000-grain weight, grain yield per plant, and harvest index. Khan and Rizwan
(2000) reported a nonadditive type of gene action for different yield and yield contributing traits,
while Lu and Myers (2011) and Jatoi et al. (2012) also reported both additive and nonadditive gene
actions in yield traits of cotton; however, biparental mating and few spells of recurrent selection
may exploit both additive and nonadditive gene action to obtain transgressive segregants for grain
yield per plant in advanced generations.
23.10 CORRELATION
The correlation is observed as the relationship between two variables. The correlation coeffi-
cient is the measure of linear association between two variables depicting the values between −1
and +1. The value of +1 correlation coefficient reveals that two variables are very closely related in
a positive linear sense, while value of −1 correlation coefficient depicts the close relationship in neg-
ative linear sense. In the same way, the value of 0 correlation coefficient shows no linear relationship
between two variables. Pearson’s product-moment correlation coefficient (r) is commonly used to
quantify the strength of the linear association between two physically independent measured vari-
ables (x and y) (Francis et al., 1999). The sample correlation coefficient is written as
Â Â
n n
(x i - x)(y i - y) (x i - x)(y i - y)
rxy = i =1
= i =1
,
(n -1)sxsy
Â Â
n n
(x i - x) 2
(y i - y) 2
i =1 i =1
where
x and y are the sample means of X and Y
sx and sy are the sample standard deviations of X and Y
This can also be written as
rxy =
Â x y - nx y =
i i Â x y -Â x Â y
n i i
,
i i
nÂ x - (Â x ) nÂ y - (Â y )
(n - 1)sxsy 2
2
2
2
i i i i

if x and y are results of measurements that contain measurement error.

The negative correlation of grain yield per plant with different days to heading and maturity
indicated that timeliness permitted the cultivars to escape drought at the end of the growing
season (Yazdi-Samadi and Hosseini, 2002). Hafsi et al. (2001) suggest six water stress–prone
environments to examine genotype x environment interaction for grain yield per plant. They
examined that under these conditions flag leaf area and peduncle length were significantly cor-
related with grain yield per plant. Shafazadeh et al. (2004) determined the response of 20 wheat
genotypes to terminal drought stress in two separate experiments. Stress tolerance index, stress
susceptibility index, geometric mean productivity, and mean productivity were calculated based
on the grain yield per plant of the genotypes at both conditions. The correlation coefficients
between stress tolerance index and grain yield per plant (under both conditions) were positive and
significant; therefore, these indices can be used to identify high-yielding and tolerant genotypes
under stressed and non stressed conditions. The relationship between yield and drought suscepti-
bility index with morphophysiological traits under both water stress and normal irrigation condi-
tions gives the momentum of genotype stress tolerance and susceptibility. Nayyar et al. (1995)
and Nagarajan and Rane (2000) reported that grain yield per plant exhibited significant and posi-
tive association with the number of grains per spike, length of spike, and 1000-grain weight to
bear the water stress. Genetic correlation coefficients were higher than phenotypic correlation
coefficients for the major yield contributing characteristics like the number of grains per spike,
length of spike, and 1000-grain weight.
The relationship of phenology and physiological traits of rain-fed wheat (Triticum aestivum) to its
yield components revealed that improvements in grain yield per plant were associated with increases
in biological yield, harvest index, grain filling rate, and grain number per spike, whereas the number
of days to physiological maturity and grain filling duration exhibited negative correlation with the
grain yield per plant. Traits such as days to anthesis, total biomass at anthesis, and grain weight were
not significantly related to grain yield per plant (Attarbashi et al., 2002; Jatoi et al., 2011). Gupta et al.
(2011) and Noorka et al. (2013b) reported the effect of water deficit on stem reserve mobilization in
diversified wheat genotypes to monitor drought-tolerant and drought-sensitive behavior. The inter-
nodes of drought-tolerant genotypes seem to enhance the translocation of stem reserves to transform
it into grains under both the normal irrigation and water stress conditions. At the onset of drought,
fructans were mobilized in more efficient ways in the internodes of tolerant cultivar.
23.11 CONCLUSIONS
It is therefore concluded that under the changing climatic conditions, farmers will definitely be the
most affected, particularly in Asia and Africa, countries at the brink of population explosion. Plant
genetic engineering is the best option to make transgene for drought resistance in desired crops. The
best practice is choosing the best parent by rigorous screening methodology using an artificially
created water stress environment. The water stress–tolerant genotypes with maximum yield genetic
potential, mechanized farming, germ plasm conservation, and technology transfer will be the guid-
ing principles to ensure food, fiber, fuel, as well as law and order security in drought-prone areas
of the world.
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24 Physiological Mechanisms
of Nitrogen Absorption
and Assimilation in Plants
under Stressful Conditions
Rama Shanker Dubey, Rajneesh Kumar Srivastava,
and Mohammad Pessarakli
CONTENTS
24.1 Introduction........................................................................................................................... 453
24.2 Nitrogen Sources, Their Uptake, and Assimilation............................................................... 454
24.2.1 Sources of Nitrogen................................................................................................... 454
24.2.2 Absorption and Assimilation of Nitrogen................................................................. 456
24.2.2.1 Nitrate Transport Systems.......................................................................... 456
24.2.2.2 Nitrate Transporters.................................................................................... 457
24.2.2.3 Reduction of Nitrate.................................................................................... 457
24.3 Nitrogen Absorption and Assimilation under Different Stresses.......................................... 459
24.3.1 Salinity....................................................................................................................... 459
24.3.2 Water Stress...............................................................................................................465
24.3.3 Light...........................................................................................................................468
24.3.4 Heat............................................................................................................................ 470
24.3.5 Chilling...................................................................................................................... 471
24.3.6 Metal Toxicity............................................................................................................ 472
24.3.7 Ultraviolet B Radiation.............................................................................................. 474
References....................................................................................................................................... 475
24.1 INTRODUCTION
Nitrogen is the most important element for plant growth and development. In the cell, it is present
as constituent of proteins, amino acids, nucleic acids, coenzymes, vitamins, chlorophylls, hormones,
and functional groups of enzymes. It is a major part of chlorophyll and the green color of plants. It
is a key constituent of nucleic acids that serve as genetic material and regulate vital processes like
growth, reproduction, and heredity. Being a principal macronutrient, N has a special place in plant
nutrition, and plants require it in large amounts. Though N is the most abundant element in our atmo-
sphere, majority of the plants cannot utilize it directly in the elemental form. An adequate supply of N
in the soil is essential to maintain proper growth and yield of crops. The availability of N in the soil is
due to N2 fixation by symbiotic and nonsymbiotic bacteria; mineralization of organic matter, manure,
and animal waste products; or application of inorganic N fertilizers. Though the natural sources can
make significant contributions to soil N levels, with the advent of modern agricultural practices, to
453
meet the needs of high-yielding varieties of crops, inorganic N fertilizers have become major input
to the soil in order to achieve required optimum yields. To meet the increasing food demand for the
growing population, keeping pace with high N requirements of the improved varieties of crops, in the
near future, excessive uses of different forms of N fertilizers are anticipated.
The air around us contains about 78% nitrogen in the form of N2 gas. However, majority of
the plants cannot utilize N directly in elemental form as nutrient. Ammonium (NH +4 ) and nitrate
(NO3- ) are the two major forms of soil N available to plants (Botella et al., 1994, 1997). Plants take
up these two forms of N from the soil, whereas other forms of N present in the soil have to be
converted into one of these two principal forms before plants can utilize them. Among NH +4 and
NO3- , NO3- is the predominant form of N available to most cultivable plants grown under normal
field conditions. Preferably in the form of NO3- , higher plants absorb N from the soil. Availability
of N in the soil is considered as a limiting factor for plant growth and crop productivity. Plants have
evolved several uptake and transport systems for optimum utilization of soil N for their growth.
Root cells ensure uptake of NO3- from the soil via nitrate transporters (NRTs); thereafter, NO3- is
assimilated into ammonium ions and amino acids within the plant tissues by the action of NO3-
assimilatory enzymes.
In the context of climatic and environmental changes, plants are often exposed to a wide range of
abiotic stresses such as soil salinity, drought, flood, heat, cold, inadequate light, presence of excessive
levels of metals in the soil, and nutrient unavailability. These conditions create considerable stress in
growing plants, directly or indirectly affect acquisition of N by the roots and its assimilation within
the plant, and ultimately influence yield and quality of the crops (Botella et al., 1994; Yao et al., 2008).
Plants have developed capacity to adapt themselves to modulate the efficiency of root N acquisi-
tion and assimilation of NO3- in the tissues with changes in both external N availability and nutritional
status of the plants (Forde, 2002). For efficient utilization of NO3- by the plants, its uptake, allocation,
and assimilation are key factors. Membrane-bound transporters and channels have been well char-
acterized, which are involved in the uptake of NO3- from the soil as well as its allocation and storage
in the tissues (Yao et al., 2008; Dechorgnat et al., 2011). Low NO3- availability in the soil induces
the activity of NO3- uptake systems of the roots (Rawat et al., 1999). Due to fall in plant N status,
the activities of genes involved in N assimilation process are induced (Christophe et al., 2011). After
being taken up by the root system, NO3- is assimilated within the plant tissues by the coordinated
action of enzymes nitrate reductase (NR) and nitrite reductase (NiR). These enzymes have been
extensively studied in the diverse plant species and have been well characterized for their physico-
chemical properties and subcellular localizations (Campbell, 1988; Eckardt, 2005). Abiotic stressful
conditions of the environment influence N nutrition in plants by inhibiting its uptake (Alam, 1999;
Pessarakli, 1999; Rabe, 1999; Meloni et al., 2004), its allocation, and assimilation within the tissues
(Dubey and Rani, 1989; Katiyar and Dubey, 1992; Richharia et al., 1997; Kumar and Dubey, 1999;
Meloni et al., 2004; Jha and Dubey, 2004, 2005; Sharma and Dubey, 2005, 2010).
In the present review chapter, we have focused on various N sources available to plants, their
modes of uptake, and assimilation within the tissues. The information related to the effects of dif-
ferent environmental stresses on N uptake and its assimilation have also been presented.
24.2 NITROGEN SOURCES, THEIR UPTAKE, AND ASSIMILATION

24.2.1 Sources of Nitrogen
The different forms of N that are available in the soil include organic nitrogen such as proteins and
amino acids, nucleic acids, inorganic nitrogen such as ammonium salts and nitrate, and synthetic
nitrogen such as urea. Certain bacteria, blue-green algae, and leguminous plants can utilize atmo-
spheric N2 and fix them into organic form. Organic N results from the breaking down of organic
matter by soil microorganisms. It is a slow process, but organic N, due to its eco-friendly nature,
builds a healthy soil, and there is no risk of plant injury due to its overapplication.
Physiological Mechanisms of Nitrogen Absorption 455
Composted manure, green manure, and animal wastes are being widely used as organic N source
by organic crop producers. The inorganic forms of N available in soil are primarily salts of NO3-
and NH +4 . Plants readily take up these two forms of soil N, NH +4 and NO3-. Urea and processed urea
are the primary source of synthetic N to the soil. A coating when applied to the area allows its slow
release. Plants can take up both organic and inorganic forms of N from the soil. At any time, a
major portion of soil total N remains in the form of organic matter and a very small fraction of it is
in inorganic form. Soil N availability as well as the concentrations of organic and inorganic forms
depends on many factors such as soil type, pH, temperature, activity of microorganisms, wind, and
precipitation (Masclaux-Daubresse et al., 2010; Wang et al., 2012b). Depending on adaptation of
plants to soil conditions, the preferred form of N differs among plant species. In aerobic soils, where
crop plants are generally grown, the inorganic N is often supplied as fertilizers in the form of NO3- ,
NH +4 , and urea, whereas in reducing soils with low pH such as in forests, organic forms of N such
as amino acids, peptides, and proteins predominate (Nasholm et al., 2009). Among the inorganic N
forms, NO3- predominates in aerobic soils, and if not taken up by the plant roots, its leaching occurs
(Strahm and Harrison, 2006). In aerobic soil conditions with soil temperatures above freezing, dif-
ferent forms of N, except nitrogen gas, are converted to NO3- by soil microorganisms. Nitrate ions
possess negative charge, similar to clay particles; therefore, they can freely move with soil water and
can be easily taken up by plant roots along with water. Although in some soils, NH +4 and NO3- are
readily available, but the roots of plants take up N largely as NO3 because NO3- freely moves within
-
the root solution due to the overall negative charge of the soil particles (Reisenauer, 1978).
The preference of plants for N forms is related to the type of N abundant in the habitat and the
plant species (Miller and Cramer, 2008). Even within the same habitat, plant species vary in their
preference for N forms. Frota and Tucker (1978a), Saad (1979), and Pessarakli et al. (1989) found that
beans (Phaseolus vulgaris L.), C3 plants, under normal as well as stressed conditions absorbed more
NO3- than NH +4 .. Due to biological N2 fixation by symbiotic association of N2-fixing organisms, free-
living soil bacteria, and blue-green algae, sometimes, NH +4 form of N may be abundant in the soil.
However, the ammonium ions in the soil are oxidized to nitrite and then to nitrate by the process of
nitrification, accomplished by soil microorganisms. NH +4 is much more abundant in soil at acidic to
neutral pH, and its uptake results in acidification of the soil. Furthermore, the nitrification process
itself has an acidification effect, which causes acidification of the soil irrespective of whether NH +4
is taken up by plant roots (Miller and Cramer, 2004). Certain plant species such as those inhabiting
acidic soils and all C4 plants (i.e., grasses) show a preference for NH +4 form of N. Due to the defi-
ciency of NO3- in acid soils and the specific physiological, metabolic, and photosynthetic pathways
of C4 plants, such plants prefer NH +4 over NO3-.
Though NH +4 is the preferred form of N for certain plants, high concentration of NH +4 becomes toxic
to the plants and interferes with various metabolic pathways in the cell. Therefore, after being taken up
by the plants, NH +4 ions are immediately assimilated into amino acids, amides, etc., in the roots. Plant
types exist those ranging from exclusively NO3- requiring to those that exclusively require NH +4 (Miller
and Cramer, 2008). Certain plants can detoxify NH3 by forming NH +4 salts of organic acids (Singh and
Sawhney, 1989). These plant species grow well on NH +4 form of N. It has been observed that majority of
plant species grow better when N is supplied in the soil as a mixture of both NO3- and NH +4 ; however,
the optimal ratios of NO3- to NH +4 form vary. The optimum concentration of NO3- and NH +4 required
by plants depends on the species of plant, age of the plant, and the pH of the growth medium (Haynes
and Goh, 1978). A proper balance of NO3- and NH +4 is of a great significance for the growth of plants.
For optimal growth, each plant species requires a desired NO3- to NH +4 ratio. This ratio also varies with
temperature, growth stage of the plant, soil properties, and pH of the root zone. Uptake of NO3- causes
increase in pH around the root zone, while uptake of NH +4 decreases it. Therefore, in order to resist
change in pH of the soil medium, it is always advisable to use N forms in an appropriate NH +4 /NO3-
ratio, depending on the cultivar, temperature, and the growth stage of the plant.
Botella et al. (1994) studied the uptake of NO3- and NH +4 by wheat plants grown in nutrient solu-
tions containing NO3- , NH +4 , and NO3- + NH +4 , with 1 mM (control) and 60 mM (saline) NaCl each.
These investigators observed that the addition of both N forms was beneficial for the plants under
saline conditions because higher N uptake resulted in better growth and development of the plants.
They concluded that the best N source for wheat growth, under saline conditions, was a mixture of
NO3- and NH +4 . Depending on the pH of the soil or nutrient medium, certain plants can absorb either
NH +4 or NO3-. Many crop plants show NH +4 toxicity, but the concentration of NH +4 that becomes toxic
varies greatly from species to species (Britto and Kronzucker, 2002). Plants vary greatly in their
relative adaptations to either NO3- or NH +4 form of N (Kronzucker et al., 1997). Only a few plant
species perform well when NH +4 is provided as sole source of N, while most of the crop plants show
severe NH +4 toxicity symptoms with NH +4 as sole N source (Findenegg, 1987). The uptake of many
inorganic cations is reduced under NH +4 nutrition, and NH +4 -fed plants take up an excess of cations
relative to anions (Forde and Clarkson, 1999). However, growth and yield parameters are often
enhanced when both NO3- and NH +4 forms of N are provided simultaneously, compared to either
NH +4 or NO3- provided alone (Gweyi-Onyango et al., 2009).
Various groups of investigators have suggested that nutrient media containing both NO3- and NH +4
forms of N in a proper combination are more suitable for the growth of cells, tissues, and whole plants
compared to the either form alone (Botella et al., 1994; Beevers and Hageman, 1980; Ramage and
Williams, 2002). Ramage and Williams (2002), while studying inorganic nitrogen requirements during
shoot organogenesis in tobacco leaf discs, observed a synergistic effect between NH +4 and NO3- inde-
pendent of culture medium pH. These workers observed shoot formation with a wide range of ratios, but
media containing 40 mM NO3- and 20 mM NH +4 in the ratio of 70:30 produced the greatest number of
shoots per explant. In many vegetable crops, NH +4 is taken up in preference to NO3- when its concentra-
tion is above 10% of the total N in the nutrient medium (Kafkafi, 1990). Especially at low root tempera-
ture, NH +4 is regarded as a safe source of N (Kafkafi, 1990). Plants show genotypic diversity for N use
efficiency. Kafkafi (1990) while evaluating 12 genotypes of pearl millet (Pennisetum glaucum) plants
for nitrogen use efficiency (NUE) observed that genotypes varied greatly for uptake, translocation, and
NUE. Genotypes with lower NUE and translocation rate showed lower grain yields.
Whether NH +4 or NO3- is absorbed by plant roots, the initial product of N assimilation is glutamine.
Other products of assimilation are different amino acids, asparagine, citrulline, allantoin, and other sol-
uble nitrogenous compounds. The assimilated products are translocated to various organs of the plants
through xylem and phloem vessels. Plants differ in tissue localization of NO3- reduction and assimila-
tion (Black et al., 2002). In some plant species (i.e., tomatoes), NO3- reduction and assimilation occurs
in roots, whereas in many others (i.e., grasses), NO3- after absorption is transported to different organs,
where it is reduced. Even within the same plant species, different tissues vary in their capacity to reduce
NO3-. Different tissues of poplar plants vary in NO3- assimilation, where very little NO3- assimilation
occurs in roots, whereas major assimilation takes place in leaves (Black et al., 2002).
24.2.2 Absorption and Assimilation of Nitrogen

Nitrogen is a key nutrient for plants and it is acquired by most of the terrestrial plants from the soil.
Commercial inorganic fertilizers added to the soil are the major source of N for the crop plants. The
availability of N in the soil is often considered an important limitation for plant growth (Miller and
Cramer, 2008). NO3- and NH +4 are the major inorganic forms of N that predominate in croplands and
are often supplied as fertilizers. The efficient use of N by plants involves several steps, including
its uptake, assimilation, translocation, and remobilization (Masclaux-Daubresse et al., 2010). Plants
have evolved multiple uptake and transport systems to optimize N use and to support their growth
with changing soil conditions.
24.2.2.1 Nitrate Transport Systems

Nitrate is a major inorganic N source for higher plants. Nitrate present in the soil must enter either
the epidermal, the cortex, or the endodermal cells before being loaded into the xylem vessels.
Multiple uptake systems have been suggested that contribute to N uptake by plant roots (Miller and
Cramer, 2008; Maathuis, 2009; Wang et al., 2012b). Two different NO3- uptake systems exist in plants
depending on the external concentration of nitrate. These uptake systems are a constitutive, low-
affinity transport system (LATS) (possibly a carrier system or an anion channel) that operates when
NO3- concentration in the soil is high (>1 mM) and an inducible high-affinity transport system (HATS)
that operates when NO3- concentration in the soil is low (<1 mM) (Dechorgnat et al., 2011). The two
transport systems coexist in plants and coordinately act to take up NO3- from the soil. The systems
are composed of constitutive and nitrate-inducible components and utilize energy provided by proton
gradients (Crawford and Glass, 1998). NO3- uptake studies have shown that LATS has Km values in
millimolar range, whereas the HATS has Km values in micromolar range (Wang et al., 2012b).
24.2.2.2 Nitrate Transporters

For uptake of NO3- from the soil and for its movement within the plant tissues, membrane-bound
transporters are required. NO3- is actively transported via members of the families of NRTs. Five
gene families have been identified in higher plants that are involved in uptake of NO3- , its allocation,
and storage within the tissues (Plett et al., 2010; Wang et al., 2012). These are nitrate transporter
1/peptide transporter (NRT1/PTR), NRT2, NRT3, chloride channel (CLC), and slow anion chan-
nel (SLAC)-associated 1 homologue 3 (SLAC1/SLAH). It is believed that the gene family NRT1
mediates LATS, whereas HATS relies on the activity of gene family NRT2 (Wang et al., 2012b). In
Arabidopsis thaliana, a chlorate-resistant 1 (CHL1) transporter has been identified that belongs to
NRT1/PTR family. This transporter, AtNRT1.1, is a protein that plays the roles of a dual-affinity
transporter as well as of a NO3- sensor to activate the expression of NO3--related genes (Ho et al.,
2009; Wang et al., 2012b). The expression of the NRT1.1 gene is inducible by nitrate. In Arabidopsis,
53 NRT1/PTR transporters exist, out of which 16 have been well characterized. Most of these NRT1
transporters belong to LATS. The second type of transporters, NRT2 transporters, has been isolated
from higher plants that transport NO3- and require a membrane protein, NAR2, for the transport
activity (Forde, 2000). NRT2 transporters are also proton-coupled transporters. In Arabidopsis,
seven NRT2 transporters have been identified that appear to be high-affinity transporters (Orsel
et al., 2002; Plett et al., 2010). Both NRT1 and NRT2 transport NO3- together with protons (Miller
et al., 2007). Besides, a third type of transporters, NRT3 (NAR2) family members, has been identi-
fied that participates in high-affinity transport (Plett et al., 2010). The fourth type of NO3- transport-
ers belongs to CLC family, some of them function as anion channels and others as anion–proton
exchangers (De et al. 2006; Barbier-Brygoo et al., 2011). In Arabidopsis, seven CLC genes have
been identified (Wang et al., 2012b). The fifth type of transporters, SLACs, has been identified
in Arabidopsis using mutants defective in stomatal closure, which show NO3- transport activity
(Wang et al., 2012b). Five SLAC genes, namely, SLAC1 and SLAH1–SLAH4 (SLAC1 homologues),
have been identified in Arabidopsis (Wang et al., 2012b).
24.2.2.3 Reduction of Nitrate

Nitrate uptake and its reduction activities in the plant tissues are coordinately regulated. After its
uptake, NO3- may be translocated from root to different organs where it is reduced to the level of
NH +4 or NO3- may be reduced in the roots itself. Primary NO3- assimilation takes place predomi-
nantly in the roots of the plants. Excess NO3- can be translocated to the vacuole in the cells, where
it can accumulate and serve as an NO3- reserve (Clarkson, 1988). The process of intracellular NO3-
translocation possibly requires a tonoplast NO3- translocator that is different from the membrane
NRTs (Redinbaugh and Campbell, 1991). The distinct NO3- translocators present at the symplasm–
xylem interface control the translocation of NO3- from root to xylem and then to different organs of
the plant (Redinbaugh and Campbell, 1991). Although the translocator proteins appear to be differ-
ent from transport proteins and are encoded by different genes, all three processes, NO3- transport,
translocation, and reduction, are coordinately regulated.
The reduction of NO3- to the level of NH +4 in the plant tissues is one of the principal routes by
which inorganic N is incorporated in organic compounds. After NO3- is taken up by the root cells
employing NRTs, the primary step in its reduction process involves the conversion of NO3- to nitrite
(NO2- ) catalyzed by the enzyme NAD(P)H–NR. The enzyme NR requires molybdenum as cofac-
tor and is highly regulated at both transcriptional and posttranslational level (Eckardt, 2005). This
is the first and rate limit step in N assimilation pathway. NR is a cytosolic and substrate-inducible
enzyme. Its activity is also induced by light and is rapidly and reversibly modulated under light/dark
transitions (Eckardt, 2005). In plant tissues, NO3- induces increase in NR activity. The activity of
NR increases in root cells in response to exogenous NO3-.
Nitrite formed in the cytosol as a result of NR-catalyzed step is translocated into the chloro-
plasts in leaves and plastids in roots. In these organelles, NO2- is reduced to ammonium (NH +4 )
Abiotic
stress
ROS Signal
transduction
Signal (?)
? transduction Proteases
Nucleus
NH4+
Citrate Isocitrate
ICDH
2-OG
Cytosol
Citrate
Proline
Malate TCA
Isocitrate
cycle
IDH
2-OG Glutamate Glutamate

Mitochondria GDH
NH4+ NH4+
Glycine Serine
NH4+ GS2 2-OG

Glutamine
GS1 GOGAT
GS2 Glutamate Chloroplast
NiR
NH4+ NO2–
NR NO3–
NO2–
NO3– soil
FIGURE 24.1 Sequence of events leading to uptake and assimilation of NO3- in plants. Soil NO3- is taken
up by plant roots via NRTs. In the cytosol, NO3- is reduced to NO2-, whereas within the chloroplasts, NO2-
is reduced to NH +4 and it is further assimilated to glutamine. Within the mitochondria, NH +4 is assimilated
to glutamate. Abiotic stresses result in increased formation of ROS, which, in turn, induce the activities of
proteases leading to increase in intracellular level of NH +4 . For the synthesis of glutamate, 2-oxo glutarate
(2-OG) is provided in mitochondria by dehydrogenation of isocitrate. Under stresses, increased synthesis of
glutamate favors increased proline synthesis. The enzymes involved in these metabolic pathways include NR,
NiR, glutamate synthase (GOGAT), cytosolic GS (GS1), chloroplastic GS (GS2), mitochondrial isocitrate
dehydrogenase (IDH), and cytosolic isocitrate dehydrogenase (ICDH).
in a reaction catalyzed by the enzyme NiR (Rosales et al., 2011). NiR is a plastidic enzyme pres-
ent in plastids/chloroplasts. Transport of NO2- from the cytosol to the chloroplast requires NO2-
transporters. Ammonium, either directly absorbed by plant roots or as a result of NO3- reduction or
originated due to photorespiration or amino acid recycling, is assimilated in the plastids/chloroplasts
into the amide amino group of glutamine in a reaction catalyzed by the enzyme glutamine synthe-
tase (GS). GS fixes NH +4 on a glutamate molecule to form glutamine by the so-called GS/GOGAT
cycle (Lea and Forde, 1994). Glutamine subsequently reacts with 2-oxoglutarate to form two mol-
ecules of glutamate, the reaction step catalyzed by the enzyme glutamine 2-oxoglutarate amino
transferase (or glutamate synthase, GOGAT). These two enzymes, GS and GOGAT, are responsible
for the assimilation of most of the NH +4 derived from NO3- reduction under normal growth condi-
tions. An alternative route of NH +4 assimilation into glutamate involves the reductive amination
of a-ketoglutarate catalyzed by a mitochondrial enzyme glutamate dehydrogenase (GDH). Other
amino acids, such as alanine and aspartic acid, are further synthesized from glutamic acid by trans-
amination reactions. Proline, an amino acid synthesized in higher amounts under stressful condi-
tion, is also synthesized from glutamine. Figure 24.1 shows an overview of the sequential events
related to NO3- uptake, its assimilation to NH +4 , and further assimilation of NH +4 into glutamine in
chloroplasts and glutamic acid within mitochondria.
24.3 NITROGEN ABSORPTION AND ASSIMILATION

UNDER DIFFERENT STRESSES
Abiotic stressful conditions of the environment such as soil salinity, drought, heat, chilling, anaero-
biosis, low light, excessive levels of metals in the soil, and gaseous pollutants adversely affect crop
growth and productivity (Gao et al., 2007). These stressful conditions cause a series of physiologi-
cal, biochemical, and molecular changes within the plants, and the plants respond to these stresses
by physiological and biochemical adjustments. The processes of N uptake, translocation, and assim-
ilation are severely affected by different types of stresses (Broadbent et al., 1988; Pessarakli and
Tucker, 1988a,b; Pessarakli et al., 1989; Pessarakli, 1991, 1999; Alam, 1993, 1999; Botella et al.,
1994, 1997; Sharma et al., 1994; Hamdia and El-Komy, 1998; Grattan and Grieve, 1999; Rabe,
1999). Reduced N assimilation capacity is one of the most common responses, which are triggered
in plants subjected to different abiotic stresses. Because the availability of nitrogenous nutrients in
the soil, their uptake, and assimilation are directly related to each other as well as to the growth and
yield of the crops, considerable efforts have been made by various groups of researchers to study the
possible implications of various stressful conditions on N nutrition in plants (Torres and Bingham,
1973; Saad, 1979; Mahajan and Sonar, 1980; Pessarakli, 1981, 1993; Pessarakli and Tucker, 1985a;
Clarkson, 1988; Botella et al., 1994, 1997; Albassam, 2001; Irshad et al., 2002; Flores et al., 2004;
Abdelgadir et al., 2005; Pessarakli et al., 2005, 2012; Bowman et al., 2006; Uygur and Yetisir, 2009;
Song and Xing, 2010). In the following sections, the influences of various environmental stresses on
the overall process of N uptake and its assimilation are summarized.
24.3.1 Salinity
Soil salinity is a major stressful condition of the environment, which reduces crop productivity in
many areas of the world. Soil salinization has become a major cause of soil degradation. The effects
of salinity are more conspicuous in arid and semiarid areas. The effect of salinity on plants varies
depending on plant species and the developmental stage of the plant (Khan et al., 1995; Marconi
et al., 2001; Ashraf et al., 2003; Shiyab et al., 2003; Ashraf and McNeilly, 2004; Marosz, 2004;
Ahmad and Jhon, 2005; An et al., 2005; Eynard et al., 2005; Pandya et al., 2005; Jiang et al., 2006;
Mustard and Renault, 2006; Ramoliya et al., 2006; Iqbal and Ashraf, 2007; Kumar et al., 2007;
Musyimi et al., 2007; Chiroma et al., 2008; Nayak et al., 2008; Dashti et al., 2009; Gama et al., 2009;
Saleh et al., 2009; Ahmad, 2010; Ahmadi et al., 2010; Bosco et al., 2010; Don et al., 2010; Patel et al.,
2010; Prasad et al., 2010; Rivelli et al., 2010; Hardikar and Pandey, 2011; Jamil et al., 2011; Pessarakli,
2011; Shekoofa et al., 2012; Shobbar et al., 2012) as well as the types and concentration of salts
(Akhavan-Kharazian, 1991; De Oliveira et al., 1998; Li et al., 1998; Vulkan-Levy et al., 1998; Alam
et al., 2002; Wilson et al., 2002; Arshi et al., 2005; Iqbal et al., 2006; Wilson and Read, 2006; Li et al.,
2009; Khosh Kholgh Sima et al., 2012b). The responses to salinity on N uptake differ in different
plant species and also depend on the type and extent of salinity (Albassam, 2001; Irshad et al., 2002;
Flores et al., 2004; Abdelgadir et al., 2005; Pessarakli et al., 2005, 2012; Bowman et al., 2006; Uygur
and Yetisir, 2009; Song and Xing, 2010). Salinization in the soil affects N uptake in majority of crop
plants, whereas in halophytes and in many salt-tolerant crop species, no significant effect of NaCl
on NO3- uptake is observed (Sharma and Gupta, 1986; Pessarakli et al., 2005; Li et al., 2009; Song
and Xing, 2010; Pessarakli et al., 2012). Barley (Hordeum vulgare L.) plants growing under saline
conditions show reduced growth (Sharma and Gupta, 1986; Pessarakli et al., 1991; Pandya et al.,
2005; Huang et al., 2006; Khosh Kholgh Sima et al., 2012a) as well as decreased N uptake (Sharma
and Gupta, 1986). In young barley seedlings, salinity severely inhibited NO3- uptake, whereas little
effect on NO3- reduction was observed (Aslam et al., 1984). In wheat (Triticum aestivum L.) plants,
reduction in growth was even more than that in barley at higher NaCl salinity levels (Pessarakli et al.,
1991; Irshad et al., 2002; Iqbal et al., 2006; Iqbal and Ashraf, 2007: Ehsanzadeh et al., 2009; Azizpour
et al., 2010; Saqib et al., 2011), and uptake of N decreased with increasing salinity (Sharma and Gupta,
1986). However, by increasing the N supply to the soil, the effect of salinity was alleviated (Sharma
and Gupta, 1986). Khalil et al. (1967) found similar results for cotton (Gossypium hirsutum L.) and
corn (Zea mays L.) plants. Soltani et al. (1990) observed that when barley seedlings were grown in
the presence of 200 mM NaCl, growth of the seedlings decreased with a concomitant reduction in the
uptake and translocation of N compared with nonsalinized seedlings.
When 1-year-old rose (Rosa spp.) plants were grown under increasing concentration of NaCl,
the uptake of NO3- was negatively affected by increase in NaCl concentration. In such plants, maxi-
mum influx (Imax) of NO3- declined from 5.1 μmol to 2.5 μmol g−1 of plant dry weight per hour
as NaCl concentration increased from zero to 65 mol m−3 (Massa et al., 2009). A kinetic study of
NO3- uptake by rose plants under saline conditions revealed that NaCl was exerting a noncom-
petitive inhibition effect or at least a mixed competitive inhibition effect on NO3- uptake (Massa
et al., 2009). Tomato (Lycopersicon esculentum L.) seedlings grown under 50 and 100 mM NaCl
salinity showed nearly 60% and 80% decreased NO3- levels, respectively, in leaves and almost a
similar decline in NO3- content in roots (Debouba et al., 2006). In another experiment, when tomato
(Solanum esculentum L.) seedlings were exposed to salinity stress treatment of 75 mM NaCl for
24 h, decrease in NO3- uptake was observed irrespective of the NO3- concentration used (Yao et al.,
2008). An analysis of the transcript levels of the transporters in salt-stressed tomato seedlings using
quantitative real-time PCR revealed a significant decline in the expression level of the transcripts for
the transporters, LeNRT1.1 and LeNRT1.2, suggesting that the downregulation of LeNRT1 could
be possibly involved for the reduced uptake of NO3- under salt stress (Yao et al., 2008). Experiments
conducted by Cordovilla and co-workers (1999) to study the effects of NaCl on the growth and
N fixation and assimilation in inoculated and KNO3-fertilized bean plants revealed that N-fixing
plants were more sensitive to salinity than were N-fertilized plants.
In seedlings of maize genotypes differing in drought tolerance when grown at −0.84 MPa NaCl
salinity, the supply of reduced N for the synthesis of amino acids and protein in the tissues was reduced,
and the effect was more pronounced in drought-sensitive genotypes than the tolerant (Mladenova,
1990). Reduced growth in terms of dry-matter production and decreased absorption of N have been
reported by several investigators for various plant species with different degrees of salt tolerance
(Torres and Bingham, 1973; Frota and Tucker, 1978b; Pessarakli and Tucker, 1985a; Dubey and Rani,
1989; Pessarakli and Huber, 1991; Pessarakli, 1993; Grattan and Grieve, 1999; Albassam, 2001; Irshad
et al., 2002; Flores et al., 2004; Abdelgadir et al., 2005; Pessarakli et al., 2005, 2012; Bowman et al.,
2006; Debouba et al., 2006; Yao et al., 2008; Uygur and Yetisir, 2009; Song and Xing, 2010).
In salt-tolerant plants, either no effect of NaCl on N uptake is observed or only high concentra-
tions of NaCl affect growth and N uptake. When 17-day-old seedlings of an important legume
Prosopis alba (algarrobo) were subjected to increasing levels (0–600 mmol L−1) of NaCl treatment,
it was observed that only the highest concentration of 600 mmol L−1 NaCl caused a reduction in
root and shoot growth and decrease in nitrate content of roots and leaves (Meloni et al., 2004).
When growth and N uptake by a number of clones of a halophytic plant species salt grass (Distichlis
spicata L.) were observed under 20 dSm−1salt stress level, using the 15N technique, almost no dif-
ference in N uptake by most of the clones was observed under salt stress compared with the control
plants (Pessarakli et al., 2012).
In halophytes, salinity either induces uptake and accumulation of NO3- or has no effect on these
processes (Flowers et al., 1977). Inorganic ions such as Na+, K+, Cl−, and NO3- get accumulated in
excess in halophytes compared with non-halophytes under saline conditions. Atriplex, Salicornia,
and Suaeda maritima plants show higher uptake of Na+, Cl−, SO2- 4 , and NO3 in saline environments
-
than under nonsaline conditions (Flowers et al., 1977). According to Flowers et al. (1977), even in
conditions of low salinity, the levels of K+, NO3- , and SO2-4 are very high in halophytes compared
with other plants (Flowers et al., 1977). High uptake of NO3- by halophytes under salinization is
related to the intrinsic properties of these plants as they are adapted to grow at high ion concentra-
tions (Flowers et al., 1977). In another halophyte Suaeda physophora, Na+ and NO3- , when present
together in the culture medium, stimulated lateral root growth, whereas in the presence of 10 mmol
L−1 NO3- in the saline medium with increase in salinity, the concentrations of NO3- and total N were
remarkably reduced in roots but were unaffected in shoots (Jun-Feng et al., 2010).
Reduced uptake of N by crop plants under saline conditions could be possibly due to more
intake of Na+ and Cl− by the roots. Increased uptake of Na+ by the plants causes nutrient imbalance
and displaces Ca2+ from the exchange sites on the membranes and cell walls (Sharma and Gupta,
1986). Chloride present at more than 100 mM in the saline medium inhibits NO3- uptake, possibly
because of increased accumulation of Cl in the roots (Ward et al., 1973). Smith (1973) observed
that NO3- uptake in barley was dependent on the internal rather than the external concentrations
of Cl. Reduced uptake of N could lead to N deficiency in plants and thus could become a limiting
factor for growth of plants under saline conditions (Ward et al., 1973). As salinity leads to N defi-
ciency, fertilization of plants growing in a saline environment with increasing doses of nitrogenous
fertilizers has proved beneficial. It minimizes salt-induced damage and apparently provides salt
tolerance (Sharma and Gupta, 1986). However, in certain crop species such as corn, rice (Oryza
sativa L.), wheat, and spinach (Spinacia oleracea L.) with excess application of nitrogenous fertil-
izers, a decrease in salt tolerance is observed (Sharma and Gupta, 1986).
Ca2+ when present in the growth medium increases NO3- uptake by plants under saline condi-
tions. Ward et al. (1973) observed that in saline medium, the uptake of NO3- by barley seedlings
increased with increase in the level of Ca2+ between 1.0 and 3 mM. Manganese and Mg2+ also
enhanced NO3- uptake in saline medium, but Ca2+ was more effective than these two ions (Ward
et al., 1973). Calcium in the saline medium possibly decreased Na+ as well as Cl− uptake and also
reduced membrane disruption in saline solutions, leading to increased NO3- uptake (Ward et al.,
1973). Calcium helps in maintaining the integrity of the root membrane; thus, its deprivation, under
salinization, decreases ion transport and NO3- uptake by disrupting the NRT that is located in the
plasmalemma of roots (Ward et al., 1973). The activity of the NRTs is increased due to the presence
of Ca2+ in the saline medium (Ward et al., 1973).
Assimilation of NO3- is reduced in plants growing in saline medium; however, salt-tolerant plants
tend to maintain higher N assimilation efficiency than the sensitive plants. The key enzyme of
NO3- assimilation, NR, which catalyzes the reduction of NO3- to NO2- , has been studied extensively
by various groups of investigators for its behavior in different plant species under salinization.
The effects of salinity on NR activity are varied and depend on the type of salinity, duration of
salt treatment, plant species, and the plant tissues studied. NR is highly sensitive to various types
of environmental stresses including salinity (Debouba et al., 2007). In many salt-sensitive plant
species, NR activity decreases under NaCl salinity (Katiyar and Dubey, 1992; Meloni et al., 2004;
Debouba et al., 2007). When 10-day-old tomato (Solanum lycopersicum) seedlings were subjected
to 100 mM NaCl stress, the activities of NR, glutamate synthase (Fd-GOGAT), and deaminating
GDH (NAD+-GDH) declined in the seedlings compared to controls and salinity caused greater
decline in enzyme activities in the leaves than the roots (Debouba et al., 2007). In the seedlings of
salt-tolerant legume Prosopis alba, 600 mmol L−1 NaCl treatment caused a significant decline in
NR activity (Meloni et al., 2004).
While studying the effects of salinity on N metabolism on wheat plants, Abdul-Kadir and Paulsen
(1982) observed decreased NR activity under salinization. In pea (Pisum sativum L.) seedlings, an
isosmotic concentration of NaCl suppressed NR activity and caused accumulation of NO3- in the
plant tissue, and in wheat seedlings, an isosmotic salinity level decreased NR activity, without sig-
nificant accumulation of NO3- in the tissues (Sharma and Gupta, 1986). In rice (Oryza sativa L.)
seedlings, decreased NR activity has been observed under salinization (Dubey et al., 1991). While
conducting pot experiments with three rice genotypes, viz., Q-31, Y-1281, and MR-219, Islam and
co-workers (2007) observed a progressive decline in NR activity with increase in salinity level in the
range 3–15 dS m−1 NaCl. When canola (Brassica napus L.) plants were exposed to 150 and 200 mM
NaCl treatment for 7–45 days in hydroponics, NR activity decreased in the leaves compared to con-
trol plants, and the decrease was more with increase in NaCl (Bybordi and Ebrahimian, 2011).
Among the enzymes of NO3- and NH +4 assimilation, NR and GS are more prone to inhibition
due to salinity. More inhibitory effect on these enzymes due to salinity is observed in salt-sensitive
cultivars than in salt tolerants when raised under similar salinity levels (Katiyar and Dubey, 1992;
Richharia et al., 1997; Kumar and Dubey, 1999). Varying behavior of GDH has been observed when
two sets of rice cultivars differing in salt tolerance are raised in saline medium (Kumar et al., 2000).
In tomato seedlings, a lesser salt stress effect is observed for NiR and GS activities relative to NR
(Debouba et al., 2007). Inhibition in the activities of NR and GS under salinity has been regarded
as one of the key contributory factors that limit the growth of rice plants under saline conditions
(Katiyar and Dubey, 1992; Kumar et al., 2000).
In field pea (Pisum sativum L.) plants subjected to salinization with NaCl and CaCl2 (1:1) for
15 days, a significant decrease in NR activity accompanied with decrease in total N as well as pro-
tein N and increase in NO3- - N and NH +4 - N was observed (Lal and Bhardwaj, 1987). According
to these investigators, NaCl as well as CaCl2 salinity impaired NO3- assimilation in pea plants,
leading to accumulation of NO3- and NH +4 in the tissues. Tewari and Singh (1991), while conduct-
ing stress studies in lentil (Lens esculenta Moench), observed that with increasing exchangeable
sodium percentage (ESP) in the cell, there appeared a continuous decrease in NR as well as NIR
activities in plants up to 60 days after sowing. Rice plants differing in salt tolerance showed varying
behaviors of NR and NIR under salinity stress (Katiyar and Dubey, 1992). While studying the mode
of N assimilation under salinization in the seedlings of two sets of rice cultivars differing in salt
tolerance, Katiyar and Dubey (1992) observed decreased NR activity in seedlings of salt-sensitive
cultivars, but not in the tolerant.
Several possible explanations have been suggested for the decreased NR activity in salt-sensitive
plants under salinity stress (Lacuesta et al., 1990). A plausible reason appears to be the inhibition
of enzyme induction under salinization (Tewari and Singh, 1991). As NR is a substrate-inducible
enzyme, under saline conditions, NO3- uptake by the plants is reduced. Chloride ions inhibit NO3-
uptake due to competition between Cl2 and NO3- for uptake by NRTs. This would lead to a decrease
in NO3- concentrations in plant tissues and/or an inactivation of NRTs by toxic effects of salt ions
(Debouba et al., 2007). Due to limited NO3- availability in the plant tissues, NR induction is sup-
pressed, which results in decreased NR activity (Lacuesta et al., 1990). In durum wheat plants,
a lower activity of NR in the leaves of high-nitrate-treated plants under salinity is attributed to a
restriction of NO3- transport to the shoot (Carillo et al., 2005). Another reason for low NR activity in
salt-stressed durum wheat seedlings is attributed to an inhibitory effect of salt on translation of NR
mRNA or an increase in protein degradation (Carillo et al., 2005).
Depending on the level of salinity and plant genotype, in certain plant species, an increase in
NR activity has been observed due to salinity (Joshi, 1987; Pandey and Srivastava, 1989; Katiyar
and Dubey, 1992). While conducting experiments on Cajanus cajan plants, Joshi (1987) observed
that NaCl salinity stimulated NR activity in the leaves of plants, while Na2SO4 salinity inhibited
enzyme activity. In Cajanus plants, a gradual increase in NR activity was observed with increase
in the level of NaCl salinity in the range 2.5–10.0 dS m−1 (Joshi, 1987). In a short-term experi-
ment using cucumber (Cucumis sativus), it was observed that a 60 min exposure to 200 mM NaCl
resulted in significant increase of the actual NR activity (measured in the presence of Mg2+)in the
roots, whereas the total enzyme activity (measured with EDTA) was not affected (Reda et al., 2011).
It is suggested that the reversible protein phosphorylation is involved in the induction of NR activity
during salt stress (Reda et al., 2011).
Rice plants differing in salt tolerance show varying levels of NR activity under saline conditions
(Lal and Bhardwaj, 1987; Pandey and Srivastava, 1989; Katiyar and Dubey, 1992). Salt-sensitive
genotypes of rice plants showed decreased NR activity under salinization, whereas an increased NR
level under salinity was observed in salt-tolerant genotypes (Pandey and Srivastava, 1989; Katiyar
and Dubey, 1992). Katiyar and Dubey (1992) observed a marked increase in in vivo NR activity
in roots as well as shoots of salt-tolerant rice cultivars CSR-I and CSR-3 with a salinity level up
to 14 dS m−1 NaCl compared with nonsalinized plants. While conducting experiments to screen
inbred corn varieties for salt tolerance, Faustino and co-workers (2000) observed that salt-tolerant
lines had higher root and leaf NR activity compared to sensitive lines when grown under saline
medium. These workers emphasized that higher NR activity could be taken as a marker for screen-
ing salt-tolerant corn varieties. The higher NR level in plant parts of salt-tolerant cultivars suggests
that salinity may promote synthesis or induction of the enzyme in such cultivars. Salt-tolerant crop
cultivars thus appear to have better adaptability to saline stress by exhibiting efficient NO3- reduc-
tion under salinization.
The GS/glutamine oxoglutarate aminotransferase (glutamate synthase, GOGAT) pathway, which
is the route of ammonia assimilation in plants under normal conditions, is adversely affected by
salinization (Miranda-Ham and Loyola-Vargas, 1988; Katiyar, 1990). Ammonium ions are incorpo-
rated into glutamine and glutamic acid by GS and GOGAT, respectively. Miranda-Ham and Loyola-
Vargas (1988) observed that when Canavalia ensiformis plants were subjected to NaCl salinity
stress, the activity of GS decreased markedly in roots as well as shoots. Winter triticale (Triticale
cv. Malno) seedlings, 21-day-old, when exposed to 100 mM NaCl in hydroponics containing 5 mM
NO3- for 14 days, showed 30% reduction in shoot GS activity compared to nonsalinized seedlings
(Kwinta and Cal, 2005; Katiyar, 1990), while studying the behavior of GOGAT in situ in two sets
of rice cultivars differing in salt tolerance, observed that a NaCl salinity level of 14 dS m−1 was
inhibitory to the enzyme. In tolerant genotypes of crop plants, GOGAT is more tolerant to NaCl
than in sensitive genotypes (Katiyar, 1990). Ten-day-old tomato (Solanum lycopersicum) seedlings,
when subjected to 100 mM NaCl stress for 10 days, showed substantial decline in leaf ferredoxin-
dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) activity (Debouba et al., 2007). Decrease
in activities of GS and GOGAT under salinization suggests possible impairment of N assimilation
and/or amino acid biosynthesis by this pathway due to salinity stress.
Under salinity stress, proteolytic activity increases that results in increased intracellular concen-
tration of ammonia in plant tissues (Lutts et al., 1999). The enzyme GDH plays an important role in
ammonia assimilation under stressful conditions including salinity by detoxifying the ammonia that
tends to accumulate under stresses. In many crop species examined, under salinization, the activity
of GDH increases (Boucaud and Billard, 1978; Rao et al., 1981; Katiyar, 1990). However, in certain
cases, it remains comparable to that in the controls (Miranda-Ham and Loyola-Vargas, 1988) or
decreases by NaCl salinity (Lal and Bhardwaj, 1987). In the obligate halophyte Suaeda maritima,
Boucaud and Billard (1978) observed an increase in GDH activity with 25 mM NaCl. Similarly,
in peanut leaves, a salinity-induced increase in GDH activity was observed by Rao et al. (1981).
Sharma and Garg (1985), while studying the amination and transamination events in wheat plants,
observed that plants grown at 8 and 16 dS m−1 NaCl showed increased activity of GDH in leaves as
well as roots. Seedlings of rice cultivars differing in salt tolerance, when raised under increasing
levels of NaCl salinity, showed a marked increase in GDH activity both in vivo as well as in vitro
compared with controls (Katiyar, 1990). In salt-tolerant rice (Oryza sativa) plants, aminating GDH
activity increases with increasing salt stress, whereas it decreases in the salt-sensitive ones (Kumar
et al., 2000), and in pea (Pisum sativum) plants, an ammonium-tolerant plant, the aminating GDH
activity in roots remains high under salinity (Lasa et al., 2002). Increased activity of GDH with sali-
nization suggests a possible role of this enzyme in ammonium assimilation under saline conditions
(Srivastava and Singh, 1987). It is suggested that saline conditions favor increased accumulation of
ammonium and related compounds. Thus, the GS/GOGAT pathway of ammonium assimilation is
impaired, and under such conditions, an increased level of GDH imparts adaptive value to plants by
detoxifying and assimilating more ammonium (Srivastava and Singh, 1987). Rice (Oryza sativa L.)
crop cultivars appear to have better adaptability to saline stress by exhibiting efficient NO3- reduc-
tion under salinization. A possible mechanism for increased aminating GDH activity in salt-stressed
plants involves increased production of reactive oxygen species (ROS) under salt stress that par-
ticipates in the signaling pathway for expression of GDH. In tobacco and grape (Vitis vinifera cv.
Sultanina) plants, subjected to salinity stress, it has been shown that salinity-generated ROS signal
induces α-GDH subunit expression, the assembly of the anionic isoforms of GDHs, and an increase
in GDH aminating activity. As a result, GDH detoxifies the high intracellular ammonia generated
by the proteolytic and deaminating activities under salinity stress (Skopelitis et al., 2006). These
results can lead to the conclusion that GDH acts as an antistress enzyme in ammonia detoxification
and production of glutamic acid for proline synthesis under salt stress (Skopelitis et al., 2006).
The content of NO3- as well as the activities of enzymes involved in reduction of NO3- and assimi-
lation of NH +4 in the plant tissues is also influenced with the span of salinity exposure, and the dif-
ferent organs of the plant may show different response. When rice seedlings were subjected to salt
stress for 4 h, salt stress strongly stimulated the expression of genes for GS (OsGS1;1 and OsGS1;3),
GOGAT (OsNADH-GOGAT), and GDH (OsGDH1, OsGDH2, OsGDH3) in both leaves and roots,
and the response of root to salt stress was more sensitive than that of leaf (Wang et al., 2012a).
However, under a prolonged salinity stress of 5 days, most of the genes involved in NH +4 assimila-
tion were downregulated by salt stress (Wang et al., 2012a). These workers concluded that salinity
exposure of rice seedlings for 5 days caused decrease in NO3- content of roots and leaves that in turn
caused downregulation of genes for NR and NH +4 assimilatory enzymes.
Various workers have examined N content of plants grown under salinity stress, and it has been
observed that N content of plant parts varies due to salinity depending on the plant species, the organs
studied, as well as the type and extent of salinity. Lal and Bhardwaj (1987) observed decreases in
the total N and protein N content of 15-day-old Pisum sativum seedlings salinized with a mixture
of NaCl and CaCl2 with 4 and 8 dS m−1 salinity. However, an increase in soluble forms of N ( NO3-
and NH +4 ) was observed with salinity. A similar decrease in the content of pea seedlings growing
at isosmotic levels (−0.1 to −0.5 MPa) of NaCl, CaCl2, and Na2SO4 salts was observed by Singh
et al. (1990). These investigators observed a decrease in N content with increasing salt stress. While
investigating the effects of salinization on nodulation and N fixation in pea plant, Siddiqui et al.
(1985) reported decreases in nodule N and total plant N and a significant reduction in the N2-fixing
efficiency of the nodules with increasing level of salinity. It is believed that salt dissolved in saline
medium interferes with N use by plants (Mokhele et al., 2012). Salinity can serve as a major factor
responsible for low N availability to the growing plants (Debouba et al., 2006). NR activity in leaves
is largely dependent on nitrate flux from roots and is severely affected by osmotic shock induced by
NaCl (Silveira et al., 2001).
In certain cases, increased N contents have been observed in various plant species subjected
to salinity by several investigators (Abdul-Kadir and Paulsen, 1982; Krishnamurthy et al., 1987;
Blom-Zandstra et al., 1988; Rabe, 1990; Pessarakli, 1999; Pessarakli et al., 2005, 2012). Sharma
et al. (1994) observed that N concentrations in grains and N uptake in grains and straw increased
with an ESP up to 25. Pessarakli and Tucker (1985b) found that the N contents of cotton shoots and
roots increased with NaCl salinity up to −0.8 MPa osmotic potential of the nutrient solution. At
the low level of salinity (−0.4 MPa osmotic potential), plants contained significantly higher total
N (Pessarakli and Tucker, 1985) as well as crude protein (Pessarakli and Tucker, 1985b) compared
with the controls (nonsalinized plants). Khalil et al. (1967) reported similar increases in the total
N concentration of cotton and corn under salt stress conditions. In Cajanus cajan, a protein-rich
leguminous crop, a salinity treatment of 10 dS m−1 NaCl caused about a 43% increase in total N
and protein content in the leaves of 3-month-old plants over controls. A similar salinity treatment
with Na2SO4 caused a decrease in N and protein content compared with controls (Joshi, 1987). It
is suggested that the two salts, NaCl and Na2SO4, show specific ion effects on the N metabolism of
Cajanus cajan (Joshi, 1987). Phaseolus vulgaris plants, when grown in greenhouse conditions and
irrigated with water containing 44, 88, and 132 mM NaCl, showed increases in the total N content
of leaves with increasing salinity (Khavan-Kharazian et al., 1991). Addition of 4 or 8 mM CaCl2 or
CaSO4 in the NaCl treatment medium further increased the leaf N content in such plants, indicat-
ing that Ca2+ addition helps in maintaining the selective permeability of the membranes (Khavan-
Kharazian et al., 1991). In many salt-tolerant plants examined, almost no difference is observed in
total N content of salt-stressed plants compared to control plants. Pessarakli and co-workers (2012)
observed almost similar total N content in different clones of 20 dSm−1 salinity-stressed halophytic
salt grass (Distichlis spicata L.) plants compared to nonsalinized controls. These observations as
well as of other workers suggest that salt stress can alter N level in plants, and this effect varies with
cultivar, organ, developmental stage, and the degree of salt stress.
24.3.2 Water Stress

Water stress, marked by water deficit in the soil environment, is one of the most serious worldwide
constraints for agriculture, more especially in arid and semiarid regions of the world. Plants are
often subjected to periods of soil and atmospheric water deficit during their life cycle. In semi-
arid tropics, the occurrence of drought or water deficit in the soil is common, whereas crop plants
of temperate and tropical regions undergo seasonal periods of water stress, especially during the
summer. Responses of plants to water stress depend on plant species, the intensity and the dura-
tion of stress, as well as on the period when it occurs during plant life cycle (Bradford and Hsiao,
1982; Bartels and Sunkar, 2005; Pessarakli et al., 2005; Chai et al., 2006; Omami and Hammes,
2006; Saadatmand et al., 2007; Koocheki et al., 2008; Asadi-kavan et al., 2009; Ahmadi et al.,
2010; Abdelhamid et al., 2011; Pessarakli, 2011; Pessarakli and Kopec, 2011; Pessarakli et al., 2011;
Emam et al., 2012). Low water potential in the soil as well as inside the plant inhibits plant growth,
reduces developmental activities of cells and tissues, decreases the uptake of essential nutrient ele-
ments, and adversely affects a range of key metabolic processes in plants including nitrogen assimi-
lation, which may ultimately result in poor growth of plants and reduction in yield (Sharma and
Dubey, 2011). Plants growing in water-stressed environments show reduced N uptake (Dubey, 1993;
Rabe, 1993; Bonos and Murphy, 1999; Carter et al., 1999; Pawelzik and Delgado, 1999; Sari-Gorla
et al., 1999; Pessarakli et al., 2005) and decreased activities of N assimilatory enzymes (Sinha and
Nicholas, 1981, Morgan, 1984, Kathju et al., 1990, Sharma and Dubey, 2005).
As NO3- and NH +4 both are water mobile, the presence of adequate amount of water is essential in
the soil environment for optimum absorption of N by plant roots (Christophe et al., 2011). Many fac-
tors are responsible for reduced uptake of N in a water-stressed environment. These include reduced
plant transpiration, low availability of ions in the soil, and alteration in active transport mechanism
and membrane permeability (Hsiao, 1973; Christophe et al., 2011). When the water potential inside
the plant declines below a threshold value, stomata closure takes place, which causes reductions
in transpiration and water transport through the plant. This, in turn, affects the roots directly so
that the roots are unable to accumulate or absorb NO3- as effectively as when transpiration is nor-
mal (Shaner and Boyer, 1976). At low water potential, the ability of roots to supply NO3- to the
transpiration stream decreases, leading to a decrease in NO3- concentration of the xylem sap (Shaner
and Boyer, 1976). Under nonstressed conditions, in a freely transpiring plant, a continuous move-
ment of NO3- from the roots to the leaves (NO3- flux) is maintained. This NO3- flux decreases during
water stress. While some workers have observed that N uptake decreases under water stress (Shaner
and Boyer, 1976), others have shown that N uptake is independent of plant transpiration (Gastal and
Saugier, 1989). The latter observations are supported by the fact that during water stress, abscisic
acid synthesis increases that regulates the activity of aquaporins leading to a control of root conduc-
tance and absorption of nutrients from the soil (Beaudette et al., 2007; Parent et al., 2009).
Water stress causes a decrease in leaf NO3- content as well as NO3- flux from the roots to the
leaves (Shaner and Boyer, 1976). When such water-stressed plants are rewatered, NO3- flux increases
but not the leaf NO3- content. While examining the uptake of nutrients by wheat varieties, Rao
and Ramamoorthy (1981) observed a 39% drop in N uptake of six improved varieties of wheat
when moisture stress was imposed at different stages of plant growth. According to these investiga-
tors, the uptake of N was affected by applied stress mainly through restricted movement of water
under such conditions. Lee and co-workers (2009), while examining the effects of water deficit on
N uptake, as quantified by 15N tracing, in white clover (Trifolium repens L.) plants, observed that
water stress caused a decline in N uptake with a concomitant downregulation of plant N fluxes.
Water-stressed plants when fertilized with more NO3- , the NO3- flux increased and plant perfor-
mance as well as grain yield improved (Kathju et al., 1990). Kathju et al. (1990) observed that when
wheat plants were grown under low (N0P0) and high (N80P80) fertility conditions and water stress
was imposed at various stages of the plant’s life cycle, increasing intensities of stress adversely
affected leaf metabolism and plant performance. However, the performance of plants was bet-
ter under high-fertility conditions at all stages with different intensities of water stress. Similar
observations by other investigators also indicate that NO3- application can partly alleviate water
stress-associated damage in plants (Lahiri, 1980; Rao and Balasubramanian, 1986). Lahiri (1980)
demonstrated that N application to the soil reduced the adverse effect of drought on dry matter and
grain yield of pearl millet. Sorghum (Sorghum halepense L.) plants, when fertilized with N, recover
faster after relief from water stress (Rao and Balasubramanian, 1986). Although fertilized plants
experienced water stress severely, they recovered from stress more quickly than unfertilized ones.
Such observations have far-reaching consequences in the sense that in dryland agriculture, where
water is a limiting factor, fertilizer application can be considered for drought mitigation manage-
ment (Rao and Balasubramanian, 1986).
Wheat cultivars differing in water stress tolerance show differences in kinetics of NO3- uptake.
The plants with high level of drought tolerance have been shown to possess such membrane NO3-
transport systems that efficiently operate under low water potential conditions, unlike drought-
sensitive cultivars. It is suggested that in drought-tolerant wheat plants, imposition of water stress
causes stress-dependent modification of the carriers and/or stress-dependent synthesis of new car-
riers, which have NO3- transport or regulatory functions during water stress (Meshcheryakov et al.,
2001). Water stress causes decrease in N2 fixation in legume plants. With the imposition of water
stress, nodule formation decreases accompanied with decreased size of nodules and decreased
nodule activity (Streeter, 2003). Under water stress conditions, the sensitivity or tolerance behavior
of nodulating bacterial strains toward drought contributes greatly to N2 fixation efficiency of nod-
ules (Djedidi et al., 2011).
The behaviors of NO3- assimilatory enzymes have been studied by various groups of worker,
in plants growing under water stress conditions. The enzyme NR has received the most attention.
The activity of NR is sensitive to the water potential of the plant and decreases with decreasing
water potential. Even under mild water stress, NR activity declines rapidly compared with other
N assimilatory enzymes (Singh and Sawhney, 1989). In various crop species examined, NR activ-
ity has often been shown to decline with water stress (Morilla et al., 1973; Plaut, 1974; Wasnik
et al., 1988). In field-grown wheat plants, imposition of water stress caused a gradual decline in NR
activity in leaves (Kathju et al., 1990). Kathju et al. (1990) observed that in wheat plants, increasing
the intensity of water stress progressively for 3–9 days, a decrease in NR activity was observed.
These investigators also reported that under both low and high NP fertility conditions, water stress
reduced NR activity at different growth stages of plants. However, activity was always greater in
highly fertilized plants than those with low fertilizer treatments. A slow decline in NR activity with
water stress may be attributed to a partially maintained NO3- flux inside the plant despite increased
stomatal resistance and decreased rate of transpiration under stress conditions.
In white clover (Trifolium repens L.) plants, water-deficit treatment significantly reduced the
maximum NR activity and assimilation of newly absorbed N in amino acids (Lee et al., 2009).
In maize plants, desiccation caused a steady decrease in NR activity with a concomitant decrease
in leaf water potential, leaf NO3- content, and NO3- flux (Morilla et al., 1973; Wasnik et al., 1988).
Water-stressed maize plants when rewatered recovered partially and showed increased NR activity
and increased NO3- flux (Shaner and Boyer, 1976). While examining NR activity in different organs
of two chickpea (Cicer arietinum L.) varieties in relation to soil moisture stress, Wasnik et al. (1988)
observed a significant reduction in leaf NR activity due to moisture stress.
Due to increase in NO3- fertilization of plants under water stress, NR activity increases. Spring
barley plants when grown under optimal water regime and under water stress and fertilized with
NO3- , in all the fertilized and unfertilized treatments, NR activity was significantly higher under
optimal water regime than in drought stress conditions as well as when fertilized with NO3- com-
pared to unfertilized controls (Krcek et al., 2008). Nitrogen fertilization alleviated adverse effects
of drought stress (Krcek et al., 2008). In wheat plants undergoing different intensities of water stress
treatment, increased nitrogen application improved nitrate uptake and caused increase in NR activ-
ity (Kathju et al., 1990).
Severe water stress causes decreased activities of NR, GS, and aminating GDH and inhibits
NO3- assimilation within the tissues (Xu and Zhou, 2005). Sharma and Dubey (2005) observed
that 15 d-grown rice seedlings when subjected to a moderate water stress level of −0.5 MPa for
24 h showed decreased level of NR max (the activity in the presence of EDTA representing maxi-
mum NR activity) but resulted in higher NR act (activity in the presence of Mg2+ representing the
non-phosphorylated NR state) and NR activation state. However, when a higher water stress level
of −2.0 MPa was imposed on the seedlings, a marked decline in the levels of both NR act and NR
max was observed, whereas NR activation state remained almost unaltered (Sharma and Dubey,
2005). These workers suggested that water stress decreased total amount of functional NR in rice
seedlings. Similarly, wheat plants subjected to soil moisture stress showed decline in NR activ-
ity compared to nonstressed plants (El-Komy et al., 2003). Genotypes of plants differing in toler-
ance toward water stress show varying level of NR activity under water stress. When pearl millet
(Pennisetum glaucum L.) genotypes differing in drought tolerance were subjected to water stress
at the reproductive growth stage (50 days after sowing) by withholding irrigation, it was observed
that water deprivation caused a significant decline in stomatal conductance as well as NR activity
(Burman et al., 2011). Among various genotypes examined, Pusa-266 and CZP-9802 showed lower
reduction in RWC as well as lesser decline in NR activity under water stress (Burman et al., 2011).
In sorghum (Sorghum bicolor L. Moench) plants, NR activity is highly sensitive to drought. When
six genotypes of sorghum differing in drought tolerance were subjected to mid-season drought and
analyzed for NR activity, leaf NR activity reached high levels at 35 days of sowing, after which it
declined (Sivaramakrishnan et al., 1988). The decline in NR activity was more in the stressed plants
compared to irrigated plants. Upon rewatering of water-stressed plants, NR activity increased sev-
eralfold in all the genotypes (Sivaramakrishnan et al., 1988).
Several explanations have suggested for decreased NR activity in plants subjected to water
stress (Morilla et al., 1973; Shaner and Boyer, 1976; Singh and Sawhney, 1989). The most plausible
explanation, suggested by Morilla et al. (1973), is that reduction in NR activity in Zea mays plants
subjected to water stress is due to a decline in the rate of synthesis of NR protein rather than its
increased rate of degradation or a direct effect of water potential on enzyme activity. According to
these investigators, desiccation of plants leads to a decrease in leaf water potential. This, in turn,
decreases NO3- flux and causes slow delivery of NO3- to the transpiration stream. Thus, movement
of NO3- to the induction site is prevented, resulting in decreased NR activity. These investigators
believe that decreased NR activity in water-stressed plants is primarily due to a decrease in NO3-
flux and not a decrease in water potential or the NO3- content of leaves (Shaner and Boyer, 1976).
Similarly, Singh and Sawhney (1989) suggested that the decline in NR activity during water stress
is due to a lowered capacity of tissues to synthesize NR protein because of degradation of polyribo-
somes to monoribosomes. More conclusive evidence is still required to ascertain whether decreased
NR activity in water-stressed plants is due to a decreased rate of enzyme synthesis or an increased
rate of enzyme degradation. Fresneau and co-workers (2007), while examining NR activity in
slowly dehydrating wheat (Triticum durum) leaves, concluded that a drought-induced decrease of
the leaf internal CO2 concentration was in part responsible for signal triggering the decrease in NR
activity and some nonstomatal factors such as decreases in mRNA levels or, in nitrate supply, could
also be responsible for decreased NR activity under water stress.
In water-stressed plants, activities of enzymes of ammonium assimilation remain high as evident
from little or no accumulation of NH +4 in the leaves of such plants (Hanson and Hitz, 1982). However,
the pathway of ammonium assimilation under stress conditions depends on the plant species, growth
stage, and the plant organs studied. It has been shown that water stress lowers the activity of GOGAT
in the root nodules of alfalfa (Medicago sativa L.) and cicer plants (Groat and Vance, 1981). In these
plants, GOGAT is more sensitive than GS to water stress. It has been observed that root nodules of
alfalfa (Medicago sativa L.) plants maintain sufficient enzyme activity for ammonia assimilation under
water stress. When the activities of enzymes of NH +4 assimilation GS, NADH-GOGAT, and NAD+-
GDH were examined in root nodules of alfalfa plants exposed to water stress, only NADH-GOGAT
activity was inhibited during drought (Becana et al., 1984). GS/GOGAT cycle of NH +4 assimilation
was fully operational in nodules of control and mildly water-stressed alfalfa plants, but the coupling
between GS and GOGAT was lost at higher level of water stress (Becana et al., 1984).
In Brassica at the flowering stage and in the shoots of Poterium, increased NADH-GDH activity
was observed with water stress (Srivastava and Singh, 1987). Zhang and co-workers (2009) observed
that in seedlings of wheat cultivars differing in drought tolerance, when exposed to increasing levels
of osmotic stress, the activity of GS varied depending on the sensitivity of the cultivars to water
stress. In drought‑resistant cv. Luohan-6, GS activity increased under low osmotic stress, while
it decreased under high osmotic stress, whereas in drought-sensitive cv. Zhoumai-18, GS activity
decreased with increasing osmotic stress. But the activities of NADH-GDH as well as NAD+-GDH
increased in both the wheat cultivars with increasing levels of osmotic stress. These workers con-
cluded that the drought resistance of wheat plants could be correlated with ammonium assimilation
resulting from the enhanced GS activity under low osmotic stress whereas due to increased NADH-
GDH activity under high osmotic stress (Zhang et al., 2009). The observations in alfalfa and chick-
pea nodules, Brassica, Poterium, and wheat plants thus suggest that GDH pathway of ammonium
assimilation becomes important under higher level of water stress.
24.3.3 Light
Light is crucial for regulation of N uptake, its translocation, and assimilation into organic com-
pounds. Decreased light intensity reduces NO3- uptake and decreases the rate of its reduction by
lowering the activities of NO3- assimilatory enzymes. Plants grown under low light intensity show
decreased NO3- uptake per gram of fresh weight production (Blom-Zandstra et al., 1988). Roots
show higher rates of NO3- uptake during the day than during the night (Rufty et al., 1981). Blom-
Zandstra et al. (1988) observed that lettuce genotypes (Lactuca sativa L.), differing in NO3- accu-
mulation, when grown under light with decreasing intensity, showed decreased NO3- uptake with
a concomitant decrease in growth. In such plants, NO3- uptake per plant decreased proportionally
even more than fresh weight production with decline in light intensity.
Increased rates of NO3- uptake by roots are observed due to diurnal variations associated with
changes in day–night or seasonal conditions (Clement et al., 1978). Interruption of the dark period
for 3 h using light of low intensity from an incandescent lamp resulted in a twofold increase in NO3-
uptake in soybean (Glycine max L.) plants compared with the day period (Raper et al., 1991). Raper
et al. (1991) suggested that the light-induced increase in NO3- uptake by plant roots is phytochrome
mediated. This, in turn, alters the permeability of plasma membranes and enhances starch deg-
radation by increasing the activity of starch-degrading enzymes. This leads to an increase in the
availability of soluble carbohydrates for translocation from shoots to roots. During the day period,
because of the metabolic activity of roots as well as greater demand for carbohydrates from the
shoot pool, translocation of carbohydrates from shoot to root is greater, which parallels the higher
uptake of NO3- by roots under daylight conditions (Raper et al., 1991).
In plant cells, the metabolically inactive pool of NO3- remains stored in vacuoles, in the form of
storage pool, which is not available for the induction of cytosolic NR (Granstedt and Huffakar, 1982).
However, it serves as osmoticum along with organic acids and sugars that are located in the vacuoles
(Blom-Zandstra et al., 1988). The metabolically active pool of NO3- is present in the cytosol (Beevers
and Hageman, 1980). It is believed that light affects the movement of NO3- from the storage to the
metabolic pool (Singh et al., 1990). Nitrate taken up in the dark accumulates largely in vacuoles, and
when such dark-kept plants are illuminated, the proportion of NO3- in the metabolic pool increases
(Singh and Sawhney, 1989). In the light, NO3- taken up by plants enters the metabolic pool, where it
is available for NR induction. Thus, the processes of NO3- uptake and NR induction are interrelated
and both are dependent on light. It was suggested by Aslam et al. (1976) that the transfer of NO3- from
the storage to the metabolic pool is mediated by phytochrome. Light thus regulates the availability
of NO3- in the metabolic pool.
In plants grown at low light intensities, NO3- predominantly accumulates in vacuoles, where it
serves as osmoticum (Blom-Zandstra et al., 1988). The accumulation of NO3- is inversely related to
the accumulation of organic compounds, and in this way, accumulating NO3- may compensate for
the shortage of photosynthates as a result of a decreased rate of photosynthesis under shade condi-
tions (Blom-Zandstra et al., 1988). Plants grown under shade thus show a twofold demand for N,
one for the metabolic pool, which after reduction can be used for protein synthesis, and the other
for the storage pool, which acts as an osmoticum (Blom-Zandstra et al., 1988). The distribution of
N between organic N and nitrate N changes in plants grown in the shade. Decreasing light inten-
sity decreases the organic N level in vacuoles and increases the nitrate N level. Lettuce genotypes
differing in the extent of NO3- accumulation, when grown under shade conditions, show increased
N concentration in the cell sap in both sets of cultivars accompanied by a decreased concentration
of organic N (Blom-Zandstra et al., 1988).
Light has a marked stimulatory effect on the reduction of NO3- by regulating the synthesis as well
as the functioning of NR. Leaves of shade-grown plants show a very low level of NR activity, but
when such plants are transferred to light, the NR activity increases severalfold (Singh and Sawhney,
1989). As with NR, in photosynthetic tissues, light plays a significant role in regulating the activity
of NIR (Singh and Sawhney, 1989).
Several regulatory mechanisms for light-mediated enhancement of NR activity have been postu-
lated. Based on the inhibitor studies and the labeling experiments, it has been suggested that light
promotes de novo synthesis of both NR and NiR. Illumination of leaves leads to increased protein
synthesis, indicating that light enhances the production of NR in leaves (Beevers and Hageman, 1980).
Certain investigators suggest that light-mediated enhancement of NR activity is due to enhanced
uptake of NO3- by plants in light (Beevers and Hageman, 1972). Light enhances the movement of
NO3- from the storage pool to the metabolic pool (Aslam et al., 1976), where NO3- becomes available
for the induction of NR activity. Sharma and Sopory (1987) observed that in maize seedlings, NR
activity increased by more than 300% on treatment with red light and kinetin. These investigators
suggested that the light-induced increase in NR activity is mediated via phytochrome. Phytochrome
action does not appear to be mediated by hormones; however, there appears to be an overlap in the
signal transduction chains of phytochrome and plant hormones (Sharma and Sopory, 1987). Some
early events of photosynthesis, such as the Hill reaction, cause redox changes in green tissues and
create favorable intracellular conditions for the synthesis of NR (Sawhney and Naik, 1972).
While unravelling the mechanism of light-induced acquisition and assimilation of NO3- in the tis-
sues, it is suggested that the activity of NRTs as well as of NR is upregulated by light (Lillo, 2008).
Besides, N metabolism is tightly coupled to photosynthesis because the enzymes NiR and GOGAT
are localized in chloroplasts and receive reducing power from photosynthetic electron transport
(Lillo, 2008). Light-dependent signaling cascades exist in higher plants that regulate NO3- acquisi-
tion and its assimilation at the transcriptional as well as posttranscriptional levels (Lillo, 2008). Such
light-dependent well-coordinated network of regulation involves the participation of phytochrome
and HY5 (long hypocotyls 5)/HYH (HY5 homologue)-dependent signaling pathways, the energy-
related AMPK (AMP-activated protein kinase) protein kinase homologue SNRK1 (sucrose non-
fermenting kinase 1-related kinase), and chloroplastic thioredoxins (Lillo, 2008). In Arabidopsis
and other higher plants, it has been shown that light causes posttranslational activation of NR by
dephosphorylation at a specific Ser residue and such activation is dependent on photosynthesis
(Heidari et al., 2011). Green plants, when transferred from darkness to light, showed activation of
NR by dephosphorylation within 20 min (Lillo, 2008). Bimolecular fluorescence complementation
studies reveal that regulatory subunits of the enzyme protein phosphatase 2A (PP2AB55) interact
with NR during such activation (Heidari et al., 2011).
24.3.4 Heat
Plants are constantly exposed to changes in temperature rhythm. Air and soil temperatures regulate
many growth and development processes of plants. When temperatures are high for sufficient time,
irreversible damage to plant processes takes place leading to extensive agricultural losses (Suzuki
and Mitller, 2006). Nitrogen uptake and assimilation are strongly related to temperature. Optimum
acquisition and assimilation of N occurs at the normal temperature. Even a small increase in soil
temperature affects the growth and nutrient uptake in plants. A rise in temperature beyond the
optimum growth temperature impairs the rate of N uptake and its assimilation by exerting a pro-
found influence on the activities of N assimilatory enzymes. In many plant species, the effects of
day–night temperatures on the uptake of various nutrients have been studied extensively (Polisetty
and Hageman, 1989; Oaks et al., 1990).
C4 plants grown under conditions of high temperature and high humidity show enhanced effi-
ciency in N use compared with C3 plants (Oaks et al., 1990). C4 plants such as corn (Zea mays L.) and
sorghum (Sorghum bicolor L.) and C3 plants such as barley, rice, wheat, and oats (Avena sativa L.)
were grown either for 7 days at 20°C or 28°C or for 3 weeks at 26°C. Greater accumulation of NO3-
was observed in C3 than in C4 plants under any of the three conditions tested (Oaks et al., 1990).
However, N supplied as NO3- was more efficiently assimilated into protein in C4 than in C3 plants
(Oaks et al., 1990). Lowering the temperature in both sets of plants, from 28°C to 20°C, caused
accumulation of NO3- as well as a lower protein-to-nitrate ratio (Oaks et al., 1990). The greater
efficiency of C4 cereals toward NO3- uptake and assimilation compared with C3 plants at all tested
temperature levels appears to be due to the highly organized cellular structure and spatial organiza-
tion of N assimilatory enzymes in C4 plants (Oaks et al., 1990).
In young corn seedlings, day–night temperatures of 30°C/30°C are regarded as optimum for NO3-
uptake (Polisetty and Hageman, 1989). Polisetty and Hageman (1989), while examining the effects
of three temperature treatments, 30°C/20°C, 30°C/30°C, and 35°C/35°C day–night temperatures,
on NO3- uptake in corn seedlings, observed that the amount of NO3- taken up during the night was
about fourfold and threefold greater for 30°C/30°C over 30°C/20°C and 35°C/35°C, respectively,
whereas during the light period, NO3- uptake increased by 1.5- and 1.3-fold, respectively. This sug-
gests that optimum NO3- uptake by corn seedlings occurs at 30°C/30°C and that either an increase
or decrease in the temperature leads to a decrease in NO3- uptake.
Assimilate partitioning in different parts of the plants is affected by increasing temperature. In

wheat, heat stress reduced N remobilization (Tahir and Nakata, 2005). Similarly, in rice, high tem-
peratures induced a decrease in N transport from shoots to the ears via the phloem (Ito et al., 2009).
In rice, the absolute N content per kernel was comparatively stable in the temperature range from
24°C/19°C to 33°C/28°C, whereas beyond this temperature, a decline in the N content of the kernels
was observed (Tashiro and Wardlaw, 1991). The varieties of rice differ in sensitivity to higher tem-
perature. Japonica varieties of rice were more sensitive than indica types during kernel development
(Yoshida and Hara, 1977). The highest concentration of N in terms of percentage of dry weight
was recorded in rice kernels in the temperature range from 33°C/28°C to 39°C/34°C (Tashiro and
Wardlaw, 1991). A decrease in the N content of shoots in soybean plants was reported by Hafeez
et al. (1991) due to an increase in temperature from 30°C to 48°C compared with the control plants
growing at 30°C. Similarly He et al. (2010), while examining the effect of air temperature on NO3-
uptake and total reduced N content in aeroponically grown lettuce plants, observed that when plants
were grown at two air temperature regimes of 28 °C/22 °C (day/night) and 36 °C/30 °C, NO3- and
total reduced N concentration of shoots were higher at 28 °C/22 °C than at 36 °C/30 °C, whereas
the roots at 36 °C/30 °C had significantly higher NO3- and total reduced N concentrations than at
28 °C/22 °C. This suggests that with elevation in air temperature, shoot N concentration decreases.
In both nonlegume and legume plants, the rate of N assimilation decreases at high tempera-
tures (Rachmilevitch et al., 2006). In legume plants, decline in N assimilation at high temperature
is attributed to decreased synthesis as well as activities of key N assimilatory enzymes NR, GS,
and GOGAT and lowered synthesis of ureides (Hungria and Vargas, 2000). The NR is sensitive
to higher temperatures. Temperatures above a certain optimum affect the level of NR in plants as
well as inhibit its activity. The NR extracted from different plants varies for its optimum incubation
temperature. When NR activity was measured in different crop species at the incubation tempera-
ture range of 25°C–60°C, highest enzyme activity was observed at 40°C in pigeon pea, cowpea,
sunflower, sesame, and sorghum; 45°C in maize; and 50°C in bajra. In all crop species examined,
NR activity declined at higher incubation temperatures (Chopra, 1983). Corn seedlings maintained
at 15°C–20°C showed six times more NR activity than at 25°C–30°C (Singh and Sawhney, 1989).
In barley seedlings, induction of NR did not take place when seedlings were maintained at 41°C.
Seedlings growing at 24°C, when transferred to 43°C, lost 70% of their NR activity (Singh and
Sawhney, 1989). In perennial grass Leymus chinensis, exposure to a temperature of 32°C caused
significant decrease in NR and GS activities with a parallel decline in plant biomass and photosyn-
thesis, as compared to the plants grown at 23°C (Xu and Zhou, 2006). Whether high temperature
causes inactivation, decreased synthesis, or increased degradation of N assimilatory enzyme, still
remains to be investigated.
Among the enzymes of ammonium assimilation, GS appears to be sensitive to higher tempera-
tures, whereas GDH is comparatively heat stable (50°C–70°C) in many plant species (Srivastava
and Singh, 1987). In crude leaf homogenates of soybean plants, apparent irreversible inactivation
temperatures for NR and NADPH-GDH appeared to be 36°C and 65°C, respectively, showing that
GDH is more heat tolerant than NR (Magalhaes et al., 1976). Stability of GDH at higher tem-
peratures appears to be of adaptational significance for plants growing at elevated temperatures as
such plants may possibly assimilate NH +4 by the GDH pathway instead of the normal GOGAT/GS
pathway.
24.3.5 Chilling
Plants exposed to low temperatures show reduced N uptake (Clarkson, 1988; Macduff and Jackson,
1991), decreased N partitioning in the young shoots (Walsh and Layzell, 1986), induced remobiliza-
tion of N from older leaves to younger ones (Walsh and Layzell, 1986), and alteration in the process
of N assimilation (Vogel and Dawson, 1991). A decrease in NO3- uptake is observed in plants with
decrease in temperature. This indicates that NO3- uptake is sensitive to temperature. In Lolium
multiflorum and Lolium perenne grasses, a decrease in the rate of NO3- uptake was observed with
short-term exposure of roots to low-temperature treatment by decreasing the temperature from 25°C
to 15°C (Clarkson, 1988). Macduff and Jackson (1991) observed that in barley plants, when the root
temperature was lowered by 3°C, maintaining a common day–night air temperature of 25°C/15°C,
NO3- uptake by the roots decreased with a concomitant decrease in the total N content of the plants.
The uptake of NH +4 was however greater than the uptake of NO3- (Macduff and Jackson, 1991). NH +4
is regarded as a safe source of N at low root temperatures, whereas it is regarded as harmful at
higher temperatures (Kafkafi, 1990).
Low root temperatures drastically affect the partitioning of N within the whole plant (Walsh
and Layzell, 1986). Walsh and Layzell (1986) reported that when 35-day-old soybean plants were
exposed to 15°C temperature for 4 days, N partitioning in the young shoots decreased 52%–61%
compared with that in control plants grown at 25°C. In treated plants, mature leaves maintained
an N level similar to that in controls. In another experiment, Rufty et al. (1981) observed a similar
N partitioning pattern in soybean plant when roots were treated with low temperature. Besides
reduced N uptake and disproportionate partitioning of N, low-temperature treatment of roots caused
remobilization of N from older leaves to the young shoots. Walsh and Layzell (1986) observed about
22% remobilization of N from mature leaves of soybean plants by 11 days of temperature treatment
at 15°C compared with N present in leaves at 4 days of treatment. It appears that the remobilized
N from older leaves supports growth of the new shoots under low-temperature stress conditions.
Increased remobilization of N to the new shoots and proportionally less N partitioning indicates
that cold-tolerant cultivars have increased partitioning of N in the shoots. This also suggests that
tolerance to low temperature can be increased by increasing the N supply to young shoots (Walsh
and Layzell, 1986).
Low-temperature treatment of roots decreased the rate of NO3- flux to the leaves and, in turn,
decreased NR activity (Beevers and Hageman, 1980). Barley and maize seedlings, when grown at
20°C for 7 days, showed a drastic reduction in NR activity compared with seedlings grown at 28°C
(Oaks et al., 1990). When Azolla caroliniana plants were exposed to 5°C in darkness for 7 days,
the activities of NR and NiR as well as total protein content declined in chilled plants compared
to control plants (Mostafa and Hassan, 2006). As NR is a substrate-inducible enzyme, the level of
NO3- in the active pool has a major role in regulating leaf NR activity. Decreased NO3- uptake or a
decline in NO3- level in roots due to chilling treatment would ultimately result in apparent decline in
NR activity. However, certain workers have observed an increased rate of NO3- uptake and increased
root NR activity at a low temperature (Vogel and Dawson, 1991). Vogel and Dawson (1991) reported
that when 2-week-old black alder (Alnus glutinosa) seedlings were exposed to chilling temperatures
of −1°C to 4°C for 2 h during the night, immediately after chilling in vivo, NR activities of roots and
shoots increased significantly compared with activities in prechilled plants. The apparent increase
in NR activity following chilling appears to be due to the increased activity of a constitutive NR
enzyme that is reported to be present in many N2-fixing plants.
24.3.6 Metal Toxicity

Anthropogenic release of many metals into the soil environment through mining, metal work indus-
tries, urban traffic, power stations, agricultural practices, waste disposal, and sewage sludge has
caused great concern for plant and human health. The nonessential metals such as Cd, Pb, As, Hg,
and Cr are major environmental pollutants and drastically affect plant growth, whereas the met-
als Cu, Zn, Mn, Ni, Fe, and Mo are essential micronutrients for plants but, at high concentration,
cause toxicity and growth inhibition in plants. Elevated levels of metals in the soil cause multiple
direct and indirect effects in plants. Metals cause plant growth to deteriorate, lead to reduced NO3-
uptake by plant roots, and have direct inhibitory effects on enzymes of N assimilation pathway.
In corn seedlings, a direct adverse effect of Cd on NO3- uptake was reported by Volk and Jackson
(1973). Industrial areas in many countries that are polluted with the heavy metals show reduced
N concentration in leaves of plants. Pahlsson (1989), while investigating the effects of pollutants
in two industrialized belts of Sweden, observed reduced N content in the leaves of polluted trees
compared with those growing in nonpolluted areas. It is suggested that the elevated level of heavy
metals in the soil has a direct deteriorative effect on the growth of finer roots and root hairs of the
plants contributing to reduced N uptake from the soil (Pahlsson, 1989). Nitrogen deficiency occurs
in plants growing in soil with a high level of metals, which also results in disturbed carbohydrate
metabolism.
Presence of Cd in the nutrient medium drastically reduced NO3- uptake. The total NO3- uptake
in bean plants treated with 50 mM Cd for 10 h was about 20% of the uptake in control plants, and
the uptake further declined when plants were treated with 100 mM Cd (Gouia et al., 2000). A major
contaminant of the environment, Pb, suppresses N assimilation in plants. Using pot culture experi-
ments with Chinese cabbage (Brassica pekinensis Rupr.), Xiong et al. (2006) showed that Pb in
the concentration of 4 and 8 mmol kg−1 dry soil caused adverse effects on N assimilation and plant
growth. Pb exposure to cabbage plants decreased shoot NO3- content and NR activity indicating
decreased N assimilation in the plants. These workers concluded that suppression of N assimilation
could be an important component of Pb toxicity effects in plants (Xiong et al., 2006).
In acid soils, Al3+ toxicity is a major problem to crop productivity. In such soils, the NH +4 form
of N predominates and NO3- availability is limited. Uptake NO3- and many essential elements get
reduced due to Al3+ excess in the soil. It has been suggested that plants differ in their sensitivity
to Al3+ and that Al3+-tolerant plants are characterized by efficient use of NO3- in the presence of NH +4 .
Such plants have the capacity to increase the pH of their growth medium (Foy et al., 1978). When
genotypes of sorghum plants differing in Al tolerance were grown with different NO3- /NH +4 ratios
(39:1, 9:1, and 3:1) with 0 or 300 μM Al in the medium, Al-sensitive cultivar ICA-Natiama showed
a greater reduction in NO3- and NH +4 uptake than the Al-tolerant cultivar SC-283 when the plants
were grown with Al3+. When the plants were grown without Al3+, the sensitive cultivar showed
greater NH +4 uptake than the tolerant one (Galvez and Clark, 1991). This shows that uptake of
NO3- and NH +4 is reduced because of Al3+ toxicity. It is suggested that differences in NO3- and NH +4
uptake by plants are associated with changes in pH of the medium. With NH +4 in solution, the pH
decreases, but it increases when NH +4 is depleted from the solution (Galvez and Clark, 1991). In
other crops also, it has been shown that high Al concentrations directly restrict NO3- uptake and
cause a deficit in N assimilation. Inside the plant, Al inhibits NR activity (Ruiz et al., 2007). Mishra
and Dubey (2011), while studying the effects of increasing levels of Ni and Al in sand cultures, on
the activities of N assimilatory enzymes in rice seedlings, observed that the seedlings of two inbred
cvs. Malviya-36 and Pant-12 were raised in medium containing 200 and 400 μM NiSO4 or 80 and
160 μM Al2(SO4)3; a marked inhibition in the activities of enzymes NR and GS was observed in
roots as well as shoots during a 5–20-day growth period. However, the activity of aminating GDH
(NADH-GDH) was stimulated due to Ni and Al treatments, deaminating GDH (NAD+-GDH) activ-
ity decreased under metal toxicities. These workers observed an increase in the activities of the
aminotransferases alanine aminotransferase (AlaAT) and aspartate amino transferase (AspAT) due
to Ni and Al treatments. It is suggested that both Ni and Al impair N assimilation in rice seedlings
by inhibiting the activities of NR and GS and that GDH appears to play a role in assimilation of
NH +4 in metal stress conditions. A higher activity of aminotransferases in metal-stressed seedlings
might be helpful in meeting higher demand of amino acids under stressed conditions (Mishra and
Dubey, 2011). It has been observed that Al toxicity decreases total amount of functional NR in rice
seedlings, and the osmolytes proline, glycine betaine, and sucrose have a direct protective action on
enzyme NR under stressful conditions (Sharma and Dubey, 2005).
Due to increasing contamination of water and soil, arsenic toxicity has become a global concern
for plant growth and productivity. Even very low concentration of As in the soil reduces crop growth
and shows adverse effects on N assimilation. Rice plants raised in sand cultures for 5–20 days under
25 and 50 μM As2O3 in the medium showed a marked decline in growth, with a concomitant decline
in the activities of the nitrate assimilatory enzymes NR, NiR, and GS, whereas the activities of
aminotransferases increased in As-stressed seedlings compared to controls (Jha and Dubey, 2004).
Arsenic exposure lowers the affinity of N assimilatory enzymes toward its substrates. The enzymes
NR and GS extracted from arsenic-exposed rice seedlings showed higher Km values compared to
the enzymes extracted from control-grown seedlings. It is suggested that the inhibition in the activi-
ties of N assimilatory enzymes in As-exposed seedlings, accompanied with decreased affinity of
the enzymes toward their substrates, would eventually lead to a marked suppression of N assimila-
tion and impaired growth of rice plants in an As-polluted environment (Jha and Dubey, 2004).
24.3.7 Ultraviolet B Radiation
Due to stratospheric ozone depletion caused by pollutants, elevated fluxes of ultraviolet B (UV-B)
radiations (280–315 nm) are reaching the earth surface, causing large photobiological effects in
plants. UV-B light is absorbed by important biomolecules such as proteins, hormones, pigments,
and nucleic acids ultimately leading to damaging effects on growing plants. Absorption and assimi-
lation of NO3- are adversely affected in plants under UV-B radiation (Cao et al., 2007). Exposure of
Vigna unguiculata, barley, and maize plants to UV-B radiation causes a marked decline in NR activ-
ity (Balakumar et al., 1999; Ghisi et al., 2002; Quaggiotti et al., 2004). In soybean plants, grown in
hydroponics, low levels of UV-B radiation caused inhibition in the activities of NR and ammonia
assimilatory enzymes GS and GOGAT, whereas the activity of GDH increased (Cao et al., 2007).
Increased GDH activity in low level UV-B-exposed plants helps in preventing ammonia toxicity in
the tissues, whereas in plants exposed to high levels of UV-B radiation, GDH activity decreases due
to a direct damaging effect of UV-B on enzyme structure (Cao et al., 2007).

Nitrogen is one of the most essential nutrients for plants. It is regarded as the single most important
factor limiting growth of crops. The major inorganic forms of N that predominate in croplands are
NO3- and NH +4 . The availability of N in the soil is limited due to adverse environmental conditions
such as salinity, water deficit, low light intensity, heat, chilling, excess levels of metals in the soil,
and UV-B radiation. These stresses reduce N uptake, cause adverse effects on N assimilation in the
tissues, and drastically affect crop yields. Plants possess multiple NO3- uptake and transport systems
to optimize N use with changing soil and environmental conditions. Depending on the soil NO3-
concentration, two NO3- uptake systems HATS and LATS operate in plants. Five gene families of
NRTs have been characterized that are involved in uptake, transport, and storage of NO3- within the
tissues.
From the soil, NO3- is taken up by plant roots via NRTs; thereafter, NO3- gets assimilated into
organic compounds by the action of NO3- assimilatory enzymes. The processes of NO3- uptake, its
translocation within the tissues, and its reduction are coordinately regulated. Most of the stressful
conditions cause decrease in NO3- uptake and inhibition in the activities of N assimilatory enzymes
NR and GS. The NR is inducible by NO3- and its activity is subject to regulation by a variety of envi-
ronmental conditions that are influenced under stresses. Environmental stresses adversely affect the
behavior of enzymes of NO3- and NH +4 assimilation. Genotypes of plants differing in stress tolerance
show varying activity behaviors of NR and other N assimilatory enzymes.
Though extensive studies have been performed to unveil the biochemical mechanisms underlying
the uptake of NO3- by plants, the process of its assimilation, and the regulation of enzymes of NO3-
assimilation, our knowledge is still insufficient to address the complexities associated with effects
of the varieties of abiotic stresses on N uptake and assimilation processes. The precise biochemi-
cal mechanisms on how adverse conditions of the environment reduce NO3- uptake and inhibit NR
activity need to be investigated in more detail. Little information is available regarding molecular
events of NO3- uptake, the NO3- sensor protein system, signal transduction of environmental NO3 ,
-
NO3 induction regulatory proteins, primary responsive genes that are transcribed and translated as
-
a result of NO3- induction, etc. Besides this, the nature of different families of NRTs, NO3- transloca-
tors, events involving overall induction of NR by NO3-, and regulation of NR and other N assimila-
tory enzymes under various environmental stresses like salinity, drought, heat, chilling, light, and
excessive levels of metals in the soil need to be examined in greater detail. In certain cases, such as
salinity and water stresses, suppression of the GS/GOGAT pathway and a sustained level of induc-
tion of the GDH pathway of ammonium assimilation are observed. Thus, the role of aminating
GDH as antistress enzyme needs to be examined under a wide range of stresses. A detail under-
standing of the physiological and molecular controls of N uptake and assimilation in crop plants
under different stressful conditions will help in identifying suitable genotypes with better N use
efficiency for cultivation in stress-prone areas.
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(Article in Chinese) 20:2406–2410.
25 Effects of Hyperosmotic
Salinity on Protein Patterns
and Enzyme Activities
Hans-Werner Koyro, B. Huchzermeyer, and Christian Zörb
CONTENTS
25.1 Introduction........................................................................................................................... 487
25.1.1 Hyperosmotic Salinity............................................................................................... 488
25.2 Metabolic Strategies to Reduce Salinity Effects................................................................... 489
25.2.1 Photosynthesis........................................................................................................... 489
25.2.1.1 Photosynthetic Electron Transport............................................................. 490
25.2.1.2 CO2 Assimilation........................................................................................ 492
25.2.1.3 Photorespiration.......................................................................................... 493
25.2.2 Compatible Solutes.................................................................................................... 494
25.2.2.1 Proline......................................................................................................... 494
25.2.2.2 Glycine Betaine........................................................................................... 496
25.2.2.3 C-Containing Osmolytes............................................................................ 497
25.2.2.4 Polyamines.................................................................................................. 497
25.2.3 Reactive Oxygen Species........................................................................................... 499
25.2.4 CO2 Fixation: C4 Pathway.......................................................................................... 501
References....................................................................................................................................... 503
25.1 INTRODUCTION
Salt is so abundant on our planet that it is a major constraint to global food crop production, jeop-
ardizing the capacity of agriculture to sustain the burgeoning human population increase (Flowers
2004). It is estimated that 20% of all cultivated land and nearly half of irrigated land is salt-affected,
greatly reducing yield well below the genetic potential. Thus, salinization is of major concern for
global food production (Pitman and Läuchli 2002). Therefore, different types of soil salinity and
their effects on plant growth have been analyzed in detail to develop breeding and cropping strate-
gies to lessen the current problem (Munns 2005).
The deviation of the salinity from the optimal conditions often leads to responses at cellular
level, such as changes in the cell cycle and cell division rate and changes in cell anatomy and pat-
terns of cell compartments (Koyro et al. 2008; Geissler et al. 2009). At the biochemical level, an
altered metabolism going along with altered protein patterns can be observed (Debez et al. 2012).
However, there are great differences in development and anatomy but also in mechanisms to
tolerate hyperosmotic salinity. For example, when grown in saline soils, dicotyledonous species
generally accumulate more NaCl in shoot tissues than monocotyledonous (especially grasses),
which led early researchers to characterize the former as “includers” and the latter as “excluders”
(O’Leary 2001).
487
This heterogeneity should be considered much more at breeding for salt resistance in crops.
This means that, for example, a transfer of knowledge from the dicotyledonous plant Thellungiella
salsuginea (an extremophile model for abiotic stress tolerance studies and, belonging to the
Brassicaceae, a close relative of Arabidopsis) to the major monocotyledonous crops makes no much
sense (Flowers 2004).
In this review, we will focus on the effects of hyperosmotic salinity on enzyme activities and
metabolite patterns. As plants differ in their strategies to achieve salt stress resistance, we will focus
on only a few examples.
25.1.1 Hyperosmotic Salinity
Salinity is a common feature close to the seashore and at inland places of natural salt seepage.
However, the development of terrestrial plants and the absolute necessity of land plants to acquire
the—in comparison to seawater—low enriched nutrients from the soil is counterbalanced under
most circumstances by higher osmotic soil potentials and lower soil salinity (Flowers et al. 2010).
The growth on well-watered soils may have even facilitated the evolution of species with relatively
low ion contents because of a reduced necessity for the uptake of ions (such as for osmotic reasons)
and may have led to the loss or reduction of (a) exclusion or discrimination in favor of K+ over Na+
(or NO3− over Cl−), (b) the ability to excrete or compartmentalize toxic ions such as Na+ or Cl− in
vacuoles, and (c) the regulation of the water balance in the plants.
For these reasons and because of improper water management practices, soil salinity became the
major threat to sustainable agriculture in areas depending on irrigation.
As freshwater resources will become limited in the near future (Pereira et al. 2002), it is neces-
sary to develop sustainable biological production systems, which can resist hyperosmotic salinity.
A precondition is the identification and/or development of salinity-resistant crops (Munns 2005).
Almost all our modern crops are derived from glycophytes, plants apparently lacking the genetic
basis for salt resistance. Most of our crops as well as the model plant Arabidopsis thaliana are
glycophytes. Halophytes are native to saline soils. Some of them, Cakile maritima (Debez et al.
2012) and Thellungiella halophila (Amtmann 2009), for instance, exhibit growth stimulation at
salt concentrations that are lethal to some of the glycophyte crops. Although the terms glyco-
phytes and halophytes inseminate the impression that there are general qualitative differences
in adaptation, in reality, things are more complex. There is a fluent passage between these two
groups and all plants rely on multiple adaptation mechanisms (see earlier text) to cope with high
salinity. Therefore, the adjustment to salinity is a complex phenomenon that is characterized
by ecological complexity, structural changes, and physiological adjustment of gene expression,
protein synthesis, stress signal transduction, or even regulation of catalytic activities of enzymes
(Chaves et al. 2009; Ruan and Teixeira da Silva 2011; Szabados et al. 2011; Koyro et al. 2013).
Further on, there is a variability of plant responses in dependence of a number of factors, including
climatic conditions and phenophases. However, it has to be pointed out in this context that both
glycophytes and halophytes exhibit substantial genetic variation for salt tolerance. Moreover, this
variation can become pronounced in acclimation, priming, and adaptation experiments (Munns
et al. 2006; Tanou et al. 2012).
Plant adaptations to salinity are of three distinct types: osmotic stress tolerance, Na+ or Cl− exclu-
sion, and the tolerance of tissue to accumulated Na+ or Cl− (Munns and Tester, 2008). Even if ter-
restrial plants are able to exclude ions from a strong saline solution, they must also adjust their water
potential to be at least as low as that of the soil in which they are growing. In saline soils, plants
have to develop low water potentials to maintain water uptake into root systems and facilitate water
transport to the shoot. Therefore, one line of responses resembles those observed subsequent to
exposure to water deficit. A second line of responses aims at compensating for ion toxicity. In most
of our crops, enzymes are operationally functional at a cytosolic K+/Na+ ratio of 10:1. When com-
paring enzymes isolated from glycophytes and halophytes, no significant differences were found on
Effects of Hyperosmotic Salinity on Protein Patterns and Enzyme Activities 489
both, with respect to their amino acid patterns and their sensitivity to enhanced ion concentrations
(Huchzermeyer et al. 2004). This indicates that halophytes do not possess salt tolerance metabolism
but use alternative mechanisms to minimize salt injury.
It was observed that Na+ and Cl− can disturb ion homeostasis, affect plant nutrient status (Saqib
et al. 2005; Koyro et al. 2011), and apparently inhibit photosynthesis when accumulated inside the
chloroplasts. This latter effect probably is based on a feedback inhibition: Photosynthetic electron
transport and chloroplast coupling factor activity are relatively insensitive to salt. But carbon metab-
olism or metabolite transfer may be affected and inhibition of photosynthesis may be based on lack
of NADP+ and ADP (Kilirats et al. 2009; Zörb et al. 2009).
Secondary events such as the overproduction of toxic molecules, reactive oxygen species (ROS),
are also contributing to the macroscopic phenomenon of salt injury. Additionally, analysis of sec-
ondary effects becomes complicated by the fact that some intermediates of metabolism, like ROS,
sugars, and sugar phosphates, are acting as messenger molecules (Kangasjärvi et al. 2012). Response
to salinity stress finally will lead to a new equilibrium at cellular and tissue levels. But this reaction
will take at different time scales depending on both stress intensity and plant development (Chaves
et al. 2009).
In the last 20 years, our basic understanding of the mechanisms underlying plant tolerance and
adaptation to saline environments has greatly improved owing to the active development of advanced
tools in molecular, genomics, and bioinformatics analyzes (Chitteti and Peng 2007; Ruan and Teixeira
de Silva 2011). However, the full potential of investigative power has not been fully exploited, because
the use of halophytes as model systems in plant salt tolerance research is largely neglected.
25.2 METABOLIC STRATEGIES TO REDUCE SALINITY EFFECTS

Plant responses to environmental stresses, including salt, (1) depend on species, (2) are complex
and polygenic traits, and (3) are controlled by genetic networks. There are several examples in
the literature (1) about responses that are positively correlated to plant growth (Munns and Tester
2008) or (2) indicators of disturbed plant metabolism (Fumagalli et al. 2009; Widodo et al. 2009).
However, observed changes of metabolite patterns may be based on (1) stress perception and
signaling resulting in changes in abundance of enzymes and thus changes in metabolic capacity
(Oh et al. 2010, Orsins et al. 2010; Pang et al. 2010; Zörb et al. 2009), (2) salt-mediated changes in
enzyme activities (Barkla et al. 2009; M’Rah et al. 2006), or (3) different availability of substrates
reducing turnover rates at respective enzymes (nutrients to be taken up via the roots, nitrate, and
phosphate, for instance, as well as CO2 supply depending on stomatal gas exchange) (Lawlor
2002). There is not enough coherent literature available about these physiological or biochemical
parameter and their interactions. In most cases, results do not allow analyzing salinity responses
in a time-resolved way or to directly align gene expression, enzyme patterns, and resulting metab-
olite patterns. As a rule, authors focus on one plant species or compare closely related species
differing in salt resistance. We will try to approach this puzzle by summarizing data on the most
obvious salt stress effects on biochemical level. We will start with the most important reaction
sequence, photosynthesis, and we will look for salt stress–mediated interactions with other meta-
bolic pathways.
25.2.1 Photosynthesis
There is a strict correlation between plant growth rate and photosynthetic activity. As shown in
Figure 25.1, primary reactions of photosynthesis are controlled (1) by gas exchange via stomata and
(2) availability (recycling) of the coenzymes NADP+ and ADP. NADP+ receives electrons from the
electron transport chain during photosynthetic light reaction. This photosynthetic electron transport
results in generation of molecular oxygen. Electron transport events are located inside chloroplast
thylakoid membranes. In parallel to NADPH formation, photosynthetic electron transport generates
Light energy
Light harvesting complex

O2 O2
Stomata
Photosynthetic electron transport CO2 CO2
NADP+ ADP + Pi ATP NADPH
Export Triose phosphate Ribulose-bis-phosphate
FIGURE 25.1 Gas exchange and availability of coenzymes are controlling efficiency of photosynthesis. In
photosynthesis, absorbed light energy is converted to the energy of chemical bonds. Gas exchange supplies
the substrate of photosynthesis, CO2, and removes the by-product, O2. On the other hand, the Calvin cycle and
futile cycles are recycling NADP+ and ADP, the coenzymes functioning as energy acceptors (of photosyn-
thetic electron transport).
at these membranes a proton motive force that is driving ATP synthesis catalyzed by the chloroplast
coupling factor, an F-type ATPase (McCarty 1992; Nelson and Yocum 2006).
Abiotic stress factors that inhibit the Calvin cycle aggravate the situation. For instance, salinity-
induced stomatal closure limits CO2 availability, while at the same time light-driven electron transport
proceeds at high rates (Lawlor 2002). Light absorption by leaves then exceeds the demand for photo-
synthesis, and the excess excitation energy leads to an overreduction of the electron transport chain.
The rate of NADPH consumption (by assimilation) will be lower as compared to the photosynthetic
electron transport rate. Lack of the acceptor NADP+ will favor the transfer of electrons to alterna-
tive acceptors (Jithesh et al. 2006). Thus, the formation of ROS is initiated by the transfer of excess
excitation energy (electrons) to O2 (Koyro et al. 2011). In summary, salinity will initiate a sequence of
reactions that have been described as abscisic acid–regulated stomatal closure in leaves, limited CO2
availability, overreduction of electron transport chain, and finally, generation of ROS causing damages
to lipid membranes. In respective experiments, salt-induced reduction of photosynthetic efficiency
will be observed. As shown in Figure 25.2, this is due to the fact that CO2 and O2 are competing at
the catalytic center of RuBisCo. With increasing O2/CO2 ratio, photorespiration will take place. Under
salt stress, this futile cycle is beneficial, because (1) it is reducing plastidal O2 concentration and,
(2) together with an active GOGAT system, it is improving recycling of NADP+ and ADP.
25.2.1.1 Photosynthetic Electron Transport

There are striking differences between Thellungiella and Arabidopsis in their abilities to buffer or
control salt-induced excessive electron flows through the photosystems. Exposure of Arabidopsis to
supraoptimal, sublethal salt concentrations resulted in stomatal closure and inhibition of CO2 fixa-
tion. This leads to an inhibition of electron transport through photosystem II (PSII), an increase in
cyclic electron flow involving only photosystem I (PSI), and increased nonphotochemical quenching
of chlorophyll fluorescence. In contrast, in Thellungiella, although gas exchange was marginally
inhibited by high salt and PSI was unaffected, there was a large increase in electron flow involving
PSII. This additional electron transport activity was found to be oxygen dependent and sensitive to
the alternative oxidase inhibitor n-propyl gallate. Moreover, PSII electron transport in Thellungiella
Ru-bis-P
O2 CO2
RubisCO
Photorespiration Calvin cycle
2-P-glycolate 3-P-glycerate 3-P-glycerate 3-P-glycerate
FIGURE 25.2 Competition of CO2 and O2 in photorespiration. A reduction of photosynthetic efficiency

(light use efficiency) is correlated with increasing O2/CO2 ratio. This effect is more obvious, if ribulose-1,5-
bisphosphate supply is low due to stress situations.
showed a reduced sensitivity to 2′-iodo-6-isopropyl-3-methyl-2′,4,4′-trinitrodiphenylether, which is

an inhibitor of both the cytochrome b6f complex and the chloroplast coupling factor (Huchzermeyer
1982; Huchzermeyer and Löhr 1983; Stepien and Johnson 2009).
At the same time, a substantial upregulation of a protein reacting with antibodies raised against
the plastid terminal oxidase (PTOX) was observed in Thellungiella. No such upregulation of termi-
nal oxidase was seen in Arabidopsis. This agrees with the finding that in salt-stressed Thellungiella,
PTOX acts as an alternative electron sink, accounting for up to 30% of total PSII electron flow
(Stepien and Johnson 2009). In order to distinguish the reaction from photorespiration, the name
chlororespiration was chosen. Chlororespiration has been identified as a respiratory electron trans-
port chain in interaction with photosynthetic electron transport involving nonphotochemical reduc-
tion as well as oxidation of plastoquinones (Joët et al. 2002). Different enzymatic activities, including
a plastid-encoded NADH dehydrogenase complex, have been reported to be involved in the nonpho-
tochemical reduction of plastoquinones. Plastidal terminal oxidase strongly accelerates the nonpho-
tochemical reoxidation of plastoquinols; this effect was inhibited by n-propyl gallate. During the
dark to light induction phase of photosynthesis at low irradiances, PTOX drives significant electron
flow to O2, thus avoiding overreduction of plastoquinones, when photosynthetic CO2 assimilation
is not yet fully induced. Joët et al. (2002) proposed that PTOX, by modulating the redox state of
intersystem electron carriers, may participate in the regulation of cyclic electron flow around PSI.
These results are indicating that an optional dissipation of redox energy may be one reason for
improved salt resistance of Thellungiella as compared to Arabidopsis. It has been demonstrated
that expression of PTOX in glycophytes renders such mutants salt resistant, while 97% reduction of
PTOX expression in Arabidopsis had no negative effect if plants were grown at low light intensity
(Fu et al. 2009). There is a general agreement that salt-induced ROS toxicity is caused by an inhib-
ited consumption of assimilates. This leads to an inhibited turnover of the Calvin cycle resulting in
insufficient recycling of NADP+ and ADP + Pi. The arguments mentioned earlier indicate that pho-
tosynthetic electron transport is inhibited, because of a lack of acceptor coenzymes. Therefore, it
might be speculated that any effect stimulating consumption of redox energy or enhancing turnover
rate of the Calvin cycle should reduce the risk of ROS production and, thus, improve salt resistance.
The finding that Thellungiella, other than Arabidopsis, obviously is capable of activating futile elec-
tron transport pathways opens new discussions. An important question is whether this is a feature
special for halophytes.
25.2.1.2 CO2 Assimilation
Salt-induced changes in abundance of enzymes involved in CO2 assimilation have been described
in several reviews already (Askari et al. 2006; Geissler et al. 2010; Koyro et al. 2013; Li et al. 2011
Pang et al. 2010; Sengupta and Majunder 2009; Wang et al. 2009; Wetson et al. 2012; Xu et al.
2010; Yu et al. 2011). Overall, there were more proteins changed in abundance in Arabidopsis
than in Thellungiella. Distinct patterns of protein changes in the two species were observed (Pang
et al. 2010). When comparing salt effects on Arabidopsis and Thellungiella, salt-induced changes
in abundance of enzymes involved in assimilation indicate that glycophytes and halophytes differ
in regulation of relevant pathways indeed. Abundance of RuBisCo activase and enzymes involved
in carbon assimilation were downregulated in Arabidopsis under salt stress, while in Thellungiella,
RuBisCo activase shows maximal abundance at 50 mM NaCl and still is upregulated by more than
a factor of 2 at 150 mM NaCl added to the growth medium (Kosová et al. 2010; Pang et al. 2010).
In Suaeda aegyptiaca leaves, RuBisCo small subunit decreased significantly after salt
concentration added to the nutrient solution was increased to 150 mM NaCl, a salt concentration
optimal for the growth of Suaeda (Askari et al. 2006). When monitoring plant performance in the
presence of varying external salt concentrations, CO2 assimilation rates of Suaeda salsa were found
to increase with salt concentration (Askari et al. 2006). This is a common observation in experi-
ments with halophytes (Li et al. 2011). Among the proteins upregulated with increasing assimilation
rate in such experiments are the D2 and D1 core proteins of PSII as well (Kosová et al. 2010).
However, a general correlation between the increase of gene expression of enzymes involved in
the Calvin cycle and salt resistance is not given! Many enzymes involved in the Calvin cycle were
downregulated under salt stress in halophytes as well as in glycophytes. In Suaeda, glyceraldehyde-
3-phosphate dehydrogenase, seduheptulose-1,7-bisphosphatase, and RuBisCo large subunit were
downregulated, for instance. However, CO2 assimilation rate increased during hyperosmotic
salinity. This indicates that the remaining amount of these enzymes is sufficient to catalyze the
Calvin cycle at the enhanced rate observed under stress. Similar contradicting observations were
made with respect to heat stress as well (Li et al. 2011). It seems to be essential to monitor in
parallel both gene expression and enzyme activity to better understand the correlation between
CO2 assimilation and salt resistance (Geissler et al. 2010). Moreover, as shown in Figure 25.3,
the analysis of the situation is complicated by transport activities among chloroplast and cytosol
compartments and leaf cells and sink tissues, respectively. Availability (local concentrations)
ATP/ADP
[Pi]
Ru-bis-P 3-P-glycerate Export
Phosphate Phosphate
Calvin cycle
translocator
RubisCo
Phosphate
activity
Starch Sucrose
Triose-P Triose-P
Chloroplast Cytosol
FIGURE 25.3 Metabolite exchange across the chloroplast envelope. The cytosolic triose-phosphate pool
will build up, if sugar export to sink tissues is inhibited. This will affect substrate concentrations inside the
chloroplast as well as apparent enzyme activities in both compartments.
of substrates and intermediates will feed back on enzyme activities and, at branching points of
metabolism, will modify probability of individual pathways to occur.
These observations hold true for dicots as well as for monocots. In rice, salt accumulation inside
the leaves is reducing photosynthetic efficiency (Abbasi and Komatsu 2004). The initial response to
a 50 mM NaCl added to the growth medium is an overexpression of several proteins. Among them
are fructose bisphosphate aldolase, the protein OEE2 of the water splitting system, and superoxide
dismutase (SOD). Under moderate salt stress, upregulation of these proteins will return to normal
within a period of 24 h subsequent to salt exposure (Abbasi and Komatsu 2004). Some of the
observed effects indicate a fine-tuning of salinity response. The subunit of the water splitting sys-
tem, for instance, was downregulated after grown at severe salinity in Triticum but upregulated at
moderate salinity in rice (Caruso et al. 2008). Ferredoxin-NADP+ oxidoreductase appeared in two
spots in 2D electrophoresis indicating protein modification (Caruso et al. 2008).
25.2.1.3 Photorespiration
When closed stomata inhibit gas exchange, internal O2/CO2 ratio of leaves will increase in the light
(Lawlor and Mitchell 1991). As O2 and CO2 are competing for binding at the catalytic center of
RuBisCo, photosynthetic efficiency will decrease with increasing stress and photorespiration will
take place. As a rule, efficiency of CO2 fixation is reduced by photorespiration (Kilirats et al. 2009;
Sharkey et al. 1986). But this phenomenon will not be observed under special adverse growth condi-
tions. This may be explained as follows (see Figure 25.2):
During photosynthesis, CO2 and Pi are converted to triose-P in the chloroplast. The triose-P is
converted to sucrose in the cytosol, or starch in the chloroplast, liberating Pi for use in subsequent CO2
fixation. The production of triose-P in the chloroplast requires electron transport, regeneration of ribu-
lose-bisphosphate, and activity of RuBisCo. All these processes are also needed for photorespiration.
If photorespiration is inhibited (by experimental reduction of the O2/CO2 ratio, for instance) in
the absence of added salt, the efficiency of photosynthesis is increased. But this stimulatory effect
is not observed in the presence of high light intensity. As photorespiration does not compete for the
reactions by which triose-P is converted into end products such as sucrose and starch, regeneration
of ribulose-1,5-bisphosphate must be the limiting factor (Sharkey et al. 1986).
Independent of the plant species (C3 plants), it could be demonstrated that efficiency of CO2 fixa-
tion becomes independent of the O2/CO2 ratio, if photosynthesis is limited by triose-P utilization
(inhibition of sucrose and/or starch synthesis) (Kilirats et al. 2009; Sharkey et al., 1986). Under such
experimental conditions, metabolite patterns of leaves resembled those observed under salinity: The
ADP/ATP ratio increased, and P-glycerate, ribulose-1,5-bisphosphate, hexose monophosphates, and
UDP-glucose concentrations were enhanced as compared to controls.
These considerations may explain why moderate salinity has little effect on primary events
of photosynthetic electron transport as monitored by chlorophyll fluorescence measurement
(Lu et al. 2003). Accordingly, it has been found in dicots that salt stress neither induces effects
on the maximal efficiency of PSII photochemistry nor affects the actual PSII efficiency, the effi-
ciency of excitation energy capture by open PSII reaction centers, photochemical quenching, and
nonphotochemical quenching (Lu et al. 2002). No significant changes were observed in the con-
tents of neoxanthin, lutein, ß-carotene, violaxanthin, antheraxanthin, zeaxanthin, and chlorophyll
a and b at hyperosmotic salinity in S. salsa plants. Thus, S. salsa shows beneficial growth at saline
conditions and is not subjected to photoinhibition, even when treated with 400 mM NaCl in full
sunlight (Lu et al. 2002). Provided that regulation mechanisms within the thylakoid membranes are
functional, salinity effects on chlorophyll fluorescence will relate to abundance of compounds of
the photosynthetic electron transport chain. In agreement with this prediction, it was observed that
the degree of photoinhibition observed in Brassica juncea var. Urvashi was paralleled by reduction
of abundance of PSII D1 protein (Mittal et al. 2012).
The same observation holds true for most monocots as well. But some results indicate that regu-
latory effects on assimilate consumption may feedback on thylakoid-located reactions in a more
pronounced way. For instance, NaCl treatment does not cause serious decrease in the Fv/Fm ratio
of Oryza sativa L. cultivar Pokkali (salt tolerant), whereas the Fv/Fm ratio of O. sativa L. cultivar
IR-28 (salt sensitive) showed a remarkable decline. Exogenous application of glycine betaine pre-
served this decline in net PSII efficiency of IR-28 (Demiral and Türkan 2006). While chlorophyll b
and carotenoid contents of IR-28 decreased under salt stress, this was not observed with salt-tolerant
Pokkali. This difference remained in the presence of glycine betaine (Demiral and Türkan 2006).
The assumption that feedback regulation is involved in such observations is supported by a more
recent finding of Jacoby et al. (2010). They showed that the ROS-scavenging pathways of mitochon-
dria determine salinity resistance of wheat varieties.
25.2.2 Compatible Solutes
In a saline environment, plant cells will lower their water potential by allowing salt intake or by
increasing internal concentrations of organic solutes. These responses are well documented in both
glycophytes and halophytes (Koyro and Huchzermeyer 2004). A third alternative, accumulation of
protein, is poorly documented in the literature and the resulting changes of cellular Donnan poten-
tials are difficult to analyze (Dainty 1962; Sentenac and Grignon 1981).
The water potential can be reduced also by the accumulation of osmotic active organic sub-
stances in the cytoplasm (Caruso et al. 2008). In contrast to inorganic ions, these compounds do
not interfere with physiological functions of enzymes and biomembranes but stabilize biological
integrity of cells. Therefore, these compounds have been named compatible solutes (Koyro et al.
2013). Depending on enzyme patterns and metabolic pathways preferred in individual plants, dif-
ferent compatible solutes have been found in plants (Caruso et al. 2008; Hasegawa 2000). Among
the compounds described in the literature are proline, glycine betaine, sugars, and poly-hydroxyls
(Szabados et al. 2011).
Overexpression of genes of metabolic pathways leading to production of compatible solutes has
been shown to improve salt resistance. These responses are well documented in both glycophytes and
halophytes (Koyro and Huchzermeyer 2004). Chemically compatible solutes can be summarized
as polyamines or poly-hydroxyls. In addition to their function in ionic and osmotic homeostasis,
they can replace water in its function to stabilize aggregates of soluble proteins and membrane–
protein interactions, respectively, and most of them are protecting from toxic by-products like ROS
(Szabados et al. 2011).
The amount of carbon skeletons and assimilated ammonia used for the synthesis of compatible
solutes can be quite high and exceed 10% of the shoot dry weight (Chen et al. 2007). Subsequent to a
salinization, osmotic adjustment based on accumulation of compatible solutes develops over several
days. Therefore, it may be questioned at what extent the observed effect depends on solute synthe-
sis, inhibited cell expansion, and salt-induced imbalance among metabolic pathways, respectively.
While Munns et al. (1995) proposed a two-phase model of salt injury, where growth is initially
reduced by osmotic stress and then by Na+ toxicity, it is difficult to assess with any confidence the
relative importance of the two mechanisms to yield reduction because they overlap.
Salt-sensitive glycophytes and salt-tolerant halophytes employ common mechanisms to cope
with salinity, and it is hypothesized that differences in salt resistance arise because of changes in
the regulation of a basic set of salt resistance genes (Kant et al. 2006). Therefore, synthesis and
accumulation of compatible solutes has been analyzed in detail in several plant species (Szabados
and Savouré 2009). Recent advances in genomics and proteomics allow the correlation of such data
with regulatory events.
25.2.2.1 Proline
As shown in Figure 25.4, proline synthesis occurs on the expense of ATP and NADP+. Thus, pro-
line synthesis contributes to recycling of these two “energy acceptors” (of photosynthetic elec-
tron transport) under stress. This helps reduce the risk of ROS production. Proline accumulation
NADP+ NADPH ADP ATP
Glutamate semialdehyde Glutamyl phosphate Glutamate
Pi
NADPH NADP+
H 2O
∆-Pyrroline-5-
Proline
carboxylate
FIGURE 25.4 Proline synthesis. Glutamate is a substrate of proline synthesis. Taking into account that
nitrate reduction and subsequent GOGAT reaction are supplying most of the organic nitrogen, proline can be
addressed “an energy sink.” Dicots are using 1/3 of photosynthetic electrons for nitrate reduction and subse-
quent glutamine synthesis under control condition. This proportion will be significantly increased, if amines
are used as compatible solutes under stress.
is one of the most common responses of plants to environmental constraints (Ghars et al. 2012).
Under optimal growth conditions, T. halophila as compared to A. thaliana contains higher lev-
els of the compatible osmolyte proline and at high salinity mainly in shoots (Genga et al. 2011).
It was shown that, depending on the intensity of stress, expression of the P5CS1 gene was
induced in leaves. Time course of proline content in the leaves complied with the dynamic
of P5CS1 gene expression, while changes in expression of the PDH gene were not significant
(Radyukina et al. 2011). However, the proline catabolism in T. salsuginea was maintained
essentially unchanged, which probably relates to the participation of proline or its degradation
products in synthesis pathways of other metabolites (Radyukina et al. 2011). The PDH enzyme
activity was even lower in T. halophila indicating repression of proline catabolism in this halo-
phyte (Kant et al. 2006).
Recently, regulation of proline accumulation has been investigated in some detail (see Figure 25.4
for orientation). Phosphate moieties of membrane lipids were labeled allowing to monitor the inosi-
tol 1,4,5-trisphosphate (IP3) pathway (Ghars et al. 2012), the effects of cytosolic Ca2+ concentration
were analyzed (Parre et al. 2007), and involvement of phospholipase C (PLC) and D (PLD) has been
documented by monitoring the effects of specific inhibitors (Darwish et al. 2009; Ghars et al. 2012).
Moreover, rates of proline synthesis and degradation have been estimated by measuring abundance
and activities, respectively, of relevant enzymes isolated from leaf extracts (Ghars et al. 2012; Kant
et al. 2006). Results from experiments with glycophytes (rice and Arabidopsis mutants differing
in salt sensitivity) and halophytes (T. halophila/salsuginea) have been compared (Darwish et al.
2009; Ghars et al. 2007, 2012; Kant et al. 2006). Ghars et al. (2012) concluded that PLC and PLD
signaling are regulating proline metabolism in opposite ways in Arabidopsis and Thellungiella. In
Thellungiella, PLC is inhibiting proline accumulation in the presence of salt concentrations that are
not inhibiting plant growth (control up to 200 mM NaCl), while PLC is stimulating proline concen-
tration in the presence of 400 mM NaCl. PLD effects could not be detected under control condi-
tions, but PLD activity significantly stimulated proline accumulation in the presence of 400 mM
NaCl (Ghars et al. 2012). When using specific inhibitors, it could be demonstrated that Ca2+ is
acting as the downstream messenger of receptor-mediated phospholipases (Parre et al. 2007). This
observation holds true in rice as well (Darwish et al. 2009). In addition to elucidation of a signaling
pathway from a putative membrane receptor to proline accumulation, two other important findings
have to be mentioned: (1) The expression of the A. thaliana ortholog of proline dehydrogenase was
not detected in T. halophila (Kant et al. 2006). Therefore, it is assumed that proline catabolism is
inhibited in T. halophila, and differences in gene expression contribute to different proline levels in
glycophytes and halophytes, respectively (Kant et al. 2006). (2) When comparing salt stress effects
on Arabidopsis mutants differing in salt tolerance, it turned out that proline accumulation does not
correlate with the degree of salt tolerance. Highest salt-induced cellular proline concentrations were
found in the mutant A. thaliana eskimo-1, which was the most salt-sensitive species tested (Ghars
et al. 2008). In agreement with this observation, it was found in experiments with rice that salt-
induced phospholipase signaling activity does not correlate with the observed differences in salt
tolerance between sensitive and tolerant cultivars (Darwish et al. 2009).
Compatible solutes are competing with plant growth for carbon skeletons. Therefore, synthesis
of these protecting molecules is beneficial as long as assimilation rate can be increased and thus
can supply both pathways in parallel. Relevant observations have been made with halophytes. In
S. aegyptiaca, upregulation of assimilation rate was accompanied by an increase in abundance
of D2 protein, S-adenosylmethionin (SAM) synthetase, sedoheptulose-1,7-bisphosphatase, CoA
methyltransferase, and the E1ß-subunit of the pyruvate dehydrogenase complex (Askari et al.
2006). Maximal concentrations of these proteins were found in the presence of NaCl concentra-
tions allowing optimal growth rate. The same observation was made with respect to maximal
internal glycine betaine content. Accumulation of D2 protein and glycine betaine was reported
to be involved in the assembly and stabilization of the PSII complex (de Vitry et al. 1989). But it
is a matter of ongoing discussions whether the concentrations of these compounds correlate with
salt tolerance.
25.2.2.2 Glycine Betaine
In addition to proline, glycine betaine is a second N-containing compatible solute. Glycine betaine
is synthesized in a two-step oxidation of choline (Figure 25.5). The first step is catalyzed by choline
monooxygenase (CMO). In many halophytes, especially in Amaranthaceae, salt treatment results
in an upregulation of this enzyme. The expression of two isoforms of CMO increased by a factor of
5.9 and 9.3, respectively (Askari et al. 2006). CMO expression is co-regulated with SAM synthase
(see Figure 25.5 for involvement of SAM in betaine synthesis), indicating that both enzymes will
contribute to betaine synthesis (Askari et al. 2006; Kosová et al. 2010).
Serine Phosphatidyl
choline
CO2
Ethanolamine
ATP
Phosphatidate
Choline
ADP
Pi O2 + 2H+ + 2 ferredox.red
P-ethanolamine
3 × SAM
2 H2O + 2 ferredox.ox
Betaine aldehyde
NAD+
3 × S-adenosyl
homocysteine
P-choline NADH
Glycine betaine
FIGURE 25.5 Synthesis of glycine betaine. There are two alternative betaine synthesis pathways. Both
of them depend on supply of reduced ferredoxin, that is, synthesis is controlled by photosynthetic electron
transport activity. Either the precursor choline can be produced on the expense of (1) phosphatidylcholine, a
compound of biomembranes, or (2) serine can be the substrate in a SAM-dependent way. This second pathway
is of special interest, because it can be directly linked to serine production during photorespiration.
25.2.2.3 C-Containing Osmolytes
In addition to N-containing compatible solutes, sugars, polyalcohols, and organic acids regularly are
found. T. halophila had a higher metabolite level both without and with 150 mM added salt as com-
pared to A. thaliana (Gong et al. 2005). Under salt stress, A. thaliana as compared to T. halophila
showed a higher accumulation of fumaric acid and mannitol (Genga et al. 2011). Similar observations
have been made with monocots as well: Two rice cultivars (Arborio, salt sensitive and Nipponbare,
salt tolerant) have been characterized with respect to their metabolic profiles under salt stress condi-
tions in in vitro experiments by Fumagalli et al. (2009). In comparison to control conditions, shoots
of both cultivars accumulated a higher amount of sucrose, threonine, valine, and lactate. Although
the two rice cultivars showed the same trend in metabolic changes during stress, they significantly
differed in the relative amount of some metabolites, namely, in the sucrose/glucose ratio and in the
glutamate/total amino acids and glutamine/total amino acids ratios. These results suggested that
both sugar and glutamine–glutamate metabolisms were differentially regulated in the two cultivars
in response to abiotic stresses. Therefore, such ratios may be more reliable measures to estimate
stress resistance of a species as compared to cellular concentrations of individual metabolites. But,
a more detailed analysis is complicated: A high glucose/sucrose ratio may either indicate that glu-
cose production from triose phosphates occurs faster as compared to sucrose production or that
sucrose export to sink organs is exceeding the production rate. Similar conclusions apply for a high
glutamine/total amino acid ratio: glutamine is the first product of ammonia assimilation. It appears
to us that kinetic analysis is necessary to better understand the described observations.
Different regulation of C- and N-containing compatible solutes leads to the question whether
polyamines and poly-hydroxyls are equal in their function. More recently, Widodo et al. (2009)
conducted an analysis of the metabolic responses to salinity stress in two barley cultivars differing
in their salt resistance. Metabolic profiles of Sahara (tolerant) and Clipper (sensitive) were compared
under normal or saline conditions in a time-course experiment (24 h, 3 and 5 weeks). In both culti-
vars, short-term (24 h) and long-term responses (5 weeks) could be distinguished: While only minor
salt effects on metabolite patterns were detected within 1 day, several metabolite concentrations
showed significant changes after 5 weeks of salt treatment. This general difference was observed in
both cultivars. An interesting observation was that, after long-term incubation, preferentially amino
acids accumulated in Clipper leaves. On the other hand, organic acids, polyalcohols, and sugars
were accumulating in leaves of the variety Sahara. The authors hypothesize that this difference is
indicating a metabolic strategy leading to salt resistance (Widodo et al. 2009).
25.2.2.4 Polyamines
Polyamines are less often analyzed as compared to the previously mentioned compatible solutes.
Polyamine levels in plants change with salinity, and enhanced total cellular polyamine concentra-
tions are paralleled by enhanced salt tolerance level. Upon salt stress, putrescine will decrease in
concentration while both spermidine and spermine will increase (Gil and Tuteja 2010). As can be
deduced from Figure 25.6a and b, extension of the polyamine molecules is energy consuming: it
helps in recycling the coenzymes ADP and NADP+. Moreover, the synthesis is catalyzed on the
expense of glutamate, a by-product of photorespiration. Polyamine synthesis therefore is compet-
ing with growth for assimilated carbon as well as nitrogen. If assimilates are available at ample
concentrations, polyamines will help stabilize biomembrane and protein structures. If assimilate
export and metabolism are inhibited, polyamine synthesis can function as a safety valve preventing
overreduction of chloroplasts (risk of ROS production). It can be expected that this consumption of
resources is strictly regulated and that fine-tuning will include preference for a certain chain length
(putrescine, spermidine, and spermine) depending on the ratio of assimilation activity versus con-
sumption of products.
In experiments with Arabidopsis, the stress-inducible gene for arginine decarboxylase, AtADC2,
is stimulated in a salt-responsive manner. This results in a salt-induced accumulation of free putres-
cine. When the gene was inactivated by an insertion mutation, endogenous putrescine concentration
NADP+ NADPH ADP ATP HS-CoA Acetyl-CoA
N-acetyl N-acetyl
glutamyl N-acetyl Glutamate
glutamyl
semialdehyde glutamate
phosphate
Pi
Glutamate
α-Ketoglutarate
Carbamoylphosphate
H2O Acetate Aspartate Fumarate
N-acetyl Ornithine Citrulline Arginine

ornithine
ATP AMP
(a) PPi
Arginine Ornithine
Arginine decarboxylase Ornithine decarboxylase
SAM Putrescine SAM
SAM decarboxylase Spermidine synthase SAM decarboxylase
Methyl- Spermidine Methyl-

propylamin-5΄- propylamin-5΄-
thioadenosine thioadenosine
Spermine synthase
Methyl-5΄- Methyl-5΄-
thioadenosine thioadenosine
Spermine
(b)
FIGURE 25.6 (a) Arginine synthesis. Arginine synthesis from glutamate is requiring supply of ATP,
NADPH, as well as acetyl-CoA and carbamoyl phosphate. Moreover, transamination reactions on the expense
of glutamate and aspartate, respectively, are involved in the pathway. Arginine is not suitable as a compatible
solute because of its buffer capacity at high pH values, but this amino acid is an important substrate of second-
ary metabolic pathways, including synthesis of secondary messengers like NO. (b) Synthesis of polyamines.
Polyamine synthesis may start with arginine as well as ornithine. Decarboxylation leads to the formation of
putrescine. SAM functions as a precursor of the propylamine donor during chain elongation producing sper-
midine and spermine, respectively.
was reduced by 25% and plants became more salt sensitive. Deletion mutants returned to stress
resistance level of the controls in presence of putrescine added to the growth medium (Lefevre et al.
2001). When comparing salinity effects on sensitive (IKP) and tolerant (Pokkali) cultivars of rice,
putrescine levels decreased in leaves with increasing salinity. The effect was more significant in
Pokkali (Gil and Tuteja 2010).
These results are in agreement with findings on drought resistance of wheat: In experiments with
Triticum aestivum, drought-resistant cultivar Yumai No. 18 has been compared to drought-sensitive
cultivar Yangmai No. 9. Gil and Tuteja (2010) observed that upon drought treatment, sensitive plants
show a significant increase of free putrescine level. Thus, the resistant plants gave higher ratios of
(free spermine plus spermidine)/free putrescine than the sensitive plants. The authors suggested that
free spermine, free spermidine, and PSI-bound putrescine facilitate drought resistance of wheat.
Given that synthesis of spermine is consuming more energy as compared to the one of putrescine, this
observation indicates that Triticum in the described experiments was suffering from surplus energy
(overreduction and risk of ROS production). Similar observations were made after applying moderate
hypo-saline shock on seaweed. It caused increase in free putrescine, free spermine, and free spermi-
dine mainly due to a decrease in transglutaminase activity together with an apparent increase in the
l-arginine-dependent polyamine synthesis (ornithine decarboxylase activity) and arginase decreased
and arginine decarboxylase activity was slightly increased (Garcia-Jimenez et al. 2007).
The connection between expression of polyamines and salt resistance is hardly understood.
Current findings indicate that putrescine is a negative regulator, while spermidine and spermine
are positive regulators of amino acid metabolism (Mattoo et al. 2010). Putrescine can increase light
energy utilization through stimulation of photophosphorylation. It is an efficient stimulator of ATP
synthesis (Ioannidis and Kotzabasis 2007). Spermidine and spermine are efficient stimulators of
nonphotochemical quenching (acting as energy sinks). Moreover, at high concentrations, they are
efficient uncouplers of photophosphorylation (Ioannidis and Kotzabasis 2007). This latter observa-
tion is in line with earlier findings that polyamines are capable of buffering the internal space of
thylakoids (Dilley et al. 1987; Junge and Polle 1986). The higher the polycationic character of the
amines, the higher is their effectiveness in PSII efficiency restoration, as well as stacking of low-
salt thylakoids. A 50 μM spermine increases Fv as efficiently as 100 μM spermidine or 1 mM of
putrescine or 1 mM of MgCl2. Increase of Fv derives mainly from the contribution of PSIIα centers
(Ioannidis and Kotzabasis 2007).
Though earlier findings have been collected from experiments with only a few plant species, it
appears to us that they are indicating a general concept of compatible solute function. We therefore
suggest applying the previously mentioned ratios of compatible solutes as a measure for plant stress
perception and tolerance. It would ease analyzing relevant experiments if it could be proven that this
concept applies with all plant species.
25.2.3 Reactive Oxygen Species

Under normal growth conditions, there is an equilibrium between the production and the scaveng-
ing of ROS, but abiotic stress factors may disturb this equilibrium, leading to a sudden increase
in intracellular levels of ROS. Under conditions that limit CO2 assimilation such as hyperosmotic
salinity, the potential rate of NADPH production often exceeds the actual rate of consumption of
reductive power. ROS are mainly by-products of processes, such as photosynthetic or respiratory
electron transport. Any reduction of the electron transport rate, especially under high light condi-
tions, will increase the risk of ROS production.
The redox potential of activated chlorophyll is more negative than the one of oxygen. Therefore,
electron transfer from activated chlorophyll can occur unless energy is abstracted from activated
chlorophyll faster than electron transfer to oxygen can occur. There are further options of ROS pro-
duction as several intermediates of the photosynthetic electron transport are radicals. Accordingly,
in the literature, the occurrence of various forms of ROS is described (Halliwell and Gutteridge
1986; Ashraf 2009).
Among the ROS damages measured in respective experiments are damage to proteins, lipids,
carbohydrates, chlorophyll, and DNA (Gill and Tuteja, 2010). Cytotoxicity may be attributed to
oxidative damage of membrane lipids (Fridovich 1986; Wise and Naylor 1987) as well as oxidation
of proteins and nucleic acids (Fridovich 1986). On the other hand, ROS can spontaneously convert
or can be turned over under the control of enzymes. Salt resistance has been found to be positively
correlated to the activity of such antioxidant defense systems (Ashraf 2009). Most of the studies on
this topic have been performed on ROS-scavenging enzymatic antioxidants, which represent the
initial defense mechanism, whereas fewer studies have been reported about the direct consequences
of oxidative stress on the plant metabolome (Møller et al. 2007).
Antioxidant defense in plants is brought about by both enzymatic and nonenzymatic systems.
Among the enzymes involved are SOD, catalase (CAT), ascorbate peroxidase (APX), peroxidase
(POD), glutathione reductase (GR), and cyanase (involved in degradation of cyanide ions that are
by-products of ethylene biosynthesis) (see papers of Askari et al. 2006; Sobhanian et al. 2011 for
reference). Experimental data indicate that, in principle, components of this network are present in
all plant species. Though activities of individual compounds are well balanced in vivo, overexpres-
sion of partners of this network can compensate to a certain degree for experimental inhibition of an
individual compound. Moreover, enzyme activities as well as abundance of nonenzyme compounds
may vary a lot among plant species. Similar observations hold true for salt stress responsiveness:
it can be observed that plant species differ in responsiveness of individual compounds of the ROS
detoxification network.
SOD and another six enzymes involved in ROS detoxification and a subunit of PSII have been
found to increase continuously with added salt concentration in S. aegyptiaca leaves (Askari et al.
2006). ROS effects on metabolism have been analyzed in Arabidopsis cell cultures by Baxter et al.
(2007). They used the redox-active quinone menadione (MD) to induce defined levels of oxidative
stress. Results indicated a significant inhibition of the TCA cycle and a diversion of carbon into the
oxidative pentose phosphate pathway. The decrease of ascorbate concomitant with the accumulation
of threonate (an ascorbate breakdown product) indicated the failure of cultured cells to recycle the
oxidized ascorbate.
In B. juncea, enzyme activities of the antioxidant defense system were found to alter in depen-
dence of the salt concentration in the order of APX > GR > CAT > SOD > POD (Verma and Mishra
2005). Two varieties differing in salt sensitivity of B. juncea have been tested. The improved perfor-
mance of the variety Bio902 under hyperosmotic salinity was accompanied by an increase in pro-
line and total protein accumulation. Moreover, catalytic activities of APX and CAT were increased
in Bio902. This salt-stimulated increase was not observed in the sensitive variety B. juncea var.
Urvashi, but in this variety, SOD activity was salt stimulated. This effect was not observed in Bio902
(Mittal et al. 2012). When the compatible solute putrescine was added to the growth medium of
B. juncea, an increase of the leaf concentrations of glutathione and carotenoids was found (Verma
and Mishra 2005). In parallel to this response, typical peroxidation effects like increased leaf MDA
content and membrane leakage were diminished (Verma and Mishra 2005).
Under moderate salinity, the magnitude of the electrolyte leakage and the level of lipid peroxida-
tion (assessed through hydroxy fatty acid content) were moderate in T. halophila and quite marked
in A. thaliana (M’Rah et al. 2007). The abundance of the CDSP32 thioredoxin, a critical component
of the defense system against oxidative damage and lipid peroxidation, was found to be higher in
T. halophila than in A. thaliana under control conditions as well as under salt treatment (M’Rah
et al. 2007). The protecting effect of this system is underlined by the observation that membrane
leakage does hardly respond to low increasing external salt concentrations in Thellungiella as long
as growth rate of this halophyte is not affected. But when NaCl concentration has passed a threshold
value, membrane leakage occurs and was found to increase with further increasing salinity, while
CDSP32 thioredoxin activity did not increase with salt concentration any more. Detoxification of
ROS obviously is among the most important features leading to salt resistance of halophytes.
With respect to breeding, it is important to know whether metabolic pathways leading to salt
resistance in halophytes are present in crops as well. Gene expression will not help if the respective
protein is not embedded in a functional environment (if substrates for an enzyme are not available
in ample concentration, for instance). Therefore, patterns of enzymes involved in ROS detoxifica-
tion have been extensively analyzed in rice as well. Four rice varieties differing in salt sensitivity
were compared by Dionisio-Sese and Tobita (1998). In their experiments, the less sensitive variety
Pokkali showed only minor salt-induced changes in POD and SOD activities and no salt effect on
MDA content (indicator of lipid peroxidation), and salt-induced membrane leakage for ions was only
slightly increased in the presence of the highest salt concentration tested (12 dS/m, about 120 mM).
In a similar approach, Demiral and Türkan (2004) found in Pokkali, in addition, an increase in the
activities of SOD, APX, CAT, and GR and a decrease of POD activity. More sensitive varieties, on
the other hand, showed significant increase of POD activity and a decrease of SOD activity. Both
MDA contents and ion leakage increased significantly. O. sativa, var. Hitomebore showed the high-
est salt sensitivity of all tested rice varieties (Dionisio-Sese and Tobita 1998). In experiments per-
formed by Demiral and Türkan (2004), the salt-sensitive rice cultivar IR-28 showed a salt-induced
increase of SOD, APX, and POD activities, whereas CAT and GR decreased. Moreover, addition to
the growth medium of glycine betaine reduced the salt stimulation of SOD, APX, CAT, and GR in
Pokkali, whereas it further increased salt stimulation of CAT and APX activities in IR-28. In both
cultivars, salt-induced lipid peroxidation levels were reduced by glycine betaine treatment.
25.2.4 CO2 Fixation: C4 Pathway

Subsequent to NaCl treatment, mostly intermediates of photosynthesis, glycolysis, and citrate cycle
decrease in concentration, while amino acid contents increased. This result has to be analyzed
in terms of metabolite flow through these pathways. Upregulation of energy formation, amino
acid synthesis, C4 photosynthesis, and detoxification might provide major successful strategies for
improving salt resistance (Sobhanian et al. 2010). In C4 photosynthesis, phosphoenolpyruvate car-
boxylase (PEPC), NADP-malic enzyme (NADP-ME), and pyruvate, phosphate dikinase (PPDK)
participate in the process of concentrating CO2. Nonphotosynthetic counterparts of these enzymes,
which are present in all plants, play important roles in the maintenance of pH and replenishment
of Krebs cycle intermediates, thereby contributing to the biosynthesis of amino acids and other
compounds and providing NADPH for biosynthesis and the antioxidant system (Doubnerová and
Ryslavá 2011). Enhanced activities of these enzymes under stress conditions such as salinity may
improve the resistance as different plant species, which is followed by the concentration increase of
stress markers such as raffinose, sucrose, trehalose, mannitol, sorbitol, proline and diverse amines,
glycine betaine, and polyamines (see earlier text). These metabolites were often found to accumu-
late under stress conditions, thereby affecting water balance. The induction of PEPC activity and its
expression following salt stress was documented in the facultative CAM plant Mesembryanthemum
crystallinum and in Umbilicus rupestris with CAM-like metabolism; it is involved in the switch
from C3 to CAM photosynthesis (Cushman and Bohnert 1992; Daniel et al. 1984).
NaCl, LiCl, and drought positively affect the accumulation of PEPC transcripts and PEPC pro-
tein in wheat roots (Gonzalez et al. 2003). A. thaliana has four genes encoding PEPC. The pres-
ence of salts, but not drought, upregulates the transcription of genes Atppc1 and Atppc3. PEPC in
chickpea nodules is affected by salt; the activity increases at 4 and 7 days after adding salt and then
declines. The response of PEPC activity to salt depends on the timing of the salt application and the
duration of this type of stress (Soussi et al. 1998).
The increased activity of PEPC and greater malate content in cold-hardened winter rye (Secale
cereale L.) leaves suggest that the increased malate content could be produced via this enzyme and
NAD-MDH (Crecelius et al. 2003). Malate can function as a vacuolar osmolyte and may also serve
as an additional sink for carbon assimilation and reducing equivalents (Crecelius et al. 2003).
The CO2 concentrating mechanism in C4 and CAM plants is also expected to contribute to
decreasing ROS generation. However, the available data support this hypothesis in CAM but not in
C4 plants (Abogadallah 2010). Specific roles of enzymatic and nonenzymatic antioxidants in rela-
tion to the oxidative load in the context of whole plant salt tolerance were discussed in the review of
Abogadallah (2010). There is a hint that photorespiration is strongly induced by salt stress in C3 but
not in C4 and CAM plants (Cushman and Bohnert 1997). It has been estimated that photorespiration
accounts for over 70% of the H2O2 produced under osmotic stress (Noctor et al. 2002). This means
that photorespiration is expected to be the major source of ROS under salt stress. Furthermore, the
daytime regeneration of CO2 by malate decarboxylation in CAM plants has been suggested to limit
the overreduction of the photosynthetic machinery under water stress and therefore reduce the gen-
eration of ROS (Griffiths 1989). If this is true, then C4 and CAM plants should produce less ROS
under salt stress as compared to C3 plants (Abogadallah 2010).
Aquaporin1 (AQP1) functions as both water and CO2 channel. In tobacco plants, the stress-
induced overexpression of NtAQP1 increases leaf net photosynthesis, mesophyll CO2 conductance,
and stomatal conductance. A putative involvement of aquaporin1 in tobacco’s C4-like photosynthe-
sis characteristics is discussed by Sade et al. (2010).
In maize as a representative of a C4 plant, it could be shown that sodium ions accumulate quickly
and excessively in chloroplasts in the first hours of moderate salt stress (Zörb et al. 2009). A change
in the chloroplast protein pattern was observed without a change in water potential of the leaves.
A proteomics approach revealed that 12 salt-responsive chloroplast proteins increased while eight
chloroplast proteins decreased. Some of the maize chloroplast proteins such as CF1ε and a Ca-sensing
receptor show a rather transient response for the first hour of salt exposure. The enhanced abundance
of the ferredoxin NADPH reductase, the 23 kDa polypeptide of the PSII, and the FtsH-like protein
might reflect mechanism to attenuate the detrimental effects of high sodium concentrations in chloro-
plasts on the photosynthetic machinery of a salt-sensitive crop plant such as maize (Zörb et al. 2009).

