PHYSIOLOGIA PLANTARUM 120: 179186.

2004
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Copyright # Physiologia Plantarum 2004

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Inhibition of photosynthesis by heat stress: the activation state of

Rubisco as a limiting factor in photosynthesis
Michael E. Salvucci* and Steven J. Crafts-Brandner
US Department of Agriculture, Agricultural Research Service, Western Cotton Research Laboratory, 4135 E. Broadway Road, Phoenix,
AZ 85040, USA
*Corresponding author, e-mail: msalvucci@wcrl.ars.usda.gov
Received 13 November 2002; revised 24 February 2003

Although the catalytic activity of Rubisco increases with temperature, the low affinity of the enzyme for CO2 and its dual
nature as an oxygenase limit the possible increase in net
photosynthesis with temperature. For cotton, comparisons of
measured rates of net photosynthesis with predicted rates that
take into account limitations imposed by the kinetic properties
of Rubisco indicate that direct inhibition of photosynthesis
occurs at temperatures higher than about 30
C. Inhibition of
photosynthesis by moderate heat stress (i.e. 3042
C) is generally attributed to reduced rates of RuBP regeneration
caused by disruption of electron transport activity, and specifically inactivation of the oxygen evolving enzymes of photosystem II. However, measurements of chlorophyll
fluorescence and metabolite levels at air-levels of CO2

indicate that electron transport activity is not limiting at

temperatures that inhibit CO2 fixation. Instead, recent
evidence shows that inhibition of net photosynthesis correlates
with a decrease in the activation state of Rubisco in both C3
and C4 plants and that this decrease in the amount of active
Rubisco can fully account for the temperature response of net
photosynthesis. Biochemically, the decrease in Rubisco activation can be attributed to: (1) more rapid de-activation of
Rubisco caused by a faster rate of dead-end product formation; and (2) slower re-activation of Rubisco by activase. The
net result is that as temperature increases activase becomes
less effective in keeping Rubisco catalytically competent. In
this opinionated review, we discuss how these processes limit
photosynthetic performance under moderate heat stress.

Introduction
Inhibition of photosynthesis by heat stress is a common
occurrence for plants in tropical and subtropical regions
and is experienced periodically by plants in the temperate
zone (Larcher 1995). In the field, heat stress often accompanies drought stress, highlighting the importance of
water availability for stress avoidance. Plants with access
to adequate water keep their stomates open at high
temperatures, using evaporative cooling to reduce their
leaf temperature. This process works extremely well in
hot, arid regions like the desert south-west of the US,
where the differential between air and foliar tempera-

tures can be greater than 10
C (Idso et al. 1982). In

fact, the higher yield of modern cultivars of Pima cotton
in these regions has been attributed to a greater capacity
for leaf cooling, a direct consequence of increased stomatal control (Radin et al. 1994, Lu et al. 1997). A
similar association between yield and canopy temperature has been described for wheat (Fischer et al. 1998).
The efficiency of leaf cooling by evapotranspiration
decreases with increasing relative humidity or when transpiration slows because of water deficit. Thus, the frequency and distribution of heat stress is likely to increase

Abbreviations PGA, phosphoglyceric acid; PSII, photosystem II; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; RuBP,
ribulose 1,5-bisphosphate.
The 2002 Annual Symposium of the Southern Section of the American Society of Plant Biologists took place at the University of Georgia on
March 4, 2002. The theme of the colloquium was Mechanisms for Regulation of Plant Metabolism. This issue of Physiologia Plantarum
contains three papers based on the invited lectures. Generous support for these lectures was provided by Pioneer Hi Bred International Inc.,
Syngenta Crop Protection, Inc., and the American Society of Plant Biologists.
Physiol. Plant. 120, 2004

