Handbook

of Seed Science
and Technology

Amarjit S. Basra, PhD

Editor

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Library of Congress Cataloging-in-Publication Data

Handbook of seed science and technology / Amarjit S. Basra, editor.

p. cm.
Includes bibliographical references and index.
ISBN-13: 978-1-56022-314-6 (hard : alk. paper)
ISBN-10: 1-56022-314-6 (hard : alk. paper)
ISBN-13: 978-1-56022-315-3 (soft. : alk. paper)
ISBN-10: 1-56022-315-4 (soft. : alk. paper)
1. Seeds. 2. Seed technology. I. Basra, Amarjit S.

SB117.H275 2005
631.5'21—dc22
2005002873
 CONTENTS

About the Editor xiii

Contributors xv
SECTION I: SEED DEVELOPMENTAL BIOLOGY
AND BIOTECHNOLOGY
Chapter 1. Molecular Control of Ovule Development 3
Sureshkumar Balasubramanian
Kay Schneitz
Introduction 3
What Are Ovules? 4
Emergence of the Ovule As a Model to Study Organogenesis 4
Ovule Specification 7
Initiation/Outgrowth of the Ovule Primordia 8
Pattern Formation 9
Cell-Cell Communications During Ovule Development 17
Morphogenesis 18
Perspectives 20
Chapter 2. Female Gametophyte Development 27
Wei-Cai Yang
Introduction 27
Organization of the Female Gametophyte 28
Archesporial Cell Formation 32
Megasporogenesis 33
Formation of the Functional Megaspore 38
Megagametogenesis 41
The Female Germ Unit 51
Genetic Control of Megagametophyte Development 52
Conclusions and Perspectives 54
Chapter 3. Cytokinins and Seed Development 63
Neil Emery
Craig Atkins
Introduction 63
 Cytokinin Biosynthesis in Plants 64
Discovery and Characterization of CKs in Developing Seeds 68
Sources of Cytokinin in Seeds 69
Developing Seeds: A Rich Mine of Cytokinin Enzymes 71
Biological Activity of Cytokinin in Developing Seeds 75
Can CK Content Be Manipulated to Alter Grain
Development? 82
Molecular Basis of a Signaling Role for Cytokinins 85
Future Prospects 86

Chapter 4. Grain Number Determination in Major

Grain Crops 95
Gustavo A. Slafer L. Gabriela Abeledo
Fernanda G. González Daniel J. Miralles
Adriana G. Kantolic Roxana Savin
Elena M. Whitechurch
Introduction 95
Grain Number Determination 96
Extrapolations to Other Major Crops 105
Concluding Remarks 114

Chapter 5. Carbon Partitioning in Developing Seed 125

Yong-Ling Ruan
Prem S. Chourey
Introduction 125
Seed Anatomy and Cellular Pathway of Sugar Transport 126
Temporal and Spatial Patterns of Carbon Partitioning 131
Key Genes Controlling Carbon Partitioning in Seeds 135
Conclusions and Future Perspectives 146

Chapter 6. Metabolic Engineering of Carbohydrate

Supply in Plant Reproductive Development 153
Marc Goetz
Thomas Roitsch
The Impact of Plant Reproduction Events on Agriculture 153
The Role of the Tapetum in Male Gametophyte
Development 154
Strategies to Generate Male Sterility in Plants 155
 Importance of Carbohydrates in Plant Growth
and Development 155
Carbohydrate Supply and Male Sterility 157
Future Perspectives 164

Chapter 7. Enhancing the Nutritive Value of Seeds

by Genetic Engineering 171
N. D. Hagan
T. J. V. Higgins
Enhancing the Nutritive Value of Seed Protein 171
Enhancing the Fatty Acid Content of Seeds 177
Enhancing the Vitamin and Mineral Content of Seeds 181
Current Limitations 186
Conclusions 187

Chapter 8. The Process of Accumulation of Seed Proteins

and the Prospects for Using Biotechnology to Improve
Crops 195
Eliot M. Herman
Introduction 195
Synthesis and Accumulation of Seed Proteins 195
The Protein Storage Vacuole 196
Protein Bodies 203
Biotechnology to Study and Change Seed Proteins 206
Identifying and Altering Molecular-Based Traits in Seeds 208

Chapter 9. Synthetic Seed Technology 227

P. Suprasanna
T. R. Ganapathi
V. A. Bapat
Introduction 227
Encapsulation Methods 229
Different Propagules Used for Synthetic Seeds 236
Applications 241
Case Studies 245
Critical Considerations 253
Limitations and Prospects 257
SECTION II: SEED DORMANCY AND GERMINATION

Chapter 10. Dormancy and Germination 271

Henk W. M. Hilhorst
Leonie Bentsink
Maarten Koornneef
Introduction 271
The Regulation of Dormancy and Germination:
A Mutant Approach 273
Dormancy and Germination: Mechanisms 283
Prospects and Challenges 290

Chapter 11. Hormonal Interactions During Seed

Dormancy Release and Germination 303
Gerhard Leubner-Metzger
Introduction 303
Abscisic Acid (ABA): A Positive Regulator of Dormancy
Induction and Maintenance, a Negative Regulator
of Germination 305
Gibberellins Release Dormancy, Promote Germination,
and Counteract ABA Effects 312
Ethylene Promotes Seed Germination and Counteracts
ABA Effects on Seeds 319
Brassinosteroids Promote Seed Germination 324
Cytokinins and Auxins 327
Conclusions and Perspectives 328

Chapter 12. Photoregulation of Seed Germination 343

Chizuko Shichijo
Osamu Tanaka
Tohru Hashimoto
Introduction 343
Phytochromes 347
Action Modes (LFR, VLFR, and HIR) of Phytochromes
in Seed Germination 353
Multiple Modes of Seed Germination and Explanation
by Action Modes of Phytochromes 358
SECTION III: SEED ECOLOGY

Chapter 13. Competition for Pollination and Seed Set 369

Beverly J. Brown
Introduction 369
Pollen Quantity 374
Pollen Quality 380

Chapter 14. Seed Size 397

Jorge Castro
José A. Hódar
José M. Gómez
Introduction 397
Setting Terminology 398
Seed Mass Variability 399
Seed-Size Determination 399
Dispersal 405
Depredation 406
Germination 407
Seedling Performance 408
Habitat Type and Plant Traits 411
Seed-Size Evolution 412
Seed Size and Global Change 414
Humans and Seed Size 415