When plants are growing in a saline habitat, it is a matter of time until salt will build up in the
cytosol, interfere with cellular ion homeostasis, and disturb metabolism. Researchers have aimed
at identifying (1) fundamental reactions inhibited by salt and (2) tolerance mechanisms allowing
plants to thrive in a saline habitat. A general understanding of these points can be the basis of suc-
cessful salt tolerance breeding.
From the current literature, it becomes obvious that inhibition of photosynthesis is the most
important salt effect. A linear correlation between inhibited CO2 assimilation rate and reduced plant
growth and development has been observed. When comparing plants differing in salt tolerance,
it can be observed that tolerant species contain enhanced levels of soluble molecules, polyols or
polyamines, that have been named compatible solutes. These compounds are diminishing, at least
to some extent, adverse salt effects on enzyme activities and membrane integrity. In this review, we
have analyzed literature on these two aspects in more detail.
When comparing responses of plant varieties differing in salt tolerance, it was found that assimila-
tion rate of tolerant plants was increasing with increasing external salt concentration. This response
was reversed when salt concentration exceeded a threshold value specific for each of the experi-
mental plants. As a rule, the critical salt concentration was slightly higher as compared to the one
resulting maximal growth rate. The primary events of photosynthesis are quite salt tolerant in all
tested plant species. This points to a feedback inhibition of photosynthesis by salt-induced inhibition
of consumption (metabolism or export) of products of the Calvin cycle. This interpretation agrees
with the observation that adverse salt effects can be diminished by P and N fertilization, for instance.
Salt stress effects are paralleled by an increase in ROS production and damage by peroxidation.
As light energy capture and subsequent photosynthetic electron transport are salt tolerant, salt will
result in an overreduction of primary electron acceptors and the probability of a direct electron
transfer from activated chlorophyll a to molecular oxygen will increase. Similar effects have been
observed in experiments with chloroplasts starved in phosphate (inhibiting recycling of NADP+ by
the Calvin cycle). ROS production in both cases, under salt stress and after Pi starvation, could be
reduced by growing plants at lower light intensity.
From experiments, comparing performance under salt stress of Arabidopsis and the more salt-
tolerant relative Thellungiella indicated differences in photosynthetic electron transport. In contrast
to the salt-sensitive Arabidopsis, Thellungiella is capable of “wasting energy” by electron transport

to a PTOX. Improved performance under salinity of Thellungiella as compared to Arabidopsis is
underlining the threat of salt-induced ROS toxicity. Efficient regulation of the terminal oxidase
activity appears to be a special feature of salt-tolerant plants.
Photorespiration is another important pathway preventing ROS production. The reaction
sequence involves enzymes located in three cellular compartments, chloroplast, peroxisome, and
mitochondrium. Moreover, in addition to carbohydrates, amino acids are involved as substrates,
and recycling of ammonia via the chloroplast GOGAT system is a major energy-consuming step.
Such complex systems are salt sensitive. The reaction sequence is stabilized in function, like other
metabolic pathways and metabolite transport, by the presence of compatible solutes. Compatible
solutes are produced on expense of primary metabolites of CO2 assimilation and the respective
reaction sequences are recycling ADP and NADP+. Thus, the synthesis of compatible solutes is
reducing the risk of ROS production. Moreover, some of the compatible solutes are known to
scavenge ROS.
With respect to the positive effects of compatible solutes, a direct correlation between their
respective concentration and a plant’s tolerance level was assumed. But when comparing accessions
of experimental plants differing in salt tolerance, a correlation between growth rate and content
of compatible solutes, at least in some cases, was not found. Total amine concentrations rather
indicated that plants were experiencing stress. With respect to stress monitoring, the amine-to-
carbohydrate ratio therefore appears to be a more reliable measure.
It is generally observed that salt stress will induce ROS production. Halophytes differ from gly-
cophytes in two ways: (1) They already contain in absence of stress enhanced levels of antioxidants
and ROS detoxifying enzymes, and (2) they respond to stress by a more significant expression of
ROS scavengers as compared to glycophytes.
It has been assumed that C4 plants as compared to C3 plants might be more protected from ROS
toxicity. In support of this assumption, C4 enzymes were found to increase in abundance under
salinity stress. It is a matter of discussion that C4 plants this way can limit photorespiration activity.
Thus, they would be able to exclude one of the major ROS producing pathways of C3 plants. But we
have to point out that kinetic studies are lacking. Data from the literature exclusively show either
the equilibrium after several days of growth or a disturbed equilibrium subsequent to a stress event.
This is not sufficient to analyze metabolic pathways. Moreover, in order to better understand the
metabolic and regulatory network of plants, we need parallel analysis of gene expression, enzyme
activities, and turnover of metabolites.
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26 Reactive Oxygen Species
Generation, Hazards, and
Defense Mechanisms
in Plants under Environmental
(Abiotic and Biotic)
Stress Conditions
Pallavi Sharma, Ambuj Bhushan Jha,
Rama Shanker Dubey, and Mohammad Pessarakli
CONTENTS
26.1 Introduction........................................................................................................................... 510
26.2 Reactive Oxygen Species: Sites of Production and Their Effects......................................... 511
26.2.1 Types of ROS............................................................................................................. 511
26.2.2 Sites of Production of ROS........................................................................................ 514
26.2.2.1 Chloroplasts................................................................................................ 514
26.2.2.2 Mitochondria............................................................................................... 515
26.2.2.3 Endoplasmic Reticulum.............................................................................. 515
26.2.2.4 Peroxisomes................................................................................................ 515
26.2.2.5 Plasma Membranes..................................................................................... 516
26.2.2.6 Cell Walls.................................................................................................... 516
26.2.2.7 Apoplast...................................................................................................... 516
26.2.3 Role of ROS as Messengers....................................................................................... 516
26.2.4 ROS and Oxidative Damage to Biomolecules........................................................... 518
26.2.4.1 Lipids.......................................................................................................... 518
26.2.4.2 Proteins....................................................................................................... 520
26.2.4.3 DNA............................................................................................................ 520
26.3 Antioxidative Defense System in Plants................................................................................ 521
26.3.1 Nonenzymatic Components of Antioxidative Defense System................................. 521
26.3.1.1 Ascorbate.................................................................................................... 521
26.3.1.2 Glutathione.................................................................................................. 522
26.3.1.3 Tocopherols................................................................................................. 523
26.3.1.4 Carotenoids................................................................................................. 524
26.3.1.5 Phenolic Compounds.................................................................................. 524
26.3.2 Enzymatic Components............................................................................................. 525
26.3.2.1 Superoxide Dismutase................................................................................ 525
26.3.2.2 Catalase....................................................................................................... 526
509
26.3.2.3 Guaiacol Peroxidase................................................................................... 526

26.3.2.4 Enzymes of Ascorbate–Glutathione Cycle................................................. 527
26.4 Overproduction of ROS under Stressful Conditions............................................................. 529
26.4.1 Drought...................................................................................................................... 529
26.4.2 Salinity....................................................................................................................... 530
26.4.3 Chilling...................................................................................................................... 531
26.4.4 Metal Toxicity............................................................................................................ 532
26.4.5 UV-B Radiations........................................................................................................ 533
26.4.6 Pathogens................................................................................................................... 534
References....................................................................................................................................... 535
26.1 INTRODUCTION
Reactive oxygen species (ROS) are produced as a normal product of plant cellular metabolism.
Various environmental stresses lead to excessive production of ROS causing progressive oxidative
damage and ultimately cell death. Despite their destructive activity, they are well-described second
messengers in a variety of cellular processes, including conferment of tolerance to various environ-
mental stresses. Whether ROS would serve as signaling molecules or could cause oxidative damage
to the tissues depends on the delicate equilibrium between ROS production and their scavenging.
Efficient scavenging of ROS produced during various environmental stresses requires the action
of several nonenzymatic as well as enzymatic antioxidants present in the tissues. In this review,
we describe the generation, sites of production, and role of ROS as messenger molecules as well as
inducers of oxidative damage. Further, the antioxidative defense mechanisms operating in the cells
for scavenging of ROS overproduced under various stressful conditions of the environment have
been discussed in detail.
An unavoidable consequence of aerobic metabolism is the production of ROS. ROS include free
radicals such as superoxide anion (Oi- 2 ), hydroxyl radical ( OH), as well as nonradical molecules like
•
hydrogen peroxide (H2O2) and singlet oxygen ( O2). Stepwise reduction of molecular oxygen (O2) by
1
high-energy exposure or electron-transfer reactions leads to production of the highly reactive ROS.
In plants, ROS are always formed by the inevitable leakage of electrons on to O2 from the electron
transport activities of chloroplasts, mitochondria, and plasma membranes or as a by-product of vari-
ous metabolic pathways localized in different cellular compartments (Foyer and Harbinson, 1994;
Foyer, 1997; del Rio et al., 2006; Blokhina and Fagerstedt, 2010; Heyno et al., 2011). Environmental
stresses such as drought, salinity, chilling, metal toxicity, ultraviolet B (UV-B) radiation, as well
as pathogen attacks lead to enhanced generation of ROS in plants due to disruption of cellular
homeostasis (Shah et al., 2001; Mittler, 2002; Sharma and Dubey, 2005; Hu et al., 2008; Han et al.,
2009; Maheshwari and Dubey, 2009; Tanou et al., 2009; Mishra et al., 2011; Srivastava and Dubey,
2011). All ROS are extremely harmful to organisms at high concentrations. When the level of ROS
exceeds the defense mechanisms, a cell is said to be in a state of oxidative stress. The enhanced pro-
duction of ROS during environmental stresses can pose a threat to cells by causing peroxidation of
lipids, oxidation of proteins, damage to nucleic acids, enzyme inhibition, activation of programmed
cell death (PCD) pathway, and ultimately leading to death of the cells (Shah et al., 2001; Mittler,
2002; Verma and Dubey, 2003; Meriga et al., 2004; Sharma and Dubey, 2005, 2007; Maheshwari
and Dubey, 2009; Mishra et al., 2011; Srivastava and Dubey, 2011).
Despite their destructive activity, ROS are well-described second messengers in a variety of
cellular processes including tolerance to environmental stresses (Desikan et al., 2001; Neill et al.,
2002; Yan et al., 2007). Whether ROS will act as damaging or signaling molecule depends on the
delicate equilibrium between ROS production and scavenging. Because of the multifunctional roles
of ROS, it is necessary for the cells to control the level of ROS tightly to avoid any oxidative injury
and not to eliminate them completely. Scavenging or detoxification of excess ROS is achieved by
Reactive Oxygen Species Generation, Hazards, and Defense Mechanisms 511
an efficient antioxidative system comprising of the nonenzymatic as well as enzymatic antioxidants

(Noctor and Foyer, 1998). The enzymatic antioxidants include superoxide dismutase (SOD), cata-
lase (CAT), guaiacol peroxidase (GPX), and enzymes of the ascorbate–glutathione (AsA–GSH)
cycle such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydro-
ascorbate reductase (DHAR), and glutathione reductase (GR) (Noctor and Foyer, 1998). Ascorbate
(AsA), glutathione (GSH), carotenoids, tocopherols, and phenolics serve as potent nonenzymatic
antioxidants within the cell. Various workers have reported increased activities of many enzymes of
the antioxidant defense system in plants to combat oxidative stress induced by various environmen-
tal stresses. Maintenance of a high antioxidant capacity to scavenge the toxic ROS has been linked
to increased tolerance of plants to these environmental stresses (Zaefyzadeh et al., 2009; Chen
et al., 2010). Considerable progress has been made in improving stress-induced oxidative stress tol-
erance in crop plants by developing transgenic lines with altered levels of antioxidants (Allen et al.,
1997a; Faize et al., 2011). Simultaneous expression of multiple antioxidant enzymes has been shown
to be more effective than single or double expression for developing transgenic plants with enhanced
tolerance to multiple environmental stresses (Lee et al., 2007). The present review focuses on types
of ROS, their site of production, and their role as messenger and inducer of oxidative stress. Further,
the role of the antioxidative defense system in combating danger posed by overproduced ROS under
stresses has been discussed in detail.
This review chapter is an expansion with some modifications of a recent invited article published
by the authors in the Focus Issue on Reactive Oxygen Species in Plants in the Journal of Botany
(Sharma et al., 2012). For more information on the ROS under stressful conditions, the readers are
also referred to another recent article on this subject published by Heidari et al. (2012).
26.2 REACTIVE OXYGEN SPECIES: SITES OF PRODUCTION

AND THEIR EFFECTS
ROS are a group of free radicals, reactive molecules, and ions that are derived from O2. It has
been estimated that about 1% of O2 consumed by plants is diverted to produce ROS (Asada and
Takahashi, 1987) in various subcellular loci such as chloroplasts, mitochondria, and peroxisomes.
ROS are well recognized for playing a dual role as both deleterious and beneficial species depend-
ing on their concentration in plants. At high concentration, ROS cause damage to biomolecules,
whereas at low/moderate concentration, it acts as second messenger in intracellular signaling cas-
cades that mediate several responses in plant cells.
26.2.1 Types of ROS
The most common ROS include 1O2, Oi- 2 , H2O2, and OH. O2 itself is a totally harmless molecule as
•
in its ground state, it has two unpaired electrons with parallel spin that makes it paramagnetic and,
hence, unlikely to participate in reactions with organic molecules unless it is activated (Apel and
Hirt, 2004). Activation of O2 may occur by two different mechanisms: (1) absorption of sufficient
energy to reverse the spin on one of the unpaired electrons and (2) stepwise monovalent reduction
(Figure 26.1). In the former, 1O2 is formed, whereas in the latter, O2 is sequentially reduced to Oi-
2 ,
H2O2, and •OH (Figure 26.1).
Electrons in the biradical form of oxygen have parallel spin. Absorption of sufficient energy
reverses the spin of one of its unpaired electrons leading to formation of singlet state in which the
two electrons have opposite spin. This activation overcomes the spin restriction and 1O2 can conse-
quently participate in reactions involving the simultaneous transfer of two electrons (divalent reduc-
tion) (Apel and Hirt, 2004). In the light, highly reactive 1O2 can be produced via triplet chlorophyll
(Chl) formation in the antenna system and in the reaction center of photosystem II (PSII) (Krieger-
Liszkay, 2005). In the antenna, insufficient energy dissipation during photosynthesis can lead to
H O
H H2O
CAT
GPX
APX e– H+ Fenton
reaction
e– e– e–
OO O O O
OO 2H+ H+
O2 H H H HO
O2– H2O2
Haber–Weiss
reaction
Energy SOD
absorption
Spontaneously
OO O O
1 1
O2 (singlet O2 (singlet
oxygen) oxygen)
FIGURE 26.1 Schematic representation of generation of ROS in plants. Activation of O2 occurs by two
different mechanisms. Stepwise monovalent reduction of O2 leads to formation of Oi- 2 /H 2 O 2, H 2O2 , and OH,
•
whereas energy transfer to O2 leads to formation of 1O2. Oi-

2 /H 2 O 2 is easily dismutated to H 2O2 either nonen-
zymatically or by SOD-catalyzed reaction to H2O2. H2O2 is converted to H2O by CAT, GPX, and APX.
formation of Chl triplet state, whereas in the reaction center, it is formed via charge recombination
of the light-induced charge pair (Krieger-Liszkay, 2005). The Chl triplet state can react with 3O2 to
give up the very highly destructive ROS 1O2:
Chl æ Light
æ æÆ 3 Chl
3
Chl + 3 O2 Æ Chl + 1 O2
Further, limited CO2 availability due to closure of stomata during various environmental stresses
such as salinity and drought favors the formation of 1O2. The lifetime of 1O2 within the cell is prob-
ably 3 μs or less (Hatz et al., 2007; Hackbarth et al., 2010). A fraction of 1O2 has been shown to be
able to diffuse over considerable distances of several hundred nanometers (nm). 1O2 can last for
4 μs in water and 100 μs in a nonpolar environment (Foyer and Harbinson, 1994). 1O2 reacts with
most of the biological molecules at near diffusion-controlled rates (Foyer and Harbinson, 1994). It
directly oxidizes protein, unsaturated fatty acids, and DNA (Wagner et al., 2004). It causes nucleic
acid modification through selective reaction with deoxyguanosine (Kasai, 1997). It is thought to be
the most important species responsible for light-induced loss of PSII activity that may trigger cell
death (Krieger-Liszkay et al., 2008). 1O2 can be quenched by β-carotene and α-tocopherol or can
react with the D1 protein of PSII as target (Krieger-Liszkay, 2005).
Due to spin restriction, molecular O2 cannot accept four electrons at a time to produce H2O. It
accepts one electron at a time, and hence during reduction of O2, stable intermediates are formed
in the stepwise fashion (Halliwell and Gutteridge, 1984). Oi- 2 is the primary ROS formed in the
cell that initiates a cascade of reactions to generate secondary ROS, either directly or prevalently
through enzyme- or metal-catalyzed processes (Valko et al., 2005) depending on the cell type
or cellular compartment. Oi- 2 is a moderately reactive, short-lived ROS with a half-life of approx.
1 μs. Oi-
2 is a nucleophilic reactant with both oxidizing and reducing properties (Halliwell, 1977).
Anionic charge of Oi- 2 inhibits its electrophilic activity toward electron-rich molecules. O 2 has been
i-
shown to oxidize enzymes containing the [4Fe–4S] clusters (aconitase or dehydratase as examples)
(Imlay, 2003) and reduce cytochrome C (McCord et al., 1977). Oi- 2 can accept one electron and two
protons to form H2O2. It is easily dismutated to H2O2 either nonenzymatically or by SOD-catalyzed

reaction to hydrogen peroxide:
2O2- + 2H + Æ H 2O2 + O2
2O2- + 2H + æ SOD
æ æÆ H 2O2 + O2
H2O2 is generated in the cells under normal as well as wide range of stressful conditions such
as drought, chilling, UV irradiation, exposure to intense light, and wounding and intrusion by
pathogens. Electron transport chain (ETC) of chloroplast, mitochondria, endoplasmic reticulum,
and plasma membrane; β-oxidation of fatty acid; and photorespiration are major sources of H2O2
generation in plant cells. Photooxidation reactions, NADPH oxidase, as well as xanthine oxidase
(XOD) also contribute to H2O2 production in plants. It is also generated in tissues requiring it as a
substrate for biosynthesis such as for lignification and suberization. H2O2 is moderately reactive and
is a relatively long-lived molecule with a half-life of 1 ms (Mittler and Zilinskas, 1991). H2O2 has no
unpaired electrons; unlike other oxygen radicals, it can readily cross biological membranes and con-
sequently can cause oxidative damage far from the site of its formation. Because H2O2 is the only
ROS that can diffuse through aquaporins in the membranes and over larger distances within the
cell (Bienert et al., 2007) and is relatively stable compared to other ROS, it has received particular
attention as a signal molecule involved in the regulation of specific biological processes and trig-
gering tolerance against various environmental stresses such as plant–pathogen interactions at low
concentration (Neill et al., 2002; Torres et al., 2002; Yan et al., 2007). At high concentration, H2O2
can oxidize the cysteine (−SH) or methionine residues (−SCH3) and inactivate enzymes by oxidiz-
ing their thiol groups, such as enzymes of the Calvin cycle, Cu/Zn-SOD, and Fe-SOD (Halliwell
and Gutteridge, 1989). When hydrogen peroxide accumulates at levels of 10 μM, the enzymes in the
Calvin cycle, such as fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, and phospho-
ribulokinase, lose 50% of their activity (Kaiser, 1979; Leegood and Walker, 1982). It also oxidizes
protein kinases, phosphatases, and transcription factors containing thiolate residues. At high con-
centrations, it orchestrates PCD (Dat et al., 2000).
Both Oi- 2 and H2O2 are only moderately reactive. The cellular damage by ROS appears to be due
to their conversion into more reactive species. The formation of •OH is dependent on both H2O2 and
Oi-
2 , and, thus, its formation is subject to inhibition by both SOD and CAT.
The Haber–Weiss reaction generates •OH from H2O2 and Oi- 2 . It consists of the following two
reactions:
Fe3+ + Oi2- Æ Fe 2+ + O2
First, Fe(III) is reduced by Oi-

2 , followed by oxidation by dihydrogen peroxide (Fenton reaction):
Fe 2+ + H 2O2 Æ Fe3+ + OH - + iOH
Net reaction:
O2i - + H 2O2 Æ iOH + OH - + O2
Metal catalysis is necessary for this reaction since the rate of uncatalyzed reaction is negligible
(Rigo et al., 1977). • OH is the most reactive among all ROS. It has a single unpaired electron;
thus, it can react with oxygen in triplet ground state. • OH interacts with all biological molecules
and causes subsequent cellular damages such as lipid peroxidation, protein damage, and mem-
brane destruction (Foyer et al., 1997). Because cells have no enzymatic mechanism to eliminate
• OH, its excess production can eventually lead to cell death (Pinto et al., 2003). Under illumi-
nation, formation of • OH by the Fenton reaction at the active site of the enzyme RbcL leads to
its fragmentation in chloroplast lysates (Ishida et al., 1997; Luo et al., 2002). The oxidation of
organic substrates by • OH may proceed by two possible reactions, either by addition of • OH to
organic molecules or abstraction of a hydrogen atom from it. Because of short lifetime and the
strongly positive redox potential (close to +2 V) of free • OH, its sites of reaction are close to its
point of formation (Elstner, 1982). In this context, organic oxygen radicals such as alkoxy, per-
oxy, semiquinones, reduced hydrogen peroxide, hydrogen peroxide–electron donor complexes
(crypto-OH), as well as metallo-oxygen complexes have been proposed as the ultimately active
species besides destructive free • OH (Elstner, 1987).
26.2.2 Sites of Production of ROS

ROS are produced in both unstressed and stressed cells at several locations in chloroplasts,
mitochondria, plasma membranes, peroxisomes, apoplast, endoplasmic reticulum, and cell walls
(Figure 26.2). ROS are always formed by the inevitable leakage of electrons on to O2 from the elec-
tron transport activities of chloroplasts, mitochondria, and plasma membranes or as a by-product of
various metabolic pathways localized in different cellular compartments.
26.2.2.1 Chloroplasts
In chloroplasts, various forms of ROS are generated from several locations. ETCs in photo-
system I (PSI) and PSII are the main sources of ROS in chloroplasts. Production of ROS by
these sources is enhanced in plants by conditions limiting CO2 fixation, such as drought, salt
and temperature stresses, as well as by the combination of these conditions with high-light
stress. Under normal conditions, the electron flow from the excited PS centers to NADP that is
reduced to NADPH that, then, enters the Calvin cycle and reduces the final electron acceptor,
CO2. In case of overloading of the ETC, due to decreased NADP supply resulting from stress
conditions, there is leakage of electron from ferredoxin to O2 , reducing it to Oi-
2 (Elstner, 1991).
This process is called the Mehler reaction:
2O2 + 2Fd red Æ 2Oi2- + 2Fd ox
Chloroplast
PSI: Electron transport chain Mitochondria
Fd, 2Fe-2S, and 4Fe-4S clusters Complex I: NADH dehydrogenase segment
PSII: Electron transport chain Complex II: Reverse electron flow to complex I
QA and QB Complex III: Ubiquinone–cytochrome region
Chlorophyll pigments Enzymes
Aconitase, 1-galactono-γ lactone, dehydrogenase
(GAL)
Cell wall Plasma membrane

Cell wall–associated peroxidase ROS Electron transporting oxidoreductases
Diamine oxidases NADPH oxidase, quinone oxidase
Endoplasmic
reticulum Peroxisome
NAD(P)H-dependent Matrix: Xanthine oxidase (XOD)
electron transport Membrane: Electron transport chain
involving Cyt P450 Apoplast flavoprotein NADH and Cyt b
Cell wall–associated Metabolic processes: Glycolate
Oxalate oxidase oxidase, fatty acid oxidation, flavin
Amine oxidases oxidases, disproportionation of O2–
radicals
FIGURE 26.2 Sites of production of ROS in plants. ROS are produced at several locations in the cell like
chloroplast, mitochondria, plasma membrane, peroxisomes, apoplast, endoplasmic reticulum, and cell wall.
Leakage of electrons to O2 may also occur from 2Fe–2S and 4Fe–4S clusters in the ETC of PSI. In
PSII, acceptor side of ETC contains QA and QB. Leakage of electron from this site to O2 contributes
to the production of Oi-
2 (Cleland and Grace, 1999).
The formation of Oi-2 by O2 reduction is a rate-limiting step. Once formed, O 2 generates more
i-
aggressive ROS. It may be protonated to HO2 on the internal, lumen membrane surface or dis-
i
mutated enzymatically (by SOD) or spontaneously to H2O2 on the external stromal membrane sur-
face. At Fe–S centers where Fe2+ is available, H2O2 may be transformed through the Fenton reaction
into the much more dangerous OH•.
26.2.2.2 Mitochondria
Mitochondria can produce ROS in several sites of ETC. In mitochondria, direct reduction of oxy-
gen to Oi- 2 occurs in the flavoprotein region of NADH dehydrogenase segment (complex I) of the
respiratory chain (Arora et al., 2002). When NAD+-linked substrates for complex I are limited,
electron transport can occur from complex II to complex I (reverse electron flow). This process has
been shown to increase ROS production at complex I and is regulated by ATP hydrolysis (Turrens,
2003). The ubiquinone–cytochrome region (complex III) of the ETC also produces Oi- 2 from oxy-
gen. It is believed that fully reduced ubiquinone donates an electron to cytochrome C1 and leaves an
unstable highly reducing ubisemiquinone radical that is favorable for the electron leakage to O2 and,
hence, to Oi-2 formation (Murphy, 2009). In plants, under normal aerobic conditions, ETC and ATP
syntheses are tightly coupled; however, various stress factors lead to inhibition and modification of
its component, leading to over reduction of electron carriers and, hence, formation of ROS (Noctor
et al., 2007; Blokhina and Fagerstedt, 2010).
Several enzymes present in mitochondrial matrix can produce ROS. Some of them produce ROS
directly, for example, aconitase, whereas some others like 1-galactono-γ lactone dehydrogenase (GAL)
are able to feed electrons to ETC (Andreyev et al., 2005; Rasmusson et al., 2008). Oi-2 is the primary
ROS formed by monovalent reduction in the ETC. It is converted quickly either by the mitochondrial
form of SOD (MnSOD) or APX into the relatively stable and membrane-permeable H2O2. H2O2 can
be further converted to extremely active hydroxyl radical (OH•) in the Fenton reaction.
26.2.2.3 Endoplasmic Reticulum
In endoplasmic reticulum, NAD(P)H-dependent electron transport involving Cyt P450 produces Oi- 2
(Mittler, 2002). Organic substrate, RH, reacts first with Cyt P450 and then is reduced by a flavopro-
tein to form a radical intermediate (Cyt P450 R−). Triplet oxygen can readily react with this radical
intermediate as each has one unpaired electron. This oxygenated complex (Cyt P450-ROO −) may be
reduced by cytochrome b, or occasionally, the complexes may decompose releasing Oi- 2 .
26.2.2.4 Peroxisomes
Peroxisomes are probably the major sites of intracellular H2O2 production, as a result of their essen-
tially oxidative type of metabolism (del Rio et al., 2006). The main metabolic processes responsible
for the generation of H2O2 in different types of peroxisomes are the glycolate oxidase reaction, the
fatty acid β-oxidation, the enzymatic reaction of flavin oxidases, and the disproportionation of Oi- 2
radicals (Baker and Graham, 2002). During photorespiration, the oxidation of glycolate by glycolate
oxidase in peroxisomes accounts for the majority of H2O2 production (Noctor et al., 2002). Like
mitochondria and chloroplasts, peroxisomes also produce Oi- 2 as a consequence of their normal
metabolism. In peroxisomes from pea leaves and watermelon cotyledons, at least, two sites of Oi- 2
generation have been identified using biochemical and electron spin resonance (ESR) spectroscopy
methods: one in the organelle matrix, the generating system being XOD, which catalyzes the oxida-
tion of xanthine or hypoxanthine to uric acid and produces Oi- 2 in the process, and another site in
the peroxisomal membranes where a small ETC composed of a flavoprotein NADH and Cyt b is
involved. Three integral peroxisomal membrane polypeptides (PMPs) with molecular masses of 18,
29, and 32 kDa were found to be involved in Oi- 2 production. While the 18 and 32 kDa PMPs use
NADH as electron donor for Oi- 2 production, the 29 kDa PMP was clearly dependent on NADPH
and was able to reduce cytochrome c with NADPH as electron donor (López-Huertas et al., 1999).
Among the three integral polypeptides, the main producer of Oi- 2 was the 18 kDa PMP that was
proposed to be a cytochrome possibly belonging to the b-type group. The PMP32 very probably cor-
responds to the MDHAR, and the third Oi- 2 -generating polypeptide, PMP29, could be related to the
peroxisomal NADPH: cytochrome P450 reductase (López-Huertas et al., 1999). The Oi- 2 produced
is subsequently converted into H2O2 by SOD.
26.2.2.5 Plasma Membranes
Electron-transporting oxidoreductases are ubiquitous at plasma membranes and lead to generation
of ROS at plasma membrane. Production of ROS was studied using EPR spin-trapping techniques
and specific dyes in isolated plasma membranes from the growing and the nongrowing zones of
hypocotyls and roots of etiolated soybean seedlings as well as coleoptiles and roots of etiolated
maize seedlings (Heyno et al., 2011). NAD(P)H mediated the production of Oi- 2 in all plasma mem-
brane samples. It was suggested that in soybean plasma membranes, Oi- 2 production could be attrib-
uted to the action of at least two enzymes, an NADPH oxidase and, in the presence of menadione, a
quinone reductase (Heyno et al., 2011). NADPH oxidase catalyzes transfer of electrons from cyto-
plasmic NADPH to O2 to form Oi- 2 . O 2 is dismutated to H 2O2 either spontaneously or by SOD activ-
i-
ity. NADPH oxidase has been proposed to play a key role in the production and accumulation of
ROS in plants under stress conditions (Torres et al., 2002; Kwak et al., 2003; Apel and Hirt, 2004).
26.2.2.6 Cell Walls
Cell walls are also regarded as active sites for ROS production. The role of cell wall–associated
peroxidase in H2O2 generation has been shown. In horseradish, peroxidase associated with isolated
cell walls catalyzes the formation of H2O2 in the presence of NADH. The reaction is stimulated by
various monophenols, especially of coniferyl alcohol. Malate dehydrogenase was found to be the
sole candidate for providing NADH (Gross, 1977). The generation of ROS by cell wall–located per-
oxidases has been shown during hypersensitive response (HR) triggered in cotton by the bacterium
Xanthomonas campestris pv. Malvacearum (Martinez et al., 1998) and potassium (K) deficiency
stress in Arabidopsis (Kim et al., 2010). Diamine oxidases are also involved in the production of
activated oxygen in the cell wall using diamine or polyamines (putrescine, spermidine, cadaverine,
etc.) to reduce a quinone that auto-oxidizes to form peroxides (Elstner, 1991).
26.2.2.7 Apoplast
Cell wall–located enzymes have been proved to be responsible for apoplastic ROS production (Apel
and Hirt, 2004; Heyno et al., 2011). The cell wall–associated oxalate oxidase, also known as ger-
min, releases H2O2 and CO2 from oxalic acid (Wojtaszek, 1997). This enzyme was reported to be
involved in apoplastic hydrogen peroxide accumulation during interactions between different cereal
species and fungi (Lane, 2002). Amine oxidase–like enzymes may contribute to defense responses
occurring in the apoplast following biotic stress, mainly through H2O2 production (Cona et al.,
2006). Amine oxidases catalyze the oxidative deamination of polyamines (i.e., putrescine, sperm-
ine, and spermidine) using FAD as a cofactor (Cona et al., 2006). Heyno and coworkers (2011),
based on their study, concluded that apoplastic OH• generation depends fully, or for the most part,
on peroxidase localized in the cell wall.
26.2.3 Role of ROS as Messengers

At low/moderate concentration, ROS have been implicated as second messengers in intracellu-
lar signaling cascades that mediate several plant responses in plant cells, including stomatal clo-
sure (Neill et al., 2002; Kwak et al., 2003; Yan et al., 2007), PCD (Bethke and Jones, 2001; Mittler
2002), gravitropism (Joo et al., 2001), and acquisition of tolerance to both biotic and abiotic stresses
Mediated
Hormones Plant responses
Abscisic acid Stomata closure
Auxin Root gravitropism
Gibberellic acid Seed germination
Messenger
ROS
at low concentration Jasmonic acid Lignin biosynthesis
Gibberellic acid Programmed cell death
Salicylic acid Hypersensitive responses
Salicylic acid Osmotic stress
FIGURE 26.3 ROS as second messengers in several plant hormone responses, including stomatal closure,
root gravitropism, seed germination, lignin biosynthesis, PCD, HRs, and osmotic stress.
(Torres et al., 2002; Miller et al., 2008). Figure 26.3 shows the role of ROS as second messenger in
hormone-mediated cellular responses in plants. Plants can sense, transduce, and translate ROS signal
into appropriate cellular responses with the help of some redox-sensitive proteins, calcium mobiliza-
tion, protein phosphorylation, and gene expression. ROS can be sensed directly also by key signaling
proteins such as a tyrosine phosphatase through oxidation of conserved cysteine residues (reviewed in
Xiong et al., 2002). ROS can also modulate the activities of many components in signaling, such as
protein phosphatases, protein kinases, and transcription factors (Cheng and Song, 2006), and commu-
nicate with other signal molecules and the pathway-forming part of the signaling network that controls
response downstream of ROS (Neill et al., 2002). The strength, lifetime, and size of the ROS-signaling
pool depend on balance between oxidant production and removal by the antioxidant. Using mutants
deficient in key ROS-scavenging enzymes, Miller and coworkers (2008) identified a signaling pathway
that is activated in cells in response to ROS accumulation. Interestingly, many of the key players in
this pathway, including different zinc finger proteins and WRKY transcription factors, are also central
regulators of abiotic stress responses involved in temperature, salinity, and osmotic stresses.
ROS are considered second messengers in the abscisic acid (ABA) transduction pathway in guard
cells (Neill et al., 2002; Yan et al., 2007). ABA-induced H2O2 is an essential signal in mediating
stomatal closure to reduce water loss through the activation of calcium-permeable channels in the
plasma membrane (Pei et al., 2000). Jannata and coworkers (2011) observed that ABA-inducible
cytosolic H2O2 elevation functions in ABA-induced stomatal closure, while constitutive increase of
H2O2 does not cause stomatal closure. Role of ROS as second messenger in root gravitropism has
been demonstrated. Based on their work, Joo and coworkers (2001) proposed that gravity induces
asymmetric movement of auxin within 60 min, and, then, the auxin stimulates ROS generation to
mediate gravitropism. Further, scavenging of ROS by antioxidants (N-acetylcysteine, ascorbic acid,
and Trolox) inhibited root gravitropism (Joo et al., 2001). ROS appear to be involved in dormancy
alleviation. In dormant barley grains under control condition, gibberellic acid (GA) signaling and
ROS content are low, while ABA signaling is high, resulting in dormancy. Exogenous H2O2 does
not appear to alter ABA biosynthesis and signaling, but has a more pronounced effect on GA sig-
naling, inducing a change in hormonal balance that results in germination (Bahin et al., 2011). ROS
have been shown to play a key role in PCD in barley aleurone cells, initiated by GA. Bethke and
Jones (2001) observed that GA-treated aleurone protoplasts are less tolerant to internally generate
or exogenously applied H2O than ABA-treated protoplasts and suggested that ROS are components
of the hormonally regulated cell death pathway in barley aleurone cells.
Plants have evolved a complex regulatory network to mediate biotic and abiotic stress responses
based on ROS synthesis, scavenging, and signaling. Transient production of ROS is detected in the
early events of plant–pathogen interactions and plays an important signaling role in pathogenesis
signal transduction regulators. This production called oxidative burst could be considered as a spe-
cific signal during the interaction process (Nanda et al., 2010). In HR, SA is thought to potentiate
ROS signaling (Klessig et al., 2000). ROS are shown to act as a second messenger for the induction
of defense genes in tomato plants in response to wounding (Orozco-Cárdenas et al., 2001). ROS
were generated near cell walls of vascular bundle cells of tomato leaves in response to wounding
and resulted H2O2 from wound-inducible polygalacturonase acted as a second messenger for the
activation of defense genes in mesophyll cells, but not for signaling pathway genes in vascular
bundle cells (Orozco-Cardenas et al., 2001).
Lignin is important for the plant’s response to environmental stress. Denness and coworkers
(2011) characterized a genetic network enabling plants to regulate lignin biosynthesis in response
to cell wall damage through dynamic interactions between jasmonic acid and ROS. ROS have been
shown to play important roles in osmotic stress, low temperature, and heavy metal signal transduc-
tion pathway (Yuasa et al., 2001; Xiong et al., 2002; Yeh et al., 2007). Genes involved in osmotic
stress signaling have been shown to be upregulated by ROS, including the transcription factor
DREB2A and a histidine kinase (Desikan et al., 2001). In Arabidopsis culture cells, it was reported
that the MAPK AtMPK6 that can be activated by low temperature and osmotic stress could also
be activated by oxidative stress (Yuasa et al., 2001). Borsani and coworkers (2001) suggested that
the increased osmotic stress tolerance of transgenic Arabidopsis expressing a salicylate hydroxy-
lase (NahG) gene might result from decreased SA-mediated ROS generation. Zhao and coworkers
(2001) reported that ROS play important roles in drought-induced ABA synthesis in plant and sug-
gested that they may be the signals through which the plant can sense the drought condition. Using
pharmacological inhibitors, it is demonstrated that metals Cd2+ and Cu2+ induce MAP kinase activa-
tion via distinct ROS-generating systems (Yeh et al., 2007).
26.2.4 ROS and Oxidative Damage to Biomolecules

Production and removal of ROS must be strictly controlled in order to avoid oxidative stress. When
the level of ROS exceeds the defense mechanisms, a cell is said to be in a state of oxidative stress.
However, the equilibrium between production and scavenging of ROS is perturbed under a num-
ber of stressful conditions such as salinity, drought, high light, toxicity due to metals, and patho-
gens. Enhanced level of ROS can cause damage to biomolecules such as lipids, proteins, and DNA
(Figure 26.4). These reactions can alter intrinsic membrane properties like fluidity, ion transport,
loss of enzyme activity, protein cross-linking, inhibition of protein synthesis, and DNA damage,
ultimately resulting in cell death.
26.2.4.1 Lipids
When ROS level reaches above threshold, enhanced lipid peroxidation takes place in both cellular
and organellar membranes, which, in turn, affect normal cellular functioning. Lipid peroxidation
aggravates the oxidative stress through production of lipid-derived radicals that themselves can
react with and damage proteins and DNA. The level of lipid peroxidation has been widely used as
an indicator of ROS-mediated damage to cell membranes under stressful conditions. Increased per-
oxidation (degradation) of lipids has been reported in plants growing under environmental stresses
(Sharma and Dubey, 2005; Han et al., 2009; Tanou et al., 2009; Mishra et al., 2011). Increase in lipid
peroxidation under these stresses parallels with increased production of ROS. Malondialdehyde
(MDA) is one of the final products of peroxidation of unsaturated fatty acids in phospholipids and is
responsible for cell membrane damage (Halliwell and Gutteridge, 1989). Two common sites of ROS
ROS
at high concentrations
Oxidative damage
Lipid Protein DNA

Site-specific amino acid Deoxyribose oxidation
Chain breakage modification
Fragmentation of the Strand breakage
Increase in membrane
fluidity and permeability peptide chain Removal of nucleotides
Aggregation of cross- Modification of bases
linked reaction products
DNA–protein cross-links
Altered electric charge
Enzyme inactivation
Increased susceptibility
of proteins to proteolysis
FIGURE 26.4 ROS-induced oxidative damage to lipids, proteins, and DNA.
attack on the phospholipid molecules are the unsaturated (double) bond between two carbon atoms
and the ester linkage between glycerol and the fatty acid. The polyunsaturated fatty acids (PUFAs)
present in membrane phospholipids are particularly sensitive to attack by ROS. A single •OH can
result in peroxidation of many PUFAs because the reactions involved in this process are part of a
cyclic chain reaction. The overall process of lipid peroxidation involves three distinct stages: initia-
tion, progression, and termination steps. The initial phase of lipid peroxidation includes activation
of O2 that is rate limiting. Oi-
2 and OH can react with methylene groups of PUFA forming conju-
•
gated dienes, lipid peroxy radicals, and hydroperoxides (Smirnoff, 1995):
PUFA - H + X i Æ PUFAi + X - H
PUFAi + O2 Æ PUFA - OOi
The peroxy radical formed is highly reactive and able to propagate the chain reaction
PUFA - OOi + PUFA - OOH Æ PUFA - OOH + PUFAi
The formation of conjugated diene occurs when free radicals attack the hydrogens of methylene
groups separating double bonds and, thereby, rearrangement of the bonds occurs (Recknagal and
Glende, 1984). The lipid hydroperoxides produced (PUFA–OOH) can undergo reductive cleavage
by reduced metals, such as Fe2+, according to the following reaction:
Fe 2+ complex + PUFA - OOH Æ Fe3+ complex + PUFA - Oi

Several reactive species including lipid alkoxyl radicals, aldehydes (malonyldialdehyde, acrolein,
and crotonaldehyde), alkanes, lipid epoxides, and alcohols can be easily formed by the decomposi-
tion of lipid hydroperoxide (Davies et al., 2000). The lipid alkoxy radical produced, (PUFA–O•), can
initiate additional chain reactions (Buettner, 1993):
PUFA - Oi + PUFA - H Æ PUFA - OH + PUFAi

Peroxidation of PUFA by ROS attack can lead to chain breakage and, thereby, increase in mem-
brane fluidity and permeability.
26.2.4.2 Proteins
The attack of ROS on proteins may cause modification of proteins in a variety of ways, some are
direct and others indirect. Direct modification involves modulation of a protein’s activity through
nitrosylation, carbonylation, disulfide bond formation, and glutathionylation. Proteins can be modi-
fied indirectly by conjugation with breakdown products of fatty acid peroxidation (Yamauchi et al.,
2008). As a consequence of excessive ROS production, site-specific amino acid modification,
fragmentation of the peptide chain, aggregation of cross-linked reaction products, altered electric
charge, and increased susceptibility of proteins to proteolysis occur. Tissues injured by oxidative
stress generally contain increased concentrations of carbonylated proteins (Moller and Kristensen,
2004; Pyngrope et al., 2012a). Protein oxidation leading to increased carbonylation has often been
considered as a significant marker of oxidative damage under stressful conditions since carbonyl
group formation requires stringent conditions than the reversible oxidation of protein thiols (Moller
and Kristensen, 2004). Enhanced modification of proteins has been reported in plants under vari-
ous stresses (Romero-Puertas et al., 2002; Sharma and Dubey, 2005; Maheshwari and Dubey, 2009;
Tanou et al., 2009; Pyngrope et al., 2012a). The amino acids in a peptide differ in their susceptibil-
ity to attack by ROS. Thiol groups and sulfur-containing amino acids are very susceptible sites
for attack by ROS. Activated oxygen can abstract an H atom from cysteine residues to form a thiyl
radical that will cross-link to second thiyl radical to form disulfide bridge. Several metals, including
Cd, Pb, and Hg, have been shown to cause the depletion of protein bound thiol groups (Stohs and
Bagchi, 1995). Oxygen also can add to a methionine to form methionine sulfoxide derivative (Brot
and Weissbach, 1982). Tyrosine is readily cross-linked to form bityrosine products in the presence
of ROS (Davies, 1987).
Oxidation of iron–sulfur centers by Oi- 2 is irreversible and leads to enzyme inactivation (Gardner
and Fridovich, 1991). In these cases, the metal (Fe) binds to a divalent cation-binding site on the
protein. The metal (Fe), then, reacts in a Fenton reaction to form a •OH that rapidly oxidizes an
amino acid residue at or near the cation-binding site of the protein (Stadtman, 1986). Oxidized pro-
teins serve as better substrates for proteolytic digestion. It has been suggested that protein oxidation
could predispose it to ubiquitination, which, in turn, would be a target for proteasomal degradation
(Cabiscol et al., 2000). The incubation of pea leaf crude extracts with increasing H2O2 concentra-
tions, Cd-treated plants, and peroxisomes purified from pea leaves showed an increase in carbonyl
content. Oxidized proteins were more efficiently degraded, and the proteolytic activity increased
20% due to the metal treatment (Romero-Puertas et al., 2002). Several studies have revealed that
after a certain degree, further damage leads to extensively cross-linked and aggregated products,
which are not only poor substrates for degradation but can also inhibit proteases to degrade other
oxidized proteins (Grune et al., 1997).
26.2.4.3 DNA
ROS are a major source of DNA damage (Imlay and Linn, 1988). ROS can cause oxidative dam-
ages to nuclear, mitochondrial, and chloroplastic DNA. DNA is cell’s genetic material and any
damage to the DNA can result in changes in the encoded proteins, which may lead to malfunctions
or complete inactivation of the encoded proteins. Oxidative attack on DNA results in deoxyribose
oxidation, strand breakage, removal of nucleotides, variety of modifications in the organic bases of
the nucleotides, and DNA–protein cross-links. Further, changes in the nucleotides of one strand can
result in the mismatches with the nucleotides in the other strand, yielding subsequent mutations.
Enhanced DNA degradation has been observed in plants exposed to various environmental stresses
such as salinity (Liu et al., 2000) and metal toxicity (Meriga et al., 2004). Both the sugar and base
moieties of DNA are susceptible to oxidation by ROS. Oxidative attack to DNA bases generally
involves •OH addition to double bonds, while sugar damage mainly results from hydrogen abstrac-
tion from deoxyribose (Dizdaroglu, 1993). The hydroxyl radical is known to react with all purine
and pyrimidine bases and, also, the deoxyribose backbone (Halliwell and Gutteridge, 1999). •OH
generates various products from the DNA bases that mainly include C-8 hydroxylation of guanine
to form 8-oxo-7,8 dehydro-2′- deoxyguanosine, hydroxymethyl urea, urea, thymine glycol, thymine,
and adenine ring-opened and saturated products (Tsuboi et al., 1998). 8-Hydroxyguanine is the most
commonly observed product. 1O2 only reacts with guanine, whereas H2O2 and Oi- 2 do not react with
bases at all (Halliwell and Aruoma, 1991; Dizdaroglu, 1993). ROS-induced DNA damages include
various mutagenic alterations as well. For example, mutation arising from selective modification of
G:C sites, especially, indicates oxidative attack on DNA by ROS. ROS attack DNA bases indirectly
through reactive products generated by ROS attack to other macromolecules such as lipid (Fink
et al., 1997).
ROS attack to DNA sugars leads to single-strand breaks. ROS abstract hydrogen atom from the
C4′ position of deoxyribose, leading to generation of a deoxyribose radical that further reacts to
produce DNA strand breakage (Evans et al., 2004). Under physiological conditions, neither H2O2
alone nor Oi-2 can cause in vitro strand breakage. Therefore, it was concluded that the toxicity associ-
ated with these ROS in vivo is most likely the result of Fenton reaction. When •OH attacks on either
DNA or proteins associated with it, DNA–protein cross-links are formed (Oleinick et al., 1987).
DNA–protein cross-links cannot be readily repaired and may be lethal if replication or transcrip-
tion precedes repair. Mitochondrial and chloroplast DNA are more susceptible to oxidative damage
than nuclear DNA due to the lack of protective protein, histones, and close locations to the ROS-
producing systems in the former (Richter, 1992). Even though repair system exists for damaged
DNA, excessive changes caused by ROS lead to permanent damage to the DNA with potentially
detrimental effects for the cell.
26.3 ANTIOXIDATIVE DEFENSE SYSTEM IN PLANTS

Plants possess complex antioxidative defense system comprising of nonenzymatic and enzymatic
components to scavenge ROS. In plant cells, specific ROS-producing and ROS-scavenging sys-
tems are found in different organelles such as chloroplasts, mitochondria, and peroxisomes. ROS-
scavenging pathways from different cellular compartments are coordinated (Pang and Wang, 2008).
Under normal conditions, potentially toxic oxygen metabolites are generated at a low level, and
there is an appropriate balance between production and quenching of ROS. The balance between
production and quenching of ROS may be perturbed by a number of adverse environmental fac-
tors, giving rise to rapid increases in intracellular ROS levels (Noctor et al., 2002; Sharma et al.,
2010), which can induce oxidative damage to lipids, proteins, and nucleic acids. In order to avoid
the oxidative damage, higher plants raise the level of endogenous antioxidant defense (Sharma et al.,
2010). Various components of antioxidative defense system involved in ROS scavenging have been
manipulated, overexpressed, or downregulated to add to the present knowledge and understanding
the role of the antioxidant systems.
26.3.1 Nonenzymatic Components of Antioxidative Defense System

Nonenzymatic components of the antioxidative defense system include the major cellular redox buf-
fers AsA and GSH (L-γ-Glutamyl-L-cysteinyl-glycine) as well as tocopherol, carotenoids, and phe-
nolic compounds. They interact with numerous cellular components and in addition to crucial roles
in defense and as enzyme cofactors, these antioxidants influence plant growth and development by
modulating processes from mitosis and cell elongation to senescence and cell death (de Pinto and
De Gara, 2004). Mutants with decreased nonenzymatic antioxidant contents have been shown to be
hypersensitive to stress (Gao and Zhang, 2008; Semchuk et al., 2009).
26.3.1.1 Ascorbate
AsA is the most abundant, low molecular weight antioxidant that has a key role in defense against
oxidative stress caused by enhanced level of ROS. AsA is considered a powerful antioxidant
because of its ability to donate electrons in a number of enzymatic and nonenzymatic reactions.
AsA has been shown to play an important role in several physiological processes in plants, includ-
ing growth, differentiation, and metabolism. The majority of the AsA pool in plants is contributed
by d-mannose/l-galactose commonly called Smirnoff–Wheeler pathway that proceeds via GDP-d-
mannose, GDP-l-galactose, l-galactose, and l-galactono-1,4-lactone (Wheeler et al., 1998). AsA
is also synthesized via uronic acid intermediates, such as d-galacturonic acid (Isherwood et al.,
1954). In this pathway, d-galacturonic acid is reduced to l-galactonic acid by galacturonic acid
reductase, which is subsequently converted to l-galactono-1,4-lactone. The l-galactono-1,4-lactone
is further oxidized to AsA by l-galactono-1,4-lactone dehydrogenase (GALDH) enzyme. It is syn-
thesized in the mitochondria by l-galactono-γ-lactone dehydrogenase and is transported to the other
cell components through a proton-electrochemical gradient or through facilitated diffusion. It is
detected in the majority of plant cell types, organelles, and apoplast (Shao et al., 2008) and is found
to be particularly abundant in photosynthetic tissues (Smirnoff et al., 2004). Most of AsA, almost
more than 90%, is localized in cytoplasm, but unlike other soluble antioxidants, a substantial por-
tion is exported to the apoplast, where it is present in millimolar concentration. Apoplastic AsA is
believed to represent the first line of defense against potentially damaging external oxidants (Barnes
et al., 2002). AsA protects critical macromolecules from oxidative damage. Under normal physi-
ological condition, AsA mostly exists in reduced state in chloroplast where it also acts as a cofactor
of violaxanthin de-epoxidase, thus, sustaining dissipation of excess excitation energy (Smirnoff,
2000). It provides membrane protection by directly reacting with Oi- 2 and H2O2 and regenerating
α-tocopherol from tocopheroxyl radical and preserves the activities of the enzymes that contain
prosthetic transition metal ions (Noctor and Foyer, 1998). AsA has a key role in the removal of H2O2
via AsA–GSH cycle (Pinto et al., 2003). Oxidation of AsA occurs in two sequential steps, first pro-
ducing monodehydroascorbate (MDHA) and subsequently dehydroascorbate (DHA). In the AsA–
GSH cycle, two molecules of AsA are utilized by APX to reduce H2O2 to water with concomitant
generation of MDHA. MDHA is a radical with a short lifetime and can spontaneously dismutate
into DHA and AsA or is reduced to AsA by NADP(H)-dependent enzyme MDHAR (Miyake and
Asada, 1994). DHA is also highly unstable at pH values greater than 6.0 and is decomposed to tar-
tarate and oxalate (Noctor and Foyer, 1998). To prevent this, DHA is rapidly reduced to AsA by the
enzyme DHAR using reducing equivalents from GSH (Asada, 1996).
AsA level has been reported to alter in response to various stresses (Hernandez et al., 2001;
Sharma and Dubey, 2005; Maheshwari and Dubey, 2009; Radyuk et al., 2009; Mishra et al., 2011;
Srivastava and Dubey, 2011). The level of AsA under environmental stresses depends on the bal-
ance between the rates and capacity of AsA biosynthesis and turnover related to antioxidant demand
(Chaves et al., 2002). Overexpression of enzymes involved in AsA biosynthesis confers abiotic
stress tolerance in plants. GDP-mannose 3′,5′-epimerase (GME) catalyzes the conversion of GDP-
d-mannose to GDP-l-galactose, an important step in the Smirnoff–Wheeler pathway of AsA bio-
synthesis in higher plants. Overexpression of two members of the GME gene family resulted in
increased accumulation of AsA and improved tolerance to abiotic stresses in tomato plants (Zhang
et al., 2011). Overexpression of strawberry d-galacturonic acid reductase that participates in AsA
biosynthetic pathway involving d-galacturonic acid as intermediate and reduces d-galacturonic acid
to l-galactonic acid leads to accumulation of AsA and enhanced abiotic stress tolerance in potato
plants (Hemavathi et al., 2009). Increased AsA content has been shown to confer oxidative stress
tolerance in Arabidopsis (Wang et al., 2010). The vtc-1 mutant, deficient in the activity of GDP-
mannose pyrophosphorylase, an enzyme found in the initial part of the AsA biosynthetic pathway
before it becomes committed to AsA synthesis (Wheeler et al., 1998), was found to be more sensi-
tive to supplementary UV-B treatment than wild-type plants (Gao and Zhang, 2008).
26.3.1.2 Glutathione
Tripeptide GSH (L-γ-Glutamyl-L-cysteinyl-glycine) is one of the crucial low molecular weight
nonprotein thiol that plays an important role in intracellular defense against ROS-induced oxida-
tive damage. It has been detected virtually in all cell compartments such as cytosol, chloroplasts,
endoplasmic reticulum, vacuoles, and mitochondria (Foyer and Noctor, 2003). GSH is synthesized in
the cytosol and chloroplasts of plant cells by compartment-specific isoforms of γ-glutamyl-cysteinyl
synthetase (γ-ECS) and glutathione synthetase (GS). The balance between GSH and glutathione
disulfide (GSSG) is a central component in maintaining cellular redox state. Due to its reducing
power, GSH plays an important role in diverse biological processes, including cell growth/division,
regulation of sulfate transport, signal transduction, conjugation of metabolites, enzymatic regula-
tion, synthesis of proteins and nucleic acids, synthesis of phytochelatins for metal chelation, detoxi-
fication of xenobiotics, and the expression of the stress-responsive genes (Foyer et al., 1997). GSH
functions as an antioxidant in many ways. It can react chemically with Oi- 2 , OH, and H2O2 and,
•
therefore, can function directly as a free radical scavenger. GSH can protect macromolecules (i.e.,
proteins, lipids, DNA) either by the formation of adducts directly with reactive electrophiles (glu-
tathiolation) or by acting as proton donor in the presence of ROS or organic free radicals, yielding
GSSG (Asada, 1994). It can participate in regeneration of another potential antioxidant AsA, via
the AsA–GSH cycle. GSH recycles AsA from its oxidized to reduced form by the enzyme DHAR
(Loewus, 1988). GSH can also reduce DHA by a nonenzymatic mechanism at pH >7 and at GSH
concentrations greater than 1 mM. This may be a significant pathway in chloroplasts, where in the
presence of light, pH remains around 8 and GSH concentration may be as high as 5 mM (Foyer and
Halliwell, 1976). Generation and maintenance of reduced GSH pool, either by de novo synthesis
or via recycling by GR, using NADPH as a cofactor and electron donor, is of vital importance for
the cell. The role of GSH in the antioxidative defense system provides a rationale for its use as a
stress marker. When apple trees were subjected to progressive drought, the initial response was
a little oxidation of the GSH pool, followed by increased GSH concentrations. When the stress
increased, GSH concentrations dropped and redox state became more oxidized, which marked the
degradation of the system (Tausz et al., 2004). Similar to drought stress, altered ratio of GSH/
GSSG has been observed in plants under various stresses like salinity (Hefny and Abdel-Kader,
2009), chilling (Radyuk et al., 2009), and metal toxicity (Sharma and Dubey, 2007; Maheshwari
and Dubey, 2009; Mishra et al., 2011; Srivastava and Dubey, 2011). Overexpression of the enzyme
GS involved in GSH biosynthesis showed no effect on GSH level and was not sufficient to improve
ozone tolerance (Strohm et al., 1999) and resistance to photoinhibition (Foyer et al., 1995) in hybrid
poplar (Populus tremula × Populas alba). However, overexpression of γ-ECS showed reduced sen-
sitivity toward cadmium stress in Indian mustard (Zhu et al., 1999) and enhanced tolerance toward
chloroacetanilide herbicides in poplar plants (Gullner et al., 2001). Eltayeb and coworkers (2010)
observed greater protection against oxidative damages imposed by various environmental stresses
in transgenic potato with higher level of reduced GSH.
26.3.1.3 Tocopherols
Tocopherols (α, β, γ, and δ) represent a group of lipophilic antioxidants involved in scavenging
of oxygen‑free radicals, lipid peroxy radicals, and 1O2 (Diplock et al., 1989). Relative antioxidant
activity of the tocopherol isomers in vivo is α > β > γ > δ, which is due to the methylation pattern
and the amount of methyl groups attached to the phenolic ring of the polar head structure (Fukuzawa
et al., 1982). Hence, α-tocopherol with its three methyl substituents has the highest antioxidant
activity of tocopherols (Kamal-Eldin and Appelqvist, 1996). Tocopherols are synthesized only by
photosynthetic organisms and are present in only green parts of plants. The tocopherol biosyn-
thetic pathway utilizes two compounds homogentisic acid (HGA) and phytyldiphosphate (PDP) as
precursors. At least five enzymes 4-hydroxyphenylpyruvate dioxygenase (HPPD), homogentisate
phytyl transferases (VTE2), 2-methyl-6-phytylbenzoquinol methyltransferase (VTE3), tocopherol
cyclase (VTE1), and γ-tocopherol methyltransferase (VTE4) are involved in the biosynthesis of
tocopherols, excluding the bypass pathway of phytyl-tail synthesis and utilization (Li et al., 2010a).
Tocopherols are known to protect lipids and other membrane components by physically quenching
and chemically reacting with O2 in chloroplasts, thus protecting the structure and function of PSII
(Ivanov, 2003). Tocopherols prevent the chain propagation step in lipid auto-oxidation that makes it
an effective free radical trap. Fully substituted benzoquinone ring and fully reduced phytyl chain of
tocopherol act as antioxidants in redox interactions with 1O2 (Fryer, 1992; Halliwell and Gutteridge,
1999). 1O2 quenching by tocopherols is highly efficient, and it is estimated that a single α-tocopherol
molecule can neutralize up to 220 1O2 molecules in vitro before being degraded (Fukuzawa et al.,
1982). Regeneration of the oxidized tocopherol back to its reduced form can be achieved by
AsA, GSH (Fryer, 1992), or coenzyme Q (Kagan et al., 2000). Accumulation of α-tocopherol has
been shown to induce tolerance to chilling, water deficit, and salinity in different plant species
(Yamaguchi-Shinozaki and Shinozaki, 1994; Munné-Bosch et al., 1999; Guo et al., 2006; Bafeel
and Ibrahim, 2008). It was found that metabolic engineering of tocopherol biosynthetic pathway
affected endogenous AsA and GSH pools in leaves. Further study suggested that expression levels of
genes encoding the enzymes of Halliwell–Asada cycle were upregulated, such as APX, DHAR, and
MDHAR (Li et al., 2010a). Mutants of Arabidopsis thaliana with T-DNA insertions in tocopherol
biosynthesis genes, tocopherol cyclase (vte1), and γ-tocopherol methyltransferase (vte4) showed
higher concentration of protein carbonyl groups and GSSG compared to the wild type, indicating the
development of oxidative stress (Semchuk et al., 2009). Transgenic rice plants with Os-VTE1 RNA
interference (OsVTE1-RNAi) were more sensitive to salt stress, whereas, in contrast, transgenic
plants overexpressing OsVTE1 (OsVTE1-OX) showed higher tolerance to salt stress (Ouyang et al.,
2011). OsVTE1-OX plants also accumulated less H2O2 than control plants.
26.3.1.4 Carotenoids
Carotenoids also belong to the group of lipophilic antioxidant and are able to detoxify various
forms of ROS (Young, 1991). Carotenoids are found in plants as well as microorganisms. In
plants, carotenoids absorb light in the region between 400 and 550 nm of the visible spectrum and
pass the captured energy to the Chl (Sieferman-Harms, 1987). As an antioxidant, they scavenge
1O to inhibit oxidative damage and quench triplet sensitizer (3Chl*) and excited Chl* molecule
2
to prevent the formation of 1O2 to protect the photosynthetic apparatus. Carotenoids also serve
as precursors to signaling molecules that influence plant development and biotic/abiotic stress
responses (Li et al., 2008). The ability of carotenoids to scavenge, prevent, or minimize the pro-
duction of triplet Chl may be accounted for by their chemical specificity. Carotenoids contain a
chain of isoprenic residues bearing numerous conjugated double bonds that allows easy energy
uptake from excited molecules and dissipation of excess energy as heat (Mittler, 2002). Gomathi
and Rakkiyapan (2011) observed that high carotenoid content favors better adaptation of sugar-
cane plants under saline condition.
26.3.1.5 Phenolic Compounds
Phenolics are diverse secondary metabolites (flavonoids, tannins, hydroxycinnamate esters, and
lignin) that possess antioxidant properties. They are abundantly found in plant tissues (Grace
and Logan, 2000). Polyphenols contain an aromatic ring with –OH or OCH3 substituents that
together contribute to their biological activity, including antioxidant action. They have been
shown to outperform well-known antioxidants, AsA and α-tocopherol, in in vitro antioxidant
assays because of their strong capacity to donate electrons or hydrogen atoms. Polyphenols can
chelate transition metal ions, can directly scavenge molecular species of active oxygen, and can
inhibit lipid peroxidation by trapping the lipid alkoxyl radical. They also modify lipid pack-
ing order and decrease fluidity of the membranes (Arora et al., 2000). These changes could
strictly hinder diffusion of free radicals and restrict peroxidative reactions. Moreover, it has
been shown that, especially, flavonoids and phenylpropanoids are oxidized by peroxidase and
act in H 2O2-scavenging, phenolic/AsA/POD system. There is some evidence of induction of
phenolic metabolism in plants as a response to multiple stresses (Michalak, 2006). Janas and
coworkers (2009) observed that ROS could serve as a common signal for acclimation to Cu 2+
stress and could cause accumulation of total phenolic compounds in dark-grown lentil roots.
A mutant A. thaliana L., having a single gene defect that led to a block in the synthesis of a
group of flavonoids, displayed a dramatic increase in sensitivity to UV-B radiation compared

with wild-type plants (Lois and Buchanan, 1994). Transgenic potato plant with increased con-
centration of flavonoid showed improved antioxidant capacity (Lukaszewicz et al., 2004).
26.3.2 Enzymatic Components
The enzymatic components of the antioxidative defense system comprise of several antioxidant
enzymes such as SOD, CAT, GPX, and enzymes of the AsA–GSH cycle APX, MDHAR, DHAR,
and GR (Noctor and Foyer, 1998). These enzymes operate in different subcellular compartments
and respond in concert when cells are exposed to oxidative stress. Table 26.1 shows various antioxi-
dant enzymes that play important role in scavenging stress-induced ROS generated in plants.
26.3.2.1 Superoxide Dismutase
SOD (1.15.1.1) plays a central role in defense against oxidative stress in all aerobic organisms
(Scandalios, 1993). The enzyme SOD belongs to the group of metalloenzymes and catalyzes the
dismutation of Oi-
2 to O2 and H2O2. It is present in most of the subcellular compartments that gen-
erate activated oxygen. Three isozymes of SOD copper/zinc SOD (Cu/Zn-SOD), manganese SOD
(Mn-SOD), and iron SOD (Fe-SOD) are reported in plants (Fridovich, 1989; Racchi et al., 2001).
All forms of SOD are nuclear encoded and targeted to their respective subcellular compartments by
an amino terminal targeting sequence (Bowler et al., 1992). Mn-SOD is localized in mitochondria,
whereas Fe-SOD is localized in chloroplasts (Jackson et al., 1978). Cu/Zn-SOD is present in three
isoforms, which are found in the cytosol, chloroplast, and peroxisome and mitochondria (Kanematsu
and Asada, 1989; Bowler et al., 1992; Bueno et al., 1995; del Rio et al., 1998). Eukaryotic Cu/Zn-SOD
TABLE 26.1
Activation of Antioxidant Enzymes in Response to Oxidative Stress Induced by Various
Environmental Stresses
Stresses Antioxidant Enzymes Plant Species References
Drought SOD, GPX, APX, MDHAR, Oryza sativa Sharma and Dubey (2005),
DHAR, and GR Pyngrope et al. (2012b)
SOD, CAT, and GPX Beta vulgaris Sayfzadeh and Rashidi (2011)
SOD, APX, and GR Triticum sativum Sgherri et al. (2000)
Salinity SOD, CAT, GPX, APX, GR O. sativa Mishra et al. (2012)
CAT, SOD, and GR Olea europaea Valderrama et al. (2006)
GPX O. sativa Mittal and Dubey (1991)
Chilling APX, MDHAR, DHAR, GR Zea mays Fryer (1998)
SOD Fragaria × ananassa Zhang et al. (2008b)
CAT Cicer arietinum L. Nazari et al. (2012)
Metals
Al SOD, GPX, and APX O. sativa Sharma and Dubey (2007),
Glycine max Cakmak and Horst (1991)
Ni SOD, GPX, and APX O. sativa Maheshwari and Dubey (2009)
As SOD, GPX, and APX O. sativa Mishra et al. (2011)
Mn SOD, GPX, APX, and GR O. sativa Srivastava and Dubey (2011)
UV-B SOD, APX, CAT, and GPX Picea asperata Han et al. (2009)
GPX and APX Arabidopsis thaliana Rao et al. (1996)
Pathogens
Odium lini fungus GPX and CAT Linum usitatissimum Ashry and Mohamed (2011)
Bean yellow mosaic virus POD, CAT, APX, and SOD Vicia faba Radwan et al. (2010)
is cyanide sensitive and presents as dimer, whereas the other two (Mn-SOD and Fe-SOD) are cyanide
insensitive and may be dimers or tetramers (Scandalios, 1993; del Rio et al., 1998).
SOD activity has been reported to increase in plants exposed to various environmental stresses,
including drought and metal toxicity (Sharma and Dubey, 2005; Mishra et al., 2011). Increased activ-
ity of SOD is often correlated with increased tolerance of the plant against environmental stresses.
It was suggested that SOD can be used as an indirect selection criterion for screening drought-
resistant plant materials (Zaefyzadeh et al., 2009). Overproduction of SOD has been reported to
result in enhanced oxidative stress tolerance in plants (Gupta et al., 1993).
26.3.2.2 Catalase
Among antioxidant enzymes, CAT (1.11.1.6) was the first enzyme to be discovered and character-
ized. It is an ubiquitous tetrameric heme-containing enzyme that catalyzes the dismutation of two
molecules of H2O2 into water and oxygen. It has high specificity for H2O2, but weak activity against
organic peroxides. Plants contain several types of H2O2-degrading enzymes; however, CATs are
unique as they do not require cellular reducing equivalent. CATs have a very fast turnover rate, but
a much lower affinity for H2O2 than APX. The peroxisomes are major sites of H2O2 production.
CAT scavenges H2O2 generated in this organelle during photorespiratory oxidation, β-oxidation
of fatty acids, and other enzyme systems such as XOD coupled to SOD (Scandalios et al., 1997;
del Río et al., 2006; Corpas et al., 2008). Though there are frequent reports of CAT being present
in cytosol, chloroplast, and mitochondria, the presence of significant CAT activity in these is less
well established (Mhamdi et al., 2010). To date, all angiosperm species studied contain three CAT
genes. Willekens et al. (1995) proposed a classification of CAT based on the expression profile of
the tobacco genes. Class I CATs are expressed in photosynthetic tissues and are regulated by light.
Class II CATs are expressed at high levels in vascular tissues, whereas Class III CATs are highly
abundant in seeds and young seedlings.
H2O2 has been implicated in many stress conditions. When cells are stressed for energy and
are rapidly generating H2O2 through catabolic processes, H2O2 is degraded by CAT in an energy-
efficient manner (Mallick and Mohn, 2000). Environmental stresses cause either enhancement or
depletion of CAT activity, depending on the intensity, duration, and type of the stress (Sharma and
Dubey, 2005; Moussa and Abdel-Aziz, 2008; Han et al., 2009). In general, stresses that reduce the
rate of protein turnover also reduce CAT activity. Stress analysis revealed increased susceptibility
of CAT-deficient plants to paraquat, salt, and ozone, but not to chilling (Willekens et al., 1997). In
transgenic tobacco plants, having 10% wild-type, CAT activity showed accumulation of GSSG and
a fourfold decrease in AsA, indicating that CAT is critical for maintaining the redox balance during
the oxidative stress (Willekens et al., 1997). Overexpression of a CAT gene from Brassica juncea
introduced into tobacco enhanced its tolerance to Cd-induced oxidative stress (Guan et al., 2009).
26.3.2.3 Guaiacol Peroxidase
GPX (EC 1.11.1.7), a heme-containing protein, preferably oxidizes aromatic electron donor such as
guaiacol and pyrogallol at the expense of H2O2. It is widely found in animals, plants, and microbes.
These enzymes have four conserved disulfide bridges and contain two structural Ca2+ ions (Schuller
et al., 1996). Many isoenzymes of GPX exist in plant tissues localized in vacuoles, the cell wall, and
the cytosol (Asada, 1992). GPX is associated with many important biosynthetic processes, including
lignification of cell wall, degradation of IAA, biosynthesis of ethylene, wound healing, and defense
against abiotic and biotic stresses (Kobayashi et al., 1996). GPXs are widely accepted as stress
enzyme. GPX can function as effective quencher of reactive intermediary forms of O2 and peroxy
radicals under stressed conditions (Vangronsveld and Clijsters, 1994). Various stressful conditions
of the environment have been shown to induce the activity of GPX (Radotic et al., 2000; Shah
et al., 2001; Verma and Dubey, 2003; Sharma and Dubey, 2005, 2007; Moussa and Abdel-Aziz,
2008; Han et al., 2009; Mishra et al., 2011). Radotic and coworkers (2000) correlated increased
activity of GPX to oxidative reactions under metal toxicity conditions and suggested its potential as
biomarker for sublethal metal toxicity in plants. Recently, Tayefi-Nasrabadi and coworkers (2011)
also concluded that greater protection of salt-tolerant safflower plants from salt-induced oxidative
damage results, at least in part, through the increase of the GPX activity, catalytic efficiency, and
induction of specific isoenzymes compared to salt-sensitive cultivar.
26.3.2.4 Enzymes of Ascorbate–Glutathione Cycle

The change in the ratio of AsA to DHA and GSH to GSSG is crucial for the cell to sense oxidative
stress and respond accordingly. The AsA–GSH cycle also referred to as Halliwell–Asada pathway
is the recycling pathway of AsA and GSH regeneration that also detoxifies H2O2. The AsA–GSH
cycle involves successive oxidation and reduction of AsA, GSH, and NADPH catalyzed by the
enzymes APX, MDHAR, DHAR, and GR. The AsA–GSH cycle is present in at least four different
subcellular locations, including the cytosol, chloroplast, mitochondria, and peroxisomes (Jimenez
et al., 1997). The AsA–GSH cycle plays an important role in combating oxidative stress induced by
environmental stresses (Pallanca and Smirnoff, 2000; Sharma and Dubey, 2005).
26.3.2.4.1 Ascorbate Peroxidase
APX (EC 1.1.11.1) is a central component of AsA–GSH cycle and plays an essential role in the
control of intracellular ROS levels. APX uses two molecules of AsA to reduce H2O2 to water with
a concomitant generation of two molecules of MDHA. APX is a member of Class I super fam-
ily of heme peroxidases (Welinder, 1992) and is regulated by redox signals and H2O2 (Patterson
and Poulos, 1995). Based on amino acid sequences, five chemically and enzymatically distinct
isoenzymes of APX have been found at different subcellular localization in higher plants. These
are cytosolic, stromal, thylakoidal, mitochondrial, and the peroxisomal isoforms (Nakano and
Asada, 1987; Jimenez et al., 1997; Madhusudhan et al., 2003; Sharma and Dubey, 2004). APX
found in organelles scavenges H2O2 produced within the organelles, whereas cytosolic APX
eliminates H2O2 produced in the cytosol, apoplast, or that diffused from organelles (Mittler and
Zilinskas, 1992). The chloroplastic and cytosolic APX isoforms are specific for AsA as electron
donor, and the cytosolic isoenzymes are less sensitive to depletion of AsA than the chloroplastic
isoenzymes, including stromal- and thylakoid-bound enzymes (Ishikawa et al., 1998; Sharma and
Dubey, 2004).
APX is regarded as one of the most widely distributed antioxidant enzymes in plant cells, and
isoforms of APX have much higher affinity for H2O2 than CAT, making APXs efficient scavengers of
H2O2 under stressful conditions (Wang et al., 1999). Many workers have reported enhanced activity of
APX in response to abiotic stresses such as drought, salinity, chilling, metal toxicity, and UV irradia-
tion (Boo and Jung, 1999; Sharma and Dubey, 2005, 2007; Han et al., 2009; Hefny and Abdel-Kader,
2009; Maheshwari and Dubey, 2009). Overexpression of a cytosolic APX gene derived from pea
(Pisum sativum L.) in transgenic tomato plants (Lycopersicon esculentum L.) ameliorated oxidative
injury induced by chilling and salt stress (Wang et al., 2005). Similarly, overexpression of the tApx
gene in either tobacco or in Arabidopsis increased tolerance to oxidative stress (Yabuta et al., 2002).
26.3.2.4.2 Monodehydroascorbate Reductase
MDHA radical produced in APX-catalyzed reaction has a short lifetime, and if not rapidly
reduced, it disproportionates to AsA and DHA (Ushimaru et al., 1997). MDHAR (1.6.5.4) is a
FAD enzyme that catalyzes the regeneration of AsA from the MDHA radical using NAD(P)H
as the electron donor (Hossain and Asada, 1985). It is the only known enzyme to use an organic
radical (MDA) as a substrate and is also capable of reducing phenoxyl radicals that are generated
by horseradish peroxidase with H2O2 (Sakihama et al., 2000). MDHAR activity is widespread in
plants. The isoenzymes of MDHAR have been reported to be present in several cellular compart-
ments such as chloroplasts (Hossain et al., 1984), cytosol, mitochondria, and peroxisomes (Dalton
et al., 1993; Jiménez et al., 1997). In chloroplasts, MDHAR could have two physiological func-
tions: the regeneration of AsA from MDHA and the mediation of the photoreduction of dioxygen
to Oi-
2 when the substrate MDHA is absent (Miyake et al., 1998). Characterization of membrane
polypeptides from pea leaf peroxisomes also revealed MDHAR to be involved in Oi- 2 generation
(López-Huertas et al., 1999).
Several studies have shown increased activity of MDHAR in plants subjected to environmental
stresses (Boo and Jung, 1999; Sharma and Dubey, 2005, 2007; Maheshwari and Dubey, 2009).
Overexpression of Arabidopsis MDHAR gene in tobacco confers enhanced tolerance to salt and
polyethylene glycol stresses (Eltayeb et al., 2007). Tomato chloroplastic MDHAR overexpressed in
transgenic Arabidopsis enhanced its tolerance to temperature and methyl viologen (MV)–mediated
oxidative stresses (Li et al., 2010b).
26.3.2.4.3 Dehydroascorbate Reductase
DHAR (EC 1.8.5.1) catalyzes the reduction of DHA to AsA using GSH as the reducing substrate
(Ushimaru et al., 1997) and, thus, plays an important role in maintaining AsA in its reduced form.
Despite the possibility of enzymatic and nonenzymatic regeneration of AsA directly from MDHA,
some DHA is always produced when AsA is oxidized in leaves and other tissues. DHA, a very
short-lived chemical, can either be hydrolyzed irreversibly to 2,3-diketogulonic acid or recycled
to AsA by DHAR. Overexpression of DHAR in tobacco leaves, maize, and potato is reported to
increase AsA content suggesting that DHAR plays important roles in determining the pool size of
AsA (Chen et al., 2003; Qin et al., 2011). DHAR is a monomeric thiol enzyme abundantly found in
dry seeds, roots, and etiolated as well as green shoots. DHAR has been purified from chloroplast
as well as nonchloroplast sources in several plant species, including spinach leaves (Hossain and
Asada, 1984) and potato tuber (Dipierro and Borraccino, 1991).
Environmental stresses such as drought, metal toxicity, and chilling increase the activity of the
DHAR in plants (Boo and Jung, 1999; Hernandez et al., 2001; Sharma and Dubey, 2005, 2007;
Yoshida et al., 2006; Maheshwari and Dubey, 2009). Consistent upregulation of the gene encod-
ing cytosolic DHAR was found in L. japonicas, which was found to be more tolerant to salt stress
than other legumes. This upregulation of DHAR was correlated to its role in AsA recycling in the
apoplast (Rubio et al., 2009). Transgenic potato overexpressing Arabidopsis cytosolic AtDHAR1
showed higher tolerance to herbicide, drought, and salt stresses (Eltayeb et al., 2011).
26.3.2.4.4 Glutathione Reductase
When acting as an antioxidant by participating in enzymatic as well as nonenzymatic oxidation–
reduction cycles, GSH is oxidized to GSSG. In the AsA–GSH cycle, GSH is oxidized in a reaction
catalyzed by DHAR. GR (EC 1.6.4.2), a NAD(P)H-dependent enzyme, catalyzes the reduction of
GSSG to GSH and, thus, maintains high cellular GSH/GSSG ratio. GR belongs to a group of flavo-
enzymes and contains an essential disulfide group (Ghisla and Massey, 1989). The catalytic mecha-
nism involves two steps: first the flavin moiety is reduced by NADPH, the flavin is oxidized, and a
redox-active disulfide bridge is reduced to produce a thiolate anion and a cysteine. The second step
involves the reduction of GSSG via thiol–disulfide interchange reactions (Ghisla and Massey, 1989).
If the reduced enzyme is not reoxidized by GSSG, it can suffer a reversible inactivation. Although
it is located in the chloroplasts, cytosol, mitochondria, and peroxisomes, around 80% of GR activ-
ity in photosynthetic tissues is accounted for by chloroplastic isoforms (Edwards et al., 1990). In
chloroplast, GSH and GR are involved in detoxification of H2O2 generated by the Mehler reaction.
Several authors have reported increased activity of GR under environmental stresses (Hernandez
et al., 2001; Sharma and Dubey, 2005, 2007; Yoshida et al., 2006; Maheshwari and Dubey, 2009).
Pastori and Trippi (1992) observed correlation between the oxidative stress resistance and activity
of GR and suggested that oxidative stress caused by paraquat or H2O2 could stimulate GR de novo
synthesis, probably at the level of translation by preexisting mRNA. Antisense-mediated depletion
of tomato chloroplast GR has been shown to enhance susceptibility to chilling stress (Shu et al.,
2011). Overexpression of GR in N. tabacum and Populus plants leads to higher foliar AsA contents
and improved tolerance to oxidative stress (Aono et al., 1993; Foyer et al., 1995).
Due to the complexity of ROS detoxification system, overexpressing one component of anti-
oxidative defense system may or may not change the capacity of the pathway as a whole (Tseng
et al., 2008; Lee et al., 2009). Several studies have shown that overexpression of combinations of
antioxidant enzymes in transgenic plants has synergistic effect on stress tolerance (Aono et al.,
1995; Kwon et al., 2002). Kwon et al. (2002) demonstrated that simultaneous expression of Cu/
Zn-SOD and APX genes in tobacco chloroplasts enhanced tolerance to MV stress compared to
expression of either of these genes alone. Similarly, enhanced tolerance to multiple environmental
stresses has been developed by simultaneous overexpression of the genes of SOD and APX in the
chloroplasts (Lim et al., 2007; Kwak et al., 2009), SOD and CAT in cytosol (Tseng et al., 2008), and
SOD and GR in cytosol (Aono et al., 1995). Further, simultaneous expression of multiple antioxidant
enzymes, such as Cu/Zn-SOD, APX, and DHAR, in chloroplasts has shown to be more effective
than single or double expression for developing transgenic plants with enhanced tolerance to mul-
tiple environmental stresses (Lee et al., 2007). Therefore, in order to achieve tolerance to multiple
environmental stresses, increased emphasis is now given to produce transgenic plants overexpress-
ing multiple antioxidants.
26.4 OVERPRODUCTION OF ROS UNDER STRESSFUL CONDITIONS

The production of ROS in plants under normal growth conditions is low. However, in response to
various environmental stresses, ROS are drastically increased in plants disturbing the normal bal-
ance of Oi-
2 , OH, and H2O2 in the intracellular environment (Sharma et al., 2010). The effects of
•
various environmental stresses such as drought, salinity, chilling, metal toxicity, UV-B radiation,
and pathogen attack on ROS production are discussed in the following text.
26.4.1 Drought
Under drought stress, ROS production is enhanced in several ways. Inhibition of carbon dioxide
(CO2) assimilation, coupled with the changes in photosystem activities and photosynthetic transport
capacity under drought stress, results in accelerated production of ROS via the chloroplast Mehler
reaction (Asada, 1999). During drought stress, CO2 fixation is limited due to stomatal closure that,
in turn, leads to reduced NADP+ regeneration through the Calvin cycle. Due to lack of electron
acceptor, over reduction of the photosynthetic ETC occurs that leads to a higher leakage of electrons
to O2 by the Mehler reaction. Biehler and Fock (1996) reported a 50% more leakage of photosyn-
thetic electrons to the Mehler reaction in drought-stressed wheat plants, compared to unstressed
plants. Photosynthetic activity is inhibited in plant tissues due to an imbalance between light capture
and its utilization under drought stress (Foyer and Noctor, 2000). Dissipation of excess light energy
in the PSII core and antenna leads to the generation of ROS that are potentially dangerous under
drought stress conditions (Foyer and Harbinson, 1994). Under drought stress, the photorespiratory
pathway is also enhanced, especially, when RUBP oxygenation is maximal due to limitation in CO2
fixation (Noctor et al., 2002). Noctor and collaborators (2002) have estimated that photorespiration
is likely to account for over 70% of the total H2O2 production under drought stress conditions.
Oi-
2 initiates a chain reaction leading to the production of more toxic radical species, which
may cause damage far in excess of the initial reaction products. Under drought stress, one of
the real threats toward the chloroplast is the production of the • OH in the thylakoids through
iron-catalyzed reduction of H2O2 by both SOD and AsA. Increased production of ROS leads to
oxidative stress in growing plants. Rice seedlings subjected to drought showed increased con-
centration of Oi- 2 , increased level of lipid peroxidation, Chl bleaching, and loss of some antioxi-
dants (AsA, GSH, α-tocopherol, and carotenoids), total soluble protein, and thiols (Boo and Jung,
1999; Sharma and Dubey, 2005). To combat the danger posed by ROS, plant possesses different
scavenging enzymes and metabolites. Enhanced activity of enzymes of antioxidative defense
system has been reported under drought stress in several plant species (Boo and Jung, 1999;
Sgherri et al., 2000; Sharma and Dubey, 2005; Sayfzadeh and Rashidi, 2011). A comparative
study of the antioxidant responses in drought-tolerant and drought-sensitive genotypes revealed
higher antioxidant capacity in tolerant genotypes. In contrast to the drought-susceptible wheat
genotype HD 2329, the drought-tolerant wheat genotype C 306 had higher APX and CAT activ-
ity, higher AsA content, and lower H2O2 and MDA content (Sairam et al., 1998). In another
study, the drought-tolerant maize genotype Giza 2 was suggested to be comparatively tolerant to
water stress compared to the drought-sensitive Trihybrid 321 owing to the lower increase in H 2O2
and MDA content along with higher increase in SOD, CAT, and POX activities (Moussa and
Abdel-Aziz, 2008). Similarly, among two apple rootstocks Malus prunifolia (drought tolerant)
and M. hupehensis (drought sensitive), M. hupehensis was more vulnerable to drought than was
M. prunifolia, resulting in larger increases in the levels of H2O2, Oi- 2 , and MDA. The activities of
SOD, POD, APX, GR, and DHAR and levels of AsA and GSH increased to a greater extent in
M. prunifolia than in M. hupehensis in response to drought (Wang et al., 2012). APX serves as an
important component of antioxidative defense system under drought (Sharma and Dubey, 2005).
In rice plants, the increase in the capacity of AsA regeneration system by de novo synthesis of
MDHAR, DHAR, and GR has been shown to be one of the primary responses to water deficit so
as to mitigate oxidative stress (Boo and Jung, 1999; Sharma and Dubey, 2005).
Pyngrope and coworkers (2012b), while examining the responses of ROS production, levels of
nonenzymatic antioxidants AsA, GSH, and their redox pool, as well as activity levels of enzymes
of the AsA–GSH cycle in seedlings of rice (Oryza sativa L.) cultivars differing in drought toler-
ance subjected to polyethylene glycol (PEG-6000)–induced water deficit in sand cultures, observed
that water deficit caused increased production of ROS such as Oi- 2 , H2O2, and HO in the tissues,
·
and the level of production was higher in the sensitive cv. Malaviya-36 than the tolerant cv. Brown
Gora. Water deficit caused reduction in AsA and GSH and decline in their redox ratios (AsA/DHA
and GSH/GSSG) with lesser decline in the seedlings of tolerant cultivar than the sensitive. Further,
these workers observed that water deficit level of −1.0 and −2.1 MPa imposed for 24–72 h caused
increased activities of the enzymes MDHAR, DHAR, APX, and GST in the seedlings of both rice
cultivars, but the increased activity levels were higher in the seedlings of drought-tolerant cultivar
than the sensitive. It was suggested by these workers that an enhanced oxidative stress tolerance by
a well-coordinated cellular redox state of AsA and GSH in reduced forms and induction of antioxi-
dant defense system by elevated activity levels of enzymes of the AsA–GSH cycle is associated with
water deficit tolerance in rice (Pyngrope et al., 2012b).
Water deficit stress causes increased carbonylation of proteins, and such proteins when accumu-
lated in the cells become toxic and also become more susceptible to proteolysis due to unfolding
of target protein domains (Pyngrope et al., 2012a). In Indica rice seedlings, the parameters such
as constitutive higher status of the antioxidative enzymes SOD, CAT, and GPX and their further
enhancement under water deficit, low level of proteolysis, and reduced level of protein carbonyls in
water deficit–stressed plant parts have been regarded as a suitable model for depicting water deficit
tolerance in Indica rice seedlings (Pyngrope et al., 2012a).
26.4.2 Salinity
Salinity stress results in an excessive generation of ROS (Hernandez et al., 2000; Tanou et al., 2009).
High salt concentrations lead to overproduction of the ROS Oi- 2 , OH, H2O2, and O2 by impairment
• 1
of the cellular electron transport within different subcellular compartments such as chloroplasts
and mitochondria, as well as from induction of metabolic pathways such as photorespiration. Salt
stress can lead to stomatal closure, which reduces CO2 availability in the leaves and inhibits car-
bon fixation that, in turn, cause exposure of chloroplasts to excessive excitation energy and over
reduction of photosynthetic electron transport system leading to enhanced generation of ROS and
induced oxidative stress. Low chloroplastic CO2/O2 ratio also favors photorespiration leading to
increased production of ROS such as H2O2 (Hernández et al., 2000). Elevated CO2 mitigates the
oxidative stress caused by salinity, involving lower ROS generation and a better maintenance of
redox homeostasis as a consequence of higher assimilation rates and lower photorespiration (Perez-
Lopez et al., 2009). Salinity-induced ROS disrupt normal metabolism through lipid peroxidation,
denaturing proteins, and nucleic acids in several plant species (Hernández et al., 2000; Tanou et al.,
2009; Karray-Bouraoui et al., 2011).
Differential genomic and proteomic screenings carried out in Physcomitrella patens plants
showed that they responded to salinity stress by upregulating a large number of genes involved in
antioxidant defense mechanism (Wang et al., 2008), suggesting that the antioxidative system may
play a crucial role in protecting cells from oxidative damage following exposure to salinity stress
in P. patens. Salinity-induced oxidative stress and possible relationship between the status of the
components of antioxidative defense system and the salt tolerance in Indica rice (O. sativa L.) geno-
types were studied by Mishra et al. (2012). Seedlings of salt-sensitive cultivar showed a substantial
increase in the rate of Oi-2 production; elevated levels of H2O2 and MDA; declined levels of thiol,
AsA, and GSH; and lower activity of antioxidant enzymes compared to salt-tolerant seedlings. It
was suggested that a higher status of antioxidants AsA and GSH and a coordinated higher activity of
the enzymes SOD, CAT, GPX, APX, and GR can serve as the major determinants in the model for
depicting salt tolerance in Indica rice seedlings (Mishra et al., 2012). Similarly, a study of the imme-
diate responses (enzymatic and nonenzymatic) to salinity-induced oxidative stress in two major rice
(O. sativa L.) cultivars, salt-sensitive Pusa Basmati 1 (PB) and salt-tolerant Pokkali (PK), revealed a
lesser extent of membrane damage (lipid peroxidation), lower levels of H2O2, higher activity of the
ROS-scavenging enzyme CAT, and enhanced levels of antioxidants like ASA and GSH in PK com-
pared to PB (Vaidyanathan et al., 2003). A comparative study using cultivated tomato L. esculentum
Mill. cv. M82 (Lem) and its wild salt-tolerant relative L. pennellii (Corr.) D’Arcy accession Atico
(Lpa) showed better protection of Lpa roots from salt-induced oxidative damage, at least partially,
from the increased activities of SOD, CAT, APX, and MDHAR and increased contents of AsA and
GSH (Shalata et al., 2001).
In salt-stressed root of Lem, a gradual increase in the membrane lipid peroxidation was observed,
whereas no change in lipid peroxidation was observed in Lpa. Salt-tolerant Plantago maritima
showed a lower level of MDA and a better protection mechanism against oxidative damage caused
by salt stress by increasing activities of SOD, CAT, GR, and APX than the salt-sensitive P. media
(Sekmen et al., 2007). NADP dehydrogenases and peroxidase have been suggested as key antioxi-
dative enzymes in olive plants under salt stress conditions (Valderrama et al., 2006). Mittal and
Dubey (1991) observed a correlation between peroxidase activity and salt tolerance in rice seed-
ling. The salinity tolerance mechanism in a salt-tolerant dicotyledonous C3 halophyte plant quinoa
(Chenopodium quinoa) involves protection of its photosynthetic machinery against oxidative stress
in developing leaves due to accumulation of organic osmolytes (Shabala et al., 2012). Similarly, it
was shown by Abbaspour (2012) that salt-stressed pistachio (Pistacia vera) plants possess protection
mechanism against salt stress–induced oxidative damage by maintaining an inherited and induced
activity of antioxidant enzymes and higher proline content that may play a role as an enzyme-
stabilizing agent under salt stress.
26.4.3 Chilling
Chilling stress is a key environmental factor limiting growth and productivity of crop plants.
Chilling leads to the overproduction of ROS by exacerbating imbalance between light absorption
and light use by inhibiting Calvin–Benson cycle activity (Logan et al., 2006), enhancing photosyn-
thetic electron flux to O2, and causing overreduction of respiratory ETC (Hu et al., 2008). Chilling
stress also causes significant reductions in rbcL and rbcS transcripts, ribulose-1,5-bisphosphate
carboxylase oxygenase (RUBISCO) content, and initial RUBISCO activity, leading to higher elec-
tron flux to O2 (Zhou et al., 2006). H2O2 accumulation in chloroplast was negatively correlated
with the initial RUBISCO activity and photosynthetic rate (Zhou et al., 2006). Chilling-induced
oxidative stress evident by increased accumulation of ROS, including H2O2 and Oi- 2 , lipid peroxi-
dation, and protein oxidation, is a significant factor in relation to chilling injury in plants (Prasad,
1997; Fryer et al., 1998; Zhang et al., 2008a). Protein carbonyl content, an indication of oxidative
damage, was increased twofold in maize seedlings when exposed to chilling temperatures (Prasad,
1997). Lipoxygenase activity as well as lipid peroxidation was increased in maize leaves during low
temperatures, suggesting that lipoxygenase-mediated peroxidation of membrane lipids contributes
to the oxidative damage occurring in chill-stressed maize leaves (Fryer et al., 1998). Responses to
chilling-induced oxidative stress include alteration in activities of enzymes of antioxidant defense
system. The activities of antioxidative enzymes APX, MDHAR, DHAR, GR, and SOD increased
during chilling periods in maize (Fryer et al., 1998) and strawberry leaves (Zhang et al., 2008b).
However, if the duration of chilling stress is too long, the defense system may not remove over-
produced ROS effectively, which may result in severe damage or even death (Zhang et al., 2008a).
Nonenzymatic antioxidants (AsA, GSH, carotenoids, and α-tocopherol) also play an important role
in cold response. Under cold stress conditions, low molecular weight antioxidants, especially, that
of reduced AsA, have been suggested to be an important component in plant cell defense (Radyuk
et al., 2009). Many comparative studies using chilling-tolerant and chilling-sensitive genotypes
have shown greater antioxidant capacity in chilling-tolerant species compared to sensitive ones
(Jahnke et al., 1991; Hodges et al., 1996; Huang and Guo, 2005). In rice, higher activities of defense
enzymes and higher content of antioxidant under stress were associated with tolerance to chilling
(Huang and Guo, 2005). The responses of antioxidative system of rice to chilling were investigated
in a tolerant cultivar, Xiangnuo-1, and a susceptible cultivar, IR-50. The electrolyte leakage and
MDA content of Xiangnuo-1 were little affected by chilling treatment, but those of IR-50 increased.
Activities of SOD, CAT, APX, GR, and AsA content of Xiangnuo-1 remained high, while those of
IR-50 decreased under chilling stress. GR activity was also found to increase within 24 h in chill-
ing-tolerant Zea diploperennis, but it decreased slightly in chilling-susceptible Z. mays cv. LG11
(Jahnke et al., 1991).
In chickpea (Cicer arietinum L.) plants, short-term acclimation to cold induced greater cold
tolerance, which was marked by low levels of MDA and electrolyte leakage index, indicating lower
lipid peroxidation and higher membrane stability compared to nonacclimated plants when the plants
were exposed to cold stress (Nazari et al., 2012). In chickpea plants, CAT appears to serve as a more
effective enzyme than GPX and APX in protecting the cells against H2O2, which accumulates in
plants on cold exposure. Such response of cell protection is more pronounced in cold-acclimated
plants than in control and nonacclimated plants (Nazari et al., 2012).
26.4.4 Metal Toxicity
The increasing levels of metals into the environment drastically affect plant growth and metabo-
lism, ultimately, leading to severe losses in crop yields (Salt et al., 1995; Mishra and Dubey, 2005).
One of the consequences of the presence of the toxic metals within the plant tissues is the formation
of ROS, which can be initiated directly or indirectly by the metals and, consequently, leading to
oxidative damage to different cell constituents (Shah et al., 2001; Gallego et al., 2002; Sharma and
Dubey, 2007; Maheshwari and Dubey, 2009; Srivastava and Dubey, 2011). Under metal stress con-
dition, net photosynthesis (Phn) decreases due to damage to photosynthetic metabolism, including
photosynthetic electron transport (Phet) (Vinit-Dunand et al., 2002). For example, copper has been
shown to negatively affect components of both the light reactions (e.g., PSII, thylakoid membrane
structure, and Chl content) (Vinit-Dunand et al., 2002) and CO2-fixation reactions (Moustakas
et al., 1994). These alterations in photosynthetic metabolism lead to overproduction of ROS such
as Oi-2 , OH, and H2O2. The induction of ROS production due to metals (cadmium and zinc) in
•
Nicotiana tabacum L. cv. Bright Yellow 2 (TBY-2) cells in suspension cultures showed proper-
ties comparable to the elicitor-induced oxidative burst in other plant cells (Zrobek-Sokolnik et al.,
2009). Redox-active metals, such as iron, copper, and chromium, undergo redox cycling producing
ROS, whereas redox-inactive metals, such as lead, cadmium, and mercury, deplete cell’s major
antioxidants, particularly thiol-containing antioxidants and enzymes (Gallego et al., 1996; Weckx
and Clijsters, 1996; Yamamoto et al., 1997; Shah et al., 2001; Verma and Dubey, 2003; Sharma and
Dubey, 2007; Maheshwari and Dubey, 2009; Srivastava and Dubey, 2011). If metal-induced produc-
tion of ROS is not adequately counterbalanced by cellular antioxidants, oxidative damage of lipids,
proteins, and nucleic acids ensues (Halliwell and Gutteridge, 1989; Dat et al., 2000; Sharma and
Dubey, 2007; Sandalio et al., 2009; Sharma and Dietz, 2009; Mishra et al., 2011; Srivastava and
Dubey, 2011). Significant enhancement in lipid peroxidation and decline in protein thiol contents
was observed when rice seedlings were subjected to Al, Ni, and Mn toxicity (Sharma and Dubey,
2007; Maheshwari and Dubey, 2009; Srivastava and Dubey, 2011).
The increased activity of antioxidative enzymes in metal-stressed plants appears to serve as an
important component of antioxidant defense mechanism of plants to combat metal-induced oxida-
tive injury (Shah et al., 2001). Responses of metal exposure to plants vary depending on plant spe-
cies, tissues, stages of development, type of metal, and its concentration. One of the key responses
includes triggering of a series of defense mechanisms that involve enzymatic and nonenzymatic
components (Gallego et al., 1996; Shah et al., 2001; Verma and Dubey, 2003; Sharma and Dubey,
2007; Maheshwari and Dubey, 2009; Mishra et al., 2011; Srivastava and Dubey, 2011). Various
groups of workers have reported increased activities of antioxidant enzymes like GPX, SOD, APX,
MDHAR, DHAR, and GR as well as nonenzymatic antioxidants in metal-treated plants and sug-
gested involvement of antioxidant defense system in the adaptive response to metal ions (Cakmak
and Horst, 1991; Shah et al., 2001; Verma and Dubey, 2003; Sharma and Dubey, 2007; Maheshwari
and Dubey, 2009; Mishra et al., 2011; Srivastava and Dubey, 2011). However, results suggest that
activation of antioxidant enzymes in response to oxidative stress induced by metals is not enough
to confer tolerance to metal accumulation. A comparative study of antioxidative response of two
maize lines differing in Al tolerance suggested that better protection of the Al-tolerant maize roots
from Al-induced oxidative damage results, at least partially, from the increased activity of their
antioxidative system. After 24 h of Al exposure, a gradual increase in the membrane lipid peroxida-
tion in Al-stressed root of the susceptible maize line was accompanied by decreased activities of
the antioxidant enzymes SOD and POD. In contrast, increased activities of the SOD and POD were
found in Al-treated roots of the tolerant maize line, in which the level of membrane lipid peroxida-
tion remained almost unchanged (Giannakoula et al., 2010). A comparative antioxidant profiling of
tolerant (TPM-1) and sensitive (TM-4) variety of Brassica juncea L. performed after exposure to
arsenate (As(V)) and arsenite (As(III)) showed in general better response of antioxidant enzymes
and the level of GSH in TPM-1 than in TM-4 (Srivastava et al., 2010). These responses presumably
allowed TPM-1 to tolerate higher As concentrations as compared with that of TM-4 (Srivastava
et al., 2010).
Many studies conducted involving metal-sensitive, metal-tolerant, mutant, transgenic, and hyper-
accumulator plants have shown that the nonenzymatic antioxidant GSH by itself and its related
metabolizing enzymes and peptides play a pivotal role in heavy metal tolerance by controlling dif-
ferent plant physiological processes, including ROS detoxification and uptake of heavy metals, its
translocation, chelation, etc. (Hossain et al., 2012).
26.4.5 UV-B Radiations
UV-B radiation on plants is now of major concern to plant biologists due to the threat to productiv-
ity in global agriculture (Blumthaler and Ambach, 1990). Enhanced UV-B significantly inhibits net
photosynthetic rate. It has been shown that UV-B treatment results in decrease in the light-saturated
rate of CO2 assimilation, accompanied by decreases in carboxylation velocity and RUBISCO con-
tent and activity (Allen et al., 1997b). He and coworkers (1993) observed marked decrease in the
ratios of variable to maximum Chl fluorescence yield and in the quantum yield of photosynthetic
oxygen evolution in pea and rice leaves. Limited CO2 assimilation due to UV-B leads to excessive
production of ROS that, in turn, cause oxidative damage in plants (Strid et al., 1994; Han et al.,
2009). Rao and coworkers (1996) suggested that UV-B exposure generates activated oxygen species
by increasing NADPH oxidase activity. Plants must adapt to the deleterious effects of UV-B radia-
tion because they are dependent on sunlight for photosynthesis and, therefore, cannot avoid expo-
sure to UV-B radiation. Plants possess antioxidative enzymatic scavengers SOD, POD, CAT, and
APX and nonenzymatic antioxidants like AsA, GSH, and carotenoids to keep the balance between
the production and removal of ROS. In Picea asperata seedlings, although enhanced UV-B (30%)
increased the efficiency of antioxidant defense system consisting of UV-B-absorbing compounds,
carotenoids, and antioxidant enzymes SOD, APX, CAT, and GPX (Han et al., 2009), it induced
overproduction of ROS and oxidative stress eventually. Peroxidase-related enzymes were found
to be preferentially induced by UV-B exposure in Arabidopsis (Rao et al., 1996). Gao and Zhang
(2008) observed that AsA-deficient mutant vtc1 was more sensitive to supplementary UV-B treat-
ment than wild-type plants and, therefore, suggested that AsA could be considered as an important
antioxidant for UV-B radiation.
26.4.6 Pathogens
One of the earliest cellular responses following successful pathogen recognition is oxidative burst
involving production of ROS. Recognition of a variety of pathogens leads to generation of Oi- 2 or
its dismutation product H2O2 in apoplast (Doke, 1983; Grant et al., 2000). Radwan and coworkers
(2010) observed higher H2O2 and MDA concentrations in Vicia faba leaves infected with bean yel-
low mosaic virus than those of the corresponding controls. Several enzymes have been implicated in
apoplastic ROS production following successful pathogen recognition. The use of inhibitors pointed
to plasma membrane NADPH oxidases and cell wall peroxidases as the two most likely biochemical
sources (Grant et al., 2000). The expression of these enzymes is induced following recognition of
bacterial and fungal pathogens (Chittoor et al., 1997; Sasaki et al., 2004). Although the primary oxi-
dative burst following pathogen recognition occurs in the apoplast, ROS can be produced in other
cellular compartments like mitochondria and chloroplast. Abdollahi and Ghahremani (2011) stud-
ied the role of chloroplasts in the interaction between Erwinia amylovora and host plants by using
uracil as chloroplast ETC inhibitor. Uracil presence significantly reduced ROS generation during
pathogen–host interaction, and ROS generation corresponded with the appearance of necrosis in
all cultivars (Abdollahi and Ghahremani, 2011). Liu and coworkers (2007) showed that activation
of the SIPK/Ntf4/WIPK cascade by pathogens actively promotes the generation of ROS in chloro-
plasts, which plays an important role in the signaling for and/or execution of HR cell death in plants.
They concluded that chloroplast burst occurs earlier than NADPH oxidase burst, and mitochondria-
generated ROS might be essential in accelerating the cell death process.
Differential regulation of antioxidant enzymes, in part mediated by SA, may contribute to
increases in ROS and activation of defenses following infection (Mittler et al., 1999; Klessig et al.,
2000). In tobacco, the reduction of CAT and APX activities resulted in plants hyperresponsive to
pathogens (Mittler et al., 1999). A significant increase in the activities of POD and CAT was observed
in leaves of flax lines infected with powdery mildew (Ashry and Mohamed, 2012). Increase in POD
activity was much pronounced in tolerant lines than susceptible lines. Enhanced activities of POD,
CAT, APX, and SOD were observed in V. faba leaves infected with bean yellow mosaic virus indi-
cating that the ROS-scavenging systems can have an important role in managing ROS generated in
response to pathogens (Radwan et al., 2010).
ROS are unavoidable by-products of normal cell metabolism. ROS are generated by electron
transport activities of the chloroplast, mitochondria, and plasma membrane or as a by-product of
various metabolic pathways localized in different cellular compartments. Under normal growth
condition, ROS production in various cell compartments is low. However, various environmental
stresses such as drought, salinity, chilling, metal toxicity, and UV-B, if prolonged over to a certain
extent, disrupt the cellular homeostasis and enhance the production of ROS. ROS play two diver-
gent roles in plants; in low concentrations, they act as signaling molecules that mediate several
plant responses in plant cells, including responses under stresses, whereas in high concentrations,
they cause exacerbating damage to cellular components. Enhanced level of ROS causes oxidative
damage to lipid, protein, and DNA leading to altered intrinsic membrane properties like fluidity,
ion transport, loss of enzyme activity, protein cross-linking, inhibition of protein synthesis, and
DNA damage, ultimately resulting in cell death. In order to avoid the oxidative damage, higher
plants possess a complex antioxidative defense system comprising of nonenzymatic and enzymatic
components. Although rapid progress has been made in recent years, there are many uncertain-
ties and gaps in our knowledge of ROS formation and their effect on plants mainly due to short
half-life and high reactivity of ROS. Study of formation and fate of ROS using advanced analytical
techniques will help in developing broader view of the role of ROS in plants. Future progress in
genomics, metabolomics, and proteomics will help in the clear understanding of biochemical net-
works involved in cellular responses to oxidative stress. An improved understanding of these will
be helpful in producing plants with inbuilt capacity of enhanced levels of tolerance to ROS using
biotechnological approach.
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27 Implications of Oxidative
Stress for Crop Growth
and Productivity
Abdul Wahid, Muhammad Farooq,
and Kadambot H.M. Siddique
CONTENTS
27.1 Introduction........................................................................................................................... 549
27.2 Diversity and Metabolism of ROS in Plants.......................................................................... 550
27.2.1 Superoxide Anion...................................................................................................... 551
27.2.2 Hydrogen Peroxide.................................................................................................... 551
27.2.3 Hydroxyl Radical....................................................................................................... 552
27.2.4 Singlet Oxygen........................................................................................................... 552
27.3 Oxidative Stress and Plant Productivity................................................................................ 553
27.4 Conclusion............................................................................................................................. 554
References....................................................................................................................................... 554
27.1 INTRODUCTION
Biological systems show the operation of metabolic phenomena in a coordinated manner. Such coor-
dination occurring among various cellular organelles is involved in the generation of reactive oxygen
species (ROS). Of the various ROS, superoxide anions (O2- ), singlet or activated oxygen (1O2), hydro-
gen peroxide (H2O2), hydroxyl ions (OH−), and activated oxygen (O2) (also referred to as prooxi-
dants) are biologically the most important. Since aerobic life originated 2.7 billion years ago, these
partially reduced forms of oxygen have been uninvited companions of molecular oxygen. Although
initially thought as toxic by-products of the metabolism, ROS are now known to play important
roles in modulating plant growth by acting as signaling molecules (Bailey-Serres and Mittler 2006).
Reactive oxygen species are involved in the regulation of growth, development, and defensive path-
ways (Dat et al. 2003) and the unfavorable induction of lipid peroxidation of biomembranes (Liu
and Huang 2000; Farooq et al. 2009). These properties make ROS versatile actors in plant biology.
There is evidence that ROS are necessary for root hair growth, where they manage the activity
of Ca2+ channels necessary for polar growth (Carol and Dolan 2006). Among ROS, H2O2 regulates
gene expression in cells such as genes encoding antioxidants (e.g., catalase, superoxide dismutase
[SOD] peroxidase), cell defense, signaling, stress proteins, and transcription factors (Hernández
et al. 2001, Pastori and Foyer 2002; Wahid et al. 2007a). Hydrogen peroxide breaks seed dormancy
in conifers and enhances heat and salt tolerance in rice (Uchida et al. 2002) and wheat (Wahid et al.
2007b). However, as a result of ROS production, peroxidation of membrane lipids and damage to
important molecules such as nucleic acids, proteins, chlorophyll, and other important macromol-
ecules occur (Foyer and Fletcher 2001; Hernández et al. 2001; Wahid et al. 2007a). Thus, a balance
between ROS production and their elimination is a prerequisite for normal growth and productivity.
549
Abiotic stress Biotic stress

(temperature, salinity (pathogens, allelopathy,
drought, nurtients, chemicals) wounding, nodulation)
Induction
Natural Optimum
production ROS growth
Oxidative stress
Reduced growth
FIGURE 27.1 Sources for ROS production in plants. Under normal conditions, ROS production and quench-
ing rates correspond with each other, thus having a profound effect on growth. Stress conditions induce higher
ROS synthesis, which surpasses their scavenging rate thus leading to damage to cellular functions.
In addition to their normal routes of production, various environmental cues induce the
production of ROS, thereby causing an imbalance in their production and dousing (Figure 27.1;
Bhattacharjee 2012). These ROS cause losses to biological membranes, both cytosolic and
those of organelles, thus disrupting cellular function (Karuppanapandian et al. 2011). Plants
have developed defense systems to reinstate the balance between the production and scaveng-
ing of ROS. These defenses are both enzymatic and nonenzymatic in nature. The efficient
operation of these systems determines the tolerance to prevailing stress conditions (Wahid
et al. 2007a).
It has been widely accepted that, irrespective of causative factors, the ultimate outcome of oxida-
tive stress is the reduction in plant growth and productivity. However, the extent of this reduction
varies between plants. This chapter presents an overview of the properties, sources, emerging roles,
and repercussions of ROS for plant growth and productivity.
27.2 DIVERSITY AND METABOLISM OF ROS IN PLANTS

Reactive oxygen species occur globally in the plant kingdom. Of those, O2- , OH, H 2O2 , and sin-
glet oxygen 1O2 are among the most important and play pivotal metabolic roles in plant biology
(Gill and Tuteja 2010; Sharma et al. 2012). Since the introduction of aerobic life on earth some
2.7 billion years ago, the production of ROS from molecular oxygen (O2) eventually became
fundamental to cellular functions (Halliwell 2006). Briefly, O2 released during photosynthesis
is nonreactive but becomes highly reactive in the form of 1O2 after gaining extra energy and O2-
after accepting electrons. The dismutation of O2- leads to the production of H 2O2 via the Fenton
reaction, while H 2O2 is either scavenged if scavengers are present or converted to OH− follow-
ing the Haber–Weiss reaction (Sharma et al. 2012). Reactive oxygen species have different half-
lives, physiological concentrations, movement within cells, and reactivity (Table 27.1), which
make them biologically quite important.
Implications of Oxidative Stress for Crop Growth and Productivity 551
TABLE 27.1
Physicochemical Characteristics of the Four Major ROS in Plants
Half-Life Physiological
ROS (s) Concentration Reactivity Movement References
Superoxide anion O ( ) -
2 10−6 ∼10−11 M Highly reactive 30 nm D’Autréaux and
Toledano (2007),
Karuppanapandian
et al. (2011)
Hydrogen peroxide 10−3 ∼10−7 M Mildly reactive 1 μm D’Autréaux and
(H2O2) Toledano (2007),
Karuppanapandian
et al. (2011)
Hydroxyl radical 10−9 Traces to 10−10 M Highly reactive 1 nm D’Autréaux and
(OH−) Toledano (2007),
Shao et al. (2008),
Karuppanapandian
et al. (2011)
Singlet oxygen (1O2) 10−5 ∼10−9 M Highly reactive 30 nm Knox and Dodge
(1985), Krieger-
Liszkay (2005),
Karuppanapandian
et al. (2011)
27.2.1 Superoxide Anion
Denoted as O2- , superoxide is a highly reactive anion and perhaps the most damaging ROS (Sharma
et al. 2012). Being short-lived, O2- can move over short distances and therefore is toxic mainly to
the site of its production. Moreover, the presence of a negative charge limits its role as a signaling
molecule (D’Autréaux and Toledano 2007). O2- is produced in a variety of cellular components due
to the activity of NADP oxidases during respiration, a large number of photo-oxidation reactions,
the Mehler reaction in the chloroplast, redox reactions of mitochondria, and photorespiration in
peroxisomes and glyoxysomes (Karuppanapandian et al. 2011).
As a resistance strategy to O2- , cells immediately eliminate it from the site of synthesis. This elim-
ination may be both enzymatic and nonenzymatic. Superoxide dismutases are a class of enzymes for
the dousing of O2- by conversion to H2O2 and O2. In addition to enzymatic dismutation of O2- , flavo-
noids (Robak and Gryglewski 1988), carotenoids especially β-carotene and lycopene (Trevithick-
Sutton et al. 2006), and sparteine, a quinolizidine alkaloid (de Pinto and Ros Barceló 1997), have
been documented.
27.2.2 Hydrogen Peroxide
A relatively more stable molecule in the ROS synthesis pathway, H2O2 is produced as a result of spon-
taneous or enzymatic dismutation of O2- , which gains one electron and two protons (Sharma et al.
2012). The common sites for production of H2O2 are the electron transport chain in mitochondria,
chloroplasts, endoplasmic reticulum, and plasmalemma. Photorespiration taking place during photo-
synthesis in three organelles (chloroplast, mitochondria, and peroxisomes) is an important source of
H2O2 (Taiz and Zeiger 2010; Karuppanapandian et al. 2011). H2O2 is mildly reactive, with a relatively
longer half-life (10 −3 s). It can diffuse over short distances and can cross and damage both protein
and lipid components of the membrane. Major targets of H2O2 damage are thiol groups of enzymes
especially those of the Calvin cycle (Sharma et al. 2012). β-Oxidation of membrane lipids, leading to
enhanced membrane permeability to solute leakage, is a well-known phenomenon triggered by H2O2
(Vellosillo et al. 2010). Conversely, when present at low concentrations, a mild reactivity and greater
diffusion of H2O2 makes it an important signaling molecule (Halliwell 2006; Möller et al. 2007).
The quenching of H2O2 is an essential component of tolerance against its toxicity. The dousing
of H2O2 in plants is both enzymatic and nonenzymatic. Catalases and peroxidases are important
enzymatic sources to scavenge H2O2. Catalase, also known as H2O2 oxidoreductase, is responsible
for dismutating H2O2 into H2O and O2. It has been documented that any reduction in the level and
activity of catalase is likely to strongly reduce H2O2 elimination from the site of its synthesis and
action (Dat et al. 2003; Sharma et al. 2012). Peroxidases are a family of compounds, such as cyto-
chrome C peroxidase and glutathione (GSH) peroxidase, which use H2O2 as a substrate for activity
and by doing so eliminate it from the system (Dat et al. 2003).
Among the nonenzymatic cellular arsenal, the most studied source is ascorbate. It is synthesized
in mitochondria to a fairly high concentration (∼50 mM) and is transported to other cellular com-
ponents. It reduces H2O2 to water in the presence of ascorbate peroxidase (Foyer and Noctor 2005a;
Shao et al. 2008). In addition to its direct role in scavenging H2O2, ascorbate participates in the
synthesis of α-tocopherol and zeaxanthin in the xanthophyll cycle, which are important scavengers
of H2O2 (Foyer and Noctor 2003, 2005b).
Glutathione—a thiol group containing nonprotein compounds—is an important agent that con-
trols H2O2 levels in the cell by cycling between the reduced (GSH) and oxidized (GSSG) states.
A high GSH/GSSG ratio is therefore desirable for ROS quenching and signaling in plants (Möller
et al. 2007). A number of phenolic compounds including quinones and flavonoids have important
roles in the scavenging of ROS including H2O2. They act as terminators of free radical chains and
can change the peroxidation of lipid kinetics by modifying their packing order (Winkel-Shirley
2002), which ultimately leads to improved membrane integrity (Blokhina et al. 2003). As a mecha-
nism of action, peroxidase enzymes oxidize flavonoids and phenylpropanoids, which help in H2O2
quenching via the phenolic/AsA/POD system (Sharma et al. 2012).
27.2.3 Hydroxyl Radical
Hydroxyl radical is produced from H2O2 following the Haber–Weiss route if the scavenging system
is not functional. It is highly reactive with a strong oxidizing potential close to its production sites,
and being too short-lived, it does not pass through the membrane. Due to its high reactivity, OH−
damages almost all biomolecules. It causes peroxidation of membrane fatty acids, oxidizes amino
acid residues near the cation-binding site, and damages DNA by reacting with purine and pyrimi-
dine as well as deoxyribose sugar (Halliwell and Gutteridge 2000; D’Autréaux and Toledano 2007;
Karuppanapandian et al. 2011; Sharma et al. 2012).
A high damaging potential of OH− prompts its rapid elimination from its site of synthesis. There
are no enzymatic systems for scavenging OH−; however, mannitol has been suggested to protect
phosphoribulokinase from being oxidized by OH− via thioredoxin, ferredoxin, and GHS synthesis
(Shen et al. 1997). In addition, flavonoids by virtue of their chemical properties are effective scav-
engers of OH− (Chen et al. 2002).
27.2.4 Singlet Oxygen
As a result of gaining extra energy by O2, 1O2 is produced by photoinhibition and electron trans-
fer in photosystem II (PS II) during photosynthesis (Karuppanapandian et al. 2011; Sharma
et al. 2012). Chlorophyll molecules act as a photosensitizer for its production (Agnez-Lima et al.
2009). 1O2 is a highly reactive ROS produced in biological systems. Although 1O2 has a very short
half-life, it has the ability to diffuse and biomembranes exhibit permeability to its movement.
Once generated in the biological system, 1O2 can readily oxidize proteins, lipids, and DNA
(Scandalio 1993; Karuppanapandian et al. 2011; Agnez-Lima et al. 2012).
The highly toxic nature of 1O2 demands its elimination as a detoxification mechanism. Unlike
the superoxide anion, there is no enzymatic machinery for its quenching. However, certain non-
enzymatic sources including carotenoids, ascorbic acid, and α-tocopherol have been reported to
be important in its scavenging. Carotenoids, in addition to their role in light harvesting, also help
in the quenching of activated chlorophyll (3Chl) and 1O2. Carotenoids, with more than nine double
bonds (such as β-carotene and lutein), play a major role in heat transfer and reduced generation
of 1O2 (Knox and Dodge 1985). α-Tocopherol is a lipid-soluble, low molecular weight compound
regarded as an important antioxidant to scavenge 1O2. It chemically reacts and physically quenches
1O from PS II in the thylakoid membrane (Shao et al. 2008; Karuppanapandian et al. 2011; Sharma
2
et al. 2012). Ascorbate, which is present in the chloroplast stroma to 2–3 mM, is also an effective
quencher of 1O2. By virtue of its 1O2 quenching ability, ascorbate can repair the damage caused to
the D1 protein of PS II in the thylakoid membrane (Knox and Dodge 1985; Krieger-Liszkay 2005).
Among the various other secondary metabolites, quinones, especially the hypencins in Hypericum
hirsutum, are important quenchers of 1O2 (Knox and Dodge 1985).
27.3 OXIDATIVE STRESS AND PLANT PRODUCTIVITY

Environmental and biotic factors are important driving elements in the enhanced production of ROS
in plant cells leading to disruption of the balance in their synthesis and quenching (Figure 27.1).
Oxidative stress changes the phase, constituent, and permeability of biomembranes. A number of
studies are available on oxidative stress, lipid peroxidation, and changes in the antioxidative enzy-
matic activities of plant species (Wahid et al. 2007a, 2009; Sharma et al. 2012).
The damage caused by ROS to cellular function is difficult to assess in empirical terms due
to the intricacy of the processes involved in ROS synthesis. However, it is possible to make some
generalizations from the environmental stresses per se (Table 27.2). The duration of exposure to
stress conditions has been regarded as yet another important factor (Karuppanapandian et al. 2011).
A few studies have shown that drought stress is the most important factor in the production of ROS
and reduced plant productivity (Farooq et al. 2009; Chugh et al. 2011). Likewise, the type of ROS
TABLE 27.2
Influence of the Generation of ROS on Growth and Yield Reduction in Some
Important Environmentally Stressed Plant Species
Environmental
ROS Stress Plant Species Damage (%)a References
Superoxide anion O2-( ) 500 mM NaCl Beta maritima 15 Bor and Turkan (2003)
Hydrogen peroxide Drought Pea 15 Moran et al. (1994)
(H2O2) Maize 40 Chugh et al. (2011)
A. thaliana 74
Hydroxyl radical Low temperature Beta vulgaris 7 Gulen et al. (2008)
(OH−) Strawberry 50 Bor and Turkan (2003)
Drought Ctenanthe setosa 20 Terzi and Kadioglu
(2006)
Singlet oxygen (1O2) Photooxidative A. thaliana 15 Triantaphylides et al.
stress (2008)
a Damage caused to growth and ultimate yield. The values were derived as the percentage reduction relative to
the respective control.
is important. Under drought stress, H2O2 is the most toxic ROS (Chugh et al. 2011) in view of its
longer half-life and ability to penetrate cellular components (Table 27.1). Temperature stress is yet
another environmental factor that hampers membrane function by making them more labile (Bor
and Turkan 2003; Wahid et al. 2007a). Sandalio et al. (2001) assigned the reduction in pea (Pisum
sativum L.) productivity to substantially changed activities of oxidative metabolism enzymes and
ultrastructure changes in the organelles under cadmium stress.
Plants deploy an antioxidative arsenal to cope with the production of ROS and reduce the dam-
age to plant growth, which is both enzymatic and nonenzymatic (Mahmood et al. 2012; Xu et al.
2013). Differences among genera, plant species, and varieties of a plant species are also important
when studying the accumulation of ROS, induction of antioxidative systems, and generalizing the
damage to growth and biomass yield. For instance, reduction in growth of Arabidopsis thaliana
under drought stress was substantially more with the generation of H2O2 than with 1O2 (Table 27.2).
Also the yield loss in pea, maize (Zea mays L.), and A. thaliana differed under drought stress
(Moran et al. 1994; Chugh et al. 2011).
27.4 CONCLUSION
Land plants as a solitary part of nature are subject to the vagaries of the environment. The gen-
eration of ROS under normal metabolism is a physiological requirement. However, adverse con-
ditions such as biotic and abiotic stresses lead to their induction, causing an imbalance in their
production and dousing. This imbalance leads to considerable yield losses, which may account for
up to 70% or more. There are four main ROS that cause oxidative stress in plants—superoxide
anion, hydrogen peroxide, hydroxyl radical, and singlet oxygen—all of which have been thor-
oughly studied. The extent of damage caused to plants by ROS is mainly related to physicochemi-
cal properties and the ability of ROS to penetrate cellular components. In addition, the prevailing
stress conditions, plant species, generation of specific ROS, and induction of antioxidative arsenal
are of paramount importance in the final growth and productivity of plants. The search for plants
better able to express ROS-scavenging systems should fetch better yields from environmentally
stressed lands.
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28 Physiological and Biophysical
Responses of Plants under Low
and Ultralow Temperatures
Jiří Zámečník and Miloš Faltus
CONTENTS
28.1 Heat and Temperature............................................................................................................ 558
28.1.1 Heat Transfer............................................................................................................. 558
28.1.2 Temperature and Heat Capacity................................................................................ 558
28.1.3 Energy of Phase Transition........................................................................................ 559
28.2 Low Temperatures................................................................................................................. 561
28.2.1 Frost Stress................................................................................................................. 561
28.2.1.1 Plant Response to Frost............................................................................... 562
28.2.1.2 Coping with Frost....................................................................................... 563
28.2.1.3 Plant Protection against Frost..................................................................... 563
28.2.2 Freezing Stress...........................................................................................................564
28.2.2.1 Freezing of Water........................................................................................564
28.2.2.2 Ice Nucleation............................................................................................. 565
28.2.2.3 Ice Crystal Growth...................................................................................... 566
28.2.2.4 Crystal Size Distribution............................................................................. 567
28.2.2.5 Coping with Freezing Stress....................................................................... 567
28.2.3 Plant Adaptations to Freezing Stress......................................................................... 569
28.2.3.1 Cold Hardening........................................................................................... 570
28.2.3.2 Osmotic Adjustment................................................................................... 571
28.2.3.3 Ice Growth Inhibitors.................................................................................. 572
28.2.3.4 Antifreezers................................................................................................ 572
28.2.3.5 Barriers to Ice Propagation......................................................................... 572
28.2.3.6 Adaptation of Herbs and Trees to Low Temperatures................................ 574
28.3 Ultralow Temperatures.......................................................................................................... 575
28.3.1 Glass Transition......................................................................................................... 575
28.3.2 Water and Glass Formation....................................................................................... 576
28.3.3 Cryoprotectants.......................................................................................................... 577
28.3.4 Plant Long-Term Storage at Ultralow Temperatures................................................. 580
Acknowledgment............................................................................................................................ 580
References....................................................................................................................................... 580
557
28.1 HEAT AND TEMPERATURE

28.1.1 Heat Transfer
Heat flows by transferring energy to adjacent molecules of any state of matter, and this transferred
energy causes an increase or decrease in temperature and internal energy of adjacent molecules.
The transfer of energy is made via molecules and they move to a region of lower temperature.
The environment acts as a heat sink that absorbs/releases the thermal energy during cooling or
freezing from the plant until equilibration with the surroundings is reached. The rate of heat flux
depends on the way of heat transfer. Heat transfer can be divided into three different categories,
depending on the way the energy transmits in and out of plants: conduction, convection, and radiation.
Conduction—Heat moves from the warmer part of the plant to its cooler part as a result of
differential temperature or between different plant parts usually connected by xylem ves-
sels filled with water, through which the plant can be in heat conduction connection.
Convection—Heat from a plant is transferred to the surrounding of plant by circulating liq-
uid or air. Convection is the most powerful mechanism of heat transfer during cooling
and freezing of biological systems. This process of energy transfer has been described as
combining the action of heat conduction, energy storage, and mixing motion in a number
of steps.
Radiation—Heat flux through space in the form of infrared radiation. Radiation of the heat
usually occurs at the samples taken from liquid nitrogen to the surroundings at room tem-
perature. Radiation does not require physical contact between objects.
28.1.2 Temperature and Heat Capacity

Temperature is the measure of how hot or cold something is; specifically, a measure of the average
kinetic energy of the particles in an object. The transfer of energy always takes place from a sub-
stance of higher temperature to that of lower temperature. Heat is the energy transferred between
objects that have different temperatures. One can use temperature to predict which way the energy
transfer is occurring. Heat is the thermal energy of the particles in a substance due to the kinetic
energy of the particles moving.
Temperature is a property of a system in thermal compensation. Heat energy is transferred
between systems with different temperatures. Heat is expressed in units of Joules (J). Older units
are calories (cal). Heat capacity of system C is a proportional constant between absorbed heat (∆Q)
and temperature change (∆T):
DQ DQ
C= = (28.1)
DT Tk - Tp

where
Tk is the final temperature
Tp is the initial temperature
The specific heat capacity (c) is defined as the amount of energy that is needed to produce a unit
of temperature change, to raise the temperature of a unit mass of one degree, and depends on the
properties of the substance:
C
c = Æ DQ = c m DT (28.2)
m
where m is the mass. Commonly used heat capacity Cp is measured at constant pressure.
The heat capacity of water is the energy (4.2 kJ kg−1 K−1) (Table 28.1) needed to raise the tem-
perature of 1 kg water by 1°C from 0°C.
Physiological and Biophysical Responses of Plants under Low and Ultralow Temperatures 559
TABLE 28.1
Specific Heat Capacity (kJ kg−1 K−1) of Water, Ice, Main Organic
Substances, and Ash from Plants
Temperature (°C) H2O Ice Protein Fat Carbohydrate Fiber Ash
40 4.1786 2.05 2.04 1.62 1.91 1.16
30 4.1785 2.04 2.02 1.60 1.90 1.15
20 4.1819 2.03 2.01 1.59 1.88 1.13
10 4.1922 2.02 2.00 1.57 1.86 1.11
0 4.2177 2.050 2.01 1.98 1.55 1.85 1.09
−10 2.000 2.00 1.97 1.53 1.83 1.07
−20 1.943 1.98 1.95 1.51 1.81 1.05
−30 1.882 1.97 1.94 1.48 1.79 1.03
−40 1.818 1.96 1.92 1.46 1.77 1.01
Note: Data for water from Anonym (2013a), and for ice from Anonym (2013b) and other data
were calculated according to published equations from Choi and Okos (1986).
28.1.3 Energy of Phase Transition

Phase transitions can be divided into two types:
1. The phase transition of the first order, when the entropy, enthalpy, and volume changes
abruptly by jump. The latent heat emits or absorbs. Melting, evaporation, and sublimation
are examples of the first-order transition.
2. The phase transition of the second order, when the first derivative of these variables (heat
capacity and expansion coefficient) changes by jump. Vitrification (glass transition) is an
example of the second-order transition.
Specific latent heat (or enthalpies or energies) has two main forms of those commonly encountered:
latent heat of fusion (melting or freezing) and latent heat of vaporization (boiling or condensing).
The energy flow from one phase to the next one according to the endothermic or exothermic reac-
tion is depending on these phase changes, for example, melting is an endothermic reaction, while
ice crystal formation is the exothermic reaction (Figures 28.1 and 28.2).
Latent heat (L) is expressed as the amount of heat accompanying the phase transition related to
the weight unit of the system:
Q
L= (28.3)
m
where
Q is the absorbed heat
m is the mass
The latent heat describes how much energy is required to change a state of matter. Latent heat can
be used by calculation to predict the effects of larger temperature changes for masses (Figure 28.1).
When converting, for example, ice to steam, heat is absorbed in three endothermic processes—
ice melting, water heating, and evaporation of water (Figure 28.1). If these processes are consecu-
tive, the absorbed heat of the process is the sum of the heat of each process.
Phase change Water Phase change Steam

solid liquid heating liquid vapor heating
100
Boiling
Heat of vaporization
Temperature (°C)
point
2260 kJ/kg
50
0 Ice melting point
Heat of melting 334 kJ/kg
–50
0 500 1000 1500 2000 2500 3000 3500 4000
Heat absorption (kJ)
FIGURE 28.1 Temperature dependence on the absorption heat of 1 kg of water at normal pressure, which
passes from the ice at −50°C to water vapor at a temperature higher than 100°C. Horizontal lines represent
phase changes, the corresponding oblique heat absorption: (a) the ice absorbs heat and increases its tem-
perature, (b) absorption of latent heat of fusion, (c) increasing the temperature of the hot water absorption,
(d) water boils and absorbs latent heat of evaporation, and (e) water vapor absorbs heat and its temperature
increases. When the temperature of the phase transitions remains unchanged, heat is consumed for the phase
transformation.
0 Freezing point Melting point
Supercooling
Temperature (°C)
AFP+ AFP–
Ice
nucleation
Recrystallization
Heat
of Freeze tolerance
crystallization
Freezing Thawing
Time
FIGURE 28.2 Temperature response of plants cooled to subzero and warmed up from subzero tempera-
tures. After supercooling at nucleation temperature, plants started to freeze. The transitions between liquid
and solid, typically involving large amounts of energy, present in sudden rise of temperature. Freeze-tolerant
and freeze-avoidance bars are in the range of temperatures. Beside the melting curve, there is schematically
expressed the size of ice crystals with (AFP+) and without (AFP−) antifreeze proteins.
Latent heat of vaporization is a heat escaping from plant by evaporation of water. This latent heat
of vaporization is specifically crucial for heat balance of plants. Water changes from liquid water to
water vapor by phase transition during evaporation and transpiration. This happens due to the dif-
ference of vapor tensions of interior of the leaf and water vapor tension of the surrounding air. The
stomata on the top and bottom of a leaf are the main regulatory element. Latent heat of evaporation/
condensation is 2260 kJ kg−1 that means for evaporation of 1 kg of 100°C hot water is needed to
absorb 2260 kJ of heat. Conversely, the condensation of steam per 1 kg of water creating at 100°C
releases 2260 kJ of heat to the surroundings.
Latent heat of vaporization of water has a great influence on the temperature of leaves. This is
the most important when water evaporates from the leaves of plants (transpiration) to maintain leaf
temperature lower than would correspond to the temperature of leaves exposed to sunlight. The low
temperature of 7°C induced rapid stomata closure in cold-tolerant Commelina communis leaves as
a defense reaction, but not in cold-sensitive tobacco leaves (Wilkinson et al., 2001). Under ambient
conditions at the alpine timberline during the winter months, Pinus cembra L. needle conductance
was almost fully suppressed, even at the highest needle temperatures, as compared to spring and
fall (Wieser, 2000).
Latent heat of melting is the energy required to melt 1 kg of the substance at its melting tempera-
ture. The example of water (Figure 28.1) clearly shows that it is needed to deliver 334 kJ for 1 kg of
ice to melt. The same situation is in the case of freezing, when for 1 kg of water to freeze, the same
energy has to be utilized. Water heat of fusion of 334 kJ kg−1 is one of the highest heats of fusion
in comparison to substances of similar molecular weight and molecular binding. From this point of
view, the water is underestimated.
Latent heat of sublimation is the heat needed to convert a solid directly into gas (vapor). An
example is the transition of the ice to water vapor. The opposite process is the process of deposition,
for example, freezing of water in the extracellular space or on the plant surface. That occurs due to
the difference between the water vapor tension over supercooled water and the water vapor tension
over extracellular ice.
The liquid phase has a higher internal energy than the solid phase. This means that the
energy must be supplied to a solid in order to melt it, and energy is released from a liquid
when it freezes. The molecules in the liquid have weaker intermolecular forces and a larger
potential energy.
The heat from the plant is losing also by air movement around the plant by convection, which
depends on the quality of the surface, the plant size, and speed of the air movement. Biologically,
the high latent heat of water is advantageous, as it allows the temperature of an organism to be
buffered against rapid changes with alterations in the surrounding environment. Water acts as the
heat reservoir for winter day/night fluctuation of surrounding temperature from above to below zero.
Water can buffer or slow down it by phase transition. The heat capacity seems to be the important
property of water at lowering temperature. The heat capacity of water is unique in comparison to
another substance of similar composition.
28.2 LOW TEMPERATURES

28.2.1 Frost Stress
There are two types of frost influencing a plant according to energy flux: radiation and advection
frost. Frost occurs in nature at temperature zero or below. Radiation frost is typical at clear night-
time skies, calm; the temperature during the day is usually above zero with inversion weather situ-
ation. During advection, frost occurs at typical windy night, cloudy conditions, and during the day,
it is usually below zero with no inversion weather. In the literature, it is distinguished between frost
and freezing. Both types of frost situations occur commonly. Advection frost usually occurs during
the winter months and the radiation frost usually during spring, less during autumn time.
Hoar frost is a radiation frost when heat is radiated into the open sky. Loss of heat causes the
plant body to become more cooler than the surrounding mass. Water vapor deposits onto the surface
as white ice crystals during hoar frost at high relative humidity of the air. It is sometimes called
white frost compared to that so-called black frost that can occur at low relative air humidity. The ice
is brittle and it is seen as a black underlay. White hoar frost is not so dangerous for plants because
ice crystal deposition on the surface releases the heat that can increase the temperature by a few
degrees centigrade.
Plant does not have to be necessarily in a frozen state during frost. Plants in a special situation
below zero can be in a supercooling state at which no ice crystals are present. This occurs in the
nature quite often at some special organs of the plants, for example, dormant buds of trees. The
supercooling is a crucial avoidance factor.
28.2.1.1 Plant Response to Frost

The first consequence of lowering the temperature is the stop of flowing of cytoplasm, which causes
the disintegration of the cytoskeleton being dependent on energy supplied during respiration. A
partial plasma membrane damage leads to the loss of its semipermeability, a fault osmotic mem-
brane function, and a reduction of turgor pressure. The decrease of turgor pressure has a tremendous
impact on stopping plant growth. A prolonged duration of this condition may result in depletion of
energy pool and, as a consequence, cell death. The long influence of low temperatures results in the
death of the whole plant.
The role of low temperatures on respiration thus in carbon balance in plants is relatively small, com-
pared to the effects of high temperatures. The reduction in respiration, expressed as a break in Arrhenius
dependence, is close to 10°C, that is, at a lower temperature, a reduction in photosynthesis occurs.
Exposure to low temperature is accompanied by excessive water stress. Freezing affects mostly
plant roots so that they reduce their hydraulic conductivity and cannot adequately supply the plant
parts transpired over the ground. The result of this imbalance is a reduction in fluid with all the
external signs of wilted plants. Root growth is reduced due to cooling at much higher temperatures
than is the critical temperature for the aboveground part of the plant. When a stronger cooling
occurs, root system is damaged. Damage of the root system is in most plant species reversible after
the cold episode. Restore functions of the root system can be dependent on the severity of cold
stress. Cold has a direct effect on the leaves, which has resulted in locked open stomata. The posi-
tive effect of ABA application to improve the water status of plants after exposure to cold suggests
a positive role of this stress hormone in the tolerance of cold stress. ABA can help also for plant
survival at ultralow temperature (Bruňáková et al., 2011).
In frost-sensitive plant, cell disruption follows the course outlined earlier, which unfolds over
about 3°C depending on a cooling curve. Membrane integrity might be lost at around −4°C as ice
formation in plasma ruptures membranes. Intracellular freezing started at about −7°C and had been
inevitably lethal due to, that is, combined effects of membrane injury, symplast dehydration, and
protein denaturation. Tissue damage following freezing can be demonstrated by the loss of differ-
ential permeability, metabolite leakage, and failure to achieve either plasmolysis or deplasmolysis.
Bathing solution of frozen/thawed tissue thus shows a sudden increase in electrical conductivity
according to freezing damage and then serves as a reliable assay for comparative frost tolerance
(Prášil and Zámečník, 1998).
Lichens are an example of organism resistance to extreme temperatures in the coldest, terrestrial
regions of the planet. Their resistance to extreme temperatures is of interest and has been the subject
of numerous investigations. When studying the impact of environmental extremes on lichens, the
specific fact that they are composed of two symbiotic organisms has to be considered. Damage to
the lichen can result from injury to either, or both, mycobiont (90%–95% dry weight basis) or photo-
biont. In general, photobiont has been found to be more sensitive to both high- and low-temperature
stresses than mycobiont (Hájek et al., 2006; Kappen et al., 1996; Matthes and Feige, 1983; Schroeter
and Scheidegger, 1995).
The mechanism of how lichen can underlay the extreme frost tolerance is not well understood
yet. In laboratory experiments, most lichens tolerated lower temperatures than appear in any natu-
ral habitat (Kappen and Lange, 1970). The main two reasons for freezing damage are mechanical
damage by the formation of intracellular ice crystals and desiccation due to withdrawal of water
from the cells during freezing. In the fully dehydrated state, lichens are extremely frost resistant
(Haranczyk et al., 2012). Gradual cooling causes a less sensitive reaction than immediate exposure
to low temperatures (Lange and Kappen, 1972). This is probably due to slow cooling, which allows
intracellular water escape and freeze with extracellular spaces. The crystals are bigger than at rapid
cooling. It may also leave plants unharmed because it can induce vitrification of water in the cells.
This mechanism is belonging to one as is believed for higher plants freezing tolerance.
28.2.1.2 Coping with Frost

The first living strategy of plants is to avoid freezing by supercooling or by lowering the melting
point of their tissues and organs. Supercooling (sometimes called more properly undercooling) is a
known effect in many plants and plant tissues, for example, parenchyma cells (Ristic and Ashworth,
1994) or whole organs, as generative buds of Ribes (Ashworth and Wisniewski, 1991) and Malus
(Bilavčík and Zámečník, 1996) or vegetative buds of Abies (Sakai, 1982). The level of water super-
cooling may go down to −40°C (Šesták, 2004). The solid, hexagonal ice has the structure that is
uncanonical, with respect to the pentagon-like symmetry of solidifying clusters of liquid water that
contains a high number of incommensurable nuclei of icosahedron and dodecahedron (Šesták and
Zámečník, 2007).
The second strategy toward a better survival during sudden cooling is called an extra-organ
freezing (Sakai, 1979, 1982). The whole plant organ (e.g., bud) is protected by survival strategy
against ice nucleation and ice spreading inside the organ. Extra-organ ice formation resulted in bud
deep supercooling following frost dehydration (Chalkerscott, 1992)—see succeeding text.
The third living strategy of plants is based on tolerating the extracellular freezing (Beurroies
et al., 2004; Bilavčík and Zámečník, 1996; Malone and Ashworth, 1991; Šesták, 2004; Warmund
et al., 1988). Survival of such plant cells/tissues is based on their tolerance to excessive dehydration
of the protoplast. In the nature, quite a few of plant tissues survive temperatures down to −40°C, and
with the techniques used for preservation of plant genetic material, they can survive temperatures
even below −150°C.
One example of the above mentioned survival strategies of plant species, while tolerating ice in
their tissues is as follows: the woody plants can exhibit extra-organ freezing in their buds, extra-
cellular freezing in their bark tissues, and supercooling in their parenchyma cells of xylem rays
(Hirsh et al., 1985).
Plants are sensitive to cold damage even at zero temperatures, while the mechanism of frost dam-
age of these plants may be different. In some plants, even when ice formed just in the extracellular
space, all cells were collapsed after thawing. Caused damage witnessed more of the lethal effect of
cellular desiccation than the formation of ice crystals. In other plants, which are susceptible to frost,
mesophyll cells were also damaged by extracellular freezing of water. Epidermal cells and cells of
the vascular sheaths were damaged by intracellular ice formation during freezing (Ashworth and
Pearce, 2002).
28.2.1.3 Plant Protection against Frost

It is particularly beneficial to choose the proper strategy for plant protection against frost. There
are many published weather models on how to predict the weather, orographic disposition, which is
helping to choose the plant protection method against frost. The methods in plant protection differ
when there are expected subzero temperatures in windy conditions without an inversion and when
it is needed to protect against a mild radiation frost for a few hours. Shortly are mentioned the ways
how to protect the plant against frost events. First of all, the species and cultivars with improved
genetic potential to be tolerant to the frost stress have to be grown at the local site supported with a
good agrotechnical practice. The active frost protection can be achieved through the plant cover by
soil, or by organic materials, or by mulches, and or by foil. The over/under plant sprinklers, micro-
sprinklers, soil irrigation (to increase heat sink), and application of artificial fog reduce heat loss
by radiation. Heaters are more economically demanding, as well as the helicopters used for mixing
cold and warm atmosphere layers.
28.2.2 Freezing Stress

Freezing is describing what happens inside the plant, in contrary to the frost stress that is describing
the situation outside the plant. A frost event becomes a freeze event when ice forms in plant tissue.
Water starts to freeze at temperatures below zero in the presence of ice nuclei. The change of water
thermodynamic status can or cannot lead to injury of plant cell, depending on plant resistance. Plant
injury depends on many factors like length of hardening, hardening temperature, and physiological
status of plants, for example, dormancy and drought resistance (Bilavčík et al., 2012). Plant injury
due to freezing stress occurred when freezing of water caused death or malfunction of the plant cells
by ice crystal growth or desiccation.
28.2.2.1 Freezing of Water

Processes associated with water freezing (particularly in conjunction with its supercooling) have
been intensively studied (Bartell, 1997; Nitsch, 2009; Šesták, 2004; Šesták and Zámečník, 2007)
for many years due to their significance for both in nature plants (Burke et al., 1974) and in human
activity (food storage) (Goff and Sahagian, 1996; Slade and Levine, 1991). In the Earth environ-
ment, water freezing phenomena are well known to be connected with the formation of snow, hail,
and freezing of various water flows.
There are many strategies, depending on ice occurrence. Most overwintering plants can tolerate
the extracellular ice. After thawing, the water from extracellular ice can be taken back into the cells
and the plants can be without any injury. Some plants tolerate extracellular ice; some try to avoid
it through processes called supercooling, production of metabolic heat, and liquid glass creation;
or some special mechanisms developed to prevent ice formation within their tissues. The second
strategy is to tolerate the low temperature without ice formation. The cells are in the supercooling
stage. Intracellular freezing is usually lethal (Wisniewski and Fuller, 1999).
Unlike most other compounds, water expands as it freezes. It is believed that a great dipole
moment in the water molecules and creation of attractions among them create such property.
Explanation comes from a better understanding that water molecules form an infinite hydrogen-
bonded network. This network with confined and structured water molecules clustering becomes
more intensive and more concentrated under decreasing temperatures.
Measurement of temperature hysteresis, thus occurring between melting and solidification of
water confined in cavities, is a standard methodology called thermoporometry (Beurroies et al.,
2004). However, the possible interference/overlapping of the temperature hysteresis, caused either by
the very small porous size or originating from water (independent) supercooling, is still a question.
The melting of a solution is determined by the concentration and type of substance dissolved in
the solution. A simple equation describes the relationship between the osmotic potential (Ψs [MPa])
of a solution in equilibrium and ice at subzero temperature (T [°C]):
T
Ys = (28.4)
1.86
where
Ψs is the osmotic potential of solution (MPa)
1.86 is the constant
A 1 molal solution of ideal nonionized solute freezes at −1.86°C and has osmotic potential of −2.27 MPa.
The freezing point of pure water is 0.01°C. The solutes in plant cells depress the freezing point
below 0°C, and it is proportional to the concentration of dissolved solute particles. The freezing-
point depression of any unknown solution can be calculated from the osmotic potential as follows
from Equation 28.4.
Conversion of the freezing-point lowering of a solution from osmotic pressure values at 0°C is
easily accomplished by the following relation:
Ys
DTm = (28.5)
1.221
where
Ψs is the osmotic potential of solution (MPa)
1.221 is the constant MPa deg−1
Since Equation 28.5 is valid for solutions at 0°C (273 K), the equation must be corrected to ambient
temperature by multiplying it by the ratio of the ambient temperature and the absolute temperature
(ambient temperature in K/273 K):
Y s 273
DTm = (28.6)
1.221 Ta
where Ta is the ambient temperature in K.

The effect of osmotic potential on the freezing-point depression also holds for nonideal solutions.
However, the freezing-point depression is nonlinear with concentration changes during dehydration.
Osmotic potential (MPa at 0°C) can be derived by the following empirical equation (Crafts et al.
1949; Lewis, 1908):
Y s = -1.206 DTm + 0.0021 DTm2 (28.7)

where ∆Tm is the freezing-point depression.
28.2.2.2 Ice Nucleation

Supercooling occurs when water solution freezes at a lower temperature than is the freezing-point
depression. Supercooling is essential for the survival of plants at low temperature stress. The ice
nuclei start the freezing process. Most common ice nuclei are ice crystals. Biological ice nucleators
like bacteria and fungi spores (Figure 28.3) can occur also on the plant surface.
Under normal atmospheric pressure, the typical liquid water can be easily supercooled
down to about −25°C, with some further requirements of purity as low as −38.1°C and with an
enhanced supercooling (such as in small droplets of ∼5 μm diameter) down to the lowest −40°C.
The lower temperature limit for water supercooling, also known as the heterogeneous freezing
point, is achieved at water activity equal to one, and the associated freezing-point depression
(Broto and Clausse, 1976) is close to −40°C. The homogeneous freezing point reduces about
twice the melting point (Equation 28.8) by salts or hydrophilic substances. The spontaneous
ice nucleation temperature is influenced by the concentration of solutes reflecting in freezing-
point depression, as shown in the following equation for homogenous ice nucleation temperature
(Sakai and Larcher, 1987):
Th = -(38.1 + 1.86 DTm ) (28.8)

Temperature (°C)
–18 –16 –14 –12 –10 –8 –6 –4 –2 0
–1
–2
Log (ice nulclei/cell or spore)

Fusarium avenaceum Fusarium tricinctum –3
–4
–5
–6
–7
Pseudomonas syringae
–8
–9
FIGURE 28.3 Biological ice nucleators as heterogeneous ice nuclei occurring on the plant surface. Ice nucle-
ation activity was tested by thermobattery with 30 thermocouples in thirty 10 μL droplets. Ice nucleation activity
is expressed as the logarithm of ice nuclei per one bacteria cell able to form a colony units or per spore of fungi.
where ∆Tm is the freezing-point depression for the solution (in K); practically, the pure water drop-
lets can be supercooled down to −38.1°C.
During freezing, an initial ice nucleus, or more nuclei with the same ice nucleation temperature,
forms from the water cluster or on impurity particles. The rate at which ice crystals grow and the size
they attain are all dependent upon the rate at which heat is removed from the immediate environ-
ment. Crystal growth and recrystallization will depend on the rate of the heat supply to the system.
Besides the common effect of crystallization during the cooling, the comparable recrystallization
may possibly occur during rewarming as well, known as a cold crystallization that can be equally
dangerous for plant cells. Simulated ice nucleation of plant samples is thus used for investigation,
and the seeding by ice crystal is the simplest method so far developed. After equilibration and at
a suitable level of freezer capable of providing the high rate of cooling, a sudden decrease of tem-
perature is applied in order to induce the ice nucleation, and such a freezing protocol is commonly
serviceable for uniform samples. This can be done at an exact temperature, which corresponds to
the exothermic event taking place after the preliminary first freezing run of control samples.
Various producers supply different companies’ freezers with various auto-seeding accesso-
ries based on the other methods such as conditioning ice nucleation by vibration and ultrasound.
Alternatively, for an entrapped certain ice nucleation, active bacteria can become applicable to affix
nucleation at a proper temperature. The slower cooling rates could be applied to give enough time
for water movement outside the cells. A suitable method of the bacterial entrapping ice nuclei is
used (Zámečník et al., 1991).
28.2.2.3 Ice Crystal Growth

Isotropic and constant growth rate of nucleation leads to the known relationship by Avrami (JMAK)
equation:
m
X = 1 - e( -Bt ) (28.9)
where
B is a temperature-dependent parameter, that includes the speed of growth and nucleation
m is a variable dependent on the dimension of growth, termed Avrami exponent
t is time of the crystal growth
For 3D growth, m has a value of 4; for the 2D growth, a value of 3; and finally, for 1D growth, a
value of 2. New nucleation nuclei can grow at the expense of the deformed matrix if the Gibbs free
energy of the system decreases.
The critical radius of nuclei during recrystallization must be such that the driving force allows
the creation of a new surface of grain boundaries:
2g
rµb2 > (28.10)
r
The size of the critical radius in meters (e.g., γ = 0.5 J m−2, b = 5 10 −10 m, μ = 4 1010 Pa)
108
r> (28.11)
r
Assuming dislocation density, ϱ = 1014 m−2 for the critical radius value of 10 −6 m. If we start from the
classical fluctuation theory, these values indicate that critical nuclei would contain about 1013 atoms
and would overcome the energy barrier at 5.1010 J per 1 mol nuclei, which represents a period of
bud formation around 107 s. There are physical factors influencing the growth rate, such as surface
tension of solution, pressure, temperature, relative crystal velocity in the solution, and the Reynolds
number.
A key factor is the rate of cooling, which is crucial for the ice growth and sizing. The ice forma-
tion in plants meets a certain temperature stress. Water relocates, at slow cooling rates, from intra-
cellular to the extracellular space, where ice crystal can form easily (Figure 28.4).
However, the ice crystals outside the plant cells are not harmful. When the rate of freezing
becomes too intensive and thus water retains not enough time to move/diffuse outside the cells, the
ice crystals start forming inside the cell, which has a lethal effect on the cell.
28.2.2.4 Crystal Size Distribution

The size of ice crystals is decreasing with drop of the temperature. The size of critical ice nuclei,
depending on a proper size of water molecules cluster, is decreasing to approximately −40°C. At
this temperature, the number of water molecules in the cluster of liquid solution is the same as the
critical cluster size for ice nucleation. This temperature is called temperature of homogenous ice
nucleation (see Figure 28.9).
In plant physiology, the recrystallization (or cold crystallization) can occur during warming
after glass transition. Two assumptions are needed for recrystallization: the ice nuclei of the proper
size and supercooled water or solution from which the ice crystals can grow to be present in plant
tissue. The relationship between time and temperature of recrystallization is dependent on the tem-
perature and heating rate and, therefore, limited to two basic questions: Does the recrystallization
occur at any temperature? And what is the minimum time needed to complete recrystallization?
Recrystallization plot is quite complex, consisting of several basic processes, whose speed varies
and depends significantly on the composition of the substances in the plant (Figures 28.2 and 28.5).
28.2.2.5 Coping with Freezing Stress

Plants surviving low temperatures below freezing can be divided into three groups:
1. Plants susceptible to a low temperature below zero are damaged by the formation of intra-
cellular ice crystals, resulting in tearing of the cells and tissue necrosis. These plants can
avoid the formation of intracellular ice by supercooling or reducing the freezing point.
2. Plants tolerating freezing temperatures by deep dehydration of protoplasm when the water
gradually freezes extracellular. This group includes perennial terrestrial plants from areas
(a) (b) (c)
(d) (e) (f )
(g) (h) (i)
FIGURE 28.4 Ice spreading, dark spots in time-lap snaps of leaf abaxial view of cold-hardened winter rape
(Brassica napus L.) leaf during cooling down to −9°C. Dark spots increase their size, from (b) to (i), according
to ice front propagation in the leaf mesophyll. The white arrow in (i) shows how the veins of low order act as a
barrier to ice spreading. Bar represents 10 mm. Note: The arrow shows the direction of ice spreading from the
first, ice crystals initiation site to around secondary vein (i). The secondary veins act as a barrier to ice spread-
ing. The plants were cultivated in semisterile conditions so the ice nucleation from bacteria can be excluded.
(a) (b)
FIGURE 28.5 The evaluation of AFP effect from apoplastic solution of winter wheat seedlings. The plants
were cultivated either at (a) nonacclimating or (b) cold-acclimating conditions. The AFPs from apoplast
stopped the growth of ice crystals by direct binding to ice crystals’ axes—smaller size of ice crystals from
cold-acclimated plants, decreased by AFPs from apoplast as a result of cold hardening acclimation, is evident.
After ice crystal regrowth at −8°C for 24 h, the ice crystals in a thin layer were photographed under Nomarski
interference contrast at −6°C. The bars represent the same size of both photos 100 μm.
with cold winters. Typical representatives of this group are winter cereals, winter rape, and
most over wintering woody species.
3. Plants tolerant to low temperatures, has the cytoplasm in an amorphous state, which is
therefore highly viscous and inhibits the growth of ice crystals. This situation has been
described in several deciduous tree species, and most likely, it is a strategy for some trees to
survive very low temperatures in combination with the aforementioned points (1) and (2).
Low temperatures determine the occurrence of organisms, depending on how intense they are
and for how long they last and how often and for how long the thawing takes place in between
episodes of freezing. The rate of cooling plant parts can predict how they will be frost damaged.
Slow freezing rate of 1°–3°C h−1 is essential for the survival at equilibrium freezing of water. The
strategy for (1) and (3) is a prerequisite for a higher rate of freezing and the low water content
in tissues.
The low temperature can act as a stimulus, which determines whether or not the plant will
develop. The same applies to dormancy (Bilavčík et al., 2012) when plants must retrieve a final
number of days with an average low temperature in order to continue in following up the ontogenetic
stages. Low temperatures affect a shift into the next ontogenetic phase despite the fact that the sum
of the limit temperatures for this transition is not yet fulfilled.
Bud dormancy is often induced by low temperatures and/or photoperiod during dormancy—for
example, tree buds fail to grow because of internal conditions. They have not sum up a sufficient
number of days with low temperature even though the external conditions have met the condition
for growth. Vernalization refers specifically to low-temperature induction of flowering. Location of
the response is probably in meristems.
Furthermore, many evaluations of the impact of low temperatures have been based on a measure-
ment of survival. The survival of each plant organ can be different. Usually, the youngest plant parts
are more low-temperature tolerant than the oldest ones. It is caused in most cases by higher adapta-
tion ability of the young plant organs to low temperature. Whether a biological system withstands a
decrease in temperature successfully or not depends on many environmental factors, physiological
status, and on genetic background.
The plant regeneration after low-temperature injury to the whole plant depends on low tempera-
ture resistance of the organ critical for survival of the whole plant. Sometimes, the critical organ
cannot be a part of the plant with the highest resistance to the low temperature, for example, some
leaves of winter cereals are more low temperature resistant than the crown that is responsible for
the whole crop regeneration. In these crops, the correlations between resistance to the low tempera-
ture and survival of the whole plant were proved many times (Waalen et al., 2011), also shown in
Equations 28.12 and 28.13 (Fowler et al., 1999).
28.2.3 Plant Adaptations to Freezing Stress

Unlike most other compounds, water expands as it freezes. It is believed that the reason for such
properties is a strong dipole moment in the water molecules and creation of attractions among them.
Enlightenment comes from a better understanding that water molecules form an infinite hydrogen-
bonded network with confined and structured clustering. The clustering becomes more intensive
and better localized under decreasing temperatures.
A question remains: What is the state of water or the state of protoplasm in the plant so that
supercooled liquid can possibly change into the form of noncrystalline solid, that is, glass? The glass
formation at the narrow temperature range is called glass transition temperature (Tg). The glass tran-
sition fortunately does not possess the volume change as the transformation to ice does. The glassy
state, found for example, in a dormant twig of poplar tree (Hirsh et al., 1985) offers the explanation
at what state of solid water can be found in frozen tissues supposing that this state occurred due to
freezing dehydration of tissues (Bartell, 1997; Šesták, 2005).
28.2.3.1 Cold Hardening

Besides defense mechanisms developed during evolution, some plants are capable of much faster
adaptation to current conditions. However, even this process, called acclimation, requires enough
time and must meet environmental conditions (primarily temperature and photoperiod) for develop-
ing the maximum possible low-temperature tolerance.
Plant species growing in temperate zone adjust in autumn the overall metabolism to the com-
ing low temperatures. It includes synthesis of cryoprotectants such as soluble sugars (sucrose,
raffinose, stachyose), sugar alcohols (sorbitol, ribitol, inositol), and low-molecular nitrogen com-
pounds (proline, glycine, betaine). These solutes together with some proteins (dehydrins, cold-
regulated proteins, heat-shock proteins) contribute to stabilization of membrane phospholipids
and membrane and cytoplasmic proteins. All these substances maintain the hydrophobic interac-
tions, and some of them even ion homeostasis scavenge the reactive oxygen species or solutes
released from the symplast and can create a thin film around the plasma membrane thus prevent-
ing ice adhesion and cell disruption (Gusta and Wisniewski, 2013; Gusta et al., 2004; Iba, 2002;
Wang et al., 2002). It is assumed that a higher content of unsaturated fatty acids predisposes
plants for tolerance to cold, but the increased rate of formation of unsaturated lipids in mem-
branes and the temperature of the phase transition of fatty acids in response to cold is just one of
a series of adaptive mechanisms (Finegold, 1986).
Frost hardiness is a dynamic and composite property of plant cells involving cell size, wall thick-
ness, osmotic pressure of cell sap, and membrane properties, all of which featured in either delaying
onset or diminishing adverse consequences of ice formation (Wolfe and Bryant, 1999). In areas
where warm periods alternate with periods when temperatures drop below zero, such as the change
of day/night temperature, the plants are periodically subjected to freezing/thawing events. These
temperature changes are gradual and therefore regularly contribute to hardening of plants. After
the first phase of hardening, which takes place at temperatures close to zero (6°C–0°C), tempera-
tures just below zero (−2°C to −6°C) have a significant impact on increasing the resistance to frost
(Jánská et al., 2011, Segeťa, 1982). Light for photosynthetic processes of forming storage substance
is essential for to the first stage of hardening (Hudečková et al., 1990) as well as substances stored in
the grain. The second phase of hardening, for which the first stage is a prerequisite, can take place
already in the dark (Segeťa, 1982). Fluctuating temperature has a greater impact on resistance to low
temperatures than the constant low temperature. The increase in resistance to frost is exponential
with higher increase in the initial hardening of plants (Equation 28.12). An increase of the resistance
to low temperatures is dependent on the ontogenetic stage. Plants are able to harden more in the
dormant state or in short photoperiod than during the growing season.
The cumulative nature of the acclimation process and a close relationship between temperature
and rate of hardening (RateH) to model daily changes in lethal temperature (LT50) (Fowler et al.,
1999):
RateH = 0.012(10 - Tc )(LT50 - LT50C ) (28.12)
where
Tc is the average daily temperature of crown for temperatures below 10°C
LT50 is the critical temperature of crown at the previous day
LT50C is the genetic coefficient depending on the cultivar (such as the winter wheat cultivar
“Norstar” that has LT50C = −24°C)
If plants are exposed to a temperature near the level causing damage, then the period during
which the plants are exposed to this temperature is critical for their survival. Temperatures above
freezing in late winter or higher temperatures in the middle of winter for a few days can cause
rapid loss of plant frost resistance. When the daily mean temperature (Tc) of the crown is colder
than −3°C and the difference between the minimum lethal temperature (LT50M) attained during
low-temperature acclimation and the crown temperature (Tc) is greater than −12°C, then the daily
change in plant tolerance to low temperatures is calculated from the following equation:
LT -T
LT50n = LT50 - 50M c
(28.13)
e -0.654 (LT50M -TC ) -3.4 7
where
LT50 is the critical temperature of the crown of the previous day
LT50n is the crown LT50 of the current day
At temperatures warmer than 3.5°C, LT50M corresponds to the calculated daily LT50. LT50M cor-
responds to a genetic factor of tolerance (LT50C) to a low temperature when full acclimation is
achieved (Fowler et al., 1999).
Damage to plants is expected when the current day temperature at depth of the crown is lower
than the calculated critical temperature. The ability to obtain the resistance to low temperatures
decreases rapidly after a period of plant dormancy. There is a close correlation between the activity
of plants in the initiation of new growth in the spring and reducing resistance to low temperatures
(Levitt, 1980). Plants are able to repair the damage and restore physiological function after partial
frost damage.
28.2.3.2 Osmotic Adjustment

The supersaturate solution can supercool until it undergoes a glass transition. This indicates that
the proximity of chemical groups, which are capable of forming hydrogen bonds, can prevent water
molecules from fusing to the ice front and participating in the crystallization process.
The ability of intracellular water to act as a solvent for the cellular constituents is of particular
biological importance, and it can be measured indirectly from the osmotic behavior of cells by van’t
Hoff’s equation:
dlnK DH
= (28.14)
dT RT 2
where
K is the equilibrium constant
T is the temperature
H is the enthalpy of the reaction
R is the gas constant
The volume of water (V) along with n moles of solute in a cell is related to the osmotic pres-
sure by the van’t Hoff equation (Equation 28.14). If the cell would behave exactly according
to this relationship, it could be described as a perfect osmometer. However, observed osmotic
response of cells does not conform this equation until V is replaced by a term rV, where r rep-
resents the fraction of intracellular water that participates in the osmotic equilibrium. Values V
or r are approximately 0.8, implying that 20% of the intracellular water is osmotically inactive
(Cooke and Kuntz, 1974). Osmotic adjustment is a change in solute content either by transport
and accumulation from other plant parts or more often by storing the photosynthesis-produced
osmotic-active substances. Changing water content per se also can occur, this is not osmotic
adjustment. The relationship of decreased osmotic potential and freezing-point depression fol-
lows from Equation 28.6.
28.2.3.3 Ice Growth Inhibitors

The three classes of substances that inhibit ice nucleation are known:
1. The first class comprises those solutes that depress the freezing point. Depressing the
freezing point of a solution also depresses the nucleation temperature—see Equation 28.8.
2. The second class comprises a number of substances that have been positively identified
as having the ability to inhibit ice nucleation without significantly affecting the freezing
point. The mechanisms for these are not known, but it seems that they are active either on
the embryonic ice crystals, preventing them from growing, or on the heterogeneous nucle-
ating sites, preventing them from acting as nucleators (Holt, 2003).
3. The third class represents the so-called antifreeze proteins (AFPs) that are produced by
plants. They lower the ice nucleation temperature of ice nucleation substances (Zámečník
and Janáček, 1992), or they prevent the growth of ice crystals (Griffith et al., 1992).
28.2.3.4 Antifreezers
Some plants produce proteins and/or carbohydrates that act as antifreezers. Others induce freezing
of extracellular water before the water can reach the cell membrane. Sudden freezing of water is far
more destructive than its slow course of action, and others engage warming via heat provided by
external crystallization yet.
AFPs bind to the surface of ice crystals, thereby influencing their growth and shape, mainly to the
a1, a2, and a3 axes—crystals are then small and spicular (Atici and Nalbantoglu, 2003; Wisniewski and
Fuller, 1999). AFPs are activated by noncolligative depression of the freezing point of a solution. It was
concluded (Griffith et al., 2005) that cold-acclimatized winter rye leaves produce multiple polypep-
tides with antifreeze activity that appear to be distinct from antifreezers produced by fish and insects.
Extraction and isolation of AFPs from winter rye (Secale cereale L.) leaves were first done by Griffith
(Griffith et al., 1992; Hassas-Roudsari and Goff, 2012; Hon et al., 1994; Zámečník and Bieblova, 1994).
In cold-acclimated leaves, AFPs inhibit the propagation of ice from the surface of a leaf. No
freezing occurs until the tissue reaches its equilibrium freezing point when ice starts to propagate
through the apoplast (Figure 28.4). After the leaves get frozen, AFPs inhibit the recrystallization of
extracellular ice (Figure 28.5). AFPs can become particularly protective by slowing ice propagation
in such situations where fluctuating temperatures cause repeated freezing and thawing of tissues.
When an insulating snow cover keeps the temperature near the melting point of the extracellular
ice, recrystallization can be promoted (Griffith et al., 2005). Although ice can recrystallize more
slowly at lower subzero temperatures. The inhibition of ice recrystallization can be a benefit for
those plants that are frozen for prolonged periods (Griffith et al., 2005).
Contrary to AFPs that have been found in insects, plants, fungi, and bacteria, thermal hysteresis
proteins (THPs) have been found in insects mainly. In plant, the THP has a low effect 0.2°C–0.4°C
(Hassas-Roudsari and Goff, 2012). These AFPs are similar to members of three classes of patho-
genesis-related proteins (Hon et al., 1995).
28.2.3.5 Barriers to Ice Propagation

The barrier against the spread of ice in the crown of cereals was demonstrated. In the crown of cereals
(in winter triticale), the spread of the ice crystal occurs twofold slowed down (Wisniewski et al., 1997;
Zámečník et al., 1994), unlike in woods, at which ice front completely stops at ice barriers (Chalkerscott,
1992). The group includes tropical and subtropical herbs and shrubs and various plants from warm tem-
perate regions. The plants tolerate frost enduring low temperatures in the supercooled state; therefore,
they eliminate ice crystals in tissues. This group includes most deciduous woody plants that have buds
covered by scales to prevent the penetration of ice crystals inside the bud—see Figure 28.6.
Three possible barrier locations were found in Azalea bud tissues (Figure 28.6). First, a heav-
ily suberized, lignified area was observed beneath the bud scales and may serve as a barrier to
Scale
Flower
Bud axis
Vegetative bud
Stem
FIGURE 28.6 Longitudinally sectioned cold hardy Azalea flower bud. Black marked area indicates the
tissue with phenolic layer in cell walls acting as ice barrier. Ice barriers are located between the base of bud
scale and bud axis, between the bud axis and flower in flower pedicel tissues of each flower, and between the
stem and bud axis. (Redrawn and printed with permission from Chalkerscott, L., Ann. Bot., 70, 409, 1992.)
penetration of ice from scale tissue into the bud axis. A second heavily lignified zone occurs radially
through the bud at the point where the bud scales originate; this zone could prevent ice nucleation
from the stem into the primordia. Finally, cell walls of the flower pedicels seem to be suberized; the
presence of this hydrophobic barrier may partly explain the sequential, rather than simultaneous,
form of low-temperature exothermal pattern seen within the given bud.
Two possible barrier locations were found in wheat tissues (Figure 28.7). The first was found
between the crown and mesocotyl. The second was situated between mesocotyl and roots. In the
Leaves
Crown
Mesocotyl
Roots
FIGURE 28.7 The barriers (black marked in the picture) to ice propagating through the plant were found in
winter wheat tissues. The barrier in wheat plant appears to be of anatomical origin. The first barrier to ice prop-
agating was found between the crown and mesocotyl. The second was situated between mesocotyl and roots.
vp
vp 1 mm
vp
Oat –12°C Barley –14°C

(a) (b)
vp vp
Wheat–14°C Rye –16°C

(c) (d)
FIGURE 28.8 Longitudinal sections of winter cereal crowns 7 days after freezing and thawing. Each species was
frozen at its respective LT50 that is indicated at the bottom of each panel. Note the severely distorted shoot apexes of
each species. Unlabeled arrows indicate regions of the crown with barrier-like freezing responses. Note the continu-
ous barrier in (a) oat that was not seen in the other species: (b) barley, (c) wheat, and (d) rye. vp—signifies areas of
vessel plugging in the lower crown. (From Livingston, D.P. III. et al., PLoS ONE, 8, e53468, 2013. With permission.)
anatomical section, the discontinuity of xylem vessels was found (Figure 28.8). From these, it is
believed that anatomical structure of the tissue is creating the two barriers to ice spreading from soil
to plant part below and above the soil.
28.2.3.6 Adaptation of Herbs and Trees to Low Temperatures

The main difference between perennial herbs and fruit trees, playing a role in their response to low
temperature, is in the distribution of their critical organs. While the organs of the main importance
for regeneration of herbs are below the ground (e.g., crown of winter cereals) (Figure 28.8) or close
to the soil surface (e.g., meristematic tissue of winter rape), buds as critical organs of fruit trees are
situated far aboveground; hence, they meet lower temperatures. Woody plants, tolerating ice crys-
tals in their tissues, have several overall strategies to stay alive during temperatures below zero. The
critical temperatures for both herbs and trees relate strongly to the phenological stages.
The limits of the natural distribution of many plants, including some deciduous fruit trees,
are related to the minimum temperature at which supercooling can occur (i.e., homogeneous
nucleation point), which is near to −40°C. Below the homogeneous nucleation point, freezing is
intracellular and lethal (Burke et al., 1976; Ikeda 1982).
28.3 ULTRALOW TEMPERATURES

Surprisingly, after certain procedures, plants become capable of endurance of ultralow tempera-
tures artificially provided by immersion in liquid nitrogen (−196°C) (Sakai, 1960). For the first
time, it was demonstrated that the willow twigs collected during extremely strong winter, addi-
tionally prefrozen at −30°C, were successfully cryopreserved in liquid nitrogen for 1 year with a
subsequent plant regeneration (Sakai, 1960). The plants cannot be commonly adapted toward such
ultralow temperatures just during their evolutionary development, because such low temperatures
cannot naturally appear during plant evolution or their lifespan. The lowest temperature measured
on the surface of the Earth was −89.2°C (at the Russian Vostok Station in Antarctica), still far above
the boiling point of nitrogen. The tolerance of plants to the ultralow temperatures does not arise
from their long-term evolution or through acclimation to the lowest temperatures on the Earth. It is
allowed by the intrinsic physiological status of their membranes, being a result of their hardening.
28.3.1 Glass Transition
Vitrification (from Latin vitreum, “glass”) is a state of a solid mater with viscosity of liquid or it is
a liquid with solid matter viscosity. From this definition, it is obvious that the glassy state of matter
has a high-viscosity threshold of 1012 Ps. The glass transition is defined by glass transition tempera-
ture (Tg): usually at the half of heat capacity change. The change of heat capacity is also typical
for glass transition. Below the glass transition temperature still deep in ultralow temperatures, the
plant is at amorphous phase without ice crystals. Above the glass transition temperature, the plant is
molten or, through the cold crystallization temperature with ice crystal formation, is frozen.
It is believed that plants can survive ultralow temperature by formation of biological glass, a bio-
logical matter excluding formation of ice crystals. So successful cryopreservation methods are those
that involve biological glass formation by dehydration or adding cryoprotectant (Reed and Uchendu,
2008; Sakai, 1966) (Table 28.2).
The two-step freezing method was successfully applied to hardy tree buds, which were recov-
ered by grafting (Forsline et al., 1998; Sakai and Nishiyama, 1978). Controlled rate of cooling is
efficient for storing suspension and callus cultures, embryogenic cultures, and in vitro shoot tips
from temperate and subtropical plants (Reed and Uchendu, 2008). In addition, cryopreservation of
cultured cells and meristems was achieved by controlled slow prefreezing to about −40°C in the
presence of a relevant cryoprotectant (Sugawara and Sakai, 1974), in which single chemicals or
their combination in various ratios (mixed in a suitable cryoprotectant cocktails) can be employed.
The advantages of controlled rate of cooling include the use of standardized procedures associated
with preprogrammed cooling rates and the use of larger samples. However, the disadvantage of
TABLE 28.2
Examples of Elements and Substances Commonly Present
in Plants That Form Easily Glasses
Type Examples
Elements P, S
Oxides SiO2, P2O5
Aqueous solutions (Na, K, Li) Cl (aq.)
Organic compounds Glycerol, glucose, fructose, sucrose
controlled rate of cooling is dependence toward the possession of a freezer capable to maintain low
temperatures of −40°C together with a controlled unit for providing low cooling rates.
The rate of warming up the plant samples is essential for a high regeneration of plant. The high
rates of warming are applied to the plant samples simply to overcome the recrystallization of ice
crystal inside the plant cell.
28.3.2 Water and Glass Formation

In all cases, glass transition temperature can be determined at various water contents. The various
water contents are the result of different dehydration methods used. For example, the sugars and
amino acids are likely to be phase separated, which means that the reactants are differing both
in the water content and the level of plasticization (Table 28.2). Though the water activity in both
phases may be the same, the reactant may go through alternative glass transitions because the glass
transitions can be at a variance with plasticization in different amorphous phases.
A decrease of water vapor in the surrounding air is possible to reach in two ways using the so-
called static and dynamic dehydration:
1. The static dehydration is the dehydration under a constant relative humidity, in which the
constancy can be reached by equilibration of plant tissues over a saturated salt solution
(where the accessible water is dissolved into the oversaturated solution). Advantage of this
sample preparation is based on the realization of certain equilibrium of the water content
as a response to the proper activity of water. The essential disadvantage is a relatively long
time for reaching the constant water content.
2. The dynamic dehydration is carried out when the air with constant air humidity flow removes
water from plant samples to equilibrate within a certain time. By moving air, the excess water
vapor is removed from the sample leaving it with a higher humidity than relative humidity
in the input. The advantage of these methods is a shorter time for removing the water from
plant samples in comparison with the static method, but it has some exceptions. The rate of
hydration depends on many coupled features, such as the ratio of sample volume to surface,
permeability of surface for water vapor, and sensitivity of plants to the dehydration speed. In
the samples dehydrated too quickly, the surface can turn out to be overdehydrated and even
can get covered by a glassy layer, which bestows a diffusion barrier to further dehydration.
When lower air humidity is used, then a shorter time is needed for dehydration but requires a
greater air flow (with a defined constant humidity). The parallel samples must be weighed for
the determination of the final volume of water inside the samples. For dehydration, a sterilized
air flow in laminar flow boxes is commonly used. Therefore, a rather complex control of the
final dehydration level yields an undesirable disadvantage of this method.
Any commencement of a state diagram for water relies on at least two sources of information:
1. Variation of glass transition temperature (Tg) as a function of water content.

