Effect of activase level and isoform on the thermotolerance of photosynthesis in Arabidopsis

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation decreases under moderate heat stress. This decrease is caused by an impairment of activase function, which is exacerbated by faster rates of Rubisco deactivation at elevated temperatures. To determine if stromal oxidation causes inhibition of activase, transgenic Arabidopsis plants expressing suboptimal amounts of either the redox-regulated 46 kDa alpha- or non-redox regulated 43 kDa beta-isoform of activase were examined. Photosynthesis, as measured by gas exchange and chlorophyll fluorescence, and Rubisco activation were inhibited to a much greater extent by moderately high temperatures in the two transgenic lines expressing suboptimal levels of the individual isoforms of activase compared with wild-type plants or transgenic plants expressing levels of the beta-isoform sufficient for wild-type rates of photosynthesis. Net photosynthesis and Rubisco activation in transgenic plants expressing suboptimal amounts of the beta-isoform of activase from the Antarctic hairgrass were even more sensitive to inhibition by moderate heat stress than in the transgenic plants containing Arabidopsis activase. The results demonstrate that photosynthesis exhibits a similar sensitivity to inhibition by moderately high temperature in plants expressing either of the two different isoforms of activase. Thus, impairment of activase function under heat stress is not caused by oxidation of the redox-sensitive sulphydryls of the alpha-isoform of activase. Instead, the results are consistent with thermal denaturation of activase under moderate heat stress, the effects of which on Rubisco activation would be enhanced when activase levels are suboptimal for photosynthesis.     

Two residues of rubisco activase involved in recognition of the Rubisco substrate

Rubisco activase is an AAA(+) protein, a superfamily with members that use a "Sensor 2" domain for substrate recognition. To determine whether the analogous domain of activase is involved in recognition of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), two chimeric activases were constructed, interchanging a Sensor 2-containing region between activases from spinach and tobacco. Spinach chimeric activase was a poor activator of both spinach and tobacco Rubisco. In contrast, tobacco chimeric activase activated spinach Rubisco far better than tobacco Rubisco, similar to spinach activase. A point mutation, K311D, in the Sensor 2 domain of the tobacco chimeric activase abolished its ability to better activate spinach Rubisco. The opposite mutation, D311K, in wild type tobacco activase produced an enzyme that activated both spinach and tobacco Rubisco, whereas a second mutation, D311K/L314V, shifted the activation preference toward spinach Rubisco. The involvement of these two residues in substrate selectivity was confirmed by introducing the analogous single and double mutations in cotton activase. The ability of the two tobacco activase mutants to activate wild type and mutant Chlamydomonas Rubiscos was also examined. Tobacco D311K activase readily activated wild type and P89R but not D94K Rubisco, whereas the tobacco L314V activase only activated D94K Rubisco. The tobacco activase double mutant D311K/L314V activated wild type Chlamydomonas Rubisco better than either the P89R or D94K Rubisco mutants, mimicking activation by spinach activase. The results identified a substrate recognition region in activase in which two residues may directly interact with two residues in Rubisco.     

Alterations in Photosystem II electron transport as revealed by thermoluminescence of Cu-poisoned chloroplasts

The Rubisco activase amino acid sequences of spinach and tobacco are 79% identical, yet the tobacco protein does not facilitate the activation of the uncarbamylated, ribulose bisphosphate bound form of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) and vice versa. In contrast, combinations of the spinach Rubisco activase with Rubisco from non-Solanaceae species and combinations of tobacco Rubisco activase with Rubisco from other Solanaceae species are almost as effective as the analogous combination. To examine the basis of the preference of an activase protein for either Solanaceae or non-Solanaceae Rubisco, several recombinant chimeric proteins were obtained by combining regions from the cDNAs of spinach and tobacco activase and expression in Escherichia coli. The chimeric proteins were analyzed for ATP hydrolysis and ability to activate spinach and tobacco Rubisco. Comparisons of Rubisco preference with composition of the various activase chimeras indicate that the major determinants of Rubisco preference seem to be localized in the carboxyl-terminal region.     