179

The dependence of net photosynthesis on the kinetics of

Rubisco
For mesothermic organisms, enzymatic activities generally increase with temperature over their physiological
range, causing an increase in the overall flux through
metabolic pathways. For example, the dark respiration
rate of a cotton leaf increases in response to temperature
with a Q10 of 1.86 between 28 and 42.5
C (Fig. 1, inset).
In contrast, net photosynthesis of a similar cotton leaf
decreases at temperatures higher than about 30
C
(Fig. 1), producing a negative Q10 between 28 and
42.5
C. The peculiar response of net photosynthesis to
temperature is caused by the complex kinetics of Rubisco
and some very unusual properties of this enzyme.
Rubisco catalyses the first step in two competing pathways, photosynthesis and photorespiration, whose rates
are determined by the rates of the carboxylase and oxygenase activities, respectively (Laing et al. 1974). The
VMax of the carboxylase activity increases with temperature, but the affinity of Rubisco for CO2 and the solubility of CO2 decrease (Laing et al. 1974, Monson et al.
1982, Jordan and Ogren 1984). When CO2 levels are
saturating, the carboxylase activity of the isolated
enzyme increases with temperature with a Q10 of 2.04
between 30 and 50
C (Fig. 2). At air-levels of CO2, the
increase in activity over the same temperature range is
much smaller, reflected in a Q10 value of only 1.51. Thus,
as temperature increases at air-levels of CO2, reduced
affinity of Rubisco for CO2 and lower solubility of CO2
offset potential increases in carboxylase activity (Berry
and Bjorkman 1980). In other words, with increasing
temperature, CO2 concentration becomes an increasingly
greater limitation on Rubisco activity, as less of the
Rubisco capacity is used for carboxylation. Another factor
180

Respiration

Net photosynthesis
(mol m2 s1)

20

3
2
1
0

30 35 40
Temperature (C)

30

35

40

45

Temperature (C)
Fig. 1. Response of net photosynthesis and dark respiration to
temperature in cotton leaves. Net photosynthesis in air was measured
at the indicated temperatures (*) or predicted from the kinetics of
Rubisco with (*) and without (&) adjustment for the measured
changes in activation state (Law and Crafts-Brandner 1999). The grey
area indicates the difference between the measured rates and rates
predicted without adjust for changes in Rubisco activation. The data
for net photosynthesis was from Crafts-Brandner and Salvucci
(2000). Calculations of net photosynthesis (Laing et al. 1974) were
based on the activity of fully activated Rubisco at 25
C determined
after extraction from cotton leaves. These rates were adjusted for
temperature based on improved measurements of the temperature
response of isolated cotton Rubisco (Fig. 2), the effects of
temperature on the other kinetic constants of Rubisco (Jordan and
Ogren 1984) and changes in the solubilities of CO2 and O2 with
temperature without adjustment for respiration. Inset, dark
respiration (~) was measured at the indicated leaf temperatures.

ATPase activity

The complex effect of temperature on photosynthesis

40

8
Rubisco activity
(Umg protein1)

with global warming, especially if higher air temperatures are accompanied by reduced precipitation.
Inhibition of photosynthesis is reversible when temperatures are slightly higher than optimal (moderate
heat stress), whereas damage to the photosynthetic apparatus is permanent under severe heat stress (Berry and
Bjorkman 1980). Since moderate heat stress occurs more
frequently and the effects are not permanent, numerous
studies have attempted to elucidate the biochemical component that is most sensitive to (i.e. first affected by) high
temperature. A broad survey of the literature reveals that
PSII is often considered the most heat-labile component
of the photosynthetic apparatus even though several
studies, including some dating back at least 20 years,
have shown that electron transport capacity is unaffected
at temperatures that inhibit CO2 fixation (discussed
below). In this review we describe how a decrease in the
activation state of Rubisco is responsible for inhibition
of photosynthesis under moderate heat stress and discuss
some new ideas and data concerning the underlying biochemical mechanism.