2. Curve-based determination of a freezing-point depression (Tm) (Equations 28.5 and 28.6)
can be completed up to the intersection with the Tg curve (Figure 28.9).
Temperature of both curves for homogenous and heterogeneous nucleation depends on water/solute
concentration. A state diagram cannot be created without detailed knowledge of water content
and the corresponding glass transition temperature; thus, the glass transition temperature must be
defined. The glass transition temperature for hyperquenched water is widely believed to lay between
−133°C and −138°C (Chang et al., 2006). The highest heat capacity at temperatures above zero has
water (Table 28.1). The same is valid for ice at temperatures below zero in comparison with other
substances occurring in plants.
Tms
e
rv
y cu
b ilit
S olu
A B Tgs
Crystal
0
Liquid
Tm
Heterogeneous ice nucleation

Temperature (°C)
Homogeneous ice nuc

leati
on
n
itio
Ice/freeze concentrated matrix
ns
tra
ass
Gl
Glass
–136
0 Concentration 1
FIGURE 28.9 Phase–state diagram of matter depending on temperature with respect to its chemical com-
position and physical state. There are two situations mostly occurred during freezing. Situation (A) is usual at
plant freezing during winter. Plants are supercooled till the freezing-point depression, and after heterogeneous
ice nucleation (e.g., by ice crystals nucleation), the water freezes in the extracellular space. The same situation
occurred also during two steps of cryopreservation method. The first step is after slow cooling, when plant cells
are dehydrated by extracellular freezing. The second step is a rapid quenching in liquid nitrogen. Situation (B)
is used to preliminary dehydrate meristematic parts of a plant. After ultra-rapid freezing in liquid nitrogen, ice
crystal formation is avoided by glass formation in the plant meristematic tissue. Where Tm is the melting point
depression, glass transition of water is approximately −136°C, Tgs is the glass transition temperature of maxi-
mum concentrated substance, and Tms is the melting point depression of maximum concentrated substance.
28.3.3 Cryoprotectants
Glass formation involving the vitrification of unique solutions (Langis and Steponkus, 1990; Sakai
et al., 1990; Yamada et al., 1991) and the encapsulation by dehydration techniques (Figure 28.10)
(Fabre and Dereuddre, 1990) was developed in the 1990s, and since then, the number of cryopre-
served species has markedly increased (Sakai et al., 2008).
An ethylene glycol–based solution applied for the glass-formation procedure was first presented by
Steponkus and colleagues (Langis and Steponkus, 1990). For dehydration of winter rye protoplasts, they
used a concentrated vitrification solution made of ethylene glycol sorbitol and bovine serum albumin
(BSA) acting at 0°C. In the same year, Sakai and colleges (Sakai et al., 1990) published another combina-
tion of chemicals useful for vitrification. These cryoprotectants help the plant samples to stabilize their
membranes and proteins after deep dehydration and alternatively act as an energy pool during the recov-
ery of plants. For a common use, dimethyl sulfoxide (DMSO) is utilized as a representative cryoprotec-
tant of the colligative group capable of penetrating inside the cells. The polysaccharides (mainly sucrose
and polyethylene glycols) are representative of nonpenetrating cryoprotectants (Tao and Li, 1986).
FIGURE 28.10 Encapsulated Allium shoot tips in alginate beads. After dehydration, the plants are prepared
for immersion into liquid nitrogen for long-term storage. Average perimeter of beads is 4 mm.
The cells of naval orange were sufficiently dehydrated with a highly concentrated plant vitrifica-
tion solution no.2 (PVS2) prior to direct plunge into liquid nitrogen. The PVS2 contains glycerol
(30% w/v), ethylene glycol (15% w/v), and DMSO (15% w/v). Since the first publication of positive
results on cryopreservation when using PVS2, this treatment has been applied to more than 200
species and varieties, which exhibited a successful storability in liquid nitrogen through vitrifica-
tion (Sakai et al., 2008). However, this solution is toxic to plants, in which the ability to survive
is just several minutes using PVS2 at room temperature. This disadvantage is overcome by keep-
ing the plant parts in PVS2 at lower (0°C) temperature prolonging its nontoxic time to an hour.
A less toxic cryoprotective solution (without DMSO) for plants is plant vitrification solution no.3
(PVS3) (Nishizawa et al., 1993). Subsequently, cryotubes containing meristems (in 0.5 mL PVS)
are directly plunged into LN at the cooling rate of about 300°C min−1, even higher. Alternatively,
meristems are put on the aluminum foil with or without a small drop of PVS. Using different types
of cryoprotectants, it can be managed to distinguish their action, at what extension the transporta-
tion takes place, and to which part of the plant tissue they are delivered (Figure 28.11).
c
B
A b
Regeneration
Time
FIGURE 28.11 Time course of regeneration of plants after exposure to liquid nitrogen in relation cryopro-
tectant treatment (curve a). The time course of regeneration of control plants without the action of ultralow
temperature (curve b). An ideal regeneration of the control plants (curve c). Potential regeneration increase
(A) by improved cryoprotocol. Potential regeneration increase (B) by improved regeneration of control plants.
Increased regeneration (A + B) represents a potential increase in plant regeneration by improvement of cryo-
protectant agents and cryoprotocol.
The optimization of this technique for cryopreservation was connected with water permeabil-
ity, which is characteristic during freezing in the presence of extracellular ice and cryoprotective
agents; for details, see Devireddy et al. (1998).
Sugars are commonly used as cryoprotectants. Their concentrations and the individual types
of sugars must be optimized toward developing cryopreservation methods (Paul et al., 2000). The
cryobiologists tested various carbohydrates in an attempt to improve the percentage of explants sur-
viving and regenerating. Unlike sugar alcohols, such as sorbitol and mannitol, which are not readily
metabolized in the cells of many plant species, sucrose is readily metabolized and is thought to
have several protective functions during dehydration and subsequent freezing. Some sugar solutions
have low glass transition temperatures and the associated time for ice formation becomes almost
not measurable in real time. Most of these sugar alcohols are not as effective cryoprotectants as
sucrose. Sucrose has been found to be the most effective cryoprotectant in other studies (Martinez
et al., 1999; Paul et al., 2000).
Sugar activities are important intermediary for cryopreservation:
1. They can decrease water content by osmotic dehydration.

2. After the influx of sugars into the cells, the osmotic potential of plant tissues gets often
decreased.
3. Osmoprotectants (sugars like sucrose) are thought to contribute to maintaining the mem-
brane integrity during dehydration and freezing processes. It is intermediated by the forma-
tion of hydrogen bonds between sugars and hydrophilic components of cellular membranes
(Taylor, 1987).
4. They act as a glue sticking the shoot tips to the carrier (e.g., aluminum foil) after dehydration.
5. They accomplish the first aid during the energy starvation throughout the plant regenera-
tion after warming.
Sugars are seen as the most usable cryoprotectants involved in plants involved in a response to
abiotic stress (mainly in drought and cold conditions). The sucrose is used also as the most accept-
able cryoprotectant added to the plant during their cryopreservation at ultralow temperatures. On
the sugar as a simple cryoprotective example, it can be shown how difficult it is to explain complex
behavior of such a simple substance and how difficult it can be to define its mixtures in the plant
tissue.
The glass transition temperature for a binary mixture—the semiempirical Couchman and
Karasz expression (Couchman and Karasz, 1978)—is only partially successful in aqueous solutions.
Although empirical, the Couchman–Karasz equation seems to be the best model to explain varying
Tg with composition. A modified Gordon–Taylor equation (MGT) (Equation 28.15) can be used to
estimate the Tg values of single-phase sucrose solutions (i.e., no ice present):
kTg2 - Cw (kTg2 - Tg1 )

Tg = + a1Cw (1 - Cw ) + a 2Cw2 (1 - Cw ) (28.15)
Cw (1 - kTg ) + kTg

where
Cw is the concentration of water at Tg (weight fraction)
Tg1 and Tg2 are the midpoints of the glass transitions for pure water and sucrose, respectively
k, α1, and α2 are constants
Here, we use 135 K for Tg1, the water glass transition temperature, 348.2 K for Tg2, the glass transi-
tion temperature of pure sucrose, and 135 K for the water taken at Tg1. The “best fit values” for the
parameters are k = 0.092, α1 = 481, and α2 = −1225 (Chang et al., 2006).
There are no attempts to apply this equation to the multicomponent plant samples, but it could
be possible under further examinations. However, the equation allows considering a mixture of
several components as one component and water as another one having essentially some practical
values. The problem still remains because it is not known how to define ∆Cp for even pure water
that the Couchman–Karasz equation uses for predicting the Tg data of the multicomponent systems.
Nevertheless, both equations were satisfactorily used for description of water content and glass
transition for such multicomponent biological material as the fruit powders (Khalloufi et al., 2000).
28.3.4 Plant Long-Term Storage at Ultralow Temperatures

Detrimental influence of man-affected environment bears, however, an injurious impact on plant
growth and endurance. On the other hand, there are also many plant species close to the edge of
their disappearance under the current deterioration effect of the changing environment or even
under a routine fatality of species vanishing. Recently, there has been vast progress in genomics and
the ongoing development of genetically modified organisms (GMO). There is a supplementary call
for the urgent need to store plants with their entire genetic information, using the cultural varieties
preferably, landraces, and original wild-plant genetic resources. Fortunately, it is not necessary to
store a whole plant, but it is enough to save its meristematic tissues like buds, even just protoplasts,
because the plant cells are totipotent. Theoretically, it is possible to regenerate the whole plant from
one cell. Therefore, it is possible to store only a small part of plants, typically a part of meristematic
tissues of vegetatively propagated plants (Zámečník et al., 2003). The plant storage at low or even
at ultralow temperatures (cryopreservation) is one of the appropriate ways on how to keep their
viability in a long-term prospect (Zámečník et al., 2007).
The glass formation in plants can be of help to control the cryogenic status in which the optimal
cooling conditions ensure a suitable cryopreservation of plant germplasm.
The process of vitrification is, however, closely associated with the anomalous behavior of liquid
water (Buitink and Leprince, 2004).
From the view of cryopreservation, the glassy state, which possesses the same volume as liquid,
occurs as the most suitable state for a long-term biological storage of germplasm. This strategy of
being in the glassy state is repeatedly exploited by plants in nature in order to withstand unfavorable
dry and cold conditions of the surrounding environment, which can be seen as a sort of miracle. For
example, plant pollen flying in the air has a potential to be in the glassy state, keeping its viability
after stigma pollination. Dormant poplar twigs can survive the low temperatures being in the glassy
status in midwinter. It is possible to add other examples of glassy state occurrence in plant species
in the nature. However, the conditions at which the glassy state is induced are not fully understood.
ACKNOWLEDGMENT
This work was partially supported by the project LD12041 of the Ministry of Education Youth and
Sports of the Czech Republic.
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29 Stress Tolerance in Some
European Resurrection
Plants (Haberlea rhodopensis
and Ramonda spp.)
Iliya Denev, Detelin Stefanov, Maria Gevezova, Ina Kirilova,
Katya Georgieva, Maya Kurteva, and Galina Panayotova
CONTENTS
29.1 Introduction........................................................................................................................... 586
29.1.1 Drought and Desiccation: A Terminology for Plant Response to Water Deficit....... 586
29.1.2 Ecological Classification of Plants Based on Their Adjustment to Environments
with Different Moisture............................................................................................. 586
29.1.2.1 Vascular (Homoiohydric) Plant Strategy.................................................... 587
29.1.2.2 Poikilohydric Strategy................................................................................ 587
29.1.3 Evolution of Desiccation Tolerance........................................................................... 587
29.2 Resurrection Plants and Protection against Dehydration...................................................... 588
29.2.1 Zeaxanthin-Independent Deactivation of Excited States of Photosynthetic
Pigments in Lichens and Mosses Is a Mechanism Limiting Damages after
Dehydration............................................................................................................... 589
29.2.2 Slow Growth and Small Elasticity Modulus Allow Decrease in Leaf Surface:
Completely Embolized Xylem Vessels Ensured Resurrection Plants to Survive
Dehydration............................................................................................................... 590
29.3 Balkan Resurrection Plants Haberlea rhodopensis and Ramonda sp.................................. 590
29.4 Photosynthesis and Desiccation Tolerance in Haberlea and Ramonda................................ 592
29.5 Other Stress Responses in Haberlea and Ramonda.............................................................. 594
29.5.1 Desiccation Tolerance of H. rhodopensis Can Be Dramatically Influenced by
Light Conditions........................................................................................................ 594
29.5.2 Dehydration of Plants at High Temperature Can Increase the Rate of Water Loss......... 594
29.5.3 Effect of Prolonged Light Deprivation...................................................................... 595
29.6 Biotechnological Perspectives for Improvement of Drought Tolerance in Crop Plants
Based on Molecular Genetics of Haberlea and Ramonda.................................................... 595
29.7 Conclusion and Future Perspectives...................................................................................... 597
References....................................................................................................................................... 598
585
29.1 INTRODUCTION
Water is an indispensable factor ensuring life on Earth. Water facilitates many vital biological
reactions by being a solvent, a transport medium, and evaporative coolant (Bohnert et al. 1995).
Organisms dispose with great ability to spread in various water environments, but the level of tol-
erance to changes in water content affects their spreading. The modern scientific study of drought
tolerance began in 1702 when Antonie van Leeuwenhoek discovered that rotifers could survive
without water for months (cited in Bernacchia and Furini 2004). Now, it is known that a few groups
of animals and a wide variety of plants can tolerate desiccation in the adult stages of their life cycle.
Water availability has been one of the major crop production-limiting factors. It continues to be
a permanent constraint to agricultural production, and understanding the mechanisms that allow
plants to tolerate drought is an important task for agriculture development. The study of desiccation-
tolerant plants started the study of Irmscher (1912) with the botanical description of several species.
Until now, physiological, biochemical, and molecular aspects of this phenomenon are extensively
investigated. In arid lands or environments where drought occurs, the mechanisms of desiccation
tolerance in plants have a long-term protective role for support plant survival until water becomes
available. The increasing knowledge on mechanisms of desiccation and drought tolerance may be
applied in the development of drought-tolerant agricultural species.
29.1.1 Drought and Desiccation: A Terminology

for Plant Response to Water Deficit
Many plant systems can survive dehydration, but to different extents. Hoekstra et al. (2001) consid-
ered drought and desiccation as tolerances to water deficit that are distinguished on the basis of the
critical water level. The tolerance to moderate dehydration can be considered as drought tolerance.
Drought is characterized with moisture content below which there is no bulk cytoplasmic water
present (∼23% water on a fresh weight basis or ∼0.3 g H2O·g DW−1). Desiccation generally refers
to the tolerance of further dehydration, when the hydration shell of molecules is gradually lost.
Desiccation tolerance includes also the ability of cells to rehydrate successfully. By this reason in
this review, we can use the term desiccation tolerance for the group of resurrection plants (see in the
following text) that appear to be the highest tolerant plants to water deficiency.
29.1.2 Ecological Classification of Plants Based on Their

Adjustment to Environments with Different Moisture
Plants dispose with great ability to spread in various water environments. On this basis, plants could
be classified by their adjustment to water content. The relationship between the water potential and
the water content is very important for such classification (Grace 1997). As the leaf loses water, the
cells reduce in volume and the solutes become more concentrated. At the same time, the pressure
exerted by the walls declines. The relationship between the water potential and the water content
differs markedly between species and may influence the ecological range of the species. A meso-
phyte plant grows without severe moisture stresses, that is, a plant unable to grow in dry places may
show a small decline in water potential during decline in relative water content, but a xerophyte,
normally growing in dry places, shows a relatively large decline while still maintaining a positive
turgor. Thus, the xerophyte is more able to extract water from the soil. Other plants that are typical
for dry environments are succulent plant with tissue of high degree of succulence. Succulence is a
thick fleshy state of herbaceous tissues due to high water content. Succulence could be referred to
the presence of a few simple morphological anatomical modifications such as increasing the volume
of cortex and pith, reducing leaf size and number, and establishing mechanisms (spines or poisons)
that protect stored water (Mauseth 2004). Some desert plants, known as phreatophytes, produce
extremely deep roots that tap the water table (Lambers et al. 2008). These plants generally have
Stress Tolerance in Some European Resurrection Plants 587
high rates of photosynthesis and transpiration with little capacity to restrict water loss or withstand
drought. The representative group of true desiccation-tolerant plants in the sense of Hoekstra and col-
laborators’ classification is the so-called resurrection plants. Resurrection plant withstands complete
dehydration and resumes functioning upon rehydration (Gaff 1971). In opposite to typical vascular
(homoiohydric) plants, resurrection ones are defined as poikilohydric (for review, see Scott 2000;
Proctor and Tuba 2002; Bernacchia and Furini 2004). Poikilohydric plants or plant parts (seeds,
pollen) can dry out without losing their capacity to function upon rehydration (Lambers et al. 2008).
There are two survival strategies among resurrection angiosperms: (1) those that lose chlorophyll and
break down their chloroplasts upon drying (poikilochlorophyllous) and (2) those that retain some or
all of their chlorophyll and chloroplast ultrastructure (homoiochlorophyllous). Hydrophyte plants
grow partly or wholly in water, while hygrophyte species typically occur on permanently moist sites.
Plants exhibit several strategies to deal with life in extremely dry environments, namely, avoid-
ance, resistance, or tolerance to desiccation (Levitt 1980). For instance, desiccation avoidance is a
distinctive feature for annuals. A typical case for adaptation strategy is succulents that have modi-
fied their morphological structures that allow them to retain most of their cellular water and resist
equilibration with the air. These adaptations allow desiccation-resistant plants to extend the range
of conditions and their habitats. The aforementioned group of resurrection plants is extremely
desiccation tolerant and can survive almost complete desiccation, usually considered as drying to
<0.1 g H2O g−1 dry mass or to 10% absolute water content or less (Alpert 2005). Generally, Proctor
and Tuba (2002) consider two main plant strategies to water deficit.
29.1.2.1 Vascular (Homoiohydric) Plant Strategy

Water is absorbed by roots from the soil at relatively high water potential followed by a low-resistance
pathway via the xylem directed to the leaves. The loss of water vapor is limited by structures such
as cuticle and regulated by the variable diffusion resistance of the stomata. This vascular plant
pattern of adaptation has limitations. It depends critically on the water availability in the soil, pho-
tosynthetic activity sustaining open stomata, and higher transpiration (Proctor and Tuba 2002). On
the other hand, transpiration is linked with the heat balance of the plant (Proctor and Tuba 2002).
29.1.2.2 Poikilohydric Strategy

Poikilohydric plants can gain and lose water rapidly, and there is no control over water loss compa-
rable to that in vascular plants (Proctor and Tuba 2002). Water conduction is typically external and
diffuse. On rehydration, essentially normal metabolism returns within minutes or hours. The capil-
lary space associated with the plant often provides a substantial reservoir of water external to the
photosynthesizing cells while still permitting relatively free gas exchange (Dilks and Proctor 1979;
Proctor and Smith 1995; Zotz et al. 2000). Most of this external water can be lost without affecting
cell water status. The response of photosynthesis to cell water content appears to be substantially
the same as in vascular plants (Proctor and Tuba 2002 and references therein).
29.1.3 Evolution of Desiccation Tolerance

In order to colonize the land, the earliest terrestrial plants must have been desiccation tolerant at
every stage of the life cycle. With the evolution of more complex vascular plants, desiccation toler-
ance was lost in vegetative tissues but was retained in reproductive tissues (Oliver et al. 2000a,b).
Hartung et al. (1998) suggest that the genetic properties required for tolerance are present in flower-
ing plants and that only some tightly regulated programs of gene expression in plant development
are required to induce the revival of a dried plant body.
The majority of terrestrial plants today are capable of producing desiccation-tolerant struc-
tures such as spores, seeds, and pollen. Consequently, water stress in vegetative tissues is
characterized with different responses depending on the aforementioned evolutionary status.
Desiccation escaping Drought stress
Annuals
Desiccation resistance Desiccation tolerance
Xeromorphyc plants Poikilohydric plants
Desiccation avoidance
Homoihydric plants
Limit water loss
Waxy stomata
Few stomata Storage of water Modified
Drought deciduous Sunken stomata desiccation
Succulents tolerance
CAM
Hedera Fleshy tuber
boxus Large hairs Trie
Curled leaves desiccation
Water uptake tolerance
Phreatophytes
Deep roots rapid water loss
trichomes
Resurrection lichens
Homoiochlorophyllous Resurrection mosses
0% chl loss—Haberlea
50% chl loss—Myrothammus
70% chl loss—Ramonda Poikilochlorophyllous
100% chl loss—Xscabrida
FIGURE 29.1 Classification of drought effects.
Such classification of water deficit responses of higher plants was summarized by Mundree
et al. (2002). Modified scheme is presented in Figure 29.1.
About 330 species of angiosperms have been found to survive desiccation (Bewley and
Krochko 1982; Gaff 1989; Porembski and Barthlott 2000; Proctor and Pence 2002; Proctor
and Tuba 2002), but no resurrection gymnosperms are known (Hartung et al. 1998). There
are both monocotyledonous plants such as Xerophyta viscosa and Sporobolus stapfianus and
dicotyledonous species such as Myrothamnus flabellifolia, Craterostigma plantagineum, and
Chamaegigas intrepidus. The latter is the unique example known of aquatic resurrection
plants (Hartung et al. 1998). Usually, they are found in habitats subjected to lengthy periods
of drought, where rainfall is extremely sporadic, particularly on rock outcrops below 2000 m
in tropical and subtropical areas, and to a lesser extent in temperate zones (Porembski and
Barthlott 2000). Typical representatives for resurrection plants in temperate zones are Haberlea
rhodopensis and Ramonda sp. (R. serbica, R. nathaliae, R. myconi, etc.). Distinctive feature
of H. rhodopensis is that the plant is saxicolous, that is, grown in rocky places and it is a deep
shade perennial plant.
29.2 RESURRECTION PLANTS AND PROTECTION AGAINST DEHYDRATION

Numerous metabolic changes have been found to occur in resurrection plants as they dehydrate
and rehydrate. Upon water loss, the decrease in cellular volume causes crowding of cytoplas-
mic components, and the cell contents become increasingly viscous, increasing the chance for
molecular interactions that can cause protein denaturation and membrane fusion (Hoekstra
et al. 2001). A broad range of compounds limits the dependence of desiccation-induced dam-
ages such as proline, glutamate, glycine betaine, mannitol, sorbitol, fructans, trehalose, sucrose,
and oligosaccharides. The sugars can act to stabilize membranes and proteins in the dry state
by maintaining hydrogen bonding within and between macromolecules (Alisson et al. 1999),
and sugars could vitrify the cell content and stabilize internal cell structure (Crowe et al. 1996).
Changes in proportions among various sugars are also important for protection against desic-
cation (Djilianov et al. 2011). The accumulation of some late embryogenesis-abundant proteins
and small heat shock proteins (HSPs) to high concentrations coincides with the acquisition of
desiccation tolerance, and they are thought to play a role in desiccation tolerance (Hoekstra
et al. 2001). However, when water dissipates from the water shell of macromolecules at a mois-
ture content <0.3 g H 2O g−1 dry weight, the hydrophobic effect responsible for structure and
function is lost. It is envisaged that sugars, especially the nonreducing disaccharides but also
the tri- and tetrasaccharides and fructans that accumulate in anhydrobiotes, can replace the
dissipating water (Hoekstra et al. 2001). There are two well-studied properties of resurrection
plants considered in the following text.
29.2.1 Zeaxanthin-Independent Deactivation of Excited States

of Photosynthetic Pigments in Lichens and Mosses Is a
Mechanism Limiting Damages after Dehydration
Many lichens, bryophytes, and a few ferns can survive in a dried state. Heber and coauthors
built up a general hypothesis, describing the bases of desiccation tolerance in lichens and mosses
related with altered photosynthesis and the development of the photooxidative processes (Heber
et al. 2007). Because photosynthesis and repair of photooxidative damage are not possible in
the absence of water, survival of desiccation-tolerant organisms, which retain their chloro-
phyll in the desiccated state, requires deactivation of excited states of photosynthetic pigments
before destructive photoreactions can occur. As energy dissipation must be faster than charge
separation, Heber and coauthors concluded that thermal deactivation of excited states occurs
within the subpicosecond, or femtosecond, time domain when water is absent. This means that
slower zeaxanthin-dependent mechanism of thermal energy dissipation of excessive excitation
(for details about the role of zeaxanthin in photoprotection, see Demmig-Adams [1990]) can-
not be the main mechanism of photoprotection of desiccated homoiochlorophyllous photoau-
totrophs because it is only weakly competitive with energy capture by photosystem (PS) II
reaction centers. The zeaxanthin-dependent mechanism dissipates energy in the antenna of PSII
(antenna-dependent quenching). In some desiccation-tolerant mosses, the site of energy dissipa-
tion was within PSII reaction centers, that is, reaction center–dependent quenching of excessive
excitation energy (for details, see Weis and Berry [1987]). During desiccation, a photoreaction
within the reaction centers stabilizes radicals such as reduced pheophytin or oxidized chloro-
phyll, which act as effective quenchers of chlorophyll fluorescence (Heber et al. 2006). The
radicals are stable as long as water is absent. They revert to the uncharged state by losing charge
upon hydration. Another zeaxanthin-independent mechanism of effective thermal energy dis-
sipation in desiccated photoautotrophs, which is independent of the presence of light, is dem-
onstrated by Heber et al. (2007). It is activated during desiccation in darkness and is thought
to involve conformational changes of a chlorophyll–protein complex, which are triggered by
desiccation. These changes are considered to form the basis of a dissipating mechanism that
is even faster than energy capture by photosynthetic reaction centers, that is, faster than a few
picoseconds (Holzwarth et al. 2006). This mechanism is present in chloro- and cyanolichens and
in desiccation-tolerant mosses. The long-wavelength quencher closely coupled to PSII identified
by Veerman et al. (2007) is capable for efficient downregulation of PSII in desiccated lichens
and fast restoration of PSII upon rehydration. Desiccation-induced conformational change in the
hypothetical long-wavelength antenna pigment–protein complex could allow certain chlorophyll
to interact at a short enough range with neighboring chlorophylls or carotenoids to form exciton-
coupled bands and thus form an efficient quencher.
29.2.2 Slow Growth and Small Elasticity Modulus Allow Decrease

in Leaf Surface: Completely Embolized Xylem Vessels Ensured
Resurrection Plants to Survive Dehydration
Resurrection plants can tolerate extreme dehydration but there was not a hypothesis describing pro-
tection against desiccation-induced damages in both poikilochlorophyllous and homoiochlorophyl-
lous resurrection plants. In earlier studies, Bewley (1979) suggested that to survive in a dehydrated
state, plants must be able to limit the damage, they must maintain the physiological integrity in the
desiccated state, and they must possess repair mechanisms to recover from the damage upon rehy-
dration. In more recent studies, Bewley and Oliver (1992) classified desiccation-tolerant plants into
two groups based on their resistance mechanism: protection of cellular integrity or cellular repair
of desiccation-induced damage.
Resurrection plants are slow-growing plants that are characterized by a small elasticity modulus
that means they contain nonrigid cell walls, and as a consequence, their leaf surface decreases dur-
ing dehydration due to cell wall folding. This mechanism minimizes drought-induced mechanical
stresses on the plasma membrane, maintaining cell-to-cell connections and allowing a rapid recov-
ery upon rewatering (Schiller et al. 1999; Farrant 2000; Vicré et al. 2004). Resurrection plants lose
their water more rapidly than their comparable nonresurrection counterparts (e.g., Deng et al. 2003).
Protection against oxidative damages can be determined by a regulatory process leading to down-
regulated photosynthetic metabolism on one hand and upregulated antioxidant systems on the other
(Farrant 2000). Furthermore, the mechanisms that trigger the heat dissipation of energy trapped
by the light-harvesting antennae (involving an increase in zeaxanthin content and probed by the
nonphotochemical quenching [NPQ] of PSII chlorophyll fluorescence [Casper et al. 1993; Augusti
et al. 2001]) appear to operate in the same way in resurrection plants as in nonresurrection plants,
while the aforementioned faster thermal dissipation of excessive excitation energy that was found
by Heber and coauthors (Heber et al. 2006, 2007) in lichens and mosses has not been demonstrated
in resurrection plants. Very recently, Strasser et al. (2010) suggest that the desiccation tolerance of
the photosynthetic machinery in H. rhodopensis is mainly based on a mechanism that leads to the
inactivation of PSII reaction centers and their transformation to heat sinks. Desiccation tolerance
allows a plant to survive equilibrium with 0% air humidity and it is preserved until water becomes
available. Upon watering, dried resurrection plants rapidly revive and become fully photosyntheti-
cally active within 24 h (Bernacchia et al. 1996). In addition, the processes of drying and rehydra-
tion cause only limited damage to plant tissues. Thus, resurrection plants have the advantage over
other species in arid environments in that they can remain quiescent during long periods of drought.
Resurrection plants have evolved structures and mechanisms to allow survival under extreme condi-
tions. The plant is in the air-dried state; the xylem vessels are completely embolized. During rehy-
dration, the xylem must be able to refill, avoiding the formation of air bubbles, in order to provide
a supply of water to the leaves. Vulnerability of xylem to the formation of air bubbles depends on
wood anatomy, particularly the size of pores in intervessel pits. Larger pores are more susceptible
to air entry than small pores.
29.3 BALKAN RESURRECTION PLANTS HABERLEA

RHODOPENSIS AND RAMONDA SP.
Resurrection plants are widely distributed; they are found in all continents except Antarctica.
According to Gaff (1989), they are mainly concentrated in arid climates such as southern Africa,
eastern South America, and western Australia, while only a few species have been found in Europe
in the Balkan Mountains. The studies on Balkan resurrection plant H. rhodopensis Friv. began
from the first investigation of Ganchev (1950), and it was investigated during the middle of the
1970s by Kimenov and collaborators (Kimenov and Jordanov 1974; Kimenov and Minkov 1975).
Detailed studies were carried out extensively in the last two decades on H. rhodopensis (Stefanov et al.
1992; Markovska et al. 1994, 1995, 1997; Jensen 1996; Muller et al. 1997; Markovska and Kimenov
1998; Markovska 1999; Alpert and Oliver 2002; Djilianov et al. 2005, 2009, 2011; Georgieva et al.
2005, 2007, 2008, 2009, 2010, 2012; Péli et al. 2005a,b, 2012; Zivkovic et al. 2005; Gechev et al. 2006,
2012a,b; Georgieva and Maslenkova 2006; Moler and Cronk 2007; Ionkova et al. 2008; Baloutzov
et al. 2009; Peeva and Cornic 2009; Yahubyan et al. 2009; Petrova et al. 2010; Strasser et al. 2010;
Berkov et al. 2011; Daskalova et al. 2011; Ebrahimi et al. 2011; Mihailova et al. 2011; Nagy-Déri
et al. 2011; Popov et al. 2011; Apostolova et al. 2012; Dell’Acqua and Schweikert 2012; Georgieva
et al. 2012b), Ramonda serbica (Stefanov et al. 1992; Markovska et al. 1994, 1997; Muller et al. 1997;
Sgherri et al. 1998, 2004; Drazic et al. 1999; Augusti et al. 2001; Alpert and Oliver 2002; Quartacci
et al. 2002; Veljovic-Jovanovic et al. 2006, 2008; Degl’Innoscenty et al. 2008; Sabovljevic et al.
2008), Ramonda myconi (L.) Rchb. (Pico et al. 2002; Toth et al. 2004, 2006; Dubreuil et al. 2008;
Siljak-Yakovlev et al. 2008), and Ramonda nathaliae Panc. et Petrov (Muller et al. 1995; Stefanovic
et al. 1997; Drazic et al. 1999; Pico and Riba 2002; Pico et al. 2002; Riba et al. 2002; Siljak-Yakovlev
et al. 2008; Radulovic et al. 2009; Jovanović et al. 2011). H. rhodopensis Friv. (Gesneriaceae—for
evolutionary relationships among genera members of this family, see Wang et al. 2010) is a popular
ornamental plant. Although nowadays it is available in most of the botanical gardens in the world,
H. rhodopensis is a tertiary relict, native for the Balkan Peninsula described in 1835 by E. Frivaldzky
from materials collected in 1834 by Hinke and Manolesku in the Rhodope Mountains in Bulgaria
between Asenovgrad and Bachkovo (Frivaldzky 1835; Andreev et al. 2006). About the history of the
discovery and typification of the H. rhodopensis, see Szelag and Somlyay (2009). It inhabits silicate
and calcareous rocky places in shady environment at high air humidity in the oak and beech belts
between 250 and 1400 m. Haberlea populations are highly abundant with distribution for Bulgaria
mainly in the Rhodope Mountains and rarely in Central Balkan region of the Balkan Mountains,
along the Osam River, eastern part of the Sredna Gora mountain.
As one of the few resurrection angiosperms, H. rhodopensis have been extensively used as a
model plant for the study of mechanisms underlying desiccation tolerance (Georgieva et al. 2008).
The physiological adaptation of H. rhodopensis leaves and roots during dehydration–rehydration
cycle is manifested as increased tolerance to water deficit in both leaves and roots (Péli et al.
2012). There was a good correlation between the root respiration and water content. Roots were
more sensitive and reacted faster to water stress than leaves, but their activity rapidly recovered
due to immediate and efficient utilization of periodic water supply. The effects of artificially
induced drought stress on photosynthesis and transpiration (Markovska et al. 1994), lipid and
sterol compositions in the leaves (Stefanov et al. 1992), sugar content (Müler et al. 1997; Djilianov
et al. 2011), and chloroplast ultrastructure (Kimenov and Minkov 1975; Markovska et al. 1995;
Georgieva et al. 2010; Nagy-Déri et al. 2011) have been extensively studied. It was found that
air-dried H. rhodopensis leaves could preserve more than 80% of their chlorophylls (Kimenov
and Jordanov 1974; Markovska et al. 1994). On this basis, H. rhodopensis is considered as a
homoiochlorophyllous species that is able to retain its photosynthetic apparatus and chlorophylls
in a recoverable form upon drought stress (Proctor and Tuba 2002). Carbohydrates (including
reducing sugars), flavonoids, tannins, phytosterols, glycosides, and saponins were discovered in
all organs of H. rhodopensis (Baloutzov et al. 2009). Ionkova et al. (2008) reported that the in
vitro-produced plants possess the same resurrection capability as the plants from nature. Leaf
extracts of the in vitro-grown plants contain three groups of biologically active substances: flavo-
noids, tannins, and polysaccharides. Total content of flavonoids showed a better correlation than
total tannins with the free radical scavenging effect. Electron microscopy of structural changes in
the chloroplast showed that the thylakoid lumen is filled with an electron-dense substance (dense
luminal substance), while the thylakoid membranes are lightly stained (Georgieva et al. 2010).
Accumulation of dense luminal substance (possibly phenolics) in the thylakoid lumen is demon-
strated and is proposed as a mechanism protecting the thylakoid membranes of H. rhodopensis
during desiccation and during recovery. Sucrose was the predominant soluble carbohydrate in
Haberlea (Muller et al. 1997), and its level steadily increased from 2% to 10% during desicca-
tion. Starch amounted to ca. 2% in control leaves and disappeared completely within 8 days of
desiccation. Considerable amounts (1%–2%, 5%) of raffinose and smaller amounts of its precur-
sor galactinol (1-α-galactosyl-myo-inositol) were present in control leaves; these carbohydrates
showed only minor changes upon desiccation. Sucrose accumulation is connected to desiccation
tolerance in Gesneriaceae; the presence of raffinose may be a preadaptation since this sugar pre-
vents crystallization of sucrose during drying. The size, shape, and number of chloroplasts in the
palisade and spongy parenchyma layers of H. rhodopensis leaves changed significantly during
desiccation and following rehydration (Nagy-Déri et al. 2011). The chloroplasts became smaller
and more rounded during desiccation and aggregated in the middle of the cell. The size and
number of chloroplasts in the palisade parenchyma cells were higher than in spongy parenchyma.
Changes in chloroplast number during desiccation and rehydration process are characteristic
features for desiccation-tolerant plants (especially in homoiochlorophyllous strategy). R. serbica
Panc. (Gesneriaceae) belongs to a small group of poikilohydric angiosperms originating from the
Balkan Peninsula (Oliver 1996), as an endemic and relict species of the Tertiary period. It inhabits
northern-positioned calcareous rocks of some gorges in Serbia and Bulgaria. It withstands severe
water shortage for more than 3 months. The plant is a perennial herbaceous and shade-adapted
plant belonging to the homoiochlorophyllous poikilohydric group of plants that preserve almost
all their chlorophyll content during dehydration (Markovska et al. 1994; Navari-Izzo et al. 1995;
Drazic et al. 1999; Augusti et al. 2001). Augusti and coworkers (2001) found the recovery of full
photosynthetic activity of R. serbica leaves after rehydration.
Jovanović and coworkers (2011) studied oxidative and antioxidative events during dehydration
and rehydration of resurrection plant R. nathaliae. Their results indicated that enhanced oxidative
status during dehydration could play an active role in inducing the desiccation adaptive response
in R. nathaliae. A critical phase (in the range of RWC between 50% and 70%) during which a
significant increase in hydrogen peroxide accumulation, lipid peroxidation, and ion leakage, accom-
panied by a general decline in antioxidative enzyme activity, takes place. This phase is designated
as a transition characterized by change in the type of stress response. The initial response, relying
mainly on the enzymatic antioxidative system, is suspended. Rapid rewatering of R. nathaliae led to
reactivation of antioxidative enzymes. It was proposed that controlled elevation of reactive oxygen
species (ROS), such as hydrogen peroxide, could be an important mechanism enabling resurrection
plants to sense dehydration and to trigger an adaptive program. These results did not correlate with
changes in oxidative metabolism in H. rhodopensis (Djilianov et al. 2011). In opposite to enhanced
oxidative status in R. nathaliae, the levels of H2O2 decreased significantly both in H. rhodopensis
and nonresurrection plant Chirita eberhardtii. That accumulation of malondialdehyde was much
more pronounced in the desiccation-tolerant H. rhodopensis than in C. eberhardtii. An accumula-
tion of total phenols in H. rhodopensis during the late stages of drying should be related with higher
antioxidant activity in opposite to decreased antioxidant activity in R. nathaliae. The total gluta-
thione concentration and ratio oxidized (GSSG)/reduced (GSH) glutathione also increased upon
complete dehydration of H. rhodopensis.
29.4 PHOTOSYNTHESIS AND DESICCATION TOLERANCE

IN HABERLEA AND RAMONDA
Recent advances in studies of photosynthesis of resurrection angiosperms indicate that photo-
chemical activities are sensitive indicators for the study of physiological state of resurrection
angiosperms during desiccation and rehydration (Yang et al. 2003). The electron micrographs
of leaf segments of H. rhodopensis displayed a large number of grana having up to 25 tightly
stacked thylakoids (Denev et al. 2012) that display shade-type morphology of chloroplasts.
Adaptation to light intensity influenced on physiology of H. rhodopensis. Sun- and shade-type
leaves of H. rhodopensis reveal different responses to desiccation (Georgieva et al. 2012). The
malondialdehyde (MDA) content in well-watered sun Haberlea plants was higher compared to
shade plants suggesting higher lipid peroxidation, but desiccation of plants at high light did not
cause additional oxidative damage as judged by the unaffected MDA content. The electrolyte
leakage from dried leaves from both shade and sun plants increased fourfold indicating similar
membrane damage. Stomata were visible only on the abaxial side of sun leaves having also higher
abundance of nonglandular trichomes. Increased trichome density and epicuticular waxes and
filaments upon desiccation could help plants to increase reflection, reduce net radiation income,
slow down the rate of water loss, and survive adverse conditions. Haberlea expresses the abil-
ity for full recovery of photosynthesis after rehydration. Georgieva et al. (2005, 2007) showed
that water loss influences fluorescence induction, thermoluminescence emission, far-red-induced
P700 oxidation, and oxygen evolution in the leaves of H. rhodopensis. Moreover, all these param-
eters nearly returned to the control levels within a few days after rehydration.
There are different phases of photosynthetic response of H. rhodopensis to desiccation. During
the first phase of desiccation, the net CO2 assimilation decline was influenced by stomatal closure
(Georgieva et al. 2007). Further lowering of net CO2 assimilation was caused by the decrease in
both stomatal conductance and the photochemical activity of PSII. Severe dehydration caused inhi-
bition of quantum yield of PSII electron transport, disappearance of thermoluminescence B band
and mainly charge recombination related to S2QA takes place. The blue and green fluorescence
emission in desiccated leaves strongly increased. It was suggested that unchanged chlorophyll con-
tent and amounts of chlorophyll–proteins, reversible modifications in PSII electron transport, and
enhanced probability for nonradiative energy dissipation during desiccation of Haberlea contribute
to drought resistance and fast recovery after rehydration (Georgieva et al. 2007). The low rate of
leaf net CO2 uptake (4–6 μmol m−2 s−1) under saturating photosynthetic photon flux densities in
normal air was also found (Peeva and Cornic 2009). Enhanced CO2 uptake at saturating CO2 and
light leads to assumption that H. rhodopensis leaves have a very low mesophyll CO2 conductance.
This explains the low rate of leaf net CO2 uptake in normal air. Experimental evidences suggest that
mesophyll conductance is not sensitive to temperature in the 20°C–35°C range. On the other hand,
at leaf relative water content of between 90% and 30%, the decrease of leaf net CO2 assimilation
during drought can be explained by a decrease of leaf CO2 diffusional conductance. Interestingly,
the effect of leaf desiccation on photosynthetic capacity, measured at very high ambient CO2 molar
ratios under saturating photosynthetic photon flux density (PPFD), is identical to that observed
for three nonresurrection C3 mesophytes. This demonstrates that the photosynthetic apparatus of
H. rhodopensis is not more resistant to desiccation when compared to other C3 plants but following
the hypothesis of Farrant and coauthors (Farrant et al. 2003). Peeva and Cornic (2009) conclude
that the main advantage of H. rhodopensis leaf in drought conditions is related to the possibil-
ity of Haberlea leaf cells to avoid mechanical stress. The changes in pigment content and some
proteins involved in the light reactions of photosynthesis of the resurrection plant H. rhodopensis
were examined in connection with desiccation (Geogieva et al. 2009). HPLC analysis revealed that
β-carotene content was only slightly enhanced in desiccated leaves compared with the control, but
the zeaxanthin level was strongly increased. Desiccation of H. rhodopensis to an air-dried state at
very low light irradiance led to a little decrease in the level of D1, D2, PsbS, and PsaA/B proteins in
thylakoids, but a relative increase in LHC polypeptides. In contrast to spinach, Haberlea thylakoids
appeared to be much more resistant to the same solubilization procedure, that is, complexes were
not separated completely and complexes of higher density were found. The photosynthetic proteins
remained comparatively stable in dried Haberlea leaves when plants were desiccated under condi-
tions similar to their natural habitat. Low light during desiccation was enough to induce a rise in the
xanthophyll zeaxanthin and β-carotene. Together with the extensive leaf shrinkage and some leaf
folding, increased zeaxanthin content and the observed shift in antenna proteins from PSII to PSI
during desiccation of Haberlea contributed to the integrity of the photosynthetic apparatus, which
is important for rapid recovery after rehydration (Geogieva et al. 2009).
R. serbica also displays zeaxanthin-independent photoprotection, involving lutein pathway that

limits photooxidative damages during desiccation. Thylakoid membranes of this species can be pro-
tected by membrane-compatible solutes such as sucrose and inorganic ions especially K+ and Cl−
(Zivkovic et al. 2005) by changes in lipid composition (Quartacci et al. 2002), as well as by activating
protective mechanisms such as higher zeaxanthin and reduced ascorbate and glutathione (Augusti
et al. 2001). The observed increase in phenolic acids during dehydration (Sgherri et al. 2004) might
also play a role in protecting Ramonda membranes from desiccation-induced damage. In vascular
homoiochlorophyllous plants, photochemical activity has been reported to be maintained longer in
the course of drying than CO2 assimilation (Schwab et al. 1989). In drying leaves of R. serbica, the
intrinsic efficiency of the PSII photochemistry and the photon yield of PSII electron transport show
strong progressive decrease (Augusti et al. 2001). Simultaneously, the fraction of excitation energy
dissipated as heat in the PSII antenna increases markedly. When plants were rewatered, photosyn-
thetic efficiency and nonradiative energy dissipation quickly recovered to their initial control values.
Recently, Degl’Innoscenty and coworkers (2008) suggested two different mechanisms of responses to
dehydration in R. serbica. In the first, lower CO2 assimilation induces excess accumulation of the pro-
tons in the thylakoid lumen thus activating a protection based on enhanced NPQ. Stronger dehydration
causes the markedly decreased electron transport rate, accompanied by reduced NPQ. In such case,
ROS formation is better prevented by mechanisms that quench chlorophyll triplet formation via lutein.
29.5 OTHER STRESS RESPONSES IN HABERLEA AND RAMONDA

29.5.1 Desiccation Tolerance of H. rhodopensis Can Be
Dramatically Influenced by Light Conditions
H. rhodopensis was very sensitive to photoinhibition because this plant is typically shade adapted
(Georgieva and Maslenkova 2006). The light intensity of 120 μmol m−2 s−1 photon flux density (PFD)
sharply decreased the quantum yield of PSII photochemistry, and it was almost fully inhibited at
350 μmol m−2 s−1 PFD. When plants were kept in darkness or at low irradiance (about 30 μmol m−2
s−1 PFD), they are able to survive desiccation below 10% water content, and their photosynthetic activ-
ity can be fully recovered after rehydration (Georgieva et al. 2005, 2007). Plants subjected during
desiccation to high irradiance (350 μmol m−2 s−1 PFD) suffer irreversible damages in the photosyn-
thetic apparatus and leaves did not recover after rehydration (Georgieva et al. 2008). Simultaneously,
the blue/red and green/red fluorescence ratios increased considerably suggesting increased synthesis
of polyphenolic compounds in high light conditions. In order to establish the causes and mechanisms
of irreversible damage of the photosynthetic apparatus due to dehydration at high irradiance and to
elucidate the mechanisms determining recovery, the changes in the structure and function of the pho-
tosynthetic apparatus of H. rhodopensis during desiccation and subsequent rehydration under medium
irradiance (ML) (100 μmol m−2 s−1 PFD; ML) were compared to changes under low irradiance (LL)
(30 μmol m−2 s−1 PFD; LL) (Georgieva et al. 2009). Upon dehydration and rehydration, the dense lumi-
nal substance (see Section 29.3) thinned and disappeared and the time course largely depending on
the illumination. While dense luminal substance persisted during desiccation and started to disappear
during late recovery under LL, it disappeared from the onset of dehydration and later was completely
lost in ML. Disappearance of dense luminal substance (possibly phenolics) during desiccation in ML
could leave the thylakoid membranes without protection, allowing oxidative damage during dehydra-
tion and the initial rehydration, thus preventing recovery of photosynthesis.
29.5.2 Dehydration of Plants at High Temperature

Can Increase the Rate of Water Loss
It was found that high temperatures can increase the water loss threefold and had a more detrimental
effect than either drought or high temperature alone (Mihailova et al. 2011). The stability of PSII
in leaves of resurrection plant H. rhodopensis to high temperature is not significantly influenced

by temperatures up to 40°C (Georgieva and Maslenkova 2006). F0 reached maximum at 50°C,
which is connected with blocking of electron transport in reaction center II. The high amount of
chlorophyll molecules retained during desiccation could be a source for potentially harmful singlet
oxygen production. When plants were desiccated at optimal (23°C) and high (38°C) temperatures,
the results showed that superoxide dismutase (SOD) activity gradually decreased, whereas catalase
activity significantly increased during desiccation of Haberlea plants under both optimal and high
temperatures (Mihailova et al. 2009). Exposure of plants to high temperature reduced the activity of
these enzymes. The enhanced activity of ascorbate peroxidase (PO) and guaiacol PO was observed
under moderate water stress, after which they declined. High temperature stress applied alone did
not influence the APX and GPX activity (Mihailova et al. 2009).
29.5.3 Effect of Prolonged Light Deprivation

Recently, the effect of light deprivation on the ultrastructure, pigment composition, and functions
of photosynthetic apparatus of the resurrection plant H. rhodopensis Friv. (Gesneriaceae) was also
studied with the aim to check stress tolerance of resurrection plant such as Haberlea to factors
other than desiccation (Denev et al. 2012). Intact plants were kept up to 15 weeks in darkness. H.
rhodopensis demonstrated extraordinary ability to preserve its photosynthetic machinery intact
despite complete absence of light. The first 4 weeks of the dark treatment were characterized by
preservation of pigment content, chloroplast ultrastructure, and by the decrease in the rate of CO2
assimilation. It was found that when detached leaves from plants deprived from light for 2 weeks
were supplied with exogenous 5-aminolevolinic acid in darkness, they accumulated both proto-
chlorophyllide and chlorophyllide, that is, synthesis of chlorophyll a precursors in darkness was
suggested (Denev et al. 2005a,b). Such behavior of H. rhodopensis in the dark could be considered
as a part of the protection mechanism to preserve photosynthetic apparatus against various stresses
in conditions of preserved chlorophyll amounts (homoiochlorophyllous plant) that is different from
that in lichens and bryophytes considered in details by Heber and coauthors (2007). Haberlea
survive long dark deprivation with preserved respiration activity and without loss of pigments that
are necessary for rapid recovery of functional activity during resurrection. The necessity from
rapid recovery could explain this unusual survival to long darkness. The signs of dark-induced
senescence were observed only after the 4th week, but the processes were unusually slow and took
another 9 weeks (from fourth till 15th week of light deprivation). This phase was characterized by
decrease of pigment content and partial disintegration of chloroplast ultrastructure. Consequently,
H. rhodopensis Friv. could be used not only to study desiccation tolerance but also to investigate
mechanisms determining tolerance to multiple stresses and the senescence processes of photosyn-
thetic apparatus.
29.6 BIOTECHNOLOGICAL PERSPECTIVES FOR IMPROVEMENT

OF DROUGHT TOLERANCE IN CROP PLANTS BASED ON
MOLECULAR GENETICS OF HABERLEA AND RAMONDA
Drought resistance is a complex trait, expression of which depends on action and interaction of
different morphological (earliness, reduced leaf area, leaf rolling, wax content, efficient rooting
system, awn, stability in yield, and reduced tillering), physiological (reduced transpiration, high
water use efficiency, stomatal closure, and osmotic adjustment), and biochemical (accumulation of
proline, polyamine, trehalose, etc.; increased nitrate reductase activity; and increased storage of
carbohydrate) characters (for review, see Mitra (2001) and references therein).
Resurrection plants are characterized by their unique ability to survive months to years without
water, lose most of the free water in their vegetative tissues, fall into anabiosis, and, upon rewatering,
quickly regain normal activity. Thus, they are fundamentally different from other drought-surviving
plants such as succulents or ephemerals, which cope with drought by maintaining higher steady-
state water potential or via a short life cycle, respectively. The existing studies on molecular level of
responses revealed molecular adaptations that enable resurrection plants to withstand long periods
of desiccation. The recent transcriptome analysis of C. plantagineum and H. rhodopensis under
drought, desiccation, and subsequent rehydration revealed common genetic pathways with other
desiccation-tolerant species as well as unique genes that might contribute to the outstanding desic-
cation tolerance of resurrection species. Some molecular responses are common for both drought
stress and desiccation; several genes that are induced during desiccation have no apparent sequence
homologies to genes of other species (Gechev et al. 2012 a,b; Georgieva et al. 2012). Thus, resurrec-
tion plants are potential sources for gene discovery because understanding the cellular mechanisms
of desiccation tolerance in this unique group of plants may enable future molecular improvement
of drought tolerance in crop plants. Metabolome and proteome investigations also can contribute to
unraveling of drought tolerance mechanisms. As it has been discussed previously, prolonged desic-
cation causes imbalance between electron transport and CO2 fixation rates. The enhanced electron
leakage to oxygen causes increased production of ROS that are considered ultimate consequence of
many environmental stresses (Elstner 1982; Smirnoff 1993) and the main reason for lipid peroxida-
tion, protein denaturation, and DNA damage (Inze and Van Montague 1995). Plants, on the other
hand, use ROS as secondary messengers in signal transduction cascades in processes as diverse
as mitosis, tropisms, and cell death, so the balance between ROS and cellular antioxidant systems
is crucial to plant development as well as defense (Noctor 2005; Gechev et al. 2006). Most of the
protective mechanisms rely on the activation of nonenzymatic and enzymatic antioxidant systems
(Alscher and Hess 1993) and are common for vegetative desiccation-sensitive (VDS) and vegetative
desiccation-tolerant (VDT) plants (Knight and Knight 2001). Some authors have suggested that the
desiccation tolerance can be to some extent a function of the plant’s ability to process ROS by main-
taining defensive mechanism or amplifying them during desiccation and rehydration (Navari-Izzo
and Rascio 1999). The enzymatic antioxidant system provides defense against ROS. It includes sev-
eral functionally interrelated enzymes such as SOD, PO, catalase, and glutathione reductase (GR).
SOD converts the destructive superoxide radical into less dangerous forms, hydrogen peroxide and
molecular oxygen (Noctor and Foyer 1998).
The SODs are classified into three groups, depending on the metal cofactor in their catalytic
center: copper/zinc SOD (Cu, Zn-SOD), manganese SOD (Mn-SOD), and iron SOD (Fe-SOD),
which are localized in different cellular compartments (Mittler 2002). PO is a widely distributed
group of enzymes that can oxidize a variety of hydrogen donors at the expense of hydrogen peroxide
(Noctor and Foyer 1998). Under mild water deficiency, the VDS plants respond with increase of the
antioxidant enzyme activity (Baisak et al. 1994; Moran et al. 1994; Van Rensburg and Kruger 1994),
whereas severe water stress reduced the activity of antioxidant enzymes (Sgherri and Navari-Izzo
1995; Schwanz et al. 1996; Balsamo et al. 2005). In contrast, the activities of the key antioxidant
enzymes were found to increase during progressive dehydration in VDT species such as X. viscosa
(Sherwin and Farrant 1998), Eragrostis nindensis (Illing et al. 2005), and R. serbica (Veljovic-
Jovanovic et al. 2006). The levels of SOD and PO in H. rhodopensis after exposure to drought
and recovery were studied by Yahubyan and coworkers (2009). It has been found that in H. rhodo-
pensis, SOD and PO were able to adjust their activity very promptly to changing water supply. In
addition, the leaves of this resurrection species retained significant activities of SOD and PO even
in air-dried state. Several isoforms of SOD were found on native protein gels—four of them were
Cu/Zn-SOD isoforms, one Mn-SOD, and one Fe-SOD (Yahubyan et al. 2009). The corresponding
cDNAs were also cloned and characterized (Apostolova et al. 2012). Seven full-length cDNAs, and
their partial genomic clones, were obtained by the combination of degenerate PCR, RT-PCR, and
RACE. The derived amino acid sequences exhibited a very high degree of similarity to cytosolic
Cu, Zn-SODs (HrCSD2, HrCSD3), chloroplastic Cu, Zn-SODs (HrCSD5), other Cu, Zn-SODs
(HrCSD4), Mn-SODs (HrMSD), and Fe-SODs (HrFSD). One cDNA turned out to be a pseudogene
(HrCSD1). The transcripts of HrCSD2, HrCSD5, HrMSD, and HrFSD were found in all organs,
while HrCSD3 and HrCSD4 were organ specific (Apostolova et al. 2012). The SOD transcripts were
present at significant levels even in air-dried leaves as it was reported first at protein level (Yahubyan
et al. 2009). Despite these successes, information on cDNA sequences of other genes involved in the
response of H. rhodopensis to desiccation, their regulation, and molecular characterization is still
lacking (Georgieva et al. 2012).
Recently, several attempts to fill these gaps were undertaken. Georgieva and coworkers (2012)
used cDNA-amplified fragment length polymorphism (cDNA-AFLP) to study differentially
expressed genes in H. rhodopensis upon desiccation. The cDNA-AFLP is an efficient, sensitive,
and reproducible technology for isolation of differentially expressed genes (Bachem et al. 1996; Ditt
et al. 2001). It does not require prior sequence information and, in contrast to microarrays, often
enables identification of novel sequences even in well-annotated species. The study revealed 20
transcripts among 33 sequenced cDNA fragments to be involved in energy metabolism, transport,
cell wall biogenesis, and signal transduction or probably transcription regulators according to their
putative function. Expression patterns of two upregulated (HrhDR8, HrhDR35) and two down-
regulated (HrhDR6, HrhDR25) transcripts were verified by sqRTPCR analysis at different stages
of water stress. The results demonstrated that two upregulated transcripts HrhDR8 and HrhDR35
encoding putative succinate dehydrogenase and xyloglucan endotransglucosylase/hydrolase (XTH),
respectively, were induced during early stage of dehydration, persist in desiccated state, and subse-
quent rehydration of the plant. About 30% of the identified Haberlea sequences show no significant
similarity to any sequence in the searched databases and were differentially regulated (Georgieva
et al. 2012). Another approach was adopted by Gechev and coworkers (2012a). They used tran-
scriptome analysis based on next-generation sequencing. Repression of photosynthetic and growth-
related genes was found together with induction of transcription factors (members of the NAC,
NF-YA, MADS box, HSF, GRAS, and WRKY families). The authors concluded that they probably
are acting as master switches of the genetic reprogramming upon drought. The induction of genes
related to sugar metabolism and signaling and genes encoding early light-inducible (ELIP), late
embryogenesis-abundant (LEA), and HSPs was also found. The genes encoding stress-protective
proteins were constitutively expressed at high levels even in unstressed controls. Significant percent-
age of the genes upregulated upon dehydration and rehydration did not have sequence homology
among annotated in plant database genes (Gechev et al. 2012a).
29.7 CONCLUSION AND FUTURE PERSPECTIVES

The anticipated climate changes are a great challenge for the worlds’ agriculture because they
can cause water scarcity in many places on Earth. The wild flora and particularly the plant tol-
erant to drought and heat can serve as sources of resistance traits for plant biotechnology and
agriculture practice. Resurrection plants such as H. rhodopensis and Ramonda sp. are consid-
ered as model plants for future investigation of intimate mechanisms of plant drought tolerance.
On the other hand, Haberlea rhodopensis contains important secondary metabolites and can
serve as a potential source of bioactive compounds with putative application in pharmacology,
veterinary medicine, and cosmetics (Djilianov et al. 2009; Berkov et al. 2011; Ebrahimi et al.
2011). H. rhodopensis possesses also anticlastogenic and radioprotective compounds according to
Popov and coworkers (Popov et al. 2010). The extract of Haberlea was used by cosmetic compa-
nies for antiaging treatments, protection from oxidation, increased skin elasticity, and enhanced
skin radiance (Dell’Acqua and Schweikert 2012). Endemic plants such as H. rhodopensis and
Ramonda sp. are also object of conservation biology (Daskalova et al. 2011). Resurrection plants
from Gesneriaceae family are included in the Red Book of Bulgaria and in the European Register
of rare, endangered, and endemic plants and are subjects of world’s conventions on the preser-
vation of biodiversity. Therefore, Haberlea and Ramonda are subjects of conservation efforts
including GPS mapping of wild habitats and creation of in vitro gene banks for preservation and
propagation and multiplication of these plants for research and reintroduction in nature (Djilianov
et al. 2009; Daskalova et al. 2011). Recently, an unusual ability of Haberlea to survive for long
periods without light was found (Denev et al. 2012), which can make it a model plant for new
investigations in the area of photobiology.
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30 Salinity and Amenity
Horticulture
Atif Riaz and Peter M. Martin
CONTENTS
30.1 Introduction...........................................................................................................................606
30.2 Sources of Salts in Soils........................................................................................................607
30.3 Processes of Salinization.......................................................................................................608
30.4 Salinity in Urban Areas.........................................................................................................608
30.4.1 Natural Salinity (Primary Salinity)...........................................................................609
30.4.2 Human-Induced Salinity (Secondary Salinity).........................................................609
30.5 Types of Salt-Affected Soils.................................................................................................. 610
30.6 Measurement of Soil Salinity................................................................................................ 612
30.7 Salinity in Water.................................................................................................................... 615
30.7.1 Classification of Poor-Quality Waters....................................................................... 615
30.8 Effects of Salinity on Plants.................................................................................................. 615
30.9 Landscape Management under Salinity................................................................................ 618
30.9.1 Use of Salt-Tolerant Plant Species............................................................................. 619
30.9.1.1 Salt-Tolerant Grasses................................................................................... 623
30.9.2 Irrigation Practices.................................................................................................... 623
30.9.2.1 Use of Suitable Irrigation Systems.............................................................. 623
30.9.2.2 Use of Reclaimed/Sewerage Water............................................................. 624
30.9.2.3 Supplemental Watering............................................................................... 624
30.9.2.4 Issue of the Use of Supplemental Water for Irrigation............................... 624
30.9.3 Agronomic Practices for Saline Landscaping........................................................... 624
30.9.3.1 Provision of Drainage............................................................................... 625
30.9.3.2 Land Preparation....................................................................................... 625
30.9.3.3 Incorporation of Amendments/Chemicals................................................ 625
30.9.3.4 Application of Mulch................................................................................ 626
30.9.3.5 Pretreatment of Seeds/Seedlings.............................................................. 626
30.9.3.6 Planting Techniques.................................................................................. 626
30.9.3.7 Fertilizer Application................................................................................ 627
30.9.3.8 Aftercare of Plantation.............................................................................. 627
30.9.3.9 Harvesting/Pruning.................................................................................. 628
30.9.3.10 Minimizing Salt Damage due to Deicing................................................. 628
References....................................................................................................................................... 628
605
30.1 INTRODUCTION
Plants in amenity horticulture are frequently exposed to environmental conditions without any pro-
tection, which can induce physiological stress and adversely affect their growth and appearance.
Over the last few decades, many reviews have been published on how plant species respond to
different environmental stress factors including drought (Munns and Tester, 2008; Farooq et al.,
2009; Riaz, 2010; Riaz et al., 2010; Ricardo, 2012), salinity (Flowers et al., 1977; Greenway and
Munns, 1980; Läuchli and Grattan, 2007; Munns, 2011; Nadeem et al., 2012), temperature extremes
(Krasensky and Jonak, 2012), and pollution (Bell et al., 2011). From these reviews, it is evident that
important advances are being made in understanding the fundamental mechanisms by which plants
adapt to stress, while at the applied level, considerable effort is being devoted to the search for effi-
cient screening methods for the identification of stress-resistant genotypes. Germplasm so identified
can ultimately be used for breeding and development of stress-resistant landscape plants (Steve and
Harold, 2000; Martin, 2004).
Among abiotic stresses, salinity caused by elevated levels of sodium chloride (NaCl) and other
soluble salts is widely recognized as the most serious environmental problem affecting urban and
rural landscapes (Wilson, 2003). This phenomenon is more evident in arid and semiarid regions
(Ghassemi et al., 1995), where evaporative demand is high and precipitation is insufficient to leach
the salts away from the root zone (Viégas et al., 2001; Zhao et al., 2007). Soil salinization is on the
increase all over the world due to removal of deep-rooted vegetative cover and the cultivation of
high evapotranspiration crops with limited applications of irrigation water, thereby allowing net
salt movement in the soil profile to be upward instead of downward. Also, the use of poor-quality
water is an important cause of salinization. Estimates of the proportion of the world’s croplands that
have increased in salt content over the last 50–60 years range from approximately 20% (Sairam and
Tyagi, 2004) to as high as 50% (Szabolcs, 1992; Flowers, 1999). According to the FAO Land and
Plant Nutrition Management Service, currently over 6% of the world’s land is sufficiently severely
affected to warrant the classification of its soils as either saline or sodic (see Table 30.1).
According to an estimate published in 1991, salinity caused a global income loss of about US$
11.4 billion per year in irrigated areas and US$ 1.2 billion per year in nonirrigated areas (Dregne
et al., 1991), whereas the cost of reclamation of salt-affected agricultural land has been estimated
conservatively to be about US$ 12 billion per annum, and this figure is expected to increase as soils
are further affected with time (Ghassemi et al., 1995).
TABLE 30.1
Regional Distribution of Salt-Affected Soils
Saline Soils Sodic Soils
Total Area
Regions Mha Mha % Mha %
Africa 1,899 39 2.0 34 1.8
Asia, the Pacific, and Australia 3,107 195 6.3 249 8.0
Europe 2,011 7 0.3 73 3.6
Latin America 2,039 61 3.0 51 2.5
Near East 1,802 92 5.1 14 0.8
North America 1,924 5 0.2 15 0.8
Total 12,781 397 3.1 434 3.4
Source: Dajic, Z., Salt stress, physiology and molecular biology of stress toler-
ance in plants, in: Rao, K.V.M., Raghavendra, A.S., and Reddy, K.J.
(eds.), Physiology and Molecular Biology of Salt Tolerance in Plant,
Springer, Dordrecht, the Netherlands, pp. 41–99, 2006.
Mha, Million hectares.
Salinity and Amenity Horticulture 607
Salinity of the soil has long been recognized as a measure of the total soluble salts in the soil,
the dominant anions usually being chloride, sulfate, and bicarbonate, while the dominant cations
are typically sodium, magnesium, and calcium. All soils contain some soluble salts: it is only when
the amounts are excessive that problems occur. Districts with low precipitation are more prone to the
development of saline soils because the rainfall is insufficient to leach down salts that accumulate in
the surface soil. In accordance with long-established terminology, salt-affected soils are categorized
as saline, sodic (alkali), or saline–sodic (saline–alkali) based on the level of accumulation of total
salts and whether soluble carbonates are present. This will be further explained in Section 30.5.
With increasing costs of acquiring freshwater, saline and other alternative water sources are
beginning to be used for irrigating large urban landscapes in many countries. Saline water sources
with salinity greater than 1000 mg/L include groundwater, reclaimed municipal effluent, and agri-
cultural drainage water, the use of which can conserve potable water and reduce irrigation budgets
(for an example, see Fox et al., 2005). However, these low-quality waters, finding their way into
drainage and runoff, can cause serious damage to downstream water users, while direct effects on
soil structure at the site of use lead to decreased infiltration rate and increased soil erosion and in
arid and semiarid districts may contribute to desertification (Banin and Fish, 1995). Alternative
water sources have for many years been recognized as a salinization threat to clean irrigated soils
(Westcot and Ayers, 1984; Miyamoto and Arturo, 2006), so their ever-increasing use is a matter
of grave concern. Water quality issues for amenity horticulture have received growing attention as
potable water sources have become scarcer and more expensive. However, it is increasingly recog-
nized that the high rates of water use in many forms of amenity horticulture place definite limits on
the extent to which these sources can be used because of the very real risk of buildup of salinity in
the soil and in the underlying groundwater.
As soluble salt levels are raised, it becomes increasingly more difficult for plants to extract water
from the soil (osmotic effects), leading to a progressive deterioration in plant growth. This osmotic
effect is sometimes referred to as physiological drought since affected plants show some visual symp-
toms similar to those of plants suffering from lack of water, but it must be remembered that there may
also be direct toxic effect from some of the salts present. In addition, elevated salt levels alter ionic
relations and have been reported to induce oxidative stress (Molassiotis et al., 2006; Silva et al., 2008)
in some species. Plant species and varieties differ markedly in salt tolerance, with very sensitive types
such as violets being killed by levels that would have little or no visible effect on carnations.
The aim of this chapter is to present an outline of the science and practice of landscape establish-
ment and landscape management on salt-affected lands in and near urban areas. Although much of
the science is relatively complex, it is hoped that the manner of presentation adopted here will be
found useful by both planners and practitioners alike. Those interested in the scientific details of
the biochemical, physiological, and anatomical responses of plants or topics such as the physico-
chemical aspects of soils and the breeding of salt-tolerant species should refer to the comprehensive
reviews listed in the first paragraph of this section.
30.2 SOURCES OF SALTS IN SOILS

Understanding the causes of salinization is a prerequisite to its management and possible mitiga-
tion in amenity horticulture. Salts in soils originate from a number of sources such as salts released
from weathering of bedrock, salts impregnated in the soil profile (e.g., in the Indus basin after the
sea receded), salts present in shallow groundwater, and salts contained in irrigation water. Although
rain always contains some dissolved salts, it is usually only in areas very close to the coast that these
additions are important in terms of soil salinity. Depending on the prevailing wind patterns, the
strip of land immediately adjoining the sea may receive large increments of salt via wind-drifted
salt spray, while large areas of low-lying coastal and estuarine land may be salinized through seawa-
ter inundation during exceptionally high tides or in times of flood. Used responsibly, the fertilizers
and soil amendments employed in amenity horticulture contribute only negligible amounts of salt.
30.3 PROCESSES OF SALINIZATION

The first process is the direct addition of salts through poor-quality irrigation water or excessive applica-
tions of soluble fertilizers, leading to an immediate rise in the salinity level of the soil water. Secondly,
salts present in the soil profile may be redistributed in such a way as to generate a zone of salinized soil
within the root range of the plants. Thus, in a deep soil profile, the salts may initially be uniformly distrib-
uted at relatively low levels or accumulated at a certain depth below the root zone, in either case, there-
fore, having little effect on plant growth. However, these salts may be redistributed by water movements
in response to climatic conditions to accumulate at toxic levels in the root zone. Shallow groundwater
and dry climate accentuate the upward movement of salts and salinization of the root zone, while heavy
irrigations, heavy rainfall, and good soil cover minimize it, especially in well-drained soils.
Bedrock or other soil parent material impregnated with salt by contact with the sea or other
saline water bodies in the prehistoric era (e.g., in the Indus basin, Pakistan, and the Wianamatta
Shale of the western Sydney region, Australia) will, under suitable climatic and topographic con-
ditions, give rise to soils having accumulations of salts at the surface or at a shallow depth. Soils
whose salt arises in this way are usually described as having natural or primary salinity. On the
other hand, salinization caused by human intervention, for example, development of surface irri-
gation, deforestation, or use of saline water for irrigation, is generally termed secondary salinity.
These processes are described in greater detail in the following section.
30.4 SALINITY IN URBAN AREAS

Amenity horticulture is mainly focused in urban and peri-urban areas, which makes it important to
understand the causes of soil salinization in these areas as distinct from the more natural processes
operating in rural districts. Urban salinity is the term applied to the accumulation of salts (often
dominated by sodium chloride and sodium sulfate) in soil and water to levels that adversely affect
natural and built assets in urban areas, including plants, animals, rivers, streams and lakes, water
supplies, landscape features, buildings, roads, and railways.
Salts are corrosive and damage infrastructure, especially reinforced concrete structures and ser-
vices containing steel or iron components and ultimately shortens their life (Figure 30.1). Included
here are roads underlain by concrete bases, bridges, railway lines, buildings, houses, pavements and
underground services such as gas, water, and drainage lines. In addition, salinity leads to the degrada-
tion of soil structure with successive loss of stability that contributes to movements of the foundations
of roads and buildings with consequent damage. In urban areas it largely increases the maintenance
FIGURE 30.1 (See color insert.) Damage to structures caused by salinity.

costs of municipal, recreational, residential, and commercial facilities. Through its effects on plant
biology and soil structure, salinity also causes degradation of sports fields, open green spaces, and
vegetation in urban parks and gardens (Slinger and Tenison, 2007) reducing their amenity values.
Salinity problems in cities and towns not originally built on saline surface soils are a result of
urban developments. Podmore (2009) highlights the role of rising water tables caused by the leaking
of water from sewerage and drainage pipes and obstruction or modification of natural surface and
subsurface drainage paths. The ever-increasing amount of impervious surface in urban areas leads to
rapid runoff after heavy rains and the concentration of the water in low-lying areas, adding further to
the influences raising the water table. Effluents from towns, intensive agriculture, and some chemical
industries also contain high levels of salts, meaning that unless the disposal of these waters is managed
appropriately, the result will be polluted waterways, rising water tables, and ultimately salinized urban
soils. In towns with originally small areas of salinized soil, increased intensity of urban development
usually leads to a significant extension of the salt-affected area by the same processes as mentioned
previously. Podmore (2009) also points out that, there is a great need to impart a greater awareness of
the all-pervasive negative effects of urban salinity on urban living, recommending more community
education programs focused on ways to minimize the raising of the water table.
30.4.1 Natural Salinity (Primary Salinity)

Various factors that may contribute to salinization of soils under natural conditions are given
as follows:
1. Weathering of surface rocks releases salt of various types, mainly chlorides and sulfates of
sodium, calcium, and magnesium, depending upon the rock type and the extent of weathering.
Among them, sodium chloride is typically the most abundant. The released salts accumulate
in groundwater as a result of water percolating through the weathering deposits.
2. Rising saline groundwater (e.g., during the rainy season) brings the naturally accumulated
salts close to the soil surface, and evapotranspiration results in water loss leaving salts con-
centrated in the upper soil, with subsequent bad effects on plant growth and soil structure.
This phenomenon is a common cause of saline soils in arid and semiarid regions of the
world where the rainfall and temperature conditions are such that salts rarely have the
opportunity to leach downward out of the plant root zone (Nazir et al., 2006). The process
is accentuated by poor soil drainage (Alan, 1994).
3. Rainfall may also carry salts dissolved during its passage through the atmosphere (cyclic
salts). Though the concentration of salts in rainfall over continental areas is very low, it can
be significant near the coasts ranging from 6 to 50 mg of salt per liter of rain. An average con-
centration of 10 mg/L can add 10 kg/ha of salt for each 100 mm of rainfall per year (Munns,
2009). Very close to the coast, the growth and development of urban gardens and landscape
plants can be directly reduced as a result of exposure to salt spray (Vernieri et al., 2010).
30.4.2 Human-Induced Salinity (Secondary Salinity)

Secondary salinization of soil, surface water, or groundwater due to human activities such as urban-
ization and agriculture can occur under both irrigated and rain-fed conditions. Secondary saliniza-
tion is accelerated by the processes listed as follows:
1. Water table rise associated with urban activities. These activities include the following:
removal of native vegetation and replacement by hard (impervious) surfaces and less water-
efficient shallow-rooted plants; excessive watering of home gardens, lawns, parks, sports
fields, and golf courses; leakage of water from sewerage and drainage pipelines; and obstruc-
tion or modification of natural surface and subsurface drainage paths by construction of
urban structures like roads, bridges, and flood/drainage control structures (McGregor, 2002).
2. Use of poor-quality natural irrigation water. In irrigated areas, higher salt concentrations can
be created in the soil by the use of irrigation water with high levels of salts (Messer, 1982).
3. Improper irrigation techniques. Inappropriate durations of application, uneven distribution
of water from an incorrectly operated sprinkler system, and ponding in unleveled fields all
contribute to salinization of the root zone where there is a shallow saline water table.
4. Anthropogenic salt inputs. These include excessively heavy applications of fertilizers
or manures (Panno et al., 2006), deicing salts used on sidewalks and roads (Buttle and
Labadia, 1999; Mason et al., 1999), and the effluent from domestic and industrial waste
management systems (Phillip et al., 2003).
5. Use of reclaimed water for irrigation. Reclaimed waters generally have higher salinity than
potable water. Although their use in urban areas is seen as desirable to conserve supplies
of potable water, unless adequate leaching fractions are used, salts will accumulate in the
root zone of plants (Schuch et al., 2008).
6. Overextraction of subsoil water. Overextraction of these waters lowers the water table and
in some situations permits the inflow of more saline water from other parts of the aquifer,
thereby causing deterioration of quality of the irrigation water. Overextraction through
tube wells in the irrigated lands of the Indus basin (Pakistan) and the Batinah coast of the
Sultanate of Oman has led to water quality reduction and rapid salinization of the surface
soil. Similar problems have been encountered in many large urban parks in lower rainfall
districts as a result of overdependence on groundwater bores.
30.5 TYPES OF SALT-AFFECTED SOILS

Salt-affected is used as the general term to describe soils showing accumulation of salts above a defined
level that may affect normal development of common plants and, more specifically, includes the soils
classified as saline, sodic, or saline–sodic, with further division into moderately and highly affected
categories by some scientists. Various definitions and nomenclature of these categories have been used
by different countries/organizations (e.g., USDA system, 1954; USSR system; European system; FAO-
UNESCO system, 1974). However, the most commonly used nomenclature and levels for a soil-based
system are those proposed by the US Salinity Laboratory (USDA, 1954). Other approaches, using dif-
ferent key criteria, include that of Rafiq (1990) who classified soils of the Indus basin from the point of
view of capability for crop production and that of Qureshi et al. (2003) who classified salt-affected soils
of the same area from the agronomic point of view. Ghafoor et al. (2004) have summarized into a con-
venient tabular form (Table 30.2) the chemical and physical properties of the three different classes of
salt-affected soils in the US Salinity Laboratory system, as defined in the text of Chapter I of the USDA
Handbook No. 60 (USDA, 1954). To this, they have added information on plant growth, amelioration
prospects, and ecological factors for each of the soil types (see Table 30.2).
Reduced to its simplest level, the saline soils are those with elevated salt levels (as indicated by
an ECe of four or more) but a soil pH of less than 8.5, while the sodic soils are those with elevated
soil pH (above 8.5, often as high as 10.5) and a salt content less than ECe 4.0. The saline–sodic soils
combine the worst features of both types, with extremely high salt and pH values.
To the casual observer, for a saline soil, the easiest approach gaining information about
the salinity distribution pattern of a site is to visit it at a time in the year when environmental
conditions are most conducive for salt accumulation on the soil surface. White crusts on the
soil surface are most obvious in the Indus basin lands during the month of March when salts
move up to the surface with capillary rise of water and are left there after water evaporates
(Figure 30.2). On the other hand, sodic soils are less obvious but in some cases can be identified
TABLE 30.2
Distinguishing Characteristics/Features of Salt-Affected Soils (USDA System)
I. Saline Soils II. Sodic Soils III. Saline–Sodic Soils
Chemical properties
Dominated by neutral soluble salts Less dominated by neutral soluble Properties of category I and II soils are
(ECe ≥ 4 dS/m), mainly chlorides and salts (ECe < 4 dS/m), contains present (aECe ≥ 4 dS/m).
sulfates of Na+, Ca2+, and Mg2+. salts capable of alkaline
hydrolysis, e.g., Na2CO3.
pHsoil < 8.5. pHsoil ≥ 8.5. pHsoil ≥ 8.5.
No well-defined relationships between Well-defined relationships between Relationships may or may not be
pHsoil and ESP or SAR. pHsoil and ESP or SAR. defined between pHsoil and bESP or
cSAR.
Ca2+ and Mg2+ in considerable Ca2+ and Mg2+ concentration is low. Ca2+ and Mg2+ concentration is low.
concentration in spite of Na+ being
the dominant cation.
Soils contain significant amount of More lime than gypsum. More lime than gypsum.
lime and gypsum.
Physical properties
In the presence of neutral soluble salts, High exchangeable Na+ (ESP) and Mostly like category II.
clays are flocculated and have stable high soil pH result in dispersion
structure. and unstable structure.
Permeability to air and water Permeability to air and water is May be like category I or II with high
comparable to normal soils. restricted and lower than that of values for salts and ESP, SAR, and
normal productive soils. soil pH.
Effect on plant growth
Adversely affected due to the osmotic Adversely affected due to poor Both the problems of categories I and
effect of excess soluble salts, physical properties because of II may exist together depending upon
resulting in reduced water availability. high Na+ and high soil pH-induced ECe and SAR/ESP/pHsoil.
dispersion.
Toxicity through specific ion effects, High soil pH leading to nutrient —
such as Na+, Cl−, and B. problems including deficiencies of
Ca2+, K+, Cu, Zn, Fe, and Mn and/
or specific ion toxicities like Na+,
Mg2+, and Mo.
Soil improvement
Removal of soluble salts through Essentially requires a source of Ca2+ Like category II.
leaching and drainage is essential to replace exchangeable Na+
without any Ca amendments. followed by leaching.
Geographic distribution
Tends to dominate under arid and Tends to dominate under semiarid Tends to dominate under semiarid and
semiarid climates. and subhumid climates. subhumid climates.
Groundwater quality
Groundwaters have potential for Low to medium electrolyte Like category II.
salinity problems. concentration with RSCs and high
SAR, i.e., potential for sodicity.
a ECe, electrical conductivity.

b ESP, exchangeable sodium percentage.
c SAR, sodium adsorption ratio.
FIGURE 30.2 Salts accumulated on soil surface.
by the paucity of plant growth associated with blackening and compactness of the soil surface
due to the reaction of the free alkali with organic matter.
General indications of salt problems in urban areas are patchy grass covers and stunted growth
of plants in the landscape. Decline of permanent plantation and dieback of shrubs and trees without
any pathogenic attack also indicate high salt stress conditions. Such observations can be verified
through random on-the-spot soil testing by field methods, by laboratory testing of soil samples, or by
more sophisticated field-survey instruments using the electromagnetic induction method (EM-38)
(Triantafilis et al., 2001) for profile salinity.
30.6 MEASUREMENT OF SOIL SALINITY

As can be seen from Table 30.2, the key measurements in the detection and classification of saline
and sodic soils are the amount of salt and the soil pH. In earlier times (e.g., Kinch, 1906), the usual
approach to measuring soil salt content was to extract a known amount of soil with distilled water,
filter, evaporate the filtrate, and record the weight of the dry residue. This time-consuming and
tedious process is subject to many assumptions and at best gives results of only approximate accu-
racy but is the origin of the method of reporting soil salinity as total soluble salts in milligrams per
kilogram of dry soil, which is still preferred by some practitioners. Following the invention, in the
1920s, of convenient conductimetric methods for assessment of the salt content of solutions, this
technique was soon applied to the measurement of the electrical conductivity (EC) of water extracts
of soil and used as a guide to the salinity level of the soil.
Because the salt concentration (and therefore the EC) of the water in the soil pores increases as
the soil dries out, it is clear that plants growing in a given soil are subjected to a cycle of variation
in salinity as the soil moisture content moves from field capacity to wilting point and back again.
To provide consistency in soil salinity measurements by the conductivity method, it is therefore
necessary to make all test measurements on extracts in which there is a definite and standard rela-
tionship between the amount of soil and the amount of water used. Starting with air-dry soil, two
rather different extraction methods have been widely used since the 1930s. The first method uses
a 1:5 soil to water ratio on a weight for weight (w/w) basis, while the second involves the addition
of water until the soil pores are completely filled with water (saturated). This is not the place to go
into the theory of the methods or their relative academic merit. In practice, the 1:5 method is now
generally preferred because of its simplicity and rapidity (no filtration needed) and the fact that in
the laboratory, the pH and EC can be measured directly on the same extract, all factors leading to
lower laboratory charges. EC values determined by this method are indicated by the notation EC1:5.
In contrast, the saturation method requires considerable skill in judging the saturation point and
requires the use of vacuum-assisted filtration of the thick slurry produced in the saturation process.
A further drawback is that although pH can be determined on the slurry or the filtrate, modern prac-
tice is to determine soil pH on extracts having a 1:5 or 1:2 soil to water ratio (w/w) and values of EC
determined by the saturation extract method are indicated by the notation ECe.
In measuring EC with either system, it is important to remember that the value is temperature
sensitive, and for accurate work, it should be corrected to a standard temperature, usually 20°C or
25°C. Some instruments incorporate a temperature probe alongside the EC probe and contain cir-
cuitry that makes the correction automatically; in others, the temperature of the extract at the time
of measuring must be noted and the correction should be applied manually.
Chapter 1 of the now classic handbook of the US Salinity Laboratory, published under the
title of Diagnosis and Improvement of Saline and Alkali Soils, Agriculture Handbook No. 60
(USDA, 1954), discusses the merits of the 1:5 and saturation extract methods. The conclusion was
reached that for specialized salinity research, the saturation method was preferable. The result
is that a very large body of data, mostly from the United States, has accumulated using the ECe
method. On the other hand, the method almost universally used today is the EC1:5 method. This
has led to a growing interest in ways of converting between the two systems. In a comprehensive
review, Shaw (1999) notes that several workers have developed empirical conversions approximat-
ing to EC1:5 × 6.5 = ECe. In an effort to develop a theoretically more rigorous conversion, Shaw
(1999) found that it was necessary to use additional factors such as the chloride content of the 1:5
extract and the approximate clay content of the soil.
Results of the EC test by either method should be presented using the preferred unit of decisie-
mens per meter (dS/m). This is numerically equivalent to millisiemens per centimeter (mS/cm) and
the older unit of millimhos per centimeter (mmho/cm). Occasionally, one encounters EC meters
calibrated in microsiemens/centimeter (μS/cm). The relations between these units are as follows:
1 dS/m = 1 mS/cm = 1 mmho/cm = 1000 μS/cm
Older references (e.g., USDA, 1954) provide many for converting ECe to approximate values for
the amount of salt per kilogram of soil and to an estimate of osmotic pressure at saturation, but it
must be remembered that these conversions are based on averages for the mixture of salts present,
something that can vary widely from aquifer to aquifer.
Testing of composite soil samples to estimate salinity will often fail to provide an accurate pic-
ture of the salinity distribution across the field. It is better to assess separately soil samples from
different parts of the field, either working on a systematic grid or selected on the basis of visual
observations of salt crusts and vegetation stand. Samples may be collected for surface soil only,
but a better understanding is gained if boreholes are run down through the soil profile and samples
taken at intervals of 10, 20, or 30 cm depending on the depth of the profile, the rate of change
of salinity with depth, and the amount of detail required. Profile sampling guided by in-the-field
testing using the EC1:5 method for salts and the Raupach color method for pH can readily be done
(Raupach and Tucker 1959) with a minimum of fuss (see Figure 30.3).
Interpretation of results for the diagnosis of a saline soil is straightforward. As per the USDA
guidelines (see Table 30.2), if the soil pH is 8.5 or lower and the ECe is 4.0 dS/m or more, the soil is
definitely saline. ECe readings between 1.5 and 3.9 dS/m are indicative of elevated salt content that
could lead to damage in very salt-sensitive plants. With the EC1:5 method, the guidelines for diag-
nosing a saline soil are pH below 8.5 and EC1:5 0.6 dS/m or greater. Damage to very salt-sensitive
plants can be expected if the EC1:5 value exceeds 0.25 dS/m (Martin & Associates Ptv. Limited,
personal communication).
FIGURE 30.3 Efficient systems for infield testing of salinity and pH.
Diagnosis of sodic (alkali) and saline/sodic soils is a little more complex. The key feature is a pH
in excess of 8.5 as a result of the presence of carbonate ions. In a sodic soil, the salt level as indicated
by EC measurements by either method is less than that of a saline soil, whereas in a saline/sodic soil,
which combines the properties of both soil types, the EC will be equal to or greater than that defined
for a saline soil. At a more advanced level, sodicity status can be confirmed by measuring the sodium
adsorption ratio (SAR) of the liquid in contact with the soil in either the saturation extract or 1:5
procedures. The SAR is concerned with the ionic balance between sodium ions on the one hand and
the divalent calcium and magnesium ions on the other as shown in the formula SAR = Na+/[(Ca2+ +
Mg2+)/2]1/2. An insufficiency of these latter two ions leads to the development of sodicity in the liquid
phase that is accompanied by an undesirable increase in the exchangeable sodium percentage (ESP)
of the cation exchange complex in the solid phase of the soil in contact with the saturation extract.
SAR and ESP are not field tests and require a well-equipped laboratory for their determination.
Indicative interpretation guidelines for the determination of sodic and saline/sodic soils based on
the ECe system are set out in Table 30.3 (USDA, 1954).
TABLE 30.3
US Salinity Laboratory Parameters for the
Classification of Saline, Sodic (Alkali), and
Saline/Sodic Soils
Type of Soil ECe, dS/m ESP SAR pHs
Saline ≥4 <15 <13.2 <8.5
Sodic <4 ≥15 ≥13.2 ≥8.5
Saline–sodic ≥4 ≥15 ≥13.2 ≥8.5
Source: USDA, Diagnosis and Improvement of Saline and

Alkali Soils, United States Salinity Laboratory
Staff, US Department of Agriculture, Washington,
DC, 1954.
Interpretation guidelines for the diagnosis of sodic and saline/sodic soils using the EC1:5 system
and the values for SAR and ESP of the liquid and solid phases, respectively, in contact with that
extract are provided by Rengasamy and Churchman (1999).
30.7 SALINITY IN WATER

Water quality can have a profound effect on plant growth. All irrigation waters contain dis-
solved salts, but their composition and concentration varies greatly depending upon the source
of the water.
30.7.1 Classification of Poor-Quality Waters

In general, water quality is assessed by measuring EC, SAR, and residual sodium carbonate (RSC),
all of which influence plant performance and soil conditions, while measurement of ions like
sodium, chloride, and boron is especially important from the point of view of potentially toxic
effects on plants. EC reflects the salinity of the water, SAR measures the tendency of the water
to raise the exchangeable sodium level of the soil (the higher the value, the worse the effects), and
RSC provides a guide to the capacity of the water to increase soil pH to excessively high levels. The
method of calculating SAR has been given in the previous section, while RSC is calculated as fol-
lows: RSC = (Ca 2 + + Mg2 + ) - (CO32 - + HCO3-1 ). A convenient classification for poor-quality water,
and one that has stood the test of time, is that drawn up by the US Salinity Laboratory (USDA,
1954) (Figure 30.4).
The following table (Table 30.4), extracted from Ghafoor et al., 2004, is based on the guidelines
for the use of irrigation waters of different qualities prepared by the US Salinity Laboratory (USDA,
1954). In both the previously given diagram and the table in the following discussion, salinity cat-
egories are indicated by the prefix C and sodicity (i.e., risk of developing elevated ESP levels in the
soil) by the prefix S.
As mentioned previously, plant species and varieties differ markedly in their capacity to grow
in salt-affected soils. Dividing plants broadly into sensitive, semisensitive, and tolerant groups with
respect to their reaction to saline water, the acceptable water quality classes for irrigation on soils of
different textures are set out as follows in Table 30.5 (Ghafoor et al., 2004).
30.8 EFFECTS OF SALINITY ON PLANTS

Plant species exhibit different morphological (Jaleel et al., 2009), physiological (Niu and
Cabrera, 2010; Sarah et al., 2010), and biochemical (Hasegawa et al., 2000) responses under
saline conditions. In general, high levels of salts in soil or irrigation water as well as in water
sprays reduce growth of plants. The reduction in plant growth varies with the level of salinity,
type of salinity, amount of specific toxic ions, management practices, and tolerance level of
the species involved. Plants generally show stunted growth and frequently twig dieback, while
the size, shape, and color of flower and leaves, as well as the overall appearance of ornamental
plants, may be badly affected (Küçükahmetler, 2002). An overview of effects of salinity on
plants is shown in Figure 30.5.
Many of the symptoms of salt injury in plants are similar to drought as both conditions are
characterized by wilting (water stress) and reduced growth. As mentioned previously, increasing
the level of salts in the soil raises the osmotic pressure of the soil water and results in reduced
water uptake even if the soil has a high moisture content. In addition, salinity may cause spe-
cific ion effects, for example, sodium (Na), chloride (Cl), and boron (B), which can be toxic
to plants if absorbed in excess, damaging both biochemical and photochemical processes of
photosynthesis (Munns and Tester, 2008), with symptoms ranging from leaf burn to necrosis
(Figure 30.6) (Munns, 2002) followed by leaf shedding and twig dieback. High levels of sodium
100 2 3 4 5 6 7 8 1000 2 3 4 5000
Very
high
30
4 28 C1–S4
26 C2–S4
High
3
24
C3–S4
22 C4–S4
C1–S3
20
Sodium adsorption ratio (SAR)
Sodium (alkali) hazard
18
C2–S3
Medium
16
2
14
C3–S3
C1–S2
12
C2–S2
10 C4–S3
8
C3–S2
6
Low
1
C4–S2
C1–S1
4
C2–S1
C3–S1
2
C4–S1
0
100 250 750 22 50
Cl
as Conductivity—μ-mho/cm (EC × 106) at 25°C.
s
1 2 3 4
Low Medium High Very high
Salinity hazard
FIGURE 30.4 Diagram for classification of irrigation water. (From USDA, Diagnosis and Improvement of
Saline and Alkali Soils, United States Salinity Laboratory Staff, US Department of Agriculture, Washington,
DC, 1954.)
decrease the availability of potassium (K) and magnesium (Mg) to plants leading to nutritional
imbalance. Other plant symptoms due to salinity problem include delay in leaf bud and flower
bud break, leaves turning bluish green and darker than normal, stunted growth, stems with
short internodes, and chlorosis of the leaves. Leaf thickness also increases due to more layers
of mesophyll and larger cell size; thus, leaves become more succulent (Ibrahim et al., 1991;
Mazher et al., 2007). In addition to these physiological disturbances, salinity and other abiotic
factors such as heavy metals can trigger oxidative stress due to accumulation of reactive oxygen
species (Gratão et al., 2005; Cavalcanti et al., 2007). Young or unestablished plants or those
suffering from other stresses are more sensitive to salinity damage than healthy, mature plants.
TABLE 30.4
Guidelines for the Use of Irrigation Waters in Terms of US Salinity Laboratory
Water Quality Classification of 1954
Salinity Classification Sodicity Classification
Low-salinity water (C1) can be used for irrigation of most Low-sodium water (S1) can be used for irrigation on
crops on most soils with little likelihood that soil salinity almost all soils with little danger of the development of
will develop. Some leaching is required, but this occurs harmful levels of exchangeable Na+. However,
under normal irrigation practices except in soils of Na+-sensitive crops such as stone-fruit trees
extremely low permeability. and avocado may accumulate injurious
concentration of Na+.
Medium-salinity water (C2) can be used if a moderate Medium-sodium water (S2) will present an appreciable Na+
amount of leaching occurs. Plants with moderate salt hazard in fine-textured soils having high CEC, especially
tolerant can be grown in most cases without special under low leaching conditions unless gypsum is present in
practices for salinity control. soils. This water may be used on coarse-textured or
organic soils of good quality.
High-salinity water (C3) cannot be used on soils with High-sodium water (S3) may produce harmful levels of
restricted drainage. Even with adequate drainage, special exchangeable Na+ in most soils and will require special
management for salinity control may be required and soil management including good drainage, high leaching,
plants with good salt tolerance should be selected. and organic matter additions. Gypsiferous soils may not
develop harmful levels of exchangeable Na+ from such
waters. Chemical amendments may be required for
replacement if exchangeable Na+, except that use of
amendments may not be feasible with waters of very
high salinity.
Very-high-salinity water (C4) is not suitable for Very-high-sodium water (S4) is generally unsatisfactory for
irrigation under ordinary conditions but may be used irrigation except at low perhaps medium salinity, where
rarely under very special circumstances. Soils must the solution of calcium from the soil or use of gypsum or
be permeable, drainage must be adequate, irrigation other amendments may make the use of these waters
water must be applied in excess to provide feasible.
considerable leaching, and very salt-tolerant crops
should be selected.
Source: Ghafoor, A. et al., Salt-Affected Soils, Principles of Management, Allied Book Centre, Lahore, Pakistan, 2004.
TABLE 30.5
Water Quality Classes and Their Suitability for Plants Grown in Soils
of Different Textures
Water Quality Class Suitable for
Soil Texture Sensitive Crops Semisensitive Crops Tolerant Crops
Clay C2S1 C3S1, C1S2 C4S1, C2S2
Loam C2S1, C2S2 C2S1, C2S2, C1S3 C4S1, C3S2, C2S3
Sandy loam C2S1, C2S2, C1S3 C4S1, C3S2, C2S3 C4S1, C4S2, C3S3
Loamy sand C3S1, C2S2, C1S3 C4S1, C3S2, C2S3, C1S4 C4S1, C4S2, C3S3, C2S4
Source: Ghafoor, A. et al., Salt-Affected Soils, Principles of Management, Allied Book Centre,
Lahore, Pakistan, 2004.
Morphological Physiological Biochemical
Alteration of Osmotic
phenological Change in gene
inhibition expression and
development
Energy protein
Succulence, Water uptake consumption synthesis
enlargement of by roots Specific Change in plant
palisade cells ion effect hormonal levels
Reduced
photosynthesis Influence on
Reduced shoot Direct toxicity cellular and
growth and dry nuclear volume
matter content;
Insolubility or
increases Reduction of
competitive absorption
root/shoot ratio enzymatic
activity
Nutritional
plant imbalance
FIGURE 30.5 Morphological, physiological, and biochemical effects of salt stress on plants. (From
Cassaniti, C. et al., The response of ornamental plants to saline irrigation water, in: Show, K.Y. and Guo, X.
(eds.), Irrigation—Water Management, Pollution and Alternative Strategies, Intech, Zagreb, Croatia,
pp. 131–158, 2012.)
FIGURE 30.6 (See color insert.) Necrotic leaves of Alstonia scholaris due to salinity.
Sodic (alkali) soils have additional negative effect on soil structure, which reduce aeration and
drainage, while the increase in soil bulk density as a result of loss of structure increases resis-
tance to root penetration.
30.9 LANDSCAPE MANAGEMENT UNDER SALINITY

The utilization of saline environments (salt-affected soils and brackish water) for successful growth
and development of crop plants is a well-established concept (see Qureshi and Barrett-Lennard,
1998; Wyn Jones et al., 2006). The same concept is equally applicable for development and main-
tenance of saline landscapes. The concept has two important components, namely, (1) selection
of suitable ornamental plant species that can withstand elevated levels of salinity and toxic ions
and (2) soil and water management practices that will maintain or lead to minimum accumulation
of salts in the root zone. In addition, use of amendments and fertilizers/manures to improve plant
growth is quite helpful for maintaining a good-quality landscape under saline environmental condi-
tions. It is emphasized that proper information on the site characteristics (especially soil conditions)
and on the quality of the water available for soil reclamation and irrigation is a prerequisite for suc-
cessful saline landscape development. The two components of saline landscaping are discussed in
the following sections.
30.9.1 Use of Salt-Tolerant Plant Species

The increasing demand for good-quality water for urban, domestic, and industrial uses has restricted
its availability for the irrigation of landscape ornamental plants, particularly in urban areas. The
alternative sources are found to be saline or reclaimed water. When the rising salinity level of the
water available for irrigation of landscape plants starts to cause problems, it is obvious that the use
of salt-tolerant ornamental plant species becomes essential, whether for residential, city, or highway
beautification. Plant species that have grown for thousands of years in naturally salt-affected areas
near any given city provide a good starting point for choosing species to try in these situations. In
cities with a frontage to the sea or a saline estuary, a careful study of the foreshore plants is sure to
provide many useful examples of species worthy of trial. Similarly, in cities with salt marshes or
salt deserts in their immediate hinterland, numerous useful species may be found. For example, in
naturally highly salt-affected district of Pakistan, Capparis decidua and Suaeda fruticosa are of
frequent occurrence and have proved of value in landscaping work in human-induced saline areas
in cities with similar climates (Figure 30.7).
Ornamental plants vary widely in their salinity tolerance and in their responses to a saline envi-
ronment (e.g., Niu and Rodriguez, 2006; Niu et al., 2007; Niu and Cabrera, 2010). The responses
of plants under salt stress may be of a morphological, anatomical, physiological, or biochemical
nature and are controlled by the genetic makeup of the plants. From the landscaper’s point of view,
salt stress responses such as changes in the overall growth pattern and development of discoloration
or necrosis of leaves or twig and branch dieback are generally undesirable and point to a need to
make use of more salt-tolerant plants at that site. However, in some cases, the stunting of growth,
reduction in size of leaves and flowers, and changes in flower color may not necessarily reduce the
landscape value of a given species provided that its survival is not threatened and the modified form
is aesthetically acceptable.
(a) (b)
FIGURE 30.7 (See color insert.) C. decidua (a) and S. fruticosa (b) are widely grown in salt-affected land-
scapes in Pakistan.
Trees, shrubs, and climbers are important parts of amenity horticulture. Salinity can affect the per-
formance of ornamental trees or shrubs by reducing their overall growth due to decreased leaf number
and decreased leaf expansion, reducing the photosynthetic area (Munns and Termaat, 1986). An impor-
tant measure of the reduction in aesthetic value of broad-leaved landscape plants is the percentage of
leaves showing necrotic areas, due to toxicity caused by high concentration of Na+ and Cl− in the leaves
(Karakas et al., 2000; Cassaniti et al., 2009). Other visible responses of woody plants in saline environ-
ments include crown dieback, lesions on the stem or trunk, and leaf scorch (Percival, 2005). In conifers,
needle tips turn brown and needles fall prematurely (Mazher et al., 2007), while with extended exposure
to saline conditions, dieback of limbs occurs followed by death of the trees (Dobson, 1991).
Studies on salt tolerance of various species of economic importance (including some that are
significant in amenity horticulture) have been carried out at the US Salinity Laboratory, Riverside,
California, from its inception in 1937. Mass and Hoffman (1977) summarized the results of nearly
40 years of these studies and developed relationships between reduction in growth or yield (relative
yield) and levels of substrate salinity. Based on the relative yield response curves, plant species
can be categorized as sensitive, moderately sensitive, moderately tolerant, and tolerant. Generally,
the salinity level at which 50% reduction in growth or yield takes place is considered as the basis
for comparison of salt tolerance of different plants. The studies conducted by the US Salinity
Laboratory were mostly done under controlled conditions in which levels of root zone salinity were
kept constant, whereas under natural conditions, the salinity levels in various parts of the root zone
are highly variable as a result of changes in soil moisture content, leaching events, and other factors.
As pointed out by Schleiff (2006), this often leads to better performance in the field than indicated
by controlled condition studies for a given species grown at the average salinity of the root zone.
Qureshi and Barrett-Lennard (1998) have described the technology for successful cultivation of
different grass, shrub, and tree species in different classes of salt-affected land in Pakistan. Their
approach involves the selection of the best fit species under the circumstances prevailing at a par-
ticular site with preference being given to local species adapted to those conditions, if available.
These authors have also proposed appropriate agronomic practices (land preparation, planting tech-
niques, irrigation practices, fertilizer application, etc.) for each species under given circumstances.
This commonsense type of approach is the key to successful saline landscaping as demonstrated in
the work of many specialists in various parts of the world. In all cases, a thorough assessment of the
climatic and soil conditions and economic constraints is used to provide the factual data on which
to base a proper decision about the most appropriate set of species and agronomic practices for a
given site. In relation to soil conditions, it is vital that a detailed survey is made on the site to reveal
the presence or absence of various growth-limiting factors in addition to salt. These factors include
soil density, presence of hardpan in the subsoil, presence of sodicity and specific toxic ions, nutrient
deficiency, and surface and subsurface drainage characteristics. The economic constraints include
availability of labor, difficulties of access to the site, availability and quality of irrigation water,
costs associated with land preparation, and cost of installation of drainage and irrigation systems.
The choice of plants for amenity horticulture is based on aesthetic and practical considerations. The
realization of an attractive landscape design depends on the active and normal growth of the species
selected rather than their mere survival as stunted and deformed specimens. The more difficult the
environment, the more limited is the number of species capable of delivering an adequate performance
under those conditions. In such situations, it has long been recognized that the use of locally adapted
species (and ecotypes or provenances within species) is advantageous (see, e.g., Hall et al., 1972). More
recently, this important concept has been emphasized in the amenity horticulture literature (Iles, 2003;
Kotzen, 2004; Savé, 2009) in relation to both biotic and abiotic stress tolerance.
By noting the limit of soil salinity that did not induce significant salt stress symptoms, plant
species used in amenity horticulture can be grouped as highly tolerant, moderately tolerant, and
sensitive. Detailed tolerance trials have been conducted with a considerable number of amenity spe-
cies, and practical experience has allowed the recognition of the salinity tolerance rating of a great
many more. In Table 30.6 in the following text, the salt tolerance of important candidate species
TABLE 30.6
Salt-Tolerant Plant Species for Horticulture (Excluding Grasses)
Salt
Common Names Botanical Names Tolerance References
Drought-tolerant trees
American holly Ilex opaca M Glen (2004)
Conocarpus Conocarpus erectus H Stephen et al. (2011),
Gilman and Watson (1993)
Crape myrtle Lagerstroemia indica M Cabrera (2009)
Japanese black pine Pinus thunbergii H Monk and Wiebe (1961),
Simini and Leone (1986)
Silver oak Grevillea robusta H Wu and Dodge (2005),
Costello et al. (2011)
Bottlebrush Callistemon viminalis M Miyamoto et al., (2004)
Arizona cypress Cupressus arizonica M
Melia Melia azedarach H Kratsch et al. (2008)
Weeping willow Salix alba H Qureshi et al. (2003),
Leucaena Leucaena leucocephala H Miyamoto et al. (2004),
Acacia spp. Acacia nilotica; Acacia H Marcar et al. (1995)
ampliceps
Eucalyptus E. camaldulensis H
Terminalia Terminalia arjuna H
Prosopis Prosopis juliflora, Prosopis H
nigra
Tamarix spp. Tamarix aphylla, Tamarix H
spicigera, Tamarix indica
Casuarina Casuarina equisetifolia H
Salt-tolerant shrubs
Azalea Rhododendron spp.a M Glen (2004),
Japanese privet Ligustrum japonicum M Cabrera (2009)
Yucca Yucca spp. H
Japanese euonymus Euonymus japonicus M
Butcher’s broom Ruscus aculeatus H
China rose Hibiscus spp.a H Knox and Schoellhorn
(2011), Li et al. (2012)
Fire bush Hamelia patens M Jarret (2003);
Smith (1995)
Hydrangea Hydrangea spp.a M Wu and Dodge (2005)
Century plant Agave americana L. H
Indian hawthorn Rhaphiolepis indica M Jull (2009), Glen (2004)
Oleander Nerium oleander H Miyamoto et al. (2004)
Rosemary Rosmarinus officinalis H
Star jasmine Trachelospermum M
jasminoides
Pittosporum Pittosporum tobira H Gilman (1999),
Wu and Dodge (2005)
Saltbush Atriplex spp. H Barson and Barrett-Lennard
(1995), Munns and Tester
(2008)
(continued)

Salt-Tolerant Plant Species for Horticulture (Excluding Grasses)
Salt
Common Names Botanical Names Tolerance References
Yellow oleander Thevetia peruviana M Black (2003)
Capparis C. decidua H Sharif and Khan (2009)
Salt-tolerant vines/climbers
Bougainvillea Bougainvillea spp. H Devitt et al. (2005)
Creeping fig Ficus pumila M Glen (2004), Wu and Dodge
(2005)
English ivy Hedera helix M
Honeysuckle Lonicera japonica M
Railroad vine Ipomoea pes-caprae H Suárez (2011)
Salt-tolerant ground covers

Agapanthus Agapanthus africanus M Russ (2007)
Blanketflower Gaillardia pulchella H
Golden Stonecrop Sedum acre H
Daylily Hemerocallis spp. M
Cast iron Aspidistra elatior M Glen (2004)
Mondo grass Ophiopogon japonicus H
Purple heart Setcreasea pallida M
Creeping juniper Juniperus horizontalis M Monk and Wiebe (1961)
Joyweed Alternanthera ficoidea H Wu and Dodge (2005)
Aloe Aloe vera H
Prickly pear cactus Opuntia compressa H
Ice plant Malephora crocea H
Red apple ice plant Drosanthemun hispidum H
Wintercreeper Euonymus fortunei H Jull (2009)
Lantana Lantana camara M Niu et al. (2007),
Wu and Dodge (2005)
Germanium Pelargonium spp. M Miyamoto et al. (2004)
Fountain grass Pennisetum setaceum M
Century plant A. americana H
Spider plant Chlorophytum comosum M
Kalanchoe Kalanchoe spp. M Bezona et al. (2001)
Gazania Gazania rigens H Niu and Rodriguez (2006)
Suaeda S. fruticosa H Hameed et al. (2012)
Salt-tolerant annuals
Coleus Solenostemon hybrids M Glen (2004)
Moss rose Portulaca grandiflora; H Kamal-Uddin et al. (2012a)
Portulaca oleracea
Blanketflower Gaillardia spp. M Costello et al. (2003), Niu and
Rodriguez (2006)
Ice plant Carpobrotus chilensis M Miyamoto et al. (2004)
Kochia Kochia scoparia H Monk and Wiebe (1961)
Petunia Petunia hybrida M
a Selected species/cultivars only.

Abbreviations: M, moderate salt tolerance; H, high salt tolerance.
(excluding grasses) for saline landscape is given together with supporting references. However,
please note that many species, especially from the local flora, can be added to the list based on
research and personal observation in the field.
30.9.1.1 Salt-Tolerant Grasses

Grasses are adapted to a wide range of soil and climatic conditions. Under saline conditions, the
range is somewhat restricted, but observation in local salt-affected areas will generally reveal
grasses suitable for general groundcover purposes, for example, Sporobolus virginicus in the
Sydney coastal region of Australia. For use as turfgrasses in urban parks, sports grounds, and
golf course fairways with salt-affected soils or saline irrigation, research over the last 30 years
has brought to light many useful species, some of which are now available in a range of bred
cultivars. Distichlis spicata, commonly called saltgrass, performs well as a warm-season turf-
grass with the ability to grow under highly saline conditions and limited water supplies (Kopec
et al., 2001a,b, 2005; Pessarakli et al., 2001a,b; Marcum et al., 2005; Pessarakli et al., 2005;
Pessarakli, 2007; Pessarakli and Kopec, 2008; Gessler and Pessarakli, 2009). Some species and
cultivars of Zoysia have also given promising results in trials (Dudeck and Peacock, 1993), while
a number of highly tolerant cultivars of the Cynodon grasses have been identified by Nadeem
et al. (2012). Seashore paspalum (Paspalum vaginatum) is a highly salt-tolerant warm-season
turfgrass that has been the subject of a great deal of research and development, with several
named cultivars now being available commercially (Dudeck and Peacock, 1985; Duncan and
Carrow, 2000; Kamal-Uddin et al., 2012b). For a comprehensive treatment of the management
of salt-affected turfgrass sites, see Carrow and Duncan (1998).
30.9.2 Irrigation Practices
Recommended irrigation practices for any area will depend upon the quality of irrigation water,
while other important considerations include amount and distribution of rainfall in that area, local
climate, appropriate irrigation system, soil characteristics, availability of freshwater, and quality of
saline water available. If brackish water needs to be used, the total salt level of the water must be
considered when developing a landscape design. For routine irrigation with saline waters, allowance
must be made for periodic leaching to a depth below the root zone to avoid accumulation of salt.
If the budget can accommodate the expense, the use of water amendments such as sulfuric acid or
gypsum will assist in maintaining a good soil structure.
When irrigation water of adequate quality is available, salts can most efficiently be leached out
from the surface soil with heavy irrigation at intervals. Keeping soil moisture levels higher between
irrigation events dilutes salt concentrations in the root zone (Cardon et al., 2007) and thereby helps
to reduce the salinity hazard for plants. To leach down salts in landscape, flood irrigation is effective
if the area is leveled, while a furrow or basin can also be created to contain the water if required, but
the basin system cannot be controlled to apply less than 75–100 mm of water per application that has
low application efficiency. With other irrigation systems like sprinklers for grasses and bubblers/
drips for trees, shrubs, and groundcovers, the best way to leach salts is to use a cycled system of
watering with minimum runoff (Vernon and Koenig, 2003).
30.9.2.1 Use of Suitable Irrigation Systems

Using a suitable irrigation system is the most important consideration in amenity horticulture.
Studies conducted to estimate the effect of drip and sprinkler irrigation systems on aesthetic quality
of landscape plants (Wu et al., 2001a,b; Miyamoto et al., 2004) have shown that generally plants are
more severely injured by saline water applied by sprinkler than by drip systems (Maas and Francois,
1982). Saline water applied by sprinklers can deposit toxic salts on plant leaves resulting in burning
and desiccation of the leaves of salt-sensitive species (Fox et al., 2005), though plant species with a
strong waxy covering on their leaves are less sensitive to aerial salts (Van Arsdel, 1996).
TABLE 30.7
Estimated Water Application Needed to Leach Salts
Percent Salt Reduction (%) Amount of Water Required
50 150 mm (6 in.)
80 300 mm (12 in.)
90 600 mm (24 in.)
Source: Cardon, G.E. et al., Managing Saline Soils, Fact Sheet No. 0.503,
Cooperative Extension, Colorado State University, Fort Collins,
CO, http://www.ext.colostate.edu/pubs/crops/00503.pdf, 2007.
30.9.2.2 Use of Reclaimed/Sewerage Water

Sewerage water is highly variable in quality depending upon whether it is coming from household
sources, or from industrial sources, or from a mixture. Additionally, the degree of treatment at the
sewage works will influence the quality. These waters need to be analyzed for salinity (EC), sodicity
(SAR and RSC), organic matter, nutrients, and expected ions that may be toxic to plants. For a study
on the use of secondary treated sewage water for turf irrigation, see Mancino and Pepper (1992),
and for its effect on some shrubs, see Gori et al. (2000). Sometimes, testing for pathogens such as
bacteria, viruses, and fungi is also necessary. Such waters may infect plants, animals, and workers
in contact with the polluted water, in which case additional treatment will be required to minimize
deleterious effects.
30.9.2.3 Supplemental Watering

Once a salinity problem is identified in the area under amenity horticulture, one of the best ways to
improve the situation is to leach the salt out of the root zone soil. This can be done only by applying
large amounts of extra water, the practice being known as supplemental watering. If soils are well-
drained, it is possible to dispose of as much as 90% of the salt, but this requires the application of
very large amounts of water as shown in Table 30.7.
The amount of water needed to remove excess salts from the soil will depend on the soil
type (drainage characteristics), the EC of soil to be achieved, and the EC of the irrigation water.
Obviously, the salt level cannot be reduced below that of the leaching water. Using excessive water to
leach salts down into the soil needs care because if the soils are imperfectly drained and the leachate
is not adequately moved down or drained out, the salts can move back up into the root zone of the
landscape plants by diffusion through the waterlogged soil and later through capillary action as the
soil dries. Naturally, plentiful rain can lessen salt damage by washing salts from the aerial portion of
the plant and leaching salts from the plant root zone depending on the condition of the soil.
30.9.2.4 Issue of the Use of Supplemental Water for Irrigation

Maintenance and development of good landscape may require supplemental irrigation for which
good-quality, poor-quality, or sewage water with varying levels of potential damage to soil and
landscape plants may be available. It is important to use these waters with care to avoid damage to
the plants. The type and amount of water that can be used safely will depend on the drainage char-
acteristics of soil, type of plant species being grown, and soil and irrigation management practices.
30.9.3 Agronomic Practices for Saline Landscaping

Proper management practices are necessary to ensure minimum salt accumulation in the root zone
and help maximum plant growth under the given saline conditions. The relevant management steps
including land preparation, use of soil and water amendments, seeds/seedling treatments, planting
methodology, irrigation practices, fertilizer application, and harvesting techniques will all affect
successful establishment and growth of species planted in saline environment. It is difficult to write
down all possible practices necessary for successful saline landscaping because the number of spe-
cies involved is large and the diversity of soil and climatic conditions is so great that each project
will have different but highly specific requirements. In what follows, all that can be done is to
provide somewhat generic recommendation and examples to assist saline landscape practitioners in
developing their own solutions for particular projects.
30.9.3.1 Provision of Drainage

Provision of good drainage is a prerequisite for development of amenity horticulture under saline
environments. Salinity and sodicity both can be controlled and managed with a well-designed
irrigation and drainage system. If the water table is shallow in the soil, it limits the leaching
of salts out of the root zone, and therefore, artificial drainage will be required. This can be
achieved through setting up a subsurface drainage system that is somewhat expensive. In areas
with a high water table, cut drainage ditches around the landscape, below the water table level,
can help to channelize and drain away water and allow the salts to leach out of plant root zone.
Drainage tiles or plastic drain pipe can also be buried in the ground for this purpose, particularly
in high clay soils (Vernon and Koenig, 2003). The drainage water must flow to some natural
drain, such as a river, stream, or other water catchment to preserve the normal hydrology of
those resources, although sometimes in dry climates where there is concern about already high
levels of salt in the river system, the regional environmental protection authority may require the
use of retention ponds and controlled release only at time of high river flow. Under no circum-
stances should the drainage water be dumped into the nearby areas. In dry climates, it must be
remembered that the beneficial effects of drainage systems in salt control will only be obtained
when the soil profile’s moisture content exceeds field capacity. In some situations, banks and
shallow surface drains in between can be used to intercept saline surface waters in wet weather
and prevent their access to the planted areas (Hamish, 2004).
30.9.3.2 Land Preparation

The land needs to be well leveled to avoid salt accumulation in raised parts and water stagnation in
the lower parts. For home lawns and turfs in sports fields, precise land leveling should be done by
planking or preferably laser leveling. For dense soil and soils having hardpan, it is essential to break
the hardpan and loosen the soil before planting. For trees and shrubs, the use of a posthole digger or
chiseling with a crowbar or similar tool can be very helpful.
At this stage, use of soil amendments like gypsum (CaSO 4 · 2H 2O) powder for sodic soils
and organic manure is beneficial (Qureshi and Barrett-Lennard, 1998; Vernon and Koenig,
2003). In warm climate areas, addition of pesticides to control termite attack is also highly
recommended before planting. Heavy irrigation is recommended before planting to leach the
salts below the immediate root zone in the case of well-drained soils, but in the case of soils
with a surface crust or a dense soil, the water will stay at the soil’s surface causing temporary
waterlogging and thus accentuating the effect of salinity unless the irrigation is intermittent
and managed with care.
30.9.3.3 Incorporation of Amendments/Chemicals

Addition of organic soil amendments such as composts and animal manures can improve soil
structure and allow salts to move through the root zone, whereas other chemicals like gypsum
(CaSO4 · 2H2O), calcium chloride (CaCl2), or humates, alone or in combination, can help to mitigate
the effect of salinity to a fair extent, especially, as explained previously, if a program of supplemen-
tal watering is used.
30.9.3.4 Application of Mulch

Lowering the evaporation rate can be another strategy to reduce the intensity of salinity in the sur-
face soil. This goal is achieved by applying organic mulch (e.g., sugarcane refuse, wood chippings,
shredded bark) to the soil surface. These mulches will gradually break down and new applications
should be made annually before the onset of the warm or dry season as applicable.
30.9.3.5 Pretreatment of Seeds/Seedlings

Direct seeding is not generally recommended but if it is unavoidable, then seed rate should be
increased. With some pretreatments such as seed priming in water or a dilute solution of calcium
chloride and scarification to break hard seed coats, seed germination and seedling establishment is
improved greatly (see Van den Beldt and Brewbaker, 1985).
Seedlings grown in well-drained bags (polythene or other materials, 7 cm diameter × 15 cm
height) are recommended as planting stock for trees and shrubs. These seedlings can be hardened
through ageing and irrigating with mild to moderate salinity levels in the irrigation water to avoid
osmotic shock at the time of transplantation. The bags containing the seedlings should be heavily
irrigated about one day before transportation to the field. At the time of planting, the bottom part of
bag should be cut away to allow roots to enter the soil without obstruction.
30.9.3.6 Planting Techniques

Irrigation furrows can be dug in the field for planting seedlings. The furrow to furrow and plant
to plant distance will vary with species and expected growth rate of plants. Placement of seed-
lings at the bottom of the furrow or along the side will depend upon the drainage characteristics
of the field. For a well-drained soil, placement at the bottom of the furrow is recommended,
whereas for a poorly drained soil, planting on the sides of the furrow is preferred (Figure 30.8).
FIGURE 30.8 Trees planted in the bottom of furrows in a well-drained soil. (From Qureshi, R.H. and
Barrett-Lennard, E.G., Saline Agriculture for Iirrigated Land in Pakistan: A Handbook, Australian Centre
for International Agricultural Research, Canberra, Australian Capital Territory, Australia, Monagraph No. 50,
p. 141, 1998.)
Do not plant in areas where runoff water collects. For roadside plantation, plant distance from
the road should be based on prevailing winds, volume and speed of traffic, and terrain. Before
planting along highways or other public roads, check with the responsible government authori-
ties who may well have definite policies about acceptable species, planting positions in relation
to underground and overhead services, and distance from the road. In the absence of any such
regulations, a general guide is that along high-speed highways, trees should be kept at least 20
or more meters from the road, while for other main roads, they should be at least 10 meters
from the road, especially if the road carries a high volume of traffic. In cities, the distance
between the edge of the road and the buildings is often only a few meters: here one needs to
choose species of a narrow canopy form that tolerate trimming to a clear trunk for a distance
of 4 or 5 m above the ground.
Time of planting can also play an important role in the success of saline landscaping. In tropical
countries with monsoonal rains, a pertinent example is the practice of planting saline areas with
trees, shrubs, and grasses during or at the end of monsoon season because at that time, salinity of the
surface soil is reduced through surface washing and downward leaching of salts. In the Indus basin,
successful large-scale planting of Eucalyptus camaldulensis and other salt-tolerant species was made
possible simply by planting at the right time, that is, toward the end of August to early September
when salts were leached down and the soil profile was moist with a better-quality shallow ground-
water. This allowed plants to establish without irrigation application at the time of planting and grow
rapidly until the cooler season began. At the end of winter, the root system of plants had already
developed well and plants survived the following hot, dry season with only one or two irrigations.
In using this wet-season planting method, it is important to remember that temporary waterlogging
may kill some species, so arrangements must be made to ensure water does not pond on the land for
a long time after heavy rains.
30.9.3.7 Fertilizer Application

Fertilizer application is expected to improve growth of plants even under saline conditions. In
small-scale or intensive cultivation such as home gardens or turf situations, farmyard manure
or composts may be used to supply major and minor plant nutrients and improve soil structure.
In selecting inorganic fertilizers for field application, special attention needs to be given to the
fact that salt-affected soils generally have a high pH of about 8, which reduces the availability
of phosphorus, calcium, and micronutrient such as Fe, Zn, Cu, and Mn. Therefore, a fertilizer
with acidic reaction and high residual acidity should be preferred. Use of ammonium sulfate
and calcium ammonium nitrate is preferred over urea as a source of nitrogen for saline soils.
Soil analysis should be used as a basis for the fertilizer program to ensure that all deficien-
cies are corrected, that money is not wasted supplying unneeded nutrients, and that toxicities
(particularly of trace elements) are avoided. When heavy dressings of fertilizer are required, it
is always advisable to use split applications rather than a single dose, as this minimizes losses
by volatilization and leaching and reduces the incidence of fertilizer burn to leaves and young
shoots. Sometimes, micronutrient deficiencies identified in the soil of the site can be addressed,
at least for several years, by dipping the seedlings in solutions or suspensions of the relevant ele-
ments before planting out. For example, where zinc is known to be deficient or marginal, dipping
the seedlings in a 1% zinc oxide suspension for half an hour will prove effective.
Excessive use of fertilizers in the more intensive types of landscape management may aggravate
salinity problem to some extent. In that case, applications of fertilizer must be stopped until the
levels of salts are reduced and the fertilizer program reviewed. If continuing heavy applications are
required for nutritional purposes, fertilizers with a low salt index (Beard, 1973) must be used.
30.9.3.8 Aftercare of Plantation

After planting in saline conditions, plants, unless highly salt tolerant, are exposed to stress due to
salinity. Aftercare aims to ensure that the plants are not subjected to additional stresses from causes
such as drought or defoliation. Newly planted trees, shrubs, and grasses need special protection to
save them from destruction due to grazing by wild and farm animals or, in urban areas, human traf-
fic and vandalism. When the plantation is well established, such care may not be necessary.
30.9.3.9 Harvesting/Pruning
Harvesting and pruning techniques need to meet the maintenance and ornamental requirements of
plants, which will vary with the species. Systematic harvesting of trees, shrubs, and grasses will keep
plants in appropriate form and may generate income to meet the maintenance expenses of the landscape.
30.9.3.10 Minimizing Salt Damage due to Deicing

Salt damage to landscape plants because of deicing salt is a common salinity issue during winter in
the colder regions of the world (Dobson, 1991; Buttle and Labadia, 1999). This kind of damage can
be minimized with a preventive care approach, where the amount of salt in the deicing product can
be reduced by substituting part of the salt with sawdust, sand, or similar materials whenever pos-
sible. Use of alternative deicing salts such as calcium magnesium acetate and calcium chloride can
also help in saving salt damage. In addition, thoughtful plant selection for such places and timely
maintenance can minimize the risk of salt injury.
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31 Physiological Responses
of Cotton (Gossypium
hirsutum L.) to Salt Stress
Mohammad Pessarakli
CONTENTS
31.1 Introduction........................................................................................................................... 635
31.2 Responses of Cotton to Salt Stress........................................................................................ 637
31.3 Results and Discussion.......................................................................................................... 639
31.3.1 Dry-Matter Production of Cotton Plants................................................................... 639
31.3.2 Nitrogen Absorption by Cotton Plants......................................................................640
31.3.2.1 Nitrogen (15N) Absorption and Concentration in Plant Tissues..................640
31.3.2.2 Total N Uptake by Plants............................................................................ 642
31.3.3 Nitrogen Metabolism and Assimilation in Cotton Plants.......................................... 642
31.3.3.1 Protein-N Content of Plants........................................................................ 642
31.3.3.2 Total Soluble-N Content of Plants.............................................................. 645
31.3.3.3 Ammonium plus Amide-N Content of Plants............................................ 647
31.3.3.4 Free Amino-N Content of Plants................................................................ 647
31.3.4 Total Water Uptake by Plants.................................................................................... 647
31.4 Summary and Conclusions....................................................................................................648
References....................................................................................................................................... 649
31.1 INTRODUCTION
No plant species or animals are immune to stress. Any species at least once during its life cycle
is subjected to stress. Nutrient uptake and utilization as well as water absorption by plants are
adversely affected under stressful conditions. Plant growth and metabolism are usually impaired
under such conditions, resulting in decreased crop yields.
Among the essential nutrient elements, nitrogen is one of the most widely limiting elements for
crop production, and when plants are subjected to stress, N uptake and utilization are likely to be
more severely affected than any other mineral nutrient. Regarding nutrient uptake and metabolism,
plant species behave differently under stressful conditions. The adverse effects of stress are usually
less severe on salt-tolerant plants such as cotton than on the salt-sensitive species such as beans.
Since the first publication of this chapter in 1994, numerous studies have been conducted on cot-
ton and the findings have already been published [1–31] that are referred to in the second edition
of this book published in 2001 [32]. Most of these studies were concerned with the cellular and
molecular aspects of this plant [3,6,7,9,10,15–18,21,25–27,29,30]. Li et al. [3] studied the effect of
salt stress on the activity of protective enzymes in cotton seedlings and concluded that the adapta-
tion of cotton seedlings to salt stress was expressed by roots, but the difference in salt tolerance
between cultivars was expressed by cotyledons. Studying stomatal density and size under salinity
stress conditions, Jafri and Ahmad [4] reported that a decrease in stomatal density under salt stress
635
was compensated by an increase in stomatal size and mesophyll surface area. Adaptation to a saline
environment was adjusted by increasing mesophyll surface area to ensure normal exchange of gases
and photosynthetic activities under the stress condition.
After publication of the second edition of the Handbook of Plant and Crop Physiology, most
recently, numerous studies have been conducted on cotton and the findings have already been pub-
lished [33–50]. Several relatively new reports have also been found on cotton during the last decade
prior to the twenty-first century [1–31,51,52].
Evaluating several stages of cotton growth and development, including seedling, preflowering,
flowering, and boll formation stages, Khan et al. [5] found that the seedling stage was the most sensi-
tive one as compared with other growth and development stages. In their study [5], the lowest seed
cotton yield was found at this stage of growth. At all growth stages, the yield of the salt-tolerant
cultivar was less affected than that of the salt-sensitive one by salinity stress. Renu and Goswami [6]
studied the activities of several enzymes at various stages of growth in cotton treated with gibberellic
acid (GA-3) and NaCl. These investigators [6] observed that nitrate reductase activity in cotyledonary
leaves decreased with salt stress and maximum activity was observed at the first stage. While salinity
invariably resulted in an increase in the cellulase and protease activities at all stages, GA-3 alone as
well as its interaction with NaCl increased the nitrate reductase activity. In another study, Renu and
Goswami [7] found that NaCl decreased the total chlorophyll (Chl) and carotenoid content in cotyle-
donary leaves of cotton. However, the carotenoid content decreased more slowly than the Chl.
Lin et al. [8] observed that with increasing NaCl concentration, the protein content in cotton
seedlings decreased, while the enzyme activity and the soluble sugar content increased. These find-
ings [8] indicated that changes in metabolism led to synthesis of large amounts of proline and sol-
uble sugars to maintain the osmotic pressure. Examining a wide range of species of various genera
in the family Malvaceae, Gorham [10] detected the zwitterionic quaternary ammonium compound
glycine betaine in all but 3 of over 100 species. In a more limited range of the species, particularly
of Gossypium, glycine betaine accumulated to concentrations sometimes in excess of 100 mM in
response to water deficit or salinity stress. In Gorham’s [10] study, glycine betaine concentrations
were highest in young tissues and accumulated to about 10% of the total nitrogen.
To improve salt tolerance in cotton, Shen et al. [11] grew this plant after the seeds were soaked in
paclobutrazol solution. They found that under salt stress, the growth rate and Chl, soluble sugar, and
proline contents of cotton seedlings grown from seed soaked in paclobutrazol solution were higher
than those of the controls. The results of these investigators [11] also showed a significant improve-
ment in the water relations of these plants. From this study [11], it was concluded that seed treatment
with paclobutrazol can mitigate the effects of salt stress and promote salt tolerance in cotton.
According to Qadir and Shams [12], in a pot culture study, imposed salinity stress had a deleteri-
ous effect on germination and vegetative growth with significant differences among the cotton gen-
otypes. Leaf area, stem thickness, and shoot and root weights decreased with increasing substrate
salinity level. Leidi and Saiz [14] studied physiological responses of two cotton cultivars previously
selected on the basis of growth under salinity. They postulated that the higher tolerance was the
result of several traits such as higher Na+ uptake and water content. These investigators [14] also
suggested that adaptation through adequate but tightly controlled ion uptake, typical of some halo-
phytes, along with efficient ion compartmentation and redistribution would result in an improved
water uptake capacity under salt stress conditions and lead to maintenance of higher growth rates.
Zhu and Zhang [15] studied antitranspiration and antigrowth activities of xylem sap of several
plants including maize, sunflower, cotton, and castor bean subjected to various stress treatments,
such as soil drying, flooding, and salinity. All xylem sap samples showed an increased concentra-
tion of proteins when plants were either soil dried, salt treated, or flooded. As a result, the protein
transportation flux in xylem sap was also increased. In an experiment conducted on a salt-sensitive
cultivar of cotton, Lin et al. [16] found greater relative reductions in root length and root fresh
weight than in hypocotyl length of seedlings grown in 75 mM NaCl. This indicates that the root was
more severely affected than the hypocotyl by the salt stress.
Physiological Responses of Cotton (Gossypium hirsutum L.) to Salt Stress 637
Banks et al. [17] studied the antioxidant response of several salt-sensitive and salt-tolerant
cotton cultivars to salt stress during fiber development. The results of their study [17] indicated
that salt treatment reduced fiber growth in all the cultivars, except the most salt-tolerant one.
Glutathione-S-transferase activity significantly increased in all the cultivars when treated with
NaCl. Regarding effects of salt stress on enzymes in cotton plant, experiments of Fowler et al. [18]
revealed increased levels of the antioxidants peroxidase, catalase, ascorbate peroxidase, super-
oxide dismutase, glutathione reductase, and glutathione-S-transferase in NaCl-stressed cotton
callus and plants.
In a greenhouse experiment, Oliveira et al. [19] studied the effects of different salinity levels of
irrigation water (0, 2000, 4000, 6000, and 8000 mg/L 70% NaCl and 30% CaCl2 solution) on ger-
mination and growth periods of different cotton cultivars. These investigators [19] found that salt
concentrations above 2000 mg/L decreased germination, vigor, and plant height, and salt concentra-
tions above 4000 mg/L decreased cotton yield and dry weight. According to Kasumov et al. [21],
salt stress stimulated root respiration by separating oxidation and phosphorylation. The antioxidant
activity of roots decreased abruptly, resulting in uncontrolled acceleration of free radical processes.
Vulkan-Levy et al. [24] carried out an experiment on the effect of water supply and salinity on
Pima cotton. These workers [24] found that an increase in water salinity caused a decrease in the
seed cotton yield and the salinity threshold increased with an increasing amount of water. Delayed
fluorescence and a decrease in intermittent amplitudes in the early stages of salt stress imposed on
cotton plants were observed by Ganieva et al. [25]. This phenomenon is an indication of a decrease
in photosystem II (PSII) activity. It may be related to damage to Chl in the PSII donor site and a
decrease in Chl b molecules leading to an increase in the Chl a/b ratio.
31.2 RESPONSES OF COTTON TO SALT STRESS

When cotton callus tissues were exposed to salt stress, Banks et al. [27] observed an increase in
the activity of antioxidant enzymes, including ascorbate peroxidase and glutathione reductase.
According to these investigators [27], these responses suggest that the upregulation of the activity of
these enzymes in response to salt stress is due to de novo transcription of the genes encoding the two
enzymes and not to translation of the existing transcripts or mobilization of existing enzyme pools.
The most recent findings on cotton plants prior to the year 2000 that were included in the second
edition of the Handbook of Plant and Crop Physiology [32] were reported by Feng et al. [28], Murray
et al. [29], Gossett et al. [30], and Rajguru et al. [31]. Feng et al. [28] studied the effects of salt stress
on vesicular–arbuscular mycorrhizal (VAM) formation and of inoculation with VAM fungi on saline
tolerance of plants, including cotton, maize, soybean, and melon, grown on soils containing NaCl.
They found that at a given NaCl level, cotton, maize, and soybean plants incubated with VAM had
a higher biomass than noninoculated plants. These investigators [28] suggested that the VAM fungi–
plant symbiosis might play an important role in survival of plants grown on saline soils. Also, inocula-
tion with VAM fungi could enhance crop production in plants grown on saline soils and reduce the
loss of plant yield caused by salt stress. Gossett et al. [30] reported that the total antioxidant enzyme
response to NaCl stress in cotton callus tissue is somewhat specific to the combined effects of Na+
and Cl− ions. Although Rajguru et al. [31] showed that salt treatment reduced ovule fresh weight in
several cotton cultivars, superoxide desmolase activity increased in most of the cultivars under the
salt stress condition. Glutathione-S-transferase activity significantly increased in all the cultivars
treated with NaCl.
Numerous reports have recently been published on cotton [33–50]. Several relatively new reports
have also been found on cotton during the last decade prior to the twenty-first century [1–31,51,52].
Li et al. [3] studied the effect of salt stress on the activity of protective enzymes in cotton seedlings
and concluded that the adaptation of cotton seedlings to salt stress was expressed by roots, but
the difference in salt tolerance between cultivars was expressed by cotyledons. Studying stomatal
density and size under salinity stress conditions, Jafri and Ahmad [4] reported that a decrease in
stomatal density under salt stress was compensated by an increase in stomatal size and mesophyll
surface area. Adaptation to a saline environment was adjusted by increasing mesophyll surface area
to ensure normal exchange of gases and photosynthetic activities under the stress condition.
Several studies indicated that decreases in plant growth and crop yields under stress conditions
have been associated with impairment of nutrient and water uptake, abnormal metabolism, and
inhibition of plant protein synthesis [32–98]. In these studies, salt and/or water stress impaired
growth and incorporation of nutrients (i.e., N) into the protein and increased accumulation of inor-
ganic N in plants. Reduction of nutrient uptake and utilization by plants was also reported by several
investigators in earlier studies [99–105]. Uptake of N and P by plants was inhibited by high NaCl
and Na2SO4 concentrations in the root medium, and the excess amount of absorbed Na+ depressed
NH +4 absorption in these studies. Absorption and metabolism of ammonium (NH +4 ) and nitrate
(NO3- ) in red kidney beans (Phaseolus vulgaris L.) were significantly reduced under salt or water
stress [103–105]. In all of the preceding studies, reduction of root permeability and the consequent
decrease in water and nutrient uptake under high electrolyte concentrations were stated as the cause
of this abnormality in water and nutrient absorption and metabolism. Nevertheless, low levels of
salts in the presence of N, P, and K stimulated growth and increased yield of cotton, Gossypium
hirsutum L. [83,84,106–109]. With further increase in salinity, dry-matter yield decreased, but it
increased with the addition of N at each salinity level. Moreover, plants continued to accumulate N
under saline conditions in spite of the reduction in yield and dry-matter production.
Soil salinity did not inhibit N absorption by Bermudagrass (Cynodon dactylon L.), a high-
salt-tolerant plant [120], and stress had little or no effect on the rate of NO3- uptake by barley
(Hordeum vulgare L.), another high-salt-tolerant crop, except at the highest osmotic pressure,
lowest osmotic potential (−0.54 MPa) of the rhizosphere [64,111]. Also, NaCl in the culture
solution did not influence NO3- uptake by tomato (Lycopersicon esculentum Mill.), a medium-
salt-tolerant plant [101].
Abdul-Kadir and Paulsen [112] reported that the soluble protein and free amino acid content
of wheat (Triticum aestivum L.) plants were not consistently affected by MgSO4, MgCl2, and
NaCl. Udovenko et al. [113,114] found that under salt stress, the nonprotein-N fraction increased
in beans, peas (Lathyrus hirsutus L.), barley, and wheat, whereas the protein-N fraction changed
irregularly. These investigators [113,114] concluded that the response of N metabolism to salt
stress is similar in plants with varying salt tolerance. An increase in the soluble-N fractions and
free amino acid levels and decrease in protein-N content of cotton plants under medium (−0.8
MPa osmotic potential) and high (−1.2 MPa osmotic potential) levels of salinity were reported
by Pessarakli and Tucker [84]. However, these investigators found that the low level of salinity
(−0.4 MPa osmotic potential) slightly enhanced dry-matter production and protein content of the
cotton plants. On the other hand, this level of salinity (−0.4 MPa osmotic potential) and lower
(−0.25 MPa osmotic potential) of the culture solution substantially decreased protein content of
red kidney beans [104,105], green beans [78,79,115,116], and alfalfa, Medicago sativa L. [77].
Impaired N metabolism and decreased protein content of a number of plants under stress condi-
tions have also been reported by several other investigators [117–123]. Rabe [85,86] and Dubey
[59,60] reviewed altered N metabolism and protein synthesis, respectively, in plants under stress-
ful conditions. These authors reported that N metabolism and protein synthesis in plant species
were severely affected under stress.
Water stress induced by Carbowax also caused a marked reduction in protein synthesis by plants
[104,105]. Although these studies were conducted on red kidney beans, a salt-sensitive plant, salt
(NaCl) stress resulted in an appreciably greater reduction in 15N incorporation in the protein fraction
than water stress created by the Carbowax treatment. These results [104,105] indicated inhibition of
N utilization caused by an ionic effect in addition to the osmotic effect of either NaCl or Carbowax.
Although investigations on the effects of salt and/or water stress on nutrient (i.e., N) absorption,
utilization, metabolism, and protein synthesis mostly indicated a reduction in the absorption rate
of N and decrease in the protein content of plants, a few controversial results make generalization
difficult. These and other inconsistent results that demonstrated either an increase or no effect on
the nutrient (i.e., N) absorption and metabolism can probably be explained as resulting from a dilu-
tion effect. This was suggested by Frota and Tucker [103,104], Saad [105], Pessarakli and Tucker
[81–84], and Pessarakli [76,107,115,116], in which plant growth was affected more than the nutrient
uptake and metabolism by salt stress, and as a result, the relative concentration of N was higher for
the stressed plants.
Although the mechanisms by which salinity stress or drought adversely affect plant growth are
still controversial, it is generally agreed that impairment of N absorption and metabolism is a criti-
cal factor. For a detailed review of the adverse effects of stress on plants and crops, readers are
referred to the most comprehensive source, the new edition [124] and the previous editions [125,126]
of the Handbook of Plant and Crop Stress.
If it could be determined at what particular stage of growth high salinity most negatively affects
plant growth and metabolism, the mechanisms by which these adverse effects occur might be iden-
tified and the detrimental effects prevented. In this regard, in addition to this work, several inves-
tigators have already attempted to study the effects of stress at different stages of plant growth
[83,84,107,109,127–148].
The purpose of this investigation was to determine the physiological effects of salt stress on
growth in terms of dry-matter production, nitrogen (15 NH +4 ) absorption and metabolism, protein
synthesis, and water uptake by cotton plants at two stages of growth.
31.3 RESULTS AND DISCUSSION

31.3.1 Dry-Matter Production of Cotton Plants
At both the vegetative and the reproductive stages of growth, salt stress (particularly at medium
and higher NaCl levels) drastically reduced dry-matter production (Table 31.1). The results of Khan
et al. [5] showing a substantial decrease in cotton yield at different stages of growth under salinity
TABLE 31.1
Dry-Matter Production of Cotton Plants Subjected to NaCl
Salinity during Vegetative and Reproductive Stages
of Growth
Plant Dry Weight/Pot (Two Plants) (g)
Treatment, Osmotic
Growth Stage Potential (MPa) Shoots Roots Total
Vegetative Control 5.42 0.93 6.35
−0.4 3.79 0.97 4.76
−0.8 2.71 0.77 3.48
−1.2 1.71 0.39 2.10
LSD (0.05)a 1.43 0.20 1.58
Reproductive Control 20.13 3.90 24.03
−0.4 16.72 3.98 20.70
−0.8 12.11 3.52 15.63
−1.2 7.60 2.42 10.02
LAD (0.05)a 4.22 0.83 4.60
Source: Pessarakli, M. and Tucker, T.C., Soil Sci. Soc. Am. J., 49, 149, 1985.
a The least significant difference among the means at the 0.05 probability level.
stress confirm this finding. The report of Qadir and Shams [12] indicating that the imposed salinity
stress had a deleterious effect on the germination and vegetative growth of cotton plants is also in
agreement with the present work. According to these investigators [12], leaf area, stem thickness,
and shoot and root weights decreased with increasing substrate salinity level. Kurth et al. [149] also
observed adverse effects of both NaCl and CaCl2 salinity on cotton growth in terms of cell enlarge-
ment and cell production. Several other investigators [2,3,8,10,15,17,19,24,31–52] reported similar
reductions in different parameters of cotton growth and development under salinity stress that sup-
port the results of the present work.
In the present work, the dry-matter production of the stressed plants was highly negatively correlated
with increasing levels of salinity at both stages of growth (r of −0.98 to −0.96). Reduction of plant growth
at higher levels of salinity has also been reported by other investigators for other salt-tolerant plants,
such as barley [64,150,151], mangrove, Avicennia marina [72], and other halophytes including Suaeda
maritima L. [152]. Several other investigators, in stress physiology, found that the growth of various plant
species substantially decreased under stressful conditions [55,56,58,61,65,66,68,69,71–82,90–97,102–
107,109,116,126–148,153–175]. The present study showed that the shoot dry weight was reduced more by
increasing salinity than the root dry weight. This is supported by the findings of several other investiga-
tors [76–82,103–105,107,115,116,125,126,150,151] and is consistent with the common knowledge in plant
physiology that plant roots under stress conditions grow more and penetrate deeper in the soil or in the
root medium in search of water and nutrients. Other studies also indicated a substantial reduction in shoot
growth under stress conditions. For example, sodium chloride stress severely decreased shoot growth of
rice, Oryza sativa L., cultivar GR-30 [176], and Lactuca sativa plants [177].
In the present study, the effect of salinity was more pronounced at the vegetative growth than
at the reproductive growth stage. Other studies have also indicated that plants at earlier stages
of growth were more sensitive to stress than those at later stages of growth [5,12,71,107,109,127–
148,169,178–182]. Abnormal plant growth was also observed in experiments using sufficient
amounts of salts other than sodium chloride [80,122,126,129,160,161] as well as under drought
stress conditions [92,129,131,146,147,183–186]. This is an indication of the adverse effects of stress
on plant growth regardless of the source of the salt or the type of the stress.
31.3.2 Nitrogen Absorption by Cotton Plants

31.3.2.1 Nitrogen (15N) Absorption and Concentration in Plant Tissues
The mean values for 15 NH +4 absorption by cotton plants for 24 h uptake time, under normal
Hoagland solution (control) and salt (NaCl) stress conditions, obtained by analyzing solution
samples indicated that low and medium levels of salinity did not significantly decrease the rate of
15N absorption (Figures 31.1 and 31.2). In fact, absorption was increased slightly at the vegetative
stage at a low salinity level (−0.4 MPa osmotic potential). Similar amounts of NaCl drastically
reduced the uptake rate of 15N in red kidney beans [103,105], green beans [76,78,79,115,116],
alfalfa [77], and eggplant, Solanum melongena L. [81]. Such differences reflect variations in the
salt tolerance of these different plant types. The high level of salinity (−1.2 MPa) appears to have
caused a substantial reduction in the N absorption rate of cotton plants. The effect of the high
salinity level on 15N uptake was more pronounced at the vegetative stage than at the reproductive
stage of growth. The values for 15N uptake obtained by total N analysis of the plant materials
(Tables 31.2 and 31.3) indicated essentially the same pattern as the solution loss data (Figures
31.1 and 31.2). Total amounts of 15N recovered in plants generally accounted for 95%–99% of the
apparent solution loss.
The concentration of 15N in roots was higher than in shoots for all treatments at both stages of
growth (Table 31.4). Adsorption of ammonium ions to the root surfaces or infusion of ions into
apparent free space within roots, as suggested by Pessarakli and Tucker [81–84] and Pessarakli
[76,107,115,116], could be a possible reason for the higher 15N concentrations in cotton roots.
10
Control
–0.4 MPa
Lost from solution (uptake) mg 15N/POT

8 –0.8 MPa
–1.2 MPa
0
2 4 8 12 16 20 24
Time (h)
FIGURE 31.1 Solution loss of 15N (uptake) by cotton plants under various NaCl salinity conditions during
the vegetative stage of growth. (From Pessarakli, M. and Tucker, T.C., Soil Sci. Soc. Am. J., 49, 149, 1985.)
15
Control
–0.4 MPa
Lost from solution (uptake) mg 15N/POT
–0.8 MPa
–1.2 MPa
10
0
2 4 8 12 16 20 24
Time (h)
FIGURE 31.2 Solution loss of 15N (uptake) by cotton plants under various NaCl salinity conditions at the begin-
ning of the reproductive stage of growth. (From Pessarakli, M. and Tucker, T.C., Soil Sci. Soc. Am. J., 49, 149, 1985.)
At both stages of growth, the 15N concentration of shoots significantly increased for salinized plants
(−0.8 MPa) compared with the controls. This can be explained as a dilution effect as suggested by
Frota and Tucker [103], Saad [105], Pessarakli and Tucker [81–84], and Pessarakli [76,115,116], as
being due to a greater reduction in plant growth than 15N absorption under stress conditions. The
relative translocation of 15N from roots to shoots was not appreciably affected by salt concentration.
TABLE 31.2
Distribution of 15N Absorbed as Ammonium in Cotton
Shoots and Roots under Different NaCl Salinity Levels
during Vegetative Stage of Growth
15N Uptake/Pot (Two Plants)
Treatment, Osmotic (mg) Uptake Time (h)
Plant Parts Potential (MPa) 6 12 24
Shoots Control 0.98 1.61 4.97
−0.4 0.82 1.71 4.78
−0.8 1.02 1.63 4.36
−1.2 0.36 0.88 1.81
LSD (0.05)a 0.43 0.49 0.45
Roots Control 0.73 1.29 3.22
−0.4 139 1.84 4.20
−0.8 0.67 1.27 3.23
−1.2 0.34 0.57 1.24
LSD (0.05)a 0.48 0.93 0.77
Total Control 1.71 2.90 8.19
−0.4 2.21 3.55 8.98
−0.8 1.69 2.90 7.59
−1.2 0.70 1.45 3.05
LSD (0.05)a 0.32 0.95 0.98
31.3.2.2 Total N Uptake by Plants

Total N uptake by plants decreased as the culture medium became more saline (Table 31.5). The reduc-
tion in total N uptake values at −0.8 MPa osmotic potential was approximately 50% and 70% of the
control values at the vegetative and the reproductive stages of growth, respectively. At the higher salin-
ity level (−1.2 MPa), the reduction in N uptake was proportionately greater at the vegetative stage than at
the reproductive stage of growth. Reduced N uptake at the vegetative stage was largely due to the reduc-
tion in dry-matter production, except at the high salinity level, where the N concentration was reduced
appreciably (Tables 31.1 and 31.5). At the reproductive stage, N concentration was essentially the same
at all salinity levels, indicating that plants had adjusted somewhat to salinity and its effect on N uptake.
The dry weights at the high salinity level were still less than 50% of the control levels, as were values
for the total N uptake. Although generally these observations are similar to the previous discussion of
15N data, some small deviations are apparent. Concentration data for 15N (Table 31.4) indicate that short-
term 15N concentrations increased in the shoots at the −0.8 MPa salinity level.
31.3.3 Nitrogen Metabolism and Assimilation in Cotton Plants

31.3.3.1 Protein-N Content of Plants
After a 24 h exposure to 15 NH +4 , the protein-15N content of plants treated with a high level of NaCl
(−1.2 MPa osmotic potential) was significantly less than in either the control, low, or medium NaCl
treatments (Table 31.6). This is in agreement with the observations of Lin et al. [8] that with increas-
ing NaCl concentration, the protein content in cotton seedlings decreased. The depressing effects
TABLE 31.3
Distribution of 15N Absorbed as Ammonium in Cotton
Shoots and Roots under Different NaCl Salinity Levels
at the Beginning of the Reproductive Stage of Growth
15N Uptake/Pot (Two Plants)
(mg) Uptake Time (h)
Treatment, Osmotic
Plant Parts Potential (MPa) 6 12 24
Shoots Control 1.77 4.11 8.57
−0.4 1.91 3.78 8.64
−0.8 1.49 3.55 8.45
−1.2 1.15 2.18 4.67
LSD (0.05)a 0.43 0.64 1.67
Roots Control 1.32 2.44 5.49
−0.4 1.62 3.07 5.68
−0.8 1.58 2.85 5.65
−1.2 1.01 1.81 3.39
LSD (0.05)a 0.39 0.65 1.80
Total Control 3.09 6.54 13.99
−0.4 3.53 6.85 14.32
−0.8 3.07 6.40 14.10
−1.2 2.16 3.99 8.06
LSD (0.05)a 0.21 0.60 1.71
TABLE 31.4
Nitrogen (15N) Concentration of Cotton Plants during
Vegetative and Reproductive Stages of Growth as
Influenced by NaCl Salinity
15N Concentration
(mg 15N/kg dry wt)

Treatment, Osmotic
Growth Stage Potential (MPa) Shoots Roots
Vegetative Control 917 3462
−0.4 1261 4330
−0.8 1609 4195
−1.2 1059 3180
LSD (0.05)a 384 586
Reproductive Control 422 1408
−0.4 517 1427
−0.8 698 1605
−1.2 615 1401
LAD (0.05)a 109 245
TABLE 31.5
Total N Uptake of Cotton Plants during Vegetative
and Reproductive Stages of Growth as Influenced
by NaCl Salinity
Total N/Pot
(Two Plants) (mg)
Treatment, Osmotic
Growth Stage Potential (MPa) Shoots Roots Total
−0.4 114.8 23.0 137.8
−0.8 89.8 17.8 107.6
−1.2 43.1 7.2 50.3
LSD (0.05)a 15.9 5.3 17.7
−0.4 333.8 66.7 400.6
−0.8 245.9 62.0 307.9
−1.2 156.5 41.6 198.1
LAD (0.05)a 96.1 15.0 105.6
Source: Pessarakli, M. and Tucker, T.C., Soil Sci. Soc. Am. J., 49,
149, 1985.
a The least significant difference among the means at the 0.05 proba-
bility level.
TABLE 31.6
Concentration of 15N Fractions and Protein-15N to Nonprotein-15N Ratio of Cotton
Shoots Influenced by NaCl Stress for Two Stages of Growth after 24 h of Uptake
Protein-15N to
Osmotic Protein-N Total Ammonium plus Free Amino Nonprotein-
Potential (MPa) (mg) Soluble-N (mg) Amide-N (mg) Acid-N (mg) 15N Ratio
Vegetative
Control 489.6 318.6 6.97 129.0 1.54
−0.4 560.9 463.9 9.37 189.1 1.21
−0.8 544.3 741.3 21.96 287.1 0.73
−1.2 230.4 523.2 13.67 273.1 0.44
LSD (0.05)a 68.3 120.4 2.10 51.9 0.24
Reproductive
Control 229.9 145.5 4.87 60.6 1.58
−0.4 232.8 204.8 5.31 65.9 1.14
−0.8 217.3 363.6 19.71 96.4 0.60
−1.2 98.8 393.6 9.64 108.1 0.25
LSD (0.05)a 46.6 76.2 1.83 11.6 0.47
Source: Pessarakli, M. and Tucker, T.C., J Plant Nutr 8,1025–1045, 1985.

of salt on the protein content of cotton plants at high levels of NaCl could be attributed to decreased
amino-N incorporation into protein as reported for red kidney beans [104,105], rice [61,100,122],
and other plants [59,60,85–87,113,114,187,188]. The decrease in polyribosome levels, as reported
for corn, Zea mays L. [187], and for barley and pea shoots [188], is probably another reason for the
decrease in protein synthesis in cotton shoots.
The low level of NaCl (−0.4 MPa osmotic potential of the nutrient solution) significantly
increased the protein content of cotton shoots at the vegetative stage of growth. This is in agree-
ment with the findings of Renu and Goswami [2] on uptake and accumulation of labeled 14C pho-
tosynthates in cotyledonary leaf of cotton treated with GA-3 under salt stress. According to these
investigators [2], the low levels of salinity stimulated 14CO2 uptake and accumulation of carbohy-
drates in the cotyledonary test, whereas high salinity decreased it. The report of Zhu and Zhang
[15] showing an increased concentration of proteins when maize, sunflower, cotton, and castor
bean plants were either soil dried, salt treated, or flooded is also in support of the present study.
However, the same level (−0.4 MPa) of salt stress substantially decreased protein synthesis in a
number of other plants with lower degrees of salt tolerance [77–79,104,105,115,116,119]. Impaired
N metabolism with the consequence of reduced protein content of several other plant species with
various degrees of salt tolerance under stress conditions has been reported by several investigators
[60,61,64,70,71,86,87,89,93,98,100,110,112–114,117,118,120–123,187,189].
Water stress is also known to impair N metabolism and reduce protein synthesis in plants
[60,86,87,89,92,104,105,119,120,188]. In addition to the osmotic effect of salt, the specific ion
effects of Na+ and/or Cl− have certainly contributed appreciably to the inhibition of 15N incorpora-
tion into protein. However, this study was not designed to distinguish specifically between osmotic
and ionic effects.
The rates of 15N incorporation into protein as measured by the concentration of 15N in the pro-
tein fraction at 6, 12, and 24 h of exposure to 15 NH +4 appear to have been influenced only by the
high level of NaCl at both growth stages (Table 31.7). The rate of incorporation was reduced at
the −1.2 MPa salt level by factors of 2.5 and 2.8 at the vegetative and reproductive stages of growth,
respectively. The rate of 15N incorporation into protein at the vegetative stage was approximately
2.5 times greater than the rate at the reproductive stage, based on the 15N concentration. The total 15N
in the protein fraction was greater at the reproductive than at the vegetative stage of growth, appar-
ently because of the much greater amount of shoot dry weight (Table 31.1). Furthermore, the total
protein-15N decreased with increased salt level at both stages of growth. This reflects the combined
effect of salt on shoot growth and 15N incorporation into protein.
31.3.3.2 Total Soluble-N Content of Plants

Soluble-N compounds that should be in an ethanol extract of plant tissues include NO3+ , NH +4 ,
amides, amino acids, amine, amino sugars, peptide, alkaloids, nucleotide, Chl, and even some fats.
Because only the NH +4 form of N was used in this investigation, 15N from 15 NH +4 exposure should
not be found in the NO3- form in the plant tissues in this study.
The total soluble-15N concentration of the plant tissues increased with NaCl concentration
at −0.8 MPa osmotic potential after 24 h exposure to 15 NH +4 at the vegetative growth stage, then
declined at −1.2 MPa (Table 31.6). At the reproductive growth stage, total soluble-15N increased
in a similar manner but did not decline at the highest salinity level. Thus, the decrease in pro-
tein-15N at the −1.2 MPa salinity level did not result from a shortage of soluble-15N compounds.
The rates of 15N incorporation into the total soluble-15N fraction as indicated by the slope of the
regression of the 15N tissue concentration versus time (Table 31.7) followed the same pattern as
described before for the 24 h uptake time. Accumulation of soluble-N compounds in plants under
stress conditions has also been reported by several other investigators for various plant species
[64,70,74–82,86,87,90,91,93–97,100,102–107,110–117,122,123,175].
TABLE 31.7
Slope (b) and Intercept (a) of the Regression Lines for
Concentration of 15N Fraction in Cotton Shoots Influenced
by NaCl Salinity versus 6, 12, and 24 h Exposure Time for
Two Stages of Growtha
Growth Stage
Treatment, Osmotic Vegetative Reproductive

15N Fraction Potential (MPa) b a b a
Protein Control 24.22 −99.08 10.38 24.65
−0.4 26.44 −81.19 10.43 23.18
−0.8 24.71 −61.10 9.36 −1.58
−1.2 9.89 −6.80 3.61 +11.56
Soluble Control 13.20 −13.70 5.14 17.53
−0.4 21.28 −58.50 0.06 −1.79
−0.8 30.64 −19.25 16.36 −28.95
−1.2 20.88 +30.25 16.17 +2.36
Ammonium plus amide Control 0.32 −0.53 0.19 −0.06
−0.4 0.40 −0.16 0.23 −0.17
−0.8 0.93 −0.49 0.80 +0.58
−1.2 0.51 +1.71 0.30 +2.52
Free amino Control 6.03 −22.79 2.65 −1.05
−0.4 9.63 −48.44 2.73 +1.13
−0.8 13.88 −58.63 4.41 −9.63
−1.2 14.24 −76.61 5.12 −15.17
Source: Pessarakli, M. and Tucker, T.C., J Plant Nutr 8:1025–1045, 1985.

a Correlation coefficient, r, values lie between 0.92 and 1.00.
The amounts of soluble-15N reflect both concentrations in the tissue and dry-matter production
(Table 31.1). Significantly less total soluble-15N was found after 24 h with −1.2 MPa salinity at
the vegetative growth stage. With the −0.8 MPa salinity, a larger amount of total soluble-15N was
observed than with other treatments. At the reproductive stage, the amounts of total soluble-15N
were equal at the high and low levels of salinity, with the intermediate salinity levels resulting in
higher quantities of total soluble-15N in the plant parts.
Although the rate of 15 NH +4 absorption was severely curtailed by high salinity at both growth
stages [83], growth was not restricted by decreased total soluble-N concentration. However, impair-
ment of soluble-N utilization at high salinity was reflected in a severe decrease in the protein
concentration of plants. It is not clear whether this lower protein concentration was a cause of
reduced growth. Growth was reduced at the lower salinity levels without a reduction in the protein
concentration.
The ratio of protein-N to nonprotein-N (soluble-N) is further evidence for the decrease in the
protein-N content of the NaCl-treated plants. A substantial decrease in the ratio of protein-15N to
nonprotein-15N was observed for the plants subjected to a high level of NaCl (−1.2 MPa osmotic
potential) compared with the controls at both stages of growth (Table 31.6). At both stages of growth,
the values for the −0.8 MPa osmotic potential of the NaCl-treated plants were significantly lower
than the controls.
31.3.3.3 Ammonium plus Amide-N Content of Plants

At both stages of growth, significantly higher concentrations of ammonium plus amide-15N accumu-
lated in the shoots of the plants subjected to NaCl stress compared with the controls (Table 31.6). The
concentration of ammonium plus amide-15 NH +4 increased with increasing salinity to a maximum
at −0.8 MPa osmotic potential. Because the absorption rate of 15 NH +4 did not change appreciably at
these salinity levels, this increased accumulation of ammonium plus amide-N must have resulted
from a reduced rate of utilization; however, reduced growth is another possible consideration. The
concentrations of ammonium plus amide-N at the −1.2 MPa stress were lower than at the −0.8 MPa
osmotic potential. These values reflect markedly reduced absorption rates at the −1.2 MPa stress
[83]. The rate of 15N utilization also decreased, allowing a higher 15N concentration than commen-
surate with absorption rate. Slopes for regressions of ammonium plus amide-15N and time of uptake
for each salinity level (Table 31.7) indicate a rate of accumulation pattern similar to the concentra-
tions indicated for the 24 h exposure time (Table 31.6).
31.3.3.4 Free Amino-N Content of Plants

Free amino acids would be expected to constitute the major portion of the total ethanol solu-
ble-N compounds from plant tissues. In this study, the amino-N and ammonium plus amide-N
accounted for 30%–55% of the total soluble-N. The ninhydrin release method for free amino-N
determination was used in this investigation. This method, however, can result in poor recoveries
of a number of amino acids [190]. In Kennedy’s [190] investigation, recoveries varied from 2% to
60% for 12 amino acids with complete recovery of 14 others. In the present study, the apparent
low recovery of amino-15N from cotton tissues by the ninhydrin release method is consistent with
the results of Kennedy [190] when all aspects of the methodology are considered. Even with the
low recovery, however, the relative effects of NaCl salinity on amino acid formation and utiliza-
tion should be valid.
After a 24 h exposure to 15 NH +4 , a higher concentration of amino-15N was found in the NaCl-stressed
plants, as compared with the controls, at both growth stages (Table 31.6). This increased concentration
was sufficient to equal or exceed the reduction in dry weight, except at the highest NaCl level (−1.2 MPa
osmotic potential), when dry weight was reduced most drastically. Yet, at the higher NaCl salinity level,
the amino-15N concentration either remained constant or increased slightly as protein concentration
values declined to less than half of the values for all other treatments. Slopes of the regression lines of
the amino acid and exposure time (Table 31.7) reflect rate of amino acid accumulation. Thus, the incor-
poration of amino acids into protein was impaired by a high level of NaCl. This level of salinity that
was required for interference with protein formation in cotton, a relatively high-salt-tolerant plant, was
much higher than reported for green beans [78,79,115,116], red kidney beans [104,105,119], soybeans,
Glycine max L. [123], peas [118], alfalfa [77], corn [89,187], rice [61,70,100,122], and wheat [112], which
all have lower degrees of salt tolerance than cotton.
31.3.4 Total Water Uptake by Plants

