Enzyme Co-Localization with Rubisco in Pea Leaf Chloroplasts

Immunocytolocalization experiments indicate that carbonic anhydrase, phosphoribulokinase, and P-glycerate kinase are near neighbors of Rubisco in the pea leaf chloroplast stroma. Direct transfer of ribulose bisphosphate and gaseous CO(2) from phosphoribulokinase and carbonic anhydrase to Rubisco, and direct transfer of P-glycerate from Rubisco to P-glycerate kinase in the chloroplast stroma, is then a possibility. Rubisco activase, responsible for the removal of inhibitory sugar phosphates that bind to the active site of Rubisco in the dark, also appears to be co-localized with Rubisco.     

Rubisco Activity: Effects of Drought Stress

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activityis modulated in vivo either by reaction with CO₂ and Mg²⁺ tocarbamylate a lysine residue in the catalytic site, or by thebinding of inhibitors within the catalytic site. Binding ofinhibitors blocks either activity or the carbamylation of thelysine residue that is essential for activity. At night, inmany species, 2-carboxyarabinitol-1-phosphate (CA1P) is formedwhich binds tightly to Rubisco, inhibiting catalytic activity.Recent work has shown that tight-binding inhibitors can alsodecrease Rubisco activity in the light and contribute to theregulation of Rubisco activity. Here we determine the influencethat such inhibitors of Rubisco exert on catalytic activityduring drought stress. In tobacco plants, ‘total Rubiscoactivity’, i.e. the activity following pre-incubationwith CO₂ and Mg²⁺, was positively correlated with leaf relativewater content. However, ‘total Rubisco activity’in extracts from leaves with low water potential increased markedlywhen tightly bound inhibitors were removed, thus increasingthe number of catalytic sites available. This suggests thatin tobacco the decrease of Rubisco activity under drought stressis not primarily the result of changes in activation by CO₂and Mg²⁺ but due rather to the presence of tight-binding inhibitors.The amounts of inhibitor present in leaves of droughted tobaccobased on the decrease in Rubisco activity per mg soluble proteinwere usually much greater than the amounts of the known inhibitors(CA1P and ‘daytime inhibitor’) that can be recoveredin acid extracts. Alternative explanations for the differencebetween maximal and total activities are discussed.     

Regulation of ribulose-1,5-bisphosphate carboxylase activity in response to diurnal changes in irradiance

The regulation of ribulose-1,5-bisphosphate (RuBP) carboxylase (Rubisco) activity and metabolite pool sizes in response to natural diurnal changes in photon flux density (PFD) was examined in three species (Phaseolus vulgaris, Beta vulgaris, and Spinacia oleracea) known to differ in the mechanisms used for this regulation. Diurnal regulation of Rubisco activity in P. vulgaris was primarily the result of metabolism of the naturally occurring tight-binding inhibitor of Rubisco, 2-carboxyarabinitol 1-phosphate (CA1P). In B. vulgaris, the regulation of Rubisco activity was the result of both changes in activation state and CA1P metabolism. In S. oleracea, Rubisco activity was regulated by a combination of changes in activation state and the binding/release of another tight binding inhibitor, probably RuBP. Despite these different mechanisms for the light regulation of Rubisco activity, the relationship between the in vivo activity of Rubisco and the PFD was the same for all three species. Rates of CA1P metabolism were thus sufficient to allow this mechanism to participate in the diurnal regulation of Rubisco activity as PFD changed at its normal rate. Furthermore, under natural conditions this regulatory mechanism was found to be important in controlling Rubisco activity over approximately the same range of PFD as did changes in activation state of the enzyme. Finally, this regulation of Rubisco activity resulted in relatively similar and saturating RuBP pool sizes for photosynthesis at all but the lowest PFD values in all three species.     

Subunit interactions of Rubisco activase: polyethylene glycol promotes self-association, stimulates ATPase and activation activities, and enhances interactions with Rubisco.