Chapter 15. Seed Predation 429

Jose M. Serrano
Juan A. Delgado
Introduction 429
Defending Seeds 431
A Hierarchical Perspective 435
The Case of Cistus ladanifer L. 440

Chapter 16. Natural Defense Mechanisms in Seeds 451

Gregory E. Welbaum
Introduction 451
Seed Defense Mechanisms 452
Summary 464
Chapter 17. Seed Protease Inhibitors 475
A. M. Harsulkar V. S. Gupta
A. P. Giri M. N. Sainani
V. V. Deshpande P. K. Ranjekar

Introduction 475
Protease Inhibitors 477
Expression of Protease Inhibitors in Seeds 480
Biological Role of Protease Inhibitors 485
Protease Inhibitor Transgenic Plants for Pest Control 489
Conclusions 490

Chapter 18. Soil Seed Banks 501

A. J. Murdoch

What Is the Soil Seed Bank? 501

Functions of Soil Seed Banks 502
Prerequisites for a Seed Bank 503
How Many Seeds Are in the Soil? 506
What Types of Seeds Are in the Soil? 510
Where Does the Seed Bank Come From? 510
What Happens to the Soil Seed Bank? 511
Impacts on Weed Management 516

Chapter 19. The Ecophysiological Basis of Weed Seed

Longevity in the Soil 521
R. S. Gallagher
E. P. Fuerst

Introduction 521
Conceptual Model for Seed Longevity 522
Resource Allocation to Seed 524
Seed Dormancy 527
Role of Seed Vigor 536
Microbial Seed Decay 542
Implications for Seed Bank Management 547
Implications for Future Research 548
SECTION IV: SEED TECHNOLOGY

Chapter 20. Seed Quality Testing 561

Sabry Elias
Introduction 561
What Is Seed Quality? 561
Is Testing the Quality of Seed Really Important? 564
Seed Purity Testing 566
Seed Viability Testing 576
Testing for Special Seed Attributes 586
Seed Vigor Testing 590
Pathological Testing of Seed 590
Testing Genetically Modified Seeds 594
Quality Assurance in Seed Testing 595
Seed Quality Testing and Seed Certification 597
Seed Testing Organizations 598

Chapter 21. Seed Vigor and Its Assessment 603

Alison A. Powell
Concept of Seed Vigor 603
Causes of Differences in Seed Vigor 606
Assessment of Seed Vigor 618
Standardization of Vigor Test Procedures 631
Current Status of Vigor Testing 632
Presentation and Interpretation of Vigor Tests 633
Application of Vigor Tests 635
Conclusions 636

Chapter 22. Diagnosis of Seedborne Pathogens 649

Emily Taylor
Jayne Bates
David Jaccoud
Introduction 649
Conventional Seed Health Diagnostic Methods 651
Molecular Methods for Seed Health Testing 659
Conclusions 670
Chapter 23. Seed Quality in Vegetable Crops 677
S. D. Doijode
Introduction 677
Factors Affecting Seed Quality 679
Improvement of Seed Quality 686
Maintenance of Seed Quality 690
Revival of Seed Quality 694
Conclusion 696
Chapter 24. Vegetable Hybrid Seed Production
in the World 703
David Tay
Introduction 703
The Gene-Control Pollination F1 Vegetable Seed
Production System 705
The Hand-Pollinated F1 Vegetable Seed Production System 705
Distribution of F1 Vegetable Seed Production in the World 706
Development of Hand-Pollinated Hybrid Vegetable Seed
Production in the World 708
F1 Hybrid Vegetable Seed Production: A Case Study
on Tomato in Taiwan 713
Future of Hand-Pollinated F1 Vegetable Hybrid Seed
Production 718
Chapter 25. Practical Hydration of Seeds of Tropical
Crops: “On-Farm” Seed Priming 719
D. Harris
A. Mottram
Chapter 26. Seed Technology in Plant Germplasm
Conservation 731
David Tay
Introduction 731
Seed Science and Technology in Gene Banks 733
Gene-Bank Management System 737
Basic Gene-Bank Design 744
Gene-Bank Research Program 745
Index 749
 Chapter 1

Molecular Control
Molecular Controlof Ovule
of Ovule Development
Development

Sureshkumar Balasubramanian
Kay Schneitz

INTRODUCTION

A flowering plant’s life cycle contains two phases: a vegetative phase

and a reproductive phase. The shoot apical meristem, which gives rise to all
the aboveground parts of a plant, produces leaves at its flanks during the
vegetative phase. Upon entry into the reproductive phase, it undergoes an ir-
reversible phase change to become the inflorescence meristem that
produces floral meristems which in turn form the flowers. External environ-
mental as well as internal genetic cues control floral induction. More than
80 genetic loci have been implicated as playing a role in the induction of
flowering. The genetics of floral induction is beyond the scope of this
chapter, and the reader should look into the wealth of information available
in several recent reviews (Levy and Dean, 1998; Simpson et al., 1999;
Simpson and Dean, 2002).
A seed plant’s flower contains two types of floral organs: the reproduc-
tive organs and the perianth organs (Esau, 1977). In a flower, stamens and
the carpels represent the male and female reproductive structures, respec-
tively. In dicots, sepals and petals are the usual perianth organs, and in
monocots, they are represented by glumes, lemma, and palea. All the floral
organs originate from the floral meristem. Molecular and genetic analysis
using mainly Arabidopsis thaliana and Antirrhinum majus has given insight
into the molecular mechanisms that control floral organ identity (Coen and
Meyerowitz, 1991; Lohmann et al., 2001; Lohmann and Weigel, 2002;

We would like to thank David Chevalier for unpublished information and Markus
Schmid for comments on the manuscript. S.B. was supported by a postdoctoral fellow-
ship from Roche during the final stages of his stay in Schneitz lab at Zurich. The work in
the Schneitz lab is supported by Swiss National Foundation, Kanton of Zurich at Zurich,
and the Deutche Forschung Gemeinschaft at Munich.
3
4 HANDBOOK OF SEED SCIENCE AND TECHNOLOGY

Sessions et al., 1998; Weigel, 1995). The gynoecium, which bears the fe-
male reproductive organs, is made of carpels, and within the carpels ovules
develop.
Ovules are the progenitors of the seeds and thus represent the major fe-
male reproductive organs. The molecular control of ovule development has
picked up since the 1990s, and a network of genes that underlie various as-
pects of ovule development is being uncovered. In this chapter, we will
summarize our present knowledge of the molecular control of ovule devel-
opment, mainly focusing on the results obtained from the molecular and ge-
netic analysis done in Arabidopsis and Petunia.