At both stages of growth (except for the −0.4 MPa osmotic potential during the reproductive stage),
salt-stressed plants absorbed significantly less water than the controls (Table 31.8). Plants at −0.4 MPa
stress did not exhibit a statistically significant difference in water uptake during the reproductive
stage of growth compared with the controls. Reduction in water absorption by plants due to salinity
stress has been reported by many investigators [15,22,24,76–83,95,103,105,107,115,116,151,191–194].
These investigators generally agreed that root permeability of plants (expressed as hydraulic con-
ductivity of the root system) was decreased significantly under salt stress. This is an explanation for
the reduction in water absorption rate and may contribute to a similar reduction in nutrient uptake
and consequently reduction in crop yield under salinity conditions.
TABLE 31.8
Influence of NaCl Salinity on Water Absorption by Cotton
Plants during the 24 h 15N Uptake Period for the Vegetative
and Reproductive Stages of Growth
Water, Uptake/Pot (Two
Plants (mL)) Uptake Time (h)
Treatment, Osmotic
Growth Stage Potential (MPa) 6 12 24
−0.4 87.5 122.5 172.5
−0.8 57.5 120.5 135.0
−1.2 40.0 70.0 87.5
LSD (0.05)a 46.0 23.4 28.2
−0.4 147.5 245.0 430.0
−0.8 130.0 167.5 215.0
−1.2 75.0 107.5 145.0
LSD (0.05)a 78.7 102.8 205.5

Cotton plants grown in normal (control) and NaCl-treated Hoagland solutions were studied at two
stages of growth (vegetative and reproductive). Plant growth in terms of dry-matter production was
measured. Nitrogen absorption (total N and 15N) and water uptake were determined. Plant parts
(shoots and roots) were analyzed separately for N content and distribution of 15N in ammonium plus
amide-N, free amino-N, total soluble-N, and protein-N after the plants were provided 15NH4NO3 in
nutrient solutions for 6, 12, and 24 h.
Dry-matter production of the cotton plants was significantly reduced by decreasing the osmotic
potential (increasing salinity) of the nutrient solution. The low and medium levels of salinity did not
have a significant effect on the 15N absorption rate, but the high salt levels caused a substantial reduc-
tion in the 15N uptake rate. The 15N concentration of the roots was higher than that of the shoots,
particularly under stress conditions. The 15N concentration in plants increased with increasing salinity
levels. The concentration of 15N in plants in terms of the ratio of plant total 15N content to dry matter
produced (mg 15N/kg dry matter) was significantly higher for moderately stressed than for control
plants. This indicates that plants continued to accumulate 15N under salt stress conditions in spite of the
reduction in dry-matter production. Total water absorbed by plants decreased linearly with increasing
salinity. This reduction was even more appreciable than the reduction in 15N absorption rate. The effect
of salinity was more pronounced at the vegetative than at the reproductive stage of growth.
The metabolism of 15N in salinized cotton plants was adversely affected under medium and high
levels of NaCl, at both vegetative and reproductive stages of growth. Significant accumulations of all
soluble-15N fractions occurred when plants were subjected to medium and high levels of NaCl com-
pared with the controls. The −0.4 MPa osmotic potential of the culture solution enhanced protein syn-
thesis at the vegetative growth stage. Only the −1.2 MPa osmotic potential significantly decreased the
protein-15N content of plants as compared with the controls and any other level of NaCl. Protein synthe-
sis was impaired by a large excess of NaCl in the nutrient solution, which inhibited NH +4 metabolism.
Consequently, under salt stress conditions of sufficient magnitude, plant growth, N absorption
and metabolism, protein synthesis, and water absorption will be altered. This will result in the
failure of plants to fully utilize nutrients and water. Salinity levels in excess of those causing drastic
interference with plant growth, nutrient (i.e., N) absorption and metabolism, and water uptake in
salt-sensitive plants such as beans do not appreciably interfere with these factors in cotton, a rela-
tively high-salt-tolerant plant. This indicates a link between salt tolerance, growth, nutrient (i.e., N)
absorption and metabolism, and water uptake. Although the contribution of osmotic and specific ion
effects cannot be distinguished from this study, it is likely that both were involved.
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32 Isoprenoid Biosynthesis in
Higher Plants and Green
Algae under Normal and
Light Stress Conditions
Parisa Heydarizadeh, Justine Marchand, Mohammad
Reza Sabzalian, Martine Bertrand, and Benoît Schoefs
CONTENTS
Abbreviations.................................................................................................................................. 655
32.1 Introduction........................................................................................................................... 656
32.2 Biosynthesis of Isoprenoids................................................................................................... 657
32.2.1 Toward GPP Biosynthesis.......................................................................................... 657
32.2.2 From GPP to Menthol Biosynthesis...........................................................................660
32.2.3 Carotenoids................................................................................................................ 662
32.2.4 From GPP to Secondary Carotenoids........................................................................ 663
32.3 Isoprenoid Protection against Diverse Stresses..................................................................... 669
32.3.1 Light Stress................................................................................................................ 669
32.3.1.1 Light Stress and Menthol Biosynthesis....................................................... 669
32.3.1.2 Light Stress and Carotenogenesis............................................................... 670
32.4 Genetic Engineering.............................................................................................................. 671
32.5 Conclusion............................................................................................................................. 671
References....................................................................................................................................... 672
ABBREVIATIONS
AAC Acetoacetyl-CoA
AACT AAC thiolase
AC Acetyl-CoA
CDP-ME 4-Diphosphocytidyl-2-C-methylerythritol
CDP-ME2P 4-Diphosphocytidyl-2-C-methyl-d-erythritol-2-phosphate
CMK CDP-ME kinase
CMS CDP-ME synthase
DMAPP Dimethylallyl diphosphate
DOXP 1-Deoxy-d-xylulose 5-phosphate
DXR DOXP reductoisomerase
DXS DOXP synthase
ER Endoplasmic reticulum
FPP Farnesyl diphosphate
655
GA-3P Glyceraldehyde-3-phosphate
GGPP Geranylgeranyl diphosphate/pyrophosphate
GPP Geranyl diphosphate
GGPPS GGPP synthase
GPPS Geranyl diphosphate synthase
HDS HMBPP synthase
HMBPP (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate
HMG-CoA 3-Hydroxy-3-methylglutaryl-CoA
HMGR HMG-CoA reductase
HMGS HMG-CoA synthase
IDI Isopentenyl/dimethylallyl diphosphate isomerase
IDS IPP/DMAPP synthase
iPD isopiperitenol dehydrogenase
IPI Isopulegone isomerase
iPR isopiperitenone reductase
iPP Isopentenyl diphosphate
L3OH Limonene-3-hydroxylase
LS Limonene synthase
MCS ME-cPP synthase
ME-cPP 2-C-methyl-d-erythritol 2,4-cyclopyrophosphate
MEP 2-C-methyl-d-erythritol-4-phosphate
MFS Menthofuran synthase
MNR Menthone–neomenthol reductase
MMR Menthone–menthol reductase
MR Menthone reductase
MVA Mevalonate
MVD Mevalonate diphosphate decarboxylase
MVK Mevalonate kinase
MVP Mevalonate-5-phosphate
MVPP Mevalonate-5-pyrophosphate
PMK Phosphomevalonate kinase
PR Pulegone reductase
32.1 INTRODUCTION
Based on their function, plant metabolites are classified into two groups: (1) primary metabolites
such as carbohydrates, sterols, carotenoids, growth regulators, the polyprenol substituents of doli-
chols, quinines, and proteins participating in nutrition and essential metabolic processes within the
plants and (2) secondary metabolites including substances in chemical defenses against herbivores
and pathogens, which influence on ecological interactions between plants and their environment
(Chappell 1995, 2002, Croteau et al. 2000). Secondary metabolites of the isoprenoid group constitute
the most diverse family of natural products present in all living organisms, which to date have more
than 40,000 identified compounds (Bohlmann and Keeling 2008, Dudareva et al. 2013). In addi-
tion, this family of compounds constitutes the most abundant biogenic volatile organic compounds
in plants. The rate of emission in these compounds is estimated to be more than 1 × 1012 kg per year
and has important role in plant growth, development, and general metabolism (Croteau et al. 2000,
Guenther et al. 2006, Bohlmann and Keeling 2008, Xing et al. 2010). The isoprenoid compounds
consist of hemiterpenes (C5; e.g., isoprene), monoterpenes (C10; e.g., menthol), sesquiterpenes (C15;
e.g., farnesol, bisabolol), diterpenes (C20; e.g., camphorene, taxol, ginkgolides), triterpenes (C30;
e.g., oleandric acid), tetraterpenes (C40; e.g., lutein, β-carotene and secondary carotenoids such as
Isoprenoid Biosynthesis in Higher Plants and Green Algae 657
astaxanthin), and polyterpenes (C5H8)n (Lichtenthaler 2010). They may be lipophilic or hydrophilic,
volatile or nonvolatile, cyclic or acyclic, and chiral or achiral (Bohlmann and Keeling 2008). They
serve in a variety of different functions in basic and specialized metabolism and they have a vari-
ety of roles in mediating antagonistic and beneficial interactions among organisms. They protect
many species of plants, animals, and microorganisms against predators, pathogens, competitors,
and environmental stresses (Gershenzon and Dudareva 2007, Lohr et al. 2012).
For many years, it was accepted that in all organisms, isoprenoids are synthesized through the
MVA pathway, and a few years ago, an alternative pathway for the biosynthesis of IPP (and DMAPP)
was identified. This novel pathway, known as the MEP pathway, is widely distributed in nature and
is present even in most eubacteria (Rodríguez-Concepción and Boronat 2013). Depending on the
biotic and abiotic stress factor(s), either the MVA or the MEP pathway contributes to the biosyn-
thesis of specific isoprenoids (Lohr et al. 2012). Almost all biotic and abiotic stress factors are able
to affect isoprenoid biosynthesis and emission. Because abiotic stresses inhibit photosynthesis and
volatile isoprenoids are mainly formed by the carbon directly derived from photosynthetic carbon
metabolism, a concurrent negative effect of abiotic stresses on photosynthesis and isoprenoids emis-
sion would be expected. However, abiotic stresses could also stimulate biosynthesis and emission
of constitutive isoprenoids (Fineschi and Loreto 2012). The primary aim of the present chapter is to
review and compare the occurrence, variation, and function of isoprenoid and carotenoid biosyn-
thesis pathways in higher plants and green algae species under normal and light-stressed conditions
and finding the key genes and enzymes associated with their biosynthesis.
32.2 BIOSYNTHESIS OF ISOPRENOIDS

32.2.1 Toward GPP Biosynthesis
Despite their diversity, isoprenoids derive from the condensation of the five-carbon compounds IPP
and its allylic isomer, DMAPP (Mahmoud and Croteau 2001, Vranová et al. 2013, Xiang et al. 2013).
The biosynthesis of IPP was first investigated by Konrad Bloch and Feodor Lynen in 1958 using ani-
mals and yeast as models (Spurgeon and Porter 1981, Lichtenthaler et al. 1997, Lichtenthaler 1999),
indicating that IPP and DMAPP are biosynthetically derived from MVA, a six-carbon compound.
Actually, MVA is a cytosolic pathway (Figure 32.1), which starts with three acetyl-CoA and produces
IPP for sterols, triterpenes, sesquiterpenes, polyterpenes, dolichol, brassinosteroids, and isoprenyl
groups (Sauret-Güeto et al. 2006, Gunatilaka 2012). This pathway operates through the participation
of six key enzymes and requires three ATP and two NADPH molecules to reach to the end products,
IPP and its isomer DMAPP (Lichtenthaler 2010). The related enzymes have been found in higher
plants (Bach et al. 1999), animals (Rodwell et al. 2000), archaea, fungi (Lombard and Moreira 2011),
and bacteria (Miziorko 2011, Takaichi 2011). Despite the fact that terrestrial plants and green algae
have a common evolutionary ancestry (Delaux et al. 2013), it is accepted that the MVA pathway is
generally absent in the common ancestor, Chlorophyta. In higher plants and green algae, however,
the pathway has not been fully identified yet in detail (Grauvogel and Petersen 2007, Lohr et al. 2012).
In 1990s, an alternative MVA-independent pathway was detected by labeling experimental mate-
rial using 13C-labeled glucose isotopomers in plants and bacteria (Flesch and Rohmer 1988, Rohmer
et al. 1993) and the first proof for the presence of MEP pathway was obtained (Schwarz and Arigoni
1999, Zhao et al. 2013). This detection demonstrated that there are two isoprenoid biosynthesis
pathways in cyanobacteria, higher plants, several green algae (Chlorophyceae has only the MEP
pathway), and some bacteria (Lichtenthaler et al. 1997, Zeidler et al. 1997, Lichtenthaler 1998, 1999,
2010, Disch et al. 1998). The two pathways are localized in different compartments: the classical ace-
tate/MVA pathway is in the cytosol and the MEP pathway in plastids. The two pathways are linked
through exchange of metabolic precursors across the plastid envelopes, and it has been demonstrated
that the transport of isoprenoid constituents proceeds exclusively in the chloroplast-to-cytosol direc-
tion, the reverse direction occurring at extremely slow rates (Vickers et al. 2009, Lichtenthaler 2010).
GA-3P GA-3P
Cytoplast + Chloroplast
PEP Pyruvate
CO2 DXS
PEP dxs
DXP
NADPH
NADP DXR
Pyruvate IspC(dxr)
MEP
CTP
CMS DOXP/MEP
AC PPi IspD(ygbp)
pathway
AACT CDP-ME
ATP CMK
ADP
MVA AAC IspE(ychB)
pathway HMGS CDP-ME2P
CMP MCS
HMG-CoA IspF(ygbB)
2NADPH HMGR ME-cPP
2NADP
NADPH HDS
MVA IspG(gcpE)
ATP MVK HMBPP
ADP IDS
NADPH
MVP IspH(lytB)
ATP PMK IPP DMAPP Prenylated metabolites
ADP IDI TPS (Cytokinins, anthraquinones)
MVPP Hemiterpenes (C5)
ATP Phytosterols GPP synthase (Isoprenes)
MVD
ADP
GPP (C10) Monoterpenes
Cytokinins DMAPP IPP Triterpenoids
IPP FPP synthase Essential oil
2X Ubiquinone, plastoquinone,
Squalene FPP (C15) Sesquiterpenes abscisic acid, prenylated proteins
IPP GGPP synthase
FPP Sesquiterpenes Saponins
Triterpenes (C30) GGPP (C20) Diterpenes
2X Homoterpenes Gibberellins, chlorophyll,
(IPP)n
(C11,C30) prenylated proteins, carotenoids
Squalene
Polyterpenes
Sterols
Polyprenols
FIGURE 32.1 Metabolic pathways of the two independent isoprenoid biosynthesis pathways in the plant
cell. (Adapted from Lichtenthaler, H.K., Annu. Rev. Plant Physiol. Plant Mol. Biol., 50, 47, 1999.)
The non-mevalonate pathway occurs in plastids and requires eight consecutive enzymes to pro-
duce IPP and DMAPP, universal basic blocks for isoprenoid biosynthesis. This pathway is initiated
by a head-to-head condensation of GA-3P and pyruvate (carbon 2 and 3) to DOXP that is catalyzed
by DXS and is already known as an intermediate not only for the biosynthesis of IPP and DMAPP
but also for thiamin and pyridoxol biosynthesis (Julliard and Douce 1991, Julliard 1992). The gene
encoding DXS, dxs, have been isolated from Escherichia coli (Sprenger et al. 1997, Lois et al.
1998), Chlamydomonas (Lichtenthaler 1999), and various plant species including Mentha piperita,
Arabidopsis thaliana, and Catharanthus roseus (Bouvier et al. 1998, Lange et al. 1998) but were
absent in animals and yeast genomes (Rodríguez-Concepción and Boronat 2002, Shanker Dubey
et al. 2003). Actually, the DXS enzyme seems to be coded by two or three genes in A. thaliana (Araki
et al. 2000), Zea mays (Cordoba et al. 2009, 2011), Oryza sativa (Kim et al. 2005), Medicago trun-
catula (Walter et al. 2000, 2002), Ginkgo biloba (Kim et al. 2006), and Pinus densiflora (Kim et al.
2009). The enzyme requires thiamine diphosphate and divalent cations such as Mg2+ or Mn2+ for its
activity (Bouvier et al. 1998, Lange et al. 1998, Estévez et al. 2000, Lois et al. 2000, Shanker Dubey
et al. 2003). Several experiments have reported that DXS plays a critical role in the synthesis of IPP
and DMAPP (Lois et al. 2000, Gong et al. 2006, Morris et al. 2006). Increasing or decreasing the
level of different final isoprenoid products (between twofold and sevenfold) including chlorophyll,
carotenoids, tocopherols, and ABA in transgenic plants expressing higher (overexpression) or lower
(antisense) DXS levels supports the rate-limiting function of DXS enzyme in this pathway in plants
and shows accumulation of numerous isoprenoid products in the cell (Lois et al. 2000, Estévez et al.
2001, Shanker Dubey et al. 2003, Gong et al. 2006, Morris et al. 2006).
In the second step of the pathway, DOXP is converted to MEP by DXR in the presence of NADPH.
DXR is the key enzyme of the pathway and has also rate-limiting roles in IPP and DMAPP biosyn-
thesis (Veau et al. 2000, Mahmoud and Croteau 2001, Carretero-Paulet et al. 2006). In transgenic
peppermint (M. piperita), overexpressing DXR led to an increase in essential oil monoterpenes in
leaf tissues compared to the wild type (Mahmoud and Croteau 2001, Shanker Dubey et al. 2003). The
related gene, ispC (formerly designated yaeM or dxr), was first isolated from E. coli (Takahashi et al.
1998). Homologous proteins have been also reported from plants, algae, bacteria, and the protozoa,
Plasmodium falciparum (Lange and Croteau 1999a, Eisenreich et al. 2004, Matsuzaki et al. 2008).
The enzyme uses Mg2+ or Mn2+ as cofactor and is inhibited by fosmidomycin (FSM), an herbicidal
substance that inhibits plant carotenoid, phytol, and isoprenoid biosynthesis (White 1978, Zeidler
et al. 1998, Lange and Croteau 1999a, Fellermeier et al. 1999, Lichtenthaler 2000, Eisenreich et al.
2004). The final result is the bleached phenotype and the failure of seedling establishment (Zeidler
et al. 1998, Carretero-Paulet et al. 2002, 2006). Treatment of unicellular green alga, Dunaliella
salina, with FSM resulted in the suppression of biosynthesis of C5 units, carotenoids, β-carotene, and
chlorophyll. There is less information regarding whether the isoprenoid pathway in other algae can be
influenced by selective inhibitors, such as mevinolin and FSM, or not (Paniagua-Michel et al. 2009).
The further step in the pathway contains the conversion of MEP to CDP-ME by CMS in the
presence of cytidine triphosphate (CTP). The enzyme has essential role for IPP and DMAPP biosyn-
thesis (Herz et al. 2000). The gene encoding the enzyme in E. coli was named ygbP and later was
renamed as ispD (Eisenreich et al. 2004). In plants, the related gene was first cloned from A. thaliana
(Rohdich et al. 2000a, Shanker Dubey et al. 2003). The similar enzyme in E. coli uses Mg2+, Mn2+,
and Co2+ ions as cofactor, whereas the plant enzyme usually uses Ni2+ ions (Eisenreicha et al. 2004).
In Arabidopsis, the enzyme requires a divalent cation, preferably Mg2+ (Rohdich et al. 2000a).
The next step of the pathway is the phosphorylation of the 2-hydroxyl group of CDP-ME into
CDP-ME2P by the CMK enzyme in the presence of adenosine triphosphate (ATP). In isolated
chromoplast of Capsicum annuum, 14C-labeled CDP-ME (the substrate for CMK) was efficiently
converted into carotenoids, suggesting the possible role of the enzyme in carotenoid biosynthesis
(Shanker Dubey et al. 2003). CMK requires Mg2+ as cofactor (Eisenreich et al. 2004).
The fifth step of the pathway is the formation of ME-cPP under the release of CMP. The reac-
tion is catalyzed by MCS. The gene encoding such enzyme in E. coli was named ygbB (renamed as
ispF) and has many putative orthologues in eubacteria as well as in A. thaliana (Herz et al. 2000),
which require Mn2+ or Mg2+ as cofactor (Eisenreich et al. 2004). The sixth step in the pathway is the
conversion of ME-cPP to HMBPP in the presence of NADPH (Rodríguez-Concepción and Boronat
2002, Shanker Dubey et al. 2003) by the enzyme HDS (Rodríguez-Concepción and Boronat 2002).
The E. coli gene coding HDS annotated as gcpE (ispG) is conserved in plants, algae, and eubacteria
but is absent in archaebacteria, yeast, and animal genomes (Rodríguez-Concepción and Boronat
2002, Matsuzaki et al. 2008). The terminal step is the conversion of HMBPP into a 5:1 mixture
of IPP and its isomer, DMAPP. The lytB gene (renamed as ispH) appears to encode the IDS
(Rodríguez-Concepción et al. 2000, 2002) and causes the branching in which IPP and DMAPP are
generated sequentially and this biochemical activity is an important difference between MVA and
non-MVA pathways (Figure 32.1) (Charon et al. 2000, Rodríguez-Concepción et al. 2000, 2002).
In general, the chromosomal replacement study has revealed that in the MEP pathway, (1) the dxs
gene has the highest impact among other isoprenoid genes on carotenoid production, (2) the IPi gene
plays a significant role in the isoprenoid biosynthesis, (3) the ispD and ispF genes appear to form
an operon, (4) CMS and MCS are rate-limiting enzymes in the isoprenoid flux, and (5) there are no
rate limitations for ispE, ispG, and ispH genes (Das et al. 2007). In Arabidopsis and other plants
(with some exceptions such as rubber trees), the transcript levels of all genes from the MEP pathway
accumulate upon the exposure to light and during the development of the first true leaves of seed-
lings (Guevara-García et al. 2005, Hsieh et al. 2008). This positive regulation by light provides an
advantage during early seedling development, which may elevate the demand for the photosynthetic
pigments derived from the pathway (Schoefs et al. 1998, Cordoba et al. 2009).
A pivotal enzyme in monoterpene metabolism is GPPS, a member of the short-chain prenyl-
transferase family catalyzing the head-to-tail condensation of one IPP and one DAMPP molecules
to give rise to GPP. Supply of GPP is critical for terpenoid yield; therefore, studies on the regula-
tion of genes in GPP biosynthesis assume central importance (Rohmer 1993, Croteau et al. 2005,
Paniagua-Michel 2009, Clastre et al. 2011, Lohr et al. 2012, Paniagua-Michel et al. 2012). GGPPS is
a central intermediate in the synthesis of plastidic isoprenoids such as monoterpenes and carotenoids.
Other terpenes contain a 15-carbon and 20-carbon backbone synthesized by FPP synthase and
GGPP synthase (GGPPS), yielding FPP and GGPP, respectively. FPP and GGPP are key substrates
for s everal important branch-point enzymes. In plants, FPP and GGPP are required for the first
committed step in the biosynthesis of sesquiterpenes and diterpenes, respectively. Geranylgeranyl
diphosphate synthase catalyzes the condensation of three molecules of IPP and one molecule of
DMAPP to produce GGPP, a 20-carbon molecule in the carotenoid pathway. Pairwise condensa-
tion of FPP and GGPP provides triisoprenoid (C30) and tetraisoprenoid (C40) biosynthesis. On the
other hand, assembly of an undefined number of C5 precursors yields polyisoprenoids (Bohlmann
and Keeling 2008, Gonzales-Vigil et al. 2012). In land plants and green algae, collectively termed
Viridiplantae (Latin name for green plants), currently, it is clear that the biosynthesis of monoter-
penes appears to be localized in plastids. In recent years, our understanding of the numerous facets
of isoprenoid metabolism in land plants has been rapidly increasing, while knowledge on the meta-
bolic network of isoprenoids in algae still lags behind (Lohr et al. 2012).
32.2.2 From GPP to Menthol Biosynthesis

Terrestrial plants and marine algae produce a variety of secondary metabolites, including monoter-
penes. The algal monoterpenes present several highly unusual characteristics, nearly always haloge-
nated, and they possess ring structures quite unusual. There are a few reports suggesting monoterpene
emission in algae, and a limited number of field studies suggest that these compounds play a role in
the defense of marine algae (Bonsang et al. 1992, Milne et al. 1995, McKay et al. 1996, Wise 2003).
Yassaa et al. (2008) have provided the first evidence for marine production of monoterpenes in nine
algae species consisting of coccolithophorids, Emiliania huxleyi; diatoms, Chaetoceros neogracilis,
Chaetoceros debilis, Fragilariopsis kerguelensis, Phaeodactylum tricornutum, and Skeletonema
costatum; chlorophyte, D. tertiolecta; and cyanobacteria, Synechococcus and Trichodesmium. Among
the phytoplanktons sampled, green algae species, D. tertiolecta, was the strongest emitter of mono-
terpenes followed by P. tricornutum, and nine monoterpenes were identified, namely, (−)-/(+)-pinene,
myrcene, (+)-camphene, (−)-sabinene, (+)-3-carene, (−)-pinene, (−)-limonene, and p-ocimene. So far,
it is not known that emissions by algal cells are a response to biotic (e.g., defense against predation) or
abiotic (e.g., temperature, injury) stresses (Yassaa et al. 2008).
In many types of terrestrial plants, monoisoprenoids are the primary volatile constituents of the
essential oils. In the genus Mentha, the peppermint (M. piperita L.) produces almost exclusively
monoterpenes bearing an oxygen function at position C3 such as (−)- or l-menthol, whereas spear-
mint types such as native spearmint (M. spicata L.) and Scotch spearmint (M. gentilis var. cardiaca)
produce almost exclusively monoterpenes bearing an oxygen function at position C6, typified by car-
vone (Lawrence 1981). The biosynthesis of (−)-menthol has been studied by Croteau and coworkers for
more than two decades, and the results were used as a model for biochemical and molecular genetic
characterization of monoisoprenoid and essential oil biosynthesis (Croteau et al. 2005). Recently,
the main and characteristic component of M. piperita essential oil, the monoisoprenoid (−)-menthol,
was investigated, and cDNAs have been identified and characterized for all enzymes from GPP
to (−)-menthol (Figure 32.2), except for isopulegone isomerase (Turner et al. 2012).
The biosynthesis of (−)-menthol from primary metabolism requires eight enzymatic steps and
proceeds from GPP. The first committed reaction catalyzes the cyclization of GPP to (−)-limonene
by the first committed enzyme of the pathway, (−)-LS, a typical monoterpene cyclase. (−)-Limonene
serves as olefinic precursor of essential oil terpenes of both peppermint and spearmint. The subse-
quent step, (−)-L3OH, using O2 and NADPH, catalyzes the allylic hydroxylation of (−)-limonene at
the three positions to form (−)-trans-isopiperitenol. Biosynthetic investigations have demonstrated
that (−)-limonene undergoes cytochrome-mediated hydroxylation at C3 to yield (−)-trans-isopiperitenol
(peppermint) or at C6 to yield (−)-trans-carveol (spearmint) (Lupien et al. 1999). Typically, car-
vone accumulates in spearmint and superior oils of peppermint containing high quantities of men-
thol, moderate amounts of menthone, and low levels of pulegone and menthofuran. The remaining
enzymes responsible for the subsequent redox transformations of isopiperitenol to menthol is pres-
ent in both peppermint and spearmint species; however, carveol is a poor substrate in these pro-
cesses (Croteau et al. 1991). In a study by Mahmoud et al. (2004), overexpression of L3OH gene
resulted in a substantial increase in the limonene content (up to 80% of the essential oil compared
to about 2% of the oil in wild-type peppermint) in the essential oil, without influence on oil yield,
OPP
IPP
OPP
+ GPPS LS L3OH iPD
OH O
OPP Geranyl (–)- trans-

(–)-Limonen (–)-isopiperitenone
diphosphate isopiperitenol
DMAPP
iPR
MFS iP1
O O O
(+)-Menthofuran (+)-Pulegone (+)-cis-isopulegone
PR PR
O O
(–)-Menthone (+)-Isomenthone
MR MR MR MR
OH HO HO
HO
(–)-Menthol (+)-Neomenthol (+)-Isomenthol (+)-Neoisomenthol
FIGURE 32.2 Metabolic pathway leading to the synthesis of (−)-menthol and related monoisoprenoids
in Mentha.
but simultaneously resulted in a decrease in (−)-menthol yield. According to this result, limonene
does not impose a negative feedback suggesting that pathway engineering can be employed to sig-
nificantly alter essential oil composition without adverse metabolic consequences in peppermint.
For the next step of the pathway, (−)-trans-isopiperitenol is converted to (−)-isopiperitenone by
(−)-trans-isopiperitenol dehydrogenase (iPD) that oxidizes the hydroxyl group on the three positions
using NAD+ followed by (−)-isopiperitenone reductase (iPR) that catalyzes the reaction with reduc-
tion of the double bond between carbons 1 and 2 using NADPH to form (+)-cis-isopulegone. It has
been reported that (+)-cis-isopulegone is the key intermediate in the conversion of (−)-isopiperitenone
to (+)-pulegone (Park et al. 1993). The next step of the pathway is isomerization of the remaining dou-
ble bond to form (+)-pulegone by (+)-cis-iPI and then (+)-PR (isopiperitenone reductase) reduces this
double bond using NADPH to form (−)-menthone. At the terminal step of the pathway, (−)-MR (men-
thone reductase) reduces the carbonyl group using NADPH to form (−)-menthol and (+)-neoisomenthol
by (−)-MMR (menthone-menthol reductase) and to (+)-neomenthol and (+)-isomenthol by (−)-MNR
(menthone-neomenthol reductase). The latter three monoterpenol isomers are minor constituents of
peppermint oil (Croteau et al. 2005). The related enzymes are localized in different parts of the
cell. GPPS and LS are localized in the leucoplasts (Turner et al. 1999, Turner and Croteau 2004),
limonene 6-hydroxylase (of spearmint) is localized in the endoplasmic reticulum, iPD is found to be
mitochondrial, and PR is localized in the cytosol (Turner and Croteau 2004). Both MMR and iPR are
also localized in the cytoplasm and nucleoplasm of the secretory cells of peltate glandular trichomes
(Turner et al. 2012). There is possibility to alter the composition and volume production of monoter-
pene and essential oil quality through transgenic manipulations (Mahmoud and Croteau 2001, 2003,
Croteau et al. 2005, Wildung and Croteau 2005, Bohlmann and Keeling 2008).
32.2.3 Carotenoids
Carotenoids are C40 natural fat-soluble, yellow, orange, or sometimes red, isoprenoid pigments and
essential components of the photosynthetic apparatus that serve two functions: light harvesting (acces-
sory pigments) or photoprotection of the chlorophyll a molecules in the photosynthetic reaction cen-
ters against photooxidation (Lichtenthaler 2012, Ruiz-Sola and Rodríguez-Concepción 2012, Xiumin
et al. 2012). There are over 750 known structurally defined carotenes that have been identified so far.
Carotenoids are also formed in several nongreen and nonphotosynthetic organisms, such as yeast, bac-
teria, and molds, to protect them against damage by light and oxygen (Lichtenthaler 2012, Ruiz-Sola
and Rodríguez-Concepción 2012). They can be synthesized from carotenoid pathway by most organ-
isms except for the animal kingdom that are generally unable to synthesis the components (Moran
and Jarvik 2010, Takaichi 2011, Lichtenthaler 2012). So far, little is known about the mechanisms that
regulate carotenoid biosynthesis (Meier et al. 2011). High carotenoid levels are found in the chloroplasts
of photosynthetic tissues, but the highest amounts of carotenoids are found in chromoplasts. Besides
chromoplasts, all other plastid types synthesize carotenoids but the level of carotenoid accumulation
varies widely among different plastid types (Ruiz-Sola and Rodríguez-Concepción 2012). Unlike chro-
moplasts that show highly diverse carotenoids, depending on the organ, species, and genetic variely,
chloroplasts have a remarkably similar carotenoid composition in all plants, with lutein (45% of the
total), β-carotene (25%–30%), violaxanthin (10%–15%), and neoxanthin (10%–15%) as the most abun-
dant carotenoids (Britton, 1993). Photosynthetic reaction centers are enriched in carotene (β-carotene),
whereas xanthophylls are most abundant in the light-harvesting processes (Ruiz-Sola and Rodríguez-
Concepción 2012).
Many different types of carotenoids are extracted from higher plants and algal species. The
largest structural variety of carotenoids is encountered in marine environments produced by micro-
scopic and macroscopic algae (Liaaen-Jensen 1991, Mimouni et al. 2012). Approximately, 30 types
of carotenoids participate in photosynthesis (primary carotenoids) and others are functional caro-
tenogenesis intermediates (secondary carotenoids).
32.2.4 From GPP to Secondary Carotenoids

The main carotenoid biosynthetic pathway was elucidated in the latter half of the twentieth
century using biochemical (from the 1960s) and molecular (from the 1980s) approaches. Major
advances in the identification of genes and enzymes of the pathway have been made from the
1990s (Ruiz-Sola and Rodríguez-Concepción 2012). In terrestrial plants, most of the caroteno-
genesis pathways and functionally related enzymes are known, but little is known among algae
(Takaichi 2011).
In the large group of green algae, the carotenoid biosynthesis is more complex. Like higher
plants, the more advanced evolutionary group of green algae, such as Charales and Zygnematales,
possesses both MVA and MEP pathways. In contrast, often single-cell organisms of green algae,
such as Chlorella, Scenedesmus, and Trebouxia, represent carotenoid and sterol biosynthesis via
the MEP pathway, and the MVA pathway is lost (Lichtenthaler 2010). Despite the importance of
these taxa, Haematococcus pluvialis (astaxanthin biosynthesis) and Dunaliella sp. (β-carotene
biosynthesis), for the commercial production, most of the studies on secondary carotenoid path-
way have been performed by these organisms (Das et al. 2007, Lemoine and Schoefs 2010,
Moulin et al. 2010).
The first committed step in carotenoid biosynthesis is a head-to-head condensation of the two C20
molecules of GGPP by phytoene synthase (PSY) (CrtB, Psy, Pys) to form phytoene (Figure 32.1).
GGPP is also the precursor for several other groups of metabolites, including chlorophyll and tocoph-
erol. PSY is a major rate-controlling carotenoid enzyme at unknown plastid sites, either in plastoglob-
uli or in stroma and thylakoid membranes (Shumskaya et al. 2012). The green algae Ostreococcus
and Micromonas possess two orthologous copies of the PSY genes, possibly indicating an ancient
gene duplication event. In contrast, higher plants possess only one class of the PSY gene and the
other gene copy is lost (Tran et al. 2009). For the next step in the pathway, phytoene undergoes four
sequential reactions to form lycopene (Farré et al. 2010, Walter and Strack 2011). This route requires
three enzymes including phytoene desaturase (PDS) (CrtP, Pds), ζ-carotene desaturase (CrtQ, Zds),
and cis-carotene isomerase (CrtH, CrtISO). In higher plants and green algae, α-carotene, β-carotene,
and their derivatives are derived from lycopene (Figure 32.3). In the next step, the carotenoid path-
way has branches at the cyclization reaction to produce carotenoids with either two β-rings (such as
β-carotene and its derivatives) or one ε- and one β-ring (such as α-carotene and lutein) (Figure 32.3).
Inability of the lycopene ε-cyclase enzyme (LCYe) to add two ε-rings to the symmetrical lycopene
is the cause of the absence of a branch leading to carotenoids with two ε-rings in most plants (except
lettuce) (Cunningham 2002). Plants contain two CrtL lycopene cyclases (LCYs), lycopene ε-cyclase
(CrtL-e, LCYe) and lycopene β-cyclase (CrtL-b, LCYB), that form enzymatic products of α-carotene
and β-carotene. Lutein and zeaxanthin are generated by the hydroxylation of these two carotenoids,
respectively. Their function is light harvesting within the antenna of photosystems I and II (PSI and
PSII) (Sheen 1991, Bradbury et al. 2012). In the absence of stress, hydroxyl groups are introduced
into β-carotene to produce zeaxanthin by β-carotene hydrolase (CrtR, CrtR-b, BCH), and zeaxanthin
epoxidase (Zep, NPQ) is responsible for producing violaxanthin through antheraxanthin, in both
higher plants and algae. Epoxidases, such as violaxanthin and zeaxanthin, are more common among
algal carotenoids (Liaaen-Jensen 1991). The first Zep cDNA was identified from tobacco (Nicotiana
plumbaginifolia) and was named as ABA2 (Marin et al. 1996). Under light stress and with the devel-
opment of a high-pH gradient across the thylakoids, the reaction is different, and the two steps of
mono-de-epoxidation reactions of the violaxanthin into zeaxanthin with antheraxanthin as an inter-
mediate are catalyzed by violaxanthin de-epoxidase (Vde) in order to dissipate the excess energy
in the form of heat from excited chlorophylls (V, Takaichi 2011). The first sequenced cDNA of Vde
was obtained from romaine lettuce (Lactuca sativa L.), and the homologous genes were sequenced
in the genome of diatoms Thalassiosira pseudonana and P. tricornutum. So far, most of the caro-
tenogenesis enzymes and genes have been found in cyanobacteria (Takaichi and Mochimaru 2007),
diatoms (Bertrand 2010), green algae (Lemoine and Schoefs 2010, Moulin et al. 2010), and higher
CHYe CrtP CrtB

ε-Carotene ζ-Carotene Phytoene Geranylgeranyl
CHYe In lettuce CrtQ diphosphate
LCYe CrtH/CrtISO
Lactucaxanthin
CrtL-e Lycopene
CrtL-b
δ-Carotene
γ-Carotene
CrtL-b
CrtL-b 3-Hydroxyechinenone
α-Carotene
β-Carotene Echinenone Canthaxanthin Adorirubin
CrtW CrtW CrtR-b
CrtR-b
α-Cryptoxanthin Zeinoxanthin CrtR-b
β-Cryptoxanthin
CrtW
CrtR-b Adonixanthin Astaxanthin
CrtW
Lutein Zeaxanthin Caloxanthin Nostoxanthin
CrtG CrtG
Zep Vde Diatoxanthin Alloxanthin
Crocoxanthin Prasinoxanthin Antheraxanthin

Loroxanthin
Zep Vde
Diadinoxanthin Heteroxanthin
Monadoxanthin Violaxanthin
Siphonaxanthin
Vaucheriaxanthin
Nsy
Peridinol Peridinin Pyrrhoxanthin
Neoxanthin
Dinoxanthin
9΄-cis neoxanthin Fucoxanthin
FIGURE 32.3 Carotenogenesis scheme and confirmed enzymes in higher plants and green algae. The
dashed part of graph could be synthesized only in some algae.
plants (Frommolt et al. 2008); however, in algal species, some of them have not been found yet
(Takaichi 2011). It seems that higher plants and algae have common carotenogenesis pathways, and
all of the enzymes and genes related to this pathway are presented in Table 32.1 (for confirmed genes
and enzymes in cyanobacteria and algae, see Takaichi 2011). No counterpart of the neoxanthin syn-
thase (Nsy) was reported in A. thaliana (Cunningham 2002).
Major carotenoids in most species of the class of the green algae, Chlorophyta (Prasinophyceae,
Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Charophyceae), and land plants consist of
β-carotene, violaxanthin, and neoxanthin (Takaichi 2011). Some carotenoids are found only in some
classes or divisions of higher plants and algae, and for this reason, they are used as chemotaxonomic
markers (Rowan 1989, Liaaen-Jensen 1990). In this respect, green algae including Euglenophyta,
Chlorarachniophyta, and Chlorophyta contain the same carotenoids, such as lutein, β-carotene,
9-cis neoxanthin, and violaxanthin (Takaichi 2011). For comparison, tomato (Lycopersicon escu-
lentum Mill.) contains prolycopene, ζ-carotene, β-carotene, or δ-carotene, and carrot (Daucus
carota L.) contains xanthophylls, ζ-carotene, β-carotene, δ-carotene, and lycopene (MacKinney
and Jenkins 1949, Buishand and Gabelman 1980, Ronen et al. 2000).
Carotenoid content pattern can change during the life cycle of plants and algae. The unicellular
green alga H. pluvialis, the most suitable source of astaxanthin (of the most important diterpenes),
accumulates this antioxidant (up to 4% by dry weight) in response to various environmental stress
conditions such as high light intensities, nitrogen limitation, and salt stress in extraplastidic lipid glob-
ules as a secondary carotenoids (Boussiba et al. 1999, Boussiba 2000, Grünewald et al. 2001, Schoefs
et al. 2001, Lemoine and Schoefs 2010, Moulin et al. 2010). The massive accumulation of astaxanthin
occurs during cyst cell formation, whereas in green vegetative phase and without stress condition,
zeaxanthin accumulation occurs (Vidhyavathi et al. 2008). In red pepper (Ca. annuum L.) when
ripening is started, chloroplast pigments, lutein, and neoxanthin are decreased, whereas β-carotene
and antheraxanthin are increased (Hornero-Mendez et al. 2000).
TABLE 32.1
Enzymes and Genes Related to Isoprenoid Pathways (MEP, Menthol Biosynthesis, Carotenogenesis) Whose Functions Have Been
Confirmed in Higher Plants and Green Algae
Gene/Synonym Green Algae
Pathway Enzyme Name Genes Plant Species References Species References
MEP DOXP synthase (DXS) dxs A. thaliana Araki et al. (2000), Estévez et al. Botryococcus Daisuke et al. (2012)
(2000) braunii
M. piperita Lange et al. (1998) Dunaliella sp. Hermin Pancasakti (2008)
Me. truncatula Walter et al. (2002) Ostreococcus Matsuzaki et al. (2008)
lucimarinus
Ca. annuum Bouvier et al. (1998) Ch. reinhardtii Matsuzaki et al. (2008)
C. roseus Chahed et al. (2000) Ostreococcus tauri Frommolt et al. (2008)
Ly. esculentum Lois et al. (2000) Volvox carteri Frommolt et al. (2008)
O. sativa Matsuzaki et al. (2008)
Picea abies Phillips et al. (2007)
Z. mays Walter et al. (2000)
G. biloba Kim et al. 2006
MEP DOXP reductoisomerase ispC (dxr) A. thaliana Carretero-Paulet et al. (2002), Os. lucimarinus Matsuzaki et al. (2008)
(DXR) Schwender et al. (1999)
Isoprenoid Biosynthesis in Higher Plants and Green Algae
M. piperita Lange and Croteau (1999a), Os. tauri Frommolt et al. (2008)
Mahmoud and Croteau (2001)
O. sativa Matsuzaki et al. (2008) Ch. reinhardtii Matsuzaki et al. (2008)
Hevea brasiliensis V. carteri Frommolt et al. (2008)
MEP CDP-ME synthase ispD (ygbP) A. thaliana Rohdich et al. (2000a) Os. lucimarinus Matsuzaki et al. (2008)
(CMS) Ca. annuum Luttgen et al. (2000) Os. tauri Frommolt et al. (2008)
O. sativa Matsuzaki et al. (2008) Ch. reinhardtii Matsuzaki et al. (2008)
V. carteri Frommolt et al. (2008)
MEP CDP-ME kinase (CMK) ispE (ychB) M. piperita Lange and Croteau (1999b) Os. lucimarinus Matsuzaki et al. (2008)
A. thaliana Frommolt et al. (2008) Os. tauri Frommolt et al. (2008)
Ly. esculentum Rohdich et al. (2000b) Ch. reinhardtii Matsuzaki et al. (2008)
O. sativa Matsuzaki et al. (2008) V. carteri Frommolt et al. (2008)
(continued)
665
666

Triticum aestivum Frommolt et al. (2008)
Hordeum vulgare Frommolt et al. (2008)
Populus trichocarpa Frommolt et al. (2008)
MEP ME-CP synthase (MCS) ispF (ygbB), MDS Mentha × piperita Dolzhenko et al. (2010) Os. lucimarinus Matsuzaki et al. (2008)
C. roseus Veau et al. (2000) Os. tauri Frommolt et al. (2008)
Ca. annuum Fellermeier et al. (2001), Herz Ch. reinhardtii Matsuzaki et al. (2008)
et al. (2000)
Narcissus Fellermeier et al. (1999)
pseudonarcissus
O. sativa Matsuzaki et al. (2008)
MEP HMBPP synthase ispG (gcpE) A. thaliana Hecht et al. (2001) Os. lucimarinus Matsuzaki et al. (2008)
(HDS) O. sativa Matsuzaki et al. (2008) Ch. reinhardtii Matsuzaki et al. (2008)
MEP HMBPP reductase (IDS/ ispH (lytB) Ca. annuum Adam et al. (2002) Os. lucimarinus Matsuzaki et al. (2008)
HDR) O. sativa Matsuzaki et al. (2008)
G. biloba Kim et al. (2008)
Pinus taeda Kim et al. (2008)
MEP Isopentenyl diphosphate IPi A. thaliana Cunningham (2002) Ch. reinhardtii Frommolt et al. (2008)
isomerase (IDI) Os. lucimarinus Frommolt et al. (2008)
Os. tauri Frommolt et al. (2008)
Menthol biosynthesis (−)-Limonene synthase LS/LC, TPS, FES M. spicata, Colby et al. (1993), Alonso et. al.
Mentha × piperita (1992)
A. thaliana, P. abies Bohlmann et al. (2000), Martin
et al. (2004)
Ocimum basilicum Iijima et al. (2004)
Menthol biosynthesis (−)-Limonene-3- L3OH, ER, PM, GI M. spicata, Turner and Croteau (2004)
hydroxylase Mentha × piperita
Mentha × piperita Lupien et al. (1999)
Perilla frutescens Mau et al. (2010)
Menthol biosynthesis (−)-trans-Isopiperitenol IPD/ISPD Mentha × piperita, Turner and Croteau (2004), Ringer
dehydrogenase M. spicata et al. (2005)
Menthol biosynthesis (−)-Isopiperitenone ISPR Mentha × piperita, Ringer et al. (2003, 2005)
reductase M. spicata
A. thaliana Ringer et al. (2005)
Menthol biosynthesis (+)-cis-Isopulegone IPL Mentha × piperita Bohlmann and Keeling (2008)
isomerase
Menthol biosynthesis (+)-Menthofuran synthesis MFS Mentha × piperita Mahmoud and Croteau (2003)
Menthol biosynthesis (+)Pulegone reductase PR Mentha × piperita Ringer et al. (2003)
Menthol biosynthesis (−)-Menthone reductase MR, MMR, MNR Mentha × piperita, Bohlmann and Keeling (2008), Davis
M. spicata et al. (2005), Ringer et al. (2005)
A. thaliana Ringer et al. (2005)
Carotenogenesis Geranylgeranyl CrtE, ggps A. thaliana Cunningham (2002)
pyrophosphate synthase
Carotenogenesis Phytoene synthase PSY, PYS, crtB A. thaliana Frommolt et al. (2008) Ch. reinhardtii McCarthy et al. (2004)
T. aestivum Frommolt et al. (2008) H. pluvialis Steinbrenner and Linden
NIES-144 (2003)
Ho. vulgare Frommolt et al. (2008) Os. lucimarinus Frommolt et al. (2008)
Po. trichocarpa Frommolt et al. (2008) Os. tauri Frommolt et al. (2008)
Ly. esculentum Frommolt et al. (2008) V. carteri Frommolt et al. (2008)
O. sativa Frommolt et al. (2008)
Isoprenoid Biosynthesis in Higher Plants and Green Algae
Carotenogenesis Phytoene desaturase PDS, crtP, CRTI A. thaliana Cunningham (2002) Ch. reinhardtii Vila et al. (2008)
Ly. esculentum Mill. Cunningham (2002) Chlorella Liu et al. (2010)
zofingiensis
ATCC 30412
H. pluvialis (SAG Vidhyavathi et al. (2008)
19-a)
Carotenogenesis ζ-Carotene desaturase ZDS, crtQ A. thaliana Cunningham (2002)
Z. mays Isaacson et al. (2004)
Carotenogenesis Lycopene β-cyclase CrtL-b, crtL, lcy-b Solanum Ronen et al. (1999)
lycopersicum
A. thaliana Cunningham (2002) D. salina CCAP Ramos et al. (2008)
19/30 Steinbrenner and Linden
H. pluvialis NIES-144 (2003)
(continued)
667
668

Carotenogenesis Carotene isomerase CrtH/CrtISO A. thaliana Cunningham (2002)
Carotenogenesis Lycopene ε-cyclase CrtL-e, lcy-e S. lycopersicum Ronen et al. (1999)
A. thaliana Fiore et al. (2012)
L. sativa romaine Cunningham and Gantt (2001)
Carotenogenesis β-Carotene hydroxylase CrtR-b, CrtR, A. thaliana Cunningham (2002) H. pluvialis Linden (1999),
BCH, CHY, NIES-144 Steinbrenner and Linden
CHYB (2001)
Carotenogenesis β-Carotene ketolase CrtW, BKT H. pluvialis Steinbrenner and Linden
NIES-144 (2001)
H. pluvialis strain Lotan and Hirschberg
34/7 (1995)
Chl. zofingiensis Huang et al. (2006)
ATCC 30412
Carotenogenesis Zeaxanthin epoxidase Zep, npq, ABA A. thaliana Cunningham (2002) Ch. reinhardtii Baroli et al. (2003)
N. plumbaginifolia Marin et al. (1996) Os. lucimarinus Frommolt et al. (2008)
Ca. annuum Hieber et al. (2000) Os. tauri Frommolt et al. (2008)
Ly. esculentum Hieber et al. (2000)
Prunus armeniaca Hieber et al. (2000)
Carotenogenesis Violaxanthin vde A. thaliana Cunningham (2002) Mantoniella Goss (2003)
de-epoxidase squamata
Os. lucimarinus Frommolt et al. (2008)
Os. tauri Frommolt et al. (2008)
Carotenogenesis β-Carotene crtG
2-hydroxylase
Carotenogenesis Neoxanthin synthase Nsy
32.3 ISOPRENOID PROTECTION AGAINST DIVERSE STRESSES

Almost all of isoprenoids have the ability to increase the tolerance of leaves to transiently high tem-
peratures and light stress, and they may also quench ozone and ROS levels inside the leaves (Loreto
and Velikova 2001, Sharkey et al. 2001, Behnke et al. 2007, Loreto and Fares 2007, Vickers et al.
2009, Lohr et al. 2012). They also reduce the formation of nitric oxide in the mesophyll and could
modulate the signaling of defense-induced biosynthetic pathways (Velikova et al. 2005). Among
the 100,000 chemical products that are known to be produced by plants, at least 1700 of these are
known to be volatile isoprenoids. Two of the most important functions of volatile isoprenoids are
protections of plant tissues from thermal and oxidative stresses (Behnke et al. 2007, Loivamäki
et al. 2007, Spinelli et al. 2011). There are key dissipation processes and mechanisms for the resis-
tance to excess energy and the other environmental stresses, which are mediated by a particular
group of carotenoids. Monoterpene emission is equivalent to 1%–2% of photosynthetic carbon fixa-
tion (Sharkey and Yeh 2001). Some enzymes positioned at key points in metabolic pathways are
ideal candidates for regulation, as their activity can affect the output of the entire pathways. These
enzymes typically share two characteristics: they catalyze (1) reactions far from equilibrium and
(2) early committed steps in the pathways. For instance, in menthol pathway and during stressful
conditions, an important diversion from the pathway to menthol is the transformation of (+)-pulegone
to menthofuran catalyzed by menthofuran synthase (MFS), and because of this, it is considered as a
stress metabolite (Croteau et al. 2005). Together, menthofuran and (+)-pulegone are described as an
off odor and accumulate to high levels (15%–20% of the oil) under stress conditions (Croteau et al.
2005, Rios-Estepa et al. 2008). Some carotenoids play similar role(s) during the stress.
32.3.1 Light Stress
Menthol and carotenoid biosynthesis involve a series of enzymes that mainly starts from MEP
pathway and each alteration in the pathway flux should be observable at the level of products
(Mahmoud and Croteau 2001, Paniagua-Michel et al. 2012). Comparative expression analysis of
the MEP pathway genes under various growing conditions shows that transcript accumulation of
these genes in plants is modulated by multiple external signals and in a coordinated manner. One
signal that impacts strongly the transcript accumulation of several genes in the MEP pathways is
light (Cordoba et al. 2009). Studies show that light stress can increase expression of some genes in
MEP pathway. For instance, dxs is more expressed under light stress (Kawoosa et al. 2010, Meier
et al. 2011). UV-B irradiation may induce upregulation of dxs, gpps, fpps, and Ipi genes that are
necessary for menthol and carotenoid biosynthesis (Dolzhenko et al. 2010, Lemoine and Schoefs
2010). Vidhyavathi et al. (2008) also reported that the expression of carotenogenic genes, PSY, PDS,
LCY, β-carotene ketolase (BKT), and β-carotene hydroxylase (CHY) were upregulated in green
algae, H. pluvialis, under nutrient stress and higher light intensity, and astaxanthin content as a
stress metabolite was increased. It is also believed that most plants respond to high light conditions
with a 1.4–2-fold increase of xanthophyll cycle carotenoids (violaxanthin, zeaxanthin, neoxanthin),
an enhanced operation of the xanthophyll cycle, and an increase of β-carotene levels (Lichtenthaler
2007, Lemoine and Schoefs 2010).
32.3.1.1 Light Stress and Menthol Biosynthesis

Light quality is an important factor for essential oil production. Menthol biosynthesis in pepper-
mint can be decreased by supplementary blue light (450 nm) because of shifting the carbon flow to
(+)-menthofuran production (Maffei and Scannerini 1999). In addition to visible light, one of the
unavoidable stress factors in natural condition of photosynthetic organisms is the exposure to UV-B
radiation (280–320 nm). However, isoprenoid production is induced by UV radiation, but not always
supplementary UV-B leads to increased isoprenoid production (Dolzhenko et al. 2010). Maffei and
Scannerini (2000) reported that additional UV-B light has negative effect on essential oil quality
with positive effect on high amounts of (+)-menthofuran. In addition, UV-A radiation (360 nm)
affects the composition of peppermint oil by increasing (+)-menthofuran content. UV-B irradiation
may induce upregulation of ipI and MR-genes and also can downregulate the expression of L3OH
and TPS genes (Dolzhenko et al. 2010).
32.3.1.2 Light Stress and Carotenogenesis

In plants, the xanthophyll cycle (the reversible interconversion of two carotenoids, violaxanthin
and zeaxanthin) has a key photoprotective role to enhance tolerance to high light but also to other
stress conditions, such as nitrogen starvation, which has not been reported previously (Cousoab
et al. 2012). The accumulation of β-carotene and zeaxanthin at high photon flux densities has been
reported in A. thaliana. In this plant, overexpression of the chyb gene that encodes CHY, an enzyme
in the zeaxanthin biosynthetic pathway, causes a specific twofold increase in the size of the xantho-
phyll cycle pool. The plants are more tolerant to conditions of high light and high temperature than
other organisms. Their stress protection is probably due to the function of zeaxanthin in preventing
oxidative damage of membranes (Davison et al. 2002).
In Chlamydomonas reinhardtii, the high increase in the transcript levels of the cytochrome-dependent
CHY and ε-carotene hydroxylase in response to high light suggests an important role of these enzymes
in regulation of xanthophyll synthesis upon light stress (Depka et al. 1998, Cousoab et al. 2012). In
Chlorella, high-irradiance stress did not increase mRNA levels of neither lycopene β-cyclase gene (lcy-b)
nor lycopene ε-cyclase gene (lcy-e), whereas the transcript levels of psy, pds, chyB, and bkt genes were
enhanced. Nevertheless, the synthesis of the secondary carotenoids astaxanthin, canthaxanthin, and
zeaxanthin was triggered, and the levels of the primary carotenoids α-carotene, lutein, violaxanthin, and
β-carotene were decreased (Cordero et al. 2012). In higher plants, LCY plays as key role under high light
stress and assists in preventing ROS-dependent damage of DNA, proteins, carbohydrates, and lipids by
reducing accumulation of β-carotene-5,6-epoxide (Bradbury et al. 2012).
In green algae, in addition of lycopene as an important intermediate, the carotenogenesis path-
way involves two key enzymes, PSY (PSY/CrtB) and CHY (CrtR-b/CHYB). The application of high
light intensities in H. pluvialis caused a transient increase in CHY mRNA and consequently astaxan-
thin accumulation (Steinbrenner and Linden 2001). Carotenogenic genes expression in H. pluvialis
including PSY, PDS, LCY, BKT, and CHY is upregulated under high light (Steinbrenner and Linden
2001, Vidhyavathi et al. 2008). In the study of Pirastru et al. (2012), Scenedesmus sp. was tolerant
to the long-term (40 days) high light stress condition due to the production of secondary carot-
enoids, such as astaxanthin and canthaxanthin. The biosynthesis of these secondary carotenoids
was highly induced after the deterioration of PSII complexes when chlorophyll synthesis and cell
division were inhibited. In the other study, exposure of Chlorococcum to high irradiance caused an
increase in the amount of xanthophyll-cycle pigments and in the carotenoid/chlorophyll ratio. As a
result of exposure to stress conditions, cell division was completely stopped, although an increase
in the biomass dry weight could be detected due to an increase in the cell size (Masojídek et al.
2000). Dunaliella sp. was exposed to high light intensity (4000 μmol−2 m−2 s−1) for 2 h and it lost its
carotenoid and chlorophyll content up to 20% and 15%, respectively. In contrast, zeaxanthin was
increased by approximately 200% (Young and Britton 1990). In this respect, it seems that carotenes
(such as α- and β-carotene) are more sensitive to photobleaching than xanthophylls (such as lutein,
zeaxanthin, neoxanthin, violaxanthin, and α- and β-cryptoxanthin), and sensitivity of chlorophyll is
ranged between carotenes and xanthophylls (Siems et al. 2002). In the other report by Chang et al.
(2013), Ch. reinhardtii was exposed to high light (3000 μmol−2 m−2 s−1), and carotenoid content was
slightly reduced during the first 30 min of illumination and strongly diminished after 60 min. The
expression of the transcripts of enzymes involved in carotenoid biosynthesis, including PSY, PDS,
and lycopene ε-cyclase (LCYE), initially increased and then decreased. These results suggest that
to ameliorate the stress, a reduction in the degree of carotenoid breakdown occurs by activation of
de novo carotenoid synthesis.
32.4 GENETIC ENGINEERING

Harnessing the powers of plant, algal, and microbial systems for economically valuable isopren-
oid production requires extensive research and completely understanding their biosynthesis and
genomics, as well as the effect of environmental and stress conditions. Metabolic engineering could
enhance plant adaptation to climate change and improve food security and nutritional value. For
instance, in transgenic peppermint, by downregulation of the MFS gene, lower amount of men-
thofuran and (+)-pulegone in the oil was produced. This led to an increase in the isoprenoid flux
through PR and ultimately more (−)-menthol content (Mahmoud and Croteau 2003). Increasing
the other enzymes involved in carotenogenesis pathway, CHY and PSY, has also an important role
during stress conditions, such as light stress. Discovery of differential gene products like PSY or
CHYB locations linked to activity and isozyme type advances the engineering potential for modify-
ing carotenoid biosynthesis (Shumskaya et al. 2012).
In the recent years, all the genes of menthol biosynthesis pathway have been isolated, cloned, and
characterized, but there are still several enzymes such as iPD, iPR, iPI, PR, and MR that need to be
explored for their metabolic engineering potential (Dolzhenko et al. 2010). On the other hand, for
carotenoid biosynthetic pathway, the catalytic steps have been described, although, the regulatory
mechanisms that control carotenoid accumulation remain poorly understood (Lee et al. 2012). With
genetic engineering or genetic modification, it is possible to manipulate the characteristics and func-
tions of the original genes of each pathway in organisms. The objective of this process is to introduce
new physiological and physical features or characteristics, change the patterns of production, and
boost the yield of metabolites including isoprenoid synthesis in plants and microorganisms.
32.5 CONCLUSION
In conclusion, the MEP pathway of plastid IPP and isoprenoid biosynthesis is well established with its
seven main enzymes and corresponding genes, but all of the enzymes and genes related to the pathway
and also the physiological roles of isoprenoids are not entirely clear (Sharkey and Yeh 2001, León and
Cordoba 2013). The regulatory mechanisms employed by plants and algae to adjust the amount and
composition of isoprenoids are being revealed. Many of the developmental and regulatory aspects of
menthol biosynthesis are known in the Mentha genus. Recently, many new carotenoids synthesis genes
have been isolated (Cheng 2006). Much of what has been learned about the carotenoid pathway in
higher plants has come from Arabidopsis and tomato and in green algae has come from H. pluvialis and
Dunaliella sp. (Cunningham 2002, Lemoine and Schoefs 2010, Moulin et al. 2010).
In the aspect of human life, isoprenoids, as the great chemical derivatives of plants and algae, are
utilized in industrial and chemical materials and could potentially even become as biofuel sources.
Discovering the biosynthesis of the most important isoprenoids and identifying the role of related
enzymes in plants and algae species is critically important to achieve modification of each path-
way and product via plant metabolic engineering and biochemical engineering of plant and micro-
bial systems. The performance of metabolically engineered higher plants and green algae species
toward essential oil or carotenoid production is encouraging, and in some cases, their production
capacity has either reached to the maximum or exceeded the production by native species. For
instance, when the mutant of unicellular green alga, Ch. reinhardtii, was exposed to nutritional
stress (N starvation), after 48 h, the carotenoid content was increased 30-fold, whereas in wild type,
the carotenoid content was increased 15-fold, demonstrating that genetic manipulation can enhance
carotenoid production (Wang et al. 2009). In tomato fruits, overexpression of lycopene β-cyclase
enzyme (LCYB) increased both β-carotene and total carotenoids (Cunningham 2002). It is impor-
tant to develop technologies that will produce isoprene in a cost-effective, environmentally friendly
way and utilizing renewable sources. Modification of the MEP pathway and its related pathways in
plants and microorganisms is necessary for the future production of chloroplast isoprenoids such as
β-carotene (provitamin A) and α-tocopherol (vitamin E).
ACKNOWLEDGMENTS
The authors thank the French Ministry for Education and Scientific Research and the University
of Le Mans for financial supports. PH is very grateful to Pres LUNAM (programme: Structuration
des coopérations internationales des laboratoires par la mobilité internationale des doctorants) and
the Collège doctoral of the University of Le Mans (programme: Aide à la cotutelle internationale
de thèse) for their financial supports.
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Part V
Physiological Responses of
Plants/Crops to Heavy Metal
Concentration and Agrichemicals
33 Formation and Action
Metal Nanoparticles in Plants
Elena Masarovičová, Katarína Král’ová,

and Smita Sachin Zinjarde
CONTENTS
Abbreviations..................................................................................................................................684
33.1 Introduction........................................................................................................................ 685
33.2 General Characterization of Nanoparticles and Metal Nanoparticles............................... 685
33.3 Experimental Methods Applied for Characterization of Metal Nanoparticles................. 686
33.3.1 Ultraviolet–Visible Spectroscopy.......................................................................... 686
33.3.2 Fourier Transform Infrared Spectroscopy............................................................ 686
33.3.3 Electron Microscopy............................................................................................. 699
33.3.4 Atomic Force Microscopy..................................................................................... 699
33.3.5 X-Ray Diffraction..................................................................................................700
33.3.6 Energy-Dispersive X-Ray Spectroscopy...............................................................700
33.3.7 X-Ray Absorption Near-Edge Structure Spectroscopy and
Extended X-Ray Absorption Fine Structure Spectroscopy...................................700
33.3.8 X-Ray Photoelectron Spectroscopy.......................................................................700
33.4 Green Synthesis of Metal Nanoparticles Using Plant Extracts: Characterization
and Determination.............................................................................................................700
33.4.1 Gold Nanoparticles................................................................................................ 701
33.4.2 Silver Nanoparticles.............................................................................................. 702
33.4.3 Palladium Nanoparticles....................................................................................... 703
33.4.4 Platinum Nanoparticles......................................................................................... 703
33.4.5 Zinc Oxide Nanoparticles..................................................................................... 704
33.4.6 Copper Nanoparticles............................................................................................ 704
33.4.7 Selenium Nanoparticles......................................................................................... 705
33.4.8 Titanium Dioxide Nanoparticles........................................................................... 705
33.5 Formation of Metal Nanoparticles in Living Plants.......................................................... 705
33.6 Uptake, Translocation, and Accumulation of Metal Nanoparticles in the Plants.............. 707
33.7 Toxicity of Metal Nanoparticles to Algae.......................................................................... 707
33.8 Effect of Metal Nanoparticles to Vascular Plants.............................................................. 711
33.8.1 Beneficial Effects of Metal Nanoparticles to Plants............................................. 711
33.8.2 Adverse Effects of Metal Nanoparticles to Plants................................................ 713
33.8.2.1 Ag Nanoparticles................................................................................... 713
33.8.2.2 ZnO Nanoparticles................................................................................ 715
683
33.8.2.3 Cu and CuO Nanoparticles................................................................... 716

33.8.2.4 Al2O3, SiO2, TiO2, and Fe3O4 Nanoparticles......................................... 717
33.8.2.5 Rare Earth Elements............................................................................. 717
33.9 Application of Metal Nanoparticles in Phytoremediation Technology............................. 718
33.10 Concluding Remarks.......................................................................................................... 718
References....................................................................................................................................... 719
ABBREVIATIONS
AFM Atomic force microscopy
CAT Catalase
Chlorophyll Chlorophyll
DLS Dynamic light scattering
EDAX Energy-dispersive x-ray analysis
EDS (or EDX) Energy-dispersive x-ray spectroscopy
EDXF Energy-dispersive x-ray fluorescence
EELS Electron energy-loss spectroscopy
EPS Extracellular polymeric substance
FESEM Field emission scanning electron microscopy
FTIR Fourier transform infrared spectroscopy
GA-XRD Glancing angle x-ray diffraction
HADDF High-angle annular dark-field imaging
HRSEM High-resolution scanning electron microscopy
HRTEM High-resolution transmission electron microscopy
ICP Inductively coupled plasma
LED Light-emitting diode
MNPs Metal nanoparticles
NIR Near-infrared spectroscopy
NPs Nanoparticles
OER Oxygen evolution rate
PET Photosynthetic electron transport
μ-PIXE Proton-induced x-ray emission or microparticle-induced x-ray emission
POD Peroxidase
QELS Quasi-elastic light scattering
PS Photosystem
RuBisCo Ribulose-1,5-bisphosphate carboxylase/oxygenase
SAED (or SAD) Selected area electron diffraction
SEM Scanning electron microscopy
SEM–EDS Scanning electron microscope–energy-dispersive spectra
SPR Surface plasmon resonance
SOD Superoxide dismutase
STEM Scanning transmission electron microscopy
TEM Transmission electron microscopy
UV–Vis Ultraviolet–visible spectroscopy
XANES X-ray absorption near-edge structure
XAS X-ray absorption spectroscopy
XPS X-ray photoemission spectroscopy
XRD X-ray diffraction analysis
XRF X-ray fluorescence
μXRF Micro-x-ray fluorescence
Metal Nanoparticles in Plants 685
33.1 INTRODUCTION
Nanomaterials with a characteristic dimension in the range of 1–100 nanometers (nm) are at the lead-
ing edge of nanoscience and nanotechnology. In recent years, nanomaterials and specifically metal
nanoparticles (MNPs) have received particular interest in diverse fields ranging from material science
to biotechnology (Huang et al. 2007). Although widespread interest in nanomaterials is recent, the con-
cept was introduced over 40 years ago. Nanomaterials have actually been produced and used by humans
for hundreds of years: for example, the beautiful ruby red color of some glass is due to gold nanopar-
ticles (AuNPs) trapped in the glass matrix. In the decorative glaze known as luster, found on some
medieval pottery, the special optical properties of the glaze arose from metallic spherical nanoparticles
(NPs) that were dispersed in the glaze in a random fashion (Das and Marsili 2011). Michael Faraday in
1857 on his pioneering work “Experimental relations of gold (and other metals) to light” (Faraday 1857)
explained the properties of this glaze. Now with advances of science and technology, the morphology of
this material, which contains metallic NPs, has been understood. Because of extremely small size and
high surface–volume ratio of NPs, the physicochemical properties of NP-containing materials are quite
different to those of the bulk materials (El-Sayed 2001). Thus, nanomaterials have potential applications
in electronics, photonics, catalysis, information storage, chemical sensing, imaging, environmental
remediation, drug delivery, and biological labeling (Huang et al. 2007).
Over the past decade, growing interest and research investigations have been focused on MNPs
(as a one type of NPs) due to their unique physicochemical properties and potential applications in
selective catalysis, sensitive sensing, and enhanced imaging (Sun and Xia 2002). Recently, noble
MNPs have also attracted attention (Su et al. 2012) due to their optical property of surface plasmon
resonance (SPR). SPR produces hot light spots due to a stronger electromagnetic field. Because of
their low toxicity, ease of delivery, and strong and tunable SPR, noble MNPs are used in the field of
the photothermal ablation therapy to treat cancer (Zhang 2010).
It is well known that plants can help in reducing global warming by absorbing CO2 during the
process of photosynthesis. In addition, since they are known to interact with different metals, they
have been used for the “green biosynthesis” of MNPs. Such bioinspired methods are dependable,
environmentally friendly, and benign. In this procedure, not only vascular plants, but also algae,
bacteria, yeasts, fungi, and actinomycetes can be used (for details, see Masarovičová and Král’ová
2013). The use of plant extracts offers several advantages over their microbial counterparts. There
are no special requirements for plant maintenance; such processes are feasible on a large scale and
the synthetic processes are often rapid. Phyto-inspired synthetic procedures are thus popular and
this chapter describes literature on plant-mediated synthesis of MNPs.
33.2 GENERAL CHARACTERIZATION OF NANOPARTICLES

AND METAL NANOPARTICLES
NP—assemblies of hundreds to thousands of atoms and a size in the range of 1–50 nm—can be
considered at first approximation as a state of matter intermediate between single atoms or mol-
ecules and bulk bodies. While the properties of atoms and molecules can be described via quantum
mechanics, the properties of bulk bodies are described by solid-state physics. The use of quan-
tum chemical models for describing NPs is demanding as a huge number of strongly interacting
atoms have to be taken into account. On the other hand, methods of solid-state physics that cannot
always be applied as NPs often demonstrate size-dependent quantum effects, for example, in SPR.
Magnetic, thermodynamic, catalytic, and other properties of NPs can also depend on their size.
Due to their small size, NPs have a very high specific surface area and dispersion, the latter being
defined as the ratio of the number of surface atoms to the total number of atoms in the particle
(Kraynov and Müller 2011). Besides size and shape, the properties of NPs are also greatly affected
by their crystal structure; thus, it is possible to tune the properties of the particle by controlling its
growth process.
Toward shape-controlled NP synthesis, molecular capping agents such as organic surfactants and
polymers have been used to direct nanocrystal growth in a face-selective fashion (Xia et al. 2009).
Despite tremendous progress, the mechanism of the shape control is not well understood, in part
due to the difficulty in defining structures and conformations of these surfactants and polymers
in solution and in systematic variation of functional groups (e.g., Wang et al. 2010). It should be
stressed that variation in reaction conditions (e.g., different pH, temperature, and time intervals,
respectively) can markedly affect NP synthesis (for details, see Tables 33.1 through 33.3).
33.3 EXPERIMENTAL METHODS APPLIED FOR CHARACTERIZATION

OF METAL NANOPARTICLES
Formation and characterization of MNPs can be monitored by several experimental techniques, such
as ultraviolet–visible (UV–Vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR), scan-
ning electron microscopy (SEM), transmission electron microscopy (TEM), high-resolution TEM
(HRTEM) and scanning TEM (STEM), atomic force microscopy (AFM), x-ray diffraction (XRD),
x-ray absorption near-edge structure (XANES) and extended x-ray absorption fine structure (EXAFS),
and x-ray photoelectron spectroscopy (XPS) (cf. Sareen 2001, Hammer 2008, Ghatak 2011).
33.3.1 Ultraviolet–Visible Spectroscopy
UV–Vis spectroscopy is a technique used to quantify the light that is absorbed and scattered by
a sample. Gold and silver plasmonic NPs have optical properties that are sensitive to size, shape,
concentration, agglomeration state, and refractive index near the NP surface, which makes UV–Vis
spectroscopy a valuable tool for identifying, characterizing, and studying nanomaterials. UV–Vis
spectrum, known as the surface plasmon absorption band, is produced by the movement of the con-
duction electrons in the particles as a consequence of the incident electric field light, which results
in a displacement of the negative and positive charges in the metal (Slistan-Grijalva et al. 2005).
When silver nanoparticles (AgNPs) aggregate, the metal particles become electronically coupled
and this coupled system has a different SPR than the individual particles. For the case of a multi-NP
aggregate, the plasmon resonance will be red-shifted to a longer wavelength than the resonance of
an individual NP, and aggregation is observable as an intensity increase in the red/infrared region
of the spectrum (Ghosh and Pal 2007).
33.3.2 Fourier Transform Infrared Spectroscopy

In FTIR, a spectrum showing molecular vibrations is obtained. It is used to identify or characterize
mainly organic material. FTIR analysis results in absorption spectra that provide information about
the chemical bonds and molecular structure of a material. The FTIR spectrum is equivalent to the
“fingerprint” of the material and can be compared with catalogued FTIR spectra to identify the
material (Griffiths and de Hasseth 2007). FTIR spectroscopy analysis is suitable to identify
the biomolecules responsible for the reduction of metal ions and capping of the bioreduced AgNPs
synthesized by using plant extract (e.g., Jain et al. 2009, Zayed et al. 2012).
From FTIR absorption spectra of water-soluble plant extract before and after reduction of metal
ions and from changes in the absorbances of absorbance bands that are known to be associated with
the stretching vibrations for certain groups, it could be concluded which compounds are mainly
responsible for the reduction of metal ions. For example, Jain et al. (2009), who used papaya fruit
extract for bioreduction of Ag+ ions to prepare AgNPs, observed before reduction absorbance bands
at 1697, 1618, 1514, 1332, and 1226 cm−1. These absorbance bands are known to be associated
with the stretching vibrations for −C C−C O, −C C− [(in-ring) aromatic], −C−C− [(in-ring)
aromatic], C−O (esters, ethers), and C−O (polyols), respectively. In particular, the 1226 cm−1 band
arose most probably from the C−O group of polyols such as hydroxyflavones and catechins, and the
TABLE 33.1
Summary of the Plant Species Used for the Preparation of Au NPs, Their Important
Characteristics, and Experimental Techniques Applied for Characterization
Plant Species Size of NPs Morphology of NPs Methods References
Achillea wilhelmsii, 70 nm Spherical UV–Vis, SEM, FTIR Andeani et al.
extract from dried (2011)
flowers
Adhatoda vasica, leaf 10–20 nm Spherical UV–Vis, XRD, Pandey et al.
extract HRTEM (2012d)
A. vera, leaf extract 15.2 nm Triangles UV–Vis–NIR, TEM Chandran et al.
(2006)
Aloe barbadensis, leaf 10–60 nm Hexagonal and triangles UV–Vis, TEM, XRD Pandey et al.
extract (at lower pH and (2012c)
temperatures)
spherical shape (at
100°C, pH 10)
Anacardium occidentale, 6.5 and 17 nm Spherical UV–Vis, FTIR, XRD, Sheny et al.
dried leaves extract HRTEM, SAED (2011)
A. occidentale, essential 36 nm Hexagonal UV–Vis, TEM, FTIR Sheny, et al.
oils extracted from fresh monodispersed (2012)
leaves
Asparagus racemosus, leaf 10–50 nm (30°C, Mostly spherical; some UV–Vis, FTIR, SEM, Pandey et al.
extract pH 8) triangular, oval, and XRD, HRTEM (2012b)
hexagonal (pH 2 at
100°C)
Azadirachta indica, leaf Not specified Mostly triangular, very UV–Vis, XRD, FTIR, Shankar et al.
broth few hexagonal shaped, TEM, EDAX (2004)
spherical
Azadirachta indica, fruit ∼70 nm Different shapes and sizes UV–Vis, TEM, XRD D’Britto et al.
leaf and bark; (2012)
Cephalandra indica,
leaves and fruits;
C. procera, leaves;
Syzygium jambolanum,
fruit seeds
Banana, peel extract ∼300 nm Triangles and hexagons; DLS, SEM–EDS, Bankar et al.
in air-dried drops, they XRD, FTIR (2010c)
formed micronetworks
and dendrite-like
structures
Barbated Skullcup, herb 10–100 nm Spherical and triangular. UV–Vis, TEM, EDX Wang et al.
extract (2009)
Benincasa hispida, seed 15 and 24 nm Spherical UV–Vis, TEM, Aromal and
extract HRTEM, XRD, Philip
SAED, FTIR (2012b)
Black tea, leaf extract ∼20 nm Mostly spherical, some UV–Vis, TEM, FTIR Begum et al.
prisms (2009)
Callistemon viminalis, leaf ∼90 nm Triangles to spherical UV–Vis, XRD, TEM, Kumar et al.
extract with increasing content QELS (2011b)
of extract
(continued)

Carthamus tinctorius L., 40–200 nm Spherical triangles UV–Vis, TEM Nagaraj et al.
flower extract 40–60 nm (2012)
∼200 nm
Cassia fistula, stem bark 55.2–98.4 nm Polydispersed rectangular UV–Vis, FTIR, SEM Daisy and
extract and triangular Saipriya
(2012)
Centella asiatica, leaf 2–22 nm Mostly spherical, some UV–Vis, TEM, XRD, Das et al.
extract (average triangular and hexagonal HRTEM, SAED, (2010)
9.3 nm) FTIR
Chenopodium album, leaf 10–30 nm Mostly spherical, some UV–Vis, TEM, XRD, Dwivedi and
extract triangular EDX, FTIR Gopal (2010)
C. zeylanicum, leaf broth 25 nm Mostly spherical and UV–Vis, TEM, XRD, Smitha et al.
some nanoprisms FTIR, (2009)
photoluminescence
study
Coleus amboinicus Lour., 20.5 ± 11.45 nm Spherical and anisotropic UV–Vis, XRD, TEM, Narayanan and
leaf extract nanostructures SEM, SAED, FTIR Sakthivel
(nanotriangles) (2010)
Coriander, leaf extract 6.75–57.91 nm Mostly spherical, UV–Vis, XRD, Narayanan and
(average: triangle, truncated EDAX, FTIR, TEM Sakthivel
20.65 ± 7.09 nm) triangles, and decahedral (2008)
Crocus sativus (saffron), 11–20 nm Mostly spherical, UV–Vis, SEM, Vijayakumar
plant extract (average occasionally triangular HRTEM, XRD, et al. (2011)
∼15 nm) FTIR
Cypress, leaf extract 5–94 nm Mostly spherical, some UV–Vis, XRD, EDX, Noruzi et al.
irregular shapes FTIR, ICP–AES, (2012)
TEM
Dalbergia sissoo, leaf 50–80 nm Mostly spherical, few UV–Vis, FTIR, TEM, Singh et al.
extract triangular and hexagonal XRD (2012)
D. kaki, leaf extract 300 nm Triangles, pentagons, ICP, EDS, SEM, Song et al.
hexagons plate TEM, AFM, FTIR (2009)
structures; most plates
disappeared at 95°C
Dioscorea bulbifera, tuber 11–30 nm Nanotriangles, UV–Vis, FESEM, Ghosh et al.
extract nanoprisms, DLS, EDX, XRD (2011)
nanotrapezoid, and
spheres
Eucalyptus camaldulensis, 1.25–17.5 nm Not specified UV–Vis, TEM, EDS Ramezani
methanol extract (average et al. (2008)
5.5 nm)
Gnidia glauca, flower ∼10 nm also NPs Mostly spherical, few UV–Vis, TEM, Ghosh et al.
extract between 50 and triangles and HRTEM, SEM, EDS, (2012)
150 nm nanohexagons XRD
Helianthus annuus, flower 30–50 nm Round, some having UV–Vis, TEM Liny et al.
extract (average 35 nm) penta- or octahedral (2012)
200 nm edges nanotriangles

Hibiscus rosa-sinensis, ∼14 nm Large number of UV–Vis, TEM, XRD, Philip (2010b)
leaf extract anisotropic particles, FTIR
e.g., triangular, with
smaller ones being
multi-branched (10 mL
extract) triangular,
hexagonal, dodecahedral,
and spherical shapes (20
mL extract) almost
spherical (30 mL extract)
Lonicera japonica, flower 2–100 nm Circular, polydispersed UV–Vis, FTIR, XRD, Nagajyothi
extract (average EDAX, SEM, et al. (2012)
8.02 nm) HRTEM
Macrotyloma uniflorum, 14–17 nm Spherical UV–Vis, TEM, Aromal et al.
plant extract HRTEM, XRD, (2012)
SAED, FTIR
Magnolia kobus, leaf 110 nm at 25°C Triangles, pentagons, ICP, EDS, SEM, Song et al.
extract hexagons, and spherical TEM, AFM, FTIR (2009)
40 nm at 95°C at 25°C and 60°C;
mostly spherical at 95°C
Mangifera indica, leaf ∼20 and 17 nm Spherical UV–Vis, TEM, XRD, Philip (2010a)
extract SAED, FTIR
Memecylon edule, leaf 20–50 nm Spherical nanospindle- UV–Vis, SEM, TEM, Elavazhagan
extract (average shaped, triangular, EDAX, FTIR and
25.2 nm) circular, hexagonal, Arunachalam
55–80 nm and spherical (2011)
Mentha piperita, leaf 150 nm Spherical UV–Vis, FTIR, MubarakAli,
extract SEM–EDS et al. (2011)
Momordica charantia, 10–100 nm Roughly spherical UV–Vis, XRD, TEM Pandey et al.
fruit peel extract (pH 10, 100°C) (2012a)
30–100 nm
(pH 10, 60°C)
Murraya koenigii, leaf ∼20 nm Nearly spherical UV–Vis, TEM, Philip et al.
extract SAED, XRD, FTIR (2011)
Ocimum sanctum, leaf ∼30 nm Hexagonal UV–Vis, TEM, XRD, Philip and
extract FTIR Unni (2011)
Panax ginseng C.A. 2–40 nm (average Spherical (without appl. UV–Vis, TEM Leonard et al.
Meyer, aqueous 16.2 nm) of NaBH4) (2011)
suspension of red 3–40 nm (average Spherical (with appl. of
ginseng root 20.1 nm) NaBH4)
Pear, fruit extract edge lengths of Triangular and hexagonal TEM, AFM, XRD, Ghodake et al.
the nanoplates assembled XPS, EDAX (2010)
nanostructures with hexagonal AuNPs
200–500 nm;
nanohexagons
thickness
12–20 nm
(continued)

Pelargonium roseum, 2.5–27.5 nm Not specified UV–Vis, TEM, EDS Ramezani
methanol extract (average et al. (2008)
7.5 nm)
Phyllanthus amarus 65–99 nm Spherical and cubic UV–Vis, SEM, XRD, Annamalai
Schum. and Thonn, leaf EDX, AFM, FTIR et al. (2011)
extract
Psidium guajava, leaf 27 ± 3 nm Mostly spherical UV–Vis, XRD, Raghunandan
extract FESEM, TEM, AFM, et al. (2009)
FTIR
Putranjiva roxburghii 24–38 nm Spherical UV–Vis, SEM, Badole and
Wall., leaf extract EDAX, XRD Dighe (2012)
Rosa × damascena, flower 10–30 nm Quasi-spherical UV–Vis, TEM, XRD, Ghoreishi et al.
extract (average FTIR (2011)
15.3 nm)
Rosa hybrida, petal extract 10 nm Face-centered cubic (fcc) UV–Vis, FTIR, XRD, Noruzi et al.
structure EDX, TEM, DLS (2011)
Rosa rugosa, leaf extract 11 nm Mostly spherical, some UV–Vis, TEM, XRD, Dubey et al.
triangular and hexagonal FTIR, EDX (2010c)
Sargassum wightii 8–12 nm Mostly thin planar UV–Vis, TEM, XRD Singaravelu
Greville, (marine alga) structures few spherical et al. (2007)
Sorbus aucuparia, leaf Average: 18 nm Spherical, triangular, and UV–Vis, TEM, XRD, Dubey et al.
extract hexagonal EDX, FTIR (2010a)
Sphaeranthus 39.1–46.4 nm Mostly spherical UV–Vis, HRTEM, Nellore et al.
amaranthoides, leaf EDX, FTIR (2012)
extract
Sugar beet pulp 10 nm (pH 9) Spherical UV–Vis, TEM, FTIR, Castro et al.
25 nm (pH 10) Nanorods SAED (2011)
15 nm (pH 11) Nanowires
Swietenia mahogani Not specified Spheroids, triangles, UV–Vis, TEM, FTIR Mondal et al.
JACQ, leaf extract and hexagons (2011)
Tagetes erecta, flower 8–10 nm > 200 nm Spherical nanotriangles UV–Vis, TEM Krishnamurthy
extract et al. (2012)
Tanacetum vulgare, fruit 11 nm Spherical and triangular UV–Vis, TEM, XRD, Dubey et al.
extract EDX, FTIR (2010b)
Terminalia catappa, leaf 10–35 nm Predominantly spherical UV–Vis, XRD, FTIR, Ankamwar
extract (average TEM (2010)
21.9 nm)
Trigonella foenum- 15–25 nm Nearly spherical TEM, XRD, FTIR, Aromal and
graecum, seed extract HRTEM, SAED Philip
(2012a)
Zingiber officinale, 5–15 nm Spherical UV–Vis, DLS, TEM Kumar, et al.
rhizome extract (2011a)
TABLE 33.2
Summary of the Plant Species Used for the Preparation of Ag NPs, Their Important
Plant Species
and Part Size of NPs Morphology of NPs Methods References
Acalypha indica, leaf 20–30 nm Spherical UV–Vis, SEM, Krishnaraj et al.
extract HRTEM, XRD, EDS (2010)
A. indica Linn., leaf 2–50 nm Almost spherical to cubical HRTEM, XRD Krishnaraj et al.
extract (average (2012)
24 nm)
Allium sativum, 7.3 ± 4.4 nm Polydispersed in nature, UV–Vis, TEM, XRD, Rastogi and
aqueous extract spherical in shape FTIR Arunachalam
(2011)
A. vera, leaf extract 15.2 nm Spherical UV–Vis –NIR, TEM Chandran et al.
(2006)
A. occidentale, leaf 15.5 nm Spherical UV–Vis, FTIR, XRD, Sheny et al.
extract HRTEM, SAED (2011)
Andrographis 28 nm Cubic and hexagonal UV–Vis, XRD, SEM, Sulochana et al.
paniculata, leaf extract FTIR (2012)
A. squamosa, aqueous 35 ± 5 nm Irregularly spherical in UV–Vis, TEM, XRD Kumar et al.
peel extract shape (2012)
Arbutus unedo, leaf 3 and 20 nm Predominately spherical UV–Vis, HRTEM, Kouvaris et al.
extract morphology, often SEAD (2012)
agglomerated into small
aggregates of 5–6 particles
Aristolochia bracteata, 3.26–10.31 nm Not specified UV–Vis, XRD, FTIR Vijaya Raj et al.
leaf extract (average (2012)
7.2 nm)
A. indica, leaf broth 5–35 nm Predominantly spherical UV–Vis, XRD, FTIR, Shankar et al.
TEM, EDAX (2004)
A. indica, leaf extract 10–37 nm Mainly spherical, also UV–Vis, TEM, SAED Khan et al. (2012)
triangle, flat, platelike
hexagonal, and some
irregular morphology in
presence of surfactant
A. indica, leaf extract 10–100 nm Not specified UV–Vis, SEM, EDS Renugadevi and
Aswini (2012)
Azardirecta indica, ∼70 nm Different shapes and sizes UV–Vis, TEM, XRD D’Britto et al.
fruit leaf and bark; (2012)
C. indica, leaves and
fruits; C. procera,
leaves;
S. jambolanum, fruit
seeds
Black tea, leaf extract ∼20 nm Mostly spheroidal, few with UV–Vis, TEM, FTIR Begum et al.
a pronounced anisotropic (2009)
morphology (nanoprisms
and nanorods)
Boswellia serrata, 7.5 ± 3.8 nm Spherical UV–Vis, TEM, XRD, Kora et al. (2012)
gum olibanum Raman spectroscopy,
extract FTIR
(continued)

Plant Species
Callicarpa maingayi, 12.40 ± Spherical UV–Vis, XRD, TEM, Shameli et al.
stem bark extract 3.27 nm SEM, EDXF, FTIR (2012)
Calotropis gigantea, 6.3–12.67 nm Spherical UV–Vis, AFM Baskaralingam
leaf extract et al. (2012)
C. procera, flower 35 nm Cubical shape UV–Vis, SEM, EDX, Babu and Prabu
extract FTIR (2011)
Camellia sinensis, leaf 2–100 nm Spherical UV–Vis, XRD, TEM, Loo et al. (2012)
extract (average FTIR
4.06 nm)
Cardiospermum 5–50 nm Skew spherical in shape UV–Vis, SEM, XRD Vishnudas et al.
halicacabum L., leaf (2012)
extract
C. fistula, leaf broth Diameters Nanowires UV–Vis, SEM, TEM, Lin et al. (2010)
50–60 nm and SAED, HRTEM,
length up to XRD, FTIR
tens of
micrometers
Catharanthus roseus, 48–67 nm Spherical UV–Vis, SEM, XRD, Mukunthan et al.
leaf extract EDX (2011)
C. album, leaf extract 10–30 nm Mostly spherical some UV–Vis, TEM, XRD, Dwivedi and
triangular EDX, FTIR Gopal (2010)
Cinnamon zeylanicum, 31 and 40 nm Quasi-spherical and small, UV–Vis, TEM, XRD, Sathishkumar
bark extract rod-shaped EDX et al. (2009b)
Cissus 42.46 nm Spherical and oval UV–Vis, XRD, FTIR, Santhoshkumar
quadrangularis, stem FESEM, EDX et al. (2012)
extract
C. quadrangularis, 5–30 nm Spherical, polydispersed UV–Vis, TEM, EDAX Renugadevi et al.
plant extract (2012)
Citrullus colocynthis 31 nm Spherical UV–Vis, FTIR, AFM Satyavani et al.
(L.) Schrader, leaf (2011)
extract
Citrus × limon Below 50 nm Nearly spherical UV–Vis, XRD, FTIR, Prathna et al.
(lemon), aqueous AFM, TEM (2011)
extract
Citrus sinensis, peel 35 nm at 25°C Spherical UV–Vis, FTIR, XRD, Kaviya et al.
extract 10 nm at 60°C EDAX, FESEM, TEM (2011)
Cleome viscosa, leaf 7–50 nm Mostly spherical and FTIR, XRD, TEM, Yamini
extract occasionally triangular SEM SudhaLakshmi
et al. (2011)
Coccinia grandis, leaf 20–30 nm Monodisperse spherical UV–Vis, XRD, SEM, Arunachalam
extract EDS, FTIR, HRTEM et al.(2012)

Plant Species
C. amboinicus Lour., 2.6–79.8 nm Anisotropic nanostructures UV–Vis, XRD, EDAX, Narayanan and
leaf extract (average of triangles, truncated TEM, FTIR Sakthivel
35.8 nm) triangles, decahedral, (2011a)
4.9–55 nm and little spherical
(average morphologies (0.5 mL
30.6 nm) extract); more spherical and
4.3–40.4 nm little anisotropic structures
(average of triangles, truncated
17.6 nm) triangles, and decahedral
(2 mL extract);
predominant isotropic
spherical NPs
(4 mL extract)
Coleus aromaticus, 40–50 nm Spherical UV–Vis, XRD, SEM, Vanaja and
leaf extract EDEX, FTIR Annadurai
(2012)
Costus igneus, leaf ∼20 nm Predominantly spherical TEM, XRD Sataraddi and
extract Nandibewoor
(2012)
Crossandra 38 nm Flake-like UV–Vis, FTIR, XRD, Kaviya et al.
infundibuliformis, FESEM, EDAX (2012)
leaf extract
D. sissoo, leaf extract 50–80 nm Spherical, few triangular UV–Vis, FTIR, TEM, Singh et al.
and hexagonal polyshaped XRD (2012)
Desmodium triflorum, 5–20 nm Predominantly spherical, UV–Vis, TEM, XRD Ahmad et al.
plant extract some oval and/or elliptical (2011)
Dioscorea batatas, Not specified Monodispersed, having UV–Vis, SEM, FTIR, Nagajyothi and
rhizome extract circular and flower shapes, XRD, EDX Lee (2011)
respectively, at 80°C
and 25°C
Eclipta, leaves 2–6 nm Almost spherical UV–Vis Jha et al. (2009)
E. prostrata, leaf 35–60 nm Spherical with a small UV–Vis, SEM, TEM, Rajakumar and
extract (average percentage of elongated XRD, FTIR Rahuman
45 nm) particles (2011b)
Elettaria 40–70 nm Spherical UV–Vis, XRD, GnanaJobitha
cardamomum, seed SEM–EDX, FTIR et al. (2012)
extract
Euphorbia hirta L., 40–50 nm Relatively spherical UV–Vis, SEM Elumalai et al.
leaf extract (2010)
Euphorbia milii, latex ∼20 nm Spherical UV–Vis, TEM de Matos et al.
(2011)
Euphorbia prostrata, 25–80 nm Rod in shape UV–Vis, XRD, FTIR, Zahir et al. (2012)
leaf extract (average SEM
52.4 nm)
Ficus benghalensis, 16 nm Monodispersed and UV–Vis, TEM–EDS, Saxena et al.
leaf extract spherical XRD (2012)
(continued)

Plant Species
Garcinia mangostana, 6–57 nm Spherical UV–Vis, TEM, FTIR Veerasamy et al.
leaf extract (average (2011)
35 nm)
Gliricidia sepium, leaf 10–50 nm Predominantly spherical UV–Vis, TEM, XRD, Rajesh et al.
broth (average FTIR (2009)
27 nm)
Glycyrrhiza glabra, 20–30 nm Nearly spherical UV–Vis, SEM, XRD, Dinesh et al.
root extract EDX, FTIR, TEM (2012)
Hevea brasiliensis, 2–100 nm Spherical UV–Vis, TEM, SAED, Guidelli et al.
natural rubber latex FTIR (2011)
Hibiscus cannabinus, 9 nm Spherical UV–Vis, FTIR, XRD, Bindhu and
leaf extract TEM Umadevi (2012)
H. rosa-sinensis, leaf ∼13 nm Nearly spherical (pH 7.5) UV–Vis, TEM, XRD, Philip (2010b)
extract FTIR
Ipomea carnea, leaf 25 and 150 nm Not specified UV–Vis, dynamic light Kiruba Daniel
extract scattering, AFM, et al. (2012)
TEM
Iresine herbstii, leaf 44–64 nm Cubic UV–Vis, SEM, EDX, Dipankar and
extract XRD, FTIR Murugan (2012)
J. curcas, latex 10–55 nm Spherical UV–Vis, HRTEM, Bar et al. (2009b)
20–40 nm Uneven shapes XRD, FTIR
49 nm
J. curcas, seed extract 15–50 nm Mostly spherical UV–Vis, HRTEM, Bar et al. (2009a)
XRD, FTIR
Lantana camara, fruit 12.55– Spherical UV–Vis, FTIR, TEM Sivakumar et al.
extract 12.99 nm (2012)
Lawsonia inermis, leaf 59.52 nm Spherical UV–Vis, XRD, FTIR, Marimuthu et al.
extract SEM, EDX (2012)
Lippia citriodora, leaf 15–30 nm Spherical UV–Vis, TEM, EDX, Cruz et al. (2010)
extract XRD, FTIR
L. japonica, flower 2–100 nm Circular, polydispersed UV–Vis, FTIR, XRD, Nagajyothi et al.
extract (average EDAX, SEM, (2012)
7.8 nm) HRTEM
Malva parviflora, leaf 19–25 nm Monophasic cubic XRD, FTIR Zayed et al.
extract (2012)
M. indica, leaf extract 20–30 nm Spherical and a few UV–Vis, TEM, XRD, Philip (2011)
rod-shaped FTIR
Mangrove plant, leaf 4–26 nm, Spherical UV–Vis, XRD, FTIR, Umashankari
bud extract mainly 4 nm HRTEM et al. (2012)
Manilkara zapota, leaf 70–140 nm Spherical and oval UV–Vis, FTIR, XRD Kamaraj et al.
extract (2012)
M. zapota, leaf extract 70–140 nm Spherical and oval UV–Vis, SEM, EDX, Rajakumar and
FTIR, XRD Rahuman
(2011a)

Plant Species
M. sativa, seed 5–51 nm Spherical or flowerlike TEM, SAED, XRD, Lukman et al.
exudates (30°C) particle clusters (at higher AFM, XPS, SEM (2011)
(average Ag concentrations),
104 nm, edge nanoplates, hexagonal
lengths of particles, and
86–108 nm) nanotriangles (predilution
12 nm of the exudate);
monodisperse NPs (pH 11)
Mentha piperita, leaf 7–50 nm Particle of small size: UV–Vis, TEM Parashar et al.
extract homogeneously spherical (2009)
Mentha piperita, plant 90 nm Spherical UV– Vis, FTIR, MubarakAli et al.
extract, SEM–EDS (2011)
M. edule, leaf extract 50–90 nm Predominantly square, also UV–Vis, SEM, TEM, Elavazhagan and
triangular EDAX, FTIR Arunachalam
(2011)
Mollugo nudicaulis, 8.3–10.2 nm Not specified UV–Vis, FTIR, XRD Anarkali et al.
entire plant extracts (average (2012)
9.3 nm)
Morinda citrifolia, 10–60 nm Spherical UV–Vis, FTIR, Sathishkumar
leaf extract (average size HRTEM, SEAD, et al. (2012)
29 nm EDAX
[HRTEM] or
27 nm [SEM])
Moringa oleifera, leaf 57 nm Spherical UV–Vis, TEM Prasad and
extract Elumalai (2011)
Mulberry, leaf extract 20–40 nm Cubical UV–Vis, SEM, XRD Awwad and
Salem (2012)
M. koenigii, leaf ∼10 nm Spherical UV–Vis, TEM, SAED, Philip et al.
extract XRD, FTIR (2011)
M. koenigii, leaf 20–40 nm Formation of various Ag UV–Vis, FTIR, Ankamwar et al.
extract (average nanostructures that include HRTEM, XRD (2012)
diameter “nanobuns” and also
30 nm) spherical NPs with defects
on the surface
Myrica esculenta, leaf 45–80 nm Different morphologies UV–Vis, XRD, TEM Phanjom et al.
extract (average (2012)
55 nm)
N. tobaccum, leaf 8 nm Not specified UV–Vis, TEM, EDAX, Prasad et al.
extract FTIR, SEAD, FTIR, (2011a)
photoluminescence
O. sanctum, leaf 10–20 nm Nearly spherical UV–Vis, TEM, XRD, Philip and Unni
extract FTIR (2011)
O. sanctum, plant 10 ± 2 nm (root) Spherical UV–Vis, XRD, TEM Ahmad et al.
extract 5 ± 1.5 nm (stem) (2010)
(continued)

Plant Species
Ocimum tenuiflorum, 25–40 nm Spherical UV–Vis, TEM, SAED Rupali et al.
leaf extract (2012)
Opuntia ficus, aqueous 8–50 nm Spheroids UV–Vis, TEM Silva-de-Hoyos
extract (average et al. (2012)
23 nm)
Padina 10–100 nm Predominantly spherical UV–Vis, XRD, SEM, Jegadeeswaran
tetrastromatica, leaf (average EDX, TEM et al. (2012)
extract 20 nm)
Papaver somniferum, 3.2 and 7.6 μm Almost spherical UV–Vis, HRSEM, Vijayaraghavan
seed extract EDAX et al. (2012a)
Papaya, fruit extract 15 nm Cubic structure UV–Vis, FTIR, XRD, Jain et al. (2009)
SEM, XRD
Parthenium, leaf ∼50 nm Assembled in very irregular UV–Vis, TEM Prashar et al.
extract shape of variable (2009)
morphology
P. amarus Schum. and 43–53 nm Spherical and cubic UV–Vis, SEM, XRD, Annamalai et al.
Thonn, leaf extract EDX, AFM, FTIR (2011)
Phytolacca, 91 nm Spherical with smooth DLS, AFM, UV–Vis, Bhattacharyya
homeopathic mother surface CD et al. (2012)
tincture
Piper betle L., leaf 17 ± 12 nm Spherical shape (80 min TEM, SAED, XRD, Rani and
extract light exposure) FTIR. XPS, Rajasekharreddy
fluorescence emission (2011)
spectra
Piper longum, leaf 17.6–41 nm Spherical UV–Vis, FTIR, SEM Jacob et al.
extract (2012)
Piper nigrum, extract 20–50 nm Spherical UV–Vis, XRD, TEM Shukla et al.
(2010)
P. nigrum, fruit extract 40–60 nm Predominantly spherical UV–Vis, SEM Mani et al. (2012)
Prosopis chilensis, 5–25 nm Mostly spherical XRD, TEM, FTIR Kandasamy et al.
plant extract (average 11.3 ± (2013)
2.1 nm)
P. guajava, leaf extract 59 nm Spherical UV–Vis, TEM Prasad et al.
(2011b)
Pongamia pinnata, 24–55 nm Spherical UV–Vis, FTIR, TEM, Panda et al.
Hemidesmus indicus, (average 37 ± EDS (2011)
Syzygium cumini, A. 11 nm)
cepa, leaf extracts
Rhizophora apiculata, 19–42 nm Spherical UV–Vis, FTIR, TEM Antony et al.
aqueous extract (2011)
Riccia, extract of 20–50 nm Cuboidal and triangular UV–Vis, SEM, EDS Kulkarni et al.
green mature thalli (2012)
R. damascena, flower 10–30 nm Almost quasi-spherical UV–Vis, TEM, XRD, Ghoreishi et al.
extract (average size FTIR (2011)
18.3 nm)

Plant Species
R. rugosa, leaf extract 12 nm Mostly spherical UV–Vis, TEM, XRD, Dubey et al.
FTIR, EDX (2010c)
Saururus chinensis, 38 nm Not specified UV–Vis, XRD, FTIR, Nagajyothi et al.
leaf extract (immediately) SEM with EDX (2011)
54 nm (after
1 day)
Sesuvium 5–20 nm Spherical XRD, FTIR, TEM Nabikhan et al.
portulacastrum L., (2010)
leaf extract
Solanum torvum, leaf 14 nm Spherical UV–Vis, FTIR, XRD, Govindaraju et al.
extract HRTEM (2010)
S. aucuparia, leaf 16 nm Spherical, triangular, UV–Vis, TEM, XRD, Dubey et al.
extract and hexagonal EDX, FTIR (2010a)
Stevia rebaudiana, 2–50 nm Spherical UV–Vis, TEM, XRD, Yilmaz et al.
leaf extract FTIR (2011)
Suaeda monoica, leaf 31 nm Spherical UV–Vis, AFM Satyavani et al.
extract (2012)
S. mahogani JACQ., — Mostly spheroidal, a few UV–Vis, TEM, FTIR Mondal et al.
leaf extract with definite anisotropic (2011)
morphology, forming
small aggregates
Switchgrass (Panicum 20–40 nm Spherical, rodlike, UV–Vis, XRD, TEM Mason et al.
virgatum), plant triangular, pentagonal, (2012)
extract and hexagonal
Syzygium aromaticum, 20–149 nm Almost spherical UV–Vis, SEM, EDAX Vijayaraghavan
extract from dried et al. (2012b)
buds
T. vulgare, fruit extract 16 nm Spherical and triangular UV–Vis, TEM, XRD, Dubey et al.
EDX, FTIR (2010b)
Trachyspermum ammi, 87–998 nm Triangular UV–Vis, HRSEM, Vijayaraghavan
seed extract EDAX et al. (2012a)
Tribulus terrestris L., 16–28 nm Spherical TEM, AFM, XRD, Gopinath et al.
dried fruit body FTIR, UV–Vis (2012)
extract
Tridax procumbens, 55 nm Not specified UV–Vis, SEM Dhanalakshmi
leaf extract and Rajendran
(2012)
Withania somnifera, 5–40 nm Spherical UV–Vis, FTIR, SEM, Nagati et al.
leaf extract TEM (2012)
Wrightia tinctoria, 19–68 nm Not specified UV–Vis, XRD, FTIR Bharani et al.
leaf extract (2012)
TABLE 33.3
Summary of the Plant Species Used for the Preparation of Other MNPs, Their Important
Plant Species Metal Size of NPs Morphology of NPs Methods References
A. barbadensis ZnO 25–40 nm Predominantly UV–Vis, FTIR, Sangeetha et al.
miller, leaf extract spherical photoluminescence, (2011)
SEM, TEM and XRD
Alpinia purpurata, ZnO ∼50 nm Not specified SEM Aswathi
rhizome extract Sreenivasan
et al. (2012)
A. squamosa, peal TiO2 23 ± 2 nm Spherical UV–Vis, XRD, SEM, Roopan et al.
extract TEM, EDS (2012b)
Catharanthus TiO2 25–110 nm Clustered and XRD, FTIR, SEM, Velayutham
roseus, leaf extract (average irregular, mostly AFM et al. (2012)
65 nm) aggregated
E. prostrata, leaf TiO2 36–68 nm Spherical clusters, FTIR, XRD, AFM and Rajakumar
extract (average quite polydisperse FESEM analysis, FTIR et al. (2012)
49.5 nm)
J. curcas L., latex TiO2 25–59 nm Spherical uneven XRD, SAED, TEM, Hudlikar et al.
extract 50–100 nm shape EDAX, FTIR (2012)
C. zeylanicum, bark Pt 25–30 nm Nano-sized Pt TEM, XRD Krishnamurthy
extract particles/aggregates et al. (2009)
C. longa, tuber Pt 25–30 nm Nano-sized Pt TEM, XRD Krishnamurthy
extracts particles/aggregates et al. (2009)
D. kaki, leaf extract Pt 12 nm (25°C) Spheres and plates UV–Vis, TEM, HRTEM, Song et al.
5 nm (95°C) EDS, XPS, FTIR (2010)
M. sativa, root Pt 3 and 100 nm Spherical TEM, μ-PIXE Bali et al.
(2010)
A. squamosa L., Pd 100 nm Spherical UV–Vis, XRD, TEM Roopan et al.
aqueous peel (2012a)
extract
Banana, peel extract Pd ∼50 nm On air drying, NPs in UV–Vis, SEM–EDS, Bankar et al.
the center and XRD, FTIR, DLS (2010b)
aggregates toward
the periphery as
dendrites
C. zeylanicum, bark Pd 15–20 nm Spherical TEM, EDX, XRD Sathishkuma
extract et al. (2009a)
Soybean (Glycine Pd ∼15 nm Spherical UV–Vis, FTIR, XRD, Petla et al.
max), leaf extract TEM (2012)
Green tea, leaf Fe 40 and 60 nm Irregular clusters TEM, SEM, EDX, XPS, Shahwan et al.
extract XRD, FTIR (2011)
C. procera L., latex Cu 15 ± 1.7 nm Spherical TEM, XRD, EDAX, Harne et al.
aqueous extract FTIR (2012)
A. barbadensis CuO 15–30 nm Versatile and UV–Vis, FTIR, XRD, Gunalan et al.
Miller, leaf extract spherical SEM, TEM, (2012)
photoluminescence
A. cepa, root Co 60 ± 10 nm Spherical, truncated, TEM, XRD Ghodake et al.
and uneven NPs (2011)
Grape, skin extract Pb 661–796 nm Spherical UV–Vis, SEM Pavani et al.
(2012)

Summary of the Plant Species Used for the Preparation of Other MNPs, Their Important
Plant Species Metal Size of NPs Morphology of NPs Methods References
Arachis hypogaea, Cr2O3 60–80 nm Hexagonal and cubic XRD, SEM, UV–Vis, Ramesh et al.
leaf extract with rough surfaces FTIR (2012)
A. wilhelmsii, flower CdO 35 nm Spherical and SEM, FTIR, UV–Vis, Andeani and
extract irregular FESEM Mohsenzadeh
(2013)
Aqueous suspension Ti/Ni 1–4 nm Structure based on an TEM, HRTEM, EELS, Schabes-
of powdered fcc-like geometry HADDF in STEM Retchkiman
milled alfalfa for the smallest et al. (2006)
clusters and with
more complex
arrays for larger
clusters
total disappearance of this band after the bioreduction may be due to the fact that the polyols are
mainly responsible for the reduction of Ag ions, whereby they themselves get oxidized to unsatu-
rated carbonyl groups leading to a broad peak at 1650 cm−1 (for reduction of Ag).
33.3.3 Electron Microscopy
SEM, TEM, and STEM are closely related techniques that use an electron beam to image a sample.
High-energy electrons incident on ultra-thin samples allow for image resolutions that are on the
order of 1–2 Å. Compared to SEM, TEM and STEM have better spatial resolution and are capable
of additional analytical measurements but require significantly more sample preparation. Using
TEM and STEM, it is also possible to characterize crystallographic phase and crystallographic
orientation (by diffraction mode experiments). TEM is a microscopy technique whereby a beam of
electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes
through. An image is formed from the interaction of the electrons transmitted through the specimen
(Goldstein et al. 2003; Rajagopal 2011; Carlton and Ferreira 2012).
HRTEM is an imaging mode of the transmission electron microscope that allows the imaging
of the crystallographic structure of a sample at an atomic scale (Fultz and Howe 2001). Because of
its high resolution, it is a valuable tool to study nanoscale properties of crystalline material such as
semiconductors and metals.
STEM techniques can provide imaging, diffraction, and spectroscopic information, either simul-
taneously or in a serial manner, of the specimen with an atomic or a sub-nanometer spatial resolu-
tion. A scanning tunneling microscope (STM) is an instrument for imaging surfaces at the atomic
level. For an STM, good resolution is considered to be 0.1 nm lateral resolution and 0.01 nm depth
resolution (Joshi et al. 2008).
33.3.4 Atomic Force Microscopy

AFM provides images with atomic or near-atomic-resolution surface topography, capable of quanti-
fying surface roughness of samples down to the angstrom scale. In addition to presenting a surface
image, AFM can also provide quantitative measurements of feature sizes, such as step heights and
other dimensions (Joshi et al. 2008).
33.3.5 X-Ray Diffraction
XRD is a versatile, nondestructive technique that reveals detailed information about the chemi-
cal composition and crystallographic structure of natural and manufactured materials (Srinivasulu
et al. 2012). When a monochromatic x-ray beam with wavelength λ is projected onto a crystalline
material at an angle θ, diffraction occurs only when the distance traveled by the rays reflected from
successive planes differs by a complete number n of wavelengths. By varying the angle θ, Bragg’s
law conditions are satisfied by different d-spacings in polycrystalline materials. Plotting the angular
positions and intensities of the resultant diffracted peaks of radiation produces a pattern, which is
characteristic of the sample. Where a mixture of different phases is present, the resultant diffracto-
gram is formed by addition of the individual patterns.
33.3.6 Energy-Dispersive X-Ray Spectroscopy

Energy-dispersive x-ray spectroscopy (EDS or EDX) is an analytical technique used for the elemental
analysis or chemical characterization of a sample. It relies on the investigation of an interaction of
some source of x-ray excitation and a sample. Its characterization capabilities are due in large part
to the fundamental principle that each element has a unique atomic structure allowing unique set of
peaks on its x-ray spectrum (Goldstein et al. 2003; Cezar et al. 2010).
33.3.7 X-Ray Absorption Near-Edge Structure Spectroscopy and

Extended X-Ray Absorption Fine Structure Spectroscopy
XANES and EXAFS spectroscopy have been used as powerful tools for studying the structures and
dynamics of the nanoscale materials. XANES measures the modulation of the absorption coeffi-
cient at a particular core level of an atom in a chemical environment. XANES has been successfully
applied to investigate the chemical bonding, electronic structure, and surface chemistry of many
nanomaterials (Zhou et al. 2007; Baker et al. 2009). It is element specific and can also be sensitive
to either the surface or the bulk of the specimen depending on the edge and the detection schemes.
EXAFS have been used extensively in the investigation of local atomic structures such as the
number and type of neighboring atoms, interatomic distances, and disorder, and it is well suited for
determining the local structures of both noncrystalline and crystalline materials (Baker et al. 2009).
33.3.8 X-Ray Photoelectron Spectroscopy

XPS utilizes photo-ionization and analysis of the kinetic energy distribution of the emitted photo-
electrons to study the composition and electronic state of the surface region of a sample. XPS uses
soft x-rays (with a photon energy of 200–2000 eV) to examine core levels (Rao and Biswas 2009).
33.4 GREEN SYNTHESIS OF METAL NANOPARTICLES USING PLANT

EXTRACTS: CHARACTERIZATION AND DETERMINATION
For “green synthesis” of different MNPs, plant leaf extracts are widely employed as reducing agents.
The NPs synthesized by this way are usually characterized by several experimental techniques to
determine mainly the size and shape of MNPs. Extracellular synthesis of MNPs using plant extracts
was comprehensively reviewed by Narayanan and Sakthivel (2011b). Preparation of gold, silver,
palladium, platinum, zinc oxide, copper, selenium, and TiO2 NPs using extracts of plant origin is
discussed briefly in the following texts. Plant species used for preparation of AuNPs (Table 33.1),
AgNPs (Table 33.2), and some other metals (ZnO, TiO2, Pt, Pd, Fe, Cu, CuO, Co, Pb Cr2O3, CdO,
Ti/Ni) NPs (Table 33.3) were summarized. In Tables 33.1 through 33.3, important characteristics of
MNPs and experimental techniques applied for characterization are also presented.
33.4.1 Gold Nanoparticles
AuNPs are some of the most extensively studied NPs because of their wide use in biodiagnos-
tics (medicine), biological and chemical sensing techniques, catalysis, imaging, optics, optical
electronics, and microelectronics. AuNPs can be easily synthesized, exhibit intense SPR, and
display high chemical as well as thermal stability (e.g., Jennings and Strouse 2007). A variety of
gold nanostructures (spheres, rods, triangles, hexagons, octagons, cubes, as well as nanowires)
can be synthesized using different physical, chemical, or biological techniques. Micropatterning
methods such as photolithography and nano-imprinting are the conventional techniques that are
used for the fabrication of microstructures (e.g., Gates et al. 2005). Many authors (e.g., Daniel and
Astruc 2004) stressed that these methods are complex, cost-intensive, and often involve multiple
steps. Thus, extensive and cost-effective micropatterning methods are an important aspect of
fundamental research in this field. According to Bankar et al. (2010c) in the recent years, the use
of natural and modified polysaccharides for the synthesis of gold microcrystals varying in size
and shape has become a popular alternative. Moreover, the use of naturally available agricultural
waste material has not been investigated so far, for such applications. Similarly to the synthesis of
AgNPs, banana peels are a classical example of such abundantly available, simple, nontoxic, eco-
friendly, “green” natural material. These authors used for synthesis of AuNPs (microcubes and
microwires) banana peel extract (BPE) powder and chloroauric acid (HAuCl4) in distilled water
as a source of gold. A variety of NPs were formed when the reaction conditions were altered with
respect to pH, BPE content, chloroauric acid concentration, and temperature of incubation. The
reaction mixture displayed vivid color and UV–Vis spectra characteristics of AuNPs. Dynamic
light scattering studies revealed that the average size of the NPs under standard synthetic condi-
tions was around 300 nm. SEM and EDS confirmed these results. A coffee ring phenomenon led
to the aggregation of the NPs into microcubes and microwire networks toward the periphery of
the air-dried samples. XRD studies of the samples revealed spectra that were characteristic for
gold. The PBE-mediated NPs displayed efficient antimicrobial activity toward most of the tested
fungal and bacterial cultures (Candida albicans BX, C. albicans BH, Shigella sp., Enterobacter
aerogenes, Klebsiella sp., Pseudomonas aeruginosa). Bankar et al. (2010c) stressed that the size
of the microcubes due to the coffee ring effect with BPE was in the range of 10–20 μm. Moreover,
the cubes formed in this study (due to the coffee ring effect) aggregated into networks that were
very long. These authors mentioned that an advantage of their study is the use of waste banana
peels rather than the edible fruit for the synthetic process. Biosynthesis of gold nanoplates and
nanocubes is a common feature. However, the formation of large microcubes (10–20 μm) and
their assembly into long networks is an unusual phenomenon that has not been hitherto reported.
Additionally, this system could also be used as a model for understanding the mechanism of
microstructure evolution mediated by biological systems.
In general, size- and shape-dependent physicochemical and optoelectronic properties of metal
and semiconductor NPs have significant relevance to catalysis, biosensing, and optics. The shape-
controlled synthesis of AuNPs has been previously studied, and the procedures often involve sur-
factants and/or seeds in solutions (e.g., Wu et al. 2005). In the paper of Ghodake et al. (2010), a
simple biosynthetic method using pear fruit extract under alkaline conditions was investigated
for producing gold nanostructures with a platelike morphology. Successfully biosynthesized
triangular and hexagonal nanoplates were observed, elegantly assembled with hexagonal NPs.
Nanostructure size, crystal nature, purity, and morphologies were characterized by TEM, AFM,
XRD, x-ray photoemission spectroscopy, and EDS. The edge lengths of the nanostructure ranged
from 200 to 500 nm. Using AFM analysis, the nanohexagons were observed to have a thickness
ranging from 12 to 20 nm. Powder prepared from pear fruit extract and chloroauric acid (HAuCl4)
was applied during the experiments. The authors proposed positive effect of pear extract, which
contains essential phytochemicals consisting of organic acids, amino acids, peptides, and proteins.
In addition, the presence of saccharides in the pear extract provides synergistic reduction power
50 µm 10 µm
FIGURE 33.1 SEM images showing the assembly of BPE-mediated AuNPs into microcubes (10–20 μm)
and long networks.
for the reduction of chloroaurate ions into AuNPs. It was found that alkaline condition appeared to
be efficient for the generation of hexagonally and triangularly shaped AuNPs. Many triangular and
hexagonal nanoplate structures were observed, and the hexagonal NPs demonstrated an assembled
nature. The corners of the gold triangles were not sharp but rather blunt, flat, or curved. The mix-
ture produced under alkaline conditions (pH 9) consisted of triangular NPs (22%), hexagonal NPs
(37%), and others (41%). This paper clearly showed that the biosynthesis of gold nanostructures
using pear fruit extract in an alkaline condition is very efficient and provides an optimal quantity
of pure nanomaterials. The mechanism for the induction of these nanostructures is postulated to
involve alkaline-responsive phytochemicals, such as amino acids, organic acids, peptides, and/or
proteins. These compounds are present in the pear fruit extract, too. The authors of the previously
mentioned paper stressed that gold nanostructures, based on the green biosynthetic method (“green
chemistry”), will play important role in future optoelectronic and biomedical device applications.
SEM images showing the assembly of BPE-mediated AuNPs into microcubes (10–20 μm) and
long networks are presented in Figure 33.1.
33.4.2 Silver Nanoparticles
AgNPs also possess unique electrical, optical, as well as biological properties and can be applied in
catalysis, biosensing, imaging, medicine, drug delivery, and nanodevice fabrication (for details, see
Jain et al. 2008). They have antimicrobial properties and are even being projected as the future gen-
eration of antimicrobial agents (e.g., Rai et al. 2009). AgNPs can be synthesized by several physi-
cal (Nair and Laurencin 2007), chemical (Zhang et al. 2008), and biological (Sharma et al. 2009)
methods. Till now, several biological systems such as bacteria, fungi, and algae have been used for
AgNPs synthesis. It was shown that naturally available agricultural wastes have not yet been inves-
tigated for AgNPs synthesis. A classical example of such an abundantly available natural material is
the banana (Musa paradisiacal) peel. The following applications of banana peels include exploita-
tion for their medicinal properties, in methanol fermentation; application as a substrate for generat-
ing fungal biomass, use in the production of lactase; and utilization as a biosorbent for heavy-metal
removal (for details, see Bankar et al. 2010a). These authors demonstrated a hitherto unreported
green biological route for the AgNPs synthesis using an extract derived from banana peels. The
NPs have been characterized by UV–Vis, SEM–EDS, XRD, and FTIR analysis. An application of
these biologically synthesized NPs as antimicrobial agents has also been studied using the following
pathogenic strains: C. albicans, Shigella sp., Klebsiella sp., and Citrobacter koseri.
SEM images of colloidal silver nanostructures formed by using BPE are presented in
Figure 33.2.
5 µm 0.5 µm
FIGURE 33.2 SEM images of colloidal silver nanostructures formed by using BPE.
33.4.3 Palladium Nanoparticles
Palladium NPs (PdNPs) have also several practical applications, but these MNPs are at present not so
commonly used compared to AuNPs or AgNPs. The most frequent application of PdNPs is in devis-
ing sensors, as catalysts, and in making active membranes (e.g., Mubeen et al. 2007). However, from
scientific aspect, PdNPs are interesting for their remarkable variability in form and shape (for details,
see Bankar et al. 2010b). PdNPs can be synthesized by chemical (Schmid 1992), electrochemical
(Li et al. 1997), or sonochemical methods (Nemamcha et al. 2006). There are also reports concern-
ing plant-based synthesis of PdNPs (cf. Yang et al. 2010), but there are no reports on their synthesis
using agricultural wastes (for details, see Bankar et al. 2010c). Alike to AuNPs or AgNPs, these
authors hypothesized that banana peel, an abundantly available agricultural waste that is composed
of polymers such lignin, hemicellulose, and pectins, could be applied in the synthesis of PdNPs, too.
In the paper of Roopan et al. (2012a), aqueous peel extract of Annona squamosa L. was used to
synthesize PdNPs in the 80 ± 5 nm range with the average particles size 100 nm. Compounds hav-
ing the aldehyde and hydroxyl group as a functional group in the structure occurring in peel extract
were found to be responsible for the reduction of Pd(II) ions and the stabilization of PdNPs. More or
less, uniformly sized PdNPs were synthesized with an average size ranging from 15 to 20 nm using
Cinnamomum zeylanicum bark extract, and terpenoids were believed to play an important role in
PdNP biosynthesis through the reduction of Pd ions (Sathishkumar et al. 2009a). To synthesize
PdNPs with the average size around 15 nm, protein-rich soybean leaf extract acting as an effective
reducing agent for palladium ions was applied, too. It was found that also amino acids occurring in
soybean leaf extract were involved in the reduction of Pd ions and they also acted as surfactants that
inhibit the rapid agglomeration (Petla et al. 2012).
Scanning electron micrographs of BPE-mediated palladium nanostructures aggregated into
microcubes and dendrites are presented in Figure 33.3.
33.4.4 Platinum Nanoparticles
Platinum NPs (PtNPs) can be synthesized using different plant extracts. For example, C. zeylanicum
bark extract and Curcuma longa tuber extract were selected for the preparation of PtNP aggregates
ranging from 25 to 30 nm (Krishnamurthy et al. 2009). It was found that high content of polyols
and antioxidants in these plants contributes to the reduction of metal ions to NPs. Moreover, it
was observed that the rate of reduction increased with increase in incubation temperature. The
eco-friendly extracellular synthesis of PtNPs from an aqueous H2PtCl6·6H2O solution and the leaf
extract of Diospyros kaki as a reducing agent has been done by Song et al. (2010). Depending on the
reaction temperature and concentrations of the leaf broth and PtCl62−, the authors prepared PtNPs
with average particle size ranging from 2 to 12 nm. In another paper (Song et al. 2009), the average
100 µm 10 µm
FIGURE 33.3 Scanning electron micrographs of BPE-mediated palladium nanostructures aggregated into
microcubes and dendrites.
particle size of AuNPs decreased from 12 nm at 25°C to 5 nm at 95°C. This could be connected with
increased reaction rates at higher temperatures and the rapid formation of metal nuclei. The results
of FTIR analysis of PtNPs indicated that PtNPs synthesized with D. kaki extract are surrounded
by some metabolites like terpenoids that have functional groups of amines, alcohols, ketones, alde-
hydes, and carboxylic acids. Based on the results of FTIR analysis as well as on the finding that the
highest rate of PtNPs synthesis occurred at temperatures as high as 95°C, it can be concluded that
PtNPs synthesis using D. kaki is not an enzyme-mediated process.
Resting cells of Shewanella algae (rod-shaped Gram-negative marine bacterium) reduced aque-
ous PtCl62− into elemental platinum within 60 min under room temperature and neutral pH condi-
tions when lactate was provided as an electron donor. Formed PtNPs were approximately 5 nm in
diameter within the periplasm of the cells (Konishi et al. 2007).
33.4.5 Zinc Oxide Nanoparticles

Aloe vera leaf extract and zinc nitrate were used by Sangeetha et al. (2011) for preparing highly
stable and spherical zinc oxide NPs (ZnO NPs). These ZnO NPs were polydispersed, predominantly
spherical, and the average size ranged from 25 to 40 nm. ZnO NPs’ size increased with increasing
A. vera leaf extract concentrations, and the TEM images at higher resolution also revealed that ZnO
NPs are not in physical contact but are separated by uniform interparticle distance. The presence of
secondary material capping with a thickness of about 10 nm (which may be assigned to bioorganic
compounds present in the leaf broth) was confirmed as well, and phenolic compounds, terpenoids,
or proteins that were bound to the surface of ZnO NPs remained on NP surface despite repeated
washing. The previously mentioned authors found that stability of ZnO NPs may be due to the free
amino and carboxylic groups that have interacted with the zinc surface, and the proteins present in
the medium prevent agglomeration and aid in the stabilization by forming a coat, covering the MNPs.
33.4.6 Copper Nanoparticles
Harne et al. (2012) prepared monodisperse copper NPs (CuNPs) using aqueous extract of
Calotropis procera L. latex. This plant extract was found to be highly efficient in stabilizing and
making these CuNPs biocompatible as these NPs were nontoxic. Monodisperse and spherical
particles with the sizes ranging from 5 to 30 nm and an average diameter of 15 ± 1.7 nm were
obtained at application of 0.5% of aqueous extract of C. procera L. latex indicating that enzymes
present in this latex were suitable for fabrication of monodisperse NPs. Based on the FTIR spec-
tra, the previously mentioned authors described that on the surface of the biosynthesized CuNPs,
using C. procera antioxidant enzymes, cysteine, protease, and tryptophan with functional groups
of amines, alcohols, ketones, aldehydes, and carboxylic acids may be absorbed.
Valodkar et al. (2011) synthesized CuNPs using stem latex of a medicinally important plant,
Euphorbia nivulia, which were stabilized and subsequently capped by peptides and terpenoids pres-
ent within the latex.
33.4.7 Selenium Nanoparticles
The uniform spherical selenium NPs (SeNPs) by adding Undaria pinnatifida polysaccharides to the
redox system of selenite and ascorbic acid were synthesized by Chen et al. (2008). While nano-Se
particles in water would easily aggregate (with particle size distributed between 788 and 1212 nm
and with an average diameter of 892 nm), the presence of U. pinnatifida polysaccharides signifi-
cantly decreased the particle size to 44–92 nm with an average diameter of 59 nm, and monodis-
persed nano-Se could keep stable at least for 3 months.
33.4.8 Titanium Dioxide Nanoparticles

Rajakumar et al. (2012) prepared titanium dioxide NPs (TiO2 NPs) using Eclipta prostrata leaf
extract. Uneven surface morphology of TiO2 NPs indicated the presence of both individual and
agglomerated NPs. Synthesized TiO2 NPs showed shape in spherical clusters, quite polydisperse,
and the size of particles ranged from 36 to 68 nm. It was found that E. prostrata leaves contained
β-amyrin, wedelolactone, triterpenoids, flavonoids, luteolin-7-O-glucoside, l-terthienyl methanol,
and stigmasterol. The analysis of FTIR spectra of the E. prostrata powder and synthesized TiO2
NPs indicated that alcohols, phenols, alkanes, primary amines, and aliphatic amines presented in
studied plant species may be participating in the process of NP synthesis.
Aqueous extract prepared from latex of Jatropha curcas L. was used by Hudlikar et al. (2012)
for synthesis of TiO2 NPs of the average size in the range of 25–100 nm. These NPs could be
divided in two broad categories: (1) having diameter from 25 to 50 nm that are mostly spherical
in shape and (2) having some larger and uneven shapes. The XRD pattern showed the presence of
broad peaks indicating that the particles have very small crystallite size and are semicrystalline
in nature. Their results indicated that (NH)−C=O groups from peptides of protein possess very
high affinity to bind to titania. Thus, the protein itself acted as an encapsulating agent, thereby
protecting NPs from agglomerization. The protein/peptide encapping the NP surface could be
removed from the NP surface.
33.5 FORMATION OF METAL NANOPARTICLES IN LIVING PLANTS

By the end of the 1990s, it was shown that gold accumulated by the plants and stored in leaf
and stem biomass can be present as discrete NPs of pure metal (Gardea-Torresdey et al.1999).
Discrete AuNPs of 2–20 nm in diameter as well as coalesced particles of 20–40 nm scale dis-
tributed through certain zones of tissue of alfalfa sprouts germinated on gold chloride–enriched
agar (320 mg kg−1) were observed using x-ray absorption spectroscopy (XAS) and TEM techniques
(Gardea-Torresdey et al. 2002).
Several plant species were used as model systems to investigate formation of metal NPs in living
plants treated with different metal salts. As mentioned earlier, Gardea-Torresdey et al. (2002) deter-
mined in shoots of Medicago sativa grown on agar substrate AuNPs with particle size ranging from 4
to 40 nm with icosahedral morphology. On the other hand, AgNPs in roots and shoots of this plant were
spherical and ranged from 2 to 20 nm (Gardea-Torresdey et al. 2003). The sizes of AuNPs of Chilopsis
linearis also grown on agar substrate were 80 nm in roots, 350 nm in stems, and 180 nm in shoots
(Rodriguez et al. 2007). Spherical AuNPs (60–20 nm) were observed in the roots and shoots of agar-
grown Sesbania drummondii (Sharma et al. 2007). In hydroponically cultivated plants of Brassica
juncea, spherical AgNPs with the mean size of 50 nm were determined in plant biomass, while in
M. sativa, the size of formed AgNPs varied from 2 to 100 nm (Harris and Bali 2008). Compared to
relatively small sizes of AgNPs observed in stems and leaves of hydroponically cultivated B. juncea
ranging from 2 to 35 nm (Haverkamp and Marshall 2009), the size of AuNPs determined in roots and
stems of this species varied from 2 to 200 nm showing various shapes: spherical, triangular, hexago-
nal, or decahedral (Bali and Harris 2010). On the other hand, Beattie and Haverkamp (2011) found
NPs of Ag and Au in the leaves, stem, roots, and cell walls of the plants at a concentration of 0.40%
Ag and 0.44% Au in the leaves, and these particles were approximately spherical with sizes ranging
from 2 to 100 nm (10–30 nm AgNPs in roots and 4–6 nm AgNPs in stems; 2–40 nm AuNPs in roots,
2–100 nm AuNPs in stems and leaves, and 100 nm AuNPs in leaf cell walls), whereby the sites of the
most abundant reduction of metal salts to NPs were the chloroplasts, regions of high reducing sugar
(glucose and fructose) content. An experiment of Starnes et al. (2010) with six hydroponically culti-
vated plants (Cucumis sativus, Helianthus annuus, Lolium multiflorum, M. sativa, Origanum vulgare,
and Trifolium pratense) treated with chloroaurate showed that variable growth conditions affected
size distribution of AuNPs, size of which varied from 1 to 50 nm, whereby particles of various shapes
(spherical, triangular, hexagonal, and rectangular) were formed.
The transformation of copper into metallic NPs in and near roots with evidence of assistance by
endomycorrhizal fungi when common wetland plants Phragmites australis and Iris pseudacorus
were grown in contaminated soil in the natural environment was reported by Manceau et al. (2008).
The researchers stated that this mode of copper biomineralization by plant roots under copper stress
may be common in oxygenated environments because the transformation occurs likely due to bio-
molecular responses to oxidative stress, similar to reactions used to abiotically synthesize Cu(0)
nanostructures of controlled size and shape.
Senapati et al. (2012) reported on the use of alga Tetraselmis kochinensis in the intracellular
synthesis of AuNPs of dimensions 5–35 nm. The particles were more concentrated upon the cell
wall than on the cytoplasmic membrane, possibly due to the reduction of metal ions by enzymes
presented in the cell wall and cytoplasmic membrane.
The study of uptake and PtNPs formation in M. sativa and B. juncea was found to be signifi-
cantly dependent on the pH. Like that, B. juncea shoots showed 26 times higher uptake at pH 3 as
compared to pH 9. Due to the action of local metabolites, the sequestered Pt(II) was reduced to
Pt(0). In vivo formation of PtNPs between 3 and 100 nm in size and of varying morphologies in the
epidermal root cells was confirmed by electron microscope images. Large quantities of particles,
the majority spherical in shape, were observed, in particular on the cell walls and organelles; how-
ever, besides spherical, triangular and irregular particles were observed using STEM. The formed
PtNPs were crystalline Pt. Higher-magnification TEM images suggested that the particles surround
the root organelles, but using proton-induced x-ray emission (μ-PIXE) spectroscopy, even distribu-
tion across all tissue systems (epidermal, cortical, and vascular) within the roots of both M. sativa
and B. juncea was confirmed (Bali et al. 2010).
Recently, a comprehensive review about biosynthesis (or phytosynthesis) of MNPs in living
plants was published by Marchiol (2012). This author mentioned that biosynthesis of MNPs is a type
of bottom-up approach where the main reaction occurring in biological material is reduction/oxidation.
The exact mechanism for the formation of MNPs in living plants is not yet known nor investi-
gated in depth. The most reliable hypothesis is that the ionic form of element must be transported
across the root membrane, then translocated into the plant and reduced to the elemental form. The
extracts of plant contain a lot of biomolecules, such as proteins/enzymes, polysaccharides, amino
acids, and vitamins, which could be used as reductants. Various organisms have been suggested as
nanofactories for intra- and extracellular synthesis of metals. The use of plants for NP synthesis is a
comparatively new and under-researched technique. Since synthesis of MNPs using plant extracts is
very cost effective, the plant extract can be used as an economic and valid alternative for the large-
scale production of MNPs. In general, biosynthesis of MNPs using vascular plants has become one
of the exciting research fields in the burgeoning area of material science. Similarly, Narayanan and
Sakthivel (2011b) emphasized that synthesis of various MNPs using plant extract is easy in down-
stream processing and in scaling up of MNPs.
33.6 UPTAKE, TRANSLOCATION, AND ACCUMULATION

OF METAL NANOPARTICLES IN THE PLANTS
Exposure of tobacco plants (Nicotiana tabacum L. cv. Xanthi) to AuNPs showed that AuNPs
entered plants through the roots and moved into the vasculature, and aggregate bodies were also
detected within root cell cytoplasm. Uptake of AuNPs was size selective as 3.5 nm AuNP spheres
were detected in plants, but 18 nm AuNPs remained agglomerated on the root outer surfaces (Sabo-
Attwood et al. 2012).
Absorption of AuNPs through root uptake was found to be size and species dependent in
experiment with aquatic macrophytes Myriophyllum simulans Orch. and Egeria densa Planch.
(submerged aquatic vascular plants) and Azolla caroliniana Willd. (free-floating aquatic fern)
treated with 4 and 18 nm AuNPs. AuNPs were confirmed in tissue by using EDS. TEM indi-
cated that 4 and 18 nm AuNPs were absorbed by A. caroliniana, whereas only 4 nm AuNPs
were absorbed by M. simulans and E. densa did not absorb AuNPs of either size. Differences
in absorption of AuNPs by plants may be explained with the salinity tolerance of each species
(Glenn et al. 2012).
Experiment with wheat (Triticum aestivum) plants exposed to 25 nm anatase TiO2 NPs neither
affected their seed germination rate nor their root elongation; however, 12 nm anatase NPs were
efficiently taken up by plant roots and localized in the parenchymal region and vascular cylinder.
Their nutrient solution to root transfer did not induce their dissolution nor localized change in their
crystalline structure (Larue et al. 2011). These results showed that TiO2 NPs can be transferred to
plants by their roots in case of an accidental release in the environment and consequently may enter
the food chain.
Study of uptake and the potential for trophic transfer of AuNPs with 5, 10, and 15 nm diameter
AuNPs using N. tabacum L. cv. Xanthi and Manduca sexta (tobacco hornworm) demonstrated tro-
phic transfer and biomagnification of AuNPs from a primary producer to a primary consumer by
mean factors of 6.2, 11.6, and 9.6 for the 5, 10, and 15 nm treatments (Judy et al. 2011).
Silver content of both shoots and roots of L. multiflorum plants treated with AgNPs increased
with increasing AgNPs exposure and the values of bioaccumulation factor for roots occurred
in the range of 25–30. The translocation factor (i.e., the ratio of accumulated Ag concentration
in the shoot to that in root) was very low, indicating that most of this silver appeared to remain
associated with the roots. Many AgNPs adsorbed to the surface of plant roots while TEM showed
the presence of particulates in AgNPs-treated roots, and using micro-x-ray fluorescence (μXRF),
silver-rich areas were determined inside the root at 50, 300, 800, and 4000 μm from the apex (Yin
et al. 2011).
Similarly, the majority of 7 and 25 nm CeNPs only loosely adhered to the root surface of cucum-
ber plants but the seedlings treated with 7 nm CeNPs showed significantly higher ceria contents in
both roots and shoots than those exposed to 25 nm Ce particles at all test concentrations (2, 20, and
200 mg L−1). Although only very limited amounts of CeNPs could be transferred from the roots to
shoots because the entry of NPs into the roots was difficult, once they have entered into the vascu-
lar cylinder, CeNPs could move smoothly to the end of the vascular bundle along with water flow
(Zhang et al. 2011).
33.7 TOXICITY OF METAL NANOPARTICLES TO ALGAE

The physicochemical properties of NPs determine their interaction with living organisms. Cells
of plants, algae, and fungi possess cell walls that constitute a primary site for interaction and a
barrier for the entrance of NPs. Mechanisms allowing NPs to pass through cell walls and mem-
branes are as yet poorly understood. However, inside cells, NPs might directly provoke alterations
of membranes and other cell structures and molecules, as well as protective mechanisms. Indirect
effects of NPs depend on their chemical and physical properties and may include physical restraints
(clogging effects), solubilization of toxic NP compounds, or production of reactive oxygen species

(ROS) (Navarro et al. 2008a).
Toxicity of nano-sized metal oxide particles to algae compared to that of larger-sized bulk par-
ticles was found to be higher when dose is expressed as mass and EC50 values are generally in the
mg L−1 range (e.g., Rogers et al. 2010; Hartmann 2011). EC50 (half maximal effective concentration)
presents a concentration of a compound where 50% of its maximal effect is observed.
Application of TiO2 NPs with particle size of 25 nm (crystalline form: mainly anatase) to green
alga Desmodesmus subspicatus resulted in inhibition of algal growth with EC50 44 mg L−1. The
measured toxic effect was caused by the TiO2 itself and not by a photocatalytic effect because no
difference in growth reduction was observed for the tests with or without preliminary illumination.
On the other hand, treatment of D. subspicatus of NPs with greater particle size (100 nm; crystal-
line form: 100% anatase) in the concentration range 0–50 mg L−1 was not toxic to algae (Hund-
Rinke and Simon 2006). The results of Lang et al. (2010) related to acute algal growth toxicity test
of undoped TiO2 NPs (crystallite size 4.5 nm) and Ag-doped TiO2 NPs (crystallite size 4.6 nm)
using D. subspicatus confirmed higher toxic effect for Ag-doped TiO2 NPs (EC50 = 4.12 mg L−1) in
comparison with undoped ones (EC50 = 7.59 mg L−1).
The effect of different concentrations of TiO2 NPs (0, 50, 100, 200, and 300 ppm) on
growth and metabolism of three species of freshwater green algae (Scenedesmus quadricauda,
Chlamydomonas moewusii, and Chlorella vulgaris) was examined by Cardinale et al. (2012).
Population growth rates were consistently reduced by TiO2 NPs, with reduction ranging from
11% to 27% depending on the species. However, the mechanisms of reduction differed among
studied species. For Chlamydomonas species, it was found that TiO2 NPs reduced both meta-
bolic rate of gross primary production (GPP) and respiration (R), but effects on GPP were stron-
ger. Consequently, carbon was respired more quickly than it was fixed that resulted in reduced
growth. In contrast, TiO2 NPs stimulated both GPP and R in Chorella; however, because R was
stimulated to a greater extent than GPP, carbon loss again exceeded fixation, leading to reduced
growth. On the other hand, for Scenedesmus species, it was confirmed that TiO2 NPs had no
significant impact on R but reduced GPP, and this pattern also caused carbon loss to exceed fixa-
tion. The results of these authors suggested that TiO2 NPs may generally suppress the growth of
pelagic algae, but these impacts are manifested through contrasting effects on species-specific
metabolic functions.
Aruoja et al. (2009) investigated toxicities of ZnO, TiO2, and CuO NPs to Pseudokirchneriella
subcapitata and found that the shading effect by NPs was negligible. It was found that bulk TiO2
(EC50 = 35.9 mg Ti L−1) and bulk CuO (EC50 = 11.55 mg Cu L−1) were less toxic than their nano-
formulations (EC50 = 5.83 mg Ti L−1 and 0.71 mg Cu L−1), while the toxicities of bulk ZnO and
ZnO NPs were both similar to that of ZnSO4 (72 h EC50 similar to 0.04 mg Zn L−1). Consequently,
in this case, the toxicity can be attributed solely to solubilized Zn(II) ions. Similarly, bioavailable
copper ions were found to exhibit toxic effects of CuO NPs to algae. On the other hand, nano-TiO2
formed characteristic aggregates entrapping algal cells that may contribute to the toxic effect of
nano-TiO2 to algae. Based on the toxicity experiments using the freshwater alga P. subcapitata, in
which comparable toxicity with a 72 h EC50 value near 60 μg Zn L−1 was determined for ZnO NPs,
bulk ZnO, and ZnCl2, toxic effect of nanoparticulate ZnO was also attributed solely to dissolved
zinc (Franklin et al. 2007).
The process of Au(III) incorporation, intracellular reduction, and Au(0) NP release in the cul-
ture medium was compared for four photosynthetic microorganisms, Klebsormidium flaccidum
and Cosmarium impressulum green algae, Euglena gracilis euglenoid, and Anabaena flos-aquae
cyanobacteria in order to design cell-based bioreactors for inorganic particles (Dahoumane et al.
2012). At low gold content, the two studied green algae showed maintained photosynthetic activity
and recovered particles (∼10 nm in size) were similar to internal colloids, indicating a full biologi-
cal control over the whole process. Under similar conditions, the euglenoid exhibited a rapid loss of
biological activity, due to the absence of protective extracellular polysaccharide. These green algae
could grow again after an adaptation period that resulted in a larger particle size dispersity but
larger reduction of the yield. However, the cyanobacteria undergo rapid cell death, due to their pro-
karyotic nature, leading to high gold incorporation rate but poor control over released particle size.
Experiments concerning growth inhibition of P. subcapitata alga by micron-size CeO2 and CeO2
NPs showed higher reduction in algal growth rate (expressed by 72 h EC50 value) due to treatment
with NPs (10.3 mg L−1) in comparison to bulk CeO2 (66 mg L−1). Because light illumination condi-
tions stimulated photocatalytic activity of CeO2 NPs, the generation of hydroxyl radicals and per-
oxidation of plant fatty acids occurred that resulted in cell–particle interaction causing membrane
damage (Rogers et al. 2010).
Application of CeO2 NPs of three different sizes (14, 20, and 29 nm) caused significant chronic
toxicity to P. subcapitata, which was found to increase with decreasing nominal particle diameter
and the difference in toxicity was connected with the difference in surface area. It is assumed that
toxicity could not be related to a direct effect of dissolved Ce or CeO2 NPs uptake or adsorption nor
to an indirect effect of nutrient depletion (by sorption to NPs) or physical light restriction (through
shading by the NPs), even though observed clustering of NPs around algal cells may locally cause a
direct or indirect effect (Van Hoecke et al. 2009).
In another experiment focused on the inhibition of the cellular function of P. subcapitata by four
different cerium oxide NPs, EC50 values ranging from 2.4 to 29.6 mg L−1 were determined, and mem-
brane disruption in algal cells as well as highly damaged algal cells were observed. The negligible
amount found dissolved in the NP suspensions could not explain the observed toxic effect of nanoceria
on the aquatic organism. While no evidence of NP uptake by cells was found, it could be suggested
that their toxic mode of action required direct contact between NPs and cells, and cell damage most
probably took place by cell wall and membrane disruption (Rodea-Palomares et al. 2011).
Growth rate of P. subcapitata was also inhibited by silica (SiO2) NPs with 12.5 and 27.0 nm
diameter, which clearly adhered to the outer cell surface, and no evidence was found for particle
uptake. Because no aggregation was observed and dissolution of the NPs was negligible, the toxicity
was attributable to the solid nanospheres (Van Hoecke et al. 2008).
In short-term toxicity of AgNPs and ionic silver (Ag+) to photosynthesis in Chlamydomonas
reinhardtii, it was found that toxicity was 18 times higher for AgNO3 than for AgNPs (in terms
of EC50) if total Ag concentration was taken into account. However, when compared as a func-
tion of the Ag+ concentration, toxicity of AgNPs appeared to be much higher than that of AgNO3.
Consequently, it can be concluded that the interaction of these particles with algae influences the
toxicity of AgNPs, which is mediated by Ag+, and that particles contributed to the toxicity as a
source of Ag+, which is formed in the presence of algae (Navarro et al. 2008b).
Using TEM, it was found that amine-coated 10 nm AuNPs were strongly adsorbed by the cell
wall of alga Scenedesmus subspicatus, leading to progressive intracellular and cell wall distur-
bances (Renault et al. 2008).
Silver-engineered NPs can be taken into the cells and accumulated inside the algal cells
of mixotrophic freshwater alga Ochromonas danica, where they exerted their toxic effects
(Miao et al. 2010). Thus, NP internalization may be an alternative pathway through which algal
growth can be influenced. Silver-engineered NPs were found to be toxic also to the marine diatom
Thalassiosira weissflogii, and toxic effects are connected with Ag+ from the oxidative dissolution
of NPs (Miao et al. 2009). It could be stressed that the secretion of polysaccharide-rich algal exo-
polymeric substances (EPS) significantly increased at increasing free Ag+, and both dissolved and
particulate polysaccharide concentrations were higher for nutrient-limited cells, coinciding with
their higher Ag+ tolerance, suggesting that EPS may be involved in Ag+ detoxification.
Exposure of C. vulgaris and Dunaliella tertiolecta to 50 nm AgNPs (0–10 mg L−1) for 24 h
resulted in strong decrease in chlorophyll content, viable algal cells, increased ROS formation,
and lipids peroxidation (Oukarroum et al. 2012a). It was found that temperature enhanced toxic
effects of AgNPs in these two green algae. When temperature conditions changed, alternations in
inhibitory effect of AgNPs were observed. Temperature increase from 25°C to 31°C induced higher
altering effects to photosystem II (PS II) quantum yield, primary photosynthetic electron trans-
port (PET), and consequently higher decrease of total photosynthetic performance if compared to
AgNPs effect alone. At the same time, negative effect was higher for D. tertiolecta compared
to C. vulgaris (Oukarroum et al. 2012b).
Study of short-term toxicity of citrate-stabilized AgNPs and ionic silver Ag(I) in ichthyotoxic
marine raphidophyte Chattonella marina showed that based on total silver mass, toxicity was much
higher for Ag(I) than for AgNPs. Cysteine, a strong Ag(I) ligand, completely removed the inhibitory
effects of Ag(I) and AgNPs on the metabolic activity of C. marina, suggesting that the toxicity of
AgNPs was due to the release of Ag(I) (He et al. 2012).
The intracellular Ag accumulation upon exposure to carbonate-coated AgNPs (0.5–10 μmol L−1,
average diameter 29 nm) and silver nitrate (20–500 nmol L −1) was examined in the green alga
C. reinhardtii at pH 7.5. It was observed that intracellular Ag (which was measured over time up
to 1 h after a wash procedure to remove Ag+ ions and AgNPs from the algal cell surface) increased
with increasing exposure time and with increasing AgNPs and AgNO3 concentrations in the expo-
sure media. Higher accumulation rate constants were assessed in the cell wall free mutant, indicat-
ing a protective role of the cell wall in limiting Ag+ uptake. However, the bioavailability of AgNPs
was calculated to be low in both strains relative to Ag+, suggesting that AgNP internalization across
the cell membrane was limited (Piccapietra et al. 2012).
Pletikapic et al. (2012) investigated the interactions of AgNPs with ubiquitous, lightly silicified
marine diatoms Cylindrotheca fusiformis and Cylindrotheca closterium and their extracellular poly-
meric substance (EPS) using AFM with a subnanometer resolution. In natural seawater, the single
AgNPs adopted truncated tetrahedron morphology with particle sizes of 10, 20, 30, and 40 nm. It was
confirmed that AgNPs penetrated the cell wall through the valve region built of silica NPs embedded
in organic matrix. The AgNPs caused a local damage inside the cell without disintegration of the cell
wall. The EPS production has been shown to increase as a feedback response to AgNPs exposure and
may contribute to detoxification mechanisms. Imaging EPS at high resolution revealed the incorpora-
tion of AgNPs and their aggregates into the EPS gel matrix, proving their detoxifying capacity.
In the marine macroalga, Ulva lactuca exposed for 48 h to different concentrations of Ag added
as AgNPs or aqueous metal (AgNO3) quenching of chlorophyll a fluorescence and accumulation of
Ag were measured to determine the resulting toxicity. Aqueous Ag was toxic at available concentra-
tions as low as about 2.5 μg L−1 and exhibited considerable accumulation that could be defined by
the Langmuir equation, which relates the coverage or adsorption of molecules on a solid surface to
gas pressure or concentration of a medium above the solid surface at a fixed temperature. AgNPs
were not phytotoxic to the macroalga at available Ag concentrations up to at least 15 μg L−1, and
metal measured in U. lactuca was attributed to a physical association of NPs at the algal surface.
It was stressed that, at higher AgNPs concentrations, a dose–response relationship was observed
that was similar to that for aqueous Ag recorded at much lower concentrations. Consequently, it can
be suggested that AgNPs are only indirectly toxic to marine algae through the dissolution of Ag+
ions into bulk seawater, albeit at concentrations orders of magnitude greater than those predicted in
the environment (Turner et al. 2012).
Under the stress of NiO NPs, growth inhibition of the microalgae C. vulgaris was observed (72 h
EC50 = 32.28 mg NiO L−1) and algal cells showed plasmolysis, cytomembrane breakage, and thyla-
koids disorder. Besides this, NiO NPs aggregated and deposited in algal culture media and the pres-
ence of algal cells accelerated aggregation of NPs. The attachment of aggregates to algal cell surface
and the presence of released ionic Ni were likely responsible for the toxic effects, but the toxicity and
bioavailability of NiO NPs to marine algae were reduced by aggregation and reduction of NiO (Gong
et al. 2011). Whereas nanoparticulate Al2O3, SiO2, and TiO2 (rutile) had no significant toxicity, nano-
ZnO and nano-TiO2 (anatase) greatly inhibited the growth of C. vulgaris with 6 d EC30 of about
20 and 30 mg L−1, respectively (Ji et al. 2011). In the concentration range below 50 mg L−1, the algal
toxicities decreased in the following order: Zn2+ > nano-ZnO > bulk-ZnO. However, at higher concen-
trations, ZnO NPs exhibited higher algal toxicity than Zn2+ ions and released less Zn2+ ions into the
culture media than bulk ZnO. These results suggested that dissolved Zn2+ ions from ZnO NPs were not
the dominant mechanism for the algal growth inhibition. The comparison of the nanotoxicities in the
presence and absence of illumination excluded shading effects of nano-ZnO and nano-TiO2 (anatase)
from the main mechanism of the nanotoxicity. However, observed large aggregates of nano-ZnO and
nano-TiO2 (anatase) entrapping and wrapping the algal cells may contribute to the nanotoxicity.
Alumina NPs were found to inhibit growth of Chlorella sp. and Scenedesmus sp. (72 h EC50 val-
ues were 45.4 and 39.35 mg L−1, respectively), and they were more toxic than bulk alumina (72 h EC50
values were 110.2 mg L−1 for Chlorella sp. and 100.4 mg L−1 for Scenedesmus sp.). In the treated
cells, a clear decrease in chlorophyll content was observed in comparison to untreated cells and this
reduction of pigment content was more notable in the case of NPs (Sadiq et al. 2011).
The study of influence of five Al2O3 nanopowders on the growth of S. quadricauda depending on
the physical and chemical properties of the alumina NPs and on the concentration of the nanopow-
der showed that the strongest algal growth-inhibiting effect was accompanied by the highest degree
of agglomeration and the highest crystallization level as well as the smallest specific surface area
(Karwowska et al. 2012).
Induction of cellular aggregation processes and deteriorative effect on chlorophyll by inducing
the photoinhibition of PS II by core–shell CuO NPs was observed by Saison et al. (2010). The dete-
rioration of photosynthesis was interpreted as being caused by the formation of ROS induced by
core–shell copper oxide NPs. However, it could be stressed that no formation of ROS was observed
when C. reinhardtii was exposed to the core without the shell or to the shell only.
In the experiment of Marsalek et al. (2012), it was confirmed that treatment with NPs of zerova-
lent iron is an effective and environmentally benign method for destroying and preventing the for-
mation of cyanobacterial water blooms. Studied NPs have multiple modes of action, including the
removal of bioavailable phosphorus, the destruction of cyanobacterial cells, and the immobilization
of microcystins, preventing their release into the water column. Ecotoxicological tests showed that
zerovalent iron NPs are highly selective agent, having an EC50 of 50 mg L−1 against cyanobacteria
that is 20–100 times lower than its EC50 for algae, daphnids, water plants, and fishes. Moreover, the
primary product of zerovalent iron NPs treatment was nontoxic and highly aggregated Fe(OH)3,
which promotes flocculation and gradual settling of the decomposed cyanobacterial biomass.
33.8 EFFECT OF METAL NANOPARTICLES TO VASCULAR PLANTS

Bioelements, to which belong some of metals such as Fe, Cu, Ca, Zn, Mn, or K, act as essential elements
responsible for the growth, development, and biochemical and physiological processes occurring
in the shoot and root of the plants. Both ionic form of these metals and nano-sized particles some
of them (MNPs) show positive and negative action to the vascular plants. In the following text,
beneficial and adverse effects of several MNPs to the most important plant features are presented.
33.8.1 Beneficial Effects of Metal Nanoparticles to Plants

It is well known that iron is an essential nutrient for plants. Treatment with nanoscale iron oxide
or ferrofluids was found to have also beneficial effects on plants indicating that such supply of iron
could be advantageous in agricultural practice. Iron oxide NPs at the concentration of 0.75 g L−1 in
the form of spray on soybean plants in field experiments increased leaf pod dry weight and pod dry
weight (Sheykhbaglou et al. 2010). The highest grain yield showing 48% increase in comparison
with control was obtained after application of 0.5 g L−1 iron oxide NPs. Nano-iron oxide was also
found to facilitate the photosynthate and iron transferring to the leaves of peanut (Liu et al. 2005).
Application of small ferrofluid concentrations (10–50 μL L−1) on the growth of Zea mays plant in
early ontogenetic stages resulted in plant length stimulation and the increase of chlorophyll a (up to
13%) as well as that of the nucleic acid level (up to 10%) in maize plantlets during their first days of
life. However, treatment with higher concentrations (100–250 μL L−1) of this ferrofluid containing
magnetic NPs coated with tetramethylammonium hydroxide led to a marked drop in chlorophyll a
level and the chlorophyll a/b ratio (about 35% decrease in both cases) (Racuciu and Creanga 2007).
Even though water-based ferrofluid addition in culture medium represents a source of iron, it could
be supposed that ferrophase NPs may have also a magnetic influence on the enzymatic structures
implied in the different stages of the photosynthesis reactions. On the other hand, application of
an aqueous dispersion of water-based magnetic fluid constituted by coating the small magnetic
NPs with perchloric acid on Z. mays plantlets slightly inhibited plant growth, and on the leaf sur-
face of plants treated with an enhanced volume fraction of aqueous magnetic fluid solution, brown
spots occurred (Racuciu and Creanga. 2009). Hence, the iron oxides provided by the magnetite
from magnetic fluid ferrophase could interfere with the complex redox reactions involved in the
photosynthesis phenomenon. The biocompatible magnetic fluids can be uptaken into whole living
plants and further can move inside using the vascular system being concentrated in specific areas
by application of magnetic gradients (Gonzalez-Melendi et al. 2008). An interesting experiment
was carried out by Racuciu et al. (2009). These researchers used two groups of seeds for cultiva-
tion of maize plants that were tested for the content of assimilatory pigments and nucleic acids:
(1) seeds germinated in the magnetic fluid presence and then exposed to electromagnetic field dur-
ing the germination process (LM–EMF samples) and (2) seeds germinated in the magnetic fluid
presence but in the lack of electromagnetic exposure (LM samples). Based on a decrease of pigment
contents in LM–EMF samples, it was supposed that the electromagnetic field exposure moment
could produce a process like that of hyperthermia, a local heating occurring due to the electromag-
netic field energy absorbed by the magnetic NPs internalized in vegetal tissue, and this local heat-
ing of the vegetal tissue could affect the redox reactions implicated in the photosynthetic process.
Regeneration reactions of the plant metabolism processes against the putative local heating of the
vegetal tissue produced by the electromagnetic field energy absorbed by the magnetic NPs internal-
ized in vegetal tissue could be responsible for a twice higher level of nucleic acids in the LM–EMF
samples in comparison with the control samples.
Beneficial effects on germination and growth of plants were also observed with other metal
NPs, mainly with TiO2, Al2O3, and ZnO NPs. Aged seeds of spinach soaked in solution of TiO2
NPs with concentrations ranging from 0.25% to 4% promoted seed germination as well as the
growth of seedlings. Treatment with 2500 mg L−1 TiO2 NPs was found to be the most effective.
Stimulation of growth of spinach (Spinacia oleracea L.) plants and acceleration of nitrogen assimi-
lation by TiO2 NPs was reported by Yang et al. (2006). Stimulation of PET resulting in increased
oxygen evolution rate (OER) in spinach chloroplasts by addition of 0.25% TiO2 (rutile) NPs and
enhanced activities of Mg2+-ATPase and chloroplast coupling factor I CF1-ATPase on the thyla-
koid membranes were observed, as well (Hong et al. 2005b), and treatment with TiO2 (rutile) NPs
protected chloroplasts from aging for long-time illumination (Hong et al. 2005a). Chloroplasts
from nano-anatase–TiO2-treated spinach exposed to visible light and UV light illumination exhib-
ited significant increase in PET, OER, and photophosphorylation (Zheng et al. 2007). Biomass
accumulation of spinach (S. oleracea) by TiO2 NPs (by 60%) was also observed (Gao et al. 2008).
Significant increase in RuBisCo activity (by up to 2.33 times in comparison to the control) in the
nano-anatase-treated spinach was recorded, while bulk TiO2 treatment had no such notable effects.
One of the molecular mechanisms of carbon reaction promoted by nano-anatase is that the nano-
anatase treatment results in the enhancement of RuBisCo mRNA amounts, the protein levels,
and activity of RuBisCo, thereby leading to the improvement of RuBisCo carboxylation and the high
rate of photosynthetic carbon reaction (Wang et al. 2008). In the 7-day-old cucumber (C. sativus)
plants, hydroponically grown with TiO2 NPs (0–4000 mg L−1), significant increase of root length
(average >300%) was estimated. It was found by μXRF that Ti was transported from the roots into
the leaf trichomes, suggesting that trichomes are possible sink or excretory system for the Ti. The
micro-XANES spectra also showed that the absorbed Ti was present as TiO2 within the cucumber
tissues, demonstrating that the TiO2 NPs were not biotransformed (Servin et al. 2012). Moreover,
the effect of TiO2 NPs on growth of Lemna minor was found to be more obvious than bulk TiO2.
The TiO2 NPs stimulated plant growth in low concentrations but inhibited plant growth at high
concentrations. The peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activity of
L. minor increased when TiO2 NPs concentration was lower than 200 mg L−1 indicating increased
elimination of accumulated ROS in plant cells. On the other hand, the SOD activity decreased when
the TiO2 NPs concentration was higher than 200 mg L−1, and the plant cell membrane encountered
serious damage from 500 mg L−1 TiO2 NPs concentration in the culture media (Song et al. 2012).
Treatment with alumina NPs (average nominal size of 20 nm) resulted in significant stimula-
tion of root length, increase in the number of fronds per colony, as well as biomass accumulation
in L. minor plants accompanied with increased photosynthetic efficiency. This enhancement of
biomass accumulation was associated with increased efficiencies in the light reactions of photosyn-
thesis (Juhel et al. 2011). Whereas alumina NPs increased the quantum yield of PS II, the maximal
quantum yield of PS II (Fv /Fm) was not affected indicating that the alumina NPs act not directly on
PS II (Nel et al. 2006). Positive influence of nano-Al2O3 (applied at 400, 2000, and 4000 mg L−1)
on root elongation of Arabidopsis thaliana could be connected with the fact that inert nano-Al2O3
could serve similar functions as nano-sized perlite, which enhances gas transfer, prevents water
loss, and hinders soil compaction (Lee et al. 2010). In the experiment with tobacco (N. tabacum)
plants (Burklew et al. 2012) exposed to 0%, 0.1%, 0.5%, and 1% Al2O3 NPs, it was observed that
the average root length and average biomass increased, but the leaf count of the seedlings signifi-
cantly decreased. Moreover, microRNAs miR395, miR397, miR398, and miR399 showed an extreme
increase in expression during exposure to 1% Al2O3 NPs as compared to the other treatments and the
control indicating that these miRNAs may play a key role in mediating plant stress responses to NP
stress in the environment by strengthening the ability of plants to withstand stress to Al2O3 NPs.
Substantial growth stimulation of Lactuca sativa plants due to treatment with silica, palladium,
gold, and copper NPs (15 days) increased the shoot/root ratio in comparison with the control (Shah
and Belozerova 2009). Study of the effect of various concentrations of ZnO NPs introduced to
the agar media in form of suspension confirmed that at certain optimum concentration, both the
seedlings of mung (Vigna radiata) and chickpea (Cicer arietinum) displayed good growth over
control, and beyond that, retardation in growth was estimated. The presence of ZnO NPs affected
the growth of mung and chickpea seedlings at different concentrations: the maximum effect was
found at 20 ppm for mung and 1 ppm for chickpea seedlings. However, beyond this concentration,
the growth was inhibited that may be attributed to the accumulation and uptake of ZnO NPs by the
roots, which was dependent on the exposure concentrations (Mahajan et al. 2011).
33.8.2 Adverse Effects of Metal Nanoparticles to Plants