The effect of polyethylene glycol (PEG) on the enzymatic and physical properties of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase was examined. In the presence of PEG, Rubisco activase exhibited higher ATPase and Rubisco activating activities, concomitant with increased apparent affinity for ATP and Rubisco. Specific ATPase activity, which was dependent on Rubisco activase concentration, was also higher in the presence of Ficoll, polyvinylpyrrolidone, and bovine serum albumin. The ability of Rubisco activase to facilitate dissociation of the tight-binding inhibitor 2-carboxyarabinitol 1-phosphate from carbamylated Rubisco was also enhanced in the presence of PEG. Mixing experiments with Rubisco activase from two different sources showed that tobacco Rubisco activase, which exhibited little activation of spinach Rubisco by itself, was inhibitory when included with spinach Rubisco activase. Polyethylene glycol improved the ability of tobacco and a mixture of tobacco plus spinach Rubisco activase to activate spinach Rubisco. Estimates based on rate zonal sedimentation and gel-filtration chromatography indicated that the apparent molecular mass of Rubisco activase was two- to fourfold higher in the presence of PEG. The increase in apparent molecular mass was consistent with the propensity of solvent-excluding reagents like PEG to promote self-association of proteins. Likewise, the change in enzymatic properties of Rubisco activase in the presence of PEG and the dependence of specific activity on protein concentration resembled changes that often accompany self-association. For Rubisco activase, high concentrations of protein in the chloroplast stroma would provide an environment conducive to self-association and cause expression of properties that would enhance its ability to function efficiently in vivo.     

Moderately High Temperatures Inhibit Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase-Mediated Activation of Rubisco

We tested the hypothesis that light activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inhibited by moderately elevated temperature through an effect on Rubisco activase. When cotton (Gossypium hirsutum L.) or wheat (Triticum aestivum L.) leaf tissue was exposed to increasing temperatures in the light, activation of Rubisco was inhibited above 35 and 30°C, respectively, and the relative inhibition was greater for wheat than for cotton. The temperature-induced inhibition of Rubisco activation was fully reversible at temperatures below 40°C. In contrast to activation state, total Rubisco activity was not affected by temperatures as high as 45°C. Nonphotochemical fluorescence quenching increased at temperatures that inhibited Rubisco activation, consistent with inhibition of Calvin cycle activity. Initial and maximal chlorophyll fluorescence were not significantly altered until temperatures exceeded 40°C. Thus, electron transport, as measured by Chl fluorescence, appeared to be more stable to moderately elevated temperatures than Rubisco activation. Western-blot analysis revealed the formation of high-molecular-weight aggregates of activase at temperatures above 40°C for both wheat and cotton when inhibition of Rubisco activation was irreversible. Physical perturbation of other soluble stromal enzymes, including Rubisco, phosphoribulokinase, and glutamine synthetase, was not detected at the elevated temperatures. Our evidence indicates that moderately elevated temperatures inhibit light activation of Rubisco via a direct effect on Rubisco activase.     

Reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase content by antisense RNA reduces photosynthesis in transgenic tobacco plants

A complementary DNA for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was cloned from tobacco (Nicotiana tabacum) and fused in the antisense orientation to the cauliflower mosaic virus 35S promoter. This antisense gene was introduced into the tobacco genome, and the resulting transgenic plants were analyzed to assess the effect of the antisense RNA on Rubisco activity and photosynthesis. The mean content of extractable Rubisco activity from the leaves of 10 antisense plants was 18% of the mean level of activity of control plants. The soluble protein content of the leaves of anti-small subunit plants was reduced by the amount equivalent to the reduction in Rubisco. There was little change in phosphoribulokinase activity, electron transport, and chlorophyll content, indicating that the loss of Rubisco did not affect these other components of photosynthesis. However, there was a significant reduction in carbonic anhydrase activity. The rate of CO₂ assimilation measured at 1000 micromoles quanta per square meter per second, 350 microbars CO₂, and 25°C was reduced by 63% (mean value) in the antisense plants and was limited by Rubisco activity over a wide range of intercellular CO₂ partial pressures (p_i). In control leaves, Rubisco activity only limited the rate of CO₂ assimilation below a p_i of 400 microbars. Despite the decrease in photosynthesis, there was no reduction in stomatal conductance in the antisense plants, and the stomata still responded to changes in p_i. The unchanged conductance and lower CO₂ assimilation resulted in a higher p_i, which was reflected in greater carbon isotope discrimination in the leaves of the antisense plants. These results suggest that stomatal function is independent of total leaf Rubisco activity.     

