Von Caemmerer

National Library Cataloguing in Publication entry
von Caernmerer, S. (Susanne).

Biochemical models of leaf photosynthesis.
Bibliography.
ISBN 064306379 X.
I. Botanical chemistry.
2. Photosynthesis - Measurement.
1.Title. (Series: Techniques in plant sciences j no. 2).
572.460723
© CSIRO 2000
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Contents
Preface vii
Acknowledgments x
The kinetics and regulation of rubisco 1
1.1 Introduction 1
1.2 Kinetics of fully activated rubisco 2
1.2.1 Definition of kinetic constants 2
1.2.2 Kinetics at saturating RuBP 3
1.2.3 Relative specificity, 5c/ o 4
1.2.4 Rubisco kinetic constants in vitro and in vivo 6
1.2.5 RuBP-limited rate of carboxylation 10
1.2.6 High rubisco site concentrations in the chloroplast 11
1.2.7 The binding of phosphorylated compounds and other ligands 14
1.3 Activation of rubisco 16
1.3.1 Activation of rubisco in the absence of RuBP 17
1.3.2 Activation of rubisco in the presence of RuBP 17
1.3.3 The carbamylation ratio of rubisco 18
1.3.4 Rubisco activase 19
1.3.4.1 A model of activase action 20
1.3.4.2 A hyperbolic response of carbamylation to activase activity 21
1.3.4.3 Binding of other ligands 22
1.3.4.4 An unknown function of activase 22
1.4 Derivations 24
1.4.1 Derivation of rate equations for activated enzyme 24
1.4.2 The rate equations at high concentration of enzyme sites 25
1.4.3 Derivation of rate equations of activation 26
1.4.4 Carbamylated enzyme sites as a fraction of total enzyme sites 27
1.4.5 Ligands other than RuBP 28
2 Modelling (3 photosynthesis 29
2.1 Introduction 29
2.2 A few simplifying assumptions 29
2.3 Stoichiometry of C3 photosynthesis 30
2.3.1 Carbon stoichiometry of the PCR and PCO cycles 30
2.3.2 The carbon path from PGA to RuBP 31
2.3.3 ATP and NADPH consumption in the PCR and PCO cycles 32
2.3.4 Thylakoid reactions 32
iv Contents
2.3.4.1 Production ofNADPH and ATP 32
2.3.4.2 Light dependence of electron transport rate 34
2.3.5 Respiration 35
2.4 Rate equations for CO 2 assimilation 35
2.4.1 RuBP-saturated CO 2 assimilation rate 35
2.4.2 RuBP-limited CO 2 assimilation rate 36
2.4.3 Export-limited CO 2 assimilation rate 38
2.4.4 Summary of rate equations 39
2.5 CO 2 partial pressure in the chloroplast 39
2.6 Parameterization of the model 42
2.6.1 Values at 25°C 42
2.6.2 Temperature dependencies 44
2.6.2.1 Rubisco and Rd 44
2.6.2.2 Temperature dependence of Jmax 45
2.7 The CO 2 compensation point 47
2.7.1 r in the absence of Rd 48
2.7.2 r in the presence of Rd 48
2.7.3 Light and temperature dependence 49
2.7.4 Measurements off. 50
2.8 CO 2 response curves 51
2.8.1 CO 2 response curves in transgenic tobacco with impaired photosynthesis 52
2.8.2 CO 2 assimilation rate at different 02 partial pressures 54
2.8.3 CO 2 assimilation rate at different irradiances 55
2.8.4 CO 2 assimilation rate at different temperatures 56
2.8.5 The initial slope of the CO 2 response curve, dAJde, 57
2.9 Light response curves 58
2.9.1 Dependence on 02 and CO 2 partial pressures 58
2.9.2 Quantum yield 60
2.10 Temperature responses 62
2.11 Does the activation state of rubisco need to be incorporated into models
of CO 2 assimilation? 62
2.12 Long-term effect of environment on photosynthesis 65
2.12.1 V emax and Jmax and the transition from rubisco to electron
transport limitation 65
2.12.2 Effect of growth temperature 68
2.12.3 Growth at elevated CO 2 68
2.13 Engineering a better rubisco 70
3 Chlorophyll fluorescence and oxygen exchange during Cg pbomsynthesis 72
3.1 Introduction 72 'J-'

3.2 Chlorophyll fluorescence and chloroplastelectmn transport 72
3.2.1 Calculating electron transport late from dLknuphyll fluorescence 73
3.2.2 Estimating electron transport late, [,..., from CO:; assimilation rate 74
3.2.3 Relationship between If/(absl) and F,,'F"" 76
3.2.4 Calculation of mesophyll conductance to CO: diffusion 78
3.2.5 Estimation of rubisco oxygenation rate from j.and I, 79
3.3 Oxygen exchange during C 3 photosynthesis - 80

3.3.1 Introduction 80
3.3.2 Basic equations 81
3.3.2.1 Total oxygen evolution 81
Contents v
3.3.2.2 Total oxygen uptake 82
3.3.2.3 The Mehler ascorbate peroxidase (MAP) reaction 83
3.3.2.4 Net o, and COz exchange 85
3.3.2.5 Estimation of rubisco carboxylation and oxygenation rates 86
3.3.3 The CO 2 dependence of Oz exchange 87
3.3.4 The Oz dependence of 02 exchange 88
3.3.5 The O 2 exchange at the compensation point 89
3.3.6 The temperature dependence of 02 exchange 90
4 Modelling (4 photosynthesis 91
4.1 Introduction 91
4.2 Basic model equations 91
4.2.1 Equations for enzyme-limited photosynthesis 94
4.2.1.1 CO 2 assimilation rate in the bundle sheath 94
4.2.1.2 Bundle-sheath CO 2 partial pressure 94
4.2.1.3 Bundle-sheath O 2 partial pressure 94
4.2.1.4 The rate of PEP carboxylation 95
4.2.1.5 Quadratic expression for the enzyme-limited CO 2 assimilation rate 95
4.2.2 Light- and electron-transport-limited photosynthesis 96
4.2.2.1 Rates of ATP and NADPH consumption 96
4.2.2.2 Partitioning of electron transport rate between C3 and C4 cycles 97
4.2.2.3 Light dependence of electron transport rate 98
4.2.2.4 Quadratic expression for electron-transport-limited CO 2
assimilation rate 98
4.2.3 Summary of equations 99
4.3 Analysis of the model 99
4.3.1 Parameterization of the model 99
4.3.2 The model at high irradiance 101
4.3.2.1 PEP carboxylase and rubisco activity 101
4.3.2.2 Bundle-sheath conductance 102
4.3.2.3 Variation of bundle-sheath conductance, PEP carboxylase
activity and rubisco with leaf age 103
4.3.2.4 CO 2 response curves 105
4.3.2.5 Oxygen sensitivity of C4 photosynthesis 109
4.3.2.6 CO 2 compensation point 110
4.3.2.7 CO 2 diffusion from intercellular air spaces to the
mesophyll cytosol 113
4.3.3 CO 2 fixation at limiting light 116
4.3.3.1 Optimal partition of electron transport 116
4.3.3.2 Leakiness 118
4.3.3.3 Quantum yield 119
4.3.4 Modelling different decarboxylation types 120
4.3.4.1 Bundle-sheath conductance 120
4.3.4.2 02 evolution in the bundle sheath 121
4.3.4.3 Energy requirements 122
5 Models of (3-(4 intermediate photosynthesis 123
5.1 Introduction 123
5.2 Basic model equations 124
5.3 Equations for photosynthesis at high irradiance 125
vi Contents
5.3.1 RUbisco-limited CO2 assimilation rate

125
5.3.2 Net 02 evolution in the bundle sheath

126
5.3.3 Quadratic expression for CO 2 assimilation rate

126
5.4 Parameterization and analysis of the model at high irradiance

5.4.1 Kinetic constants 127
5.4.2 Respiration 127
127
5.4.3 Partitioning of photorespiration

128
5.4.4 Bundle-sheath conductance

5.5 The glycine shuttle 128
129
5.5.1 Bundle-sheath rubisco activity and conductance

5.5.2 CO 2 compensation point 130
131
5.5.3 CO 2 and 02 responses of CO 2 assimilation rate

132
5.6 The contribution of C4 photosynthesis

5.6.1 CO 2 compensation point 133
134
5.6.2 CO 2 and 02 responses of CO 2 assimilation

135
5.7 Energy requirements of CJ-C 4 photosynthesis

135
5.7.1 Rates of ATP and NADP consumption

5.7.2 Electron transport rate 135
137
5.7.3 Light-dependent CO2 assimilation rate

5.8 Conclusion 138
139
6 Concluding remarks
140
Appendix List of symbols
References
141
145
Preface
Increasing concerns about global climate change have revived research interests in all aspects of
carbon exchange. Natural ecosystems form an important part of the global carbon balance as
sinks for atmospheric CO 2 , Interest in predicting net primary productivity has restored interest
in leaf photosynthetic models to predict and assess changes in photosynthetic CO 2 assimilation
in different environments. Photosynthetic processes of leaves have a remarkable influence on
our global atmosphere. Seasonal and latitudinal variations in the carbon isotope ratio of atmo
spheric CO 2 relate to rubisco's preference for l2C0 2 rather than l3C02. The oxygen isotope
composition of atmospheric CO 2 is influenced by the amount of carbonic anhydrase in the
chloroplast of C 3 species and the mesophyll cytosol of C4 species (Francey and Tans 1987; Yakir
et at. 1992; Farquhar et al. 1993). This book deals exclusively with the photosynthetic processes
,.. of leaves. The models discussed are based on the underlying biochemical processes of photo
synthesis and were designed to help in the interpretation of leaf gas-exchange measurements.
However, because of their simplicity they have also proved valuable as submodels in a variety of
other larger scale applications such as canopy photosynthesis and climate models.
At present, the techniques of genetic and molecular biology, which allow the modulation of
individual plant characters, enable new questions to be asked in ecophysiology about photosyn
thesis and plant growth. The steady-state leaf-photosynthetic models have become an invaluable
guide for the analysis of such genetic manipulation, where they are frequently used in conjunction
with gas-exchange measurements to provide in vivo estimates of biochemical parameters.
Leaf gas-exchange measurements were first developed in the late 1950s. Penman and Schofield
(1951) put the theories of diffusion of CO 2 and water vapour through stomata on a firm physical
basis. Gaastra took up their ideas in the 1950s and modern analytical gas exchange is often attrib
uted to his seminal work (Gaastra 1959). His work was a landmark because it examined CO 2
assimilation and water vapour exchange rates of individual leaves under different environmental
conditions, and he distinguished between stomatal and internal resistances. Gaastra at the time
concluded that the rate of CO 2 uptake was completely limited by diffusion processes at low CO 2
partial pressures and that biochemical processes became important only at high CO 2 partial pres
sures. Thus, gas-exchange studies focused initially on physical limitations to diffusion. Based on
Gaastra's ideas, early models of leaf gas exchange had been developed as analogues of electrical
resistances, and this proved useful in making a distinction between stomatal and mesophyillimita
tions on CO 2 assimilation. Mesophyll, or 'residual', resistance was a collective term that embodied
non-stomatal diffusive factors, and included both physical and biochemical constraints.
In Australia, particularly, there was a great interest in determining the relative importance of
stomatal and mesophyll resistance in limiting CO 2 assimilation rates under adverse conditions of
high temperature and frequent water stresses (Bierhuizen and Slatyer 1964;Troughton and Slatyer
1969). It was not long, however, before persuasive arguments were being brought forward to show
that leaf biochemistry had an important influence on the rate of CO 2 fixation, even at low CO 2
partial pressures. For example, Bjorkman and Holmgren (1963) made careful gas-exchange mea
surements of sun and shade ecotypes of Solidago, and noted a strong correlation between
viii Preface
photosynthetic rate measured at high irradiance and ambient CO 2 and the nitrogen content of
leaves, and later related it to different concentrations of rubisco. Furthermore, following earlier
discoveries of the 02 sensitivity ofphotosynthesis, viz. an enhancement of CO 2 assimilation rate at
low 02' Gauhl and Bjorkman (1969) showed very elegantly that, while oxygen partial pressures
did affect CO 2 assimilation rate, water vapour exchange was not affected (i.e, stomata had not
responded). Clearly, the increase in CO 2 assimilation rates seen with a decrease in 02 partial pres
sures could not be explained by a limitation on CO 2 diffusion. Mathematical models of leaf
photosynthesis based on Gaastra's resistance equation could not accommodate this O 2 sensitivity
of CO 2 assimilation. They were quickly superseded by the development of more biochemical
models in the early 1970s. The discoveries by Bowes et aL (1971) that rubisco was responsible for
both carboxylation and oxygenation of ribulose-1,5--bisphosphate put rubisco in the limelight.
Laing et al. (1974) and Peisker (1974) were first to compare the gas exchange of leaves with the in
vitro kinetics of rubisco.
In this book rubisco takes centre stage. Although there are many chloroplast components
essential for the operation of photosynthesis, successful mathematical descriptions of photosyn
thesis are inevitably linked to rate equations of rubisco carboxylation and oxygenation. Chapter 1
thus deals with the kinetic properties of rubisco and these equations form the basis for the bio
chemical models presented in this book. In Chapter 1, in vitro and in vivo responses of rubisco are
compared and analysed. Since the leaf photosynthetic models are based on rubisco's kinetic prop
erties they have also proved a useful tool for examining the in vivo activity of rubisco. This is taken
up in the later part of the chapter where transgenic plants with impaired photosynthetic proper
ties are used to unravel the mysteries of in vivo regulation of rubisco.
Chapter 2 is a straightforward treatment of the now frequently used photosynthesis model of
Farquhar et al. (1980). The chapter contains many examples of applications of the model to the
analysis of transgenic plants with altered photosynthetic properties. It identifies some of the exist
ing gaps in our knowledge, which need to be addressed because of the present need to model
photosynthesis with respect to global climate change.
Chlorophyll fluorescence has emerged as a powerful, non-destructive tool for the analysis of
photosynthesis and is providing insights into chloroplast electron transport rates. It is particularly
useful as a field measure of photosynthetic performance and has thus stimulated considerable
interest in comparisons with photosynthetic CO 2 exchange. In Chapter 3 a comparison is made
between the use of measurements of chlorophyll fluorescence to estimate chloroplast electron
transport rate and estimates made from gas-exchange measurements. Furthermore, the model of
Farquhar et al. (1980) is used to derive rate equations for the O 2 exchange that occurs during C3
photosynthesis.
Though the C 3 pathway of photosynthesis dominates most of the terrestrial ecosystem, the C4
pathway of photosynthesis is important in certain agricultural and natural ecosystems and
accounts for as much as 20% of global carbon fixation. The C 4 pathway is common amongst spe
cies native to tropical and subtropical grasslands. It took some very energetic grinding of C4leaves
before rubisco was recognized as a key player in the C 4 photosynthetic pathway (Hatch 1997;
Osmond 1997). It is now well recognized that the C4 photosynthetic pathway functions as a CO 2
concentrating mechanism that provides rubisco, located in the bundle sheath, with a high CO 2
atmosphere where it can function at near CO 2 saturation with minimal oxygenase activity. This
requires the cooperation between mesophyll and bundle-sheath cells, and the involvement of two
cell types has complicated biochemical analysis. Here, the photosynthetic models provide an
important quantitative tool to predict bundle-sheath function.
The fifth chapter discusses biochemical models of leaf photosynthesis of C 3-C4 intermediate
species. Different biochemical variants give rise to the syndrome of C 3-C4 intermediacy, but all
such plants have a C 4-like leaf anatomy. C 3-C4 species are sometimes considered to be evolu
tionary intermediates between C 3 and C 4 species. The pathways revolve around efficient
Preface ix
refixation of photorespiratory CO 2 , Their leaf gas exchange shows a reduced oxygen sensitivity
in comparison with that of C 3 species and improved photosynthetic rates at low CO 2 partial
pressure. Since many of the details of these pathways remain unexplored the photosynthetic
models are, of necessity, experimental. Perhaps this chapter provides the best examples of how
the biochemical models presented in this book can aid in the formulation of ideas. Each photo
synthesis model provides a set of hypotheses brought together in a quantitative form that can be
used to design and interpret experiments.
Acknowledgments
I wish to thank C. Barry Osmond for inviting me to contribute to this series of Techniques in
Plant Sciences. I have greatly enjoyed this opportunity and appreciate the encouragement
and support he has provided throughout my scientific career. With his never-ending enthusi
asm for science he has been a source of inspiration for me. I had the great fortune to have
Graham D. Farquhar as my PhD supervisor and have been irrevocably influenced by his rig
orous approach to science. I am fortunate to be able to work within the stimulating environ
ment of the Molecular Plant Physiology Group at the Research School of Biological Science. I
am indebted to John Andrews for many fascinating discussions on the mechanism and regu
lation of rubisco. I thank Murray Badger, John Andrews and Dean Price for the opportunity
to collaborate on the analysis of transgenic plants with impaired photosynthesis. Lastly I
would like to thank John R. Evans for his friendship. I am thankful for his helpful, energetic
and apposite criticisms.
The kinetics and regulation of rubisco
1.1 INTRODUCTION
Today all mechanistic models of leaf photosynthesis are based on the kinetic properties of
ribulose-l,5-bisphosphate carboxylase-oxygenase (rubisco, EC 4.1.1.39). The importance of
rubisco in determining the rate of photosynthesis had been inferred early on from correla
tions between photosynthetic rate and the amount of rubisco protein in leaves (Bjorkman
1968; Wareing et al. 1968; Bowes et al. 1972). After the oxygenase activity of rubisco had been
demonstrated by Bowes et al. (1971), Laing et al. (1974) and Peisker (1974) showed that the
oxygen sensitivity of photosynthetic CO 2 assimilation could be explained from rubisco's
kinetic properties. They presented the first kinetic equations of the ribulose-l,5-bisphos
phate (RuBP) saturated rates of rubisco and used them to formulate rate equations for CO 2
assimilation.
Rubisco is located in the chloroplast stroma were it catalyses the competing reactions of the
carboxylation or the oxygenation of RuBP. The carboxylation of RuBP is the first step of the
photosynthetic carbon reduction (PCR) cycle and the carboxylation of one molecule of RuBP
yields two molecules of 3-phosphoglycerate (PGA). The oxygenation of one molecule of
RuBP gives one molecule of PGA and one molecule of 2-phosphoglycolate (PGly), which leads to
the necessary recycling of carbon skeletons in the photorespiratory (PCO) cycle. Rubisco is a slow
catalyst and the leaves of C 3 plants allocate as much as a quarter of their total leaf nitrogen to
rubisco, making it one of the world's most abundant proteins (Kung 1976; Ellis 1979). Its pivotal
role in carbon assimilation of plants has made it one of the most widely studied plant enzymes
(Jensen and Bahr 1977; Andrews and Lorimer 1987; Gutteridge 1990; Gutteridge and Gatenby
1995; Cleland et al. 1998).
Two main forms of rubisco exist in photosynthetic organisms. The simpler form of the
enzyme is composed of two identical subunits, with a molecular weight of 52 kDa, and is found in
photosynthetic bacteria, the most studied of these being the rubisco of Rhodospirillum rubrum. In
higher plants the rubisco holoenzyme has a molecular weight of approximately 550 kDa and con
sists of eight large subunits (LSu) and eight small subunits (SSu). A single chloroplastic gene
encodes the large subunit, whereas a family of nuclear genes encodes the small subunits. The reac
tion sites are located on the large subunits and there are eight catalytic sites per holoenzyme
(Andrews and Lorimer 1987).
To function, rubisco needs to be activated by CO 2 and magnesium. The CO 2 binds to a specific
lysine residue in the active site. This carbamate formation is then followed by the addition of mag
nesium (Laing and Christeller 1976; Lorimer et al. 1976; Andrews and Lorimer 1987; Cleland et al.
1998). It has been shown that the activator CO 2 is distinct from the substrate CO 2 that is fixed dur
ing catalysis (Lorimer 1979; Miziorko 1979). Although the activation of rubisco by CO 2 and
magnesium is essential, it is not clear how chloroplast CO 2 and magnesium concentration affect
the in vivo regulation of rubisco activity. A second major chloroplast protein, rubisco activase, is
1
2 Biochemical Models of Leaf Photosynthesis
involved in and essential for the in vivo activation of rubisco; however, its mode of action is still
poorly understood (for review see Portis 1992; Andrews et al. 1995; Salvucci and Ogren 1996).
This chapter includes a discussion and derivation of the kinetic equations of rubisco car
boxylation and oxygenation that are used in the photosynthetic models of the other chapters.
Peisker (1974) and Farquhar (1979) have described these equations in some detail. The rate
equations of the fully activated rubisco enzyme are discussed first and the process of activation
is examined later.
The basic rubisco equations have formed the foundation of photosynthetic models. Improved
gas-exchange and biochemical techniques now allow us to use these models to explore the regula
tion of rubisco in vivo. For example, gas-exchange measurements on transgenic tobacco with
reduced amounts of rubisco have been used to derive rubisco kinetic constants in vivo (von
Caemmerer et al. 1994). Careful quantitative comparisons of CO 2 assimilation rates measured by
gas exchange and measurements of in vitro activities of rubisco aided by our model equations have
pointed to a yet unknown role of rubisco activase in vivo (Mate et al. 1993; Andrews et al. 1995;
Mate et al. 1996). Some of these issues are explored in the second half of this chapter.
1.2 KINETICS OF FULLY ACTIVATED RUBISCO
1.2.1 Definition of kinetic constants

The derivations in this book are based on the assumption that the catalytic sites act indepen
dently; in other words, cooperativity or anti-cooperativity of rubisco sites is not considered.
One must therefore keep in mind that the model may not be applicable in all circumstances.
On the other hand, in the following examples we see that some success can be had with these
simple assumptions.
The scheme in Fig. 1.1 describes the sequence of activation and catalysis of rubisco. E stands
for free enzyme site, ER for the unproductive enzyme site and RuBP complex; EC, ECM and the
subsequent complexes are the carbamylated enzyme sites. ECMRC and ECMRO represent all cat
alytic intermediates of carboxylation and oxygenation. In detailed analysis of the catalytic
mechanism these can be further subdivided (Andrews and Lorimer 1987). The total number of
enzyme sites E, is given by the sum of all the complexes and:
E, = E + ER + EC + ECM + ECMR + ECMRC + ECMRO (1.1)
In the first instance where the fully carbamylated enzyme is considered, it is assumed that the
concentrations of E, ER, and EC are negligible. The Michaelis-Menten constants for the car
boxylase (K[) and the oxygenase (Ko ) are defined as follows:
(1.2)
(1.3)
and
(1.4)
is the dissociation constant of RuBP. All rate constants are denoted by k and the subscripts
are the numbers shown in Fig. 1.1. The scheme assumes that the catalytic mechanism is
ordered with RuBP binding first (Pierce et al. 1986; Andrews and Lorimer 1987). Because of
Activation Catalysis
Activase assisted
\
\
, ECMRC
~ f(A)
lY/lOli 9C
~ 3C 5M /7R
ER -------. E
+--
-------.
+-- EC -------.
+- ECM ~ ECMR
~
8
4
2R 6
f(A) 13
i1120
14 ECMRO
Figure 1.1 The reaction sequence assumed to be involved in the activation and catalysis of rubisco. E, C. 0, M,
R stand for rubisco enzyme site, C02' 02' Mg2 + and RuBP respectively. The concentrations arethose of the free
species. Edenotes free enzyme sites, ER denotes uncarbamylated enzyme sites with RuBP bound. ECM and EC
denote the carbamylated enzyme sites with and without magnesium. ECMR represents enzyme sites with
carbamylated rubisco and RuBP bound. ECMRC and ECMRO represent all catalytic intermediates of carboxylation
and oxygenation. f(A) denotes a complex function positively correlated with activase activitythat describes
activase-mediated dissociation of ER and ECMR. (Redrawn from Mate et al. 1996.)
this, the Michaelis-Menten constant for RuBP is a composite defined later by Eq. (1.16)
(Farquhar 1979).
The catalytic turnover rate for carboxylation is defined as kccat = k 11 and the units are moles
CO 2/((mole of enzyme sites) s). The catalytic turnover rate for oxygenation is kocat = k]4"
The rate of carboxylation is given by:
V c = kcca,ECMRC (1.5)
and the maximal carboxylation rate is proportional to the total concentration of enzyme sites
and is given by:
V cmax = kcca,E, (1.6)
Similarly, the rate of oxygenation is given by:
Va = kaca,ECMRO (1.7)
and the maximal oxygenation rate is:
V om nx kaca,E, (1.8)
1.2.2 Kinetics at saturating RuBP

In many in vitro studies of rubisco kinetics, RuBP is added in saturating concentrations, and
RuBP is also often present in saturating amounts in the chloroplast (Perchorowicz et al. 1981;
von Caemmerer and Edmondson 1986). The equations for the RuBP-saturated carboxylation
and oxygenation rates are therefore amongst the most important ones. The substrates CO 2
and 02 each behave as competitive inhibitors of the oxygenase and the carboxylase reactions
respectively (Bowes and Ogren 1972; Badger and Andrews 1974; Laing et al. 1974; Peisker
1974). In both cases the competition is linearly competitive and this is consistent with the
interaction of CO 2 and 02 at a common enzyme site. The equation for the rate of carboxyla
tion, Vel at saturating RuBP has a typical Michaelis-Menten form with respect to its substrate
CO 2 and:
(1.9)
In this chapter we denote the carboxylation rate in general as V, and the RuBP-saturated rate
by We (Farquhar and von Caemmerer 1982). V em ax is unaffected by 02 concentration; how
ever, the effective Michaelis-Menten constant in the presence of 02 is given by Ke(l + O/Ko)'
which increases linearly with O 2 partial pressure. At low CO 2 partial pressures when C <<
K e( 1 + 0/K o ) ' We can be approximated by:
(1.10)
The rate equation for carboxylation given by Eq. (1.9) is a standard rate equation of an
enzyme reaction in the presence of a competitive inhibitor and derivations can be found in
most standard texts on enzyme kinetics (e.g. Fersht 1984). In Section 1.4 the equations for
the carboxylation and oxygenation rates are derived from the scheme outlined in Fig. 1.1.
The analogous equation for the RuBP-saturated rate of oxygenation, Woo is given by:
(1.11)
Figure 1.2 shows the modelled dependence of carboxylation rate and oxygenation rate on
CO 2 at different 02 partial pressures. The double reciprocal plot of liVe against l/C is also
shown. The fact that all lines intersect at one point on the Y-axis shows that V emax is the same
at the different 02 partial pressures. The fans of intersections on the X-axis give the various
values of -lI(Ke(l + O/Ko)) at the different 02 partial pressures. A similar double reciprocal
plot can be constructed for the oxygenase rate.
Equations (1.9) and (1.11) are two fundamental equations used in all mechanistic models
described in this book, be it C3 , C 4 or CrC 4 photosynthetic models. Farquhar and von Caem
merer (1982) have reviewed the use of these equations.
1.2.3 Relative specificity. Sc/o

The ratio of the carboxylation to oxygenation rate can be obtained from Eqs (1.9) and (1.11),
and is given by:
(1.12)
(Laing et al. 1974). The term in parentheses is called the relative specificity or the CO 2/0 2
specificity (Sclo)' of rubisco, i.e.
V em ax x,
s.), = -K -V (1.13 )
c oil/ax
Sclo is the ratio of the carboxylase to oxygenase rate when CO 2 and 02 are present at equal
concentrations or partial pressures. The Sclo values of different higher plant enzymes are very
similar, whereas rubiscos from cyanobacteria and algae have lower values (Jordan and Ogren
a 25 b I I I I I I I I
100
20
~ 80 ~
g ~ 15
>~ 60 >"
4
o '0 10
t'2- 40
'-' ~
:;;,.~
=
:;;,." 5
20
o o I ,z :=::-r- I I r 1
o 2000 4000 o
2000 4000
CO 2 (ubar)
CO 2 (ubar)
0.2
:;;,.'
-- 0.1
0.0
0.00 0.01 0.02
I/C0 2 (I/~bar)
Figure 1.2 (a, b) Modelled rates of carboxylation and oxygenation versus CO 2 partial pressures at oxygen
partial pressures of (1) a mbar, (2) 100mbar, (3) 200mbar, (4) 500 mbar and (5) 1000 mbar. Both rates are
expressed as a fraction of the maximum carboxylation rate. The kinetic constants used are: Ke = 258 ubar,
Ko = 171 mbar, and ValVe =0.255 (von Caemmerer et al. 1994). (c) Double reciprocal plotsof 11Ve against 11 Cat
various 02 partial pressures (see parts a.b). The lines intersect the Y-axis at 11Vemax and the X-axis at
-1/(Ke(1 + OIKJ).
1981). However the increase in specificity in the higher plant enzyme has been at the expense
of the catalytic turnover rate of the carboxylase, kecat (Andrews and Lorimer 1987; Badger
and Andrews 1987; Morell et al. 1992). Careful in vitro measurements of Se/o at 25°C have
shown only minor variations amongst C 3 species (Kane et al. 1994). The higher plant values
of Table 1.1 show that at equal dissolved concentrations of O 2 and CO 2 the carboxylation rate
would be 77-90 times the rate of oxygenation. For the photosynthetic modelling the values
of Sclo in the gas phase are more useful and these are simply derived by multiplying by the
ratio of solubilities of O 2 and CO 2 (Table 1.1). In the gas phase at equal partial pressures of
O 2 and CO 2 the carboxylation rate is 2000-3000 times the oxygenation rate because the solu
bility of O 2 is much less than that of CO 2 , The relative specificity of rubisco determines the
CO 2 compensation point of CO 2 assimilation (the CO 2 partial pressure at which no net
exchange of CO 2 occurs) in C 3 species. Laisk (1977) pioneered a technique to determine Sclo
from gas-exchange measurements and this is discussed in Section 2.7.
Table 1.1 COzlO z specificity, Sclo of rubiscos from different plants at 25°C
Plant type Sclo (M/M) Sclo (barlbar)

C 3 plants 77-90 2064-2412
C 4 plants 76 1554-2037
green algae 60 1608
cyanobacteria 48 1286
photosynthetic bacteria 10-20 268-536
Collated from Jordan and Ogren (1981, 1983), Kane et al. (1994). To convert from concentration to partial pressures,
solubilities of 0.0334 mol (L bar) " and 0.00126 mol (L bar) " were used for CO 2 and 02 respectively.
1.2.4 Rubisco kinetic constants in vitro and in vivo

An important part of the parameterization of the C 3 model in Chapter 2 is the kinetic con
stant of rubisco and Table 1.2 lists a range of in vitro values. The K, value of rubisco has been
measured frequently, but K; has been measured much less frequently. It is, however, also an
important input into the C 3 model since it affects the value of KJ 1 + DIKo ) at present ambi
ent partial pressures of 02' Figure 1.3 shows the diversity of K, values measured for different
species in a frequency plot. The data, taken from the tables ofYeoh et al. (1980, 1981) show
for example clear differences between the K, values for C 3 and C 4 species.
Von Caemmerer et al. (1994) made in vivo determinations of rubisco kinetic constants with
transgenic tobacco that had reduced amounts of rubisco. In these plants CO 2 assimilation rate at
high light is always limited by the amount of rubisco such that the gas exchange reflects the
rubisco RuBP-saturated rate. They first used carbon isotope techniques to quantify the internal
diffusive resistance of the leaves so that chloroplast CO 2 partial pressure could be estimated.
Figure 1.4 shows CO 2 response curves at five different 02 partial pressures. To highlight the hyper
bolic nature of the curves, gross CO 2 assimilation rate (A + Rd ) is also shown as a function of
40
CJ C3
>,
o
c
Q)
::l
0
Q)
.....
30
20
- C4
CAM
~ aquatic
LL
10
o
10 20 30 40 50 60 70
<(IlM)
Figure 1.3 Distribution of Michaelis-Menten constant for CO 2, Kc for differentspecies. Data are taken from the
tables of Yeoh et al. (1980, 1981).
'e::t::
30
a °2
as
+-'
....co
c ";"
o c'}')
§ 'E 20
0
E 50%
E
(f)
(f)
CO - :::t
10
d"
o
o 500 1000 1500

Intercelluar CO 2 partial pressure
Gil (ubar)
ex::""
+ b
< 30
Q)
+-'
....
CO
c ";".
0 (f)
.-
+-'
CO
C\l
20
E
E 0
(f) E
0
(f)
CO
C\l -- :::t
10
o
(f)
(f)
....0 o
0>
o 500 1000 1500
Chloroplast CO 2 partial pressure -1.
Gc-l*, (ubar)
Figure 1.4 (a) CO 2 assimilation rate, A, of a transgenic tobacco leafwith reduced amount of rubisco asa
=,
function of intercellular CO 2 partial pressure, C; at five different 02 partial pressures (A., 2%; 5%; _, 21 %;
: , 35%; e, 50%). Measurements were made at an irradiance of 1500 urnol quanta m-2 S-1 and a leaf
temperature of 25°C. (b) The data are redrawn asgross CO 2 assimilation (A + Rd ) versus the CO 2 partial pressure
at the site of carboxylation minus I". (Cc - r.) to show the hyperbolic nature of the curves. The lines in (a) and (b)
arethe predicted A with the kinetic constants of Table 1.3and Cc was calculated from Cc = Ci - AlO.3, where 0.3
was the measured CO 2 transfer resistance between intercellular airspace and chloroplasts. Twenty-one percent 02
is equivalent to 200 mbar in Canberra. (Redrawn from von Caemmerer et al. 1994.)
Figure 1.5 (a) The apparent Ke (C02) asa function of the partial pressure of 02' Estimates were derived from
Eadie-Hofstee plots of CO 2 response curves similarto those shown in Fig. 1.4. Different symbols denote
measurements on different leaves. (b) Vemaxas a function of the partial pressure of 02' Vemax values were
normalized by dividing by the mean values for each leaf (ranging from 35 to 42 urnol m-2 S-I). Estimates were
derived from Eadie-Hofstee plots of CO 2 response curves like those shown in Fig. 1.4. Different symbols denote
measurements on different leaves. (Redrawn from von Caemmerer et al. 1994.)
(C e - L), where L is the CO 2 compensation point in the absence of mitochondrial respiration

and is uniquely related to Sclo (Section 2.7). These measurements confirmed that V emax was inde
pendent of O 2 partial pressure (Fig. 1.5). They also observed the expected linear relationship of
KeO + OIKo) on O 2 partial pressure (Fig. 1.5). This technique can be further exploited to derive in
vivo temperature dependencies for the kinetic constants, which are needed to improve our predic
tions of photosynthetic productivity in a global climate of rising temperatures (Long 1991). At
present there are only very few measurements of temperature dependencies of rubisco kinetic
constants (for discussion see Section 2.6.2).
The greatest uncertainty in the kinetic constants is in the ratio of VomaxlVemax Badger and co
workers determined both carboxylase and oxygenase activity and obtained a value of 0.22
(Table 1.2; Badger and Andrews 1974; Badger and Collatz 1977). Makino et al. (988) calculated
values of 0.29 and 0.33 in wheat and rice from measurements of carboxylase and oxygenase
Table 1.2 In vitro kinetic constants of rubisco from several C 3 species
K c (1 + OIKo ) (ubar)
c Reference
Species s; (f.lM) x, (ubar) s, (flM) x, (mbar) at 200 mbar O 2 VomaxlVemax Sclo
_~.
- - - - - - - - _ ... - ~-_._---, .. _----~-,."
.._ . _ - - - - - - - - _ ..
__._-,._.__._.... _-_ - - - - _..
_-----,--_._".
Atriplex gtabriuscula 21 629 328 260 1113 0.18 85 Badger and Colla tz (J 977)
Spinacia oleracea 13.6 407 354 280" 698 0.22 120 Badger and Andrews (1974)
15.2 b 455 196 156 1038 0.22 60
Glycine max 9 269 430 341" 427 0.58 82 Jordan and Ogren (1981)
Tetragonium expansa 13 389 600 476" 552 0.55 81
Si olcracea 14 419 480 381" 639 0.43 80
l.olium perenne 16 479 500 397" 720 0.38 80
Nicotiana tabocum II 329 650 516" 456 0.77 77
Si oleracca 11 329 500 397" 495 0.52 88 Jordan and Ogren (1984)
Triticum acstivuni 11.2 335 383 304 555 0.29 120 Makino et al. (1988)
Oryza saliva 8.0 239 335 266 418 0.33 128
Nicotiana tabacum 11.5 344 222 176 713 0.24 81 Whitney et al. (1999)
- - - - - - -
All values were measured at 25°C. To convert K,. and K" values from concentration to partial pressures, solubilities for CO 2 01'0.0334 mol (L bar)' ' and for 02 01'0.00126 mol (L bar):"
were used.
K, values by Badger and Collatz (1977) and Badger and Andrews (1974) were measu red in IOU niM Hepes pH 8.3. The values had been calculated with a pK" of 6.37 and were recalculated
here with a pK" 01'6.12. Measurements by Jordan and Ogren (1981,1984) were made with 50 mM Bicinc buffer pH 8.3 and a pK, 01'6.23 used. Measurements by Makino et al. (1988) --i
:::T
were made in 100 mM Bicinc pH 8.15 and a pK" of 6.12 was used. m
A
" Measured as KJ02) ::;'
b Measured as KJC0 2 ) ~
;:;.
c Sdo values by Jordan and Ogren (1981,1984) and Whitney et al. (1999) were derived from simultaneous measurements of carboxylation and oxygenation. Other Sdo values were
'"
ClJ
calculated from the individual constants given in the table. :::J
0..
ro
LC
<::
§I
o'
:::J
S,
2
0
Vi'
n
o
\C
Table 1.3 Rubisco kinetic constants for tobacco calculated from gas-exchange analysis by von
Caemmerer et at. (1994)
K, 258 ± 50 ubar (8.6 ± 1.7 flM) 404 ± 51 ubar (13.5 ± 1.7 flM)
x, 171 mbar (215 flM) 248 mbar (313 flM)
K, (1 +O/Ko ) at 200 mbar O 2 560 pbar (18.7 flM) 730 pbar (24.4 flM)
r. 38.6 pbar 36.9 ubar
97.5 102
0.255 0.226
K cat (mol CO2 (molsites)"! S-l 3.53 ± 0.18 3.64 ± 0.22
K cat (mol CO2 (molsites)-l S-l (in vitro) 2.87 ± 0.08
Toconvertvalues from partialpressures to concentrations, solubilities for CO2 and O2 given in Table 1.1 wereused.
Atmospheric pressure in Canberraaverages 953 mbar.
Constantswerecalculated from measurements of CO2 assimilation rate at different intercellular CO2 and O2 partial
pressures usingthe measured CO2 transferconductance, gj (column 1) to calculate chloroplast CO2 partialpressure,
or with the assumptionthat gj wasinfinite and that chloroplastic CO2 partial pressure was the sameas intercellular
CO2 partialpressure (column 2). Measurements weremadeon transgenic tobacco with reduced amount of rubisco
(Figs 1.4, 1.5).
activity. However, Jordan and Ogren (1981,1984) calculated this value from their measurements
of Sci,,' K, and K" and arrived at a higher value of 0.54. In vitro measurements by Whitney et al.
(1999) in tobacco are similar to the in vivo measurements (Tables 1.2 and 1.3). In vitro values of
k eeat vary widely. The in vivo estimates in Table 1.3 are comparable to higher in vitro estimates
reported for wheat of 3.0-3.8 mol CO 2 (mol sites):" S-1 (Evans and Seemann 1984; Makino et al.
1988) and Chenopodium album (Sage etal. 1995).
1.2.5 RuBP-limited rate of carboxylation

Badger and Collatz (1977) derived equations for carboxylation and oxygenation when RuBP
is not saturating for the special cases of saturating CO 2 or 02 respectively. Farquhar (1979)
derived the more general equations for when RuBP is not saturating and showed that in this
case:
W_R_
v. eR + s,
(1.14)
R_
V = W_ (1.15)
" "R + s,
where We and W" are the RuBP-saturated rates of carboxylation and oxygenation given by Eqs
(1.9) and (1.11) and R is the concentration of free RuBP. The effective Michaelis-Menten
constant for RuBP, K r , is given by:
(1.16)
where Kre (= k lllk 7 ) and Kro (= k141k 7 ) are the Michaelis-Menten constants for RuBP in the
presence of saturating CO 2 and 02 respectively, and K ir is the dissociation constant for the
activated enzyme-RuBP complex (Eq. 1.4). The effective Michaelis-Menten constant for
RuBP, K» is dependent on CO 2 and 02 concentration; however, Farquhar (1979) suggested
that this dependence is slight and can be ignored. Full derivations of these equations from
the scheme in Fig. 1.1 are given in Section 104. In vitro measurements of the Michaelis-Menten
constant for RuBP are usually made at CO 2 saturation. At CO 2 saturation K r , == K rc It
is experimentally not as easy to measure K ro' since 02 saturation cannot be achieved; how
ever, Badger and Collatz (1977) confirmed that, as expected from the reaction scheme,
Kr / x; == keca/kocat·
1.2.6 High rubisco site concentrations in the chloroplast
In vitro kinetics are usually derived with the assumption that the enzyme site concentration is
negligible in solution compared to the substrate concentrations. Estimates of rubisco site concen
tration in the chloroplast stroma, however, range from 2 to 5 mM and are of the same order of
magnitude as RuBP concentrations (Jensen and Bahr 1977; Badger et al. 1984; Evans et al. 1994).
Thus in the chloroplast a large amount of RuBP is bound to rubisco sites and the standard
Michaelis-Menten kinetics do not apply. Peisker (1974) was the first to take these high rubisco
concentrations into account in his kinetic equations; however, he assumed that the enzyme con
centration would exceed the concentration of total RuBP, which we now know is not usually the
case. Farquhar (1979) derived the more general solution, and predicted quadratic equations relat
ing carboxylase and oxygenase rate to R t , the total (free and enzyme-bound) RuBP concentration.
These equations are given here and are derived in Section 104.
Ve)2 - (VWee)[ 1 + R
(We E; + Kr'
t J u,
E + E; == 0 ( 1.17)
t
V )2- (Vo)[
(-w, O
- u, Kr'J +-
1+-+- R- t
0 ( 1.18)
w, E e, e,
t
The exact solution for the rate of carboxylation is then given by:
v, _ 1{( 1 + -
- - - Rt+K
-r. ) - ( Rt+K
1+ - R t}
-r. ) - 4- (1.19)
We 2 Et s, Et
and the solution for the oxygenase rate is analogous.

In these equations carboxylation and oxygenation rates are expressed as a fraction of the respec
tive RuBP-saturated rates (Eqs 1.9 and 1.11). The kinetics of rubisco with respect to free and bound
RuBP are analogous to that which would occur if a tight binding substrate were present (Farquhar
1979). The earlier description (Eqs 1.14 and 1.15) of the dependence of carboxylation and oxygen
ation on R remains valid if these refer to free not enzyme-bound RuBP. However, experimentally, it
is difficult to measure free RuBP from leaves or chloroplasts. The dependence of carboxylation rate
on free RuBP, R, as well as on free and enzyme-bound RuBP, Rt , is illustrated in Fig. 1.6.
Figure 1.6a shows the typical Michaelis-Menten dependence (a rectangular hyperbolic
response) of carboxylation rate on free RuBP. The Michaelis-Menten constant for RuBP, K r " for
the carboxylase is small at approximately 20 J.lM (Badger and Collatz 1977; Yeoh et al. 1981) and
the carboxylation rate is close to RuBP saturation at 200 J.lM RuBP. In contrast, the dependence of
carboxylation rate on total RuBP concentration is predicted to increase linearly with total RuBP,
R t , until R, equals rubisco site concentration, and then saturates abruptly (Fig. 1.6b). Thus, at an
enzyme site concentration of 2 mM, RuBP saturation occurs at 200 J.lM free RuBP and at above
2 mM total RuBP. The abruptness of the transition from RuBP-limited to RuBP-saturated rate in
Eq. (1.19) is dependent on the magnitude of K r ,. If K r , == 0 the two solutions to Eq. (1.17) are V/We
== 1 or V/We == RIEt . Because K r , is small the carboxylation rate (Eq. 1.19) can be closely approxi
mated by:
1.0 a
0.8
=s:<>
0.6
--
::::.,<>
0.4
0.2
0.0
0.0 0.1 0.2 0.3 0.4
free RuBP, R (,..tM)
8
1.0
0.8
v/w
c c
6 -
.....
CD
CD
=s:<> J)
0.6 . c
---
. . ..
::::.,<> 4 OJ
"'U
R
0.4 "" "" ::0
/. ""
/.
23
0.2 :s::
0.0 0
0 2 4 6 8
total RuBP, R t (mM)
Figure 1.6 (a) Modelled carboxylation rate, Ve (expressed asa fraction of the RuBP-saturated carboxylation
rate, We) as a function of free RuBP, R. Kr, = 20 IJM. (b) The dependence of carboxylation rate, Ve (expressed as a
fraction of the RuBP-saturated rate, We) on free and enzyme-bound RuBP, R; The enzyme siteconcentration is
2 mM, and the Kr, =20 IJM (solid lines) or 200 IJM (dashed lines). Also shown is the dependence of free RuBP on
R; (Adapted from Farquhar 1979.)
v, = W,·min{I,R,JE,}, (1.20)
where min { } denotes 'minimum of'.