It has already been stressed that due to rapid proliferation of new materials (likely to become a
source of engineered NPs to the environment), it is indispensable to investigate their possible eco-
toxicological impacts. Cells of plants, algae, and fungi possess cell walls that constitute a primary
site for interaction and a barrier for the entrance of NPs. According to Navarro et al. (2008a), NPs
may induce the formation of new and large-size pores and routes for the internalization of large
NPs through cell walls. Thus, inside the cells, NPs might directly provoke alterations of membranes
and other cell structures and molecules, which ultimately could result in adverse effects on plants.
33.8.2.1 Ag Nanoparticles
Recently, Musante and White (2012) found that AgNPs reduced biomass and transpiration rate of
Cucurbita pepo plants by 66%–84% when compared with bulk Ag and the Ag ion concentration
was 4.4–10 times greater in NPs than in bulk particle solutions. Similar results (Hawthorne et al.
2012) were obtained at application of 250 and 750 mg L−1 AgNPs when reduced zucchini biomass
and transpiration rate by 49%–91% compared to equivalent bulk Ag were observed and the shoot
Ag content did not differ based on particle size or concentration. Using root-tip cells of Allium
cepa as an indicator organism for the study of cytotoxic and genotoxic impacts of AgNPs (below
100 nm size), it was found that with increasing concentration of applied AgNPs (0–100 ppm), the
mitotic index decreased from 60% (control) to 28% (100 ppm AgNPs) and the AgNPs impaired
stages of cell division causing chromatin bridge, stickiness, disturbed metaphase, multiple chro-
mosomal breaks, and cell disintegration (Kumari et al. 2009). Patlolla et al. (2012) with root-tip
meristem of Vicia faba investigated the genotoxicity of AgNPs under GENE-TOX-modified test
conditions. In these experiments, AgNPs exposure significantly also increased (p < 0.05) the num-
ber of chromosomal aberrations, micronuclei, and decreased the mitotic index in exposed groups
compared to control.
Gubbins et al. (2011) observed the inhibition of L. minor L. plant growth after exposure to small
(similar to 20 nm) and larger (similar to 100 nm) AgNPs at low concentrations (5 μg L−1). This effect
was more acute with a longer exposure time. In the experiment with L. multiflorum seedlings (Yin
et al. 2011), it was found that AgNPs inhibited growth, but root and shoot Ag content increased with
increasing AgNPs exposures. Exposed to 40 mg L−1 gum arabic-coated AgNPs, seedlings failed to
develop root hairs and had highly vacuolated and collapsed cortical cells and broken epidermis and
root cap. Such effects were not observable in seedlings exposed to identical concentrations of AgNO3
or supernatants of ultracentrifuged AgNP solutions. However, AgNP toxicity was affected by total NP
surface area with smaller AgNPs (6 nm) more strongly affecting growth than did similar concentra-
tions of larger (25 nm) NPs for a given mass. Silver speciation within exposed roots documented by
x-ray spectromicroscopy suggested that silver is oxidized within plant tissues. Consequently, it could
be assumed that growth inhibition and cell damage can be directly attributed either to the NPs them-
selves or to the ability of AgNPs to deliver dissolved Ag to critical biotic receptors. Inhibitions of root
growth was observed at 1000, 1200, and 1600 μg mL−1 AgNPs for Oryza sativa, Brassica campestris,
and Vigna radiata, respectively. Comparative inhibitions of roots by ions were found to be 4500, 6000,
and 3000 μg mL−1, respectively (Mazumdar and Ahmed 2011).
In the seed germination tests with Lycopersicum esculentum and Z. mays (Ravindran et al.
2012), it was shown that AgNPs exhibited more toxicity than equivalent silver ion concentration
by inhibiting root length and germination in a concentration-dependent manner. At the same time,
L. esculentum was found to be more sensitive to the treatments than Z. mays. On the other hand,
morphological studies under SEM showed that no severe toxic effects were observed in hydroponi-
cally grown Bacopa monnieri (Linn.) Wettst. treated with AgNPs, while structural aberrations were
observed in the light microscopic evaluation of root and stem anatomy (Krishnaraj et al. 2012).
The period during which a cut flower or cut foliage retains its appearance in a vase is called “vase
life.” Short-lived cut Acacia holosericea foliage was used in experiments when three chemically
different nano-silver (nano-Ag) formulations (neutral, acidic, and ionic) were applied as vase (lower
concentrations) or pulse (higher concentrations) solutions to evaluate their potential to extend the
vase life. The found results shown significant extension of the vase life of A. holosericea obtained
with the following treatments: neutral nano-Ag as a 4 mg L−1 vase solution or as a 40 mg L−1 24 h
pulse treatment and acidic nano-Ag as a 0.5 mg L−1 vase solution or as a 5 mg L−1 24 h pulse treat-
ment. Vase life extensions over the deionized water controls were associated with better mainte-
nance of relative fresh weight and vase water uptake, suppression of bacterial growth in the vase
water and stem end, and delaying stem blockage. In contrast, ionic nano-Ag applied as a 0.5 or
1 mg L−1 vase solution treatment or as a 5 or 10 mg L−1 pulse treatment caused severe phytotoxicity
to cut A. holosericea stems. These results of Liu et al. (2012) suggested that nano-Ag treatments,
especially neutral nano-Ag and acidic nano-Ag pulse treatments, could be a potential postharvest
technology for commercial application to cut A. holosericea.
Application of AgNPs and AgNO3 on aquatic plant duckweed (Spirodela polyrhiza) in the experi-
ments of Jiang et al. (2012) confirmed that Ag content in plant tissue increased significantly with
higher concentrations of AgNPs and AgNO3, which was accompanied with significant decrease of
plant biomass, disintegration of S. polyrhiza colonies, and with root abscission. Significant decrease of
plant tissue nitrate nitrogen content, chlorophyll a content, chlorophyll a/b ratio, as well as chlorophyll
fluorescence (Fv /Fm) and changes in soluble carbohydrate and proline content were estimated after
both AgNPs and AgNO3 treatment. The values of median effective concentration (EC50) for Chl a and
phosphate content showed that AgNO3 was more toxic than AgNPs (EC50 values: 16.10 ± 0.75 vs.
7.96 ± 0.81 and 17.33 ± 4.47 vs. 9.14 ± 2.89 mg Ag L−1, respectively), whereas based on dry weight
EC50 values, AgNPs were found to be more toxic than AgNO3 (13.39 ± 1.06 vs. 17.67± 1.16 mg Ag L−1).
33.8.2.2 ZnO Nanoparticles
Study of the impact of ZnO NPs on the root growth, root apical meristem mitosis, and mitotic
aberrations of garlic (Allium sativum L.) showed that ZnO NPs caused a concentration-dependent
inhibition of root length. Treatment with 50 mg L−1 ZnO NPs for 24 h resulted in complete inhibi-
tion of root growth, while the 50% inhibitory concentration (EC50) was estimated to be 15 mg L−1
(Shaymurat et al. 2012). After ZnO NPs application, the decrease of mitosis index in a concentration-
and time-dependent manner and induction of several kinds of mitotic aberrations, mainly chromo-
some stickiness, bridges, breakages, and laggings, were observed. Total percentage of abnormal
cells increased with the increase of ZnO NPs concentration and the prolongation of treatment time.
Kumari et al. (2011) also observed an increase of chromosomal aberration index in A. cepa roots
of hydroponically cultivated plants with the increasing concentrations of ZnO NPs. Moreover, the
frequency of micronucleated cells was higher in ZnO NP-treated cells as compared to control and
the effect of ZnO NPs on lipid peroxidation was evident at all the concentrations (25, 50, 75, and
100 μg mL−1) compared to bulk ZnO. The TEM image confirmed internalization of ZnO NP-like
particles and SEM image of treated A. cepa demonstrated that the internalized NPs agglomerated
depending on the physicochemical environment inside the cell.
Application of 400, 2000, and 4000 mg L−1 of ZnO NPs inhibited seed germination, root elonga-
tion, and number of leaves of A. thaliana (Lee et al. 2010). NPs exerted higher toxicity than larger
(micron-sized) particles at equivalent concentrations. Since significant inhibition was observed by
the smaller, monodisperse nano-ZnO particles (44.4 nm), it could be suggested that the transport
of soluble nutrients as well as small particles to the embryo was facilitated by intracellular spaces
(<10 mm) in seed coat parenchyma filled with aqueous media. Because complete (100%) inhibition
of seed germination was obtained only with the application of 500 mg L−1 of soluble Zn, that is, at
concentration, which was one order of magnitude higher than the amount released by toxic levels
of ZnO NPs, the phytotoxicity contributed not only to the dissolved metal species but also to the
particles themselves.
Treatment of hydroponically cultivated C. sativus seedlings with Zn2+ as well as ZnO NPs (size
of 50 nm) resulted in plant biomass reduction, and the corresponding EC50 values were as fol-
lows: 629 mg L−1 (ZnO NPs) and 262 mg L−1 (Zn2+), respectively (Kim et al. 2012). More effective
aggregation of NPs was observed in the nutrient solutions in comparison with the deionized water
and the size of the aggregated Zn and ZnO NPs was found to be 500 nm. The NPs crossed the cell
membrane and formed agglomerates, either with themselves or with other cellular materials within
the cells, and induced toxic effects within the cell. High Zn accumulation in C. sativus due to ZnO
NP treatments indicates high defense mechanism of this plant to this toxicant, and increased metal
uptake by plants is probably connected with the ability of root exudates to change the property and
behavior of ZnO NPs in solution. Increased antioxidant enzyme (SOD, CAT, and POD) activities in
plant root tissues exposed to ZnO NPs were observed, as well.
De La Rosa et al. (2011) studied phytotoxicity of ZnO NPs at germination stage of the
following desert plants: blue palo verde (Parkinsonia florida), velvet mesquite (Prosopis
juliflora-velutina), and tumbleweed (Salsola tragus). Whereas germination was not significantly
affected in any of the three plants species, application of 4000 mg ZnO NPs L−1 reduced root
elongation in blue palo verde by 16%, with respect to control; tumbleweed root size diminished
by 14% and 16% at ZnO NP levels of 500 and 2000 mg L−1, respectively; and velvet mesquite
root length was reduced at all ZnO NP concentrations used in this study (EC50 was between
1000 and 2000 mg L −1). Results found by XAS demonstrated that ZnO NPs were biotrans-
formed on/within the root and Zn was present as Zn(II) in the all investigated desert plant species.
Boonyanitipong et al. (2011) reported detrimental effects of ZnO NPs on rice (O. sativa L.) roots
resulting in stunt roots length and reduced number of roots, while germination of seeds was not
affected. The study of inhibition of the growth of mung (V. radiata) and chickpea (C. arietinum)
seedlings by ZnO NPs (Mahajan et al. 2011) carried out in plant agar media to prevent precipita-
tion of water-insoluble NPs also showed that beyond 20 ppm for mung (V. radiata) and 1 ppm
for gram (C. arietinum) seedlings, the growth of plants was inhibited, while at lower NP concen-
trations, plant growth stimulation was observed. Absorption of NPs by seedlings root was also
detected by inductive coupled plasma/atomic emission spectroscopy.
Phytotoxic effects of cobalt and ZnO NPs on roots of hydroponically cultivated A. cepa (onion
bulbs) were investigated by Ghodake et al. (2011) who found that with increasing concentrations
of the NPs (from 5 to 20 μg mL−1), the elongation of the roots related to the control plants was
severely inhibited by both the metal oxide NPs. The shapes of applied metal oxide NPs differed
one from others: cobalt oxide NPs were found to be of spherical, truncated, and uneven nature
with an average size of approximately 60–10 nm, while most of ZnO NPs were rod-shaped,
spherical, or hexagonal with sizes ranging between approximately 50 and 100 nm, and the par-
ticles were found to be clustered. During the exposure of the A. cepa roots to both metal oxide
NPs, aggregation and precipitation occurred, probably due to the interaction of these NPs with
unknown extracellular biomolecules. Massive adsorption of cobalt oxide NPs into the root system
was the reason of resulting phytotoxicity. On the other hand, damaging effects of ZnO NPs were
connected with their severe accumulation in both the cellular and the chromosomal modules.
Thus, it could be supposed that the cobalt oxide NPs could block the water channels through
adsorption and the ZnO NPs possibly penetrate radically into the onion roots and spoil the whole
cellular metabolism and stages of cell division.
33.8.2.3 Cu and CuO Nanoparticles

Investigation of copper toxicity to C. pepo plants showed that both bulk particles and CuNPs were
highly phytotoxic causing 60%–70% reduction of growth and transpiration rate relative to untreated
controls. Concentration of Cu ion was 1.4–4.4 times greater in bulk than NPs-amended solutions
(Musante and White 2012). Similar results related to toxic effects on growth and transpiration rate
of zucchini plants due to treatment with CuNPs were obtained by Hawthorne et al. (2012) who con-
firmed phytotoxic effects for both bulk and CuNPs and observed that the pH of NP Cu solution was
significantly higher than that of bulk Cu.
Wang et al. (2012) studied the toxicity of CuO NPs to maize (Z. mays L.) and their transport and
redistribution in the plant. Treatment with 100 mg L−1 CuO showed no effect on germination, but
this concentration inhibited the growth of maize plants. On the other hand, dissolved Cu2+ ions and
CuO bulk particles had no obvious impact on maize growth. Using TEM and EDS techniques, it
was found that CuO NPs were present in xylem sap, and they were transported from roots into the
shoots through the xylem. However, split-root experiments and HRTEM observation confirmed that
CuO NPs could also translocate from shoots back into the roots through phloem, and during
this translocation CuO NPs could be reduced from Cu(II) to Cu(I). It should be stressed that this
direct evidence for the bioaccumulation and biotransformation of CuO NPs (20–40 nm) in maize
has significant implications on the potential risk of NPs and food safety.
According to Kim et al. (2012), exposure of hydroponically cultivated C. sativus seedlings to
Cu2+ as well as to CuNPs and CuO NPs of the size of 50 nm resulted in plant biomass reduc-
tion; corresponding EC50 values were as follows: 333 mg L −1 (CuNPs), 376 mg L −1 (CuO NPs),
14 mg L−1 (Cu2+). It was observed that NPs were more greatly aggregated in the nutrient solutions
than in the deionized water. It seems that NPs crossed the cell membrane and formed agglomerates,
either with themselves or with other cellular materials within the cells because both NPs increased
the Cu concentrations in C. sativus tissues and the presence of NPs or NP aggregates within the cell
resulted in toxic effects. Increased antioxidant enzyme (SOD, CAT, and POD) activities in plant root
tissues exposed to CuO NPs were estimated, as well.
Study of growth inhibition of duckweed (Landoltia punctata) treated with CuO NPs and compa-
rable doses of soluble Cu showed that concentrations causing the 50% growth inhibition were found
to be 0.6 mg L−1 soluble copper or 1.0 mg L−1 CuO NPs that released only 0.16 mg L−1 soluble Cu
into growth medium (Shi et al. 2011). Whereas the level of chlorophyll in plants was not affected
after application of 0.2 mg L−1 soluble Cu, treatment with 1.0 mg L−1 CuO NPs resulted in signifi-
cant decrease of this assimilation pigment in plants. These findings could be connected with the fact
that the Cu content of fronds exposed to CuO NPs was four times higher than in fronds exposed to
an equivalent dose of soluble copper.
Higher sensitivity of Phaseolus radiatus to CuNPs in comparison with the effects of nanoscale
Cu particles on T. aestivum was confirmed in experiments of Lee et al. (2008) who found 2-d
median effective concentrations 335 mg L−1 for P. radiatus and 570 mg L−1 for T. aestivum. The
apparent toxicity clearly resulted from CuNPs because cupric ions released from CuNPs had negli-
gible effects. With increasing concentration of CuNPs, bioaccumulation was enhanced and agglom-
eration of particles was observed in the cells using TEM and EDS.
In Elodea densa (Planch) plants treated with Cu2+ ions and CuNPs, observed lipid peroxidation
reached 120% and 180% of the control, respectively. Due to more active accumulation of CuNPs
by plants, CAT and SOD activities in plants treated with NPs increased by a factor of 1.5–2.0 and
photosynthesis was suppressed at a concentration of 1.0 mg L−1, while Cu2+ ions reduced photosyn-
thesis already at a concentration of 0.5 mg L−1 (Nekrasova et al. 2011). Induction of DNA damage
in agricultural and grassland plants by CuO NPs was for the first time reported by Atha et al. (2012)
who observed significant accumulation of oxidatively modified, mutagenic DNA lesions and strong
plant growth inhibition in radish (Raphanus sativus), perennial ryegrass (Lolium perenne), and
annual ryegrass (Lolium rigidum) under controlled laboratory conditions.
33.8.2.4 Al2O3, SiO2, TiO2, and Fe3O4 Nanoparticles

Lee et al. (2010) published that phytotoxic effects of three metal oxide NPs (Al2O3 NPs, SiO2 NPs,
and Fe3O4 NPs) applied at three different concentrations (400, 2000, and 4000 mg L−1) related to
seed germination, root elongation, and number of leaves of A. thaliana decreased in the following
order: Fe3O4 NPs > SiO2 NPs > Al2O3 NPs. In the experiment of Burklew et al. (2012), Al2O3 NPs
were found to have a negative effect on the growth and development of tobacco (N. tabacum) seed-
lings. The authors also suggested that miRNA may play a role in the ability of plants to withstand
stress to Al2O3 NPs in the environment.
In cucumber plants, Fe3O4 and TiO2 NPs negatively affected seed germination rate, root elonga-
tion, and germination index (Mushtaq 2011). Effects of TiO2 NPs on seed germination and root elon-
gation applying at concentration range from 0.2 to 4.0 parts per thousand in the plants Z. mays L.
and Vicia narbonensis L. resulted in delayed germination progression for the first 24 h in both
materials, while root elongation was affected only after treatment with the higher nano-TiO2 con-
centration. Further significant effects were also detected showing mitotic index reduction and
concentration-dependent increase in the aberration emergence that evidenced a nano-TiO2-induced
genotoxic effect for both species. The results published by Giorgetti et al. (2011) showed that Fe3O4
NPs of 6 nm diameter dispensed at doses ranging from 2.01 to 33.5 mg L−1 to carrot in vitro cultures
slightly affected growth, mitotic index, and dedifferentiation in cell cultures of Daucus carota L.
at exposure from 2.01 to 6.70 mg L−1. However, the authors stressed that at the concentration close
to the 20.10 mg L−1, cell growth and relative mitotic index dropped dramatically and stopped com-
pletely at concentration of 33.5 mg L−1. These doses entirely blocked embryo formation when treat-
ments were done along somatic embryogenesis induction.
33.8.2.5 Rare Earth Elements

Zhang et al. (2012) studied the phytotoxicity of nanoparticulate Yb2O3, bulk Yb2O3, and YbCl3 · 6H2O
to cucumber plants and found that the decrease of biomass was evident at the lowest concentration
(0.32 mg L−1) when exposed to Yb2O3 NPs, while at the highest concentration, the most severe
inhibition was from YbCl3. The inhibition depended on the actual amount of toxic Yb uptake by
the cucumber plants. In the intercellular regions of the roots, Yb2O3 particles and YbCl3 were all
transformed to YbPO4 and similar results were obtained in assessing the phytotoxicity of La2O3
NPs to cucumber plants in which LaCl3 was also studied as a reference toxicant (Ma et al. 2011). In
these experiments, both La2O3 NPs and LaCl3 were transformed to needlelike LaPO4 nanoclusters
in the intercellular regions of the cucumber roots. Significant enhancement of the dissolution of
La2O3 NPs by acetic acid, which was observed in in vitro experiments, suggested that the dissolu-
tion of NPs at the root surface induced by the organic acids extruded from root cells could play an
important role in the phytotoxicity of La2O3 NPs.
33.9 APPLICATION OF METAL NANOPARTICLES

IN PHYTOREMEDIATION TECHNOLOGY
Some principles of phytoremediation have been considered useful to be applied in metal nano-
technology. While many studies have looked at metal uptake by plants, particularly with regard to
phytoremediation and hyperaccumulation, and a few have distinguished between elemental metal
deposition and metal salt accumulation, quantification of the proportion of metal salts converted to
MNPs has only rarely been addressed. On the other hand, powerful investigation techniques have
only become available in the last decade, allowing more detailed studies on metal compartmental-
ization in plant tissues (for details, see Marchiol 2012).
In recently appeared paper (Qu et al. 2011) a new route for the recycling of metals in plant
sources using green metallic oxide NP synthesis is provided. In this paper, authors mentioned that
there are not only different kinds of metal hyperaccumulators in the world, but metals in plants
are also a resource that can be recycled, lowering the need for metal mines. Using renewable
natural products, such as these metal sources, it offers numerous benefits ranging from increased
environmental safety to further exploitation of existing plant and natural products. In experi-
ments with wild plant species Physalis alkekengi L., classified as a Zn hyperaccumulator, syn-
thesis of ZnO NPs was observed. The XRD pattern and EDS spectrum clearly confirmed that
ZnO NPs showed a polydisperse behavior, crystalline or triangular character, and a mean size of
72.5 nm. These results indicated both that P. alkekengi could be used for the remediation of zinc-
contaminated soils and the methodical procedure of synthesizing ZnO NPs from Zn hyperac-
cumulator plants constitutes a new insight into the recycling of metals in plant sources. However,
to realize the transformation of Zn in plants to ZnO NPs, two problems must be solved. The first
problem is the extraction of Zn from plants, and the second one is the separation of other metals
from the plants during the extraction process to acquire pure ZnO NPs. The previously mentioned
authors stressed that ZnO NPs were synthesized using P. alkekengi, which grew healthily at Zn
levels from 50 to 5000 mg kg−1 in soils. The plants incorporated Zn into the shoots 235–10,980
mg kg−1 dry mass during 12 weeks. In conclusion, it should be stressed that to achieve large
practical application of NPs in phytoremediation technology, the further development of green
metallic oxide NPs, like the ZnO NPs, is needed.
MNPs are attracting attention for many technological applications as catalysts, in optical materials,
medical treatments, sensors, and energy storage and transmission. The great interest in MNPs is a
result of their unique properties, mainly electronic, magnetic, and optical. The controlled synthesis
of the size and shape of MNPs is essential in order to explore their unique chemical and physical
features. Currently, there are various chemical and physical synthetic methods used for synthesis
of MNPs as well as several experimental techniques aimed at controlling their size and shape.
However, most of these methods utilize toxic and expensive chemicals, and problems are often
experienced with stability and agglomeration of particles. Therefore, synthesis of MNPs based on
biological material (“green synthesis” as an environmentally benign process) including bacteria,
algae, and vascular plants (mainly metallophytes) is preferred. In biological methods for preparation
of MNPs, mainly leaf reductants occurring in leaf extracts are used. However, MNPs can be formed
also directly in living plants by reduction of the metal ions absorbed as a soluble salt, indicating that
plants are a suitable vehicle for their production. Besides MNP formation and determination, action
of MNPs on living organisms is important, too. Nevertheless, it should be stressed that MNPs’
impact on human and environmental health remains still unclear.
ACKNOWLEDGMENTS
This chapter was in part financially supported by Sanofi Aventis Pharma Slovakia. This chapter
is sincerely dedicated to Assoc. Prof. Dr. Vladimír Bella and Mgr. Erika Zámečníková from
St. Elisabeth Oncology Institute in Bratislava, Slovakia.
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34 Arsenic Toxicity and Tolerance
Mechanisms in Crop Plants
Pallavi Sharma, Ambuj Bhushan Jha,
and Rama Shanker Dubey
CONTENTS
34.1 Introduction........................................................................................................................... 733
34.2 Arsenic as Toxic Metal.......................................................................................................... 735
34.2.1 Toxic Species of Arsenic........................................................................................... 735
34.2.2 Sources of Arsenic to the Soil................................................................................... 737
34.2.3 Uptake of Arsenic by Plants...................................................................................... 739
34.2.3.1 Uptake of Arsenite...................................................................................... 741
34.2.3.2 Uptake of Arsenate..................................................................................... 742
34.2.4 Symptoms of Arsenic Toxicity.................................................................................. 743
34.3 Metabolic Alterations in Arsenic-Stressed Plants................................................................. 745
34.4 Oxidative Stress and Antioxidative Defense under Arsenic Toxicity................................... 749
34.4.1 Nonenzymatic Antioxidants...................................................................................... 752
34.4.2 Enzymatic Antioxidants............................................................................................ 753
34.5 Arsenic Tolerance Mechanisms in Plants.............................................................................. 756
34.5.1 Suppression of High-Affinity Phosphate/Arsenate Transport................................... 758
34.5.2 Reduction of Arsenate to Arsenite............................................................................ 758
34.5.3 Increased Synthesis of Glutathione and Phytochelatins............................................ 759
34.5.4 Arsenic Sequestration in the Vacuoles...................................................................... 760
34.5.5 Arsenic Efflux............................................................................................................ 760
34.5.6 Methylation and Volatilization.................................................................................. 761
34.6 Strategies for Developing Arsenic Tolerance in Plants......................................................... 762
34.7 Phytoremediation of Arsenic-Polluted Soil........................................................................... 765
34.8 Conclusions and Future Prospects......................................................................................... 768
References....................................................................................................................................... 768
34.1 INTRODUCTION
Arsenic (As) is a ubiquitous element present in the environment. Although metalloid, it is often
grouped among the toxic metals. It is the 20th most abundant element in the Earth’s crust, with an
average concentration ranging from 1.5 to 5 mg/kg (Cullen and Reimer, 1989). In noncontaminated
soils, arsenic concentrations typically range from 0.2 to 40 mg/kg (World Health Organization,
1981), whereas in contaminated soils, arsenic concentrations as high as 100–2500 mg/kg have been
reported (World Health Organization, 1981; Diaz-Barriga et al., 1993; Vaughan, 1993). Arsenic can
enter soil through both natural processes such as weathering and erosion of arsenic-bearing rocks
(Alloway, 1990; Yan-Chu, 1994) and anthropological activities such as mining, smelting of ores,
coal combustion, release of arsenic-laden liquid and solid wastes from industrial plants, irrigation
with arsenic-contaminated water, and use of arsenic-based pesticides, herbicides, and fertilizers
733
(reviewed by Smith et al., 1998; Mahimairaja et al., 2005). Arsenic is a significant contaminant
of soils and groundwater in many regions of the world including Argentina, Bangladesh, Chile,
Mexico, China, Hungary, India, and Vietnam (Mahimairaja et al., 2005 and references therein), and
the situation is worst in the densely populated floodplains and river deltas of South and Southeast
Asia (Nordstrom, 2002; Brammer and Ravenscroft, 2009). Arsenic leads to stimulation of plant
growth at low concentrations (Woolson et al., 1971; Carbonell-Barrachina et al., 1997; Miteva,
2002; Garg and Singla, 2011); however, at high concentrations, it not only exerts toxic effects on
plants and animals but may pose severe health complications for humans and animals.
Arsenic exists in the form of different chemical species that can be classified as inorganic
(arsenite [As3+] and arsenate [As5+]) and organic (e.g., monomethylarsenate [MMA], dimethylarse-
nate [DMA], arsenobetaines, arsenocholines, and arsenosugars) species. Plants and soils contain
predominately inorganic arsenic species with lower levels of organic arsenic species (Bowell, 1994;
Koch et al., 2000; Kuehnelt et al., 2000; Geiszinger et al., 2002; Meharg and Hartley-Whitaker,
2002; Bissen and Frimmel, 2003; Szakova et al., 2005). Interconversion among arsenic species is
regulated by biotic and abiotic processes (Takamatsu et al., 1982; Meharg and Hartley-Whitaker,
2002). Reduction–oxidation (redox) reactions mediated by soil microorganisms are suggested to
be responsible for the origin of organic forms of arsenic (Pongratz, 1998; Geiszinger et al., 2002;
Huang and Matzner, 2007). Whether plants synthesize these organic species is not known. The
amount of arsenic absorbed by plants depends on many factors including plant species and geno-
types (Liebig, 1966; Bernal and Peterson, 1975; Wildung et al., 1981; Chaturvedi, 2006; Abbas and
Meharg, 2008), concentration and forms of arsenic in the soil (National Academy of Sciences, 1977;
Carbonell-Barrachina et al., 1997; Tu and Ma, 2002; Chaturvedi, 2006; Abbas and Meharg, 2008),
and soil properties such as soil acidity, redox potential, clay content, organic matter, amounts of
phosphorus (P), iron oxide (Fe2O3), and other ions and soil microorganisms (Rumberg et al., 1960;
Dickens and Hiltbold, 1967; Von Endt et al., 1968; Johnson and Hiltbold, 1969; Woolson et al., 1973;
Thornton, 1994; Smith et al., 2002; Tu and Ma, 2003). Aquaglyceroporins mediate uptake of As3+
and undissociated methylated arsenic species in plants (Meharg and Jardine, 2003; Li et al., 2009),
whereas As5+, which acts as a phosphate analog, competes with phosphate for the phosphate uptake
system (Asher and Reay, 1979; Ullrich-Eberius et al., 1989). The Michaelis–Menten model can
describe the uptake kinetics of As3+ as well as As5+ (Abedin et al., 2002b; Abbas and Meharg, 2008).
Due to its high affinity to bind with sulfhydryl groups, As3+ can disrupt the structure and func-
tions of proteins involved in cellular metabolism (Meharg and Hartley-Whitaker, 2002; Ali et al.,
2009), whereas being a phosphate analog, As5+ can substitute inorganic phosphate in a plethora of
biochemical processes including oxidative phosphorylation (Dixon, 1997). MMA and DMA have
been suggested to block protein synthesis in plants (Sckerl and Frans, 1969). Although inorganic
forms of arsenic are generally considered more toxic compared to organic forms (Marin et al.,
1992; Meharg and Hartley-Whittaker, 2002), in several plant species including tomato (Burlo et al.,
1999; Tlustos et al., 2006), radish (Rhapanus sativus) (Carbonell-Barrachina et al., 1999a), turnip
(Brassica napus) (Carbonell-Barrachina et al., 1999b), and Spartina species (Carbonell-Barrachina
et al., 1998a), DMA and MMA have been found to be more toxic than the inorganic forms.
At higher concentrations, arsenic adversely affects a range of key metabolic processes in plants
such as respiration (Chen and Liu, 1993; Liu and Wang, 2002; Shao et al., 2011), nitrogen metabo-
lism (Jha and Dubey, 2004a; Chang et al., 2006; Singh et al., 2009), carbohydrate metabolism
(Miteva and Merakchiyska, 2002; Jha and Dubey, 2004b, 2005), phosphate metabolism (Dixon,
1997; Mishra and Dubey, 2008), thiol metabolism (Srivastava et al., 2009; Tripathi et al., 2012), as
well as RNA and protein metabolism (Mishra and Dubey, 2006) that may result in poor growth,
reduced yield, and ultimately death of the plant (Meharg and Hartley-Whitaker, 2002; Tu and Ma,
2002). Arsenic is a redox-active metal, and exposure of plants to the inorganic as well as organic
form of arsenic has been reported to result in the excessive production of reactive oxygen species
(ROS) such as superoxide anion O2 i- and hydrogen peroxide (H2O2), causing progressive oxidative
damage in plants (Hartley-Whitaker et al., 2001a; Mascher et al., 2002; Gunes et al., 2009; Mishra
Arsenic Toxicity and Tolerance Mechanisms in Crop Plants 735
et al., 2011a). Plants respond to arsenic treatment by altering the levels of many enzymatic and
nonenzymatic antioxidants, which may serve as important components in mitigating As3+-induced
oxidative damage (Mishra et al., 2011a).
A variety of tolerance and resistance mechanisms including suppression of the high-affinity phos-
phate uptake system, a major pathway for As5+ entry (Meharg and Macnair, 1991, 1992), reduction
of As5+ to As3+ (Pickering et al., 2000; Dhankher et al., 2002), and sequestration of As3+ in roots by
complex formation with thiols, particularly phytochelatins (PCs) (Pickering et al., 2000; Schmoger
et al., 2000; Hartley-Whitaker et al., 2001b), have been reported in arsenic non-hyperaccumulating
plants that help them survive in arsenic-enriched environments. In contrast to non-hyperaccumulat-
ing plant species, which have a threshold concentration of phytotoxicity between 5 and 100 mg/kg
dry weight (Kabata-Pendias and Pendias, 1992), arsenic hyperaccumulating ferns such as Chinese
brake fern (Pteris vittata) possess a remarkable ability to tolerate exceedingly high concentrations
(>10,000 mg/kg) of arsenic in the fronds (Tu and Ma, 2002; Wang et al., 2002). Strategies usually
include reduction of As5+ to As3+ in cytoplasm, chelation of As3+ by PCs (Pickering et al., 2000;
Li et al., 2004), and sequestration of free As3+ or arsenic–PC complex in vacuole (Lombi et al., 2002;
Pickering et al., 2006; Indriolo et al., 2010). Arsenic hyperaccumulator plants take up and seques-
ter exceptional concentrations of arsenic in the aboveground parts and hence offer a great promise
for phytoremediation of arsenic, which is a less expensive, more effective, and an environmentally
friendly alternative to physicochemical remediation of arsenic (Rathinasabapathi et al., 2006).
Plant breeding and genetic modification methods can be employed to develop arsenic tolerance
in plants. Strategies for developing arsenic tolerance in plants usually include limiting the uptake
of arsenic or enhancing the detoxification reduction of As5+ to As3+ in cytoplasm, chelation of As3+
by PCs, and eventual sequestration of As3+ or arsenic–PC complexes into vacuoles to be stored.
As3+ extrusion and arsenic volatilization might also be helpful. Very few studies report the devel-
opment of arsenic-tolerant plants using overexpression of single or two genes suggesting that arse-
nic tolerance in crops may require a multifaceted approach. This chapter presents an overview of
arsenic toxicity and tolerance mechanisms in plants. Strategies for developing arsenic tolerance in
plants and phytoremediation of arsenic-polluted soil are also discussed.
34.2 ARSENIC AS TOXIC METAL

Although metalloid, arsenic is typically considered as toxic metal and shares many toxic charac-
teristics with other heavy metals like lead and mercury. It exists under the form of various chemi-
cal species that differ in bioavailability, mobility, toxicity, biotransformation, and physicochemical
behavior (Maeda, 1994). Excess arsenic in soil can arise from both natural and anthropogenic
sources. Plants absorb arsenic from soil through their roots. The phytotoxicity of arsenic varies
with the plant species, the soil arsenic levels, soil characteristics, and the type of arsenic species
(Horswell and Speir, 2006). For many crops, arsenic toxic levels range from 2 to 50 mg/kg dry soil,
and arsenic concentrations between 0.1 and 5.0 mg/kg shoot dry matter are considered toxic to the
plants (Wauchope, 1983). Toxic species of arsenic, their sources to the soil, uptake mechanism, and
symptoms of arsenic toxicity in plants are discussed briefly in this section.
34.2.1 Toxic Species of Arsenic

Arsenic exists in the form of various chemical species in soil and plants, which can be classified
as inorganic and organic forms. Arsenic can occur in five oxidation states as +5 (As5+), +3 (As3+),
+1 (arsonium metal), 0 (arsenic), and −3 (arsine). The speciation and solubility of arsenic are sensi-
tive to redox condition and pH of the soil that in turn affects its bioavailability, toxicity, and mobility
(Figure 34.1). Arsenic speciation is suggested to be more important than the arsenic concentration
in solution in determining the phytotoxic effects of arsenic to tomato (Lycopersicum esculentum)
Soil properties
pH
Plant species Clay content
Presence of other ions
Redox potential
Concentration
of arsenic in the soil
Arsenic speciation
and solubility
Arsenic
phytoavailability
and phytotoxicity
FIGURE 34.1 Factors determining arsenic availability and toxicity in plants. Speciation and solubility of
arsenic are sensitive to soil properties such as pH of the environment, clay content, redox conditions, and other
ions. Soil properties, concentration of arsenic in soil, and plant species determine arsenic phytoavailability
and phytotoxicity.
plants (Burlo et al., 1999). Interconversion between arsenic species is regulated by biotic and abiotic
processes (Takamatsu et al., 1982; Meharg and Hartley-Whitaker, 2002).
Inorganic arsenic compounds are most abundant in soil as well as plants (Bowell, 1994;
Kuehnelt et al., 2000; Geiszinger et al., 2002; Bissen and Frimmel, 2003; Szakova et al., 2005)
and include pentavalent As5+ (occurring as H2AsO4− and HAsSO2− 4 in most environments) and the
trivalent As3+ (As2O3), which dissolves as As(OH)3. Under aerobic conditions, As5+ is favored over
As3+, whereas reverse is true for anaerobic condition. In non-hyperaccumulator plants such as
tomato and rice (Oryza sativa), predominant form of arsenic in the roots was found to be As3+
(Xu et al., 2007; Zhao et al., 2010a). In hyperaccumulator plant Chinese brake fern, As5+ was
found to be predominant species in roots (Zhao et al., 2002; Poynton et al., 2004; Pickering et al.,
2006; Mathews et al., 2010; Huang et al., 2011), whereas As3+ was found to be the dominant arse-
nic species in the fronds (Lombi et al., 2002; Webb et al., 2003; Mathews et al., 2010), rhizomes
(Mathews et al., 2010), and the xylem sap (Su et al., 2008). Huang and coworkers (2011) concluded
that As5+ is transformed in the roots and rhizomes to As3+ and is then efficiently translocated
to the fronds by xylem. Organic arsenic can exist in many forms such as methylated species,
MMA [CH3AsO(OH)2], DMA [(CH3)2AsO(OH)], tetramethylarsonium ion (TMA(+)), arsenobeta-
ine, arsenosugars, and arsenolipids. Soil microorganism–mediated redox reactions have been sug-
gested to be responsible for the origin of these organic forms of arsenic in soil (Pongratz, 1998;
Geiszinger et al., 2002; Huang and Matzner, 2007). In plants growing on arsenic-contaminated
soil, arsenic form was predominantly inorganic with lower levels of organic arsenic species (Koch
et al., 2000; Kuehnelt et al., 2000; Geiszinger et al., 2002; Meharg and Hartley-Whitaker, 2002),
but whether plants synthesize these organic species is not known.
The degree of bioavailability, mobility, and toxicity of arsenic species varies according to the
plant species. Inorganic forms of arsenic are generally considered more toxic compared to organic
forms (Marin et al., 1992; Meharg and Hartley-Whittaker, 2002). In general, As3+ is considered
more toxic to plants compared to As5+. As3+ inhibits cellular function by reacting with the sulfhydryl
groups of enzymes and proteins (Ullrich-Eberius et al., 1989), whereas As5+, which is an analog of
the macronutrient phosphate, disrupts metabolism by replacing inorganic phosphate in a plethora
of biochemical processes (Meharg and Hartley-Whitaker, 2002). A study of the relative toxicity of
As3+ and As5+ on germination and early seedling growth of rice revealed that As3+ was more toxic
than As5+ for rice seed germination, whereas reduction of root tolerance index caused by As5+ was
higher than that of As3+ (Abedin and Meharg, 2002). In wheat (Triticum aestivum), both As5+ and
As3+ were found to adversely affect germination, seedling growth, and amylolytic activity; however,
As3+ decreased all the endpoints more remarkably than As5+ (Liu et al., 2005). In maize (Zea mays)
seedlings, As5+ was suggested to be more toxic compared to As3+ on the basis of maximum root
growth and tolerance index (Abbas and Meharg, 2008).
MMA and DMA, present as anions in soils, appeared to be less toxic than As3+ or As5+ to plants
and blocked protein synthesis in plants (Sckerl and Frans, 1969). Relative toxicity of arsenic spe-
cies was in order of As5+ > As3+ ≫ DMA in maize seedlings (Abbas and Meharg, 2008). However,
summarization of the data on the phytotoxicity of arsenic from the literature revealed that the
inorganic sources are less toxic than the organic sources (Sheppard, 1992). Geometric means of soil
arsenic concentrations toxic for plants were reported to be 13 mg/kg for organic sources, whereas
for inorganic sources, it was 60 mg/kg (Sheppard, 1992). DMA and MMA have been found to be
more toxic than the inorganic forms in several plant species such as tomato (Burlo et al., 1999;
Tlustos et al., 2006), radish (R. sativus) (Carbonell-Barrachina et al., 1999a), turnip (B. napus)
(Carbonell-Barrachina et al., 1999b), and Spartina species (Carbonell-Barrachina et al., 1998a).
Significantly greater toxicity of DMA than inorganic As5+ was observed in the arsenic hyperaccu-
mulators Chinese brake and Cretan brake ferns (Pteris cretica) and an arsenic-tolerant plant China
grass (Boehmeria nivea) (Huang et al., 2008). MMA was found to be the most toxic arsenic form
in rice (Marin et al., 1992). The higher degree of MMA translocation in the plant was suggested to
be responsible for larger intrinsic toxicity of MMA (Marin et al., 1992). Similarly, higher degree of
upward translocation has been suggested to contribute to the observed greater toxicity of MMA and
DMA in tomato plants leading to lower fruit yields (Burlo et al., 1999).
While arsenobetaine and arsenosugars are considered to be relatively nontoxic compared with inor-
ganic arsenic species (Kaise et al., 1985; Agency for Toxic Substances and Disease Registry, 2007),
biotransformations of these organic species can result in toxic arsenicals. Demethylation of DMA and
arsenobetaine in Oa (forest floor) and fen extracts was evidenced by both decreased concentrations of
DMA and arsenobetaine and apparently increased concentrations of metabolites, for example, DMA
for arsenobetaine and MMA for DMA demethylation (Huang et al., 2007). Rapid demethylation of
arsenobetaine occurs probably via the pathway arsenobetaine → dimethylarsenoylacetate → DMA,
followed by slow demethylation of DMA → MMA → inorganic arsenic species in organic soils
(Huang et al., 2007). A decomposition pathway of arsenosugars in soil amended with seaweed has
been proposed by Castlehouse and coworkers (2003) in which the arsenosugars are transformed to
DMA and further to inorganic arsenic without appreciable amounts of MMA.
34.2.2 Sources of Arsenic to the Soil

Concentration of arsenic in the soil typically ranges from 0.2 to 40 mg/kg in noncontaminated
soils (World Health Organization, 1981) to as high as 100 to 2500 mg/kg in contaminated soils
(World Health Organization, 1981; Diaz-Barriga et al., 1993; Vaughan, 1993). Arsenic can enter
soil through both natural processes and anthropological activities (Frank et al., 1976; Ball et al.,
1983; Bhumbla and Keefler, 1994; Peryea and Creger, 1994; Yan-Chu, 1994; Smith et al., 1998;
Mandal and Suzuki, 2002; Foley and Ayuso, 2008; Neihardt et al., 2012; Solgi et al., 2012). Major
sources of arsenic to the soil are shown in Figure 34.2. Primary source of arsenic in soil is the par-
ent materials from which it is derived (Yan-Chu, 1994). Arsenic is released into soils by natural
weathering and erosion processes of arsenic-bearing rocks and minerals. Variation in soil parent
material results in differences in arsenic content of the soil, for example, the lowest concentration
Weathering
Natural and erosion
processes of arsenic-
bearing rocks
Sources of Discharge of
arsenic to the industrial waste
soil
Combustion of
fossil fuels
especially coal
Mining and
smelting of arsenic-
containing ore
Anthropogenic
activities Irrigation with
arsenic-
contaminated water
Use of arsenical
pesticides,
herbicides, and
fertilizers
Use of arsenic-
containing wood
preservatives
FIGURE 34.2 Arsenic enters soil through both natural processes such as weathering and erosion of arsenic-
bearing rocks and anthropological activities such as discharge of industrial waste, combustion of fossil fuels
especially coal, mining and smelting of arsenic-containing ores, irrigation with arsenic-contaminated water,
and use of arsenical pesticides, herbicides, fertilizers, and wood preservatives.
of arsenic is found in sandy soils and those derived from granites, whereas larger concentrations
are found in alluvial and organic soils (Kabata-Pendias and Pendias, 1984). The mean concen-
trations of arsenic in igneous rocks range from 1.5 to 3.0 mg/kg, whereas in sedimentary rocks,
it ranges from 1.7 to 400 mg/kg (Smith et al., 1998). Arsenic released due to weathering process is
translocated and gets accumulated in colloidal fractions, resulting in higher level of arsenic in soil
than parent rock (Alloway, 1990; Yan-Chu, 1994). Fe hydroxides and sulfides in sedimentary rocks
coprecipitate arsenic, and soils derived from such arsenic-enriched rocks may contain as high as
20–30 mg arsenic/kg (Zou, 1986). Arsenic is also being introduced into the soil through various
anthropogenic activities such as discharge of industrial wastes; combustion of fossil fuels especially
coal, mining, and smelting of arsenic-containing ore; irrigation with arsenic-contaminated water;
and use of arsenical pesticides, herbicides, and wood preservatives (Nriagu and Pacyna, 1988;
Smith et al., 1998). Nriagu and Pacyna (1988) estimated global input of arsenic to soils by human
activities in the range of 52,000–112,000 tons/year. Major sources of arsenic discharged onto land
belong to the category of wastage of commercial products (about 40%), coal fly ash and bottom fly
ash (about 22%), mine tailings (about 16%), and atmospheric fallout from the production of steel
(about 13%) (Nriagu and Pacyna, 1988).
Industry plants that release arsenic-laden liquid and solid wastes are considered as a potential
source of soil arsenic contamination. Arsenic is used extensively in the manufacturing of ceramic
and glass, electronics, pigments and antifouling agents, cosmetics, fireworks, textiles, paper, metal
adhesives, ammunition, Cu-based alloys, pesticides, and wood preservatives (Leonard, 1991; Alam
et al., 2002). Indiscriminate discharge of industrial effluents from the manufacturing of Paris Green
(copper acetoarsenite) resulted in the contamination of soil and groundwater in residential area of
Calcutta, India (Chatterjee and Mukherjee, 1999). Recently, Solgi and coworkers (2012) found high
concentration of arsenic in soil due to industrial activities in the three industrial states in Arak, Iran.
Arsenic, widely distributed in Pb, Zn, Au, and Cu ores, can be released in soil during mining and
smelting process. Ecosystems nearby smelters can get contaminated by the flue gases and particu-
late matter released from smelting operations (Adriano, 2001). Ball and coworkers (1983) investi-
gated the extent of arsenic contamination from a Utah copper smelter, United States, and found that
arsenic soil contamination was evident up to 10 km from the smelter and arsenic had also leached
through the soil. In coal, arsenic concentrations generally vary from 2 to 82 mg/kg, depending on
geological origin (Adriano et al., 1980). Disposal of fly and bottom ashes generated from coal com-
bustion often leads to arsenic contamination of soil and water (Beretka and Nelson, 1994).
Soil contamination with arsenic also occurs through the arsenic-contaminated groundwater being
used for irrigation. In the Hetao Plain, part of China’s Inner Mongolia Autonomous Region, irriga-
tion of sunflower (Helianthus annuus) and maize field for 3 years with saline well water, with ele-
vated arsenic concentrations (154 and 238 μg/L), led to annual arsenic input of 120 and 186 mg/m2,
respectively, in the soil (Neihardt et al., 2012). Arsenic is present in many pesticides, herbicides, and
fertilizers. Historically, arsenic-containing pesticides such as lead arsenate (PbAsO4), calcium arse-
nate (CaAsO4), magnesium arsenate (MgAsO4), zinc arsenate (ZnAsO4), zinc arsenite [Zn(AsO2)2],
and Paris Green [Cu(CH3COO)2.3Cu(AsO2)2] are extensively being used in orchards and have thus
contributed to soil arsenic contamination in many parts of the world (Frank et al., 1976; Merry et al.,
1983; Peryea and Creger, 1994; Alloway, 1995). Arsenic concentrations in PbAsO4-contaminated
soils are high enough to be of potential environmental concern. Peryea and Creger (1994) studied
the vertical distribution of arsenic in six contaminated orchard soils in the State of Washington,
United States, and observed that arsenic was restricted to the upper 40 cm of soil, with concentra-
tions ranging from 0.77 to 4.85 mmol/kg. Similarly, Frank and coworkers (1976) also reported
high concentrations of arsenic in apple orchards to which PbAsO4 sprays were applied for periods
ranging from 5 to 70 years. An increase of 7.4–121 mg arsenic/kg was observed after 70 years of
PbAsO4 use. Use of organoarsenical herbicides such as monosodium methanearsonate (MSMA)
and disodium methanearsonate (DSMA) also leads to soil arsenic contamination (Robinson, 1975;
Gilmore and Wells, 1980; Smith et al., 1998). Fixation studies have shown that methylarsonate is
strongly absorbed, and in soil, it may be slowly oxidized to inorganic As5+ (Dickens and Hiltbold,
1967). Plots receiving 72, 144, and 288 kg/ha of MMA for 5 years showed significant increases in
arsenic residues (Robinson, 1975). Soil arsenic contamination also results from continuous applica-
tion of fertilizers that contain trace levels of arsenic (McLaughlin et al., 1996). Arsenic is used for
the long-term protection of wood. The two most common arsenic-containing wood preservatives
are chromated copper arsenate (CCA) and ammonium and copper arsenate (ACA). Increased con-
centrations of arsenic have been observed in soil close to CCA-treated decks and fences (Stilwell
and Gorny, 1997; Chirenje et al., 2003).
34.2.3 Uptake of Arsenic by Plants

Arsenic uptake has been well characterized in plants. Absorption of arsenic by plants is influenced
by many factors including plants species (Liebig, 1966; Walsh and Keeney, 1975; Mahmud et al.,
2006; Singh and Ma, 2006; Rofkar et al., 2007), the concentration and species of arsenic in the
soil (National Academy of Sciences, 1977; Marin et al., 1992; Mascher et al., 2002; Tu and Ma,
2002; Jha and Dubey, 2004a; Mahmud et al., 2006; Singh and Ma, 2006), soil properties such as
pH and clay content (Dickens and Hiltbold, 1967; Von Endt et al., 1968; Johnson and Hiltbold,
1969), redox potential (Marin et al., 1993), and the presence of other ions (Rumberg et al., 1960;
Woolson et al., 1973; Khattak et al., 1991). Factors determining arsenic availability and toxicity in
plants are shown in Figure 34.1. A wide range of variation exists among plant species for arsenic
absorption. A comparative study of Chinese brake fern, an arsenic hyperaccumulator, and slender
brake fern (Pteris ensiformis), a nonarsenic hyperaccumulator, revealed 20 times greater arsenic
concentration in Chinese brake fern than that of slender brake fern (Singh and Ma, 2006). In
another comparative study, castor oil plant (Ricinus communis) accumulated more arsenic than
buckwheat (Fagopyrum esculentum) (Mahmud et al., 2006). With an increase in arsenic con-
centration in the growth medium, a concomitant increase in the uptake of arsenic was noticed in
several plant species including red clover (Trifolium pratense) (Mascher et al., 2002), rice (Jha and
Dubey, 2004a), and Chinese brake fern (Tu and Ma, 2002). Under 50 μM arsenic treatment, arsenic
accumulation in roots and shoots of 20 days grown rice seedlings was more than double of the
arsenic accumulation in the roots and shoots of 25 μM arsenic-treated seedlings (Jha and Dubey,
2004a). In roots, accumulation of arsenic was almost seven times of that present in the nutrient
medium. Low translocation of arsenic from roots to shoots was observed in rice seedlings, as the
concentration of arsenic in the shoots was only up to 21% of that present in roots (Jha and Dubey,
2004a). Similar to rice, tomato plants also accumulated arsenic mainly in the root system (85% of
total arsenic) and only relatively low quantities were translocated to shoots (14%) and fruit (1%)
(Burlo et al., 1999). However, in arsenic hyperaccumulator plant, Chinese brake fern, most of the
arsenic taken up from soil was translocated to the fronds (90%), in which arsenic concentration
increased with frond age (Tu et al., 2002a). Arsenic concentration of 7230 mg/kg was observed
in the fronds of Chinese brake fern after 20 weeks with a bioconcentration factor (ratio of plant
arsenic concentration to water-soluble arsenic in soil) of 1450 and a translocation factor (ratio
of arsenic concentration in shoot to that in root) of 24 (Tu et al., 2002a).
Arsenic species differ in their solubility and mobility. Further, the degree of bioavailability of
arsenic species varies according to the plant species. Studies have shown that the order of arsenic
species availability was DMA < As5+ < MMA < As3+ in rice (Marin et al., 1992), DMA < MMA ≪
As5+ ≈ As3+ in tomato (Burlo et al., 1999), DMA ≪ MMA < As5+ < As3+ in marsh grass (Spartina
alterniflora) (Carbonell-Barrachina et al., 1998b), MMA < DMA < As3+ < As5+ in turnip (Carbonell-
Barrachina et al., 1999a), and DMA < As3+ < As5+ < MMA in radish (Carbonell-Barrachina et al.,
1999a). Upon absorption, DMA is readily translocated to shoots compared to inorganic arsenic and
MMA that are mainly accumulated in the roots of Spartina species (Carbonell-Barrachina et al.,
1998a) and rice (Marin et al., 1992), whereas both MMA and DMA have a greater upward translo-
cation than inorganic arsenic in tomato (Burlo et al., 1999). Soil properties such as pH, redox poten-
tial, clay content, organic matter, amounts of P and other ions, and soil microorganisms (Rumberg
et al., 1960; Dickens and Hiltbold, 1967; Von Endt et al., 1968; Johnson and Hiltbold, 1969; Woolson
et al., 1973; Thornton, 1994; Smith et al., 2002; Tu and Ma, 2003) affect arsenic speciation and
solubility, thereby determining arsenic phytoavailability and phytotoxicity (Marin et al., 1993).
Quazi and coworkers (2011) observed that arsenic availability to rice was mainly influenced by soil
physicochemical properties. For soil with the lowest arsenic retention capacity, highest arsenic con-
centration was found in the leachate as well as in the plant tissue, whereas soils with higher arsenic
retention capacity had higher arsenic concentrations in the surface soil that resulted in the inhibition
of plant growth. In rice plants, arsenic phytotoxicity depends on the type of soil and it is highest in
loamy sand and lowest on silty clay loam (Woolson et al., 1973). Arsenic is found to be five times
more toxic to plants in sand (mean = 40 mg/kg) than in clay soils (mean = 200 mg/kg) (Sheppard,
1992). In anaerobic soils, arsenic mostly exists as inorganic As3+, whereas under aerobic conditions,
As5+ predominates. Marin and coworkers (1993) observed higher dissolved arsenic concentrations
with decrease in pH. Most of the arsenic was present as As3+, when soil redox potential dropped
below 0 mV, whereas both As3+ and As5+ were present under more oxidizing conditions. Amended
MMA was found to be approximately two times more phytoavailable than the indigenous inorganic
arsenic forms and increased with decreasing pH and redox potential (Marin et al., 1993).
Increasing concentrations of P cause decrease in the amount of As5+ uptake by plants from the
nutrient solution (Rumburg et al., 1960). In soils containing low amounts of Fe (<100 mmol/kg),
the presence of P (0.16 mmol/L) greatly decreased As5+ sorption, whereas in soils with high Fe con-
tent (>800 mmol/kg), a similar amount of P had little effect on the amount of As5+ adsorbed by soils
(Smith et al., 2002). It is suggested that P and As5+ competes for soil sorption sites, through either
the higher affinity or the effect of mass action of the increasing concentration of P in solution (Smith
et al., 2002). The amount of As3+ sorbed decreased in the presence of P in the low-sorbing Alfisol and
high-affinity Oxisol (Smith et al., 2002). As5+ sorption increases in the presence of calcium ion (Ca2+)
compared with sodium ion (Na+) and is manifested through changes in the surface charge charac-
teristics of the soils (Smith et al., 2002). Using MSMA as a model compound to study the influence
of soil microorganisms on organic arsenical, Von Endt and coworkers (1968) found that 7%–10% of
MSMA–C14 was degraded in nonsterile soil compared with 0.7% in steam-sterilized control.
34.2.3.1 Uptake of Arsenite

As3+ is the predominant form of arsenic in anaerobic soils. In plants, As3+ uptake follows
Michaelis–Menten kinetics suggesting that transport of As3+ is an active process, which requires
an energy supply as a driving force, and selective binding sites (Abedin et al., 2002b; Chen et al.,
2005; Zhang et al., 2009). The K m values determined for rice, pea (Pisum sativum), and wheat
plants were within the same order of magnitude: 0.18, 0.34, and 0.51 mM, respectively (Meharg
and Jardine, 2003).
In Escherichia coli, yeast, and humans, aquaporins that allow passage of neutral molecules such
as glycerol can transport As3+ (reviewed by Bhattacharjee and Rosen, 2007). In addition to the
aquaporins, As3+ influx in yeast also involves hexose permeases (Liu et al., 2004b). In higher plants,
glycerol competed with As3+ for transport in a dose-dependent manner, indicating that As3+ and
glycerol uptake mechanisms were the same (Meharg and Jardine, 2003). Further, antimonite (Sb3+),
an As3+ analog that is transported into Saccharomyces cerevisiae cells by aquaporins, also com-
peted with As3+ transport in a dose-dependent manner, providing further evidence that As3+ is trans-
ported into rice roots via glycerol transporting channels (Meharg and Jardine, 2003). On the basis
of sequence similarity and localization, plant aquaporins are classified into four major subfami-
lies: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin 26–like
intrinsic membrane proteins (NIPs), and small and basic intrinsic proteins (SIPs) (Chaumont et al.,
2005). NIP has been shown to be involved in As3+ transport. To date, there is no report of As3+ per-
meability in the PIP, TIP, and SIP channel proteins in plants. It has been proposed that the substrate
selectivity of aquaporins is mainly influenced by two pore constrictions, one formed by the highly
conserved asparagine (Asn)–proline (Pro)–alanine (NPA) boxes and the other by aromatic/arginine
(ar/R) selectivity filter (Wallace et al., 2006; Maurel et al., 2008). On the basis of the sequence simi-
larity of the ar/R constriction region, the plant NIP subfamily can be further divided into groups I,
II, and III. Group I NIPs are permeable to water, glycerol, and lactic acid (Dean et al., 1999; Choi
and Roberts, 2007). Group II NIPs have larger pore size than those of the NIP I and have perme-
ability for larger solutes such as urea, formamide (Wallace and Roberts, 2005), and boric acid
(Takano et al., 2006) but only very low permeability for water (Wallace and Roberts, 2004; Mitani
et al., 2008). Among all NIP subgroups, NIP III proteins possess the largest constriction size and
allow larger solutes such as silicic acid to permeate (Ma et al., 2006, 2008; Zhao et al., 2010a,b). It
appears that transport of As3+ is not controlled by the ar/R selectivity filter as members of all three
NIP subgroups were shown to conduct As3+.
Expression of several NIP isoforms including AtNIP1;1, AtNIP2;1, AtNIP5;1, AtNIP6;1, and
AtNIP7;1 from Arabidopsis (Arabidopsis thaliana); OsNIP1;1, OsNIP2;1, OsNIP2;2, and OsNIP3;2
from rice; and LjNIP5;1 and LjNIP6;1 from Lotus (Lotus japonicus) in a S. cerevisiae ∆fps1 mutant
that is resistant to As3+ revealed that cells expressing members of the NIP II subgroup (AtNIP5;1,
AtNIP6;1, AtNIP7;1, OsNIP3;2, LjNIP5;1, and LjNIP6;1) and the NIP III subgroup (OsNIP2;1
and OsNIP2;2) facilitated As3+ transport across cell membranes and regained wild-type sensitiv-
ity to the arsenic (Bienert et al., 2008). No difference in sensitivity to arsenic was observed in
cells expressing members of the NIP I subgroup (AtNIP1;1, AtNIP2;1, and OsNIP1;1) compared to
empty vector control (Bienert et al., 2008). Similarly, Isayenkov and Maathuis (2008) also suggested
the role of AtNIP7;1, a member of the NIP II subgroup, in mediating As3+ uptake and transport
in plants. In contrast to the findings of Bienert and coworkers (2008), AtNIP1;1, a member of the
NIP I subgroup, has also been shown to be involved in As3+ uptake by roots, and loss of its function
led to reduced total arsenic and increased plant tolerance to As3+ (Kamiya et al., 2009). In rice,
mutation in OsNIP2;1 (Lsi1, a silicon influx transporter) expressed at the distal side of exodermis
and endodermis cell layers led to significantly decreased uptake of As3+, whereas mutation in sili-
con efflux transporter Lsi2 expressed proximal of exodermis and endodermis cells decreased As3+
transport to the xylem and accumulation in shoots and grain (Ma et al., 2008). Lsi1 also mediates
silicic acid influx. Silicic acid supply reduces As3+ uptake of rice, which was shown to be due to a
decreased expression of Lsi1 and Lsi2 during continuous high silicic acid supply (Ma et al., 2006,
2007). Although both As3+ and silicic acid are taken up via the same pathway, the K m value for
silicic acid is about 40 times higher than the K m value reported for As3+ (Abedin et al., 2002b; Chen
et al., 2005).
34.2.3.2 Uptake of Arsenate

Arsenate predominates in aerobic soils and is readily taken up by plant roots. In plants simi-
lar to As3+ uptake, the uptake of As5+ also follows the Michaelis–Menten equation (Abedin
et al., 2002b; Abbas and Meharg, 2008), suggesting the presence of a transporter. The K m
value of 0.0157 mM has been reported for As5+ in rice plants. As5+ is a chemical analog of
phosphate with regard to membrane transport. As5+ competes as a substrate for the phosphate
uptake system in a wide variety of plant species including non-hyperaccumulator plants such
as Arabidopsis, rice, and hyperaccumulator plants such as Chinese brake fern (Wang et al.,
2002). The uptake mechanism involves cotransport of phosphate or As5+ and protons, with
stoichiometry of at least 2H+ for each H 2PO 4 or H 2AsO 4 (Ullrich-Eberius et al., 1989). The
first evidence of competitive inhibition of As5+ uptake by phosphate in plants was presented
by Asher and Reay (1979). Phosphate was found to be a powerful inhibitor of both the initial
and the steady-state phases of As5+ uptake, whereas As5+ was a comparatively mild inhibitor of
steady-state phosphate uptake at similar concentrations (Asher and Reay, 1979). A less marked
effect of As5+ on phosphate uptake (Meharg and MacNair, 1990; Dunlop et al., 1997) led to the
hypothesis that these transporters have a higher affinity toward phosphate compared to As5+
(Asher and Reay, 1979; Ullrich-Eberius et al., 1989; Meharg et al., 1994). Reduced uptake of
As5+ is supposed to be a well-known mechanism of As5+ resistance employed by many plant
species, which is achieved through a suppression of the high-affinity phosphate/As5+ uptake
system. Meharg and Macnair (1990, 1992) demonstrated that As5+ resistance in the common
velvet grass (Holcus lanatus) was due to suppression of the high-affinity H 2PO 4 uptake sys-
tem, which led to reduced uptake of both phosphate and As5+. Arabidopsis double mutants,
defective in two phosphate transporters, Pht1;1 and Pht1;4, which play a significant role in
phosphate acquisition from both low- and high-P environments, showed enhanced resistance
to As5+ compared to the wild type, indicating that Pht1;1 and Pht1;4 mediate As5+ uptake (Shin
et al., 2004). Further, Arabidopsis mutant defective in phosphate transporter traffic facilitator 1
(PHF1) showed impaired trafficking of the Pht1;1 protein from the endoplasmic reticulum to
the plasma membrane and was much more resistant to As5+ than the wild type, supporting a role
of Pht1;1 in As5+ uptake (Gonzalez et al., 2005). A rice mutant defective in OsPHF1 lost much
of the ability to take up phosphate and As5+ and to transport them from roots to shoots, whereas
transgenic rice overexpressing either the phosphate transporter OsPht1;8 (OsPT8) or the tran-
scription factor OsPHR2 (for phosphate starvation response 2) showed enhanced abilities of
phosphate and As5+ uptake and translocation (Wu et al., 2011). OsPT8 was found to have a high
affinity for both phosphate and As5+, and its overexpression increased the maximum influx by
three- to fivefold (Wu et al., 2011).
34.2.4 Symptoms of Arsenic Toxicity

The symptoms of arsenic phytotoxicity vary with available fraction of arsenic and plant species but
toxicity symptoms are similar in majority of the plants (Figure 34.3). Plants that are not tolerant to
arsenic, when exposed to excess arsenic, exhibit toxicity symptoms such as poor seed germination
(Abedin and Meharg, 2002; Liu et al., 2005; Li et al., 2007), reduced root and shoot growth (Cox
et al., 1996; Abedin and Meharg, 2002; Shaibur and Kawai, 2009), decreased plant height (Marin
et al., 1992; Carbonell-Barrachina et al., 1995; Abedin et al., 2002a; Rahman et al., 2007), decreased
tillering (Kang et al., 1996; Rahman et al., 2004), inhibited leaf formation (Shaibur and Kawai,
2009), wilting and necrosis of leaf margins (Zhang et al., 2009), chlorosis (Mascher et al., 2002;
Singh et al., 2006), nutrient deficiencies (Shaibur et al., 2006; Shaibur and Kawai, 2010), biomass
inhibition (Zhang et al., 2009), lower fruit and grain yield (Carbonell-Barrachina et al., 1995; Kang
et al., 1996; Abedin et al., 2002a; Horswell and Speir, 2006; Zhang et al., 2009), and sometimes
death of the plant (Baker et al., 1976; Marin et al., 1992). Toxicity symptom can be correlated with
soluble arsenic concentrations in the range of 2–50 mg/kg dry soil for many crops (Wauchope, 1983).
In contrast, no visual symptoms of toxicity are observed in some arsenic hyperaccumulators such
as Chinese brake fern plants, after As5+ exposure up to 300 μM for 10 days (Srivastava et al., 2005).
Seed germination is the first and most critical stage in seedling establishment, determining suc-
cessful crop production (Almansouri et al., 2001). Plants grown in presence of As5+ as well as As3+
show reduced seed germination and growth (Abedin and Meharg, 2002; Jha and Dubey, 2004a;
Liu et al., 2005; Rahman et al., 2012). Varietal differences are observed among the varieties of
crops in response to As3+ and As5+ exposure (Abedin and Meharg, 2002; Rahman et al., 2012).
As3+ is observed to be more toxic than As5+ for rice seed germination (Abedin and Meharg, 2002).
Jha and Dubey (2005) observed inhibition in activities of amylolytic enzymes in germinating rice
Poor seed
germination
Reduction
Death in root and
shoot
growth
Lower fruit Nutrient

and grain deficiencies
yield
Arsenic
toxicity
symptoms
Decrease
Chlorosis in plant
height
Wilting and
necrosis of Decrease in
leaf Inhibition tillering
margins of leaf
formation
FIGURE 34.3 Toxicity symptoms in plants due to excess-arsenic exposure. Plants that are not tolerant to
arsenic, when exposed to excess arsenic, exhibit toxicity symptoms such as poor seed germination, marked
reduction in root and shoot growth, decrease in plant height, decrease in tillering, inhibition of leaf formation,
wilting and necrosis of leaf margins, chlorosis, nutrient deficiencies, biomass inhibition, lower fruit and grain
yield, and sometimes death.
seeds and suggested that this might contribute to delayed mobilization of endospermic starch, which
could affect germination of seeds in arsenic-polluted environment. Rahman and coworkers (2012)
observed a significant decrease in the moisture content of rice seeds with increasing As5+ concen-
trations and suggested that As5+ reduces seed germination by reducing the activities of enzymes
involved in the degradation of reserve materials stored in the endosperm (Liu et al., 2005), normal
metabolic processes, and early seedling development (Mishra and Dubey, 2006). Arsenic exposure
results in reduced root and shoot growth in plants (Cox et al., 1996; Abedin and Meharg, 2002;
Shaibur and Kawai, 2009; Choudhury et al., 2011). As3+ was found to be more toxic than As5+
in terms of root length and shoot height (Coddington, 1986; Liu et al., 2005). Arsenic-toxicity
response was found to be higher in shoot of Japanese mustard spinach (Brassica rapa) compared
to root. In Japanese mustard spinach, a significant repression in shoot growth was observed at 33.5
and 67 μM arsenic exposures, whereas arsenic limited root elongation and lateral root formation
at 67 μM level (Shaibur and Kawai, 2009). Choudhury and coworkers (2011) observed arsenic led
damage to the root epidermal cells and aerenchymatous cortex, reduction in root growth, formation
of fewer and short root hairs, and stubby and brittle roots. Arsenic led reduction in root growth is
partially attributed to rapid disruption of plasma membrane structure. Tuan and coworkers (2008)
showed that As5+ concentrations greater than 0.1 mM caused fluidization of algal cells and artificial
liposomes formation due to the binding and substitution of As5+ groups for phosphates or the choline
heads on their membrane surface. In the presence of arsenic, plants may also suffer from nutrient
deficiencies, which may not be direct effects of arsenic toxicity itself but a consequence of reduced
root growth leading to reduction of the root surface area available for uptake. Shaibur and Kawai
(2010) observed that arsenic limited the nutritional quality of Japanese mustard spinach by hamper-
ing the concentrations and accumulations of nutrient elements P, Ca, Fe, K, Mg, Mn, Cu, and Zn
in plant tissues. In rice seedlings, the concentrations of K, Mg, Fe, Mn, Zn, and Cu decreased sig-
nificantly in shoots under 26.8 μmol/L arsenic treatment; however, the concentration of P increased
in shoots at 6.7 and 13.4 pmol/L arsenic levels, indicating a cooperative rather than antagonistic
relationship (Shaibur et al., 2006). Arsenic and Fe concentrations increased in roots at higher arse-
nic treatments (Shaibur et al., 2006). Seedlings supplied with arsenic developed a reddish staining
along the root in the presence of 67 μM arsenic, suggesting the formation of Fe precipitates or Fe
plaque (Batty and Younger, 2003).
Both chlorophyll a and b contents decrease in leaves of plants with increase in soil arsenic concen-
trations (Stoeva et al., 2003; Rahman et al., 2007; Choudhury et al., 2011). In rice plants, chlorophyll
contents decreased significantly with a concomitant reduction in growth and yield of the plants sug-
gesting that arsenic toxicity affects the photosynthesis, which ultimately results in the reduction of
rice growth and yield (Rahman et al., 2007). A decrease in variable to maximum fluorescence ratio
(Fv /Fm) was also observed in maize plants exposed to arsenic indicating decreased photosynthetic
efficiency due to arsenic exposure (Stoeva et al., 2003). Shaibur and coworkers (2006) observed
chlorosis in the young leaves, necrosis in the older leaves, and burning and curling of leaf tips due
to arsenic toxicity. Arsenic toxicity–induced chlorosis symptoms in the younger leaves of rice seed-
ling are suggested to be due to decreased chlorophyll content (Shaibur et al., 2006). Leaf number
and width of leaf blade also decreased with arsenic toxicity (Shaibur et al., 2006). Fergusson (1990)
observed reddish coloration of leaves due to arsenic toxicity.
Increased arsenic concentration in the soil leads to biomass reduction. In wheat plants, increased
arsenic concentrations in the soil led to more pronounced reductions in root biomass than in any
other plant part (Zhang et al., 2009). Biomass inhibition followed the order: roots > stems > spikes
(Zhang et al., 2009). Tomato plants grown in soils with high arsenic background concentrations
(100–130 mg/kg) showed leaf dieback from the tip and poor quality of fruit set (Fergus, 1955).
Yan-Chu (1994) observed a rice yield reduction of 10% at relatively lower arsenic concentra-
tions of 13 and 23 mg/kg soil. Arsenic-affected rice plants exhibit straighthead disease in which
increased sterility reduces yield (Wells and Glilmour, 1977). Rahman and coworkers (2008) also
observed a close association between arsenic concentration and straighthead of rice that led to
sterile florets with distorted lemma and palea, reduced plant height, tillering, panicle length, and
grain yield. Straighthead caused approximately 17%–100% sterile florate/spikelet formation and
about 16%–100% loss of rice grain yield (Rahman et al., 2008).
34.3 METABOLIC ALTERATIONS IN ARSENIC-STRESSED PLANTS

Arsenic interferes with a range of key metabolic processes in plants such as photosynthesis, respira-
tion, nitrogen assimilation, and synthesis of proteins and nucleic acids, which may ultimately result
in poor growth and reduction in crop yield. The two forms of inorganic arsenic species, As5+ as well
as As3+, disrupt plant metabolism, but through distinct mechanisms. As5+, being a phosphate analog,
can substitute inorganic phosphate in a plethora of biochemical processes. For example, depend-
ing on the Ca2+ levels, As5+ can be used by the ATP synthase more efficiently than phosphate-
producing ADP–As5+ (adenosine diphosphate) which unlike ATP becomes rapidly hydrolyzed and
unable to form stable high-energy compounds, thus affecting oxidative phosphorylation (Wickes
and Wiskich, 1975; Moore et al., 1983; Moreno-Sanchez, 1985). As3+ has a high binding affinity
for sulfhydryl groups of proteins, leading to disruption of protein structure and functions involved
in various cellular metabolisms. As3+ also binds and rapidly depletes glutathione (GSH), leading to
excessive production of ROS (Mylona et al., 1998). Organic forms of arsenic, MMA and DMA, have
been suggested to block protein synthesis in plants (Sckerl and Frans, 1969). To what extent plants
suffer from arsenic exposure is species and cultivar dependent and also dependent on readjustment
of several metabolic pathways (reviewed in Finnegan and Chen, 2012). Alteration of the activities of
enzymes involved in various metabolic pathways due to variable arsenic treatment in different plant
species is shown in Table 34.1.
Arsenic has been shown to perturb carbohydrate metabolism and its partitioning in growing
plants (Jha and Dubey, 2004b, 2005). It inhibits assimilation and transformation of carbon (Chang
et al., 2006); decreases chlorophyll content, chlorophyll fluorescence ratio Fv /Fm, and photosynthetic
rates (Miteva and Merakchiyska 2002; Stoeva and Bineva 2003; Stoeva et al., 2003; Chang et al.,
2006; Rahman et al., 2007); alters the activities of enzymes involved in starch and sugar metabo-
lism (Jha and Dubey, 2004b, 2005; Liu et al., 2005); and results in accumulation of soluble sugars
(Jha and Dubey, 2004b, 2005; Chang et al., 2006) in plants. In young plants of maize, Stoeva
and coworkers (2003) observed about a 20% decrease in the rate of CO2 fixation and a signifi-
cant decrease in PS2 activity. Arsenic also damages the chloroplast membrane and disorganizes
membrane structure (Miteva and Merakchiyska, 2002). Significant correlations have been observed
between reduced chlorophyll content and rice growth and yield due to arsenic toxicity suggesting
that arsenic affects the photosynthesis that ultimately results in the reduction of crop growth and
yield (Rahman et al., 2007). Protein expression profile of rice leaves under arsenic stress revealed
that expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) larger subunit sig-
nificantly decreased under arsenic stress (Ahsan et al., 2010). The downregulation of RuBisCO and
chloroplast 29 kDa ribonucleoproteins was suggested to be the possible causes of the decreased
photosynthetic rate under arsenic stress (Ahsan et al., 2010). The arsenic-tolerant monocotyledon
bent grass (Agrostis tenuis) also showed decreased amounts of RuBisCO large and small subunits
when exposed to As5+ or As3+ (Duquesnoy et al., 2009). In frond of arsenic hyperaccumulator plant
Chinese brake fern, As5+ predominantly affected proteins belonging to the photosynthesis and
carbon fixation group including RuBisCO large and small subunits and RuBisCO activase (Bona
et al., 2010). Proteins related to sugar metabolism and bioenergetics group were also significantly
influenced. Malate dehydrogenase (MDH) was upregulated, whereas triosephosphate isomerase,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase, pyruvate dehydro-
genase, aspartate (Asp) aminotransferase, and sedoheptulose-1,7-bisphosphatase were downregu-
lated (Bona et al., 2010). GAPDH, which plays key roles in photosynthesis and glycolysis, was found
to be upregulated in leaves but downregulated in roots (Ahsan et al., 2008) of rice plants treated
with As5+, suggesting that sugar metabolism during arsenic stress is highly active in the leaf when
TABLE 34.1
Alteration in the Activities of Enzymes Involved in Various Metabolic Pathways
due to Arsenic Treatments in Different Plant Speciesa
Metabolic
Pathways Enzyme Activities Altered Arsenic-Treated Plant Material Reference
Carbohydrate α-Amylase and β-amylase T. aestivum seeds germinated for Liu et al.
metabolism 6 days under 4–16 mg/L (2005)
Na2HAsO4·7H2O or NaAsO2
α-Amylase, β-amylase, starch Endosperms and embryo axes of Jha and Dubey
phosphorylase, and acid invertase germinating O. sativa seeds treated (2005)
with 25 and 50 μM As2O3 for 24, 48,
72, 96, and 120 h
α-Amylase, β-amylase, starch Roots and shoots of O. sativa Jha and Dubey
phosphorylase, acid invertase, seedlings treated with 25 and 50 μM (2004b)
sucrose phosphate synthase, and As2O3 for 5, 10, 15, and 20 days
sucrose synthase
Nitrogen NR, NiR, GS, aminotransferases, Endosperms and embryo axes of Jha and Dubey
metabolism aminating and deaminating GDHs germinating O. sativa seeds treated (2004c)
with 25 and 50 μM As2O3 for 24, 48,
72, 96, and 120 h
NR, NiR, GS, aminotransferases, Roots and shoots of O. sativa Jha and Dubey
aminating and deaminating GDHs seedlings treated with 25 and 50 μM (2004a)
As2O3 for 5, 10, 15, and 20 days
NR and NiR Roots, rhizomes, and fronds of Singh et al.
4-month-old plant of P. vittata and (2009)
P. ensiformis grown hydroponically for
2 weeks and then treated with 150 or
300 μM of Na2HAsO4 for 7 days
Protein Protease, leucine aminopeptidase, Roots and shoots of O. sativa Mishra and
metabolism and carboxypeptidase seedlings treated with 25 and 50 μM Dubey (2006)
RNA RNase Roots and shoots of O. sativa Mishra and
metabolism seedlings treated with 25 and 50 μM Dubey (2006)
Thiol Cysteine synthase, serine Shoots of B. juncea seedlings treated Srivastava et al.
metabolism acetyltransferase, γ-ECS, GR, GST, with 50 μM and 500 μM Na2HAsO4 (2009)
and γ-glutamyl transpeptidase and 25 μM and 250 μM NaAsO2 for
7 or 15 days
Serine acetyltransferase, cysteine Roots and shoots of O. sativa Tripathi et al.
synthase, γ-glutamylcysteine seedlings treated with 10, 20, and (2012)
synthetase, γ-glutamyl 50 μM Na2HAsO4 for 7 days after
transpeptidase, GST, and PCS 10 days of growth in hydroponics
without treatment
Phosphate Acid phosphatase, alkaline Roots and shoots of O. sativa Mishra and
metabolism phosphatase, inorganic seedlings treated with 25 and 50 μM Dubey (2008)
pyrophosphatase, mitochondrial As2O3 for 5, 10, 15, and 20 days
ATPase, and chloroplastic isoform
of ATPase
a For the details of alterations in enzyme activity levels, please see the text.
compared to the roots (Ahsan et al., 2010). As glycolytic enzymes play major role in sugar metabo-
lism, increased expression of GAPDH due to arsenic treatment reflects altered patterns of carbon
flux in response to reduced photosynthesis, presumably due to the use of photoassimilates directly
from the chloroplast for mitochondrial respiration (Ahsan et al., 2010).
Marked decline in the activities of amylolytic enzymes alpha-amylase, beta-amylase in endo-
sperms, as well as embryo axes was observed in As3+-treated germinating rice seeds (Jha and
Dubey, 2005). It was suggested that the decline in the activities of amylolytic enzymes might con-
tribute to delayed mobilization of endospermic starch that could affect germination of seeds in
arsenic-polluted environment (Jha and Dubey, 2005). Induced acid invertase activity and increased
sugar accumulation observed in embryo axes of As3+-treated germinating rice seeds were suggested
to serve as possible components for adaptation mechanism of rice seedlings grown under arsenic-
containing medium. In arsenic-treated rice seedling, the content of reducing, nonreducing, and total
soluble sugars increased and activities of α-amylase, β-amylase, and sucrose phosphate synthase
declined, whereas activities of starch phosphorylase, acid invertase, and sucrose synthase were
elevated (Jha and Dubey, 2004b), indicating that in rice seedlings, arsenic toxicity causes perturba-
tions in carbohydrate metabolism leading to the accumulation of soluble sugars by altering activities
of sugar metabolizing enzymes. Liu and coworkers (2005) observed significantly depressed total
amylase, α-amylase, and β-amylase activity in seeds of six wheat varieties germinated under As3+
or As5+ (4–16 mg/L). As3+ decreased all the endpoints more remarkably than As5+.
Arsenic toxicity has been reported to alter respiration rate in several plant species including rice
(Chen and Liu, 1993), soybean (Glycine max) (Liu and Wang, 2002), and wheat (Shao et al., 2011).
High levels of As3+ caused inhibition of respiration in rice plants (Chen and Liu, 1993). In wheat
roots, increased respiratory rate was observed at arsenic concentrations lower than 1 mg/L, but
a decreasing trend was observed at higher concentrations (Shao et al., 2011). In shoots, however,
respiratory rate increased gradually. It is suggested that respiration rate is altered in plants in the
presence of arsenic due to synthesis of highly unstable ADP–As5+ (Moore et al., 1983), which may
lead to uncoupling of ATP synthesis from electron transport (Wickes and Wiskich, 1975). The levels
of some important enzymes in respiratory process such as cytochrome oxidase (COD), isocitrate
dehydrogenase (IDH), and MDH isoenzymes are induced in shoots and roots with increasing con-
centrations of arsenic. Shao and coworkers (2011) concluded that arsenic could change the expres-
sion of these enzymes, thereby affecting respiration and eventually leading to physiological changes
in wheat.
Arsenic toxicity also alters nitrogen metabolism and decreases the nitrogen assimilation capac-
ity of plants (Jha and Dubey, 2004a; Chang et al., 2006). Symbiotic N2 fixation has been shown to
be sensitive to As5+ treatment in alfalfa (Medicago sativa) root systems supporting well-established
N2-fixing symbiosis with rhizobia (Porter and Sheridan, 1981). Pajuelo and coworkers (2008)
observed that As3+ negatively affected Sinorhizobium–alfalfa symbiotic interaction. As3+ concen-
tration of 25–35 μM caused a 75% decrease in the total number of nodules, due to a 90% reduction
in the number of rhizobial infections. This effect was associated to root hair damage and a shorter
infective root zone. Molecular mechanism underlying this toxic effect was examined by Lafuente
and coworkers (2010). They used reverse transcription–polymerase chain reaction (RT–PCR) and
real-time RT–PCR to analyze various markers for the different events leading to nodule formation.
A significant decrease in the expression of four early nodulins, the genes coding the Nod factor
receptor (NORK), the transcription factor NIN (for nodule inception), and the markers for infection
progression (N6) and nodule organogenesis (Enod2), was observed, especially from days 1–5 after
the inoculation (Lafuente et al., 2010). The expression of a marker for nitrogen fixation (Legbrc)
was also reduced, probably due to the reduction in nodule number induced by arsenic (Lafuente
et al., 2010). These results suggest that arsenic affects the expression of nodulation genes associ-
ated with the processes that take place in the epidermis and the outer cortical cells and that the
expression of genes associated with events that take place in the inner cortical cells is less affected
(Lafuente et al., 2010).
Arsenic toxicity causes a marked decline in the activities of nitrogen assimilatory enzymes,
nitrate reductase (NR), nitrite reductase (NiR), and glutamine synthetase (GS) in germinating rice
seeds (Jha and Dubey, 2004c) and growing seedlings (Jha and Dubey, 2004a), whereas the activi-
ties of glutamate dehydrogenase (GDHs) and aminotransferases get elevated. It is suggested that
reduced assimilation of NO3− in arsenic-treated germinating rice seeds and seedlings may lead to
reduced seedling vigor and impaired growth. Lower amount of GS protein was also observed in
rice roots treated with As5+ compared to control (Ahsan et al., 2008). Increased activity of GDH
and aminotransferase appears to provide adaptational significance to the seeds germinating under
arsenic-polluted conditions (Jha and Dubey, 2004c). NR and NiR activities were reduced more in
the roots, rhizomes, and fronds of As5+-treated plants of Chinese brake fern (arsenic hyperaccumulator)
than in slender brake fern (nonarsenic hyperaccumulator). This effect was accompanied by similar
decreases in tissue NO3− concentrations suggesting that decrease in NR activity is the consequence
of the reduced NO−3 uptake and translocation in arsenic-exposed plants (Singh et al., 2009).
Arsenic exposure leads to changes in amino acid and protein content in plants. Content of free
amino acid pool was found to decrease in arsenic-treated rice seedlings (Mishra and Dubey, 2006) but
increased in arsenic-treated flue-cured tobacco (Nicotiana tabacum) plants during the middle and late
growth stages (Chang et al., 2006). In rice grains, total amino acid content was positively correlated
with arsenic accumulation (Dwivedi et al., 2010). Changes of glutamate (Glu), Asn, Asp, and Pro levels
also showed a positive linear dependency with increase in arsenic concentration in spinach (Spinacia
oleracea) plants (Pavlík et al., 2010). Accumulation of Pro in the tissues of plants growing under As3+
toxicity appears to serve as osmolyte and enzyme protectant (Mishra and Dubey, 2006). Transcriptome
analysis revealed that genes involved in transport of amino acids, peptides, and oligopeptides were dif-
ferentially expressed in rice seedlings challenged with arsenic (Norton et al., 2008; Chakrabarty et al.,
2009). Increased protein content due to arsenic treatment was observed in flue-cured tobacco during
the middle and late growth stages (Chang et al., 2006) and in rice seedlings (Mishra and Dubey, 2006),
whereas decreased total plant protein content due to arsenic was observed in slender brake fern and
Chinese brake fern (Singh et al., 2006). In duckweed (Lemna minor) plants, total protein increased at
low As5+ supply but decreased at high arsenic supply (Duman et al., 2010). Arsenic treatment also led
to decreased soluble protein in maize plants (Stoeva et al., 2003). Arsenic alters activities of proteases
and aminopeptidases in plants. Mishra and Dubey (2006) observed marked inhibition in activities
of protease and leucine (Leu) aminopeptidase in arsenic-treated rice seedling, whereas the activity
level of carboxypeptidase was enhanced. Results suggest that arsenic exposure impairs hydrolysis of
proteins in rice seedlings due to inhibition of protease activities (Mishra and Dubey, 2006). Pepper
and coworkers (1988) observed increase in the length of the cell cycle and frequency of G1 and G2 cells
along with partial inhibition of Leu uptake and its incorporation into protein in onion (Allium cepa)
root meristems. It was proposed that the increase in the frequency of cells in G1 and G2 is a conse-
quence of the partial inhibition of protein synthesis observed and/or a result of arsenolysis of phospho-
proteins necessary for the cell cycle transit (Pepper et al., 1988).
In situ As3+ treatment caused increased level of RNA, marked inhibition in activities of ribo-
nuclease (RNase), and significant alteration in isoform pattern of RNase in arsenic-treated rice
seedlings compared to unexposed seedlings. Results suggest that arsenic exposure impairs
hydrolysis of RNA in rice seedlings due to inhibition in activity of RNases (Mishra and Dubey,
2006). Arsenic exposure also leads to chromosomal abnormalities in plants. The most frequent
abnormalities involved disturbance of the spindles mainly due to its known affinity for thiol
groups (Bandopadhyay and Maity, 1995). Frequency of chromosomal abnormalities in barley
(Hordeum vulgare) seeds was found to be directly proportional to the concentration of arsenic salts
(Bandyopadhyay and Maity, 1995). When assessing the genotoxicity of arsenic, Yi and coworkers
(2007) observed arsenic-induced concentration-dependent micronuclei formation, mitotic index
inhibition, and mitotic delay in onion root tips.
Sulfur metabolism is also affected by arsenic in plants. Plants assimilate sulfate to form cys-
teine (Cys) for the synthesis of GSH in two ATP-dependent steps: In the first, rate-limiting step,
γ-glutamylcysteine (γEC) is synthesized by γ-glutamylcysteine synthetase (γ-ECS) using Cys

and γ-glutamic acid (γ-Glu) as substrates followed by the synthesis of GSH by GSH synthetase,
using γ-ECS and glycine (Gly) as a substrate. Binding of As3+ with GSH and PCs is an important
mechanism in the detoxification of arsenic. Plants exposed to arsenic substantially increase the
synthesis of GSH and PCs and the polymers of GSH (Schmoger et al., 2000; Gupta et al., 2004;
Srivastava et al., 2007; Sung et al., 2009; Mishra et al., 2011a) and require adequate supplies of
GSH-building blocks Glu, Cys, and Gly, possibly due to their utilization in PC synthesis. Sulfate
transporter gene has been shown to be upregulated by As3+ (Chakrabarty et al., 2009; Srivastava
et al., 2009) as well as As5+ (Norton et al., 2008) supporting the increased need of sulfur for
biosynthesis of GSH and PC. Arsenic toxicity leads to decreased cellular Cys pools (Sung et al.,
2009) and depletion of GSH pools (De Vos et al., 1992; Sneller et al., 1999; Hartley-Whitaker
et al., 2001b; Srivastava et al., 2009). However, in contrast to arsenic-sensitive variety of Indian
mustard (Brassica juncea), increased levels of both Cys and GSH were observed in an arsenic-
tolerant variety of Indian mustard (Srivastava et al., 2009). Increased levels of both Cys and GSH
in the arsenic-tolerant Indian mustard variety were accompanied with an induction in the activi-
ties of Cys synthase, serine acetyltransferase, and γ-ECS, indicating that the induction of sulfur
metabolism may be involved in increasing arsenic tolerance in plants. Enhanced transcriptional
activation of γ-ECS and GSH synthetase, involved in GSH biosynthesis, has also been observed
in Arabidopsis plants exposed to arsenic (Sung et al., 2009). In response to arsenic, plants induce
the synthesis of PCs, the polymers of GSH, through the enzyme phytochelatin synthase (PCS)
(Grill et al., 1985; Cobbet and Goldsbrough, 2002). PCS transcript level was found to be higher in
Indian mustard arsenic-tolerant variety Pusa Bold as compared to arsenic-sensitive variety Varuna
due to As3+ treatment (Gupta et al., 2009). Expression of a gene 5′-adenylsulfate reductase, which
catalyzes the second step of reduction of sulfate to sulfite and ferredoxin, a key redox protein used
as a reductant in conversion of sulfite to sulfide by sulfite reductase, has been shown to be elevated
in Arabidopsis in response to As5+ supply (Abercrombie et al., 2008).
Due to strong chemical similarity between As5+ and phosphate, As5+ can interfere with phos-
phate metabolism by replacing phosphate in ATP synthesis and/or in various phosphorolysis reac-
tions (Dixon, 1997). As3+ stress results in the repression of genes involved in phosphate acquisition,
redistribution, and phosphorylation (Abercrombie et al., 2008). As3+ toxicity has been shown to
lower phosphate pool and alter the activities of key phosphohydrolytic enzymes in As3+-treated rice
seedlings, which might contribute to metabolic perturbations and decreased growth of rice plants
in an As3+-polluted environment (Mishra and Dubey, 2008). Application of 50 μM As3+ in situ
resulted in 34%–77% inhibition of acid phosphatase activity in roots and about 38%–50% inhibi-
tion of activity in shoots of 15–20-day-old seedlings. Similarly, alkaline phosphatase activity was
also inhibited in shoots under in situ As3+ toxicity. Mishra and Dubey (2008) observed varietal as
well as organ-specific differences in inorganic pyrophosphatase activity in response to in situ As3+
treatment. A moderately toxic in situ As3+ level of 25 μM as well as a highly toxic level of 50 μM
inhibited mitochondrial-ATPase activity, whereas 25 μM As3+ stimulated the chloroplastic isoform
of ATPase, but at a higher level (50 μM), As3+ was inhibitory (Mishra and Dubey, 2008). The
arsenic-tolerant monocotyledon bent grass also showed decreased amounts of mitochondrial ATP
synthase when exposed to As5+ or As3+ (Duquesnoy et al., 2009).
34.4 OXIDATIVE STRESS AND ANTIOXIDATIVE

DEFENSE UNDER ARSENIC TOXICITY
There is significant evidence that exposure of plants to inorganic as well as organic forms of arsenic
results in the excessive production of ROS such as O2i- , •OH, and H2O2, causing increased oxidative
damage to plants (Hartley-Whitaker et al., 2001a; Meharg and Hartley-Whitaker, 2002; Mishra
et al., 2011a). Arsenic is a redox-active metal, and in mammalian cells, arsenic-induced production
of ROS has been linked with conversion of As5+ to As3+ (Flora, 1999; Huang et al., 2004); however,
little is known about the mechanisms by which arsenic induces ROS generation in plants. Mylona
and coworkers (1998) proposed that intraconversion of arsenic molecules from one ionic form to
the other and inhibition of the mitochondrial electron transport chain by arsenic may be involved
in arsenic-induced production of ROS in plants. Reduction of As5+ to As3+, a process that read-
ily occurs in plants, has been suggested to result in the production of ROS in plants (Meharg and
Hartley-Whitaker, 2002). Cytochrome/COD mediates rapid reduction of As5+ to As3+ using oxy-
gen as a final electron acceptor in mitochondria and chloroplasts (Tamaki and Frankberger, 1992).
Reaction of COD with oxygen can lead to generation of Oi- 2 (Elstner, 1982). It has been suggested
that arsenic may potentially be further metabolized to methylated species leading to ROS genera-
tion (Zaman and Pardini, 1996). However, increase in ROS may also be the result of either depletion
of GSH or inhibition of antioxidant enzymes (Abercrombie et al., 2008). Like other environmental
stress, arsenic has also been suggested to create conditions in the thylakoids, where the energy level
exceeds the amount that can be dissipated by the metabolic pathways of the chloroplast, leading to
enhanced production of ROS (Dat et al., 2000; Singh et al., 2006).
A cell is said to be in a state of oxidative stress when the level of ROS exceeds the ability of
antioxidant defense mechanisms to quench them. Enhanced level of these ROS causes oxidative
damage to biomolecules such as membrane lipids, proteins, enzymes, nucleic acids, and chlo-
roplast pigments. Oxidative damage due to arsenic-induced ROS formation in plants has been
reported in a wide range of plant species including rice (Mishra et al., 2011a; Tripathi et al.,
2012), Chinese brake fern (Singh et al., 2006), wheat (Li et al., 2007), maize (Stoeva et al.,
2003), and mung bean (Phaseolus aureus) (Singh et al., 2007). Enhanced production of ROS
in plants due to arsenic exposure can stimulate a chain-like peroxidation of polyunsaturated
fatty acids of lipid bilayer in cellular membranes leading to enhanced electrolyte leakage and
amount of thiobarbituric acid–reactive substances (TBARSs) (Hartley-Whitaker et al., 2001a;
Mascher et al., 2002; Singh et al., 2007; Shri et al., 2009; Mishra et al., 2011a). Two contrast-
ing rice genotypes, one tolerant (Triguna) and one sensitive (IET-4786) variety, exposed to
50 μM As5+ for 7 days, showed significant increase in the levels of Oi- 2
, H 2O2 , malondialdehyde
(MDA), electrical conductivity (EC), and nitric oxide (NO) in roots. However, induction was
more prominent in sensitive rice variety for the oxidative stress–related parameters (Tripathi
et al., 2012). Mishra and coworkers (2011a) also observed increased formation of ROS (Oi- 2
,
H 2O2) and elevated level of lipid peroxidation in 25 and 50 μM As3+ -stressed seedlings of two
indica rice cvs. Malviya-36 and Pant-12. Arsenic hyperaccumulator Chinese brake fern as well
as non-hyperaccumulator slender brake fern showed increased concentration of H 2O2 at 133 and
276 μM As5+ exposure compared to the control (Singh et al., 2006). After exposure to 267 μM
As5+, the H 2O2 concentration in slender brake fern reached 220% of the control after 1 day com-
pared to 175% after 10 days exposure in Chinese brake fern. After exposure to 133 and 276 μM
As5+ for 10 days, the rate of increase in lipid peroxidation was 102% and 177% in Chinese brake
fern compared to 132% and 203% in slender brake fern. It is suggested that high lipid peroxi-
dation coupled with high H 2O2 might be responsible for the damage of chloroplast, decrease
in plant biomass, and inhibited chlorophyll and protein concentrations in slender brake fern
(Singh et al., 2006).
Induction of oxidative stress is suggested to be a key component underlying arsenic toxicity
in plants (Requejo and Tena, 2005). Plants respond to arsenic treatment by altering the levels of
many enzymatic [superoxide dismutase (SOD; EC 1.15.1.1.), catalase (CAT; 1.11.1.6), guaiacol per-
oxidase (GPX; EC 1.11.1.7), glutathione-S-transferase (GST; E.C. 2.5.1.18), ascorbate peroxidase
(APX; EC 1.1.11.1), monodehydroascorbate reductase (MDHAR; 1.6.5.4), dehydroascorbate reduc-
tase (DHAR; EC 1.8.5.1), and glutathione reductase (GR: EC 1.6.4.2) (Noctor and Foyer, 1998)]
and nonenzymatic [ascorbate (AsA), GSH (γ-glutamyl–cysteinyl–glycine), tocopherol, carotenoids,
and phenolic compounds] antioxidants, which may serve as important components in mitigating
arsenic-induced oxidative damage (Mishra et al., 2011a). Effects of variable arsenic treatments on
TABLE 34.2
Effects of Variable Arsenic Treatments on ROS Production, Oxidative Damage,
and Alteration in the Level of Antioxidants in Different Plantsa
ROS Oxidative
Arsenic-Treated Plants Production Damage Antioxidant Altered Reference
Roots and shoots of O. sativa seedlings Oi-
2 , H2O2 Lipid peroxidation SOD, APX, chl-APX, Mishra et al.
treated with 25 and 50 μM As2O3 for GPX, CAT, MDHAR, (2000a)
5, 10, 15, and 20 days GR, AsA, and GSH
Roots and shoots of O. sativa seedlings O2i-, H2O2 Lipid peroxidation, NADPH oxidase and Tripathi et al.
treated with 10, 20, and 50 μM ion leakage, and AAO, AsA and GSH (2012)
Na2HAsO4 for 7 days after 10 days of nitric oxide
growth in hydroponics without treatment production
Roots and shoots of O. sativa seedlings Not determined Lipid peroxidation SOD, APX, POD, and Shri et al.
treated with 50 and 100 μM GR (2009)
NaAsO2/100 and 500 μM Na2HAsO4
for 10 days
Roots and shoots of O. sativa seedlings Not determined Not determined SOD, APX, GPX, Rai et al.
treated with 10, 20, and 50 μM CAT, MDHAR, (2011)
Na2HAsO4/5, 10, and 25 μM NaAsO2 DHAR, and GR
for 7 days after 10 days of growth in
hydroponics without treatment
Leaves of T. aestivum seedlings treated O2i- Lipid peroxidation SOD, APX, POD, Li et al.
with 0.5, 1, 5, 15, and 20 mg/kg CAT, and AsA (2007)
NaAsO2 for 7 days
Roots of Z. mays seedlings treated with Not determined Lipid peroxidation POD Stoeva et al.
2 and 5 mg dm−3 Na3AsO4 for 5 days (2005)
after 15 days of plant emergence
Scutella of germinating Z. mays embryo Not determined Not determined SOD, CAT, and GST Mylona et al.
(4 days post inbibition) seeds treated (1998)
with Na2HAsO4 or NaAsO2 at 0.01,
0.1, 1, and 10 mM for 24 h
Leaves of 20-day-old B. juncea plants H2O2 Lipid peroxidation SOD, APX, GR, AsA, Khan et al.
exposed to 5 and 25 μM Na3AsO4 for and GSH (2009)
96 h in hydroponic culture
Fronds of 4-month-old P. vittata and H2O2 Lipid peroxidation AsA, GSH, and Singh et al.
P. ensiformis plants treated with 133 or carotenoids (2006)
267 μM Na2HAsO4·7H2O for 1, 5, or
10 days
Frond, rhizome, and root tissues of Not determined Lipid peroxidation SOD, APX, and CAT Srivastava
4-month-old P. vittata, P. ensiformis, et al. (2005)
and N. exaltata plants grown
hydroponically for 2 weeks and then
treated with 150 or 300 μM of arsenic
Na2HAsO4·7H2O for 10 days
a For the details of alterations in the level of antioxidants, please see the text.
ROS production, oxidative damage, and antioxidants in various plant species have been shown in
Table 34.2. In Chinese brake fern, though both enzymatic and nonenzymatic antioxidants play
significant roles in arsenic detoxification and hyperaccumulation, the former plays a major role at
low arsenic exposure (≤20 mg/kg), whereas the latter plays a more important role at high arsenic
exposure (50–200 mg/kg) (Cao, 2004).
34.4.1 Nonenzymatic Antioxidants
Nonenzymatic components of the antioxidative defense system include the major cellular redox
buffers AsA and GSH, as well as tocopherol, carotenoids, and phenolic compounds, which play
a key role in defense against oxidative stress. AsA can donate electrons in a number of enzymatic
and nonenzymatic reactions and is therefore considered as powerful antioxidant. It is present in the
majority of plant cell types, organelles, and apoplasts (Shao et al., 2008) and is found to be par-
ticularly abundant in photosynthetic tissues (Smirnoff et al., 2004). It plays key role in removal of
H2O2 via the AsA–GSH cycle (Pinto et al., 2003). In the AsA–GSH cycle, two molecules of AsA
are utilized by APX to reduce H2O2 to water with concomitant generation of monodehydroascor-
bate (MDHA). MDHA is a radical with a short life time and can spontaneously dismutate into
dehydroascorbate (DHA) and AsA or is reduced to AsA by NADP(H)-dependent enzyme MDHAR
(Miyake and Asada, 1994). DHA is also highly unstable at pH values greater than 6.0 and is decom-
posed to tartrate and oxalate (Noctor and Foyer, 1998). To prevent this, DHA is rapidly reduced to
AsA by the enzyme DHAR using reducing equivalents from GSH (Asada, 1996). Reduced GSH,
one of the crucial nonprotein thiol, has been detected virtually in all cell compartments such as
cytosol, chloroplasts, endoplasmic reticulum, vacuoles, and mitochondria (Foyer and Noctor, 2003).
In addition to its participation in the regeneration of another potential antioxidant AsA, via the
AsA–GSH cycle, GSH can react chemically with O2 i-, •OH, and H2O2 and, therefore, can function
directly as a free radical scavenger.
An enhanced level of AsA and/or GSH in response to arsenic toxicity has been observed in
rice (Ahsan et al., 2008; Mishra et al., 2011a), Indian mustard (Khan et al., 2009), and cucumber
(Cucumis sativus) (Czech et al., 2008) plants. Increase in AsA and GSH concentration was depen-
dent on exposure time (Khan et al., 2009) and irrespective of the variation in the level of DHA and
glutathione disulphide (GSSG) in rice seedlings subjected to arsenic toxicity, which in turn influ-
enced redox ratios of AsA/DHA and GSH/GSSG. Czech and coworkers (2008) studied the effect
of As5+ on ion efflux, H2O2 concentration, AsA concentration, ascorbate oxidase (AAO) activity,
and MDA formation and concluded that AsA protects the plants by preventing lipid peroxidation
so the cucumber hypocotyls remain turgid, and the inhibition of growth is much smaller. Mishra
and coworkers (2011a) observed higher levels of AsA and DHA and decline in AsA/DHA ratio
in arsenic-treated rice seedlings compared to controls during 5–20 days growth period. Seedlings
exposed to 50 μM As3+ for 20 days showed about 50%–133% increase in AsA level in roots and about
14%–46% increase in AsA level in shoots compared to controls (Mishra et al., 2011a). Total GSH
content in rice roots during As5+ stress responds in a dose-dependent manner and plays a central role
in protecting cells against arsenic stress (Ahsan et al., 2008). Mishra and coworkers (2011a) observed
higher GSH level in arsenic-treated rice seedlings compared to controls during 5–20 days growth
period. Seedlings exposed to 50 μM As3+ for 20 days showed about 62%–77% increase in GSH
level in roots and about 25%–32% increase in GSH level in shoots (Mishra et al., 2011a). The level
of GSSG was higher in As3+-stressed seedlings compared to controls up to the 10th day of growth,
whereas during the later growth period, that is, at 15–20 days, GSSG level declined. The ratio of
GSH/GSSG declined in the roots during 5–10 days of As3+ treatment, whereas during 15–20 days,
this ratio increased in both roots and shoots (Mishra et al., 2011a). A significant increase in GSH
content has also been observed in As-tolerant plants like hydrilla (Hydrilla verticillata) upon As5+ as
well as As3+ exposure (Srivastava et al., 2007). Increase in the concentration of GSH was found to be
more pronounced in hydrilla plants exposed to As3+ than As5+ (Srivastava et al., 2007). About 112%
increase in GSH concentration was observed in hydrilla plants exposed to As5+ at 10 μM after 7 days,
while in As3+-exposed plants, the maximum increase of 349% was noticed after 4 days at 1 μM.
Prolonged exposure to higher concentrations of both As5+ and As3+ caused depletion in GSH levels.
Decrease in GSH content was also observed in arsenic-treated common velvet grass and red clover
plants (Hartley-Whitaker et al., 2001b; Mascher et al., 2002). Shri and coworkers (2009) reported
that GSH and Cys supplementation resulted in elimination of oxidative stress and restoration of the
growth of rice seedling exposed to arsenic. The levels of AsA and GSH and their reduced/oxidized
ratios in the fronds of Chinese brake fern (arsenic hyperaccumulator) appear to be much higher than
slender brake fern (arsenic nonaccumulator), indicating that Chinese brake fern has an inherently
greater antioxidant potential than slender brake fern and protection from oxidative damage due to
a greater level of AsA–GSH pool is associated with arsenic tolerance in arsenic hyperaccumulator
Chinese brake fern (Singh et al., 2006). The lower levels of antioxidant compounds (AsA and GSH)
in slender brake fern than Chinese brake fern are consistent with its higher ROS production and
lower ROS scavenging ability under similar arsenic exposure (Singh et al., 2006).
Carotenoids are a class of fat-soluble pigments that are able to detoxify various forms of ROS
(Young, 1991). These are located in thylakoid membranes of chloroplasts where they have a critical
role in light-harvesting and membrane-associated antioxidant activity (Siefermann-Harms, 1987).
They scavenge 1O2 to inhibit oxidative damage and quench triplet sensitizer (3Chl*) and excited
chlorophyll (Chl*) molecule to prevent the formation of 1O2 in order to protect the photosynthetic
apparatus (Mittler, 2002). Arsenic exposure has been reported to decrease carotenoid content in
maize (Stoeva et al., 2003), red clover (Mascher et al., 2002), pea (Srinivas et al., 2001), oat (Avena
sativa) (Stoeva and Bineva, 2003), bean (Phaseolus vulgaris) (Stoeva et al., 2005), and slender brake
fern (Singh et al., 2006) and increase in common reed (Phragmites australis) (Ghassemzadeh et al.,
2008) and Chinese brake fern (Singh et al., 2006). The possible reason for the decline in carot-
enoids concentration under As5+ stress has been suggested to be due to arsenic-mediated breakage
and swelling of the thylakoid membrane and accumulation of starch in chloroplasts (Simola, 1977).
A comparative study of Chinese brake fern and slender brake fern revealed that arsenic exposure
leads to increased concentrations of carotenoids in Chinese brake fern but causes decrease in slen-
der brake fern (Singh et al., 2006), which is consistent with greater exposure to ROS and lower
scavenging ability of slender brake fern compared to Chinese brake fern. Increased concentration
of carotenoids in arsenic-tolerant plants suggests that carotenoids appear to play an important role
in combating arsenic-induced oxidative stress.
34.4.2 Enzymatic Antioxidants
The enzymatic components of the antioxidative defense system comprise of several antioxidant
enzymes such as SOD, CAT, GPX, and GSTs and enzymes of the AsA–GSH cycle such as APX,
MDHAR, DHAR, and GR (Noctor and Foyer, 1998). These enzymes operate in different subcellu-
lar compartments and respond in concert when cells are exposed to oxidative stress. Increase in the
activity of various antioxidant enzymes was found to be more pronounced in hydrilla plants exposed
to As5+ than As3+ (Srivastava et al., 2007). The enzyme SOD belongs to the group of metalloen-
zymes and catalyzes the dismutation of O2i- to O2 and H2O2. It plays a central role in the protection
against oxygen toxicity in aerobic organisms (Scandalios, 1993). It is present in most of the subcel-
lular compartments that produce ROS. Three isozymes of SOD, copper/zinc SOD (Cu/Zn-SOD),
manganese SOD (Mn-SOD), and iron SOD (Fe-SOD) have been identified and characterized in
plants (Fridovich, 1989; Racchi et al., 2001). Cu/Zn-SOD is located in the cytosol, chloroplast, per-
oxisome, and mitochondria (Kanematsu and Asada, 1989; Bowler et al., 1992; Bueno et al., 1995;
del Rio et al., 1998). Mn-SOD is localized in mitochondria, whereas Fe-SOD is localized in chloro-
plasts (Jackson et al., 1978). The arsenic treatments significantly increased the activities of SOD in
Indian mustard (Khan et al., 2009), mung bean (Singh et al., 2007), rice (Shri et al., 2009; Mishra
et al., 2011a), maize (Mylona et al., 1998; Requejo and Tena, 2005), and red clover (Mascher et al.,
2002), suggesting that it may serve as an important component in mitigating arsenic-induced oxi-
dative stress in plants. Activities of total SOD and its isoforms such as Mn/Fe-SOD and Mn-SOD
were found to be higher in As3+-treated rice seedlings compared to controls (Mishra et al., 2011a).
Seedlings grown in the presence of 50 μM As3+ for 20 days showed about 60% increase in total SOD
activity, 50%–55% increase in Mn/Fe-SOD activity, and 44%–60% increase in Mn-SOD activity
in roots (Mishra et al., 2011a). In shoots of rice seedlings, 42%–55% increase in total SOD activ-
ity, 56%–68% increase in Mn/Fe-SOD activity, and 50%–70% increase in Mn-SOD activity were
observed due to arsenic exposure compared to unexposed control plants (Mishra et al., 2011a).
Mylona and coworkers (1998) studied the effect of As5+ as well as As3+ in developing embryos
and young leaves of maize and observed that arsenic exposure triggered responses of SOD genes,
which were expressed in a tissue-, developmental stage-, and isoenzyme-specific form. Proteomic
analysis reveals that As5+ and As3+ lead to induction of SOD isoforms in maize roots and that
cytosolic Cu/Zn-SODs are highly responsive enzymes involved in cellular homeostasis (Requejo
and Tena, 2005). In addition to two major Cu/Zn-SOD isozymes, the capacity of Mn-SOD also
increased in response to As5+ in red clover (Mascher et al., 2002). Abercrombie and coworkers
(2008) employed whole-genome oligonucleotide microarrays to investigate the transcriptional
responses of Arabidopsis plants to As5+ stress and observed the induction of Cu/Zn-SOD and strong
suppression of Fe-SOD. Srivastava and coworkers (2005) observed significant increase in SOD
activities of the frond, rhizome, and root tissues in arsenic hyperaccumulator Chinese brake fern
exposed to As5+. The SOD activities in the roots and fronds of arsenic nonaccumulator ferns slender
brake fern and Boston fern (Nephrolepis exaltata) also increased compared with their respective
controls. However, in the rhizome tissues, no significant increase in SOD activity was observed.
Higher constitutive SOD activity was observed in the fronds of Chinese brake fern, followed by
slender brake fern and Boston fern.
CAT, another antioxidative enzyme, is a ubiquitously present tetrameric hemeprotein that cata-
lyzes the dismutation of two molecules of H2O2 into water and oxygen. Unlike other H2O2-degrading
enzymes, CATs do not require cellular reducing equivalent. Though there are frequent reports of
CAT being present in cytosol, chloroplast, and mitochondria, CAT activity is mainly associated
with peroxisomes (major sites of H2O2 production) (Scandalios et al., 1997; Mhamdi et al., 2010).
Increased activity of CAT under arsenic toxicity has been reported in rice (Mishra et al., 2011a),
maize (Mylona et al., 1998), and Indian mustard (Khan et al., 2009), whereas in mung bean, CAT
activity declined with As exposure (Singh et al., 2007). In rice seedlings, As3+ treatment, regardless
of the dose (25 or 50 μM) used, did not significantly influence CAT activity at 5 or 10 days of expo-
sure; however, a prolonged As3+ treatment for 15–20 days caused a significant increase in enzyme
activity (Mishra et al., 2011a). Higher activity of CAT has also been observed in arsenic-tolerant
Chinese brake fern compared to arsenic-sensitive slender brake fern and Boston fern (Srivastava
et al., 2005). In Chinese brake fern, CAT activity was activated by 200 μM As5+ up to 300% com-
pared to the control, whereas only 133% increase was observed for slender brake fern (Kertulis-
Tartar et al., 2009). It is suggested that increased CAT activity in the fronds of Chinese brake fern
in response to arsenic exposure may allow the fern to more efficiently scavenge arsenic-induced
overproduced H2O2 as a result of reduction of As5+ to As3+ (Kertulis-Tartar et al., 2009).
GPXs are the members of a large family of peroxidases. These are heme-containing proteins
and oxidize several substrates, preferably aromatic electron donors such as guaiacol and pyrogallol
in the presence of H2O2 (Penel et al., 1992; Vianello et al., 1997). GPXs exist as multiple isozymes
in plant tissues and are localized in vacuoles, mitochondria, the cell wall, and the cytosol (Asada,
1992; Prasad et al., 1995). GPXs are widely accepted as stress enzymes in plants. Arsenic treat-
ments significantly increase GPX activity in wheat (Li et al., 2007), mung bean (Singh et al., 2007),
and rice (Shri et al., 2009; Mishra et al., 2011a) plants. Mishra and coworkers (2011a) reported a
consistent increase in the activity of GPX in rice seedlings with increase in As3+ concentration
as well as treatment length. As3+-led increase in GPX activity was greater in roots than in shoots
(Mishra et al., 2011a). Rice seedlings grown for 20 days in the presence of 50 μM As3+ showed about
115%–128% increase in GPX activity in roots and about 70%–80% increased activity in shoots. Shri
and coworkers (2009) also reported increased GPX activity in roots of rice seedlings exposed to
100 μM As3+ and 500 μM As5+. However, no significant change in the activity of GPX was observed
in response to As5+ or As3+ in shoots (Shri et al., 2009). Dwivedi and coworkers (2010) observed
either decrease or no significant change in the activities of GPX in rice genotypes exposed to As5+.
It was suggested that this might be due to less availability of H2O2 because of its efficient breakdown
in the AsA–GSH cycle or due to inactivation of enzyme directly by arsenic or ROS. GPX activity is
reported to significantly increase in frond and root tissues of arsenic-treated slender brake fern and
Boston fern but not Chinese brake fern, suggesting that GPX serves as an intrinsic defense tool to
resist oxidative damage in slender brake fern and Boston fern (Srivastava et al., 2005).
APX is a member of the class I superfamily of heme peroxidases (Welinder, 1992) and plays a
significant role in the removal or detoxification of H2O2 in plant tissues. It is a central component of
the AsA–GSH cycle. APX uses two molecules of AsA to reduce H2O2 to water with a concomitant
generation of two molecules of MDHA. Five isoforms of APX (cytosolic, stromal, thylakoidal,
mitochondrial, and peroxisomal) have been identified in plants (Nakano and Asada, 1987; Jimenez
et al., 1997; Madhusudhan et al., 2003; Sharma and Dubey, 2004). The function of cytosolic APX is
probably to eliminate H2O2 produced in cytosol or apoplast and that has diffused from organelles,
whereas APX found in organelles scavenges H2O2 produced within the organelles (Mittler and
Zilinskas, 1992). Increased APX activity has been reported in Indian mustard (Khan et al., 2009),
rice (Shri et al., 2009; Mishra et al., 2011a), maize (Miteva and Peycheva, 1999), and beans (Stoeva
et al., 2005) exposed to arsenic. Mishra and coworkers (2011a) observed higher APX as well as
chloroplastic APX (chl-APX) activity in As3+-treated rice seedlings compared to controls. Rice
seedlings exposed to 50 μM As3+ for 20 days showed 33%–62% increased APX activity in roots and
75%–200% increased activity in shoots. A consistent increase in chl-APX activity was noted from
5 to 20 days of growth, and seedlings grown for 20 days in the presence of 50 μM As3+ showed
86%–100% increased chl-APX activity compared to controls. The arsenic hyperaccumulator,
Chinese brake fern, and nonaccumulator slender brake fern demonstrated a significant increase
in APX activity in its frond, rhizome, and root tissues at 300 μM of As5+ exposure. However, no
significant increase in the activity of APX was observed in the case of frond tissues of the sensitive
species, Boston fern (Srivastava et al., 2005).
GSH reductase, an NAD(P)H-dependent enzyme, catalyzes the reduction of GSSG to GSH and
hence maintains the GSH levels for most of the functions in the cell. As an antioxidant, GSH partici-
pates in enzymatic as well as nonenzymatic redox cycles and gets oxidized to GSSG. In the AsA–
GSH cycle, GSH is oxidized to GSSG in a reaction catalyzed by DHAR. Although it is located in the
chloroplasts, cytosol, mitochondria, and peroxisomes, around 80% of GR activity in photosynthetic
tissues is accounted for by chloroplastic isoforms (Edwards et al., 1990). In chloroplast, GSH and
GR are involved in detoxification of H2O2 generated by the Mehler reaction. The arsenic treatments
significantly increase the activity of GR in Indian mustard (Khan et al., 2009), mung bean (Singh
et al., 2007), rice (Ahsan et al., 2008; Shri et al., 2009; Mishra et al., 2011a), and water hyssop
(Bacopa monnieri) (Mishra et al., 2011b) plants. About 30%–122% increased GR activity in roots
and 120%–125% increased activity in shoots were observed in rice seedlings exposed to 50 μM As3+
for 20 days compared to controls (Mishra et al., 2011a). Rice cultivars Triguna, IR-36, and PNR-519
responded positively to arsenic stress due to significant induction in GR activity in these cultivars,
providing sufficient GSH to support the antioxidant system, namely, the AsA–GSH cycle (Rai et al.,
2011). New isoforms of GR were induced during As3+ stress in rice (Shri et al., 2009). It is suggested
that the enhanced demand for GSH in response to arsenic-induced oxidative stress might be met by
the increased GR activity and elevated GSH turnover (Shri et al., 2009). Srivastava and coworkers
(2005) observed significantly higher GR activity in the rhizome tissues of slender brake fern than
Chinese brake and Boston ferns. There was no significant increase in GR activity in the frond and
root tissues of Chinese brake fern at 300 μM As5+ (Srivastava et al., 2005). Similarly, As5+ concen-
trations up to 3 mM did not inhibit GR activity in the fronds of Chinese brake fern or slender brake
fern (Kertulis-Tartar et al., 2009). At 1 mM As3+, GR activity was inhibited approximately by 64%
in both ferns (Kertulis-Tartar et al., 2009).
MDHAR is a FAD enzyme that catalyzes the regeneration of AsA from the MDHA radical using
NAD(P)H as the electron donor (Hossain and Asada, 1985). The isoenzymes of MDHAR have
been reported to exist in several cellular compartments such as chloroplasts (Hossain et al., 1984),
cytosol, mitochondria, and peroxisomes (Dalton et al., 1993; Jimenez et al., 1997). During 10–20
days, a concomitant increase in MDHAR enzyme activity was observed with increase in the con-
centration of As3+ treatment in the seedlings of rice cultivars. Seedlings grown for 20 days in the
presence of 50 μM As3+ showed 22%–34% increase in MDHAR activity in roots and about 9%–61%
increased activity in shoots compared to controls (Mishra et al., 2011a). MDHAR activity increased
up to 20 μM in tolerant rice cultivars Triguna, IR-36, and PNR-519 followed by a decline at 50 μM.
Sensitive rice cultivar IET-4786 showed a concentration-dependent decline upon exposure to both
As5+ and As3+ (Rai et al., 2011).
DHAR catalyzes the reduction of DHA to AsA using GSH as the reducing substrate (Ushimaru
et al., 1997) and, thus, plays an important role in maintaining AsA level in its reduced form. The
activity of DHAR declined in the rice seedlings during 5–10 days of growth with As3+ treatment,
but during the later growth period of 15–20 days, As3+-treated seedlings showed increased DHAR
activity compared to controls (Mishra et al., 2011a). Seedlings exposed to 50 μM As3+ for 20 days
showed about 42%–79% increased DHAR activity in roots and about 50%–115% increased activ-
ity in shoots compared to the activity in controls (Mishra et al., 2011a). Exposure of both As5+ and
As3+ to tolerant rice cultivars Triguna, IR-36, and PNR-519 increased DHAR activity up to 20 μM
followed by a decline at 50 μM, whereas in sensitive rice cultivar IET-4786, DHAR activity showed
a concentration-dependent decline (Rai et al., 2011).
GSTs are a superfamily of multifunctional, dimeric enzymes, performing a range of functional
roles using GSH as a cosubstrate or coenzyme (Moons, 2005; Ghelfi et al., 2011). Based on simi-
larities in sequence and gene organization, six functional classes of GSTs, namely, phi (F), tau (U),
zeta (Z), theta (T), lambda (L), and DHAR, have been identified in plants (Dixon et al., 2002;
Moons, 2005). It has been demonstrated that in maize and mesquite (Prosopis sp.), GST activity is
stimulated upon exposure to arsenic (Mylona et al., 1998; Mokgalaka-Matlala et al., 2009). Mylona
and coworkers (1998) observed triggered responses of GST genes in maize upon As3+ as well as
As5+ exposure. GST genes are expressed in a tissue-, developmental stage-, and isoenzyme-specific
form, in developing embryos and young leaves. GST genes were found to be upregulated during
As3+ exposure in arsenic-tolerant rice cultivars Triguna, PNR-519, and IR-36 but downregulated in
arsenic-sensitive rice cultivar IET-4786. In case of As5+ stress, GST genes were upregulated in all
the rice cultivars examined (Rai et al., 2011).
34.5 ARSENIC TOLERANCE MECHANISMS IN PLANTS

Arsenic is a nonessential element for plants and its high concentration in soil leads to phytotox-
icity. Many species of crop plants typically suffer from phytotoxicity when arsenic concentration
reaches 5–20 mg/kg dry weight in shoots (Kabata-Pendias and Pendias, 1992). Plants that are not
tolerant to arsenic show various toxic symptoms including decrease in plant growth and crop yield
(Carbonell-Barrachina et al., 1995). In contrast, the growth of arsenic hyperaccumulating plants
such as Chinese brake fern is not compromised even at exceedingly high (>10,000 mg/kg dry
weight) concentrations of arsenic in the fronds (Ma et al., 2001; Tu et al., 2002a). The tolerance and
hyperaccumulation ability of Chinese brake fern is considered as a constitutive property (Bondada
and Ma, 2003; Wei and Chen, 2006). Several other ferns including Pityrogramma calomelanos
(Visoottiviseth et al., 2002), P. cretica, Pteris longifolia, Pteris umbrosa (Zhao et al., 2002), Pteris
multifida (Du et al., 2005), and Pteris nervosa (Chen et al., 2003) have been reported to hyperaccu-
mulate arsenic with no visible symptoms of injury. High resistance to arsenic has also been observed
in a number of plant species growing on arsenic-contaminated soils including Agrostis capillaris
(Porter and Peterson, 1975), Andropogon scoparius (Rocovich and West, 1975), Plantago lanceolata
(Pollard, 1980), Agrostis castellana, Agrostis delicatula (De Koe and Jacques, 1993), Deschampsia
cespitosa (Meharg and Macnair, 1991), H. lanatus (Macnair and Cumbes, 1987), Silene vulgaris
(Paliouris and Hutchinson, 1991), Cynodon dactylon (Jonnalagadda and Nenzou, 1997), Paspalum
tuberosum, Spergularia grandis (Bech et al., 1997), and Calluna vulgaris (Sharples et al., 2000).
S. vulgaris (Paliouris and Hutchinson, 1991) and P. lanceolata (Pollard, 1980) plants collected from

uncontaminated soils also exhibited resistance to arsenic. Analysis and comparison of responses of
plants growing on arsenic-contaminated site with uncontaminated site and various hypertolerant and
hyperaccumulator plants with nontolerant and nonaccumulator plants have provided valuable infor-
mation about the mechanisms of arsenic tolerance and detoxification (Dhankher et al., 2002; Ellis
et al., 2006; Srivastava et al., 2012).
A variety of tolerance and resistance mechanisms including avoidance or exclusion, which
minimizes the cellular accumulation of metals, and tolerance, which allows plants to survive
while accumulating high concentrations of metals, have been identified in plants (Figure 34.4).
In arsenic non-hyperaccumulating plants, resistance to environmental arsenic has been shown to
involve a decreased uptake of As5+ due to suppression of the high-affinity phosphate uptake sys-
tem (Meharg and Macnair, 1991, 1992); detoxification of As5+ by reduction to As3+, which sub-
sequently complexes with thiols, particularly PCs, and remains sequestered in roots (Pickering
et al., 2000; Schmoger et al., 2000; Hartley-Whitaker et al., 2001b; Dhankher et al., 2002); and
rapid efflux of As5+ and As3+ from plant roots back to the medium (Xu et al., 2007; Zhang et al.,
2008b; Logoteta et al., 2009). In hyperaccumulator plants, tolerance appears to involve increased
As5+ uptake, decreased As3+ –thiol complexation and As3+ efflux to the external medium, greatly
enhanced xylem translocation, and vacuolar sequestration of As3+ in fronds (Su et al., 2008;
Zhao et al., 2009). The present section describes the mechanisms associated with arsenic toler-
ance and detoxification in plants.
Suppression of
high-affinity
phosphate/arsenate
transport
Methylation and Reduction of

volatilization arsenate to arsenite
Arsenic tolerance
mechanisms in
non-hyperaccumulating plants
Arsenic efflux Synthesis of thiols
Arsenic
sequestration in the
vacuoles
FIGURE 34.4 Arsenic tolerance mechanisms in arsenic non-hyperaccumulating plants. In arsenic non-
hyperaccumulating plants, resistance to environmental arsenic involves decreased uptake of arsenate due to
the suppression of the high-affinity phosphate uptake system; detoxification of arsenate by its reduction to
arsenite, which subsequently complexes with thiols, particularly PCs, and remains sequestered in roots; and
rapid efflux of arsenate and arsenite from roots back to the medium. Whether arsenic methylation and volatil-
ization occur in vivo in higher plants is still a matter of debate.
34.5.1 Suppression of High-Affinity Phosphate/Arsenate Transport

Decreased As5+ uptake is considered as the major mechanism of naturally selected As5+ hypertoler-
ance in plants (Bleeker et al., 2003, 2006). AsTol, an As5+ tolerance gene identified on chromosome
six in rice inbred lines of Bala × Azucena, was shown to be related to another quantitative trait locus
(QTL), which is known to affect P uptake (Dasgupta et al., 2004). As As5+ is a chemical analog of
phosphate, it is easily incorporated into plant cells through the high-affinity phosphate transport
system. Therefore, constitutive suppression of high-affinity phosphate/As5+ transport might be an
important mechanism to avoid arsenic toxicity. However, only a few plants are known that can sup-
press phosphate transport system. Meharg and Macnair (1990) investigated the mechanism of As5+
tolerance in the velvet grass and showed that tolerance was achieved, at least in part by adaptation
of the phosphate uptake system. The high-affinity phosphate uptake system is inducible under low
plant phosphate status, thus increasing plant phosphate status tolerance by decreasing As5+ influx in
velvet grass (Meharg, 1994). Superior arsenic tolerance in the Portuguese broom (Cytisus striatus)
plants grown in arsenic-contaminated sites compared to uncontaminated site has also been attrib-
uted to reduced As5+ uptake through suppression of phosphate transporter activity (Bleeker et al.,
2003). Arabidopsis mutants defective in phosphate transport were also found to be more tolerant to
As5+ (Shin et al., 2004; Catarecha et al., 2007).
34.5.2 Reduction of Arsenate to Arsenite

As part of arsenic detoxification, the majority of As5+ is reduced to As3+ by the enzyme arsenate
reductase concomitantly oxidizing GSH to its oxidized form GSSG. It was suggested that all plants
may reduce As5+ to As3+; however, significant differences exist between Chinese brake fern and arse-
nic non-hyperaccumulating plants (Dhankher et al., 2002; Ellis et al., 2006). As5+ is reduced to As3+
that then forms a complex with thiolate ligands and remains sequestered in the roots of non-hyperac-
cumulating plants, Indian mustard (Pickering et al., 2000) and Arabidopsis (Dhankher et al., 2002).
Silencing the arsenate reductase gene AtACR2 in the root of Arabidopsis led to hyperaccumulation
of arsenic in the shoot (Dhankher et al., 2006), whereas Arabidopsis plants overexpressing E. coli
arsenate reductase gene, arsC, under the control of a light-induced soybean RuBisCO promoter
(SRS1p) strongly expressed ArsC protein in leaves, but not in roots, and were consequently hyper-
sensitive to As5+ (Dhankher et al., 2002). Coexpression of E. coli γ-ECS gene with arsC resulted
in higher tolerance and accumulation of arsenic (Dhankher et al., 2002). A comparative study of
As5+-hypertolerant velvet grass ecotype and nontolerant ecotype revealed increased expression of
As5+-inducible arsenate reductase (HlAsr) gene in hypertolerant ecotype. Further, Arabidopsis asr
T-DNA insertion mutants showed enhanced As5+ sensitivity and increased arsenic accumulation in
the roots, whereas overexpression of the AtASR cDNA in Arabidopsis was found to increase the
tolerance of plants to As5+ (Bleeker et al., 2006). In addition to decreased uptake, enhanced expres-
sion of HlASR was suggested to act as an additional determinant of As5+ hypertolerance and arsenic
transport in velvet grass. Also, enhanced PC-based sequestration in As5+-hypertolerant velvet grass
was suggested to be not due to enhanced capacity of PC synthesis as such but due to increased arse-
nate reductase activity (Bleeker et al., 2006).
In hyperaccumulating plant Chinese brake fern, arsenic is efficiently taken up from the soil
and is rapidly transported to the shoot in the xylem mainly as As5+ (Kertulis-Tartar et al., 2005).
Speciation analysis using x-ray absorption spectroscopy and high-performance liquid chromatography–
inductively coupled plasma mass spectrometry (HPLC–ICP-MS) revealed that arsenic is finally
stored as free As3+ in the fronds (Lombi et al., 2002; Wang et al., 2002; Webb et al., 2003; Zhao
et al., 2003; Huang et al., 2004). As5+ reduction was suggested to involve arsenic–thiolate species
surrounding veins as intermediates (Pickering et al., 2006). However, this model was not supported
by studies (Duan et al., 2005; Mathews et al., 2010). Duan and coworkers (2005) detected arsenate
reductase activity exclusively in the roots of Chinese brake fern and, because most of the arsenic
exists as As3+ in the fronds, concluded that arsenic is translocated in its reduced form. Mathews and
coworkers (2010) reported that As5+ reduction occurs mainly in the rhizomes where As3+ accounts
for 68%–71% and is limited in roots where it accounts for 7%–8%. Huang and coworkers (2011)
concluded that As5+ is transformed in the roots and rhizomes to As3+ and is then efficiently translo-
cated to the fronds by xylem.
34.5.3 Increased Synthesis of Glutathione and Phytochelatins

Complexation of As3+ with GSH and PCs, the polymers of GSH, is an important mechanism
employed by plants to detoxify arsenic. Plants exposed to arsenic substantially increase the syn-
thesis of GSH and PCs (Gupta et al., 2004; Grill et al., 2006; Srivastava et al., 2007; Mishra et al.,
2011a). Both As5+ and As3+ efficiently induce the biosynthesis of PCs (Schmoger et al., 2000). PCs
are synthesized from GSH by the action of enzyme glutamylcysteine dipeptidyl transpeptidase,
trivially named as phytochelatin synthase (PCS), which is constitutively expressed in plants but
may be regulated at transcriptional and translational levels by metals and metalloids (Grill et al.,
1985; Cobbett, 2000; Cobbet and Goldsbrough, 2002). Semiquantitative RT–PCR analysis of PCS
showed more expression of its transcript in Indian mustard tolerant variety Pusa Bold as compared
to Varuna due to As3+ stress. Increased tolerance of Pusa Bold compared to Varuna toward arse-
nic has been attributed at least in part to the upregulation of PCS transcripts (Gupta et al., 2009).
The level of PCs and PCS activity were found to be more pronounced in tolerant rice genotype
Triguna, compared to sensitive genotype IET-4786 (Tripathi et al., 2012). In Triguna, harmonized
responses of thiol metabolism were suggested to be responsible for arsenic tolerance in contrast to
IET-4786 showing its susceptible nature toward arsenic exposure. PCS-deficient Arabidopsis plants
were found to be hypersensitive to As5+, showing that PCs are absolutely required for As5+ tolerance
in these species (Howden et al., 1995; Ha et al., 1999).
Increased PC synthesis has been observed in response to arsenic exposure in nontolerant, nonac-
cumulator, as well as hypertolerant and hyperaccumulator plants (Grill et al., 2006; Tripathi et al.,
2007; Gupta et al., 2008). The rapid induction of the metal-binding PCs has been observed in cell
suspension cultures of snake root (Rauvolfia serpentina), in seedlings of Arabidopsis, and in enzyme
preparations of bladder campion (S. vulgaris) upon challenge to arsenicals (Schmoger et al., 2000).
Formation of arsenic complexes with GSH and PCs has been demonstrated in various plant spe-
cies like Arabidopsis (Schmoger et al., 2000), snake root (Schmoger et al., 2000), bladder campion
(Schmoger et al., 2000), velvet grass (Raab et al., 2004), Cretan brake fern (Raab et al., 2004), sun-
flower (H. annuus) (Raab et al., 2005), and Indian mustard (Pickering et al., 2000; Montes-Bayon
et al., 2004), and the species complexing is As3+ exclusively. The maximum amount of arsenic
chelated by PCs was found to be about 39% in hydrilla plants exposed to As3+ (at 10 μM) for 4 days
and up to 35% when exposed to 50 μM As5+ for 4 days (Srivastava et al., 2007). Raab and coworkers
(2004) demonstrated the presence of arsenic–PC and GSH–arsenic–PC complexes in roots, leaves,
and stems of arsenic-exposed sunflower. In arsenic-exposed sunflower, 14 different arsenic com-
plexes were detected in roots, 4 in stems, and 6 in leaves. Arsenic bound to arsenic–PC species in
roots included As3+–PC3, GS–As3+–PC2, the complex of As3+ with two (γ-Glu–Cys)2–Gly molecules
[As3+–(PC2)2], and monomethylarsonic PC-2 or (γ-Glu–Cys)2–Gly CH3As(MMA(III)–PC2) (Raab
et al., 2005). The complex of As3+ with (γ-Glu–Cys)3–Gly (As3+–PC3) and the complex of As3+ with
GSH and (γ-Glu–Cys)2–Gly (GS–As3+–PC2) were present in all samples (roots, stems, and leaves)
taken from plants exposed to arsenic. Thirteen percent of arsenic was present in PC complexes for
velvet grass (Raab et al., 2004).
In wild-type Arabidopsis roots, 69% of arsenic was complexed as As3+–PC4, As3+–PC3, and
As –(PC2)2 (Liu et al., 2010). A high percentage (>50%) of arsenic in the tissues of an arsenic-
3+
accumulating, hypertolerant Brassica, Isatis capadocica, plants was complexed with PC, and it
was argued that it is a constitutive, rather than an adaptive, mechanism of tolerance (Karimi et al.,
2009). In duckweed Asian watermeal (Wolffia globosa) plants, 74% of the arsenic accumulated
was complexed with PCs, with As3+–PC4 and As3+–PC3 being the main species. L-buthionine
sulfoximine (BSO), a specific inhibitor of γ–ECS, completely suppressed the synthesis of PCs and
the formation of As3+–PC complexes and resulted in decreased arsenic accumulation and arsenic
tolerance in velvet grass, both in As5+-hypertolerant and non-hypertolerant ecotypes (Bleeker et al.,
2006), and Asian watermeal plants (Zhang et al., 2012). These results demonstrate an important role
of PCs in arsenic tolerance in these plants.
PCs have been suggested to play a limited role in the arsenic hypertolerance in Chinese brake
fern (Zhao et al., 2003; Zhang et al., 2004). Synthesis of PC is induced upon exposure to As5+ in
Chinese brake fern, with PC2 as major detected species (Zhang and Cai, 2003; Zhao et al., 2003;
Zhang et al., 2004). In roots and shoots, arsenic concentration is correlated significantly with PC2
synthesis (Zhao et al., 2003). The molar ratio of PC–SH to arsenic was 0.03 and 0.09 for roots and
shoots, respectively, suggesting that only a small proportion (1%–3%) of the arsenic in Chinese
brake fern can be complexed with PCs (Zhao et al., 2003). Raab and coworkers (2004) observed
that arsenic hyperaccumulator Cretan brake fern synthesizes only PC2 and forms dominantly the
GS–As3+–PC2 complex and only 1% of arsenic is present in the PC complex.
34.5.4 Arsenic Sequestration in the Vacuoles

Arsenic sequestration in the vacuoles of plant tissues represents a crucial step in arsenic d etoxification.
Using energy-dispersive x-ray microanalyses (EDXA), it has been shown that arsenic is primarily
located in the upper and lower epidermal cells, probably in the vacuoles (Lombi et al., 2002). It is
proposed that plants can reduce As5+ to As3+ and store As3+–thiol complexes within the cell vacuoles
(Pickering et al., 2000). Acidic conditions present in the vacuoles have been suggested to increase
stability of such complexes by preventing reoxidation of As3+ and concomitant release of As3+ from
PC complexes (Sneller et al., 1999; Meharg and Hartley-Whittaker, 2002). Vacuolar uptake of PC–
metal(loid) complexes has been suggested to be mediated by ATP-binding cassette transporters (ABC
transporters) in yeast (Ortiz et al., 1992, 1995). In plants, long-sought and major vacuolar PC trans-
porters AtABCC1 and AtABCC2 have been identified recently (Song et al., 2010). Arabidopsis plants
were extremely sensitive to arsenic and arsenic-based herbicides in the absence of these two trans-
porters due to very low residual As3+–PC2 transport activity in vacuoles. In PC-producing S. cere-
visiae, heterologous expression of these ABCC transporters led to pronounced As3+–PC2 transport
activity in membrane vesicles and enhanced arsenic tolerance and accumulation (Song et al., 2010).
In arsenic hyperaccumulating species, such as Chinese brake fern, arsenic is not immobilized in
the roots, but is instead rapidly transported as As3+ through the xylem to the fronds (Lombi et al.,
2002; Pickering et al., 2006; Su et al., 2008). As arsenic is mostly present as inorganic As3+ and
only a small proportion of the arsenic can be complexed with PCs in hyperaccumulating and toler-
ant species Chinese brake fern, California plantain (Plantago erecta), and velvet grass (Mendoza-
Cozatl et al., 2005; Clemens, 2006; Rea, 2007; Verbruggen et al., 2009), it is suggested that plants
might have vacuolar As3+ transporters, possibly with similarities to bacterial As3+ extrusion pumps
(Tripathi et al., 2007). It has been shown that PvACR3, a homolog of the yeast ScACR3p protein
(a plasma membrane protein responsible for the efflux of As3+ from the yeast cell), is involved in
the vacuolar sequestration of As3+ in Chinese brake fern (Indriolo et al., 2010). Instead of delivering
the As3+ to the outside of the cell, PvACR3 resides on the vacuolar membrane and transports the
As3+ into the vacuole (Indriolo et al., 2010). Single-copy ACR3 genes are found in moss, lycophytes,
ferns, and gymnosperms, but not in angiosperms, which may help explain the lack of arsenic hyper-
accumulating capacity among the angiosperms (Indriolo et al., 2010).
34.5.5 Arsenic Efflux

Efflux of As3+ and As5+ from plant roots back to the medium is another strategy employed by
the plants to avoid arsenic toxicity. It has been suggested that arsenic efflux may be the general
mechanism for arsenic tolerance in plants (Zhang et al., 2008b; Logoteta et al., 2009). When
supplied with As5+, plant roots extrude a substantial amount of As3+ to the external medium
through as yet unidentified pathways. Rapid reduction of As5+ to As3+ and active efflux of
some part of As3+ have been shown in tomato, rice (Xu et al., 2007), Arabidopsis, velvet grass,
wheat, barley, and maize (Su et al., 2008). In tomato roots, initially, efflux of As3+ was faster
than that of As5+ and thereafter As5+ efflux exceeded that of As3+ (Xu et al., 2007). In arsenic-
accumulating fern, Azolla caroliniana and Azolla filiculoides, the amount of arsenic efflux
was proportional to the amount of arsenic accumulation and 10 times more As5+ was effluxed
than As3+ (Zhang et al., 2008b). To test whether As3+ efflux is a component of the adaptive
As5+ tolerance in velvet grass, Logoteta and coworkers (2009) exposed tolerant and nontolerant
phenotypes of velvet grass to different arsenic concentrations. Most of the As5+ taken up by
tolerant and nontolerant phenotypes was effluxed to the medium as As3+ within 2–24 h showing
that As3+ efflux is not adaptively enhanced in the tolerant phenotype velvet grass, but it could
be a basal tolerance mechanism to greatly decrease cellular arsenic burden in both phenotypes.
During the 24 h exposure to 5 μm As5+, rice roots extruded As3+ to the external medium rapidly,
accounting for 60%–90% of the As5+ uptake. Aquaporins have been thought to be the candidate
transporters responsible for As3+ efflux in plants (Bienert et al., 2008; Isayenkov and Maathuis,
2008; Zhao et al., 2009). A rice mutant defective in Lsi1 (OsNIP2;1, an aquaporin channel)
extruded significantly less As3+ than the wild-type rice and, as a result, accumulated more
As3+ in the roots, suggesting that Lsi1 plays a role in As3+ efflux in rice roots exposed to As5+.
However, this pathway accounts for only 15%–20% of the total efflux, suggesting the existence
of other efflux transporters (Zhao et al., 2010a).
Although arsenic hyperaccumulator Chinese brake fern possesses a highly efficient efflux sys-
tem for As3+ for xylem loading, little efflux of As3+ was observed to the external medium (Su et al.,
2008). In the arsenic efflux experiment conducted by Huang and coworkers (2011), As5+ efflux was
also observed with As3+ efflux in the external solution after 24 h of As5+ uptake. Low rates of As5+
efflux, ranging from 5.4% to 16.1% of the previously absorbed arsenic, indicated that a low rate of
As5+ efflux to the external solution is also a characteristic of Chinese brake fern, as was reported
with As3+ efflux.
34.5.6 Methylation and Volatilization

Members of the class of arsenite-S-adenosylmethyltransferase enzymes catalyze As3+ methyla-
tion to a variety of mono-, di-, and trimethylated species, some of which are less toxic than
As3+ itself. Though S-adenosylmethionine methyltransferases (arsM) have been well charac-
terized in cyanobacteria and algae, no methyltransferase gene has been identified in plants.
Nissen and Benson (1982) observed that methylation and reduction of As3+ to methanearsonic
acid, methanearsinic acid, and dimethylarsinic acid were apparent only in phosphate-deficient
tomato plants, whereas in lower and higher freshwater plants, for example, Nitella tenuissima
and Lemna minima, a considerable amount of arsenic was methylated. These workers suggested
that freshwater but not terrestrial plants have evolved mechanisms for rapid detoxication of As5+,
As3+, and other toxic arsenic species. In several studies where plants were fed only inorganic
arsenic in hydroponic culture, small amounts of methylated arsenic were detected in plant tis-
sues or xylem sap (Quaghebeur and Rengel, 2003; Mihucz et al., 2005; Raab et al., 2005; Xu
et al., 2007). It has been suggested that methylated arsenicals in plants probably originate from
microorganisms present in soils and in the rhizosphere. Recently, Lomax and coworkers (2012)
suggested that plants are unable to methylate inorganic arsenic and instead take up methylated
arsenic produced by microorganisms as methylated arsenicals were not found in rice, tomato,
and red clover exposed to inorganic arsenic under axenic conditions. Due to little information
available regarding arsenic methylation in higher plants, whether arsenic methylation and vola-
tilization occur in vivo in higher plants is still a matter of controversy.
34.6 STRATEGIES FOR DEVELOPING ARSENIC TOLERANCE IN PLANTS

Development of arsenic tolerance in plants depends largely on our understanding of arsenic uptake,
detoxification, and efflux mechanisms operating inside plant cells. Arsenic tolerance in plants is
modulated by a network of functional genes, their expression patterns, and temporal/spatial coor-
dination. Strategies for developing arsenic tolerance in plants usually include limiting the uptake of
arsenic, enhancing the detoxification and reduction of As5+ to As3+ in cytoplasm, chelation of As3+
by PCs, and eventual sequestration of As3+ or arsenic–PC complexes in the vacuoles. As3+ extrusion
and arsenic volatilization might also be helpful in minimizing arsenic load efflux. Plant breeding
and genetic engineering techniques have been suggested to be useful in development of arsenic
tolerance in plants (Dhankher et al., 2002; Dasgupta et al., 2004). QTL analyses can be used to iden-
tify genes involved in arsenic uptake, accumulation, and tolerance in plants, which can be used for
breeding arsenic-tolerant plants. In rice, a total of four QTLs including one QTL on chromosome 2
for arsenic concentrations in shoots at seedling stage, one on chromosome 3 for arsenic concentra-
tions in roots at seedling stage, and two QTLs on chromosomes 6 and 8 for arsenic concentrations
in grains at maturity have been identified. QTL on chromosome 8 is found to be colocated for arse-
nic concentrations in grain at maturity and shoot P concentrations at seedling stage (Zhang et al.,
2008a). In maize, 11 QTLs including 3 QTLs mapped on chromosomes 1, 5, and 8, respectively,
for arsenic concentration in leaves, 2 QTLs in the bracts, 3 QTLs for arsenic concentration in the
stems, and three QTLs on chromosomes 3, 5, and 7, respectively, were identified for kernels (Ding
et al., 2011). The results implied that different molecular mechanisms control arsenic accumula-
tion in different tissues in maize. QTLs identified in rice (Zhang et al., 2008a) and maize (Ding
et al., 2011) will be useful for selecting inbred lines and hybrids with low arsenic concentration
in their tissues and hence will be useful in breeding for arsenic-tolerant plants. By screening 108
recombinant inbred lines of the Bala × Azucena rice mapping population, a major gene, AsTol, was
identified, which was mapped on chromosome 6 and colocalized with a phosphate uptake QTL in
another population of rice. Characterization of this gene has been suggested to be valuable for inves-
tigations into the physiological and molecular mechanisms underlying arsenic tolerance in plants
and may lead to strategies aimed at breeding for arsenic tolerance (Dasgupta et al., 2004).
Manipulation of genes involved in arsenic uptake, reduction of As5+ to As3+, increased accumu-
lation or synthesis of PCs and/or GSH, chelation of As3+, vacuolar and compartmentalization of
complexed As3+ or even free As3+, and arsenic volatilization are suggested to be good candidates
for developing genetically modified arsenic-tolerant plants. Very few studies report the develop-
ment of arsenic-tolerant plants by manipulating single or two genes (Table 34.3). Improving arse-
nic tolerance in crop plants requires a multifaceted approach. Arsenic in the environment mainly
exists in two inorganic oxidation states, As5+ and As3+, which enter plant cells via phosphate trans-
porters and aquaglyceroporins, respectively. These transporters provide useful tools for genetic
engineering of plants with enhanced arsenic tolerance. As phosphate transporters are main routes
of As5+ uptake in plants, suppression of high-affinity phosphate/As5+ transport appears to be an
important mechanism to limit arsenic uptake in plants. Arabidopsis mutants defective in phos-
phate transport were found to be more tolerant to As5+ (Shin et al., 2004; Catarecha et al., 2007).
However, suppression of high-affinity phosphate/As5+ transport will inhibit uptake of P also. P is
an essential element for plant and its deficiency severely limits crop yield. Hence, limiting arsenic
uptake by inhibiting phosphate transporter might not be suitable for crop plants. Arsenic and P act
as competitive inhibitors for each other; hence, high levels of phosphate might prove beneficial.
Nonresistant plants can be made more resistant to As5+ by raising their P status, which in turn leads
to lower levels of As5+ accumulation through suppression of phosphate/As5+ uptake (Meharg et al.,
1994; Meharg and Hartley-Whitaker, 2002). In velvet grass, increasing plant phosphate status
increases tolerance by decreasing As5+ influx (Meharg, 1994). Lee and coworkers (2003) isolated
Arabidopsis mutant arsenic-resistant 1 (ars1) with increased tolerance to As5+. They attributed the
tolerance in ars1 to increased phosphate uptake in ars1.
TABLE 34.3
Arsenic Tolerance and Arsenic Accumulation in Transgenic Plants Overexpressing Various Enzymes Involved
in Detoxification, Methylation, and Efflux of Arsenic
Enzyme/ Transgenic Arsenic Tolerance Arsenic Accumulation
Transporter Gene Origin Plant of Transgenic Plant in Transgenic Plant Reference
PCS AtPCS1 A. thaliana B. juncea Growth of seedlings less No increase in the Gasic and Korban (2007)
affected and had longer roots accumulation of arsenic in the
compared to wild-type plants aboveground tissues
PCS and γ-ECS AsPCS1 A. sativum, A. thaliana Root growth greater than Higher accumulation of arsenic Guo et al. (2008)
with GSH1 S. cerevisiae single-gene transgenic and in aboveground vegetation of
wild-type plants dual transformed lines
γ-ECS and arsenate γ-ECS with E. coli A. thaliana Greater fresh shoot weight than Higher concentrations of Dhankher et al. (2002)
reductase arsC wild-type or single-gene arsenic in shoots than
transgenic plants wild-type or single-gene
transgenic plants
PCS and vacuolar AsPCS1 A. sativum, A. thaliana Longer root than wild-type and Higher concentrations of Guo et al. (2012)
transporter with YCF1 S. cerevisiae single-gene transgenic plants arsenic in leaves than
wild-type plants
PCS and vacuolar AtPCS1 with A. thaliana A. thaliana Better growth and increased Not determined Song et al. (2010)
Arsenic Toxicity and Tolerance Mechanisms in Crop Plants
transporter AtABCC1 shoot weight compared to

wild-type and single-gene
transgenic plants
Arsenite efflux ScAcr3 S. cerevisiae O. sativa Not determined Lower arsenic concentrations Duan et al. (2012)
transporter in both roots and shoots than
wild-type plants
Arsenite efflux ACR3 S. cerevisiae A. thaliana Increased protoplast viability Tissue arsenic levels hardly Ali et al. (2012)
transporter and comparable or better affected in roots and shoots
growth/fresh weight compared compared to wild-type plants
to wild-type plants
As(III)-S- arsM R. palustris O. sativa No obvious differences in No significant difference in Meng et al. (2011)
adenosylmethionine appearance, growth, and arsenic accumulation in roots
methyltransferase biomass between transgenic and shoots between transgenic
and nontransgenic and wild-type plants
763
Manipulation of aquaglyceroporins involved in uptake of As3+ provides opportunities to increase

arsenic tolerance in plants. Increased tolerance to As3+ was observed in Arabidopsis mutant that
lacks aquaglyceroporins NIP7;1 (Isayenkov and Maathuis, 2008). Similarly, Kamiya and cowork-
ers (2009) also observed that disruption of NIP1;1 function leads to enhanced As3+ tolerance in
plants. Uptake of As3+ in rice is mediated by the silicic acid transporters OsLsi1 (a silicon influx
transporter) and OsLsi2 (responsible for the efflux of silicic acid and As3+ from exo- and endoder-
mal cells toward the stele) (Ma et al., 2008). Due to lower selectivity of OsLsi1 for As3+ than for
silicic acid, it may be difficult to control As3+ uptake by modifying OsLsi1 without affecting silicic
acid uptake (Mitani-Ueno et al., 2011). It was suggested that manipulation of other transporters for
As3+ (e.g., OsLsi2) may provide more promising opportunities to reduce arsenic accumulation in
plants (Mitani-Ueno et al., 2011). Iron plaque provides a further means to modulate arsenic uptake
and hence arsenic tolerance in plants. Arsenic uptake by rice seedlings is altered by iron plaque on
root surface (Liu et al., 2004a). In plants grown in solution without P, arsenic concentrations were
significantly higher in iron plaque and significantly lower in shoots compared to plants grown in
P-supplemented solution, indicating that iron plaque might sequestrate arsenic and consequently
reduce the translocation of arsenic from roots to shoots (Liu et al., 2004a).
Complexation of arsenic with thiols including PCs and GSH constitutes the first step in their
detoxification. Therefore, increased thiol production can be used as a method to enhance arsenic tol-
erance in plants. Transgenic plants overexpressing the genes of thiol (Cys, GSH, or PC) biosynthesis
pathway showed enhanced tolerance to arsenic due to increased thiol synthesis (Gasic and Korban
2007; Guo et al., 2008; Wojas et al., 2010; LeBlanc et al., 2011). Overexpression of Arabidopsis
AtPCS1 gene, encoding PCS in Indian mustard, increased its tolerance to arsenic but did not enhance
arsenic accumulation probably because PC synthesis is also limited by the production of GSH (Gasic
and Korban, 2007). Guo and coworkers (2008) observed that the overexpression of AtPCS1 and
GSH1, encoding γ-ECS, the rate-limiting step in GSH biosynthesis, simultaneously in Arabidopsis,
led to elevated total PC production and increased arsenic tolerance and accumulation in transgenic
Arabidopsis plants. Eastern cottonwood (Populus deltoides) plants expressing ECS had elevated
thiol group levels (consistent with increased ECS activity) and enhanced growth compared to control
under arsenic toxicity. Furthermore, roots of ECS-expressing plants accumulated significantly more
arsenic than control roots (approximately twice as much), while shoots accumulated significantly
less arsenic than control shoots (approximately two-thirds as much) (LeBlanc et al., 2011).
Plant vacuoles act as final detoxification stores for arsenic (Song et al., 2010). As3+ forms com-
plex with PC and the complexes get sequestered in vacuole. As5+, which is the major species taken
up by plant cells under aerobic conditions, cannot form complexes with PCs as it has no affinity
for thiol groups. Therefore, the reduction of As5+ to As3+ inside the cytoplasm, which can then
form complexes with PCs, plays an important role in the detoxification of arsenic in higher plants
(Jocelyn, 1972; Quaghebeur and Rengel, 2005). Arabidopsis plants transformed with the gene for
the E. coli arsenate reductase gene, arsC, expressed from a light-responsive transcription factor
strongly expressed ArsC protein in leaves, but not roots, and the plants were consequently hypersen-
sitive to As5+. Hypersensitivity to arsenic in these plants was suggested to be possibly due to higher
toxicity of product, As3+, compared to substrate, As5+, without sufficient thiols to form the arsenic
GSH conjugates. Coexpression of E. coli γ-ECS gene with arsC resulted in higher tolerance and
accumulation of arsenic (Dhankher et al., 2002).
Attempts have also been made to manipulate arsenic chelation and vacuolar compartmentaliza-
tion process simultaneously for arsenic detoxification and tolerance in plants. Overexpression of
vacuolar transporter has been suggested to increase plants’ ability to pump heavy metals into a safe
compartment while requiring only a small amount of transporters rather than a large amount of che-
lating peptide material (Tong et al., 2004). Transgenic Arabidopsis plants simultaneously overex-
pressing AsPCS1 derived from garlic (Allium sativum) and yeast vacuolar transporter YCF1 derived
from baker’s yeast showed increased tolerance and accumulation of arsenic (Guo et al., 2012).
Similarly, Arabidopsis plants overexpressing AtPCS1 and major vacuolar PC transporter AtABCC1
showed increased arsenic tolerance compared to wild-type plants or plants overexpressing AtPCS1
or AtABCC1 independently (Song et al., 2010).
The rapid As3+ efflux by roots may contribute to an important mechanism of arsenic detoxifi-
cation in plants, and enhancing arsenic efflux by roots would be a potential strategy to increase
arsenic tolerance in plants (Tripathi et al., 2007; Verbruggen et al., 2009; Zhao et al., 2009; Zhu
and Rosen, 2009). In S. cerevisiae, ScAcr3p is involved in extrusion of As3+. However, no ScAcr3p
homolog has been identified in plants. Overexpression of ScAcr3 in rice led to significantly higher
arsenic efflux activities and about 30% lower arsenic concentrations in shoots and roots compared
to control (Duan et al., 2012). Wild-type and nip7;1 (which carries a loss of function in AtNIP7;1)
Arabidopsis plant expressing S. cerevisiae As3+ efflux transporter ACR3 showed increased toler-
ance to As3+ and As5+ and a greater capacity for As5+ efflux but did not lower arsenic tissue levels
(Ali et al., 2012). As some of the methylated arsenic species are less toxic than As3+ itself, there is
possibility of engineering arsenic methylation and volatilization for increasing arsenic tolerance
in plants. ArsM, which converts As3+ into various methylated metabolites that are less toxic than
As3+ itself, has been identified in mammals and microbes (Thomas et al., 2004) but not in plants.
Expression of an arsM gene from the soil bacterium Rhodopseudomonas palustris in Japonica rice
cv. Nipponbare resulted in production of methylated arsenic species including MMA and DMA.
After 12 days exposure to As3+, the transgenic rice gave off 10-fold greater volatile arsenicals
(Meng et al., 2011). However, no significant differences were observed in biomass or total arsenic
accumulation between the transgenic rice and the nontransgenic control after exposure to As3+ or As5+.
Ye and coworkers (2012) proposed that integration of various components influencing biomethyl-
ation may be essential in developing smart photosynthetic organisms with an enhanced and sensi-
tive biomethylation capacity.
34.7 PHYTOREMEDIATION OF ARSENIC-POLLUTED SOIL