0.4
0.2
0

20
40
60
Temperature (C)

0
0

10

20

30

40

50

Temperature (C)
Fig. 2. Effect of CO2 concentration on the response of Rubisco
activity to temperature. The carboxylase activity of isolated cotton
Rubisco was measured at the indicated temperatures in the presence
of 1 (*) or 30 mM (*) NaHCO3 at pH 8. Assays containing 1 mM
NaHCO3 also contained 1000 Wilbur-Anderson units of carbonic
anhydrase. Rubisco was fully carbamylated prior to assay. Inset: the
response of the ATPase activity (U mg protein1) of recombinant
cotton activase (both forms) to temperature (~).
Physiol. Plant. 120, 2004

reducing net photosynthesis at higher temperatures is loss

of CO2 from photorespiration. Photorespiratory activity
increases with temperature because of decreases in both
the relative specificity of Rubisco for CO2 compared with
O2 and the relative solubility of CO2 compared to O2
(Jordan and Ogren 1984).
Predicted versus observed effects inhibition of
photosynthesis at high temperature
The effect of temperature on the kinetic constants of
isolated Rubisco can be used to predict the response of
photosynthesis to temperature (Fig. 1). At 28
C, the optimal leaf temperature for photosynthesis in cotton, the
predicted rate of net photosynthesis is similar to the
measured rate. However, as temperature increases, the
measured rate becomes increasingly lower than the predicted rate (Fig. 1, grey area). This difference represents
inhibition of net photosynthesis since it cannot be
accounted for by changes in the solubilities of CO2 and
O2 and the kinetic properties of Rubisco. For cotton
plants grown at 28
C, inhibition of net photosynthesis
in air occurs at leaf temperatures above about 30
C. At
35
C, a leaf temperature that is not unreasonably high
for a plant such as cotton that grows in hot, arid regions
(Lu et al. 1997), the loss of photosynthetic potential is
about 16%. Consequently, inhibition of net photosynthesis under even moderate heat stress could represent a
significant loss of carbon to the plant, especially if
integrated over the entire canopy and throughout the
growing season.
Inhibition of net photosynthesis by moderate heat
stress has often been attributed to the adverse effects of
temperature on components of the photosynthetic electron transport, particularly the water-splitting reactions
of PSII. Reduced rates of electron transport activity
would slow the rate of RuBP regeneration below the
rate of consumption, causing a decrease in net photosynthesis similar to that shown in Fig. 1. Indeed, measurements of electron transport activity clearly show that
PSII activity is inhibited at high temperatures (cf.
Havaux 1993, Rokka et al. 2000). However, the relationship between net photosynthesis and electron transport
activity is often compromised by the use of extremely
high CO2 concentrations for the measurement of leaf
photosynthesis in an O2 electrode (Havaux 1993,
Murakami et al. 2000, Yamasaki et al. 2002). Very high
CO2 concentrations reduce the activation state of Rubisco
(Woo and Wong 1983, Salvucci et al. 1986) making
RuBP consumption by Rubisco limiting for photosynthesis even in non-heat-stressed leaves (Crafts-Brandner
and Salvucci 2000).
When net photosynthesis and chlorophyll fluorescence
are measured at air-levels of CO2 in an intact system,
electron transport capacity is unaffected at temperatures
that inhibit CO2 fixation (Weis 1981a, Bilger et al. 1987,
Law and Crafts-Brandner 1999, Crafts-Brandner and
Law 2000). For example, inhibition of net photosynthesis in cotton occurs above about 30
C (Fig. 1), whereas
Physiol. Plant. 120, 2004