WHAT ARE OVULES?

Ovules are the female reproductive organs of seed plants (Esau, 1977).
An ovule typically has three parts: the nucellus, the chalaza, and the
funiculus. The nucellus at the distal end harbors the megaspore mother cell
(mmc) that will give rise to the embryo sac. From the central region, re-
ferred to as chalaza, integuments originate and develop to envelop the grow-
ing embryo sac. The integuments eventually function as a seed coat. The
number of integuments that are present can vary from species to species.
Based on the number of the integuments, the ovules can be unitegmic
(which have a single integument, e.g., Petunia) or bitegmic (which have two
integuments, e.g., Arabidopsis) (Esau, 1977). At the proximal end, the
funiculus connects the ovule to the placenta (Esau, 1977). Upon double fer-
tilization, the ovule becomes the site of seed formation. Ovules have a char-
acteristic shape as well. For example, Arabidopsis ovules show a character-
istic curvature referred to as anatrophy. The anatrophy of the Arabidopsis
ovules is a result of the differential initiation and growth of the outer integu-
ment along the adaxial-abaxial axis (adaxial: adjacent to the meristem;
abaxial: away from the meristem). The outer integument initiates at the
abaxial side and grows more on the abaxial side compared to the adaxial
side. This leads to a curvature in the ovule.

EMERGENCE OF THE OVULE AS A MODEL

TO STUDY ORGANOGENESIS

Since the 1990s, ovules have been recognized as a very good model to
study organogenesis (Gasser et al., 1998; Grossniklaus and Schneitz, 1998;
Schneitz, Balasubramanian, and Schiefthaler, 1998; Chevalier et al., 2001).
Several factors make ovules a suitable system to study organ development.
 Molecular Control of Ovule Development 5

First, ovules have a simple structure of a characteristic size and shape, pro-
viding a simple model to study organogenesis. Second, a defect in ovule de-
velopment usually results in sterility, allowing easy identification of mu-
tants in genetic screens. Third, all the ovules within a gynoecium follow a
stereotypic developmental pattern. This allows for large numbers of ovules
that can be studied, which is very useful in statistical analysis. Fourth, even
mature ovules have only a few cell layers with a limited number of cell
types, which allows for easy cytological inspection of ovules with organic
clearing methods. Furthermore, being the progenitor of seeds, understand-
ing the biology of ovule development has major agronomical implications.

Basic Aspects of Organogenesis

Organogenesis, the formation of an organ from a group of undifferenti-

ated cells, is a precisely controlled process. This process includes several
aspects: specification, initiation and outgrowth, pattern formation, and
morphogenesis. These processes can theoretically be separated, though in
reality they are interdependent and may occur simultaneously. During spec-
ification, a group of cells is set aside from a mass of undifferentiated cells to
form an organ. This is followed by the initiation of organ formation and the
outgrowth of the primordia. Pattern formation lays down the basic struc-
tural plan of the organ. Pattern formation refers to the process in which the
cell activities are organized in a nonrandom manner to give rise to a definite
pattern during development. Defined cell division and differentiation pat-
terns during morphogenesis lead to a mature organ of the correct size and
shape. Almost every organ formation would involve these basic aspects that
occur repeatedly during development, though the mechanisms by which
these processes occur might differ with respect to individual organs.

An Overview of Wild-Type Ovule Development

Ovule development in wild-type Arabidopsis is very well documented

and is divided into distinct stages that can be distinguished by visible mor-
phological markers (Schneitz et al., 1995). Ovules are formed within the
carpel from the placental tissue as fingerlike protrusions around stage 1.
Around stage 2-I, one hypodermal cell enlarges and differentiates into the
mmc (see Figure 1.1, part B). After the specification of the mmc, the inner
and outer integuments initiate from the epidermis of the chalaza, succes-
sively (see Figure 1.1, parts A and B). The outer integument shows asym-
metry in its initiation and growth. It initiates and grows more on the abaxial
side leading to the anatrophy of the Arabidopsis ovule. Simultaneously, the
6 HANDBOOK OF SEED SCIENCE AND TECHNOLOGY

FIGURE 1.1. Ovule development in wild-type (Ler) Arabidopsis. A and C: Scan-

ning electron micrographs (SEMs). B and D: Vertical optical sections through
whole mount ovules. Stages: A and B: 2-III; C: 4-V; and D: Late 3-IV. nu-nucellus;
ii-inner integument; oi-outer integument; mmc-megaspore mother cell; fu-
funiculus; pt-pollen tube; cc-central cell; et-endothelium; ec-egg cell; vs-vascular
strand; syn-synergids. The initiation of the outer integument at the abaxial side is
visible (A). Note the presence of the megaspore mother cell (B) which develops
into an embryo sac (D). Scale bars, 20 mM.

mmc undergoes meiosis and one of the meiotic products continues its de-
velopment to form finally a seven-celled polygonum type embryo sac (see
Figure 1.1, part D) (Maheswari, 1950). The growing integuments eventu-
ally envelop the embryo sac (see Figure 1.1, parts C and D).
 Molecular Control of Ovule Development 7