Arsenic is one of the most hazardous pollutants of the biosphere (Kramer, 2000). Arsenic-
contaminated sites can be remediated by ex situ physical and chemical techniques; however,
these techniques can be very expensive, which could damage soil structure, ecology, and soil
productivity (Salt et al., 1995). Phytoremediation is a plant-based, less expensive, more effective,
and environmentally friendly alternative to physicochemical remediation. There are several fac-
tors that can affect the efficiency of phytoremediation of arsenic-contaminated soils, including
plant species, arsenic availability for the hyperaccumulator plants, physical and chemical proper-
ties of soil, soil amendments, and soil microorganisms (Yan et al., 2010). Basically, the strategy
of phytoremediation can be divided into five fundamental processes including phytoextraction,
stabilization, immobilization, volatilization, and rhizofiltration (Salt et al., 1998; Wenzel et al.,
1999). Numerous studies associated with arsenic phytoremediation, including phytoextraction,
phytostabilization, and phytovolatilization on arsenic-contaminated areas, can be found in the
literature (Ma et al., 2001; Sakakibara et al., 2007). Phytoextraction is the use of hyperaccumula-
tor plants to take up and translocate metal contaminants from soil to the aboveground portions,
whereas phytostabilization is based on the use of plants, especially roots or plant exudates,
to stabilize, demobilize, and bind the contaminants in the soil matrix, thereby reducing their
bioavailability (Cunningham et al., 1995; Wenzel et al., 1999; Pilon-Smits and Freeman, 2006;
Padmavathiamma and Li, 2007). Phytovolatilization is the most controversial phytoremediation
technology and involves the production of toxic volatile compounds and their emission from
plants (Sakakibara et al., 2007).
Arsenic hyperaccumulator plants are used in phytoextraction of arsenic from soil. In contrast
to arsenic non-hyperaccumulating plants, hyperaccumulators do not restrict uptake of arsenic by
the roots; instead arsenic is taken up and translocated immediately from soil to the aboveground
portions. Chinese brake fern is the best-known arsenic hyperaccumulator plant (Ma et al., 2001).
Chinese brake fern can accumulate extremely large concentrations of arsenic in its aboveground
biomass (fronds) and thus has potential application in phytoremediation of arsenic-contaminated

sites. When grown in areas with elevated levels of arsenic, more than 1% of the dry weight of
Chinese brake fern frond was found to be arsenic (Tongbin et al., 2002; Wang et al., 2002). Arsenic
concentration in Chinese brake fern fronds growing in soil spiked with 1,500 parts/million (ppm)
arsenic increased from 29.4 to 15,861 ppm in 2 weeks (Ma et al., 2001). Tu and coworkers (2002a)
observed arsenic concentration of 7230 mg/kg in the fronds of Chinese brake fern after 20 weeks
with a translocation factor (ratio of arsenic concentration in shoot to that in root) of 24 and a biocon-
centration factor (ratio of plant arsenic concentration to water-soluble arsenic in soil) of 1450. Most
of the arsenic taken up from soil was translocated to the fronds (90%), in which arsenic concentra-
tion increased with frond age (Tu et al., 2002a). Old fronds accumulated as much as 13,800 mg
arsenic/kg. About 26% of the original soil arsenic was removed by the fern after 20 weeks of growth
(Tu et al., 2002a). Chinese brake fern has numerous other desirable characteristics such as versatility
and hardiness, large biomass, vigorous perennial growth habit, extensive root system, fast growth,
easy to reproduce, resistant to disease and pests, high arsenic accumulation rate, and diverse eco-
logical niche with high pH that make it ideal for phytoremediation of arsenic-contaminated soil and
water (Bondada and Ma, 2003).
Hyperaccumulator plants have evolved a variety of tolerance and resistance mechanisms that
allow plants to accumulate extremely high concentration of metals. Mechanisms of arsenic hyper-
accumulation by Chinese brake fern (Ma et al., 2001) is believed to involve uptake of As3+ and
As5+ by the roots (Wang et al., 2002; Fayiga et al., 2005), translocation of As3+ and As5+ from the
roots to fronds (Kertulis-Tartar et al., 2005; Singh and Ma, 2006), reduction of As5+ to As3+ in the
fronds (Bondada et al., 2004; Tu et al., 2004), and transportation of As3+ into vacuoles for stor-
age (Lombi et al., 2002). Lack of As3+ –PC complexation in Chinese brake fern is suggested to be
one of the important reasons for the highly efficient translocation of arsenic from roots to fronds
(Su et al., 2008; Zhao et al., 2009). The tolerance and hyperaccumulation ability of Chinese brake
fern is considered as a constitutive property (Bondada and Ma, 2003; Wei and Chen, 2006). The
growth of these plants is not compromised due to extremely high levels of accumulating arsenic.
It has been suggested that ferns that exhibit arsenic hyperaccumulation arrived relatively late in
terms of fern evolution, as this character is not exhibited by primitive ferns or their allies (Meharg,
2003). Tu and coworkers (2002b) suggested that phosphate application may be an important strat-
egy for efficient use of Chinese brake fern to phytoremediate arsenic-contaminated soils. A pre-
liminary kinetics of uptake characteristic of arsenic by Chinese brake fern indicated that roots
with low arsenic concentration and high P/arsenic ratio exhibited increased affinity to, and high
influx rate of, arsenic (Tu et al., 2002b). Jankong and coworkers (2007) studied the effect of P fer-
tilizer and rhizosphere bacteria on arsenic accumulation by the silverback fern (P. calomelanos).
Both P fertilizer and rhizobacteria were found to significantly increase plant biomass and arsenic
accumulation of silverback fern in both greenhouse and under field conditions suggesting that
P fertilizer and rhizosphere bacteria enhance arsenic phytoextraction. Arsenic accumulation in
food crops such as rice is of major concern. Effective stripping of bioavailable arsenic from con-
taminated paddy soils by arsenic hyperaccumulator Chinese brake fern reduced arsenic uptake
by rice (Ye et al., 2011). Ding and coworkers (2011) suggested that maize plants that accumulate
large concentrations of arsenic and follow the leaves > stems > bracts > kernels trend of arsenic
concentration could also be a useful model plant for phytoremediation of arsenic-contaminated
paddy soil (Ding et al., 2011).
In phytoextraction method, aboveground biomass needs to be harvested periodically for dis-
posal in some safe facility. In phytostabilization, contaminants are immobilized in the soil through
absorption and accumulation by root, absorption onto roots, or precipitation within the rhizosphere;
therefore, arsenic is not accumulated in the aboveground vegetative biomass, thus reducing or elimi-
nating the need to address disposal issue and the risk of integration of toxic elements into the food
chain of animal species (Madejon et al., 2002). Mendez and Maier (2008) suggested that phytoex-
traction, due to high implementation costs and long time frames, will be more suitable for sites that
have high land values and for which metal removal is required, whereas phytostabilization, due to
lower costs and easier implementation, could be a more common approach. Important characteris-
tics of a plant to be useful for phytostabilization include arsenic tolerance and low levels of arsenic
accumulation, as well as the ability to limit arsenic availability (King et al., 2008). Many plant
species including C. dactylon, Sorghum halepense (Madejon et al., 2002), Salix, Populus, Alnus
(French et al., 2006), Lupinus albus (Vazquez et al., 2006), Eucalyptus cladocalyx (King et al.,
2008), Cistus salviifolius (Abreu et al., 2012), and Opuntia lasiacantha (Santos-Jallath et al., 2012)
were found to be suitable for phytostabilization of arsenic. White lupin (L. albus) reduced the con-
centrations of soluble arsenic in the soil in the vicinity of a spill from the Aznalcóllar pyrite mine in
southern Spain and accumulated significant quantities of it in the roots and root nodules (Vazquez
et al., 2006). Eucalyptus species (E. cladocalyx, Eucalyptus melliodora, Eucalyptus polybractea,
and Eucalyptus viridis) used for phytostabilization of arsenic from gold mine tailings were found
to be tolerant to high levels of arsenic to which they were exposed and accumulated little arsenic
in aboveground biomass and none in the young foliage. The presence of these trees did not affect
arsenic availability and pH of the soil (King et al., 2008). Of the four Eucalyptus species studied,
E. cladocalyx was suggested to be an ideal candidate for the phytostabilization of arsenic (King
et al., 2008). Transformation and volatilization of arsenic are well studied in bacteria; however, little
is known about the process of volatilization in plants (Bentely and Chasteen, 2002; Qin et al., 2006).
The enzyme arsM catalyzes the formation of a number of methylated intermediates from As3+, with
volatile trimethylarsine (TMA) as the end product (Qin et al., 2006). Sakakibara and coworkers
(2007) collected vapor samples to determine volatilization of arsenic compounds from fronds of
Chinese brake fern grown in arsenic-polluted soil. The percent of arsenic components in one vapor
sample was found to be 37% for arsenite and 63% for arsenate. However, a large amount of arsenic
released from the contaminated site into the atmosphere by the fern may cause a secondary arsenic
contamination to the surrounding environments (Sakakibara et al., 2007).
Genetic engineering can substantially improve the efficiency and versatility of phytoremediation
(Zhu and Rosen, 2009). Genes involved in arsenic transport, reduction of As5+ to As3+, translocation
of arsenic from roots to shoots, increased accumulation or synthesis of PCs and/or GSH, chelation
of As3+, vacuolar compartmentalization of complexed As3+ or even free As3+, and arsenic volatil-
ization are suggested to be good candidates for genetically enhanced phytoremediation strategies
(Dhankher et al., 2006; Meng et al., 2011; Ye et al., 2012). As many gene products are involved in
the process of tolerance and hyperaccumulation of arsenic, single genes and multigenic engineering
approaches may be applied to enhance the efficiency of phytoremediation (Dhankher et al., 2002;
Tripathi et al., 2007; Zhu and Rosen, 2009). Arsenate reductase converts As5+ to As3+ in roots and
thus plays a role in immobilizing arsenic belowground. Dhankher and coworkers (2006) suggested
that blocking this activity by reducing expression of arsenate reductase (ACR2) homologs in plants
would lead to enhanced mobilization of As5+ in aboveground parts. Thus, arsenate reductase can
play a vital role in the phytoremediation of environmental arsenic contamination (Dhankher et al.,
2006). Simultaneous overexpression of AsPCS1 from garlic (A. sativum), which encodes PCS and
GSH1 from S. cerevisiae, which encodes γ-ECS, the rate-limiting step in GSH biosynthesis, led to
elevated total PC production in transgenic Arabidopsis. When treated with arsenic (As3+/As5+), dual-
gene transgenic lines had the longest roots and the highest accumulation of arsenic than single-gene
transgenic lines and wild type (Guo et al., 2008). The increase in arsenic tolerance and accumula-
tion was also observed in tobacco plants overexpressing AtPCS1 from Arabidopsis and CePCS
from Caenorhabditis elegans. These genes were suggested to be promising candidates for plant
engineering for phytoremediation (Wojas et al., 2010). Attempts have also been made to manipulate
arsenic chelation and vacuolar compartmentalization process for arsenic detoxification and toler-
ance in plants. Dual-gene transgenic lines accumulated over 2–10-fold cadmium/As3+ and 2–3-fold
As5+ than wild type or plants expressing AsPCS1 or YCF1 alone. Such stacking of modified genes
involved in chelation of toxic metals and vacuolar compartmentalization represents a highly prom-
ising tool for their use in phytoremediation efforts (Guo et al., 2012).
34.8 CONCLUSIONS AND FUTURE PROSPECTS

Arsenic is a well-known toxic metalloid that is present in the form of various chemical species. It
can enter soil through both natural processes and anthropological activities. Absorption of arsenic
by plants is influenced by many factors including plants species, the concentration of arsenic in the
soil, soil properties such as pH and clay content, and the presence of other ions. Aquaglyceroporins
mediate uptake of As3+ and undissociated methylated arsenic species in plants, whereas being a
phosphate analog, As5+ is taken up by phosphate transporter. Phytotoxicity of arsenic species is
affected considerably by the chemical form in which it occurs in the soil and concentration of the
metalloid. The primary cause of As3+ toxicity is its interference with a variety of enzymes because
it has high affinity to sulfhydryl groups found on many enzymes, whereas being a phosphate analog,
As5+ can substitute inorganic phosphate in a plethora of biochemical processes, thus affecting key
metabolic processes in the cell. Arsenic toxicity is also mediated by oxidative stress by overproduc-
ing ROS and altering antioxidant defenses in plant tissues, which can be detected by oxidative modi-
fication of lipids, proteins, and nucleic acids. At elevated arsenic concentrations, biomass production
and yields of various plant species are reduced significantly. A variety of tolerance and resistance
mechanisms including avoidance or exclusion, which minimizes the cellular accumulation of met-
als, and tolerance, which allows plants to survive while accumulating high concentrations of metals,
have been identified in plants. These include decreased uptake of As5+ due to suppression of the
high-affinity phosphate uptake system; detoxification of As5+ by reduction to As3+, which subse-
quently complexes with thiols, particularly PCs, and remains sequestered in roots; and rapid efflux
of As5+ and As3+ from plant roots back to the medium. Whether arsenic methylation and volatiliza-
tion occur in vivo in higher plants is still a matter of debate. Arsenic hyperaccumulator plants take
up and sequester exceptionally high concentrations of arsenic in the aboveground parts and hence
offer a great promise to phytoremediation of arsenic. Tolerance in these plants appears to involve
increased As5+ uptake, decreased As3+–thiol complexation and As3+ efflux to the external medium,
greatly enhanced xylem translocation of As3+, and vacuolar sequestration of As3+ in fronds.
Considerable progress has been made during recent years in our understanding of the mecha-
nisms underlying uptake of arsenic species in plants; however, much remains to be explored related
to the diversity of arsenic uptake, complex transportation behaviors, and regulation of the uptake
of different arsenic chemical forms and its transformations in various plant species. Mechanism
of exceedingly efficient root to shoot translocation of arsenic remains to be elucidated in arsenic
hyperaccumulator species. A better understanding of the mechanisms responsible for arsenic resis-
tance, accumulation, transport, and toxicity in plants is needed for production of arsenic-resistant
plants for safe cropping and phytoremediation. Plant breeding techniques and genetic engineering
approaches have been employed to enhance arsenic tolerance in plants. Using transgenic approach,
to produce arsenic-tolerant crop plants, efforts have been largely focused on introduction of single
or dual genes involved in detoxification, sequestration, and efflux of arsenic in plants; however, to
make real progress, a multifaceted transgenic approach using multiple gene targets is needed.
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Part VI
Physiology of Plant/Crop
Genetics and Development
35 Small RNAs in Crop Response
to Temperature Stress
Noncoding RNAs in Plants
Galina Yahubyan, Elena Apostolova,
Ivan Minkov, and Vesselin Baev
CONTENTS
35.1 Plant Small RNAs.................................................................................................................. 785
35.2 Small RNAs Are Involved in Plant Stress Response............................................................ 786
35.3 Temperature-Responsive Small RNAs of Crop Plants.......................................................... 786
35.3.1 Rice............................................................................................................................ 786
35.3.2 Wheat......................................................................................................................... 787
35.3.3 Chinese Cabbage....................................................................................................... 788
35.3.4 Other Crop Small RNAs............................................................................................ 789
35.4 Conclusions............................................................................................................................ 789
References....................................................................................................................................... 792
35.1 PLANT SMALL RNAs

Plant small RNAs, which range in size from approximately 20–30 nucleotides (nt), can be distin-
guished both by their biogenesis and mode of action. They include microRNAs (miRNAs) and
small interfering RNAs (siRNAs) such as repeat-associated siRNAs (ra-siRNAs), trans-acting
siRNAs (ta-siRNAs), and natural antisense transcript siRNAs (nat-siRNAs) (Vaucheret 2006). Each
type of small RNA is unique with respect to length, the plant DICER-like (DCL) endonucleases that
are involved in its biogenesis pathway, and the ARGONAUTE (AGO) proteins that are small RNA
guides for target silencing. Most siRNAs target the same locus they were derived from, except for
miRNAs and ta-siRNAs, which target mRNAs from different loci. All small RNA molecules may
function as negative regulators of gene expression (Ruiz-Ferrer and Voinnet 2009).
miRNAs are the most abundantly expressed and well-studied class of small RNAs in plants.
They are typically 21 nt in length and their biogenesis involves DCL1 (Jones-Rhoades et al. 2006).
miRNAs are generated through the PolII/DCL1 pathway from single-stranded RNA precursors that
form hairpin structures. Though the guide and passenger strands of miRNA are the most predomi-
nant species from a precursor, high-throughput sequencing (HTS) data show that there are often
low-frequency positional and length variants from miRNA hairpin structures (Meyers et al. 2008).
miRNAs are loaded into AGO1 and act upon target mRNAs in a sequence-dependent manner. They
share perfect or nearly perfect complementarity with their targets (Rhoades et al. 2002) and regulate
their expression by either an RNA cleavage mechanism or translational repression (Hutvagner and
Zamore 2002; Llave et al. 2002; Sunkar et al. 2007).
In general, ra-siRNAs are 21–24 nt in length and their biogenesis involves DCL2, DCL3, and
DCL4 (Kasschau et al. 2007; Xie et al. 2004). These small RNAs are mainly loaded into AGO3
and are known to be involved in DNA and histone methylation (Xie et al. 2004; Zheng et al. 2007;
785
Zilberman et al. 2003). ta-siRNAs are phased 21 nt RNA molecules whose production involves only
DCL4 and is triggered by miRNA-directed cleavage of the TAS transcripts. Further ta-siRNAs are
loaded into AGO1 and, like miRNAs, promote guide sequence-specific cleavage of their targeted
gene transcripts (Allen et al. 2005; Axtell et al. 2006). The biogenesis of 21 and 24 nt nat-siRNAs
involves one of DCL1, DCL2, and DCL3, and a subgroup of nat-siRNAs is dependent on RDR2
and PolIV (Borsani et al. 2005; Katiyar-Agarwal et al. 2006; Zhang et al. 2012). A new category of
small RNAs ranging from 30 to 40 nt in size, referred to as long siRNAs (lsiRNAs), has been added
to the list recently (Katiyar-Agarwal et al. 2007).
35.2 SMALL RNAs ARE INVOLVED IN PLANT STRESS RESPONSE

In plants, miRNAs and siRNAs function as negative regulators of gene expression (Voinnet 2009).
Main targets of miRNA-controlled gene regulation in plants are transcription factors that can poten-
tially participate in most developmental processes (Jones-Rhoades et al. 2006). A growing number
of studies demonstrate that miRNAs are vital not only for plant growth and development but for
plant adaptation to stress conditions as well. The role of siRNAs in plant stress response had become
evident even when they were originally discovered in plants undergoing infection with viruses
(or transgene silencing) (Hamilton and Baulcombe 1999; Zamore et al. 2000). In 2003, Szittya and
collaborators analyzed Nicotiana benthamiana protoplasts transfected with Cymbidium ringspot
virus and showed that virus- and transgene-induced RNA silencing was dependent on temperature.
The authors found that the accumulation of siRNAs, but not of miRNAs, was lower under cold
stress and speculated that the activity of some DCL enzymes could be affected in these conditions
(Szittya et al. 2003). Zhu and co-workers (Sunkar et al. 2007) supposed two possible modes of small
RNA-guided target gene regulations under abiotic stress. Stress-induced small RNAs might target
negative regulators of stress tolerance for enhanced suppression, while stress-repressed small RNAs
likely target positive regulators of stress tolerance resulting in the accumulation of gene products.
In field, crop plants can tolerate a broad range of daily and seasonal changes in the temperature.
However, dramatic shifts in temperature beyond genotypic thresholds result in low- or high-temper-
ature stress. Plants cope with temperature stress by altering cellular structures and functions and
by reprogramming gene expression profiles of their vegetative and reproductive organs (Fowler and
Thomashow 2002; Frank et al. 2009; Kreps et al. 2002; Larkindale and Vierling 2008). Genome-
wide transcriptome studies revealed specific temperature-depending responses. For example, high
temperature triggers an increased expression of heat stress transcription factors (HSFs), while low
temperature promotes the expression of cold-specific transcription factors (Hu et al. 2009; Jung
et al. 2012; Qin et al. 2008). With the HTS techniques becoming more and more accessible, a great
number of species-specific small RNAs have been identified in crops to be involved in plant response
to temperature stress conditions.
35.3 TEMPERATURE-RESPONSIVE SMALL RNAs OF CROP PLANTS

35.3.1 Rice
In rice (Oryza sativa), there are 491 annotated miRNAs according to the miRBase database (ver-
sion 17; www.mirbase.org). A total of 43 miRNAs showed significant changes in their abundance
under drought, cold, and salt stress in rice inflorescences (Barrera-Figueroa et al. 2012). Majority
(12 out of 15) of cold-regulated miRNAs were upregulated (miR393, miR394, miR396, miR529,
miR530-3p, miR1856, miR2275d,e,c, and miR2871), and that was the first report, which revealed
the importance of miRNAs for rice cold stress response.
Jeong and collaborators (2011) identified a new miRNA, miRNA912q, which powerfully upregu-
lated in rice panicles under cold stress. The miRNA912q was inversely correlated with its computa-
tionally predicted target—calcineurin B-like protein-interacting protein kinase. The authors found
Small RNAs in Crop Response to Temperature Stress Noncoding RNAs in Plants 787
miR1425 to be downregulated by cold and correlated it with induced fertility restorer genes neces-
sary for enhancing cold tolerance in rice (Jeong et al. 2011).
There are a number of examples of opposing expression levels of miRNAs during different stress
conditions in plants. The following example illustrates this very well—miR168, miR170/miR171,
and miR319 are upregulated in Arabidopsis but are downregulated in rice in response to cold stress
(Sunkar et al. 2012). During the cold treatment, members of the miR167 and miR319 families
showed similar profiles, while members of the miR171 family showed diverse expression patterns.
It was suggested that different members of the same miRNA family might perform different func-
tions (Lv et al. 2010).
Using microarray, Lv and collaborators (2010) detected 18 rice miRNAs, which responded
rapidly to cold stress. Of them, 12 miRNAs, belonging to 10 different families, were downregu-
lated. The rest of the miRNAs, corresponding to five families, were upregulated. miR1850 and
miR1868 were upregulated at 9 h and were downregulated at the last time point (24 h). miR166 k
and miR166 m were upregulated at 6 h and restored to a normal expression level at the end of
stress treatment. The expression levels of the most cold-responsive miRNAs were altered after
6 h in the cold (Lv et al. 2010).
Usually, ra-siRNA biogenesis requires DCL3 and RNA-dependent RNA polymerase 2. In rice,
Yan and collaborators (2011) have identified MITE-derived ra-siRNAs, siR441 and siR446, which
are encoded by three siR441 loci (SIR441a-c) and one siR446 locus (SIR446), respectively. The two
ra-siRNAs are processed by OsDCL3a but independent of OsRDR2, indicating that they are gener-
ated from single-stranded stem-loop precursors.
The expression profile of siR441 and siR446 was determined in 10-day-old rice seedlings under
cold, drought, salt, or abscisic acid (ABA) treatment conditions. RNA gel blot analyses showed
that those treatments resulted in an increased accumulation of all ra-siRNA precursors (to differ-
ent extents) and in a decreased production of mature siR441 and siR446, especially at 12 h after
the treatments. The authors consider this stress-induced defective processing as a consequence of
increased precursor accumulation per se. Functional examinations indicate that siR441 and siR446
are positive regulators of rice ABA signaling and tolerance to cold and other types of abiotic stress,
possibly by regulating the expression of the F-box domain gene—MAIF1 (Yan et al. 2011).
35.3.2 Wheat
In wheat (Triticum aestivum), a total of 58 miRNAs, which comprise 43 miRNA families, have
been cloned; however, their expression level in response to abiotic and biotic stress is still unknown.
With the development of HTS technology, it became possible to discover several species-specific
or lowly expressed miRNAs and different members in the same miRNA family. By using Solexa
HTS, Xin and collaborators (2010) identified a diverse set of wheat small RNAs that are respon-
sive to powdery mildew infection and heat stress. A total of 51 known conserved miRNAs and
81 new identified miRNAs were reported, increasing the number of wheat miRNA families from
43 to 132. The authors show that only five miRNAs were conserved in different species, suggesting
that wheat appears to have evolved species-specific miRNAs. Therefore, their study revealed that
wheat genome encoded more nonconserved miRNA families than conserved miRNA families. It
has been proposed that these nonconserved miRNAs presumably emerged and dissipated in short
evolutionary time scales (Fahlgren et al. 2007; Sunkar et al. 2008).
It was reported that miR172 was significantly decreased in heat stress conditions with 1.5-fold
changes, and eight miRNAs, including miR156, miR159, miR160, miR166, miR168, miR169,
miR827, and miR2005, were upregulated with the highest expression change of 2.9-fold for miR168
(Xin et al. 2010). Furthermore, Northern blot analysis was performed to determine the expression
patterns of nine miRNAs in heat-tolerant genotype TAM107 and heat-susceptible genotype Chinese
Spring (CS) after heat treatment for 0.5, 1, and 2 h and returning to normal growth condition. The
results revealed that expression of miR156 was upregulated in both TAM107 and CS genotypes after
heat treatment, miR159 was downregulated only in CS genotype after 2 h heat treatment, miR166
was upregulated only in CS genotype after 2 h heat treatment, and miR393 and Ta-miR2002 were
upregulated only in CS genotype after 0.5 h heat treatment. It was also noted that expression of
some miRNAs, such as miR156 and miR393, was significantly changed at 0.5 h after heat treat-
ment, suggesting that the expression of some miRNA is responsive to the very short heat treatment.
In wheat breeding, the male sterility of thermosensitive genic male sterile (TGMS) lines of wheat
is very important for the utilization of heterosis in wheat breeding. The early phase of anther devel-
opment is especially susceptible to cold stress. Deep sequencing of small RNA libraries obtained
from spike tissues of the TGMS line under cold stress identified several miRNAs and one ta-siRNA,
namely, ta-siRNA-auxin-responsive factor (ARF), to be cold stress responsive (Tang et al. 2012).
Cold treatment downregulated miR167, miR172, miR393, miR396, and miR444 and had no effect
on miR156/miR157, miR165/miR166, miR169, and miR319 (Tang et al. 2012).
The noncoding TAS3 transcripts of ta-siRNA-ARF are the targets of miR390, and cleaved TAS3
gives rise to ta-siRNAs that target transcripts in the ARF family (Allen et al. 2005; Axtell et al.
2006). Tang and collaborators (2012) showed that the expression of ta-siRNA-ARF was dramati-
cally depressed at an early stage of spike development (L1.5 stage) during cold stress. The authors
predicted that phased siRNAs were derived from two TAS3 loci that are targeted by tae-miR390
and speculated that during cold stress, the abnormal decline of ta-siRNA-ARF levels contributes to
the aberrant development of the anther in the TGMS line through the negative regulation of ARFs.
Other small RNAs are also important components in temperature stress response of wheat.
Yao and collaborators (2010) have reported four siRNAs that substantially change their expression
in wheat seedlings in response to different abiotic factors, including cold (4°C for 2 h) and heat
(40°C for 2 h). siRNA002061_0636_3054.1 is strongly downregulated by heat, salt, and drought;
siRNA005047_0654_1904.1 is greatly upregulated by cold stress and downregulated by heat, salt,
and drought; siRNA080621_1340_0098.1 is slightly upregulated by cold and downregulated by
heat but not by salt and drought; siRNA007927_0100_2975.1 is downregulated by cold, salt, and
drought but not by heat stress (Yao et al. 2010).
35.3.3 Chinese Cabbage
Chinese cabbage (Brassica rapa ssp. chinensis) is an important vegetable crop that is closely related to
the model plant Arabidopsis thaliana (Snowdon 2007). The species is quite sensitive to numerous abi-
otic stresses, including heat stress. Therefore, losses in the yield and quality of that crop may occur due
to such stress conditions especially in warm regions within the summertime. So far, progress has been
made in the sequencing and annotating of the B. rapa genome, making promise to conduct a genome-
wide survey of small RNAs and miRNAs in B. rapa. Discovery and annotation of heat-responsive
miRNAs can provide the opportunity to understand mechanisms of plant response to heat stress.
Very few miRNAs have been reported in Chinese cabbage. A genome-wide profiling of small
RNAs in B. rapa is necessary for the genome-wide annotation of MIRNA genes. By comparing
the known miRNAs and the miRNA precursors, Yu and collaborators (2012) were able to identify
35 miRNA families conserved between Chinese cabbage and Arabidopsis. The conserved miRNAs
in Chinese cabbage are identical to their members in Arabidopsis. The authors also reported that
many MIRNA genes in B. rapa show far more copies than in Arabidopsis (Yu et al. 2012). In total,
22 miRNAs from the conserved miRNA families in Brassica show one or two nucleotide substitu-
tions compared with those of Arabidopsis. These findings also overlap with previously reported
conserved miRNAs in Arabidopsis (He et al. 2008; Hsieh et al. 2009; Kutter et al. 2007).
Yu and collaborators (2012) conducted a deep sequencing of two libraries constructed from rice
grown in control and heat stress conditions. Among heat-responsive miRNAs, bra-miR398a and
bra-miR398b were downregulated with more than 10- and 3-fold changes, respectively; bra-miR398a
and bra-miR398b are derived from two homologous precursors of miR398 and have one mismatch in
sequence. The homologue of bra-miR398 in Arabidopsis responds to oxidative stress by regulating
its target—Cu/Zn superoxide dismutase 1 (CSD1) (Sunkar et al. 2006). To confirm the heat response
of bra-miR398, the authors performed Northern blotting of miR398 and real-time PCR of the tar-
get gene. The accumulation of both, bra-miR398a and bra-miR398b, in the heat stress sample was
much lower than in the control sample. On the contrary, the expression of BracCSD1, as a target
gene of bra-miR398, was higher in the heat stress library. The authors also reported that miR156h
and miR156g were especially upregulated in contrast to miR156a–f and miR399 is repressed and
miR167 is induced by heat stress, consistent with a report in wheat (Xin et al. 2010).
Yu et al. (2012) also discovered new miRNA gene models. Among the novel miRNAs, bra-miR5714
and bra-miR5726 were upregulated, and bra-miR5716 and bra-miR1885b.3 were downregulated
under heat stress conditions. Under such type of stress, the accumulations of bra-miR1885b.3, bra-
miR1885b.3*, and bra-miR1885b.2* were also repressed severely. Another potential heat-responsive
miRNA is bra-miR5718. Under heat stress, its accumulation was increased by more than 1.5-fold.
Chinese cabbage develops a great number of green leaves rich in chloroplasts. Wang and col-
laborators (2011) compared the small RNA libraries, which have been produced by next-generation
sequencing (NGS) from nontreated and heat-treated samples, and analyzed the proportion of small
RNAs that were matched to the chloroplast genome. The chloroplast small RNAs include those
derived from mRNA, rRNA, tRNA, and intergenic RNA. Heat treatment resulted in decreased
accumulation of chloroplast small RNAs from rRNA, tRNA, and mRNA and in significant increase
of small RNAs produced from chloroplast intergenic regions (Wang et al. 2011).
35.3.4 Other Crop Small RNAs

Using a newly modified comparative genome strategy, Xie and collaborators (2011) identified a
total of 202 potential miRNAs in potato (Solanum tuberosum). On the basis of Gene Ontology and
Kyoto Encyclopedia of Genes and Genomes pathway analyses, they defined biological processes in
which targets of identified miRNAs are involved and participation of the targets in metabolism net-
works. 16 miRNAs of identified 202 (miR157, miR159, miR166, miR397, miR403, miR414, miR415,
miR477, miR530, miR821, miR865, miR1426, miR1435, miR1514, miR1522, and miR1533) were
involved in cold response in potatoes (Xie et al. 2011). Based on the identified target genes (cold-
induced plasma membrane proteins) in peanut (miR13) and radish (rsa-miRn62 and rsa-miRn33)
miRNAs, the involvement of relevant miRNAs in response to cold stress was suggested (Chi et al.
2012; Xu et al. 2013). Yan-du and collaborators (2008) identified potential miRNAs by searching
in grapevine sequence databases, and after detailed bioinformatics analysis, they predict miRNA
targets. The target of Vvi-miR157 was found to be calcineurin B-like protein 9, which belongs to
the group of calcium sensors that are involved in stress response including cold (Yan-du et al. 2008).
The expression pattern of miR319 in sugarcane (Saccharum sp. cv SP70-1143) characterized it as
a positive regulator in cold stress responses. This upregulation (roots and shoots) was validated by
stem-loop RT-PCR (Thiebaut et al. 2012). Two cassava varieties (SC124 and C4) naturally grown in
different ecological conditions displayed opposite miRNA expression profiles under cold and drought
stress conditions (Zeng et al. 2010).
35.4 CONCLUSIONS
The evidences presented in this chapter disclosed important roles of a diverse set of small RNAs in
temperature response of crop plants. By now, great efforts were made in characterizing the involve-
ment of conserved and species-specific miRNAs in stress response. Future studies are likely to iden-
tify other types of small RNAs that take part in response to diverse stress factors. Our unpublished
data from NGS analysis of temperature-treated Arabidopsis reveal that low and high temperatures
bring about completely different genome-wide small RNA profiles and affect the sRNA production
from particular sets of protein-coding genes. What the genome-wide small RNA response of crop
species to temperature stress is remains to be answered.
TABLE 35.1
Appendix
Species miRNA Organ specifciity (Putative)Target Reference
Low-temperature stress
Vitis vinifera miR157 — Squamosa promoter binding Lu et al., 2008
protein-like 2/SBP,
Calcineurin B-like protein 9
Oryza sativa miR156/157 ↓; — squamosa promoter binding Tang et al., 2012;
miR156k ↓ proteins Lv et al., 2010
miR165/miR166 ↑; — HD-Zip proteinS Tang et al., 2012;
miR166k ↑; Lv et al., 2010
miR166m ↑
miR167a/b/c ↓ — auxin response factor Lv et al., 2010
miR168 ↓; — PINHEAD protein Tang et al., 2012;
miR168b ↓ Lv et al., 2010
miR169 ↓; — CCAAT-binding proteins Tang et al., 2012;
miR169e,f,h ↓ Lv et al., 2010
miR170/miR171 ↓ — scarecrow-like proteins Lv et al., 2010
miR319a/b ↓ — TCP family transcription Lv et al., 2010
factors, GAMYB
miR393 ↑ inflorescences MYB family transcription Barrera-Figueroa
factor et al., 2012
miR394 ↑ inflorescences F-box domain containing Barrera-Figueroa
protein et al., 2012
miR396 ↑ inflorescences Growth regulating factor Barrera-Figueroa
et al., 2012
miR444 ↓; — MADS-box transcription Tang et al., 2012;
miR444a.1 ↓ factor Lv et al., 2010
miR529 ↑ inflorescences SBP-box gene family Barrera-Figueroa
et al., 2012
miR530-3p ↑ inflorescences Hairpin-induced protein 1 Barrera-Figueroa
domain containing protein et al., 2012
miR535 ↑ — — Lv et al., 2010
miR809 ↓ — Glutaredoxin 2, putative Barrera-Figueroa
et al., 2012
miR810b.2 ↑ — — Barrera-Figueroa
et al., 2012
miR812q ↑ leaf and panicle Calcineurin B-Like (CBL) Jeong et al., 2011
protein interacting protein
kinase
miR1320 ↓ Clathrin assembly protein; Lv et al., 2010
Fucosyltransferase 7;
Remorin
miR1425 ↓ panicle RF1 (fertility restorer) Jeong et al., 2011
miR1435 ↑ — B3 DNA binding domain Lv et al., 2010
containing protein;
UDP-glucosyl transferase
miR1850 ↑ — Diverse targets Lv et al., 2010
(at 9 h) ↓ (at 24 h)
miR1856 ↑ inflorescences — Barrera-Figueroa
et al., 2012

Appendix
miR1866 ↓ inflorescences Hypotethical protein Barrera-Figueroa
et al., 2012
miR1868 ↑ — — Lv et al., 2010
(at 9 h) ↓ (at 24 h)
miR1874-3p ↓ — — Barrera-Figueroa
et al., 2012
miR1876 ↓ — Histone deacetylase 6 Lv et al., 2010
miR1877 ↓ — — Barrera-Figueroa
et al., 2012
miR1884b ↓ — Diverse targets Lv et al., 2010
miR2275c,d,e ↑ inflorescences — Barrera-Figueroa
et al., 2012
miR2871 ↑ inflorescences Glycosyltransferase family Barrera-Figueroa
protein et al., 2012
osa-cand042 ↓ inflorescences — Barrera-Figueroa
et al., 2012
osa-cand052 ↓ inflorescences — Barrera-Figueroa
et al., 2012
osa-cand066 ↑ — LTPL8 - Protease inhibitor Barrera-Figueroa
et al., 2012
osa-cand084 ↓ — Transposon protein Barrera-Figueroa
et al., 2012
Arachis miR13 cold induced plasma Chi et al., 2011
hypogaea membrane protein
[Jatropha curcas]
Solanum miR157, miR159, BG351037, BG597134, Xie et al., 2011
tuberosum miR166, BI432893, BQ113871,
miR397, BQ505460, CK273636,
miR403, CK274744, CK274767,
miR414, CK276957, CK851489,
miR415, CV430574, CV471905,
miR477, CV495613, CV496519,
miR530, DN922105, DN937994,
miR821, DN941590, DR034630,
miR865, DR036093, DR036285,
miR1426, DV623655, DV623954,
miR1435, FG549224
miR1514,
miR1522,
miR1533
Raphanus miRn62; miRn33 rare cold inducible plasma Xu et al., 2013
sativus membrane protein
(Saccharum sp. miR319 ↑ roots and shoots — Thiebaut et al.,
Cv SP70-1143) 2012
Triticum miR167 ↓ spike tissues of the ARF Tang et al., 2012
aestivum TGMS line
miR172 ↓ AP2
(continued)

Appendix
miR393 ↓ Transport inhibitor response
1-like protein
miR396 ↓ GRF
miR444 ↓ —
Manihot miR156a; — — Zeng et al., 2010
esculenta miR159a;
miR160a;
miR162a;
miR165/
miR166a;
miR167a;
miR170/
miR171a;
miR395a;
miR396a;
miR397a
High-temperature stress
Triticum miR172 ↓ — — Xin at al., 2010
aestivum miR156 ↑
miR159 ↑
miR160 ↑
miR166 ↑
miR168 ↑
miR169 ↑
miR827 ↑
miR2005 ↑
Brassicarapa miR156h ↑ BracPAP10. BracCSD1 — Yu et al., 2012
miR5714 ↑
miR5718 ↑
miR5726 ↑
miR398a ↓
miR398b ↓
miR399b ↓
miR827 ↓
miR5716 ↓
miR1885b.3 ↓
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Part VII
Bioinformatics and Using Computer
Modeling in Plant Physiology
36 Comparative Genomics of
Grass Genomes Using CoGe
Eric Lyons, Matthew D. Bomhoff, Shannon L. Oliver,
and Andrew J. Lenards
CONTENTS
36.1 Introduction........................................................................................................................... 797
36.2 Welcome to CoGe.................................................................................................................. 798
36.2.1 To Access CoGe and Load a Genome into CoGe.....................................................800
36.3 Navigating CoGe and Finding Genomes of Interest.............................................................802
36.3.1 To Search for a Genome in CoGe..............................................................................802
36.4 Whole Genome Comparisons Using SynMap and Syntenic Dotplots.................................. 805
36.4.1 To Generate a Self–Self Syntenic Dotplot of Foxtail Millet.....................................806
36.5 Identifying Orthologs through Cross-Species Syntenic Dotplots.........................................807
36.5.1 To Generate Syntenic Dotplots between Foxtail Millet and Sorghum...................... 810
36.6 Microsynteny Analysis of Syntenic Regions......................................................................... 810
36.6.1 To Perform Microsyntenic Analysis in Order to Identify Conserved
Noncoding Sequences................................................................................................ 812
36.7 Conclusions............................................................................................................................ 814
References....................................................................................................................................... 815
36.1 INTRODUCTION
In many modern biological research environments, science is driven by data-intensive applications
and the rate of generating data is no longer limiting. Rather, the processes of transforming those
high volumes of data into applied information, knowledge, and wisdom have become analytical
bottlenecks.1 This transformative process relies on computational systems and domain knowledge
of trained scientists. However, the size of these datasets often scales beyond the computational
resources easily available to many research groups, and the requisite knowledge to utilize the most
effective hardware and software is not always readily available.2 Thus, while many research groups
can easily generate large datasets, analyzing them is often difficult and time consuming. Parallel
to this problem are both the ability of researchers to quickly and easily integrate publicly available
data with their prepublication restricted data and their ability to make their data publicly available
post-publication.3 Fortunately, substantial advances have been made to provide cyberinfrastructure4
(CI; computing resources and knowledge to use them) for life science researchers.
This chapter focuses on using the online comparative genomics platform CoGe (http://
genomevolution.org)5 to analyze and compare grass genomes, which is powered by the cyberin-
frastructure available through the iPlant Collaborative,6 to integrate and analyze genomic data. For
each stage of the analytical process, links will be provided to regenerate that stage of the analysis.
Readers are encouraged to perform the analyses presented in the chapter and to use this chapter as
a resource for repeating similar analyses with their own genomes and datasets of interest.
797
High-throughput sequencing (HTS) epitomizes this dichotomy of opportunity and challenges

embodied by data-intensive life science. HTS has revolutionized biology by enabling researchers
to make many measurements of a biological system simultaneously; HTS exponentially decreases
in cost each year while yielding unprecedented increases in throughput.7 For example, the first
draft of the human genome cost $2.7 billion and took approximately 10 years to complete,8 while
today an equivalent amount of DNA sequencing data can be produced for less than $10,000 in
10 days.9 Genomes may be sequenced, transcriptomes characterized, and polymorphisms uncov-
ered. HTS applications include two general classes: genome sequencing, which is used for assem-
bling a draft-level genome sequence and characterizing genetic diversity, and functional genomics
and genetic studies, which are used to understand the state and activity of a genome. The latter
class of studies include transcriptomics (identifying functional genomic transcripts), expression
profiling (quantitating the relative amounts of transcripts produced under various environmental
and developmental conditions), DNA–protein occupancy (ChIP-seq), polymorphism identification
(characterizing genetic diversity such as SNPs and CNVs of a population of individuals), QTL
identification (identifying regions of the genome involved with various organismal phenotypes),
and epigenetics (understanding chromatin states by histone occupancy and DNA methylation).
Researchers employ a variety of these techniques to understand the functional behavior of a genome
by assigning numerical measurements (e.g., expression value) or state measurements (e.g., SNP)
to a region of a genome. Such data are generally known as quantitative genomic measurements.
While new omic technologies revolutionized the life sciences by liberating high-throughput
quantitative data acquisition, there exist substantial challenges in transforming those data into
usable knowledge.1 There is a critical need for easy-to-use computational resources that facilitate
management, analysis, and visualization of genomic data.10 For agriculture, these analytical plat-
forms must enable researchers to accelerate their ability to improve agronomically important traits.
Inherent are the ubiquitous needs of researchers to be in charge of their data, share them with col-
laborators, integrate them with existing public datasets, easily scale their analyses as their datasets
grow, visualize and interact with the results of their analyses, and quickly move data between vari-
ous computational platforms that may be better suited for a particular set of analyses.11
CoGe is a web-based comparative genomic system, providing over 30 interconnected applica-
tions to analyze, compare, and visualize nearly 20,000 genomes from all domains of life in any state
of assembly or annotation. CoGe’s tools provide single gene,12 multiple gene,13 whole genome,14 and
multiple-genome analyses.15 In addition, these tools employ interactive visualizations of complex
data.16 All data files produced by CoGe are easily exported and downloaded by researchers17; each
tool in CoGe tracks provenance by creating a unique URL that will regenerate an analysis exactly
as configured by the researcher including the original data, selected parameters, and visualization
customizations. These URLs are saved internally and provide researchers with their analytical his-
tory, enabling them to find their previously run analysis. CoGe also has an advanced user-data man-
agement system that permits researchers to add public or private genomes and functional genomic
experiments, keep them private, and share them with collaborators.18 Researchers may also create
“notebooks” in which they can store collections of genomes, experiments, and sets of important
genes along with metadata about those collections that include text descriptions, links to external
resources, links to CoGe analyses, and images.
36.2 WELCOME TO COGE

CoGe is available online at http://genomevolution.org. Its homepage provides an overview of the num-
ber of organisms, genomes, genomic features, and annotations available, links to tutorials and infor-
mation on getting started, and links to the major starting points of tools in CoGe (Figure 36.1a). There
is a set of drop-down menus located on the upper right of each page of CoGe that provides a set of
links to these resources as well. People may use CoGe anonymously, but will only be able to access
public data. In order to access private data, researchers must log into CoGe using their iPlant account.
Comparative Genomics of Grass Genomes Using CoGe 799
(a)
(b)
FIGURE 36.1 CoGe’s homepage and user profile page. (a) The homepage provides an overview of data in
CoGe and links to popular tools and information. (b) For people with CoGe user accounts, the user profile
page provides a place to organize and share data, load in new data, and get an overview of information. Data
marked with ® denote restricted/private data.
Logging into CoGe enables a set of additional features such as adding private genomes and experi-
ments, sharing those data with other users, creating notebooks, and tracking previously run analyses.
If one is logged into CoGe, there are two places where data and analyses may be managed. The menu
bar has a link to My Profile and a drop-down to My Stuff. The user profile page (Figure 36.1b) is where
people can easily organize and share their data, get access to their analytical history, and add new data
to CoGe.
The following exercise will provide an introduction to CoGe’s homepage and user profile
page. Of specific interest, it will walk through the process of loading a genome into CoGe. While
genome sequencing is attainable for nearly every organism, the resulting assemblies are usually
of draft quality and consist of many contigs and scaffolds. CoGe is designed to handle these
types of genomes; however, there is a limit of 100,000 contigs per genome at this time. For this
exercise, a link will be provided to a de novo assembled genome from a strain of Escherichia coli.
This is used so that the reader may move quickly through this exercise, but more complex
genomes may be substituted.
It is important to note that extensive documentation exists for CoGe and is accessible through
the Help button located in the menu bar. CoGepedia is CoGe’s documentation wiki and is a good
place to search for topics. There is also a link to Page Docs in the CoGepedia documentation and
is an excellent place to get more information on how to use a particular tool. For any questions that
are not covered in the documentation, there is a link to CoGe’s forums (hosted by iPlant) where
researchers can view previously discussed topics and start new ones.
36.2.1 To Access CoGe and Load a Genome into CoGe

1. Go to http://genomevolution.org.
2. If a user account is needed, follow these directions: http://genomevolution.org/r/4sr8
a. Requires an iPlant account.
b. If you have an iPlant account, simply use those credentials to log into CoGe.
3. To log into CoGe, click the sign-in link at the top of the page.
a. The sign-in link will send you to iPlant’s Authentication Service. After you authenti-
cate at iPlant, you will be sent back to CoGe.
b. The first time you log in, CoGe will create an internal account using a minimal
amount of information obtained from iPlant: your first name, last name, iPlant user-
name, and e-mail address. Your e-mail address is used to send e-mail notifications
from analyses and general information about the system (e.g., scheduled downtime
for maintenance).
4. Next, go to the user profile page by clicking the link to My Profile located in the menu bar
(quick link: http://genomevolution.org/r/62k7).
5. Create a notebook to store items by clicking on the Create button on the left side of the
page, and select New Notebook. This will cause a popup window to appear where you can
enter a name and description for the notebook. The type of notebook helps you organize
your data. For example, if you want to have a notebook focused on your favorite genomes
for searching with BLAST,19 you can specify the type genome. When you are finished,
click the red Create Notebook button. The popup window will close and the notebook will
appear in My Stuff.
a. To edit what is in the notebook, click on the notebook to go to NotebookView where
you can modify the name and description, add annotations about the notebook,
and add genomes, experiments, features, and other notebooks (quick link: http://
genomevolution.org/r/8l3l).
6. Next, you are going to load a genome into CoGe. From the user profile page, click the
Create button on the left side of the page, and select LoadGenome. This will send you to
CoGe’s genome loading page (quick link: http://genomevolution.org/r/5xic).
7. In LoadGenome, you will need to specify the following information about your genome:
a. Organism: You can search for organism by name in CoGe’s system by using the text
box. However, if your organism has not been previously entered, you can create a new
organism by clicking the New button beside the text box. This will create a popup win-
dow that will let you search National Center for Biotechnology Information (NCBI) for
your organism. This feature is included to capture NCBI’s taxonomic description of an
organism. If needed, you can make further edits to the organism’s name and taxonomy.
Click the red Create button to create that organism.
b. Version: Provide a version of your genome (e.g., 0.1).
c. Type: Specify the type of genomic sequence. Most genomes come unmasked, but
some may be masked where repeat sequences have been converted to N or X. If
your sequence type is not listed, you may create a new one by clicking the New
button.
d. Source: From where did this sequence come? Several common sources are available
(e.g., NCBI, JCVI), but you may create a new source by clicking the New button.
e. Restricted: Check this box if the genome is going to be restricted or uncheck to make
the genome available to the public. It is checked (private) by default.
8. Next, add sequence data files for your genome. These must be in FASTA format. There are
several ways to import sequence data to CoGe:
a. Integration with your iPlant Data Store: If you are familiar with the iPlant Data Store,
just put your sequence files in your coge_data directory to make them accessible to
CoGe. You will want to use this option if you are integrating large genomes (>200 MB)
and must use this option for very large genomes (>2 GB). For more information on how
to use this system, please see http://genomevolution.org/r/8l3q.
b. FTP/HTTP: If your genome is already available through a website or FTP site, you
may paste in the URL for the file or directory. You can optionally specify a username
and password for accessing basic authentication protected sites. You may retrieve large
sequence files (>200 MB) over FTP.
c. NCBI: If your genome is available through NCBI, you may enter in a list of NCBI
or GenBank accessions. CoGe will automatically retrieve those entries and load the
genome (including gene models and annotations, if provided in the GenBank file).
d. Upload: You may upload files directly from your computer. This works well for smaller
files as HTTP is not designed for moving files larger than 2 GB.
9. For the purposes of this exercise, you may use this small genome:
a. Organism: Escherichia coli K12 strain K-12 substrain NCM4139
b. Version: 0.1
c. Type: Unmasked
d. Source: CoGe Team
e. Restricted: Checked
f. FASTA file for the genome may be fetched by FTP/HTTP from http://genomevolution.
org/r/8l9t
10. Click the red LoadGenome button to load the genome. This step may take a while depend-
ing on the size of the genome. A status update will be posed in a popup window. When
loading has completed, you will see an icon representing whether or not the genome loaded
(thumbs-up equals success).
11. To share this genome with other users of CoGe, go to your user profile by following the
My Profile link in the menu bar (quick link: http://genomevolution.org/r/62k7).
12. Restricted data will be denoted with a “®” symbol before their name.
13. Select the genome by checking the box next to its name.
14. When one or more items are checked, click the icon that looks like a person at the top
of the list.
15. This will cause a popup window to appear with the title Share Items. This will show the list
of users and groups that have access to the genome. New users or groups may be added by
searching for them by name and then selecting them. When you add a new user or group,
you can specify if they may only read (access) the data or if they may modify it (editor),
which includes granting other user’s access.
16. Add this genome to your previously created notebook by selecting the checkbox next to
the genome and clicking on the folder icon at the top of the list (located next to the person
icon for sharing). This will cause a popup window to appear that lets you search and select
a notebook by name.
17. Once the notebook has been selected, click the red Add Items button.
18. Alternatively, you may add this genome to your previously created notebook by clicking on
that notebook within your user profile page. This will take you to NotebookView.
19. To add this genome, click the Add Items button. A popup window will appear that has sev-
eral tabs for selecting data. Data that you have added to CoGe will be under the My Stuff tab.
With that tab selected, search for your genome, select it, and then click Add Selected Items.
20. You may share this notebook by the same procedure used to share the genome from your
user profile page.
36.3 NAVIGATING COGE AND FINDING GENOMES OF INTEREST

The ability to compare private and public genomic data is essential. The first step in this process is
to find organisms and genomes of interest. CoGe’s main tool for finding organisms and genomes is
called OrganismView. OrganismView permits researchers to search for organisms by name and taxo-
nomic description. If an organism matches, it then displays a tiered set of information in two columns.
The left column displays matching sets of data found in CoGe and the right column displays detailed
information about the selected set of data. Organized from top to bottom are organisms that match
your search term, genomes for the selected organism, datasets comprising the selected genome, and
chromosomes for the selected dataset. OrganismView then provides a set of links to get more informa-
tion for each tier of data. These links include searching popular websites, accessing additional tools
in CoGe with the selected data preloaded, and global genome analytics such as codon usage tables.
This exercise will use OrganismView to search for the genome of foxtail millet, Setaria italica,20
and extract additional information about the genome. Figure 36.2 shows a search for organisms con-
taining the word foxtail in OrganismView, selecting the organism that matches Setaria italica, and
getting additional information about one of the associated genomes. For the selected genome, the list
of annotated genomic features, a histogram of GC content for coding sequences (CDSs), a histogram
of GC content for the wobble position of codons, an amino acid usage table, and a codon usage table
were generated. All of these charts and tables are calculated on the fly and can provide unique insights
into the composition of genomes. For example, the histograms of GC content for coding and wobble
(third codon position) sequences are bimodal, which are particular features of grass genomes.21
36.3.1 To Search for a Genome in CoGe

1. Go to OrganismView. OrganismView is accessible from any page in CoGe by clicking on
Tools from the menu bar (quick link: http://genomevolution.org/r/48px).
2. Type foxtail in the box next to Organism Name. CoGe will automatically search for match-
ing organism names. From the returned list of organisms, select Setaria italica (quick link:
http://genomevolution.org/r/8lx0).
3. The Organism information box contains the name and taxonomic description of foxtail mil-
let and links to additional information. For example, you can look up the organism at Google,
NCBI, and Wikipedia. Each part of the taxonomic description will autopopulate the description
search box in OrganismView with the selected term. This provides a one-click mechanism for
(b) (c)
(a)
(d)
FIGURE 36.2 Organism view, CoGe’s tool for searching for genomes and getting an overview of their
genomic content. Shown is the genome for foxtail millet with various information boxes showing histograms
of GC content and codon usage tables. This gene may be accessed in CoGe at http://genomevolution.org/r/5d5i.
(a) GC content histograms, (b) genome information box, (c) links for generating histograms of GC
content, and (d) links for protein specific information.
quickly searching for related organisms in CoGe at a particular phylogenetic depth (this is also
the reason why having accurate and complete NCBI taxonomic descriptions is essential for all
organisms in CoGe). There are also links to additional tools in CoGe, including OrganismView.
This provides a way to easily save a link to a particular view of data for future use.
4. Next, you will see several genomes associated with foxtail millet. CoGe can store an unlim-
ited number of genomes in any state of assembly or annotation. This includes different
versions of the primary sequence as well as different treatments of the primary sequence,
for example, different assemblies of the genome as well as masked and unmasked versions
of the sequence. In the example shown in Figure 36.2, there are two different versions of
unmasked genomes and the same version of the genome masked by two different methods
(quick link: http://genomevolution.org/r/5d5i).
5. Selecting a genome causes the data for that genome to be shown in the following text. The
Genome information box provides an overview of the number of chromosomes, scaffolds,
or contigs that comprise the genome, the type of sequence, the length of the genome, and
links to get additional information about the genome. Most of this information is dynami-
cally calculated and will be automatically generated for smaller genomes. Otherwise, a
link will appear that may be clicked to generate the information. For example, Click for
percent GC content will calculate the GC content of the genome.
6. Directly below the Genome information box is the Dataset information box. This provides
an overview of the datasets comprising the genome. A dataset corresponds to each of the
original data files used to load a genome. It is important to note that genomes are often dis-
tributed as several files and this tracks the provenance of those files, including the source of
the genome, the name of the original data file, the versions of the data file, and, oftentimes,
a link to the original data file. In addition, there is a summary of the sequence data derived
from the dataset including chromosome count and total length of sequence. Note: The term
chromosome is used in CoGe to denote chromosomes, scaffolds, and contigs.
7. Below Dataset information is Chromosome information, which gives a summary of the
sequence contained for a specific chromosome. Due to the large number of chromosomes
(scaffolds/contigs) that may be contained within a dataset, OrganismView only lists the
first 20 for system performance reasons.
8. Inside of the Genome information box are many important links. The next section will go
through some of these.
9. If you would like to download the genome’s sequence and annotation files, the links next to
Download let you export the sequence in FASTA format and the annotations as GFF, TBL,
and BED formats.
10. If you click the link at the bottom of the box labeled Click for Features (Figure 36.2a),
a table of genomic features will appear next in the right of the Genome information box
(Figure 36.2b). In CoGe, the phrase genomic feature is used to denote a region for a
genome for which we have additional information such as a gene, mRNA, CDS, and repeat
(quick link: http://genomevolution.org/r/8m0v).
11. Once the genomic feature table appears, there are links to generate GC content histograms
for each feature type (Figure 36.2c) as well as links for generating tables and charts for
protein CDSs if they have been annotated for a genome (Figure 36.2d).
12. To generate the GC content histogram for CDS sequences, click the link % GC Hist
located next to CDS in the feature table. This will cause a popup window to appear with
the title Histogram of GC content for CDSs. Since this histogram is dynamically gen-
erated, it may take several minutes to be generated (depending on the number of CDS
features).
13. To generate a similar histogram for the GC content of wobble codon positions, click the
link Histogram of wobble codon GC content located at the bottom of the feature table.
14. To generate amino acid and codon usage tables, click on the appropriately named links at
the bottom of the feature table.
15. If you want to create a list of genomes, you can search for each genome and click Add to
Genome List found at the bottom of Genome information. A popup window will appear
titled Genome List that lets you edit your growing list of genomes. Once you have added
all your genomes of interest, you can create a genome list from them by clicking Send to
GenomeList. This will send you to CoGe’s tool GenomeList, which lets you get informa-
tion about all the genomes in the list, export those data, and create a notebook of those
genomes.
16. Once a genome of interest has been found, the next step is to use it in another of CoGe’s
analytical tools. Links are provided to popular tools such as SynMap, for whole genome
syntenic comparisons, and CoGeBlast for search of a genome with sequence data. When
those tools are loaded, the selected genome will be autopopulated. The next section will
use SynMap for performing a whole genome comparison of foxtail millet to sorghum
(quick link: http://genomevolution.org/r/8m1h).
36.4 WHOLE GENOME COMPARISONS USING

SYNMAP AND SYNTENIC DOTPLOTS
All angiosperms share an evolutionary history that contains paleopolyploidy events,22 and the grass
lineage has evidence for at least three of these events.23 Whole genome comparisons are useful for
characterizing such events within and between genomes by identifying syntenic regions. Synteny,
in a genomic context, refers to genomic regions that are derived from a common ancestor and is
often inferred by the identification of collinear homologous gene among genomic regions. CoGe’s
tool SynMap provides an interface for identifying syntenic genes between a pair of genomes and
displaying the results in an interactive dotplot.
Figure 36.3 shows a self–self syntenic dotplot for foxtail millet. In this plot, each axis represents
the foxtail millet genome with each horizontal and vertical grey line separating adjacent chromo-
somes/scaffolds. The pileup of grey lines at the top and right edge of the dotplot is many small
contigs that were not assembled into the larger chromosome-sized scaffolds. The protein CDSs of
this genome were compared against themselves using the algorithm LAST.24 LAST is one of many
BLAST-like algorithms that perform rapid searches of large sequence datasets.
Such a whole genome comparison generates a lot of potential homologous gene pairs; the results
are processed to filter repetitive sequences and screen low-scoring sequence matches. All poten-
tial homologous gene pairs are then fed into the algorithm DAGchainer25 to identify collinear
y-axis organism: Setaria italica (foxtail millet) (v2.1)

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x-axis: Setaria italica (foxtail millet) (v2.1)
FIGURE 36.3 (See color insert.) Syntenic dotplot of a self–self analysis of foxtail millet using SynMap.
Green dots are syntenic gene pairs identified through a collinear arrangement. These are derived from
the most recent whole genome duplication event in this lineage. Analysis may be regenerated at http://
genomevolution.org/r/8m4c.
homologous gene pairs. These gene pairs are plotted as green dots in the dotplot and represent
intragenomic syntenic regions. Because the majority of the genome is covered by intragenomic syn-
tenic regions, it is inferred that these regions are derived from an ancient whole genome duplication
event. The age distribution of these gene pairs will be shown in a subsequent example. They are
indeed from the same age class as determined by synonymous mutation rate estimation. The green
line running along the 45° axis represents the self–self comparison of this genome against itself.
36.4.1 To Generate a Self–Self Syntenic Dotplot of Foxtail Millet

1. Find foxtail millet using OrganismView (quick link: http://genomevolution.org/r/5d5i).
2. Follow the link to SynMap in the Genome information section (quick link: http://
genomevolution.org/r/8m1h).
3. When SynMap loads, you will see that foxtail millet has been autopopulated for the two
organisms under the Select Organisms tab.
4. Note: SynMap, as with many tools in CoGe, reuses a common theme for configuring and running:
A. Tabs
a. One tab for selecting data
b. One tab for modifying the analysis
c. One tab for modifying the result’s visualization
B. Running the analysis: A big red button that says Run or Generate.
C. Links to regenerate an analysis: Once an analysis has completed, there will be a link
provided that can be used to regenerate the analysis exactly as configured at any point
in the future.
5. By default, SynMap will order chromosomes by size. For the dotplot shown in Figure 36.3,
the chromosomes were ordered by name. To change this option, select the Display Options
tab and then select Name in the menu for Sort Chromosomes by.
6. Run SynMap by clicking the red button labeled Generate SynMap.
7. Based on the options selected, CoGe will run the following analytical workflow:
A. Extract sequences
B. Run the whole genome comparison
C. Filter tandem duplicates
D. Filter low-quality matches
E. Identify collinear gene pairs
F. Filter syntenic regions by syntenic depth (more on this topic later)
G. Calculate synonymous and nonsynonymous mutation values
H. Generate an interactive dotplot
Since all of these analyses are calculated on the fly, it may take awhile for the results to be
returned. While an analysis is running, SynMap will display an update as to how far the
entire analysis has progressed. CoGe has an analytical results caching system that saves
previously run analyses for a period of time and then fetches those results if the same
analysis is run again. This means that if an entire analytical workflow has been previously
run, the results are returned very quickly. Similarly, if a previously run workflow has one
step changed, only the new parts of the workflow are run.
8. When the results of SynMap are returned, an interactive dotplot will be displayed near the
top of the page and below it are links for downloading each file generated by the workflow.
This includes a tab-delimited file of all the syntenic gene pairs. Details as to the contents
and the formats of these files can be found on the documentation page for SynMap (quick
link: http://genomevolution.org/r/488j).
9. Of particular importance is the link for regenerating the analysis. If you are logged into CoGe,
this link is automatically added to your analytical history, which may be accessed through
your user profile page or by clicking My Stuff from the menu bar and selecting History.
10. If you wish to modify the analysis, make the necessary changes and click the red Generate
SynMap button to rerun the analysis.
A. For example, to change the size of the dotplot, perform the following:
a. Select the Display Options tab.
b. Set the Master image width to 500.
c. Click Generate SynMap.
d. See quick link http://genomevolution.org/r/8m4n.
36.5 IDENTIFYING ORTHOLOGS THROUGH

CROSS-SPECIES SYNTENIC DOTPLOTS
One aim of comparative genomics is to identify orthologous genes between organisms. The infer-
ence of evolutionary orthology helps transfer functional information of genes between species and
is often used to transfer knowledge about a gene from a well-studied organism to a less well-studied
one. Before whole genome sequencing, this process of transferring information from a functionally
characterized gene (or gene product) to another gene used sequence similarity to identify homolo-
gous genes. However, since genes may be duplicated by a variety of means (e.g., whole genome
duplication, tandem duplication, and transposition duplication) and these duplicates may be under
different evolutionary constraints, using sequence similarity alone often resulted in many annota-
tion errors.26 Fortunately, enforcing orthology between genes to help transfer annotations decreases
errors significantly, and genomic synteny is a standard method to define orthology.27
Since SynMap identifies syntenic genes between two genomes, it may be used to identify orthol-
ogous genes. However, if the genomes being compared share an evolutionary history with poly-
ploidy events, syntenic regions may be derived either from the divergence of the lineages (orthologs)
or from shared polyploidy events (out-paralogs). Since the self–self syntenic dotplot of foxtail millet
revealed strong evidence for an ancient whole genome duplication event in its lineage (Figure 36.3),
cross-species comparisons to other grasses will yield orthologous and out-paralogous syntenic
regions. Thus, in order to determine orthology between genes from two species of grass, it is impor-
tant to be able to identify and remove those syntenic regions derived from shared polyploidy events.
Figure 36.4a shows a syntenic dotplot between foxtail millet and sorghum.28 For many genomic
regions in each genome, there are two syntenic regions (Figure 36.4a; red dashed lines). One of these
syntenic regions is orthologous and derived from the divergence of the lineages; the other one is
out-paralogous and derived from a shared whole genome duplication. Each of these events (lineage
divergence and whole genome duplication) contemporaneously affected all genes in the genome, but
happened at different times. As such, the relative ages of genes derived from these events are different,
and this difference may be detectable. One popular method for determining the relative divergence age
of a pair of genes is to measure the synonymous mutation rate (Ks), which is an estimate of the number
of synonymous mutations that have occurred per synonymous site between a pair of genes.29 Since
many synonymous mutations are neutral and do not affect the function of the gene’s CDS, the number
of these events for a synonymous site is a proxy for estimating the age. If Ks values are estimated for
all syntenic gene pairs between foxtail millet and sorghum, genes in syntenic regions derived from
different evolutionary events should have different effective Ks values.
Figure 36.4b shows the same syntenic dotplot between foxtail millet and sorghum as shown in
Figure 36.4a, except that syntenic gene pairs are colored by the Ks value. These colors are deter-
mined by the histogram of log10-transformed Ks values shown in Figure 36.4c, with smaller Ks
values on the left and larger Ks values on the right, representing younger and older gene pairs,
respectively. The peak on the far right of the Ks histogram represents gene pairs with non-log trans-
formed Ks values of 50–100 synonymous substitutions per synonymous site. These results represent
noise in the analysis that may be due to bad gene models, incorrectly called syntenic genes, pseudo-
genes, and errors in the primary sequence.
y-axis organism: Sorghum bicolor (v1.4) y-axis organism: Sorghum bicolor (v1.4)
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(a) x-axis: Setaria italica (foxtail millet) (v2.1)

(b) x-axis: Setaria italica (foxtail millet) (v2.1) S ca
Mean: 0.0048 median –0.2284

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(c) log 10() substitution per site for Ks
FIGURE 36.4 (See color insert.) Syntenic dotplots of a foxtail millet (x-axis) versus sorghum (y-axis) using
SynMap. (a) Syntenic gene pairs are colored green. Note that a given region of either genome is syntenic
to two regions in the other genome (red dashed line). This is due to one syntenic region being orthologous
and one being out-paralogous. Note the large gaps in some syntenic regions (blue arrow). This is a cen-
tromere in sorghum. Results may be regenerated here: http://genomevolution.org/r/8m2u. (b) Syntenic gene
pairs are colored by their synonymous mutation values (Ks). Purple gene pairs are younger than cyan gene
pairs and represent orthologous and out-paralogous relationships, respectively. Results may be regenerated
here: http://genomevolution.org/r/8m2v. (c) Histogram of Ks values shown used in the dotplot from (b).
The syntenic regions in Figure 36.4b fall into two general color classes: younger, purple regions
and older, cyan regions. Since the divergence of these lineages postdated their shared whole genome
duplication event, the purple syntenic regions are orthologous, while the cyan regions are out-paral-
ogous. Note that several sorghum orthologous syntenic regions have a large gap (Figure 36.4; blue
arrows). Also, the ends of the syntenic regions near the gap appear to have a change in gene fre-
quency, giving these syntenic regions a sigmoidal appearance. These regions likely represent cen-
tromeric and peri-centromeric regions.
SynMap has many options for modifying the visualization of the final dotplot. One of these
options changes the axis metrics from nucleotides to genes, thereby normalizing the relative size
of the dotplot based on the number of annotated genes and reducing the visual effects of varying
distance between genes. Figure 36.5a shows the same analysis as used in Figure 36.4b, expect that
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(a) x-axis: Setaria italica (foxtail millet) (v2.1) (b) x-axis: Setaria italica (foxtail millet) (v2.1) Sc
y-axis: Sorghum bicolor (v1.4): 2 (4.483 bp)
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FIGURE 36.5 Syntenic dotplots of a foxtail millet (x-axis) versus sorghum (y-axis) using SynMap. Syntenic
gene pairs colored by their Ks values as described in Figure 36.4. (a) Axis metrics are in genes as opposed
to nucleotides as seen in Figure 36.4. Results may be regenerated here: http://genomevolution.org/r/8m33.
(b) Only orthologous syntenic regions are retained after screening for a 1:1 syntenic depth relationship. Results
may be regenerated here: http://genomevolution.org/r/8m4d. (c) Zoomed-in view of chromosome 2 dotplot
between both organisms. Crosshairs are on a syntenic gene pair that is used for the microsynteny analysis as
shown in Figure 36.6.
the axis metrics are in genes instead of nucleotides. This has the effect of straightening the lines
of syntenic regions near centromeres as well as removing much of the gap caused by gene-poor
centromeric regions.30
SynMap also has the option to filter syntenic regions by coverage and depth in order to retain stron-
ger scoring syntenic regions. Such filtering by syntenic depth provides a method for removing non-
orthologous syntenic regions since orthologous syntenic regions will usually have a stronger signal of
synteny than out-paralogous syntenic regions. The reason for this is due to fractionation of gene content
following polyploidy, reducing the number of syntenic gene pairs, while orthologous syntenic regions
retain a high percentage of syntenic gene pairs.13 Figure 36.5b shows the results of a syntenic depth filter
applied to the syntenic dotplot shown in Figure 36.5a that retains the highest scoring syntenic regions
giving a one-to-one coverage between the two genomes. This results in orthologous syntenic regions
being retained, while nearly all other syntenic regions are filtered from the results.
36.5.1 To Generate Syntenic Dotplots between Foxtail Millet and Sorghum

1. Start with the self–self syntenic dotplot of foxtail millet used in the previous exercise
(quick link: http://genomevolution.org/r/8m4c).
2. In the Select Organism tab, type sorghum into the name search box for one of the organisms
and select Sorghum bicolor (id331) from the list of available organisms. Several different
genomes of sorghum are available. As a general rule, genomes with CDS annotations will
be analyzed more quickly than those without annotations. This is due to repeat sequences
that are often found in non-CDS. If CDSs are not available, genomes that have been masked
of repeats will be analyzed more quickly than unmasked genomes. If unmasked genomic
sequences are compared for large genomes that contain a lot of repeat sequences (e.g., plants),
the various steps of the analysis may take days to weeks to complete due to the number of
matches found in repeat sequences (quick link: http://genomevolution.org/r/8md9).
3. To order the chromosomes by name in the dotplot, select the Display Options tab and
choose Name for Sort Chromosomes by.
4. Click Generate SynMap to run the analysis (quick link: http://genomevolution.org/r/8m2u).
5. If the dotplot is too big to fit on your screen, you may change the size of it by selecting the
Display Options tab and setting Master image width to 500. This will adjust the width of
the image to 500 pixels and then dynamically scale the height of the image based on the
ratio of sizes between the genomes selected (quick link: http://genomevolution.org/r/8mdf).
6. To color syntenic gene pairs by their synonymous mutation values (Ks), click the Analysis
Options tab and select Synonymous (Ks) for CodeML. This will use CodeML31 to calculate the
Ks values of all identified syntenic gene pairs (quick link: http://genomevolution.org/r/8m2v).
7. To change the dotplot axis metrics from nucleotides to genes, click the Display Options tab
and select Genes for Dotplot axis metric (quick link: http://genomevolution.org/r/8m33).
8. To screen for orthologous regions only in the dotplot, click the Analysis Options tab and
select Quota Align for Syntenic Depth. Next, make sure that the ratio of coverage depth for
the two genomes is 1:1 (quick link: http://genomevolution.org/r/8m4d).
9. Rerun the analysis by clicking the Generate SynMap button.
10. If you want to download the orthologous gene pairs with Ks and Kn values, click on the
link Results with synonymous/nonsynonymous rate values.
36.6 MICROSYNTENY ANALYSIS OF SYNTENIC REGIONS

Once syntenic genes are identified, it is often useful to perform microsynteny analyses on these
regions in order to identify conserved gene pairs, patterns of gene loss, problematic gene models,
and conserved non-CDSs. SynMap’s dotplots are interactive and provide an interface to zoom into
various regions and then link those to another tool in CoGe, GEvo, for microsynteny analyses.
When SynMap’s dotplot loads, there are crosshairs that track mouse movement on the dotplot. If
the crosshairs are clicked when on a chromosome–chromosome comparison, a close-up dotplot of
that pair of chromosomes will be generated. Figure 36.5c shows a close-up of chromosome 2 from
foxtail millet and sorghum generated from Figure 36.5b. This dotplot shows not only the syntenic
gene pairs plotted in purple but also all gene pairs that had sequence similarity (grey dots). This
dotplot is also interactive and when the crosshairs move over a dot representing a gene pair, they
change color from blue to red. When red, SynMap will display the annotation information for the
pair of genes. Also, if clicked, SynMap will launch GEvo and automatically anchor a microsynteny
analysis on the pair of selected genes.
GEvo provides an interface for specifying and comparing multiple genomic regions within and
among genomes. Its interface is similar to SynMap’s with three main tabs for specifying data, con-
figuring the analysis, and modifying the result visualization. Genomic regions are specified by
entering a gene name from CoGe, retrieving data from NCBI by GenBank accession, or pasting
a sequence directly into a submission box and then specifying the amount of upstream and down-
stream sequence. While there are several algorithms available for comparing sequence data, the two
most frequently used algorithms are local alignment search tool (LASTZ)32 and basic local align-
ment search tool-nucleotide databases (BLASTN),19 which identify large and small blocks of similar
sequence, respectively. LASTZ is used for identifying synteny and characterizing gene retention pat-
terns, while BLASTN is used for identifying conserved non-CDSs and characterizing gene models.5
Figure 36.6a shows the microsynteny analysis of orthologous regions between foxtail millet
and sorghum generated by GEvo after being linked from SynMap (Figure 36.5c). When GEvo
loads, each gene is used to anchor a genomic region from each genome with an addition of 50 kb
of upstream and downstream sequence. By default, LASTZ is selected as the sequence compari-
son algorithm. When run, GEvo extracts the requested sequence, compares them, and displays
the results in an interactive viewer (Figure 36.6a). Each genomic region is shown as a panel with
gene models above and below a dash line that separates the top and bottom strands of DNA. The
genes used to anchor the analysis have their CDS colored yellow. Regions of sequence similarity
are drawn as colored boxes (pink, in Figure 36.6) above and below the gene models if they are in
the same (++) or opposite (+−) orientation, respectively. Matched regions (i.e., HSPs) may be visu-
ally connected with a transparent wedge. From GEvo’s results, it is easy to see that these genomic
regions have a strong signal of synteny due to the collinear arrangement of homologous genes
(as evidenced by sequence similarity). Also, this view permits the rapid identification of unique
features in each region such as lineage-specific genes (Figure 36.6a; blue arrows) as well as tandem
gene duplications (Figure 36.6a; magenta dashed box).
The interface to GEvo is dynamic, and slider bars located at the ends of the genomic panels may
be used to narrow in on a region of interest for higher-resolution analysis. Figure 36.6b shows a
detailed analysis of a gene pair with the neighboring sequence extracted from Figure 36.6a (dashed
blue lines). This gene’s annotations describe it as having transporter activity in the chloroplast enve-
lope. Here, BLASTN was used to compare the regions in order to identify putative conserved non-
CDSs (Figure 36.6b; orange arrows) and evaluate concordance of gene models between species.
While many putative conserved non-CDSs exist in and around these genes, they may be due to
neutral carry-over. Also, the gene models are not in concordance between these regions. In order to
further explore both the conserved non-CDS and nonconcordance of gene models, an orthologous
gene from an out-group genome needs to be integrated in the analysis. Figure 36.7 shows the inclu-
sion of the orthologous gene from rice (Oryza sativa japonica33) into the analysis. Here, it is clear
that the gene model in foxtail millet is likely in error due to the concordance between sorghum’s
and rice’s gene models and the high degree of sequence conservation over annotated CDS. Also,
there are several conserved non-CDSs with collinear positioning among these genes. While most
are intronic, there is a set 5′ conserved sequences found among all the genes that may be involved in
the regulation of these genes.34 However, there appears a gene transposed between these conserved
non-CDSs and the orthologous gene in the rice lineage. If this was a gene of interest, these types
(a)
(b)
(c)
FIGURE 36.6 Microsynteny analyses using GEvo of syntenic regions from foxtail millet and sorghum. Each
comparison has two panels: the top is foxtail millet and the bottom is sorghum. Each panel represents a genomic
region with the dashed line separating the top and bottom strands of DNA. Gene models are shown as composite
colored arrows with thicker (green/yellow) regions being protein CDSs, slightly thinner (blue) being transcribed
regions, and the full extent of the gene as the thinnest. Lighter colored (yellow) CDS denotes the genes that were
used to anchor these regions when linked by SynMap (Figure 36.5b). Boxes above and below gene models repre-
sent regions of sequence similarity and have transparent wedges connecting them. (a) Comparison using BLASTZ
as the alignment algorithm. In the top and middle panel, arrows point to genes unique to one organism. The
dashed trapezoid encompasses a tandem gene duplication in sorghum. Analysis can be regenerated here: http://
genomevolution.org/r/8m35. (b) Comparison using BLASTN as the alignment algorithm using a subsequence
of the region shown in (a) (denoted by dashed lines). Note that BLASTN permits the identification of conserved
non-CDSs that fall 5′, 3′, and intronic. Additionally, this comparison shows that the gene models are not in agree-
ment between these organisms. Color figure analysis can be regenerated here: http://genomevolution.org/r/8m37.
of comparative analysis help transfer functional annotations between species, identify putative cis-
regulatory sequences, and characterize gene model problems.
36.6.1 To Perform Microsyntenic Analysis in Order

to Identify Conserved Noncoding Sequences
1. Start with a syntenic dotplot from SynMap that has been screened for orthologous regions
between foxtail millet and sorghum (quick link: http://genomevolution.org/r/8m4d).
2. Click on a chromosome–chromosome comparison in the dotplot that has orthologous syn-
teny (e.g., Figure 36.5c).
3. Use the crosshairs to navigate over a syntenic gene pair. When the crosshairs turn red,
click on the gene pair. This will link to GEvo with that pair of genes preloaded (quick link:
http://genomevolution.org/r/8m35).
4. When GEvo loads, you may adjust the extent of genomic region analyzed by modifying the
amount of left and right sequence in each sequence submission box or using the text box next
to Apply distance to all CoGe submissions found below the sequence submission boxes.
(a)
(b)
(c)
FIGURE 36.7 Microsynteny analysis of orthologous genes from foxtail millet (a), sorghum (b), and rice (c).
Details of genomic regions are described in Figure 36.6. BLASTN used to identify regions of sequence simi-
larity and those regions showing shared conserved non-CDSs among these genes have been connected with
transparent wedges. Results may be regenerated here: http://genomevolution.org/r/8mf5.
5. To run the analysis, click the red Run GEvo Analysis (quick link: http://genomevolution.
org/r/8m35).
6. When the results are returned, clicking on a gene will get its annotation to appear in a
popup window. Clicking on a region of sequence similarity will generate a transpar-
ent wedge connecting it to its partner region and get an overview of its information
to appear in a popup window. If you wish to get detailed information about a region
of sequence similarity, follow the link in the popup window to full annotation. This
will provide the entire sequence as well as a detailed view of the alignment. For addi-
tional information on how to use GEvo’s interactive viewer, see http://genomevolution.
org/r/48z4.
7. To get a detailed microsyntenic analysis of a gene, drag the slider bars located at the ends
of each genomic panel to border the gene you would like to analyze and include some
sequence 5’ and 3’ of the gene. If you are interested in analyzing the structure of a gene
model or want to identify conserved non-CDSs, you will want to change the sequence
comparison algorithm to BLASTN.
8. To change the sequence comparison algorithm to BLASTN, click on the Algorithm tab and
select BLASTN: Small Regions from the menu located next to Alignment Algorithm. The
default settings for BLASTN use a dynamic e-value cutoff that is tuned to identify plant
conserved non-CDSs. You may turn this off by setting the Spike sequence length to 0 and
then setting an e-value cutoff (quick link: http://genomevolution.org/r/8m37).
9. To rerun the analysis with the new settings, click Run GEvo Analysis (quick link: http://
genomevolution.org/r/8m37).
10. In the example shown in Figure 36.7, an orthologous gene from rice was added to a GEvo
analysis between rice and sorghum in order to help determine which gene model may be
in error and which conserved non-CDSs are deeply conserved. CoGe has tools to find such
genes quickly and the following steps walk through that process.
11. To find the ortholog to a gene shown in GEvo in a different organism, first click on a gene
to get its annotation to appear in a popup window. For this example, a gene from sorghum
was used.
12. In the annotation for the gene, click on the link SynFind found in CoGe Links (quick link:
http://genomevolution.org/r/8mfz).
a. SynFind is a tool in CoGe that takes a gene from one genome and then finds all syn-
tenic regions in multiple other genomes, regardless if the gene is present in the other
genomes.
13. When SynFind loads, search for a rice genome by typing rice in Organism Name under
Specify Organisms and selecting Oryza sativa japonica (Rice). Select version 7, unmasked
of its genome and click Add to add it to the list of genomes to search (quick link: http://
genomevolution.org/r/8mg0).
14. Run SynFind by clicking the red Run SynFind button.
15. When the results of SynFind are returned, two syntenic genes will likely be identified. One
is orthologous to the sorghum gene, and the other one is out-paralogous to the sorghum
gene. The orthologous one will have a higher synteny score. Copy that gene name.
16. Return to your GEvo analysis with the pair of genes from foxtail millet and sorghum.
From the Sequence Submission tab, click the Add Sequence button. This will cause a new
sequence submission box to appear.
17. Paste the name of the rice gene into the text box next to Name in the new sequence submis-
sion box (quick link: http://genomevolution.org/r/8mf3).
18. By default, 10 kb of upstream and downstream sequence is added. You may modify this as
needed. Click Run GEvo Analysis to rerun the analysis (quick link: http://genomevolution.
org/r/8mf3).
19. When the results are returned, modify the amount of sequence analyzed by adjusting the
slider bars or adding more sequence in order to get a better view of interesting features
(quick link: http://genomevolution.org/r/8mf5).
20. Once the amount of sequence has been adjusted for all three regions, you can com-
pare gene models and identify conserved non-CDSs across all three regions. If more
orthologous genes are to be added to the analysis, you may repeat these steps with
SynFind.
36.7 CONCLUSIONS
Every scientist will have the opportunity to sequence the genome of any organism. However, in
order to transform those data into knowledge, a variety of analytical tools are needed to compare
them to other genomes. Equally important is the need for tools to facilitate the management of
genomic data by keeping them private and securely sharing them with collaborators. This chapter
walks through the process of using the comparative genomic platform CoGe to add and share
new genomic data and use a variety of its tools for analyzing a genome and comparing it to a
variety of other genomes. These analytical examples spanned analyzing the whole genome down
to individual gene models. Intragenomic whole genome comparisons identified syntenic regions
derived from paleopolyploidy. Intergenomic whole genome comparisons identified orthologous
and out-paralogous syntenic region. Integration of syntenic analyses with synonymous mutation
analyses characterized the evolutionary history of syntenic genes permitting the unambiguous
identification of orthologous genes. Finally, microsyntenic analyses identified putative regulatory
conserved non-CDSs and erroneous gene models. The examples shown are a partial sampling of
all that can be done with CoGe, and more examples and walk-through tutorials are available at
http://genomevolution.org/r/4a3.
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Part VIII
Plants/Crops Growth Responses
to Environmental Factors
and Climatic Changes
37 Carbon Dioxide, Climate
Change, and Crops in the
Twenty-First Century
The Dawn of a New World
Glen M. MacDonald
CONTENTS
37.1 Introduction........................................................................................................................... 819
37.2 Crop Contribution to Greenhouse Gases............................................................................... 820
37.3 Impacts of CO2 Fertilization on Crop Plants......................................................................... 821
37.4 Impacts of Increasing CO2 and Climate Change on Agricultural Production...................... 823
37.5 Conclusions............................................................................................................................ 826
References....................................................................................................................................... 826
37.1 INTRODUCTION
Considerations of crop physiology in the twenty-first century must take into account the ongoing
and pervasive influences of anthropogenic increases in carbon dioxide and resulting climate change.
Similarly, attempts to predict the impacts of increasing CO2 and climatic change on future crop
yields must include a refined understanding of physiological responses to CO2 fertilization and the
influence of this on crop responses to warmer temperatures and moisture stress. Finally, nitrogen,
one of the most applied nutrients, produces a powerful greenhouse gas, N2O, and this must be con-
sidered when developing agricultural strategies to feed the planet’s burgeoning human population.
In many ways, twenty-first-century crop sciences have to contend with a new and different world
relative to the past 10,000 years of agriculture.
Due to human activity, including fossil fuel combustion and land-cover alterations, the amount
of CO2 in the atmosphere has increased from approximately 280 ppm v in the eighteenth century
to 400 ppm v levels as of May 2013 (IPCC 2007; http://keelingcurve.ucsd.edu/). Since the publica-
tion of the 2007 Intergovernmental Panel on Climate Change Assessment (IPCC 2007), greenhouse
gas emission scenarios and climate models for the twenty-first century have been further developed
and refined. Although estimates of future increases in greenhouse gases are predicated upon tech-
nological, social, and economic factors that are difficult to predict, it is estimated by the newly
developed Representative Concentration Pathway 8.5 (RCP 8.5) scenario that without substantial
mitigation efforts and technological advances, levels of CO2 will double compared to today and
reach over 800 ppm v by 2100. Increased radiative forcing induced by greenhouse gases will reach
values of 8.5 W m−2 in 2100 (Riahi et al. 2007; Jones et al. in press). This could produce an increase
in average global temperature of slightly more than 4°C (Knutti and Sedláček 2013). Even if the
most optimistic projection of greenhouse gas emissions is employed, as represented by the RCP 2.6
scenario, levels of CO2 would peak in the mid-century and remain at levels over 400 ppm v at 2100
(van Vuuren et al. 2007; Jones et al. in press). Although it has been suggested that projected fossil
819
fuel production and emissions may be overestimated in such scenarios (e.g., Höök et al. 2010), it is
notable that the observed emissions and concentrations of CO2 over the past decade have been near
the upper end of earlier scenario estimates (Manning et al. 2010). If one were to choose an interme-
diate scenario with some global stabilization targets, such as represented by the RCP 6.0 scenario,
CO2 concentrations would reach 650 ppm v by 2100 and the average global temperature would
increase by about 2.5°C (Hijioka et al. 2008; Knutti and Sedláček 2013; Jones et al. in press). It
seems clear that increased CO2 fertilization and rising temperature are inescapable over the twenty-
first century (IPCC, 2007).
This chapter examines some of the relationships between crops and global greenhouse gas
balances, with a particular focus on N2O, the physiological impacts of increased CO2 on crop
photosynthesis, heat response and response to moisture stress, and the potential impacts of the
twenty-first-century climate change on global agricultural productivity.
37.2 CROP CONTRIBUTION TO GREENHOUSE GASES

Crops both are affected by and affect greenhouse gas concentrations and climate change
(Easterling et al. 2007; Smith et al. 2007). Arable lands total over 1400 million ha. Between 1981
and 2002, the global land area under permanent crop cultivation increased from 104 to 130 mil-
lion ha, with almost all of this increase (26 million ha) occurring in developing countries (Smith
et al. 2007). At present, agriculture contributes about 10%–12% of total anthropogenic emissions of
greenhouse gases (equivalent to 5.1–6.1 GtCO2 year−1). This is emitted mainly in the form of CH4
and N2O (Smith et al. 2007). The IPCC estimates that agriculture, including livestock, is responsible
for 60% of N2O and about 50% of CH4 emissions (Smith et al. 2007). Agricultural crop emissions
of CO2 are mainly derived from the microbial decay of plant and soil organic matter and from bio-
mass burning (Smith et al. 2007). Crop CH4 emissions largely derive from rice paddy cultivation
(Smith et al. 2007; Snyder et al. 2009). Crops also take up atmospheric CO2 through photosynthe-
sis, and although there is a large active exchange, the resulting net CO2 emissions are only around
0.04 GtCO2 year−1 (Smith et al. 2007). The production of N2O is caused largely by microbial trans-
formation of N in soils and by biomass burning (Smith et al. 2007; Snyder et al. 2009). The former
is enhanced where fertilization leads to N concentrations in excess of crop requirements (Smith
et al. 2007). The production of N2O by agriculture is particularly acute under warm and moist soil
conditions that promote microbial activity (Snyder et al. 2009).
To accommodate the increase of human population to nine billion people by 2050, it is likely that
cereal production will have to double. The response of this in terms of CO2 and CH4 emissions may
be negligible, but the agricultural yields for cereals and other crops needed will likely require greater
N fertilization and increase the amounts of N2O emitted (Smith et al. 2007). Maintaining adequate
leaf concentrations of N in common C3 and C4 crop plants such as wheat, barley, corn, soybeans, and
sorghum typically requires 30–60 g kg−1 of leaf mass (Fageria 2010). At present, atmospheric N2O
concentrations are around 324 ppb v compared to preindustrial levels of 270 ppb v (IPCC 2007;
US Department of Energy http://cdiac.ornl.gov/pns/current_ghg.html). Tellingly, emissions of N2O
increased by nearly 17% between 1990 and 2005 and are projected to further increase by 35%–60%
by 2030 (Smith et al. 2007). N2O is of particular concern because it is approximately 298 times more
effective as a greenhouse gas than is CO2 (IPCC 2007).
N2O arises from soil zone nitrification that occurs when bacteria such as Nitrosomonas oxidize
NH4 and transform it to NO2 that is in turn transformed to NO3 by Nitrobacter and Nitrospira
bacteria (Norton 2009). N2O and NO are resulting by-products, ranging from 0.04% to 0.45% of
N applied (Snyder et al. 2009). Denitrification occurs when NO3 is transformed to N2. A small part
of the N is emitted as N2O (Snyder et al. 2009). The fraction of N fertilizer that is ultimately emitted
as N2O is highly variable from place to place and season to season due to differences in soil type,
oxygen and ammonium content, temperature, and moisture (Snyder et al. 2009). Spikes in emissions
occur when aerated soils are moistened, or previously frozen soils thaw and become warm and
Carbon Dioxide, Climate Change, and Crops in the Twenty-First Century 821
moist (Snyder et al. 2009). A general emission factor of 0.01 has been suggested with an uncertainty
range of 0.003–0.03. This seems small, but due to the efficiency of N2O as a greenhouse gas, it is
equivalent to the production of 4.65 kg of CO2 kg−1 of N applied, with an uncertainty of 1.4–14.0
dependent upon field conditions (Snyder et al. 2009).
The effectiveness of N2O as a greenhouse gas makes increases in N application to agricultural
crops and resulting elevations in N2O emissions of particular concern with some troubling potential
consequences for greenhouse gas mitigation strategies. For example, cropping and biofertilization
techniques that increase organic carbon stocks in soils may reduce atmospheric CO2, but some
strategies such as the application of biogas to fields may produce substantial increases in N2O pro-
duction and negate the C storage gains (Jaeger et al. 2013). The production and use of biofuels has
been touted as a means to decrease CO2 emissions from the transportation sector. However, when
the extra N2O emission resulting from production of biofuels such as rapeseed biodiesel and maize
bioethanol is taken into account, the CO2 equivalency of the emissions can contribute more to global
greenhouse gas emissions than the use of the biofuel mitigates (Crutzen et al. 2008).
37.3 IMPACTS OF CO2 FERTILIZATION ON CROP PLANTS

While increasing CO2 is the leading driver of climate change, it is also a central resource for plants.
Increasing levels of CO2 can influence both stomatal conductance and net photosynthetic rate
simultaneously. This produces improved water use efficiency (WUE) and net photosynthetic gain.
The Free-Air Carbon Dioxide Enrichment (FACE) experiments have greatly increased our knowl-
edge of plant physiological responses to the levels of CO2 anticipated over the twenty-first century
(e.g., Long et al. 2004; Ainsworth and Long 2005; Ainsworth et al. 2008; Leakey et al. 2009). In
most C3 and C4 plants, analyzed stomatal conductance has been shown to decrease in direct propor-
tion with increasing levels of CO2 (Ainsworth and Long 2005; Bernacchi et al. 2007; Kruijt et al.
2008). Concentrations of CO2 within leaves are typically 10%–20% less than the atmosphere. As
concentrations of CO2 in the air increase, the amount of gaseous exchange via the stomata needed
to ensure adequate CO2 for photosynthesis and to offset respiration decreases. In turn, this pro-
duces decreased transpiration from photosynthetic surfaces and lessens water demand by the plant
(Farquhar and Sharkey 1982). On short time scales of minutes and hours, the photosynthetic tissue
of plants responds to heightened availability of CO2 by stomatal closure and this lessens water losses
by transpiration (Katul et al. 2010). On the longer time scales of leaf and stem growth, epidural
properties of stomatal density and stomatal dimension decrease under prolonged high concentra-
tions of CO2 (Woodward 1987; Lake et al. 2002; Wagner et al. 2004; Lammertsma et al. 2011).
Analysis of stomatal properties from preserved preindustrial leaves shows that during past periods
when atmospheric concentrations of CO2 were lower, stomatal densities were greater compared to
modern leaves (Woodward 1987; Wagner et al. 2004). A recent study of common plant species from
Florida found that over the past 150 years, there has been a 34% reduction in maximum stomatal
conductance per 100 ppm v of increased CO2. This was a result of both decreased stomatal density
and changing pore size at maximal stomatal opening (Lammertsma et al. 2011).
Experimental studies show that for winter wheat, increases in CO2 from 400 to 800 mmol m−2 s−1
can cause decreases in stomatal conductance by 40%–50%. These results are consistent with mea-
surements from other studies of wheat and a wide variety of other crops that show decreases in
stomatal conductance on the order of 17%–59% when CO2 is increased from current ambient levels
to concentrations ranging from 550 to 700 ppm v (Table 37.1). These results are also similar for a
wide variety of noncrop plants (Kruijt et al. 2008).
Increasing levels of CO2 can also produce elevated rates of photosynthesis relative to respiration
in C3 crop plants. This is related to ribulose bisphosphate (RuBP). Higher levels of CO2 promote
carboxylation and inhibit oxygenation and the decreased oxygenation lowers photorespiration rates
(Long et al. 2004). At a temperature of 25°C, an increase in CO2 concentrations to 550 mmol mol−1
increases RuBP-saturated photosynthesis by 34%. This effect becomes more pronounced under higher
TABLE 37.1
Observed Decreases in Stomatal Conductance
with Increasing CO2
CO2 (ppm v) ∆ Conductance (%)
C3 crops
Barley 680 −52
Barley 700 −33
Beans 700 −38
Grass 550 −22.2
Grass 700 −33
Legume 550 −22.9
Potato 680 −59
Potato 700 −32
Soy 680 −23
Soy 700 −25
Wheat 550 −30
Wheat 600 −17
Wheat 680 −22
C4 crops
Grass 550 −24.9
Maize 680 −37
Source: Data extracted from Kruijt, B. et al., J. Hydrol., 349,

257, 2008.
temperatures (de Pury and Farquhar 1997; Long et al. 2004). Because current atmospheric concentra-
tions of CO2 are already sufficient to provide for optimal primary carboxylation in C4 plants, those
crops will not benefit in the same manner as C3 crops. C4 crops such as maize will, however, benefit
from the additional atmospheric CO2 in cases where CO2 acquisition is strongly curtailed by decreased
stomatal conductance due to moisture stress during droughts. The decreased demands for stomatal
conductance that both C3 and C4 plants experience under elevated CO2 conditions (Table 37.1) would
lessen moisture loss for C4 plants and yet still allow for sufficient uptake of CO2 to meet photosynthesis
requirements (Long et al. 2004; Leakey et al. 2006; Vu and Allen 2009; Markelz et al. 2011).
The decreased stomatal densities in C3 and C4 plants, and the increased rates of photosynthe-
sis and decreased photorespiration in C3 plants, produced by elevated levels of CO2 present some
interesting synergies. The most obvious is the increase in WUE through increased photosynthesis/
decreased photorespiration and/or decreased transpiration. Experimental and modeling studies of
increasing WUE for crops under CO2 conditions of ∼400 to ∼800 mmol m−2 s−1 indicate significant
increases in WUE and yields due to CO2 fertilization. In an experimental treatment of potatoes,
WUE values ranged from 5 to 14 and yields ranged from 11 to 30 kg m−3 for ambient and elevated
CO2 conditions, respectively. Compared to typical potato yields, this represents a potential doubling
or more of ultimate crop productivity due to increased CO2 (Fleisher et al. 2008). Similarly, in
experiments with maize, it was found that up to 20%–35% less water was used under elevated CO2
conditions than under the ambient conditions (Chun et al. 2011). Although elevated CO2 can lead
to increased total leaf area and resulting higher ecosystem transpiration rates, these results seem
to indicate that the negative impacts of this effect are less than the positives due to increased WUE
(Fleisher et al. 2008).
When increased CO2 is coupled with a warmer climate, additional synergy might come from the
increase in the boundary layer and leaf temperatures produced by climate warming and decreased
rates of transpiration resulting from decreased stomatal conductance (Bernacchi et al. 2007). This
synergy may arise because increased leaf temperatures will further promote the effect of increased
CO2 on RuBP-saturated photosynthesis. However, this supposition cannot be universally applied.
From a meta-analysis of experiments pertaining to the response of plants to simultaneous increases
in CO2 and temperature, it was concluded that for C3 plants, there was a positive response in photo-
synthesis when CO2 was elevated to 560 mmol mol−1 and temperatures increased by 1.4°C to >8.0°C
compared to the response of plants to a similar temperature increase under ambient CO2 conditions
of 400 mmol mol−1. However, C4 plants showed no positive response to increased CO2 at ambient
temperatures, a positive response at 1.4°C–6.0°C, increased temperatures, and a negative response
to temperature increases >8.0°C (Wang et al. 2012). In contrast, the highest positive response from
the C3 plants was at the >8.0°C level of warming (Wang et al. 2012).
Nitrogen requirements may play a role in the heat stress tolerance of C3 and C4 plants at elevated
levels of CO2 (Wang et al. 2012). Demand for N at higher temperatures may be partially offset in
C3 plants by increased RuBisCO efficiency under elevated CO2. Reductions in RuBisCO content
of 31% to >50% have been reported for C3 plants under conditions of elevated CO2 (Leakey et al.
2009). About 25% to as high as 50% of leaf N is tied up in RuBisCO. Thus, decreased RuBisCO
requirements due to high CO2 concentrations can have a strong effect on crop N demands (Leakey
et al. 2009). The negative response of C4 plants to the combination of elevated CO2 and highly
elevated temperature (>8.0°C) noted previously could be due to N limitations leading to impaired
synthesis of photosynthetic enzymes and protecting systems. In C3 plants, this is compensated for at
high CO2 concentrations and high temperatures by decreased photorespiration (Wang et al. 2012). In
an analysis of the interactive effects of moisture stress, N supply, and elevated CO2 on net photosyn-
thesis on maize, the plants were grown at CO2 concentrations of ∼385 and 550 mmol mol−1 and with
high and low N supplies (168 kg N ha−1 fertilizer and no N fertilizer) (Markelz et al. 2011). Typical
of C4 plants, in the absence of water stress, there was no stimulation of net photosynthesis as a result
of increased CO2 and this was irrespective of N availability. During drought conditions, however,
elevated levels of CO2 ameliorated the stomatal and nonstomatal impacts of moisture stress on net
photosynthesis. Limiting N also exacerbated the impacts of moisture stress on the maize, but during
drought, the magnitude of amelioration produced by elevated CO2 was the same for N-limited and
non-N-limited plants (Markelz et al. 2011). It has been suggested from this and other experimental
studies that elevated levels of CO2 are unlikely to provide added photosynthetic gains for maize and
other C4 crops despite the warmer temperatures of the twenty-first century except in cases where
moisture stress is ameliorated by decreased demands on stomatal conductance (Leakey et al. 2006;
Markelz et al. 2011).
37.4 IMPACTS OF INCREASING CO2 AND CLIMATE

CHANGE ON AGRICULTURAL PRODUCTION
Confidently predicting the impacts of increasing CO2 and climate changes on crop yields over
the twenty-first century remains difficult. Although the atmospheric concentrations of CO2 and
other greenhouse gases will increase at similar magnitudes across the globe, the climatic impacts
of increasing greenhouse gases will display considerable geographic heterogeneity (IPCC 2007;
Knutti and Sedláček 2013; Chadwick et al. in press; Jones et al. in press). Precipitation as well
as temperature will change and some regions will become significantly more arid, while other
regions may experience greater rainfall (IPCC 2007; Knutti and Sedláček 2013; Chadwick et al.
in press). In some regions, decreases in precipitation will couple with increased evapotranspira-
tion rates to greatly increase crop moisture stress. In addition, it is not only the means of variables
such as daily temperature or annual precipitation that will change but factors such as diurnal
temperature differences, start, close, and duration of the summer growing season, climatic vari-
ability, and the frequency and magnitude of extreme weather events (IPCC 2007; Field et al. 2012).
Finally, regional climates will develop that due to their seasonal combinations of temperature,
precipitation, and photosynthetically active radiation have no analogue in historical climatic con-
ditions. It has been estimated that with moderate continuation of greenhouse gas emission rates,
about 63% of the United States will have climatic conditions that have no modern analogue in the
country today (Saxon et al. 2005).
The last time there were persistent atmospheric CO2 levels in the range of 400–500 ppm v was
in the Pliocene, some 3 million years ago (Pagani et al. 2010). This means that all crop plants were
domesticated from ancestral wild varieties and have subsequently been selectively bred for almost
their entire history under ambient CO2 levels, which have already been exceeded by today’s con-
centrations. For example, it has been suggested from theoretical studies that the current form of
RuBisCO in C3 crop plants has evolved for optimal adaptation to CO2 levels 220 ppm v typical of
the long-term average over the past 25 million years (Long and Ort 2010). In terms of nonanalogue
climates, elevated CO2 levels, and the evolutionary physiological adaptations of current crop plants,
we have entered a new world.
In the absence of increasing temperatures and decreased precipitation, the 2007 IPCC Assessment
concluded that CO2 fertilization would produce increased crop yields, but the magnitude depends
upon factors such as photosynthetic pathway and management practices related to such factors as
water provision and N application (Easterling et al. 2007). Based on the assessment of several
important crop species, it was determined that at CO2 concentrations of 550 ppm v, increases in
crop yields in the range of 10%–20% might be experienced for C3 crops and 0%–10% for C4 plants
(Easterling et al. 2007). However, the estimates of the impact of the increasing levels of CO2 are
not realistic in the absence of consideration of changing temperatures and precipitation. When CO2
fertilization and climatic changes were taken into account, the IPCC Assessment concluded that at
higher latitudes, the increase in direct CO2 fertilization coupled with modest warming in the range
of 1°C–3°C could increase yields, if precipitation changes do not impart moisture stresses. However,
in low-latitude regions, such warming would produce declines in yields for most cereals. Greater
warming than this would produce widespread declines across latitudes according to the assessment
(Easterling et al. 2007). This conclusion was based in part on an analysis of 69 individual studies
of climate change and crop yield response (Figure 37.1). However, the studies were not uniform in
how they projected or modeled the impacts of increasing temperatures, precipitation changes, and
CO2 (Easterling et al. 2007). There is clearly much scatter, and thus much uncertainty conveyed, in
the graphical summation of the studies considered by the IPCC (Figure 37.1). The IPCC identified a
number of key unknowns related to increasing CO2, resulting climate change and crop yields. They
noted in particular that there are insufficient experimental data for the effects of CO2 fertilization
and changes in temperature and moisture on many noncereal crops. More generally, the saturation
levels at which CO2 fertilization will no longer have a positive effect and what controls these thresh-
olds remain an area in need of further research (Easterling et al. 2007).
Since publication of the IPCC Fourth Assessment in 2007, there have been a number of new
studies on analysis and projection of CO2 and climate change impacts on crop yields. An impor-
tant recent study analyzed climatic trends and crop yield data for the period 1980–2008. In many
regions, the magnitude of the trend in increasing temperatures exceeded one standard deviation of
long-term variability. Over the same period, CO2 increased by 47 ppm v (Lobell et al. 2011). The
study concluded that despite CO2 fertilization, coupled with improved technology and other factors,
maize and wheat production fell by 3.5% and 5.5%, respectively. China and Brazil experienced
the greatest climate-related declines in maize production, while Russia experienced the greatest
declines in wheat production. Rice yields showed little change and soybean yields were regionally
variable with no significant global change (Lobell et al. 2011). From a modeling analysis of crop
yields, the global statistical impact of increasing CO2 at current levels was found to be negligible for
maize and on the order of only 0.3% for wheat, rice, and soybean. On the other hand, the negative
impacts of increasing temperatures over the period ranged from −4.9% for wheat, −3.1% for maize,
and −0.8% for soybean. Rice had a minor positive response of 0.1 (Lobell et al. 2011).
60 60
40 40
20 20
% Yield change
0 0
–20 –20
–40 –40
–60 –60
0 1 2 3 4 5 6 0 1 2 3 4 5 6
(a) Mean local temperature change (°C) (b) Mean local temperature change (°C)
60 60
40 40
% Yield change
20 20
0 0
–20 –20
–40 –40
–60 –60
0 1 2 3 4 5 6 0 1 2 3 4 5 6
(c) Mean local temperature change (°C) (d) Mean local temperature change (°C)
60 60
40 40
20 20
% Yield change
0 0
–20 –20
–40 –40
–60 –60
0 1 2 3 4 5 6 0 1 2 3 4 5 6
(e) Mean local temperature change (°C) (f ) Mean local temperature change (°C)
FIGURE 37.1 Graphical summary of 69 published studies of the response of cereal yields to temperature
change. (a) Maize, mid to high latitude. (b) Maize, low latitude. (c) Wheat, mid to high latitude. (d) Wheat, low
latitude. (e) Rice, mid to high latitude. (f) Rice, low latitude. (From Easterling, W.E. et al., Climate change:
Impacts, adaptation and vulnerability, Contribution of Working Group II to the Fourth Assessment Report of
the Intergovernmental Panel on Climate Change. Food, Fibre and Forest Products, Cambridge University
Press, Cambridge, U.K., pp. 273–313, 2007, Fig. 5.2, p. 286. With permission from the Intergovernmental
Panel on Climate Change.)
37.5 CONCLUSIONS
Taken together, crop physiological experiments and modeling studies of crop response to increasing
CO2 and changing climate point out the potential significant impacts of increasing CO2 and related
changes in climate. There is a need for extensive experimental data on the range of physiological
responses that crop plants will have to interacting effects of simultaneous variations in CO2, tem-
perature, and moisture stress. In this regard, the marked differences in the responses of the C3 and
C4 photosynthetic pathways to CO2 fertilization become of paramount importance. The complexity
of the reaction of crops to increasing CO2 and climatic changes is concisely articulated in a recent
article by Long and Ort (2010). At this time, much of the experimental data used to confront these
issues come from chamber experiments. Whole-field experimentation in representative agricultural
regions, climates, and soils is much needed (Long and Ort 2010). Available experimental studies of
the complex interactions of CO2 enrichment, warming, moisture stress, and nutrients such as N have
been focused on the midlatitudes of the northern hemisphere, and not enough field study has been
conducted in the tropics and high latitudes (Leakey et al. 2012). Finally, the emission of N2O from
agriculture is a potential positive feedback to climate change. How the demand for N fertilization
may change with changing levels of CO2 and changing climate is a critical question. The interactions
between the impacts of elevated temperatures, moisture stress, and CO2 with N supply are complex
and require further experimental study. Understanding and efficiently adapting N demands to mini-
mize N2O emissions while at the same time maintaining adequate food supplies is one of the great
challenges for crop sciences in the twenty-first century.
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Part IX
Future Promises: Plants and Crops
Adaptation and Biotechnological
Aspects of Plants/Crops Improvement
38 Adaptable, Metabolically Flexible,
CAM Plants as Crops
and Highly Productive Cultivars

Karina E.J. Trípodi, Florencio E. Podestá, Carlos M. Figueroa,
Claudia V. Piattoni, Alberto A. Iglesias, and Valeria E. Perotti
CONTENTS
38.1 CAM Pathway of Carbon Fixation........................................................................................ 831
38.1.1 Introduction............................................................................................................... 831
38.1.2 Core Processes of Photosynthetic Carbon Fixation: C3 and C4 Plants...................... 832
38.1.3 CAM Plants: General Description............................................................................. 833
38.2 Biochemistry of CAM........................................................................................................... 835
38.2.1 Night Period Reactions.............................................................................................. 835
38.2.2 Day Reactions............................................................................................................ 837
38.3 Regulation.............................................................................................................................. 839
38.3.1 Transcriptional Regulation........................................................................................ 839
38.3.2 Posttranscriptional and Translational Regulation......................................................840
38.3.3 Posttranslational Regulation......................................................................................840
38.3.4 Tonoplast....................................................................................................................840
38.4 Evolution and Taxonomic Distribution..................................................................................840
38.5 Productivity and Use of CAM Plants as Crops..................................................................... 841
38.6 Ecophysiology........................................................................................................................ 841
38.7 Conclusions and Perspectives................................................................................................ 842
References....................................................................................................................................... 843
38.1 CAM PATHWAY OF CARBON FIXATION

38.1.1 Introduction
The term photosynthesis describes the process by which some living beings can meet their energy
and nutrient needs using solar radiation and inorganic matter. One of the prominent features of
photosynthetic organisms is their ability to transform little reactive inorganic molecules, CO2 and
H2O mainly, in reduced organic compounds that can be used as building blocks for all living things.
Photosynthesis is a widespread phenomenon on Earth and is present in a variety of habitats and
organisms. This remarkable adaptability allows photosynthetic organisms to thrive in the all oceans
and most of land habitats, from tropical forests to deserts.
It is hard to think of a desert without almost immediately evoke the shape of a cactus, and it is
equally hard to imagine at first glance how these plants can survive the tremendous aridity of their
habitat. This chapter addresses a biochemical adaptation, the Crassulacean acid metabolism (CAM),
831
FIGURE 38.1 Three CAM plant species growing in the Parque Nacional de las Quijadas (Province of San
Luis, Argentina), with a rainfall lower than 200 mm per year. It shows a cactus, Trichocereus candicans (1);
a terrestrial bromeliad, Bromeliad hieronymi (2); and an epiphytic bromeliad, Tillandsia aizoides (3).
that cactus and other similar plants put into play to survive where most other plants cannot (Figure 38.1).
It will also describe how CAM plants can achieve high biomass productivity without most of the
requirements of the more common C3 and C4 crops.
38.1.2 Core Processes of Photosynthetic Carbon Fixation: C3 and C4 Plants

C3 plants are restricted to a single metabolic pathway of assimilation of CO2, the pentose phosphate
reductive cycle (PPRC), in which atmospheric CO2 is used directly as a source of carbon, and have
a single photosynthetic cell type (Figure 38.2). The PPRC comprises a series of light-dependent
reactions occurring in the chloroplast and starting with the carboxylation of a 5-carbon molecule to
yield a 3-carbon intermediate (Edwards and Walker 1983; Iglesias and Podestá 2005). Most plants
are within this group, among which wheat, rice, and most trees can be accounted.
The carboxylation reaction involves the addition of a molecule of CO2 to ribulose bisphosphate to
yield two molecules of 3-phosphoglycerate. This step is catalyzed by ribulose bisphosphate carboxylase/
oxygenase (RuBisCO), which is one of the control points of the photosynthetic process (Edwards and
Walker 1983; Iglesias and Podestá 2005). This enzyme is active only during the light period and has the
additional ability to oxidize ribulose bisphosphate, generating a cycle of photorespiration involving C2
intermediates (Tolbert 1997) (Figure 38.3). Photorespiration results in a net loss of carbon and energy at
a rate that might affect the photosynthetic performance of the plant. The oxygenase activity of RuBisCO
depends on the relative concentrations of O2 and CO2 in the cell, which in turn are governed by tempera-
ture. Thus, the higher the temperature, the lower the ratio CO2/O2 and the higher the photorespiration
rate, with the concomitant detrimental effect on the carbon fixation capacity of the plant (Tolbert 1997).
It becomes clear then that any factor leading to an elevation of the CO2/O2 ratio will result in
an abatement of photorespiration and an enhancement of photosynthetic yield. Another group of
plants, termed C4, has evolved auxiliary systems that minimize the C2 metabolism by increasing the
CO2/O2 ratio inside the chloroplast (Edwards and Walker 1983; Edwards et al. 2001). In C4 photo-
synthesis, an additional carboxylation reaction, located in the cytosol, is present. This reaction is not
affected by O2 and yields as a 4-carbon organic acid as product hence the denomination of the group
CAM Plants as Crops 833
C3 plants
C3 C6
RuBisCo PPRC Starch

CO2
C5
C4 plants
Mesophyll cell Vascular bundle
C4 C4
PEPC
CO2
HCO–3
C3 C3
PPRC
FIGURE 38.2 Schematic representation of C3 and C4 metabolisms.
Carbon fixation:
CO2 Photosynthesis
Ribulose-1,5-P2 RuBisCO
Carbon loss:
O2 Photorespiration
FIGURE 38.3 Scheme of RuBisCO’s carboxylase and oxygenase activities that give rise to either the photo-
assimilatory of photorespiratory pathways.
(Figure 38.2). This compound is used as a temporary reserve of fixed CO2. The reaction is catalyzed
by a HCO3- -requiring highly regulated enzyme that uses phosphoenolpyruvate (PEP) as acceptor and
is known as PEP carboxylase (PEPCase) (Chollet et al. 1996). Most C4 plants require two cell types
that spatially separate the initial (PEPCase-dependent) and definitive (RuBisCO-dependent) CO2
fixation (Edwards et al. 2001). Mesophyll cells contain PEPCase, but not RuBisCO, and surronds (as
an anatomical barrier against oxygen) the vascular shealth cells that contains the RuBisCo and the
complete PPRC. The net result is an increase in the concentration of CO2 in the vascular sheath that
almost completely prevents photorespiration (Edwards and Walker 1983; Iglesias and Podestá 2005).
Additionally, due to the greater efficiency in the absorption of CO2, the stomatal opening is reduced,
thereby mitigating loss of water. Thus, C4 plants, among which corn and sugar cane are included, can
develop in high-temperature, and intensively irradiated environments (Edwards and Walker 1983).
The two-cell architecture typical of most C4 plants is not essential for C4 photosynthesis. As has been
demonstrated in recent years, single-cell C4 photosynthesis can exist as well (Edwards et al. 2004).
38.1.3 CAM Plants: General Description

CAM-bearing plants, as those displaying C4 photosynthesis, can survive in habitats with high light-
ing and reduced irrigation, although the latter is particularly applicable to CAM species. Indeed,
CAM is a remarkable adaptation to life under stress, as illustrated by the fact that many succulent
species that display this metabolism live in a desert habitat. However, the CAM phenomenon is not
restricted to arid environments and can be found in aquatic plants or shade plants in tropical forests
in which the stress can occur due to lack of CO2. CAM is also present in epiphytes (orchids, air
plants) that grow on substrates with limited or nil water retention capacity.
CAM is characterized by the ability of certain plants to assimilate CO2 in the dark, yielding 4-carbon
organic acids. This feature is the basis of other characteristics of CAM that were described almost two
centuries ago, being the most conspicuous the cycles of daily deacidification–acidification and carbo-
hydrate turnover and, in some cases, some degree of succulence in leaves (low surface to volume ratio,
which helps in reducing exposure of tissues to the environment). CAM plants have achieved their spe-
cialization within a single cell type, temporarily separating the PPRC and RuBisCO activities from an
ancillary metabolic pathway of CO2 fixation that depends on PEPCase, as in C4 plants. One of the pecu-
liarities of CAM is that CO2 uptake is nocturnal, in contrast to C3 and C4 plants, in which this event is
restricted to the day hours. This feature allows CAM plants to exchange gases with the environment at
night, minimizing water loss by the high daytime temperatures that occur in desert environments. One
of the major challenges surrounding CAM-related research is the elucidation of the molecular machinery
that controls the rhythm of these circadian oscillations (Boxall et al. 2005).
The emergence of the CAM biochemistry in itself does not involve the advent of new enzyme
activities; rather, specific preexisting enzyme family members have been recruited (Cushman and
Bohnert 1999), and in some cases, CAM-type isoforms with special regulatory properties have
evolved. CAM requires an active metabolite exchange between organelles, and consequentially
special transport systems are present, and of course, due to the temporal separation of the carbox-
ylase activities, the stomata movement is subject to a different control. Being CAM a polyphyletic
phenomenon, it is not unusual to find variations in the many different CAM species.
The study of the CAM phenomenon then covers various fields of plant biochemistry. The follow-
ing describes the more important aspects of this metabolism that reflect the most prominent adapta-
tions to stress developed by these plants. The event that initiates the nocturnal phase of the cycle is
the CO2 fixation catalyzed by PEPCase that produces oxaloacetate (OAA), which is then reduced to
malate (Figure 38.4). This acid is the main responsible for the acidification of nocturnal CAM plant
leaves, although other organic acids such as citric acid may also be accumulated. Malate is stored
in the vacuole by a passive process coupled to an active proton pumping into the organelle. This
process is reversed during the day, in which the malic acid leaves the vacuole and CO2 is obtained
by decarboxylation to be definitively fixed by PPRC. The rest of the organic molecule is converted
into sugars by gluconeogenesis or breathed through the tricarboxylic acid cycle.
Vacuole
Malic acid
Malate Cytosol Malate

MDH/PEPCK
MDH PPRC or
Chloroplast CO2
OAA ME(NAD/NADP)
Starch Pyruvate/PEP
PEPC PEP
– 3P-glycerate
HCO3
CO2
Stomata (open) Stomata (closed)
CO2
FIGURE 38.4 Schematic representation of CAM metabolism.

Phase I II III IV
PEPCase PEPCase RuBisCO RuBisCO
RuBisCO PEPCase
tion
mila
t
en
nt
assi
co
cid
2
ca
CO
ali
M
18:00 06:00 18:00

Time
FIGURE 38.5 CAM phases. CAM plants present phases clearly distinguishable through the gas exchange
pattern and the accumulation of organic acids, mainly malic acid. The graph shows those phases and the cor-
relation with the main carboxylation reaction during the daily cycle.
The cycle of reciprocating carbohydrate, organic acids, and gas exchange can be presented as a
phase diagram that clearly portrays the main biochemical processes as they distribute along the day
(Figure 38.5). While this is the general mechanism by which CAM plants operate, there are sev-
eral variations among the many species that show this metabolism. For example, the carbohydrate
source for the synthesis of PEP can be chloroplastic or vacuolar involving soluble sugars or starch,
respectively, thus defining a major subdivision between different CAM plants (Black et al. 1996).
Other differences are related to the mechanism of malate decarboxylation, as this can occur in the
cytosol or in mitochondria by the action of different types of decarboxylases.
Additionally, CAM may be a constitutive or inducible feature in different species (Winter et al.
2008). Stimuli that naturally induce a transition from a C3 or C4 metabolism to CAM are varied
and include water stress or salt stress and temperature (Cushman and Borland 2002). The great
variability among different CAM plants has been the subject of intense research, and many of the
fundamental processes governing this path of carbon assimilation have begun to be elucidated.
There are two phenomena associated with CAM that appear under specific circumstances. One
of them is CAM idling. Some CAM plants under severe water stress show diurnal variations in acid
content but do not perform gas exchange. The complete stomatal closure prevents loss of water, and
the respired CO2 is refixed and recycled through CAM. This allows the plant to survive until favor-
able conditions appear. The other is CAM cycling, which some inducible succulents show as they
prepare to switch to full CAM. CAM cycling is characterized by daily organic acid fluctuations but
no nocturnal CO2 uptake.
38.2 BIOCHEMISTRY OF CAM

38.2.1 Night Period Reactions
CAM begins with the reaction catalyzed by PEPCase that incorporates CO2 to yield a 4-carbon
acid, OAA:
PEP + HCO3- Æ OAA + Pi

PEPCase is key to the overall regulation of the metabolic pathway and differs in several aspects
with respect to RuBisCO, for example, the use of HCO3- instead of CO2 without using biotin. The
presence of carbonic anhydrase in the cytosol of CAM plants makes the HCO3- concentration opti-
mal for the carboxylation reaction. PEPCase is a typical multimeric regulatory enzyme of cytosolic
localization. It has been the subject of intense research in the last 20 years because of its central role
in regulating the carbon flux in C4 and CAM metabolisms and its anapleurotic function in C3 species
(Plaxton and Podestá 2006).
CAM PEPCase is regulated at transcriptional and posttranslational levels (Nimmo 2000).
The transcriptional control regulates the amount of PEPCase protein through changes in the
abundance of mRNA, as observed for CAM facultative species as Kalanchoë blossfeldiana or
Mesembryanthemum crystallinum (Schmitt 1990; Taybi et al. 1995). In fact, expression levels of
PEPCase can vary from 2 to 20 times with the transition of an inducible plant to a CAM status.
mRNA can start accumulating 2 or 3 h after the stress event that initiates the metabolic conversion
(Schmitt 1990). However, this type of control is restricted to the induction of CAM and not the
control of the circadian oscillations. In fact, the only CAM-related gene to undergo daily fluctua-
tions is PPCK, which codes for PEPCase kinase (PPCK) (Taybi et al. 2000; Mallona et al. 2011).
Although the mechanism involved has not been completely unveiled, it seems probable that the
levels of abscisic acid are partially involved in the induction of CAM (and PEPCase) during water
stress (Taybi et al. 1995). MYB transcription factors are thought to be involved in the salt-inducible
gene expression (Cushman and Bohnert 1999).
PEPCase activity is controlled by a phosphorylation–dephosphorylation process and has become
a model system for the study of metabolic regulation in plants (Nimmo 2000). Unlike the C4 plants
enzyme, the phosphorylated form of CAM plants, less susceptible to inhibition by malate but more
responsive to the allosteric activator Glc-6-P, abounds during the night period (Bakrim et al. 2001).
Phosphorylation of PEPCase occurs on a single serine (Ser) residue located at the N-terminus of the
protein. The phosphorylatable Ser is located in the I/DR/KxxSIDAQL/MR sequence common to all
plant-type PEPCases, suggesting that the regulation by phosphorylation of the enzyme occurs in the
many diverse physiological contexts where PEPCase is involved. However, this motif is absent in
cyanobacterial- and plant bacterial-type PEPCases (Chollet et al. 1996; Sánchez et al. 2003). During
the daylight period, PEPCase is predominantly dephosphorylated and more sensitive to feedback
inhibition by malate, although total extractable activity may differ much from values observed in
the dark. Being malate the responsible for the cyclic acid flow between the vacuole and cytosol of
CAM plants, it is not difficult to understand why the temporal regulation of PEPCase is coordi-
nated with the circadian rhythm of CAM plants that show high malate synthesis at night and high
decarboxylation during the day, avoiding a futile cycle of carboxylation/decarboxylation.
The phosphorylation state of PEPCase depends on the levels of PPCK. PPCK is highly specific
and belongs to the family of Ca2+/calmodulin-dependent kinases (CDPK), although its activity is
independent of Ca2+. As described earlier, PEPCK is under the control of a circadian oscillator
(Carter et al. 1991), a homolog to that found in Arabidopsis thaliana but different enough to be
considered CAM type (Mallona et al. 2011). Interestingly, PPCK regulation is almost exclusively
at the level of gene expression, through rapid changes in the rate of synthesis and high turnover
rates (Hartwell et al. 1999; Taybi et al. 2000). Circadian regulation of PPCK expression can also
be affected by the metabolic state of the leaf (Borland et al. 1999). Therefore, it is the integration
of circadian signals modulating metabolism and finally the abundance of the mRNA of the kinase
what ultimately determine the activation state of PEPCase during the day/night cycle, since dephos-
phorylation by protein phosphatase 2A has a constant activity during the 24 h cycle (Carter et al.
1991, 1996; Cushman 2001).
The OAA produced by PEPCase is then reduced to malate by means of a malate dehydroge-
nase (MDH). The MDH reaction may in principle occur in the cytosol or in mitochondria. CAM
induction increases the levels of the cytosolic, NAD-dependent, isoform of MDH and its proper-
ties are compatible with a role in the reduction of OAA (Cushman and Bohnert 1999; Trípodi and
Podestá 2003). It is also suggestive that MDH can form protein–protein associations with PEPCase
in K. blossfeldiana and Ananas comosus (Queiroz-Claret and Queiroz 1992). This interaction may
provide a means to effectively channel an unstable metabolite such as OAA directly to the next reac-
tion site and implies that the reduction reaction is cytosolic.
The enormous amounts of malate produced overnight must be efficiently removed from the cyto-
sol to prevent inhibition of PEPCase and a drop in cytosolic pH to critical levels. To achieve this,
malate is transported to the vacuole by a two components mechanism: a passive transport of malate
and active transport of H+ (Luttge et al. 2000; Pantoja and Smith 2002). Indeed, malate would
be in equilibrium across the tonoplast, but at the low internal pH of the vacuole acid, it becomes
malic acid, which cannot cross through the tonoplast. The transport of malate, during which the
charge balance is maintained by the influx of H+, is thus thermodynamically favored. Malate influx
involves a specific vacuolar membrane transporter that exhibits a high degree of rectification, that
is, transport is in one direction (Cheffings et al. 1997; Luttge 2000). H+ translocation is mediated by
a H+-ATPase (V-ATPase) and a H+-PPase (Cheffings et al. 1997), although it is believed that the first
is the most important in plant leaf photosynthesis conducting CAM.
The H+-ATPase is of the same type as that found in other eukaryotic organelles acid, a complex
of at least nine different subunits forming the ball and stem structure typical of proton translocator
enzymes. The expression levels of the H+-ATPase are linked to the induction of CAM in
M. crystallinum (Cushman and Bohnert 1999). It is not clear yet whether the H+-PPase has an equally
important role in the transport of H+. The cytosolic acidification caused by the carboxylation of PEP
drives H+-ATPase activity and initiates the movement of protons into the vacuole. This process con-
tinues until the electrochemical potential across the tonoplast is high enough to stop the pumping of
H+. Computer simulations strongly suggest that the tonoplast could be the main switch governing the
circadian oscillator (Luttge 2000).
38.2.2 Day Reactions

Malate efflux from the vacuole is a process that has not been revealed as yet. So far, it is clear that
cell turgor is a key factor in initiating the efflux process (Luttge 2000). The output of malate during
the day period may follow different ways. If it is through a transporter, then a separate H+ channel
must exist to maintain charge balance. At very low vacuolar pH values, the apolar H2malate0 could
flow out of the tonoplast. It is not clear if the forms Hmalate−1 and malate−2 could also contribute
to the output of this metabolite. Cytosolic malate is the source of CO2 for diurnal fixation. The
mechanism of decarboxylation determines subtypes of CAM plants (Winter and Smith 1996), but
most species have more than one of the three possible decarboxylating enzymes (Black et al. 1996).
These reactions can be catalyzed by NADP- or NAD-dependent MEs in the cytosol or mitochon-
dria, respectively (Figure 38.6). MEs have been extensively studied and characterized in C3 and C4
NAD(P)+ NAD(P)H
Malate Pyruvate
1
NAD(P)+ ATP
2 4
CO2
NAD(P)H AMP + Pi
3
Oxaloacetate PEP
ATP ADP
FIGURE 38.6 Reactions involved during daytime decarboxylation of malate. The enzymes are (1) ME,
(2) MDH, (3) pyruvate phosphate dikinase, (4) PEP carboxykinase.
plants but only partially in CAM species (Falcone Ferreyra et al. 2003). The transcript levels and
activity of a specific CAM form of an NADP-ME from Aloë vera and M. crystallinum increase up
to 12 times during the induction of CAM (Saitou et al. 1992, 1994). NADP-ME in CAM plants is
located in the cytosol, unlike what happens in C3 plants. So far, the relative contribution of each ME
during daytime decarboxylation of malate is unknown. The mitochondrial NAD-ME is activated
during the day by acetyl-CoA, fructose-1,6-bisphosphate (Fru-1,6-P2), and pyruvate. Alternatively,
malate can be oxidized to OAA by MDH, and then the OAA can be decarboxylated by a PEP car-
boxykinase in the cytosol (Figure 38.6).
Despite its abundance in species such as A. vera and the bromeliad A. comosus, PEP carboxyki-
nase study has been hampered by the extreme lability of the enzyme to proteolytic cleavage of the
N-terminus (Leegood and Walker 2003; Martin et al. 2007, 2011). The pineapple enzyme exhibits
properties that suggest it may be functional only during the daytime, but has not shown signifi-
cant regulatory properties. This is certainly intriguing, since it coexists with the cytosolic enzyme
PEPCase, and the simultaneous action of both can lead to a futile cycle in which ATP is hydrolyzed.
Moreover, there is evidence that an N-terminal truncated form could be functional in this plant
(Martín et al. 2011). Although PEP carboxykinase is phosphorylated during the night, only indi-
rect evidence exists that posttranslational modification affects its kinetic properties, as neither the
phosphorylated nor dephosphorylated enzymes have been purified and cannot be assayed separately
(Chen et al. 2002b). PEP carboxykinase-type CAM species usually contain variable amounts of
NAD- and NAD(P)-ME, but ME types usually do not show PEP carboxykinase activity (Cushman
and Bohnert 1999).
Whatever is the decarboxylation mechanism involved, the CO2 released is incorporated into
PPRC. The PEP or pyruvate generated serves as a building block for the biosynthesis of carbohy-
drates. When pyruvate is the resulting metabolite, the carbon skeleton is recovered for metabolism
by the action of pyruvate phosphate dikinase. This enzyme uses ATP and Pi to generate PEP, AMP,
and PPi:
Pyruvate + ATP + Pi Æ PEP + AMP + PPi

This reaction is unidirectional in vivo, and the PEP produced can be transformed to hexose phos-
phates by gluconeogenesis and later incorporated into sucrose or starch. The pyruvate phosphate
dikinase in CAM plants can be cytosolic or chloroplastic (Kondo et al. 2000). So far, no known
regulation mechanism has been found that controls this enzyme activity in CAM plants.
Toward the end of the day, the process begins to reverse from a C3 carbon assimilation into
a C4 -based biochemistry. At this time, carbohydrates start to be degraded to provide PEP. The
process of PEP generation for the purpose of serving as acceptor for CO2 is one of the primary
sources of the reciprocal changes in carbohydrate and malic acid in CAM plants. The type of
carbohydrate used in PEP synthesis in turn determines another subdivision between CAM plants
(Black et al. 1996). Indeed, both starch and soluble sugars can be mobilized overnight to provide
PEP (Black et al. 1996).
In plants that degrade starch, reserve carbohydrates must be converted into soluble intermedi-
ates before they can reach the cytosol. Chloroplasts of M. crystallinum export dihydroxyacetone
phosphate, 3-phosphoglycerate, glucose-6-phosphate (Glc-6-P), glucose, and maltose (Hausler et al.
2000). The transport of Glc-6-P, which is selectively induced in M. crystallinum by salt stress, is
reduced by up to 70% during illumination (Neuhaus and Schulte 1996; Kore-eda and Kanai 1997).
This is consistent with the progress of activities of cytosolic glycolytic enzymes, such as glyceralde-
hyde-3-phosphate dehydrogenase, 3-fosfoglycerokinase, enolase, and phosphoglyceromutase during
the induction of CAM. A second group of CAM plants are those that use soluble carbohydrates as
a source of PEP, such as pineapple and some species of the genera Clusia and Aloë. Metabolized
carbohydrates are glucose, fructose, and sucrose (Black et al. 1996).
Information on possible differences in the degradative pathways in plants with different carbo-
hydrate reserves is scarce. There is a need for a thorough study on the regulation of glycolysis in
CAM, for instance, at the level of the interconversion of Fru-6-P to Fru-1,6-P2 and the coordinated
regulation of pyruvate kinase and PEPCase. As mentioned earlier, the effect on the induction of
CAM triose phosphate metabolism-related enzymes has been established, but no such effects have
been observed for other regulatory enzymes of glycolysis, such as the ATP-dependent (PFK) or
PPi-dependent (PFP) phosphofructokinases or pyruvate kinase. PFP levels in various CAM spe-
cies often exceed by a factor of 20 or more those of PFK (Trípodi and Podestá 1997). Suggestively,
studies in 45 species of Peperomia show that PFP levels are higher in species that perform “CAM
cycling” or CAM and that this trend is parallel to that of PEPCase, a typical enzyme of this metabo-
lism (Ting et al. 1996). In pineapple, contrary to most plants, PFP contains a single polypeptide
having immunological identity with the ß-subunit of the enzyme from potato, even when activa-
tion by Fru-2,6-P2 is maintained (Trípodi and Podestá 1997). Its glycolytic activity (though not the
gluconeogenic) and activation by Fru-2,6-P2 are strongly modulated by pH. These properties and
the increase in the intracellular night effector are consistent with the role of PFP in the glycolytic
breakdown of carbohydrates to PEP overnight (Trípodi and Podestá 1997).
In pineapple, the cytosolic activity of fructose bisphosphatase is greater than the chloroplastic,
whereas the opposite occurs in M. crystallinum. This difference appears to be related to the content
of Fru-2,6-P2, which inhibits the cytosolic isoenzyme but not to the chloroplastic. At the same time,
it was established that Fru-2,6-P2 levels are very low in plants that mainly degrade starch while
abundant in those that degrade extrachloroplastic carbohydrate (Fahrendorf et al. 1987; Chen et al.
2002a). Nevertheless, no rule can be established in this matter, since the variations found in other
plants are more complex.
Research into RuBisCO regulation in CAM plants has shown that the enzyme starts being acti-
vated early in phase II, reaching a maximum (more than 70%) during phase II. Maximal activity
is reached at the beginning of phase IV, which coincides with the restart of atmospheric CO2
uptake (Griffiths et al. 2002). These studies show that RuBisCO activity is limited when PEPCase
is active. The delay in the activation of RuBisCO seems to be due to a delayed accumulation of
RuBisCO activase, which reaches its higher levels near noon (Maxwell et al. 1999; Griffiths et al.
2002). It has been found that modification of the phases by drought or switching to a N2 atmosphere
(which disrupts normal metabolism) changes RuBisCO’s activation pattern to a more C3-like one
(Griffiths et al. 2002).
38.3 REGULATION
38.3.1 Transcriptional Regulation
In facultative species, CAM can be induced as a result of certain environmental stimuli such as
water stress, photoperiod length, or the application of growth regulators (Cushman and Bohnert
1999; Cushman and Borland 2002; Dodd et al. 2002; Lüttge 2004; Osmond et al. 2008). Most stud-
ies on the subject have been carried out in M. crystallinum, in which CAM can also be induced by
salt stress. The primary level of control is transcription. Transcripts can start accumulating a few
hours after the stimuli have been applied. The degree of transcriptional activation depends on the
nature of the stress and the developmental stage of the plant.
Genes involved in the response include enzymes from glycolysis, gluconeogenesis, c arboxylases,
and decarboxylases and those proteins involved in biocompatible solute synthesis, metabolite trans-
port, and signal transduction (Cushman and Bohnert 1999; Dodd et al. 2002; Winter et al. 2008).
Until now, some elements have been identified that regulate the expression of various genes in
response to light and the abscisic acid. For instance, the presence of silencers and activators of tran-
scription has been reported to exist in the promoter regions of the genes encoding the PEPCase and
glyceraldehyde-3-phosphate dehydrogenase (Schaeffer et al. 1995).
38.3.2 Posttranscriptional and Translational Regulation

Steady-state levels of transcripts depend on both the rate of transcription and the rate of degradation
of the mRNA. While the messenger for the PEPCase increases its stability against stress, transcripts
encoding the small subunit of RuBisCO decline, probably due to destabilization of the messenger,
since the rate of transcription is not changed. In the case of the enolase, there are increased levels
of protein while increasing both the rate of transcription of the gene and the transcript abundance.
This is most likely a case of translational regulation.
38.3.3 Posttranslational Regulation
There are various ways by which posttranslational regulation may be carried out: reversible phos-
phorylation mechanisms, presence of positive and negative effectors, protein degradation, and
reduction of sulfhydryl groups, among others. This option further increases the plasticity of CAM
plants, allowing them to survive against the most diverse environmental changes. Several cases of
posttranslational regulation have been described during the description of the biochemistry of CAM
plants (e.g., regulation of PEPCase by phosphorylation).
38.3.4 Tonoplast
The vacuole plays a dynamic role in the regulation of carbon metabolism enabling photosynthetic
assimilation of CO2 in CAM plants. It not only maintains ion homeostasis and cellular pH, but
it also has the ability to store secondary metabolites and cytotoxic compounds and to sequester
hydrolases and proteases. In strict correlation to the importance of their function in plants CAM, the
vacuole can cover slightly more than 95% of the cell volume, whereas the cytoplasm and cell wall
occupy 1% and 2%, respectively.
Temperature affects the CAM function, so that there is no endogenous rhythm at tempera-
tures outside the range of 8°C–29°C, although some plants can acclimate to recover rhythmicity
after severe thermal extremes. The crystalline state of the tonoplast is decisive in this process. At
high temperatures, the disordered structure allows tonoplast acid efflux, while lower temperatures
promote a high state of arrangement that prevents the exit of malate to the cytosol. The effect of
acclimation is increase or decrease, as appropriate, the threshold temperature at which the change
of state occurs in the crystalline structure of the tonoplast. Molecular modeling studies have sug-
gested that the surface of the membrane lipids varies due to conformational changes suffered by
some proteins (Luttge 2000). A rise in temperature causes no apparent changes in the membrane
until reaching a critical value (between 26°C and 34°C), which promotes a phase transition from an
ordered state to a disordered one altering membrane permeability.
38.4 EVOLUTION AND TAXONOMIC DISTRIBUTION

The oldest fossil records belong to members of the family Agavaceae (15 million years) and the
genus Opuntia (40,000 years). CAM has most likely evolved from C3 photosynthesis, and in a
polyphyletic way, that is, it has arisen independently several times in different families and even
within the same family. CAM is present in 33 families of vascular plants, both between monocots
(Agavaceae, Asphodelaceae, Bromeliaceae, and Orchidaceae) and between eudicots (Aizoaceae,
Asclepiadaceae, Cactaceae, Crassulaceae, and Euphorbiaceae). There are aquatic plants with CAM
(Isoetes). Including the family Orchidaceae, CAM plants constitute 6% of the vascular plants known
to date, which is approximately 16,000 species.
38.5 PRODUCTIVITY AND USE OF CAM PLANTS AS CROPS

The productivity index can be analyzed as the interplay of several factors, of which the most impor-
tant are night temperature, water availability, and light intensity received. Among CAM species,
agaves and cacti are the most productive crops. In Mexico, annual biomass production of agave, used
in the manufacture of tequila, can range between 26 and 30 metric tons per hectare (Mg ha−1 year−1).
Given that the yield of C3 and C4 crops is about 40 and 50 Mg ha−1 year−1, respectively, the values
found for CAM plants are considerably high. Aside of its use in the production of tequila, agaves are
cultivated for its fiber, with a total production of 45 × 104 Mg in 2011 (FAO 2013).
In the case of pineapple, values of 35 Mg ha−1 are within the usual range of performance. In
more favorable environmental conditions, these figures may be even higher. Pineapple is the biggest
CAM crop, second to bananas in the ranking of tropical fruits. World production in 2011 was close
to 17 × 106 Mg, with most of the production located in Central and South America (Brazil, Costa
Rica, and México) and Asia (Thailand, Philippines, and China) (FAO 2013).
Prickly pear (Opuntia ficus-indica), along with maize, played an important role in the agricultural
economy of Central America in pre-Columbian times (AnayaPérez 2001). Today, it is a widely culti-
vated crop in arid and semiarid regions, serving as a food complement for cattle and goats. These gener-
ally small plantations also allow the use of fresh fruit for human consumption or small-scale farming.
The area covered by Opuntia plantations worldwide is close to 9 × 105 ha (Reynolds and Arias 2001).
Cladodes also serve as fodder when drought causes grasses availability to decline, although spines must
be burned by torching to allow access of cattle to the food. O. ficus-indica productivity, nutritional
requirements, and ecophysiology have been widely studied, which makes this plant a good model to
predict the use of arid habitat-tolerant species (Nobel 2001). Predictions of maximal productivity and
field assays reveal a production of up to 50 Mg ha−1 year −1. This value, however, is obtained under
optimal cultivation conditions, that is, artificial irrigation and nutrient supply (N and P) (Nobel 2001).
Opuntia is also a salt-sensitive plant, so salty soils can drastically reduce growth. Phosphate
requirements for optimal growth are low, but the P content of the edible tissue is also low when con-
sidering cattle nutritional needs. N content is also low. A rise in atmospheric CO2 could potentially
benefit Opuntia in terms of biomass accumulation, but experimental work also shows that when
ambient CO2 increases, the N content of the plant decreases (Nobel 2001). So, in terms of its use as
forage, Opuntia should be considered as a key ingredient to supplement the diet of domestic animals
because it provides water and is a good source of carbohydrates and fiber. However, a medium–low
protein content (of which 1%–2% is digestible) prescribes it from being a primary fodder.
In addition to its use as forage, O. ficus-indica produces highly valued fruits (prickly pear, tuna,
or higo chumbo in Spanish) in many Latin American countries but also in the United States, Israel,
Italy, and South Africa. Despite technical problems related to the spines that the fruit bears, in
many places this can be a profitable crop, even more than more traditional fruit (Mizrahi et al.
2010). Moreover, these authors report that in a selected semidesertic location in Israel, mango
(a C3 fruit tree) can be produced with an irrigation equivalent to 2400 mm year−1, while only 400 mm
are required for cactus in the same place (Mizrahi et al. 2010).
38.6 ECOPHYSIOLOGY
As mentioned earlier, in addition to water supply, factors such as the decline in the availability of CO2
in aquatic habitats due to poor diffusion or lack of solar radiation due to shadowing by the canopy in
the dense forest environments determined alternative paths for the development of CAM. In the genus
Isoetes, which contains species of aquatic habitat, the development of CAM allows CO2 fixation at
night, when other aquatic plants are respiring, using the liberated CO2 that would otherwise be lost.
Epiphytic bromeliads, like orchids and Tillandsia, which often set in the foliage of tropical trees, do
not always get the proper amount of sunlight in their leaves but have amazing adaptive mechanisms.
Its greater photosynthetic efficiency at low irradiances allows better use of the scarce sunlight that
reaches its leaf surface. Moreover, under high light intensities, they show greater resistance to pho-
todamage. This last feature is potentially beneficial in habitats in which usually several types of
stress converge.
At present, the atmospheric concentration of CO2 has doubled that of preindustrial times due to
the high rate of deforestation and the burning of fossil fuels. As a result, there has been an increase
in the photosynthetic capacity of C3 and C4 plants. In CAM plants, the phenomenon has been less
studied, although it has been possible to describe some changes at the morphological, anatomical,
and biochemical levels that would relate to the rise in the concentration of CO2. Among them are an
increase in biomass chlorenchyma (Opuntia and Agave), with a thicker and therefore greater amount
of photosynthetic tissue, less stomatal frequency that avoids excessive perspiration and development
of thicker epicuticular layers, bringing about an enhanced sunlight reflection (Nobel et al. 1996;
Drennan and Nobel 2000). It was also reported that doubling the atmospheric CO2 concentration
increases net CO2 uptake and that this reflects increases in CO2 fixation by both RuBisCO and
PEPCase (Drennan and Nobel 2000). This trait is the result of higher levels of activated RuBisCO
and lower K m for PEPCase, while protein levels for both enzymes and chlorophyll content are actu-
ally reduced (Drennan and Nobel 2000). In Cotyledon species (C. orbiculata and C. paniculata),
gradients in the content of chlorophyll and carotenoid in leaves offer this plants a higher capacity
to reflect sunlight, which would make them more resistant in case their habitats would turn more
irradiated (Barker et al. 1997).
Another feature that is beneficial in drought periods is the accumulation of organic acids, because
it acts as an osmotic, favoring soil water intake. Moreover, it can also protect the plant against pho-
todamage, since the release of large amounts of CO2 by decarboxylation avoids overreduction of the
photosynthetic electron chain transporters.
38.7 CONCLUSIONS AND PERSPECTIVES

The CAM syndrome has been found in leaves, stems, and roots of a variety of genus, as well as in
the most diverse habitats (in terrestrial, aquatic, epiphytes and tropical, subtropical, and temper-
ate plants), which gives account of the great phenotypic plasticity granted this metabolism. It is
important to consider CAM crop species in the context of the already more than incipient global
climate changes, probably involving the occurrence of prolonged drought in some regions and even
desertification of fertile zones. CAM plants provide crops with minimal nutritional requirements,
which can survive with little or no irrigation and a high inherent resistance to high temperatures
and radiation levels.
Currently, the economic importance of CAM plants pales in contrast to the traditional C3 or C4
crops, but some species have a high market value. In products for human consumption, pineapple
and Agave tequilana are the most important, followed by ornamentals, of which the most impor-
tant are the orchids. Many garden plants are CAM, including several of the genera Kalanchoë,
Aptenia, Crassula, and Mesembryanthemum (also used in the setting of dunes). However, as has
been described above, there are CAM species whose annual productivities are comparable to those
obtained for C3 and C4 crops. This encourages thinking about the possibility of using such crops as
biomass producers, and even as bioproducers of metabolites such as malic and citric acid, widely
used in the food industry. The cultivation of these species in arid, little usable land for other types
of farming could also help preserve and improve soil quality.
A still not much explored field is the study of CAM plants in phytoremediation. The existence
of members belonging to the families Crassulaceae, Euphorbiaceae, and Asteraceae that are able to
accumulate heavy metals (Ni, Cu, and Co) has been reported (Lu et al. 2008; Küpper et al. 2009).
Despite the large storage capacity of the tonoplast in CAM, there is still no information about the
role of these plants in hyperaccumulation processes or other detoxification of harmful compounds.
ACKNOWLEDGMENTS
The authors acknowledge support from CONICET (PIP 2519 to FEP and AAI), FONCyT (PICT
2011 01122 to FEP; PICT 2008 1754 to AAI and PICT 2011 1986 to CVP), and UNL (CAI+D Redes
and Orientado to AAI). All the authors are members of the investigator career from CONICET.
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39 Advances in Improving
Adaptation of Common
Bean and Brachiaria
Forage Grasses to Abiotic
Stresses in the Tropics
Idupulapati Madhusudana Rao
CONTENTS
39.1 Introduction........................................................................................................................... 847
39.2 Major Abiotic Constraints for Crop and Forage Production in the Tropics.......................... 849
39.2.1 Edaphic Constraints................................................................................................... 849
39.2.2 Climatic Constraints.................................................................................................. 851
39.3 Adaptation of Common Bean to Abiotic Constraints............................................................ 853
39.3.1 Low Phosphorus Availability in Soil......................................................................... 853
39.3.2 Soil Acidity and Aluminum Toxicity........................................................................ 854
39.3.3 Drought...................................................................................................................... 857
39.3.4 High Temperature...................................................................................................... 859
39.3.5 Multiple Stress Resistance......................................................................................... 861
39.4 Adaptation of Brachiaria Forage Grasses to Abiotic Constraints........................................ 863
39.4.1 Soil Acidity and Aluminum Toxicity........................................................................ 865
39.4.2 Low Phosphorus Availability in Soil......................................................................... 868
39.4.3 Drought...................................................................................................................... 869
39.4.4 Waterlogging.............................................................................................................. 870
39.4.5 Multiple Stress Resistance......................................................................................... 871
39.5 Conclusions and Future Perspectives.................................................................................... 872
References....................................................................................................................................... 875
39.1 INTRODUCTION
Agriculture occupies about 38% of the Earth’s terrestrial surface. Croplands cover 1.53 billion hect-
ares, while pastures cover another 3.38 billion hectares (Foley et al., 2011). Between 1985 and 2005,
the world’s croplands and pastures expanded by 154 million hectares with a net redistribution of
agricultural land toward the tropics, with implications for food production, food security, and the
environment. Over one billion people are hungry and living in poverty, and 75% of them live in
rural areas and depend on agriculture for their livelihoods, with the majority relying on small-scale
crop–livestock systems. To meet the food needs of the expected 9.2 billion people in 2050, agri-
cultural production has to increase by 70% compared to what it was in 2000. About 90% of this
847
expansion will be through production intensification (i.e., increase in output per unit area), and 10%
will be from area expansion mainly in sub-Saharan Africa and Latin America (FAO, 2010).
Plants are adapted to edaphic and climatic conditions. The term “adaptation” can be used to
define processes conferred by genetic attributes that serve to “fit” the plant to the ambient condi-
tions of temperature, light, and mineral and water availability. Adaptedness is the degree to which
an organism is able to live and reproduce in a given set of environments, the state of being adapted,
and adaptation is the process of becoming adapted or more adapted (Allard, 1988). For both crop
and wild species, adaptedness means matching the successive developmental stages to climatic and
edaphic resources and, where unfavorable extremes are unavoidable, minimizing their coincidence
with more vulnerable growth stages (Roberts et al., 1993). Adaptation implies changes in population
genetic pools in response to environmental changes.
Edaphic and climatic stress factors are the two sets of abiotic stress factors plants have to cope
with. These factors influence plant adaptation in different ways, but these factors are not totally
independent either of each other or in their effects on plants. This was illustrated by the ability
of plants to withstand high-temperature stress that is also partly regulated by the soil water avail-
ability (Burke, 1990; Mittler, 2006). Edaphic stress factors change slowly in the course of time but
sometimes very fast in space. On the other hand, climatic stress factors can change very fast in short
periods of time but usually much more slowly in space.
Adaptedness is both complexly inherited and much affected by the environment, and conse-
quently, the genetic and physiological mechanisms that have led to improvements in adaptedness
have been difficult to identify and to quantify. Understanding the mechanisms by which plants
adapt to changes in environmental conditions is critical for creating efficient strategies to develop
stress-resistant cultivars for sustainable intensification of production systems (Rao, 2001). Improved
adaptation of a crop to its environment can be achieved by two general approaches (Evans, 1993):
the growth environment may be altered or the plant genotype may be improved. Often, a combined
approach is the most effective.
Plant growth is closely related to the assimilation of carbon, the element’s partitioning into dif-
ferent plant structures, and its loss through respiration, all of which must be accompanied by water
and nutrient uptake. Assimilated carbon enters a pool of carbohydrates, and from there, it is used
either in respiration or in the growth of assimilatory and supportive structures. Partitioning of dry
matter into leaves has a positive feedback on plant productivity because of its effects on total leaf
area, but it inevitably increases demand for nutrients and water under conditions where too few
carbohydrates are available for root growth. These simultaneous parallel requirements need to be
balanced by the plants (Evans, 1997).
Abiotic stresses such as drought, heat, low soil fertility, soil acidity, or soil salinity could cause
extensive losses to global agricultural productivity and thereby impacting food security, particu-
larly in the developing countries. Resource-poor farmers are facing a range of challenges such as
variability in weather patterns induced by global climate change, soil acidity and low soil fertility
due to nutrient depletion, and combinations of different abiotic stress factors. It was shown that
the response of plants to a combination of two different abiotic stresses is unique and cannot be
directly extrapolated from the response of plants to each of the different stresses applied individu-
ally (Mittler, 2006). A comparison made by Mittler (2006) of all major US disasters between 1980
and 2004 indicated that a combination of drought and heat caused an excess of $120 billion in dam-
ages compared to $20 billion in damages caused by drought not accompanied by heat stress, over
the same period.
A change of focus is needed in plant-stress research in order to understand the nature of multiple
stress responses and to create avenues for developing plants that are resistant to multiple stresses yet
maintain high yields (Atkinson and Urwin, 2012). A combination of approaches is also needed to
significantly improve the abiotic stress tolerance of crops in the field (Mittler and Blumwald, 2010).
Transcriptome responses to combinations of stresses in Arabidopsis revealed that some 61% of the
transcriptome changes in response to double stresses were not predictable from the responses to
Advance of Common Bean and Brachiaria to Abiotic Stresses 849
single stress treatments (Rasmussen et al., 2013). Plants prioritized between potentially antagonistic
responses for only 5%–10% of the responding transcripts indicating that plants have evolved to
cope with combinations of stresses and, therefore, may be bred to endure them. Recent advances
indicated that specialized plant membrane transporters can be used to enhance yields of staple
crops, increase nutrient content, and increase resistance to key stresses, including phosphorus (P)
deficiency, aluminum (Al) toxicity, and soil salinity, which in turn can expand available arable land
(Schroeder et al., 2013).
Several authors reviewed research efforts on improving plant adaptation to different edaphic
and climatic stress factors (Blum, 1983; Evans, 1984; Ludlow and Muchow, 1990; Marschner, 1991;
Hall, 1992, 2004; Noble and Rogers, 1993; Ashraf, 1994, 2010; Loss and Siddique, 1994; Amzallag
and Lerner, 1995; Lynch and Beebe, 1995; Subbarao et al., 1995; Boyer, 1996; Sanderson et al.,
1997; Turner, 1997; Lambers et al., 1998; Joshi, 1999; Rao et al., 1999a; Miflin, 2000; Rao, 2001;
Chaves and Oliveira, 2004; Ishitani et al., 2004, 2011; Kochian et al., 2004; Lynch and St.Clair,
2004; Boote and Sinclair, 2006; Miklas et al., 2006; Mittler, 2006; Ainsworth and Ort, 2010; Beebe
et al., 2010, 2011, 2013a,b; Blum, 2010; Hirayama and Shinozaki, 2010; Horst et al., 2010; Mittler
and Blumwald, 2010; Ramaekers et al., 2010; Lynch, 2011; Richardson et al., 2011; Sinclair, 2011;
Atkinson and Urwin, 2012; Bailey-Serres et al., 2012; Beebe, 2012; Cossani and Reynolds, 2012;
George et al., 2012; Mir et al., 2012; Omae et al., 2012; Silva, 2012; Veneklaas et al., 2012; Tardieu,
2013; Yang et al., 2013). In general, many of these reviews discussed physiological processes rela-
tive to whole-plant stress tolerance.
The most successful approaches to improving crop and forage adaptation to abiotic stresses have
historically used field-based evaluations to identify tolerant cultivars, followed by breeding and
selection of genotypes that combine performance in stressful environments with other desirable
plant attributes (Rao, 2001; Humphreys, 2005). Progress in the improvement of crops and forages
depends on identifying plant attributes that contribute to tolerance to major abiotic constraints
and the development of rapid and reliable screening methods (Rao, 2001). An essential part of
germplasm improvement is to identify morphological, physiological, and biochemical mechanisms
by which plants adapt to abiotic stress conditions. Defining specific mechanisms of adaptation to
abiotic stress factors contributes to the development of high-throughput phenotyping protocols on
which the efficiency of genetic improvement programs depends. The phenotype of an organism
is fundamentally a manifestation of a genotype’s interaction with the environment (Cobb et al.,
2013). Identification of genes that control abiotic stress tolerance in food and feed crops requires
innovations that enable high-throughput and accurate measurements of shoot and root development
through time. Recently, Topp et al. (2013) demonstrated the ability of a semiautomated 3D in vivo
imaging and digital phenotyping pipeline to interrogate the quantitative genetic basis of root system
growth in a rice biparental mapping population (Bala x Azucena). Their imaging and analytical
platform provides a means to identify genes with high potential for improving root traits and agro-
nomic qualities of crops.
In this chapter, I have attempted to review the progress in improving common bean and
Brachiaria forage grasses for their adaptation to major abiotic stresses in the tropics. This chapter
reports advances for the past 12 years building on the research approach used and progress reported
by Rao (2001).
39.2 MAJOR ABIOTIC CONSTRAINTS FOR CROP

AND FORAGE PRODUCTION IN THE TROPICS
39.2.1 Edaphic Constraints
Edaphic constraints (mainly mineral deficiencies such as P and nitrogen [N] and toxicities such as Al
and manganese [Mn]) are probably the biggest single cause of a persistent yield gap between poten-
tial and realized crop and forage productivity, particularly in developing countries in the tropics
(Rao, 2001). General symptoms of crop plants due to mineral deficiencies or toxicities include poor
emergence; slow growth; seedling and adult plant stunting; leaf yellowing, chlorosis, and bronzing;
reduced overall growth and dry-matter production; delayed and prolonged flowering and maturity;
excessive flower and pod abortion; low harvest index (HI); reduced seed weight; deformed and
discolored seeds; and severe yield loss (Singh et al., 2003). Root growth, development, and distribu-
tion across the soil profile may also be adversely affected (Fawole et al., 1982; George et al., 2012).
Two major edaphic constraints in the tropics are low P availability and acid soil–induced
Al toxicity. P deficiency is widespread, covering an area estimated at over 2 billion hectares
(Fairhurst et al., 1999). P deficiency is considered as a major limiting factor for plant growth and
crop productivity, especially in the tropics and subtropics (Rao et al., 1999a; Vance et al., 2003).
Each year, millions of tons of phosphate are being mined from finite ground deposits, and based
on the growing demand for fertilizers to feed an increasing population, it has been estimated that
global P reserves will last for around 5–100 years or even only until 2050 (Cordell et al., 2009).
Compared to other major nutrients, P is by far the least mobile and least available to plants in most
soil conditions (Schachtman et al., 1998; Hinsinger, 2001). Most soils that have little P available
for the plant do contain considerable amounts of P, but a large proportion is bound to different soil
constituents, forming complexes of limited availability (Fairhust et al., 1999; Driessen et al., 2001).
These soils impose agronomic and economic constraints. Application of P fertilizer is common
practice and necessary if agricultural productivity is not to be seriously limited. Annual P appli-
cations are needed to sustain crop yields (Sanchez, 1976). In recent years, fertilizer prices have
risen substantially, making widespread adoption of fertilizer application at optimal levels more
difficult for resource-poor farmers, particularly in Africa (Sanginga and Woolmer 2009; Lunze
et al., 2011). Transport costs compound the cost of fertilizer inputs by as much as four times in
Africa. Thus, improving fertilizer use efficiency of crops and forages through genetic adaptation to
low-to-moderate levels of applied P is critical for smallholder agriculture (Lunze et al., 2011; Beebe
et al., 2013a). Improved cultivars with genetic adaptation to low-P soils may be a viable alternative
or complement to P fertilization, particularly for crop–livestock systems in the tropics (Rao, 2001).
Plants adapted to low P availability in soil activate a range of mechanisms that result either in
increased acquisition of P from the soil or in a more efficient use of the internal P (Rao et al., 1999a;
Vance et al., 2003; Lynch, 2011; Richardson et al., 2011; Veneklaas et al., 2012). This topic has been
reviewed by several researchers (Vance et al., 2003; Lambers et al., 2006; Lynch and Brown, 2006;
Lynch, 2007, 2011; Richardson et al., 2009; Ramaekers et al., 2010). P efficiency is defined as the
ability to grow and yield in soils with reduced P availability (Lynch, 2011). P acquisition efficiency
refers to the plant’s ability to acquire greater amounts of P per unit root length, while P use effi-
ciency refers to the plant’s ability to produce yield per unit of acquired P from soil. With a given
P supply in soil, P acquisition per plant might be improved in at least three ways: (1) with a root
system that provides greater contact with P; (2) a greater uptake per unit of root, due to enhanced
uptake mechanisms; and/or (3) an ability to use insoluble organic or inorganic P forms that are rela-
tively unavailable or poorly available to plants. Association with arbuscular mycorrhizae (AM) sig-
nificantly affects each of these attributes (Rao, 2001; Vance et al., 2003). Recently, Gamuyao et al.
(2012) showed that the protein kinase Pstol1 from traditional rice confers tolerance to P deficiency.
The factors that contribute to acid soil infertility and subsequent stunted plant growth in acid soils
are complex. In low-pH soils, it is not usually hydrogen ion toxicity that affects plant growth but
rather other toxicities, such as Al and manganese (Mn), and deficiencies of P, nitrogen (N), potas-
sium (K), calcium (Ca), magnesium (Mg), sulfur (S), zinc (Zn), and molybdenum (Mo) (Rao et al.,
1993; Rao and Cramer, 2003). Aluminum phytotoxicity is the primary limitation to agricultural
production on these acid soils (Rao et al., 1993; Shen et al., 2004). At soil pH values of 5 or below,
toxic forms of Al are solubilized into the soil solution (Foy, 1974). Excess level of toxic Al mainly
inhibits the root growth of plants (Delhaize and Ryan, 1995). Since crop growth largely depends
on the ability of roots to explore the soil and absorb water and nutrients, factors that restrict the
development of the root system will reduce crop and forage yields (Goldman et al. 1989; Rao, 2001;
Trachsel et al., 2010; Kell, 2011). Although acid soils can be made productive through application
of lime and other inputs such as P fertilizers, liming is not an economically realistic alternative in
many developing countries, particularly in the tropics and subtropics. This is because the high cost
is beyond the ability of low-input resource-poor farmers. Existing information clearly indicates that
long-term, sustainable crop and forage production on acid soils requires both Al-resistant cultivars
and appropriate agronomic practices.
The primary and earliest symptom of Al toxicity is a rapid (within minutes) inhibition of root
elongation (Ryan et al., 1993; Sivaguru and Horst, 1998; Kollmeier et al., 2000). The distal part of
the transition zone (TZ) in the root apex was identified as the primary site of action of toxic Al ions
(Sivaguru and Horst, 1998). Research conducted over the past two decades on the physiology, genet-
ics, and molecular biology of plant Al resistance and toxicity has shown that Al resistance can be
achieved by mechanisms that exclude Al from the root apex (Al exclusion) and/or by mechanisms
that enable plants to tolerate Al in the symplasm (Al tolerance) (Barcelo and Poschenreider, 2002;
Garvin and Carver, 2003; Horst et al., 2010; Kochian et al., 2004; Vitorello et al., 2005).
39.2.2 Climatic Constraints
It is widely recognized that some of the most important impacts of global climate change will be
felt among “subsistence” or “smallholder” farmers in developing countries (Morton, 2007). There
are around 500 million smallholder farms in the world, and these farms provide up to 80% of the
food consumed in sub-Saharan Africa and Asia. An analysis based on statistical crop models and
climate projections for 2030 from 20 general circulation models suggested that South Asia and
Southern Africa will suffer negative impacts on several important food security crops in 12 food-
insecure regions (Lobell et al., 2008). Developing countries will experience unprecedented heat
stress because of global climate change (Battisti and Naylor 2009; IPCC, 2012). In Africa alone,
climate change is expected to impact on 75–250 million more people to increased water stress by
2020. An additional 40–170 million more people will be undernourished as a direct consequence of
climate change (Evans, 2009). Fifteen major farming systems affected by drought stress and pov-
erty, especially in South Asia, the Sahel, and Eastern and Southern Africa, were identified as targets
for international development (Hyman et al., 2008). From a crop improvement perspective, the
physiological responses that allow plants to be most productive in these environments that will be
affected by climate change and variability must be better defined, and the genetic factors that con-
trol these responses must be discovered and introgressed into modern varieties (Yadav et al., 2011).
Climate change is multifaceted, yet the current constraints on time, space, and money mean
that too often, experiments are limited in scope and not validated in field settings (Gleadow et al.,
2013). The strategies for devising solutions to address climate change will vary across geographical
regions as the types and magnitudes of the problems will vary (Mba et al., 2012). Although rainfall
is expected to increase globally overall, some places will actually be receiving less annual rainfall,
while the seasonality of rains and hence the timing of the cultivation of crops will also change.
The frequencies of occurrence and durations of the extreme weather events are also expected to
increase. The greatest demand for yield increases as population continues to increase will be in the
developing countries of the world (Tester and Langridge, 2010). Thus, success in producing more
food under worsening climatic conditions and with a severely constrained natural resource base
depends on enhanced physiological and agronomic efficiencies contributing more yield per unit of
input (Keating et al., 2013). Developing climate-smart and low soil fertility-adapted crop and forage
genotypes that are capable of producing “more with less” will be critically important to develop
eco-efficient agricultural systems for the poor. Cultivation of high-yielding, well-adapted, input
use-efficient, and climate-resilient crop and forage varieties should be a critical component of the
innovative interventions to attain global food security and environmental sustainability.
Drought is defined as a limited moisture supply that causes a reduction in plant production
(Blum, 2010). It results from restricted water supply and high evaporation rate. Plant water deficit
(drought) may occur as a consequence of a decline in soil water availability developing during
the crop-growing season or may result from intermittent drought spells. The timing, intensity, and
duration of stress are critical to determine the effects and impact of drought on crop and forage
productivity and quality. Mechanisms by which plants resist drought include escape, dehydration
avoidance, and desiccation tolerance. Tolerance to severe drought can be critical in natural dryland
ecosystems but has little relevance to increasing or stabilizing crop and forage productivity. Thus,
increasing crop and forage yield in drought-prone areas requires the optimization of the physiologi-
cal processes involved in plant response to soil dehydration.
Dehydration avoidance is defined as the plant’s ability to maintain its water status under condi-
tions of soil water deficits, which can be achieved through either increased water uptake by roots
or reduced transpiration through stomatal control. Desiccation tolerance and water use efficiency
(WUE) are likely to be very different from escape or avoidance mechanisms (e.g., large root sys-
tem) and thus require quite different strategies to elucidate and utilize them. Similarly, induced
mechanisms must be treated differently from constitutive mechanisms. WUE is the ratio of total
dry matter to evapotranspiration and other losses from the system of water that is not transpired
through the plant. An increase in transpiration efficiency (TE) or a reduction in soil evaporation
will increase WUE (Blum, 2010). But TE is only a concern under water-limited conditions, and
in that, the real challenge is to increase the effective use of water for transpiration when water
is limiting (Blum, 2009). Recently, Sinclair (2012) concluded that TE is not a variable that can
be easily resolved for use in many breeding programs. He suggested that component traits con-
tributing to TE need to be studied to increase the effective use of available water through the
growing season to ultimately maximize growth and yield of the crop. High TE (defined as the
ratio of accumulated plant mass to water transpired) is obtained by partial stomatal closure, and
consequently by a decrease of transpiration, that often leads to lower crop and forage yields.
Developments in infrared thermography could provide new and feasible screening methods for
detecting genetic variation in the stomatal response to water deficit in controlled environments
and in the field (Munns et al., 2010).
According to the Intergovernmental Panel on Climate Change (IPCC) A1B scenario, contin-
ued emissions of greenhouse gases (GHG) will increase annual temperatures by 2.5°C–4.3°C in
important crop-growing regions of the world by 2080–2099 (Christensen et al., 2007). The most
robust feature of global warming in agricultural areas will continue to be temperature increases
(Lobell and Gourdji, 2012). Many countries have average temperatures that currently exceed the
optimal temperature for crop production. This highlights the importance of adapting crops to a
warmer environment. Cossani and Reynolds (2012) described the physiological processes and
traits for which there is evidence that genetic enhancement could improve adaptation of wheat
to heat stress. They showed that cooler canopy temperature has some common genetic basis
under both heat and drought stress, and it is strongly associated with grain yield under both
environments. Ruiz-Vera et al. (2013) tested the interaction between high temperature (+3.5°C)
and elevated CO2 (approximately 585 μmol mol−1) on soybean under field conditions and showed
that global warming can negate the expected CO2 stimulation in photosynthesis and productivity
for soybean grown in the Midwestern United States. This study also suggested that the potential
is limited for elevated CO2 to mitigate the influence of rising temperatures on photosynthesis,
growth, and yields of C3 crops.
Higher than optimal temperatures during reproductive stages of a crop could lead to signif-
icant impacts beyond shortening the duration of grain filling (Ainsworth and Ort, 2010). High-
temperature stress that affects any of the reproductive processes (e.g., pollen viability, female
gametogenesis, pollen–pistil interaction, fertilization, grain formation, grain filling) can severely
reduce yield even when the seasonal average temperature is within a favorable range. This makes
reproductive growth stage as the most critical in determining the response of crop yield to high
temperatures. Small increases in temperature above the optimum can very negatively affect pollen
viability via a range of mechanisms that will lead to yield penalties (Hedhly et al., 2008).
39.3 ADAPTATION OF COMMON BEAN TO ABIOTIC CONSTRAINTS