the Fv : Fm ratio, the fluorescence parameter commonly

used for estimating PSII capacity, is relatively constant
at temperatures between 25 and 40
C (Law and CraftsBrandner 1999). In contrast, three measures of thylakoid
energization, non-photochemical chlorophyll fluorescence quenching, the electrochromic absorption shift
and light scattering, all are affected when CO2 fixation
is inhibited by moderate heat stress, indicating that the
energy supply for photosynthesis is not being utilized by
the Calvin Cycle (Weis 1981a, Bilger et al. 1987, Law and
Crafts-Brandner 1999). That the rate of net photosynthesis in heat stressed leaves increases with increasing
CO2 concentration even under non-photorespiratory
conditions provides another indication that the capacity
for electron transport and RuBP regeneration is in excess
under moderate heat stress (Crafts-Brandner and Law
2000). This conclusion is supported by direct measurements of metabolite levels (Kobza and Edwards 1987,
Law and Crafts-Brandner 1999) and 14C labelling patterns (Weis 1981a), which show that RuBP actually
increases in concentration and PGA decreases when net
photosynthesis is inhibited by moderate heat stress.
These responses are characteristic of a limitation in the
Calvin Cycle and specifically a reduced level of Rubisco
activation.

Temperature effects on the activation state of Rubisco

Each higher plant-type Rubisco holoenzyme contains
eight active sites that appear to function independently
of one another (reviewed in Spreitzer and Salvucci 2002).
The activation state of Rubisco represents the portion
of these sites that is catalytically competent. Rapid
extraction and assay of Rubisco has shown that the
activation state of Rubisco changes in response to light,
CO2 and other environmental factors (Perchorowicz et al.
1981, Sage et al. 1988, Kanechi et al. 1996, Feller et al.
1998). Measurements conducted in the early 1980s by
Weis (1981a, b) and later by Kobza and Edwards
(1987) showed that the activation state of Rubisco
decreased at moderately high temperatures. In fact,
these studies concluded that this decrease was the main
cause of photosynthetic inhibition under moderate heat
stress. At the time, a decrease in the activation state of
Rubisco was thought to reflect a loss of carbamylation
due to changes in stromal pH and Mg21 concentration.
Consequently, the effects of temperature were generally
attributed to changes in the stromal ion concentration
brought about by unspecified changes in the properties of
the thylakoid membranes (Weis 1981c). Since then, we have
gained a much better understanding of the biochemical
basis for changes in Rubisco activation state, including
the involvement of Rubisco activase in the process.
Recent investigations from our laboratory have
re-examined the involvement of RuBP regeneration and
Rubisco activation in the inhibition of net photosynthesis by moderate heat stress in both wheat and cotton
(Feller et al. 1998, Law and Crafts-Brandner 1999).
181

Measurements of chlorophyll fluorescence and metabolite

levels at air-levels of CO2 confirm that the potential
yield of PSII reaction centres and the availability of
RuBP do not decrease at temperatures that inhibit net
photosynthesis (Law and Crafts-Brandner 1999). In contrast, the activation state of Rubisco decreases under
moderate heat stress, accounting for the decrease in net
photosynthesis (Law and Crafts-Brandner 1999, CraftsBrandner and Law 2000, Crafts-Brandner and Salvucci
2000). In fact, the response of net photosynthesis to
temperature can be predicted from the kinetic properties
of Rubisco if the VMax terms are adjusted for the measured changes in activation state (Fig. 1, see also CraftsBrandner and Salvucci 2000).

Biochemical mechanism for changes in activation state

Detailed biochemical and structural studies have shown
that three interrelated properties of Rubisco impact its
activation state (Fig. 3). The first is carbamylation of
Rubisco active sites by spontaneous addition of (nonsubstrate) CO2 to an active site lysine (Lorimer 1979).
Carbamylation of this lysine is required for activity since
the carbamate participates in metal binding and proton
abstraction (Cleland et al. 1998). The second property is
related to specific conformational changes that accompany the binding of sugar-P (Schreuder et al. 1993).
These changes, which take place during the normal
course of catalysis, involve extension and retraction of
loop 6 of the large subunit.