Data supporting the use of Eqs (1.17) and (1.19) have been obtained for Chlamydomonas and
spinach cells by Collatz (1978). Collatz measured CO 2 assimilation rate and total RuBP pools at
various irradiances in an oxygen electrode. Mott et al. (1984), in an elegant set of experiments,
validated the above equations with measurements of CO 2 assimilation rate and RuBP pool sizes
on whole leaves. They examined the kinetics of the response of CO 2 assimilation and RuBP pool
sizes after a step change in irradiance from saturating irradiance to rate-limiting irradiances in
Xanthium strumarium. The activation state of rubisco changed with irradiance. However, since
rubisco requires several minutes to deactivate after a reduction in irradiance, they were able to
-..........
(fJ
<;J
E o
20 o e e
o 0
E ---------------------------
)....---
--
--GL~
:::t o -- _---~-----
(J) 15 ............
+-'
eel
.....
c
o 10
n;
E
(fJ
(fJ 5
eel
C\J
o
o o
o 40 80 120
-2
RuBP (umol m )
Figure 1.7 CO 2 assimilation rate as a function of RuBP pools in wild-type (e) and transgenic tobacco with an
antisense construct to glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH, 0). Measurementswere made
at 1.5 mmol quanta m-2 S-1, an ambient CO 2 partial pressure of 350 ubar and a leaf temperature of 25°C (data
redrawn from Price et al. 1995). The curves depict relationships between A and free and enzyme-bound RuBP
pool predicted by Eq. (1.19). The rubisco site concentration, E, = 2.0 mM, = 20 urnol m-2, and the Km(RuBP),
Kr,= 0.2 mM (solid line), Kr,= 0.6 mM (dashed line) or K( = 0 (dotted line). RuBP-saturated CO 2 assimilation rate
was taken as 20 urnol m-2 S-l and Rd =2 urnol m-2 S-1. The mean rubisco site concentration of the data set was
20 umol m-2 S-1,
examine the dependence of CO 2 assimilation rate on RuBP pool sizes immediately after a change
of irradiance and therefore at a constant rubisco activation state. Transgenic tobacco plants with
an antisense reduction in chloroplastic glyceraldehyde-3-phosphate dehydrogenase activity (anti
GAPDH plants) have reduced rates of RuBP regeneration and RuBP content without concomitant
changes in rubisco activation. The relationship between assimilation rate and RuBP pools gener
ated from wild-type and anti-GAPDH plants is also consistent with the model proposed by
Farquhar (1979), especially when the K r , is increased to take into account the competitive inhibi
tion by PGA (Fig. 1.7).
There has been some debate over how much RuBP is required to saturate rubisco sites. Several
studies have suggested that RuBP needs to be more than 1.5-2 times the rubisco site concentration
(Seemann and Sharkey 1986; von Caemmerer and Edmondson 1986; Seemann et al. 1987; Price et
al. 1995). The RuBP and PGA pools vary drastically and in opposite directions with CO 2 partial
pressure, with little change in the total esterified phosphate pool. This is probably governed by the
strict exchange of triose phosphate and phosphate by the phosphate translocator at the chloro
plast envelope. In vivo the RuBP pool is very large at low CO 2 partial pressures and low at high
CO 2 partial pressures and, under these conditions, PGA pools are also very high (Fig. 1.8; See
mann and Sharkey 1986; von Caemmerer and Edmondson 1986; Seemann et al. 1987). RuBP and
PGA change much less with changing irradiance since several of the PCR cycle enzymes are light
regulated (Fig. 1.9; Stitt 1996). Curiously, in all species examined, RuBP pool size rarely dropped
below rubisco site concentration even at high CO 2 partial pressure, except in transgenic tobacco
with reduced amount of GAPDH activity (Ruuska et al. 2000a), Figure 1.10 shows the modelled
relationship between CO 2 assimilation rate and RuBP content at two CO 2 partial pressures and
Raphanus sativus
Q)
Cii 60
•• • • • • • • • •
..... 100
c: 50
o • 80
~ '\IE 40
E (5 30 ",- • • • • 60
~ E 20 40
Cll E,
0« 10 20
o o o
___ 150 •• •• • 300
• • 1=
'\I~ • ~
n,
~
100 • •
It • ·C 200
3
"U
G)
a: ~
0
50
• 100
r(, »
o
• • o
o 600
~ • 1=
••
..... 1.5 I\)
«
(9
n,
--eo
n,
;:,
a:
1.0
0.5
••
•
••
• • •• •
•
•
400
200
3"U
+
"U
G)
~ ~
»
0.0 • o
o 400 800 o 400 800
Intercellular CO 2 partial pressure (ubar)
Figure 1.8 CO 2 assimilation rate, rubisco activation and RuBP and PGA pool sizes in Raphanus sativus (radish)
at different intercellular CO 2 partial pressures. Measurements were made at a leaf temperature of 25°C and an
irradiance of 1.4 mmol quanta m-2 S-1. Each point represents a measurement made on a different leaf.The arrow
points to measurements made at an ambient CO 2 partial pressure of 340 ubar. E, denotes the estimated rubisco
site concentration. (Redrawn from von Caemmerer and Edmondson 1986.)
data from wild-type and transgenic tobacco with reduced amounts of GAPDH activity. The slope
of the relationship between CO 2 assimilation rate, A, and RuBP is greater and saturates at higher
values of A at higher CO 2 partial pressures as We increases with increasing CO 2 ,
1.2.7 The binding of phosphorylated compounds and other ligands

The interaction of carbamylated and non-carbamylated rubisco sites with RuBP, phosphory
lated compounds and other ligands is very complex (Andrews and Lorimer 1987). Badger
and Lorimer (1981) developed a simplifying scheme that could account for many of the
diverse experimental observations. The basic principle underlying their model was that
ligands could bind to both carbamylated and uncarbamylated rubisco sites in a competitive
manner with respect to RuBP. This would affect catalysis, as carbamylated sites bound with
phosphorylated compounds other than RuBP would reduce the carboxylation and oxygen
ation capacity. On the other hand, binding of effectors could also affect the activation pro
cess depending on the ratio of their dissociation constants for the carbamylated and
uncarbamylated enzyme complexes (Badger and Lorimer 1981). The model was based on the
assumption that the eight catalytic sites of the enzyme acted independently of the other
seven, although there is some in vitro evidence that this is not always the case (for discussion
see Andrews and Lorimer 1987; Servaites and Geiger 1995). At present there is very little
information on how the dissociation constants of various ligands change with increased site
occupancy and I leave this for future extensions of the model.
The kinetics and regulation of rubisco 1S
Raphanus sativus
, ..
Q)
30" '" , "
"§ 100 ::IJ
I::
o ';"
".;i en 20
• 80
I::
0
iii'
Cll N o
E 'E 60 -0
'iii "0
CIl
Cll
ON
E
::t
10 40
20
~PJ
~(")
-
<'
~
o'
o
150
Or- ' , , '11' , , I 'I o ::::J
• • ••
100 • 200_
0-
CO
')IE
_ • •• ••
1=
3
o
"'tI
G)
::::J
a: E
0
50 • 1003"
J
}>
~
o I' , , "II' "'I o
o
• • •• • 400
. .
~ ••
I\)
• • • _ ::IJ
« •• ••
1= I::
0.5
r ,. ••• 3 OJ
•.'
CJ
200
--
0
0-
CO
Q.
3
J
"'tI
:t
::::J
a: 0.0 •
II I I I
•
II I I I ! I II
o
G)
}>
0.0 0.5 1.0 1.5 2.00.0 0.5 1.0 1.5 2.0

2s·1
Irradiance (urnol quanta m· )
Figure 1.9 CO 2 assimilation rate, rubisco activation and RuBP and PGA pool sizes in Raphanus sativus (radish)
at differentirradiances. Measurements were made at a leaf temperature of 25°C and at an ambientCO2 partial
pressure of 340 pbar. Each point represents a measurement made on a different leaf. Et denotes the estimated
rubisco site concentration. (Redrawn from von Caemmerer and Edmondson 1986.)
Table 1.4 Competitive inhibitors with respect to RuBP (K[, = 20 flM)

- - - - - - - - - - - - - - - - - - - - - -
InhibItor K p (flM) at 25°C
6-phosphogluconate 8.5
fructose lo-bisphosphate 40
NADPH 70
3-phosphoglycerate 840
inorganic phosphate 900
sulfate 630
Data taken from Badger and Lorimer (1981).
Von Caemmerer and Farquhar (1985) included the effects of phosphorylated compounds
other than RuBP binding to enzyme sites. They showed that the competitive interaction of other
ligands (P t , P 2 ••• ) with RuBP at the catalytic site modifies the Michaelis-Menten constant for
RuBPto:
x.. = Kr,(l+PJKpl+P2IKp2+"') (1.21)
(see Section 1.4.5 for derivations). A number of the PCR cycle intermediates and compounds
such as NADPH are competitive inhibitors with respect to RuBP. However, most are present
.. 30
0
C)'
en
E 25 0
0
0
E
--
::l
Q)
ro
20
•
.... 15 0
c
0
:;::;
ro 10
• •
E
en
en 5
ro
(\J
0
o 0
0 40 80 120 160
Figure 1.10 Assimilation rate, A. as a function of RuBP content in wild-type (e, .) andtransgenic tobacco with
an antisense construct to glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH, 0, 0). Measurements were
made at 1.0 mmol quanta m-2 S-1, a leaftemperature of 25°C andtwo ambient CO 2 partial pressures that resulted
in mean intercellular CO 2 partial pressures of 260 (0, e) and 470 ubar(0, .). (Data aretaken from Ruuska
1998.) The curves depict relationships between A andfree and enzyme-bound RuBP pool predicted by Eqs (1.19)
and (2.19). The rubisco site concentration, E, = 2.0 mM, = 20 urnol m-2, and Kr = 0.2 mM. RuBP-saturated CO 2
assimilation rate wastaken as 18 urnol m-2 S-l at Cj = 260 ubar andcalculated to be 1.5 times that at
Cj = 470 ubarwith the model parameters of Table 1.3. Rdwas 2 umol m-2 S-l.
at only low concentration and therefore do not cause a large increase in K, •. PGA, which can
be present at high concentrations (12 mM) has a Kp of 0.9 mM (Table 1.4; Badger and
Lorimer 1981 and references therein). The value of K r , = 20 flM is then increased to 286 uM
but is still not very large relative to the existing RuBP concentrations and the impact is
shown in Figs 1.7 and 1.10. It is perhaps worth noting that the binding constants have only
been measured under standard in vitro assay conditions and very little is known about their
temperature dependencies or variation with pH. There are some indications for example that
the Kp increases at lower pH (Prinsely et al. 1986). In vivo the fact that RuBP and sugar phos
phates chelate magnesium has also to be taken into account (von Caemmerer and Edmond
son 1986 and references therein).
1.3 ACTIVATION OF RlIBISCO

For catalysis rubisco needs to be activated with CO 2 and Mg 2 + (Laing and Christeller 1976;
Lorimer et al. 1976; Christeller and Laing 1978, 1979). The activation of rubisco involves the
slow reversible reaction of a molecule of CO 2 with a lysine residue of the reaction site. The
formation of the carbamate is followed by the rapid addition of Mg 2 + to create a ternary
complex. The activator CO 2 molecule is different to the substrate CO 2 and does not get fixed
during catalysis (Lorimer 1979; Miziorko 1979). To be fully active the enzyme requires a
molecule of CO 2 and Mg 2 + bound to each of the eight catalytic sites.
Although the activation of rubisco by CO 2 and Mg2+ is essential it is not clear how chloroplast
CO z and magnesium concentrations take part in the in vivo regulation of rubisco. The regulation
of rubisco activity by light was shown early on with isolated chloroplasts (Jensen and Bahr 1977)
and in intact leaves (Perchorowicz et al. 1981; von Caemmerer and Edmondson 1986; Fig. 1.9).
Light-dependent changes in stromal pH and Mgz+ seemed to provide a suitable mechanism for
the light regulation (Jensen and Bahr 1977), but changes in stromal pH and Mgz+ become
saturated at very low irradiances compared to the activation state of rubisco, which detracted
from this early hypothesis. Subsequent work has shown that the regulation of rubisco is far more
complex and involves the protein rubisco activase, whose function is as yet poorly understood
(Salvucci et al. 1985; Portis 1990,1992; Salvucci and Ogren 1996).
The following section traces a somewhat historic path, discussing first the equations that
describe the carboxylase rate when the enzyme has been activated in vitro in the absence of RuBP.
The scheme of Fig. 1.1 is then used to derive equations for rubisco activation in the presence of
RuBP. This scheme serves to illustrate the inconsistencies that exist between in vitro measure
ments and in vivo functions and how these may be addressed by the function of rubisco activase.
1.3.1 Activation of rubisco in the absence of RuBP

The kinetic process of activation by CO z and Mgz+ has been well characterized in vitro for
purified rubisco from several plant and microbial sources (Laing and Christeller 1976;
Christeller and Laing 1978; Lorimer et al. 1976; Badger 1980). To activate rubisco in vitro it
has been customary to pre-incubate the enzyme in the presence of CO 2 and Mgz+ and then
measure the activity at saturating CO z, Mgz+ and RuBP. When activating in the absence of
RuBP the fraction of carbamylated rubisco is given by:
EC+ECM CM
(1.22)
E+EC+ECM CM + KeKdC 1 + Kd/ M)
This expression is analogous to the expression derived by Lorimer et al. (1976). The term in
parentheses in the denominator is there because it is assumed that under in vitro assay condi
tions EC will rapidly bind Mgz+ and be counted as part of the carbamylated enzyme. The car
boxylation rate, VC' measured in the assay with saturating CO z and RuBP, is given by:
CM
v, = VcmnxCM + r,«, (1.23)
K, (= k41k 3) and Kd (= k6lk s) are the dissociation constants of the enzyme site CO z complex,
EC, and the enzyme site CO z/M g2+ complex, ECM, respectively and M refers to free magne
sium (Fig. 1.1 and Section 1.4). Table 1.5 lists some measured values of K, and K d or K~d
from the literature. It was clear early on that the activation state of rubisco in vivo could not
be solely regulated by chloroplast concentrations of CO z and Mgz+ (Lorimer et al. 1976). At
an ambient CO z concentration of 8 JlM and free magnesium concentrations between 5 and
10 mM the product of these concentrations is below the required Michaelts-Menten constant
for activation, K~d' and rubisco is predicted to be essentially inactive. This of course con
trasts with experiments which showed that fully activated rubisco could be obtained from
illuminated leaves after rapid extraction (Figs 1.8 and 1.9; Taylor and Terry 1984; Brooks
1986; Sharkey et al. 1986a; von Caemmerer and Edmondson 1986).
1.3.2 Activation of rubisco in the presence of RuBP

Equation (1.23) applies when rubisco is activated in vitro in the absence of RuBP. In vivo activa
tion needs to occur in the presence of RuBP and Laing and Christeller (1976) showed that RuBP
could bind to the inactive enzyme sites, E, and the CO z and Mgz+ activated enzyme sites, ECM,
and proposed a scheme similar to that shown in Fig. 1.1. Farquhar (1979) used this scheme to
Table 1.5 Values of equilibration constants for CO 2 and Mg 2+ activation of rubisco in vitro
Species K.,Kd (flM)2 s, (flM) K d (flM) Reference

---------------
Glycine max 1.93 X 105 91 1130 Laing and Christeller 1976'

1.03
1.33
Spinacia oleracea 1.81 X 105 309 529 Lorimer et al. 1976b
1.46 X 105
Rhodospirillum rubrum 0.54 X 105 600 90 Christeller and Laing 1978 c
Spinacia oleracea 450 Jordan and Ogren 1983d
Nicotiana tabacum 490
Glycine max 900
Amaranthus hybridus 580
Zea mays 900
Aphanizomen flos-aquae 3150
Rhodosptrillum rubrum 1050
Oryza sativa 1.9-2.4 X 105 Makino et al. 1988 e
Experimental conditions: a 0.05M Tris HCl pH 7.9, 18°C; b 0.05M Tris HCl pH 8,2, 10°C; c 0.05 M Tris HCl pH 8.3,
19°C; d 0.05M Bieine pH 8.22, 25°C; e 0.05M Bieine pH 8.2, 25°C
derive the rate equations of carboxylation and oxygenation of the partially activated enzyme in
the presence of RuBP and the equation of the carboxylation rate is given by:
C CM R
( 1.24)
V, = V cnIa x C + K c ( 1 + 0/K o) CM + KeKdKrJ s; R + K r"
where Kr"is the Michaelis-Menten constant of RuBP of the partially activated enzyme, which
has a dependence on CO 2 and Mg 2+,
CM + (C + Ke)K
K" = K .--,-------=--d (1.25 )
r r CM + KeKdKrJ Kf
K, (= k tlk 2 ) is the dissociation constant of the inactive enzyme site RuBP complex, ER. The
larger the value the looser the binding of RuBP to the inactive site (Section 1.4).
At high concentrations of enzyme sites carboxylation and oxygenation of the partially acti
vated enzyme are given by Eqs (1.17) and (1.18) with Kr,replaced by Kr"and the RuBP-saturated
rate, We' of the partially activated enzyme is given by:
(1.26)
The expression of Wo is analogous (Farquhar 1979).
1.3.3 The carbamylation ratio of rubisco

It is possible to measure both rubisco activity and the carbamylation state of rubisco in leaf
extracts (Butz and Sharkey 1989). Rubisco activity is measured in leaf extracts from the rate
of 14C02 incorporation into acid-stable products immediately after extraction or after pre
incubation of the extract with CO 2 and magnesium to achieve full carbamylation. The car
bamylation state can be measured with 14C labelled carboxyarabinitol-l,5-bisphosphate
(CABP). CABP is a reaction intermediate analogue that binds tightly and stoichiometrically
100
----
~
'-'
0
80 ~ -: K/Kr , = 100
u
.,...,
VJ
.D 60
...
;:i
"0
Q.)
...... 40
ro
.......
;>-.
20~
S K/Kr , = 1
ro
.D
...ro
U I ~~ K/Kr , = 0.01
0 , , , , , ,
0 1 2 3 4 5 6
Total RuBP concentration, s, (mM)
Figure 1.11 Predicted carbamylation status of rubisco as a function of RuBP concentration (freeplus bound
Rubisco, Rt ) at three different ratiosof KflKr" The curves shownwere calculated using Eqs (1.27) and (1 .64) in
Section 1.4. Total rubisco site concentration, Et = 2 mM, M = 5 mM, C = 811M, K( = 20 11M, Kd =1600 11M and
KeKd = 160 000 11M2 (Table 1.4; Laing and Christeller 1976; Lorimer et al. 1976). (Redrawn from Mate et al. 1996.)
to rubisco sites. Furthermore, since CABP binds significantly more tightly to carbamylated
rubisco sites, it is possible to exchange [14CjCABP from non-carbamylated sites with an
excess of unlabelled CABP and quantify the number of carbamylated sites (Collatz et al.
1979; Butz and Sharkey 1989). The carbamylation ratio of rubisco can be derived from the
equations given in Section 1.4 and:
EC+ECM+ECMR+ECMRC+ECMRO
Et
(1.27)
K d)) / ( 1+-
Kr'( 1+-
( 1+-
s,- +KeK
K r'( 1 + - -d) +KeKdK
--
r')
R M R M CM CM Kf
A brief examination of the above equations shows, that in this particular scheme, the ratio of
KIKr, is very important for establishing the activation state of rubisco. It is well known that
the presence of RuBP inhibits activation of rubisco in vitro (Jordan and Chollet 1983; Portis
1990). Kfhas been measured to be 2 nM, which is an order of magnitude less than measure
ments of K r , around 2-20 11M depending on whether chelation of RuBP and Mg2+ has been
taken into account (Laing and Christeller 1976; Jordan and Chollet 1983). The dependence
of the carbamylation ratio on RuBP concentration is shown at different ratios of KI K r, in
Fig. 1.11. The model predicts that rubisco can be fully carbamylated only at high ratios of
KIKr,and that carbamylation is strongly dependent on RuBP concentration There is some
in vitro evidence for this prediction (Portis et al. 1995). At ratios of KI K r, less than 1 rubisco
carbamylation decreases with increasing RuBP (Mate et al. 1996).
1.3.4 Rubisco activase
The isolation and characterization of a high CO 2 requiring mutant of Arabidopsis thaliana
(Somerville et al. 1982; Salvucci et al. 1985) provided a major breakthrough in our under
standing of rubisco regulation. This mutant had a defect in the in vivo activation process,
which was not apparent in vitro with rubisco purified from the mutant. This led to the iden
tification of the chloroplast protein, rubisco activase, which was absent in the mutant. In
vitro experiments have shown that rubisco can be activated in the presence of RuBP, activase
and ATP; however, activase does not affect in vitro activation in the absence of RuBP or other
ligands (Streusand and Portis 1987; Portis 1992). This has led to the hypothesis that one of
the roles of activase is to prevent the binding of RuBP and other ligands to the inactive
rubisco sites. Activase activity requires ATP and activase itself appears to be light regulated,
although the mechanism is unclear (Campbell and Ogren 1992; Portis 1992; Salvucci and
Ogren 1996; Zhang and Portis 1999; Ruuska et al. 2000a).
1.3.4.1 A model of activase action
Andrews et al. (1995) and Mate et al. (1996) proposed a model for the mechanism of activase
action. They suggested that when activase is activated by ATP hydrolysis, it can recognize
rubisco with a closed loop protein structure that occurs when active site loops close over a
tight binding ligand. They suggested that activase binds selectively to this conformation and
in doing so causes the loops to open, releasing the ligand and returning activase to its inac
tive form. The closed loop complexes can occur with both carbamylated (ECM) and uncar
bamylated (E) rubisco complexes. The unassisted rate at which they release their ligands is
very slow. However, when the ligand is the substrate, RuBP, catalysis provides another rapid
means of opening loops for the ECMR complex that does not require the assistance of acti
vase. Therefore, activase induces a much larger increase in the rate of opening of the unpro
ductive ER complex than it will in the rate of opening of the catalytically competent,
carbamylated complexes. This difference provides the opportunity for rubisco's carbamyla
tion status to respond to the concentration of RuBP as well as to the activity of activase. Mate
et al. (1996) used the scheme outlined in Fig. 1.1 to formulate this model. Since the model
proposes that activase can recognize only closed complexes, activase can have no effect on the
rate at which those complexes are formed and the influence is restricted to promoting disso
ciation of the complexes. The thick arrows in Fig. 1.1 indicate this. Mate et al. (1996) rede
fined Kfas:
(1.28)
where f(A) is a complex function positively correlated with activase activity that describes
activase-mediated dissociation of the ER and the ECMR complex. Kf now defines the ratio of
E.RIER in the steady state and K r , is modified to:
(1.29)
It can be demonstrated mathematically that activase action causes an increase in the ratio
Kri r;since:
(1.30)
If activase interacts only with uncarbamylated sites (i.e. the f(A) term disappears from the
denominator), the increase in Kri K» is obvious. However, the way activase facilitates the
release of the nocturnal inhibitor, 2' -carboxy-D-arabinitol-l-phosphate (CAIP), from ECM
(Gutteridge et al. 1986; Robinson and Portis 1988; Servaites 1990) suggests that activase
interacts at both carbamylated and uncarbamylated sites. In this case both the numerator
and the denominator increase with activase activity. Nevertheless, the increase in the numer
ator will be proportionally larger since in the denominator increases in f(A) are scaled
against the potentially larger catalytic release term kllC/Kc + k 140IK", whereas in the numer
15
- -r:
Q)
a....3
e 00
r:
c
0 en 10
:;: C\J 0
a3
E 0
E 0
'wen E
a3 E, 5
N
0
0
0
I e
;!2
0
80
-----
c
0
:;:;
60
a3
>
E 40
a3
.0
....
a3
0 20
0
0 10 20 30 40 50
Activase content (mg rn" )
Figure 1.12 CO 2 assimilation rate and rubisco carbamylation for control tobacco leaves (e) and leavesof
tobacco with an antisense constructagainst rubisco adivase ( , ). Measurements were made at 1.5 mmol quanta
m-2 S-1, an ambient CO 2 partial pressure of 350 ubar and a leaf temperature of 25°C. (Data are redrawnfrom
Mate et al. 1996.)
ator it is scaled in comparison only with the small unassisted release term k 1 . The increase in
KffK r , in Fig. 1.11 can be interpreted as an increase in activase activity.
1.3.4.2 A hyperbolic response of carbamylation to activase activity

Studies with activase-deficient transgenic tobacco have shown that the amount of activase
can be substantially reduced before rubisco carbamylation state and steady-state CO 2 assimi
lation are affected (Fig. 1.12; Hammond et al. 1995; Jiang et al. 1994; Mate et al. 1996). Under
these conditions, RuBP content was always saturating and the equation for rub isco carbamy
lation (Eq. 1.27) can be simplified to:
ECMR+ECMRC+ECMRO K/K r ,
(1.31)
ER + ECMR +ECMRC+ECMRO K,I x; + KeKdl CM
which has a hyperbolic dependence on KffK; (Fig. 1.13) The relationship between carbamy
lation and activase content and between carbamylation and KI K r , share a strikingly similar
hyperbolic character. If the dependence of activase activity on the concentration of ER and
ECMR can be approximated by a first-order reaction the hyperbolic dependence shown in
Fig. 1.13 would be inherent in the basic mechanism of activase action. The model also pre
dicts a linear relationship between the rate of activation and activase activity. When activase
100
»<;
~
'-./
80
0
U
o:
.,....;
,D
:::i
;...,
60
"d
....
-
tl)
('j 40
>-.
8('j
,D
;...,
20
('j
U
0
0 20 40 60 80 100
K/Kr ,
Figure 1.13 Predicted carbamylation status of rubisco asa function of Kfl K,' at a saturating RuBP
concentration (Rt = 6 mM). It is assumed that activase increases KflK," The curve shown wascalculated using Eqs
(1.58) and (1.59) in Section 1.4. Otherconditions were as for Fig. 1.12.
is limiting, the initial rate of activation (-dERldt) will be a linear function of k] + f(A). Ham
mond and co-workers have shown that there is indeed a linear relationship between the rate
of rubisco activation in response to a step change in irradiance and the amount of activase
(Hammond et al. 1995, 1998), giving some support to such a simple model. However, it
needs to be recognized that at present all experiments with transgenic tobacco with reduced
amount of activase have relied on western blots for quantification of the amount of activase
protein and that no good assay for activase activity from leaf extracts exists at present. A
recent report suggests that the light regulation of activase in Arabidopsis occurs via the
thioredoxin pathway (Zhang and Portis 1999). There are also indications from in vivo studies
with transgenic tobacco with reduced amount of chloroplast bf complex that a similar mech
anism may modulate activase activity in tobacco (Ruuska et al. 2000a).
1.3.4.3 Binding of other ligands
So far the only ligand considered has been RuBP. The presence of phosphorylated com
pounds in in vitro assays affects the activation process (Badger and Lorimer 1981; McCurry
et al. 1981) and von Caemmerer and Farquhar (1985) modelled this. The model of activase
action could also be extended to include other ligands and in Section 1.4 an equation is
derived for the carbamylation ratio in the presence of other phosphorylated compounds (Eq.
1.77). Jordan et al. (1983) measured the dissociation constants for a number of phosphory
lated effectors to the carbamylated and uncarbamylated rubisco sites. Activase has been
shown to aid in the release of these ligands (Portis 1990). From our model considerations it
is unlikely that activase could affect the ratio of EPIECMP (where P stands for a ligand other
than RuBP) since there will be no catalytic release for ECMP.
1.3.4.4 An unknown function of activase

The model discussed here has provided a useful framework for considering the interac
tions between activase and rubisco. It has been demonstrated that carbamylation would be
promoted even if activase were to act equally at carbamylated and uncarbamylated sites.
-- 8°1 • • ",
0~
c
0
0.0::;
• •
(lj
>.
E
(lj
.0
"
(lj
0
-~
~
°
Q)Q)
+-' +-'
b
•
.g~oo
.§ 0.8 ~ • • ••
(lj.o
•-
E"
'--0
:::::l
•
~Q)
(lj
N>,
co 0.4
°E
O(lj
.0
"
(lj
2 0.0
0 40 80 120160200240
Activase (mg rn")
Figure 1.14 (a) Rubisco carbamylation and (b) CO 2 assimilation rate per carbamylated rubisco sites versus
activase content for controltobacco leaves (.) and leavesof tobacco with an antisense construct against activase
(0). Gas-exchange measurements were made at 1.5 mmol quanta m-2 s', an ambient CO 2 partial pressure of
350 pbar and a leaf temperature of 25°C after which the leaves were rapidly freeze clamped. (Data are redrawn
from He et al. 1997).
However, this model should be viewed as only a first attempt in understanding rubisco
activase interactions. Studies with transgenic tobacco with reduced amounts of activase
have shown that in vivo activase does not only affect the carbamylation state of rubisco, but
may also affect the catalytic turnover rate of carbamylated sites (Fig. 1.14; He et al. 1997).
These studies relied on careful quantitative comparisons of measured CO 2 assimilation
rate with measurements of rubisco carbamylation state and in vitro activities. He et al.
(1997) found that, although in vivo rubisco activity was reduced per carbamylated sites in
tobacco with reduced amount of activase, the in vitro extractable initial activities were sim
ilar to wild type when expressed on a carbamylated site basis. This ruled out the presence
of tight binding inhibitors, unless these inhibitors were destroyed during extraction. The
possibility of the presence of an unknown loose binder at high concentration remains. It

also raises the question as to whether activase is involved in the process of catalysis per se,
and this is not part of the current model.
1.4 DERIVATIONS
1.4.1 Derivation of rate equations for activated enzyme

These derivations follow those of Farquhar (1979) and the scheme outlined in Fig. 1.1. The
total enzyme site concentration E, is given by:
E, = E + ER + EC + ECM + ECMR + ECMRC + ECMRO (1.32)
At first we assume that rubisco is fully activated and that the concentration of uncarbamyl
ated sites is zero such that E, = ECM + ECMR + ECMRC + ECMRO. All rate constants are
denoted by k and the subscripts are the numbers shown in Fig. 1.1. The Michaelis-Menten
.. k ll + k lO k
constants for CO 2 and 02 are defined In Section 1.2.1 as K, = k ' K; = -----:k-----:
I4+k 13
9 12
respectively. Furthermore K j r = k s/ k 7 , K r c = k ll / k 7 and K r o = k 14 / k 7 . The catalytic turnover rate
for carboxylation is defined as kcwt = k ll and the catalytic turnover rate for oxygenation is
kowt = k 14 •
(1.33 )
( 1.34)
(1.35)
(1.36)
In the steady state the concentration of enzyme complexes is constant and the following rate
equations can be formed:
d
-ECMRC
dt
= 0 = (kgC)ECMR - (k ll + klO)ECMRC (1.3 7)
d
-ECMRO
dt
= 0 = (k 120)ECMR - (k 14 + k 13)ECMRO (1.38)
(1.39)
These yield the following equalities. From Eq. (1.37) it can be derived that:
K
ECMR = ~ECMRC (1.40)
and from Eqs (1.38) and (l.40) one derives that:
ECMRO = K c 0 ECMRC (1041)

CKo
From Eqs (1.39) to (1.41) one obtains an expression for ECM:
ECM = {KiTK c + K TC+ KToK e }ECMRC

R C R R CKo
° (1.42)
Substituting into Eq. (1.32) then gives:
E, = {(1 + x, KeO) s; 1( KcO)}

C + C K + R KiT C + KTe + KTOC K ECMRC (1.43 )
o o
and
( K K0)
E t = {l+K T,IR} 1+ ~+ ~Ko ECMRC (1.44)
where
(K Ti + KTCCx; + KTOKO )
o
Kr' (1.45 )
(1+ x,C+ x,0)
and
K,
ECM = ;(ECMR+ECMRC+ECMRO) ( 1.46)
Equation (1.44) is an expression for ECMRC that can be substituted into Eq. (1.33). Together
with Eq. (1.34) and some regrouping it can be shown that:
CVcm ax R R
V. = = W.-- (1.47)
c C+Ke(l+OIKo)R+K T, 'R+Kr'
Equation (1.47) is Eq. (1.14). When R is large it degenerates to Eq. (1.9). The equations for
the oxygenase activity can be derived in an analogous manner.
1.4.2 The rate equations at high concentration of enzyme sites

The carboxylation rate, V e, and the oxygenation rate, Va' have a typical Michaelis-Menten
dependence on free RuBP, R. However, in vivo rubisco site concentration is large and of a
similar magnitude as the total (free and rubisco-bound) RuBP pool. Since in experiments
with leaves we can only measure total chloroplast RuBP, it is of interest to express the
carboxylation and oxygenation rates as functions of the total (free and enzyme-bound)
RuBP. When rubisco is fully active, the total RuBP pool, R p is given by:
R, = R+ECMR+ECMRC+ECMRO (1.48)
This can also be rewritten as:
R, = R + E,-ECM (1.49)
ECM can be eliminated from Eq. (1.49) with the use of the following expression, which is
obtained by combining Eqs (1.42), (1.43) and (1.45):
Et
ECM= --~-::-:- (1.50)
1 + RI(Kr, )
This then gives a quadratic expression for R:
(1.51)
From Eq. (1.47) it can be shown that R = (K,,(V/W,))/(1 - V/W,) which, upon substitu
tion into Eq. (1.51), gives an expression for VJW( as a function of Rt :
(1.52 )
The exact solution to this quadratic equation is:
-v, -_ -
We
1{(
2
1+ - + K,,)
Rt -
Et
- - (1+ - + K,,)2
Rt -
Et
Rt }
- - 4-
e,
(1.53)
However because Kr , << E, the solution to the quadratic can be closely approximated by:
v. = min{ W, W{~) } (1.54)
where 'min' means 'the minimum of'.
1.4.3 Derivation of rate equations of activation

Rate equations similar to Eqs (1.37) to (1.39) can be set up for each of the enzyme com
plexes.
(1.55)
(1.56)
(1.57)
and, in the steady state, the following equalities hold:
ER = E· RIKr (1.58)
EC = E· CIK, (1.59)
ECM=EC·MIKd (1.60)
where r, x,
= k]/k2 , = k4 / i, and x, = k6 / i;
The derivation of V( for the partially activated enzyme (Eq. 1.24) is analogous to the deriva
tion shown above for the fully active enzyme. With the incorporation of the additional
enzyme complexes:
(1.61)
and with some rearranging:
v =W CM _R_ (1.62)
C C CM + KeKdK r,/ Kf R + K»
where
KKK)
K,I+---.E+_'_d
r ( M CM
K r" - (1.63 )
( r;«,
1 + - - ·K-' )
r
CM Kf
\ Using the equalities above, free RuBP can be related to total RuBP in a similar manner as
before and it can be shown that:
2
R +R(Et-Rt+Kr,,)-RtKr" =0 ( 1.64)
1.4.4 Carbamylated enzyme sites as a fraction of total enzyme sites

The total enzyme site concentration is given by Eq. (1.32). The concentrations of all enzyme
complexes can be expressed as a function of the ECM complex using the above equations and
it can be shown that:
R x, KeK d KeKd R }
(1.65 )
t, = ECM{ 1 + K r, + M + CM + CM K
f
Similarly the sum of the concentrations of carbamylated enzyme complexes is given by:
EC + ECM + ECMR + ECMRC + ECMRO = ECM{ 1 +..!i + K d} (1.66)

x, M
The ratio between the concentrations of carbamylated complexes and total enzyme is
obtained by dividing the two equations and multiplying the numerator and denominator by
~,IR:
EC+ECM+ECMR+ECMRC+ECMRO
(1+ ~' ( 1 + ~))
( 1.67)
Et «,- +KeK
Kr'( 1 + d) KeKdKr')
( 1+-
R M
-- +
CM
--
CM r;
In the absence of RuBP tR, = R = 0), Eq. (1.67) simplifies to:
EC+ECM CM
(1.68)
E+EC+ECM CM + K eK,/(1 + K/M)
which is analogous to the expression derived by Lorimer et al. (1976) for in vitro activation of
mbisco in the absence of RuBP.
At saturating RuBP, Eq. (1.67) simplifies to:
ECMR +ECMRC+ ECMRO CM KrlKr'

(1.69)
ER+ECMR+ECMRC+ECMRO CM + KeKdK r./ s, x.r«; + KeK,/CM
which predicts a hyperbolic dependence of the carbamylation ratio on the ratio KJK,..
1.4.5 ligands other than RuBP

Phosphorylated compounds other than RuBP also bind to E and ECM and it can be shown
that, if P is a ligand:
(1.70 )
and
ECMP = ~ECM (1.71)

Kp
such that
EP _ s,«,«,
(1.72)
ECMP - CM K pe
where K pe and K p are the dissociation constants of the EP and ECMP complexes, respectively
(von Caemmerer and Farquhar 1985).
When the enzyme is fully carbamylated:
E, = ECM + ECMP + ECMR + ECMRC + ECMRPO (1.73)
and
P (x; s; s; 0)
C + «; + x.; C K
ECMP = 0 ECMRC (1.74)
Kp R
Substituting into Eq. (1.73) as before:
E
t R RK
r; r;
x, s, P )( 1+-+--
= (1+-+- C C x,
0) ECMRC
, (1.75)
p
Equation (1.75) is used to substitute for ECMRC in Eq. (1.33) and, together with Eq. (1.34):
( 1.76)
When the enzyme is not fully carbamylated the effect of phosphorylated compounds on the
carbamylation ratio is given by:
EC+ECM+ECMP+EGWR+ECMRC+ECMRO
Et
(1.77)
2
Modelling (3 photosynthesis
2.1 INTRODUCTION
In this chapter the C3 photosynthesis model first described by Farquhar et al. (1980), von Caem
merer and Farquhar (1981) and Farquhar and von Caemmerer (1982) is discussed with the help
of some of the experimental data that can be used to critically review the model. This model is
widely used and many different versions exist (e.g. Kirschbaum and Farquhar 1984; Harley et al.
1985; Harley and Sharkey 1991; for review see Cheeseman and Lexa 1996). The model has also
been incorporated as a submodel into other models (see for example Leuning 1990; Sage 1990;
Collatz et al. 1991; Gross et al. 1991; McMurtrie et al. 1992; Sellers et al. 1992; Leuning et al. 1995;
Lloyd et al. 1995; de Pury and Farquhar 1997; Pearcy et al. 1997).
There are many different ways that photosynthetic models can be constructed and the format
they take will be determined to a large extent by the questions one would like to answer. The
model by Farquhar et al. (1980) was set up to describe the steady-state CO 2 exchange of leaves
measured by gas exchange. The aim was to provide a mechanistic model for responses of photo
synthesis in the intact leaf to light, CO 2, 02 and temperature. It followed and updated earlier
models by Hall and Bjorkman (1975), Peisker (1974) and Berry and Farquhar (1978) and related
the biochemistry of CO 2 assimilation to gas exchange.
2.2 A FEW SIMPLIFYING ASSUMPTIONS

Simplicity is often the key to making a model useful. This requires careful consideration of
the detail needed to be incorporated into the model and what can safely be ignored for first
approximations without much loss of accuracy. It is important to keep the number of param
eters that have to be assigned to a minimum. Many of the decisions made can be experimen
tally tested. A photosynthesis model thus provides a set of hypotheses brought together in a
quantitative form that can be used to interpret experiments.
CO 2 exchange takes place in the chloroplasts and mitochondria of leaf mesophyll and palisade
cells. To obtain rates of CO 2 assimilation of the leaf it is necessary to add the individual contribu
tions from these organelles across the leaf. The gradients of temperature and O 2 within the C 3 leaf
are likely to be unimportant. Small gradients of CO 2 may develop across the leaf and the greatest
uncertainty will be the distribution of light within the leaf. Nevertheless, many leaves are thin
enough for these not to be a major issue and, like the original model (Farquhar et al. 1980), our
initial model development assumes the leaf to be one large bag providing a uniform environment
to identical chloroplasts. However, patchy stomatal responses and the imaging of chlorophyll flu
orescence across leaves has shown that under certain conditions photosynthesis across leaves can
be non-uniform and this will affect the interpretation of gas-exchange measurements (Downton
et al. 1988; Terashima et al. 1988; Terashima 1992; Cardon et al. 1994; Genty and Meyer 1994;
Siebke and Weis 1995).
29
~
~
<. • (I+$)RuBP ~
(1+$) Ru5P
~GUbiSC~<$ O I I 0.5 ~~~PH I
1-0.5$
CH 20
rl
triose-P
(
2PGA
\--+$PGlY
$PGA ~
2
~ ~
ijW'5$ ATP
~I '\
$ glycine NH +
2
<Q.5$~1~
0.5$PGA~
~
~
Figure 2.1 Stoichiometry of the photosynthetic carbon reduction and photorespiratory cycles (<IJ denotes the
ratio of oxygenation to carboxylation).
2.3 STOICHIOMETRY OF (3 PHOTOSYNTHESIS
2.3.1 Carbon stoichiometry of the PCR and pca cycles

Rubisco, located in the chloroplast stroma, catalyses the competing reactions of the carboxy
lation and the oxygenation of RuBP. The carboxylation of RuBP is the first step of the PCR
cycle and the carboxylation of 1 mol of RuBP leads to the formation of 2 mol of PGA. The
oxygenation of 1 mol of RuBP, on the other hand, leads to the formation of 1 mol of PGA and
1 mol of phosphoglycolate (PGly). We assume that the recycling of 1 mol of PGly in the PCO
cycle results in the release of 0.5 mol of CO 2 (Fig. 2.1). It has been suggested that the release
may be less than 0.5 mol and there continue to be reports of complete oxidation of glycolate
as proposed by Zeltich (see Hanson and Peterson 1986; Zelitch 1989; Harley and Sharkey
1991) .
Some mitochondrial respiration associated with the tricarboxylic acid (TCA) cycle continues
in the light, although not necessarily at the rate that occurs in the dark (Brooks and Farquhar
1985; Atkin et al. 1998; Hoefnagel et al. 1998). Thus we can write the net rate of CO 2 uptake as:
(2.1)
where A denotes net CO 2 assimilation rate, V, and V o are the carboxylase and oxygenase rates
of rubisco and R d denotes day respiration, which comprises mitochondrial CO 2 release
occurring in the light other than that of photorespiration. Equation (2.1) can be rewritten in
a simpler form as:
(2.2)
where <p is the ratio of oxygenation to carboxylation rates, VoIVe. Equation (2.2) says that, for
each carboxylation, <p oxygenations occur which release 0.5<p CO 2 , This and other important
stoichiometries of the PCR cycle are summarized in Fig. 2.1. For example, because <p oxygen
ations take place per carboxylation, the steady-state rate of RuBP consumption has to be
(l + <p) times the rate of carboxylation:
Modelling (3 photosynthesis 31
.
(1~) RuBP (C5)
Figure 2.2 Regeneration of ribulose-1 ,5-bisphosphate (RuBP) from 3-phosphoglycerate (PGA). Details included
are: (1) the phosphorylation of 3PGA by AlP to form 1,3-bisPGA catalysed by 3-phosphoglycerokinase; (2)
reduction of 1,3-bisPGA to 3-phosphoglyceraldehyde (GAP), catalysed by 3-phosphoglyceraldehyde
dehydrogenase; (3) isomerization of GAP to dihydroxyacetone phosphate (DHAP), catalysed by triose phosphate
isomerase; (4) DHAP exportfrom the chloroplast; (5)combination of GAP and DHAP to fructose-1 ,6-bisphosphate
(FBP) by aldolase; (6) hydrolysis of phosphate from FBP to fructose-6-phosphate (F6P) by FBP phosphatase; (7)
transfer of upper two carbons of F6P to GAP to form xylulose-5-phosphate (Xu5P)and erythrose-4-phosphate
(E4P). catalysed by transketolase; (8)combination of E4P andGAP to form sedoheptulose-l ,7-bisphosphate (SBP)
by aldolase; (9) hydrolysis to sedoheptulose-7-phosphate (S7P) by SBP phosphatase; (10) transfer of upper two
carbons to GAP to form ribose-5-phosphate (R5P) andXu5P by transketolase; (11) isomerization of Xu5P to
ribulose-5-phosphate (Ru5P) by an epimerase; (12) differentisomerization of R5P to Ru5P; (13) conversion of
Ru5P to RuBP by phosphoribulokinase. is the ratio of oxygenation to carboxylation.
the rate of RuBP regeneration = (1 + <\» V( (2.3)
The carboxylation of 1 mol of RuBP produces 2 mol of PGA, and the oxygenation of 1 mol of
RuBP produces 1 mol of PGA and 1 mol of PGly. One mol of PGly in turn gives rise to 0.5
mol of PGA and thus:
the rate ofPGA production = (2 + 1.5<\» V c (2.4)
From Eq. (2.2) it can also be deduced that, per carboxylation, 1 - 0.5<\> carbons can be
exported from the cycle to produce starch and sucrose.
2.3.2 The carbon path from PGA to RuBP

There are many enzymatic steps involved in the regeneration of RuBP from PGA and these
are outlined stoichiometrically in Fig. 2.2. Most reactions are reversible, apart from those
32 Biochemical Modelsof Leaf Photosynthesis
catalysed by fructose bisphosphatase (FBPase) and sedoheptulose bisphosphatase (SBPase)

(Stitt 1996). Figure 2.2 can be used to calculate the steady-state flux through any of the
metabolites. For example, it can be seen that FBPase and SBPase have to run at only one
third of the rate of RuBP regeneration (Farquhar and von Caemmerer 1982; Brooks and Far
quhar 1985; Sharkey 1985). Details of the enzymes of the regeneration path are not included
in the basic C 3 model as it is assumed that they are not rate limiting (Farquhar and von Cae
mmerer 1982). Recent results with transgenic tobacco with antisense reductions to GAPDH,
FBPase, SBPase and phosphoribulokinase have confirmed that this is a reasonable assump
tion under most circumstances (Kossmann et al. 1994; Paul et al. 1995; Price et al. 1995;
Harrison et al. 1998). Several of the PCR cycle enzymes are, however, redox regulated by the
thioredoxin ferredoxin system (Stitt 1996) and since their activities reflect the amount of.
reduced ferredoxin they may co-limit the rate of RuBP regeneration with electron transport
(Farquhar and von Caemmerer 1982).
2.3.3 ATP and NADPH consumption in the PCR and pca cycles
The regeneration of RuBP requires energy in the form of ATP and NADPH. The rates of ATP
and NADPH consumption are calculated from the amount required to regenerate RuBP at
the rate of (l + <1» V c' In the regeneration of RuBP from PGA, PGA is first phosphorylated
and then reduced, requiring one ATP and one NADPH. The subsequent phosphorylation of
ribulose-5-phosphate to RuBP requires a further ATP. The regeneration of 0.5 mol of PGA
from 1 mol of PGly produced during oxygenation requires 0.5 mol of ATP in the final step.
The events that follow oxygenation also include the release and refixation of 0.5 mol of
ammonia (Keys et al. 1978; Woo et al. 1978). The refixation of 0.5 mol of ammonia requires 1
mol of reduced ferredoxin, which in terms of electron transport is equivalent to 0.5 mol of
NADPH, and 0.5 mol of ATP (Fig. 2.1). Summing these requirements we find that:
the rate ofNADPH consumption = (2 + 2<1» V, (2.5)
and
the rate of ATP consumption (3 + 3.5<1»Vc (2.6)
(Farquhar and von Caemmerer 1982).
2.3.4 Thylakoid reactions