Rubisco, Rubisco activase, and global climate change

Global warming and the rise in atmospheric CO(2) will increase the operating temperature of leaves in coming decades, often well above the thermal optimum for photosynthesis. Presently, there is controversy over the limiting processes controlling photosynthesis at elevated temperature. Leading models propose that the reduction in photosynthesis at elevated temperature is a function of either declining capacity of electron transport to regenerate RuBP, or reductions in the capacity of Rubisco activase to maintain Rubisco in an active configuration. Identifying which of these processes is the principal limitation at elevated temperature is complicated because each may be regulated in response to a limitation in the other. Biochemical and gas exchange assessments can disentangle these photosynthetic limitations; however, comprehensive assessments are often difficult and, for many species, virtually impossible. It is proposed that measurement of the initial slope of the CO(2) response of photosynthesis (the A/C(i) response) can be a useful means to screen for Rubisco activase limitations. This is because a reduction in the Rubisco activation state should be most apparent at low CO(2) when Rubisco capacity is generally limiting. In sweet potato, spinach, and tobacco, the initial slope of the A/C(i) response shows no evidence of activase limitations at high temperature, as the slope can be accurately modelled using the kinetic parameters of fully activated Rubisco. In black spruce (Picea mariana), a reduction in the initial slope above 30 degrees C cannot be explained by the known kinetics of fully activated Rubisco, indicating that activase may be limiting at high temperatures. Because black spruce is the dominant species in the boreal forest of North America, Rubisco activase may be an unusually important factor determining the response of the boreal biome to climate change.     

Sensitivity of photosynthesis in a C4 plant,maize, to heat stress

Our objective was to determine the sensitivity of components of the photosynthetic apparatus of maize (Zea mays), a C4 plant, to high temperature stress. Net photosynthesis (Pn) was inhibited at leaf temperatures above 38 degrees C, and the inhibition was much more severe when the temperature was increased rapidly rather than gradually. Transpiration rate increased progressively with leaf temperature, indicating that inhibition was not associated with stomatal closure. Nonphotochemical fluorescence quenching (qN) increased at leaf temperatures above 30 degrees C, indicating increased thylakoid energization even at temperatures that did not inhibit Pn. Compared with CO(2) assimilation, the maximum quantum yield of photosystem II (F(v)/F(m)) was relatively insensitive to leaf temperatures up to 45 degrees C. The activation state of phosphoenolpyruvate carboxylase decreased marginally at leaf temperatures above 40 degrees C, and the activity of pyruvate phosphate dikinase was insensitive to temperature up to 45 degrees C. The activation state of Rubisco decreased at temperatures exceeding 32.5 degrees C, with nearly complete inactivation at 45 degrees C. Levels of 3-phosphoglyceric acid and ribulose-1,5-bisphosphate decreased and increased, respectively, as leaf temperature increased, consistent with the decrease in Rubisco activation. When leaf temperature was increased gradually, Rubisco activation acclimated in a similar manner as Pn, and acclimation was associated with the expression of a new activase polypeptide. Rates of Pn calculated solely from the kinetics of Rubisco were remarkably similar to measured rates if the calculation included adjustment for temperature effects on Rubisco activation. We conclude that inactivation of Rubisco was the primary constraint on the rate of Pn of maize leaves as leaf temperature increased above 30 degrees C.     

The Effects of Chilling in the Light on Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activation in Tomato (Lycopersicon esculentum Mill.)

Photosynthesis rate, ribulsoe-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation state, and ribulose bisphosphate concentration were reduced after exposing tomato (Lycopersicon esculentum Mill.) plants to light at 4[deg]C for 6 h. Analysis of lysed and reconsituted chloroplasts showed that activity of the thylakoid membrane was inhibited and that Rubisco, Rubisco activase, and other soluble factors were not affected. Leaf photosynthesis rates and the ability of chilled thylakoid membranes to promote Rubisco activation recovered after 24 h at 25[deg]C. Thylakoid membranes from control tomato plants were as effective as spinach thylakoids in activating spinach Rubisco in the presence of spinach Rubisco activase. This observation is in sharp contrast to the poor ability of spinach Rubisco activase to activate tomato Rubisco (Z.-Y. Wang, G.W. Snyder, B.D. Esau, A.R. Portis, and W.L. Ogren [1992] Plant Physiol 100: 1858-1862). The ability of thylakoids from chilled tomato plants to activate Rubisco in the assay system was greatly inhibited compared to control plants. These experiments indicate that chilling tomato plants at 4[deg]C interferes with photosynthetic carbon metabolism at two sites, thioredoxin/ferredoxin reduction (G.F. Sassenrath, D.R. Ort, and A.R. Portis, Jr. [1990] Arch Biochem Biophys 282: 302-308), which limits bisphosphatase activity, and Rubisco activase, which reduces Rubisco activation state.     