Extension of loop 6 across the active site, together

with a repositioning of the N-terminal domain of an
adjacent large subunit, closes the site by effectively
shielding it from solvent (Schreuder et al. 1993). Cleavage
of the C-C bond of the reaction intermediate in the
last phase of catalysis allows loop 6 to retract, which
makes the site accessible to solvent, allowing the release
of product and the binding of another substrate molecule
(Duff et al. 2000). Interestingly, closure of Rubisco active
sites can also be triggered by the binding of RuBP to
uncarbamylated sites or the binding of certain naturally
occurring sugar-P inhibitors to either carbamylated or
uncarbamylated sites. This binding causes dead-end inhibition because without catalysis and, specifically, C-C
cleavage these sites are very slow to open (Duff et al.
2000). A third and related property of Rubisco that
impacts its activation state is the formation of inhibitory
sugar-P by misprotonation of RuBP. This reaction
occurs directly at the active site while the site is in the
closed conformation, converting RuBP to either
xylulose bisphosphate or 3-ketoarabinitol bisphosphate
(Edmondson et al. 1990). Both of these so-called fallover or catalytic misfire products inactivate Rubisco by
binding tightly to the active-site and stabilizing the site in
the closed conformation (Duff et al. 2000 and references
therein). Thus, as shown in Fig. 3, the activation state of
Rubisco decreases when sites lose their carbamate (1), or
when they become sequestered in the closed conformation because of binding of RuBP to decarbamylated sites
(2), binding of inhibitory sugar-P to carbamylated sites

Fig. 3. Scheme for de-activation and re-activation of Rubisco. Rubisco active sites are represented by the yellow (closed conformation) or blue
(open conformation) boxes and include loop 6 in red. In the open conformation, loop 6 is retracted, making the site accessible to solvent. In the
normal course of catalysis, the enzyme active site is carbamylated by CO2 (1), binds Mg21, and then binds RuBP. RuBP binding triggers
extension of loop 6, producing the closed conformation, which opens following product formation (substrate CO2 is not shown). The
formation of dead-end complexes by the binding of RuBP to decarbamylated sites (2), binding of inhibitors like 2-carboxyarabinitol 1phosphate to carbamylated sites (3) or formation and binding of the catalytic misfire product, 3-ketoarabinitol bisphosphate, at the site (4) is
indicated by the dashed arrows. In all three cases, these complexes stabilize the site in a closed conformation. The action of activase plus ATP in
opening closed sites is indicated by the solid blue arrows. The formation of catalytic misfire products (asterisk) is accelerated at high
temperatures, while the opening of the closed conformation by activase (blue arrows) is inhibited.

182

Physiol. Plant. 120, 2004

(3), or the formation (i.e. by catalytic misfire) and binding of substrate analogs directly at the site (4).
To overcome dead-end inhibition of Rubisco, plants
contain Rubisco activase, an ATPase that releases tightbinding sugar-P from Rubisco active sites (reviewed in
Portis 2002, Spreitzer and Salvucci 2002). Activase rescues Rubisco sites from dead-end inhibition by promoting ATP-dependent conformational changes that open
closed sites, making them more accessible to solvent
and facilitating the dissociation of inhibitory sugar-P.
Activase is an AAA1 protein, a member of a superset
of proteins related to the AAA family that includes a
wide variety of proteins with chaperone-like functions
(Portis 2002, Spreitzer and Salvucci 2002). As with
other AAA1 proteins activase functions as a molecular
chaperone, switching the conformation of Rubisco from
inactive (closed) to active (open). Because of the dependence on ATP hydrolysis (Robinson and Portis 1989),
the controlled switching of Rubisco active-sites from the
closed to open conformation by activase forms the basis
for the regulation of Rubisco by light. Without activase,
photosynthesis is severely impaired because Rubisco sites
become sequestered in an inactive form (Salvucci et al.
1986).