Common bean (Phaseolus vulgaris L.) is the most important food legume for human consumption,
and it is an important source of protein (∼22%), vitamins (folate), and minerals (Ca, Cu, Fe, Mg, Mn,
and Zn) for human diets especially in developing countries in the tropics (Broughton et al., 2003;
Beebe, 2012). Annual production, including both dry and snap beans, exceeds 21 million metric
tons (Miklas et al., 2006). It is now grown extensively in all major continental areas in diverse
environments and agricultural conditions (Chacón et al., 2005). Its production spans from 52°N to
32°S latitude and from near sea level to elevations of more than 3000 masl (meters above sea level)
(Schoonhoven and Voysest, 1991). It has two major gene pools, the Andean and the Mesoamerican,
based on their centers of origin in South and Central America, respectively (Gepts and Debouck,
1991; Blair et al., 2006). Within these two gene pools, there are a total of six races, including three
Mesoamerican (Mesoamerica, Durango, and Jalisco) and three Andean (Peru, Nueva Granada, and
Chile) (Singh et al., 1991a,b). An additional Mesoamerican race has been designated Guatemala,
which includes certain climbing beans from Central America (Beebe et al., 2000).
A majority of bean production occurs under low-input agriculture on small-scale farms in devel-
oping countries particularly in Latin America and Africa (Beebe, 2012). Research to genetically
enhance common bean is complicated by the diversity of edaphic and climatic conditions under
which the crop is grown, compounded by highly specific local preferences for particular grain types
or colors. However, great progress has been achieved in developing genotypes resistant to several
biotic and abiotic constraints (Schoonhoven and Voysest, 1991; Rao, 2001; Beebe, 2012). While the
success in improving genetic adaptation to major abiotic constraints has been substantial, progress
in improving yield potential has been limited indeed. Different perspectives on the reasons why
are given in previous reviews (Singh, 1992; Graham and Ranalli, 1997; Singh, 2001; Beaver and
Osorno, 2009; Beebe, 2012).
In most tropical countries, abiotic stress factors limit on-farm yields and cause national average
values below 1 Mg ha−1. Edaphic constraints are the most widespread limitations and, in contrast to
drought, are present every year (Thung and Rao, 1999; Beebe, 2012). Among the edaphic stresses,
P deficiency is the primary constraint to common bean production in the tropics and subtropics,
limiting seed yield on at least 60% of the bean-producing areas of Latin America and Africa (CIAT,
1992; Wortmann et al., 1998). Other edaphic constraints include low levels of N and toxicities of
Al and Mn associated with acid soil together with low Ca availability (Rao, 2001). About 40% of
the bean-growing area is affected by Al toxicity, resulting in decrease of grain yield between 30%
and 60% (Thung and Rao, 1999; Rao, 2001). About 60% of common beans produced worldwide
are grown in regions subjected to water stress, making drought the second largest contributor to
yield reduction after the incidence of diseases (White and Singh, 1991a; Thung and Rao, 1999). The
crop needs significant improvement in abiotic stress resistance to reduce the dependence of small
farmers on purchased inputs. Drought severely limits yields in highland Mexico and Northeast
Brazil while occasionally causing major yield losses in other traditional bean-production regions
of Central America and Eastern and Southern Africa (Wortmann et al., 1998; Beebe, 2012). Heat
stress limits the cultivation of beans in tropical lowlands of Central and South America and Africa
(Beebe et al., 2011).
39.3.1 Low Phosphorus Availability in Soil

More than 7000 bean germplasm accessions were evaluated at Centro Internacional de Agricultura
Tropical (CIAT) for soil constraints, especially low P availability. Lynch and Beebe (1995) hypoth-
esized that the existing genetic variation for P efficiency in bean germplasm, especially variation
that is agronomically useful, is largely due to variation in P acquisition efficiency rather than P
use efficiency. Relationship between geographic origin and response to low P supply in soil in a
selection of 364 genotypes indicated highly significant variation in P efficiency among genotypes
in all growth habits (Beebe et al., 1997). It was also found that wild beans usually performed rela-
tively poorly, indicating that P-efficiency traits in common bean have been acquired during or after
domestication. Studies on plant nutrition indicated that plant adaptation to low-P soils is not specific
to soil type in common bean and that results derived on one soil type may be extrapolated to other
soils (Yan et al., 1995a,b). Mesoamerican and Andean germplasm responded differently to P avail-
ability in soil. Mesoamerican types were more responsive to added P in terms of seed yield.
Long-term research efforts were made to define root phenes (traits) and their role for enhanced
soil exploration and P acquisition (Lynch, 2011). The extent of topsoil foraging was identified
as an important P acquisition mechanism (Bonser et al., 1996; Liao et al., 2001; Ho et al., 2005;
Lynch, 2007, 2011). Root phenes that were associated with topsoil foraging include shallower
growth angles of axial roots, enhanced adventitious rooting, a greater number of axial roots,
and greater dispersion of lateral roots (Lynch, 2011). In common bean, basal root whorl number
(BRWN) varies among genotypes from one to four, with each whorl typically generating four
basal roots. Uppermost whorls produce basal roots with shallower growth angle, while lower
whorls produce roots of progressively steeper angle. Greater BRWN could increase soil explo-
ration by increasing the vertical range of root deployment (Lynch, 2011). In addition to topsoil
foraging, reducing metabolic cost of soil exploration through root cortical aerenchyma formation
and root etiolation and increase in root hair length and density also contribute to greater P acqui-
sition from low-P soils (Lynch, 2011).
Using a mapping population of recombinant inbred lines (RILs), quantitative trait loci (QTL)
were identified that contribute to enhanced P acquisition. These include at least six for specific
root length and six for root length in the field (Beebe et al., 2006), six for traits associated with
root hairs (Yan et al., 2004), three for basal root growth angle (Liao et al., 2004), and with some
regions in common for different traits. One of the parents of this RIL population (G19833) is being
sequenced as a reference genome of the BeanCAP (http://www.beancap.org/). This can open up the
opportunities to define the genetic control of root structure in common bean (Beebe, 2012). Twelve
regions associated with adventitious root development were also identified using a second mapping
population of common bean (Ochoa et al., 2006). Recently, a comparative study using two common
bean genotypes with contrasting response to P deficiency (P-tolerant BAT 477 and P-sensitive DOR
364) indicated variations in the microRNA 399-mediated PvPHO2 regulation within the PvPHR1
transcription factor-signaling pathway (Ramírez et al., 2013). Results showed that higher PvPHO2,
resulting from less-efficient PvmiR399-mediated mRNA degradation, in DOR 364 would result
in increased PvPHO2-mediated degradation of P-responsive proteins, such as P transporter PHT1,
which would cause a decrease in P content and P use efficiency in P-sensitive DOR 364.
While increase in P acquisition through root phenes contributes to enhanced biomass produc-
tion, superior remobilization of photosynthate from vegetative structures to grain has also been
shown to contribute to yield under low-P conditions (Rao, 2001; Beebe et al., 2009; 2013a). One of
the small-seeded germplasm accessions, G21212, expresses this trait (Rao et al., 2001), and some
lines selected for drought resistance also yielded well under P-limited conditions due to superior
photosynthate remobilization capacity (Beebe et al., 2008, 2013a). These lines also showed lower
amounts of P in the grain compared with less-adapted lines. Improvements in P distribution within
the plant may be possible by increased remobilization from tissues that no longer need it (e.g.,
senescing leaves) and reduced partitioning of P to developing grains. These changes could prolong
and enhance the productive use of P in photosynthesis and have nutritional and environmental ben-
efits (Veneklaas et al., 2012).
39.3.2 Soil Acidity and Aluminum Toxicity

Soil acidity and the associated toxicities, especially Al and Mn, are serious problems in several bean-
producing regions of the tropics. In Latin America, Brazil has the greatest bean area cultivated on
acid soil with use of lime to correct for Al toxicity (Pereira de Oliveira and Thung, 1988). In Africa,
acid soil is found in the central region of the continent (Wortmann et al., 1998). Developing bean
genotypes tolerant to acid soil conditions is an ecologically friendly, energy-conserving, and eco-
nomical solution for resource-poor farmers in the tropics (Rangel et al., 2005). Genetic variation
exists for acid soil adaptation among common bean genotypes (Rao, 2001; Rao et al., 2004). These
genotypic differences in seed yield could be related to differences in resistance to Al and acquisition
and utilization of nutrients for transport of photoassimilates to developing seeds. Field screening
for Al resistance would seem to be the most desirable approach, because it best approximates the
intended cropping environment. In practice, however, reliable ranking of genotypes in the field has
been difficult. This is mainly because exchangeable Al levels may not be uniform and also environ-
mental factors could interact with soil Al to mask the expression of Al resistance. Thus, it is neces-
sary to combine field screening with greenhouse screening techniques based on physiological traits
of Al resistance (Butare et al., 2011, 2012).
Common bean is very sensitive to low pH (4.3) with large genotypic differences in proton sen-
sitivity (Rangel et al., 2005). Increasing the pH to 4.5, the Ca2+ concentration to 5 mM, and adding
0.5 mM KCl to the solution fully prevented proton toxicity in all cultivars and allowed to identify
differences in Al resistance among 28 cultivars. Genotypic differences were quantified based on
inhibition of root elongation at 20 μM Al during 36 h of treatment. Several researchers found sig-
nificant genotypic differences in Al resistance in common bean based on Al-inhibited root elonga-
tion in nutrient solution (Foy, 1988; Massot et al., 1999; Rangel et al., 2005, 2007; Manrique et al.,
2006; Blair et al., 2009; López-Marín et al., 2009). Plant resistance to Al stress is a key component
of an appropriate and effective integrated approach for farmers with low income in Africa and Latin
America. Along with diverse germplasm and an appropriate breeding program, a reliable screening
procedure for Al stress is one of the most important tools required to effectively develop Al-resistant
cultivars (Butare et al., 2011).
In greenhouse trials, Andean cultivar ICA Quimbaya (Quimbaya), among other Andean geno-
types, was superior in Al-toxic nutrient solution and in acid soil studies, while bred line VAX 1
is highly sensitive (Rangel et al., 2009; Butare et al., 2011). However, in the field ICA, Quimbaya is
highly sensitive, and VAX 1 is superior due to its abundant and shallow root system that permits it to
acquire nutrients, especially P, and to escape Al toxicity in lower soil strata. The contrast between
field and controlled conditions is not fully understood, but the field situation is clearly more complex
and must be considered in evaluations (Beebe et al., 2009).
Using hydroponic screening method, several Andean (G 19833, BRB 191, and G 5273) and
Mesoamerican (G 1261, MAR 1, DOR 714, FEB 190, G 11015, G 3513, A 774, and G 855) genotypes
were identified as Al resistant based on the relationship between the % inhibition of total root length
and the % increase of average root diameter per plant (Manrique et al., 2006). Some accessions of
Phaseolus coccineus (scarlet runner bean) were found to be more resistant to Al in solution than
common bean (CIAT, 2005; Butare et al., 2011). Screening of 30 germplasm accessions of scarlet
runner bean resulted in identification of three accessions (G 35341, G 35266, G 35025) with a high
level of Al resistance (CIAT, 2005).
The use of a combination of hydroponic and greenhouse soil-based evaluations could identify
acid soil-tolerant genotypes. A technique using plastic tubes packed with high Al saturation soil
was developed at CIAT to rank genotypes for Al-toxic acid soil tolerance based on differences in
root development and distribution (Butare et al., 2011). The technique allows evaluation of effects
of Al toxicity on plant growth (shoot and root development) in conditions similar to the field. The
methodology is reproducible since it employs a known acid soil with high Al saturation from the
target area. It permits characterizing the depth of the primary root penetration, extraction of roots
for quantification of root distribution across soil depth, and total root length in soil with a uniform
bulk density.
Differential genotypic response to Al stress contributes to identification of new sources of Al
resistance as well as improved understanding of mechanisms of Al resistance in common bean
(Rangel et al., 2005, 2007, 2009, 2010; Blair et al., 2009; Lopéz-Marín et al., 2009). Mechanisms
of Al resistance in common bean were defined using Al-resistant Quimbaya (Quimbaya) and
Al-sensitive VAX 1. The analysis of spatial growth profiles revealed that the initial inhibition of
root elongation by Al resulted from a generalized effect along the entire elongation zone (EZ)
(Rangel et al., 2007). Localized application of Al to specific zones of the root apex showed
that application of Al to the transition zone (TZ) resulted in root growth inhibition to the same
extent as if the whole root tip would have been treated with Al indicating that the TZ is the
most Al-sensitive apical root zone. Application of Al to the EZ also reduced root growth in
both Al-resistant and Al-sensitive genotypes, though to a lesser extent than when applied to the
TZ, indicating that not only the TZ but also the entire EZ need to be protected from Al injury
(Rangel et al., 2007).
The binding state and cellular distribution of Al in the root apices were monitored during the
initial inhibition of root elongation and subsequent recovery in Al-resistant genotype Quimbaya
(Rangel et al., 2009). Results showed that the root elongation rate was significantly negatively cor-
related with the free apoplastic and the stable-bound, not citrate-exchangeable cell wall Al rep-
resenting the most important Al fraction in the root apex (80%), but not with the symplastic and
the labile-bound, citrate-exchangeable cell wall Al. Main conclusion from this work is that the
induced and sustained Al resistance in the Al-resistant genotype Quimbaya is mediated by reducing
the stable-bound Al in the apoplast, thus allowing cell elongation and division to resume (Rangel
et al., 2010). Greater accumulation of Al in the symplastic fraction in genotype Quimbaya might be
explained by the greater cell volume and thus vacuoles typical for genotypes of Andean origin in
comparison to the Mesoamerican origin of VAX 1.
The kinetics of citrate exudation from root tips offered the most consistent explanation for
the response in root elongation and Al uptake of both Al-resistant and Al-sensitive genotypes of
common bean to Al treatment (Rangel et al., 2010). The Al-induced citrate exudation pattern is
characteristic for a Pattern II response, with a 4–5 h lag phase, which corresponded to the period
of maximum Al accumulation in the root apices and root growth inhibition. Initial Al-dependent
efflux of citrate from the root tips in both genotypes was not regulated by the internal citrate levels
in the roots but required the expression of an organic anion permease in the plasma membrane
(PM). Transient recovery from initial Al stress was found to be dependent on the capacity to utilize
internal citrate pools (Al-resistant Quimbaya) or enhanced citrate synthesis (Al-sensitive VAX 1)
that was associated with a reduced cytosolic turnover of citrate.
Resistance to Al in common bean was attributed to the release of citrate by the root apex (Rangel
et al. 2010), and the expression of a citrate transporter multidrug and toxin extrusion (MATE) family
protein gene was crucial for citrate exudation (Eticha et al. 2010). Although the MATE gene expres-
sion was a prerequisite for citrate exudation and Al resistance, genotypic difference in Al resistance
in common bean was mainly dependent on the capacity to sustain the synthesis of citrate for main-
taining the cytosolic citrate pool that enables exudation (Eticha et al., 2010; Rangel et al., 2010). The
initial Al-induced inhibition of root elongation in both Al-resistant (Quimbaya) and Al-sensitive
(VAX 1) genotypes was correlated with the expression of the 1-aminocyclopropane-1-carboxylic
acid oxidase (ACCO) gene (Eticha et al., 2010).
Higher levels of Al resistance were sought in P. coccineus in a field screening and were subse-
quently confirmed in Al-toxic nutrient solution and in acid soil (Butare et al., 2011). A selection
G35346-3Q expressed excellent root development in all three evaluations and appeared to offer a
resistance mechanism that could be selected readily in any of these systems. However, when crossed
to SER 16, a drought-resistant and Al-sensitive genotype, only very few progenies behaved similarly
to G35346-3Q, while most expressed one or another trait of the drought-resistant parent (Butare
et al., 2012). Resistance was apparently complex and the result of multiple traits that segregated
among the progenies. This is probably indicative that multiple traits are required to confront an
acid soil complex, of which Al resistance is one component. While resistance sources for individual
stresses may be employed in breeding, it may be necessary to combine these multiple traits and
subject populations to relevant selection pressure in soil.
39.3.3 Drought
Increased adaptation of common bean genotypes to soil water deficits would contribute to both
stability and expansion of production in drought-prone environments such as Northeast Brazil and
the central highlands of Mexico. Bean cultivars adapted to drought would require less water for
irrigation and would therefore contribute to the conservation of an important natural resource. The
effects of drought on common bean are dependent on the intensity, type, and duration of the stress
(White and Izquierdo, 1991; Terán and Singh, 2002a,b; Muñoz-Perea et al., 2006; Beebe et al.,
2010). Development of drought-adapted bean varieties is an important strategy to minimize crop
failure and improve food security in bean-growing regions in the face of climate change (Beebe
et al., 2011).
Primary selection criterion in improving drought resistance in common bean has been yield
under stress, with or without reference to yield under nonstressed conditions (Singh, 1992). This
approach has contributed to significant progress, but a broader understanding of the physiology of
drought response is considered as key for identifying useful selection criteria beyond yield per se
(Beebe, 2012). The optimal plant response to deal with drought stress will differ depending upon
the type or pattern of drought, which is especially variable in the tropics due to variation in rainfall
patterns within shorter distances of a few kilometers. Four patterns of drought have been defined:
late initiation of rains, early cessation of rains or terminal drought, intermittent drought, or low
rainfall throughout the season (Beebe et al., 2013b). Matching plant traits to environments marked
by a given pattern of drought, or to environments with patterns that can vary from year to year, is
a significant challenge.
Common bean has evolved several mechanisms to maintain plant water status within reason-
able limits for normal metabolic functioning under drought stress (Beebe et al., 2013b). Significant
research efforts were made in improving common bean adaptation to drought for the past three
decades (White and Singh, 1991a,b; Singh, 1995; Schneider et al., 1997a,b; Ramirez-Vallejo
and Kelly, 1998; Rao, 2001; Teran and Singh, 2002a,b; Hall, 2004; Beebe et al., 2008, 2013b;
Beebe, 2012). These involve studying the effects of drought stress on plant growth, development,
and seed yield; evaluating physiological traits related to underlying mechanisms of adaptation to
drought; developing field screening methods; evaluating and identifying sources of drought toler-
ance in germplasm; and genetic improvement through intraspecific crosses (Beebe et al., 2013b).
Moderate to severe drought stress in common bean is known to reduce canopy biomass (CB) and
seed yield, HI, number of pods and seeds, seed weight, and days to maturity (Nielsen and Nelson,
1998; Ramirez-Vallejo and Kelly, 1998; Teran and Singh 2002a; Nunez-Barrios et al., 2005; Beebe
et al., 2008; Habibi, 2011; Rao et al., 2013a). Earliness, deep rooting, and greater ability to partition
photosynthates to grain have been identified as key mechanisms contributing to improved drought
resistance in common bean (Sponchiado et al., 1989; White and Singh, 1991a; Rao, 2011; Rao et al.,
2013a; Klaedtke et al., 2012; Beebe et al., 2013a,b).
Smallholder farmers are seeking for early maturing varieties that not only avoid drought but
also can enter the market sooner when grain prices can be high. But early maturity can reduce
yield; each day of reduction in growth cycle can result in a loss of 74 kg ha−1 (White and Singh,
1991b). However, recent breeding efforts for improving drought resistance resulted in small-seeded
Mesoamerican lines (coded as SER and SEN) up to 36% greater yield d−1 in favorable environments
(Beebe et al., 2008). Some of these lines were found to be superior in WUE under greenhouse or
field conditions (Ramirez Builes et al., 2011; Beebe et al., 2013a).
Deep rooting in response to soil drying can enhance access to soil moisture and contributes to
drought tolerance when soil profile can hold greater levels of soil moisture in deeper soil layers
(Sponchiado et al., 1989; Beebe et al., 2013b). Drought-resistant bean genotypes could extend
their roots to 1.2 m depth in drought environments, whereas the sensitive genotypes could not
extend their roots beyond 0.8 m. Common bean root systems also show considerable architec-
tural variation among species, among genotypes of given species, and even with different parts
of a single root system (Lynch and Beebe, 1995; Lynch, 2011). BAT 477 was identified as a
promising source of the deep rooting trait (Sponchiado et al., 1989), and recent efforts to evaluate
variation under well-watered or drought stress conditions for root traits among parents and RIL
population from BAT 477 crossed with a small red-seeded genotype (DOR 364) indicated that
QTL for rooting depth measured in a greenhouse screening technique with soil cylinders did not
correspond to QTL for yield under field conditions (Rao et al., 2006a; Polania et al., 2008a,b;
Blair et al., 2011; Asfaw and Blair, 2012).
Photosynthate remobilization to grain could be particularly important during terminal drought
to permit grain fill as stress becomes increasingly acute at the end of the season. It is a trait with
wide utility under multiple patterns of drought and even under other types of stress and optimal con-
ditions and in conditions of low availability of soil P (Beebe et al., 2008, 2013a,b; Beebe, 2012). This
mechanism can contribute to improved drought resistance in shallower soils of hillside agroecosys-
tem where the possibility for deep rooting is limited. Studies on multienvironment QTL analysis for
photosynthate remobilization traits in common bean under drought stress using RIL population of
DOR 364 and BAT 477 indicated that QTL for grain yield and photosynthate remobilization traits
did not overlap with any of the rooting depth or rooting pattern QTL detected for this population
(Asfaw and Blair, 2012; Asfaw et al., 2012). Genotypes that could combine the three traits (earliness,
deep rooting, and photosynthate mobilization) could be more resilient to smallholder farm condi-
tions to minimize risk from climate change and low soil fertility.
Combining races Durango and Mesoamerica has been a consistent source of improved drought
resistance in common bean for lowland tropical environments (Terán and Singh, 2002b; Frahm
et al., 2004; Ishitani et al., 2004; Beebe et al., 2008). In highland environments of Mexico, cv. Pinto
Villa performed better (Acosta-Gallegos and White, 1995). Beebe et al. (2008) found that selection
for drought resistance improves yield potential and plant efficiency across different environments
(e.g., nonstress, low P stress) and suggested that selection under drought stress reveals genes that
correct inefficiencies (excessive vegetative growth) inherited from the wild P. vulgaris and that are
key to yield improvement of common bean.
Different domesticated species within the Phaseolus genus express contrasting degrees of HI.
For example, interspecific lines derived from crosses between common bean and species from its
secondary gene pool (Phaseolus dumosus, P. coccineus) tend to have excessive vegetative growth
and low grain yield, while drought-adapted common bean lines produce lower shoot biomass but
high grain yield (Beebe, 2012). This was attributed to greater mobilization of photosynthates to
grain development, concluding that differences in biomass partitioning to grain play a key role in
improving drought stress of common bean (Beebe et al., 2008; Klaedtke et al., 2012). Tepary bean
(Phaseolus acutifolius A. Gray) originated in the deserts of Mexico and Southwestern United States
and is well adapted to drought stress. Drought tolerance mechanisms in tepary include deep rooting
to avoid dehydration, small leaves for reduced water use, and stomatal control but not with osmotic
adjustment (Mohamed et al., 2005; Beebe et al., 2013b). Greenhouse evaluation under drought stress
using soil cylinders revealed that it has fine, profusely branched roots that access limited water
reserves rapidly (Butare et al., 2011). Tepary bean has much less tendency to excessive vegetative
growth than common bean, but there is limited comparative analysis of biomass partitioning differ-
ences between these two species under drought stress (Rao et al., 2013a). But quantifying genotypic
differences in HI in common bean has been difficult due to leaf fall during grain filling. Developing
methodology to dissect HI into successive components to track the photosynthate mobilization from
vegetative organs to pod and grain could help the breeders to identify genotypes that are better
adapted to drought stress (Rao et al., 2009; Beebe et al., 2013b; Rao et al., 2013a).
Attempts were made to dissect HI into three component traits including pod partitioning index
(PPI), stem biomass reduction (SBR), and pod harvest index (PHI) (Rao et al., 2009; Beebe et al.,
2013b; Rao et al., 2013a). Genotypic differences in these three traits or component shoot phenes
could indicate the extent of photosynthate mobilization as assessed by sink strength (PPI), stem
reserve mobilization (SBR), and grain filling (PHI). PPI is measured as the value of pod biomass
at harvest relative to CB value at mid-pod filling growth stage that is assumed to be the time that
reflects the maximum vigor of the genotype. This measurement is useful to assess the genotypic dif-
ferences in sink strength. The measure of SBR at harvest relative to stem biomass at mid-pod filling
indicates the extent of stem reserve mobilization to grain filling. The measure of PHI (measured at
harvest) indicates the extent of photosynthate mobilization from pod wall to seed. Pod wall is the
source of carbon for the developing seed (Turner et al., 2005). These same traits could also contrib-
ute to greater yield potential under optimum (nonstress) conditions (Beebe et al., 2008). Thus, an
accelerated photosynthate (products of photosynthesis) partitioning toward the reproductive struc-
tures could be an important mechanism for improved grain yield under both drought stress and
nonstress conditions (Rao, 2001; Rosales-Serna et al., 2004; Rao et al., 2006, 2009, 2013a; Beebe
et al., 2008). Genotypes that combine these three plant attributes with greater values of CB or shoot
vigor should outperform the other genotypes under drought stress conditions.
In common bean, maintaining remobilization of photosynthate to grain under stress for better
HI has been identified as an important drought resistance mechanism (Rao et al., 2000; Rosales-
Serna et al., 2004; Rao et al., 2007a, 2009, 2013a; Beebe et al., 2008; Assefa, 2010; Beebe et al.,
2013). SEA 15, a line derived from race Durango parentage (Beebe et al., 2008), displays this
mechanism, as does G21212, a landrace of race Mesoamerica (Rao et al., 2000). Both PPI and
PHI correlated with yield under drought and frequently under favorable conditions, although PHI
presented the most consistent correlations (Rao et al., 2007, 2009). Field evaluation of 121 RILs of
the cross MD 23–24 x SEA 5 over three seasons of intermittent drought stress resulted in identifica-
tion of two lines (MR 81, MR 25) that were superior in their yield under stress, and their superior
performance was associated with higher values of CB, PHI, HI, and seed number per area (Polania
et al., 2008c). Field evaluation of 78 advanced inbred lines of another cross ICA Bunsi x SXB 405
under drought stress and irrigated conditions in two seasons at Melkassa, Ethiopia, resulted in iden-
tification of two genotypes (G87, G80) that performed better than a standard check (Awash Melka)
(Assefa et al., 2013). The values of PHI were reduced in drought-sensitive genotypes and increased
in drought-resistant genotypes indicating the importance of remobilization of photosynthates from
pod wall to seed. Significant correlations of PHI with yield and fair heritability in drought indicated
that PHI would be an effective selection criterion for identifying genotypes with improved drought
resistance. Results from this study also indicated that correlated gain in drought yield from selec-
tion for PHI would be greater than direct yield selection, due to much better heritability of PHI, and
would also contribute to irrigated yield.
For the past few years, we conducted several field trials for testing the hypothesis that the supe-
rior level of drought resistance observed in some genotypes of common bean and tepary bean could
be associated with greater mobilization of photosynthates from vegetative structures (leaves and
stems) to developing pods and from pod walls to grain (Rao et al., 2009; Klaedtke et al., 2012;
Assefa et al., 2013; Beebe et al., 2013a,b; Rao et al., 2013a). We found that CB, PPI, SBR, and PHI
are four important traits to improve the efficiency of breeding programs to select superior genotypes
of common bean under drought stress. Because of the simplicity in measurement, PHI should be
particularly useful as a selection criterion (Assefa et al., 2013) for improving both yield potential and
drought resistance if the breeder combines it with visual evaluations of shoot vigor and pod load.
Higher values of CB at mid-pod filling under drought stress could be related to greater root vigor
and deeper rooting ability that facilitates canopy cooling and moderate crop growth rate. Further
research work is needed to test these relationships under different type (intermittent or terminal),
intensity (moderate or severe), and duration of drought stress.
39.3.4 High Temperature
Beans are grown over a wide range of latitudes with mean air temperature of 14°C–35°C. The
Andean gene pool typically adapts best at mid-high altitudes (1400–2800 masl) or cooler climates,
with race Nueva Granada more heat tolerant than race Peru. The Mesoamerican gene pool adapts
to higher temperatures at low (400 masl) to mid-high altitudes (2000 masl), and Mesoamerican race
Durango adapts to the semiarid highlands of Mexico. Given its mid-to-high altitude origin, bean is
sensitive to high temperatures (Porch and Jahn, 2001). High temperatures can aggravate terminal
drought stress in lowland environments (Graham and Ranalli, 1997). Breeders have focused on
improving heat tolerance to expand bean production in the lowland tropics of Central America and
the Caribbean and made significant progress in developing common bean cultivars with improved
levels of heat tolerance (Rosas et al., 2000, 2003).
Reproductive growth in beans is initiated by flowering and a period of pod and seed set, and the
number of pods or seeds that a bean crop community produces is determined during this period,
and this number determines, in large part, the final grain yield (Hall, 2004). Several researchers
have examined the effects of genotype and environmental stress on reproductive development and
seed yield in common bean (Izquierdo and Hosfield, 1987; Sage and Webster, 1987; Monterroso and
Wien, 1990; Konsens et al., 1991; Ofir et al., 1993; Gross and Kigel, 1994; Shonnard and Gepts,
1994; Nakano et al., 1998; Suzuki et al., 2003; Tsukaguchi et al., 2003; Rainey and Griffiths, 2003).
As a self-pollinating crop, common bean produces many flowers and only a limited number of
them develop into pods. A large proportion of flowers or young pods are abscissed by the effects of
different factors. Reported value of average reproductive abscission was 48%, the tendency being
more marked in determinate bush types than in indeterminate types (Izquierdo and Hosfield, 1987).
Abscission ratio of reproductive organs throughout the flowering and pod setting periods ranged
from 48% to 82% (Tanaka and Fujita, 1979; Izquierdo and Hosfield, 1981).
Changes in temperature affect development during the reproductive growth stage in common
bean (Porch and Hall, 2013). High temperatures during this stage result in a reduction in pod and
seed set due to enhanced abscission of flower buds, flowers, and pods (Monterroso and Wien,
1990; Konsens et al., 1991). High temperature negatively impacts pollen–stigma interaction, pollen
germination, pollen tube growth, and fertilization (Weaver and Timm, 1988; Gross and Kigel, 1994),
with the lowest pod set observed in plants exposed to high temperature for 1–6 days prior to anthesis
(Graham and Ranalli, 1997). Lower pod and seed set after exposure to high temperature during
day/night (32°C/27°C) result from combined effects of lower pollen viability (evaluated by pod and
seed set resulting from reciprocal hand pollination) and impaired female performance in a large
proportion of the flowers (Gross and Kigel, 1994). Flowers that were affected by high temperature
(32°C/28°C for 24 h) at 8–11 days before anthesis showed a highly positive correlation between
pod set and pollen stainability (Suzuki et al., 2001). Thus, high-temperature-induced water deficit
at the time of anthesis could adversely affect the development and function of reproductive organs
(Tsukaguchi et al., 2003). Snap bean cultivars with a smaller midday drop in leaf water content
showed a higher pod setting ratio and consequently had higher yield than the plants with a larger
midday drop in leaf water content (Omae et al., 2005).
High temperature affects not only reproductive development but also several physiological pro-
cesses including photosynthesis and translocation of photosynthates to grain (Hall, 2004). Higher
biomass allocation to pods and higher pod set in branches, which vary with the cultivar and tem-
perature, play an important role in achieving a higher HI in the heat-tolerant compared to the
heat-sensitive cultivars (Omae et al., 2006, 2007). Day temperatures higher than 30°C or night
temperatures higher than 20°C could result in yield reduction (Rainey and Griffiths, 2005a,b; Porch,
2006; Porch et al., 2008). High night temperatures at flowering (and to a lesser degree, high day
temperatures) cause flower and pod abortion, reduced pollen viability, impaired pollen tube forma-
tion in the styles, and reduced seed size (Gross and Kigel, 1994; Porch and Jahn, 2001; Hall, 2004).
Although night temperatures in excess of 20°C can cause serious problems of pollen fertility and
pollination, recent analyses revealed that nocturnal temperatures still seldom reach critical levels,
but that day temperatures may actually be more limiting (Beebe et al., 2013a). Acclimation to occa-
sional high night temperatures may be a genotypic resistance mechanism (Hall, 1992; Wahid et al.,
2007). Pollen-based methods developed to evaluate heat tolerance in soybeans (Salem et al., 2007)
could be useful to screen bean genotypes for tolerance to nocturnal heat stress. There is a need to
(1) improve our understanding of the physiological and/or biochemical mechanisms causing pollen
and ovule sterility that lead to lower seed set and (2) incorporate temperature functions into bean
models to determine potential seed set under stress conditions.
39.3.5 Multiple Stress Resistance

Multiple abiotic stress factors often co-occur in farmers’ fields. Roots that are stunted by Al toxic-
ity are inefficient in absorbing both nutrients and water (Mossor-Pietraszewska, 2001). Al-resistant
plants may be more drought resistant and require lower inputs of lime and P fertilizer than less-
resistant genotypes (Little, 1988). On many acid soils of the tropics, variability in rainfall distri-
bution and longer dry spells during the main growing period of crops are becoming increasingly
important yield-limiting factors (Tang et al., 2001; Welcker et al., 2005; Beebe et al., 2011) with the
change in global climate. As a result, crop plants are needed that are tolerant of both drought and
Al toxicity. In the Cerrados region of Brazil (205 million hectares), soils are highly acidic with toxic
levels of Al with a long (up to 5 months) dry season. In the common bean-growing regions of the
Cerrados of Brazil, it is estimated that 80% of the area is affected by intermittent drought stress.
In the bean-growing acid soils of the Andean region (26%) with its bimodal rainfall distribution,
intermittent drought stress is also very common. In tropical Africa, soil acidity and Al toxicity are
intense in Rwanda, eastern Congo, and northeast Zambia and moderate in southern and western
Congo, Zambia, Tanzania, Mozambique, and Malawi. Drought and Al overlap on both sides of the
Rift Valley in Kenya, Congo, Rwanda, and Tanzania and intermittently between 5° and 15° south
latitude in Angola, Zambia, Congo, and Malawi.
Root phenes play a major role in improving tolerance to several abiotic stress factors: drought,
low P availability, and Al toxicity. Genetic diversity exists in common bean for root traits (Lynch
and Beebe, 1995; Lynch, 2007, 2011; Butare et al., 2011, 2012), and matching the root system to
the environment will be a particular research challenge for the future. Different root systems will
be required for different soil environments. Shallow root system will be beneficial to maximize P
acquisition in a low-P soil (Ho et al., 2005; Nord and Lynch, 2009; Lynch, 2011; Beebe, 2012). Deep
root system will improve water acquisition under drought stress (Sponchiado et al., 1989), particu-
larly in deep soils. Greater numbers of root tips will improve Ca uptake, while exudation of organic
acids will serve as a defense mechanism against Al toxicity or as an efficient mechanism of P acqui-
sition (George et al., 2012; Yang et al., 2013). A dimorphic root system that combines shallow roots
with deep roots may be more suited for improving abiotic stress tolerance in common bean.
Most bean improvement efforts for abiotic stress have been focused on individual stresses. But a
few studies considered the effects of multiple stress conditions. Butare et al. (2011) found that roots
of P. coccineus accession G35346-3Q developed better than those of common bean under com-
bined stress of acid soil toxicity and drought. A few interspecific progenies expressed tolerance to
combined stress (S. Beebe, unpublished results). Shallow root development in common bean favors
P acquisition, while deeper root development is preferable for accessing moisture and improving
drought resistance (Ho et al., 2005; Henry et al., 2010). Yang et al. (2010) characterized the com-
bined effects of Al toxicity and drought stress on root growth, with special emphasis on Al/drought
interaction in the root apex of common bean. Using polyethylene glycol (PEG) to simulate osmotic
stress (OS) or drought stress, they found that OS enhances Al resistance by inhibiting Al accumula-
tion in the root apices of the Al-sensitive genotype (VAX-1). This alleviation of Al toxicity was found
to be related to the alteration of cell wall porosity resulting from PEG-induced dehydration of the
root apoplast. Physiological and molecular analysis of PEG-induced reduction of Al accumulation
in the root tips of common bean indicated that genes related to cell wall assembly and modification
(XTHs, BEG, and HRGP) play important roles in the PEG-induced decrease in cell wall porosity,
leading to reduced Al accumulation in root tips (Yang et al., 2011). Studies on short-term effects
of Al toxicity and drought stress on root growth in acid, Al-toxic soil showed that in soil, drought
alleviates Al injury, but Al renders the root apex more drought sensitive, particularly by impacting
the gene regulatory network involved in ABA signal transduction and cross talk with other phyto-
hormones that is necessary for maintaining root growth under drought stress (Yang et al., 2012).
Interactions of a particular stress with other factors in the environment, especially other stresses,
complicate the selection for stress tolerance (Beebe et al., 2013a; Yang et al., 2013). Drought
stress can interact with higher temperatures or by poor soil fertility that limits root development.
Identifying the critical interactions and incorporating these into a selection program is perhaps the
most challenging aspect of improving adaptation to abiotic stress factors.
A modeling exercise was carried out to estimate the distribution and relative importance of cli-
matic constraints to bean yields and the potential value of genetic improvement (Beebe et al., 2011).
Breeding drought resistance into common bean could improve suitability of some 3.9 million hect-
ares of current bean area (the equivalent of 31% more area classified as >80% suitable) and would
add another 6.7 million hectares of area that is currently not suitable. Heat tolerance could benefit
7.2 million hectares (some of which would also benefit by drought tolerance) and could increase
highly suitable areas (>80%) by some 54%. Thus, both drought resistance and heat tolerance are
important objectives for genetic improvement. Although heat tolerance may offer wider impact,
drought causes great year-to-year yield variability and can induce famines and must also be given
priority. These analyses refer to likely changes in rainfall and temperature, with predicted effects on
current and potential distribution of bean production. Understanding the biological effects of these
changes on specific constraints will aid bean breeders to design breeding strategies to confront
climate change. White et al. (2011) reviewed 221 peer-reviewed papers that used crop simulation
models to examine diverse aspects of how climate change might affect agricultural systems and
suggested a coordinated crop, climate, and soil resource would allow researchers to focus on under-
lying science.
Our own experience indicates that in cases of combined stress of poor soil fertility and drought,
addressing problems of soil fertility will be beneficial to drought resistance, presumably through
better root development (Beebe et al., 2013a). Lines expressing tolerance to both low P and drought
have been developed from crosses among elite parents for each individual stress, expressing a sig-
nificant advantage over an elite commercial check (Tio Canela) and modest advantage over the
respective elite checks for each stress. P use efficiency will be a useful parameter to identify bean
genotypes for use on P-deficient soils or those suffering from Al toxicity. Some breeding lines per-
formed better than the check lines under combined stress conditions of drought and low P and under
Al toxicity. In rainfed agriculture, WUE will be a useful parameter to identify eco-efficient bean
genotypes with higher values of agronomic WUE (Beebe et al., 2013a). Several breeding lines were
identified to be superior to the checks in their efficiency to use water to produce grain yield. One of
the small-seeded red lines, SER 78, was outstanding in its ability to use water under drought stress.
The values of agronomic WUE observed for the bred lines were similar to the values reported in
other studies on common bean (Muñoz-Perea et al., 2007; Builes et al., 2011). Lines developed with
improved drought resistance also need to be combined with tolerance to higher temperatures as well
as excess soil moisture. These combinations can not only improve yield stability in farmers’ fields
but also can minimize total crop failure in extreme environmental conditions.
An ideotype suggested for improved yield under multiple abiotic constraints such as low P avail-
ability, drought, and Al toxicity must combine root and shoot traits both for increased biomass
production and for enhanced photosynthate remobilization to grain (Beebe et al., 2009, 2013a; Rao
et al., 2009; Beebe, 2012). A general framework suggested for this would be an extended vegeta-
tive phase (delayed flowering) to favor P acquisition and biomass accumulation (Nord et al., 2011)
together with improved symbiotic nitrogen fixation under stress (Devi et al., 2013), and specific root
traits can be incorporated to further promote nutrient acquisition (Beebe, 2012). Once flowering is
initiated, the shift to reproductive processes should be decisive (Beebe, 2012; Beebe et al., 2013a).
The reproductive shift is often weak in common bean, which readily reverts back to a vegetative
phase under environmental stimuli such as rainfall late in the growth cycle. While this ideotype
could be useful under a range of bean-growing environments, it would be especially relevant to
addressing abiotic stresses that are prevalent in the smallholder farms of the tropics. Such an ideo-
type could require combining traits from different species of Phaseolus that could optimize the
expression of different traits.
39.4 ADAPTATION OF BRACHIARIA FORAGE

GRASSES TO ABIOTIC CONSTRAINTS
Livestock production contributes to the livelihoods of more than 800 million people, and it is also
the most important agricultural land use in the world with grasslands covering 25% of the Earth’s
land surface (Steinfield et al., 2006). Livestock production offers one of the few rapidly growing
markets that poor, rural people can join even if they lack substantial amounts of land, training,
and capital (Delgado et al., 1999). In the tropics, forage/grassland-based crop–livestock systems
represent about 70% of agricultural land use. Over the past 30 years, meat and milk consumption
in developing countries has grown three times as fast as in developed countries with an additional
market value of US$155 billion. Smallholder mixed crop–livestock systems provide over 50% of
the world’s meat and over 90% of its milk. These are the most important livestock systems in
developing countries (Herrero et al., 2010). Across the developing world, people keep livestock to
earn income, increase their crop production, store wealth, and feed their families. The analyses on
the roles of livestock in developing countries indicated that livestock play a significant role in rural
livelihoods and the economies, and to provide these benefits, the sector uses a significant amount of
land, water, biomass, and other resources and emits a considerable quantity of GHG (Herrero et al.,
2013). The challenge is how to manage complex trade-offs to enable livestock’s positive impacts to
be realized while minimizing and mitigating negative ones, including threats to the health of people
and the environment (Smith et al., 2013). Contrary to much conventional thinking, forage-based
sustainable intensification of livestock production has huge potential to generate environmental
benefits while also improving rural livelihoods (Peters et al., 2013).
A major constraint to livestock production in the tropics is the quantity and quality of forage
production. Overgrazing and a lack of suitable forage options that are better adapted to biotic (pests
and diseases) and abiotic (edaphic and climatic) stress factors contribute to low productivity (Miles
et al., 2004). Nutrient depletion and improper management of pastures lead to pasture degradation
and limit livestock production (Fisher et al., 1996).
Tropical forage grasses and legumes as key components of eco-efficient agriculture have major
implications for alleviating poverty, improving food security, and protecting the environment (CIAT,
2009; Peters et al., 2013). Forage grass or legume is a complex crop, and its value for agriculture must
be assessed in terms of the quantity and quality of downstream livestock products (milk, meat). The
most widely sown forage grasses in tropical America are Brachiaria grasses (Miles et al., 2004; Valle
and Pagliarini, 2009), and in Brazil alone, about 80 million hectares are planted to Brachiaria pas-
tures (Macedo, 2005). These pastures make significant contribution to farmers’ incomes by increasing
animal productivity by 5–10 times with respect to native savanna vegetation in the tropical areas of
Latin America (Lascano, 1991; Rao et al., 1993). The excellent adaptation of Brachiaria grasses to
low-fertility soils has contributed to their use for extensive, permanent, low-input pastures but also in
more intensively managed pastures. Although rotation of annual cropping with grazed pasture is not
commonly practiced, it is increasingly becoming an option for farmers in tropical America follow-
ing the example of Brazil (Miles et al. 2004; Rao et al., 2011). In Latin America, superior Brachiaria
grasses have been widely adopted with large economic benefits (Holmann, 2009). In Brazil, for
example, they are believed to be as large as $4 billion, while in Colombia, they are thought to exceed
$1 billion. Holmann et al. (2004) studied the adoption of Brachiaria grasses from 1990 to 2003 in
several Central American countries and Mexico and estimated the value of the additional production
of milk and beef due to the adoption of Brachiaria grasses to be around $1 billion.
Commercial cultivars of Brachiaria grasses selected from natural germplasm showed defects
such as sensitivity to spittlebugs, poor edaphic adaptation, or inadequate seed filling. No single
cultivar has combined all the desirable attributes (Rao, 2001; Miles et al., 2004). Sowing vast areas
of tropical American savannas to two major cultivars (signalgrass and palisadegrass) resulted in
millions of hectares of degraded pastures due to inadequate fertilizer application and overgrazing.
New Brachiaria grass cultivars that are more productive and of better quality are needed to restore
degraded pastures and to intensify livestock production in integrated crop–livestock systems, even
if these cultivars require higher inputs. Long-term persistence of pasture grasses under grazing will
be less important, and building an arable layer and improving soil conditions will be more impor-
tant in intensive systems of annual crop–pasture rotation where no-till or minimum tillage is prac-
ticed for crop production (Guimaraes et al., 2004; Miles et al. 2004; Rao et al., 2004; Amézquita
et al., 2007).
An important research strategy of CIAT has been exploiting the natural variability of forage
germplasm to identify tropical grass species adapted to the various agroecosystems in acid soil
regions. Breeding efforts started in the late 1980s at CIAT and Empresa Brasileira de Pesquisa
Agropecuária (EMBRAPA) for developing interspecific hybrids to combine traits of three parental
species: acid soil adaptation of signalgrass (Brachiaria decumbens) and spittlebug resistance of
palisadegrass (Brachiaria brizantha), both tetraploid apomicts, and sexual reproduction of a tetra-
ploidized biotype of ruzigrass (Brachiaria ruziziensis) (Miles et al., 2004, 2006; Souza Sobrinho
and Aud, 2009; Valle and Pagliarini, 2009). Combining genes of the two apomictic species is pos-
sible, and fully sexual germplasm-containing genes from the three species including B. ruziziensis
have been developed (Rao et al., 1998; Ishitani et al., 2004; Miles et al., 2004, 2006). From these
breeding efforts, three commercial cultivars have been released (Mulato, Mulato 2, and Cayman).
These three superior Brachiaria-bred cultivars combine high productivity, nutritional quality, resis-
tance to spittlebugs, dry season tolerance, and adaptation to infertile acid soils (Miles et al., 2004,
2006; Argel et al., 2005, 2007; Ricaurte et al., 2007a,b). But both Mulato and Mulato 2 are not toler-
ant to waterlogging conditions (Rincón et al., 2008). Recently, CIAT initiated efforts to breed for
superior Brachiaria humidicola hybrids. These hybrids are required to diversify pastures in poorly
drained or occasionally waterlogged soils that are estimated to cover about 7% of the cerrados
(savanna) region of Central Brazil (ca. 17 million hectares) and major cattle production regions in
the Amazon (Ricci et al., 2011).
There is limited knowledge on the environmental adaptation of Brachiaria grasses. Some
attempts were made to understand the physiological and biochemical bases of their adaptation to
edaphic and climatic constraints (Fisher and Kerridge, 1996; Rao et al., 1996; Rao 2001; Miles
et al., 2004; Louw-Gaume 2009, Louw-Gaume et al., 2010a,b; Watanabe et al., 2011). Commercial
use is generally restricted to tropical regions that are below 2000 masl, an annual rainfall of more
than 600 mm, and at least a 5-month rainy season (Bogdan, 1977; Thomas and Grof, 1986). Both
high and low temperatures affect the growth of Brachiaria grasses. Increase in temperature to an
optimum between 30°C and 35°C markedly improves both shoot and root growth, and most species
grow very well at 35°C and some even up to 38°C (McWilliam, 1978; Ludlow, 1980). Growth is
substantially reduced with low temperature, and little growth occurs below 15°C, particularly with
ruzigrass that is adapted to lowland tropics but develops severe chlorosis at 20°C/20°C day/night
temperature (Ludlow and Wilson, 1970) and dies at 20°C/6°C (Mannetje and Pritchard, 1974).
However, Miles et al. (2004) indicated that Brachiaria grasses seem to stimulate flowering under
lower temperature at higher altitudes (1800 masl).
Rao (2001) considered the selection or breeding of tropical forages adapted to abiotic stress
factors as the most viable approach for increasing pasture and livestock productivity that could
contribute to food security in the tropics, as the use of lime to improve soil chemical properties is
not economically feasible because the returns in terms of forage yield and quality are low (Miles
et al. 2004). Rao et al. (1998) observed wide genetic diversity in plant attributes in Brachiaria
genetic recombinants for tolerance to infertile acid soils and emphasized that this diversity is useful
for breeding programs to develop superior Brachiaria genotypes. Among the Brachiaria grasses,
B. decumbens cv. Basilisk (signalgrass) is highly adapted to infertile acid soils, that is, tolerant to
high Al saturation and low P and low Ca supply in soil (Louw-Gaume, 2009, Louw-Gaume et al.,
2010; Rao et al., 1995, 1996a; Wenzl et al., 2001, 2003). However, signalgrass is sensitive to spittle-
bugs, a major insect pest (Miles et al., 2006). B. brizantha cv. Marandu (palisadegrass) is resistant
to spittlebugs, adapted to seasonal drought stress, and highly responsive to fertilizer application, but
it is not well adapted to low-fertility acid soils (Miles et al., 2004, 2006). B. ruziziensis cv. Kennedy
(ruzigrass) is sensitive to spittlebugs, performs better in well-drained fertile soils, and has high for-
age quality, but is poorly adapted to low-fertility acid soils (Miles et al., 2004).
Significant progress in defining the specific physiological and biochemical mechanisms that are
associated with greater adaptation to abiotic stress factors will contribute to developing rapid and
reliable screening methods to select the phenotypes and to develop molecular markers for marker-
assisted breeding of Brachiaria grasses. Efficient screening methodologies are required to recover
the desired traits through stepwise accumulation of favorable alleles over repeated cycles of selec-
tion and recombination (Rao, 2001; Rao et al., 2006, 2012; Wenzl et al., 2006). Edaphic adapta-
tion is particularly difficult to select for because it is only manifest in the persistence of pastures
over several growing seasons. Developing superior Brachiaria cultivars from the ongoing breeding
programs that combine the desirable attributes including adaptation to major biotic and abiotic
constraints, forage quality, and seed production will facilitate developing forage components for
sustainable intensification of crop–livestock systems in the tropics (Rao, 2001; Miles et al., 2004,
2006; Louw-Gaume et al., 2010).
In addition to their adaptation to major abiotic constraints and ability to accumulate large
amounts of carbon in deeper soil layers through vigorous root growth and distribution (Fisher et al.,
1994, Fisher, 2009; Rao, 1998), Brachiaria grasses are also capable of inhibiting nitrification pro-
cess in soil (Subbarao et al., 2009). This phenomenon observed with Brachiaria grasses is termed
as biological nitrification inhibition (BNI). Up to 70% of the nitrogen (N) fertilizers applied to
agricultural systems are lost due to nitrification and denitrification. Nitrification is a microbiological
process that generates nitrate (NO3−) and promotes the losses of N fertilizers by leaching and deni-
trification (Subbarao et al., 2006). Nitrification and denitrification are the only known biological
processes that generate nitrous oxide (N2O), a powerful GHG contributing to global warming. There
is an urgent need to suppress nitrification process in soil to improve N recovery and N use efficiency
of agricultural systems and to mitigate climate change (Subbarao et al., 2012). Some Brachiaria
grasses such as B. humidicola CIAT 679 can suppress soil nitrification by releasing brachialactone
from roots, thereby reducing N2O emissions (Subbarao et al., 2009). There is a wide range in the
BNI capacity of Brachiaria grasses (Subbarao et al., 2007). If BNI activity of a Brachiaria pasture
were to carry over to a subsequent crop, it might improve the crop’s N economy, especially for crops
fertilized with substantial amounts of N (Subbarao et al., 2013). This exciting possibility is currently
being researched and could result in cropping systems with low nitrification and low N2O emissions
that could be economically profitable and ecologically sustainable (Rao et al., 2013b).
Progress made in defining the mechanisms of adaptation of Brachiaria grasses to major abiotic
stress factors including low-fertility acid soils (resistance to high Al and tolerance to low P avail-
ability), drought, and waterlogging is described in the following text.
39.4.1 Soil Acidity and Aluminum Toxicity

In the past, edaphic adaptation of Brachiaria grasses was exclusively evaluated by quantifying for-
age yield and pasture persistence in field trials (Rao et al., 1996; Miles et al., 2004). These trials have
resulted in the release of several well-adapted cultivars such as Basilisk, Tully, and Llanero (Miles
et al., 2004). Forage yield on low-fertility acid soils is mainly limited by Al toxicity and P deficiency
(Rao et al., 1993). In addition, other soil acidity–related stresses, such as proton and Mn toxicity, and
other nutrient deficiencies including N, Mg, Ca, and Mo are also important c onstraints. Acid soil–
adapted genotypes typically had root and shoot attributes that facilitated acquisition and/or efficient
use of key nutrients (N, P, and Ca) in a low-pH, high-Al soil (Rao et al., 1996, 1998; Li et al., 1997;
Rao, 2001; Wenzl et al., 2001, 2002a,b, 2003; Ishitani et al., 2004; Miles et al., 2004; Nanamori
et al., 2004; Wagatsuma et al., 2005a,b; Begum et al., 2006; Haussler et al., 2006; Watanabe et al.,
2006; Wenzl et al., 2006). These include (1) maintenance of root growth at the expense of shoot
growth, (2) acquisition and use of N (both forms, nitrate and ammonium) (e.g., B. humidicola),
(3) ability to acquire N through associative biological fixation (B. decumbens), (4) ability to acquire
P through an extensive root system and association with vesicular–arbuscular mycorrhizae, and
(5) development of an extensively branched root system (more root tips), which facilitates greater
acquisition of Ca (B. ruziziensis). Significant progress was also made to demonstrate genotypic
variation among Brachiaria grasses for Al resistance.
Using a nutrient solution that simulates the ionic composition of soil solutions extracted from
two oxisols collected in the Colombian Llanos, Wenzl et al. (2003) found that the relative growth of
seedlings in this solution (compared to unstressed conditions) ranked the three parental genotypes
of the Brachiaria breeding program (signalgrass, palisadegrass, and ruzigrass) the same as they
were ranked in field trials based on pasture persistence over several growing seasons. Al sensitivity
of ruzigrass (least adapted) increased disproportionately with low nutrient supply. Well-adapted sig-
nalgrass tolerated an approximately fivefold higher level of Al than poorly adapted ruzigrass, even
though the resistance of ruzigrass was comparable to that of wheat, triticale, and maize genotypes
that were previously classified as Al resistant (Wenzl et al., 2001).
The precise mechanism of Al-induced inhibition of root elongation is yet to be defined, and it con-
tinues to be a matter of debate whether the primary lesions of Al toxicity are apoplastic or symplastic
(Horst et al., 2010). Although there is evidence for symplastic lesions of Al toxicity, the protection of
the root apoplast appears to be a prerequisite for Al resistance in Al-excluder and Al-accumulator
plants. The very high level of Al resistance found in signalgrass has prompted a series of experiments
to determine the physiological and biochemical basis of resistance (Wenzl et al., 2001). The close
relationship found between Al accumulation in root apices and inhibition of root growth indicated
that exclusion mechanisms might contribute to Al resistance (Wenzl et al., 2001; Wagatsuma et al.,
2005a). However, secretion of organic acids and phosphate at root apices was clearly not the main Al
exclusion/resistance mechanism in signalgrass (Wenzl et al., 2001). Two major observations led to this
conclusion: (1) root apices of signalgrass secreted quantities of organic acids only moderately larger
than those of ruzigrass, and (2) organic acid and phosphate efflux rates from signalgrass root apices
were 3.4- to 30-fold lower than those of Al-resistant genotypes of buckwheat, maize, and wheat, which
are severalfold more sensitive to Al than signalgrass (Wenzl et al., 2001). Thus, other mechanism(s)
may be responsible for exclusion of Al from root apices of signalgrass.
Both Al-resistant signalgrass and less-resistant ruzigrass accumulated high concentrations of Al
in roots (Wenzl et al., 2002a). About two-thirds of the total Al was complexed by soluble low molec-
ular weight ligands, suggesting that it had been taken up into the symplasm. This conclusion was
confirmed by a 27Al nuclear magnetic resonance (NMR) spectral analysis of the Brachiaria hybrid
cv. Mulato, which showed that Al in the root symplasm was present as a complex with ligand(s)
(Watanabe et al., 2006). In many Al-accumulator species, leaves and roots with high concentration
of Al are detoxified by organic ligands (Watanabe et al., 2011) such as citric acid, malic acid, trans-
aconitic acid, oxalic acid, and 1,3-di-O-trans-feruloylquinic acid, a chlorogenic acid analogue pre-
viously isolated from Brachiaria grass roots (Wenzl et al., 2000). These ligands could act as a sink
for Al ions in mature roots because very little Al was translocated to shoots (Wenzl et al., 2002a).
Root apices accumulated significantly larger amounts of citric, malic, trans-aconitic, and oxalic
acid than mature root sections, indicating a role for organic acids in the internal detoxification of Al
in Brachiaria grass root apices (the most Al-sensitive site). However, they do not account fully for
the superior resistance level of signalgrass (Wenzl et al., 2002a).
A separate line of evidence suggests that Al resistance of signalgrass may only be a facet of a
more generic resistance mechanism to inorganic cations. Wenzl et al. (2004) found that the differ-
ence in Al resistance between signalgrass and ruzigrass coincided with a similar difference in resis-
tance to a range of trivalent lanthanide cations and some divalent cations. It appears that resistance
to a range of different toxicants can be more easily achieved through an exclusion mechanism
than through an internal, detoxification mechanism. Further research work indicated a possible
role for root PM negativity and/or PM composition in high level of Al resistance observed in sig-
nalgrass (Wagatsuma et al., 2005a,b). Signalgrass was the most Al-resistant and least methylene
blue-stainable plant compared with ruzigrass and 16 different cultivars belonging to eight other
plant species (Wagatsuma et al., 2005b). Treatment with Al for short duration resulted in less per-
meability of PM of root apices of signalgrass than those of ruzigrass (Wagatsuma et al., 2005a). It
is possible that at least three major changes in PM may contribute to superior level of Al resistance
in B. decumbens: (1) higher proportion of sterol relative to phospholipid, (2) higher concentration of
phenolic compounds in cytosol, and (3) higher inclusion of phenolic compounds in PM lipid layer in
the root apex. These changes may contribute to an extremely strong PM lipid layer that plays a key
role in exclusion of Al and the high level of Al resistance in B. decumbens. A major challenge is to
demonstrate the existence of phenolic compounds in PM lipid layer (Watanabe et al., 2011).
Arroyave et al. (2011) used scanning electron microscope/energy-dispersive spectrometry, con-
focal fluorescence microscopy, and optical microscopy of lumogallion- or morin-stained roots to
characterize Al resistance mechanism in signalgrass. They found that Al-induced changes in root
epidermal cell patterning is a distinctive feature of high level of resistance to Al in signalgrass.
They showed that roots of signalgrass accumulated less Al than those of palisadegrass and the sites
of Al accumulation in signalgrass corresponded to root hairs. They found an Al-induced increase
of soluble phenolic substance, chlorogenic acid, in signalgrass but not in palisadegrass. They have
indicated a possible role for chlorogenic acid as a primer for changes in root epidermal cell pattern-
ing that may contribute to hyperresistance to Al in signalgrass.
Studies on mechanisms of Al resistance in Brachiaria grasses were conducted using seedlings.
The selection scheme of the Brachiaria breeding program at CIAT is based on the simultaneous
assessment of a set of hybrids for a variety of traits including edaphic adaptation, insect and dis-
ease resistance, nutritional quality, and seed production (Miles et al., 2004). All phenotypic assays,
therefore, need to be based on vegetative propagules (rooted stem cuttings) rather than seedlings.
Hence, the seedling-based Al resistance assay (Wenzl et al., 2001) was modified to be suitable
for the adventitious roots of stem cuttings, by increasing the concentration of Al (200 μM AlCl3,
200 μM CaCl2, pH 4.2) and simultaneously quantifying the intrinsic root vigor of each genotype in
a solution containing only 200 μM CaCl2 (pH 4.2) (Wenzl et al., 2006). The concurrent assessment
of root length in the Al-free solution revealed a large amount of genetic variation for root vigor in
the absence of nutrients: the root system of the best segregants was more than eight times longer
than that of poorly adapted ruzigrass (Wenzl et al., 2006). Vigorous root growth should improve a
plant’s nutrient-foraging ability (particularly for immobile nutrients such as P) and was previously
identified as associated with pasture persistence (Rao et al., 1996). Root vigor, therefore, emerged
as another selection target in the context of edaphic adaptation and was easily incorporated into the
breeding program through our solution culture technique.
Implementation of a simplified version of this screening method for the past 12 years allowed
simultaneous assessment of Al resistance and root vigor based on visual inspection and contrib-
uted to breeding progress toward improving edaphic adaptation. Several well-adapted Brachiaria
hybrids that had been preselected for spittlebug resistance were identified (Rao et al., 2006). Results
from the evaluation of a number of populations for Al resistance clearly indicated that resistance is
improving with each breeding cycle, illustrating the genetic gain achieved through recurrent selec-
tion (Miles et al., 2004; Rao et al., 2006b; Ricaurte et al., 2008). A group of nine sexual and six
apomictic hybrids were identified with greater Al resistance (Ricaurte et al., 2008). Field evaluation
of 15 Brachiaria hybrids together with three parents and four checks over 3 years at a location in
the Colombian Llanos with infertile acid soil and no maintenance fertilizer resulted in identifica-
tion of cultivar Mulato 2 and two hybrids (BR02NO/0465, BR02NO/1728) that were superior to
other hybrids (Ricaurte et al., 2007a). These hybrids need to be further evaluated in multiple sites
for livestock production under grazing and cut-and-carry forage feeding systems.
39.4.2 Low Phosphorus Availability in Soil

Low P availability in soil is a major constraint to productivity of Brachiaria grasses and these
grasses with P deficiency exhibit multiple adaptive responses (Rao, 2001). An understanding of
plant mechanisms underlying tolerance to low P stress would permit efficient development of low
P-adapted germplasm. In addition, the identification of genetic differences in P efficiency by plants
can be used in a breeding program aiming to develop cultivars more tolerant to low P availability.
P-efficient cultivars integrate different traits and mechanisms that contribute to adaptation to low
P availability (Rao et al., 1999a; Ramaekers et al., 2010; Lynch, 2011). P efficiency of Brachiaria
grasses can be related to an internal low P requirement, usually assessed as the plant P concentra-
tion, or the forage yield obtained per unit of P taken up (P use efficiency). P efficiency may also be
due to an increased ability to take up P (P uptake efficiency), which is related either to an extension
of the root system (higher root production, root hairs, or association with mycorrhiza) or increased
uptake per unit root surface (usually due to secretion of P-dissolving compounds). Breeding for
tolerance to low P needs to take into account specific soil and environment characteristics, as the
effectiveness at which the various mechanisms operate will differ depending on the P speciation in
the soil (Louw-Gaume et al., 2010).
Brachiaria grasses have higher P efficiency (lower internal P requirements) than other grasses
because they are able not only to acquire P with their extensive root systems but also to use the
acquired P more efficiently for growth and metabolism (Rao et al., 1996a,c, 1997, 1999a–c).
However, knowledge on mechanisms of P use efficiency in Brachiaria grasses is limited. Shoot and
root growth of Brachiaria grasses is responsive to P fertilization, and forage yield increases follow-
ing P applications have been reported in field (Rao et al., 1998) and in pot experiments (Rao et al.,
1996c). Rao et al. (1998) evaluated 55 Brachiaria genotypes, including signalgrass and ruzigrass, in
the field. Ruzigrass was the least efficient in acquiring P and N and also the least persistent in the
short term (i.e., 5.5 months after pasture establishment) when biomass production was compared.
Under P-deficient conditions, the Brachiaria grasses improve their P acquisition by greater root
growth, uptake efficiency, and ability to use poorly plant available P (Rao et al., 1999b,c, 2001a;
Louw-Gaume et al., 2010). Although Brachiaria grasses have much lower internal requirements for
P than do other grasses, they also show interspecific differences (Rao et al., 1996a).
To determine adaptive responses of Brachiaria grasses to low P supply, Louw-Gaume et al.
(2010) used a nutrient culture system with hydroxyapatite suspended in a dialysis pouch that permits
the slow and constant release of phosphate into nutrient solution that simulates low P availability
in soil. This system was tested and implemented for comparative analyses of morphophysiological
and biochemical responses of signalgrass and ruzigrass at different levels of P supply. They found
that both grasses increased biomass allocation to roots when grown at low P supply. Ruzigrass also
increased lateral root growth when grown at low P supply, while lateral root growth of signalgrass
was unaffected by P supply. Signalgrass grew more slowly and had higher tissue mass density.
Signalgrass also had higher plant carbon concentrations and higher C to N ratios. Ruzigrass was
more nutrient responsive and showed strong biomass accumulation in response to P fertilization, but
could not maintain its growth rate under conditions of very low P availability. In order to cope with
P limitation, ruzigrass showed higher levels of phenotypic plasticity. Signalgrass was able to match
nutrient uptake with nutrient demand, relying on mechanisms to maintain phosphate homeostasis
including those for more optimal partitioning of P between shoot and root.
Results on comparison of signalgrass with ruzigrass under low P supply indicated a relationship
between decreasing plant P concentrations and increasing rates of exudation of oxalate and acid phos-
phatase (APase) from roots (Louw-Gaume, 2009). The two grasses differed in the temporal dynamics
of these responses as the faster-growing ruzigrass developed P deficiency at an earlier developmental
stage. Oxalate was the dominant exuded organic acid anion for signalgrass, and cell wall–associated
APase was strongly induced for ruzigrass when both grasses reached critical plant P concentrations
of 0.1%. In addition, roots of both grasses had higher APase and phytase activities when grown at low
P supply. However, phytases represented only a small proportion of the total pool of APase for both
grasses. These comparative studies indicated that phosphohydrolases were induced by P deficiency as
a P recycling system in Brachiaria grasses. Li et al. (1997) also reported enhanced secretion of phy-
tases, a subtype of APase that hydrolyzes P from phytate, by signalgrass during P limitation.
Low P supply decreases P status of plant tissue and markedly reduces leaf expansion and changes
leaf photosynthetic carbon metabolism (Rychter and Rao, 2005). Nanamori et al. (2004) quantified
the effects of P deficiency on the enzymatic activities of phosphohydrolases and on carbon metabo-
lism of Brachiaria hybrid cv. Mulato. They found that P deficiency induced the activity of phos-
phohydrolases and enhanced the partitioning of photosynthate into amino acids and organic acids
at the expense of carbohydrates. These metabolic adjustments appeared to generate a larger pool of
inorganic P than in other species such as rice, which apparently stimulated P turnover and enabled
the hybrid to use P more efficiently. Begum et al. (2006) showed that, when grown on a low-P soil,
the Brachiaria hybrid cv. Mulato had a higher P use efficiency than wheat or rice. In contrast to
wheat and rice, P use efficiency of the Brachiaria hybrid increased under more severe P deficiency
and soil acidity. The Brachiaria hybrid synthesized larger amounts of organic acids such as oxalate
and fumarate in leaves. P deficiency further increased accumulation of organic acids, presumably
as a consequence of a two-fold increase in leaf phosphoenolpyruvate carboxylase (PEPC) activity
and a decreased malate inhibition ratio of the enzyme. This PEPC stimulation was even more pro-
nounced in Brachiaria roots, where it could play a role in the synthesis of organic acids exuded by
roots to aid P acquisition from poorly soluble sources such as Al phosphate.
39.4.3 Drought
Brachiaria grasses are not noted for superior tolerance to drought, although B. brizantha cv.
Marandu performs well in Central Brazil with 700 mm annual precipitation and a 5-month dry sea-
son (Soares Filho, 1994). The ex situ collections of the commercial species contain accessions from
sites with more than 590 mm annual precipitation and less than 7-month dry season (Keller-Grein
et al., 1996). Under semiarid environments, other grasses such as buffelgrass (Pennisetum ciliare
[L.] Link) could be superior to Brachiaria grasses but may require soils of higher fertility.
Seasonal drought is a dominant feature of tropical savanna environments (Baruch and Fisher,
1991), and both quantity and quality of forage production can be markedly affected (Baruch
1994a,b). Brachiaria grasses differ in drought tolerance; palisadegrass and signalgrass are the most
tolerant (CIAT, 1978; Fisher and Kerridge, 1996; Pizarro et al., 1996). Under field conditions, signal-
grass was superior to guineagrass (Jones et al., 1980). Signalgrass plants tolerated leaf water poten-
tial of −13.0 MPa with a maximum value for osmotic adjustment of 0.9 MPa (Baruch and Fisher,
1991). Results from a comparative study of four important C4 forage grasses (Andropogon gayanus,
Hyparrhenia rufa, Echinochloa polystachya, and Brachiaria mutica) indicated that A. gayanus
and H. rufa are relatively more tolerant to drought (Baruch, 1994a). The higher level of drought
tolerance of A. gayanus was attributed to its positive rate of photosynthesis under lower leaf water
potential than in the other three species (Baruch, 1994b).
Polania et al. (2009b) evaluated a set of 71 Brachiaria hybrids for their drought resistance under
greenhouse conditions and identified one hybrid (BR06/1932) that was outstanding in its drought
adaptation as determined by green leaf biomass proportion to total leaf biomass, though its water
use for growth was greater than that of other hybrids. Another hybrid (BR06/1922) was identified
to be capable of producing greater green leaf biomass proportion to total leaf biomass with moder-
ate use of soil water. These hybrids need to be tested under field conditions for further evaluation.
Drought tolerance of 15 palisadegrass accessions was compared with that of two koroniviagrass
accessions and one signalgrass accession during a 6-month dry season (CIAT, 1998). The high-
est yielding accession during the dry season, palisadegrass CIAT 16305, maintained greater N
concentration in forage tissue. Superior dry season performance was associated with lower levels
of shoot mineral concentration, particularly Ca. Field evaluation for tolerance to dry season of 15
Brachiaria hybrids together with three parents and four checks over a 3-year period at a location in
the Colombian Llanos with infertile acid soil with no maintenance fertilizer identified four hybrids
superior to the other hybrids based on green forage yield and nutrient acquisition (Ricaurte et al.,
2007b). These hybrids need to be further evaluated in other locations under grazing for their dry
season tolerance.
39.4.4 Waterlogging
Soils with poor drainage (hydromorphic) are found in about 11.3% of agricultural land in Latin
America where physiography promotes flooding, high groundwater tables, or stagnant surface water
(waterlogging) (Wood et al., 2000). Waterlogging causes hypoxia through drastic reduction in oxy-
gen diffusion into the soil. Hypoxia reduces root aerobic respiration and the absorption of minerals
and water. Plants adapt to waterlogging conditions with traits and mechanisms that improve root
aeration such as production of aerenchyma, development of adventitious roots, and stem and leaf
elongation (Evans, 2003; Jackson and Colmer, 2005). During the rainy season, Brachiaria pas-
tures occasionally experience waterlogging conditions that severely limit pasture productivity and
therefore livestock production. Waterlogging in pastures is caused by the combination of intensive
rains and poor soil drainage. Overgrazing, soil compaction, and pasture degradation can markedly
decrease the natural drainage of soils, making them prone to intermittent periods of flooding or
waterlogging. The Marandu palisadegrass “death syndrome” observed in the north of Brazil has
been a major issue for livestock production in tropical America (Dias-Filho, 2006). This syndrome
is attributed to very low tolerance of Marandu to excess soil water that adversely affects plant
metabolism, resulting in predisposition to biotic stress factors such as fungal diseases. Caetano and
Dias-Filho (2008) recommended that the research programs aiming to release new grass cultivars
should prioritize screening for tolerance to waterlogging.
Several researchers evaluated tolerance of Brachiaria grasses to waterlogging (Baruch, 1994a,b;
Baruch and Merida, 1995; Dias-Filho and Carvalho, 2000; Dias-Filho, 2002; Rao et al., 2007b;
Caetano and Dias-Filho, 2008). Palisadegrass is sensitive, signalgrass is moderately tolerant, and
koroniviagrass is tolerant to waterlogging (Dias-Filho and Carvalho, 2000). But koroniviagrass
has low forage quality that limits animal performance (Lascano and Euclides, 1996). Studies on
morphophysiological responses (leaf elongation rate, relative growth rate, root dry mass production,
net photosynthesis, stomatal conductance, and transpiration) of six Brachiaria grasses to root zone
flooding indicated that B. brizantha cv. Arapoty was most tolerant, while a ruzigrass accession was
most sensitive (Caetano and Dias-Filho, 2008).
Under greenhouse conditions, palisadegrass CIAT 26110 (cv. Toledo) was adapted to waterlog-
ging conditions by developing aerenchyma tissue in the root cortex and also by developing adventi-
tious roots from the lower nodes (CIAT, 1997). Aerenchymas are cortical air spaces that provide
a low-resistance internal pathway for the movement of O2 from the shoots to the roots, where it is
consumed in respiration and could also partially oxidize the rhizosphere. Cardoso et al. (2009)
measured phenotypic differences in formation of aerenchyma among six Brachiaria grasses

(two accessions of koroniviagrass: CIAT 679 and CIAT 6133, ruzigrass, cv. Toledo, cv. Marandu,
cv. Mulato 2). They found that all grasses responded to waterlogging by increasing the formation of
root aerenchyma and suggested that the higher percentage of aerenchyma formation and its suppos-
edly constitutive nature in the two highly waterlogging-tolerant accessions of koroniviagrass should
be of adaptive advantage during waterlogging stress.
Cardoso et al. (2013a) conducted an outdoor soil cylinder study using seven Brachiaria grass
cultivars with different levels of tolerance to waterlogging with an objective to quantify differ-
ences in their adaptive responses to hypoxic conditions based on alcohol dehydrogenase (ADH)
activity and formation of aerenchyma in root tissue. When plants were subjected to 3 and 10 days
of hypoxia, root ADH activity increased. Although waterlogging-tolerant cultivars showed higher
ADH activities, these values were not significantly different to explain variation among cultivars.
For all seven cultivars, 10 days of hypoxia resulted in lower values of ADH than 3 days of hypoxia.
Root aerenchyma development increased with increased time of exposure to hypoxic conditions.
Tolerant genotypes (e.g., B. humidicola CIAT 679) showed higher values of aerenchyma formation
than the other cultivars. Lower values of root ADH after 10 days indicated that O2 diffusion in roots
was improved presumably by increased formation of root aerenchyma. Results from this study
indicated that short-term adaptive responses in Brachiaria grasses to hypoxic conditions in the
root tissue are accompanied by a switch to fermentative catabolism, whereas longer-term response
includes an extensive root aerenchyma formation that could substantially improve root aeration to
facilitate root elongation.
Developing hybrids of Brachiaria with higher forage quality combined with waterlogging toler-
ance can improve meat and milk production and mitigate the impacts of climate change in the humid
tropics. CIAT has developed a screening method to evaluate waterlogging tolerance in Brachiaria
grasses (Rao et al., 2007b). Using this method, Rincón et al. (2008) evaluated a set of 71 Brachiaria
hybrids for their tolerance to waterlogging and identified four hybrids that were superior to the other
hybrids in their level of tolerance to waterlogging. The superior performance of these hybrids was
based on greater values of green leaf biomass proportion to total leaf biomass, green leaf biomass,
green leaf area, leaf chlorophyll content, and photosynthetic efficiency and lower values of dead leaf
biomass. These promising hybrids need to be tested under field conditions for further evaluation.
Cardoso et al. (2013b) evaluated 71 promising hybrids derived from three Brachiaria species
(B. ruziziensis, B. brizantha, and B. decumbens) for their tolerance to waterlogging using plants
grown in pots. Four hybrids were identified as superior in waterlogging tolerance. Their superiority
was based on greater values of green leaf biomass production, proportion of green leaf to total leaf
biomass, green leaf area, leaf chlorophyll content, photosynthetic efficiency, and on less amount
of dead leaf biomass per plant. These hybrids together with previously selected hybrids and germ-
plasm accessions are being field-tested for waterlogging tolerance in collaboration with the National
Agricultural Research Institutions and farmers in Colombia, Nicaragua, and Panama.
39.4.5 Multiple Stress Resistance

Aluminum toxicity and P deficiency tend to occur in parallel in infertile acid soils, largely because
Al forms insoluble precipitates with phosphate (Rao et al., 1993; Rao and Cramer, 2003). Chemical
interactions between Al and P within plant tissues are commonly considered an important second-
ary effect of Al toxicity. In Brachiaria grasses, Al had no effect on P concentrations in root apices
of Al-resistant signalgrass but led to a severe decline in apices of ruzigrass, thus suggesting an
Al-induced inhibition of acropetal P transport in roots (Wenzl et al., 2002b). Immobilization of P by
Al within plant tissues could be prevented through compartmentalization of Al in vacuoles (roots,
shoots) or inhibition of root-to-shoot translocation of Al. Alternatively, a range of metabolic adjust-
ments and accelerated P recycling could increase the efficiency with which P is taken up and/or used
in Al-stressed plants.
Hoyos et al. (2008) determined differences among six Brachiaria grasses (Marandu and Toledo
palisadegrass, signalgrass, ruzigrass, and hybrid cultivars Mulato and Mulato 2) in regulation of
water use, WUE, and growth when grown under individual and combined stress conditions of
drought and Al toxicity in pots under greenhouse conditions. Among the six Brachiaria grasses,
signalgrass and cv. Toledo were superior in their ability to tolerate the combined stresses of drought
and Al toxicity. The superior performance of these two grasses was attributed to a delay in stomatal
closure combined with efficient use of the moisture stored in the soil during the soil drying process.
Ruzigrass and cv. Marandu were found to be sensitive to the combined stresses due to early stomatal
closure that impacted their ability to fix carbon and produce forage biomass. Cultivars Mulato and
Mulato 2 demonstrated greater demand for water with their higher growth rate and moderate ability
to adjust to the decreasing soil moisture.
Polania et al. (2009a) determined differences among 11 Brachiaria grasses (including eight
hybrids) in root growth responses to individual and combined stress factors of drought and low soil
fertility using soil cylinders that permit quantification of root distribution at different soil depths
(0–80 cm). They found that three hybrids were superior in their total root length across soil depth
under combined stress conditions.
39.5 CONCLUSIONS AND FUTURE PERSPECTIVES

This review showed that considerable progress is being made in improving genetic adaptation of
common bean and Brachiaria forage grasses to major abiotic constraints in the tropics. It also
highlighted the role of physiological studies for improving genetic adaptation to major abiotic con-
straints. Crop physiology has been called “the retrospective science” by one plant breeder because
physiologists elucidate what the breeders have already achieved (Evans, 1998). This is because
the links between physiology and genetics have not been well established. This situation is likely
to change in the future, when knowledge of plant physiological processes will become extremely
important in screening for and measuring phenotypic traits.
Common bean is not well adapted to major edaphic (low P and Al toxicity) and climatic (drought,
high temperature) constraints. In the tropics, the crop is cultivated largely by resource-poor farmers,
often on soils that are deficient in N and P. Both edaphic and climatic constraints cause severe yield
losses. These constraints are widespread, often intense, and occur every year in the case of heat
and soil problems. Climate change and variability causing more intense droughts will adversely
affect important production regions in Mexico, Central America, the Caribbean, and Southern
Africa. Genetic improvement for resistance to major abiotic constraints will have significant and
wide impact. Application of fertilizers that improve productivity through enhanced plant vigor,
root growth, and access to soil moisture, or use of irrigation to overcome water deficits during crop
development, are seldom viable options for small-scale farmers. While development programs and
policies to subsidize such inputs merit consideration, crop improvement through plant breeding will
probably be the keystone of adapting beans to low-fertility soils of the tropics in the era of climate
change and variability.
Developing the right root system to cope with soil problems and drought stress in each produc-
tion environment will be a major research challenge. Deep rooting is an inherited response that
differs among common bean genotypes exposed to drought stress under different soil conditions.
Significant genetic variability is found within common bean for P acquisition efficiency, P use
efficiency, and agronomic WUE. Several bred lines have been developed that combine adaptation
to drought, low P, and Al-toxic acid soil conditions. Phosphorus-efficient common bean genotypes
develop more adventitious roots, shallower basal roots, and longer, denser root hairs. Traits includ-
ing root hair length, adventitious rooting, and basal root growth angle under low P availability were
shown to be under control of multiple QTL. Architectural trade-offs for P and water acquisition
have been demonstrated for root growth angle. Genotypes with deeper basal roots had superior
growth under water stress, while genotypes with shallower basal roots have superior growth under
low P conditions.
Mechanisms of Al resistance in common bean were defined using Al-resistant Quimbaya and
Al-sensitive VAX 1. It was shown that the induced and sustained Al resistance in the Al-resistant
genotype Quimbaya is mediated by reducing the stable-bound Al in the apoplast, thus allowing cell
elongation and division to resume. Resistance to Al in common bean is attributed to the release of
citrate by the root apex and the expression of a citrate transporter MATE gene is crucial for citrate
exudation. Although the MATE gene expression was a prerequisite for citrate exudation and Al
resistance, genotypic difference in Al resistance in common bean was mainly dependent on the
capacity to sustain the synthesis of citrate for maintaining the cytosolic citrate pool that enables
exudation. The initial Al-induced inhibition of root elongation in both Al-resistant and Al-sensitive
genotypes was correlated with the expression of the ACCO gene. Higher levels of Al resistance were
observed in P. coccineus. The secondary and tertiary gene pools of common bean cover a range of
environments from cool moist highlands to hot semiarid regions and will be important resources for
the genetic improvement of common bean for adaptation to multiple abiotic stresses.
Phenological adjustment, plant–water relations, photosynthetic parameters, and shoot growth
are all related to reproductive responses and thus may play an important role in heat and drought
tolerance. Different adaptive morphological and physiological traits and mechanisms may be
associated with growth, biomass partitioning, and yield of common bean under different drought
stress conditions. Deep rooting ability was identified as a key mechanism for improving drought
resistance. Biomass partitioning from vegetative structures to pod and from pod wall to seed was
found to vary among genotypes and to be a useful approach to identify drought-resistant geno-
types. PHI is considered as one of the key partitioning indices that indicate the extent of remobili-
zation of photosynthates to seed. Strong positive associations were found between PHI and grain
yield under drought stress and nonstress conditions. PHI has been suggested as a useful selection
criterion for improving resistance to drought. It presents quite acceptable heritability, and much
better than that of grain yield, and selection for PHI can lead to even better yield gains than
selection for drought yield per se and with positive effects on yield in favorable environments.
While more detailed phenotyping protocols might be applied to smaller numbers of genotypes
(e.g., candidates for use as parental materials), PHI could be a useful and practical selection tool
for bean breeders where a large number of early generation materials need to be evaluated before
performing yield trials and final selection.
Heat-tolerant cultivars have higher biomass allocation to pods and higher pod set in branches. The
number of pods per plant strongly correlates with seed yield. Large differences were found among
common bean genotypes in occurrence of reproductive organs such as flowers, pods, seeds, and their
abscission. Therefore, selection of common bean genotypes that compensate for seed yield losses
from abscission of flower buds, flowers, and young pods may contribute to stable seed yield under
stressful conditions. New common bean genotypes should be considered for their potential expres-
sion of reduced abscission and higher yields. In addition to yield-related characteristics, flowering,
pod, and seed-setting performance should be considered as selection criteria in common bean breed-
ing programs. General characteristics of flower production, flower and pod abortion, and pod and
seed set in common beans are well known. However, they have generally not been included into mod-
els to provide a better understanding of the determination of pod and seed number per unit area or
per plant. Detailed studies in the future on modeling the dynamics of flowering, pod and seed setting,
and reproductive success in common bean under individual, combined, and multiple abiotic stress
conditions could be helpful to explain unknown complex systems at these critical growth periods.
Studies on physiological and biochemical mechanisms of adaptation of Brachiaria grasses to
abiotic stresses indicated the higher level of resistance to Al and tolerance to low P supply in signal-
grass. This was mainly attributed to greater ability of Al complexation and Al localization in roots,
less upward translocation of Al to shoot tissue, and improved P utilization efficiency. Signalgrass
was also found to be superior in its adaptation to combined stress factors of drought and acid soil
stress. Tolerance to waterlogging was outstanding in koroniviagrass with a constitutive expression
of cortical aerenchyma formation in roots. Identifying the genes responsible for these superior traits
of Brachiaria grasses is a major objective for future research. The genetic control of stress tolerance
is often complex, requiring the combination of several different mechanisms in order to achieve
significantly elevated levels of stress tolerance. In these cases, gene discovery and efficient marker-
assisted pyramiding technologies will be important.
Significant progress has been made in developing screening methods to evaluate phenotypic
differences in tolerance to acid soil, drought, and waterlogging conditions. Using these screening
methods, it is possible to make further advances in breeding or agronomic evaluation of Brachiaria
grasses for improved adaptation to abiotic stresses. A major challenge is to define the interactions
between different abiotic stress factors so that cultivars with multiple abiotic stress tolerance can be
developed through breeding. Investigations into morphophysiological and biochemical mechanisms
of tolerance to abiotic stresses that could explain differences in field performance between Brachiaria
grass genotypes suggest that the key to the detection of subtle changes in growth is to be familiar
with whole-plant development and plasticity responses to stress conditions. Selection of Brachiaria
grasses for extensive agricultural systems should be based on plant traits associated with plant
survival and persistence that are observed with signalgrass, while nutrient-responsive Brachiaria
hybrids with traits that are found in ruzigrass might be suitable for intensive agropastoral systems as
well as cut-and-carry systems for smallholders in which productive potential for higher forage yield
is desired for livestock production.
Brachiaria grasses can be considered as climate-smart and low soil fertility-adapted forage
grasses because of their ability not only to adapt to drought and waterlogging stresses in the field
but also to accumulate large amounts of carbon in soil due to high root turnover and to inhibit nitri-
fication in soil and reduce nitrous oxide emissions. Considerable genotypic variation was observed
in BNI capacity among Brachiaria grasses indicating potential for the genetic improvement of
BNI capacity by selection and genetic recombination. The perennial ryegrass GenomeZipper-based
comparative genomics with Brachiaria grasses enables the maximum use of the significant invest-
ments that have been made in establishing genomic resources for model species (Pfeifer et al.,
2013), allowing us to accelerate genomic research on Brachiaria grasses.
Combining multiple stress resistance will require multidisciplinary approaches. We are using
a multidisciplinary approach at CIAT to cross-validate and integrate information from breeding,
agronomy, physiology, soil science, plant nutrition, and molecular genetics. Collaboration between
breeders and physiologists contributes to define appropriate traits and mechanisms that can serve
as selection criteria for breeding and to help design selection schemes and methods to address
multiple stresses (Rao, 2001; Beebe et al., 2011; Rao et al., 2011). Experts in genomics, transgen-
ics, and bioinformatics contribute to define which genes and mechanisms are most promising so
that an aggressive program of recurrent selection will be possible to improve multiple stress resis-
tance. Physiological and molecular studies on RILs of beans and Brachiaria hybrids provide a path
toward the identification of physiological mechanisms and genomic regions contributing to stress
resistance, which in turn could lead to the isolation of genes contributing to individual and multiple
abiotic stress resistance.
A broader view is needed to implement an integrated multidisciplinary approach to make prog-
ress in breeding for multiple abiotic stress resistance. An integrated improvement of resistance
to abiotic stresses is likely to be more productive than considering them in isolation (Yang et al.,
2013). The aim is to explore genomic advances to strike a balance between internal use of carbon
resources within the plant and the benefits gained from such investments in terms of fraction of
photosynthates that can be efficiently translocated to economically important products. Recently,
the “SWEET” sugar transporters were identified, and these are PM proteins located in the phloem
parenchyma, a cell type inside the veins that exports sucrose to SUT1 sugar loaders (Chen et al.,
2012). Since the translocation rates and relative distribution of carbon critically determine the crop
yield, further research work is needed to define the role of these SWEET proteins in improving
multiple stress tolerance.
The challenge is to identify QTLs of major effect that are independent of the particular genetic
background and clone the genes in the QTL. The ability of next-generation sequencing and advanced
metabolic profiling to co-sequence or co-screen a large number of F2 or RILs coupled with statisti-
cal linkage analysis could improve the efficiency of molecular breeding for improved adaptation to
multiple abiotic stress factors. A combination of genomic selection and genome-wide association
studies and next-generation mapping populations will improve our ability to connect phenotypes
and genotypes, and genomic selection can take advantage of these data for rapid selection and
breeding (Lorenz et al., 2011; Morell et al., 2012; Heyes et al., 2013). The socioeconomic impact of
use of multiple stress-adapted crop and forage cultivars would be immense in terms of increased
food production, more efficient use of purchased inputs, and improved integration of crop–livestock
systems benefiting both agriculture and the environment.
ACKNOWLEDGMENTS
The ideas presented here were developed from discussions with many colleagues and collabora-
tors, and I wish to thank them for their inputs. Particular thanks go to Drs. Steve Beebe, John
Miles, Manabu Ishitani, Walter Horst, Emmanuel Frossard, and Tadao Wagatsuma. I gratefully
acknowledge the contributions of past and present colleagues of the Bean Program and Tropical
Forages Program at CIAT. I also thank CIAT’s collaborators from several national programs in
Latin America and Africa for their valuable contributions to selection and breeding efforts over
the years. I am grateful to Jaumer Ricaurte, José Polania, Gonzalo Borrero, Mariela Rivera,
and Juan de la Cruz Jimenez for their research support. This work was partially funded by the
German Ministry of Economic Cooperation and Development, Bill and Melinda Gates Foundation,
Colombian Ministry of Agriculture and Rural Development, Commission for Developmental Issues
of the Austrian Academy of Sciences, Swiss Center for International Agriculture and Swiss Agency
for Development and Cooperation, FONTAGRO, and Swedish International Development Agency.
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40 Improving Maize Production
under Drought Stress
Traits, Screening Methods,
and Environments
Gerald N. De La Fuente, Ivan Barrero, Seth C. Murray,
Tom Isakeit, and Michael V. Kolomiets
CONTENTS
40.1 Introduction........................................................................................................................... 892
40.1.1 Drought Tolerance versus Drought Avoidance.......................................................... 892
40.1.2 Drought Tolerance versus Water Use Efficiency....................................................... 893
40.2 Stages of the Maize Drought Response................................................................................. 894
40.2.1 Seedling Drought Tolerance...................................................................................... 894
40.2.2 Drought Tolerance during the Vegetative Stages...................................................... 895
40.2.3 Maize Flowering Physiology Is Critical to Tolerate Drought.................................... 895
40.2.3.1 Anthesis Silking Interval............................................................................ 895
40.2.4 Post-Flowering Drought Tolerance............................................................................ 897
40.2.5 Water Is Necessary for Photosynthesis, Not Kernel Filling...................................... 897
40.3 Root Traits Involved in Drought Tolerance........................................................................... 898
40.3.1 Root Distribution, Structure, and Development........................................................ 898
40.3.2 Root Extraction of Soil Moisture at Low Saturation Levels..................................... 899
40.3.3 Metabolic Efficiency of Maize Roots........................................................................ 899
40.4 Leaf Traits Involved in Drought Tolerance...........................................................................900
40.4.1 Maize Leaf Rolling: Highly Visible but Controversial for Drought..........................900
40.4.2 Leaf Cuticle and Cuticular Wax: Layers of Protection............................................. 901
40.5 Biochemical Interactions in Drought..................................................................................... 901
40.5.1 Drought Stress Is Accompanied by Rapid Lipid Peroxidation.................................. 901
40.5.2 Production of Reactive Oxygen Species Is a Biochemical Hallmark
of Drought Stress.......................................................................................................902
40.5.3 Hormones Play an Important Role in Drought and Whole Plant Physiology...........903
40.5.4 ABA Is a Major Hormone Implicated in the Adaptation to Drought Stress.............903
40.5.5 Ethylene Reduces Growth under Drought Stress......................................................903
40.5.6 Role of Methyl Jasmonate and Jasmonic Acid in Drought Tolerance.......................904
40.5.6.1 Phenotyping of Biochemical Properties.....................................................904
40.6 Using Physiological Traits for Crop Improvement................................................................904
40.6.1 Environments for Screening and Selection...............................................................905
40.6.2 Methods of Selection and Traits Utilized in the Conventional Selection
of Drought-Tolerant Maize........................................................................................908
40.6.3 Direct Selection of Nonreproductive Secondary Traits.............................................908
891
40.7 Genomics, Gene Discovery, and Marker-Assisted Selection................................................908

40.8 Conclusions............................................................................................................................909
References.......................................................................................................................................909
40.1 INTRODUCTION
Nutrient and water availability are the two most limiting resources in crop production (Lea and
Azevedo, 2006; Moser et al., 2006). Nutrient application has benefitted from advancements in pre-
cision agriculture techniques. Producers are now able to customize applications by specifying the
blend ratio of nutrients and by precisely controlling rates of application based on data from the
previous years’ yield and soil sampling results (Miao et al., 2007). Water and irrigation, however,
because of the large quantities needed, still pose problems to producers in regions that receive less
than adequate rainfall, despite advances in technology and management practices (Sadler et al.,
2005). In Kansas, Nebraska, and Texas, maize uses 53–97 cm of water per year depending on water
supply, hybrid, maturity, and soil type (Howell et al., 1998). These regions historically have also had
the most erratic rainfall patterns.
A global climate change of only 1°C is estimated to have little effect on agricultural production;
however, this change, and the added demands of a growing and wealthier population, is expected
to put a strain on water resources around the world (Hare, 2005). Water competition between urban
and agricultural use will continue to increase, putting stress on maize production areas that are
highly dependent upon available surface and subsurface water supplies. These increased demands
will most likely force a choice between water and calories for certain areas of the world where
maize production is dependent upon supplemental irrigation.
Describing drought stress can be difficult, but is important to define for crop improvement objec-
tives. The term “drought” can carry many meanings depending on the situation in which it is used.
For example, while a given region may not be in a drought over an entire year, a dry season during
that year may lead to drought conditions. Here we define drought (also referred to as agricultural
drought) as the time point when the amount of moisture in the soil no longer meets the needs of the
crop (Mannocchi et al., 2009). Throughout the growing season, there are often time periods where the
soil moisture drops below the needs of the crop. Seasonal water use of the maize plant fluctuates and
follows the pattern of increasing through the vegetative stages, peaking at flowering, and decreasing
through grain fill and plant maturity (Figure 40.1). Yield components are determined throughout the
growing season and especially from V8 (eighth leaf stage) to dent stage. Thus, moisture stress has a
large window in which it can affect yield, so improvement of maize lines for multiple time periods of
drought stress is one of the most critical areas of cultivar development (Bruce et al., 2002).
An assortment of physiological mechanisms for coping with drought stress and classical breeding
techniques have been investigated and used. Additional modern breeding methods are now being uti-
lized to improve the ability of maize to cope with drought stress. However, the ideotype that is needed
for the particular target area is not always clear from the literature, as stages of drought tolerance are
complex (Blum, 2005). In addition, even in years where drought is not a serious problem on a large
scale, producers with sandier soils that do not retain water could see localized drought symptoms, even
with adequate rainfall. Because periods of drought conditions are expected to increase both locally
and globally, drought-tolerant lines of maize will not only aid in weathering stresses of the natural
environment, such as timely rainfall and the ability of the soil to hold water, but also aid with the
stresses of inevitable failure of irrigation equipment such as wellhead pumps and pivots.
40.1.1 Drought Tolerance versus Drought Avoidance

Herein we will define drought terms relevant to the discussion presented. For further discussion of
terminology, the reader is directed to Levitt (1980) and a comprehensive review by Blum (2005) on
drought resistance and water use efficiency (WUE). Drought resistance across the plant kingdom is
Improving Maize Production under Drought Stress 893
Average daily water use of maize

0.3
Silking 90°F–99°F
Tassel Blister kernel
0.25 emergence 80°F–89°F
Early dent
0.2 70°F–79°F
Water use (in.s/day)
Dent
8th leaf 60°F–69°F
0.15
Black layer
3rd leaf 50°F–59°F

0.1
0.05
0
0 2 4 6 8 10 12 14 16 18
Weeks after emergence
FIGURE 40.1 Average daily water use of maize at different temperature ranges from emergence to senes-
cence. Water use increases rapidly during late vegetative stages, peaks at flowering, and then decreases
through grain fill and maturity. (From Killen, M., Modification of the Checkbook Method of Irrigation
Scheduling for Use in Minnesota, Design Project, Agricultural Engineering Department, University
of Minnesota, Minneapolis, MN, 1984; Wright, J., Average water use for corn in inches/day, Irrigation
Scheduling Checkbook Method, University of Minnesota, Minneapolis, MN, 2002, Extension http://www.
extension.umn.edu/distribution/cropsystems/components/DC1322_02.html#crop, accessed December 12, 2011.)
a rare phenomenon. Water is essential to plant growth, and no plant can fully resist extended periods
of time without water uptake. However, there are multiple mechanisms that allow plants to complete
a life cycle even when the locality may suffer from drought conditions. Plants that can complete
their life cycle in a short period of time allowing them to reproduce during the wet season or before
exhausting stored soil moisture exhibit what is known as “drought escape” (Yue et al., 2006). Plants
that have adapted to an environment that follows the rainy and dry season pattern by flowering and
maturing during the wet season can be classified as “drought resistant.” In contrast, plants that can
withstand periods of minimal water supply without excessive damage or loss of function are classi-
fied as “drought tolerant.” Drought tolerance is separate from drought resistance, and there are likely
different physiological and genetic mechanisms involved. Drought tolerance can be subdivided into
two categories: dehydration avoidance and dehydration tolerance (Levitt, 1980). Dehydration avoid-
ance is defined as the plant’s capacity to sustain high plant water status or cellular hydration under
the effects of drought, while dehydration tolerance is defined as the relative capacity to sustain or
conserve plant function in a dehydrated state (Levitt, 1980; Blum, 2005). Drought escape by mecha-
nisms of early flowering is not a feasible trait to breed for, as this is inefficient for total photosyn-
thetic capture and yield when the growing season is long and weather patterns are highly variable.
40.1.2 Drought Tolerance versus Water Use Efficiency

WUE is a term that is commonly mentioned in discussions of crop improvement for drought con-
ditions. However, a very important distinction needs to be made between the concepts of drought
tolerance and WUE. Similar to drought tolerance, the definition of WUE fluctuates depending on
the context. In its strictest sense, WUE is the reciprocal of the transpiration ratio, defined as the total
quantity of water transpired over the amount of carbon dioxide used by photosynthesis (Taiz and
Zeiger, 2006). In other words, how much carbon can the plant take up per volume of water used?
Therefore, WUE can be considered when drought is not present. A recent argument has also been
made that the effective use of water and not WUE is the true target of breeding for drought tolerance
(Blum, 2009). In other words, yield in crops is driven by transpiration, and thus an effort should be
made to extract as much water as possible to drive photosynthesis and transpiration versus using
only part of the available water and distributing it within the plant in the most efficient way.
40.2 STAGES OF THE MAIZE DROUGHT RESPONSE

Historically, grain yield components for maize have been established and vary among studies with
respect to the type and timing of stresses. For maize, grain yield per unit land area can be broken
down into four major components, each having a critical developmental stage. Plant density or
population per acre is established first during the growing season and reflects seedling drought tol-
erance. Ears per plant, while mostly no longer variable in elite maize hybrids, play a role in inbred
and open-pollinated (OP) varieties of maize and are established in the vegetative stages of plant
development. Kernel row number per ear is a major component of yield potential set by the maize
plant before anthesis. Kernel number per row, across the length of the ear, is a function of several
stages of development and can fluctuate with stress levels after kernel row number is established
(e.g., pollination success and aborted kernels, both discussed later) (Hoeft et al., 2000).
40.2.1 Seedling Drought Tolerance

Except in unique cases, field-based drought stress during the seedling stage is rarely observed
because moisture is typically adequate after the fallow season, and planting into dry soil is rare.
Because plant density has become one of the most important factors for yield per land area in
maize, stand establishment is critical to final crop yield and reaching full yield potential (Tuberosa
and Salvi, 2009; Grassini et al., 2011). Therefore, pre- and post emergence drought tolerance, while
normally unseen, is an important trait on rare occasions that saves producers money by eliminating
the need for replanting a poor stand early in the season, thereby also preventing potential losses
from a late-planted crop. Simple screening techniques are commonly used such as plant recovery
after rewatering in the lab setting. Measurement of this trait can be conducted as a live versus dead
plant ratio but is subject to excessive measurement error (Meeks et al., 2013). More complicated
objective techniques have been investigated such as membrane stability measurements (Blum and
Evercon, 1981) but are both time consuming and expensive. A novel technique variant pioneered by
the International Maize and Wheat Improvement Center (CIMMYT) takes advantage of the plant
recovery overnight in the field (Banzinger et al., 1996). In this method, plants are scored for their
recovery before daytime heating induces leaf rolling again. Plants that do not recover overnight are
considered dead since the inability to regain turgor overnight is indicative of severe physiological
failures. Correlations between this method and the traditional method of re-irrigation and dead plant
counts are high and significant. While this technique reduces phenotyping time by not having to re-
irrigate, field testing allows for typically large environmental variations that can reduce heritability
estimates.
In an attempt to remove environmental variation, Meeks et al. (2013) screened maize seedlings
for drought tolerance in a greenhouse setting using a method that had previously been successful in
cowpea (Singh et al., 1999) and cotton (Longenberger et al., 2006). The objectives of these green-
house studies were to create a quick and easy method to screen large numbers of lines for drought
tolerance and correlate this to predictions of yield under drought. While the cowpea and cotton
studies were successful in differentiating drought-tolerant and drought-susceptible lines, the study
with maize had much lower success in predicting field-based drought tolerance, possibly due to the
extreme physiological changes that accompany the transition from juvenile to adult phase (Revilla
et al., 2002, 2004; Chandler and Tracy, 2007; Riedeman and Tracy, 2010).
40.2.2 Drought Tolerance during the Vegetative Stages

While not usually thought of as a part of drought adaptation and certainly not the most crucial, the
vegetative stages of maize (V3–V12) are nonetheless important for obtaining maximum yield poten-
tial. As will be discussed later, photosynthate produced prior to anthesis only accounts for ∼10% of
that present in the mature grain (Swank et al., 1982; Simmons and Jones, 1985). This suggests that
mild-to-moderate stress during the vegetative states will not affect kernel number directly through
decreased photosynthesis. A major yield component, however, the number of kernel rows, is deter-
mined around the V8 stage with the initiation of the ear shoot (Hoeft et al., 2000). If moisture stress,
or other stress, is present during the initiation of the ear shoot, kernel row number may decrease as
a response. The number of kernel rows has been shown to have significant additive genetic effects,
and it is likely that through artificial selection over the past ∼80 years, the genes controlling kernel
number have become fixed in elite germplasm (Daniel, 1963; Lu et al., 2011a). Despite its quantita-
tive inheritance, the number of kernel rows is only slightly affected by environmental stresses dur-
ing development, when compared to other traits and once established kernel row number is fixed
and always an even number (Emerson and Smith, 1950; Daniel, 1963). The number of kernels per
row can be further modified by the plant based on moisture and other stress conditions all the way
up to and after flowering.
40.2.3 Maize Flowering Physiology Is Critical to Tolerate Drought

Maize has monoecious and imperfect flowers with male and female organs located on the same
plant in different positions. The result of imperfect flowers is that pollen is forced to travel from
the tassel (contains anthers), located at the top of the plant, to the silks (stigmas) that protrude
from the developing ear shoot. This floral anatomy of maize heightens the effects that water
stresses have on plant development and ultimately yield (Westgate, 1997). The vertical distance
that pollen must travel to reach the silks can range from 1 to 1.3 m, and pollen is generally moved
by the wind; therefore, the pollen and silks must be exposed to a desiccating environment (Aylor
et al., 2003).
Pollen shed is theoretically adequate to pollinate all of the kernels on the developing ear shoot.
Older maize varieties can produce up to 25–50 million pollen grains (Kiesselbach, 1999; Burris,
2001), and an average-sized modern hybrid tassel produces 2–5 million (Burris, 2001) while the
average number of ovaries on the shoot ranges from 750 to 1000 (Aylor et al., 2003). Thus, pollen
density is far more than adequate to pollinate all of the embryos under favorable environmental
conditions, and pollen viability has been shown to be unaffected by water deficit (Herrero and
Johnson, 1981; Hall et al., 1982; Schoper et al., 1986). For maximum yield in maize, it is essential
to have successful pollination of the maximum number of kernels possible since kernel number per
unit field area has been found as an important yield component (Otegui et al., 1995). One of the
most important traits observed in maize flowering is delay in silk emergence (silking) with respect
to pollen shed (anthesis), as a result of stress. This phenomenon, termed the anthesis silking interval
(ASI), has been studied extensively (Bolaños and Edmeades, 1996; Westgate, 1997).
40.2.3.1 Anthesis Silking Interval

Increased ASI has been shown to have significant effects on reducing yield across a variety of cul-
tivars, hybrids, and environments (Bolaños and Edmeades, 1996; Westgate, 1997; Chapman and
Edmeades, 1999; Monneveux et al., 2006). There are several different effects as well as different
responses that the ASI has to varying types and levels of stress. The ASI can take on both negative
and positive values. Negative values arise from the silks emerging before pollen shed, known as
protogyny and is rare, while positive values arise from the silks emerging after pollen shed, known
as protandry and is much more common. Drought stress has little effect on time to pollen shed, but
will increasingly delay silking, as the severity of drought stress increases at critical times (Bolaños
and Edmeades, 1996). This follows the accepted dogma of Bateman’s principle that more energy is
required from the female than the male to produce viable offspring (Burd, 1994). It is important to
note that evidence has shown that ASI is not an indicator of the water status of the plant but rather an
indicator of the amount of biomass being produced in the developing ear (Chapman and Edmeades,
1999; Monneveux et al., 2006). When other abiotic stresses other than drought are present, the
maize plant will also respond by delaying silking. The delayed development of silks has been pri-
marily linked to a decrease in silk cell elongation rate (Herrero and Johnson, 1981). Silk elongation
occurs through a four-phase process of cell division and tissue expansion that occurs along its entire
length. Water deficit affects tissue expansion significantly more than cell division, leading to smaller
silk cell size at the cessation of growth (Faud-Hassan et al., 2008). Thus, the silk elongation rate and
its effects on ASI are highly dependent on the water status of the plant. The lengthening of the ASI
causes floral asynchrony that can lead to bareness (loss of kernel set, leading to an incompletely pol-
linated ear), which greatly decreases yield. While the causes of the ASI have been well established,
a significant knowledge gap exists in the understanding of the mechanism and genes regulating it
(Setter et al., 2011). Further research into this relationship could provide new avenues to search for
quantitative variation.
The effects of floral asynchrony (as measured by ASI) on reducing seed set are not limited only
to the loss of viable pollen. An early hypothesis for improvement of drought tolerance in maize
was to use incremental or varying maturities so that the window of pollen shed was larger, in an
effort to provide a pollen source for later emerging silks (Fischer et al., 1982). However, the addition
of pollen to silks that have emerged later than the tassels does not improve kernel number. Silks
that asynchronously flower significantly after pollen shed are permanently damaged and cannot be
recovered (Westgate and Boyer, 1986; Otegui et al., 1995).
Lengthening the duration of pollen shed within a plant has not been investigated as an avenue for
drought improvement to our knowledge, but variation has been observed. The trend for tassel size
selection has been a decrease in size over time (Fischer et al., 1987), reducing the amount of pollen
produced per plant. Minimum pollen densities for full kernel set have been determined (Uribelarrea
et al., 2002; Westgate et al., 2003), but not under drought conditions. Could lengthening the duration
of pollen shed effectively pollinate late emerging silks? No studies have been conducted addressing
this, but speculation based on other work cautions against this strategy. If the ASI lengthens beyond
4 days under drought stress, then silk elongation rate quickly drops to zero (Anderson et al., 2004).
However, this study only investigated four hybrids and even within these four existed variations for
kernel set, pollen shed duration, silk growth rate, and senescence time. It may well be that quanti-
tative variation exists for these traits that could be exploited for improvement. For the time being,
however, focus should be on keeping the ASI as short as possible, decreasing bareness and thus
increasing yield. If the plant is able to synchronize its flowering, then the next obstacle that must be
overcome is the receptivity of the silks under stress.
While the viability and amount of pollen shed are not affected by drought, the receptivity of the
silks to pollen is affected by drought (Schoper et al. 1986; Bassetti and Westgate, 1993). Receptivity
to pollen can decrease even after the pollen has germinated and begun to move down the silk.
Senescence of the silk causes it to collapse around the growing pollen tube, ceasing its growth
and consequently, not allowing it to fertilize the ovary. Bassetti and Westgate (1993) found that
this senescence and, thus, loss of receptivity occurred only when decreases in silk water potential
occurred over 4 days after the emergence of the first silk. When stress was induced more than 4 days
after first silk, senescence of the silks were accelerated, leading to decreases in silk receptivity.
Additionally, pollen tube growth in silks with low water potentials is slower, which allows time for
the silk base to collapse and cut off the pollen from the ovary. Bassetti and Westgate (1993) argued,
however, that it is unlikely in a field situation silk senescence would significantly harm yield, since
pollination from surrounding plants occurs long before any silks senesce if ASI is short. The most
likely reason for a loss in kernel set in a field setting after pollination is from abortion of the devel-
oping kernel.
FIGURE 40.2 Incomplete tip pollinations and kernel abortion as seen on harvested ears from a drought-
stressed field plot experiment in College Station, TX. Failed pollinations are likely from silks that emerged
after pollen shed ceased. Water stress through the grain fill period caused a further reduction in grain yield by
causing aborted kernels in the apical region of the ear.
40.2.4 Post-Flowering Drought Tolerance

If the sequence of events and conditions discussed so far are successful, namely, good stand and
vegetative development, synchronous development of male and female floral organs, favorable pol-
len shed, pollen remaining viable and traveling down to the silks, and silks staying receptive and
healthy enough to allow the germinated pollen to grow down to the embryo, fertilization is suc-
cessful. However, kernel number is not dependent solely on successful fertilization. The plant now
enters the second, longer part of the period critical for yield that is affected by water deficit, the
2 weeks after silking (Shaw, 1974). Kernel number at this point is no longer potential kernel num-
ber, which is a function of the number of spikelets (unfertilized embryos) in the developing ear, but
rather, the number of developing kernels. As a response to environmental triggers, gene expression
patterns affecting metabolism and growth now regulate the number of kernels on the ear (Hayano-
Kanashiro et al., 2009; Wei et al., 2009). A spikelet that has been pollinated, is developing, and
has the potential to reach maturity but is allowed to die may then be considered an aborted kernel
(Hanft and Jones, 1986) and can be seen in Figure 40.2. Kernel number per plant has been related to
plant photosynthesis (Edmeades and Daynard, 1979), which can then be decreased by water deficit
(Pelleschi et al., 1997). Water deficit increases until it reaches a threshold where photosynthetic rate
decreases, which varies among genotypes (Pelleschi et al., 1997).
40.2.5 Water Is Necessary for Photosynthesis, Not Kernel Filling

Artificial infusion of water-stressed plants with a liquid culture medium helps maintain kernel num-
ber, while artificial infusion with water has no beneficial effect on kernel number (Boyle et al.,
1991). While intuitive, this reminds us that decreased water availability in the plant is not the
direct cause of yield loss. The decrease in water availability in the plant affects photosynthesis and
decreases the amount of photosynthate available to the developing ear. Kernels that developed on
the water-infused stressed plants only had kernels on the basal or mid region of the ear, and aborted
kernels usually occur in the apical region of the ear (Boyle et al., 1991 and Figure 40.2). This sug-
gests that filled kernel number is ultimately a function of the amount of available carbohydrates
(Boyle et al., 1991; McLaughlin and Boyer, 2004), which can be moved to supply the developing
kernels. However, an inhibition of photosynthesis because of drought-induced stress decreases the
amount of available carbohydrates, and the low “sink strength” of the ear during pollination also
limits the amount of nutrients moving into the ear (Lafitte and Edmeades, 1995; Zinselmeier et al.,
1995; Westgate, 1997; Setter et al., 2001). Nitrate and carbohydrate tracking studies have shown that
the most photosynthate is moved from the leaves to the ear sometime between 26 and 34 days after
anthesis (Swank et al., 1982; Simmons and Jones, 1985) and was mostly generated in the plant after
silking (Swank et al., 1982). As a consequence, reduction in photosynthesis after silking greatly
reduces grain yield. A full ear of successful pollinations cannot be supported by a drought-stressed
plant. Thus, the plant’s response is to abort the development of kernels keeping only the ones it
can support with its current available resources reducing the number of kernels on the ear, which
reduces yield. These responses appear to occur once the plant has no other physiological way to
uptake more water and the genetic mechanism remains unknown. The key to preventing drought-
induced kernel abortion, and overall drought stress, is to therefore delay, during grain fill, the time
point when the amount of moisture in the soil no longer meets the needs of the crop. Maize has a
number of physiological responses and traits that maximize available moisture uptake, which are
mostly heritable and can be used for breeding.
40.3 ROOT TRAITS INVOLVED IN DROUGHT TOLERANCE

Beneath the soil surface, out of view, is the most important organ of the maize plant when consider-
ing water deficiency. The root not only serves as the source of water uptake for the plant but also
plays a crucial role in the signaling of the drought response to the stem, leaves, and reproductive
organs. It is suggested that changes in root system architecture (more compact root systems), not
changes in leaf angle, have allowed for the increase in plant density tolerance of maize (Hammer
et al., 2009). This reinforces that root systems are a major component of maize adaptation. Roots
have been called the “new frontier of drought research” (Pennisi, 2008) and represent a critical part
of plant physiology that remains very difficult to explore on a large scale. Root traits to improve
drought tolerance include root distribution, structure and development, root metabolic efficiency,
and osmotic adjustment.
40.3.1 Root Distribution, Structure, and Development

Roots can exhibit beneficial traits and physiological responses to water deficit. Maize roots con-
tinue to grow even when environmental conditions inhibit the growth of shoots (Zhu et al., 2010).
However, this exploration of the soil profile for moisture is metabolically costly to the plant. While
intuitive, a common observation is that in maize, the rooting depth of drought-tolerant lines is
greater than the rooting depth of drought-susceptible lines (Bruce et al., 2002; Hund et al., 2009).
When compared to temperate germplasm, tropical maize germplasm generally has root systems
with less lateral root structure in the upper portion of the soil profile but with larger and deeper roots
in the lower soil profile (Hund et al., 2009). Tropical growing regions of the world, which account
for about 70 million hectares of production, experience more drought and drought-like conditions
than temperate growing regions (Pingali, 2001), which likely contributed to their evolution of these
root systems and the temperate-derived lines losing them. Deeper roots allow the plant to access
deeper supplies of moisture. Larger roots allow increased water flow, explained by models of xylem
water transport in which the volumetric flow rate of water through the root is proportional to the
fourth power of the radius of the vessel (Taiz and Zeiger, 2006). Root distribution, however, can be
costly to the maize plant, as nutrient distribution in the soil, especially nonmobile nutrients, might
not follow the same pattern as water distribution, causing the plant to be starved of nutrients that are
held only in the uppermost levels of the soil profile (Ho et al., 2005). Deeper exploration of the soil
profile requires energy that can add up to over 50% of daily metabolic production (Lambers et al.,
2002). Therefore, decreasing the subsoil root sink demand during the reproductive period could
allow better allocation of metabolites to the ear (Zhu et al., 2010).
Root development, structure, and distribution, however, should be carefully considered in the
context of how environmental factors will affect them as they will have serious effects on whole
plant health and yield. For example, a hybrid or variety with a root system that develops deeply in
the soil profile would be poorly suited to land that is irrigated with a center pivot, as the water only
penetrates the uppermost portions of the soil profile (Musick et al., 1988). The opposite is true for
furrow irrigated land, which soaks deeply into the soil profile and will hold water deeper for sig-
nificantly longer than a pivot-irrigated field (Musick et al., 1988). An intriguing question can now
be posed. Most of the literature points to studies done on seedling root structure and development
(Voetberg and Sharp, 1991; Bohn et al., 2006), since it is much easier to phenotype seedlings at a
large scale as opposed to mature maize plants. So, what effect does early-season water stress have
on the root system later in the season? Suppose a field is water stressed and the roots develop deeper,
earlier in the season as has been documented (Stasovski and Peterson, 1991). Does this cause the
mature plants to become more drought tolerant later in the season? This environmental effect, along
with known differences between juvenile and adult plants, would have serious complications on
selections made at flowering. A substantial knowledge gap is present concerning the structure and
distribution of mature plant roots. Imaging technologies are helping to create noninvasive tech-
niques to examine plant roots (Bohn et al., 2006; Grift et al., 2011; Surovy et al., 2011); however,
more efforts should be made to understand the relationships between late-season and early-season
root structure. Once the plant has exhausted its ability to explore the soil, or exploration of the soil
profile yields no more moisture, genetic mechanisms in the plant signal the next response: osmotic
adjustment.
40.3.2 Root Extraction of Soil Moisture at Low Saturation Levels

As soil moisture decreases, soil water potential decreases due to the decrease in matric potential:
the attraction of water to a solid surface in the soil (Brady and Weil, 2004). In order for the plant to
be able to take up water adsorbed to the soil surfaces, root cell water potential must be lower than
that of the soil water to create the gradient needed to move the water, or else there is no water flow
into the plant. Maize, like many other plants, decreases osmotic potential by accumulating solutes
in root cells lowering its water potential, allowing flow along the gradient into the plant again
(Morgan, 1984; Bolaños and Edmeades, 1991; Taiz and Zeiger, 2006). Osmotic adjustment in maize
is localized to the elongation zone of the root (Ogawa and Yamauchi, 2006) but has mixed findings
for use in drought tolerance. Bolaños and Edmeades (1991) concluded that osmotic adjustment was
poorly correlated with yield under drought across many tropical and temperate lines. However,
more recently, Chimenti et al. (2006) found detectable differences in yield between lines selected
for both low and high capacities for osmotic adjustment. The authors posed an important question
when they compared their results to those of Bolaños and Edmeades (1991): are there lines that
exhibit constitutive expression of osmotic adjustment while others are drought inducible? This sug-
gests important distinctions between the environment for which the variety/line is being developed
and the origin of material used for improvement.
40.3.3 Metabolic Efficiency of Maize Roots

Roots do not photosynthesize and are a plant photosynthesis sink (Kriedemann et al., 1976; Barnett
and Pearce, 1983) so increasing their metabolic efficiency increases the photosynthate available
elsewhere. Efforts should be made to find traits and genotypes that not only have deep roots with
osmotic adjustment but have roots that are metabolically efficient in their growth (Liedgens and
Richner, 2001). One trait that increases the metabolic efficiency of maize roots is an expansion
of the cortical aerenchyma. Expansion decreases root metabolism and allows an increase in root
growth and water uptake (Zhu et al., 2010). These aforementioned changes in root structure are also
aided by other traits, which occur at a molecular level. Cell wall loosening proteins such as expan-
sins respond to drought by maintaining the elongation rate of the roots under the stress conditions,
allowing the plant to continue root growth in an effort to reach soil moisture (Wu and Cosgrove,
2000). Wu and Cosgrove (2000) also demonstrated that responses that occur in the cell walls of root
cells are complicated, as are techniques of measurement for the different variables in question and
until easier to phenotype it will be difficult to select for.
40.4 LEAF TRAITS INVOLVED IN DROUGHT TOLERANCE

Leaves in maize are very diverse and one of the most easily observed tissues in the growing plant,
increasing opportunities for rapid phenotyping. Leaf length, width, angle, erectness, and color can
be seen readily, while leaf thickness, stomate density, waxiness, and stomatal conductance can be
measured using instruments. The majority of leaf traits tend to be heritable (Flint-Garcia et al.,
2005; Hung et al., 2011), and since leaves are easily accessible, unlike roots, they are prime targets
for measurements and speculative observations on the drought response by researchers and farmers
alike.
40.4.1 Maize Leaf Rolling: Highly Visible but Controversial for Drought

The most visible indicator of the water status of maize is leaf rolling, in which the leaf curls trans-
versely along the edges (Figure 40.3). Leaf rolling has been associated with leaf conductance and
decreased transpiration rates in maize and other crops such as rice (Sobrado, 1987; Kadioglu et al.,
2012). The loss of leaf conductance and decrease of transpiration decrease the rate of photosynthe-
sis, thus decreasing the productivity of the plant. Periods of water deficit cause leaf rolling in maize
and help to reduce water loss where stomatal closure is incomplete (Fernandez and Castrillo, 1999).
In other words, when the plant cannot close all of the stomata, water is still being lost to the environ-
ment, and the capacity to roll its leaves allows the plant to reduce the continued losses. Leaf rolling
also decreases the amount of radiation that is intercepted by the leaves (Kadioglu and Terzi, 2007).
Transpiration rates are reduced in maize through the creation of a microclimate near the rolled leaf
9:00 a.m. 1:00 p.m.

Overnight recovery of turgor Leaf rolling
FIGURE 40.3 Leaf rolling in a drought-stressed field experiment in College Station, TX. Turgor was
regained overnight (a) but was lost again by midafternoon (b). This leaf rolling was likely caused by both
water stress and high incident radiation, which makes leaf rolling a difficult target for drought-based selection.
surface (Oppenheimer, 1960), along with the rapid reduction of effective leaf area (Clarke, 1986).
This has also been shown in maize’s close relative, sorghum (Matthews et al., 1990). Unlike senes-
cence, leaf rolling is a temporary and reversible process, allowing the leaves to unroll once turgor is
reestablished (Begg, 1980). One problem with using leaf rolling as a trait is that it also can appear
with high incident radiation and/or heat stress and might confound estimates of drought adaptation
(Cartwright et al., 2001; Kadioglu et al., 2012). No studies, to our knowledge, have definitely tested
leaf rolling; developments of isogenic hybrids would be a valuable resource to test this trait.
40.4.2 Leaf Cuticle and Cuticular Wax: Layers of Protection

Another conserved mechanism that maize and other higher plants have for desiccation protection
is a cuticle that covers the surface of aboveground plant tissue with a thin layer of material, primar-
ily lipids (Holloway, 1982). The cuticle plays a fundamental protective role against loss of water
especially when the stomata have already closed because of stress (Jenks and Ashworth, 1999).
Furthermore, when the stomata are closed, the epidermal transpiration is inversely proportional to
the cuticle’s thickness or weight per unit area (DeLucia and Berlyn, 1984; Jenks et al., 1994). This
inverse relationship has also been shown in maize leaves measured in the dark (when stomata are
presumably closed) and suggests, along with other studies (Hajibagheri et al., 1983; DeLucia and
Berlyn, 1984), that a thicker cuticle may reduce plant epidermal conductance to water vapor at all
times (Ristic and Jenks, 2002). The leaf cuticle is comprised of a number of layers. The outermost
layer of the cuticle, known as the epicuticular wax layer, is a mixture of long-chained hydrocarbons,
esters, fatty acids, alcohols, and aldehydes (Bianchi and Avato, 1984). The presence of an epicuticu-
lar wax layer has been suggested to benefit drought tolerance in many crops (Bondada et al., 1996;
Luo, 2010). While cuticular wax weight by leaf area is a heritable trait with variation in both maize
inbreds and hybrids, it has unfortunately not been well correlated with epidermal water losses, yield,
or yield stability under stress (Ristic and Jenks, 2002, Meeks et al., 2012). However, it is possible
that the variables are much more complex and could involve polar pores and their distribution, or
wax composition along the leaf and between the cutin meshworks, and deserve future investigation.
40.5 BIOCHEMICAL INTERACTIONS IN DROUGHT

40.5.1 Drought Stress Is Accompanied by Rapid Lipid Peroxidation
Lipid peroxidation occurs during free radical damage to cell membranes under drought, cold, or
high salinity stress (Shalata and Tal, 1998; Hara et al., 2003; Türkan et al., 2005; DaCosta and
Huang, 2007; Anjum et al., 2012). Lipid peroxidation induces changes in the lipid composition of
the membrane, which affect the structural and functional properties of cell membranes (Smirnoff,
1993). Lipid peroxidation is caused by the accumulation of reactive oxygen species (ROS) during
abiotic stress. Singlet oxygen (1O2), superoxide radical (O2-), hydrogen peroxide (H2O2), and the
hydroxyl radical (OH) are the principal ROS in plants (Cruz de Carvalho, 2008). ROS play a dual
role in the abiotic stress response; based on their concentration, they can participate in the stress-
signaling pathways, which contributes to the stress acclimation and proper physiological and meta-
bolic responses. On the other hand, if ROS accumulation reaches high levels, they can initiate an
oxidative burst that will lead to cell death (Cruz de Carvalho, 2008).
Besides ROS-mediated nonenzymatic membrane lipid peroxidation, several enzymes also con-
tribute to this lipid modification process. One of the principal enzymes in lipid peroxidation is
lipoxygenases (LOX). This family of proteins is responsible for the dioxygenation of polyunsatu-
rated fatty acids containing a cis, cis-1,4-pentadiene backbone either free or esterified to complex
membrane lipids (Feussner and Wasternack, 2002; Porta and Rocha-Sosa, 2002; Liavonchanka and
Feussner, 2006; Christensen and Kolomiets, 2011). In case of free fatty acids as the substrates, the
primary product of this reaction is hydroperoxy fatty acids, which are highly reactive compounds
toxic to cells. Therefore, these intermediaries are rapidly metabolized into a vast array of more
stable oxidized lipids called oxylipins. The best studied oxylipins include the phytohormone jas-
monic acid, conjugate dienoic acids, and green leaf volatiles and aldehydes such as malondialde-
hyde (MDA) (Blée, 2002).
40.5.2 Production of Reactive Oxygen Species

Is a Biochemical Hallmark of Drought Stress
Depending on the extent and duration of stomatal closure caused by water deficit, water balance
in the leaves and fixation of carbon dioxide (CO2) is affected. The lack of CO2 fixation reduces the
regeneration of NADP+ by the Calvin cycle, which triggers a major leakage of electrons from the
photosynthetic electron transport chain to O2 by the Mehler reaction, generating superoxide (O2-)
and hydrogen peroxide (H2O2) (Smirnoff, 1993).
ROS are produced naturally by the chloroplast during photosynthesis and photorespiration. The
mitochondrial electron transport chain is also responsible for ROS generation. ROS are increased
significantly under drought stress, which represent a risk to the chloroplast and thylakoid membranes
because of their high content of polyunsaturated fatty acids (Smirnoff, 1993). Lipid peroxides repre-
sent a risk to the cell membrane integrity as is described by studies showing a positive relationship
between the concentration of MDA and reduced electron transport capacity. Singlet oxygen and
hydroxyl radical are the most toxic ROS, and these are minimally produced under normal condi-
tions because of their capacity to oxidize lipids, DNA, and RNA. Under drought conditions, the real
threat to the thylakoid membranes is the production of the hydroxyl radical by the Mehler reaction
through “iron-catalyzed” reduction of (H2O2) by the superoxide dismutase (SOD) and ascorbate.
Plants are natural producers of ROS and have developed scavenging systems that protect cells
against ROS-mediated cellular damage. The major enzymes of this scavenging system are SOD,
metabolites and enzymes of the ascorbate–glutathione cycle, and catalase (CAT). Different studies
have given contradictory evidence regarding the role of each of these systems in scavenging ROS.
However, it is well established that there is a positive relationship between the level of induction of
these antioxidant systems and the degree of drought tolerance (Quartacci and Navari-Izzo, 1992;
Pastori and Trippi, 1993; Sofo et al., 2004; Moussa and Abdel-Aziz, 2008; Torres-Franklin et al.,
2008; Kakumanu et al., 2012; Pal et al., 2012). Similarly, studies have shown that enzymes from the
glutathione metabolism, glutathione-S-transferase (GST), and glutathione peroxidase (GPX) path-
ways are induced under drought stress. GPX has the capacity to scavenge H2O2 and lipid hydroper-
oxides, which further protect the cellular membranes under drought stress (Sasaki-Sekimoto et al.,
2005; Torres-Franklin et al., 2008). Antioxidants also play a role under drought stress in scavenging
ROS and preventing membrane damage. Alpha-tocopherol has been shown to accumulate under
drought stress. This antioxidant has the ability to scavenge the most dangerous ROS including lipid
peroxyl radicals, hybroxyl radicals, and singlet oxygen (Munné-Bosch, 2005). Another strategy of
the plant during drought stress is shifting the production of H2O2 to the peroxisomes to prevent
formation of hydroxyl radicals by the Mehler reaction. This is accomplished by the increase in
the photorespiration rate by the RuBisCO enzyme, which produces glycolate. Glycolate is trans-
ported to peroxisomes, where it is oxidized to produce glycine, a precursor of glutathione and H2O2
(Cruz de Carvalho, 2008).
Diverse ROS molecules can be measured, but most are relatively unstable and accumulate for
relatively short periods of time, and have been proven challenging to measure at the scale necessary
for breeding applications (Shulaev and Oliver, 2006). Furthermore, diverse ROS species are often
located in different subcellular compartments, requiring sophisticated and expensive detection
methods, which are impractical in high-throughput screening. Differences in specific antioxidants
or their enzymes are more practical to measure by flash freezing and homogenizing tissue and using
analytical chemistry (Fu and Huang, 2001, Stapleton and Walbot, 1994) but are relatively low throughput.
A higher-throughput method relevant to evaluate ROS damage from stress is to measure electrolyte
leakage (Blum and Ebercon, 1981). However, electrolyte leakage is a crude approximation of dam-
age and cannot pinpoint the specific mechanisms that lead to cellular damage. Thus, there is a need
for a robust and high-throughput quantitative analysis of ROS and antioxidants.
40.5.3 Hormones Play an Important Role in Drought and Whole Plant Physiology

Hormones allow the plant to grow and coordinate physiological responses to diverse biotic and abi-
otic environmental stresses. Phytohormones, abscisic acid (ABA), auxins, cytokinins, jasmonates
(JA), and salicylic acid, among others, serve as the mobile molecular signals that regulate timing,
duration, and type of response to a specific stress (Wang et al., 2008). Among these hormones,
indole-3-acetic acid, cytokinin, polyamines, zeatin, gibberellin3, and ABA have been the most
clearly shown to affect drought, while the other hormones have not been well investigated. Recent
evidence suggests that other hormones such as ethylene and JAs play a critical role in plant adapta-
tion to drought (Golldack et al., 2011). Because of large pleiotropic effects of diverse hormones on
many important traits, transgenic manipulation of hormone levels themselves may not be the best
target to improve drought tolerance in plants. Instead, it is commonly accepted that downstream
genes regulated by specific hormones may prove to be more useful targets for improvement.
40.5.4 ABA Is a Major Hormone Implicated in the Adaptation to Drought Stress

ABA is one of the most important and extensively studied hormones with respect to drought adap-
tation (Cutler et al., 2010). ABA accumulation in higher plants is thought to act as an early signal
for the initiation of processes involved in adaptation to drought (Hartung and Davies, 1991; Bray,
1993). Under water stress, ABA accumulates in the roots and aboveground parts of the plant and is
involved in the regulation of the growth and the opening and closing of the stomata (Sauter et al.,
2001; Schroeder et al., 2003; Taiz and Zeiger, 2006; Ren et al., 2007; Sirichandra et al., 2009).
A major function of rapid induction of ABA is to reduce the amount of water lost through
transpiration (e.g., closing of the stomata) and greater uptake of water through the roots (Setter,
1997). ABA has been shown to decrease the relative growth rates of leaves and increase the growth
rates of roots, thereby increasing the root-to-shoot ratio (Sharp et al., 1994). ABA increases hydrau-
lic conductance for water movement, thereby enhancing the transport of water from the roots to the
leaves (Zhang et al., 1995). Wilting of plants is usually accompanied by an increase in ABA levels,
but correlation versus causation has not be determined (Taylor, 1991). ABA is also one of the best
understood components in the initiation of the aforementioned process of osmotic adjustment (Ober
and Sharp, 2003). ABA has been shown to regulate changes in efflux and uptake of ions and anions.
Recent studies shed light on the importance of nitric oxide (NO) and hydrogen peroxide in affecting
potassium uptake and the efflux channels in the guard cells (Hetherington and Woodward, 2003;
Yan et al., 2007; Sirichandra et al., 2009; Kim et al., 2010; Wilkinson and Davies, 2010). A single
nucleotide polymorphism (SNP) was statistically associated with ABA levels within maize silks
during water stress, suggesting that the gene containing the SNP might be involved in the regulation
of the ASI (Setter et al., 2011).
40.5.5 Ethylene Reduces Growth under Drought Stress

Ethylene plays an important role in plants under drought response. Ethylene synthesis begins in
root in response to water deficit. Similar to ABA, ethylene is a mobile phytohormone that trav-
els from roots to shoots. Water deficit in the roots triggers biosynthesis of the ethylene precur-
sor 1-aminocyclopropane-1-carboxylic acid (ACC) that is transported via xylem sap to shoots
where the enzyme ACC oxidase (ACO) synthesizes ethylene in shoots (Gagne et al., 2004;
Young et al., 2004; Manavella et al., 2006; Wilkinson and Davies, 2010). Different studies
using mutant and transgenic plants have shed light on the role of ethylene during drought stress.
One of the most relevant studies was the study by Gagne et al. (2004) that shows that ethylene-
insensitive Arabidopsis mutants exhibited improved leaf growth. ACC synthase mutants also
reveal a possible role of ethylene in decreasing photosynthetic ability under drought condi-
tions (Young et al., 2004). Recent transcriptome analyses showed that ethylene biosynthesis
and signaling genes are upregulated during drought stress (Skirycz et al., 2011). More sup-
porting evidence for the role of ethylene in drought stress was published by Manavella et al.
(2006), who characterized the transcription factor Hahb-4 in sunflower. Hahb-4 is a member
of the subfamily I of HD-Zip proteins that is regulated transcriptionally during water stress.
This study showed that overexpression of this transcription factor repressed the biosynthesis
of ethylene. Consequently, transgenic plants that overexpressed Hahb-4 displayed delayed leaf
senescence, which allowed sunflower to maintain photosynthetic activity for a longer time
under drought conditions. These results suggest that Hahb-4 is involved in the regulation of
ethylene-dependent genes (Manavella et al., 2006).
40.5.6 Role of Methyl Jasmonate and Jasmonic Acid in Drought Tolerance

JA and its volatile derivative, methyl JA (MeJA), have been extensively studied. Their function in
plant development and response to biotic stresses is well established (Creelman and Mullet, 1997;
Blée, 2002). By contrast, the role of JAs during drought stress is not yet clear. A molecular and phys-
iological analysis of drought stress in Arabidopsis revealed that JAs are involved in stomata closure
in early drought responses and homeostasis of plant cell in late drought response, which contribute
to yield under drought stress (Suhita et al., 2004; Harb et al., 2010). Furthermore, other studies have
shown that JA synthesis and antioxidant activity increases under drought (Sasaki-Sekimoto et al.,
2005). Similar results were obtained in a study in soybeans. The authors demonstrate that the exog-
enous application of MeJA increases the activity of antioxidant enzymes such as SOD, peroxidases
(POD), and CAT. This study also showed that the concentration of proline, which is known for
its contribution to maintaining osmotic balance, was increased in soybeans as well (Anjum et al.,
2011). Other studies have found conflicting results with the possible role of JAs in sustaining yield
under drought conditions. A study in rice and peanuts found that the application of exogenous JA
decreased yield (Kim et al., 2009a,b).
40.5.6.1 Phenotyping of Biochemical Properties

Much like measuring of ROS and antioxidants, large scale phenotyping of plant hormones is
difficult. Most previous work has focused on the phenotyping of responses to plant hormones,
and much of the work has been conducted in climate-controlled greenhouses or growth cham-
bers. To detect changes or genetic variation in plant hormones, even the most recent methods
require flash freezing and the use of HPLC and mass spectrometry and only result in the mea-
surement of some hormones (Forcat et al., 2008). Hormones also present the complication that
their measurement is tissue and time point specific making inferences limited to the growth
stage and experimental conditions used. It is important to better understand these hormones,
the role they play in drought stress, and the genetic diversity of production and response, but
until more high-throughput methods can be identified, they are unlikely to be useful for field-
based phenotyping.
40.6 USING PHYSIOLOGICAL TRAITS FOR CROP IMPROVEMENT

Now that the traits and adaptive mechanisms of the maize plant have been discussed, we will revisit
some of these, along with others used as selection criteria, from the perspective of crop improve-
ment. The basic research involved in most of the aforementioned studies has been critical to the
understanding of the mechanisms of drought tolerance and avoidance; however, understanding
Screening environments
Early Full
season Manageable? season
Is screening Late Repeatable? Partial Is germplasm
environment season season cultivatable in
appropriate for screening
trait? Yield environment?
potential
Traits Germplasm
Selectable? Adapted? Pleiotropy
Roots Heritable? Available?
Leaves Epistasis
Flowering Linkage Markers
Hormones
Seed set Inbred Open pollinated
Does germplasm segregate for

appropriate trait of interest?
FIGURE 40.4 The drought stress triangle. Many different factors, each interacting in a complex manner
with the others, contribute to drought tolerance in maize. Careful consideration of the target environment,
screening environment, germplasm used, molecular tools used, and traits screened and selected must be done
in order for an improvement to be made.
alone is not adequate for crop improvement. Many of these traits can have deleterious pleiotropic
effects and their interactions are unknown. Additionally, some traits are likely conserved across the
species, and we are unlikely to identify any substantial or useful natural genetic variation. Previous
studies have investigated some drought tolerance traits and calculated heritability estimates (Table 40.1).
Heritability estimates are, not surprisingly, varied for the different traits considered, likely due to
large measurement error and high environmental variances under stress conditions. The interac-
tions between these traits, the germplasm in which they are being screened, and the screening
environment are highly complex. We summarize these interactions as the “drought stress triangle”
(Figure 40.4). Each of the three major factors must be carefully considered when an experiment is
being designed to ensure that confounding effects are minimized. Interdisciplinary cooperation
between basic research and field breeding has and will continue to determine whether the afore-
mentioned traits and adaptive mechanisms are beneficial as selection criteria for the improvement
of maize to tolerate drought conditions.
40.6.1 Environments for Screening and Selection

Many different methods have been used to screen and select maize for drought traits. To our
knowledge, no comprehensive study has been undertaken to look at the advantages and disadvan-
tages of these methods, largely due to the difficulty in measuring each phenotype. It is clear that
one of the most important aspects to genetic gain in selection procedures for drought tolerance
is the proper management of the timing, duration, and intensity of the water stress (Bruce et al.,
2002). Each treatment serves the purpose of exposing the genetic variation for the traits evalu-
ated in such a way that the plants would experience similar conditions in a farmer’s field. One of
the more successful institutional examples in breeding maize for drought tolerance, as defined
by citations and imitation, is the work of CIMMYT, which first initiated breeding specifically
for drought tolerance in their tropical germplasm in 1975. The method described by Edmeades
et al. (1987), a maize physiologist with CIMMYT, consisted of growing maize under three dif-
ferent controlled water regimes: no moisture stress, stress during grain filling, and full-season
stress. Selections of the top 30%–40% of full-sib families were made for eight cycles using the following
TABLE 40.1
Reported Heritabilities of Traits Involved in the Improvement of Maize for Drought
Tolerance
Developmental Stage Reported Screening Estimated
Trait for Phenotyping Environment Heritability References
Seedling traits
Seedling drought Seedling Germination/post- 0.07–0.10 Meeks et al. (2013)
tolerance germination stress
(greenhouse)
Leaf traits
Cuticular wax on a Flowering Full season 0.58 Meeks et al. (2012)
weight basis
Leaf rolling Flowering Flowering and through 0.56 Bolaños and Edmeades
grain fill (1996)
Flowering traits
Anthesis Flowering Flowering through 0.68a Sari-Gorla et al. (1999)
maturity
Silking Flowering Flowering through 0.58a Sari-Gorla et al. (1999)
maturity
ASI Flowering Flowering and through 0.60 Bolaños and Edmeades
grain fill (1996)
ASI Flowering Flowering through 0.40a Sari-Gorla et al. (1999)
maturity
Root traits
ABA levels Flowering At flowering 0.43 Setter et al. (2011)
Lateral root elongation Seedling Post-germination stress 0.57 Ruta et al. (2010)
(growth chamber)
Lateral root length Seedling Post-germination stress 0.60 Ruta et al. (2010)
(growth chamber)
Osmotic adjustment Flowering Flowering and through 0.46b Bolaños and Edmeades
grain fill (1991)
Ear traits
50-kernel weight Maturity (harvested 2 weeks before anthesis 0.94a Frova et al. (1999)
ears) through plant maturity
Ear length Maturity (harvested 2 weeks before anthesis 0.86a Frova et al. (1999)
ears) through plant maturity
Note: Heritabilities range from 0.07 to 0.94. Not surprisingly, those traits that are more influenced by the stress environment
and prone to measurement error such as the ASI and osmotic adjustment are lower.
a Measure across well-watered and water-stressed environments.
b Realized heritability h2 = R/S, where R = response to selection and S = selection differential.
criterion: maintenance of days to anthesis, maintenance of yield under drought stress, and a short-
ening of the ASI. Bruce et al. (2002) notes that in the process of this selection and subsequent
reporting, CIMMYT created an ideotype that most others have used when breeding for drought
tolerance: high grain yield, short ASI, low level of leaf senescence under intermediate water stress
while maintaining yield stability, and small tassels and upright leaves under well-watered condi-
tions. It is important to remember to also make observations in a favorable environment, ensuring
that the yield potential of lines being moved forward in selection is relatively high. This ensures
that selection does not favor plants that only perform well under drought and have low potential
in favorable environments (Edmeades et al., 1994).
Selection for drought-associated traits needs to be conducted in carefully controlled envi-
ronments to minimize additional effects that may occur. For example, selecting against kernel
abortion could involve providing sufficient moisture up to and including flowering and then with-
holding supplemental irrigation after pollination at different levels and for different durations to
evaluate the ability of the lines to maintain kernel set. The question that arises at this stage of
selection is what trait(s) are actually being selected for or against when kernel abortion is being
evaluated. In other words, is there selection pressure exhibited on a gene, or group of genes, that
directly controls kernel abortion? Or is the selection against kernel abortion simply an indirect
method for selection of lines with superior alleles for other secondary traits such as improved
root systems or flowering physiology because of epistatic effects and/or pleiotropic effects present?
When considering environments for selection, even if irrigation could be strictly controlled, rela-
tive maturities and days to flowering will have confounding effects on the final analyses. This
could occur if the breeder decides to impose stress only at flowering and not during grain fill, and
the duration of flowering time spans 20 days from the earliest line to the latest line. Table 40.2 out-
lines ways to manage this. It is likely that a well-established breeding program will have a target
maturity for the majority of its material, but this might not always be the case especially when
extreme genetic variation is created introgressing more exotic drought-tolerant material. Relevant
genetic gains are dependent upon the skill in reproducing an environment that is identical or
similar to the one from the previous selection cycle and adapted to the trait being investigated.
Similarly, if this selection environment is different or artificial from what the grower will experi-
ence, the gain is not useful.
TABLE 40.2
Methods for Dealing with the Confounding Factor of Flowering Time Differences in
Diverse Germplasm Screened for Drought Tolerance
Advantages Disadvantages
(1) Ignore flowering time Simplest method, no additional Could be imposing stress/selection on trait of
differences. work needed. interest in material flowering at one point in time
while material flowering at other times outside of
stress/selection may escape causing erroneous
conclusions to be drawn about the trait
(2) Measure flowering time, Simple. Measurement necessary Could still be imposing stress/selection on the
harvest all material at same for ASI also. Easy post-harvest wrong trait. Significant loss in statistical power.
time, and use flowering correction, plant and harvest all Cannot truly separate effect but can determine
time to partition out samples at the same time, how much error it is causing
variation. consistent with traditional field
management.
(3) S
tagger drought stress Stress is imposed at correct time Difficult to manage and requires expensive
based on factor (i.e., for each plot, which treats all monitoring equipment and intensive irrigation
maturity/days after anthesis) material alike. management
(4) Classify material into Virtually eliminates differences in Cannot make comparisons between different
specific maturity groups material. Stress is imposed at maturity groups. Makes management of the
based on target adaptation same physiological stage across program more difficult as more space/time/
and test each group all maturities. Should put stress analysis is needed
separately. on same trait.
Note: Stress at flowering and the 2 weeks after is crucial, and germplasm may vary more than 2 weeks for flowering time.
40.6.2 Methods of Selection and Traits Utilized in the

Conventional Selection of Drought-Tolerant Maize
Drought can occur at many stages and with various intensities. Because of the inherently large
genotype by season, genotype by location, and genotype by microenvironment interactions present,
mass selection of plants within populations is not a beneficial method for making selections resistant
to drought (Jackson et al., 1996) especially if flowering time differences are not taken into account
(Table 40.2). For areas of the world that still grow open-pollinated varieties and for organizations
like CIMMYT, population improvement techniques such as reciprocal recurrent selection are popu-
lar and have been successful (Bolaños and Edmeades, 1993). These have been used to develop new
populations such as the well-known Tuxpeno Sequia, DTP1, and DTP2 (Monneveux et al., 2006).
From such populations, superior lines can be selected and then inbred for use as parents for hybrid
combinations following the pedigree or other breeding methods.
Quantification of and selection for drought tolerance in maize is usually calculated and carried out by
measuring decreases in yield per unit area in a drought-stressed treatment plot versus a control plot, since
the most economically undesirable response of maize to drought stress is a reduction in yield (Bruce
et al., 2002). However, since the heritability of grain yield under drought conditions is very low, even rela-
tive to low heritability of grain yield under well-watered conditions, selection based only on grain yield
is not highly beneficial (Monneveux et al., 2008). For this reason, secondary traits have become a major
focus in the progress of selection. Lafitte et al. (2003) defines secondary traits as plant characteristics
other than grain yield that provide additional information about how the plant performs under a given
environment. A good secondary trait should be associated with grain yield under drought conditions,
have genetic diversity, and be highly heritable. Low cost and ease of measurement are also beneficial for
secondary traits, and developing these methods remains an important area for future studies. Among
secondary traits ASI is likely the most commonly used. Decreases in the heritability of grain yield in
water-stressed environments are accompanied by increases in the heritability and genetic variance of
the ASI and ears per plant (Bolaños and Edmeades, 1996). In other words, as the effect of asynchronous
flowering becomes more predictable, yield becomes less predictable. Bolaños and Edmeades (1996) also
describe that in order to maximize the genetic gain per cycle, the environment must be carefully man-
aged to create a mean ASI of at least 5 days and limit irrigation to drop the mean ears per plant below 0.7.
While intuitive, in their effort to elucidate the degree of impact that the ASI has on the drought tolerance
of maize, Bolaños and Edmeades (1996) concluded that progress with secondary traits is limited until
grain yield under stressed environments can be established. Remember that ASI is not an indicator of the
water status of the plant but has effects that are directly related to (current or past) water stress. A sensible
first step when initiating breeding for drought tolerance is to shorten the ASI in order to reveal genetic
diversity for other traits (Bolaños and Edmeades, 1996).
40.6.3 Direct Selection of Nonreproductive Secondary Traits

Phenotypic selection of secondary traits related to plant water status such as leaf rolling, rooting
depth, and leaf wax content can be measured relatively easily and for minimal cost. The effort can
be made here to complete the ideotype described earlier by selecting for lines that exhibit low leaf
senescence, extensive leaf rolling, and deep roots. Secondary traits involved with physiological pro-
cesses can also be evaluated (e.g., ABA and osmotic adjustment). Looking at these conclusions and
at the challenges presented, there seems to be a significant gap between the understanding of the
physiology of the drought response and the genetic information used for selection.
40.7 GENOMICS, GENE DISCOVERY, AND MARKER-ASSISTED SELECTION

While advances in genomics technology have been remarkable over the past 15 years, the complex-
ity of the drought response and its many pathways has made accurate phenotyping a major chal-
lenge (Tuberosa and Salvi, 2006). Failure to accurately phenotype renders a quantitative trait locus
(QTL) study useless, and QTL for very small effects or those with high genotype by environment
interactions are very difficult to detect (Beavis et al., 1994). This establishes the continual need for
conventional breeding and breeders, as their skill and experience with plants is absolutely essential
for the successful application of genomics to breeding for drought tolerance.
While recent QTL studies have been numerous and successful in identifying significant QTL (Lu
et al., 2010, 2011b; Messmer et al., 2011), many are focused on the ASI (Beavis et al., 1994; Zehr et al.,
1994; Ribaut et al., 1996; Liu et al., 2010) and are outside the scope of this review. Despite the inher-
ent environmental influences on the expression of QTL that have been linked to drought tolerance and
especially ASI (Liu et al., 2010), there could be benefits from the use of marker-assisted selection (MAS),
which can eliminate the need to reproduce the given environment in which the initial phenotype was
seen (Ribaut et al., 1996). However, if the QTL is in trans-linkage with a QTL expressed in other envi-
ronments and in the opposite direction, this approach of using MAS alone may be counterproductive.
Once the ASI has been shortened sufficiently, lines being evaluated for their drought tolerance
should begin to show genetic diversity for other secondary traits that are measureable and can
be selected. Banziger et al. (2000) nicely outlines that if the genetics underlying the phenotype
observed in the field cannot be properly identified, there is no way to use molecular techniques to
aid in selection.
40.8 CONCLUSIONS
Water availability will continue to limit production of maize in the southern United States and
worldwide. Drought tolerance is a highly quantitative and complex trait involving many genes from
many different pathways. Basic research should be targeted toward understanding of the pathways
in an effort to find additional targets for selection. Physiology efforts should develop methods to
rapidly and inexpensively quantify the importance of secondary traits. Breeding efforts should be
divided and focused on the specific target adaptation, as the target environment and relative timing
of drought stress is crucial. Pleiotropy and epistatic effects are likely to hamper efforts to create the
ideal drought-tolerant phenotype, as pathways involved in ear shoot development, root development,
ASI, kernel abortion, and the other traits mentioned in this review are likely interconnected through
common hormones or precursors. Linkage of these genes with small effects further restricts prog-
ress without impractically large populations.
A problem outlined by Banziger et al. (2000) is that phenotypic selection for improvement of
drought tolerance has not improved WUE of lines due in part to the limitations of methodology
used. It has been argued that in classical breeding, selection for secondary traits involving leaves
and roots becomes unfruitful as the association between these secondary traits and yield is signifi-
cantly reduced over the course of continuous selection. As the fields of plant breeding and genetics
move from classical phenotypic selection toward increased reliance on molecular techniques, paral-
lel efforts to make selection more efficient and to improve the phenotypes screened and selected
for will be just as important as identifying specific genes. Even with new technology, traditional
field breeding techniques will still be necessary to create variability to be evaluated, to regulate the
stress environment, and to search for new traits useful to screen lines for drought tolerance. The
sheer complexity of the drought stress response and its many pathways gives hope that there are still
many unexplored natural traits and genetic variation present that can be used to help create new,
more robust, drought-tolerant ideotypes.
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41 New Approaches to
Turfgrass Nutrition
Humic Substances and
Mycorrhizal Inoculation
Ali Nikbakht and Mohammad Pessarakli
CONTENTS
41.1 Introduction........................................................................................................................... 917
41.2 Humic Substances.................................................................................................................. 918
41.3 Mycorrhizal Inoculation........................................................................................................ 918
41.4 Mode of Action of Humic Substances and Mycorrhizal Inoculation.................................... 919
41.4.1 Nutrient Uptake......................................................................................................... 919
41.4.2 Plant Growth, Root Development, and Architecture................................................. 921
41.4.3 Plant Quality.............................................................................................................. 923
41.4.4 Stress Alleviation....................................................................................................... 923
41.4.5 Other Beneficial and Physiological Effects...............................................................925
41.5 Conclusions............................................................................................................................925
References.......................................................................................................................................926
41.1 INTRODUCTION
The use of turfgrasses plays an important role in designing urban areas and sport facilities.
The major goal of turfgrass management programs that needs to be carried out properly is to pro-
duce beautiful, appealing, attractive, and healthy turfgrasses. On the other hand, turfgrass areas
are designed to resist traffic. Turfgrass nutrition, which through adequate fertilization, is one of the
most important components of a turfgrass management program. Fertilization greatly affects turf
quality, including color, density, and uniformity. Turfgrass managers need to meet two apparently
opposite demands and typically use high-nutrient fertilizers in an attempt to maximize turfgrass
quality: (1) the demands of the users (athletes need to play and enjoy high-quality turfgrasses and
amblers desire to see a well-manicured turf) and (2) ecological concerns. In addition, fertilized turf-
grasses normally compete with weed invasions more efficiently and are able to recover from dam-
ages imposed by stresses than improper fertilized turfs. However, high-input fertilization programs
normally lead to some side effects. From an environmental point of view, high toxicity to soil, high
leaching potential, and suppressing impact on soil microorganisms are important issues. On the
other hand, this may lead to an increase in maintenance costs by using more chemical fertilizers.
A careful and economic use of fertilizers and other inputs becomes increasingly important with
regard to increasing serious environmental problems. Improving efficiency of nutrients can reduce
these environmental concerns and economic losses and have beneficial effects on plant growth
917
(Christians, 2007). Hence, new approaches must consider low-input turfgrass management systems.
Sustainable turfgrass systems rely on natural processes to maintain acceptable turfgrass quality
and follow long-term solutions rather than reactive measures at system levels. These systems must
be ecologically sound, economically viable, and socially responsible (Harrier and Watson, 2004).
Humic substances (HSs) and mycorrhizal inoculation are discussed in this chapter as two potential
approaches toward these goals.
41.2 HUMIC SUBSTANCES
HSs, including fulvic acid (FA) and humic acid (HA), naturally occurring polymeric organic
compounds, are produced by the decay of organic materials, and are found in soil, peat, and lig-
nites (Sharif et al., 2002). The chemical properties of HS are known to vary considerably from FA
to HA and their fractions. FA is reported to be larger in total acidity and carboxyl group content
and is assumed to be considerably higher in adsorption and cation exchange capacity (CEC) than
HA (Schnitzer and Khan, 1972; Stevenson, 1994; Tan, 2003). FA is water soluble under all pH
conditions, while HA is less water soluble in high pH (Schnitzer, 1990). HA is the main segment
of HS and the most active component of soil and compost organic matter (Ferrara et al., 2007).
These compounds are the products of decomposition of plant tissues and are predominantly
derived from lignified cell walls (Turkmen et al., 2004). These materials have several benefits
such as phytohormone-like activities (Fike et al., 2001; Nikbakht et al., 2008; Pizzeghello et al.,
2001) that directly and indirectly have stimulating effects on the physiological processes of plant
growth (Yang et al., 2004). It is reported that HAs have beneficial effects on nutrient uptake by
plants and are particularly important for the transport and availability of micronutrients (Bohme
and Thi Lua, 1997). Hypotheses that account for the stimulatory effects of HA are numerous,
the most convincing of which is a direct action on the plant that is hormonal in nature, together
with an indirect action on the metabolism of microorganisms, and the dynamics of uptake of
soil nutrients and substrate physical conditions, through positive effects on seed germination,
seedling growth, root growth, and shoot development (Cacco and Dell’Agnolla, 1984; Chen and
Aviad, 1990; Piccolo et al., 1991).
41.3 MYCORRHIZAL INOCULATION
Under natural conditions, about 90% of all plant species form a symbiotic association with arbuscu-
lar mycorrhizal fungi (AMF) (Brundrett, 2002; Smith and Read, 1997). Generally, the importance
of mycorrhizal relationship is ignored, because mycorrhizal association occurs underground and
is not visible. They are important soil microorganisms and are considered as obligate symbiotic
biotrops, which rely on the carbon provided by the host plant (Siddiqui and Pichtel, 2008). AM
symbiosis provides many benefits to host plants, including enhanced plant growth (Kim et al., 2010),
mineral nutrition (George, 2000), and resistance to abiotic stresses, including drought (Abbaspour
et al., 2012; Auge, 2001) and salinity (Sharifi et al., 2007), when compared to similar noncolonized
(non-AMF) plants. In this symbiotic association, the fungus colonizes the plant’s root hairs by
entering the cortex cells and acts as an extension of the root system (Muchovej, 2001). Mycorrhizal
fungi association have been found in most turfgrass species, including creeping bentgrass (Agrostis
palustris), velvet bentgrass (Agrostis canina), annual bluegrass (Poa annua), fescues (Festuca
rubra), perennial ryegrass (Lolium perenne L.), wheatgrasses (Agropyron spp.), and some other
grasses. The most abundant mycorrhizal fungi species reported were Glomus, Acaulospora, and
Entrophospora (Kafi et al., 2013; Koske et al., 1997; Skálová and Vosátka, 1998). Some researchers
noted that mycorrhizal fungi naturally colonize new greens turf without being added as an inocu-
lum. This means special effort should be made to conserve this naturally occurring colonization
(Gemma et al., 1997b).
New Approaches to Turfgrass Nutrition 919
41.4 MODE OF ACTION OF HUMIC SUBSTANCES