OVULE SPECIFICATION

Although several genetic screens were done in different laboratories, no

single mutant that fails to form an ovule could be isolated, suggesting that
redundant factors are involved in the specification of an ovule. Tissue cul-
ture experiments in tobacco have revealed that the commitment to ovule
identity follows a progressive pattern (Evans and Malmberg, 1989). Placen-
tal explants cultured in vitro showed that depending on the developmental
age, the placentae can differentiate into either carpels or ovules. Although,
under in vivo conditions, these placentae would have normally committed
to form ovules, they seem to get to this commitment in a progressive way:
first to a carpel and then to an ovule. At least two loci, Mgr3 and Mgr9, af-
fect this commitment to ovule identity in tobacco (Evans and Malmberg,
1989). These loci have not yet been characterized at a molecular level.
What is the molecular nature of genes that confer ovule identity? With
the exception of AP2, all the ABC genes, which confer the identities to the
floral organs, encode MADS domain transcription factors (Goto and Mey-
erowitz, 1994; Jack et al., 1992; Mandel, Gustafson-Brown, et al., 1992;
Yanofsky et al., 1990). Not surprisingly, two MADS domain proteins,
FLORAL BINDING PROTEIN 7 (FBP7) and FBP11 in Petunia, are ex-
pressed in the ovule primordium and play a role in the commitment to ovule
identity (Angenent et al., 1995; Cheng et al., 2000; Colombo et al., 1995).
Simultaneous cosuppression of FBP7 and FBP11 results in carpelloid
structures in place of ovules, suggesting their involvement in the specifica-
tion of the ovule. The ectopic expression of FBP11 leads to ectopic ovule
formation, corroborating its role in ovule specification (Colombo et al.,
1995).
Several MADS box genes in Arabidopsis are expressed in ovules (Rouns-
ley et al., 1995). Although the exact orthologues of FBP7 and FBP11 in
Arabidopsis are not known, AGL11 is a likely candidate based on sequence
comparisons. Recently a loss-of-function allele of agl11 has been isolated.
Seeds formed in this mutant referred to as seedstick (stk) fail to detach from
the placenta (Pinyopich et al., 2001). STK is closely related to two other
MADS box genes, SHATTERPROOF 1 (SHP1) and SHP2, which show an
expression pattern in the ovule similar to that of STK. A triple mutant of stk
shp1 shp 2 forms carpelloid tissue in place of ovules, showing the redundant
involvement of STK, SHP 1, and SHP 2 in the specification of an ovule
(Pinyopich et al., 2001). SHP genes are negatively regulated by another
MADS box gene, FRUITFULL, (FUL), in the carpel (Ferrandiz et al., 2000),
and the carpelloid tissue that is formed in the stk shp1 shp2 triple mutant
shows an ectopic expression of FUL (Pinyopich et al., 2001).
8 HANDBOOK OF SEED SCIENCE AND TECHNOLOGY

Another MADS box gene that is expressed in ovules is AGAMOUS (AG)

(Yanofsky et al., 1990). It has been difficult to study the role of AG in ovule
development due to its early function that leads to a floral phenotype in ag
mutants. The overexpression of the AG orthologue from Brassica napus
causes homeotic conversion of sepals into carpels which bear carpelloid
ovules (Mandel, Bowman, et al., 1992) in tobacco. These organs show re-
semblances to the mgr mutants described by Evans and Malmberg (1989).
This suggests an inhibitory role of AG in ovule specification. Contrary to
this, the A function gene AP2 promotes ovule identity as several loss-of-
function alleles of ap2 show conversion of ovules into carpelloid structures
(Modrusan, Reiser, Feldmann, et al., 1994). However, the ap2 ag double
mutants form normal ovules (Bowman et al., 1991), although the percent-
age of normal ovules might be less in these double mutants compared to the
single mutants (Western and Haughn, 1999). This suggests that neither AG
nor AP2 is absolutely required for the commitment to ovule fate.

INITIATION/OUTGROWTH OF THE OVULE PRIMORDIA

We refer to the emergence of the ovule primordia, as fingerlike protru-

sions, from the placental tissue as “outgrowth.” Genetic analysis identified
three loci that are involved in this process (Broadhvest et al., 2000;
Schneitz, Baker, et al., 1998). In wild-type Arabidopsis, approximately 50
ovules develop within a gynoecium, which are spaced in a characteristic
way to form interdigitating ovule primordia. aintegumenta (ant) mutants
form fewer ovules that are distantly spaced compared to the wild type
(Baker et al., 1997; Elliott et al., 1996; Klucher et al., 1996). ANT encodes a
protein that belongs to the AP2/EREBP family of transcription factors
(Okamuro et al., 1997; Riechmann and Meyerowitz, 1998). The ovules of
ant mutants lack integuments and often show a reduction in the outgrowth
of the ovule primordium. They do not form a viable embryo sac and, there-
fore, are sterile (Baker et al., 1997; Elliott et al., 1996; Klucher et al., 1996;
Schneitz et al., 1997). ANT has pleiotropic roles in flower development and
plays a role in controlling organ size (Krizek, 1999; Mizukami and Fischer,
2000). The expression pattern of ANT suggests that it is involved in cell pro-
liferation and growth control.
In spite of the absence of ANT, few ovules still develop in ant mutants,
suggesting the involvement of redundant factors. Plants that are mutant for
huellenloss (hll) form fewer ovules that are smaller and more distantly
spaced than in the wild type, similar to ant (Schneitz, Baker, et al., 1998;
Schneitz et al., 1997). While the ant mutants show defects in many parts of
the plant, the hll phenotype is restricted to ovules. The ovules of hll mutants
 Molecular Control of Ovule Development 9

lack integuments and often show cell death in the distal region of the ovule
primordium. They appear small and short, suggesting a role for HLL in the
outgrowth of the ovule primordium. Interestingly, the ant hll double mu-
tants show a drastic reduction in the ovule primordium compared to that of
either ant or hll single mutants. The analysis of these short outgrowths
using molecular markers revealed that the morphological units that are
present in an ovule can form independently of each other (Schneitz, Baker,
et al., 1998). HLL encodes a mitochondrial ribosomal protein (Skinner et al.,
2001), indicating the importance of energy requirements during organo-
genesis.
SHORT INTEGUMENT 2 (SIN2) is another locus that has been impli-
cated in the outgrowth of the ovule primordium. Ovules of sin2 mutants
show a phenotype similar to that of hll mutants (Broadhvest et al., 2000). In-
teguments initiate normally in ovules of sin2 mutants but are arrested in
their growth shortly after initiation, implying a role for SIN2 in the integu-
ment outgrowth. sin2 hll double mutants produce ovules that are arrested in
their outgrowth and appear even shorter than the ant hll double mutants
(Broadhvest et al., 2000). sin2 mutants also form fewer ovules compared to
the wild type, indicating a role for SIN2 in the initiation of the ovule
primordia. Taken together, these results indicate that ANT, HLL, and SIN2
redundantly control the initiation and outgrowth of the ovule primordium
through coordinating the energy requirements with cell proliferation.