Exceptional Sensitivity of Rubisco Activase to Thermal Denaturation in Vitro and in Vivo

Heat stress inhibits photosynthesis by reducing the activation of Rubisco by Rubisco activase. To determine if loss of activase function is caused by protein denaturation, the thermal stability of activase was examined in vitro and in vivo and compared with the stabilities of two other soluble chloroplast proteins. Isolated activase exhibited a temperature optimum for ATP hydrolysis of 44 degrees C compared with > or =60 degrees C for carboxylation by Rubisco. Light scattering showed that unfolding/aggregation occurred at 45 degrees C and 37 degrees C for activase in the presence and absence of ATPgammaS, respectively, and at 65 degrees C for Rubisco. Addition of chemically denatured rhodanese to heat-treated activase trapped partially folded activase in an insoluble complex at treatment temperatures that were similar to those that caused increased light scattering and loss of activity. To examine thermal stability in vivo, heat-treated tobacco (Nicotiana rustica cv Pulmila) protoplasts and chloroplasts were lysed with detergent in the presence of rhodanese and the amount of target protein that aggregated was determined by immunoblotting. The results of these experiments showed that thermal denaturation of activase in vivo occurred at temperatures similar to those that denatured isolated activase and far below those required to denature Rubisco or phosphoribulokinase. Edman degradation analysis of aggregated proteins from tobacco and pea (Pisum sativum cv "Little Marvel") chloroplasts showed that activase was the major protein that denatured in response to heat stress. Thus, loss of activase activity during heat stress is caused by an exceptional sensitivity of the protein to thermal denaturation and is responsible, in part, for deactivation of Rubisco.     

Regulation of Rubisco activity in vivo

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is not able to achieve and maintain adequate CO₂ and Mg²⁺ activation under physiological conditions. Higher plants and green algae contain Rubisco activase, a soluble protein which not only facilitates Rubisco activation in situ but also regulates enzyme activity in response to irradiance and other factors. Regulation of Rubisco activity by modulation of activation state coordinates the rate of CO₂ fixation with the rate of substrate regeneration. This regulation may be required to ensure that the levels of photosynthetic metabolites in the chloroplast are optimal for photosynthesis under a variety of environrmental conditions. Some plant species also appear to regulate Rubisco activity by synthesizing 2-carboxyarabinitol 1-phosphate, an inhibitor of Rubisco in the dark. This inhibitor may function primarily as a regulator of metabolite binding in the dark rather than as a modulator of Rubisco activity in the light.     

Low sink demand limits photosynthesis under P(i) deficiency.

The role of the demand for carbon assimilates (the 'sink') in regulating photosynthetic carbon assimilation (Pn: the 'source') in response to phosphate (P(i)) deficiency was examined in tobacco (Nicotiana tabacum L.). P(i) supply was maintained or withdrawn from plants, and in both treatments the source/sink ratio was decreased in some plants by darkening all but two source leaves (partially darkened plants). The remaining plants were kept fully illuminated. P(i)-sufficient plants showed little variation in rate of Pn, amounts of P(i) or phosphorylated intermediates. Withdrawal of P(i) decreased Pn by 75% under the growing conditions and at both low and high internal CO2 concentration. Concomitantly, P(i), phosphorylated intermediates and ATP contents decreased and starch increased. RuBP and activity of phosphoribulokinase closely matched the changes in Pn, but Rubisco activity remained high. Partial darkening P(i)-deficient plants delayed the loss of photosynthetic activity; Rubisco and phosphoribulokinase activities and amounts of sucrose and metabolites, particularly RuBP and G6P, were higher than in fully illuminated Pi-deficient plants. Rates of sucrose export from leaves were more than 2-fold greater than in fully illuminated P(i)-deficient plants. Greater sucrose synthesis, facilitated by increased G6P content, an activator of SPS, would recycle P(i) from the cytosol back to the chloroplast, maintaining ATP, RuBP and hence Pn. It is concluded that low sink strength imposes the primary limitation on photosynthesis in P(i)-deficient plants which restricts sucrose export and sucrose synthesis imposing an end-product synthesis limitation of photosynthesis.     