Effect of temperature on de-activation of Rubisco and

its reversal by activase
Under steady-state conditions the activation state of
Rubisco in leaves represents a dynamic balance between
the rates of Rubisco de-activation and re-activation (i.e.
opening of the closed conformation) by activase. The
impact of temperature on these processes has only
recently been examined, but the results indicate that, as
temperature increases, the balance shifts towards
de-activation. In the absence of activase, the rate at
which Rubisco de-activates under catalytic conditions
increases with temperature (Crafts-Brandner and Salvucci
2000). As discussed above, loss of activation could involve
loss of carbamate, either alone or in conjunction with
binding of RuBP to decarbamylated sites, or the formation of misfire products on carbamylated sites.
To our knowledge, the effect of temperature on the
exchange of the carbamate has not been thoroughly

investigated, although the results of early experiments

on Rubisco carbamylation clearly showed that, in the
absence of RuBP, sites decarbamylate faster at higher
temperature (Lorimer 1979). More recently, Portis et al.
(1995) have shown that loss of the carbamate is extremely
slow when RuBP levels are saturating (see also Table 1).
To understand the mechanism of Rubisco de-activation
at high temperature, the effect of temperature on the
carbamylation status of Rubisco was measured after
incubating the fully carbamylated enzyme at air-levels
of CO2 and with saturating levels of RuBP. The
results show that, while there was some loss of the carbamate at higher temperatures, decarbamylation failed
to account for the decrease in Rubisco activity (Table 1).
Instead, this result, namely a decrease in Rubisco activity
without a corresponding loss of the carbamate, indicates
the formation and accumulation of misfire products.
Thus, it appears that as temperature increases, Rubisco
de-activates under catalytic conditions because of
increased rates of misfire product formation.
Under non-stress conditions, activase maintains
Rubisco in an active form by converting Rubisco from
the closed to the open conformation, thereby loosening
the binding of tightly bound sugar-P. In vitro measurements have shown that as temperature increases, the
ability of activase to maintain Rubisco in an active
form fails to keep pace with a faster rate of Rubisco
de-activation (Crafts-Brandner and Salvucci 2000). Presumably, de-activation occurs at high temperature
because activase activity is insufficient to offset the faster
rates of catalytic misfire product formation. The effect is
dependent on the concentration of activase with higher
amounts of activase counteracting the faster rates of
Rubisco de-activation at high temperature (CraftsBrandner and Salvucci 2000). The shift towards Rubisco
de-activation at higher temperatures is not caused by
changes in the availability of ATP, since de-activation
occurs in vitro even with saturating ATP and an ATP
regenerating system. In vivo, measurements of ATP and
other metabolites, as well as measurements of chlorophyll fluorescence and light scattering, indicate that
chloroplast ATP levels do not decrease under moderate
heat stress (Weis 1981a, Bilger et al. 1987, Law and
Crafts-Brandner 1999). Furthermore, the consensus

Table 1. Effect of temperature on carbamylation level and the catalytic competency of Rubisco. Isolated cotton Rubisco (0.5 mg ml1) was
incubated for 1.75 min under the indicated conditions as described previously (Crafts-Brandner and Salvucci 2000), before removal of an
aliquot for measurement of carbamylation or Rubisco activity at 30
C. The carbamylation status was determined by trapping 14CO2 with
carboxyarabinitol bisphosphate (Edmondson et al. 1990). Catalytically competent active sites were determined by measuring Rubisco activity
(Crafts-Brandner and Salvucci 2000). aProduced by incubation with 10 mM NaHCO3 at 25
C for 20 min (specific activity
2.5 U mg protein1). bProduced by incubation with 1 mM NaHCO3 at 45
C for 20 min. cCatalytic conditions included 1 mM NaHCO3 and
6 mM RuBP.
Conditions
Fully carbamylated controla
Partially carbamylated controlb
25
C Catalytic conditionsc
35
C Catalytic conditions
45
C Catalytic conditions

Physiol. Plant. 120, 2004

Carbamylated sites
(mol mol1)

Catalytically competent sites

(mol mol1)