PATTERN FORMATION

Pattern formation is the process by which the cell activities are organized
in a nonrandom manner with respect to its position in a three-dimensional
space. In a mature ovule, one can clearly distinguish two axes of polarity:
the proximal-distal axis and the adaxial-abaxial axis (see Figure 1.2).

Proximal-Distal Pattern Formation

Along the proximal-distal axis (also referred to as chalazal-micropylar

axis), three morphological units are present in an ovule (Esau, 1977;
Schneitz et al., 1995): the nucellus at the distal end, chalaza in the central re-
gion, and the funiculus at the proximal end connecting the ovule to the pla-
centa. This morphological pattern is typical of seed plants (Esau, 1977). It
has been proposed that a corresponding initial prepattern is laid down
which ultimately results in this structured arrangement along the proximal-
distal axis (Schneitz et al., 1995).
10 HANDBOOK OF SEED SCIENCE AND TECHNOLOGY

FIGURE 1.2. Schematic representation showing the different axes of polarity

during ovule development in Arabidopsis thaliana. Note the onset of the outer
integument at the abaxial side. Nu-nucellus; Ch-chalaza; Fu-funiculus; ii-inner
integument; oi-outer integument; ad-adaxial; ab-abaxial. The dotted lines in the
chalaza represent the possible subdivision of the chalaza that would give rise to
the inner and the outer integuments.

Several lines of evidence support such a model of prepatterning. Several

mutants show phenotypes restricted to distinct morphological units, which
suggests the independence of each of these units. bell (bel1) mutants show a
phenotype restricted to the chalaza and bear ovules that have bulbous out-
growths in place of integuments (Modrusan, Reiser, Fischer, et al., 1994;
Robinson-Beers et al., 1992) (see Figure 1.3, part A). At very late stages in
development, these outgrowths often show carpelloid features (Modrusan,
Reiser, Feldmann, et al., 1994). BEL1 was suggested to negatively regulate
AG expression. Thus, the bel1 phenotype could be due to an ectopic AG ex-
pression in the ovule (Ray et al., 1994). This view is not supported by the
fact that AG and BEL1 have overlapping expression domains in the ovule
(Reiser et al., 1995). The molecular cloning of BEL1 revealed that it en-
codes a homeodomain containing transcription factor (Reiser et al., 1995).
Homeodomain proteins have been identified in animals as well as in plants
and play vital roles in pattern formation (Wolpert et al., 1998). BEL1 is ex-
pressed throughout the ovule primordia at the initial stages and gets re-
stricted to the central region around stage 2-I (Reiser et al., 1995) (see Fig-
ure 1.3, part B) before the initiation of the integuments and thereby marks
the central region at a molecular level. The expression pattern of BEL1 and
the biochemical nature of BEL1 protein make BEL1 a candidate gene in-
volved in pattern formation during ovule development. However, bel1 mu-
tants still show morphological distinctions along the proximal-distal axis.
 FIGURE 1.3. The ovule phenotypes of mutants and the expression patterns of the corresponding genes in the ovule.
A, C, E, and G: SEMs of ant, bel1, ino, and nzz, respectively. B, D, F, and H: Expression patterns of ANT, BEL1, INO,
and NZZ, respectively, in ovules of wild-type Arabidopsis. Stages: B and D: 2-I; F: 2-III; H: 2-IV; G: 3-IV; A, C, and E: 4-V.
Note the absence of INO expression in the tip cell that undergoes initial division to give rise to the two cell layers of the
outer integument (arrow F). Note a rare occassion of highly reduced integuments in nzz (arrow G). oi-outer integument.
Scale bars = 20 mM.
11
12 HANDBOOK OF SEED SCIENCE AND TECHNOLOGY

The outgrowths appear only from the chalaza, and the chalazal restricted
BEL1 expression can be observed in bel1-1460, an ems-induced mutant
allele. This suggests that bel1 mutants still retain some chalazal identity.
The ant mutants show severe reduction in integument growth (see Figure
1.3, part C). ANT encodes a protein that belongs to the AP2 family of tran-
scription factors. The expression pattern of ANT in the ovule is very similar
to that of BEL1. ANT is initially expressed throughout the ovule primordia.
Around stage 2-I, ANT expression is excluded from the nucellus and the
proximal region, and molecularly marks the central region (Elliott et al.,
1996; Klucher et al., 1996) (see Figure 1.3, part D). Later in development,
ANT is expressed in the developing integuments and the growing region
(distal) of the funiculus.
The inner no outer (ino) mutants bear ovules that lack the outer integu-
ment (Baker et al., 1997; Schneitz et al., 1997) (see Figure 1.3, part E) and
INO plays a vital role in the adaxial-abaxial pattern formation. The defects
in the ino mutants are restricted to the proximal chalaza. From the flanks of
the distal chalaza, the inner integument initiates and develops normally.
Thus, the phenotype of ino mutants allows us to divide the central region
into two subdomains, one that forms the inner integument and one that
forms the outer integument (Schneitz, Balasubramanian, and Schiefthaler,
1998). Then what regulates the boundary between these two subdomains?
This process seems to be controlled by ABERRANT TESTA SHAPE (ATS),
since ats mutants show defects in the maintenance of the boundary between
the inner and outer integuments (Léon-Kloosterziel et al., 1994). The
ovules of ats mutants initiate both integuments normally as in wild type, but
later in development, the inner cell layer of the outer integument and the
outer cell layer of the inner integument fuse (K.S, unpublished). Conse-
quently, at later stages, these integuments behave as a single integument
(Léon-Kloosterziel et al., 1994). In addition to these mutants, several
enhancer trap lines show region-restricted expression patterns (Grossnik-
laus and Schneitz, 1998), suggesting the existence of these pattern elements
along this axis.
Recent studies have shown that NOZZLE (NZZ, also known as SPL
[SPOROCYTELESS]), a gene encoding a novel protein, plays a vital role in
patterning along the proximal-distal and the adaxial-abaxial axis (Bala-
subramanian and Schneitz, 2000, 2002; Schiefthaler et al., 1999; Yang
et al., 1999). nzz mutants show defects throughout the ovule (see Figure 1.3,
part G). In ovules of nzz mutants, the nucellus is reduced, the integuments
show temporal alterations in their initiation and are often reduced, and the
funiculus is enlarged. The expression pattern of NZZ matches with its phe-
notype (see Figure 1.3, part H). How does NZZ function during proximal-
distal pattern formation? The best clue comes from the analysis of nzz bel
 Molecular Control of Ovule Development 13