AND MYCORRHIZAL INOCULATION
41.4.1 Nutrient Uptake
The most considerable effect of both HSs and mycorrhizal symbiosis is improving nutrient uptake
(Daneshvar et al., 2013; Giasson et al., 2006; Nikbakht et al., 2008). It is shown that addition of
HA increased maize nitrogen (N) accumulation over control with no significant differences within
the treatments of different levels of HA applied (Sharif et al., 2002). It is suggested that a higher
phosphorous (P) availability is a consequence of some protection given by HAs against precipita-
tion (Chen and Aviad, 1990). Some researchers have concluded that HA may enhance the uptake of
P indirectly by complex formation with Fe (David et al., 1994). It is reported that HA from wheat
straw leachate can inhibit the formation of insoluble Ca phosphates and, thus, may enhance P and
Ca bioavailability (Grossl and Inskeep, 1991). Although some researchers reported that HA applica-
tion improves the uptake of most elements, including N, P, K, Mg, Ca, Fe, and Zn (Fagbenro and
Agboola, 1993), it is also reported that HA may decrease the absorption of some elements, espe-
cially in higher concentrations. In a study of pepper plants irrigated with solutions containing 8,
80, and 100 mg L−1 HA, it was found that there were an increase in the uptake of N, P, and Mg and
a decrease in the uptake of K and Ca in higher concentrations (Sanchez-Conde and Ortega, 1968).
HA has the ability to increase the permeability of cell membranes (Valdrighi et al., 1996) by its
hormone-like activities (Zhang et al., 2003a), which seem to increase with increasing HA concen-
tration. In a study on the effect of HA on Ca absorption in tomato hydroponic production, it was
reported that HA enhanced Ca uptake, but it was also mentioned that greater concentrations of HA
decreased Ca uptake (Turkmen et al., 2004). High concentrations of HA in wheat hydroponic grow-
ing resulted in HA–Ca flocculation (Grossl and Inskeep, 1991). It seems that the contrasting results
on the effect of HS on plant nutrition might be partially related to different soil, media, origination
of HS, and also the species treated by HS. It was reported that nutrient uptake was not affected
significantly by the applications of solid and liquid HA forms on strawberry plants (Pilanali and
Kaplan, 2003). These researchers (Pilanali and Kaplan, 2003) concluded that because of having
excessive calcareous soil in the experimental site, uptake of nutrients in strawberry plants was not
affected significantly. It is believed that a major benefit of HA in agricultural systems is its ability to
form complex metal ions and it can form aqueous complexes with micronutrients, though not to the
same extent as many synthetic chelating agents (Aiken et al., 1985). However, it is suggested that the
lowering of the uptake might be caused by complex substance formations of Fe, Mn, Zn, and Cu by
the HS (Kreij and Basar, 1995). Some other researchers suggested another possible HA mechanism
in improving Fe uptake. For instance, it is proven that the reduction of Fe3+ to Fe2+ by HA was a
possibility to explain a higher Fe availability for plants, as shown in the Adani et al. (1998) study.
According to Sanchez-Sanchez et al. (2002), the addition of HS to Fe-EDDHA improved Fe uptake
by lemon trees. Therefore, it is suggested that HS could be used as an inexpensive chelating agent in
order to increase Fe uptake. Other mechanisms suggested by other researchers show that the organic
acids such as citric, oxalic, tartaric, and α-ketoglutaric acid typically present in root exudates can
extract Fe from humic complexes (Pietramellara and Piccolo, 1991). DeKock (1955) proposed that
without HA, Fe would form insoluble precipitation with P. Excessive P was proven to interfere with
Zn uptake as well as translocation and metabolism (Mills and Jones, 1996). This supports the find-
ings that Zn accumulation significantly decreased with an increase in P uptake up to 1000 mg L−1
in gerbera (Gerbera jamesonii L.) leaves (Nikbakht et al., 2008).
It was reported that the enhancement in root growth and nutrient uptake, which is obtained by
incorporating pig manure vermicompost and food waste vermicompost, was due to their HA con-
tents, not nutrient changes caused by composts (Atiyeh et al., 2002). This means that having just a
high nutrient concentration in the media cannot always guarantee enough nutrient uptake and the
states should also be optimized to enhance nutrient uptake that is in agreement with our findings.
There are some reports showing the beneficial effect of HS on enhancing turfgrass nutrient uptake.
For example, in our recent work on perennial ryegrass (L. perenne), different concentrations of
HA (0, 100, 400, and 1000 mg L−1) were applied monthly as foliar application, and results showed
that leaf P, potassium (K), and zinc (Zn) contents were not affected by HA, while 100 mg L−1
HA improved N and Fe contents, which are the most important elements affecting turfgrass color
(Daneshvar et al., 2013). It was reported that HA had no influence on the concentrations of P, K, Fe,
Mo, and Zn in creeping bentgrass (Agrostis stolonifera), although the tissue concentrations of Mg,
Mn, and sulfur (S) were increased (Liu et al., 1998). In a study, creeping bentgrass (A. stolonifera)
was grown in both sand and a hydroponic system, and it was shown that spraying HA from differ-
ent sources increased P content in plants grown in sand culture by 3%–5%, but not in hydroponic
system. Some investigators documented that HA might have limited promoting effects when plants
are receiving adequate nutrients (Cooper et al., 1998; Daneshvar et al., 2013). Some other research-
ers also believe that HA improved P uptake in ryegrass only in the case of very low P availability
(Bidegain et al., 2000). The results of a comprehensive work showed that HS can reduce the stan-
dard fertilizer program input in creeping bentgrass (A. stolonifera) (Zhang et al., 2003a). It seems
that HS spray can maximize the efficient use of some nutrients, reduce fertilizer costs, and help
release those plant nutrients presently bound in minerals and salts, especially when incorporated in
soil (El-ghamry et al., 2009). As it was stated earlier, the leaching of major nutrients occurs from
sand-based root zones. It was reported that when HA was applied to the turf (A. stolonifera) at a
rate of 5 L ha−1, it quickly reduced nitrate, but not phosphate, leaching from the root zone. This has
been attributed to the high CEC of HS. However, it did not affect the nutritional status of the leaf
tissue (Hunter and Anders, 2004).
It is believed that the most important factor facilitating symbiosis between AMF and its host
plant is an exchange of nutrients. The host plant provides carbon for the AMF and in return fungus
delivers nutritional elements to the plant (Siddiqui and Pichtel, 2008). The extra radical mycelium
of AMF absorbs phosphate, ammonium, nitrate, micronutrients, and amino acids from the soil
(Hodge et al., 2001). It should be noted that under heavily fertilized conditions that normally occur
in high-input agricultural systems such as intensive turfgrass management, the performance of colo-
nized plants can fall below that of noncolonized ones. This is because the cost of colonization is up
to 20% more than total carbohydrate produced by the host plant (Janos, 2007). Some researchers
believe that AM species can be used to inoculate plants to compensate for Zn and P deficiencies
under P and Zn deficient soils (Ortas et al., 2011). The result of a comprehensive 4-year study on
the beneficial effect of AMF on putting greens proved that P fertilization rate affected how well the
vesicular AMF (VAMF) performed. The most vigorous mycorrhizal turfs were those that received
frequent applications of a low-P fertilizer solution rather than too high or too low P concentrations in
which mycorrhizae did not improve the plant growth (Gemma et al., 1997a,b). In a study, the effect
of AMF colonization by Glomus mosseae on creeping bentgrass (A. stolonifera L.) and Kentucky
bluegrass (Poa pratensis L.) showed that the AM grass symbiosis could be successfully employed
as a method for the reduction of fertilizer input in the environment (Charest et al., 1997). It should
be noted that the response of AMF colonization could be species dependent. For instance, the
responses of six wheatgrass (Agropyron spp.) cultivars to mycorrhizal inoculation resulted in dif-
ferent P concentrations in the plant leaves. Inoculation increased leaf P concentration in four of the
six cultivars; however, N concentration was not significantly affected by mycorrhizal inoculation
for any of the cultivars (Di Jian and Allen, 1991). It was reported that differences in plant growth
were largely due to changes in root morphology and mycorrhizal colonization in plants with differ-
ent P fertilization regimes (Gahoonia et al., 1999; Yan et al., 1995). In our recent work, when both
HA spray and AMF colonization by Glomus intraradices and G. mosseae tested on L. perenne
simultaneously, neither HA treatments nor mycorrhizal inoculation affected N and Fe contents of
the leaves. However, P, K, and Zn concentrations were improved by AMF inoculation. More roots
were colonized by G. intraradices than G. mosseae. With the help of AMF, a host plant can obtain
more nutrients and plant resistance to undesirable condition can be enhanced (Cheng et al., 2007;
Gaur et al., 2000; Kim et al., 2010). Our results showed significant differences in the mycorrhizal
species responses in ryegrass nutrition. It was shown that G. intraradices was more efficient in
promoting some elements uptake into turfgrass plants than G. mosseae (Daneshvar, 2010). It seems
AMF colonization, especially under low-input fertilizer conditions, can be an appropriate method
to enhance nutrient uptake by turfgrasses. However, further investigation is needed to cast a light on
different dimensions of optimum turfgrasses colonization in open fields.
41.4.2 Plant Growth, Root Development, and Architecture

It was reported by many researchers that HSs influence plant growth and generally improve the
quality of the crops (Nikbakht et al., 2008). However, there are reports showing that this effect is
more evident on root growth, especially when HSs are incorporated into soil or media. For instance,
a study on gerbera showed a greater effect of HS on root growth than that of the shoots, indicating
probably greater resource allocation to the roots than the leaves (Adani et al., 1998; Atiyeh et al.,
2002; Nikbakht et al., 2008; Turkmen et al., 2004). In a study, wheat (Triticum aestivum) root
and shoot growths were compared when the plants were grown in water alone and in a complete
(Hoagland) nutrient solution. Each solution was supplemented with 50 mg L−1 HA. The results
showed a 58% increase in root growth when HA was added to water alone. Addition of HA to
plants growing in nutrient solution increased root growth approximately 25% compared with plants
growing in nutrient solution alone (Vaughan and Malcolm, 1995). It was shown that incorporated
humate in creeping bentgrass (A. stolonifera L.) grown in sand culture stimulated a 45% increase in
root mass compared with non-treated turf (Cooper et al., 1998). Higher concentrations of HA can
stimulate root growth and produce highly branched root system, and this may result in an increase
in surface area, which could facilitate more efficient nutrient absorption (Nikbakht et al., 2008).
Atiyeh et al. (2002) reported that the enhancement in growth is due to HA ability in improving
nutrient uptake by roots. This improvement could be attributed partially to the enhancement in the
root growth. The further alternative explanation on root growth could be the hormone-like mode of
action of HA. It is shown that extracted plant growth regulators such as indoleacetic acid, gibberel-
lins, and cytokinins from HA derived from vermicompost had significant effects on plant growth,
including root development (Atiyeh et al., 2002). Increased rooting following HA application was
found in many crops, including tomato (Lycopersicon esculentum) (David et al., 1994), maize (Zea
mays) (Canellas et al., 2002), and Kentucky bluegrass (P. pratensis) (Zhang et al., 2003b). Moreover,
the positive effects of HS on plant growth and productivity, which seem to be concentration-related,
could be mainly due to the hormone-like activities of the HS (Chen and Aviad, 1990; Vaughan
et al., 1985; Zhang and Ervin, 2004; Zhang and Schmidt, 2000; Zhang et al., 2003a). Some studies
have shown a more intense effect on root development triggered by FAs than HAs (Chen and Aviad,
1990). Reduced growth associated with high HS levels is often attributed to reduced micronutrient
availability to plant roots. It could be possibly due to excessive ligands and physical properties of
the medium becoming unfavorable for growth (Ayuso et al., 1996; Dudley et al., 2004; Rauthan and
Schnitzer, 1981).
Liu et al. (1998) reported that HA solution had no effect on root regrowth and actually
reduced root length at a low concentration, but 400 mg L −1 HA visually produced more devel-
oped root mass. The findings of other researchers also indicate that incorporation of granular
HA into the root zone produced significantly longer roots and greater root mass deeper in the
root zone (Cooper et al., 1998). In our recent work on perennial ryegrass (L. perenne L.), the
roots of plants that received 100 mg L −1 HA through spraying produced more developed roots
and grew more than the roots of plants that received higher concentrations (Daneshvar et al.,
2013). HA application in our study had little effect on root parameters in higher rates, and lower
concentration was more effective. Consequently, although HA foliar application could improve
root architecture, it had little effect on root growth of the plants. The optimal levels of humic
materials to enhance root and shoot growth are 50–300 mg L −1 (Chen and Aviad, 1990), which
is very species dependent. Previous works have demonstrated that exogenous applications of HA
may be causing endogenous shifts in the balance of hormones, increasing cytokinins, IAA, and
ABA levels, while decreasing gibberellins (Zhang et al., 2003a). Katkat et al. (2009) showed that
HA increased dry matter weight in wheat at low concentrations. However, species behavior may
be different in response to HA application. This might be the reason that foliar application dif-
ferently affects plant growth and development. Also, some researchers showed that spray appli-
cation of HA on plants had no effect on root growth. Perhaps, this problem was related with no
tangent HA with roots (Cooper et al., 1998; Liu et al., 1998). It was found that HAs were effec-
tive on root architecture and growth of creeping bentgrass (A. stolonifera) and also increased
the plant resistance to drought stress (Hunter and Anders, 2004). In an attempt to improve the
establishment of Kentucky bluegrass (P. pratensis L.) sod, HA from peat (47 g m−2) and HA
from leonardite (58 g m−2) were foliar-applied every 2 weeks. The results showed that both treat-
ments significantly increased root mass (73% and 34%, respectively) and root strength (Ervin
et al., 2008). This shows that the origin of HA can also be important on root growth (Ervin
et al., 2008). The result of applying HA on bentgrass (A. stolonifera) showed that it improved
root architecture and growth of the growing plants (Hunter and Anders, 2004). To sum up, it
seems HS can be successfully applied to improve the root growth. The most appropriate rate and
method of application should be optimized locally, which could be spraying or incorporating the
HS into the soil/media.
There are many reports showing the improving effect of AMF colonization on general growth
of plant species. However, this enhancement could be related to aboveground, underground, or
both parts of a plant. The enhancement of the host plant roots is one of the major effects of AMF
colonization. It is described that the fungal hyphae extend beyond the host root system, allowing
a greater soil volume to be exploited for nutrient uptake (Siddiqui and Pichtel, 2008). Mycorrhizal
dependency (plant response to colonization with respect to nutrient acquisition and plant growth)
has been shown to change both between crop species and within species (Kafkas and Ortas, 2009).
The formation of mycorrhizae induces great changes in the physiology of the roots, in the internal
morphology of plant, and in the soil surrounding the roots (Martin et al., 2007). However, there
are some reports showing that the infection has decreased root biomass, for instance, for some
cultivars of Agropyron. Interestingly, this reduction in root biomass has not led to general growth
decline (Di Jian and Allen, 1991). The result of a work on creeping bentgrass (A. stolonifera L.) and
Kentucky bluegrass (P. pratensis L.) infected by G. mosseae implied that root density was not sig-
nificantly altered by mycorrhizal colonization. However, mycorrhizal turfgrasses tended to produce
more aboveground biomass over time (Charest et al., 1997). In our study, ryegrass (L. perenne L.)
growth improved by AMF inoculation. It was observed that inoculation with AMF resulted in a
higher growth (height, fresh and dry weights) than control treatments. AM inoculation resulted in
improved root architecture rather than root biomass production. It was shown that there were some
differences between fungi species, and the plants inoculated with G. intraradices had the largest
response in establishment when inoculated at seedling stage compared to inoculated plants with
Glomus etunicatum at the same stage (Pelletier and Dionne, 2004). This increase in plant growth
parameters might be firstly attributed to the stimulatory effect on nutrient uptake and then the result
of enhanced plant growth regulator production in plants that have a strong stimulatory impact on
plant growth (Artursson et al., 2006). In sum, it seems that the effect of AM colonization on turf-
grass roots is more dedicated to root architecture, rather than root biomass. This might be related
to the fact that most turfgrasses have fibrous developed root under normal soil conditions. The
results of our recent work suggest the importance of further investigation to understand the impact
of HS nature and AMF interactions and their effects on plant growth. It should be considered that
a decrease in plant height in turfgrasses, while plants are experiencing an improvement in biomass,
could be of economic importance. Any treatment controlling turfgrass height would lead to a reduc-
tion in mowing frequency (Christians, 2007). This will result in substantial savings in turfgrass
management expenses.
41.4.3 Plant Quality
Plant quality is a concept that is most attributed to the ornamental plants. As far as turfgrasses are
concerned, the quality, which is a measure of aesthetics, consists of four components, including
turfgrass density, uniformity, texture, and color. Turfgrasses must be properly fertilized to keep
their best color and remain healthy. It should be considered that normal high-quality color should
not be always dark green as some species are naturally yellow-green or blue-green. The nutrient
will influence turfgrass quality, growth, color, and stress tolerance (Christians, 2007). Meanwhile,
in regard to turfgrass color, the most important nutrients are N and Mg, which are the main compo-
nents of chlorophyll molecule in leaves. Although Fe is not a part of the chlorophyll molecule, it is
one of the nutrients essential for chlorophyll synthesis. On the other hand, chlorophyll is the mole-
cule that performs photosynthesis and sufficient amount of this compound in the leaves is necessary
for plant maintenance, health, and quality. Many treatments aim to elevate chlorophyll content in
the leaves. Enhancement of chlorophyll content with application of HS in nutrient solutions or foliar
spray has been reported (Ferrara et al., 2007; Vaughan and Malcolm, 1995). However, in our previ-
ous work, we observed no significant effect on chlorophyll content by spraying HA on perennial
ryegrass (L. perenne L.) (Daneshvar et al., 2013). These results were contradicting with the reported
results, indicating improved chlorophyll content in creeping bentgrass (A. stolonifera) following HS
application (Chen et al., 1999). These investigators (Chen et al., 1999) explained that HA-mediated
maintenance of Fe and Zn at sufficient levels is the key factor in elevating chlorophyll content in
the leaves. However, there are some reports showing that HA was not effective on chlorophyll con-
tent in creeping bentgrass (A. stolonifera), but could enhance net photosynthesis (Liu et al., 1998).
Some researchers showed that no differences were observed in the chlorophyll content of the turf
with any HS treatment suggesting that turf color and visual quality are not enhanced by HS (Van
Dyke et al., 2009). On the contrary, some researchers showed that chlorophyll content significantly
increased by the application of HA interacted with amino acids (El-ghamry et al., 2009). It was
demonstrated that chlorophyll content of perennial ryegrass (L. perenne L.) grown in amended soil
with 5%–100% composted sewage sludge was greatly improved (Cheng et al., 2007). It seems that
the effect of HA on chlorophyll content and photosynthesis is species and concentration dependent.
The reports on the effect of mycorrhizal inoculation on chlorophyll content of turfgrasses are
rare. However, there are some contradictory reports on chlorophyll content of different species. Our
recent work shows that mycorrhizal inoculations with G. intraradices and G. mosseae significantly
increased turfgrass visual quality compared to noninoculated control. The best visual quality was
recorded in pots inoculated with G. mosseae. Plants inoculated by G. intraradices showed 15%
increase in total chlorophyll content. Total chlorophyll content increased when plants were colo-
nized by AMF, especially G. intraradices. Higher total chlorophyll content may be due to changes
in the plant metabolism (Kim et al., 2010) and might result in enhanced plant growth and biomass
production (Daneshvar, 2010; Kohler et al., 2007). There are some reports indicating that the chlo-
rophyll contents of creeping bentgrass (A. stolonifera L.) and Kentucky bluegrass (P. pratensis L.)
were not significantly altered by mycorrhizal colonization in either grass species (Charest et al.,
1997). Some researchers believe that the concentration of chlorophyll is higher in mycorrhizal than
nonmycorrhizal plants under drought stress that is shown in lettuce (Lactuca sativa) (Baslam and
Goicoechea, 2012). The same finding is reported for maize (Z. mays L.) (Zhu et al., 2012).
41.4.4 Stress Alleviation
Both biotic and abiotic stresses affect growth and quality of turfgrasses. Traffic is the most signifi-
cant stress on sport fields, which is a specific stress to turfgrasses. Hence, many efforts have been
made to alleviate these stresses on turfgrasses. Meanwhile, application of HS and AMF is consid-
ered to increase resistance to the stresses. In a comprehensive work, it is well documented that the
foliar application of HA alone or in combination with seaweed extracts (SWE) improved creeping
bentgrass (A. stolonifera L.) resistance to drought stress. The researchers believe that this effect is
mainly associated with the hormonal components, not the improved nutrient uptake by HA (Zhang
and Ervin, 2004). They showed that two mechanisms might be involved in resistance to drought
stress following HA application. The first one, which is hormonal in nature, describes that HA con-
tains significant amounts of cytokinins that appear to be associated with increased leaf cytokinin
levels. The second one is the protective mechanism induced by higher levels of antioxidant activity
containing higher α-tocopherol and superoxide dismutase (SOD) content in the leaves of bentgrass
(A. stolonifera L.) induced by these compounds (Zhang and Ervin, 2004; Zhang et al., 2003a). The
same result is reported on Kentucky bluegrass (P. pratensis L.) subjected to drought stress. Foliar
application of HA plus SWE increased α-tocopherol, ascorbic acid, β-carotene content, and SOD
activity, especially under low soil moisture (Zhang et al., 2003a). These compounds increase over-
all physiological health of the plant and are associated with protection of the photosynthetic appa-
ratus and alleviation of heat damage to both shoots and roots (Zhang and Ervin, 2004). Reports
on the alleviation of all kinds of stresses following the application of HA are rare, but the results
reported on other crops indicate the potential of these compounds to improve the general health
of plants through activation on antioxidant activity in plants. For instance, it is documented that
HA from leonardite could increase SOD and peroxidase (POD) content of the gerbera plants even
under half-strength nutritional program (Nikbakht, 2007). Increased leaf antioxidant activity may
improve abiotic stress tolerance, including drought tolerance by scavenging reactive oxygen spe-
cies and protecting membrane integrity (Smirnoff, 1995). It is also implied that foliar application
of HA + SWE may reduce shipment heat injury and improve post-transplant rooting and quality
of tall fescue (Festuca arundinacea Schreb.) sod and tolerance to stress during storage (Zhang
et al., 2003c). It was demonstrated that HA applied to creeping bentgrass (A. stolonifera) at a rate
of 5 L ha−1 improved the plant’s resistance to drought (Zhang and Ervin, 2004). HA application
at 150 mg m−2 during summer abiotic stresses was beneficial to creeping bentgrass (A. palustris
Huds.) (Zhang et al., 2002).
It is well documented that the AMF not only increase the rate of nutrient transfer from the
roots to the host plant, but they also increase resistance to biotic and abiotic stresses (Martin et al.,
2007). The literature presents numerous reports on the AMF tolerance and adaptation to stresses.
Resistance refers to the ability of microorganisms to withstand the effects of stresses usually effec-
tive against them, while tolerance refers to the ability of microorganisms to adapt to the persistent
presence of the stresses (Giasson et al., 2008). These improving effects are considerable in the
case of heavy-metal stress alleviation, which is important when a turfgrass field is constructed
in a polluted soil. The mechanisms are summarized as (1) P nutrition by activating P absorption,
(2) chemical precipitation of heavy metals in the soil, (3) tissue dilution due to increased shoot and
root biomass, (4) hyphal sequestration of metal, and (5) root immobilization of metals (Giasson
et al., 2008). The results of mycorrhizal symbiosis influence on L. sativa physiological response
under drought stress on lettuce showed that AMF colonization improved the accumulation of anti-
oxidant compounds, mainly carotenoids and anthocyanins (Baslam and Goicoechea, 2012). The
influences of AMF on maize (Z. mays L.) plants exposed to drought stress for 4 weeks showed
that, although drought stress significantly decreased AM colonization, the results implied that AM
symbiosis alleviates the toxic effect of drought stress via improving photosynthesis and water status
of maize plants (Zhu et al., 2012). Similarly, it is demonstrated that AM symbiosis protects maize
plants against low-temperature stress through improving the water status and photosynthetic capac-
ity (Zhu et al., 2010). As far as turfgrasses are concerned, it is reported that application of microbial
inoculants and SWE treatments into the root zone of creeping bentgrass (A. stolonifera L.) signifi-
cantly alleviated stress side effects (Butler and Hunter, 2008; Butler et al., 2007). It was also shown
that a lawn mixture of Kentucky bluegrass (P. pratensis L.), red fescue (F. rubra L.), and peren-
nial ryegrass (L. perenne L.) inoculated with G. intraradices established more quickly than non-
AMF-treated turfgrasses (Pelletier and Dionne, 2004). The results of a study on creeping bentgrass
(A. palustris cv. Penncross) and velvet bentgrass (A. canina cv. Kingstown) showed considerable
effect of AMF on drought resistance of turfgrasses. In a greenhouse study, turf without mycorrhizae
began wilting after 3 days, but mycorrhizal plants were wilted after 5 days (Gemma et al., 1997a).
In the same way, protection against drought stress was conferred by G. intraradices when turf was
grown under conditions of low-phosphorus fertilization; however, the benefits disappeared when the
P concentration of the fertilizer was quadrupled (Gemma et al., 1997a). To put it briefly, it seems that
HS and AMF colonization solely or in combination with other plant-enhancing treatments, includ-
ing natural plant growth regulators (such as SWE), would be beneficial to alleviate and overcome
environmental stresses during the stress period.
41.4.5 Other Beneficial and Physiological Effects

The beneficial effects of HS and AMF colonization are not limited to the already mentioned influ-
ences. Several researchers have noted that both HS treatments and AMF association influence different
physiological aspects of plants; however, the physiological process might be different. The following
are the most important reports on the role of HS and AMF colonization in turfgrass management.
In an interesting study, AMF was successfully applied to control P. annua, a major weed in golf
putting greens. In greens where AMF were relatively high, P. annua was rare. Furthermore, when the
fungi were common, abundance of the main turfgrass (A. stolonifera) was greater. On the other hand,
where mycorrhizal inoculum was added to a golf green, the colonization level of A. stolonifera roots
and A. stolonifera abundance were enhanced. AMF might have altered the balance of competition
between the two grasses or the fungi may have directly reduced the growth of P. annua. It shows that
AMF have a great potential to be a much more environmentally sound method of controlling weeds
(P. annua in this case) in sports turf than the currently used chemicals (Bary et al., 2005; Gange
et al., 1999). Further investigations are needed to show the applicability of this method to control
other weeds in sport turfgrasses. There are also some reports showing that inoculation of turfgrasses
with AMF could enhance the establishment of turfgrasses. For instance, a report shows that the
establishment improved and enhanced when a lawn mixture of Kentucky bluegrass (P. pratensis L.),
red fescue (F. rubra L.), and perennial ryegrass (L. perenne L.) was inoculated with two Glomus spe-
cies. Turfgrass inoculated with G. intraradices at rates between 40 and 60 mL m−2 established more
quickly than turfgrass inoculated with G. etunicatum (Pelletier and Dionne, 2004). Another study
suggests that the establishment of the AM grass symbiosis, creeping bentgrass (A. stolonifera L.) and
Kentucky bluegrass (P. pratensis L.), not only improved the establishment, but also could be consid-
ered as a potential agent for the reduction of fertilizer input in the environment (Charest et al., 1997).
As mentioned earlier, it is proven that spraying HA could improve rooting of Kentucky bluegrass
(P. pratensis), which is a primary grass species used for athletic fields leading to better establishment.
This is a main concern during sod establishment, especially in sand-based sport fields (Ervin et al.,
2008). It was observed that no AM activity was found in newly constructed sand-based root zones,
suggesting that root colonization could be of potential use for newly constructed turfgrass fields
and pitches (Butler and Hunter, 2008). In summary, it seems that the successful application of both
AMF and HA could be a useful and environmentally sound management process in any turfgrass
management program in the future. However, more investigations might be needed to clarify the
most feasible method of application for widespread usage during turfgrass establishment, renovation,
and maintenance. The synergistic effect of AMF inoculation with other plant-enhancing compounds
could be a matter of interest for further investigations.
41.5 CONCLUSIONS
• New approaches in turfgrass management must consider low-input turfgrass management
systems to reduce environmental impacts and lower turfgrass maintenance costs.
• The most considerable effect of both HSs and mycorrhizal symbiosis is improving soil
mineral nutrient uptake.
• HSs have beneficial effects on nutrient uptake by plants and are particularly important for
the transport and availability of micronutrients.
• Mycorrhizal symbiosis provides many benefits to host plants, including enhanced plant
growth and health and resistance to abiotic stresses.
• Mycorrhizal symbiosis, especially under low-input fertilizer conditions, can be an appro-
priate method to enhance nutrient uptake by turfgrasses.
• The effect of AM colonization on turfgrasses roots is more dedicated to root architecture,
rather than root biomass.
• Both HS application and AM symbiosis alleviate the adverse effects of stresses such as
drought, heat, and salinity partly via antioxidant protective systems. On the other hand,
HA contains significant amounts of cytokinins that appear to be associated with increased
leaf cytokinin levels and improved stress resistance.
• HS and AMF colonization solely or in combination with other plant-enhancing treatments,
including natural plant growth regulators, would be beneficial to alleviate and overcome
environmental stresses during the stress period.
• AMF and HA applications could be considered as a useful and environmentally sound
management process in any turfgrass management program.
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42 Use of Sewage in Agriculture
and Related Activities
Manal El-Zohri, Awatief F. Hifney,
Taha Ramadan, and Refat Abdel-Basset
CONTENTS
42.1 Overview and Prospective..................................................................................................... 931
42.2 Definition and Characteristics of Wastewater and Sludge..................................................... 933
42.3 Sewage Uses in Agriculture and Aquaculture....................................................................... 934
42.3.1 In Agriculture............................................................................................................ 934
42.3.1.1 Effect of Sewage Application on Soil and Plant......................................... 935
42.3.1.2 Case Studies for Sewage Application in Agriculture................................. 939
42.3.2 In Aquaculture........................................................................................................... 947
42.3.2.1 Case Studies of Sewage Application in Aquacultures................................948
42.3.3 Other Beneficial Uses of Sewage Effluents............................................................... 949
42.3.3.1 Formation or Recharge of Underground Water Reservoirs
in Deserts........................................................................................ 949
42.3.3.2 Thermal Value with Subsequent Implications on Land and Air................ 949
42.3.3.3 As Adsorbents............................................................................................. 952
42.4 Most Common Drawbacks of Sewage Application............................................................... 953
42.4.1 Pathogenic Micro- and Macroorganisms.................................................................. 953
42.4.2 Organic Pollutants..................................................................................................... 953
42.4.3 Pharmaceuticals and Other Compounds................................................................... 954
42.4.4 Potentially Toxic Heavy Metals................................................................................. 954
42.4.5 Soil Water Repellency............................................................................................... 955
42.5 Sewage Treatments................................................................................................................ 955
42.5.1 Physical Unit Operations........................................................................................... 956
42.5.2 Chemical Unit Processes........................................................................................... 956
42.5.3 Biological Unit Processes.......................................................................................... 956
42.5.3.1 Bacteria in Water Purification.................................................................... 956
42.5.3.2 Algae in Water Purification........................................................................ 957
42.6 Conclusion............................................................................................................................. 958
References....................................................................................................................................... 958
42.1 OVERVIEW AND PROSPECTIVE

Sewage water is a complex mixture in terms of chemical, biological, and physical characteristics.
It is part of the most renewable wastewaters, while it should not be, at least in arid and semiarid
countries. A trogonal problem of aridity, increasing population, and mismanagement of natural
resources exists in many areas of the world, predominantly in the southern hemisphere and politi-
cally referred to as developing countries. Increasing population per se imposes the adoption of
new strategies for handling wastewater including sewage water more efficiently, especially when
931
combined with shortage of food resources. Green areas not only provide crops for food and fodder
but also enrich the global atmosphere with oxygen. Plants also absorb oxides of carbon, nitrogen,
and sulfur (COX, NX, and SX, respectively), thus cleaning the atmosphere and making it healthier.
Subsequently, greening any area on earth by cultivating any kind of green cell must be of global
concern, not neglected as a national issue.
Using wastewater in agriculture might play a role in solving the predicted energy crisis.
Carbohydrate-accumulating plants (sugarcane, wheat, rice, potatoes, corn, etc.) and oil-accumulating
plants (corn, sunflower, olive, etc.) might be cultivated for bioethanol or biodiesel production, or for
methane and hydrogen production, or pyrolyzed. If concerns on human health from wastewater-
irrigated crops are high at a global level, inedible plants (forest trees, grasses, etc.) must replace aridity.
Planting Jatropha and Jojoba, for example, to produce bio-oil in desert lands using treated wastewater
(TWW) is part of a very large forestation scheme in Egypt. Therefore, the Ministry of Agriculture and
Land Reclamation and the private sector are taking up cultivation of such plants in many sites of the
country, especially in the south (Abdel Wahaab and Omar, 2011). Algal ponds, lakes, or lagoons are to
be set up or constructed deliberately to transform low-quality water (e.g., sewage) and low-quality land
(e.g., sand) into valuable resources. Algae grow very quickly, tolerate a wide range of harsh conditions,
and perform gas exchange (CO2 and O2) much more efficiently than higher plants. Their contribution,
with other ocean hydrophytes, may approach at least 75% in the stabilization of atmospheric CO2/O2.
Algae have been quite popular in biotechnological research since a long time, and this popularity is
increasing. Algae are being extensively and intensively studied for the production of biofuels (biohy-
drogen, biodiesel, and methane) and pharmaceuticals. Furthermore, they can participate in purify-
ing or amending wastewater by their documented ability to accumulate minerals (e.g., phosphorous,
nitrates, and heavy metals). In addition, their photosynthetic oxygen kills the anaerobic bacteria and
germs that are mostly harmful to the living cells as well as to the environment. In aquacultures, the
organic nature of and the oxygen produced by algae are important for raising fish.
Deserts are arid areas found in both temperate and tropical regions, mainly between 15° and 40°
north and south of the equator, with perceptibly distinct characteristics. Of particular concern in this
chapter are the temperature, pH, and permeability to water. Arid soils of these deserts are extremely
dry and hot, and their sandy soil is highly permeable to water. Hence, we hypothesize that the great
majority of germs in sewage water would be killed quickly by the combined action of the extreme condi
tions of solar radiation, high temperature, and desiccation. The extreme permeability of the sandy
soil leaks water quickly into the underground, that is, the surface soil where germs most probably
attach becomes very dry in an extraordinarily short time, another lethal effect on germs and coli-
forms abundant in sewage water. In Egypt, for example, or in southern latitudes, the atmospheric
temperature goes up to 40°C and more with bright sunshine during the day; the highest temperature
prevails over several months in a year. Unfortunately, most of the scientific research, investigation,
reporting, consciousness, and strict legislations are dominant in industrial and developed countries
that are mostly located in the north. Therefore, most of the research is conducted in cold, humid
environments, in clay soils with low permeability and high water holding capacity due its richness
in organic matter (OM). The characteristic features of arid environments, that is, humidity, tempera-
ture, pH, permeability, and other edaphic factors (compared with wet environments), were the major
motivators for this chapter.
One of the most critical steps in any reuse program is to protect public health, especially that
of workers and consumers. To this end, it is most important to eliminate any infectious agents or
pathogenic organisms that may be present in the wastewater. For some reuse applications, such as
irrigation of nonfood crop plants, secondary treatment of sewage water may be acceptable. For other
applications, further disinfection by methods such as chlorination or ozonization may be necessary.
In desert lands, water leaks form underground reservoirs or enrich existing ones. The water that
travels to the underground gets filtered while permeating soil layers (vadose zone till the a quifers).
However, there is some concern that harmful chemicals present in the soil might pollute this water.
Also, even after separating the sludge and other solids, sewage water will contain suspended impurities
Use of Sewage in Agriculture and Related Activities 933
of organic nature. Such suspended particles exert a dual impact on sand soil: the first is the improve-
ment in water holding capacity of sandy soil, which renders it more cultivable by edible or inedible
plants; the second impact, which may arise due to prolonged disposal of sewage effluent, is the block-
age of the capillary pores between sand particles, leading to bad aeration and gas exchange, which
become lethal to roots in the form of hypoxia or even anoxia, making it less cultivable. The sewage
effluents either continue to dispose in the same area or are in another area depending on the local
situation and available alternatives for the decision makers. Aridity is characterized by alkalinity.
The alkaline nature of the desert sand soils is of particular importance in the context of poor nutri-
ents, bioavailability, and toxicity for plants growing in sewage water. On average, soils with higher
OM contents have lower pH (Cooperative Extension, solutions to soil problems ii. high pH [alkaline
soil] reviewed 2010 Utah State University). Thus, it is advised to amend the alkalinity of the arid soil
by supplementing OM and elemental sulfur or applying acid fertilizers such as ammonium sulfate.
Subsequently, alkalinity of arid soils may become self-treated by the OM contained in sewage efflu-
ents. Also, some common garden plants have been suggested that adapt to soil pH of neutral–alkaline
(7.0–8.0) such as asparagus, beets, cabbage, cauliflower, celery, carrot, lettuce, parsley, and spinach.
Alkalinity, in addition, is an important factor limiting heavy metal availability for plant roots since
solubility of the most of them decreases at high pHs.
The reuse of water is becoming a must, especially in arid regions. The scarcity of water resources
is expected to become more severe in the coming years, primarily because of the growing consump-
tion of water and also because of climate change (World Bank, 2009). Therefore, the increasing
demand for clean (or at least safe) water sets the necessity of appropriate technology and management
of the available and already endangered water resources. Industrial and domestic wastewater reuse
is now considered a basic component of integrated water management, especially in countries in the
arid and semiarid regions of the world (Lazarova and Asano, 2005), like Egypt and most other Arab
countries. In Egypt, for instance, the entire population, along with their activities (e.g., industry),
inhabits the delta and the valley of river Nile, which accounts to only about 5%, whereas the rest of
the country is almost arid or has salt-affected soils devoid of life. Furthermore, the area of arable land
available to feed this population is shrinking due to expansion of housing, significant loss of topsoil
and groundwater (desertification), and other reasons. Estimates suggest about 1000 km2 is lost annu-
ally due to desertification in Africa alone (Jones, 1997).
Minerals contained in sewage water and sludge might play a vital role in redistributing min-
eral elements, thus compensating for depletion by repeated cultivation. At the same time, however,
foreign elements are introduced into the soil, which might ultimately appear in the food chain as
they accumulate in plants and animals. Risks in using sewage water and sludge in agriculture and
drawbacks of long-term application on soil, plants, animals, and humans do exist and are described
in the chapter. However, prevention is not the solution; treatment and management techniques for
improving hygienic properties must be developed. Sewage sludge, in addition to being used as a soil
fertilizer, is used in energy generation (hydrogen, methane, heat, etc.), road paving, adsorbents, and
other uses described briefly in this chapter. Beneficial and safer alternatives of sewage uses, espe-
cially in energy generation, are also summarized.
42.2 DEFINITION AND CHARACTERISTICS OF WASTEWATER AND SLUDGE

Wastewater is water used by either residential or nonresidential communities. About 99%–99.94%
of wastewater is water, and the rest is solid waste (Martijn and Huibers, 2001; Vigneswaran and
Sundaravadivel, 2004). Unless properly treated, wastewater can harm public health and the environ-
ment. However, it is defined as liquid wastes normally collected in a sewer system and processed in
a treatment plant. Wastewater from residential areas comprises two components:
• Blackwater, which is toilet discharge

• Greywater, which is shower, washing machine, and kitchen sink discharge
On the other hand, sewage sludge is defined by the US Environmental Protection Agency (US EPA,
1993) as the primarily organic solid product yielded by municipal wastewater treatment processes
that can be beneficially recycled for soil amendments. It is defined by Victorian EPA (2000) as sta-
bilized organic solids derived from biological treatment processes, which can be managed safely to
sustainably utilize their nutrient, soil conditioning, energy, or other values.
Sludge is a heterogeneous matter, varying in composition from city to city or even day by day.
Municipal sewage sludge consists of organic material, mainly dead bacterial cells, and inorganic
components in the form of various oxides and salts (Code of PAUSS, 1989; Standards of UDSS,
1993; Renner, 2000). The composition of sewage sludge is mainly dependent upon the population
density and that population’s habits, in addition to the proportion and nature of the community’s
industrial base. Concentrations of plant macronutrients in sewage sludge vary widely; averaging
median concentrations of a typical sewage sludge would contain 3.2% N, 1.4% P, and 0.23% K.
Except for K, these nutrient values are similar to those of animal manures (Hue, 1992). Potassium
content of sewage sludge is inherently low because most K compounds are water soluble and remain
in the sewage effluent or in the aqueous fraction during sludge dewatering. By the same mechanism,
a portion of inorganic N, particularly NH4, which is enriched during sludge digestion, can be lost.
However, organic N by far is the largest fraction (50%–90%) of the total N in any sludge. Unlike N,
only 10%–30% of the total P in anaerobic sludge is organic P (Sommers et al., 1976). The remaining
P is inorganic, in the form of Ca, Fe, and Al phosphates, and P is sorbent on amorphous material of
Fe, Al, and Mn hydrous oxides (Sommers, 1977).
Sewage may vary considerably in composition and strength from place to place owing to marked
differences in the dietary habits of the people, composition of trade waste, and water consumption.
The strength of sewage is determined by the amount of O2 required to oxidize completely the OM
and ammonia present in it. Chemical and biological oxygen demands (COD and BOD, respectively)
are strong and easy assessing factors for water purity. Also, there are variations in composition
between domestic and industrial sewage; the latter contains more pollutants in terms of heavy met-
als, bacterial load, and other toxic ingredients, while the domestic sewage is very rich in anaerobes
when it is raw but gradually transforms to enriched water after treatment. Sewage contains liv-
ing matter, especially bacteria and protozoa. Domestic sewage has been reported to contain about
250–400 ppm of organic carbon and 80–120 ppm of total nitrogen, resulting in a C/N ratio of
around 3:1. Industrial sewage may contain more organic carbon and hence may have a higher C/N
ratio. Nitrogen in sewage is present partly as organically bound element and partly as ammoniacal
nitrogen (Balkrishna, 2007).
42.3 SEWAGE USES IN AGRICULTURE AND AQUACULTURE

42.3.1 In Agriculture
Large-scale urbanization and industrialization is leading to production of huge quantities of
effluents all over the world. Industrial and domestic effluents are either used or disposed on land
for irrigation purposes that have both pros and cons. Sewage effluents from municipal origin are
rich in OM and also contain appreciable amounts of major and micronutrients that are essential
for crop growth (Feign et al., 1991; Pescod, 1992; Gupta et al., 1998; Brar et al., 2002; WHO,
2005). Accordingly, nutrient levels of soils are expected to improve considerably with continuous
irrigation with sewage that will reduce reliance on chemical fertilizers to enhance crop produc-
tion (Narwal et al., 1993; Brar et al., 2002; Wang et al., 2007). Generally, reusing wastewater for
irrigation in agricultural cultivation is a common practice in many countries, especially in arid
and semiarid regions. Rassoul (2006) reported that the supply of water is being augmented by
domestic wastewater reuse in countries like Saudi Arabia, Kuwait, Tunisia, Jordan, and Yemen.
Even in areas where wastewater is not the sole water source for agricultural irrigation, farmers still
prefer using sewage for irrigation by reason of its nutritive value, which reduces expenditure on
mineral fertilizers (Pescod, 1992; Lazarova and Asano, 2005). In arid and semiarid areas, waste-
water irrigation may significantly increase farm production. At a flow of 140 L per capita per day,
100,000 people would generate about 5 Mm3 of wastewater per year, enough to irrigate 1,000 ha
(hectares) at a rate of 5,000 m3/ha/year, using efficient irrigation methods. With inefficient meth-
ods, this amount of water could still irrigate 250–500 ha in arid regions. This 5 Mm3 of wastewater
contains about 250,000 kg of nitrogen, 50,000 kg of phosphorous, and 150,000 kg of potassium
(World Bank, 1994).
Hence, reusing domestic and industrial wastewater in agriculture for irrigating crops and agri-
cultural lands appears to be a lucrative option and is on the rise especially in peri-urban areas.
Among the other advantages of reusing wastewater is its convenient disposal of discharge water
in addition to adding valuable plant nutrients and OM to the soil. According to Murcott (1995),
the ideal characteristics of wastewater useful in crop irrigation are as follows: (1) high organic and
nutrient content (especially N and P), (2) low pathogen content, and (3) low contents of metal and
toxic organic compounds.
However, depending on the source of the sewage, wastewater may contain potentially harm-
ful components such as heavy metals and pathogens, which can accumulate in soil and biological
systems and prove to be hazardous (Rattan et al., 2005). These hazards may limit long-term use
of effluents for agricultural purposes as a likelihood of phytotoxicity and environmental effects.
In fact, the long-term impact of irrigation with poor-quality water on physicochemical properties
of soils has been reported to depend upon the interaction of several factors like the unique water
quality limiting parameters and site-specific crop, soil, and climatic conditions (Minhas and Gupta,
1992). Therefore, when sewage is used on land for irrigation purposes, the problems associated with
its use should be considered (Emongor and Ramolemana, 2004).
42.3.1.1 Effect of Sewage Application on Soil and Plant

Wastewater is recognized to have a direct effect on soil chemical properties. Several studies have
reported an increase in the water retention capacity of soils following sludge application. Sewage
sludge affects soil water retention either through the direct effect of its particles or through an
indirect effect on other physical properties such as bulk density, porosity, and pore size distribution
(Chang et al., 1993; Miller and Gardiner, 1998). In addition, the high temperature and arid climate
of the Sahara (the highest surface temperatures in the world) that accelerate the decomposition rate
of sewage sludge must also be taken into consideration. Zeid and Askar (1987) reported that the
application of sewage sludge to sandy and highly calcareous soils improved its physical properties
and productivity.
The addition of relatively high rates of sludge increases the cation-exchange capacity (CEC)
of soils (Epstein et al., 1976; Mitchell et al., 1978; Kladivko and Nelson, 1979; Soon, 1981). This
increase in CEC results in additional cation-binding sites that retain essential plant nutrients within
the rooting zone. Possibly the CEC increase causes more complexation of heavy metals making
them unavailable for plant uptake (Kladivko and Nelson, 1979).
The accumulation of heavy metals within plants grown in wastewater or sludge-amended soils
is easily measured. However, the quantity of metals originally present in sewage and eventually
absorbed by the plant root is difficult to assess (McGrath et al., 1995). The reactions with metals,
which occur after irrigation by industrial and domestic wastewater or sludge incorporation into
soil and after an extended time has passed, require further investigation. When wastewater is con-
tinuously used as the sole source of irrigation for field crops in arid regions, excessive amounts of
nutrients and toxic chemical substances could simultaneously be applied to the soil-plant system.
This would cause unfavorable effects on productivity and quality parameters of the crops and the
soil (Vazquezmontiel et al., 1996). Consequently, mismanagement of wastewater irrigation would
create environmental and health problems to the ecosystem and human beings (Mohammad and
Ayadi, 2004). Therefore, several countries have produced guidelines that regulate wastewater and
sewage sludge reuse on the basis of risk to public health and the environment (Tables 42.1 and 42.2).
TABLE 42.1
Maximum Permissible Concentrations of Heavy Metals
in Wastewater (mg/L) in Some Selected Countries Where
Wastewater Is Used in Agriculture
Country Cd Cu Pb Ni Zn Fe Hg
Canada 0.01 0.2–1.0 — 0.2 1.0–5.0 — —
Taiwan 1 0.01 — 0.02 5 0.2 0.1
Hungary 0.2 0.02 0.1 — 10 2 1
China 0.05 0.005 — 0.02 0.5 1 —
FAOa 0.01 0.2 5 0.2 2 5 0.001
WHO 0.01 0.2 5 0.2 2 5 0.001
Tunisia 0.01 0.01 — 0.05 5 0.5 1
Nigeriaa 0.01 0.2–1.0 5 0.2 5 — —
Pakistan 0.01 0.35 0.04 0.14 — 0.22 —
Kuwait 0.01 0.2 0.5 0.2 2 5 0.002
Oman 0.01 0.05 0.1 0.1 5 1 0.001
Jordan 0.01 0.2 5 0.2 5 5 0.002
Tunisia 0.01 0.5 0.1 0.2 5 5 0.001
Syriab 0.01 0.2 — 0.2 2 5 —
Syriac 0.1 1 1 2 4 — 0.01
Saudi Arabia 0.01 0.4 0.1 0.2 2 2 0.001
Yemend 0.01 0.2 5 0.5 2 5 0.001
Yemene 1 5 0.6 5 15 50 0.01
Egypt (G1)f 0.01 — 5 0.5 — 1 0.001
Egypt (G2)f 0.005 0.5 1 0.2 2 1 0.001
a FAO, 1985; CCREM (Canadian water quality Guidelines, 1987; FEPA,

1991).
b Syrian Arab Organization for Standardization and Metrology: reclaimed
wastewater for irrigation use, Ministerial Decision 2752/72, June 13, 2003.
c Syrian Arab Organization for Standardization and Metrology: reclaimed
wastewater for irrigation use. Industrial wastewater before discharged into
the common public sewer network (S.N.S: 2752/2003).
d,e YMWE, 1999. Yemen’s Ministry of Water and Environment. Guidelines of
treated wastewater for irrigation use. Industrial wastewater before dis-
charged into the common public sewer network. Sana’a, Republic of Yemen.
f Ministry of Housing and Public Utilities and New Communities
(MHPUNC). 2005. Egyptian Code No. 501/2005 for the Safe Use of
Treated Waste Water for the Agriculture Sector.
Wastewater, also, affects supply of macro- and micronutrients for plant growth, soil pH, soil
buffer capacity, and soil electric conductivity (EC). Data present in the literature about the effects
of wastewater or sludge on the soil or plants are contradictory. This is mostly due to the chemical
composition of the wastewater or sludge, characteristics of the soil, period and quantity of applica-
tion, and ecological conditions of the region, especially those affecting the efficiency of decomposi-
tion and of the cultivated plant species. Schipper et al. (1996) found that soil pH increased following
long-term wastewater irrigation, and they attributed such increase to the chemistry and high content
of basic cations such as Na, Ca, and Mg in the wastewater applied. Mohammad and Mazahreh
(2003) at the end of the growing season found that soil pH was significantly decreased when waste-
water was applied. They attributed this decrease to the high content of ammonium in wastewater,
TABLE 42.2
Maximum Permissible Concentrations of Heavy Metals in Sewage Sludge
(mg/kg Dry wt.) in Some Selected Countries where Land Application Is Practiced
Country Cd Cu Pb Ni Zn Hg
EU a 20–40 1000–1750 750–1000 300–400 2500–4000 16–25
EUb 10 1000 750 300 2500 10
US EPAc Ceiling Co. 85 4300 840 420 7500 57
Except Q 39 1500 300 420 2800 17
NJ state 21 1500 300 420 2800 17
Italy 20 1000 750 300 2500 10
Netherlands 1.25 75 100 30 300 4.75
Norway 4 1000 100 80 1500 5
Spain pH < 7 20 1000 750 300 2500 16
pH > 7 40 1750 1200 400 4000 25
Sweden 2 600 100 50 800 2.5
Switzerland 5 600 500 80 2000 5
Germany pH > 6 10 800 900 200 2500 8
pH 5–6 5 800 900 200 2000 8
France Reference 20 1000 800 200 3000 10
Limit 40 2000 1600 400 6000 20
Austria 5 400 400 80 1600 7
Belgium Flanders 12 750 600 100 2500 10
Walloon 10 600 500 100 2500 10
Ontario Present 34 1700 1100 420 4200 11
Canada Long term 4 380 220 80 840 1.4
New Zealand 100–250 300 60 300–600 1
Australia 1 100 150–300 60 200 1
Chinad pH < 6.5 5 250 300 100 500 5
pH > 7 20 500 1000 200 1000 15
Egypte 39 1500 300 420 2800 17
Omanf pH < 7.0 20 1000 1000 300 3000 20
Yemeng 20 1000 750 300 2500 16
a EC Council Directive (1986).

b Draft EC Council Directive (2000).
c Sewage sludge meeting the concentration requirements stipulated is deemed exception quality as the
cumulative pollutant loading limits are not likely exceeded, if the land application is properly managed.
d People’s Republic of China, National Standards, GB 4284-84.
e Oman. 1993. Wastewater reuse and discharge. Ministry of Regional Municipalities and Environment.
Ministerial Decision 145/93, June 1993.
f Ministry of Housing and Public Utilities and New Communities (MHPUNC). 2005. Egyptian Code No.
214/1997 for the Safe Use of Treated Waste Water for the Agriculture Sector
g YMWE, (1999): Yemen’s Ministry of Water and Environment. Guidelines of treated wastewater for irriga-
tion. Sana’a, Republic of Yemen.
the nitrification of which would serve as a source of hydrogen ions, thus causing a decrease in
soil pH. Other researchers also found that soil pH decreased with wastewater irrigation due to the
oxidation of organic compounds and nitrification of ammonium (Hayes et al., 1990; Vazquezmontiel
et al., 1996; Mohammad and Mazahreh, 2003).
The relationships between the long-term wastewater irrigation and increasing some nutrients
in the soil have been discussed in many previous studies. It was found that wastewater irrigation
increased the level of soil salinity due to its salt content. Continuous buildup of salts in the topsoil
will adversely affect the activity of the soil microorganisms (Garcia and Hernandez, 1996), plant
growth, and soil productivity (Papadopoulos, 1995). In addition, extractable phosphorus was higher
in soils irrigated with wastewater than in soils irrigated with freshwater or rain water (Day et al.,
1979). Monnett et al. (1996) attributed the increase in soil N, P, and K accumulated in the soil
with wastewater application to the original contents of these nutrients in the wastewater applied.
Concentrations of these nutrients were found high in the top soils and then decreased within depth,
and the increase in top soils was proportional to its content in the wastewater used (Nyamangara
and Mzezewa, 2000; Brar et al., 2002). Mohammad et al. (2007) found that long-term wastewater
irrigation increased salts, OM, and plant nutrients in the soil. Essential nutrients (total N, NO3, P,
and K) were higher in plants grown in soils irrigated with wastewater. Plant Cu, Zn, Fe, and Mn
increased with 2 years of wastewater irrigation and then reduced in the long run. Plant Pb and Cd
increased with wastewater irrigation, and their levels became higher the longer the period of waste-
water irrigation.
Day et al. (1979) found that N recovery in plants irrigated with wastewater was higher than
the N recovery in plants grown with well water. These results were attributed to the significant
increase in soil nitrogen with wastewater irrigation compared with the control. On the other
hand, Papadopoulos and Stylianou (1988) reported that during the third irrigation season for
trickle irrigation cotton (Gossypium hirsutum L.), the NO3 –N in leaf petioles were greater
with the treated effluent supplemented with no nitrogen, also in lamina; NO3 –N was greater
at sampling of the lower N level. Hussain and Al-Saati (1999) found that essential nutrients
(total N, NO3, P, and K) were higher in plants grown in soils irrigated with wastewater for
different periods. In addition, nitrate concentration increased with longer period of waste-
water irrigation. Similarly, phosphorus concentration in barley shoot increased significantly
as wastewater irrigation increased. Other researchers have reported an increase in P and K
uptake by the plants irrigated with TWW (Papadopoulos and Stylianou, 1988; Mohammad
and Mazahreh, 2003).
The aforementioned macronutrients are essential for plant nutrition although some are
required by plants in relatively smaller amounts compared with others. In addition, Brar et al.
(2002) and Mohammad and Mazahreh (2003) reported an increase in micronutrients uptake by
the roots and that transported to leaves of plants irrigated with sewage water compared to that
irrigated with groundwater. Based on the comparison between lands irrigated with and without
sewage, McGrath et al. (1988) reported that metals accumulated are caused by sewage discharges.
The investigation of Xian (1989) showed that the level of heavy metals in the soil may rise lead
level in plants; however, it is not usually possible to find out a close connection between the heavy
metal concentrations of the plants and soil, because of the metal bioavailability in the soil that
depends on a number of different factors such as plant growth and metal distribution in different
parts of the plants. Abdel-Sabour et al. (1996) indicated that the use of sewage effluent for 12 and
50 years resulted in an increase in soil Zn content by about 10–14-fold. Also, Tahoun and Abd
El-Bary (1997) mentioned that the concentrations of some heavy metals in the sewage effluent
take the following order: Fe > Pb > Zn > Mn > Co > Cd = Ni = Cu. Meanwhile, the sewage efflu-
ent water contains measurable quantities of Zn, Fe, Ni, and Cd. Ibrahim et al. (1992) observed
that Mn, Cu, Zn, Cd, Pb, Ni, and Co increased in the upper soil layer (0–10 cm) due to using sew-
age water for irrigation. El-Hassanin et al. (1993) indicated that prolonging the irrigation periods
was associated with significant increases in the total and available forms of Pb, Cd, Zn, and B.
However, they concluded that the concentrations of these elements have not accumulated to toxic
levels even after 67 years with sewage irrigation.
Mohammad and Mazahreh (2003) reported an increase in soil Fe and Mn with wastewa-
ter irrigation and no response with regard to soil Cu and Zn. On the other hand, Mancino and
Pepper (1992) found no effect on soil micronutrients. Saha et al. (2010) found that sewage irriga-
tion increased available N, P, and K contents in surface soil significantly by about 11.4%, 44%,
and 17% more, respectively, in sewage-irrigated root zone soils than groundwater-irrigated soils.
Cu and Zn were accumulated significantly in the upper 25–30 cm of soil with wastewater irrigation
(Lawes, 1993). In long-term wastewater irrigation (80 years), Christina Siebe (1998) found that the
contents of soil Cu, Zn, Mn, Fe, Pb, and Cd increased compared to the soil irrigated with potable
water. Similar results were obtained by Brar et al. (2002). However, Mohammad and Mazahreh
(2003) mentioned that the concentrations of soil Pb and Cd were not affected significantly by
wastewater irrigation.
As can be seen, several studies have addressed how irrigation with TWW affects the chemical
properties of clay soils (Heidarpour et al., 2007) and how reusing it affects heavy metal contents
in the soil (Mireles et al., 2004; Mapanda et al., 2005; Rattan et al., 2005). However, only few
researches have focused on how irrigation by TWW affects the fertility of sandy soils (Patterson and
Chapman, 1998) and salt concentrations (Bredai et al., 1996; Lazarova, 1999). Recently, phytoreme-
diation, especially phytoextraction, has received increasing attention as a promising cost-effective
alternative to conventional engineering-based remediation methods (Salt et al.,1998). Success of
phytoextraction depends on metal concentration in plant shoots, adequate plant yield, and the bio-
available fraction of heavy metals in the rooting medium (Grcman et al., 2001). An alternative to
discharging secondary TWW into surface waters is land application, where the soil acts as a tertiary
filter. Previous studies have found that TWW irrigation can be beneficial in a number of different
ways: provide water and nutrients to plants, enhance groundwater recharge, and reduce impacts on
surface water quality (Parizek et al., 1967; Toze, 2006).
The foregoing research findings clearly indicate inconsistent impact of wastewater irrigation on
soil micronutrients. However, advantages of using wastewater in irrigation can be summarized as
follows:
1. Sewage water can be used as a water source, to conserve freshwater sources, and as a sup-
plement to fertilizers because it contains valuable amounts of OM and high nutrient value.
2. As it contains OM, it improves soil water retention capacity, permits easier root penetra-
tion, and reduces water runoff and soil erosion.
3. Due to the fact that the majority of water treatment plants are located in or near farmer’s
area, it can be easily obtained at a little or no cost.
4. It improves the environment by eliminating or reducing discharge to surface waters, and
improves the economic efficiency of investments in wastewater disposal and irrigation,
mainly near cities and towns where sewerage systems exist.
5. It installs new underground water reservoirs or recharge the existing ones.
42.3.1.2 Case Studies for Sewage Application in Agriculture

The application of sewage (sludge and effluents) has been long practiced by many communities for
fertilization and watering. Rapid population growth increasingly generates pressure on existing
cultivated land and other resources and induces migration to the marginal land of arid and semi-
arid areas in many developing countries, such as Tanzania, Sudan, Egypt, and Mexico (Darkoh,
1982; Bilsborrow and Delargy, 1991; Ericson et al., 1999). Population migration to those arid and
semiarid areas increases the problems of water shortage and worsens the situation of land degrada-
tion and in turn causes severe problems of poverty, social instability, and population health threats
(Moench, 2002).
42.3.1.2.1 California
Beneficial use of wastewater has been practiced in California, where reclaimed wastewater has
been increasingly used for landscape irrigation in urban areas and for groundwater recharge. Two
hundred reclamation plants throughout California produce considerable volumes of treated efflu-
ent and save 0.759 Mm3/day of freshwater. Reclaimed wastewater used for spray irrigation of food
crops is at all times adequately disinfected, oxidized, coagulated, clarified, filtered wastewater with
a bacteriological quality such that the 7-day median number of coliform organisms does not exceed
2.2 per 100 mL and the number of coliform organisms does not exceed 23 per 100 mL in more than
one sample within any 30-day period. Exceptions to these quality requirements may be allowed by
the State Department of Health on case-by-case basis where the food crop is to undergo extensive
commercial physical or chemical processing sufficient to destroy pathogenic agents before human
consumption. For irrigation of fodder, fiber, and seed crops, wastewater has received primary treat-
ment only. However, reclaimed wastewater used to irrigate pasture to which milking cows or goats
have access was at all times adequately disinfected and oxidized to achieve a median number of
coliform organisms not exceeding 23 per 100 mL over 7 days.
The quality requirements for irrigation of golf courses, cemeteries, freeway landscapes, and
landscapes in other areas where the public has a similar access or exposure are as follows: the efflu-
ent should at all times be disinfected and oxidized to a median number of coliforms not exceeding
23 per 100 mL over 7 days and a number of coliforms not exceeding 240 per 100 mL in any two
consecutive samples. More stringent quality requirements are applied to reclaimed wastewater used
to irrigate parks, playgrounds, schoolyards, and other areas where the public has similar access or
exposure. For groundwater recharge of domestic water supply aquifers by surface spreading, the
reclaimed wastewater must be at all times of a quality that is safe for public health.
Sheikh et al. (1990) conducted a 10-year Monterey wastewater reclamation study for agricul-
ture. The study was designed to evaluate the safety and feasibility of irrigating food crops (many
eaten raw) with reclaimed municipal wastewater. Demonstration fields at Castroville in the lower
Salinas Valley, California, were used to study full-scale farm practices using reclaimed munici-
pal wastewater. Analysis of plant edible tissues for heavy metals proved that there was no consis-
tently significant difference between concentrations in plants irrigated with reclaimed wastewater
effluents and in those irrigated with well water. In addition, the metal content of artichoke tissues
from neighboring fields showed no relationship with distance from the site of the plots. Analysis
of residual tissues produced results similar to those for edible tissues except for accumulation of
zinc (higher in edible tissues for all vegetables studied) and cadmium (higher in residual tissues).
42.3.1.2.2 Egypt
According to Abdel Wahaab (2011), the amount of TWW in Egypt equals 3.5 billion cubic meters
(BCM)/year, which is sufficient to cultivate 850,000 feddans (=357,000 ha). Some of this water is
drained into canals and drains in Cairo area and Nile Delta, while the rest of the TWW is used to
cultivate forests, some crops, and plants such as sunflower, jatropha, khaya, casuarina, and castor.
The available land for irrigation by reused TWW is 160,000 feddans in different governorates
(15,000 cultivated). There are 28 man-made forests in Egypt; out of them, 21 are irrigated with
treated sewage water. Table 42.3 lists forests where Jatropha and Jojoba are cultivated.
Wastewater use is an old practice in Egypt. It has been used since 1930 in sandy soil areas like
Al Gabal Al Asfar and Abu Rawash, near Cairo. Work to formulate a master plan for water resources
development and use in Egypt was started in 1977. The Ministry of Irrigation, in collaboration with
the United Nations Development Programme (UNDP) and the World Bank, completed the first
phase of this activity in 1981 (Arar, 1989). After that, interest in the use of TWW as a substitute for
freshwater in irrigation has accelerated.
Egypt produces an estimated 5.5–6.5 BCM a year of wastewater. Of that amount, about 2.97
BCM/year is treated (Abdel Wahaab, 2011). According to Ghazy et al. (2009), the dry sludge pro-
duction rate of the activated sludge treatment plant systems in Egypt is considered at 0.225 kg/m3 of
TWW, and the production rates of the other WWTP types are assumed at 0.05–0.22 kg/m3 accord-
ing to Metcalf and Eddy (2003). However, the quantity of sewage sludge produced from all cities in
Egypt throughout the year 2011 was approximately 668 × 103 ton. If this amount of sludge is applied
to 20% of reclaimed sandy deserts (about 35 kg/m2), it will (according to our estimation) improve
the fertility and water retention of about 4600 feddans (=1932 ha) each year. Ramadan et al. (2003)
TABLE 42.3
Egyptian National Program for Safe Use of Treated Sewage Water for Afforestation
Governorate (Site) Discharge (m3/Day) Forest Area (ha) Irrigation Methoda Planted Tree Species
Bani-Swief (El-Wasta) 10,000 210.0 D Jatropha, Khaya
Assiut (Assiut) 28,000 16.8 D Jatropha, Khaya
Sohag (Gharb) 28,000 420.0 MF, D Jatropha, Khaya
Sohag (Shark) 94 420.0 MF, D Jatropha, Khaya
Luxor (Luxor) 30,000 710.0 MS, D Jatropha, Khaya, Sesban,
Eucalyptus, Acacia
saligna, Morus
Aswan (El-Alaky) 30,000 693.0 MF, D Jatropha, Jojoba, Khaya,
Castor
New Valley (Mount) 17,000 126.0 MS, D Jatropha, Jojoba
Sources: Adapted from Ministry of State for Environmental Affairs (MSEA), Annual Report (Egypt), 2006; Zalesny, R.S.
et al., Int. J. Phytoremed., 13, 102, 2011; Abdel Wahaab, R. Wastewater Reuse in Egypt: Opportunities and Challenges
(Part I). Expert Consultation on Wastewater Management in the Arab World, May 22–24, 2011, Dubai-UAE.
a Irrigation method deployed; D, drip irrigation; MF, modified flood irrigation; MS, modified surface irrigation.
found that applying sewage sludge at higher levels (40%–50%) dramatically increased the soil water
retention capacity, specially sludge with high OM content. Abu Zuhri (2004) applied sewage sludge
from four governorates in Egypt (Cairo, Alexandria, Assiut, and the New Valley), which are known
for a great variation in their industrial activities, and followed their impact on sand soil and two
crop plants. The results indicated that the water-holding capacity of the sandy soil increased as a
result of sludge application. The available water for plants increased and the wilting point (WP) of
Helianthus annuus and Sorghum bicolor delayed in comparison with sandy soil. Also, an increase
in dry weight, chlorophyll, and protein content was obvious in the investigated plants, and this was
mostly proportional to the added sludge. Both of the permanent WP of H. annuus L. and the field
holding capacity (FC) were increased proportionally by increasing the percentage of sludge in the
soil (Figure 42.1).
50
Field holding capacity

40
Water content (% of dry soil)
30
Available water
20
10 Permanent wilting point
Unavailable water
0
Control 10% 20% 30% 40% 50%
Sludge level
FIGURE 42.1 Increasing the available water for plants of H. annuus and S. bicolor cultivated on sandy
soil amended with different levels of sludge. (Modified from Ramadan, T. et al., Bull. Fac. Sci. Assiut Univ.,
32, 277, 2003.)
Many scientific works in Egypt were conducted to analyze the sewage sludge collected
from different locations (El-Keiy, 1983; El-Sokkary, 1993; Aboulroos et al.,1996; Derar and
Eid, 1996; Badawy and Helal, 1997; El-Gendi et al.,1997; Badawy and El-Motaium, 1999;
Badawy and El-Motaium, 2002). The data showed that the sewage sludge contains appreciable
amounts of N, P, and K and has significant inorganic fertilizer replacement value for these major
plant nutrients.
Analyses of sludge samples from the four wastewater treatment plants indicated concentrations
of heavy metals to be very low (Abu Zuhri, 2004). Also, Smith et al. (1995, 1999) reported that the
Egyptian sewage sludge relatively contains a low concentration of heavy metals. Concentrations
of Cd, Cu, Ni, Pb, and Zn in most sludge samples studied were found to be less than 10% of the
permissible limits of heavy metals regulated by the Egyptian government. Higher concentrations of
Cu and Zn were reported in Alexandria and Cairo sludge, but still less than the permissible limits.
However, only the few water-soluble fraction will be available to plants. According to Che Fauziah
et al. (2002), less than 5% of the total heavy metals (Ni, Cu, Zn, Cd, and Pb) in sewage sludge were
found to be water-soluble.
42.3.1.2.3 France
Waste resources are estimated as 3300 m3/capita year above the stress level (World Resources,
2000–2001). Nevertheless, during the last 10 years, there has been a 20% increase in water demand
(mainly for agriculture and resort areas) coupled with a series of drought years. In more than
one-third of the country, water tables are falling as the autumn and winter rains are no longer
making up for the amounts drawn off in spring and summer, a situation that forced authorities to
impose restrictions on water use. Presently, there are at least 30 water reuse projects in France,
half of which use reclaimed water for agricultural irrigation covering more than 3000 ha; the other
15 projects use reclaimed water for golf courses and urban area irrigation (Angelakis et al., 2003,
2005; US-EPA, 2004).
The irrigation of crops with wastewater is a very long-standing practice on a number of sites in
France. The best example is probably the region around Paris. Until 1940, the only method for treat-
ing and disposing the wastewater of the Greater Paris conurbation was by irrigating vegetable crops
sold on the market. This practice was uninterrupted until 2005 (Juanicó and Salgot, 2005) around
AcheÁ res, where some of the wastewater is reused after simple screening and settling, but it was
decided to discontinue this practice.
Another example of diluting surface water with wastewater is in Aubergenville, in Paris, where
the Seine River, which is 25% wastewater effluent, is treated and used to recharge the drinking
water aquifer.
The Clermont-Ferrand recycling scheme implemented in 1999, in which 10,000 m3/day of efflu-
ents treated by activated sludge followed by maturation ponds are used for irrigation of over 700 ha
of maize, was considered to be one of the largest projects in Europe. One of the first examples in
France of integrated water management with water reuse is on Noirmoutier Island. Wastewater treat-
ment on the island is achieved through two treatment plants with a total capacity of 6100 m3/day.
The plants have activated sludge systems followed by maturation ponds for storage and disinfection.
Thirty percent of the TWW (0.33 MCM/year) is used for the irrigation of 500 ha of vegetable crops.
The direct disposal of wastewater was in principle limited to crops whose products were cooked
before consumption. However, there is some evidence that the water was also used to irrigate small
private plots for self-consumption with no control over what was grown. There is no recorded evi-
dence of adverse health effects (Angelakis et al., 1999).
In the project for 2000 inhabitants at Villefranque near Biarritz in the Pyrenees, after single-
stage treatment, the water becomes particularly well suited for reuse in farming with no sanitary
risk. The project also includes industrial wastewater reuse after purification to supply cooling water,
wash water, or even process water after sophisticated complementary treatment is quite widely
developed in France.
42.3.1.2.4 India
Several studies concerning sewage reuse in agriculture in India confirm that domestic sewage can
effectively increase water resources for irrigation but there is a need for continuous monitoring of
the concentrations of potentially toxic elements in soil, plants, and groundwater. For example, Yadav
et al. (2002) reported that the use of sewage for irrigation in various proportions improved the OM
to 1.24%–1.78% and fertility status of soils especially down to a distance of 1 km along the disposal
channel. Available N buildup was 95 kg/ha, total N 2908 kg/ha, available P 58 kg/ha, total P 2015 kg/ha,
available K 305 kg/ha, and total K 4712 kg/ha in surface 0.15 m soil. Vertical distribution of these
parameters also varied, with most accumulations occurring in 0.3 m of the surface. Traces of NO3–N
(up to 2.8 mg/L), Pb (up to 0.35 mg/L), and Mn (up to 0.23 mg/L) could also be observed in well
waters near the disposal point, thus indicating initiation of groundwater contamination. However, the
contents of heavy metals in crops sampled from the area were below the permissible critical levels.
Changes in nutrient contents of soils were reflected in uptake by winter (wheat, berseem) and
summer (rice, sorghum) crops growing at the studied sites. Higher contents of N, P, K, and Na were
monitored in their shoots when grown in soils irrigated with sewage water, and there was constant
decline in their concentrations in samples drawn from fields lying at a distance from the disposal
point and the channel. The contents of heavy metals also declined with increasing distance of sam-
pling from the disposal pump.
42.3.1.2.5 Iran
Iran is a predominantly arid country. Shortage of freshwater, especially in the arid and semi-
arid regions of southeastern Iran, is the main barrier for proper management of soil resources,
which would maximize cropping intensity and produce higher crop yields (Salehi et al., 2008).
Asgharipour and Azizmoghaddam (2012) found that the applied municipal sewage effluent con-
tained higher levels of micronutrients and macronutrients and exhibited greater degrees of electric
conductivity compared to well water. Because of the small scale of industrial activities in Zabol (the
region in which the study was conducted), the amount of heavy metals in the sewage was negligible
(below the limits set for irrigation water in agricultural lands), and thus these contaminants would
not be severely detrimental to crop growth.
The experimental results indicated that irrigation of plants with raw or diluted (50%) sewage
stimulates the measured growth and productivity parameters of the test plants. These stimulations
were attributed to the presence of high levels of essential nutrients such as N, P, and OM in waste-
water. Additional fertilizers are required to obtain higher levels of millet productivity with sewage
farming. The results suggested that nutrients contained in sewage water irrigation did not have any
significant harmful effect on crop productivity. In contrast, these nutrients proved beneficial to soil
fertility and test plants productivity and quality.
42.3.1.2.6 Kuwait
Untreated sewage has been used for many years to irrigate forestry projects far from the inhabited
areas of Kuwait. Following extensive studies by health and scientific committees within the country
and by international consultants and organizations, namely, the World Health Organization (WHO)
and the Food and Agriculture Organization (FAO), the government of Kuwait decided to proceed
with a program of sewage treatment and effluent use. The government’s strategy to implement the
Effluent Utilization Project was to give the highest priority to the development of irrigated agricul-
ture by intensive cultivation in enclosed farm complexes, together with environmental forestry in
large areas of low-density, low-water-demand tree plantations.
The ultimate project design provides for the development of 2700 ha of intensive agriculture and
9000 ha of environmental forestry (Agriculture Affairs and Fish Resources Authority, Kuwait). In
1985, the treated effluent was used to irrigate fodder plants, field crops, fruit trees, and vegetables.
Vegetables that are to be cooked before consumption were irrigated in various methods but not by
sprinkler; vegetables that are eaten raw were irrigated with tertiary-treated sewage effluent by drip
irrigation with soil mulching.
The yield of green alfalfa was 100 tons/ha per year and the total production from the agricultural
irrigation project, using primarily treated sewage effluent, was 34,000 tons of vegetables and green
fodder plants, including dehydrated alfalfa and barley straw. At this production level, a reasonable
supply of some vegetables was made available to the local market, the total demand for green alfalfa
for animals was satisfied, and some of the needs for dehydrated fodder were met.
For forestry irrigation, the systems include storage tanks and pumping stations incorporating
fertilizer injection. Treated effluent is supplied via control points to blocks of forestry. Up to 1988,
only environmental protection forestry has been developed although there was potential to produce
high yields of commercial forestry using treated effluent irrigation (Cobham and Johnson, 1988).
In Kuwait, the decision was taken to exclude all amenity uses for the treated effluent and to
restrict agricultural use to safe crops. Furthermore, areas of tree and shrub planting and the agri-
cultural farm were to be fenced to prevent public access. An efficient monitoring system for the
treated effluent, the soil, and the crops has been implemented since the experimental farm was
initiated. Even the tertiary-treated effluent is not to be used to irrigate salad greens or strawberries.
No outbreaks of infectious diseases have occurred since this procedure began in 1976. The impact
of treated effluent-irrigated vegetables on the consumer has not been possible to assess because no
segregation of vegetables produced in this way is effected in the market.
42.3.1.2.7 Mexico
Sewage from Mexico City, mixed with variable proportions of surface water collected in reservoirs
within a basin, has enabled farmers in the Mezquital Valley to provide agricultural products for the
capital city. Hence, the concentrations of chemical constituents and pathogenic organisms in the
irrigation water vary spatially and temporally. Large impounding reservoirs providing relatively
long retention times for wastewater serve as treatment devices, settling out solids and reducing
pathogen levels. No treatment of sewage is provided before it is transported 60 km from Mexico
City to Irrigation District, and, clearly, little improvement in fecal coliform levels has occurred
before sewage is applied as irrigation water. In trying to achieve public health protection, reliance
is placed on the application of crop restrictions rather than wastewater treatment. Every year, each
farmer specifies the crops that will be planted and irrigates with water allocated by the Irrigation
District. The Ministry of Health sets the basic rules for crop restriction, and the district’s directing
committee specifies in detail the crops that may not be cultivated under its jurisdiction (Strauss and
Blumenthal, 1989). In Irrigation District, banned crops are lettuce, cabbage, beet, coriander, radish,
carrot, spinach, and parsley. Adherence to these restrictions is monitored mainly by the district’s
canal and gate operators, who are in close contact with the farmers. Maize, beans, chili, and green
tomatoes, which form the staple food for the majority of the population, do not fall under these
restrictions, and neither does alfalfa, an important fodder crop in the area.
During the agricultural year 1983–1984, 52,175 ha in Irrigation District was harvested to produce
2,226,599 tons of food crops, with a value of more than US $33 million. The yields of the crops were
greater than those obtained 10 years before, except for pasture, and it is believed that fertility conditions,
measured on the basis of productivity, are better than before. In addition, it is thought that the high con-
tent of OM and plant nutrients in wastewater has improved the physical and chemical properties of the
shallow soils in the district. The high rate of application of irrigation water has increased soil OM and
systematically leached the soils, preventing the accumulation of soluble salts (Sanchez Duron, 1988).
Mexican experience with raw wastewater irrigation suggests that successful enforcement of crop restric-
tion has provided health protection for the general public, including crop consumers.
42.3.1.2.8 Saudi Arabia
In Saudi Arabia, water demand has exceeded the reliable supply of surface water and renewable
groundwater that led to the utilization of low-quality water for irrigated agriculture. According
to Al Omron et al. (2012), a case study was undertaken to assess the long-term effect of sewage
irrigation on some soil properties and heavy metal concentrations in the soils of the date palm at
Al-Ahsa Governorate, Saudi Arabia. The results pertaining to irrigation water analysis indicated
that sewage effluents were found to contain higher content of Pb, Zn, Cu, Co, Cr, As, Cd, Fe, Mn,
and Ni than well water. On the other hand, data emphasized the role of sewage effluent irrigation
on increasing heavy metals as well as OM contents in the soil samples when compared with the
respective values found in the soil irrigated with well water. The soil salinity ranged from 3.58
to 20.7 dS/m with an average of 7.9 dS/m due to irrigation with well water, while the respective
soil salinity due to irrigation for long period with the treated sewage effluent ranged from 2.5 to
3.69 dS/m with an average of 2.8 dS/m. On an average, the soil pH dropped by 0.3 U as a result
of sewage irrigation. Long-term sewage irrigation resulted in significant buildup of total concen-
trations of Zn (130%), Pb (55%), Fe (82%), Ni (84%), Mn (30%), Cu (40%), Cr (75%), Co (78%),
and As (67%) in sewage-irrigated soil samples over adjacent well water–irrigated soil samples.
However, Cd did not show any significant changes.
The results of this case study were in agreement with the findings of Rattan et al. (2001). As it
is well known, pH and soil organic carbon (SOC) are the most important indicators of soil quality;
the latter also acts as a storehouse of the plant nutrients and plays a major role in nutrient cycling.
Besides, some estimates show that an increase in the SOC content by 0.01% could lead to carbon
sequestration being equal to the annual increase in atmospheric CO2–C (Lal et al., 1998).
42.3.1.2.9 Tunisia
Wastewater use in agriculture has been practiced for several decades in Tunisia and is now an inte-
gral part of the national water resources strategy. The use of treated effluents is seasonal in Tunisia
(spring and summer), and the effluent is often mixed with groundwater before being used to irrigate
citrus and olive trees, forage crops, cotton, golf courses, and hotel lawns.
In the period 1981–1987, the Ministries of Agriculture and Public Health, with assistance from
UNDP, carried out studies designed to assess the effects of using TWW and dried, digested sewage
sludge on crop productivity and on the hygienic quality of crops and soil. TWWs and dried, digested
sludge from the La Cherguia (Tunis) and Nabeul (SE4) activated sludge plants were used in the
studies, while irrigation with groundwater was used as the control. At La Soukra, tests were con-
ducted on sorghum (Sorghum vulgare) and pepper (Capsicum annuum) using flood irrigation and
furrow irrigation, respectively, whereas clementine and orange trees were irrigated at Oued Souhil
(Nabeul). In order to assess the long-term effects (more than 20 years) of irrigation with TWW,
investigations were carried out on the perimeter area of La Soukra (Bahri, 1988).
The characteristics of the TWW from La Cherguia and sewage sludge from Soukra and Nabeul
indicated that the effluent contains moderate to high salinity but presents no alkalization risk, and
trace element concentrations are below toxicity thresholds. The sewage sludge had a fertilizing
potential due to the presence of minerals and OM, but was of variable consistency. Evaluation of the
fertilizing value of the effluent in relation to crop uptake suggests that the mean summer irrigation
volume of 6000 m3/ha would provide an excess of nitrogen (N) and potassium (K2O) but a deficit of
phosphorus (P2O5). The fertilizing value of 30 tons dry weight of sewage sludge per hectare would
be an excess of N and P2O5 and a deficit of K2O. The application of treated effluent and sludge would
balance the fertilizing elements but would provide an excess over crop requirements. Excess nitrogen
would be of concern from the point of view of crop growth and in relation to groundwater pollution.
The application of TWWs and sewage sludge at the La Soukra and Oued Souhil experimen-
tal stations, where the soils are alluvial and sandy-clayey to sandy, has not adversely affected
the physical or bacterial quality of the soils. However, the chemical quality of the soils varied
considerably, with an increase in electric conductivity and a transformation of the geochemical
characteristics of the soil solution from bicarbonate–calcium to chloride–sulfate–sodium (Bahri,
1988). Trace elements concentrated in the surface layer of soil, particularly zinc (Zn), lead (Pb),
and copper (Cu), but did not increase to phytotoxic levels in the short term of the study period.
Rational use of sewage sludge would require standards to be developed for the specific soils,
based on limiting concentrations of trace elements.
The use of TWW resulted in annual and perennial crop yields higher than yields produced by
groundwater irrigation. Sewage sludge application increased the production of sorghum and pepper
and resulted in crops containing higher concentrations of N, P, and K and some minor elements
(Fe, Zn, and Cu). Bacterial contamination of citrus fruit picked from the ground irrigated with
TWW or fertilized with sewage sludge was significantly higher than the level of contamination of
fruit picked from trees. Natural bacterial die-off on sorghum plants was more rapid in summer than
in autumn. Tests on pepper did not indicate particular contamination of the fruit.
Irrigation with TWWs was not found to have an adverse effect on the chemical and bacteriologi-
cal quality of shallow groundwater, although the initial contamination of wells was relatively high
and subject to seasonal variation. Investigations on the peripheral area of La Soukra did not indicate
significant impacts on soils, crops, or groundwater.
42.3.1.2.10 Yemen
The Republic of Yemen is an example of a restless political situation which has an impact on all
human activities. The few existing treatment plants treat totally about 92,000 m3 per day that
amounts to 33.5 million m3 per year (UN, 2003). The farmers in Yemen, living near the disposal
sites of urban wastewater, especially in some of the large cities (such as Sana’a, Taiz, and Ibb), are
already practicing the reuse of nontreated or partially treated wastewater, and this causes a high
number of transmissible diseases to both humans and animals. The risk is high in Yemen because
most wastewater treatment plants are suffering from the lack of maintenance and operational con-
trol. On the other hand, Yemen is facing a water crisis due to the rapid decline of water resources
resulting from overexploitation of groundwater for crop irrigation (agriculture consumes more than
90% of renewable water resources). The provision of wastewater treatment inevitably results in the
production of treated effluent and sludge. These are valuable resources for agricultural irrigation
and soil fertilization, particularly under the conditions of Yemen; the current water-scarce condi-
tions emphasize the need and urgency of reusing all TWW.
Unfortunately, none of the wastewater projects in Yemen have considered how effluent and
sludge should be reused safely (Hall and Ebaid, 2008). However, any attempt to save water, increase
the area of arable land, and increase crop yield with good quality will be a matter of life or death for
the populations facing such complex challenges of poverty. In Yemen, raw sewages and wastewater
are used as such, or after mixing with precipitated water, in the irrigation of crops and vegetables
(Figure 42.2). According to the amount of precipitation and amount and source of sewages, the
composition and concentrations of different compounds and metals in used wastewater greatly vary.
The effect of irrigation by domestic and industrial wastewater on characteristics of soil, plant’s
growth, and uptake of nutritive and heavy metals in some economic plants was studied at Taiz University,
Yemen (Sultan, 2010). Four sites were chosen for this study where wastewater is being used in irriga-
tion, namely, inside and outside Taiz wastewater treatment plant, industrial wastewater at Taiz, and Ibb
wastewater treatment plant. In addition, the fields irrigated by underground water of wells at Southern
Upland Agricultural Research Station, Taiz, were chosen as a control. Two monocotyledonous species
Zea mays and S. bicolor and three dicotyledonous species Portulaca oleracea, Cucurbita pepo, and
Solanum tuberosum were studied. The results indicated that concentrations of all major ions (Na+, K+,
Ca2+, Mg2+, Cl−, NO3−, SO42−, and PO43−) in soils irrigated with wastewater were significantly higher than
that in soils irrigated with groundwater. In winter, the concentrations of all these ions in surface soils
were significantly higher than that estimated in summer (the rainy season), and vice versa for subsurface
soils. The highest concentrations of Fe, Zn, Cu, Pb, and Cd were found in the soil where Taiz wastewater
was used in irrigation, while concentration of Ni was high in the soil irrigated with industrial wastewater.
Irrigation with wastewater resulted in increasing soluble sugars and soluble proteins in all studied spe-
cies. Concentrations of major ions in leaves of all plants irrigated with different wastewater were signifi-
cantly higher than that in plants irrigated with underground water.
(a) (b)
(c)
FIGURE 42.2 Discharging of wastewater into the wadi Mitum at Ibb, Yemen, directly without any treatment
(a), where a large quantity of sludge is accumulated on the soil surface of cultivated lands due to long-term
application (b). This untreated wastewater is used for irrigation of potatoes near wadi Mitum (c).
42.3.2 In Aquaculture
In aquacultures, fish may feed solids of municipal sewage effluents directly or indirectly after
bacterial decomposition of sewage particles, increasing bioavailability of minerals and carbon diox-
ide for phytoplanktons. Zooplanktons graze phytoplanktons and fish utilize either of the intermedi-
ates. Phytoplanktons participate in aquacultures not only by their organic body as fodder but also as
the oxygen producers for fish and other respiring organisms in the aquaculture. Imbalance of oxygen
evolved mainly by phytoplankton (and other aquatic macrophytes) in relation to consumption is a
known causal agent of fish mortality in sewage-fed aquacultures. Unionized ammonia (NH3) is also
toxic to fish in the concentration range of 0.2–2.0 mg/L (Alabaster and Lloyd 1980). Thus, a com-
pletely integrated and dynamic food chain and ecosystem occurs in aquacultures.
A wide range of fish species live and flourish in aquacultures, including common carp (Cyprinùs
carpio), Indian major carps (Catla catlax, Cirrhinus mrigala, and Labeo rohita), Chinese silver carp
(Hypophthalmichthys molitrix), bighead carp (Aristichthys nobilis), grass carp (Ctenopharyngodon
idella), crucian carp (Carassius auratus), Nile carp (Osteochilus hasseltii), tilapia (Oreochromis spp.),
milkfish (Chanos chanos), catfish (Pangasius spp.), kissing gourami (Helostoma temminckii),
giant gourami (Osphronemus goramy), silver barb (Puntius gonionotus), and freshwater prawn
(Macrobrachium lanchesteri) (Pescod, 1992). The selection reflects local culture rather than fish opti-
mally suited to such environments. Edwards (1990) gives a thorough review of the current knowledge
on the various fish species that can be cultured in ponds fed with human waste. In a successful aqua-
culture system, there must be both an organismic balance, to produce an optimal supply of natural
food at all levels, and a chemical balance, to ensure sufficient oxygen supply for the growth of fish
and their natural food organisms and to minimize the buildup of toxic metabolic products (Colman
and Edwards, 1987). A wide range of yields have been reported from waste-fed aquaculture systems,
for example, 2–6 tons/ha year in Indonesia, 2.7–9.3 tons/ha year in China, and 3.5–7.8 tons/ha year
in Taiwan. Management of fish ponds can have a significant effect on fish yields, but the maximum
attainable yield in practice is of the order of 10–12 tons/ha year (Edwards, 1990).
Fish raised in aquacultures are usually contaminated with heavy metals, bacteria, viruses, and
coliforms. Major portion of chemical and biological contaminants are usually found in inedible parts,
for example, scales, gills, and digestive tract (Strauss, 1985), whereas a minimum of contaminants was
reported in the edible muscles. However, it is generally believed that holding human waste-fed fish in
clean-water ponds for several weeks at the end of the growing cycle will remove residual objectionable
odors and pathogens and provide fish acceptable for market (Khalil and Hussein, 1997).
42.3.2.1 Case Studies of Sewage Application in Aquacultures

42.3.2.1.1 Egypt
Fish farming has virtually become the main hope of the Egyptian government for achieving its optimal
animal-protein production targets. However, because of the shortage of water resources, the executing
authorities have prohibited the use of freshwater and even drainage water, in some regions, for aquacul-
ture. For this reason and in line with the country policy to control pollution, the aquaculture authorities
directed their efforts for obtaining new water resources toward recycling and the reuse of wastewater.
Zenein wastewater treatment plant, one of six units, has been constructed through the Greater Cairo
Wastewater Project in 1990. It treats about 330,000 m3/day, using a biological oxidation technique
(Khalil and Hussein, 1997). Many studies tried to evaluate the reuse of the primary- and secondary-
treated effluent of this plant in aquaculture. Moreover, the studies also aimed to evaluate potential public
health threats associated with this practice from both pathogens and toxic chemicals.
According to Khalil and Hussein (1997), the primary- and secondary-treated waste effluents
were successfully used to grow the Nile tilapia (Oreochromis niloticus L.). The growth rate of fish
reared in treated wastewater was significantly higher than that of fish reared in the natural habitat.
Bacterial loads in fish organs were higher in the gills followed by the intestine and the skin and
finally the edible muscles. The total aerobic bacterial count was very low (9.3 × 102 g−1) in the edible
muscles of fish grown in secondary-treated effluent, which complies with the WHO guidelines (less
than l05 g−1). Salmonella, Shigella, and Staphylococcus were completely absent in all fish samples
examined (Easa et al., 1995). The highest concentrations of heavy metals were found in liver tis-
sues, followed by intestine and gills and then muscles. Also, accumulated levels were within the
acceptable limits when compared with the international legal standards for hazardous elements in
fish and fishery products. In conclusion, the chemical and bacterial studies revealed no evidence of
any public health hazard associated with wastewater reuse in aquaculture, especially that subjected
to secondary treatment, as the quality of fish meets the standards recommended by the International
Commission on Microbiological Specifications for Foods (ICMSF) and the WHO (1989).
42.3.2.1.2 India
Acting on the principle that sewage is not just wastewater but also a source of nutrients, an experimental
plant in operation treats sewage with aquatic weeds and fish in the city of Cuttack, West Bengal, India.
The use of municipal wastewater to fertilize ponds began in Kolkata (formerly Calcutta) in the 1930s;
the city now has perhaps the largest wastewater-fed aquaculture system in the world. One million liters
of primary treated sewage a day is deposited first in ponds containing duckweed, then in ponds stocked
with carp and prawns. After 5 days, water quality has improved to the point where it may be used for
agriculture, although not for drinking (Jana, 1998). Taking ideas from these practices, aquaculture is
being proposed and standardized as a tool for treatment of domestic sewage (CIFA, 1998).
The waste recycling system that has evolved in Kolkata city involves garbage-based vegetable
farms, wastewater-fed fishponds, paddy fields using fish pond effluent, and sewage-fed brackish
water aquaculture. The practice of using pond effluent for paddy cultivation is of a relatively recent
origin (Nandeesha, 2002).
Although fecal coliforms were present in the Kolkata system and in the guts and gills of fish
fed on sewage, none were found in fish muscles. The sale of fish fattened in sewage ponds for
8–12 months almost offsets the operating cost of the plant.
Kolkata, now having 11 million inhabitants, discharges an average of 1,100 million liters of
municipal sewage every day through more than 14,000 km of drains and canals. West Bengal is
perhaps the pioneering state where there are 130 sewage-fed fish farms covering an area of 4000 ha
and supplying more than 8000 tons of fish per year to city consumers (Jana, 1998).
42.3.2.1.3 Indonesia
In Malang (East Java, Indonesia), there have been positive health benefits from the installation of
a sewage-based aquaculture system (Harris, 2000). The impetus for building this system was that
five children in the community died from diarrhea. A group of women in the community banded
together and pushed for some form of sanitation system to be installed. Over a period of 2 years,
Agus Guntaro, the neighborhood head, led the community in the building of a simple sewerage
network and treatment system. There are currently 67 houses in the community connected to this
system. The treatment system consists of a septic tank followed by a series of seven tanks through
which the water passes. The first two tanks are desludged every 3 months; the sludge from these
tanks is then dried and sold as compost. The final tank is stocked with Ikan lele, the local catfish
that is harvested and sold for human consumption.
The effluent from this treatment system contains 60 mg/L biochemical oxygen demand (BOD) and
23 mg/L suspended solids (UNDP-World Bank, 1999). The BOD level is above the 20 mg/L required
in most developed countries. It should be noted that subsequent replications of this system are produc-
ing effluent of a lower standard. The main reason for this appears to be a lack of technical knowledge,
with systems being underdesigned for the load they will be receiving. Thus, this system is a step in the
learning process of how sewage-based aquaculture systems can be used effectively and safely.
42.3.3 Other Beneficial Uses of Sewage Effluents

42.3.3.1 Formation or Recharge of Underground Water Reservoirs in Deserts
Where soil and groundwater conditions are favorable for artificial recharge of groundwater
through infiltration basins, a high degree of upgrading can be achieved by allowing partially
treated sewage effluent to infiltrate into the soil and move down to the groundwater. The vadose
zone (somewhat incorrectly termed unsaturated zone) then acts as a natural filter and can remove
essentially all suspended solids, biodegradable materials, bacteria, viruses, and other microor-
ganisms. Significant reductions in nitrogen, phosphorus, and heavy metal concentrations can also
be achieved (Pescod, 1992).
After the sewage, treated in passage through the vadose zone, has reached the groundwater, it
is usually allowed to flow some distance through the aquifer before it is collected. This additional
movement through the aquifer can produce further purification (removal of microorganisms, pre-
cipitation of phosphates, adsorption of synthetic organics, etc.) of the sewage. Therefore, soil and
aquifer are used as natural treatment systems called as soil-aquifer treatment (SAT) systems. SAT
is, essentially, a low-technology, advanced wastewater treatment system. It also has an aesthetic
advantage over conventionally treated sewage in that water recovered from an SAT system is not
only clear and odor free but it comes from a well or drain or via natural drainage to a stream or low
area, rather than from a sewer or sewage treatment plant (STP). Thus, the water has lost its connota-
tion of sewage, and the public see water more as coming out of the ground (groundwater) than as
sewage effluent. This could be an important factor in the public acceptance of sewage reuse schemes
(Pescod, 1992).
42.3.3.2 Thermal Value with Subsequent Implications on Land and Air

The utilization of sewage sludge in agriculture and land reclamation is a principle option (discussed
earlier). Since 1998, European legislation (UWWTD) prohibited sea disposal of municipal sludge,
in order to protect the marine environment (Ødegaard et al., 2002), and so did many other countries.
An alternative was sludge incineration or simply landfilling. Indeed, landfilling of sludge is a loss
of a massive amount of latent energy and implies pollution and thus should be prohibited as well
(the authors’ standpoint). Sewage sludge is a type of biomass fuel and its calorific value is almost
equal to that of brown coal. Therefore, the extraction of energy content and other novel uses of
sludge are also worthy alternatives. The selection of either of these options depends on several fac-
tors (locality, culture, legislations, and economic circumstances). Thermal processes are the oxida-
tion of the organic part of the sludge, leaving only the ash component for final disposal. Over the
recent decades, several technologies have been developed in the market for the thermal processing
of sewage sludge. Mono- and co-combustion of sewage sludge is perhaps the most established pro-
cesses to extract thermal content of sludge, with mono-combustion being even more predominant
(Fytili and Zabaniotou, 2008). These are diverse and include, for example, pyrolysis, incineration,
wet oxidation, gasification, or methanogenesis as well as the utilization of sewage sludge in cement
manufacture as a co-fuel.
The extraction of thermal content of sludge is accompanied by the production of a metal-
concentrated ash (its final fate is to the soils), aerosols, and toxic or greenhouse gases (being incor-
porated to the atmosphere). Since metals and gases affect plant physiology and crop production, a
brief description of the different techniques of thermal extraction of sludge is provided.
42.3.3.2.1 Incineration
Incineration offers the possibility of recovering the energy content of sludge although it does
not constitute a complete disposal method since approximately 30% of the solids remain as ash
(Malerius and Werther, 2003). In addition to the heat output, incineration has advantages of consid-
erable reduction in sludge volume (the final sludge volume after incineration is approximately 10%
of that after dewatering) and thermal destruction of toxic organic compounds (Khiari et al., 2004).
Lundin et al. (2004) reported that the amount of sludge being incinerated in Denmark has already
reached 24% of the sludge produced, 20% in France, 15% in Belgium, and 14% in Germany, while
in the United States and Japan, the it has increased to 25% and 55%, respectively. The resulting
sewage sludge ash (SSA) from incineration might be highly toxic because of its concentrated metal
content. It can be placed in controlled landfills or used in construction to improve certain properties
of building materials (Kääntee et al., 2004). SSA has been used to manufacture bricks, to incorpo-
rate into concrete mixtures and as a fine aggregate in mortar.
Another major constraint in the widespread use of incineration is the public concern about pos-
sible harmful emissions that would be a potential source of various pollutants, and thus care must
be taken during its disposal. Emission results from sludge burn include dioxins and furans, NOx,
N2O, SO2, HCl, HF, and CxHy, which are of great concern as hazardous pollutants (Johnson, 1994).
Despite the noticeable content of sewage sludge in nitrogen, the conversion ratio of fuel N–NOx is
less than 5%, and net emissions of NOx are in very low levels (Zhao et al., 1994). Therefore, devel-
opment of new technologies for controlling gaseous emissions and safe handling of ash reducing
costs are needed.
The majority of energy released during thermal processes is consumed in reducing the amount of
moisture (Khiari et al., 2004; Dennis et al., 2005), and these routes are generally considered to be self-
sufficient in energy (cost/benefit, balance). The situation is very much different in arid soils (e.g., Egypt)
where natural conditions may strongly replace energy costs for drying the sludge. The temperature is
high, the atmosphere is dry, the weather is windy, and the soil is sandy, that is, highly permeable; all these
factors enhance the evaporation of sludge water content (the authors’ own point of view).
42.3.3.2.2 Wet Oxidation
Wet oxidation of sewage sludge is included in the category of thermal processes. It takes place in
an aqueous phase at temperatures of 150°C–330°C and pressures of 1–22 MPa using pure or atmo-
spheric oxygen. The high temperature is needed to prevent boiling at the temperatures required
for the process (Hall, 1995). During the process, organic content of sewage sludge is thermally
degraded, hydrolyzed, oxidized, and converted to carbon dioxide, water, and nitrogen. The whole
process occurs in two distinctive regimes (Werther and Ogada, 1999): The first occurs at subcriti-
cal conditions, which is at a temperature below 374°C and a pressure of 10 MPa. The second is at
supercritical conditions, below 374°C and a pressure of 21.8 Pa.
42.3.3.2.3 Pyrolysis
Pyrolysis is the process through which organic substances are thermally decomposed in an oxy-
gen-free atmosphere, at temperatures varying in the range 300°C–900°C. In other words, pyroly-
sis involves heating of sewage sludge in an inert atmosphere with the consequent release of OM
and its potential recycling. Khiari et al. (2004) described the pyrolysis process as having three
stages:
1. Vaporization of volatile materials.

2. Primary decomposition of nonvolatile components, resulting in the production of solid
char, tar, and gases.
3. The char may undergo secondary higher pyrolysis. In this stage, many of the hydrocarbons
and aromatic compounds are found in the final volatile phase. Compared to conventional
heating in electrical furnaces or fluidized bed reactors, microwave treatment saves time
and energy for the same level of pyrolysis. This technique appears to be less polluting than
conventional methods (incineration, combustion), as it concentrates the heavy metals in a
solid carbonaceous residue, so its leaching is not that crucial as that in ashes from incin-
eration (Menendez et al., 2002). The reactions that take place are thermal cracking and
condensation reactions. Compared to combustion, which is highly exothermic, pyrolysis
is rather endothermic, on the order of 100 kJ/kg (Khiari et al., 2004). The major fractions
that are formed after thermal degradation of the sludge in an inert atmosphere or vacuum
are gases and liquids (Casanova et al., 1997). The noncondensable gas fraction contains
mainly hydrogen, methane, carbon monoxide, carbon dioxide, and several other gases in
smaller concentrations (Chu et al., 2001). The liquid fraction consists of a tar and/or oil,
which contains substances such as acetic acid, acetone, and methanol. Pyrolysis gas can be
used as fuel, as well as the char, while pyrolysis oil can be used as raw material in chemical
industries, even as fuel.
42.3.3.2.4 Gasification
Gasification is the thermal process during which carbonaceous content of sewage sludge is con-
verted to combustible gas and ash in a net-reducing atmosphere. Compared to incineration, gas-
ification, due to the fact that it is a net chemically reductive process, can prevent problems such
as the need for supplemental fuel, emissions of sulfur oxides, nitrogen oxides, heavy metals, and
fly ash and the potential production of chlorinated dibenzodioxins and dibenzofurans (Marrero
et al., 2003). As stated in the literature (Marrero et al., 2003), gasification is a series of complex
sequential chemical and thermal subprocesses. The total process is actually energetically self-
sustaining, and usually in steady conditions, and no energy input is necessary. During the gasifi-
cation process, sewage sludge undergoes a complex physical and chemical change, starting with
drying or removal of water contained as moisture. The dried sewage sludge is then pyrolyzed or
thermally decomposed. In the final step, the pyrolysis products, condensable and noncondensable
vapors and char, undergo gasification, where they are concurrently oxidized and then reduced to
permanent gases at the reduction zone. In the drying zone, sewage sludge descends into the gas-
ifier, and moisture is evaporated using the heat generated in the zones below. The rate of drying
depends on the surface area of the fuel, the recirculation velocity, the relative humidity of these
gases, and temperature differences between the feed and hot gases as well as internal diffusivity
of moisture within the fuel. Characteristically, sludge with less than 15% moisture loses all mois-
ture in this zone (Dogru et al., 2002).
According to Hamiltom (2000), the combustible gases produced during gasification are of high
quality for power generation to produce heat for sludge drying. Once again, we assume that phasing
water content out of wet sludge can be achieved with a low cost by utilizing the nature of aridity in
the southern hemisphere. The bottom line of Dogru et al.’s (2002) work is that small-scale gasifiers
can make an important contribution to the economy of rural areas where sewage sludge is abun-
dantly produced.
The negative impact of gasification process is that a significant amount of aerosols is generated,
giving rise to problems of some volatile elements entering into the vapor phase.
42.3.3.2.5 Methanogenesis and Hydrogen

Methanogenesis of sewage sludge does have an impact on plant physiology but within the frame
of anthropogenic production of greenhouse gases. Also, the residual ash after transformation of
organic compounds is highly rich in metal content and subsequently might be toxic to agricul-
tural soils. Methanogenesis is a very old method used to produce methane (CH4) from organic
wastes. Methane, in this case, is referred to as biogas instead of the oil-derivative natural gas.
The process might occur in highly reduced (surplus hydrogen) anoxic (devoid of oxygen) envi-
ronment by the activity of methanogens (methane-producing bacteria) from the Archaea domain.
Recently, however, Angel et al. (2011) recorded activation of methanogenesis in arid biological
soil crusts despite the presence of oxygen.
Researchers are developing a wide range of advanced processes for producing hydrogen econom-
ically from sustainable resources. Almost all of the hydrogen produced today is by steam reforming
of natural gas, and for the near term, this method of production will continue to dominate. Hydrogen
can also be produced by thermal gasification of biomass such as forestry by-products, straw, munici-
pal solid waste, and sewage sludge (Knoef, 2000; Vierrath and Greil, 2001). The use of biomass
and wastes as energy sources and/or the supply of hydrogen will constitute a practical alternative
for fossil fuels and resulting hazardous gases. Researchers from the United Kingdom and Turkey
have investigated the hydrogen production potential of sewage sludge, by applying downdraft gas-
ification technique (Aznar et al., 1998; Mathieu and Dubuisson, 2002). Wet sewage sludge can be
assumed as one of the most common feedstock to manufacture hydrogen gas (gasification) all over
the world (Dogru et al., 2002); under high temperatures, the waste breaks down to gas. The gas
consists mainly of H2, CO, and methane (CH4). Steam is then introduced to reform CH4 –H2 and CO.
CO is then put through the shift process to attain a higher level of hydrogen. The by-product of this
process is CO2, but such CO2 from biomass is considered neutral with respect to greenhouse gas,
as it does not increase the CO2 concentration in the atmosphere. The mixed gas can also be used in
fuel cells for electricity production.
42.3.3.3 As Adsorbents
Agricultural application of sewage sludge as a fertilizer is questionable particularly if high levels of
heavy metals exist (Renner, 2000). In addition to the previous surveyed uses of sewage sludge as a
fertilizer and energy source is its conversion into adsorbents. This is one of the efficient and envi-
ronmentally friendly utilizations of sewage sludge. Several patents have proposed carbonization of
sewage sludge (Kemmer et al., 1971; Messman and Nickerson, 1975; Sutherland, 1976; Lewis, 1977;
Abe et al., 1994) and application of carbonized material to remove organics in the final stages of
water cleaning (Lewis, 1977) and chlorinated compounds (Kemmer et al., 1971). Sludge-derived
adsorbents have also been tested to remove acidic gases such as sulfur dioxide and hydrogen sulfide.
Adsorbents obtained by pyrolysis of sewage sludge can be considered as complex pseudocomposite
materials. It has been recently shown that by simple pyrolysis of sewage sludge–derived fertilizer,
terrene, an exceptionally good adsorbent for removal of sulfur-containing gases, can be obtained.
Their removal capacity is twice that of coconut shell–based activated carbon (Bashkova et al.,
2001). The extraordinary performance of these materials was attributed to the specific combination
of inorganic oxides of such metals as iron, copper, zinc, and calcium.
42.4 MOST COMMON DRAWBACKS OF SEWAGE APPLICATION

A brief description of some important risks to agriculture and environment that might arise from
applying sewage effluent to arable lands is given in this section.
42.4.1 Pathogenic Micro- and Macroorganisms

Most illnesses that arise from contact of a host with sewage are caused by pathogens. Sewage sludge
contains pathogenic bacteria, viruses, protozoa, and other parasitic helminths that can give rise to
potential hazards to the health of humans, animals, and plants. The most common bacterial patho-
gens in sewage sludge are Salmonella, Shigella, and Campylobacter (Gerba, 1983). Salmonella can
cause salmonellosis; Shigella, dysentery; and Campylobacter, gastroenteritis. A WHO (1981) report
on the risk to health of microbes in sewage sludge applied to land identified salmonellae and Taenia
as being of greatest concern.
More than 110 different viruses may be present in raw sewage and sludge, and the number is
increasing (Gerba, 1983; Abo Soliman, 1997). Enteroviruses, which include poliovirus, echovi-
rus, coxsackievirus, and hepatitis virus, can cause diseases from meningitis to infectious hep-
atitis. Provirus and adenovirus may cause respiratory infection. Viruses tend to sorb strongly
onto sludge, often causing their number to be undercounted (Gaus et al., 1991). Over 90% of
total waste-borne viruses are either inactivated or adsorbed onto sludge during a treatment pro-
cess (Metro, 1983). Sorbed viruses remain active and may survive longer than free viruses in
wastewater. However, infection is only possible when the virus is separated from sludge particles
(Gerba and Bitton, 1984).
Of the common protozoa that may be found in wastewater and sewage sludge, only three spe-
cies are of major significance for disease transmission to humans: Entamoeba histolytica, Giardia
lamblia, and Balantidium coli (Gerba, 1983). All three can cause mild to severe diarrhea. Eggs of
Helminth parasites (intestinal warms), including Ascaris lumbricoides (round worm), Ancylostoma
duodenale (hookworm), Trichuris trichiura (whipworm), and Taenia saginata (tapeworm), tend to
settle with sludge solids during primary wastewater treatment (Gaus et al., 1991). These organisms
are of particular concern because they can survive many forms of sludge treatment and they can
infect humans and animals even at small numbers. The numbers of pathogenic and parasitic organ-
isms in sludge can be significantly reduced before application to the land by appropriate sludge
treatment, and the potential health risk is further reduced by the effects of climate, soil microor-
ganisms, and time after the sludge is applied to the soil. Nevertheless, in the case of certain crops,
limitations on planting, grazing, and harvesting are necessary. Growth and survival of pathogens is
temperature dependent and is thus expected to differ in hot and cold habitats.
42.4.2 Organic Pollutants
Sewage sludge contains hazardous organic pollutants that are of great public concern because they
may enter the food chain. The range of organic compounds known to exist in these matrices is
extensive and diverse and is potentially transferred to sludge- and compost-amended agricultural
soils. From a review of the literature, IAEA (2002) reported that 332 organic pollutants, potentially
hazardous to human or environmental health, were identified in sewage sludge in Germany, 42 of
which are regularly detected in sludge by researchers. Many of the organic compounds (>300),
designated by the European Commission as priority pollutants due to their potentially toxic effects,
are known to occur in sludge. Of these, the halogenated aromatics, like polychlorinated biphenyls
(PCBs), furans, dioxins, and polycyclic aromatic hydrocarbons (PAHs) are generally regarded as
the most critical.
In developing the standards for the use or disposal of sewage sludge (US EPA, 1993), the
US EPA screened 200 pollutants and selected 18 organic pollutants of principal concern for further
evaluation by the pathway risk analysis of environmental exposure (US EPA, 1992). The selec-
tion criteria consider frequency of occurrence, aquatic toxicity, phytotoxicity, human health effects,
domestic and wildlife effects, and plant uptake. Compounds with a high propensity to degrade or
volatilize from soil are generally considered of less concern in sludge-amended agricultural land.
42.4.3 Pharmaceuticals and Other Compounds

Pharmaceuticals such as synthetic hormones, personal care products, and remainders of drugs are
detected in wastewater. Even in trace amounts, concerns about the adverse effects of these com-
pounds, on animal and human health, have been growing for decades. Certain compounds called
endocrine disruptors may disrupt processes in humans that are controlled by hormones, including
development, growth, and reproduction. These compounds are already thought to be causing cancer
and genetic defects in fish. For example, estrogens in environmental waters come primarily from
untreated and not biologically treated domestic wastewaters directly discharged into surface waters.
STP effluents with primarily domestic inputs are strongly suspected to be an important source of
natural and synthetic estrogens contaminating the aquatic environment. Even a few ng/L of some of
these substances can provoke reproductive disturbances in fish.
In particular, effects and risks associated with the presence in surface waters of chemicals having
estrogenic activity (denominated as endocrine disrupters) and thus able to interfere with the endo-
crine/reproductive functions in wild fish has been the object of studies and investigations in several
countries. Starting from 1980, several conjectures and experiments with caged fishes have raised
the possibility that effluents of STPs receiving primarily domestic inputs are one of the sources
of endocrine disrupters. Desbrow et al. (1998) have indicated natural and synthetic estrogens as
candidate compounds to contribute to effluents’ estrogenic activity of STPs located in urban areas.
In vitro studies have shown that exposure of fishes to 1–10 ng/L of 17â-estradiol (Routledge et al.,
1998) and 0.1 ng/L of the synthetic birth control contraceptive 17R-ethinylestradiol (Purdom, et al.,
1994) provokes feminization in some species of male wild fishes. Although steroid conjugates do
not possess a direct biological activity, they can act as precursor hormone reservoirs able to be
reconverted to free steroids by bacteria in the environment. Based on daily excretion of estrogens
by humans, dilution factor, and previous observations by other authors (Stumpf et al., 1996; Johnson
et al., 2000), ng/L levels of estrogens are expected to be present in aqueous environmental samples.
42.4.4 Potentially Toxic Heavy Metals

The concentrations and type of heavy metals in sludge are among the pivotal factors for sludge
utilization in land application because of their potential to enter the human food chain via edible
crops. These wastewaters carry appreciable amounts of trace toxic metals (Brar et al., 2002; Yadav
et al., 2002). Although the total content of heavy metals in the sludge within agricultural soils is a
potential risk for human safety, the mobile contents of the metals are an immediate risk due to their
incorporation in to the human food chain via growing plants and contamination of the groundwater
while moving downward through the soil profile (Aboulroos et al., 1996; Badawy and EI-Motaium,
2003). Tiller (1986) and Rattan et al. (2001) stated that crops raised on the metal-contaminated soils
accumulate metals in quantities excessive enough to cause clinical problems both to animals and
human beings consuming these metal-rich plants.
Concentrations of heavy metals in sewage sludge may vary widely, depending on the sludge
origin (Rattan et al., 2002). Although the concentration of heavy metals in sewage effluents is
low, the long-term use of these wastewaters on agricultural lands often results in the buildup
of these metals at elevated levels in soils (Anikwe and Nwobodo, 2002; Rattan et al., 2002;
Chiroma et al., 2003; Mapanda et al., 2005; Karatas et al., 2006). Extent of buildup of metals in
wastewater-irrigated soils depends on the period of irrigation (Bansal et al., 1992; Palaniswami
and Sree Ramulu, 1994).
Because of their low dynamics, heavy metals possibly accumulate in the lower parts of the plant
such as the root and stem. Nevertheless, Madejo’n et al. (2006) reported the presence of some heavy
metals in the leaves of olive and holm oak trees. In fact, the quantity of element absorption by a
plant depends on several factors, including the total quantity of the elements applied through sewage
application, the soil properties, and the type of plant (Sharma et al., 2007).
Due to the physical–chemical processes involved in activated wastewater treatment, sludge tends
to accumulate heavy metals existing in the effluent (Hsiau and Lo, 1998). The mobility of trace
metals, their bioavailability, and related eco-toxicity to plants depend strongly on their specific
chemical forms or ways of binding (Fuentes et al., 2004). Their potential accumulation in human
tissues and bio-magnifications through the food chain create both human health and environmental
concerns (Krogmann et al., 1999).
42.4.5 Soil Water Repellency

Irrigation with treated sewage effluent has been found to have positive impacts on soil fertility (Speir
et al., 1999; Yadav et al., 2002; Mohammad and Mazahreh, 2003) and to improve or maintain soil
physical properties (Magesan et al., 1999; Agassi et al., 2003). Certain environmentally detrimental
aspects of effluent irrigation have also been documented in field studies, including enhanced down-
ward transportation of pesticides (Graber et al., 1995), increased soil sodicity (Balks et al., 1998),
and excessive nitrate leaching (Magesan et al., 1998; Snow et al., 1999). In addition, irrigation with
sewage effluents leads to changes in physical characteristics of the soil. These include decreased
hydraulic conductivity and blockage of pores by suspended material (Vinten et al., 1983; Levy et al.,
1999) that is referred to as water repellency.
Wallach et al. (2005) documented the development of soil water repellency under irrigation with
secondary-treated sewage effluent in field soils. Soil water repellency affected the uniformity of
moisture content distribution in the soil profile following irrigation events in the summer and rain-
fall events in the winter. The main impacts of soil water repellency are reduced infiltration capac-
ity, increased overland flow and soil erosion, development of fingered flow in structural or textural
preferential flow paths, and creation of unstable, irregular wetting fronts (Robinson, 1999; McLeod
et al., 2001). Hydrophobicity-induced fingered flow can lead to considerable variations in water
content in a water-repellent soil, which can lead to poor seed germination and plant growth (Wallis
and Horne, 1992). Additionally, water flow in preferential pathways has been found to cause acceler-
ated leaching of surface-applied agrochemicals and salts, resulting in increased risks to underlying
groundwater (Ritsema and Dekker, 1998; Blackwell, 2000; Graber et al., 2001).
The increase in water retention more than 50% of soil dry weight may have waterlogging effect on the
plant growth. The soil, due to high levels of organic matter and water content, becomes less permeable
with a low percolation rate, and hence limits gas exchange between soils and air by which the concentra-
tion of oxygen reduced in the soil (Ponnamperuma, 1984). According to Jackson and Drew (1984), these
conditions will reduce plant growth and dry matter accumulation. In this respect, Ramadan et al. (2003)
found that the growth of some plant species such as H. annuus was affected more by high levels of sludge
than others such as S. bicolor. They ascribed such difference may be due to the morphology of Sorghum
root system where the fibrous roots occupy the well-oxygenated surface soil. However, the application
of sewage sludge to reclaimed desert must be under control, and preliminary studies should focus on the
plant species to be cultivated and the physicochemical properties of the soil under reclamation.
42.5 SEWAGE TREATMENTS
Treatment of sewage effluent intends to normalize its characteristics. The degree of treatment, that is,
normalization, can be assessed by comparing the characteristics of wastewater with the required ones.
The acceptable characteristics of normal water vary greatly depending on knowledge, awareness, and
economic level that are of very low standards in many regions of the world. Wastewater treatment
involves removal of nonbiological matter followed by breakdown of complex organic compounds into
simpler compounds that are stable and nuisance-free. The individual methods involved in removal of
contaminants are usually classified as physical unit operations, chemical unit processes, and biological
unit processes. These unit operations and unit processes can be grouped together to provide various
levels of treatment such as primary, secondary, tertiary, and quaternary treatments. The scientific basis
of the various operation units is given briefly in the following discussion.
42.5.1 Physical Unit Operations

Treatment methods in which the application of physical forces predominates are known as physical
unit operations. Screening, mixing, flocculation, sedimentation, floatation, filtration, and gas trans-
fer are examples of physical unit operations.
42.5.2 Chemical Unit Processes

Chemical unit processes involve the addition of chemicals for the removal or conversion of contami-
nants. Precipitation and adsorption are the most common examples used in wastewater treatment.
Chemical precipitation treatment is accomplished by producing a chemical precipitate; the settled
precipitate, in most cases, will contain both the constituents that may have reacted with the added
chemicals and the constituents that were swept out of the wastewater as the precipitate settled.
Adsorption involves the removal of specific compounds from the wastewater on solid surfaces using
the forces of attraction between bodies.
42.5.3 Biological Unit Processes

Biological treatment is applied primarily to remove the biodegradable organic substances (colloidal
or dissolved) in wastewater. Basically, these substances are converted into gases that can evolve
to the atmosphere or incorporate into biological cell tissues. Biological treatment is also used to
remove nutrients (nitrogen and phosphorus) in wastewater. Microorganisms, principally bacteria,
metabolize organic material and inorganic ions present in wastewater during growth, whereas algae
that perform oxygenic photosynthesis are the principal source of oxygen for bacteria. Communities
of prokaryotic microorganisms present in activated sludge (bacteria-enriched sludge) or biofilm
reactors are responsible for most of the carbon and nutrient removal from sewage and thus represent
the core component of every biological wastewater treatment plant. By contrast, mass occurrence of
certain bacterial species can also be detrimental for sewage treatment by negatively influencing the
settling properties of activated sludge in the secondary clarifiers, by contributing to the formation of
foam, or by outcompeting microorganisms required for nutrient removal (Wagner and Loy, 2002).
In another concept, water treatment is classified into primary, tertiary, and quaternary processes.
It begins with the removal of nondegradable suspended solids (e.g., plastics) in the primary treatment
process. Secondary treatment is composed of three steps: activated sludge (flocculation), biological
filtration, and waste stabilization. In the activated sludge or flocculation process, the sewage is aer-
ated by diffused air or by mechanical means. The activated sludge (or biological) contains the micro-
organisms that remove the soluble and insoluble OM in the sewage by a combination of adsorption
and oxidation or assimilation. Aeration supplies the sludge microorganisms with oxygen and keeps
the system in suspension. After a suitable contact time, the sludge is separated from the sewage efflu-
ent in a settling tank. Some of the settled sludge is returned for aeration along with new sewage.
42.5.3.1 Bacteria in Water Purification

Bacteria may be aerobic, anaerobic, or facultative. Aerobic bacteria require oxygen for life support,
whereas anaerobes can sustain life without oxygen. Facultative bacteria have the capability of living
with or without oxygen. In the typical STP, oxygen is added to improve the functioning of aerobic
bacteria and to assist them in maintaining superiority over the anaerobes. Agitation, settling, pH,
and other controllable factors are carefully considered and employed as a means of maximizing the
potential of bacterial reduction of organic in the wastewater. The addition of the proper, specifically
cultured bacteria seems to be the least expensive and most generally reliable way to accomplish
desirable results.
Aerobic bacteria consume organic wastes and oxygen, and they expel water and carbon dioxide
as waste products. These bacteria play a vital part in ecosystems such as wetlands because they cre-
ate the carbon dioxide needed for micro- and macro-hydrophytes plants to thrive and help maintain
water levels depleted by evaporation. In wastewater plants, the water undergoes an involved filter-
ing process before entering the holding ponds and receives chemical disinfection before leaving the
plant.
Anaerobic bacteria operate in closed systems such as septic tanks. They consume organic wastes
and excrete methane and hydrogen sulfide gas. The wastewater enters the septic tank, where it sits
as it separates into a scum layer, middle layer of clean water, and a sludge layer. The bacteria work
in the sludge layer, eating as much of the edible portions of the waste as possible. The inedible rem-
nants are pumped periodically. The water in the middle layer still contains harmful bacteria and
viruses, so it requires further filtering. This water enters leach lines, a snaking trail of perforated
pipes under the ground, where the soil filters the water as it percolates into the groundwater supply
or evaporates into the air.
42.5.3.2 Algae in Water Purification

Algae, performing oxygenic photosynthesis, play a remarkable role in the treatment of municipal
wastewater. This is universal and may be much more efficient in tropical regions where temperature
is warm and sunlight is optimum. Algae have an indispensible role in oxygen/carbon dioxide recy-
cling with a net sequestration of CO2 (greenhouse gas). Algae have also a potential for nitrogen and
phosphorous uptake into the cells, thus acting as fertilizers by mineral recycling. Simultaneously,
they accumulate heavy metals. Moreover, algae also remove pathogens from the domestic waste-
water (Lau et al., 1994; Luz et al., 2004; Muñoz and Guieysse, 2006). Therefore, algae exert mul-
tiple effects in cleansing sewage effluents. The algal biomass produced from the treatment can be
harvested and then could be converted through various methods in to biofuels. Anaerobic digestion
to biogas, transesterification of lipids to biodiesel, fermentation of carbohydrate to bioethanol, and
pyrolysis are examples of algae-based biofuel (Mata et al., 2010). Oxidation ponds treat wastewater
naturally without adding any chemicals. The main goal of these ponds is to increase the production
of oxygen so that aerobic conditions prevail throughout the depth of the pond. Oxidative algal ponds
do not require any external source of oxygen as it produces its own oxygen simultaneously with
consumption of CO2 produced by bacteria, thus reducing the carbon load on environment. In oxida-
tion ponds, algae have a symbiotic relationship with bacteria. They provide the necessary oxygen
to aerobic bacteria to biodegrade the organic contaminants of wastewater while in turn they utilize
carbon dioxide from bacteria and energy from sunlight for the synthesis of carbohydrates.
But algae do not limit their relation of interaction to a simple oxygen and carbon dioxide exchange
(Luz et al., 2004; Muñoz and Guieysse, 2006; Shi et al., 2007). Algae have harmful effects on the
activity of bacteria too by increasing the dissolved oxygen and pH or even by the secretion of inhibi-
tory metabolites. Algae-based wastewater treatment system in oxidation ponds is cost-effective.
It has a low energy cost and lower operation costs, and in the end, algal biomass can be used
for the production of biofuels (Oswald, 1987, 1988; Ogbonna et al., 2000). It was concluded that
algae-based treatment systems can significantly reduce the ecological footprint (Groenlund et al.,
2004). The high nutrient levels in sewage act as fertilizers and cause the number of algae to bloom.
Algal blooms are rapid increases in the population of phytoplankton algae or single-celled plants
that serve as an important food source to other organisms. Oxidation ponds do not require special
skills or expertise for handling so their operation and management is easier than advance mecha-
nized treatment facilities. Treatment of wastewater in oxidation ponds in the presence of algae is an
efficient and economical way of treating wastewater. Oxidation ponds require no external source
of energy, that is, aerators and pumps, rather they utilize natural source of light, that is, sun, and
produce oxygen by algae during photosynthesis that facilitates the growth of bacteria in an optimum
way. Thus, they are economically feasible for treating wastewater.
42.6 CONCLUSION
• High heavy metal content in municipal sewage is inconsistent at all studies and seemingly
not a universal fact. Therefore, some reports denounce the risk of heavy metals down to a
rumor rather than a scientific fact, particularly in effluents, not mixed with industrial dis-
charges. It can be hypothesized that even cumulative effects may be safer than believed as
heavy metals being diluted by continual culturing.
• Arid vs. wet soils should be considered differentially with regard to sewage water use. In
wetlands, groundwater contamination might be possible due to continuous capillary water
between soil particles. Arid lands suffer drought, high temperature, high evaporation, and
high permeability that collectively would break continually of capillarity and thus heavy
metal leakage. Arid characteristics may overcome other negativities of sewage effluents
(e.g., parasites).
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43 Molecular Biologists’,
Water and Crops
Physiologists’, and Plant Breeders’

Approach in the Context
of Evergreen Revolution
Ijaz Rasool Noorka and J.S. Heslop-Harrison
CONTENTS
43.1 Civilization and Food Provision at Crossroads................................................................... 967
43.2 Green Revolution................................................................................................................. 968
43.3 Halving World Hunger by 2015........................................................................................... 968
43.4 Vulnerability, Population Change, and Global Dynamic Pressure..................................... 969
43.5 Food Security Concerns: Food Demand and Supply—The Greatest Challenge
in Africa and Asia under a Burgeoning Population............................................................ 969
43.6 Adoption of Water Conservation Technologies and Irrigation Scheduling........................ 970
43.7 Water, Wheat, Man, and Genetic Variability...................................................................... 970
43.8 Trans-boundary Framework Agreements........................................................................... 971
43.9 Second Green Revolution (Evergreen Revolution) by Conservation of Maximum
Genetic Potential................................................................................................................. 971
43.10 Positional Breeding for Drought Tolerance......................................................................... 971
43.11 Marker-Assisted and Molecular Breeding.......................................................................... 972
43.12 Conclusions......................................................................................................................... 974
Acknowledgment............................................................................................................................ 974
References....................................................................................................................................... 974
43.1 CIVILIZATION AND FOOD PROVISION AT CROSSROADS

Humankind is reaching a series of crossroads of factors such as the following:
• Resources: Fixed nitrogen, phosphate, water (fossil aquifers/rain), soil, land area (soil loss,
desertification, urbanization), sunlight, erosion, resource depletion
• Climate: Cold, heat, CO2, gaseous exchange, stomatal density, and behavior
• Fuel: Wood/biomass, biofuels, fossil fuels, and nuclear fuel
• Abiotic stress and biotic stress
• Gene pool: Biodiversity
• Genes, breeding, agronomy, and crop protection chemicals
• Human population growth (reproduction/longevity), population urbanization, more meat/
fat diets, and invasive species
• Post-harvest losses and post-consumer losses
967
Packaging, transport, and intersections are posing a challenge to food provision. Our civilization
has become divided into much privileged elite and alienated penurious fragments. Although the
Green Revolution won the battle against widespread hunger and ensured sufficient food provision for
some decades, to sustain its pace needs regular inputs from all corners of the world. Among many
alarming issues is the fragmentation of productive land due to population pressure, particularly in
Asia and Africa. Even as a big part of the rural society is being transformed into landless poor, a
large number of people—rich and poor—in developing countries are choosing to forsake an agricul-
tural lifestyle and move to cities, just as occurred 200 years ago in Europe. Political instability and
landlords’ and capitalists’ hold in politics has made the situation worse for proper planning, causing
under execution of plans in most populated parts of the world. Additionally, the regular occurrence of
disasters is a threat to humanity; although these may be caused by natural forces, their consequences
are regularly exacerbated by choices made by humankind.
High-level planning for development and control is a challenge, and is often faced by tragedies of
the commons: for example, strip grazing is much more (ranging to infinitely more!) productive and
sustainable, but to the individual, raking the grass meristems from the dust gives better immediate
return. Even, over a generation, the development of coastal areas and flood plains, or canalization of
rivers and drainage programs, is seen as an advantage, but it sets up some sorts of catastrophe over a
century (Wilfried, 2007). These planning conflicts are shared by both industrialized and developing
nations: individuals want to build on river flood plains and near ocean beaches, be it Bangladesh or
the United States. Violent conflicts; abundant illnesses; occasional floods, famines, or epidemics; and
prevailing hunger especially in less developed countries have killed hundreds of thousands and others
are in grave danger. These are part of the conventional views of the geo tectonic, climate, or biological
factors arising in nature (Wisner, 2004; Smith et al., 2006). However, Platt (1999) and Oppenheimer
(2003) considered it the human response, physical trauma, economic, and political consequences.
43.2 GREEN REVOLUTION
The widespread application of science-based food production involving both genetic improvement of
crops and better agronomy is the consequence of the Green Revolution of the 1960s, which revital-
ized the agricultural economy from a stagnant situation. International germplasm development and
testing networks such as CIMMYT played a constructive role with free exchange of genetic material
to develop a new era of plant genetics by introducing high-yielding, semi dwarf wheat and rice vari-
eties, which paved the way for the Green Revolution. The newly developed varieties achieved their
genetic yield potential by systematic changes in water management and crop yield improvement by
flexible planting dates, fertilizer response, and weed and pest control. In Asia, these species matured
much earlier than traditional landraces, thus permitting double and triple cropping (increasingly wide-
spread in the second millennium). Pakistan is the cradle of the earliest known civilization in South
Asia, the Indus Valley Civilization, and one of the sites of origin of plant domestication (Vavilov,
1992). Pakistan has the mighty river Indus, which is its lifeline, and the country’s irrigation system
mainly runs on replenished, rainfed water supply from the Indus and its tributaries rather than by
exploitation of artesian water basins. The per capita share of water can be used as an indicator to mea-
sure the level of welfare and prosperity in a country. Appropriate use of water is critical in achieving
the Millennium Development Goals, particularly with respect to poverty alleviation, sustainability,
and health goals (http://www.un.org/millenniumgoals/).
43.3 HALVING WORLD HUNGER BY 2015

In the 1996, World Food Summit (WFS), the global community, comprising 139 heads of state and
others, agreed to halve the number of hungry people, that is, down to 400 million, by 2015. The goal
was endorsed in September 2000 at the UN Millennium Summit, but actually we failed due to lack
of political will and focus: the conjunction of government ministers from developed and developing
Water and Crops 969
countries representing an urban elite (supported by economists measuring development and growth
from a strictly industrial perspective) arguably failed to realize that agriculture underpinned their
nations (IPCC, 2007). Even FAO estimates that if the current rate of reduction persists, eradication
of hunger will not be achieved until 2050; in addition, population growth, water availability, and
climate change factors are highly neglected that no reliable model exists to address these issues.
However, proper adaptation of food production and water resource development, underpinned by
sufficient research and development activities, with efficient farm-to-fork packaging and transport
systems, and proper outreach programs, may curtail world hunger in a sustainable manner.
43.4 VULNERABILITY, POPULATION CHANGE,

AND GLOBAL DYNAMIC PRESSURE
What is vulnerability? It is the state of being prone or too susceptible to any damage or injury. It is
worth attempting to consider and refine this commonsense meaning in relation to natural hazards. An
extension involves the capacity to anticipate, cope with, resist, and recover from the impact of a natural
hazard’s harmful effects. It is the pressure of population growth and lack of “modernization” of the econ-
omy and other institutions that drive human activities (Anderson, 2000). In 2000, the world population
crossed the six billion mark, yet only 100 years ago it was under two billion. The UN prediction is that
it will cross nine billion in 20 years. This burgeoning population requires food, fiber, and fuel in mas-
sive quantities (IPCC, 2007). This significant global dynamic pressure has caused destruction of forest,
degradation of soil, and depletion of aquifer, which has directly displaced the national policies to favor
food production (Ngo, 2001). The global environmental change has caused climatic hazards. The United
Nations Development Programme (UNDP) has stated that the research and development achieved over
the past decades is under threat to slow down/have its existing pace reversed by the advent of climate
change. Morrow (1999) has described an emerging food production system coupled with the dangers
of water scarcity, food security, and public health. The impacts of climate change may be a rise in sea
level, periodic and unadorned droughts, and severe heat waves; to add to this, unexpected floods/rainfall
may put an additional 600 million people in the danger of malnutrition, totaling 1.8 billion people being
affected (Brown, 1999; UNDP, 2008). Due to water shortage, production of the most common consum-
able cereal, wheat, has decreased 20%–40% in Asian countries such as Pakistan (Noorka et al., 2013).
Hence, water allocated for necessary agriculture will increase competition and scarcity among crops as
well other societal stakeholders (Khaliq et al., 2009).
43.5 FOOD SECURITY CONCERNS: FOOD DEMAND

AND SUPPLY—THE GREATEST CHALLENGE IN AFRICA
AND ASIA UNDER A BURGEONING POPULATION
Food security is defined as “a situation when all people, at all times, have physical, social, and
economic access to sufficient, safe, and nutritious food that meets their dietary needs and food
preferences for an active and healthy life.” It is a combined monetary and non monetary resource,
which allows independent and frequent access to desirable, safe, and quality food (Schmidhuber
and Tubiello, 2007). Since centuries, agriculture has remained as the backbone of many countries
by contributing a major part to the GDP, attracting foreign exchange, and generating employment.
But under the threat of an increasing population and onset of biotic and abiotic stresses, the food
demand and supply chain has been much affected. Current (2010s) cereal production is about 1.8
billion tons per year, which is equally divided between wheat, rice, and maize. Some estimates
suggest that an additional one billion ton of cereal grain will be needed annually by 2030, an
overall 50% increase in world cereal production in 2000, and perhaps a doubling of production
by 2050 to (1) meet the needs of an increased population, (2) have fewer people go hungry, and
(3) equalize the increased meat consumption. Improvements in agricultural systems can bring
Africa and Asia out of poverty (Shah et al., 2008; Wiggins, 2008). Part of the increase in grain
availability can be achieved by using minimal additional production resources by reducing post
harvest losses.
43.6 ADOPTION OF WATER CONSERVATION TECHNOLOGIES

AND IRRIGATION SCHEDULING
It is true that a fast-growing population and shrinking resources in developing countries have diverted
the world’s attention away from efficient water conservation techniques (Noorka and Shahid, 2013).
Freshwater resources throughout the world are depleting in both aquifers and surface flows. The main
causes seem to be lack of rainfall and unpredictable onslaught of drought and climatic conditions.
However, economic pressure on farmers can be minimized by efficient spadework and introducing
farmer-friendly technologies such as cost-benefit ratio, precision agriculture, irrigation scheduling, water
recycling (Grenoble, 2008), weather forecasting, introduction of growth models (Wurbs, 1994; Ali et al.,
2012), water probes, on-site sensors, climate change prediction, evapo-transpiration data, landscape irri-
gation, soil profiling and grading or terracing, economical seed bed preparation and proper designing,
need-based crop protection measures and efficient composting, zero-till planting into existing stubble
or leaf cover (herbicide-tolerant crops) and rapid establishment of crops after planting, planting of trees
or hedging to reduce both wind and evaporation, and improvement of water percolation or movement
through the soil. However, on-farm water management will remain the focal and critical component
in the process of food production (Noorka, 2011) to mitigate both climate and socioeconomic pres-
sures in the coming decades. All should be well aware that water availability and increasing demand in
agriculture as well as industry will push every society toward optimal water management (Bates et al.,
2008; Ahmed et al., 2011). The adoption of varieties and species of crops resistant to drought, heat, and
temperature stress by the use of conventional breeding or biotechnology and marker-assisted techniques
may lead toward modification, super domestication, and climate change proofing. However, the issue of
fertile land fragmentation needs the urgent attention of governments to help poor smallholders to adopt
appropriate farming technologies (Ali et al. 2012).
Irrigation scheduling is a farmer-friendly technique that maximizes irrigation efficiency by
applying water needed by crop per developmental stage. It determines the exact amount of water to
be applied at the right time. There is a need for a legal framework for water use: to not exploit aqui-
fers until they run out and are not replenished and not use all the water from rivers so that there is
no availability of water downstream. Water treatment from urban or industrial areas, reuse of water
for drinking, and “grey” or “black” qualities also need to be considered in the policy framework for
water availability and utilization. As well as genotype effects, field topology and irrigation schedul-
ing are critical for crop yield, conservation of soil moisture, and efficient use of irrigation water.
The importance of terraces has been recognized by farmers for thousands of years, like agriculture
itself, which was apparently an independent discovery of several civilizations, although they are still
underused in parts of the world both in grazing and croplands.
43.7 WATER, WHEAT, MAN, AND GENETIC VARIABILITY

The history of water, wheat, human being, wheat varieties, periodic water stress conditions, and
adverse climatic conditions runs side by side (Noorka et al., 2013a). By efficient screening, potential
genotypes may be selected (Noorka and Khaliq, 2007; Noorka et al., 2013c) that can be crossed and
utilized in succeeding generations to ensure food security. Suitable genotypes pave the way for good
crop stands and sustainable yield. Along with water conservation techniques and proficient irriga-
tion scheduling, study of wheat screening results is needed to get promising genotypes (Chang and
Loresto, 1986; Noorka and Khaliq, 2007; Raza et al., 2012) for further use in succeeding genera-
tions. According to International Water Management Institute (IWMI) (2009), under the prevailing
water shortage, many developing nations would have to import more than a quarter of their food
Water and Crops 971
items by 2050 without a substantial change to adopt arid and rainfed agricultural practices.
Although two-thirds of the planet’s surface is water, 97% of this water is saline or brackish and
unusable for field-based agriculture. There are prospects for increased marine agriculture with algae
(from several kingdoms) as primary producers and technological approaches to use brackish water
with appropriate crop genotypes. For field crops, desalination cost is uneconomical in most parts of
the world. So we have to depend on only the remaining 3%, out of which 2% is in the form of snow
or ice and located at remote regions; thus, we have to make do with the remaining 1% of the earth’s
freshwater to meet our daily needs and for proper plant growth (Zwart and Bastiaanssen, 2004).
Water stress results in immediate changes, including stomatal closure, reduced photosynthesis, and
delayed wilting. Longer-term changes include adaptation to chronic drought stress and features that
are a consequence of the stress, such as those in plant morphology, leading to reduction of plant
organs and yield (Mary et al., 2001; Noorka et al., 2007, 2009a).
43.8 TRANS-BOUNDARY FRAMEWORK AGREEMENTS

Under the changing climatic system, the only hope is strengthening of early-warning and prediction
models to overcome drought and flood by employing small-scale irrigation and water harvesting
schemes in the fertile, arid, and semi arid areas of the world by resource management practices,
because the increased water demand and decreased supply for agriculture, industry, and domestic
needs in the future decades are predicted challenges in implementing trans boundary framework
agreements to resolve potential conflicts.
43.9 SECOND GREEN REVOLUTION (EVERGREEN REVOLUTION)

BY CONSERVATION OF MAXIMUM GENETIC POTENTIAL
In the Green Revolution of the 1960s, good dwarf gene incorporation (increasing harvest index and
reducing loss through lodging), fertilizer responsiveness, increase in arable areas, and agronomic and
management improvement fed billions of people, which was indeed a great achievement. In the
twenty-first century, to feed the expanding world population and conserve dwindling resources, a
“Blue Revolution” to particularly ensure water availability, a “Brown Revolution” to save degraded
fertile soil, a “White Revolution” to provide fresh milk, and a “Gene Revolution” to conserve genetic
resources are needed. These will help us complement the “Evergreen Revolution.”
It is time to recognize the combined efforts of conventional plant breeders, physiologists, envi-
ronmentalists, agronomists, biotechnologists, and molecular biologists and cyto geneticists to make
significant contributions to enhance food production and quality to add caloric consumption as well
as to prevent/cure (Araus et al., 2002) ailments, for example, anemia, blindness, sugar, cholesterol,
cancer, etc. Efforts toward genetic resistance to insects, pests, and diseases, and tolerance to certain
herbicides (Gianessi, 2002); the success of hybrid rice in China; hybrid wheat, maize, Bt cotton, and
vegetables to withstand biotic and abiotic stresses to relieve billions of people from “stress condi-
tions” are also worth recognition.
43.10 POSITIONAL BREEDING FOR DROUGHT TOLERANCE

Notably, based on the results from heterosis, heritability, and quality traits such as increase in pro-
tein under water stress conditions (Austin et al., 1977, with respect to findings with high grain
nitrogen protein, rather than water and yield) in wheat with different yields (Afiah et al., 2000;
Farshadfar et al., 2000; Memon et al., 2007; Noorka et al., 2009b), plant traits associated with
water stress tolerance additionally having high ranges of heritability with additive genetic effects
(Noorka and Teixeira da Silva, 2012) would be the source for effective breeding and early selection
to cope with water stress problems. It is therefore suggested that potential genotypes be studied and
attempts be made to accumulate favorable genes for stability of characteristics under wider ranges
of environmental stress, and following further inbreeding, with a viable breeding strategy.
In the future, it will be valuable to further dissect the response of plants to irrigation and rainfed
conditions at the physiological level as well as to assay the morphological components of the yield
studied here. Root morphology and establishment of plants under different soil moisture regimes
should also be assessed. Nevertheless, it is clear that the experimental set-up here was suitable to
assess differential responses to irrigation regimes; it is likely that year-to-year differences will make
assessment of genetic differences more robust as, for example, differences in rainy periods made
biomass quantitative trait loci mapping more robust in Lolium forage grass lines (Ma et al., 2006;
Balint et al., 2007, 2009; Collins et al., 2008; Anhalt et al., 2009; Chenu et al., 2009). However,
employment of certain mathematical models and the growing concept of molecular biology to study
the physiological, physico chemical, agronomical, and omics of parental and recombinant lines
may provide well-designed and practical data to seek a viable genomic picture (Marino et al., 2009;
Gupta et al., 2010).
43.11 MARKER-ASSISTED AND MOLECULAR BREEDING

Plant breeding uses genetic diversity within genes and generates new combinations of genes to gener-
ate plant varieties with novel and improved characteristics. In advanced breeding programs, the man-
tra of “cross the best with the best” to obtain a better variety has been successful, relying primarily
on generating new combinations of desirable genes. Sources of new genetic diversity have also been
regularly used by breeders to introduce new alleles with desirable characteristics into new varieties.
Some of such introductions have been remarkably successful: in the case of wheat, the 1BL/1RS trans-
location is widespread globally, introducing a chromosome arm from rye with diverse advantages.
Diversity of different genes and alleles of a single gene are required for breeding. Within crops,
it is often necessary to go outside the current varieties and into the gene pool of older varieties or
their wild relatives to find desirable alleles. These can take many years to introduce by crossing into
varieties since the desirable allele may be linked to poor genes and recombination may be required.
Other sources of variation include mutation breeding (http://nucleus.iaea.org/CIR/CIR/MVD.html),
which involves the generation of new gene alleles by mutation. Sometimes, genes for desired char-
acteristics may not be present in the close relatives of a species and may require transferring from
more distant species: perhaps the best example is the lack of genes producing pro-vitamin A in rice
(leading to blindness disorders in 250 million children a year, of whom more than 1% die). Here,
genes need to be transferred from Narcissus (daffodil) and Erwinia to generate “golden rice” with
the desired characteristics (http://www.goldenrice.org). Finally, there are considerable future pros-
pects for making synthetic genes, most likely in the form of small changes to existing genes but
based on knowledge of the proteins and regulatory elements, for example, to make enzymes that
remain stable at higher temperatures or alter the rate and timing of gene expression. Phenotypes of
crops have been used for selection since the beginning of agriculture. The initial changes required
for crop domestication include a high amount of the harvestable product, and for seed-crops, non-
dispersal of the seeds when ripe and rapid, uniform germination when planted. However, there are
relatively few easily measurable agronomic and morphological characteristics, which can be scored,
particularly within varieties of a crop, and these often show complex interactions with the environ-
ment (growth conditions). Analysis of the DNA of crops now allows direct examination of genes
and measurement of genetic diversity. These data are valuable for (1) identifying sources of useful
diversity and (2) selecting diversity in material for breeding in the right combinations, without the
requirement for extensive trialing and study of segregation, for example, of stacked biotic stress
resistance traits (Nichols et al., 2013). The genetic effects on quantitative trait loci are increasingly
being understood, enabling selection for complex traits based on DNA analysis.
It is important that appropriate marker technologies are applied to address questions on a germ-
plasm collection or breeding lines. Thus, approaches would be different for maize or rice with
fully sequenced genomes, with tens of thousands of accessions, compared with an orphan crop
with a minimal genetics background and perhaps out-breeding characteristics or a long generation time.
Water and Crops 973
A breeding program involving millions of progeny (as in, say, wheat) will use different marker
approaches than one with tens or fewer (as would be the case in banana).
All DNA-based markers eventually measure polymorphisms in the DNA sequence, but different
markers assess these polymorphisms using different hybridization, amplification, or sequencing
approaches. Table 43.1 shows a list of the range of the types of markers that have been used in crop
breeding and biodiversity measurements.
TABLE 43.1
First-, Second-, and New-Generation DNA Markers
Year Acronym Nomenclature Reference
1974 RFLP Restriction-fragment length polymorphism Grodzicker et al.. (1974)
1985 VNTR Variable-number tandem repeat Jeffreys et al. (1985)
1986 ASO Allele-specific oligonucleotide Saiki et al. (1986)
1988 AS-PCR Allele-specific polymerase chain reaction Landegren et al. (1988)
1988 OP Oligonucleotide polymorphism Beckmann (1988)
1989 SSCP Single-stranded conformational Orita et al. (1989)
Polymorphism
1989 STS Sequence-tagged site Olsen et al. (1989)
1990 RAPD Randomly amplified polymorphic DNA Williams et al. (1990)
1990 AP-PCR Arbitrarily primed polymerase chain reaction Welsh and McClelland (1990)
1990 STMS Sequence-tagged microsatellite sites Beckmann and Soller (1990)
1991 RLGS Restriction landmark genome scanning Hatada et al. (1991)
1992 CAPS Cleaved amplified polymorphic sequence Akopyanz et al. (1992)
1992 DOP-PCR Degenerate oligonucleotide primer-PCR Telenius (1992)
1992 SSR Simple sequence repeats Akkaya et al. (1992)
1993 MAAP Multiple arbitrary amplicon profiling Caetano-Anollés et al. (1993)
1993 SCAR Sequence characterized amplified region Paran and Michelmore (1993)
1994 ISSR Inter simple sequence repeats Zietkiewicz et al. (1994)
1994 SAMPL Selective amplification of microsatellite polymorphic loci Morgante and Vogel (1994)
1994 SNP Single-nucleotide polymorphisms Jordan and Humphries (1994)
1995 AFLP (SRFA) Amplified-fragment length polymorphism (selective Vos et al. (1995)
restriction fragment amplification)
1995 ASAP Allele-specific associated primers
1996 CFLP Cleavase fragment length polymorphism Gu et al. (1995)
1996 ISTR Inverse sequence-tagged repeats Brow et al. (1996)
1997 DAMD-PCR Directed amplification of mini satellite DNA-PCR Rohde (1996)
1997 S-SAP Sequence-specific amplified polymorphism Bebeli et al. (1997)
1998 RBIP Retrotransposon-based insertional polymorphism
1999 IRAP Inter-retrotransposon-amplified polymorphism Waugh et al. (1997)
1999 REMAP Retrotransposon-microsatellite-amplified polymorphism Flavell et al. (1998)
1999 MSAP Methylation-sensitive amplification polymorphism Kalendar et al. (1999)
2000 MITE Miniature inverted-repeat transposable element Kalendar et al. (1999)
2000 TE-AFLP Three-endonuclease AFLP —
2001 IMP Inter-MITE polymorphisms Casa et al. (2000)
2001 SRAP Sequence-related amplified polymorphism van der Wurff et al. (2000)
2001 DArT Diversity array technology Chang et al. (2001)
Array markers Array-comparative genomic hybridization Li and Quiros (2001)
Whole-genome DNA sequencing, re sequencing of different varieties Jaccoud et al. (2001)
sequencing-based E.g., Affymetrix or Agilent
SRAP
2010 RNA seq RNA sequencing to identify expressed genes Severin et al. (2010)
43.12 CONCLUSIONS
There is dire need for better understanding of water use efficiency and, particularly, for research
and development to uplift rainfed agricultural systems, which drive production of biotic and abi-
otic resistant crop genotypes that may give high yield with adaptive performance under adverse
climatic conditions. Furthermore, researchers in collaboration with farmers need to experiment
with various types of water conservation techniques, screening methodologies, irrigation sched-
uling, genetic modification, and farmer-friendly technologies. In the last century, crop improve-
ments by genetics and yield increases from suitable agronomy played approximately equal roles
in the enhancement of productivity. More widespread application of best practices in agronomy,
enabling crops grown under conditions where they can approach their yield potential, will cer-
tainly come, but there is a need to reduce agrochemical inputs and water usage. We can therefore
expect increasing genetic contribution to yield and quality through introduction of better genetic
characteristics. Promoting crop insurance to produce enough food for the coming generations
with less water without compromising food security under the changing set of climatic condi-
tions is a critical aim. This century is awaiting a set of revolutions, that is, “Blue,” “Brown,”
“White,” and “Gene” revolutions, to achieve “Evergreen revolution.” Indeed, food security is
closely linked with law-and-order situations that not only shake a country or region, but the
entire globe.
ACKNOWLEDGMENT
The authors are grateful to the Higher Education Commission (HEC), Islamabad, Pakistan, for
sponsoring the post doctoral fellowship and the stay at University of Leicester, United Kingdom,
which made it possible to complete the chapter.
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Cellularization
Syncytium
Antipodals
Central
Embryo sac vacuole
Primary
endosperm Zygote
nucleus nucleus Embryo
3n, 3c 2n, 2c
0 1 2 3 DAP
(a)
AI
SAI
CSEn
6C 12C
3C 3C Pe
Nu 6C 3C 24C
6C Em
Pe 48C
En 12C
Em 96C
TC
G1 PI
M Mitosis G
G2 S
Endoreduplication
S
PCD
Mitotic index
Average DNA content
Nuclei number
Fresh weight
Nuclei number
Mitotic index
Average DNA content
Fresh weight
4 6 8 10 12 15 20 DAP
(b)
FIGURE 1.3 Cell cycle regulation during maize endosperm development. (a) Following double fertilization,
early endosperm development involves acytokinetic mitoses starting with the triploid primary endosperm
nucleus, which results in a syncytium surrounding the central vacuole within the embryo sac. At around 3
days after pollination (DAP), the endosperm becomes cellularized through an open-ended alveolation process
toward the central vacuole until cellularization is complete. (b) After cellularization from about 4 DAP, the
endosperm develops through a phase of mitotic cell proliferation, followed (from around by 8 to 10 DAP)
by endoreduplication (as shown by flow-cytometric profiles), and by programmed cell death (PCD) (starting
around 16 DAP). The endoreduplication phase and the last part of the cell division phase coincide with a
dramatic growth of the endosperm and the synthesis and accumulation of storage compounds. The graph at
the bottom illustrates trends in endosperm fresh weight, nuclei number, mitotic index, and mean DNA content
(C value). Al, Aleurone; CSEn, central starchy endosperm; Em, embryo; En, endosperm; Nu, nucellus; Pe,
pericarp; Pl, placentochalaza; SAl, subaleurone layer; TC, transfer cells. (Reproduced in part from Larkins,
B.A. et al., J. Exp. Bot., 52, 183, 2001; Sabelli, P.A. et al., Maydica, 40, 485, 2005b. With permission from
Oxford University Press and Maydica.)
Acres of Non-Federal Grazing Land, 2007
Each dot represents

25,000 acres
Pastureland
Rangeland
Grazed Forest Land
Federal Land
Hawaii
(no data) There were approximately 583.9 million acres of
Pacific Basin Non-Federal Grazing Land in the conterminous
United States in 2007. This includes approximately
(no data) 409.1 million acres of rangeland, 118.6 million
Northern acres of Pastureland, and 56.1 million acres of
Marianas grazed forest land. Note that the dots do not represent
actual feature locations or points.
Guam The dots are distributed randomly
within each area — in this map
state and 8-digit hydrologic unit.
American Samoa
Map ID: 11039
Data Source:
2007 National Resources Inventory Puerto Rico and US Virgin Islands
Alaska US Department of Agriculture, Natural Resources Conservation Service (no data)
(no data) Map Source:
US Department of Agriculture, Natural Resources Conservation Service
Resources Inventory and Assessment Division, Washington, DC, December 2009
FIGURE 22.1 Distribution of pastureland across the United States in 1997 (1 acre = 0.4047 ha). (Adapted with permission from Izaurralde, R.C. et al., Agron. J.,
103(2), 371, 2011.)
Tall fescue in June Tall fescue in August
Tall fescue in September Tall fescue in November
FIGURE 22.2 Tall fescue grazing plots at the Samuel Roberts Noble Foundation, Ardmore, Oklahoma,
in 2006. Grasses went to complete dormant in August and September during hot and dry weather and returned to
full green growth in November. Through dormancy, forage grasses can escape drought stress by reducing water
need until soil water is replenished through irrigation or precipitation. (Pictures taken by M. Anowarul Islam.)
FIGURE 30.1 Damage to structures caused by salinity.
FIGURE 30.6 Necrotic leaves of Alstonia scholaris due to salinity.
(a) (b)
FIGURE 30.7 C. decidua (a) and S. fruticosa (b) are widely grown in salt-affected landscapes in Pakistan.
y-axis organism: Setaria italica (foxtail millet) (v2.1)
9
ld_
o
aff
Sc
8 _
old
aff
Sc
_7
old
aff
Sc
_6
old
aff
Sc
_5
old
aff
Sc
_4
old
aff
Sc
_3
old
aff
Sc
_2
old
aff
Sc
_1
old
aff
Sc
_1
_3
_4
_2
_6
_8
_9
_7
_5
old
old
old
old
old
old
old
old
old
aff
aff
aff
aff
aff
aff
aff
aff
aff
Sc
Sc
Sc
Sc
Sc
Sc
Sc
Sc
Sc
x-axis: Setaria italica (foxtail millet) (v2.1)
FIGURE 36.3 Syntenic dotplot of a self–self analysis of foxtail millet using SynMap. Green dots are syn-
tenic gene pairs identified through a collinear arrangement. These are derived from the most recent whole
genome duplication event in this lineage. Analysis may be regenerated at http://genomevolution.org/r/8m4c.
10
10
9
9
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
_1
_3
_4
_1
9
_2
_2
_3
_4
_5
_5
Sca d_6
Sca _7
ld_
ld_
ld_
ld_
ld_
ld_
ld
ld
ld
ld
ld
ld
ld
ld
ld
ld
ld
l
ffo
ffo
ffo
ffo
ffo
ffo
ffo
ffo
ffo
ffo
ffo
ffo
ffo
ffo
ffo
ffo
ffo
ffo
S ca
S ca
S ca
S ca
S ca
S ca
S ca
S ca
S ca
S ca
S ca
S ca
S ca
S ca
S ca
S ca
(a) x-axis: Setaria italica (foxtail millet) (v2.1) (b) x-axis: Setaria italica (foxtail millet) (v2.1)
Mean: 0.0048 median –0.2284

2000
1800
Younger <––> Older
1600
Orthologs
1400
1200
Counts
1000
Out-paralogs
800
Noise
600
400
200
0
–1.24987
–1.07252
–0.89517
–0.71783
–0.54048
–0.36313
–0.18578
–0.00843
0.16892
0.34627
0.52361
0.70096
0.87831
1.056
1.233
1.410
1.588
1.765
1.942
2.120
2.262
(c) log 10() substitution per site for Ks
FIGURE 36.4 Syntenic dotplots of a foxtail millet (x-axis) versus sorghum (y-axis) using SynMap. (a)
Syntenic gene pairs are colored green. Note that a given region of either genome is syntenic to two regions
in the other genome (red dashed line). This is due to one syntenic region being orthologous and one being
out-paralogous. Note the large gaps in some syntenic regions (blue arrow). This is a centromere in sorghum.
Results may be regenerated here: http://genomevolution.org/r/8m2u. (b) Syntenic gene pairs are colored by their
synonymous mutation values (Ks). Purple gene pairs are younger than cyan gene pairs and represent ortholo-
gous and out-paralogous relationships, respectively. Results may be regenerated here: http://genomevolution.
org/r/8m2v. (c) Histogram of Ks values shown used in the dotplot from (b).
CROP SCIENCE
THIRD EDITION
Handbook of
PLANT AND CROP PHYSIOLOGY
Continuous discoveries in plant and crop physiology have resulted in an abundance of new
information since the publication of the second edition of the Handbook of Plant and
Crop Physiology, necessitating a new edition to cover the latest advances in the field. Like its
predecessors, the Third Edition offers a unique, complete collection of topics in plant and crop
physiology, serving as an up-to-date resource in the field. This edition contains more than 90 percent
new material, and the remaining 10 percent has been updated and substantially revised.
Divided into nine parts to make the information more accessible, this handbook covers the physiology
of plant and crop growth and development, cellular and molecular aspects, and production
processes. It addresses the physiological responses of plants and crops to environmental stresses,
heavy metals, and agrichemicals; presents findings on small RNAs in response to temperature stress;
and discusses the use of bioinformatics in plant/crop physiology. The book deals with the impacts
of rising CO2 levels and climate change on plant/crop growth, development, and production. It
also offers guidance on plants and crops that can be successfully cultivated under more stressful
conditions, presented in six chapters that examine alleviation of future food security issues.
With contributions from 105 scientists from 17 countries, this book provides a comprehensive
resource for research and for university courses, covering plant physiological processes ranging from
the cellular level to whole plants. The content provided can be used to plan, implement, and evaluate
strategies for dealing with plant and crop physiology problems. This edition includes numerous
tables, figures, and illustrations to facilitate comprehension of the material as well as thousands of
index words to further increase accessibility to the desired information.
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THIRD EDITION

Agricultural Engineering Robert M. Peart, University of Florida, Gainesville

Crops Mohammad Pessarakli, University of Arizona, Tucson

Environment Kenneth G. Cassman, University of Nebraska, Lincoln

Irrigation and Hydrology Donald R. Nielsen, University of California, Davis

Plants L. David Kuykendall, U.S. Department of Agriculture,

Soils Jean-Marc Bollag, Pennsylvania State University,

Soil Biochemistry, Volume 1, edited by A. D. McLaren and G. H. Peterson

Boca Raton London New York

CRC Press is an imprint of the

No claim to original U.S. Government works

International Standard Book Number-13: 978-1-4665-5329-3 (eBook - PDF)

Part I Physiology of Plant/Crop Growth

Chapter 2 Seed Dormancy, Germination, and Seedling Recruitment in Weedy Setaria............ 33

Chapter 3 Alterations in Structural Organization Affect the Functional Ability

Chapter 4 Photoperiodic Control of Flowering in Plants........................................................... 121

Chapter 5 Role of Alternative Respiratory Pathway in Plants: Some Metabolic

Chapter 6 Growth Orientation of Underground Shoots: Stolons and Rhizomes

Chapter 7 Structure and Metabolism of Underground Shoots in Perennial

Chapter 8 Evaluating and Managing Crops Water Requirement............................................... 179

Part II Cellular and Molecular Aspects of Plant/Crop

Chapter 9 Biochemistry and Physiology of Carbon Partitioning in Crop Plants...................... 193

Chapter 10 Role of Nitric Oxide in Plant Development.............................................................. 217

Chapter 11 Mitochondria in Plant Physiology............................................................................. 227

Chapter 12 Signaling Molecules Involved in the Postharvest Stress Response of Plants:

Part III Plant/Crop Physiology and Physiological Aspects

Chapter 14 Physiology of Grain Development in Cereals........................................................... 301

Chapter 16 Physiology of Crop Productivity in Cold Climate.................................................... 333

Chapter 17 Rates of Processes of Essential Plant Nutrients........................................................ 343

Part IV Physiological Responses of Plants/Crops under

Chapter 19 Role of Polyamines in Plant Abiotic Stress Responses............................................. 369

Chapter 21 Drought Resistance in Small Grain Cereal Crops....................................................405

Chapter 22 Drought Physiology of Forage Crops........................................................................ 427

Chapter 23 Effect of Drought/Water Stress and Adaptation to Unintended Consequences

Chapter 24 Physiological Mechanisms of Nitrogen Absorption and Assimilation in Plants

Chapter 26 Reactive Oxygen Species Generation, Hazards, and Defense Mechanisms

Chapter 29 Stress Tolerance in Some European Resurrection Plants

Chapter 30 Salinity and Amenity Horticulture...........................................................................605

Part V Physiological Responses of Plants/Crops to Heavy

Chapter 33 Metal Nanoparticles in Plants: Formation and Action.............................................. 683

Chapter 34 Arsenic Toxicity and Tolerance Mechanisms in Crop Plants................................... 733

Part VI Physiology of Plant/Crop Genetics and Development

Chapter 35 Small RNAs in Crop Response to Temperature Stress Noncoding RNAs

Part VII Bioinformatics and Using Computer Modeling

Chapter 36 Comparative Genomics of Grass Genomes Using CoGe.......................................... 797

Part VIII Plants/Crops Growth Responses to Environmental

Part IX Future Promises: Plants and Crops Adaptation and

Chapter 39 Advances in Improving Adaptation of Common Bean and Brachiaria Forage

Chapter 41 New Approaches to Turfgrass Nutrition: Humic Substances

Chapter 42 Use of Sewage in Agriculture and Related Activities............................................... 931

Mohammad Pessarakli, PhD

Emilia L. Apostolova Thomas W. Crawford, Jr.

Vesselin Baev Gerald N. De La Fuente

Rama Shanker Dubey Elena V. Garmash

Moustafa Eldakak Katya Georgieva

and Maria Gevezova

Yoshie Hanzawa Alberto A. Iglesias

Shahryar F. Kianian Eric Lyons

Michael V. Kolomiets Alexander M. Markarov (deceased)

A.N. Misra Valeria E. Perotti

Idupulapati Madhusudana Rao Kadambot H.M. Siddique

Atif Riaz Jas Singh

Nad’a Wilhelmová Jiří Zámečník

Galina Yahubyan Christian Zörb

Part I Physiology of Plant/Crop Growth

Part II Cellular and Molecular Aspects of Plant/Crop

Part III Plant/Crop Physiology and Physiological Aspects

Part IV Physiological Responses of Plants/Crops under

Part V Physiological Responses of Plants/Crops to Heavy

Part VI Physiology of Plant/Crop Genetics and Development

Part VII Bioinformatics and Using Computer Modeling

Part VIII Plants/Crops Growth Responses to Environmental

Part IX Future Promises: Plants and Crops Adaptation and

1.1 INTRODUCTION—CROP YIELD: A CELL CYCLE PERSPECTIVE

1.2 CORE MOLECULAR CONTROL OF THE CELL CYCLE: AN OVERVIEW

1.2.1 Mitotic Cell Cycle

1.2.1.1 Regulation of the G1/S-Phase Transition

1.2.2 Alternative Cell Cycles: Asymmetric Cell Division and Endoreduplication

1.2.2.2 Endoreduplication Cell Cycle

1.2.2.2.1 Specific Regulation of Endoreduplication

1.3 ENDOREDUPLICATION AS A POTENTIAL YIELD DETERMINANT

1.3.1 Endoreduplication and Tomato Fruit Development

1.3.2 Endoreduplication and Cereal Endosperm Development

1.3.3 Endoreduplication and Biotrophic Interactions

1.3.3.1 Endoreduplication and Symbiotic Interactions

1.3.3.2 Endoreduplication and Parasitic/Pathogenic Interactions

1.4 REGULATION OF PLANT BIOMASS AND ARCHITECTURE

1.4.4 Root Development and Architecture

1.5 PLANT TISSUE CULTURE

1.6 CONTRIBUTION OF CELL CYCLE MANIPULATION

1.7 DOES THE CELL CYCLE DETERMINE YIELD? CONCLUDING REMARKS

2.1 NATURE OF WEED SEEDS AND SEEDLINGS

2.1.3 General Nature of Weeds, the Specific Nature of Weedy Setaria

2.2 NATURE OF WEEDY SETARIA SEED–SEEDLING LIFE HISTORY

2.2.1 Seed –Seedling Life History

2.2.1.2 Germination and Seedling Emergence

2.2.2 Nature of Setaria: Spatial Structure and Temporal Behavior

2.2.3 Setaria Model System of Exemplar Species

2.3 SETARIA SPATIAL STRUCTURE

2.3.1 Plant Morphological Structure

2.3.2.2 Population Genetic Structure

2.3.2.2.1 Local Population (Deme)