0.993  0.83
0.276  0.029
1.077  0.071
1.064  0.046
0.860  0.047

1  0.02
0.293  0.001
0.727  0.006
0.583  0.021
0.455  0.011

183

opinion is that non-cyclic electron transport activity

increases in response to temperature (Weis 1985, Bukhov
et al. 2000), which would stimulate ATP synthesis under
moderate heat stress.
A likely cause of reduced Rubisco activation at high
temperature is activases low temperature optimum and
thermal lability. For example, the ATPase activity of
cotton activase exhibits a temperature optimum of
about 34
C (Fig. 2, inset), about 20
C lower than the
temperature optimum of Rubisco. Although not strictly
coupled to Rubisco activation, ATPase activity is
required for Rubisco activation and both activities
change in parallel in response to temperature and other
conditions (Robinson and Portis 1989, Portis 2002).
Thus, it is reasonable to assume that the ability of activase to maintain Rubisco in the active form diminishes
with temperature because its activities (i.e. ATPase and
Rubisco activation) do not continue to increase with
temperature above a relatively low optimum to offset
the faster rates of Rubisco de-activation. Measurements
of protein aggregation have shown that loss of ATPase
activity at temperatures above the optimum may be
caused by thermal denaturation of activase (Feller et al.
1998, Salvucci et al. 2001). In fact, compared with other
stromal enzymes, activase is exceptionally sensitive to
thermal denaturation even in situ, aggregating into insoluble complexes in response to even moderate heat stress
(Feller et al. 1998, Rokka et al. 2000, Salvucci et al. 2001).
For activase, maximum rates of ATP hydrolysis and
Rubisco activation occur when the enzyme forms a
higher ordered oligomer, with association of as many as
16 subunits (Portis 2002, Spreitzer and Salvucci 2002).
Higher temperatures generally promote dissociation of
oligomers, which could contribute to loss of activity even
before denaturation and non-specific aggregation occurs.
High temperature could also alter the structure or fluidity of the interacting domains on activase and/or
Rubisco, impairing the ability of activase to interact
productively with Rubisco. Disruption of the physical
interaction between activase and Rubisco could occur
because of subunit dissociation, in which case loss of
ATPase activity and Rubisco activation would be simultaneous. Alternatively, the domains involved in the physical interactions could be affected before the subunits
dissociate, at temperatures that cause negligible inhibition of ATPase activity. Elucidating the effect of temperature on activation activity will require direct
measurement of the actual physical interaction between
activase and Rubisco.

Adaptation of photosynthesis and heat stress forms of

activase
The temperature optimum for net photosynthesis
changes to a limited extent depending upon the conditions used for growth (Berry and Bjorkman 1980 and
references therein). Induction of heat shock proteins and
other types of adaptive responses are undoubtedly involved
184

in acclimation to higher growth temperatures, affording

protection to the various components of the photosynthetic apparatus (Preczewski et al. 2000, Burke 2001).
When heat stress is imposed gradually, de-activation of
Rubisco still accompanies inhibition of net photosynthesis,
but higher temperatures are required for inhibition and
de-activation (Crafts-Brandner and Law 2000). In cotton,
maize, and wheat, prolonged heat stress induces synthesis
of specific heat stress forms of activase (Sanchez de
Jimenez et al. 1995, Law and Crafts-Brandner 2001,
Law et al. 2001), which in cotton appear to be translated
from a distinct mRNA (Law et al. 2001).
In many plants, activase is composed of two polypeptides that are the products of alternative splicing of a
single pre-mRNA (Spreitzer and Salvucci 2002). In some
species, the two isoforms of activase differ markedly in
their thermal stability both in vivo (Rokka et al. 2001,
Salvucci et al. 2001) and in vitro (Crafts-Brandner et al.
1997). Heat stress is known to affect alternative splicing
of certain plant genes (Lazar and Goodman 2000,
Kinoshita et al. 2001), and could change the splicing
pattern of activase, culminating in the expression of a
unique product(s) derived from this gene. Considering
the exceptional sensitivity of activase to heat inactivation
(Feller et al. 1998, Salvucci et al. 2001), a change in the
splicing pattern of the activase gene in response to heat
stress could provide a mechanism for increasing the
thermal stability of activase by altering the polypeptide
composition of the holoenzyme (Crafts-Brandner et al.
1997, Law et al. 2001).