double mutants (Balasubramanian and Schneitz, 2000). The nzz bel double
mutants produce ovules that lack any integuments and have an extra-long
funiculus. No sign of the presence of the central region can be seen in these
double mutants, and the tissue that is formed in the central region resembles
that of funiculus as seen by the epidermal cell morphology (see Figure 1.4,
part A). Furthermore, INO expression, which is usually detected in only in
few cells of the chalaza in wild type, cannot be detected in these double mu-
tants, suggesting the absence of the chalazal identity of the tissue. Taken to-
gether, these results suggest that in the absence of NZZ and BEL1 chalazal
identity is lost and the tissue is replaced by funiculus, perhaps through a de-
fault pathway. Therefore, NZZ and BEL1 function redundantly to specify
the chalazal identity.
How does NZZ affect the nucellar development? A reduced nucellus is
observed in the ovules of nzz mutants. This reduction in the nucellus is over-
come in ant and ino mutant backgrounds, suggesting that NZZ functions
antagonistically with ANT and INO in the nucellus. An ectopic distal ex-
pression can be observed for ANT as well as BEL1. Though a distal mis-
expression of INO is not observed in nzz mutants, an early onset of INO ex-
pression can be seen in nzz mutants. This early onset of INO expression
interferes in the development of the nucellus. Furthermore, the expression
of INO in nzz mutants appears to be distally shifted by a few cells. This
seems to result in a distal shift of the central region, leading to a reduced
nucellus and an enlarged funiculus. The role of NZZ in adaxial-abaxial pat-
terning and in coordinating the patterning along these two axes is discussed
in the next section.
Is the development of these morphological units dependent on each
other? The analysis of ant hll double mutants provides a clue on this. The
ant hll double mutants form shorter ovules (Schneitz, Baker, et al., 1998) in
which the primordium outgrowth is arrested at different stages. The small-
est primordia in ant hll double mutants show nucellar identity as seen by the
presence of the mmc. When the primordia are bigger, in addition to the
nucellus they form the chalaza as seen by the proximally restricted expres-
sion of BEL1. This suggests that the outgrowth of the primordium takes
place in a sequential manner, with the nucellus being the first to be formed
(Schneitz, Baker, et al., 1998). This analysis also suggests that the forma-
tion of nucellar pattern element is independent of chalaza or funiculus,
since nucellus can be formed in their absence (Schneitz, Baker, et al., 1998).
Likewise, the chalaza can also be formed in the absence of funiculus. How-
ever, whether the formation of the nucellus is an obligate requirement for
the formation of the chalazal and funicular pattern elements remains to be
tested.
14

FIGURE 1.4. SEMs and expression analysis in double mutants. A and B: Ovule phenotype of the nzz bel1 and the nzz
ats double mutants. C: Expression pattern of INO in nzz ats double mutants. Stages: A and B, 4-V; C, 2-II. Note the pres-
ence of bulbous cells that bear a funicular-cell morphology in the central region of the nzz bel1 double mutants (arrow
A). Note the abaxial growth of the outer integument in nzz ats double mutants (arrow B) and the chalazal ectopic
expression of INO in these mutants (arrow C).
 Molecular Control of Ovule Development 15

Adaxial-Abaxial Pattern Formation

The mature ovule shows a polarity along the adaxial-abaxial axis. Dur-
ing the initial stages of development (until stage 2-III), the ovule does not
exhibit any polarity along the adaxial-abaxial axis in its morphology
(Schneitz et al., 1995). The polarity is visually established with the initia-
tion of the outer integument at stage 2-III. The outer integument initiates at
the abaxial side of the central region (Robinson-Beers et al., 1992). This is
followed by an asymmetric growth of the outer integument along the
adaxial-abaxial axis. The outer integument grows more on the abaxial side
compared to the adaxial side and forces a curvature in the ovule that results
in its anatropous shape. The nucellus and funiculus also show adaxial-
abaxial polarity later in development (Schneitz et al., 1995). What is the
molecular control of the formation of this axis?
A couple of mutants have been isolated that show alterations in the
adaxial-abaxial axis. ino mutants fail to form the outer integument and do
not show a typical anatropous ovule (Baker et al., 1997; Schneitz et al.,
1997) (see Figure 1.3, part E). The cloning of INO revealed that it encodes a
protein that belongs to the recently described YABBY family of putative
transcription factors (Villanueva et al., 1999). The members of the YABBY
family are involved in specifying the abaxial cell fate and are expressed in
the abaxial side of the emerging lateral organs (Bowman, 2000; Siegfried et
al., 1999). INO is expressed in a few cells at the abaxial, central epidermis
that give rise to the outer integument (Villanueva et al., 1999) (see Figure
1.3, part F). The biochemical nature of INO and its abaxially restricted ex-
pression pattern suggest a role for INO in adaxial-abaxial pattern formation.
The SUPERMAN (SUP) locus has been implicated in the adaxial-abaxial
pattern formation in the ovules, since the ovules of sup mutants show a sym-
metric growth of the outer integument resulting in a nonanatropous ovule
(Gaiser et al., 1995; Schneitz et al., 1997). sup was initially isolated as a
floral mutant that shows additional stamens at the expense of carpels, and
SUP encodes a zinc-finger transcription factor (Sakai et al., 1995). During
flower development, SUP plays a cadastral role at the boundary between the
stamens and carpels (Bowman et al., 1992; Sakai et al., 1995). SUP is likely
to play a similar cadastral role in ovules as well as restricting INO expres-
sion to the abaxial epidermis. INO expression can be detected throughout
the central region of sup mutants (Balasubramanian and Schneitz, 2002;
Villanueva et al., 1999).
Apart from SUP and INO, two more loci play a role in adaxial-abaxial
pattern formation during ovule development. The ovules of nzz ats double
mutants show a sup-like phenotype, suggesting the involvement of these
16 HANDBOOK OF SEED SCIENCE AND TECHNOLOGY