2.3.4. 1 Production of NADPH andATP
The electron transport and the concomitant proton transfer in the chloroplast thylakoids
produce NADPH and ATP. Three discrete multi-protein complexes that span the thylakoid
membrane participate in this process: the photosystem II complex (PS II), the cytochrome bf
complex and the photosystem I complex (PS 1). Mobile pools of plastoquinone (PQ), and
plastocyanine (PC) link these complexes. The protons deposited in the thylakoid lumen by
the splitting of water at PS II and by the PQ pool generate electrochemical energy for the syn
thesis of ATP by the ATP synthase, the coupling factor. The thylakoids of most higher plants
are structurally differentiated into appressed and non-appressed regions of membrane sacs.
The ATP synthase and PS I are to be found in the non-appressed regions, which are in con
tact with the chloroplast stroma. Most of the PS II complexes are located in the appressed
region of the thylakoids and the cytochrome bf complexes are uniformly distributed through
both regions (Anderson and Andersson 1988).
The reduction of NADP+ to NADPH + H+ requires the transfer of two electrons through the
whole chain electron transport which in turn requires four photons, two at each photosystem, and
NADPH+H+
~
3 or 4 H+
2t04W NAD
ATP
Stroma P
Thylakoid
P700
2e
Lumen
~J
2 to 4 H+
H,O
~2W
1/20, 2 photons
Cy hf
PSII-LHC Q-cycle PSI-LHC ATP-synthase
complex
Figure 2.3 Stoichiometry of thylakoid electron transport.
during the process 1/2° 2 is evolved. The rate of whole chain electron transport, J, required for the
PCR and pco cycles can be calculated from the rate ofNADPH consumption (Eq. 2.5):
J = (4 + 4<jJ) V c (2.7)
The enzyme that catalyses the production of NADPH is NADP+ ferredoxin reductase, which
occurs at the reducing side of PS I (Fig. 2.3).
Although it is well established that the light-driven electron transport is coupled to the trans
fer of protons across the thylakoid membrane into the lumen, neither the stoichiometry of the
H+/e- nor the number of protons required for ATP synthesis are known with certainty and may
well be somewhat flexible. The ratios between H+ transfer and ATP production are reported as 3
or 4 (Junge 1977; Davenport and McCarty 1986; Kobayashi et al. 1995a,b; Junge 1997; Haraux and
de Kouchkovsky 1998). Table 2.1 lists the various stoichiometries shown in Fig. 2.3. In previous
models (Farquhar and von Caemmerer 1982) three protons were assumed in which case the rate
of proton production required to satisfy the rate of ATP consumption of the PCR and the pco
cycles is:
rate of proton production = (9 + 10.5<jJ) V( (2.8)
In the case of four protons the requirement increases to:
rate of proton production = (12 + 14<jJ) V c (2.9)
Farquhar and von Caernmerer (1982) assumed that the movement of two electrons through
the whole electron transport chain results in the accumulation of four protons in the thyla
koid space, two from the splitting of a H 20 molecule and two from the oxidation of the PQ
pool formed as a consequence of the transfer of the electrons from water. It is not quite clear
how the cytochrome bf complex mediates this oxidation. If the electron passes straight to PS
I the H+/e- is 2; if only one proton is transferred to PS I and the other is shuttled back to the
PQ pool and is re-used, the H+/e- is 3. This is the Q cycle proposed by Mitchell (1977)
(Table 2.1; Fig. 2.3). The existence of a Q cycle (Mitchell 1977; Rich 1988; Ort 1991) in the
Table 2.1 Stoichiometries of the chloroplastic electron transport chain

H+/
Electron transport Quanta e- H+ NADPH H+/ e- quanta ATP e-/ATP Quanta/ATP
Wholechain 4 2 4 2 1.33 3 1.5 3

(1) (2) (4)
Wholechain + Q cycle 4 2 6 3 1.5 2 2

( 1.5) (1.33) (2.66)
Cyclic through PS I 4 4 4 1.33 3 3
(1) (4) (4)
Cyclic through PS I + Q cycle 4 4 8 2 2 2.66 1.5 1.5

(2) (2) (2)
3 Assuming 3H+/ATP. Numbers in brackets assume 4H+/ATP.
cytochrome bf complex is often suggested and different H+/e- ratios have been measured
(Hope et al. 1985; Kobayashi et al. 1995a,b).
If the proton production is by whole chain transport alone and H+/e- is 2 and we assume a
ratio of 3H+/ATP then the electron transport rate required to meet the necessary ATP production
for the PCR and PCO cycles is:
J = (4.5 + 5.25<1»Vc (2.10)
If the Q cycle operates (H+ / e- = 3), the whole chain electron transport rate would need to be
only:
J = (3 + 3.5<1»Vc (2.11 )
In both cases there is a discrepancy between the whole chain electron transport rates
required for NADPH and ATP production. In the case of H+/e- = 2 there is a shortfall in ATP
production of (0.5 + 1.25<1» V c' which could be accomplished by electron transport via the
Mehler reactions where O 2 serves as electron acceptor (see also Fig. 3.9, page 84). Nitrate
reduction also requires whole chain electron transport. Alternatively cyclic electron flow
through PS I can also make up for the shortfall in whole chain electron flow. The cyclic
flow has a H+/e- = 1 and requires only one photon (Farquhar and von Caemmerer 1982).
If the Q cycle operates (Ht/e" = 3) and we assume a ratio of 4H+/ATP, the requirement for
whole chain electron transport is:
J = (4 + 4.66<1>)Vc (2.12)
and the discrepancy is reduced to a shortfall of (0.66<1» V c' which is much smaller. These
uncertainties cannot be resolved at present and it must be kept in mind that the ratios may
well be flexible. In the following we shall assume that the rate of NADPH production is more
limiting and use Eq. (2.7).
2.3.4.2 Light dependence of electron transport rate

A relationship between the electron transport, J, and the absorbed irradiance is also required.
At present all the equations used are empirical and the most frequently used expression is:
(2.13)
where 12 is the useful light absorbed by PS II, Jmax is the maximum electron transport and 8 is
an empirical curvature factor and 0.7 is a good average value (Evans 1989).12 is related to
incident irradiance I by:
12 = 1xabs(l-f)/2 (2.14)
The absorptance (abs) of leaves is commonly about 0.85 and f corrects for spectral quality of
the light (~0.15; Evans 1987). Ogren and Evans (1993) give a detailed discussion on the
parameters of Eq. (2.13). The 2 is in the denominator, because each photosystem absorbs half
the available light (Eq. 2.14). The equation can be solved for J as follows:
Jm ax - JU2 + JmaJ -
2
12 + 481 2Jm ax (2.15 )
~
J= 28
2.3.5 Respiration
We denote as day respiration, R d , all CO 2 evolution from cells within the leaf, which is addi
tional to the CO 2 evolution due to the photorespiratory cycle. The extent to which this respi
ration occurs in the light is still being debated (Graham and Chapman 1979; Brooks and
Farquhar 1985; Atkin et al. 1998). The best estimates of day respiration have come from gas
exchange measurements at low CO 2 partial pressure (Laisk 1977, 1998; Peisker 1981; Brooks
and Farquhar 1985; Atkin et al. 1998; Hoefnagel et al. 1998). These measurements have
shown that mitochondrial respiration during the day can be less than respiration measured
in the dark. Experiments by Azcon- Bieto et al. (1983) have shown that dark respiration itself
is dependent on the assimilation rate of the previous light periods and may not be constant.
2.4 RATE EQUATIONS FOR CO 2 ASSIMILATION

The basis of the model is given by Eqs (2.1) and (2.2). To complete the model the dependen
cies of rubisco on CO 2 , 02' irradiance and temperature need to be added. To do this some of
the equations developed in Chapter 1 are used. Farquhar (1979) showed that, because of the
high concentration of rubisco in the chloroplast, rubisco carboxylation rate could be
described by either its RuBP-saturated rate or by the RuBP-limited rate. RuBP supply in turn
is linked to the regeneration of RuBP, which in turn is dependent on the supply of ATP and
NADPH from the chloroplastic electron transport chain. These kinetics of the concentrated
rubisco in the chloroplast suggested the simple binary formulation of CO 2 assimilation rate
as either RuBP saturated, which would occur when irradiance is high or CO 2 partial pressure
is low, or as limited by the rate of RuBP regeneration, which would occur when irradiance is
low and/or CO 2 partial pressure is high.
2.4.1 RuBP-saturated CO 2 assimilation rate

To write an equation for the RuBP-saturated rate of CO 2 assimilation the equations devel
oped in Chapter 1 are used. There it was shown that the ratio of oxygenation to carboxyla
tion rate, <jJ, is determined solely by the kinetic constants of rubisco (Chapter 1, Eqs 1.12 and
1.13), and that:
<jJ =
VA (V
V = T
omax Kc)O
V em ax C=
(1)0C
Se/o
(2.16)
c
where V emax ' Vornax ' K; and K o are the maximal rates and the Michaelis-Menten constants of
carboxylation and oxygenation respectively. Sclo is the relative specificity of rubisco and
C and 0 are the chloroplastic CO 2 and O 2 partial pressures. Inspection of Eq. (2.1) shows
that when R d = 0, A = 0 when <l> = 2. The chloroplast CO 2 partial pressure at which this occurs
has been named L (Laisk 1977; Laisk and Oja 1998) and from Eq. (2.16) it follows that:
(2.17)
and
2L
<l> = C
(2.18)
Substituting this into Eq. (2.2) one can show that:
A = (l - L/ C) V, - R d (2.19)
Further substitution of the equation for the RuBP-saturated carboxylation rate
( V, = CVcmax" . ld s:
Eq. 1.9 )) Yle
C+Ke(l+O/Ke)
(2.20)
This is the RuBP-saturated rate of CO 2 assimilation. Because of its dependence on the

maximum rubisco activity, V emax ' A c is also often called the rubisco-limited rate of CO 2
assimilation.
The rubisco-limited rate of CO 2 assimilation suggests that the dependence of CO 2 assimila
tion on chloroplast CO 2 partial pressure should have a Michaelis-Menten form. However, as
noted by several researchers (e.g. Laisk and Oja 1974; Lilley and Walker 1975; Ku and Edwards
1977a), CO 2 assimilation rate saturates more quickly than can be predicted from rubisco kinetics
alone. An example of this is shown for a tobacco leaf in Fig. 2.4. The K, of rubisco at 21 % O 2 is 570
ubar so that the CO 2 assimilation rate predicted by the rubisco-limited rate is only at half its max
imal rate at that intercellular CO 2 partial pressure. The actual CO 2 assimilation rate is, however,
almost saturated, indicative of other limitations. In the transgenic tobacco with an antisense con
struct to the small subunit of rubisco, the amount of rubisco per leaf area has been reduced with
little alteration to other chloroplast components. In this case the rate of CO 2 assimilation can be
modelled solely by the rubisco-limited rate (Fig. 2.4).
2.4.2 RuBP-limited CO 2 assimilation rate

Laisk and Oja (1974) first elegantly demonstrated that the CO 2 assimilation rate at high
intercellular CO 2 partial pressure is limited by the supply of RuBP. They exposed leaves to
low CO 2 partial pressures to allow the accumulation of high RuBP pools and then briefly
exposed the leaf to a pulse of high CO 2 , The CO 2 assimilation rate briefly exceeded the
steady-state rate by several-fold, thus contrasting the RuBP-saturated and RuBP-limited rates
at high intercellular CO 2 partial pressure where RuBP pools were expected to be low in com
parison to pools at low intercellular CO 2 partial pressure. Development of a gas-exchange
chamber connected to a freeze clamp apparatus allowed measurements of metabolite pools
to be made under a variety of environmental conditions, together with measurements of CO 2
assimilation rates (Badger et al. 1984; von Caemmerer and Edmondson 1986). These
combined measurements of RuBP pools and CO 2 assimilation rates confirm that CO 2
Modelling C3 photosynthesis 37
-.
en
~ •
~e
C)'
E 30 e
0
E
-"C..:::i.
-Q.)
.~
c
....
20
0
+=0
~
10
en
en
~
C\l
0 0
o 400 600 800
0 200
Intercellular CO 2 partial pressure
(ubar)
Figure 2.4 CO 2 assimilation rate, A. as a function of intercellular CO 2 partial pressure for a leafof a wild-type
tobacco (e) and a transgenic tobacco with reduced amount of rubisco ( _). Measurements were made at an
irradiance of 1 mmol quanta m-2 S-l and a leaf temperature of 25°C. The solid line is the CO 2 assimilation rate
predicted from the RuBP-saturated rate of rubisco. The data points for the wild typedeviate from the predicted
rate at high CO 2 partial pressure, indicative of other limitations to CO 2 assimilation ratethere. The lines are
calculated with Eq. (2.20) with the kineticconstants fromTable 2.3.The chloroplast CO 2 partial pressure, Ce, was
calculated from C(= Ci- Algi' where gi= 0.3 mol m-2 S-1 bar' wasthe measured CO 2 transfer resistance between
intercellular airspace and chloroplasts. (Redrawn from von Caemmerer et at. 1994.)
assimilation becomes limited by the rate of RuBP regeneration as RuBP pools decline at high
CO 2 partial pressures (Fig. 2.5).
Different approaches have been used to mathematically incorporate the limitation by RuBP
regeneration. The initial model by Farquhar et al. (1980) calculated chloroplastic RuBP pools and
from that derived the RuBP-limited rate. The calculation of the RuBP pool depended on a com
plex link between electron transport rate and rate of PGA reduction. This form was quickly
abandoned for the following simple format which based the rate of RuBP regeneration directly on
the electron transport rate (von Caemmerer and Farquhar 1981; Farquhar and von Caemmerer
1982 and for a detailed discussion on the merits of this formulation see Collatz et al. 1990).
If J is the electron transport at a given irradiance (Eq. 2.15) then the rate of carboxylation that
can be supported by the electron transport can be calculated from Eqs (2.7) or (2.12) as:
v = _J_ _ J (2.21)
( 4+4<jl - 4+8L/C
or
V = J _ J (2.22)
( 4+4.66<jl - 4+9.3L/C
Figure 2.5 CO 2 assimilation rate (e) and RuBP pools (= ) of radish leaves as a function of intercellular CO 2
partial pressure. Measurements were made at an irradiance of 1400 umol quanta m-2 S-1 and a leaftemperature
of 25°C (von Caemmerer and Edmondson 1986).
depending on whether the NADPH or AIP requirement is considered. Because of the uncer
tainty of the H+/e- ratio and the H+/AIP ratio the NADPH-limited version is used here. Sub
stituting Eq. (2.21) into Eq. (2.19) yields an expression for the RuBP regeneration-limited, or
electron-transport-limited rate of CO 2 assimilation:
(C-L)J
A
1
= 4C+ 8L
-R
d
(2.23 )
Here the assumption is made that the enzymes involved in the regeneration of RuBP, as for
example FBPase or SBPase, are not rate-limiting the regeneration of RuBP. Justification for
this was given in Farquhar and von Caemmerer (1982) (also see discussion in Section 2.3.2).
2.4.3 Export-limited CO 2 assimilation rate

At high CO 2 partial pressure, in particular in combination with high irradiance, or low O 2
partial pressure or at low temperatures, the rate of CO 2 assimilation can sometimes be lim
ited by the rate at which triose phosphates are utilized in the synthesis of starch and sucrose
(Sharkey 1985; Sharkey et al. 1986a,b; Leegood and Furbank 1986; Labate and Leegood 1988;
Sharkey and Vanderveer 1989; Harley and Sharkey 1991; Harley et al. 1992a,b). Because
phosphate is exchanged stoichiometrically with triose phosphate at the chloroplast envelope,

the release of phosphate in the chloroplast presumably becomes limiting to photophosphory
lation and CO 2 assimilation rate is given by:
Ap := 3Tp - R" (2.24)
where Tp is the rate of triose phosphate export from the chloroplast (Farquhar and von
Caemmerer 1982; Sharkey 1985). Under these conditions A is insensitive to changes in CO 2
and 02 partial pressures.
Frequently, however, a decline in CO 2 assimilation rate is seen at high CO 2 partial pressures,
particularly at low 02 partial pressure. This effect cannot be explained by the simple phosphate
limitation proposed above. To explain these observations Harley and Sharkey (1991) invoked a
net release of phosphate in the chloroplast through the photorespiratory cycle. They hypothesized
that a fraction of the glycolate carbon which leaves the chloroplast and is recycled by the photo
respiratory cycle to glycerate does not return to the chloroplast but is converted to glycine and
used in amino acid synthesis. In this case phosphate normally used in the conversion from glycer
ate to PGA is made available for photophosphorylation, thus stimulating RuBP regeneration. As
half a glycerate is produced per oxygenation, which consumes one phosphate, the usual rate of
phosphate consumption of VJ3 - Vj6 is then reduced by aVo12, where a < a > 1 is the fraction of
glycolate carbon not returned to the chloroplast. Under phosphate limitation this consumption
must equal the release of phosphate by triose phosphate export, Tp ' and:
v, ov, «ev, T
- - - - - - := (2.25)
362 p
By combining this equation with Eq. (2.2), A is then given by:
A:= (C-L)(3Tp ) -R
(2.26)
p (C-(1+3a/2)L) "
The phosphate limitation is particularly relevant to studies where plants are grown under
elevated CO 2 partial pressure (Harley et al. 1992b; Sage 1994).
2.4.4 Summary of rate equations

The above equations (Eqs 2.20, 2.23 and 2.23 or 2.24) describe the basic C 3 model with:
A := min{A o Ai' A p } (2.27)
which says that A proceeds at a minimum of the three rates A" A j , A p ' This is illustrated in
Fig. 2.6 where the solid line shows the actual rate of CO 2 assimilation, the dashed line is
rubisco-limited rate A[, the dotted line is the electron-transport-limited rate Ai' and the
phosphate-limited rate A p (given by Eq. 2.24) is shown at high CO 2 partial pressures. It is
important to note that, when A is limited by the electron transport rate or the capacity for
RuBP regeneration, A still continues to increase with increasing CO 2 partial pressure as
energy is diverted from oxygenation to carboxylation. When A is limited by triose phosphate
limitation, however, the rate becomes insensitive to CO 2 and 02 unless glycerate is not being
returned to the chloroplast, in which case it may have an inverse dependence (not shown, but
see Harley and Sharkey 1991).
2.5 CO 2 PARTIAL PRESSURE IN THE CHLOROPLAST

To examine the biochemistry of photosynthesis in leaves, ideally one would like to measure
CO 2 assimilation rate in relation to chloroplast CO 2 partial pressures, as this is the CO 2
'w /
~E 40 "" . d
"" atiOn \iTl1\te
"0 sp regener .
o ,," r-
U
~
E /
'-'
:::t /.
Phosphate limited
20
o 200 400
600 800
Chloroplastic CO 2 partial pressure, Cc
(ubar)
Figure 2.6 Modelled rate of CO 2 assimilation asa function of chloroplast CO 2 partial pressure. The rubisco
limited (RuBP-saturated) rate of CO 2 assimilation has a dashed line extension at highCO 2, The electron-transport
(RuBP-regeneration)-limited rate has a dotted line extension at low CO 2, The solid curve represents the minimum
rate that is the rate of CO 2 assimilation. Also shown isa phosphate limitation at high CO 2, Model parameters
were: Vcmax == 100 urnol m-2 s', Kc == 259 ubar, Ko == 179mbar, F. ==38.6 ubar , J == 170umol m-2 S-1, T == 11.8
urnol m-2 S-l and Rd == 1 urnol m-2 S-1 (Table 2.3.)
pressure determining the rubisco carboxylation. It has become common practice to calculate
the CO 2 partial pressure in the substomatal cavities (frequently referred to as intercellular
CO 2 partial pressure) from water vapour exchange measurements and this has become a
standard reference CO 2 (von Caemmerer and Farquhar 1981). It eliminates an important
variability, because stomatal conductance varies as stomata themselves respond to CO 2 and
irradiance. The response of stomata to environmental variables is complex and the precise
mechanisms are not well understood. A discussion is outside the scope of this chapter but it
is useful to realize that in C 3 species the ratio of intercellular to ambient CO 2 partial pressure
is usually around 0.75 (Wong et al. 1979, 1985). This ratio is relatively constant with varia
tion in CO 2 partial pressure and irradiance but varies with relative humidity and oxygen par
tial pressure for example. At ambient CO 2 pressure and high irradiance the CO 2 gradient
from the air surrounding the leaf to the substomatal cavities is approximately 85 ubar (for
review see Evans and von Caemmerer 1996).
It is clear that during CO 2 uptake a CO 2 gradient must also exist from the substomatal cavities
to the intercellular airspaces and across the cell wall plasma membrane and chloroplast mem
branes. Unlike stomatal conductance, which fluctuates as stomata open and close, the CO 2
transfer conductance from the substomatal cavities to the sites of carboxylation in the chloroplast,
gi' (or inversely the CO 2 transfer resistance, r.) appears to be constant for a leaf since it is to a large
degree related to the anatomy of the leaf (Evans and von Caemmerer 1996). The CO 2 transfer
resistance, r i , can be written as the sum of two components, the intercellular airspace resistance,
rias' and the liquid-phase resistance through the cell wall membranes and chloroplast, rl iq'
Although some theoretical considerations have been made (Laisk 1970; Peisker and Apel 1975;
Yocum and Lommen 1975; Raven and Glidewell 1981), ri has been difficult to quantify.
Hall (1971) incorporated a CO 2 transfer resistance from the intercellular spaces to the sites of
carboxylation in early photosynthesis models. Hall assumed that the major resistance, r i , would be
across the cell wall. He calculated the resistance by taking the mean thickness of the cell wall (L1d)
and a tortuosity factor F of 2 (which describes the mean distance travelled to the shortest distance
travelled, since the liquid path across the cell wall is probably not in straight lines). The quotient
(Sm) of the mesophyll cell area exposed to intercellular airspace to projected leaf area was used to
scale the resistance to the leaf area and:
(L1d)F
r= - - (2.28)
I DS I11
where D is the diffusivity of CO 2 in liquid water (1.98 x 10- 9 m ' S-I). Hall estimated L1d for
Atriplex patula at between 2 and 3 x 10- 9 m and calculated ri at between 10 and 20 s m'. This
converts to a resistance on a leaf area basis of 0.25 to 0.5 m? s mol-lor a conductance, gi'
between 4 and 2 mol m- 2 S-I. At a CO 2 assimilation rate of 20 urnol m- 2 S-1 this gives a drop
of CO 2 partial pressure of between 5 and 10 ubar. This gradient was considered to be small
and subsequent models frequently did not include a resistance term (for example see Farqu
har and von Caemmerer 1982 and Parkhurst et al. 1988 for discussion).
There was renewed interest in quantifying this term when Evans et al. (1986) demonstrated
that short-term measurements of carbon isotope discrimination could be used to estimate the
CO" transfer conductance. This technique relies on the fact that carbon isotope discrimination
occurring during dissolution and diffusion of CO" through liquid differs from the carbon isotope
discrimination during rubisco carboxylation. When the CO 2 flux and therefore the CO 2 gradient
from substomatal cavities is varied by varying irradiance, carbon isotope discrimination also var
ies, which allows the calculation of gi' This technique was used by von Caemmerer and Evans
(1991) and by Lloyd et al. (1992). Both groups found that gi correlated with photosynthetic capac
ity. Figure 2.7 shows data obtained with tobacco. Here the values of gi ranged from 0.2 to
0.55 mol rn" S-I bar:", giving a CO" gradient of approximately 50 ubar, Other studies using a vari
ety of techniques have confirmed these values (Lloyd et al. 1992; Loreto et al. 1992; Epron et al.
1995; for review see Evans and von Caemmerer 1996).
The carbon isotope technique does not allow the conductance to be resolved into its air
phase and liquid-phase components. Von Caemmerer and Evans (1991) suggested that in many
herbaceous species the resistance across the membrane interfaces at the plasmalemma and chlo
roplast membranes may be the most important. They hypothesized that gi should be more
closely related to the quotient, Sf' of chloroplast surface area appressed to intercellular airspaces
to projected leaf area rather than the quotient of mesophyll surface area to projected area, Sm' ini
tially used by Hall (1971). However this relationship is unlikely to be unique and different
relationships may be observed in species where airspace resistance makes a greater contribution
to the overall resistance. These questions are presently being actively researched and a better
understanding will evolve.
The explicit inclusion of a CO" transfer conductance, gi' from the substomatal cavities to the
carboxylation sites leads to a quadratic relationship between CO" assimilation rate, A and the
intercellular CO 2 partial pressure, Ci. The relationship between A and gi is given by:
A = g;(C,- CJ (2.29)
and, solving for C, and combining with Eq. (2.20) or Eq. (2.23) (where C now stands for Cc) '
one obtains the following two quadratic equations:
I 0.6 I I I
.....
ro
.D
I
N
VJ 0.5 •
•
I
E • •
0
C'l
0.4
• •• ••
U
'0
l-
•
• •
E
'-'
0.3 I-
~
• •• •
c5
u
I::
ro
• •• ••
...... 0.2 l-
u
::l
"'0
I::
0
u 0.1 I -
-aI::
.....
<l)
...... I I I
I::
...... 0.0
0 10 20 30 40 50
1s-1
CO 2 assimilation rate, A (umol m- )
Figure 2.7 The relationship between the CO 2 transfer conductance, gj, and photosynthetic capacity for a
number of C3-species. Photosynthetic capacity was measured as CO 2 assimilation rate at 1000 prnol quanta
m-2 S-1 at 25°C, 350 ubar and ambientCO 2 partial pressure and 21 % 02' The units of conductance depend on the
units used for CO 2 , Dissolution of CO 2 depends on the partial pressure of CO 2 and conductance therefore has the
units mol m-2 S-1 bar'. For airspace conductance, the units could be mol m-2 s' if CO 2 is given asa molefraction
(see Harley et al. 1992a). Data taken from von Caemmerer and Evans (1991).
A~ -A,{g.(C j + KcC1 + O/KJ) + V cmax + R a} (2.30)

+ gj{ Vcmax(Cj - I",') - Ra(C j + KcC1 + O/K G
) ) } o
and
Af -Aj{g(C j + 2L) + J/4 + Rei} (2.31 )

+ gj{(Cj - L)J/ 4 - Rei(Cj + 2f.)} =0
The presence of a significant internal diffusion resistance to CO 2 affects both the quantitative
relationship between CO 2 assimilation rate and maximal rubisco activity (Section 2.8.5) and
the shape of the CO 2 response curve (Section 2.12.1).
2.6 PARAMETERIZATION OF THE MODEL
2.6.1 Values at 25°C

In this model many of the parameters can be assigned a priori, leaving only a few key vari
ables. For example, it is well established that the kinetic constants of rubisco are very similar
amongst C 3 species such that the same K C) K; and L can be used. Farquhar et al. (1980) used
constants derived from in vitro measurements by Badger and Andrews (1974) and Badger
and Collatz (1977). In more recent in vitro measurements, values of K( have been lower and
data sets by Jordan and Ogren (1981, 1984) and Makino et al. (1988) have also been used.
Brooks and Farquhar (1985) have made detailed measurements of T; for spinach. Various
30
-
~en 25 ~
~
-6
20 l J /9;=0.05
2
:01 m- 5- bar"
1
-Q)
ctl 15
....
c
0
:;:::::
~ 10
'E
en
en
ctl 5
0 '"
500 1000 1500

Figure 2.8 CO 2 assimilation rate, A, as a function of intercellular CO 2 partial pressure, C;, for a leafof tobacco
(Nicotiana tabacum) (e) and Kalanchoe daigremontiana (_) (during C3 photosynthesis in phase VI of the CAM
cycle) with the same number of rubisco sites (18prnol m-2) but differentconductances to internal CO 2 diffusion,
g;. The arrows point to assimilation rates at ambient CO 2 partial pressures and the slopes of the arrows givethe
stomatal conductances. Measurements were made at high light anda leaftemperature of 25°C. The lines predict
the rubisco-limited CO 2 assimilation rate with the kinetic constants from Table 2.2 and C, was calculated from C;
= C;- Algi' Data are redrawn from Mate et al. (1996) and Maxwell et al. (1997).
choices have been made in papers such as Kirschbaum and Farquhar (1984), Collatz et ai.
(1991), Harley and Sharkey (1991), von Caemmerer and Evans (1991). Table 1.2 lists exam
ples of in vitro measurements, where both K, and K o have been measured. Von Caemmerer et
al. (1994) used transgenic tobacco with reduced amounts of rubisco to determine rubisco
kinetic constants in vivo (Table 1.3, Figs 1.4 and 1.5). They estimated constants in two ways:
firstly, by calculating the chloroplast partial pressure with measured values of gi' and
secondly, on the basis of C;, by assuming that g; was infinite (Table 1.3). These values were in
good agreement with the more recent in vitro measurements (Whitney et at. 1999).
The only rubisco parameter that has to be newly assigned is the maximal rubisco activity,
Vern ax' V em ax is dependent on the amount of rubisco protein present in the leaf. Rubisco has a
molecular weight of 550 kDa and eight catalytic sites per molecule. Thus, with a catalytic turnover
rate of 3.5 S-1 per site (Table 1.2), 1 g m- 2 of rubisco has a V emax of 51 umol m ? s-1 when rubisco
sites are fully carbamylated.
To parameterize the RuBP regeneration-limited rate of CO 2 assimilation, L and the light
dependence of electron transport need to be considered (Eq. 2.13). Again, values of absorptance
and the curvature factor 8 can be assigned, as was done in Section 2.3.4.2. The only parameter that
needs to be newly assigned is the maximum electron transport rate Jm,u' J max depends on the
amount of thylakoid components such as the cytochrome bf complex for example and is depen
dent on the amount of thylakoid protein present. Jmax has the units of umol electrons m- 2 S-1 and
the ratio of Jmax to V emax is usually between 1.5 and 2 (Wullschleger 1993; Watanabe et al. 1994; de
Pury and Farquhar 1997; Walcroft et at. 1997). Variations of Jmax and V em ax with plant growth con
ditions are discussed in Section 2.12.
Rd , the mitochondrial respiration other than that resulting from photorespiration, is also
scaled with photosynthetic capacity and Rd = 0.01 to 0.02Vcmax (Section 2.7).
The good correlation between CO 2 assimilation rate and internal CO 2 transfer conductance
(Fig. 2.7) suggests that the CO 2 transfer conductance can perhaps also be scaled to V cmax by gj =
O.0045Vcmax (Evans and von Caemmerer 1996). However, some exceptions can be found. For
example the obligate Crassu1acean acid metabolism (CAM) plant Kalanchoe daigremontiana,
which can operate in the C 3 mode during phase IV of the CAM cycle, has a very low internal con
ductance to CO 2 diffusion. Although leaves have about the same amount of rubisco as tobacco
leaves, CO 2 assimilation rate is only half that of a tobacco leaf at ambient CO 2 partial pressure
(Fig. 2.8; Maxwell et al. 1997). Furthermore, Makino et al. (1994a) noted that when rice was
grown at different temperatures the relationship between CO 2 assimilation rate and extractable.
rubisco activity changed, and also attributed the difference to an increase in internal resistance to
CO 2 diffusion at low temperatures.
2.6.2 Temperature dependencies

2.6.2.7 Rubisco and Rd
The temperature dependencies of rubisco's carboxylation and oxygenation rates are reflected
in the temperature dependency of the CO 2 assimilation rates of leaves (Bjorkman and Pearcy
1971; Bjorkman et al. 1980). The need for accurate estimates of the temperature dependen
cies of rubisco kinetic parameters has become apparent as mathematical modellers try to
predict the impact of increasing global CO 2 concentrations and temperatures (Bowes 1991;
Long 1991; McMurtrie and Wang 1993; Walcroft et al. 1997).
The temperature dependence of the kinetic constants can be described by an Arrhenius func
tion of the form:
Parameter(T) = Parameter(25°C)exp[(25 - T)E/(298R(273 + T))] (2.32)
where R (8.314 J K-l mol") is the universal gas constant and T is temperature in °C (Badger
and Collatz 1977). The activation energy (E) represents the kinetic energy of substrate
required for the reaction to proceed. Many reactions in photosynthesis are reversible and
activation energies of forward and reverse reactions can differ. Because of this, the activation
energies of net reactions can vary with the ratio of forward and reverse reactions and E may
not necessarily be constant over a wide temperature range. For example, E is known to
increase at lower temperatures in some cases (Badger and Collatz 1977; Table 2.2). Using the
Arrhenius function to describe temperature dependencies of the photosynthetic processes is
thus a semi-empirical approach, but allows for easy comparison between studies. This is not
as easy with polynomial fits (Kirschbaum and Farquhar 1984; Brooks and Farquhar 1985).
The Q lO function is also frequently used to describe temperature responses (Woodrow and
Berry 1988; Collatz et al. 1991). The Q lO value is the relative increase in reaction rate over a 10°C
temperature range and it is specified at a particular temperature.
Parameter(T) = Parameter(25 °C)QIO[(T - 25)/10] (2.33)
Table 2.3 and Fig. 2.9 give the photosynthetic parameters and their activation energies and
QlO used in this chapter. Table 2.2 reviews the activation energies (E) of rubisco kinetic con
stants determined in in vitro experiments. There are only two complete data sets measured
on C 3 plants by Badger and Collatz (1977) on rubisco from Atriplex and by Jordan and Ogren
(1984) from spinach rubisco. The specificity factor of rubisco decreases with increasing tem
perature. This is because the reaction mechanism of rubisco involves a 2,3-enediol intermedi
ate and its reaction with O 2 has a higher free energy of activation than the reaction with CO 2
(Andrews and Lorimer 1987). That is, the oxygenation rate increases more rapidly with tem
perature than the rate of carboxylation (Fig. 2.9).
Table 2.2 Activation energies E (J mol:") of rubisco kinetic constants
Species s, s, V cmax V amax Scla Reference
Glycine max (15-35°C) 53381 8279 Laing et al. (1974)
Spinacia oleracea (5-35°C) 54393 79497 Badger and Andrews (1974)
Atriplex glabriuscula Badger and Collatz (1977)
(above 15°C) 59414 35983 64853 59414 -18000

(below 15°C) 10962 35983 103765 103347 -73219
Spinacia oleracea (7-35°C) 81655 15632 74350 37566 -29239 Jordan and Ogren (1984)
Model plant 59356 35948 58520 58520 -23408 Farquhar et al. (1980)
Values of parameters can be calculated at any temperature from the following equation:
Parameter = Parameter(25°C)exp[ (T - 298)E/(298RT)],
where R (8.314 ) K- 1 mol:") is the universal gas constant and T is temperature in degrees Kelvin (K).
The Qlo(25°C) = exp(l3.6 x IO--6E).
Table 2.3 Photosynthetic parameters at 25°C and their activation energies E (kl mol:")
Parameter Value E (kJ rnol") QIO(25°C)d
t; (ubar) 260 or 404" 59.36 b 2.24

K a (mbar) 179 or 248" 35.94 1.63
T. (ubar ) 38.6 or 37" 23.4 1.37
V emax (umol rn? S-I) 80 c 58.52 2.21
Varnax (umol m-2 S-I) 0.25 X V em ax 58.52 2.21
Rd (urnol m- 2 S-I) 0.01-0.02 X ifemax 66.4 2.46
Jmax (umol m-2 s-I) 1.5-2 X 'f Ol Ia X
37 1.65
H (kJ mol") 220
5 (J K-I mol") 710
., (von Caemmerer et al. 1994). The first value is appropriate when an internal diffusion conductance is included; the
second value should be used if the internal conductance is not included and C, is assumed to equal C,.
b Activation energies used by Farquhar et al. (1980).
c Varies depending on the photosynthetic capacity of the leaf.
d QIO(25°C) = exp(l3.6 x IO-'E).
°
Throughout this book the values of chloroplastic CO 2 and 2> K; and K o> are given in units of
partial pressure. The chemical activity of a dissolved gas is proportional to its gas-phase (vapour)
pressure and thus the partial pressure of a gas existing in equilibrium with that in solution is a bet
ter measure of its chemical activity then dissolved concentrations (Badger and Collatz 1977). The
main difference between the use of gas-phase units of partial pressure and that of dissolved con
centrations (11M) arises when temperature dependencies are considered. The temperature
dependencies of reaction rates measured at constant gas-phase pressure relate more closely to the
temperature dependencies of the reaction mechanisms rather than also including the temperature
dependencies of the solubility of the gaseous substrates.
2.6.2.2 Temperature dependence ofJmax
The dependence on temperature of light saturated maximal electron transport f max has been
measured in in vitro systems with isolated thylakoids (Armond et al. 1978; Bjorkman et al.
1980; Sage et al. 1995). It appears that the temperature response of whole chain electron
transport is closely linked to the properties of the thylakoid membrane such as membrane
viscosity and lipid composition (Nolan and Smillie 1976; Pike and Berry 1980; Berry and
500
~
a
'ICf) 400
N
E
I 300
V cmax
"0 200
E V omax
-6 100
.....
co
.0
3000 b
-6
~u ~ , ,t
~ m2000 Kc(1 + O/Ko) ,,
~o .0 ,,
~ E ,,-,,'«;
~ ~1000 /
/
,.
~ 0
Ko
8
c
'I
Cf)
6
N
I
E 4
Rd /
"0 /
/
E 2 /
-:::l
0
0 10 20 30 40 50
Leaf temperature, T(°C)
Figure 2.9 Temperature response of rubisco kinetic constants and day respiration, Rd' Curves arecalculated
with Arrhenius equation (2.32) and the values and activation energies given in Table 2.3. Kc {1 + O/K o ) is
calculated at 200 mbar and gj = 00.
Bjorkman 1980; Raison et al. 1980). Acclimation of the thylakoid membrane can occur when
plants are grown at different temperatures, shifting the temperature optimum (Berry and
Bjorkman 1980; Bjorkman et al. 1980; Sage et al. 1995). The temperature response of whole
chain electron transport is therefore an important parameter in modelling gas exchange. Far
quhar et al. (1980) used the data of Nolan and Smillie (1976) to determine the parameters E,
5 and H in the following expression, shown in Fig. 2.10:
( T _ 298)E)[ 1 + exp(29~:8~ H)J

J max = Jmax (25°C)exp
.
( 298RT (ST _ H)
(2.34)
[1 + exp RT J
The equation is a simplified version of equations developed by Johnson et al. (1942) and
Sharpe and De Michelle (1977) to describe the effect of temperature on enzyme inactivation.
Tenhunen et al. (1976) and Hall (1979) originally used this equation. 5 and H are parameters
affecting the rate at high and low temperature respectively. (For discussion see Hall 1979 and
><
160
l1J
""":lE:
to ~
0..'en
en 120
ro '"I
C
.... E
~ ~
o
....
0~
- u U
+"
80
Ql ~
Q) (1)
c <5
:::J E
E ~
'x 40
ro
:2:
o
o 10 20 30 40 50
Leaf temperature tC)
Figure 2.10 Temperature response of maximal electron transport Jmax (Eq. 2.34). Jmax at 25°( was 120 prnol
electrons m-2 S-I. Other details aregiven in Table 2.3.
Farquhar and von Caemmerer 1982, but note that equation 36 in Farquhar et al. 1980 and
equation 16.33 in Farquhar and von Caemmerer 1982 contain typographical errors.) With vari
ations in 5, Hand E the curve can easily be fitted to measured responses of f m ax '
Kirschbaum and Farquhar (1984) calculated electron transport rates from measurements of
CO 2 assimilation at high CO 2 partial pressure and found reasonable agreements between mea
sured and modelled rates. Harley et al. (l992b) measured photosynthesis in cotton at different
temperatures and used a different function which agreed well with Eq. (2.34). Because the tem
perature optima may shift in plants grown under different environmental regimes it is useful to
have an equation for the temperature optima:
T opt = H[S+Rln(H/E-1)] (2.35)
June et al. (1997) derived a dependence for electron transport rate, l, from chlorophyll fluo
rescence measurements and opted for a simpler empirical formulation which makes it easy to
adjust the temperature optimum:
fmaxC T ) = f maxT exp[-(T- T opt )2) (2.36)

orr 2
20
where 0 is the value of (T - Top t ) at which f rna) f m ax ( Top t ) = 0.6065.
2.7 THE CO 2 COMPENSATION POINT

Woodrow and Berry (1988) pointed out that it takes a leap of faith to presume that the
physiological response of intact leaves can be interpreted from biochemical studies and
suggested that the CO 2 compensation point, r, may help us make that leap. The CO 2 com
pensation point is the CO 2 partial pressure at which net CO 2 assimilation is zero. This could
occur when stomata are completely closed. r is readily measured by illuminating a leaf in a
closed space and measuring the equilibrium CO 2 concentration and thus it has become a
"'C 4
<ll
CiS
....
o 20 40 60
Chloroplastic CO 2 partial pressure
c;(ubar)
Figure 2.11 CO 2 assimilation rate at low chloroplast CO 2 partial pressure. The relationship between ['., rand
Rd is shown. The solid line depicts the rubisco-limited and the dottedlinethe RuBP regeneration-limited
assimilation rate.
well-characterized experimental parameter. Laing et al. (1974) and Peisker (1974) were
amongst the first to measure the compensation point under different 02 partial pressures
and to relate it to the relative specificity of rubisco.
2.7.1 r in the absence of Rd

The connection of the compensation point to rubisco is most readily established if we
momentarily ignore mitochondrial respiration other than that related to photorespiration
(i.e. Rei = 0). Under these conditions CO 2 assimilation rate is zero when <!> = 2 (Eq. 2.2). Solv
ing for C when <!> = 2 in Eq. (2.16) shows that L, the CO 2 compensation point in the absence
of day respiration, is given by:
(2.37)
L is linearly dependent on 02 partial pressure as discussed by Laing et al. (1974), Peisker

(1974), Peisker and Apel (1975a) and Laisk (1977). L is that chloroplastic CO 2 partial pres
sure at which the rate of carboxylation equals the rate of photorespiratory CO 2 release.
2.7.2 r in the presence of Rd
In the presence of Rei the compensation point is given by:
r = L + K e(1 + 01 Ko)RdlVemox
(2.38)
1 - RalVcmax
(Farquhar et al. 1980; Tenhunen et al. 1980). r is also linearly dependent on 02 which can be
seen more readily when it is written in the following form:
r = (0. 5I S c/o + (KeRd)/(KoVema)) 0 + K e(Ral Ve1l1oJ

(2.39)
1 - Ral V emax 1 - Ral V emax
400 I I I
'2'
ell
.0
:::i
~ 300
-c
'0
a.
c 200
o
:;::::;
ell
CJ)
c
~ 100
E
o
o
0'" o
o I I ! I I I
o 100 200 300 400 500

02 (mbar)
Figure 2.12 The dependence of the CO 2 compensation point, F, on 02 partial pressure in wild-type (e) and
transgenic tobacco with reduced amount of rubisco (anti-rubisco, .). The dependence of r on 02 partial pressure
was modelled for wild typewith Vcmax = 70 and Rd = 3 umolm-2 s' andfor anti-rubisco plant with Vcmax = 7 and
Rd = 1 umol m-2 S-l and the values for kineticconstants given in Table 2.3. (Adapted from Ruuska et al. 2000b.)
Both the slope of this relationship and the intercept depend on the ratio RdlVcmax' Figure 2.11
illustrates the relationship between 1., I" and Rd' Peisker et al. (1981) showed that ontoge
netic changes in I" correlated with changes in the ratio of RdlVcmax' Changes in this ratio were
also used by Azcon- Bieto et al. (1981) to explain seasonal variation in I" in Lolium perenne. A
change in this ratio can also explain ontogenetic differences in I" observed by Catsky and
Ticha (1979). Figure 2.12 illustrates the impact the ratio of RdlVcmax has on I" in wild-type
and transgenic tobacco where rubisco has been reduced with an antisense construct to the
nuclear encoded small subunit of rubisco (Hudson et al. 1992). The anti-rubisco plants had
only 10% of wild-type rubisco, while respiration was not reduced to the same degree. This
led to a significant increase in I" at all 02 partial pressures. The dependence of I" on the ratio
Vcma)R d is excellent in vivo evidence demonstrating that mitochondrial respiration contin
ues in the light.
2.7.3 Light and temperature dependence

Usually I" is measured under high irradiance where the RuBP-saturated rate of rubisco limits
the CO 2 assimilation rate. However, Eq. (2.38) cannot explain the light dependence of I" seen
at very low irradiances (Catsky and Ticha 1979; Brooks and Farquhar 1985) and illustrated in
Fig. 2.13. Two hypotheses to explain this light dependence have been advanced. Laisk (1977)
suggested that it was perhaps due to an increase in Rd at low irradiance. Farquhar et al.
(1980) suggested that at low irradiance RuBP regeneration rate could become rate limiting
and under these conditions (using Eq. 2.23) I" is given by:
r = _1._(_8R_d_1!_+_1) (2.40)
1 - 4R dl!
The light dependence is more pronounced at high temperatures as the ratio Rdl! increases.
160 ,----,-------.--------,-----,-------,
..-..
~
Cll o
.0
.6 o
i--. 120
c
'0
c..
c 80
o
cu
(J)
c
CD
c.. 40
E
o
o
0'"
() 0
o 10 20 30 40 50
Leaf temperature, T (C)
Figure 2.13 The dependence of the compensation point, C on temperature and irradiance at ( - ) 90, (+) 160,
(_) 330 and (e) 1000 urnol quanta m-2 S-l in Phaseolus vulgaris (data redrawn from von Caemmerer 1981). The
curve is calculated from Eq. (2.38) with Vcmax = 100 urnol m-2 S-l and Rd = 1.2 urnol m-2 S-l at 25°C. Other
parameters are taken from Table 2.3.
- 3 r-----,-..,-----r----,--,-------,-...,-----r-r--,
'(/)
'"'E
"0
E 2
E,
Intercellular CO 2 partial pressure, C, (ubar)