C4 photosynthesis at low temperature. A study using transgenic plants with reduced amounts of Rubisco

C(4) plants are rare in the cool climates characteristic of high latitudes and elevations, but the reasons for this are unclear. We tested the hypothesis that CO(2) fixation by Rubisco is the rate-limiting step during C(4) photosynthesis at cool temperatures. We measured photosynthesis and chlorophyll fluorescence from 6 degrees C to 40 degrees C, and in vitro Rubisco and phosphoenolpyruvate carboxylase activity from 0 degrees C to 42 degrees C, in Flaveria bidentis modified by an antisense construct (targeted to the nuclear-encoded small subunit of Rubisco, anti-RbcS) to have 49% and 32% of the wild-type Rubisco content. Photosynthesis was reduced at all temperatures in the anti-Rbcs plants, but the thermal optimum for photosynthesis (35 degrees C) did not differ. The in vitro turnover rate (kcat) of fully carbamylated Rubisco was 3.8 mol mol(-)(1) s(-)(1) at 24 degrees C, regardless of genotype. The in vitro kcat (Rubisco Vcmax per catalytic site) and in vivo kcat (gross photosynthesis per Rubisco catalytic site) were the same below 20 degrees C, but at warmer temperatures, the in vitro capacity of the enzyme exceeded the realized rate of photosynthesis. The quantum requirement of CO(2) assimilation increased below 25 degrees C in all genotypes, suggesting greater leakage of CO(2) from the bundle sheath. The Rubisco flux control coefficient was 0.68 at the thermal optimum and increased to 0.99 at 6 degrees C. Our results thus demonstrate that Rubisco capacity is a principle control over the rate of C(4) photosynthesis at low temperatures. On the basis of these results, we propose that the lack of C(4) success in cool climates reflects a constraint imposed by having less Rubisco than their C(3) competitors.     

Free and Membrane-Bound Multienzyme Complexes with Calvin Cycle Activities in Cotton Leaves

Free and membrane-bound forms of Calvin-cycle multienzyme complexes with a mol wt of 520 ± 20 kD and 640 ± 25 kD, respectively, were isolated from the cotton (Gossypium hirsutum L.) leaves. Both complexes exhibited the following enzymatic activities: ribose phosphate isomerase, phosphoribulokinase, ribulose bisphosphate carboxylase (Rubisco), phosphoglycerate kinase, and glyceraldehyde phosphate dehydrogenase. The activities of the membrane-bound multienzyme complex were significantly higher than the activities of the free complex. This difference was especially pronounced in the case of carboxylase activity. An increase in the enzymatic activity of membrane-bound multienzyme complex in comparison with the free complex is presumably due to the different number of their constituent parts. Another possible cause is the membrane-level regulation of the functional activity of the enzymes composing the complex.     

Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis Is Caused by Accumulation of a Slow, Tight-Binding Inhibitor at the Catalytic Site

The slow inactivation which accompanies catalysis by higher-plant ribulose-P₂ carboxylase-oxygenase (Rubisco) in vitro was only partially reversed when the enzyme was gel filtered to remove small molecules. However, gel filtration or dialysis in the presence of high SO^2-₄ concentrations induced full recovery. This suggests that the inactivation is caused by a tight-binding inhibitor whose effective affinity is reduced by competition with SO^2-₄ ions, which are known to bind at the catalytic site. The involvement of an inhibitor was confirmed by observations that supernatants obtained after acid-precipitation of inactivated Rubisco were inhibitory when applied to fresh enzyme. The inhibitor bound slowly and tightly and showed strong negative cooperativity. The inhibitor was moderately unstable at pH 8.3, decaying with a halflife of several hours, but was more stable at pH 2. It was destroyed by phosphatase treatment but not by H₂O₂ or o-phenylenediamine, compounds which react with vicinal dicarbonyl groups. It did not contain a carbon atom derived from substrate CO₂. Possibilities concerning the identity, genesis, and physiological relevance of this inhibitor are discussed.     