The temperature response of photosynthesis in C4

plants
The temperature optimum of photosynthesis in C4 plants
is generally higher than in C3 plants, resembling the
response of C3 plants at high CO2 (Berry and Bjorkman
1980). In C4 plants, the CO2 concentration at the site of
Rubisco is elevated, which reduces photorespiration and
minimizes the effects of lower CO2 solubility and affinity
on Rubisco at higher temperatures. Because of the high
internal CO2 concentration and the marked increase in
Rubisco VMax with increasing temperature, Sage (2002)
concluded that Rubisco activity should not be limiting
for C4 photosynthesis when temperatures are optimal or
above optimal for photosynthesis. However, for the C4
plant maize, the activation state of Rubisco decreased
markedly at temperatures above the optimum for photosynthesis in concert with increased levels of RuBP and
decreased levels of PGA (Crafts-Brandner and Salvucci
2002). As in C3 plants, the measured effect of temperature on net photosynthesis in maize could be predicted
from the kinetic properties of Rubisco and the gas solubilities if the VMax terms were adjusted for the measured
changes in the activation state of Rubisco. These results
clearly indicate that Rubisco and not RuBP regeneration
limit net photosynthesis at moderately high temperatures, even in a plant with C4 photosynthesis.
Physiol. Plant. 120, 2004

The importance of the Rubisco activation state for

predicting the response of photosynthesis to
environmental change
Understanding the response of photosynthesis to
changing environmental conditions is of paramount
importance for predicting the effects of global climate
change on plant productivity. Accurate predictions of
plant growth require valid models of photosynthesis
that are biochemically based. In the natural environment, the rate of photosynthesis is influenced primarily
by temperature, the amount of light intercepted, and the
availability of water and nutrients. We now know that
the amount of Rubisco available for photosynthesis (i.e.
the activation state) changes in a predictable way to a
host of environmental conditions, including temperature
and CO2 concentration. By adjusting the kinetics of
Rubisco for changes in activation state, it is possible to
accurately predict the response of photosynthesis to
temperature and also to CO2 and O2 (Crafts-Brandner
and Salvucci 2000).
Changes in Rubisco activation are sometimes regarded
as a secondary consequence of other limitations (Sage
1990, Farquhar et al. 2001), and therefore are not incorporated into most models of photosynthesis (Bernacchi
et al. 2001). This view ignores two important facts. First,
changes in the activation state of Rubisco can be caused
by a direct effect on the properties of Rubisco and/or
activase, as in the case of high temperature. Second, even
when changes in Rubisco activation are a consequence of
limitations elsewhere in photosynthesis, the activation
state eventually becomes limiting or co-limiting for net
photosynthesis in the steady-state, making it an accurate
predictor of net photosynthesis. Considering that all
models of photosynthesis are based on the kinetic properties of Rubisco, we feel the predictive power of these
models and their validity would improve by recognizing
that changes in Rubisco activation state represent an
important and measurable biochemical limitation to
photosynthesis under natural conditions.
Acknowledgements The 2002 Annual Symposium of the Southern
Section of the American Society of Plant Biologists took place at
the University of Georgia on March 4, 2002. The theme of the
colloquium was Mechanisms for Regulation of Plant Metabolism.
This issue of Physiologia Plantarum contains three papers based on
the invited lectures. Generous support for these lectures was provided by Pioneer Hi Bred International Inc., Syngenta Crop Protection, Inc., and the American Society of Plant Biologists.

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