Figure 2.14 CO 2 assimilation rate, A. of an anti-rubisco tobacco leaf asa function of intercellular CO 2 partial
pressure, Ci. Measurements were made at irradiances of C) 66, (e) 116, ( - ) 160 and (T) 550 urnol quanta
m-2 S-l, and at a leaf temperature of 25°C. Lines werefitted by leastsquares regression and the mean of the
coordinates of their intersections weretaken as estimates of C. and -Rd' Data are redrawn from von Caemmerer
et al. (1994).
2.7.4 Measurements of I';
Laisk (1977) pointed out that T, can be estimated from measurements of CO 2 response
curves at low irradiance. Equation (2.23) shows that, even at different values of I, C = T;
when A = -Rd' An example of a measurement of F; is shown in Fig. 2.14. Note how I"
increases with decreasing irradiance as predicted by Eq. (2.40). Brooks and Farquhar (1985)
'sn 4 i i i i
~
E ,,
<5 A(C c) , /
1 2
, , ,,
,
<::(
, ,,
,
",or
co....a) 01
c
g -n.
~
'E -2 ,,
'w
(/)
,,
co -4 ,,,' C. f. r
ON ,,
o
o 20 40 60
Chloroplastic (C c) or intercellular (C i)
CO 2 partial pressure (ubar)
Figure 2.15 CO 2 assimilation rateversus chloroplastic, A(CJ or intercellular, A(C). CO 2 partial pressure. The
relationship between c., r., rand -R d is shown.
used this technique to measure T, at different temperatures for spinach leaves and they found
good agreement with the temperature dependence of Sclo measured by Jordan and Ogren
(1984). Equation (2.17) suggests that T, may be a good in vivo measure of Sclo' Evans and
Loreto (2000) have collated a table of the various measurements made on different species.
However, von Caemmerer et al. (1994) pointed out that the C, where the CO 2 response
curves intersect, c., is slightly lower than the actual T, because of the effect of the internal
diffusion resistance from the intercellular airspace to the chloroplast. They derived from Eqs
(2.30) or (2.31) that:
T. = C+ + R d / gj (2.41)
where C. is the intercellular CO 2 partial pressure at which A = -R d ; that is T, is greater than

C. (Table 1.2, Fig. 2.15). In a fully expanded tobacco leaf there was only a 2 ubar difference
between T, and C. (von Caemmerer et al. 1994; Table 1.3). However, in young, not fully
expanded tobacco leaves, where internal airspaces have not yet developed and chloroplasts
are not yet aligned with the cell walls, C. can be up to 15 ubar less than T; (Lippert, unpub
lished data). Figure 2.15 illustrates the relationship between T, and C. when CO 2 assimilation
is plotted as a function of intercellular as well as chloroplastic CO 2 partial pressure.
2.8 CO 2 RESPONSE CURVES

The response of CO 2 assimilation to intercellular CO 2 partial pressure has proved to be one
of the most useful diagnostic tools in the study of photosynthesis and a large amount of
experimental data is available. To examine the biochemistry of photosynthesis ideally one
would like to measure CO 2 assimilation as a function of chloroplastic CO 2 partial pressure.
However, the drop in CO 2 partial pressure from intercellular airspace to the chloroplast is
difficult to measure and was initially presumed to be small. Most experimental data have
related CO 2 assimilation to intercellular CO 2 partial pressure. Von Caemmerer and Farquhar
(1981) measured the CO 2 response curves of CO 2 assimilation in Phaseolus vulgaris under a
60
a
... , 0
- en 40 0
'0
.<>
')I
O'
E O'
"'5 O'
{j
E 20 J6
2
(I)
Cil
....
c: 0
0
c d
~
E 'o
en 40
en <>..::
Cil anti-rubisco 1
0 '" .•... [].•.........o·· .<i
0 20 ~ .:-»
·6
anti-rubisco 2
0
0 400 800 1200 16000 400 800 1200 1600

Figure 2.16 Comparison between steady-state (solid symbols) and transient (open symbols) rates of CO 2
assimilation at different intercellular CO 2 partial pressures, C, in (a) wild-type tobacco, (b) two transgenic
tobaccos with reduced amounts of rubisco and (e), (d) two transgenic tobaccos with reduced amounts of GAPDH.
Measurementswere made at an irradiance of 1000 urnol quanta m-2 S-1, leaf temperature of 25°C and 02
concentration of 1-2%. Thetransient values were obtained after generating a large pool of RuBP by keeping the
leaf at zero CO 2 for a few seconds, and then transferring it rapidly to each C;
variety of environmental perturbations and used these to test model predictions. More
recently, with the advent of antisense RNA technology, it has been possible to reduce the
amounts of specific photosynthetic proteins (Rodermel et al. 1988). Transgenic tobacco
plants with impaired photosynthesis have also been useful in testing model predictions.
2.8.1 CO 2 response curves in transgenic tobacco with impaired photosynthesis

Laisk and Oja (1974) developed a two-channel gas-exchange system that can record rapid
transients in CO 2 exchange rates (Laisk and Oja 1998). Maximal RuBP pools were generated
in leaves by exposing them briefly to very low CO 2 partial pressures, after which they were
transferred to varying CO 2 concentrations, and momentarily maximal CO 2 assimilation rates
were measured within the first 2-3 s. This gives a comparison between the transient (RuBP
saturated) and steady-state rates that can be used to assess the RuBP regeneration versus
rubisco limitation of CO 2 assimilation.
Figure 2.16 gives an example of these measurements in wild-type and transgenic tobaccos
with an antisense RNA directed against rub isco small subunit (anti-rubisco plants) or chloro
plast glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH plants) (Hudson et al. 1992;
Price et al. 1995; Ruuska et al. 1998). In wild-type tobaccos the transient (RuBP-saturated)
CO 2 assimilation rates exceeded steady-state rates when C, was 300 ubar or greater, indicating
the transfer to RuBP limitation of CO 2 assimilation above this C i . Transient assimilation rates
up to 2.5 times the maximum steady-state rate could be measured under the high light
Modelling e3 photosynthesis 53
a
30
0;
20
en
":'
E
(5
E 10
~
<:(
- Q)
ctl
~
s:::
0
I L,/ Phaseolus vulgaris
+:::
.!Q
0
30 Lb 19 mbar 02
·E --
~,:/
·00
en
ctl
0 '" 20
/,'/
0 I
/ 200 mbar 02
J
I
J
I
10 ,I
o 1'1 "I ! ! , I
o 200 400 600

c; (ubar)
Figure 2.17 (a) CO 2 assimilation rate, A versus intercellularCO 2 partial pressure, C;, in Phaseolus vulgaris at
200 mbar 02 (0) and 19 mbar 02 (e) at 28°C and an irradiance of 1500 umol quanta m-2 S-1. (b) Modelled
response of A versus C, at 200 and 19 mbar 02. The dotted lines and their extensions represent the RuBP
saturated rates of CO 2 assimilation and the dashed lines and their extensions represent the RuBP-limited rate of
CO 2 assimilation. (At 28°C Vcmax = 152 urnol m-2 S·1, lmax = 214 urnol m-2 S-l, K, = 513 ubar, Ko = 286 mbar and
r. = 40.6 pbar.)
conditions (Fig. 2.16a). This provides unequivocal evidence that the CO 2 assimilation rates at
high CO 2 partial pressures and high light are limited by the supply of RuBP. In anti-rubisco
plants with 40 and 10% of wild-type levels of rubisco there was no difference between tran
sient and steady-state rates, confirming that the amount of rubisco was limiting and that CO 2
assimilation was RuBP saturated, even at high C, (Fig. 2.16b). In plants with reduced activity
of chloroplast GAPDH enzyme, the RuBP regeneration capacity is decreased and as a conse
quence RuBP pools drop dramatically, as shown by Price et al. (1995). However, it was
possible to collect substantial RuBP pools also in these plants to induce high transient CO 2
assimilation rates. The shifting balance between rubisco and RuBP regeneration is demon
strated in Figs 2.16c and d: the anti-GAPDH plant in (d) with severely reduced GAPDH
-
')'
en 25
E
"'5 20
:::i.
'-'
<:( 15 /
/
/
/
Q)
(;j , /
..... /
/
/
C 10 , /
0 r
,' ,,
~ "
E "/""
5 ",",
'iii ",
en ",
eu r,
I,
0
N
•
I,
o 0
0 200 400 600
Intercellular CO 2 partial pressure, C;
(ubar)
Figure 2.18 Modelled response of A versus C, at irradiances of (1) 1000, (2) 500 and (3) 300 urnol quanta
m-2 S-I, The dotted line and itsextension isthe RuBP-saturated rate, Ac The dashed lines and their extensions are
the RuBP-limited rates, Aj . Vcmax = 80 urnol m-2 S-I, Jmax = 120 urnol m-2 S-I,
activity and steady-state CO 2 assimilation rate expressed the difference between transient and
steady-state values at clearly lower Ci (about 100 ubar) than the anti-GAPDH plant in (c),
which had only a small reduction in GAPDH activity. These experiments have demonstrated
that the balance between the two limiting factors can be changed by genetic manipulation of
photosynthetic machinery (Ruuska et aI. 1998).
2.8.2 CO 2 assimilation rate at different O2 partial pressures

Plants do not experience large fluctuations in 02 partial pressure other than naturally occur
ring altitudinal gradients (Korner et al. 1991). However, because of the dual function of
rubisco as an oxygenase and a carboxylase, varying 02 partial pressures has been one of the
most interesting experimental tools for elucidating the mechanisms of CO 2 assimilation.
CO 2 assimilation rate is dependent on the partial pressure of 02 both at low and high partial
pressures of CO 2 (Fig. 2.17). The modelled curves show how the RuBP-saturated and limited
rates are affected by 02 partial pressures. The increase in the RuBP-saturated rate of CO 2
assimilation rate with the reduction in 02 partial pressure is the result of two different
effects: firstly there is a decrease of the apparent Michaelis-Menten constant for CO 2 of
rubisco, K/l + 0/ K o ) ' through decreased competition by 02 and, secondly, there is a reduc
tion of photorespiratory CO 2 release in the PCO cycle which is manifest in a reduction of L
and r. At 200 mbar 02 when the RuBP regeneration rate is limiting, an increase in CO 2
assimilation rate occurs with increasing CO 2 partial pressure as energy is diverted away from
oxygenation to carboxylation. At low 02' when almost no oxygenations take place, CO 2
assimilation is completely saturated with CO 2 when RuBP regeneration capacity is limiting
the rate (Fig. 2.17). CO 2 assimilation is sometimes seen to decline at high CO 2 and low 02
partial pressures (Fig. 2.17; Woo and Wong 1983). At present no satisfying explanation exists
for this effect and it is difficult to model. A triose phosphate limitation as discussed by Harley
and Sharkey (1991) and in Section 2.4.3 can predict a decline in CO 2 assimilation rate with
increasing CO 2 partial pressure at ambient 02 partial pressure, but predicts a constant rate at
30 , a
, •
..-..
en
C)l
E
"0
20
10
l ~-.... ........ -
...- k"---~
__ A--- __ A
-~
E
.6
"::(
2 0
...ell 30 ~ b
c
0
~
'E
'00
en 20
ell
N
0
o
10 I
. ---- A
o I', I I I
o 200 400 600

C; (ubar)
Figure 2.19 CO 2 assimilation rate, A, versus intercellular CO 2 partial pressure, C;, at irradiances of (~) 300,
(_) 500 and (e) 1000 urnol quantam-2 S-1 in (a) wild-type and (b) transgenic tobacco with reduced amounts of
rubisco, Measurements were made at a leaf temperature of 2soc.
low 02 partial pressure. For a more detailed discussion of this phenomenon see Sharkey
(1985) and Sage and Sharkey (1987).
2.8.3 CO 2 assimilation rate at different irradiances

Irradiance is one of the most rapidly varying environmental factors encountered by the plant
and an understanding of the response of CO 2 assimilation rate to irradiance is therefore of
vital importance. Figures 2.18 and 2.19 show modelled and measured CO 2 responses curves
of assimilation rate at three irradiances. It can be predicted from the model that when CO 2
assimilation rate is given by the RuBP-saturated rate, A c' it is independent of irradiance and
the results show that this is indeed the case at low intercellular CO 2 partial pressures for
wild-type tobacco. The CO 2 assimilation rate of transgenic tobacco with reduced amount of
rubisco was independent of irradiance, confirming that rubisco was RuBP saturated at all
CO 2 partial pressures measured. Evans (1986) also found the initial slope of the CO 2
response curve to be independent of irradiance above 800 umol quanta m- 2 S-1 (Fig. 2.20a).
Sage et al.(1990) found the initial slope to be independent of irradiance above 400 and
500 umol quanta m- 2 S-1 in Phaseolus vulgaris and Chenopodium album (Fig. 2.20b). Brooks
a
QbQ_C'6'0 {fp>cf:60
~~ 'CP 0
0.8 o
o 0
o
0.4 f
ocg
0.0 I I
b
...
0.8
....
...
... ...• •• ... •
• .'
0.4
....•
~
I I I
0.0
0 500 1000 1500 2000
-2
Irradiance ( urnol quanta m 5'1)
Figure 2.20 Initial slope of the CO 2 response curve of CO 2 assimilation rateversus irradiance. (a) In wheat
genotypes. Measurements were made at a leaftemperature of 23°C and normalized by division with the mean
initial slope measured at an irradiance of 1000urnol m-2 S-l (data taken from Evans 1986). (b) In Chenopodium
album (e) and Phaseolus vulgaris (A). Measurements were made at a leaftemperature of 24-26°C and
normalized as above. (Data taken from Sage et al. 1990b.)
and Farquhar (1985) came to a similar conclusion for spinach. Together with the results in
Fig. 2.16 it confirms that C 3 photosynthesis can readily be described by either an RuBP
saturated or RuBP-limited CO 2 assimilation rate.
In this basic model of C 3 photosynthesis described by Eq. (2.27) it has been assumed that
rubisco is fully activated. It is well known that rubisco's activation state is modulated by irradiance
at ambient CO 2 (von Caemmerer and Edmondson 1986 and references therein; Fig. 1.9). The
results just discussed suggest that rubisco is fully activated at low CO 2 partial pressures when the
initial slope is independent of irradiance. (The in vivo regulation of rubisco activation is discussed
further in Section 2.11). The model correctly predicts the strong dependence of CO 2 assimilation
rate on irradiance at high CO 2 partial pressures when Ai determines CO 2 assimilation rate.
2.8.4 CO 2 assimilation rate at different temperatures
Figure 2.21 shows CO 2 response curves measured in Eucalyptus pauciflora at different leaf tem
peratures (Kirschbaum and Farquhar 1984). The initial slope does not change much with tem
perature because of the similar activation energies of K; and V cmax (Table 2.2; Fig. 2.9). Similar
results have been observed for a wide variety of C3 species (Ku and Edwards 1977b; von Caern
merer and Farquhar 1981; Woo and Wong 1983; Harley et al. 1985; Sage and Sharkey 1987; Sage
et al. 1990a; Sage et al. 1995). However, the compensation point increases with temperature
(Fig. 2.13). The RuBP regeneration-limited rate increases with temperature in this example but
is likely to decrease at higher temperatures (von Caemmerer and Farquhar 1981).
'";"
en I Eucalyptus pauciflora
')J
30
E
0
E
2,
Q) 20
Cii
....
c
0
~
E 10
'eenn
ell
N
0
o 0
0 200 400 600
C, (ubar)
Figure 2.21 CO 2 assimilation rate, A, versus intercellular CO 2 partial pressure, C, at leaf temperatures of
(A) 18°C, ( _) 25°C and (e) 32°C in Eucalyptus pauciflora. (Data are taken from Kirschbaum and Farquhar 1984.)
2.8.5 The initial slope of the CO 2 response curve, dAfdCi

Farquhar et al. (980) differentiated Eq. (2.20) to obtain:
dA L + K e ( 1 + 0/ K o )
(2.42 )
dC = V,mt/x[ C + K ( 1 + 0/ K ) ]2
e o
The slope, sometimes called 'rnesophyll conductance' or 'carboxylation efficiency' (Ku and
Edwards 1977a), should only have a small dependence on CO 2 since K, is relatively large. At L:
dA _ V emax
(2.43)
dC - L + K,O + 0/ K a )
Von Caemmerer and Farquhar (1981) estimated dA/ d C, from gas-exchange measurements of
CO 2 response curves in Phaseolus vulgaris and compared these to in vitro measurements of
rubisco activity made on the same leaves, and found good agreement with Eq. (2.43)
(Fig. 2.22). Evans (1986) and Evans and Terishima (988) found that the relationship
between initial slope and in vitro rubisco activity was somewhat curvilinear in wheat and
spinach and suggested that a limitation of internal CO 2 diffusion may be responsible. That
this can be the case was illustrated by the comparison of CO 2 fixation in a tobacco and a Kal
anchoe leaf (Fig. 2.8). Differentiating Eq. (2.30) yields an expression of d/vl d C, including gi
and:
dA _ gJVemax- (A + R d ) )
(2.44)
dC; - (g;( C; + K c ( 1 + 0/ K a ) ) + V oll ax - R d - 2A)
If this expression is evaluated at L ,
dA giVemax
(2.45)
sc, g;(L + K,( 1 + 0/ K o ) ) + V emax - u,
"-
ell
Phaseolus vulgaris
.0
::::i.
'en 0.3
')'
E • •
•• •••
(5
E
E: 0.2
~-
~ 0.1
ai
o,
o2
en
~ o. 0 "--------'---'-----'----'-------'-----'---~'--------'-----'--------'--~
C 0 50 100 150 200 250
2
In vitro rubisco activity (urnol m· S·1)
Figure 2.22 Initial slope of the CO 2 response curve of CO 2 assimilation rate versus in vitro rubisco activity in
Phaseolus vulgaris. Measurements were made at 28DC andthe source of variation in rubisco activitywas leaf age,
nitrogen nutrition and growth irradiance. (Data aretaken from von Caemmerer and Farquhar 1981.) The line
shows the relationship predicted by Eq. (2.43) with r. == 42.4, Kc == 330 ubarand Ko == 207 mbarat 28D e.
(von Caemmerer et al. 1994) and the extent of the curvature is dependent on the ratio
v.:».
The reciprocal of Eq. (2.45) was derived by Peisker and Apel (1975). They showed that the
mesophyll resistance (1lg m = 1/(dAldCi ) , which is the sum of the CO 2 transfer resistance and the
residual carboxylation resistance, is linearly dependent on 02 partial pressure as was observed by
Ku and Edwards (1977a).
(2.46)
The intercept is a function of gj' Vcmax - Rd and K c; however, given the difficulty in obtaining
accurate estimates of V cmax ' it may not be such a useful tool to extract estimates of gi'
The temperature dependence of dAldC i is largely determined by the temperature dependen
cies of rub isco kinetic constants. Using the temperature dependencies of Table 2.3 the model
predicts only a slight increase with temperature (von Caemmerer and Farquhar 1981; Farquhar
and von Caemmerer 1982). This was experimentally verified for a number of C 3 species (Sage et
al. 1995 and references therein). Furthermore it has to be borne in mind that the intial slope of the
CO 2 response curves is not necesarily rubisco limited at very high or low temperature, where it is
quite possible that the CO 2 assimilation rate is RuBP-regeneration limited at most CO 2 partial
pressures. At present little is known about a possible temperature dependence of S:
2.9 LIGHT RESPONSE CURVES
2.9.1 Dependence on 02 and CO 2 partial pressures

The rate of CO 2 assimilation at different irradiances has been widely studied (Bjorkman
1981), but the response of CO 2 assimilation to irradiance is not always as easy to interpret as
the CO 2 response. Although Wong et al. (1979) have shown that Cj does not vary much with
irradiance, there are subtle differences. Nevertheless, the response of CO 2 assimilation to
irradiance also reflects the mesophyll capacity to assimilate CO 2 ,
'"
~
(f)
E 30
(5
E
:i
"l: 20 2
OJ
"§ , , , , , , '. ' , ' , , , , , , , ' ,,~~~~==--..:-c.-'-------------1
c
,Q
" , '~L-J
§ 10
'E
'iii
(f)
Cll
OJ
o
()
500 1000 1500 2000

Irradiance, I (urnot quanta rn" S,l)
Figure 2,23 Modelled rate of CO 2 assimilation (solid line)asa function of irradiance at 02 partialpressures of
(1)20 and (2) 200 mbar 02 and Cc =250ubar. The dotted line andits extension and the dotted arrow indicate the
°
rubisco-limited rates at 200 and 20 mbar 2, The dashed line and its extension is the RuBP-regeneration-limited
°
rate at 200mbar 2, At 20 mbar 02' CO 2 assimilation rate is RuBP-regeneration limited at all irradiances shown
(other parameters aregiven in Table 2.3).
~ 30
'00
~E
Cc = 500 ubar
"6
E
.E, 20 _ I
~ Q) ~"""""/"""'-'--"" ----------
= 250 ubar
"§ Cc
c
..g 10
~ ---------
'E
'en
~ Cc = 100 ubar
ON
o
500 1000 1500 2000
Irradiance, I, (IlmOI quanta m,2 s ,1)
Figure 2.24 Modelled rate of CO 2 assimilation (solid line) as a function of irradiance at three chloroplast CO 2
partial pressures, C[' 25°C and 200 mbar 02' The dotted lines and their extensions arethe rubisco-limited rates.
The dashed lines andtheir extensions arethe RuBP-regeneration-limited rates. Other parameters are given
in Table 2.3.
Figure 2.23 illustrates the irradiance dependence of CO 2 assimilation rate given by the model.
At 20 mbar 02 A is RuBP-regeneration limited at all irradiances and the possible rubisco-limited
°
rate is indicated with an arrow. At 200 mbar 2, A is much less as energy is required in the PCO
cycle. Here, A is RuBP-regeneration limited up to 1000 umol quanta m- 2 S·1 and becomes rubisco
limited at higher irradiances. The transition between the two limitations is, however, difficult to
pinpoint experimentally.
";
..
I I
(/)
<)I
E .•...................•
20 ,
.'
(5 f-
. 79
E
:::1.
.0····0 0 ·0 20
10 f -
~
c::
o '0'"
~ ,:"~ 0 .··0···0-········0 15
1:'P••'
-
o ff I I
o 400 800 1200 1600

-2 .,
Irradiance, I (urnol quanta m s)
Figure 2.25 CO 2 assimilation rate as a function of irradiance for wild-type tobacco (e) and transgenic tobacco
with reduced amount of rubisco (. , =;
numbers givemaximal rubisco activities (prnol m-2 s-l)).(Data from
Lauerer et al. 1993.)
Figure 2.24 shows modelled light response curves of A at different CO 2 partial pressures. As
CO 2 partial pressure increases, the transition from RuBP regeneration to rubisco limitation
occurs at progressively higher irradiances. This means that the irradiance at which light saturation
of CO 2 assimilation occurs is dependent on other environmental variables such as CO 2 and tem
perature, a point that is frequently overlooked when experimenters discuss light-saturated CO 2
assimilation. Figure 2.25 shows light response curves of wild-type and transgenic tobacco with
reduced amounts of rubisco. Similar to a decrease in CO 2 partial pressure, as rubisco capacity is
decreased light saturation of CO 2 assimilation also occurs at lower irradiances.
2.9.2 Quantum yield

Farquhar and von Caemmerer (1982) give detailed equations for the calculation of quantum
yield, considering various options for the NADPH and ATP requirements. Here only the
equation based on the requirement for NADPH is presented. At low irradiance, CO 2 assimi
lation rate is given by:
(C-L)]
-4....,.C-+-8-=r=-. - Rd (2.47)
and
]= I x abs(l- f) (2.48)
2
(Section 2.3.4.2) and therefore:
(C - L) I x abs(l- f)
A j=4C+8L 2 -R d (2.49)
The quantum yield is defined as the initial slope of the relationship between assimilation
rate, A and irradiance, I and thus on the basis of the NADPH requirement:
0.10
C?
~
"E 0.08~
ctl 20 mbar 02
32 :::::l
(j) C
'>, ~
E '- 0.06
:J 0
C
ell
Eco
:J 0
00 0.04
"0 200 mbar 02
-E
0.02
I
0.00
0 10 20 30 40 50
Leaf temperature CC)
0.10
0.08
ctl
"E
ctl
-0 :::::l
<ii c 0.06
's, "7
E "0
:J
C E
co
ell
:J 0 0.04
0 0
"0
E
0.02
0.00
0 200 400 600 800
Chloroplastic CO, partial pressure, Cc
(ubar)
Figure 2.26 (a) Modelled quantum yield (Eq. (2.50)) asa function of temperature at a chloroplast CO 2 partial
pressure of Cc = 250 ubar, at 200and20 mbar 02 at 25°C. (b) Modelled quantum yield (Eq. (2.50)) asa function
c,
of at 200and 20 mbar 02 at 25°C.
dA (C-L)
(limit, I ~ 0) dI = 8C + 16L (1- f)abs (2.50)
This predicts a maximum quantum yield of 0.125 at high CO 2 or low 02' when f = 0 and
abs = 1. With f = 0.15 and abs = 0.85 this reduces to 0.09. Equation (2.50) has several
deficiencies. It ignores other requirements of the chloroplast for ATP and NADPH outside
the PCR and PCO cycles, including additional amino acid biosynthesis, lipid metabolism and
nitrate reduction, which lower the quantum yield (Raven 1972). Furthermore, the quan
tum yield is usually measured at a finite irradiance, often between 50 and 150 urnol m- 2 S-I,
where J may not necessarily be linearly related to 1. The CO 2 , 02 and temperature depen
dence of the quantum yield reflects the competition of the PCR and PCO cycles for limit
ing NADPH and ATP, which is governed by Land Sclo (Fig. 2.26) as was shown by
Ehleringer and Bjorkman (1977).
2.10 TEMPERATURE RESPONSES

Figure 2.27a shows the temperature response of CO 2 assimilation rate, A, at ambient CO 2
predicted by the model. At lower temperatures, A is limited by the amount of rubisco,
whereas at higher temperatures A is electron-transport limited. An increase in mitochondrial
respiration rate at high temperatures leads to a steeper decline in A, Ai and A( than would
otherwise occur. The steep decline at temperatures greater than 30°C, which is largely driven
by the temperature dependence of Jmax ' needs to be treated with some caution. It may be due
to irreversible inhibition in the in vitro system, from which the function was derived. Trans
genic plants with electron transport rate impaired by the reduction of the chloroplastic cyto
chrome bf content are yet to be exploited to derive new in vivo temperature dependencies of
the electron transport chain (Price et al. 1995, 1998). Results in the literature suggest that the
decline in CO 2 assimilation at high temperature may not be entirely due to an electron trans
port limitation. For example, RuBP pools have been shown to increase and rubisco activation
to decrease under these conditions, pointing to possible limitations in the dark reactions
(Weis 1981a,b; Kobza and Edwards 1987; Labate and Leegood 1988; Weis and Berry 1988;
Sage et al. 1995). Furthermore, the inactivation of rubisco at high temperature has been
linked to inactivation of activase activity (Feller et al. 1998). However, it remains to be shown
whether the activity of the PCR cycle enzymes and activase activity are merely modulated by
a reduced electron transport rate or whether they constitute the primary limitation to CO 2
assimilation rate. Although they may not always reflect the underlying biochemistry at high
temperatures, the temperature dependencies from Farquhar et al. (1980) used seem widely
applicable. For example, Kirschbaum and Farquhar (1984) determined the temperature
dependence of electron transport by calculating J from A as outlined in Section 2.10.2 for
Eucalyptus pauciflora. They observed good agreement with modelled values. For rubisco they
estimated an activation energy of 45 kl mol:". Other estimates have been reviewed by Wal
croft et al. (1997). The temperature dependencies for the model have been estimated in a
number of studies that have used the model to simulate field data (Harley et al. 1986, 1992b;
Harley and Baldocchi 1995; Leuning 1995; Leuning et al. 1997; Walcroft et al. 1997).
The temperature optimum of CO 2 assimilation rate depends on environmental conditions
such as CO 2 partial pressure and irradiance, the optimum being more pronounced at high irradi
ance and/or high CO 2 (Fig. 2.27b,c). At high CO 2 the rate of photorespiration is reduced, thereby
extending the temperature range where positive CO 2 fixation occurs.
2.11 DOES THE ACTIVATION STATE OF RUBISCO NEED TO BE INCORPORATED

INTO MODELS OF CO 2 ASSIMILATION?
The simple answer is 'no', since, during steady-state measurements, rubisco seldom appears
to be rate limiting when not fully active (Mott et al. 1984; Sharkey 1989, 1990; Sage 1990).
Rubisco activation state is modulated by light and with CO 2 (Figs 1.7 and 1.8; Jensen and
Bahr 1977; Bahr and Jensen 1978; Perchorowicz et al. 1981; von Caemmerer and Edmondson
1986; Seemann 1989; Sage 1990; Sage et al. 1990; Sassenrath-Cole et al. 1994; Sage et al.
25 I a
20 .-.:'\
15 f- , ,.,.....
A. ,-'
., )'
10 f- ,-/'
,,
'"
5 f - --
" '"Rd
0
~CJ)
'"'E -5 b I
• __ ~c
o 20
E: 15
-Q)
CO
....
c
10
o
'';::;
.£Q 5
E
'(j) 0
CJ)
co
o'" -5
o
c
20 I 1= 1000 Il mOI rn" S·1
_ ..........
.r-.....
15
10
5
0
-5
0 10 20 30 40 50
Leaf temperature (DC)
Figure 2.27 (a) Modelled rubisco-limited (Au dotted line) and electron-transport-limited (Ai' dashed line) and
net CO 2 assimilation rate (A) as a function of leaftemperature at 230 ubarCO 2 partial pressure and 1000umol
quanta m-2 s'. Also shown isthe temperature response of mitochondrial respiration, Ref (b) Modelled CO 2
assimilation rate, A (solid line), and rubisco-limited CO 2 assimilation rate (Ac' dotted line) as a function of
temperature at different CO 2 partial pressures and an irradiance of 1000umol quanta m-2 s". (c) Modelled CO2
assimilation rate, A. (solid line) and rubisco-limited CO 2 assimilation rate (Ac' dotted line) as a function of
temperature at different irradiances and a (of 250 ubar. Other parameters are as given inTable 2.3.
1995). Reduced rubisco activation is observed at CO 2 partial pressures close to the CO 2 com
pensation point and sometimes at high CO 2 partial pressures (von Caemmerer and Edmond
son 1986; Sage et al. 1988; Vu et al. 1997), Sage (1990) and Sage et al. (1990) suggested that
64 BiochemicallVlodels of Leaf Photosynthesis
~,/
a ~,/
,/
Q) 30 ,/
n;
... ",
",-"
~
",-
c: .-.. ",-
0
§ ')'
(J)
20 '" 2
'E E
(J) 0
(J)
ell E
~ 10
0 '"
o
a b
100
c: 80
.Q
n;
> 60
t5ell .-..
::!2.
0 ~ 40
o(J)
.s::::l 20
a::
0
0 100 200 300 400 500
(ubar)
Figure 2.28 (a) Modelled CO 2 assimilation rates asa function of intercellular CO 2 partial pressure, at two
electron transport rates: (1) J = 130 urnol m-2 S-l and (2) J = 90 urnol m-2 S-1. Also shown is the rubisco-limited
rate (dotted line and its extension). (b) Rubisco activation that is required to support electron-transport-limited
CO 2 assimilation rate (Eq. (2.51)) for the scenarios shown in (a).
rubisco's activation state was lowered only under conditions where RuBP regeneration rate
was limiting CO 2 assimilation rate and therefore it does not need to be considered in most
cases. Sage (1990) used Eqs (2.20) and (2.23) to calculate the required activation state of
rubisco when CO 2 assimilation rate is electron transport limited and:
C + K c {1 + OIKo ) J
% activation = C
4 + 8.
r V cm ox
100 (2.51)
An example of this calculation is shown in Fig. 2.28. There is some support for this hypoth
esis provided by the experiments of Mott et al. (1984), von Caemmerer and Edmondson
(1986) and Sage et al. (1990). The fact that the initial slope of the CO 2 response curves does
not change with irradiance over a wide range (Fig. 2.20) suggests that rubisco is fully acti
vated at low irradiance and low CO 2 partial pressure when rubisco capacity limits the rate of
CO 2 assimilation. This highlights the fact that rubisco carbamylation is no simple function
of irradiance. The suggestion of Sage (1990) offers a better working hypothesis, although not
a mechanistic link. However, Eq. (2.51) may well be a useful way to examine changes in
rubisco activation with temperature. Increasing temperature can also deactivate rubisco
(Kobza and Edwards 1987; Weis and Berry 1988). Studies by Feller et al. (1998) have
- ( j) 40
'l'
.>
E
"'6 30
E
E,
/
•
./
OJ
"§ 20
c
-~
.>
.:
0
§
10
E
'iii
(j)
ctl
a'" 0 0 100 200 300 400 500

o
- ( j)
'l'
"'6
E 20
/. •
E ........---.
---
E,
~
s •
~
~--.
c
10
0
§ _A-A A A
'E
'iii
(j)
ctl 0
a'"
o 0 500 1000 1500 2000
Irradiance, I (urnol quanta rn" s')

Figure 2.29 CO 2 assimilation rate asa function of intercellular CO 2 partial pressure or irradiance for Phaseolus
vulgaris grown with 12 mM (e), 4 mM (_) and 0.6 mM (A) nitrate nutrition. Measurements wereeithermade at
28°C and an irradiance of 1500 urnol m-2 S-l and varying ambient CO 2 partial pressure or at CO 2 partial pressure
of 330 ubarand varying irradiance. (Data aretaken from von Caemmerer 1981.)
identified a loss of activase activity as the reason. Work with the transgenic tobacco with
reduced amount of GAPDH activity shows no decrease in rubisco activation state (Price et al.
1995). Thus reduction in RuBP regeneration rate through inhibition of PCR cycle activity or
reduction of electron transport rate per se is not sufficient. However, rubisco activation was
reduced in transgenic tobacco with impaired electron transport rate through an antisense
construct to the Rieske iron sulfur polypeptide of the bf complex (Price et al. 1998; Ruuska
et al. 2000a).
2.12 LONG-TERM EFFECT OF ENVIRONMENT ON PHOTOSYNTHESIS
2.12.1 Vcmax and Jmax and the transition from rubisco to electron transport limitation
CO 2 assimilation by leaves is affected by plant nutrition, the light and temperature environ
ment during growth and leaf age (Bjorkman et al. 1972; Seemann et al. 1981; von Caemmerer
and Farquhar 1981, 1984; Wong et al. 1985; Hidema et al. 1991). For example, Fig. 2.29
shows CO 2 and light response curves of CO 2 assimilation rate typical for plants grown with
30
a ...
... ... 0
• "'0
00
')'
f
... •... I • 00 e. •
E
(5
.3
E 20 •
Q) ~~
~ ew ,
c
0 CIa. q.
~ o 00 •
I
E 10
00
00
00
(Ij
<11
0'" O. 0
I
~
U
0
0 40 80 120 160
In vitro rubisco activity (urnol m"2 5"')
30
b
')'00
1
• •0 0 • •
E • 011
•
0 20 <9 •
°co • •
E
..
2;
2
~
00
0
•
O}
c
0
0
~ 10 I
00
E 00
00
00
II o Ito
(Ij
0'" I
0
•
o
• 0 50 100 150 200 250

In vitro electron transport rate (urnol electrons m"2 s "1)
Figure 2.30 CO 2 assimilation rate versus in vitro rubisco activity and in vitro electron transport rate for
Phaseolus vulgaris grown under different environmental conditions: light environment (= ), nitrogen nutrition (_)
and defoliation (A). Measurements were made at 28°C and an irradiance of 1500 urnolm-2 S-1 and an ambient
CO 2 partial pressure of 330 ubar. (Data are taken from von Caemmerer and Farquhar 1981.)
different nitrogen nutrition (Farquhar and von Caemmerer 1981; Evans 1983; Makino et al.
1985, 1994b, 1997a). Von Caemmerer and Farquhar (1981, 1984), showed that these changes
could be correlated with changes in the in vitro rubisco activity and ill vitro electron trans
port rates (Fig. 2.30) and suggested that these changes could be modelled simply by changes
in V em ax and Jmax ' They also noted that, in all the different growth conditions they examined,
V emax and Jmax remained closely coupled. This has been confirmed in many other studies (e.g.
Thompson et al. 1992; Wullschleger 1993; Leuning 1997; Poorter and Evans 1998). Only
'Cf)
<)l
9j = infinite
E
"'6
E
20 -,
E:
o::c
Q)
(tj ,-, 0 I -2-1
",,9j = .3 rno m s
.....
c 10
o
.~
E
'00
Cf)
ttl
0'"
o
o I " I ! ! 1
o 100 200 300 400 500

Intercellular CO 2 partial pressure C,
(ubar)
Figure 2.31 Theintracellular CO 2 partial pressure, C,. at which the transition from rubisco to electron transport
limitation of CO 2 assimilation rate occurs isaffectedbythe internal diffusion conductance gj. CO 2 assimilation rate
versus C, is modelled with the same valuesof Vcmax and Jmax but two different valuesfor gj' Otherparametersare
as given inTable 2.3.
transgenic manipulation of the photosynthetic machineries in tobacco has achieved gross

imbalances (Fig. 2.16; Quick et al. 1991a,b, 1992; Stitt et al. 1991; Hudson et al. 1992; Price et
aI.1995,1998;RuuskaetaI.1998).
The balance between V cmax and Jmax determines the chloroplastic and intercellular CO 2 partial
pressure at which the transition from rubisco to electron transport limitation occurs. The chloro
plast CO 2 partial pressure is given by:
C = Kc(1+0IK,,)J/4Vcmax-2L
(2.52)
c 1 - JI( 4 VC/I",x)
(Farquhar and von Caemmerer 1982). The intercellular CO 2 partial pressure at which the
transition occurs exceeds the chloroplastic CO 2 partial pressure by Ci = Cc + Alg; The chlo
roplast CO 2 partial pressure at which the transition occurs varies in the short term with irra
diance and temperature but, because Jmax and V cmax are closely correlated, it appears to be
somewhat conserved with different growth conditions (von Caemmerer and Farquhar 1981).
Wullschleger et al. (1993) and Leuning (1997) examined CO 2 response curves in the litera
ture and concluded that the transitional C j was also similar amongst different species. How
ever, the transition is not always easy to identify experimentally. At low light or at higher
temperatures, for example, CO 2 assimilation rate can be limited by the capacity for RuBP
regeneration, even at low CO 2 partial pressures, and this is difficult to determine from
steady-state measurements. The techniques for transient measurements of CO 2 assimilation
rates developed by Laisk and Oja (1998) provide a much better way of identifying the transi
tional CO 2 partial pressure (Fig. 2.16).
It is important to note that the magnitude of the internal diffusion conductance, gi' will affect
estimates of J and Vcm"x made from CO 2 response curves. This is illustrated in Fig. 2.31 where two
CO 2 response curves are shown with identical biochemical parameters but different values of gj
(see also Fig. 2.8). The fact that very little variation in the transitional CO 2 partial pressure is
25
'en
20
15
«
-
Q)
eel
.... 10
10 20 30 40 50
Leaf temperature (DC)
Figure 2.32 Modelled CO 2 assimilation rate, A, as a function of temperature for two differentratios of
Vemaxllmax (solid line, 80/200; dashed line, 100/120) at an irradiance of 1000 prnol quanta m-2 S-l anda CO 2
partial pressure of 250 ubar,
observed (Wullschleger et al. 1993; Leuning 1997) may in part be due to the close correlation that
is also usually observed between photosynthetic capacity and gi (von Caemmerer and Evans 1991;
Evans and von Caemmerer 1996).
2.12.2 Effect of growth temperature

Acclimation may occur when plants are grown at different temperatures and the temperature
optima can shift to higher temperatures for plants grown at high temperatures (Bjorkman and
Pearcy 1971; Armond et al. 1978; Berry and Bjorkman 1980; Bjorkman et al. 1980; Badger et al.
1982). It appears that the temperature response of whole chain electron transport is closely
linked to the properties of the thylakoid membrane such as membrane viscosity and lipid com
position (Nolan and Smillie 1976; Raison and Berry 1979). Equation (2.34) may need to be
adjusted for plants grown at different temperatures. However, it may also be that the ratio
Vemax/fmax changes with different temperature regimes. For example, it has been noted that
rubisco content increases in plants grown at low temperatures (Hurry et al. 1994). Figure 2.32
demonstrates that small increases in temperature optima can result from an increase in the
ratio of Jmax to V emax (Farquhar and von Caemmerer 1982). A detailed analysis of optimal nitro
gen allocation with respect to temperature acclimation is given by Hidosaka (1997).
2.12.3 Growth at elevated CO 2

In an attempt to understand the effects of predicted increases in atmospheric CO 2 on the
carbon exchange in the terrestrial biosphere, many studies have examined the effects of long
term growth at elevated CO 2 on CO 2 assimilation and rubisco function. Several recent reviews
have summarized the present knowledge on rubisco (Bowes 1991; Long 1991; Sage 1994; Woo
drow 1994; Drake et al. 1997). Depending on growth conditions and species, both decreases in
rubisco content per leaf area and/or reductions in rubisco activation states have been observed.
However, in some species no change in either parameter occurs (Drake et al. 1997). It has been
frequently pointed out that at elevated CO 2 partial pressures much less rubisco activity is
required for the same electron transport capacity (Fig. 2.33; von Caemmerer and Farquhar
~ / ,
'en -2 1 /
N Vcmax=100 urnol m s // 75,' ,
'E /
60
o 30 /
/
E I
E, I
I
<:( I
s 20 /
/
, J=140 urnol rn" S·1
~ /
c
o
§
E 10
'eenn
ell
ON
o
200 400 600 800 1000
Intercellular CO 2 partial pressure, C;
(ubar)
Figure 2.33 With increasing C, less rubisco is required to co-limitelectron-transport-limited CO 2 assimilation

rates. Rubisco-limited rates (dashed lines) werecalculated from Eq. (2.20) and the electron-transport-limited rate
(solid line) was calculated from Eq. (2.23). Other parameters are given in Table 2.3.
1.0 ,,.-,r---,----,-----,-----,-----,-,
o 0)
:;;~
~ c 0.8
.- 0
::J'
crCtl
0)=
. . . E 0.6
0'
U en
en en
:0 CO
::J N
..... 0 0.4
0°
c
o c
:;::::;.£9
co c 0.2
U en
..... 0
LLU
0.0 I I I I ! I I
400 600 800 1000 1200 1400

Ambient CO 2 partial pressure (ubar)
Figure 2.34 Amount of rubisco (relative to that present at 350 ubar) required to maintain the same CO2
assimilation rate as at 350 ubar at different CO 2 partial pressures. Results werecalculated with the helpof
Eq. (2.20) and parameters given in Table 2.3.
1984). Woodrow (1994) and Drake et al. (1997) calculated the fraction of rubisco required to
maintain constant CO 2 assimilation rates at different CO 2 partial pressure for various tempera
tures (Fig. 2.34). Much less is required, particularly at the higher temperatures. However, there
is no evidence that plants are able to adjust the balance between rubisco and electron transport
capacity to suit environmental conditions other than perhaps with temperature (Sage 1994;
Sage et al. 1995; Hidosaka and Hirose 1998). Experiments with transgenic rice with reduced
en ~ 500
a
'l'
(5
E
30 0 0
.c
-6
I-. 400
b
•
E C
-6 20 '0 300
c.
OJ c
~ .Q
c 10 Cil 200
.Q
en
c
~ OJ
c. 100
:~
en
0 E
0
en o
co ON
ON 0 400 800 1200 100 200 300 400
U
U Intercellular CO2 partial pressure (ubar) 02 (mbar)
Figure 2.35 (a) The response of CO 2 assimilation rateto intercellular CO 2 partial pressure, C; for leaves of
control tobacco plants (0) and two chloroplast transformants with a mutant rubisco where Leu-334 was changed
to a Val (., .). Solid lines are modelled CO 2 assimilation rates. For the control Vemax = 90 urnol m-2 S-1, Ke =
404 ubar, Ko = 248 mbar, r. = 37 ubar and Rd = 3 prnol m-2 S-1. For the transformants Vemax = 37 urnol m-2 S-l,
K; = 318ubar, Ko = 55.6 mbar, I', = 140pbar and Rd = 2.5 urnol m-2 s'. (b) The CO 2 compensation point as a
function of 02 partial pressure for control leaves (0, :J) and transformants (., .). Solid lines are the CO 2
compensation points modelled using Eq. (2.38) with the parameter values given in a. (Adapted from Whitney
et al. 1999.)
amounts of rubisco suggested that transgenic rice with 65% of wild-type rubisco levels was
more efficient in nitrogen use and may provide opportunities for crop improvement for growth
at elevated CO 2 (Makino et al. 1997b).
2.13 ENGINEERING A BETTER RUBISCO