Developmental changes in the activities of enzymes of free multienzyme complexes involved in the Benson-Calvin cycle

The electrophoretically homogenous preparations of free multienzyme complexes involved in the Benson-Calvin cycle with mol wt of 520 ± 20 and 240 ± 10 kD were isolated from 15–25-day-old cotton (Gossypium hirsutum L.) leaves of the same story (the third and the fourth upper leaves). Enzyme preparations were obtained at three stages of plant development: at the stage of 6–8 true leaves, during flower-bud formation, and during flowering. Comparison studies of developmental changes in activities of ribose phosphate isomerase (RPI), phosphoribulokinase (PRK), and Rubisco were carried out. RPI and PRK activities of multienzyme complexes differed insignificantly (by 2.3% on the average). Significant changes were observed in Rubisco activity (by 30% on the average). The optimum enzymatic activities of these complexes as well as the highest photosynthetic rate were revealed at the stage of reproductive organ formation. This correlation indicates the major role of multienzyme complexes involved in the Benson-Calvin cycle in the developmental control of photosynthesis and epigenesis.     

The absence of Rubisco activase activity in total wheat leaf extracts is recovered in the purified protein

When desalted extracts of soluble protein from dark-adaptedwheat leaves were assayed for ribulose-1, 5-bisphosphate carboxylase/oxygenase(Rubisco) activase activity in the presence of 1 mM ATP andan ATP-regenerating system, very little ATP-dependent activationof RuBP-inactivated Rubisco was found. In extracts from light-adaptedleaves a very similar pattern of Rubisco activation was observedexcept that the overall level of Rubisco activity was much lowerthan in the extracts from dark-adapted leaves. These featureswere apparent both at low (120µg per ml) and high (640µg per ml) protein concentrations. We were unable to demonstrateRubisco activase activity in crude leaf extracts. Consequently,in order to establish that Rubisco activase was present in wheatleaf extracts the wheat leaf protein was purified to homogeneity.The identity of the protein was confirmed with antibodies tothe spinach enzyme, ATPase activity and activase-mediated releaseof the inhibitor, carboxyara-binitol-1-phosphate (CA1P) fromthe tertiary Rubisco complex. The pure wheat Rubisco activaserelieved the CA1P-induced inhibition of Rubisco activity. Rubiscoactivase had no significant effect on the affinity of wheatRubisco for the substrate, ribulose-1, 5-bisphosphate (RuBP). Key words: Rubisco activase, Rubisco, regulation     

Dependence of photosynthesis of sunflower and maize leaves on phosphate supply, ribulose-1,5-bisphosphate carboxylase/oxygenase activity, and ribulose-1,5-bisphosphate pool size

Sunflower (Helianthus annuus L. cv Asmer) and maize (Zea mays L. cv Eta) plants were grown under controlled environmental conditions with a nutrient solution containing 0, 0.5, or 10 millimolar inorganic phosphate. Phosphate-deficient leaves had lower photosynthetic rates at ambient and saturating CO₂ and much smaller carboxylation efficiencies than those of plants grown with ample phosphate. In addition, phosphate-deficient leaves contained smaller quantities of total soluble proteins and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) per unit area, although the relative proportions of these components remained unchanged. The specific activity of Rubisco (estimated in the crude extracts of leaves) was significantly reduced by phosphate deficiency in sunflower but not in maize. Thus, there was a strong dependence of carboxylation efficiency and CO₂-saturated photosynthetic rate on Rubisco activity only in sunflower. Phosphate deficiency decreased the 3-phosphoglycerate and ribulose-1,5-bisphosphate (RuBP) contents of the leaf in both species. The ratio of 3-phosphoglycerate to RuBP decreased in sunflower but increased in maize with phosphate deficiency. The calculated concentrations of RuBP and RuBP-binding sites in the chloroplast stroma decreased markedly with phosphate deficiency. The ratio of the stromal concentration of RuBP to that of RuBP-binding sites decreased in sunflower but was not affected in maize with phosphate deficiency. We suggest that a decrease in this ratio made the RuBP-binding sites more vulnerable to blockage or inactivation by tight-binding metabolites/inhibitors, causing a decrease in the initial specific activity of Rubisco in the crude extract from phosphate-deficient sunflower leaves. However, the decrease in Rubisco specific activity was much less than the decrease in the RuBP content in the leaf and its concentration in the stroma. A large ratio of RuBP to RuBP-binding sites may have maintained the Rubisco-specific activity in phosphate-deficient maize leaves. We conclude that the effect of phosphate deficiency is more on RuBP regeneration than on Rubisco activity in both sunflower and maize.     