two loci in the outgrowth of the outer integument. Similar to sup, INO ex-
pression can be detected throughout the central region in ovules of the nzz
ats double mutants. Neither of the single mutants of nzz or ats show a
chalazal misexpression of INO, highlighting the redundant functions of
NZZ and ATS in restricting the INO expression to the abaxial epidermis.
How does SUP relate to nzz and ats? Currently this remains an open ques-
tion. The defects in nzz ats double mutants seem to be even more pro-
nounced than in sup single mutants, since the alteration in INO expression
can be seen at an earlier stage in these double mutants compared to sup sin-
gle mutants. This suggests that NZZ and ATS may function earlier than SUP
(Balasubramanian and Schneitz, 2002).

Proximal-Distal and Abaxial-Adaxial Pattern Formation

Is Temporally Linked

Very little is known about how the pattern formation along various axes
and growth are linked during plant development. Our present knowledge on
ovule development throws some light on this aspect of plant development.
The analysis of the nzz mutant phenotype shows that an early onset of the
adaxial-abaxial axis interferes with the development along the proximal-
distal axis. This situation in analogous with the situation in limb develop-
ment in vertebrates. An early establishement of the anterior-posterior axis
in the chick limb interferes with proximal-distal development. Thereofore,
not only a spatial but also a proper temporal expression of genes is required
for the proper growth and development of the organ. How is this achieved
during ovule development at a molecular level? It appears that NZZ plays a
vital role in linking patterning along two axes of symmetry during ovule
development.
How does NZZ link proximal-distal and adaxial-abaxial pattern forma-
tion? A subtle but important aspect of NZZ function is the temporal negative
regulation of INO expression. The onset of INO expression is the first sign
of the adaxial-abaxial polarity establishment in the chalaza. In nzz mutants,
INO is turned on earlier, and consequently, the outer integument initiates
earlier than the inner integument at an incorrect time. Contrary to this, in ant
mutants the onset of INO expression is delayed. Furthermore, ANT and NZZ
function antagonistic to each other during ovule development. Taken to-
gether, these data suggest that the correct timing of the onset of INO expres-
sion requires coordinated function of ANT and NZZ. It may be recalled that
NZZ functions together in BEL1 in the specification of the chalaza. During
this time, by negatively regulating INO expression in a temporal manner
through its antagonistic interactions with ANT, NZZ ensures that the
 Molecular Control of Ovule Development 17

adaxial-abaxial axis is established at a correct time. Thus, it links the pat-

tern formation along both these axes. The early onset of INO expression in
nzz interferes with the development along the proximal-distal axis.

NZZ Couples Pattern Formation and Growth

During Ovule Development

How is growth inter-linked with pattern formation? ANT plays a vital

role in growth control during plant development. Plants that ectopically ex-
press ANT produce larger floral organs, showing that the level of ANT ex-
pression is crucial for the proper size of an organ (Krizek, 1999; Mizukami
and Fischer, 2000). NZZ functions antagonistically with ANT during ovule
development. An ectopic expression of ANT leads to a partial nzz pheno-
copy (Krizek, 1999). These findings suggest that NZZ plays a vital role in
regulating the growth of the ovule through its interactions with ANT. Thus,
NZZ—by playing a vital role in pattern formation and growth both in tem-
poral and spatial manner—couples pattern formation and growth along the
proximal-distal and the adaxial-abaxial axis during ovule development
(Balasubramanian and Schneitz, 2002).

CELL-CELL COMMUNICATIONS
DURING OVULE DEVELOPMENT

Although the morphological units can be formed independently of each

other, cell-cell communications still are necessary for the growth and devel-
opment of the ovule. WUSCHEL (WUS), a gene encoding a homeodomain
protein, is expressed in the nucellus; plants that are defective for wus show
an ovule phenotype similar to ANT, suggesting a WUS function in integu-
ment development (Gross-Hardt et al., 2002). WUS expression, however, is
restricted to the nucellus. Thus, there must be some communication be-
tween the nucellus and integuments through WUS that is necessary for the
proper development of the integuments (Gross-Hardt et al., 2002).
In nzz mutants one can observe an early onset of INO expression, but spa-
tially INO is still expressed in the chalazal region. Then how does it inter-
fere with the development of the nucellus? Interestingly, in wild type INO is
expressed only in the abaxial cell layer of the outer integument (Bala-
subramanian and Schneitz, 2000). Thus, the expression pattern and the nzz
and ino mutant phenotypes suggest a non–cell-autonomous fuction of INO.
Non–cell-autonomous mode of action has also been suggested for other
YABBY genes.
18 HANDBOOK OF SEED SCIENCE AND TECHNOLOGY

Among the isolated ovule mutants, several mutants lack the integuments.
These mutants never go on to make a viable embryo sac, suggesting that
signals from the integuments are necessary for the proper growth and devel-
opment of the embryo sac. This is true even in mutants that have a partially
enveloped embryo sac. This suggests that proper embryo sac development
also requires proper integument development. Embryo sac development is
arrested at various stages in these mutants, suggesting the need for commu-
nication between integument development and embryo sac development
(Baker et al., 1997; Schneitz et al., 1997).

MORPHOGENESIS

After an initial framework is set up by pattern formation, proper cell divi-

sion and cell elongation occur that lead to an organ of a particular size and
shape. This process is referred to as morphogenesis. In ovule development,
morphogenesis essentially occurs due to the growth and development of
the integuments. The integument morphogenesis can be divided into two
phases: (1) initiation of integuments and (2) integument growth and devel-
opment.