A rigorous test of the model has been the use of Eqs (2.20) and (2.38) to examine in vivo
function of a mutated rubisco. Whitney et al. (1999) have used chloroplast transformation
technology to generate transgenic tobacco plants with mutated rubisco where a specific resi
due in the active site (Leu-334) was altered to a Val. These plants grew only slowly and had to
be raised under elevated CO 2 partial pressure. Figure 2.35 shows a comparison of CO 2
response curves of wild-type and mutant plants. The lowered CO 2 assimilation rate and
increased compensation points are clearly evident. Figure 2.35 also shows the increased 02
dependence of the CO 2 compensation point. A comparison of gas-exchange analysis of the
kinetic parameters with in vitro measurements showed close agreement (Whitney et al.
1999). These plants had reduced Sclo' reduced catalytic turnover rates as well as reduced
values of Ko (see Fig. 2.35 caption).
Andrews and Lorimer (1987) suggested thatthe power of modern molecular genetics could be
used to engineer a better rubisco. Whitney et al. (1999) have demonstrated that precise molecular
genetic interventions are indeed possible. Furthermore, the comparison between kinetic con
stants inferred from in vitro and in vivo experiments has demonstrated the accuracy of the C3
model at predicting rubisco function.
Thus the model can be used in thought experiments to examine the likely outcomes and
benefits of designer rubiscos. One such example is given. Figure 2.36 depicts hypothetical CO 2
response curves. In Fig. 2.36a a hypothetical C 3 rubisco is replaced with a hypothetical C 4 rubisco
that has the same relative specificity, Sclo but the catalytic turnover is doubled and both K[ and K;
have increased. The negative slope of the line drawn from the 350 ubar on the X-axis graphically
depicts stomatal conductance and intersects the CO 2 response curves at the operational C, (since
a
30 ~
C4 Rubisco
~
(f)
20
I \
~
E
(5
E 10
6
Q)
§
c 0
.Q
I b
to 30
(f)
(f)
co
N 20
0
o
10
0
0 100 200 300 400 500
(ubar)
Figure 2.36 (a) Comparison of CO 2 assimilation rate modelled with C3 rubisco kinetic constants and
hypothetical C4 kineticconstants. (C 3: Vemax =' 80 urnol m-2 s'. J =' 130 urnol m-2 s'. Ke =' 260 ubar,
Ka =' 179 mbar, F. =' 37 ubar: C4 : Vemax =' 160 prnol m-2 S-1, J =' 130 urnol m-2 S-l, K; =' 650 ubar, Ko =' 450 mbar,
1 * =' 37 ubar. The increase in Vemax assumes an increase in catalytic turnover rate for the same amount of rubisco
sites.) The lines drawnfrom 350 ubar on the X-axis depict stomatal conductance and point to resulting
intercellular CO 2 partial pressures and CO 2 assimilation rates. (b) Comparison of CO 2 assimilation rate modelled
with C3 rubisco kineticconstants and hypothetical C4 rubisco with improved Sdasuch that 1* =' 25 ubar (other
constants as in a).
g = Af( C" - Ci ) ) . With the same stomatal conductance, the leaf with the C 4 rubisco does not have
a higher CO 2 fixation rate because, at this Ci , A is limited by the electron transport rate. However,
the leaf could maintain a higher rate of CO 2 assimilation at lower C, and therefore close its sto
mata more (indicated by the dotted line drawn from the X-axis). At high irradiance this could
improve both nitrogen use and water use efficiency. But, although there are advantages under
conditions of high irradiance, no benefit would accrue under low light since there has been no
change in Sclo and r. (Eq. 2.23).
In Fig. 2.36b the C 3 rubisco is replaced with the hypothetical C 4 rubisco with improved
Sclo' The improvement in Sclo has led to a decrease in CO 2 compensation point and increased
the electron-transport-limited rate of CO 2 assimilation as more of the available ATP and
NADPH is partitioned towards carboxylation rather than oxygenation. This leaf can do
considerably better at the same stomatal conductance as well as at lower stomatal conduc
tance, even at high irradiance. Furthermore, improvements in Sclo confer advantages in the
quantum yield region (Eqs 2.23 and 2.50).
3
Chlorophyll fluorescence and oxygen exchange
during C3 photosynthesis
3.1 INTRODUCTION
The previous two chapters have dealt with CO 2 fixation during C 3 photosynthesis. In this
chapter the accompanying chlorophyll fluorescence and 02 exchange characteristics are con
sidered. Fluorescence signals are widely used to assess the functioning of PS II activity
(Schreiber et al. 1986; Krause and Weis 1991) and can also be used to measure photosynthetic
activity (Genty et al. 1989; Harbinson et al. 1990; Seaton and Walker 1990; Edwards and
Baker 1993; Laisk and Oja 1998). In particular, combined measurements of CO 2 exchange
and steady-state chlorophyll fluorescence have added new insights into the relationship
between chloroplast electron transport rates and carbon metabolism. This chapter does not
deal with the techniques of chlorophyll fluorescence itself but focuses on these applications.
Both O 2 evolution and O 2 uptake also take place during C3 photosynthesis (Volk and Jackson
1964; Berry et al. 1978; Canvin et al. 1980; Badger 1985). There are several 02 uptake processes but
02 evolution is derived entirely from the water-splitting reactions at PS II. This makes it possible
with isotopically labelled 02 (usually 1802) and mass spectrometry to distinguish between O 2 evo
lution at PS II (usually 1602) and 02 uptake processes (Mehler and Brown 1952; Canvin et al.
1980; Radmer and Ollinger 1980). The equations required for the interpretation of the O 2
exchange processes are discussed in the second half of this chapter.
Both measurements of chlorophyll fluorescence and 02 exchange can be used for the estima
tion of chloroplast electron transport rate in vivo. Chloroplast electron transport supplies the
chloroplast with reductant (principally reduced ferredoxin and NADPH) and ATP. The majority
of NADPH and ATP is used to power PCR and PCO cycle activity; however, there are also addi
tional pathways in the chloroplasts that use either reduced ferredoxin or NADPH. For example,
some reductant is required for the synthesis of amino acids and lipids and other synthetic pro
cesses, and NADPH can be exported out of the chloroplast via a malate shuttle (Scheibe 1987).
PS I can also transfer electrons, either directly or via reduced ferredoxin, to 02 in a process termed
°
the Mehler reaction (Mehler 1951). This photoreduction of 2 produces highly reactive superox
ide radicals, which are rapidly detoxified by the ascorbate peroxidase pathway, which further
consumes reducing equivalents (Badger 1985; Asada and Takahashi 1987). The magnitude and
function of electron flow from PS I to O 2 continues to be debated. The equations presented in this
chapter aid in the quantitative analysis of these processes in vivo.
3.2 CHLOROPHYLL FLUORESCENCE AND CHLOROPLAST ELECTRON TRANSPORT

Chlorophyll a fluorescence is a non-invasive optical tool with high diagnostic value in photo
synthesis research (Krause and Weis 1991; Bolhar-Nordenkampf and Oquist 1993). Genty et
al. (1989) showed that electron flow through PS II per unit quantum flux could be calculated
72
Chlorophyll fluorescence and oxygen exchange during C3 photosynthesis 73
Fill
r:
F,
Fa'
Figure 3.1 Definition of characteristicchlorophyll fluorescence measurements, Fa is the fluorescence yield of

a dark-adapted leaf and Fa' is the fluorescence yield of a light-adapted leaf measured in the presence of far-red
light only. Fm is the maximal fluorescence during a saturating light pulse of a dark-adapted leaf and Fm, the
maximal fluorescence duringa saturating light pulse of a leaf in the light. Fs is the fluorescence during steady
state photosynthesis, <j>P511, the electron flow through P5 II per unit quantum flux, is defined as (Fm, - Fs)IFm" in
accordance with the method of Genty et st. (1989), Non-photochemical quenching, qN' is calculated as
(Fm- Fm,)/(Fm- Fa.), Photochemical quenching, qp, is calculated as (Fm, - Fs)J(Fm' - Fd ),
from chlorophyll fluorescence measurements. This provided three different means of esti
mating chloroplast whole chain electron transport from intact leaves: from chlorophyll fluo
rescence, from measurements of 160 2 evolution and calculated from CO 2 assimilation rates.
The comparisons of these different estimates have been used in various ways to estimate
extra electron transport not associated with PCR and pco cycle activity, to calculate the
chloroplast CO 2 partial pressure, or to calculate the rates of photo respiration.
3.2.1 Calculating electron transport rate from chlorophyll fluorescence

Different fluorescence quenching parameters can be determined using pulse-modulated flu
orometry with a brief saturated light pulse (Schreiber et al. 1986; van Kooten and Snel 1990)
and these are briefly defined in Fig. 3.1. Genty et al. (1989) showed that the electron flow
through PS II per unit quantum flux, <l>PS II, was given by (F1I1 , - FJIF m" where Fm,is the flu
orescence during a brief saturating light pulse and F, is the fluorescence during steady-state
photosynthesis. This has opened up the opportunity to calculate chloroplast electron trans
port from measurements of <l>PS II (Edwards and Baker 1993).
Whole chain electron transport, Ir,
can be approximated by:
lr = ~absI(PS II) = ~absI(l- F,IF m,) (3.1)
where I is the incident irradiance, abs is the absorptance of the leaf and ~ is the fraction of
absorbed irradiance that reaches PS II. The relationship is an approximation only and breaks
down at high values of <l>PS II (Seaton and Walker 1990; Schreiber et al. 1995). In what fol
lows, a value of ~ = 0.48 is used (see Fig. 3.3) but Laisk and Loreto (1996) suggest that the
value may vary between 0.45 and 0.5.
Whole chain electron transport, lr, measured by fluorescence, measures all electron transport,
that is electron transport in support of photosynthesis, 1,1 = (4 + 4<1» V" other synthetic processes
such as nitrate reduction UNit) and light-dependent 02 uptake by the Mehler ascorbate peroxidase
reaction UAJA P ) (see Section 3.3.2.3 for detailed discussion). If lex = hfAP + lNit:
I, = L; + lex (3.2)
where lex denotes all non-photosynthetic electron transport.

..- 30
en
')'
E a erfb b.
0.3
"0
Q:)R b. 0
E ~ b. •
E: 20 0b. •
<;(
<Ii 0b. 0.2
.....
cd
.... b.
§
C
10
°
fP. 0.1
'E b.
'00
en
cd
a '" 0.0
o "----'IL.'--_--'--------'_----'-_-'------'-_-'-_.L..-----'-----'
200 400 600 800 1000

(ubar)
Figure 3.2 Simultaneous measurements of chlorophyll fluorescence and CO 2 assimilation rate, A (el, as
a function of C, for a tobacco leaf. Measurements were made at 1000 urnol quanta m-2 S-l and a leaftemperature
of 25°C and 21 % 02' Chlorophyll fluorescence parameter (j>PS II (. ) was measured on the adaxial surface. The
rate of electron transport, JA /4 (_). was calculated from the CO 2 assimilation rate A by estimating chloroplast
CO 2 partial pressure from Cc= (- Algjwith an internal diffusion conductance of gj= OJ mol m-2 s'. (Data from
Hudson et al. 1992.)
3.2.2 Estimating electron transport rate, lA' from CO 2 assimilation rate

CO 2 assimilation rate measurements can be used to calculate chloroplast electron transport
rates required for PCR and pco cycle activity by rearranging Eq. (2.23):
(A+R d)(4C+8L)
(3.3)
L:> (C-L)
(von Caemmerer and Farquhar 1981). fA has the units of umol electrons m ? S-1 and f A/4
gives the oxygen evolution rate at PS II associated with PCR and pco cycle activity. (Note
that the subscript A has been added in this chapter to distinguish the different estimates of
electron transport.) Equation (3.3) gives the NADPH-limited rate. The estimate of fA will
vary according to the equation selected from Section 2.3.4. The ATP-limited electron trans
port rate required, assuming 4H+/ATP and 3H+/e- (Eq. 2.12), is given by:
(A + R d )( 4C + 9.33L)
t, = (C-L) (3.4)
Assuming 3H+/ATP and 2H+/e- (Eq. 2.10):
(A+R d)(4.5C+ lO.5L)

t, = (C-L) (3.5)
Figure 3.2 shows the CO 2 response curve of a tobacco leaf together with the calculated elec
tron transport rate. As expected, f A /4 is constant at high CO 2 partial pressures when electron
transport is limiting A and this is one way of establishing electron transport limitation (von
Caemmerer and Farquhar 1981). At low CO 2 partial pressure, rubisco limits the actual
~ 150
E ~(j)
.g E a
"C Ql
Ql (5
til
.~ [ 100
00 .....,~
Ql
Ql ai"
~
Q
t:
0
C
Ql
Q
(j)
•
.~.
0 Ql
(j) 50
c 0
g :J
:;:::
C 's,
e
t>
.J::.
0 y = 4.99 + 1.077x (r' = 0.977)
0
Ql
m .Q
.J::.
0
Q 0 50 100 150
Electron transport rate calculated from
CO 2 assimilation rates, JA (urnol e' m'2s'1)
0.5 ,,-------,----,-------,------.--------,
b y = 0.45 + 0.48x (r' = 0.977)
0.4 I""
0.3
~
CJ)
..Q
~
.....,
0.2 •~
0.1
0.0 I I I I I "
0.0 0.2 0.4 0.6 0.8 1.0

F/Fm = 1 -lj> PS II
Figure 3.3 (a) Correlation between electron transport rate calculated from gas-exchange measurements, lA'
and from chlorophyll fluorescence, l" in tobacco. Measurements were made at 2% 2 , a leaftemperature of 25°C °
andvarying irradiance and CO 2 partial pressures. lA was calculated from Eq. (3.3). l, was calculated from Eq. (3.1)
with ~ = 0.48, The absorptance of the leafwas 0.85. (b) The same data were used to calculate the relationship
between Fs / Fm' and lA/tabs!).
electron transport rate. The fluorescence measurements of <j>PS II closely follow the estimated
electron transport rate, demonstrating the feedback of limited CO 2 fixation rates on electron
transport rates. The similarity between the shape of this curve and CO 2 assimilation rate, A
at low 02 in Fig. 2.18 is also apparent. At low 02 partial pressures little energy is required for
the PCO cycle such that A = J,4/4 under these conditions.
Figure 3.3a shows the correlation between f" calculated from gas exchange and J; calculated
from the fluorescence measurement for measurements made at low O 2 partial pressure and vari
ous CO 2 partial pressures and irradiances in tobacco. This close correlation under non
photorespiratory conditions has been demonstrated many times (e.g. Genty et al. 1989; Edwards
and Baker 1993; Ghashghaie and Cornie 1994). Figure 3.4 shows similar measurements made in
'I
en
')' 200
E
E .$
.g ecen
"0 Ii
w t5w
Cil Qj 150
E
.~ a
w E
2 2:
-
~
~
o
o..c
...:; 100
w.
U
en W
c u
co en
.:=c ~
50
0
e~
t5::
w
- >.
w..c::
0..
.Q
..c::
o 50 100 150 200
Electron transport rate calculated from
CO2 assimilation rates, J{ (umol electrons m-2 S-1)
Figure 3.4 Correlation between electron transport rate calculated from gas-exchange measurements, lA' and
from fluorescence, If, when measured at different O2 and CO 2 partial pressures as shown in Fig. 3.3 (2% O2,
closed symbols; 20% O2, hatched symbols; 40% O2, open symbols). Measurements were made on leaves of wild
type(circles) and transgenic tobacco with reduced amount of rubisco (40% ofwild-type rubisco, squares and 10%
of wild-type rubisco, triangles) at an irradiance of 850 urnol m-2 S-I, a leaftemperature of 25°C and calculations
as described in Fig. 3.3.The rate of electron transport was calculated from the CO 2 assimilation rates, A, with
Eq. (3.3) byestimating chloroplast CO 2 partial pressure from Cc = Ci- Algiwithan internal diffusion conductance
of gi= 0.3 mol m-2 S-I. Leaf absorptance was measured foreach leafand varied between 0.8 and 0.87. (Data are
redrawn from Ruuska et al. 2000b.)
wild-type and transgenic tobacco with reduced amounts of rub isco at various CO 2 and 02 partial
pressures. The good correlation between lr and JA under these conditions illustrates that Eq. (3.3)
can correctly account for energy used in the PCO cycle when measurements are made at different
02 partial pressures.
3.2.3 Relationship between l(/(absf) and FJFm,

Laisk and Loreto (1996) preferred to graph Eq. (3.1) as:
(3.6)
since FsIFm, = 1 - <jlPS II (Fig. 3.3b). This relationship has a slope of~. It intersects the Y-axis
at (J/absI)= ~ and (J/abs!) = 0 when F)F m, = 1 (Laisk and Oja 1998). If Eq. (3.2) is used to
substitute for Jf one can see the effect that extra non-photosynthetic electron transport has
on the relationship between photosynthetic electron transport JA and F) Fm ,.
(3.7)
If the extra electron transport is constant over the different measuring conditions (such as light
and CO 2 ) used to generate the relationship it will cause a parallel shift in the line. The intercept
on the Y-axis is given by (~- Je)(absI)). The X-axis intercept is given by (I - Jexl(~absI)). This for
mulation is useful at low 02 partial pressures for calibration purposes; at higher 02 partial pres
sure the estimates of JA depend on the accurate estimates of chloroplast CO 2 partial pressure.
---"--~
Chlorophyll fluorescence andoxygen exchange during C3 photosynthesis 77
150
'Ul a
•
'I
E E
a~ Ul
C
--0 .;:;
a
Q) () 100
-co-
Q)
:::; ~
' ;E a
~ ~ e_/,
Q)
"§ J-
t:' Q)
a ()
0..
Ul
c
Q)
50 e
C ()
co
~
Ul
Q)
~ 0
a :::J
nO+::
> y ~ 0.81 + 0.845x (r' ~ 0.985)
-Q) .c
W 0..
o
(;
:E 50 100 150
o
2s")
4 X 1602 evolution rates (umot m-
'Ul
160 I
'I b
E E 140 I
~
a Ul
C
--0 .;:;
a
Q) () 120
-co-Q)
E Q)
' ;:::; 0 100
Ul
Q) ::i
E
Q)
"§-;
t:'ai
a ()
0..
Ul
C
c
Q)
()
80
60 f V"
co
~
Ul
Q) A
~ 0 "
a :::J 40 A
:e=
Q)
W-§.
>
20
... ..Ai
a y= 3.562 + 0.885x (r' = 0.981)
(;
:E
o
50 100 150
4 x gross CO 2 assimilation rates
2s-')
4(A + Rd) (IlmOI m
Figure 3.5 Correlation between electron transport rate estimated from measurements of chlorophyll
fluorescence andfrom (a) the rate of 160 2 evolution and (b) electron transport ratecalculated from CO 2
assimilation rates. Measurements were made simultaneously on a tobacco leafdisc at 25°C, 2% O2 and irradiance
of 1000 urnol m-2 S-1 andvarious CO 2 partial pressures. Jf was calculated asdescribed in Fig. 3.3. (Data are
redrawn from Ruuska et al. 2000b.)
Figure 3.5a compares simultaneous measurements of electron transport rate measured as 160 2
evolution at PS II and electron transport measured by chlorophyll fluorescence. These measure
ments were first made by Genty et al. (1992). Both of these measures should capture all whole
chain electron transport and the close correlation is evident with the correlation passing close to
the origin. This appears to be the best way to test the relationship between fluorescence and elec
tron transport rate. Figure 3.sb compares the electron transport rate measured by chlorophyll
c
.2
Cti 4
'E
'00
(Jj
ctl
0'"
o
20 40 60 80
C,- Cc (ubar)
Figure 3.6 An example ofthe estimation of gj from Eq. (3.9) fora tobacco leaf. Measurements were made at
600 urnol quanta m-2 S-l and various CO 2 partial pressures at 25°(,
fluorescence with that calculated from the concomitant CO 2 fixation rates. The measurements
were made at the same time as those in Fig. 3.5a. This relationship and that of Fig. 3.4 both also
have only a very small intercept, suggesting that in these tobacco leaves electron transport to other
electron acceptors not associated with photosynthetic CO 2 assimilation is small.
3.2.4 Calculation of mesophyll conductance to CO 2 diffusion

Measurements of carbon isotope discrimination have shown that there is a significant drop
in CO 2 partial pressure from intercellular airspace to the sites of rubisco carboxylation
(Evans et al. 1986; von Caemmerer and Evans 1991; Evans and von Caemmerer 1996; Section
2.5). Concurrent measurements of gas exchange and chlorophyll fluorescence have also been
used to estimate CO 2 partial pressure at the sites of rubisco carboxylation (Di Marco et al.
1990; Harley et al. 1992a; Loreto et al. 1992; Evans and von Caemmerer 1996). Several tech
niques have been used and Harley et al. (1992a) and Evans and von Caemmerer (1996)
reviewed these.
Equation (2.23) can be rearranged to obtain an explicit expression for chloroplast CO 2 partial
pressure:
C = LUA + 8(A + Ra » (3.8)

c J,{-4(A+R a)
Concurrent measurements of chlorophyll fluorescence and gas exchange give values for A, R a
and Jf' If it is assumed that Jf = JA or one uses the relationship between Jf and JA at low 02 par
tial pressures as a calibration curve (Fig. 3.3) to obtain values for JA , one can calculate Cc with
the further assumption of a value for L. The CO 2 transfer conductance, gil can be estimated
from the slope of the relationship of A versus (C, - CJ since:
(3.9)
An example is given for a tobacco leaf in Fig. 3.6. The difficulty of this technique lies in esti
mating JA from Jf (Eq. 3.2) and quantifying the amount of electron transport not involved in
support of PCR and PCO cycle activity. Loreto et al. (1992) have used the relationship
--~--~_.. . -
~ln
C\I
I
160
E E
o (/J
.;: c
"0
_0 )U
e
ell 0) 120
"SQi
u_
- 0
~ E
0) ::::t.
§~ 80
-"')
o 0..0)
u)
(/J
C ell
ell ...
... c
-C ',=
0 40
e.!l1
13
0)
'E
.
- (/J
W (/J
ell
C\J
oo 40 80 120 160
Electron transport rate estimated from
chlorophyll fluorescence, J( (urnol electrons m-2 S-1)
Figure 3.7 Correlation betweenelectron transport rate calculated from fluorescence, Jr, and from gas exchange
on the basis of C, with an estimate of gi (open symbols) or with the assumption that C, = C, (closed symbols) for
Kalanchoe daigremontiana during phase IV of the CAM photosynthetic cycle. Irradiances were 170 (circles) and
600 (squares) umol quanta m-2 S-1. (Data taken from Maxwell et al. 1997.)
It
between fA and measured at low 02 as a calibration curve for fA (Fig. 3.3) and taken the
values of the Y-intercept as an estimate of extra electron transport. However, it is unclear
whether one can really assume that this extra electron transport is constant across different
CO 2 partial pressures or irradiances used to generate the calibration curves. Exact estimates
of absorbed irradiance and ~ are also crucial. There is also some concern that in thick leaves
errors can arise because chlorophyll fluorescence and CO 2 measurements are an average of
different chloroplast populations. Nevertheless, the fluorescence method has led to very sim
ilar estimates of gi to the ones obtained from carbon isotope measurements (Loreto et al.
1992; Evans and von Caemmerer 1996; Evans and Loreto 2000).
The importance of estimating chloroplast CO 2 partial pressure correctly before comparing
It
estimates of f" and to calculate extra electron transport from Eq. (3.2) is illustrated in the fol
lowing example. Kalanchoe daigremontiana is an obligate CAM species. Under well-watered
conditions, C 3 photosynthesis during phase IV of the CAM cycle can contribute substantially to
its daily carbon again. However, the leaf anatomy required for CAM results in a low internal con
ductance to CO 2 diffusion (Maxwell et al. 1997; see also Fig. 2.8). Figure 3.7 shows the
It
comparison between and fA calculated assuming that C, = Cc' or with the estimated internal con
ductance' gi' Estimates of fA based on the assumption that Cc = Ci ' are substantially less than those
based on estimated chloroplast CO 2 partial pressures, because the electron transport rate required
for photorespiration is underestimated.
3.2.5 Estimation of rubisco oxygenation rate from if and i A

Cornie and Briantais (1991), Comic and Ghashghaie (1991) and Ghashghaie and Cornie
(1994) have used combined measurements of chlorophyll fluorescence and CO 2 exchange to
calculate rubisco oxygenation rate and the ratio of rubisco oxygenation to carboxylation
under a variety of environmental conditions. In a similar way to the estimation of chloroplast
CO 2 partial pressure, one has to either assume that Jf = JA or correlate Jf and JA under non
photorespiratory conditions. So, from Eq. (2.7):
(3.10)
and since
(3.11)
solving for V, and substituting into Eq. (3.10) gives
(3.12)
and
(3.13)
Similarly,
(3.14)
since from Chapter 2, Eq. (2.18)
(3.15)
An additional assumption of T, will also give an estimate of chloroplast CO 2 partial pressure,

or an independent estimate of C c will provide an in vivo estimate of rubisco's relative specific
ity, Sclo (Eq. 2.17). Cornie and Briantais (1991) and Cornie and Ghashghaie (1991) have used
these techniques to estimate chloroplastic CO 2 partial pressure in drought-stressed leaves
and demonstrated that the reduction in CO 2 assimilation rate can largely be explained by a
reduction in CO 2 partial pressure. However, they had to assume that there was no electron
transport rate to other electron sinks.
In summary, the comparisons of electron transport calculated from gas exchange and fluores
cence are interesting. However, the demonstration by von Caemmerer and Evans (1991) and
Maxwell et al. (1997) that chloroplast CO 2 partial pressure can be substantially less than intercel
lular CO 2 partial pressure has complicated the analysis of estimating extra electron transport not
associated with CO 2 fixation. Conversely, uncertainties of the magnitude and variations in extra
electron transport make it difficult to confidently estimate chloroplast CO 2 partial pressure from
chlorophyll fluorescence measurements.
3.3 OXYGEN EXCHANGE DURING (3 PHOTOSYNTHESIS
3.3.1 Introduction
Both 02 evolution and O 2 uptake take place during C 3 photosynthesis (Volk and Jackson
1964; Berry et al. 1978; Canvin et al. 1980; Badger 1985). 02 evolution is derived entirely
from the water-splitting reactions at PS II, but there are several O 2 uptake processes. These
are the oxygen uptake by rubisco and the associated 02 consumption by glycolate oxidase in
the PCO cycle, the Mehler ascorbate-peroxidase (MAP) reaction (Mehler 1951; Asada and
°
Takahashi 1987), which results in direct photo-oxidation of 2 , and 02 uptake associated
with mitochondrial respiration in the light. The stoichiometry of the CO 2 and 02 fluxes is
such that approximately 1 mol of 02 is evolved for each mol of CO 2 fixed. Net 02 exchange is
frequently measured with simple O 2 electrodes (Walker 1987, 1989).
Oxygen exchange studies have been helped by the fact that 02 evolution at PS II is derived
from the water splitting and there is no discrimination such that the isotopic composition of O 2 is
that of the water from which it is derived (Guy et al. 1993; Yakir et al. 1992). The O 2 uptake pro
cesses consume 02 in proportion to the isotopic abundance of 02 in the surrounding air. Thus it
has been possible, with isotopically labelled O 2 (usually 1802) and mass spectrometry, to distin
guish between 02 evolution (usually 1602) and 02 uptake processes (Mehler and Brown 1952;
Canvin et al. 1980; Gerbaud and Andre 1980; Radmer and Ollinger 1980). These measurements
have also been combined with measurements of chlorophyll fluorescence (Genty et al. 1992;
Maxwell et al. 1998).
Here the basic 02 exchange equations are derived from the C 3 model and compared to results
of mass spectrometric gas-exchange measurements of 16 0 2 and 18 0 2 exchange.
3.3.2 Basic equations

3.3.2.1 Total oxygen evolution
The reduction of one NADP requires the transfer of two electrons through the whole chain
electron transport. During this process ~02 is evolved. Thus the evolution of 1 mol of 02
requires the transfer of 4 mol of electrons. In Chapter 2, the rate of whole chain electron
transport required to support NADPH consumption by the PCR and PCO cycles during CO 2
fixation was shown to be:
JA = 4V(+4Va = (4+8L/C)Vc (3.16)
The total 02 evolution (in support of PCR and PCO cycle activity) at PS II derived from the
splitting of H 2 0 is therefore:
EA O = JA / 4 = V c+ Va = (1 + 2L/C)V( (3.17)
If we consider the ATP requirements of the PCR and the PCO cycles there are several options.
If we assume that ATP requirements are met by whole chain electron transport and that 4
mol H+ are required for the synthesis of 1 mol ATP and that 3H+ are produced per electron
(this assumes the operation of a Q cycle, see Section 2.3.4.1) then:
JA = 4Vc+4.66Va = (4+9.32L/C)V( (3.18)
and the total oxygen evolution is given by:
£.40 = (1 + 2.33L/ C) V( (3.19)
With this assumption there is only a small difference between the electron transport rate
required for NADPH consumption and that required for ATP consumption.
If we assume that three protons are required per ATP and no Q cycle activity (2H+/e-) then:
1.4 = 4.5 V( + 5.25 v, = (4.5 + 10.5rJ C) V( (3.20)
and
£.40 = (1.l25+2.625L/C)V( (3.21)
If we assume three protons per ATP and the operation of the Q cycle (3H+/e-):
EA O = (0.75 + l.75L/C)Vc (3.22)

In this case the rate of 02 evolution required to satisfy the ATP requirements is less than that
for the NADPH requirements (Eq. 3.17). Equation (3.17) has been used to describe the O 2
evolution required to sustain PCR and PCO cycle activity.
Following the basic model outlined in Chapter 2, it is assumed that the carboxylation rate, V c'
can be either RuBP saturated or limited by the rate of RuBP regeneration and/or electron trans
port rate. This gives two expressions for the rate of 02 evolution: the rubisco-limited rate is
derived by substituting Eq. (1.9) into Eq. (3.17):
(3.23)
The electron-transport-limited rate is derived by solving Eq. (3.16) for Vc and substituting
into Eq. (3.17):
Ei = l/4 (3.24)
(3.25)
The 02 evolution described above does not include whole chain electron transport to other
electron acceptors, such as nitrate reduction or 02' The magnitude of these fluxes may be as
high as 10% of the total and is likely to be variable (for example it may be greater in young
leaves; Farquhar 1988). With this extra oxygen evolution Eex = Je) 4 then the total rate of
oxygen evolution is:
(3.26)
3.3.2.2 Total oxygen uptake

Rubisco oxygenase activity is a major component of the leaf's oxygen uptake processes. One
mol of 02 is consumed per mol of RuBP oxygenated and a further half mol of 02 is con
sumed in the PCO cycle by glycolate oxidase in the peroxisomes (Badger 1985). Thus the
rubisco-linked O 2 uptake processes are given by 1.5 Va = 31. VJc.
Mitochondrial respiration is likely to continue in the light, which also consumes 02 (Hoefna
gel et al. 1998). In the simulations of this chapter the rate of mitochondrial 02 consumption has
been set equal to the rate of mitochondrial CO 2 evolution (Rd ) . Less certain is the occurrence of
the MAP reaction, which results in the direct photoreduction of 02 (see Section 3.3.2.3).
The total 02 consumption rate is given by:
(3.27)
As before, there are two equations: one for the RuBP-saturated and one for the RuBP-limited
oxygenation rate. When rubisco is limiting:
(3.28)
and when RuBP regeneration is limiting:
(3.29)
as before.T, is given by Eq. (2.16) and:
(3.30)
CD
202-+ 2H+ -------- ... H 20 2 + 02
~r
o
,
', .2HP
¥- --(t)--' "
02 +4H+ Fd,ec,
.,
MDH ASCA
4e-
" ® 'f
[ PSII • PSI I 4 Fd
I
ox
'\
H+
"
............
_-----a.
I I ,
\ , NADP+
" " 2 Fd red NADPH
2Hp
,
,-------- ....
,,
NADP++H+
Figure 3.8 Schematic representation of the MAP reaction (solid lines) and associated scavenging reactions
(dashed lines). ASCA, ascorbate; MDH, monohydroascorbate; (1) superoxide dismutase; (2) ascorbate peroxidase;
(3) various MDH reducing reactions.
3.3.2.3 The Mehlerascorbate peroxidase (MAP) reaction

In the absence of other electron acceptors, electrons can be transferred from PS I to oxygen
to form superoxide radicals (Mehler 1951; Badger 1985; Foyer 1997). These superoxide radi
cals are rapidly detoxified by the MAP pathway, which further consumes reducing equiva
lents (Badger 1985; Asada and Takahashi 1987; Asada 1994, 1996, 1999). The schematic
drawing in Fig. 3.8 shows the stoichiometry involved. First, superoxide dismutase, localized
in the thylakoid membrane, catalyses the dismutation of superoxide into hydrogen peroxide
and oxygen (Fig. 3.8). Hydrogen peroxide also has damaging effects on many enzymes and is
removed by oxidation of ascorbate (ASCA) to monodehydroascorbate (MDA) by ascorbate
peroxidase. There are several ways, enzymatic and non-enzymatic, that MDA can be reduced
to ASCA via reduced ferredoxin or NADPH-dependent reactions (Badger 1985; Asada 1999;
Foyer 1997). The MAP reaction and the associated scavenging mechanisms give no net
exchange of any of the reactants involved, but importantly also require whole chain electron
transport to ferredoxin and the reduction ofNADP+ (Badger 1985; Fig. 3.8). The oxygen sto
ichiometry is such that for every 2 mol of O 2 consumed in the MAP reaction, 1 mol of 02 is
°
released by superoxide dismutase and 1 mol of 2 is produced through the splitting of water
at PS II.
Of all the extra electron sinks, the MAP reaction has attracted the most attention. Neverthe
less, at the moment the occurrence and significance of MAP reaction in vivo is unclear (for review
see Osmond and Grace 1995). Mass-spectrometric measurements of mesophyll cells (Furbank et
al. 1982) or whole leaves (Canvin et al. 1980; Gerbaud and Andre 1980) have reported O 2 uptake
in light, which could not be accounted for by rubisco oxygenation or mitochondrial respiration.
However, Kent et al. (1992) and Ruuska et al. (2000b) did not record any extra 02 consumption in
their measurements. It is thought that the MAP reaction occurs especially when the ferredoxin
pool is highly reduced, thus allowing linear electron flow to continue under limited NADPH con
sumption, as may occur at low CO 2 partial pressures (Neubauer and Yamamoto 1992). It has also
been suggested that the MAP reaction can assist in development and maintenance of high ~pH,
which in turn enhances non-radiative dissipation of light energy and can protect light reactions
1.8
....
Q)
Q)
a
0..
Q)
~
C
co 0
to... :.;::::; 1.7
C 0..
0 E
li ::::l
(/)
E c0 1.6
::::l
(/) o
C I
8 a..
a..
f-«
«z
° 1.5
1.3
"c
c,
<C
-:::"' 1.2 3H+/ATP, no Q cycle

i'o
....,"'
1.1
4H+/ATP, Q cycle
1.0 '-----_---'---_-----''-----_~_~_ _
o 200 400 600 800 1000
(ubar)
Figure 3.9 (a) Ratio of the rates of AlP and NADPH consumption of the PCR and peo cycles at different
chloroplastic CO 2 partial pressures (Eqs 2.5and 2.6). (b) Ratio of the associated electron transport rates required
to satisfy the AlP (iATP) and NADPH (iNADPH) consumption of the PCR and peo cycles assuming either 3WIAlP
and no Q cycle or 4WIAlP and Q cycle activity.
from photodamage (Schreiber and Neubauer 1990; Neubauer and Yamamoto 1992; Bjorkman
and Demmig-Adams 1995).
It has also been thought that alternative electron sinks such as 02 are necessary to enable AIP
production without NADP reduction to meet the different AIP and NADPH requirements of the
PCR and PCO cycles (Foyer et al. 1990; for review see Noctor and Foyer 1998). However, the need
°
for extra electron transport to 2 depends very much on the stoichiometries used to calculate AIP
production. The ratio of the rates of AIP consumption to NADPH consumption ranges from 1.75
to 1.5 with varying CO 2 partial pressure (Fig. 3.9a, Eqs 2.5, 2.6 and 2.18). The effect this has on the
electron transport rate requirements can be calculated from the ratios of Eqs (3.16) and (3.20) or
Eqs (3.16) and (3.18). When it is assumed that three protons per AIP are required and 2H+ are
transferred per electron, the shortfall in electron transport rate from NADPH reduction can be as
high as 30% at low CO 2 partial pressure and declines to 12% at high CO 2 partial pressure (Fig.
3.9b). However, if the Q cycle is assumed to operate and it is assumed that four protons per AIP
are required the shortfall is much less, with an extra requirement of 10% at low CO 2 partial pres
sures, with no requirement at high CO 2 (Fig. 3.9b).
It has been suggested that 02 uptake at PS I could meet the differential needs for AIP and
NADPH demand and the required 02 uptake can be calculated from equations in Section 3.3.2.
Assuming 4H+/AIP and 3H+/e- the difference between Eqs (3.18) and (3.16) requires a rate of O 2
uptake at PS I of:
(3.31)
a
25 r
oe 0
20
15
r-
m
"1
E 10
0
E
.6 5 is' ~ b.
m
Q)
100
~ lb
Q)
OJ
t:
ell
s: 80
U
x ~ 0
Q)
0''' 60 I /0
0
"0
t:
ell 40
0'" I'#' / ~~
2:~ , ~----~ 1
500 1000 1500
External CO 2 partial pressure ( ubar )
Figure 3.10 Effect of CO 2 on 02 exchange in Helianthus annuus (sunflower) at irradiances of (a) 300and
(b) 1800urnol m-2 S-1 and a leaf temperature of 28°C. 160 2 evolution (0). 180 2 uptake (~), net 02 evolution (e)
and net CO 2 assimilation rate(A). Data aretaken from Badger (1985).
and, assuming 3H+/ATP and 2H+/e-:
U MAP = (0.5 + 2.5~ )Vc (3.32)
If the complete MAP reaction occurs as outlined in Fig. 3.8, this requires an extra electron
transport of rVIAP = 2 U MA p. Oxygen is evolved at a rate of 1/2 U MA P at PS II and released at a
rate of 112U,V1AP by superoxide dismutase (Fig. 3.8) such that the total O 2 evolution associated
with MAP reaction equals UiV1AP'
Nevertheless, there are other mechanisms by which the chloroplast may meet the conflicting
ATP and NADPH requirements. The electron flow in support of nitrate assimilation also reduced
the disparity and may be sufficient. Other mechanisms such as cyclic electron transport may also
operate.
3.3.2.4 Net O2 andCO2 exchange
The net O 2 exchange is the sum of Eqs (3.26) and (3.27). Together with Eq. (3.17) and:
Eex = INi' U \HP

- 4 + .. (3.33 )
i.; = (1 + C2L) v, + i.:

4 + U.\.fAP - [(3L)
C v, + l
U M A P + Rd...J
(3.34)
= (1 - L)
C V, - R + i:4 d
30 ,-----.-----,-...-----,---,------r-,-----_
a
20
r----------------
Of--L-+--+--f---+-+----j---+-----"-I
b /-----------
/
/
/
20 I
I
I
I
··f.•....••
10 .
R, __
O'------L---'-----'----'---------'-----'----------'-----'--------..J
o 200 400 600 800
Cc (ubar)
Figure 3.11 Modelled response of CO 2 and 02 exchange at irradiances of (a) 200 urnol quanta m-2 s' and
(b) 1500urnol quanta m-2 s'. Model equations werecalculated with the standard parameters at 25°C. UMAP and
Eex = 0 and Rd =1 urnot m-2 S-1. Vcmax =80 urnol m-2 S-1, Jmax = 120umol m-2 S-1. 02 uptake is represented by a
dotted line, total 02 evolution by a dashed line and net 02 evolution and net CO 2 assimilation by a solid line.
This compares with a net CO 2 exchange of:
(3.35)
It has been assumed here that mitochondrial 02 uptake equals mitochondrial CO 2 release,
although this need not always be the case. Net 02 exchange may thus marginally exceed net
CO 2 exchange but this is rarely observed (Canvin et al. 1980; Badger 1985). However, no
detailed investigations have been made on young leaves where one might expect amino acid
synthesis to be high. The elemental composition of plant material indicates that, integrated
over time, 02 evolution exceeds CO 2 uptake by 11% (McDermitt and Loomis 1981; Farquhar
1988). The MAP reaction is associated with equal amounts of 02 evolution and uptake so
that it is not seen as a discrepancy between net CO 2 and 02 exchange.
3.3.2.5 Estimation of rubisco carboxylation andoxygenation rates
Similar to the approach taken in Section 3.2.5, measurements of 16 0 2 evolution and 1802
uptake can, with some assumptions, be used to estimate the rate of rubisco carboxylation
and oxygenation and chloroplastic CO 2 partial pressure (Renou et al. 1990; Kent et al. 1992;
Tourneaux and Peltier 1995). From Eq. (3.27):
(3.36)
- r
a
30 EAO
en
<'i
E 20
0
E
~ r
en
Q) 10 ~ u
"§
Q)
C)
c
ell 0
s: I b
(J
x
Q)
cv
30 t--,---.. ___E AO
0
o
"0 20 I
c <, II ---~_.-
ell
0 '" »:
10 ~ ,/
, »:
.;~;
-
~J'
,
oL
0 200 400 600 800
02 (mbar)
Figure 3.12 Modelled 02 response of CO 2 and 02 exchange at (a) 1000and(b) 250 ubarCO 2 at an irradiance
of 1000umol m-2 S-l. Model equations were calculated with the standard parameters at 25°C. UMAP and Eex = 0
and Rd = 1 urnol m-2 S-1. Vcmax = 80 urno] m-2 S-l, lmax = 120 urnol m-2 s".
and from Eqs (3.17) and (3.26):
IN;'
V c = ET o - UM A P - 4"" - Va (3.37)
Usually, R d is estimated from the measurements of R d in darkness and UM A P and I ni / 4 are

assumed to be zero such that ETa = EA O and:
2
V, = B w - (3.38)
3(U-Rd )
and Ccis then calculated from Eq. (3.15) (Tourneaux and Peltier 1995).
3.3.3 The CO2 dependence of 02 exchange

Figure 3.10 shows an example of 1602 and 1802 uptake for a sunflower leaf in response to
changing CO 2 partial pressure. There is little difference between the net 02 and CO 2
exchange at both irradiances. At low light the 160 2 evolution is constant, indicative of an
electron transport limitation, whereas at high light it declines at low CO 2 , as CO 2 assimila
tion is rubisco limited. 02 uptake declines in both cases with increasing CO 2, reflecting the
inhibition of oxygenase activity. The substantial 02 consumption at high external CO 2 is
probably still oxygenase-dependent 02 uptake since stomatal closure at high CO 2 can lead to
substantially lower internal CO 2 partial pressure. The pattern of 160 2 evolution is similar to
the pattern of calculated electron transport rate and that of <l>PS II (Fig. 3.2). The model of 02
exchange mimics these results (Fig. 3.11). It predicts that both 02 evolution and uptake
processes alternate from being rubisco limited at low CO 2 and high light to being driven by
20
t-. .,<
ro '7~ 15 . r
r<
•• <
aJ r
Cl
"!cn .
,"
r
->
C
C1l
E r
/
s:
o (5 10 ,
,,
r:
X E
aJ 3
0'" 5
0
2.5
2.0
----------
~o 1.5
,
---~c 1.0 , ,
0.5 "
0.0
100
Co
75
.0
3 50
t-.
25
0
0 100 200 300 400 500
02 (mbar)
Figure 3.13 Modelled 02 response of CO 2 and 02 exchange at the compensation point at an irradiance of 1000
urnol m-2 s'. Model equations werecalculated with the standard parameters at 25°C. Vemax = 80 urnol m-2 s',
lmax = 120urnol m-2 s', UMAP and Eex = 0 and Rd = 1 urnol m-2 S-1 (dashed line). Rd = 0 urnol m-2 S-l (solid line).
Also shown are the ratio of oxygenation to carboxylation, VolVe and the compensation point, F.
electron transport limitation at low light and/or high CO 2, The model predicts substantial 02
uptake at 800 ubar CO 2 with the present kinetic constants of rubisco. The arrow points to the
contribution of mitochondrial respiration to 02 uptake. The model does not mimic the
decline in 02 uptake at low CO 2 in Fig. 3.10. This decline may have been related to changes in
stomatal movement or changes in rubisco activation at low CO 2 (von Caemmerer and
Edmondson 1986; Figs 1.7 and 1.8)
3.3.4 The 02 dependence of 02 exchange

The 02 dependence of O 2 exchange in this model reflects the kinetics of rubisco oxygenase
since a MAP component was not added to the model in these simulations (Fig. 3.12). At high
CO 2 , where electron transport limits the rate of O 2 exchange, and O 2 evolution is constant,
02 uptake increases with increasing 02 partial pressure as energy is diverted from rubisco
carboxylation to oxygenation and net 02 and CO 2 exchange decline. At lower CO 2 partial
pressure, O 2 exchange is electron-transport-limited at low 02 and becomes rubisco-limited
at higher 02 partial pressure. The transition from one limitation to another makes it difficult
to derive rubisco kinetic constants from these curves in wild-type plants. However, trans
genic tobacco with reduced amount of rubisco could be used very effectively to examine
rubisco oxygenase activity as has already been done for the CO 2 exchange measurements
(von Caemmerer et al. 1994; Chapter 1).
40
a
30
Ac
20
10
-en 0
~
E I b / Eco
E 30
~
Q)
Cl
t ·
c 20
co /,
J\
..c
o
x
Q)
E~o.- /./,
10 .
0 '"
"0
C
co
0
0 '"
o
30 l C Uc .:
20
10
Figure 3.14 Modelled temperature response of CO 2 and 02 exchange at 250 ubarCO 2 and an irradiance of
1000 urnol m-2 s'. Model equations were calculated with the standard parameters at 25°C. UMAP and Eex = 0 and
Rd = 1 urnol m-2 S-1. Vcmax = 80 urnol m-2 s', lmax= 120 urnol m-2 S-I. (a) Net CO 2 and 02 exchange and
mitochondrial respiration rate, (b) 02 evolution and (c) total 02 uptake and mitochondrial 02 uptake. The dotted
line and their extensions are the rubisco-limited rates; the dashed lines and their extensions arethe electron
transport-limited rates.
3.3.5 The O2 exchange at the compensation point

Figure 3.13 shows the expected 02 evolution and uptake pattern at the compensation point
with and without mitochondrial respiration. By definition the net exchange is zero and both
uptake and evolution increase with increasing 02 as the CO 2 compensation point increases
linearly with 02' In the absence of mitochondrial respiration, r = L and ValVe = 2 at all 02
partial pressures since VolVe = 2L/C[' Since there is no net flux at the compensation point,
ambient CO 2 equals chloroplastic CO 2 partial pressure. Many of the gas-exchange systems
where 160 2 and 180 2 are studied are closed systems, where the compensation point is easily
measured (Canvin et al. 1980). It is, however, important to note that in the presence of mito
chondrial respiration the ratio of VjVe is less than 2 and dependent on 02 partial pressure
(Fig. 3.13). Since Ce = Ca at the compensation point the calculations described in Section
3.3.2.4 can be used to estimate Land Sclo'
3.3.6 The temperature dependence of 02 exchange