Atomic resolution x-ray structure of the substrate recognition domain of higher plant ribulose-bisphosphate carboxylase/oxygenase (Rubisco) activase

The rapid release of tight-binding inhibitors from dead-end ribulose-bisphosphate carboxylase/oxygenase (Rubisco) complexes requires the activity of Rubisco activase, an AAA+ ATPase that utilizes chemo-mechanical energy to catalyze the reactivation of Rubisco. Activase is thought to play a central role in coordinating the rate of CO₂ fixation with the light reactions of photosynthesis. Here, we present a 1.9 Å crystal structure of the C-domain core of creosote activase. The fold consists of a canonical four-helix bundle, from which a paddle-like extension protrudes that entails a nine-turn helix lined by an irregularly structured peptide strand. The residues Lys-313 and Val-316 involved in the species-specific recognition of Rubisco are located near the tip of the paddle. An ionic bond between Lys-313 and Glu-309 appears to stabilize the glycine-rich end of the helix. Structural superpositions onto the distant homolog FtsH imply that the paddles extend away from the hexameric toroid in a fan-like fashion, such that the hydrophobic sides of each blade bearing Trp-302 are facing inward and the polar sides bearing Lys-313 and Val-316 are facing outward. Therefore, we speculate that upon binding, the activase paddles embrace the Rubisco cylinder by placing their hydrophobic patches near the partner protein. This model suggests that conformational adjustments at the remote end of the paddle may relate to selectivity in recognition, rather than specific ionic contacts involving Lys-313. Additionally, the superpositions predict that the catalytically critical Arg-293 does not interact with the bound nucleotide. Hypothetical ring-ring stacking and peptide threading models for Rubisco reactivation are briefly discussed.     

New roads lead to Rubisco in archaebacteria

The discovery of the CO(2)-fixing enzyme Rubisco in the Archaebacteria has presented a conundrum in that they apparently lack the gene for phosphoribulokinase, which is required to generate Rubisco's substrate ribulose 1,5-bisphosphate (RuBP). However, two groups have now demonstrated novel RuBP synthesis pathways, demystifying Rubisco's non-autotrophic and perhaps ancient role. A new CO(2) fixing role for Rubisco, which is distinct from the globally dominant Calvin cycle, is providing important clues furthering our understanding of the evolution of autotrophy. This perspective is strengthened by the additional recognition in this commentary that some Rubisco-containing Archaea do also contain PRK and may represent an interesting autotrophic evolutionary transition. Supplementary material for this article can be found on the BioEssays website (http://www.interscience.wiley.com/jpages/0265-9247/suppmat/index.html).     

The temperature response of photosynthesis in tobacco with reduced amounts of Rubisco

The reasons for the decline in net CO₂ assimilation ( A ) above its thermal optimum are controversial. We tested the hypothesis that increasing the ratio of Rubisco activase to Rubisco catalytic site concentration would increase the activation state of Rubisco at high temperatures. We measured photosynthetic gas exchange, in vivo electron transport ( J ) and the activation state of Rubisco between 15 and 45 °C, at 38 and 76 Pa ambient CO₂, in wild-type (WT) and anti- rbc S tobacco. The Rubisco content of the anti- rbc S lines was 30% (S7-1) or 6% (S7-2) of WT, but activase levels were the same in the three genotypes. Anti- rbc S plants had lower A than WT at all temperatures, but had a similar thermal optimum for photosynthesis as WT at both CO₂ levels. In WT plants, Rubisco was fully activated at 32 °C, but the activation state declined to 64% at 42 °C. By contrast, the activation state of Rubisco was above 90% in the S7-1 line, between 15 and 42 °C. Both A and J declined about 20% from T _opt to the highest measurement temperatures in WT and the S7-1 line, but this was fully reversed after a 20 min recovery at 35 °C. At 76 Pa CO₂, predicted rates of RuBP regeneration-limited photosynthesis corresponded with measured A in WT tobacco at all temperatures, and in S7-1 tobacco above 40 °C. Our observations are consistent with the hypothesis that the high temperature decline in A in the WT is because of an RuBP regeneration limitation, rather than the capacity of Rubisco activase to maintain high Rubisco activation state.