Initiation of Integuments

As mentioned earlier, several loci, including ANT, BEL, WUS, INO,

HLL, SIN2, ATS, and UNICORN (Gasser et al., 1998; Schneitz et al., 1997),
are involved in early aspects of integument development. The requirement
of ANT, HLL, and SIN2 at different stages of ovule development suggests
that these factors are involved in a basic machinery that is repeatedly re-
quired throughout development. BEL1 is also expressed in the developing
integuments, suggesting its involvement in this process. WUS presents a
unique case highlighting the necessity of signals from the nucellus for in-
tegument development. Thus, WUS seem to function in a non–cell-autono-
mous way (Gross-Hardt et al., 2002). Apart from specifying the adaxial-
abaxial axis, INO is also required for the growth and development of the
outer integument. During integument development, INO is expressed in a
few cells at the growing end of the outer integument (Villanueva et al.,
1999). It has been suggested that proper juxtaposition of adaxial-abaxial
signals is necessary for the outgrowth of the lateral organs (Waites and Hud-
son, 1995). INO expression is observed in only the abaxial cell layer of the
outer integument, and in ino mutants, due to the absence of INO, this juxta-
position of the adaxial-abaxial signals fails to take place, leading to an ab-
sence of integument outgrowth. ats single mutants show an alteration in
 Molecular Control of Ovule Development 19

seed shape due to the fusion of the two integuments (Léon-Kloosterziel

et al., 1994). While all these genes have a positive effect on integument
growth, UCN may have a negative effect. The ovules of ucn mutants form a
bulge, which is suggested to be an ectopic integument (Schneitz et al.,
1997). Therefore, UCN could be a negative regulator of integument out-
growth.

Integument Growth and Development

Several mutants have been isolated that show alterations in the growth
and development of integuments, without affecting their initiation and out-
growth (Schneitz et al., 1997). These mutants often show pleiotropic de-
fects throughout the whole plant, suggesting the involvement of the corre-
sponding genes in a basic function during plant development. This group of
mutants includes strubbelig (sub), blasig (bag), mollig (mog), laelli (lal),
sup, leunig (lug) (Schneitz et al., 1997), tso1 (Hauser et al., 2000; Liu et al.,
1997; Song et al., 2000), short integument 1 (sin1) (Lang et al., 1994), and
tousled (tsl) (Roe et al., 1993). Some of these mutants are characterized at
the molecular level. sub mutants show protrusions arising from the outer in-
tegument at about stage 3-I. Apart from this, sub mutants often have ovules
that are arrested in integument development (Schneitz et al., 1997). SUB
has been cloned, and it encodes a putative leucine-rich repeat receptor
kinase (Chevalier and Schneitz, unpublished). Apart from its function in
ovules, SUB has a wider role in plant development. SUB has a role in con-
trolling the meristem size and development since sub mutants show alter-
ations in the size and shape of the SAM (Chevalier and Schneitz, unpub-
lished). Another locus that encodes a kinase is TSL (Roe et al., 1993). tsl
mutants have reduced or missing floral organs similar to sub mutants. tsl
mutants also show a phenotype similar to that of lal (discussed next; Roe,
Nemhauser, and Zambryski, 1997) with reduced outer and protruding inner
integuments (Roe, Durfee, et al., 1997; Roe, Nemhauser, and Zambryski,
1997; Roe et al., 1993). The phenotype of tsl and sub and the biochemical
nature of the corresponding gene products show the importance of cell sig-
nalling during morphogenesis and plant development.
LUG was initially reported to be a cadastral gene that regulates the ex-
pression of AG in the floral primordia (Liu and Meyerowitz, 1995). Isola-
tion of new alleles of lug hinted at a function of LUG in ovule development
(Schneitz et al., 1997). lug mutants lack the embryo sac and can often pro-
duce a protruding inner integument (Schneitz et al., 1997). LUG has been
cloned, and it encodes a putative transcriptional corepressor (Conner and
Liu, 2000). TSO1 also encodes a possible corepressor (Hauser et al., 2000;
20 HANDBOOK OF SEED SCIENCE AND TECHNOLOGY

Song et al., 2000). tso1 plants have ovules that show a phenotype similar to
that of lug. The double mutants of tso1 lug show a synergistic effect, sug-
gesting that these genes might function redundantly during ovule develop-
ment. TSO1 has been implicated in regulating cell division and directional
cell expansion (Hauser et al., 1998; Liu et al., 1997).
lal and sin1 show similar phenotypes in the ovule (Ray et al., 1996;
Schneitz et al., 1997). They have reduced outer integument, and the nucel-
lus, covered by the inner integument, appears protruded. SIN1 has wider
roles compared to LAL since the defects in lal mutants are restricted to the
ovule compared to sin1 mutants which show defects in other parts of the
plant as well (Lang et al., 1994; Ray et al., 1994, 1996; Robinson-Beers
et al., 1992). Preliminary analyses of bag and mog show that their gene
products also regulate the late morphogenesis during ovule development
(Schneitz et al., 1997).

PERSPECTIVES

It has been an exciting decade for people studying ovule development.

Although much information was obtained during the 1990s, many new
questions have also emerged. In spite of all the work, the molecular mecha-
nisms have not been specified at the protein level. The genetic analysis has
clearly laid out a foundation on which further experiments can be built. The
biochemical function of NZZ protein remains elusive. The nuclear localiza-
tion and the computational biochemical analysis suggests NZZ to be a puta-
tive orphan transcription factor (Schiefthaler et al., 1999; Yang et al., 1999).
But the molecular basis of NZZ function is not clearly understood.
Genetic analysis had generated further interest in ATS, and the cloning of
ATS is ongoing. Understanding the molecular nature of ATS shall provide
additional insight into the regulation of adaxial-abaxial patterning and how
the NZZ/ATS/SUP triad functions. Many loci that affect early ovule devel-
opment encode transcription factors, but the target genes or the upstream
activators for these loci have not been elucidated, although few potential
candidates are being dissected through genetic analysis. Further molecular
experiments are needed to address these questions. Several models have
been proposed for ovule development, but none of them has been tested at a
molecular level. While SUB and TSL kinases have been cloned, the ligand
for SUB or the substrate for SUB and TSL have not yet been identified. On
the whole one would expect to see a wealth of information coming out
rather soon, and a bright time is surely ahead for understanding ovule devel-
opment.
 Molecular Control of Ovule Development 21

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