The ratio of oxygenase to carboxylase increases with increasing 02 partial pressure (Chapters
1 and 5; Fig. 5.3, page 128) and this is apparent in the temperature dependence shown in Fig.
3.14. In Fig. 3.14, net CO 2 and 02 exchange, as well as total 02 evolution and consumptions,
are shown. Also shown are the rubisco- and electron-transport-limited rates. Rubisco limits
CO 2 and 02 exchange at lower temperatures whereas the electron transport limits exchange
at higher temperatures. There the shape of the curve is determined by the temperature
dependence of electron transport (Fig. 2.10, page 47). The electron transport limitation
effects a decrease in total 02 consumption; even so, the ratio of oxygenase to carboxylase
activity continues to increase. The unusual shape of the 02 uptake curve is the result of
increasing respiratory O 2 uptake with increasing temperature. An increase of respiratory CO 2
evolution is also the reason for the decrease of rubisco-limited net CO 2 exchange (Fig. 3.14a).
4
Modelling (4 photosynthesis
4.1 INTRODUCTION
C 4 photosynthesis requires the coordinated functioning of mesophyll and bundle-sheath
cells of leaves and is characterized by a CO 2 concentrating mechanism which allows rubisco,
located in the bundle-sheath cells, to operate at high CO 2 partial pressures. This overcomes
the low affinity rubisco has for CO 2 and largely inhibits its oxygenation reaction, reducing
photorespiration rates. In the mesophyll, CO 2 is initially fixed by PEP carboxylase into C 4
acids, which are then decarboxylated in the bundle sheath to supply CO 2 for rubisco. The
coordinated functioning of C 4 photosynthesis requires a very specialized leaf anatomy where
photosynthetic cells are organized in two concentric cylinders. Thin-walled mesophyll cells
adjacent to intercellular airspace radiate from thick-walled bundle-sheath cells, adjacent to
the vasculature (Hatch 1987). Both the structure of the bundle-sheath wall (which has a low
permeability to CO 2 ) and the relative biochemical capacities of the C 3 cycle in the bundle
sheath and C 4 acid cycle (which operates across the mesophyll bundle-sheath interface) con
tribute to the high CO 2 partial pressure in the bundle sheath.
The mathematical modelling of the C4 pathway is not as frequently used as that of the C3
photosynthetic pathway. The complexity inherent in the two compartments of the C 4 photosyn
thetic mechanism is necessarily reflected in the complexity of accurate C 4 models. Nevertheless,
many of the gas-exchange characteristics observed with intact leaves have been predicted using
models based on biochemical and anatomical characteristics. The biochemical model considered
in this chapter is based on those of Berry and Farquhar (1978) and Peisker (1979) (see also Peisker
1986 and Peisker and Henderson 1992). These two models were very similar in their basic design
and differ only in details. Collatz et al. (1992) have modified the model by Berry and Farquhar
(1978) so that it could be coupled to a stomatal model. He and Edwards (1996) have used these
models to estimate diffusive resistances of the bundle sheath.
Here we build upon these previous models and test the resultant model by exploring the
relationships between gas-exchange characteristics and leaf biochemistry as described by von Cae
mmerer and Furbank (1999).
4.2 BASIC MODEL EQUATIONS

Figure 4.1 shows a schematic representation of the proposed carbon fluxes in C 4 photo
synthesis. C 4 acids are generated by CO 2 fixation by PEP carboxylase in the mesophyll
cytosol, then diffuse to the bundle-sheath cells where they are decarboxylated. Rubisco and
the complete C 3 photosynthetic pathway are located in the bundle-sheath cells, bounded by a
relatively gas-tight cell wall such that the C 3 cycle relies almost entirely on C 4 acid decarbox
ylation as its source of CO 2 ,
91
mesophyll cell bundle sheath cell
rjJL m ....-----1f--
T ,PEP
HCO~Y_C_l_e -+..
Vp
Figure 4.1 Schematic representation of the main features of the C4 model. CO 2 diffuses into the mesophyll cell
where it is converted to HC0 3- andfixed by phosphoenonyruvate (PEP) carboxylase at the rate Vp. It is assumed
that C4 acid decarboxylation in the bundle sheath occurs at the same rate. In the bundle sheath, CO 2 released by
C4 acid decarboxylation either leaks back to the mesophyll (L) or is fixed by rubisco (Ve) in the photosynthetic
carbon reduction (PCR) cycle. In the photosynthetic carbon oxidation (PCO) cycle, CO 2 is released at half the rate
of rubisco oxygenations (Vo)' CO 2 is also released in the mesophyll and bundle sheath from mitochondrial
respiration (Rm, Rs ) not involved in photorespiration.
The net rate of CO 2 fixation for C 4 photosynthesis can be given by two equations. The first
describes rubisco carboxylation in the bundle sheath. Since all carbon fixed into sugars ultimately
must be fixed by rubisco, overall CO 2 assimilation, A, can be given by:
(4.1 )
where V[ and Vo are the rates of rubisco carboxylation and oxygenation and R d is the rate of
mitochondrial respiration not associated with photorespiration. This equation is identical to
Eq. (2.1).
Mitochondrial respiration may occur in the mesophyll as well as in the bundle sheath. As
rubisco may more readily refix CO 2 released in the bundle sheath, R d is described by its mesophyll
and bundle-sheath components:
Rd = Rill + R, (4.2)
Because the bundle-sheath compartment is a semi-closed system and is dependent for its
supply of CO 2 on the decarboxylation of C 4 acids formed in the mesophyll cells, the CO 2
assimilation rate, A, can also be written in terms of the mesophyll reactions as:
(4.3)
where V p is the rate of PEP carboxylation, R m is the mitochondrial respiration occurring in

the mesophyll and L is the rate of CO 2 leakage from the bundle sheath to the mesophyll (Fig.
4.1). The leakage, L, is given by:
(4.4)
where gs is the physical conductance to CO 2 leakage and is determined by the properties of

the bundle-sheath cell wall; Cs and C m are the bundle-sheath and mesophyll CO 2 partial
PEP carboxylation rate
~'"
c:e
....
<l,)
eo
§ Assimilation rate
..<::
o
x
<l,)
d"
u
(C, - Cm )
-"'--
c, C,
Mesophyll, Cm , or bundle-sheath, C
s '
CO 2 partial pressure
Figure4.2 Graphical summary of the main features ofthe C4 photosynthetic pathway. The two curves are the
rate of phosphoenolpyruvate (PEP) carboxylation, Vp, as a function of mesophyll CO 2 partial pressure, Cm' and
CO 2 assimilation rate, A, as a function of bundle-sheath CO 2 partial pressure, C5 • The slope of the arrow
connecting the two curves gives the bundle-sheath conductance g5 =LI{C5- Cm) and the leak rate L= Vp - A.
pressures. It is assumed that there is a negligible amount of HCO 3- leakage from the bundle
sheath since the HC0 3 - pool should be small due to the absence of carbonic anhydrase activ
ity in these cells (Farquhar 1983; Jenkins et al. 1989; Ludwig et al. 1997).
The C 4 cycle consumes additional energy during the regeneration of PEP, and leakage of CO 2
from the bundle sheath is an energy cost to the leaf. This represents a compromise between retain
ing CO 2, allowing efflux of 02 and permitting metabolites to diffuse in and out at rates fast
enough to support the rate of CO 2 fixation (Hatch and Osmond 1976; Raven 1977). The CO 2
leakage depends upon the balance between the rates of PEP carboxylation and rubisco activity and
the conductance of the bundle sheath to CO 2, Leakiness (<1» defines leakage as a fraction of the
rate of PEP carboxylation and thus describes the efficiency of the C 4 cycle:
<1> = L/Vp (4.5)
A related term 'overcycling' has also frequently been used (Jenkins et al. 1989; Furbank et al.
1990). Overcycling defines leakage as a fraction of CO 2 assimilation rate and gives the frac
tion by which the flux through the C 4 acid cycle has to exceed net CO 2 assimilation rate:
L (Vp - (A + Rill))
Overcycling =A A
(4.6)
Figure 4.2 gives a graphical summary of the basic properties of C 4 photosynthesis (Eqs 4.3
and 4.4). The rate of PEP carboxylation, Vp ' is indicated at a particular mesophyll CO 2 partial
pressure, Cm' The slope of the arrow connecting the rate of PEP carboxylation with bundle
sheath CO 2 assimilation rate, A, gives the magnitude of the bundle-sheath conductance
(since by definition gs = L/(C, - e",)). The leak rate L is given by Vp - A. This graphical
summary is used later in Figs 4.5 and 4.6 to illustrate the effect of different bundle-sheath
conductances or photosynthetic capacities of the bundle sheath.
4.2.1 Equations for enzyme-limited photosynthesis

Many important features of the C 4 model can be examined with the enzyme-limited rates,
which are presumed to be appropriate under conditions of high irradiance. This is consid
ered first before discussing the more complex light- and electron-transport-limited photo
synthesis.
4.2.1.1 CO2 assimilation rate in the bundle sheath
As is the case in C 3 models of photosynthesis (Chapters 1 and 2; Farquhar et al. 1980; Farqu
har and von Caemmerer 1982) rubisco carboxylation at high irradiance can be described by
its RuBP-saturated rate:
(4.7)
where O, is the 02 partial pressure in the bundle sheath. Following the oxygenation of 1 mol
of RuBP, 0.5 mol of CO 2 is evolved in the photorespiratory pathway and the ratio of oxygen
ation to carboxylation can be expressed as:
(4.8)
where L is the CO 2 compensation point in a C 3 plant in the absence of other mitochondrial

respiration, and:
(4.9)
where the term in parentheses is the reciprocal of rubisco specificity, Sclo' In what follows the 02
dependence of L is highlighted since the 02 partial pressure in the bundle sheath may vary.
The rubisco-limited rate of CO 2 assimilation can be given by:
(4.10)
which is comparable with Eq. (2.20). The RuBP-regeneration or electron-transport-limited

rate is discussed in Section 4.2.2.
4.2.1.2 Bundle-sheath CO2 partial pressure
To derive an overall expression for CO 2 assimilation rate as a function of mesophyll CO 2 and
02 partial pressures, C m and Om' one needs to derive an expression for Cs and 0 s, Equation
(4.10) can be used to derive an expression for Cs:
(4.11 )
This equation is analogous to the equation for the C 3 compensation point (Eq. 2.38). If V cm ax
could be estimated accurately from biochemical measurements together with A it would pro
vide a means of estimating bundle-sheath CO 2 partial pressure. One can also obtain an
expression for Cs from Eq. (4.3):
Cs (4.12)
4.2.1.3 Bundle-sheath 02 partial pressure

PS II activity and 02 evolution in the bundle sheath varies widely amongst the C4 species. Some
NADP-malic enzyme (NADP-ME) species such as Zea mays and Sorghum bieolor have little or
none, whereas NADP-ME dicots and NAD and phosphoenolpyruvate carboxykinase (PCK) spe
cies can have high PS II activity (Chapman et al. 1980; Hatch 1987; Pfundel et al. 1996; Pfundel
and Pfeffer 1997). Because the bundle sheath is a fairly gas-tight compartment, this has implica
tions for the steady-state 02 partial pressure of the bundle sheath (Raven 1977; Berry and Farqu
har 1978). Following Berry and Farquhar (1978), it is assumed that the net 02 evolution, Eo' in the
bundle-sheath cells equals its leakage, Lo' out of the bundle sheath, that is:
Eo = L; = go(Os - Om) (4.13)
The conductance to leakage of 02 across the bundle sheath, go' can be related to the conduc
tance to CO 2 by way of the ratio of diffusivities and solubilities by:
go = g/(Do,So,)/(Dco,Sco,)) (4.14 )
where D oz and D e 0 2 are the diffusivities for 02 and CO 2 in water, respectively and SOz and
Seoz are the respective Henry constants such that:
go = 0.047g, (4.15 )
at 25°C (Berry and Farquhar 1978; Farquhar 1983). If Eo = aA, where a (0 < a> 1) denotes
the fraction of 02 evolution occurring in the bundle sheath, then:
o, = ~+0111
0.047 s.
(4.16)
4.2.1.4 The rateof PEP carboxylation

Like Berry and Farquhar (1978), it is assumed that a steady-state balance exists between the
rate of PEP carboxylation and the release of C 4 acids in the bundle sheath. Furthermore, it is
assumed that PEP carboxylation provides the rate-limiting step and not, for example, the
rate of hydration of CO 2 by carbonic anhydrase. As PEP carboxylase utilizes HC0 3- rather
than CO 2, hydration of CO 2 is really the first step in carbon fixation in C 4 species (Hatch and
Burnell 1990).
When CO 2 is limiting, the rate of PEP carboxylation is given by a Michaelis-Menten equation:
CI11Vpmax
(4.17)
Vp = c, + K p
where V pmax is the maximum PEP carboxylation rate and K p is the Michaelis-Menten con
stant for CO 2, This assumes that the substrate PEP is saturating under these conditions.
When the rate of PEP regeneration is limiting, for example by the capacity of pyruvate ortho
phosphate dikinase (PPDK), then:
V p = V pr (4.18)
where Vpr is a constant (Peisker 1986; Peisker and Henderson 1992) and:
Vp = rrnn
. {CmVpmax
C
V }
' pr (4.19)
I11+ Kp
4.2.1.5 Quadratic expression for the enzyme-limited CO2 assimilation rate

To obtain an overall rate equation for CO 2 assimilation as a function of the mesophyll CO 2
and 02 partial pressures (C m and Om) one combines Eqs (4.10), (4.11) and (4.16). The result
ing expression is a quadratic of the form:
,
aA~ + bAe + C =0 (4.20)
where
2
Ac =- b - Jb
2a
- 4ac
(4.21)
and
a i;
a = 1--- (4.22)
0.047 x,
b = -{ (Vp - e; + gsC I11 ) + (Vemax - R d ) + g,(Ke( 1 + Oml K o ) ) (4.23)
+ (0.~47(Y' v.., + RdKJK o) ) }
(4.24)
Equation (4.20) can be approximated by:
A e = min {( V p + g,C", - R",), (V emax - R d ) } (4.25)
where 'min { }' stands for 'minimum of'.

At low CO 2 partial pressures, COz assimilation rate is given by:
(4.26)
and linearly related to the maximum PEP carboxylase activity, Vp m ax ' The product gsCm is the
inward diffusion of CO 2 into the bundle sheath and, because gs is low (0.003 mol m- 2 S-1), the
flux is only 0.3 umol m- z s-1 at a Cm of 100 ubar and can thus be ignored. At high CO 2 partial
pressures, CO 2 assimilation rate is given by either the maximal rubisco activity, V emax' or the
rate of PEP regeneration (Vpr ) '
4.2.2 Light- and electron-transport-limited photosynthesis

4.2.2.1 Rates of ATP andNADPH consumption
The energy requirements for the regeneration of RuBP in the bundle sheath are the same as
in a C 3leaf (Section 2.3.3; Farquhar et al. 1980; Farquhar and von Caemmerer 1982). There
is, however, the additional cost of 2 mol of ATP for the regeneration of 1 mol of PEP from
pyruvate in the mesophyll such that:
RateofATPconsumption = 2Vp+(3+7y.O/CJV, (4.27)
where (7y.0,lCJ V e is the energy requirement due to photorespiration (since VjVc =

2y.OjCJ (Berry and Farquhar 1978; Peisker 1988; Collatz et al. 1992). In the PCK type C 4
species some of the ATP for PEP regeneration may come from the mitochondria such that the
photosynthetic requirement may be less (see discussion in Section 4.3.4; Burnell and Hatch
1988; Carnal et al. 1993).
There is no net NADPH requirement by the C 4 cycle itself, although, for example, in
NADP-ME species NADPH consumed in the production of malate from oxaloacetate in the
mesophyll is released in the bundle sheath during decarboxylation (Hatch and Osmond 1976).
This may have implications for the behaviour of C 4 photosynthesis under fluctuating light envi
ronments (Krall and Pearcy 1993). The rate of NADP consumption is given by the requirement of
the PCR and PCO cycles:
Rate ofNADP consumption = (2 + 4y.O/C,)Vs (4.28)
It is important to note that in most situations C, is probably sufficiently large that the term
4y.O/Cs can be ignored, but it does become relevant at low mesophyll CO 2 partial pressures,
or at very low light (Siebke et al. 1997).
4.2.2.2 Partitioning of electron transport rate between (3 and (4 cycles
NADPH and ATP are produced by chloroplast electron transport. The reduction of NADPH+
to NADPH + H+ requires the transfer of two electrons through the whole chain electron
transport, which in turn requires two photons each at PS II and PS I (Table 2.1). The genera
tion of ATP can be coupled to the proton production via whole chain electron transport, or
ATP can be generated via cyclic electron transport around PS 1. Table 2.1 gives the various
stoichiometries.
As discussed above, PS II activity in the bundle sheath varies amongst C 4 species with different
C 4 decarboxylation types. Presumably, when PS II is deficient or absent from the bundle-sheath
chloroplasts, some ATP is generated via cyclic photophosphorylation and 50% of the NADPH
required for the reduction of PGA is derived from NADPH generated by NADP+ malic enzyme
(Chapman et al. 1980). The remainder of the PGA must be exported to the mesophyll chloroplast
where it is reduced and then returned to the bundle sheath (Hatch and Osmond 1976). Measure
ments of metabolite pools of Amaranthus edulis, an NAD+ malic enzyme species, having PS II
activity in the bundle sheath, suggest that it may also export a part of PGA to the mesophyll for
reduction (Leegood and von Caemmerer 1988). It appears therefore that energy is shared between
mesophyll and bundle-sheath cells.
Here, a very simple approach has been taken and electron transport is modelled as a whole,
allocating a different fraction of it to the C 4 and C 3 cycles rather than compartmentalizing it to
mesophyll and bundle-sheath chloroplasts (Peisker 1988). That is, whole chain electron transport:
t, = Jill + t, (4.29)
and J m = x], and l, = (1 - x)J t where 0 < x > 1. Because, at most, two out of five ATP are
required in the mesophyll, x '" 0.4 (Eq, 4.27). Peisker (1988) has modelled the optimization
of x at low light in some detail.
As discussed in Chapter 2, various options exist for the calculation of the ATP requirement.
Assuming the ATP requirements shown in Table 2.1, and a stoichiometry of 3H+ required per
ATP produced, an expression for the whole chain electron transport required for C 4 acid regen
eration can be derived. However, as the efficiency of proton partitioning through the cytochrome
bf complex is also uncertain, again two cases are considered, one where photophosphorylation
operates without Q cycle activity and another where it operates with Q cycle activity (Eqs 4.30
and 4.31 respectively).
Jm 3Vp (4.30)
Jill 2Vp (4.31 )
Similarly the whole chain electron transport rates required for the C 3 cycle are:
Is = 4.5(1 + 7y.O/(3C,))Vc (4.32)

in the absence of the Q cycle activity. With the Q cycle activity the rate is:
I, = 3(1 + 7y.O/(3C,»V, (4.33)
If 4H+ are required per ATP generated, it seems necessary to also have a functional Q cycle.
In this case:
(4.34)
and
J, = 4(1 + 7y.O/(3C,»)V, (4.35)
4.2.2.3 Lightdependence of electron transport rate

The relationship between the electron transport, J, and the absorbed irradiance that is used
here is the same as that used in Chapter 2 (Section 2.3.4.2.) in the C 3 model where:
(4.36)
and 12 is the photosynthetically useful light absorbed by PS II and Jmax is the maximum elec
tron transport and 8 is an empirical curvature factor (see Section 2.3.4.2 for details).
4.2.2.4 Quadratic expression for electron-trenspott-limited CO2 assimilation rate
Here, we will assume that an obligatory Q cycle operates (i.e. Eqs 4.31 and 4.33 are used)
From Eqs (4.1), (4.3), (4.4) and (4.29) one can derive two equations for an electron-trans
port-limited CO 2 assimilation rate:
(4.37)
and from Eqs (2.19) and (4.33):
(1 - y.O/ C s )( 1 - x)J, _ R
(4.38)
Aj = 3(1 + 7y.O/(3C,») d
Equation (4.38) is similar to the equation describing the electron-transport-limited rate of

C 3 photosynthesis (Chapter 2). The bundle-sheath CO 2 partial pressure under these condi
tions is given by:
C = (y.Os)(7/3(A j + R d ) + (1 - x)J/3)
(4.39)
5 (1- x)J,/3 - (A j + R d )
Combining Eqs (4.16), (4.37) and (4.38) then yields a quadratic expression of the form:
2
aA j + bA j + c = 0 (4.40)
where
(4.41 )
and
7y.a
a 1- ----, (4.42)
3 x 0.047
b - {( X
2 m 'm 3
Y*Om
- l t - R + g C ) + ((1 - x)I t - R a) + g (7
'3
)
-- + -ay.
-
0.047
(0 -3x)It + -7R3 a)} (4.43)
c -
_((Xlt_
2 Rm+g,C m
)((I-X)It_
3 Ra
))_ g,y.Om ((1-X)It
3
7Ra)
+ 3 (4.44)
Equation (4.41) can be approximated by:
Ai = . {(XI,
mtn 2 - R m + gsCm ) , ((1-X)I,
3 - R a)} (4.45)
where 'min { }' stands for 'minimum of'. Sometimes, when the equations are used to fit gas
exchange measurements, it is sufficient to use:
(l-x)I, -R
AJ = 3 a (4.46)
with appropriate adjustments to produce the correct quantum yields (Collatz et al. 1992.)
If a proton requirement of 4H+ per ATP and the operation of a Q cycle is assumed then xftl2 is
replaced by xftl2.66 and (1 - 3 )ftI3 by (1 - x)It/4 in the above equations.
4.2.3 Summary of equations

Equations (4.20) and (4.41) are the two basic equations of the C 4 model and:
A = min{A c, A) (4.47)
Peisker and Henderson (1992) pointed out that either the enzyme activity or the substrate
regeneration rate can limit both rubisco and PEP carboxylase reactions and that in theory
four types of combinations of rate limitations are possible. In the way the electron-transport
limited equations are presented here, it is assumed that light or the electron transport capac
ity limit both PEP and RuBP regeneration rates simultaneously. Furthermore, in the model
of C 3 photosynthesis by Farquhar et al. (1980) and von Caemmerer and Farquhar (1981) it
was assumed that the limitation of RuBP regeneration could be adequately modelled by an
electron transport limitation without consideration of limitations by other PCR cycle
enzymes. In the case of C 4 photosynthesis the possibility that PEP regeneration may also be
limited by the enzyme activity of enzymes such as PPDK at high irradiance continues to be
discussed, which is why such a limitation has been included in Section 4.2.1.4.
4.3 ANALYSIS OF THE MODEL
4.3.1 Parameterization of the model

Many of the model's parameters can be assigned a priori (Table 4.1), leaving only key vari
ables like V cmax' V pmax' Vpr> g, and I max to be assigned.
It is important to note that the kinetic constants of rubisco from C 4 species differ from those
of C3 species (Badger et al. 1974; Yeoh et al. 1980, 1981; Jordan and Ogren 1981, 1983; Seemann et
al. 1984; Badger and Andrews 1987; Wessinger et al. 1989; Hudson et al. 1990). This was shown in
Fig. 1.3. K, can be between 1.5 and 3 times the C 3 value. The K o has been measured less frequently
but also appears to be greater (Badger et al. 1974; Jordan and Ogren 1981; Badger and Andrews
1987). There seems, however, to be relatively little difference in the relative specificity of rubisco
between C3 and C 4 species, although no extensive measurements exist for C 4 species (Jordan and
Table 4.1 Photosynthetic parameters (at 25°C) used in the model

Parameter Value Description
60 umol m- 2 S-1 or variable Maximumrubisco activity

650 ubar' Michaelis constant of rubisco for CO 2
450 mbar' Michaelis constant of rubisco for 02
y- 0.000193 (0.5/2590)b 0.5/(5c/ o) ' half the reciprocal of rubiscospecificity
120 urnol m-2 S-1 or variable MaximumPEP carboxylase activity
80 umol m" S-1 or variable PEPregeneration rate
80 ubar- Michaelis constant of PEP carboxylase for CO 2
3 mmol m-2 S-1 Bundle-sheath conductanceto CO 2
Bundle-sheath conductanceto 02
0.01 V cmax Leafmitochondrialrespiration
RI11 0.5R d Mesophyll mitochondrialrespiration
ex O<ex<1 Fractionof PS II activity in the bundle sheath
x 0.4 Partitioningfactor of electron transport rate
400 flmol electrons m-2 S-1 or variable Maximal electrontransport rate
a 2.5 times
the in vivo values of tobacco (von Caemmerer et al. 1994) given in the caption to Fig. 4.3.
b von Caemmerer et at. (1994)
c Bauwe (1986)
d Farquhar 1983
Ogren 1981; Kane et al. 1994). The kinetic constants used here are derived from values measured
for tobacco in vivo (von Caemmerer et al. 1994) by multiplying both the K m CO 2 and 02 for
tobacco by 2.5 and using the same relative specificity as for tobacco (Table 2.3). Seemann et al.
(1984) estimated the catalytic turnover rate, k cat' to be 1.2 times that of C 3 rubisco. We have used a
kcat of 4 S·1 as a conversion factor from rubisco catalytic site concentration to maximal activity
(however, it may well be even greater).
Figure 4.3 illustrates the difference in CO 2 assimilation rate as a function of bundle-sheath
CO 2 partial pressure (Eq. 4.10) between this hypothetical C 4 rubisco and the tobacco C 3 rubisco.
In the case of the C 4 rubisco the V cmax is greater per rubisco protein because of the higher k cat but
saturation occurs at higher CO 2 partial pressures due to the larger value of K, (Table 4.1). The dif
ference between C, and C 4 at low CO 2 partial pressures is exacerbated when the higher bundle
sheath 02 partial pressures for C 4 species that do evolve 02 in the bundle sheath are taken into
account (Fig. 4.3). It is thus important to realize that bundle-sheath CO 2 partial pressures
required to saturate a C 4 rubisco are higher than what one would predict on the basis of C 3 pho
tosynthesis. If C 4 species evolved from C 3 ancestors with rubisco with a high affinity for CO 2 , this
trait was apparently lost with the development of a CO 2 concentrating mechanism.
For PEP carboxylase, a K m CO 2 of 80 ubar has been chosen (Bauwe 1986). The maximal activ
ities of PEP carboxylase and rubisco and bundle-sheath conductance will vary with leaf age and
environmental growth conditions. However, the relative scaling of these capacities of the meso
phyll and bundle sheath is also of vital importance, as it will greatly affect bundle-sheath CO 2 and
02 partial pressures and leakiness. A ratio of 2 for Vpma)Vcmax has been chosen for standard out
puts but see further discussion below (Table 4.1). The bundle-sheath conductance used is 3 mmol
m- 2 s·1, which is greater than estimates by Furbank and Hatch (1989), Jenkins et al. (1989) and
Brown and Byrd (1993) but less than estimates by He and Edwards (1996) (see table 4 in He and
Edwards 1996).
It is difficult to know how to scale leaf mitochondrial respiration with photosynthetic capacity.
In C 3 models it has usually been scaled with rubisco and that means that the CO 2 compensation
60 I C4 Vemax
-... -
c
Q)
<13
0 en
50
-----C, V cme x

+:i ~ 40
~ E C
3
'E (5 "/
'eenn E 30 I
/
<13 ~ I
I
20
0 '" I
o I
I
10 I
I
o l.
I
I I I I I I ! I
o 2000 4000 6000 8000 10000

Bundle-sheath CO 2 partial pressure
Cs (ubar)
Figure 4.3 Comparison of rubisco-limited CO 2 assimilation rates, A. (Eq. 4.10) as a function of bundle-sheath
CO 2, C; with kinetic parameters for a C4 and C3 rubisco respectively at a bundle-sheath O2 partialpressure of 200
mbar (solid lines). Constants for the C4 rubisco are given in Table 4.1. For the C3 rubisco k cat = 3 S-1 such that Vcmax
= 45 urnol m-2 S-1, K, = 260 pbar, Ko = 179mbar, y. = 0.000193 = 0.5/2590 (von Caemmerer et al. 1994). A is
also shown for the C4 rubisco at a bundle-sheath O2 partial pressure of 500 mbar (dashed line).
60 . . 60
<:(
Q)
50 " "" 50 o
c
...
iU
o ')'en
- 40 /
/
/ ""
40
fJ>
3'
C"
~ E I III
'E "6 30 I
/
30 ~
en E
I -&
en -.=;, I
ctl 20 /
/ 20 -;g
~
ao'" 10 "" 10
0 0
0 60 120 180 240 300
PEP carboxylase activity
Vpmax (urnol m·2s")
Figure 4.4 CO 2 assimilation rate, A (solid line) as a function of maximum PEP carboxylase activity, VpmaX' at a
mesophyll CO 2 partial pressure, Cm = 100 ubar, Also shown arebundle-sheath CO 2 partial pressure, Cs' (dotted
line) and leakiness, <\J (dashed line). Maximal rubisco activity, Vcmax = 60 prnol m-2 s', mitochondrial respiration
Rd and a. were assumed to be zero. Other constants are as in Table 4.1.
point remains unchanged. Justification for this was derived from a loose correlation between R d
and leaf protein content. We have also scaled R d to rubisco here (Table 4.1).
4.3.2 The model at high irradiance

4.3.2.1 PEP carboxylase andrubisco activity
The model is very sensitive to the balance between V cm ax and Vpm ax ' Figure 4.4 examines the
PEP carboxylase activities required for the efficient use of a rubisco activity of 60 umol m- 2 s", at
g s' bundle-sheath
conductance
Assimilation rate
(C,-C)
Mesophyll, Cm' or bundle-sheath, C"
Figure 4.5 The effect of a decrease in bundle-sheath conductance on CO 2 assimilation rate, A; leakrate, L; and
bundle-sheath CO 2 partialpressure. Other details aregiven in Fig. 4.2 caption.
a mesophyll CO 2 partial pressure, Cm' of 100 ubar. This takes into account that C 4 plants
operate at lower intercellular CO 2 partial pressures than C 3 plants and 100 ubar is commonly
observed at ambient CO 2 partial pressures of 350 ubar, At this em' and PEP carboxylase
activities above 120 umol m- 2 S-I, CO 2 assimilation rate saturates and bundle-sheath CO 2
partial pressure and leakiness, $, increase rapidly. Vp max amd V cm ax have frequently been mea
sured in response to leaf age and nitrogen nutrition. Generally, measured ratios range from 2
to 8 (Usuda 1984; Hunt et al. 1985; Sage et al. 1987) and some exceptionally high ratios of 14
to 20 were observed by Wong et al. (1985). It is clear that in vivo ratios beyond 4 result in very
high bundle-sheath CO 2 partial pressures and high values of leakiness (Fig. 4.4).
There are several reasons one can suggest for the high ratios of Vpmax to V cmax measured in leaf
extracts. PEP carboxylase is a highly regulated enzyme with many of the metabolites such as
malate acting as negative effectors (Doncaster and Leegood 1987), and thus it is plausible that the
in vivo V pmax is down-regulated relative to the in vitro measurements. It is also possible that PEP is
not saturating at low CO 2 partial pressures since PEP pools have been shown to increase with
increasing CO 2 partial pressures (Leegood and von Caemmerer 1988, 1989). As a result, it is
important to bear in mind that the rate equation for PEP carboxylase (Eq. 4.17) may be an over
simplification which may need modification in future. Another possibility, which is discussed in a
later section, is the effect a diffusion limitation on the passage of CO 2 from the intercellular
airspace to the mesophyll cytoplasm has on the requirement for PEP carboxylase activity. Several
studies have reported significant changes in the ratio of PEP carboxylase and rubisco with leaf age
and nitrogen nutrition, which should have important consequences for the gas-exchange charac
teristics of these leaves (Meinzer and Saliendra 1997).
4.3.2.2 Bundle-sheath conductance
A low conductance to CO 2 diffusion across the bundle sheath is an essential feature of the C 4
pathway. It effectively eliminates CO 2 exchange with the normal atmosphere such that
C 4 acid decarboxylation is the major source of CO 2 in that compartment. For example,
-
C)J
en
60 ~ I 15
E A .........
1
0 50
E cf!.
6-
c
<:t:
_Q)..
c
ro .-
....
.
0
"';:::
.0
..c
.~

40r
30 :
.. '
c,
........ .'
.......
""TO
()
'""3
0'
0
'';::::;
~
C
Q)
OJ
20 .
..... 5
~
'E x>.
en 0 10
en
o~
ro 02 inhibition
I
0 '" I I 1
0
o 0 200 400 600 800 1000
2s
Bundle sheath resistance, rs (m mar')
Figure 4.6 CO 2 assimilation rate, A, as a function of bundle-sheath resistance. r5 =1/g5 • at a mesophyll CO 2

partial pressure, em = 100ubar, and 200mbar (solid line) and 20 mbar (dotted line) mesophyll oxygen partial
pressures. Also shown is the bundle-sheath CO 2 partial pressure at both oxygen partial pressures and the %
oxygen inhibition which is calculated from 100 x (A(20) - A(200))/A(200)).
inhibition of PEP carboxylase with an inhibitor reduced CO 2 assimilation rate almost com
pletely at ambient CO 2 partial pressures (Furbank and Hatch 1989; Jenkins 1989; Brown
1997). The conductance of CO 2 diffusion across the mesophyll bundle-sheath interface has
been measured for different C 4 species by various techniques. Estimates range from 0.6 to
2.5 mmol m- 2 sol on a leaf area basis which correspond to resistances between 400 and
1600 m? SI mol " (Jenkins et al. 1989; Brown and Byrd 1993). Figure 4.5 shows that decreas
ing bundle-sheath conductance (moving from the solid arrow to the dashed arrow) increases
CO 2 assimilation rate, decreases the leak rate and increases bundle-sheath CO 2 partial pres
sures. Figure 4.6 shows CO 2 assimilation rate under standard conditions as a function of
bundle-sheath resistance (l/g,). The low oxygen sensitivity, a characteristic of the C 4 photo
synthetic pathway, is a good test for the bundle-sheath resistance required to adequately
reflect the C 4 syndrome at ambient CO 2 partial pressure. He and Edwards (1996), who used
this modelling approach, have, however, estimated very low bundle-sheath resistances
between 15 and 90 m-' SI mol- l for Zea mays from measurements made by Dai et al. (1993,
1995) on leaves of different ages. For the standard outputs here, a bundle-sheath conduc
tance of 3 mmol m- 2 S-I (resistance of 333 m? s mol:") was chosen.
4.3.2.3 Variation of bundle-sheath conductance, PEP carboxylase activity andrubisco with leafage
It is well known that PEP carboxylase activity (VpmaJ and rubisco activity (Vona,) vary with leaf
age, nitrogen nutrition and light environment during the growth of plants (Usuda 1984; Hunt
et al. 1985; Wong et al. 1985; Sage et al. 1987; Meinzer and Saliendra 1997). At present little is
known about variations of morphological characters such as bundle-sheath conductance with
leaf age and growth conditions. Bundle-sheath conductance is the product of the diffusion con
ductance across the bundle-sheath cell wall interface and the bundle-sheath surface area. Only a
few measurements of bundle-sheath surface area per leaf area have been made, ranging between
0.6 and 2.16 m-' m- 2 (Apel and Peisker 1978) and 1.13 and 3.1 rrr' m ? (Brown and Byrd 1993). It
is likely that bundle-sheath surface area per leaf area may be larger in young expanding leaves
relative to mature leaves, leading to greater values of gs in young leaves. If S, remains constant in
Q)
'§
c a ..;.
./
.,.,; ."
0 en 60
§ E
')'
./
E "'6
en E 40
en
ctl ..=;
N
a 20
o
0
15
10
5 ~ ~ .. "
-"",:,.-._~-:.=.'.::.: -
...""'"-
0
1.0
-e 0.8
en
en 0.6
Q)
c
..l<:
ctl 0.4
Q)
...J
0.2
0.0
0 20 40 60 80
Rubisco activity, Vcmax(~mol m" 5.
1
)
Figure 4.7 (a) CO 2 assimilation rate, (b) bundle-sheath CO 2 partial pressure and (c) leakiness, <1>, at different
maximal rubisco activities, Vemax at (1)(solid lines) a constant maximal PEP carboxylase activity, Vpmax' of 90 urnol
m-2 s' anda bundle-sheath conductance of 3 mmol m-2 S-1; (2) (dashed lines) a constant ratio of Vema)Vpmaxof
1.8and 95= 50V emax; (3) (dotted lines) a constant ratio of VemaxlVpmaxof 1.8and 95= 3 mmol m-2 S-1. Mesophyll
CO 2 partial pressure, em' was 100 ubar, the fraction of 02 evolution in the bundle sheath, ex = 0, and
mitochondrial respiration, Rd = O.
fully expanded mature leaves as PEP carboxylase and rubisco activity decline with leaf age this
should have important consequences for the integrated functioning of the C 4 pathway. Measure
ments of bundle-sheath surface area and conductance are required for leaves of different ages and
plants grown under different environmental conditions.
Figure 4.7 examines this issue with three different scenarios. In the first scenario V emax is var
ied at a constant PEP carboxylase activity and bundle-sheath conductance. In this case a reduction
in V cmax results in an increase in bundle-sheath CO 2 partial pressure and leakiness as PEP carbox
ylase activity exceeds the demands of rubisco carboxylation for CO 2, This is graphically illustrated
in Fig. 4.8. This scenario was obtained when rubisco activity was artificially reduced by an anti
sense construct directed at the rubisco small subunits of Flaveria bidentis (Fur bank et al. 1996; von
Caemmerer et al. 1997). The fact that rubisco was reduced as a fraction of total soluble protein
confirmed that the antisense had been specific. In this case the decrease in CO 2 assimilation rate
was accompanied by an increase in dry matter carbon isotope discrimination (Fig. 4.9;
co
~... Assimilation rate
Il)
IOJl
~ <, ,,\ Reduction
..0 ,, ofrubisco
U
><:
Il)
d"
.,
u
(C,-C,)
~
Cm c,
Mesophyll, Cm' or bundle-sheath, C"

Figure 4.8 The effect of a decrease in rubisco activity on CO 2 assimilation rate, A; leak rate, L; and bundle
sheath CO2 partial pressure. Other details are given in the legend of Fig. 4.2.
von Caemmerer et al. 1997). Increases in carbon isotope discrimination during C 4 photosynthesis
can be indicative of an increase in leakiness, depending on the ratio of intercellular to ambient
CO 2 (Farquhar 1983), which in this case is most likely the result of increasing bundle-sheath CO 2
partial pressure.
The second and third scenarios in Fig. 4.7 look at ways rubisco activity may be changing with
the ageing of leaves or for plants grown at different nitrogen nutrition. In the second scenario
Venit'" Vpmax and gs are perfectly scaled. This results in a constant bundle-sheath CO 2 partial pres
sure and leakiness at the different rubisco activities. Henderson et al. (1994) measured CO 2
assimilation rate and leakiness (via short-term measurements of carbon isotope discrimination)
in Sorghum bicolor grown at different nitrogen nutrition. They observed no change in leakiness
over a wide range of CO 2 assimilation rates (Fig. 4.10), indicating that the balance between meso
phyll and bundle-sheath capacity was maintained. Meinzer and Saliendra (1997) studied changes
in CO 2 assimilation rate with leaf age along the length of sugarcane leaves. They observed a con
stant ratio of maximal rubisco and PEP carboxylase activity. Using gas-exchange measurements
and measurements of dry matter carbon isotope discrimination they concluded that there was
also no change in leakiness in these leaves.
In the third senario, V emax and Vp max are perfectly scaled and g, is kept constant. This results in
an increase in bundle-sheath CO 2 partial pressure as the CO 2 concentrating capacity increases
with increasing biochemical activity. At low V ernax and Vp nlt" (i.e, in older leaves) leakiness is high.
4.3.2.4 COz response curves
In C 3 species, CO 2 response curves have been useful in the analysis of photosynthetic capaci
ties (von Caemmerer and Farquhar 1981, chapter 2). In this section we examine how Vp max '
V em ax > and gs affect the shape of the CO 2 response curves of C 4 photosynthesis. C 4 photosyn
thesis is characterized by low CO 2 compensation points and CO 2 response curves that satu
rate abruptly (Fig. 4.11) and the model has these characteristics (Eqs 4.20 and 4.25). The
initial slope of the CO 2 response curve is proportional to the PEP carboxylase activity,
whereas the saturated rate is proportional to rubisco activity because in this simulation PEP
106 Biochemical Modelsof Leaf Photosynthesis
40
~
Q) I I I I •
Cil a
.....
c
0
-
en
32 -
4B~!. -
§ ~
E 24
- -
E (5
'iii 0
en E 16 - 0

(1j E, g
0 '" 8 1- -
o 0
I I I I I I
0
b
0
I-
<]
.....
~rn
2 6 I-
Cil
E
%!2J ••.,. ~
6.
~ 5 I-
o
4 I I I ••
I I I
0.15 I-
C • 6._
10 .~:
0.10
~D
I -
0.05 I- 0
00
I I I I I I
0.00
o 2 4 6 8 10 12
Rubisco sites (urnol m")

Figure 4.9 (a) CO 2 assimilation rates, (b) dry matter carbon isotope discriminations and (c) the ratio of rubisco
to soluble protein in control (closed symbols) and transgenic Flaveria bidentis with reduced amount of rubisco
(open symbols). Measurementswere made at an ambient CO 2 partial pressure of 350 ubar and 2000 umol quanta
m-2 S-1 and a leaf temperature of 25°C. Thedecline in rubisco as a fractionof soluble protein confirms the specific
reduction in rubisco. (Data are redrawn from von Caemmerer et al. 1997.)
carboxylase activity was not limited by PEP regeneration capacity. Indeed, CO 2 assimilation
rate is almost equal to the maximal rubisco activity (see Eq. 4.25). Since PEP carboxylase
activity is not limited, leakiness and bundle-sheath CO 2 partial pressures continue to
increase with increasing CO 2, This is unlikely to be the case in vivo. Henderson et al. (1992)
demonstrated with short-term measurements of carbon isotope discrimination that leaki
ness appeared constant over a range of CO 2 partial pressures (Fig. 4.12).
In Fig. 4.13 PEP carboxylase activity has been limited by the capacity of PEP regeneration at
high CO 2 , Under these conditions bundle-sheath CO 2 partial pressure and leakiness saturate. It is
clearly very difficult to distinguish between a rubisco and a PEP regeneration limitation at high
CO 2, The non-linearity of the relationship between CO 2 assimilation rate and rubisco content in
transgenic Flaveria bidentis may indicate a PEP regeneration limitation in the control plants
(Fig. 4.9). Low maximal extractable activities of pyruvate Pi dikinase have been used to infer its
possible role in limiting CO 2 assimilation rate at high irradiance (Sugiyama and Hirayama 1983;
Usuda 1984). Although interesting from a biochemical viewpoint, in many modelling situations it
/-
50
Q)
a
ca....
oc: ';"
:;::
ell
-
(;'oJ(/)
'
45
40
•• • •
= E
E -0
.- ••
~ E 35
ell E
~ •
N
0
o 30
"'.
--'-
0.4 ' b
-e
•..•
en 0.3
--------...-
(/)
~
Q)
c:
ell 0.2
•
Q)
....J
• •
0.1
0.0
40 60 80 100 120
Leaf nitrogen (mmol m")

Figure 4.10 Theeffectof plant nitrogen nutrition on (a) CO 2 assimilation rate and (b) leakiness, <», in Sorghum
bicolor L. Leakiness was calculated from concurrent measurements of carbon isotope discrimination and gas
exchange from the equation II = 4.4 + (28.2<» - 9.6)(;lCa according to Henderson et al. (1992). Measurements
were made at an irradiance of 1600 urnol quanta m-2 S-1, ambient CO 2, Ca = 330 ubar and a leaftemperatureof
29°C. The lines are drawn by eye and the data are taken from Henderson et el. (1994).
will be sufficient to model the regeneration limitations via the electron transport limitations dis
cussed in Section 4.2.2.
The model predicts that maximal rubisco activity affects only the saturated part of the CO 2
response curve. Gas-exchange measurements on transgenic Flaveria bidentis with reduced
amounts of rubisco confirm this (Fig. 4.14; Furbank et al. 1996; von Caemmerer et al. 1997).
These plants have unaltered PEP carboxylase activities in vitro and the fact that the initial slope is
not affecte d by the drastic reductions in rubisco activity indicates that activity of PEP carboxylase
in vivo is unaffected by altered rubisco activity in the bundle sheath.
Different maximal PEP carboxylase activities affect primarily the initial slope of the CO 2
response curves. However, when PEP carboxylase activity is very low there is also a reduction in
the saturated rate of CO 2 assimilation, due to the fact that the rubisco in the bundle sheath is
not completely saturated with CO 2 (Fig. 4.15 and see also Fig. 4.4). Variations in the initial
slope with variations in PEP carboxylase activity have frequently been demonstrated with
changes in leaf age or nitrogen nutrition; however, they always occur with concurrent changes
in rubisco activity (Hunt et al. 1985; Sage et al. 1987). Recently, Dever et al. (1995) and Dever et
al. (1997) have generated mutants of Amaranthus edulis with reduced PEP carboxylase activity,
but with no changes in rubisco activity. These plants show similar gas-exchange characteristics
to those predicted in Fig. 4.15.
Variations in the rate of PEP regeneration affect the CO 2-saturated rate of CO 2 assimilation in
a similar way to changes in rubisco activity and it would be difficult to distinguish between these
a /
lfJ
/
'1' I
E 60
. J. ... . . __ . . . . . . . •
I
0 I
E I
E, I
I
'<
40
'E" , I
-:::.'"
-:::.0.. 20
-c
0 20
b
--
0.4
15 OJ
-e- , c
,- ::J
rJi 0.3 / ()Q:
lfJ / '" (1)
CD
c
:s;;: I
I
10'3 ~
ell
CD
-l
0.2
J
I
I
I 0-(1)
..,
~::J'

III III
I 5 0
0.1 / NO
1/
0.0 0
o 100 200 300 400 500
Mesophyll CO 2 partial pressure
em (ubar)
Figure 4.11 (a) CO 2 assimilation rate, A (Eq. 4.20, solid line), as a function of mesophyll CO 2 partial pressure,
Cm' Also shown are the rates of PEP saturated PEP carboxylation, Vp (dashed line), and maximal rubisco activity,
Vcmax (dotted line) (see Eq. 4.25). At high Cm' Vcmax limits A. In this simulation the fraction of O2 evolution in the
bundle sheath, a, is 0, and mitochondrial respiration, Rd , is 0. Other parameters are as given in Table4.1.
(b) Leakiness, <1>, as a function of mesophyll CO 2 partial pressure, Cm (dashed line) and bundle-sheath CO 2 partial
pressure, ({solid line).
two limitations. PEP regeneration at high irradiance can be limited by the capacity of enzymes of
the C 4 cycle such as PPDK, NADP-malate dehydrogenase, for example, or by the capacity of the
chloroplastic electron transport chains. Measurements of the CO 2 response curves of Zea mays at
two irradiances (Leegood and von Caemmerer 1989) are consistent with a limitation to PEP
regeneration, since the initial slopes remain independent of irradiance. CO 2 response curves for
transgenic Flaveria bidentis with reduced activities of NADP-malate dehydrogenase (Trevanion
et ill. 1997) also show reduced COrsaturated rates of CO 2 assimilation, which are consistent with
this hypothesis.
A factor that affects the curvature of the CO 2 response curve is bundle-sheath conductance
{gJ, as it is a key determinant of the bundle-sheath CO 2 partial pressure. Although variations in
the saturation characteristics have been noted between young and old leaves and between species
(Pearcy et al. 1982), no systematic studies have yet correlated the shape of the CO 2 response curves
with variations in gs' Ludwig et al. (l997) analysed transgenic Flaveria bidentis that ectopically
expressed tobacco carbonic anhydrase in the bundle-sheath cells. They found that this led to
increased leakage of inorganic carbon out of the bundle sheath. This was because carbonic anhy
drase, normally absent from bundle-sheath cells, would increase the conversion of CO 2 to HC0 3
and hence the steady-state concentration of HC0 3 - in the bundle sheath of the transgenic plants,
which would in turn increase the total leak of inorganic carbon.
s
~
.§OC:;-
a;'l'E 40
=
.~ ~
ell::i
60 ~ a
l, ~
I
..
I~O.
_0
• ° •••

0'" ~ 20
o
~
0.4 ~ b
, ."..
-e- 0.3
C
~
ui
CfJ
Ql
ell
Ql
0.2
•
•••
...J
0.1
0.0
a 100 200 300 400
C, (ubar)
Figure 4.12 (a) CO 2 assimilation rateasa function of intercellular CO2 partial pressure, C; (open and closed
symbols) in Amaranthus edulis. For some measurements (closed symbols) carbon isotope discrimination was
measured concurrently with gas exchange. (b) Leakiness, <jJ, as a function of intercellular CO 2 partial pressure, C,
Leakiness was calculated from concurrent measurements of carbon isotope discrimination andgas exchange from
the equation a = 4.4 + (28.2<jJ - 9.6) C;!Ca according to Henderson et al. (1992) (closed symbols). Measurements
were made at an irradiance of 1600 urnol quanta m-2 S-l and a leaftemperature of 29°C. (Data areredrawn from
Henderson et al. 1992.)
4.3.2.5 Oxygen sensitivity of C4 photosynthesis

The low O 2 sensitivity of CO 2 assimilation rate is a key characteristic of the C 4 pathway. PEP
carboxylase itself is not sensitive to 02 partial pressures and the lack of O 2 sensitivity is
attributed to the high bundle-sheath CO 2 partial pressures and favourable C0 2/0 2 ratios
which inhibit photorespiration (Hatch and Osmond 1976; Hatch 1987). Figures 4.17 and
4.18 support this, showing a decrease in 02 sensitivity with increasing bundle-sheath resis
tance and bundle-sheath CO 2 partial pressure. Note that at each bundle-sheath conductance,
bundle-sheath CO 2 partial pressure increases with increasing 02 partial pressure because
rubisco carboxylation is inhibited by increasing 02 partial pressure. He and Edwards (1996)
used the 02 dependence of CO 2 assimilation rate together with in vitro measurements of PEP
carboxylase and rubisco activity to estimate bundle-sheath resistances. They estimated very
low bundle-sheath resistances between 15 and 90 m? SI mol:'. They also observed a decline in
CO 2 assimilation rate at very low 02 partial pressure, which is not a feature of this model (He
and Edwards 1996; Maroco et al. 1997).
It is important to note that a high bundle-sheath CO 2 partial pressure is not a prerequisite for
the lack of 02 sensitivity, when bundle-sheath resistance is high. This was first pointed out by
Berry and Farquhar (1978) and is illustrated in Fig. 4.19. Figure 4.19 examines in more detail CO 2
assimilation rate at low mesophyll CO 2 partial pressure (0 to 40 ubar) and two 02 partial
100
a
u;
'l' Vp
E 80
(5
E A
2, 60
s'"'"
),." 40
),.Q
«0 20
0 20
b
OJ
-e
0.3 15 c
:J
(")9:
enu; Cs <n
~cn
(1)
Q)
c: 0.2 10 30'"(1)::::r
:.;;2 III III
CIl ~=r
Q)
...J
o
0.1 5 0
ro
0.0 0
o 100 200 300 400 500
Cm (ubar)
Figure 4.13 (a) CO 2 assimilation rate, A (Eq. 4.20; solid line), asa function of mesophyll CO 2 partial pressure,
Cm' Also shown arethe rates of PEP carboxylation, Vp , which are limited by PEP regeneration at 80 urnol m-2 s'.
Maximal rubisco activityis Vcmax = 60 prnol m-2 s'. At high Cm' A is limited by the rate of PEP regeneration. The
dashed line shows A when PEP regeneration is not limiting. In this simulation the fraction of 02 evolution in the
bundle sheath, ex, is 0, and mitochondrial respiration, Rd , is O. Other parameters areas given in Table 4.1.
(b) Leakiness, <1>, and bundle-sheath CO 2 partial pressure, C5 ' as a function of mesophyll CO 2 partial pressure, Cm'
when PEP regeneration is limiting at a rateof 80 urnol m-2 s'.
pressures. Under these conditions CO 2 assimilation rate is limited by PEP carboxylation rate in
the model. At the CO 2 compensation point the predicted bundle-sheath CO 2 partial pressure is
typical of C3 compensation points, increasing rapidly with increasing mesophyll CO 2 partial pres
sure. There is an appreciable amount of photorespiration occurring at 200 mbar 02 compared to
20 mbar 02 under these conditions, which is not reflected in differences in CO 2 assimilation rates.
Instead, because the bundle-sheath compartment is almost gas tight, bundle-sheath CO 2 partial
pressure changes in response to changes in 02 partial pressure rather than the CO 2 assimilation
rate itself (Fig. 4.19). Although the ratio of bundle-sheath 02 and CO 2 partial pressure uniquely
defines the ratio of oxygenase to carboxylase rate (Eq. 4.8), CO 2 assimilation rate is also depen
dent on bundle-sheath CO 2 partial pressure per se (Eq. 4.10).
4.3.2.6 CO2 compensation point
C 4 is characterized by very low CO 2 compensation points, a criterion that has been used to
identify plants with the C 4 photosynthetic pathway. An expression for the compensation
point can be derived from Eq. (4.20) together with Eq. (4.17) by solving Eq. (4.20) for A = O.
The equation is quadratic and given by:
o (4.48)
- CIl
')l
E
40
"0
E
630
Q)
§ ~=-w - Q] '"'tf o 0
c
0
20
~
.~
CIl
10
CIl
C'il
0 '" 0
o 0 100 200 300 400 500
(ubar)
Figure 4.14 CO 2 assimilation rate asa function of intercellular CO 2 partial pressure, Cm' in Flaveria bidentis in
control plants (closed symbols) andtransgenic plants with reduced amountof rubisco dueto an antisense
construct targeted to the small subunitof rubisco (open symbols). Different symbols represent measurements
made on different leaves. (Data are redrawn from von Caemmerer et al. 1997.) Measurements were made at an
irradiance of 2000 urnol quanta m-2 s', and a leaf temperature of 25°C. The control leaves had a mean rubisco
site concentration of 9.7 urnol m-2 andthe transgenic leaves 4 urnol m-2. The lines aremodelled CO 2 assimilation
rates asa function of mesophyll CO 2 partial pressure, Cm' at maximal rubisco activities Vcmax = 73 and 25.4 urnol
m-2 s' and Vpmax = 105 urnol m-2 S-I; other parameters aretakenfrom Table 4.1, and Rd and a = O.
where
r =Y_>_0_m_+_K_c(_1_+_0_n...,.../_K_o)_(R_d_I_V_cl11_ax) (4.49)
s 1 + RdlVcmax
is the bundle-sheath CO 2 partial pressure at the compensation point (Eq. 4.11). Note that by
definition 0 s = Om at the compensation point.
This equation for I" was first derived by Peisker (1974, 1979) but without the inclusion of a
respiratory term. When c, « Kp Eq. (4.17) can be approximated by V p = VpmaxCmlKp which
gives a simpler solution where:
gsr s+ R m
(4.50)
I" = Vp mux IKp +g,
and since Vpma)Kp » gs' I" can be approximated by:
g
I" = - Kpr , RmKp
'--'+-- (4.51)
V pmax V pmax
This equation highlights the linear dependence of ron gs and K/Vpmax' It also points out the
linear dependence that I" has on O 2 but the slope is small so that it is difficult to measure
(Fig. 5.11, page 135). Figure 4.20 shows the expected dependence of I" on bundle-sheath con
ductance at various conditions. Mitochondrial respiration increases the compensation point
and that is apparent in very young C 4 leaves where compensation points can be high, but
where the CO 2 response curve suggests a well-integrated C 4 pathway (Ghannoum et al.
1998). An 80% reduction in PEP activity results in a substantial increase in the compensation
112 Biochemical Models of LeafPhotosynthesis
a
60
40
en
')'
E
(5
E 20
..=;,
s
~
c
o
~ b
60
E
'00
en
ell
d" 40 2
o
3
20 I-t----.d..-------------j
OL--------'--------'-----'---...J..-------J
o 100 200 300 400 500
em (ubar)
Figure 4.15 CO 2 assimilation rate as a function of mesophyll CO 2 partial pressure, Cm' at (a) various maximum
PEP carboxylase activities, Vpmax, and no PEP regeneration limitation (Vpmax = (1) 180, (2) 120 and (3) 80 urnot
m-2 s'): (b) different rates of PEP regeneration (PEP regeneration rates = (1) 60, (2) 40 and (3) 20 urnol m-2 s').
Otherconstants are taken from Table 4.1, and Rd and a = O.
-
')'
en
E 60
(5
E
..=;,
~40
c
0
~
E
'00
20
en
ell
cv
0
o
0 100 200 300
400 500
em (ubar)
Figure 4.16 CO 2 assimilation rate as a function of mesophyll CO 2 partial pressure ,Cm' at bundle-sheath
conductances of (1) 0.3, (2) 3 and (3) 9 mmol m-2 S-I. Other constants aretaken from Table 4.1, and Rd and a = O.
Q)
co.... 70
-
c:
o ~-
._ . en
co ')' E
60
E
"0
en
en E 50
co E
N
0 40
o
N
30
0 12
o
..c:
m'i::"
Q) co 8
..c:.o
en E
dJ
=0 4
c: L ____________
:::l
CD
o
.Q
c:
co en
.::
- 1.5
0- "0 1.0
en E
....
Q)
0
0
0 E 0.5
..c: E
[L
0.0
o 100 200 300 400 500
Mesophyll 02 partial pressure
Om (mbar)
Figure 4.17 CO 2 assimilation rate, bundle-sheath CO 2 partial pressure, and photorespiration rate asa function
of mesophyll 02 partial pressures at bundle-sheath conductances of 1 (dotted line), 3 (solid line) and 10 mmol
m-2 S-1 (dashed line). Mesophyll partial pressure, em' is 100ubar: otherconstants are taken from Table 4.1, and
Rdand a = O.
point which should be apparent in Amaranthus mutants with reduced amounts of PEP
carboxylase activity (Dever 1997).
4.3.2.7 CO2 diffusion from intercellular airspaces to the mesophyll cytosol
Recent studies have shown that there is a significant resistance to diffusion of CO 2 from the
intercellular airspace to the chloroplast in C3 species where it is possible to quantify this
internal conductance, gi' with measurements of carbon isotope discrimination (Section 2.5;
Evans and von Caemmerer 1996). In tobacco it was shown that g, was correlated with the
chloroplast surface area appressing intercellular airspace (Evans et al. 1994). In C 4 species the
diffusion path for CO 2 is from the intercellular airspace to the cytoplasm where PEP carboxy
lase and carbonic anhydrase reside. Therefore, gi is likely to be proportional to mesophyll
surface area exposed to intercellular airspace in these plants. Unfortunately, techniques used
to measure gi in C3 species cannot be used for C 4 species (for review see Evans and
<:('2
_ ell 100
moO
CilE
~ 0 95
o II
'§ E:
=E_a 90
'iii ell
~~ 85
"'0
O~
O~
80 '------'-----..L---L----'-----L-----'-----'-------'-----l._
o 100 200 300 400 500

Mesophyll O2 partial pressure
Om (mbar)
Figure 4.18 Changes in CO 2 assimilation ratewith mesophyll O2 partial pressure at bundle-sheath
conductances of 1 (dotted line), 3 (solid line) and 10 mmol m-2 S-l (dashed line). CO 2 assimilation rateis
expressed as a percentage of the rate at zero O2 partial pressure. Calculations are the same as in Fig. 4.17.
~x a
-a:
30
s 0... ~en
§ § ')IE 20
'E .~
(5
(/).:
en c.
ell
",Q)
en ~ 10
o
0(5
<5
s:
c.
o f---:--+--+----j---I
0'" 1500
o
1000
500
o f-=----t----+--t------1
c
60
"
40 ""
20
0~~==t====c:==:J
o 10 20 30 40
em( ubar )
Figure 4.19 (a) Modelled CO 2 assimilation rate, A. and photo respiration rate at low mesophyll CO 2 partial
pressure, Cm' and a mesophyll O2 partial pressure of 20 (dashed lines) and 200 mbar (solid lines). Also shown are
(b) bundle-sheath CO 2 partial pressures, CS' and (c) the ratio of bundle-sheath CO 2 and O2 partial pressures.
16
-...III
..0 14 ~ /
/
»: /
~ /
/
.... 12 /
/
/
C /
'0 10 /
c.. /
/
C
/
.Q 8 /
iii /
en /
c 6 /
Q) /
c.. /
E /
0 4
o
N
0 2
o
0
0.00 0.02 0.04 0.06 0.08 0.10
Bundle-sheath conductance, 9 5
(mol rn" S,l)
Figure 4.20 Dependence of the CO2 compensation point on bundle-sheath conductance at 200 mbar 02 partial
pressure and Rd = 1 urnol m-2 S-1 (solid line), with Rd = a (dotted line), at 500 mbar 02 and Rd = 1 prnol m-2 S-1
(dashed line), at 200 mbar 02 and Rd = 1 urnol m-2 S-1 and Vpmax = 24IJmoi m-2 S-1 (dashed and dotted line).
Otherparameters used aregiven in Table 4.1.
von Caemmerer 1996). At present there is no reliable estimate of gj, nor an estimate of the
drop in CO 2 partial pressure from intercellular airspace, C j to that of the mesophyll, Cm' C,
and C m are related in the following equation:
A=gj(Cj-Cm) (4.52 )
Incorporating Eq. (4.52) into Eq. (4.20) results in a cubic expression which is not easily
solved. It can be incorporated into Eq. (4.41), giving a slightly more complex quadratic. In
the case of the initial slope of the CO 2 response curve one can again use Eq. (4.26) and,
ignoring the term g,C m and combining it with Eq. (4.52), a quadratic similar to the one given
for C 3leaves is obtained (von Caemmerer and Evans 1991).
)
A - - A[gj( C, + K p) + V pmax - R m] + g,l Vpl1laxCj - R m ( C j + K p)] = 0 (4.53 )
The first derivative with respect to C j at Ci = 0 is given by:
dA _ gjVpmax
(4.54)
dC j gjK p + V pmax
Pfeffer and Peisker (1995, 1998) used this equation, together with measurements of PEP car
boxylase activity and initial slope (dA/dC), to estimate gj in plants grown under different
light intensities. However, they conceded that their estimate of gj = 0.87 mol m- 2 S-1 is based
on the assumption that gj does not change with growth conditions. In C 3 plants gj has been
found, by several authors, to be positively correlated with photosynthetic capacity (Evans
and von Caemmerer 1996) and this may also be true for C 4 leaves.
60 ,------,--,-----r---,--------,
-----
Q)
;'
(tj ;'
~ ;'
c:: ";
I
0
:;:J
CIl 40 I
I
C)I
~ E I
'E "0 I
I
'iii E I
I
CIl
ro .6 20 I
I
0 '" I
o I
OL-_---L.-_ _. l - - _ - - - - - L_ _---'----_-----.J
o 100 200 300 400 500
C; (ubar )
Figure 4.21 The effectof a limited conductance to CO 2 diffusion, g;, between intercellular airspace and
mesophyll cytoplasm on the shape of the CO 2 response curve underenzyme-limited conditions. Parameters are as
in Table 4.1 and Rd = 0 and a = O. g; = 0.5 mol m-2 S-1 (dashed line) or g; = (solid line). 00
Figure 4.21 illustrates the effect gi has on the initial slope of the CO 2 response curve. The curve
was generated from Eq. (4.20), deriving C, from A and Cm with the help of Eq. (4.52). The pres
ence of a substantial resistance to CO 2 diffusion across the mesophyll cell wall provides one reason
why measured ratios of V pmax and VCm<lX may need to be greater than 2.
4.3.3 CO 2 fixation at limiting light

4.3.3. 1 Optimal partition of electron transport
Figures 4.22 and 4.23 show typical modelled light response curves of CO 2 assimilation. The
shapes of the curves are determined by Eq. (4.36). Figure 4.22 shows the light response curve
of CO 2 assimilation rate when electron transport is partitioned optimally between C4 and C 3
cycles. The solutions were found by an optimization procedure but can also be calculated
from the first and second derivatives of Eq. (4.41) (Peisker 1988). It is noteworthy that the
fraction of electron transport allocated to the C 4 cycle, x, equals 0.404 over a wide range of
irradiances but drops at very low irradiance. Under low light the bundle-sheath CO 2 partial
pressure is close to the mesophyll CO 2 partial pressure and electron transport is required for
recycling of photorespiratory CO 2 , The optimal partitioning increases from 0.404 to 0.417 if
oxygen is evolved in the bundle sheath (a = 1). It also increases with increasing bundle
sheath conductance, reaching 0.415 at gs = 0.01 mol m ? S-I.
For all further modelling a constant value of x = 0.4 is used. Figure 4.23 shows a typical light
response curve for CO 2 assimilation rate under these conditions. Bundle-sheath CO 2 partial pres
sure increases with increasing irradiance whereas leakiness, <\>, is predicted to decrease. These results
are in accordance with experimental measurements of bundle-sheath CO 2 concentrations (Furbank
and Hatch 1987). It is interesting to note that an optimal partitioning would have predicted a
decrease in leakiness at very low irradiance (data not shown). The rise in leakiness at low irradiance
with a constant x is, however, consistent with experimental evidence (Henderson et al. 1992).
C4 photosynthesis is characterized by light response curves that saturate only at very high irra
diance. An example of this is shown for a Flaveria bidentis wild-type plant (Fig. 4.24). Transgenic
Flaveria bidentis plants where the amount of rubisco has been reduced through antisense saturate
at much lower irradiances (Fig. 4.24; Furbank et al. 1996; Siebke et al. 1997). The model easily
reproduces these limitations. Saturation at lower irradiance would also occur at lower CO 2 partial
'(J)
60 0.6
')IE
"0
E x
-=!: 40 /' ~ 0.4
<:l::
Q)
"§ N'" 40 ><

c:: 'E
o ""6
~ 20 ~ 20
0.2
'E "l:
'00
(J) 0.2 0.4 0,6 0,8 1.0
11l
x
ON
V I I I '0.0
o 500 1000 1500 2000
Irradiance (urnol quanta rn" 5,1)
Figure 4.22 Light response of CO 2 assimilation rate, A, with optimal partitioning, x, of electron transport
between C4 and C3 cycles at a mesophyll CO 2 partial pressure, Cm' of 100ubar, Parameters aregiven in Table 4.1
except that Rd = 0 and a = O. Inset: CO 2 assimilation rate, A, at 2000 urnol quanta m-2 S-l as a function of the
fraction, x; of electron transport partitioned to the C4 cycle.
60 I a
Q)
'§
c:';
0 Cfl 40
'+= N
~ 'E
E
'iii "'6
Cfl E
11l .E,
N 20
0
0
0 2500
b OJ
c
0.4 2000 5
CD
-e en
Cfl"
Cfl
Q)
0.3 1500 ffi
03
C ::T
~
al
Q)
0.2 1000 ~8
...J
<\>
<n\)
0.1 f-/
0.0 I
------
I I , I
'0
500
1=
cr
~
0 500 1000 1500 2000
lrradiance (urnol quanta rn" S,1)
Figure 4.23 (a) Light response of CO 2 assimilation rate, A, with a constant partitioning, x = 0.4, of electron
transport between the C4 and C3 cycles at a mesophyll CO 2 concentration, Cm' of 100 ubar, lmax= 400 urnol
electrons m-2 s'. Rd = 0 and a = O. (b) The bundle-sheath CO 2 concentration and leakiness under the
conditions in (a),
50
a
40
30
en
"i
E 20
(5
E
-3 10
OJ
'§
c 0
0
~ b
'E 50
'iii
en
C1l
N 40
0
(J
30
20
10
0 500 1000 1500 2000
Irradiance (umol quanta m"2 sol)

Figure 4.24 (a) Comparison between modelled light response curves and those measured in control (closed
symbols) andtransgenic Flaveria bidentis with reduced amounts of rubisco (open symbols). Data are redrawn
from Siebke et al. (1997) and measurements were made at an ambient CO 2 partial pressure of 350 ubaranda leaf
temperature of 25°C. Rubisco site concentrations were 10.5, 5 and 3 urnol m-2 for control andtransgenic leaves
respectively. (b) Modelled curves showthe effectof a rubisco limitation at high irradiance. Parameters are given
in Table 4.1 except that Rd = 0 and c. = 0,
pressures where A may be limited by PEP carboxylase activity, or because of limitations to the
regeneration of PEP (Eq. 4.47).
4.3.3.2 Leakiness
Leakiness, <1>, was defined by Eq. (4.5) as the ratio of the rate of CO 2 leakage out of the bundle
sheath to the rate of PEP carboxylation. Leakiness defines the efficiency of the CO 2 concentrating
function of the C4 pathway and has become an important parameter in the description of C 4 .
photosynthesis. It can only be measured indirectly and estimates range from 10 to 40% (Farquhar
1983; Krall and Edwards 1990; Henderson et al. 1992; Hatch et al. 1995). Measurements of carbon
isotope discrimination during CO 2 exchange (Henderson et al. 1992) and measurements of
chlorophyll fluorescence and CO 2 exchange (Krall and Edwards 1990; Edwards and Baker 1993;
Oberhuber and Edwards 1993) suggest that the C3 and the C4 cycles are regulated such that 
remains constant over a range of CO 2 partial pressures, irradiances and temperatures. In the
model, is determined by the bundle-sheath conductance to CO 2 and the CO 2 gradient between
bundle sheath and mesophyll (Eq. 4.4). This gradient in turn is dependent on the relative capaci
ties of PEP carboxylase or rubisco at high irradiance or the electron transport capacities at low
and moderate irradiances (Fig. 4.23). The model has no feedback regulation from the C3 cycle to
the C4 cycle so that when rubisco becomes limiting at high CO 2 partial pressures (Fig. 4.11) the
model predicts a rapid increase in unless PEP regeneration is also curtailed (Fig. 4.13).
Although there appears to be little variation in with short-term environmental perturbation in
many species, carbon isotope and chlorophyll fluorescence studies on transgenic Flaveria bidentis
with artificially reduced amounts of rub isco have shown that does increase in these circum
stances (Siebke et al. 1997; von Caemmerer et al. 1997). These results show that the relative
amounts of rub isco and C4 cycle enzymes are important in affecting leakiness. However, caution
is needed when using the model to estimate leakiness.
4.3.3.3 Quantum yield
Traditionally, quantum yield has been measured as the initial slope of the light response
curve of CO 2 fixation. The quantum yield of PS II can also be measured at other irradiances
with the use of chlorophyll fluorescence (Genty et al. 1989). Quantum yield amongst
unstressed C 3 plants varies little across a wide range of species when measured under stan
dard CO 2, 02 and temperature (see Ehleringer and Pearcy 1983; Hatch 1987). C 4 plants, on
the other hand, show considerable interspecific variation, ranging from 0.05 to 0.069 across
32 C 4 species (Ehleringer and Pearcy 1983). It has been suggested that this range of quantum
yields might reflect interspecific variation in leakiness (Ehleringer and Pearcy 1983; Farquhar
1983; Furbank et al. 1990). Considering that there are three subpathways responsible for the
C 4 photosynthetic mechanism, one might expect some trends in the energy requirements and
hence quantum yields between decarboxylation types and there is some evidence for such a
correlation. However, the largest differences observed are between NAD-malic enzyme
(NAD-ME) dicot types and the remainder of the C 4 monocots and dicots, where the former
have average quantum yields of around 0.053 compared to 0.0625 for the latter (Ehleringer
and Pearcy 1983). It is interesting to note that there appears to be no consistent correlation
between measured quantum yields and leak rates estimated either by carbon isotope discrim
ination or radio-isotope methods (Hatch et al. 1995).
Despite the reservations outlined above, it is possible to model the effect of leakiness on the
quantum requirement of CO 2 fixation in C 4 plants and this has been done on at least two occa
sions (Farquhar 1983; Furbank et al. 1990). Furbank et al. (1990) constructed a model that
predicts the quantum requirement of photosynthesis from a given leakiness, based on the ATP
and NADPH requirements of the mesophyll reactions. Because the increased energy demands
result in an increased ATP requirement per net CO 2 fixed, the following approximation can be
derived from Eq. (4.27):
ATP requirement = (21(1- <1» + 3)(A + R d ) (4.55)
With certain assumptions, this ATP requirement can be related to quantum requirement per
net CO 2 fixed (Table 2.1, page 34). A key assumption is the number of protons partitioned
per electron transported in the chloroplast electron transport chain, affecting the number of
ATP produced per quanta absorbed (see Table 4.1). A second key assumption in this model is
the number of protons, which must be partitioned per ATP, produced by the thylakoid
ATPase. The two most common values reported in the literature are 3 and 4 (Table 2.1,
page 34). Furbank et al. (1990) and Hatch (1992) calculated the relationship between quan
tum requirement and leakiness and compared this with measured quantum yields for C 3 and
C 4 . They concluded that the only way to account for the low measured values for quantum
requirement in C 4 monocots is to assume the operation of the Q cycle. They proposed that
the C 4 mechanism could not have evolved in its absence (Furbank et al. 1990; Hatch 1992).
The treatment described above is, of course, a slight oversimplification as it does not include
the possibility that at low light intensities some photorespiratory activity might occur, although,
even then, photorespiration is unlikely to be a large component. However, Berry and Farquhar
(1978) noted that in their model the light response is concave upwards at low irradiance so that
:J
g.sc
~
16
E gJ
.z 0
~.2 14
:J eel
°0E
--------------------------------
12
10 L----'------l._...1------'--_L-----l.------'-_--L------'---------l
o 200 400 600 800 1000

Bundle-sheath resistance
(m2 s marl)
Figure 4.25 Quantum requirement of CO 2 assimilation as a function of bundle-sheath resistance at mesophyll

concentrations of 20 (dashed line) and 200 mbar O2 (solid line) and a mesophyll CO 2 concentration of 300 ubar.
Calculations assume thefull operation of a Q cycle and 3W perATP. Parameters aregiven in Table 4.1 except that
Rd = 0 and a = O.
there is no unique slope as there is in C 3 models. This is because in the model <\> varies at low light
and bundle-sheath CO 2 partial pressures may be sufficiently low for some photorespiration to
occur (Figs 4.22 and 4.23). Figure 4.25 shows the quantum requirement calculated at 50 umol
quanta m- 2 S-1 of incident light as a function of bundle-sheath resistance. The quantum require
ment drops as bundle-sheath resistance increases, and above 250 m- s mol" there would be little
experimentally detectable variation in quantum yield. This would support the notion that, even
with the wide variations of CO 2 leak rates from bundle-sheath cells that are reported in the litera
ture, they are unlikely to account for the observed variations in quantum yield between C 4 species.
°
However, the model does also predict a small 2 and CO 2 sensitivity at low irradiance as low light
limits the accumulation of bundle-sheath CO 2 , Low bundle-sheath inorganic carbon concentra
tions have been observed experimentally under such conditions (Furbank and Hatch 1987).
Peisker, who modelled the optimization of energy balance between the C3 and the C4 cycles in
detail at low irradiance, also pointed out this oxygen dependence (Peisker 1988).
4.3.4 Modelling different decarboxylation types

4.3.4.1 Bundle-sheath conductance
C 4 species have been subdivided into three biochemical types based on the enzymes catalys
ing the decarboxylation of C 4 acids in the bundle sheath: NAD-ME, NADP-ME and PCK
(Hatch 1987). These biochemical variations are accompanied by a suite of anatomical fea
tures such as the presence or absence of a suberized lamella in the cell wall between bundle
sheath and mesophyll cells, and the centripetal or centrifugal location of chloroplasts in
bundle-sheath cells (Hatch 1987). There is inferential evidence that this suberized lamella is
responsible for a lower bundle-sheath conductance (Hatch and Osmond 1976) but at present
there is no clear experimental confirmation of this. Species lacking suberization almost
invariably have centripetally located chloroplasts, creating a longer liquid diffusion path, as
Hattersley and Browning (1981) suggested, which may overcome this shortcoming (Furbank
and Hatch 1989; Jenkins et al. 1989). On the other hand, variations in dry matter carbon
Q)
~ 60 '
oc ';" ~
:6:i en
<u <)I
=
.~
E
0
00
00 E 50
III ::1.
N~
o
o
40
0
N
Ib
o 15
s:
ca--=-
Q) III
.r::.o 10
00 E
~-
"C
c 5
::::l Ie
CO
N
0 1200
.r::
iii~
Q) "
s:
00.0
III 800
I
Q)~
E
=ac 400
::::l
CO
0
0.0 0.2 0.4 0.6 0.8 1.0
ex
Figure 4.26 The effectof varying the proportion of 02 evolution occurring in the bundle-sheath chloroplast (ex)
on (a) CO 2 assimilation rate, (b) bundle-sheath CO 2, Cs' and(c) 02 partial pressure, o,at bundle-sheath
conductances of 1 (dashed line) and 3 mmol m-2 S-l (solid line). Other parameters are asin Table 4.1.
isotope discrimination suggest that systematic differences in leakiness may exist between the
different biochemical subtypes (Hattersley 1982). Either bundle-sheath conductance or the
ratio of PEP carboxylase to rubisco activity could be responsible for these differences (Hend
erson et al. 1992). With the current level of knowledge it is not easy to refine the model to
account for such decarboxylation-type specific characteristics.
4.3.4.2 02 evolution in thebundle sheath

One factor which can be readily tailored to an individual species or subtype is ex, which
apportions the amount of oxygen evolution that occurs in the bundle sheath relative to the
mesophyll chloroplasts. Some NADP-ME monocots such as sorghum and maize have very
little PS 11 activity in the bundle sheath, while higher levels occur in other grass and
NADP-ME dicots. The NAD-ME and PCK types have bundle-sheath PS 11 activities similar
to C 3 species. For maize and sorghum, ex will be zero (Eq. 4.16), whereas it will approach or
even exceed 0.5 in many other cases. PCK-type species have additional mitochondrial 02
uptake because of the involvement of mitochondrial oxidative phosphorylation in the provi
sion of ATP for the PEP carboxykinase reaction in the bundle sheath (Section 4.3.4.2; Fur
bank and Badger 1982; Carnal et al. 1993; Hatch 1997).
Apart from the consequences on movement of PGA to the mesophyll for reduction (which is
difficult to model), the major effect of changes in ex will be bundle-sheath 02 partial pressure. This
is shown in Fig. 4.26 which plots the effect of a varying ex between 0 and 1 on CO 2 assimilation
rate and the bundle-sheath CO 2 and 02 partial pressures. The model predicts that bundle-sheath
02 partial pressure can be several-fold greater than ambient 02 partial pressure because bundle
sheath conductance to 02 is modelled to be 20-fold less than that to CO 2 due to the low solubility
of 02 (Eq. 4.15; Berry and Farquhar 1978; Farquhar 1983).
4.3.4.3 Energy requirements
Because of the different biochemical mechanisms used for decarboxylation in the three C 4
subtypes, one might predict differences in the energy balance and quantum requirements
between these types. For example, a unique feature of the PCK types is the involvement of
mitochondrial oxidative phosphorylation in the provision of ATP for the PEP carboxykinase
reaction in the bundle sheath (Hatch 1987; Carnal et al. 1993). In these species, some malate
is transported directly to the bundle sheath, is decarboxylated in mitochondria to give CO 2
and NADH, with ATP being generated during the subsequent oxidation of NADH.
Minimally, assuming 3 ATP are produced per NADH oxidized in mitochondrial electron
transport, one-quarter of the C 4 acids decarboxylated would have to follow this route to fully
support the ATP requirements of oxaloacetate decarboxylation. Such a partitioning between
the two pathways is supported by measurements of relative amounts of PEP carboxykinase
and NAD-ME in PCK types and from in vitro studies which suggest, however, that there may
be some flexibility in this process (Hatch 1987; Carnal et al. 1993). For the PCK types the
ATP requirement is therefore given by:
ATP requirement ={ 2 + 3}(A + R d ) (4.56)

(1 + 11)(1- <\»
and the NADPH requirement by:
NADPH requirement ={ 1 + 2}(A + R d ) (4.57)

(1+11)(1-<\»
where 11 is the mol of ATP produced in the mitochondria per mol of NADPH. If 11 = 3, 3.5
ATP and 2.25 NADPH are required from thylakoid electron transport per CO 2 fixed (assum
ing no CO 2 leak) (von Caemmerer and Furbank 1999). Converting this ATP requirement to
quanta required, and assuming no Q cycle, the basal energy requirement for PCK types is
around 14 quanta per CO 2 fixed, compared to 18.3 for NAD-ME types using the same
assumptions. This large difference is dependent upon the value taken for 11, which in turn
depends on the ATP to oxygen ratio for mitochondrial respiration (i.e. its 'efficiency'), in an
analogous fashion to the ATP/2e- ratio for thylakoid electron transport. Differences of 4
quanta per CO 2 fixed would be easily observed in vivo; however, PCK-type quantum require
ments do lie at the lower end of values observed for C 4 plants by Ehleringer and Pearcy
(1983) - 15.6 for PCK, compared to 18.9 for NAD-ME dicots and 16.6 for NAD-ME grasses
- but similar to values obtained for NADP-ME grasses (15.4). The reason for the lack of
large differences in practice may be interspecific variability in the other factors discussed
above which affect the efficiency of the C 4 processes.
5
Models of (3-(4 intermediate photosynthesis
5.1 INTRODUCTION
Species with C 3-C4 intermediate photosynthetic pathways have been identified and described
in several genera across various families including representatives of monocotyledons and
dicotyledons (Monson et al. 1984; Peisker 1986; Rajendrudu et al. 1986; Edwards and Ku
1987; Monson and Moore 1989; Brown and Hattersley 1989). Although different biochemical
variants give rise to the syndrome of C 3-C 4 intermediacy, all such plants have a Kranz- or C 4
like leaf anatomy. Their CO 2 uptake shows a reduced oxygen sensitivity in comparison with
that of C3 species and the CO 2 compensation point is intermediate between those measured
for C 3 and C 4 species (Edwards and Ku 1987; Brown and Hattersley 1989). This is illustrated
in Fig. 5.1 with CO 2 response curves typical of a C j , C 3-C 4 , and a C 4 Flaveria species.
For most C j - C 4 species the reduced oxygen sensitivities and the low CO 2 compensation points
are thought to be the result of efficient recycling of photorespiratory CO 2 in the bundle sheath
(Brown 1980; Monson et al. 1984). Studies that have shown that most or all of glycine decarboxylase
is localized within the bundle-sheath cells in several intermediate species in the genera Moricandia,
Panicum and Elaveria support this hypothesis (Hylton et al. 1988; Rawsthorne et al. 1988, 1989;
Rawsthorne 1992; Morgan et al. 1993).
-
Q)
ctl
....
c T";''-'"
40
30
,•
~AA-_-A
....---. .
..
0 CIl
+==
ctl
=E
N
'
_E
1 •
/.
20
CIl
CIl
0
E /
/ ~ -----~~
.
ctl :::i. ~/~
OJ - ~-~
0
0
10
/ ~
/::/~
tr/·J
0
0 100 200 300 400 500
c,
(ubar)
Figure 5.1 (02 assimilation rate as a function of intercellular (02 partial pressure, C;, for Flaveria bidentis ((4;
.A.), F. floridana ((3-(4; e) and F. pringlei ((3; =). Measurements were made at an irradiance of 1000urnol
quanta m-2 s' and a leaftemperature of 25°C.
123
In addition, evidence of a C 4-like biochemistry has been shown for some Flaveria species (Ku
et al. 1983; Rumpho et al. 1984; Monson et a1.1986; Moore et al. 1987). Different C rC 4 interme
diates in this family fix between 15 to 85% of atmospheric CO 2 into C 4 acids during short-term
exposure to HC0 2; however, transfer of label to the C 3 cycle does not in many cases occur at the
rates normally observed in C 4 species (Monson et al. 1986). Furthermore, in all of the Fiaveria
C 3-C4 intermediates, both rubisco and PEP carboxylase are not entirely compartmentalized
between mesophyll and bundle-sheath cells, as is observed in C4 species (Bauwe 1984; Reed and
Chollett 1985; Moore et al. 1988). There is also a group of Fiaveria species that has been called a
C4-like species. They had previously been classified as C 4 species, but investigations have shown
that they possess small amounts of rub isco in the mesophyll cells, which leads to some CO 2 being
fixed directly by rubisco (Monson et al. 1987; Moore et al. 1989).
The photosynthetic properties of these C 3-C4 intermediate species are fascinating because
they are viewed as possible evolutionary intermediates between the C 3 and C4 photosynthetic
pathways (Peisker 1986; Brown and Bouton 1993).
5.2 BASIC MODEL EQUATIONS

The earliest models of C 3-C4 intermediate photosynthesis assumed a partial C 4 cycle and did
not consider recycling of photorespiratory CO 2 in the bundle sheath (Peisker and Bauwe
1984; Peisker 1985; Peisker 1986). Von Caemmerer (1989) demonstrated with the use of a
mathematical model that the recycling of photorespiratory CO 2 in the bundle sheath could
account for many of the observed gas-exchange characteristics. The model presented in this
chapter is a synthesis of the earlier models by Peisker and co-workers and that by von Caern
merer (1989,1992).
A schematic representation of the proposed carbon fluxes is given in Fig. 5.2. A complete C 3
cycle is assumed to operate in both the mesophyll and bundle-sheath cells. Some or all of the gly
cine formed in the mesophyll following oxygenation of RuBP is assumed to diffuse to the bundle
sheath cells. There the glycine is decarboxylated and the photorespiratory CO 2 released supplies
rubisco in the bundle sheath with CO 2 , In some cases additional CO 2 may be supplied via a C 4
cycle. As for C 4 species, it is assumed that there is a substantial resistance to CO 2 diffusion across
the bundle-sheath cell wall.
Since all carbon fixation is ultimately the result of rubisco carboxylation the overall rate of net
CO 2 fixation, A, can be written as the sum of mesophyll CO 2 assimilation, Am' and bundle-sheath
CO 2 assimilation, As:
(5.1 )
where
(5.2)
and
A, = V,-,-0.5V",-R, (5.3)
Vern and V es are the rubisco carboxylation rates of rubisco localized in the mesophyll or the bundle
sheath respectively. 0.5 Vom and 0.5 Vos are the photorespiratory CO 2 releases resulting from meso
phyll and bundle-sheath rubisco oxygenations and R m and R, are the rates of other mitochondrial
respiratory CO 2 release in the two compartments not associated with photorespiration.
The net CO 2 assimilation rate of the bundle sheath, As, is also given by:
(5.4)
Models of CrC 4 intermediate photosynthesis 125
CHp
G~YCine.~ ~/r
°2 -.
\ °2
PCO:=r=J PCO PCR
J Serine
6 -.o
2
CO 2 CO
\\ r l = C O
~
RCO, C, /
mesophyll cell bundle-sheath cell
Figure 5.2 Schematic representation of carbon metabolism in C3-C4 intermediate species. CO 2 fixation bythe
photosynthetic carbon reduction (PCR) cycle occurs in both mesophyll and bundle-sheath cells. CO2 is supplied to
the bundle sheath via decarboxylation of glycine from both mesophyll and bundle-sheath photosynthetic carbon
oxidation (PCO) cycles, and in somespecies additionally via a C4 cycle.
Vp is the rate of PEP carboxylation and ~ is the fraction of photorespiratory CO 2 resulting

from mesophyll oxygenations, which is released in the bundle sheath. L is the rate of CO 2
leakage from the bundle sheath. Land Vp have been defined in Chapter 4 (Eqs 4.4 and 4.17).
Combining Eqs (5.1)-(5.4) one obtains a second equation for total CO 2 assimilation rate.
A = VClI1-(1-~)0.5Vom+Vp-L-R." (5.5)
Some of the intermediates, such as Moricandia arvensis (Rawsthorne et al. 1988) and
Panicum milioides (Edwards and Ku 1987) have no functional C 4 acid cycle and are described
without the operation of a C 4 cycle (Vp = 0). If ~ = 1 there is no photorespiratory CO 2 release
in the mesophyll cells. Flaveria intermediates are best described by the full scheme. Hylton et
al. (1988) have shown that glycine decarboxylase is solely located in the bundle sheath in
some of the Flaveria species, which makes the operation of the glycine shuttle likely. CO 2
fixation into C 4 acids had previously been demonstrated (e.g. Monson et al. 1986). However,
the C 4 cycle may not be fully functional and some of the CO 2 fixed by PEP carboxylase may
be decarboxylated in the mesophyll. If all CO 2 fixed by PEP carboxylase is decarboxylated in
the mesophyll then, effectively, Vp = O. Here, Vp stands for PEP carboxylations that are
decarboxylated in the bundle sheath.
5.3 EQUATIONS FOR PHOTOSYNTHESIS AT HIGH IRRADIANCE
5.3.1 Rubisco-Iimited CO 2 assimilation rate

The rubisco-limited CO 2 assimilation rates can be found by substituting the RUEP-saturated
rubisco carboxylation rates into Eqs (5.2) and (5.3):
A - (C.,,-y.O.,,)Vmmax -R
(5.6)
m C m+Kc(1+0n/Ko) //I
and
(5.7)
in an analogous manner to the derivations of Section 4.2.1.1.
5.3.2 Net 02 evolution in the bundle sheath

The granal nature of bundle-sheath chloroplasts of C 3-C 4 intermediate species indicates that
they are capable of PS II activity (Edwards and Ku 1987; Pfundel and Pfeffer 1997). As for C 4
species, this has implications for the steady-state O 2 partial pressure of the bundle sheath.
The net O 2 evolution in the bundle-sheath cells depends upon the balance of O 2 evolution at
PS II and the various O 2 consuming processes. In particular, the balance depends on how the
glycine shuttle is perceived to operate. Here, the solution, which generates no extra O 2 con
sumption based upon the glycine shuttle to the bundle sheath, has been chosen.
Briefly the integrated carbon reduction and oxidation cycle in the bundle-sheath cells con
sumes NADPH at the rate of 2V's + 2Vos which results in an O 2 evolution rate of V es + Vos' Oxygen
is consumed by the bundle sheath, during glycolate oxidation (resulting from glycolate produced
in the bundle sheath) and by mitochondrial respiration at the rate of 1.5Vos + R; In Moricandia
arvensis, enzymes necessary for the synthesis of glycine are present in both bundle-sheath and
mesophyll compartments and it is therefore likely that glycine rather than glycolate is the com
pound that diffuses to the bundle sheath (Rawsthorne et al. 1988). It is assumed that carbon
skeletons are returned to the mesophyll. Therefore PCA derived from mesophyll glycine is
reduced in the mesophyll such that, in addition, no extra O 2 evolution rate occurs in the bundle
sheath. In summary:
(5.8)
and
A
as --"-+0
0.047gs m
(5.9)
5.3.3 Quadratic expression for CO 2 assimilation rate

Under enzyme-limited conditions the solution for As is a function of mesophyll CO 2 and O 2
partial pressure is similar to that for C 4 species. That is:
As (5.10)
and
a (5.11)
b -1 (V, + ~O.5 V,m + g,Cm) + (V,m" - R,)

(5.12)
(r.Vsmax+ ~CRs)l
+ gs(Kc(1 + 0",/ Ko + » 0.047 0

Models of (3-(4 intermediate photosynthesis 127
Table 5.1 Photosynthetic parameters (at 25°C) used in the model
Parameter Value Description
V mmax 120 umol m- 2 S-1 Maximum rubisco activity in mesophyll cells
~l"l1/ax 10 umol m? S-1 Maximum rubisco activity in bundle-sheath cells

Ke 404 ubar" Michaelis constant of rubisco for CO 2
Ko 248 mbar" Michaelis constant of rubisco for O2
y. 0.00193 (0.5/2590)a 1/(2Sclo)' where Sclo is the rubisco specificity
VpmlU: 40 umol m-2 s-) or 0 Maximum PEP carboxylase activity

Kp 80 ubar"
s, I mmol m-2 S-1 Bundle-sheath conductance to CO 2
go 0.047g,C Bundle-sheath conductance to O 2 (Eq. 4.15)

R,j 0.01V emax Leaf mitochondrial respiration
R,n 0.8R d Mesophyll mitochondrial respiration
I; 0<1;<1 Fraction of mesophyll derived photorespiration occurring in
the bundle sheath
0< a < I
, In vivo values of tobacco (von Caemmerer et al. 1994; see also Table 2.3).
b Bauwe (1986)
c Farquhar (1983)
c = (Vsmax - R,)( Vp + ~0.5 V om + gsC",) - [Vsmaxgsy.Om + Rsg,Kc(l + On/Ko)] (5.13)
where
C In V pmax-+
)0
~
y.O",Vmn",x
Vp + ~0.5 V om -
_
(5.14)
c, + Kp c, + K/l + 0m/Ko)
Together with Eq. (5.6) this gives a solution for A.
5.4 PARAMETERIZATION AND ANALYSIS OF THE MODEL AT HIGH IRRADIANCE

A summary of standard values used in the model outputs is given in Table 5.1. As many parame
ters as possible have been assigned from values in the literature. However, a photosynthetic model
of different C 3-C4 intermediate pathways is especially designed for testing ideas rather than
empirical fitting. Parameters such as ~ (the fraction of mesophyll-derived glycine decarboxylated
in the bundle sheath), the partitioning of rubisco between mesophyll and bundle sheath and the
amount of C 4 cycle activity are important variables in this context.
5.4.1 Kinetic constants

The kinetic constants for rubisco chosen are the same as those taken for the C 3 model since
studies on rubisco of C 3-C4 intermediates show them to be C 3-like (Table 5.1; Wessinger
et al. 1989; Hudson et al. 1990). The maximum rubisco activities are likely to vary with leaf
age and environmental growth conditions and the partitioning between mesophyll and
bundle sheath is discussed below.
5.4.2 Respiration
Mitochondrial respiration rates (R m , R) have been somewhat arbitrarily assigned to meso
phyll and bundle-sheath compartments in accordance with the rubisco partitioning, since
little is known.
2.0
;" 1.5
;:0
In
S 1.0
c
0
~
's,
x 0.5
0
..c
;U
o
.8 0.0
c 0 100 200 300 400
0
~ Mesophyll CO 2 partial pressure

'0..
en em (ubar)
<D
0 1.5
15
.s::: b
a.
en
<D
'@ 1.0
<D
-5
'0
0
.~ 0.5
a:
o.0 '-------'--------L----'-------'-_'-------'-------'-------'--------'-_
o 10 20 30 40 50
Leaf temperature (C)
Figure 5.3 The ratio of photorespiration to carboxylation rate (a) as a function of mesophyll (0 2 partial
pressure, Cm' at 25°(, (b) asa function of temperature at different Cm of (1) 50 ubar, (2) 100 ubarand (3)
250 ubar, Kinetic constants of rubisco used arethoseof Table 5.1 and the activation energies are given in
Table 2.3.
5.4.3 Partitioning of photorespiration

The parameter ~ determines the fraction of the mesophyll-derived photorespiratory CO 2
respired in the bundle-sheath cells. Evidence by Rawsthorne et al. (1988) has shown that in
Moricandia arvensis all of the glycine decarboxylase is located in the bundle sheath. Similar
results have been found for Panicum milioides, Flaverta Iinearis and F.floridana (Hylton et al.
1988). ~ has been set at 1 in all simulations shown here unless otherwise indicated.
5.4.4 Bundle-sheath conductance

A low bundle-sheath conductance to CO 2 diffusion is an essential feature for a CO 2
concentrating mechanism in the bundle sheath. On a leaf area basis the conductance is the
product of bundle-sheath surface area and the conductance across the bundle-sheath cell
wall interface. Brown et al. (1983) showed that Panicum C rC 4 intermediates had a well
developed vascular bundle sheath and that the number of plasmodesmata were similar to
that of C 4 Panicum species and more than in the C 3 Panicum species. It has been assumed
that the conductance across the interface may well be similar to that in C 4 species. However,
given that the cross-sectional leaf area occupied by the bundle sheath is less in many of the
intermediate species compared to C 4 species (Brown and Hattersley 1989), it is likely that the
bundle-sheath surface area is also less. For the standard outputs a standard bundle-sheath
40
a C3
"*...
c ~~
30 I C3 - C. --
o '(J)
~ 'l'E
'E
._ -0 20
(J) E
(J) :::i
ttl ~
0'" 10
o
o
1.4 I b
;;,
~ 1.2
o'"
I", 1.0
«
0.8
0.6
o 20 40
Bundle-sheath rubisco activity
2s·1
(umol m· )
Figure 5.4 (a) CO 2 assimilation rate, A. of the (3-(4 photosynthetic pathway with no (4 cycle activity asa
function of bundle-sheath rubisco activity at two mesophyll CO 2 partial pressures, Cm of 250 ubar (top) and
100 ubar (bottom). Mesophyll rubisco activity = 120 urnol m-2 S-1 and otherparameters arethe same as in
Table 5.1, This rate is compared to (02 assimilation of (3 photosynthesis with an equal (mesophyll plus bundle
sheath) rubisco activity. (b) The ratio of A of (3-(4 to (3 photosynthesis shown in (a) asa function of bundle
sheath rubisco activity at two mesophyll (02 partial pressures, Cm of 100 ubar(top)and 250 ubar (bottom).
conductance of 1 mmol m- 2 S-1 (Table 5.1) has been chosen, which is a little less than what
was used in the C 4 model in Chapter 4.
5.5 THE GLYCINE SHUTTLE

Many of the known C 3-C4 intermediate species have no detectable C 4-cycle activity and in
this section it is shown that the recycling of photorespir atory CO 2 in the bundle sheath is suf
ficient to explain the major features of C rC 4 intermediacy. Monson (1989) and Schuster and
Monson (1990) pointed out that photorespiratory recycling may present the intermediate
species with a photosynthetic advantage at high temperatures or under low intercellular CO 2
partial pressures which might occur in xeric environments from which many of the species
derive. Figure 5.3 shows the magnitude of photorespiratory CO 2 release as a fraction of
carboxylation rate at different mesophyll CO 2 partial pressures and leaf temperatures. This
fraction is the maximum of photo respiratory CO 2 release that can be refixed by the bundle
sheath if there were no leakage of CO 2 , The ratio equals 1 at L and decreases with increasing
CO 2 partial pressure. At a mesophyll CO 2 partial pressure, em = 250 ubar (a common value
for these species at ambient CO 2 partial pressures of 340 ubar) and a leaf temperature of
25°C the photorespirator y CO 2 release is 14% of carboxylation. It increases to 23% at a leaf
40 ,--------,----,----,-----,----.--,
a
Q)
c3c,
~ 30 == _
c .,
o If)
::ffi ~
'E E 20
'iii (5
If)
Cll
E
::1.
c3c,
0'" ~
() 10"
, '--------------1
"0
C
Cll 2:
::::l
If)
b
lii If)
..Q
::1. 2: 1000
C.
0'" Cii
750
a~:eCll
() c.
.s:: 500
til lii
Q)
..Q
E /
a,
.s:: I
If)
I
Q) 0'" 250
'6 ON
C
::::l
m 0
0 500 1000 1500
Bundle-sheath resistance
2
(m s ' mar')
Figure 5.5 (a) CO 2 assimilation rate, A, of the (r(4 photosynthetic pathway with no (4 cycle activityasa
function of bundle-sheath conductance. Other parameters are given in Table 5.1. This is compared to A of (3 leaf
where rubisco activityequals the total mesophyll and bundle-sheath activity. (b) Bundle-sheath (02 and 02
partial pressures, (and 05,respectively at em = 250 ubar(dashed line) and 100 ubar(solid line).
temperature of 40°C, but is of course higher at lower mesophyll CO 2 partial pressures

(Fig. 5.3). Thus this pathway has only a limited capacity (dictated by mesophyll rubisco
oxygenase activity) to enhance CO 2 fixation rates at present atmospheric CO 2 partial pres
sures. The following section discusses the choices made for bundle-sheath rubisco activity
and conductance and then examines how successfully the model describes the known charac
teristics of C 3-C4 intermediacy.
5.5.1 Bundle-sheath rubisco activity and conductance

Figure 5.4 examines the dependence of CO 2 assimilation rate on the amount of bundle
sheath rubisco activity. Also shown is the CO 2 assimilation rate of a C 3 leaf with the same
total amount of rubisco (mesophyll + bundle sheath). CO 2 assimilation rate is greater in the
C 3-C4 intermediate when approximately 10% of the rubisco is allocated to the bundle sheath
and there is not sufficient photo respiratory CO 2 release at ambient CO 2 to support much
more rubisco in the bundle sheath. At low CO 2 partial pressures the enhancement in the CO 2
assimilation rate of the C 3-C4 intermediate over that of C 3 photosynthesis is greater but
again the response also saturates at low bundle-sheath rubisco concentrations.
A high bundle-sheath resistance (l/gJ helps concentrate CO 2 in that compartment and this
is shown in Fig. 5.5. A high resistance is essential to concentrate CO 2 in the bundle sheath, given
the low capacity of CO 2 supply by the photorespiratory CO 2 release. The simulations do not
contain a respiratory component, which would also contribute to the bundle-sheath CO 2 par
tial pressure. However, at high temperatures the pathway may also serve to fix CO 2 respired in
the bundle sheath.
Models of (r(4 intermediate photosynthesis 131
160 1----.--
~ ~
«i
..c
o o~ -
o °200
2
400
partial press ure (mbar)
600
Figure 5.6 The response of (0 2 compensation point to O2 partial pressure for Glycine max (e; (3)' Moricandia
arvensis ( .• ; (r(4)' and Panicum milioides (....; (3-(4) and Spartina pectinata (.; (4)' Data areredrawn from
Holaday. et al. (1982). Measurements were made at 25°( and 600 urnol quanta m-2 S-1,
100
«i
.0 80
2
.....
'E.
'0 60
a.
0
.~
40
(/J
c
Q)
a.
E
~ / -: /'" , , b
0
o
20
ON
o ol/~~I I I
o 100 200 300 400 500
Mesophyll 02 partial pressure, Om (mbar)
Figure 5.7 The modelled dependence of the (0 2 compensation point, T; on mesophyll O2 partial pressure for (3
photosynthesis, and for (3-(4 intermediate photosynthesis with no (4 cycle activity. (a) Standard conditions of
Table 5.1, (b) with Vsmax reduced to 5 urnol m-2 S-1, (c) with 9s increased to 10 mmol m-2 s'. (d) with ~ = 0.5.
Mitochondrial respiration was assumed to be zero in all cases.
5.5.2 CO 2 compensation point

The low CO 2 compensation point and its non-linear dependence on 02 partial pressure first
identified C rC 4 intermediate species. In C 3 species the CO 2 compensation point increases
linearly with 02 partial pressure (Forrester et al. 1966; Bjorkman 1970; Peisker and Apel
1977), which can be explained by considering only the kinetics of rubisco (Section 2.7,
page 47; Laing et al. 1974; Azcon-Bieto et al. 1981,1986). In C 3-C4 intermediate species such
as Panicum milioides and Moricandia arvensis non-linear relationships have been observed
(see, for example, Keck and Ogren 1976; Quebedeaux and Chollet 1977; Apel 1980; Holaday
')'
en
1200
-Co
E 40 1000 E,
"'6 c, "
E " " " A(C,)
(J'
~ 30 800
'<:(
~ 20
Q)
A(C 3 - cJ
600 °o1ii'"
..c
c Q)
0 400 ..c
en
~ 10 ~
'E 200 "'C
C
en :::J
en 1:0
eel
'"
a
400
°o
100 200 300
em (ubar)
Figure 5.8 Modelled response of rubisco-limited (02 assimilation, A. to mesophyll (02 partial pressure, (m' of
(3-(4 intermediate photosynthesis with no (4 cycle activity. Rubisco activities are 120and 10 urnol m-2 S-1 in the
mesophyll and bundle-sheath compartment respectively. It is assumed that all glycine is decarboxylated in the
bundle sheath (~ = 1). bundle-sheath conductance, gs = 1 mmol m-2 S-1, and Rm = Rs = O. Also shown arethe
bundle-sheath (0 2 partial pressure, (5' and bundle-sheath (02 assimilation, As' The (3-(4 photosynthetic (02
assimilation rate is compared to a (3 rubisco-limited ratewith an equal rubisco activity of 130 urnol m-2 S-1.
et al. 1982; Hattersley et al. 1986; Hunt et al. 1987). An example is shown in Fig. 5.6. The
C 3-C4 model with the glycine shuttle alone is capable of predicting low CO 2 compensation
points at ambient oxygen as well as a curvilinear response to 02 partial pressure (Fig. 5.7).
This contrasts with the diphasic response predicted by the model of Peisker and Bauwe
(1984) which depended on a partial C 4 cycle (see also Fig. 5.11). The equation for the com
pensation point can be derived from Eqs (5.10)-(5.14) but is cubic in nature. It can be
reduced to a quadratic by approximating both V em and Vp with linear dependencies on CO 2
partial pressure. The actual values were obtained by an iterative procedure.
At low 02 partial pressures the compensation point is low as CO 2 released to the bundle sheath
is rapidly refixed with little leakage of CO 2 out of the bundle sheath. As the rate of photorespira
tion increases with increasing 02 partial pressure, bundle-sheath rubisco becomes saturated with
bundle-sheath CO 2 and the leakage rate out of the bundle sheath rises and I" increases. An
increase in the amount of bundle-sheath rubisco keeps I" low until higher 02 partial pressure.
(Fig. 5.7, lines a and b). Bundle-sheath conductance influences the relationship by changing the
leak rate out of the bundle sheath (Fig. 5.7, lines a and c). Changing the proportion (~) of photo
respiratory CO 2 released in the bundle-sheath cells affects I" by the amount of CO 2 lost directly
from the mesophyll cells (Fig. 5.7, lines a and d).
A further characteristic of I" in many C3-C4 intermediates is a strong dependence on irradiance
(e.g. Brown and Morgan 1980; Holaday et al. 1982). At low irradiances I" can be almost as high as in
C3 species but then declines rapidly with increasing irradiance, which has not been modelled here.
5.5.3 CO 2 and 02 responses of CO 2 assimilation rate
In Fig. 5.8 the CO 2 assimilation rate, A, bundle-sheath CO 2 assimilation rate, As' and bundle
sheath CO 2 partial pressure, Cs' are shown as a function of mesophyll CO 2 partial pressure,
Cm • Also shown is the rate of C 3 photosynthetic CO 2 assimilation for an equivalent amount

of rubisco (mesophyll + bundle sheath). For simplicity this simulation contains no limita
tions on RuBP regeneration capacity so no saturation occurs at higher C m . RuBP regenera
tion limitations are discussed in Section 5.7. At low mesophyll CO 2 partial pressure, C m ' the
60
<l:
$'
50 ~a,
~ ~-
c 'w 40 c"(,
~ ~E
'E (5
.~
ctl
§.
~
30 r " -,
C,-C.
20
ON
o 10 c:---- _
o
100 t b--'------~----LI--~,~ I
I
0" 80
11
at
60
co
<l:
'0 40
v. 20
<l: C,
o I I !
o 200 400 600 800 1000

Mesophyll 02 partial pressure Om (mbar)
Figure 5.9 Response of (02 assimilation rate ofthe (3 and the (r(4 photosynthetic pathways (without (4
cycle activity) to oxygen partial pressure, Om' Modelled parameters are as in Fig. 5.4.
response correctly reflects experimental measurements. The expression for the initial slope is
somewhat complex but it is clear from a comparison of A for the C rC 4 and the C3 species at
low Cm that a higher assimilation rate is achieved for the same amount of rubisco. As yet no
thorough study on the relationship between rubisco content and CO 2 assimilation rate has
been made for C3 and C3-C 4 intermediate species.
One of the most distinctive features of the C rC 4 intermediate model generated solely by a
glycine shuttle is the predicted change in bundle-sheath CO 2 partial pressure, CS ' Bundle-sheath
CO 2 partial pressure, C" is predicted to be highest at low mesophyll CO 2 partial pressures, where
the rate of photorespiration is high and declines with increasing Cm. This is opposite to the C4
model where Cs increases with increasing Cm' as PEP carboxylation rate increases (Fig. 4.11; Berry
and Farquhar 1978). Direct measurements of the inorganic carbon pools in the bundle sheath of
C4 species have confirmed these C 4 predictions (Furbank and Hatch 1987). Similar measurements
in the genus Flaveria by Moore et al. (1987) have confirmed that CO 2 is concentrated in the
bundle sheath at ambient CO 2 , but the CO 2 dependence has not been examined.
Figure 5.9 shows the dependence of CO 2 assimilation rate on 02 partial pressure. This is com
pared with the 02 dependence of C3 photosynthesis with the same amount of rubisco. For the
CrC 4 intermediate model the rate at 500 mbar 02 was 400/0 of that at 0 mbar 02' whilst for the C3
model it was 28%. These values are in good quantitative agreement with measurements made by
Keck and Ogren (1976) but direct comparison is difficult, as changes in intercellular CO 2 partial
pressures were not recorded.
5.6 THE CONTRIBUTION OF (4 PHOTOSYNTHESIS

Evidence of some involvement of C 4-like biochemistry has been shown mainly for Flaveria spe
cies (Ku et al. 1983; Rumpho et al. 1984; Monson et al. 1986; Moore et al. 1987; Brown et al.
1991). Different C rC 4 intermediates in this family fix between 15 and 85% of atmospheric
I I
160
I
f-
120 I
80 I
401-
0/.
/O~Jt..-- Jt../
~?- 'V-'V
o lile-:t::::=-~'iiil--~-)l~--=:::J-~-'crf::---------:=:r:==""":Y.-----lI_-.J
o 200 400 600
02 partial pressure (mbar)
Figure 5.10 The response of the (02 compensation point to 02 partial pressure for (3' (4 and (3-(4 Flaveria
species F. cronquistii ((3; ), F. pringlei ((3; e). F. sonorensis ((3-(4; .A.), F. floridana ((3-(4; ) and F. trinervia
((4; +). Data are taken from Ku et al. (1991). Measurements were made at 300 ( and 1150 urnol quanta m-2 S-1.
CO 2 into C 4 acids during short-term exposure to 14C02; however, transfer of label to the C 3
cycle in many cases does not occur at the rates normally observed in C 4 species (Monson et al.
1986). Furthermore, in all of the Flaveria C3-C4 intermediates, both rubisco and PEP carboxy
lase are also not entirely compartmentalized between mesophyll and bundle-sheath cells, as is
observed in C 4 species (Bauwe 1984; Reed and Chollet 1985; Moore et al. 1988). There is also a
group of Flaveria species that have been called C 4-like species. They had previously been classi
fied as C 4 species, but investigations have shown that they possess small amounts of rubisco in
the mesophyll cells which leads to some CO 2 being fixed directly by rubisco (Monson et al.
1987; Moore et al. 1989).
The basic equations of Section 5.2 are set up to accommodate these variations and can be used
to test ideas. For example, a partial C 4 cycle has implications for the carbon isotope discrimination
of C3-C4 intermediates. The model that relies only on photorespiratory recycling predicts C 3-like
carbon isotope ratios, whereas even a small amount of C 4 cycle activity that results in carbon fix
ation should result in lower carbon isotope ratios (von Caemmerer 1992).
5.6.1 CO 2 compensation point

Figure 5.10 shows some examples of the response of the CO 2 compensation point to 02 partial
pressure for several Flaveria species. Figure 5.11 compares CO 2 compensation points modelled
solely with the addition of a C 4 photosynthetic path (solid lines) to the compensation points
resulting from the incorporation of a glycine shuttle. It is interesting to note that quite high
PEP carboxylase activities are required to generate low CO 2 compensation points at low 02
with the addition of a C 4 pathway alone. Peisker and Bauwe (1984) suggested that the increase
at higher 02 partial pressures was the result of low PEP regeneration capacities. The compari
son shows that photorespiratory recycling is more effective at generating low CO 2 compensa
tion points. For simplicity the model outputs have not included a respiratory component,
although it is incorporated in the equations. The effects of mitochondrial respiration on the C 3
compensation point were shown in Fig. 2.12 (page 49). The impact is similar for leaves of
C 3-C4 intermediate species and probably accounts for variations observed with leaf age and
growth conditions. The high CO 2 compensation points at low 02 of Flaveria cronquistii and F.
sonorensis may be the result of a high mitochondrial respiration rate (Fig. 5.10).
100
/
ro
.0 /
/
~ 80 /-
/
~ /
C
C3 /-
/
C
"0 /
0.. 60 /
/
C /
0 /
~
en /
/
C 40 /-
/-
Ql
0.. /
/
E /-
0 /
U
20 /-
0'" /-
/
o /
/
/
0
0 100 200 300 400 500
Mesophyll 02 partial pressure, Om (mbar)
Figure 5.11 The modelled dependence of the (02 compensation point, T, on mesophyll 02 partial pressure for
(3 photosynthesis, and for (3-(4 intermediate photosynthesis with (4 cycle activityand without a glycine shuttle
(~ = 0) (a) at the standard conditions of Table 5.1 and with Vpmax 40 urnol m-2 s', (b) with PEP regeneration
limitedto 4.4 urnol m-2 s', (c) with PEP regeneration limited to 2.87 urnol m-2 S-1. Mitochondrial respiration was
assumed to be zero in all cases. Dashed line (1) is the same as (a) in Fig. 5.7. Dashed line (2) has an additional (4
pathway with a Vpmax = 10 umol m-2 S-I.
5.6.2 CO 2 and 02 responses of CO 2 assimilation

Figure 5.12 gives an example of a CO 2 response curve of CO 2 assimilation rate for the C rC 4
model with a C 4 pathway but no photorespiratory recycling. With this model bundle-sheath
CO 2 partial pressure is predicted to increase with increasing mesophyll CO 2 partial pressure,
whereas it decreased in the model that relied solely on the recycling of photorespiratory CO 2
(Fig. 5.8). The equations are general enough so that they can also be used to model the
C 4-like C 3-C 4 intermediate species by reducing the mesophyll rubisco capacity and increas
ing both mesophyll PEP carboxylase activity and bundle-sheath rubisco activity. However,
the present model does not incorporate the light and electron transport limitations. Section
5.7 briefly discusses how this might be added. Figure 5.13 demonstrates that a partial C.
pathway also reduces the O 2 sensitivity of CO 2 assimilation. The extent of the reduction is
very much dependent on the amount of C 4 cycle activity.
Several authors have considered the significance of C 3-C4 intermediate species in the evolu
tion of C 4 species (Peisker 1986; Monson and Moore 1989). Furthermore, hybrids between C 4 and
C,-C 4 intermediate Flaveria species have been generated and studied (Brown and Bouton 1993).
Peisker (1986) used his model of C 3-C4 photosynthesis to map a possible evolutionary path of C 4
photosynthesis. The combined model proposed in this chapter can also easily be parameterized to
represent C3 , C rC 4 intermediates with photo respiratory recycling, C3-C4 intermediates with
photorespiratory recycling and just a little bit of C4 photosynthesis as well as a full C 4 pathway, and
examples of this are given in Fig. 5.14.
5.7 ENERGY REQUIREMENTS OF (3-(4 PHOTOSYNTHESIS
5.7.1 Rates of AlP and NADP consumption

Chlorophyll fluorescence measurements have been used to examine the energy requirements
of C3-C4
intermediates (Dai et al. 1996) and it is therefore of interest to examine the energy
requirements of C 3-C4 intermediates. C 3-C. intermediates are not a single, distinct group,
2500
<:( 40 -...
co
Q) 2000 .0
~
...
Cil
c
a
-
~
en 30
C
1500 O~
.s
~ E
o
'E "'6 20 .s::::.
en E 1000 Cil
en ~ Q)
co .s::::.
en
0''' 10 A.s 500 Q)
o ............ -- =0
c
:::>
0 0 CO
0 100 200 300 400
em (ubar)
Figure 5.12 Modelled response of (0 2 assimilation, A to mesophyll (0 2 partial pressure, em' of (3-(4
intermediate photosynthesis with (4 cycle activityand no glycine shuttle (I; = 0). Rubisco activities are 120and
10 urnol m-2 S-l in the mesophyll and bundle-sheath compartment respectively. Vpmax = 10 urnol m-2 S-1; bundle
sheath conductance, 95= 1 mmol m-2 s': and Rm = R5 = O. Also shown arethe bundle-sheath (0 2 partial
pressure, (, and bundle-sheath (0 2 assimilation, As' The (r(4 photosynthetic (02 assimilation rateis compared
to a (3 rubisco-limited rate with an equal rubisco activity of 130 prnol m-2 S-1.
60
a em = 250 ubar
<:(
50 ,,
i
c
a
~ -
en
~
40 \
\
~ E
\
,,
'E "'6 30
en E ""
en ~ "
co 20
ON
o
10
120
b
0 100
II
10
80
0 ~
,,
Cil ,
<:( 60
~ 40
~
<:(
20
0
0 200 400 600 800 1000
Mesophyll 02 partial pressure Om (mbar)
Figure 5.13 Response of (0 2 assimilation rateof the (3 (dashed line)andthe (r(4 (solid line) photosynthetic
pathways (with (4 cycle activity) to oxygen partial pressure, Om' Modelled parameters areas in Fig. 5.12.
50
')'
en
E c.
<5 40 /:'~/-;-;:;:;~
E c3 - c .
~
Q)
'§ 30 d
c
0 ,/~ ","', ,,> .r>
~ 20 C
'E ,/,"'b"'<'" ' 3
'wen /,/ "~",,
i:
ro 10
ON
0
Figure 5.14 Modelled response of CO 2 assimilation, A, to mesophyll (02 partial pressure, em' of (3' (r(4
intermediate photosynthesis without a (4 cycle, with (4 cycle activity and (4 photosynthesis. (a) Vcmax = 120and
10 prnol m-2 S-1 in the mesophyll andbundle-sheath compartment respectively and Vpmax = 0 urnol m-2 S-1.
(b) Vcmax = 100 and 20 urnol m-2 S-l in the mesophyll and bundle-sheath compartment respectively and Vpmax
10 urnol m-2 s'. (c) Vcmax = 80 and 20 urnol m-2 S-l in the mesophyll and bundle-sheath compartment
respectively and Vpmax = 30 urnol m-2 s'. (d) Vcmax = 20and 50 umol m-2 S-l in the mesophyll andbundle-sheath
compartment respectively and Vpmax = 50 urnol m-2 s'. ((4) V,max = 0 and 50 urnol m-2 S-l in the mesophyll and
bundle-sheath compartment respectively and Vpmax = 100 urnol m-2 S·l. ((3) Vcmax = 130 urnol m-2 s' and Vpmax =
ournol m-2 S-l. In all cases bundle-sheath conductance, gs=1 mmol m-2 s'. and Rm= Rs= 0, ~ =1.
but rather they have various degrees of C 4 anatomical or biochemical characteristics. The
degree to which the intermediacy is due to photorespiratory recycling or a C 4 cycle pathway
will alter the requirements for ATP versus NADPH. C 4 cycle activity has a requirement of 2
ATP per PEP carboxylation. The general rate of ATP consumption is:
Rate ofATP consumption = 2Vp + (3 + 7y.0Il1ICltJVcm + (3 + 7y.O/C,)Vcs (5.15 )
where, as before (7yO/C)Vc is the energy requirement due to photorespiration (since V/Ve =
2yO/C and 3.5 mol of ATP are required per mol of 02 oxygenated by rubisco). The rate of
NADPH consumption is given by:
Rate ofNADPH consumption = (2 + 4y.OIl,lC cm + (2 + 4y.O/Cs)Vcs

II.)V
(5.16)
It is possible to use the equations of Section 5.3 to calculate the ATP and NADPH require
ment for CO 2 assimilation rate at different CO 2 and 02 partial pressures. It is shown in
Fig. 5.15 that recycling of photorespiratory CO 2 in the bundle sheath leads to energy saving
for the C 3-C4 intermediate pathway (without a C 4 cycle) compared to a C 3 pathway. This is
because the bundle-sheath rubisco of the C 3-C 4 intermediate pathway functions at elevated
CO 2 with reduced oxygenase activity. This is particularly marked at lower CO 2 partial pres
sures and also pertinent at higher temperatures where photorespiration is increased.
5.7.2 Electron transport rate
One can take an approach similar to the C 4 model and assume that the energy can be shared
between mesophyll and bundle-sheath compartments. That is, whole chain electron trans
port is given by:
t, = Jill + t, (5.17)
1:
Q) 0
E
""
o
c: (5 20
.~ E
:) .......
eTo..
Q)I
~«
0..
I- a
«5 10
100 200 300 400

Mesophyll 02 partial pressure (mbar)
Figure 5.15 AlP requirement per (02 fixed for a (3 and (3-(4 intermediate photosynthesis (without (4 cycle
activity) at a mesophyll (02 partial pressure of Cm= 100 ubar at different 02 partial pressures. Modelling
parameters are the same as in Fig. 5.8.
Taking the ATP requirement and assuming that the Q cycle operates and that 4 mol of pro
tons are required per mol ATP, whole chain electron transport required is given by:
(5.18)
and
l, = 4(1 + 7y.0/(3C,))Vcs (5.19)
5.7.3 Light-dependent CO 2 assimilation rate

To obtain an overall rate equation for CO 2 assimilation as a function of the mesophyll CO 2
and 02 partial pressures, C m and Om' one proceeds as in Chapter 4 and solves a quadratic
expression for As of the form:
aA~ + bAs + c =0 (5.20)
where
A (5.21)
and
Y-::-'-=
a = 1 _3_7...c...
x 0.047
(5.22)
b = - {(S+gC Y.O m - - (Js- +7R-s') }

--R. ) +g (7- - ) +ay.
)+ (Js (5.23 )
s 4 '11/ 3 0.047 4 53
(5,24)
Models of CrC 4 intermediate photosynthesis 139
and S stands for CO 2 supply rate to the bundle sheath and is given by:
S =S y.OmJmc + Jmp (5.25)

4( c, + 7y'/(3C",)) 2.66
Again, the energy needs to be partitioned between mesophyll and bundle-sheath compart
ments and Jm = xJI and Js = (1 - x)J t , where 0 < x < 1 and Jm = Jmc + Jmp" It is likely that a large
fraction of the energy is required in the mesophyll and that the value for x is high. Finally:
A = A s + Am'
(C II1-Y'Om)J mc -R
where Am (5.26)
4(C m+7y./(3C II1 ) ) m
5.8 CONCLUSION
The models of C3-C4 photosynthesis cater for a diverse set of species, and it is therefore diffi
cult to give a standard set of parameters, or explore the many interesting questions that
remain. It is at present difficult to know how to model the light and electron transport
dependence for these species. The strong light dependence of the CO 2 compensation point
(Ku et al. 1991) suggests that the C 3-C4 intermediate pathway may not be effective at low
light and this deserves further investigation.
The quantitative analysis shows that the glycine shuttle is an effective way to refix photorespira
tory CO 2, It does, however, remain difficult to resolve, from measurements of CO 2 assimilation
alone, the contribution of a partial C 4 cycle. However, the models have been used to predict car
bon isotope discrimination of C rC 4 intermediates (von Caemmerer 1992). The equations of
Section 5.3 can also be used to develop equations of 02 exchange along the lines used in Chapter
3. For example, plants that function solely with a glycine shuttle should have high rates of O 2
uptake at low CO 2 partial pressures, compared to species that utilize a C 4 cycle, and this may prove
to be a valuable new experimental technique.
6
Concluding remarks
This book has dealt exclusively with the photosynthetic processes of leaves. Leaf gas
exchange measurements remain useful in the elucidation of biochemical processes of photo
synthesis. Highly sophisticated portable gas-exchange systems have made it easier to take
snapshots of the photosynthetic process in different ecosystems. For example, these measure
ments form an important part of many studies in free air CO 2 enrichments (FACE) sites and
aid in the analysis of long-term plant responses to CO 2 enrichment.
The biochemical models described in this book can serve two distinct functions. They allow
simple analysis of field data, providing information on underlying biochemical processes.
Together with genetic manipulation of photosynthetic processes they provide a tool for the quan
titative mechanistic analysis of photosynthetic regulation. The interplay between model
presentation and examples derived from various sources illustrates how these models can be used
and developed.
The process of constructing a model frequently serves to highlight gaps in our knowledge and
understanding. The biochemical models discussed in this book invite further development in a
number of areas. In Chapter 1, for example, the mechanistic understanding of activase action
remains incomplete. However, recent insights have come from physiological measurements on
whole leaves and could not have been obtained with in vitro experiments alone. In this case the C 3
model of photosynthesis has provided the backbone facilitating the careful quantitative compari
sons between gas exchange and in vitro measurements. The ability to express mutant rubiscos
through chloroplast transformation techniques in higher plants invites the use of the photosyn
thetic models to capture their kinetic idiosyncrasies. All the models allow 'what if' questions to be
asked (Section 2.13). The light dependence of the electron transport rate has remained empirical
and has not been further developed after an initial attempt at a mechanistic basis (Farquhar and
von Caemmerer 1981). Molecular manipulation of the photosynthetic electron chain may help
with further development in this area.
The equations for the oxygen exchange occurring during photosynthesis should be extended
to C4 and C rC 4 photosynthetic models. In the case of C rC 4 photosynthesis, O 2 uptake measure
ments could be particularly useful in distinguishing between C rC 4 species that use the glycine
shuttle compared to those with a large amount of CO 2 fixation into C4 acids, as the 02 uptake pro
cesses will reflect the amount of mesophyll rubisco. Aquatic plants such as hydrilla that can, under
certain circumstances, exhibit C 4-like photosynthesis within single cells (Bowes and Salvucci
1984; Reiskind et al. 1997) provide a new challenge for the modelling of these pathways. It is per
plexing that CO 2 concentrations can be elevated in the chloroplasts of these cells.
Although the building blocks exist in the current C 3 and C4 models, detailed biochemical
models of the CAM photosynthetic pathways remain to be formulated. Here the development of
equations for 02 and CO 2 exchange should be of particular use in resolving questions about the
energy metabolism of these pathways (Osmond et al. 1996; Maxwell et al. 1998).
140
Appendix List of symbols
A (prnol m? S-1) Rate of CO 2 assimilation

A( (umol m- 2 S-I) Rubisco-limited rate of CO 2 assimilation
Ai (prnol m- 2 S-1) Electron-transport-limited rate of CO 2 assimilation
Am (pmol m- 2 S-1) Rate of CO 2 assimilation in the mesophyll of C 3-C4
intermediates (Chapter 5)
A p (umol m' S-I) Triose phosphate export-limited rate of CO 2 assimilation
As (prnol m ? S-I) Rate of CO 2 assimilation in the bundle sheath of C 3-C4
a Fraction of PS II activity in the bundle sheath (Chapter 4)
~ Fraction of absorbed irradiance absorbed by PS II
C (ubar ) CO 2 partial pressure
C( (ubar ) Chloroplastic CO 2 partial pressure
Ci(ll bar) Intercellular airspace CO 2 partial pressure
c, (ubar) Mesophyll CO 2 partial pressure
C, (ubar ) Bundle-sheath CO 2 partial pressure
C (ubar ) Intercellular CO 2 partial pressure at which C( = T,
D eoz Diffusivity of CO 2 in air (1.93 x 10- 9 rrr' s-1 at 25°C)
D oz Diffusivity of O 2 in air (2.43 x 10- 9 m' S-1 at 25°C)
EA O (prnol m-2 S-1) 02 evolution required for the PCR and PCO cycle activity
(Chapter 3)
EOmol- 1)
Activation energy (Chapter 2)
E (mM or urnol m' S-1)
Free rubisco catalytic site (Chapter 1)
E( (umol m- 2 S-I)
Rubisco-limited 02 evolution (Chapter 3)
Eex Oxygen evolution associated with extra electron transport
(Chapter 3)
Ej (umol m? S-I) RuBP regeneration-limited 02 evolution (Chapter 3)
Eo (prnol m- 2 S-1) Net 02 evolution (Chapters 3 and 4)
E TO (umol m? S-1) Total 02 evolution (Chapter 3)
E, (mM or umol m- 2 S-I) Total rubisco catalytic sites
EC Rubisco catalytic sites CO 2 complex
ECM Rubisco catalytic sites CO 2 magnesium complex
ECMR Rubisco catalytic sites CO 2 magnesium RuBP complex
ECMRC and ECMRO All catalytic intermediates of carboxylation and oxygenation
EP and ECMP Uncarbamylated and carbamylated rubisco site ligand
complex
ER Rubisco catalytic sites RuBP complex
11 Mol of ATP produced in mitochondria per mol of NADPH
(Chapter 4)
141
f Spectral light quality correction factor

F Tortuosity factor of diffusion (Eq. 2.2)
Fm Maximal chlorophyll fluorescence during a saturating light
pulse of a dark-adapted leaf
Maximal chlorophyll fluorescence during a saturating light
pulse of a leaf in the light
Chlorophyll fluorescence of a dark-adapted leaf
Chlorophyll fluorescence of a light-adapted leaf measured in
the presence of far-red light only
Fs
Chlorophyll fluorescence in the light
gi (mol m? S-I)
Conductance for CO 2 diffusion from intercellular airspace to
site of carboxylation
go (mol m ? S-I)
Bundle-sheath conductance to 02
gs (mol m- 2 S-I)
Bundle-sheath conductance to CO 2
t Half of the reciprocal of rubisco specificity (O,S/Sclo)
HOmol- 1)
220000 (Eq. 2.34)
1 (umol quanta m- 2 S-I)
Incident irradiance
12 (prnol quanta m? S-I)
Photosynthetically active irradiance absorbed by PS II
J (urnol electrons m- 2 S-l)
Electron transport rate (Chapter 2)
JA (umol electrons m- 2 S-I)
Electron transport rate req uired for the PCR and PCO cycle
activity (Chapter 3)
Jex (urnol electrons m- 2 s") Electron transport rate not associated with PCR and PCO
cycle activity (lex zz h"it + hlAP; Chapter 3)
Electron transport rate calculated from chlorophyll
fluorescence measurements (Chapter 3)
Electron transport rate to support C 4 cycle activity
(Chapter 4)
hlAP (prnol electrons m- 2 S-I)
Jm ax (prnol electrons m- 2 S-I)
°
Electron transport rate to 2 (Chapter 3)
Maximal electron transport rate
h.:it (umol electrons m- 2 S-I) Electron transport associated with nitrate reduction and
other synthetic processes
Total electron transport rate (to support C 3 and C 4 cycle
activity; Chapter 4)
Electron transport rate to support C 3 cycle activity
(Chapter 4)
«, (pbar ) Michaelis-Menten constant of rubisco for COo
K ccat (S-I) Catalytic turnover rate of rubisco carboxylation
K f (11M) Dissociation constant of the ER complex (also see Eq. 1.28).
s; ()lM) Dissociation constant of the ECMR complex
K o (mbar ) Michaelis-Menten constant of rubisco for 02
K ocat (S-1) Catalytic turnover rate of rubisco oxygenation
K p (ubar ) Michaelis-Menten constant of PEP carboxylase for CO 2
(Chapter 4)
Dissociation constant for EP or ECMP complex of rubisco
(Chapter I)
Michaelis-Menten constant of rubisco for RuBP
Michaelis-Menten constant of rubisco for RuBP at
saturating CO 2
Michaelis-Menten constant of rubisco for RuBP at
saturating 02
Appendix List of symbols 143
L (umol m- 2 S-I) Leak rate of CO 2 out of the bundle sheath

La (urnol m-' 5- 1) Leak rate of 02 out of the bundle sheath
a (ubar) 02 partial pressure
am (ubar ) 02 partial pressure in the mesophyll
as (ubar] 02 partial pressure in the bundle sheath
<\l ~,/V[, the ratio of oxygenation to carboxylation rate
(Chapter 2)
<\l Leakiness of the bundle sheath (Chapters 4 and 5)
<\lPS II Electron flow through PS II per unit quantum flux
R (J K- I rnol "] Gas constant (8.314 J K- 1 mol)
R Free RuBP (Chapter 1)
Rt
Total (free and rubisco-bound) RuBP
R d (umol m- 2 S-I)
Mitochondrial respiration in the light
Tj (rn? s ' mol:") Resistance to CO 2 diffusion from intercellular airspace to site
of carboxylation (llg)
R m (urnol m ? S-I) Mitochondrial respiration in the mesophyll
R, (umol m- 2 S-I) Mitochondrial respiration in the bundle sheath
T (rn? Sl mol"] Bundle-sheath resistance (l I g)
s
5 (J K- I molL) 710 (Eq. 2.34)
s.; Rubisco C0 2/0 2 specificity
Sm Ratio of mesophyll surface area exposed to intercellular
airspace to projected leaf area
Se02 Solubility coefficient of CO 2 in water (0.0334 mol (L bar"]
at 25°C)
So.' Solubility coefficient of 02 in water (0.00126 mol (L bar:")
at 25°C)
T (DC) Temperature
Tp (umol m? s") Rate of triose phosphate export
L (ubar) C 3 compensation point in the abscence of mitochondrial
respiration
U (umol m- 2 S-I) Oxygen uptake rate
U, (umol m- 2 S-I) Rubisco-limited oxygen uptake rate
Uj (umol m- 2 S-I) RuBP regeneration -limited oxygen uptake rate
UMA P Oxygen uptake rate of the Mehler ascorbate peroxidase
reaction at PS 1.
V e (umol m- 2 S-I) Rubisco carboxylation rate
V em (umol m- 2 S-I) Rubisco carboxylation rate in mesophyll of C 3-C4
V em ax (urnol m' s") Maximal rubisco carboxylation rate
V(5 (urnol m- 2 s-l) Rubisco carboxylation rate in the bundle sheath of C 3-C4
~, (umol m- 2 S-I) Rubisco oxygenation rate
Vam (umol m- 2 S-I) Rubisco oxygenation rate in mesophyll of C 3-C4
Vas (umol m- 2 S-l) Rubisco oxygenation rate in the bundle sheath of C 3-C4
Vamax (urnol m- 2 s") Maximal rubisco oxygenation rate
Vp (umol m ? S-I)
Vpm ax (urnol rn' S-I)
Maximal PEP carboxylation rate
Vpr (umol m- 2 S-I) PEP regeneration rate
W[ (prnol m- 2 S-I) RuBP-saturated rubisco carboxylation rate

WO (umol m- 2 S-I) RuBP-saturated rubisco oxygenation rate
~ Fraction of photorespiratory CO 2 derived from mesophyll
oxygenations that is released in the bundle sheath
(Chapter 5)
x Partitioning factor of electron transport rate between the C 4
and C 3 cycles (Chapter 4)
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National Library Cataloguing in Publication entry

von Caernmerer, S. (Susanne).

1.Title. (Series: Techniques in plant sciences j no. 2).

This book is available from:

lei: (03) 9662 7666 Int: +(613) 96627666

Printed in Australia by Brown Prior Anderson

The kinetics and regulation of rubisco 1

1.2 Kinetics of fully activated rubisco 2

1.2.1 Definition of kinetic constants 2

1.2.2 Kinetics at saturating RuBP 3

1.2.3 Relative specificity, 5c/ o 4

1.2.4 Rubisco kinetic constants in vitro and in vivo 6

1.2.5 RuBP-limited rate of carboxylation 10

1.2.6 High rubisco site concentrations in the chloroplast 11

1.2.7 The binding of phosphorylated compounds and other ligands 14

1.3 Activation of rubisco 16

1.3.1 Activation of rubisco in the absence of RuBP 17

1.3.2 Activation of rubisco in the presence of RuBP 17

1.3.3 The carbamylation ratio of rubisco 18

1.3.4 Rubisco activase 19

1.3.4.1 A model of activase action 20

1.3.4.2 A hyperbolic response of carbamylation to activase activity 21

1.3.4.3 Binding of other ligands 22

1.3.4.4 An unknown function of activase 22

1.4.1 Derivation of rate equations for activated enzyme 24

1.4.2 The rate equations at high concentration of enzyme sites 25

1.4.3 Derivation of rate equations of activation 26

1.4.4 Carbamylated enzyme sites as a fraction of total enzyme sites 27

1.4.5 Ligands other than RuBP 28

2.2 A few simplifying assumptions 29

2.3 Stoichiometry of C3 photosynthesis 30

2.3.1 Carbon stoichiometry of the PCR and PCO cycles 30

2.3.2 The carbon path from PGA to RuBP 31

2.3.4 Thylakoid reactions 32

2.3.4.1 Production ofNADPH and ATP 32

2.3.4.2 Light dependence of electron transport rate 34

2.4 Rate equations for CO 2 assimilation 35

2.4.1 RuBP-saturated CO 2 assimilation rate 35

2.4.2 RuBP-limited CO 2 assimilation rate 36

2.4.3 Export-limited CO 2 assimilation rate 38

2.4.4 Summary of rate equations 39

2.5 CO 2 partial pressure in the chloroplast 39

2.6 Parameterization of the model 42

2.6.1 Values at 25°C 42

2.6.2 Temperature dependencies 44

2.6.2.1 Rubisco and Rd 44

2.6.2.2 Temperature dependence of Jmax 45

2.7 The CO 2 compensation point 47

2.7.1 r in the absence of Rd 48

2.7.2 r in the presence of Rd 48

2.7.3 Light and temperature dependence 49

2.7.4 Measurements off. 50

2.8 CO 2 response curves 51

2.8.1 CO 2 response curves in transgenic tobacco with impaired photosynthesis 52

2.8.2 CO 2 assimilation rate at different 02 partial pressures 54

2.8.3 CO 2 assimilation rate at different irradiances 55

2.8.4 CO 2 assimilation rate at different temperatures 56

2.8.5 The initial slope of the CO 2 response curve, dAJde, 57

2.9 Light response curves 58

2.9.1 Dependence on 02 and CO 2 partial pressures 58

2.9.2 Quantum yield 60

2.10 Temperature responses 62

2.12 Long-term effect of environment on photosynthesis 65

2.12.2 Effect of growth temperature 68