Genetic engineering of the biosynthesis of glycinebetaine enhances photosynthesis against high temperature stress in transgenic tobacco plants

Genetically engineered tobacco (Nicotiana tabacum) with the ability to synthesis glycinebetaine was established by introducing the BADH gene for betaine aldehyde dehydrogenase from spinach (Spinacia oleracea). The genetic engineering enabled the plants to accumulate glycinebetaine mainly in chloroplasts and resulted in enhanced tolerance to high temperature stress during growth of young seedlings. Moreover, CO2 assimilation of transgenic plants was significantly more tolerant to high temperatures than that of wild-type plants. The analyses of chlorophyll fluorescence and the activation of Rubisco indicated that the enhancement of photosynthesis to high temperatures was not related to the function of photosystem II but to the Rubisco activase-mediated activation of Rubisco. Western-blotting analyses showed that high temperature stress led to the association of Rubisco activase with the thylakoid membranes from the stroma fractions. However, such an association was much more pronounced in wild-type plants than in transgenic plants. The results in this study suggest that under high temperature stress, glycinebetaine maintains the activation of Rubisco by preventing the sequestration of Rubisco activase to the thylakoid membranes from the soluble stroma fractions and thus enhances the tolerance of CO2 assimilation to high temperature stress. The results seem to suggest that engineering of the biosynthesis of glycinebetaine by transformation with the BADH gene might be an effective method for enhancing high temperature tolerance of plants.     

Species-dependent variation in the interaction of substrate-bound ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) and rubisco activase

Purified spinach (Spinacea oleracea L.) and barley (Hordeum vulgare L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase supported 50 to 100% activation of substrate-bound Rubisco from spinach, barley, wheat (Triticum aestivum L.), soybean (Glycine max L.), pea (Pisum sativum L.), Arabidopsis thaliana, maize (Zea mays L.), and Chlamydomonas reinhardtii but supported only 10 to 35% activation of Rubisco from three Solanaceae species, tobacco (Nicotiana tabacum L.), petunia (Petunia hybrida L.), and tomato (Lycopersicon esculentum L.). Conversely, purified tobacco and petunia Rubisco activase catalyzed 75 to 100% activation of substrate-bound Rubisco from the three Solanacee species but only 10 to 25% activation of substrate-bound Rubisco from the other species. Thus, the interaction between substrate-bound Rubisco and Rubisco activase is species dependent. The species dependence observed is consistent with phylogenetic relationships previously derived from plant morphological characteristics and from nucleotide and amino acid sequence comparisons of the two Rubisco subunits. Species dependence in the Rubisco-Rubisco activase interaction and the absence of major anomalies in the deduced amino acid sequence of tobacco Rubisco activase compared to sequences in non-Solanaceae species suggest that Rubisco and Rubisco activase may have coevolved such that amino acid changes that have arisen by evolutionary divergence in one of these enzymes through spontaneous mutation or selection pressure have led to compensatory changes in the other enzyme.     

The activity of Rubisco��s molecular chaperone, Rubisco activase, in leaf extracts

Rubisco frequently undergoes unproductive interactions with its sugar-phosphate substrate that stabilize active sites in an inactive conformation. Restoring catalytic competence to these sites requires the "molecular chiropractic" activity of Rubisco activase (activase). To make the study of activase more routine and physiologically relevant, an assay was devised for measuring activase activity in leaf extracts based on the ATP-dependent activation of inactive Rubisco. Control experiments with an Arabidopsis activase-deficient mutant confirmed that the rate of Rubisco activation was dependent on the concentration of activase in the extracts. Activase catalyzed Rubisco activation at rates equivalent to 9-14% catalytic sites per min in desalted extracts of Arabidopsis, camelina, tobacco, cotton, and wheat. Faster rates were observed in a transgenic line of Arabidopsis that expresses only the β-isoform of activase, whereas no activity was detected in a line that expresses only the α-isoform. Activase activity was also low or undetectable in rice, maize, and Chlamydomonas, revealing differences in the stability of the enzyme in different species. These differences are discussed in terms of the ability of activase subunits to remain associated or to reassociate into active oligomers when the stromal milieu is diluted by extraction. Finally, the temperature response of activase activity in leaf extracts differed for Arabidopsis, camelina, tobacco, and cotton, corresponding to the respective temperature responses of photosynthesis for each species. These results confirmed the exceptional thermal lability of activase at physiological ratios of activase to Rubisco.     

Temperature dependence of photosynthesis in Arabidopsis plants with modifications in Rubisco activase and membrane fluidity

Net photosynthesis (Pn) is reversibly inhibited at moderately high temperature. To investigate this further, we examined the effects of heat stress on Arabidopsis plants in which Rubisco activase or thylakoid membrane fluidity has been modified. During heating leaves from 25 to 40 degrees C at 250 ppm CO2 and 1% O2, the wild-type (WT), plants expressing the 43 kDa isoform only (rwt43), and plants accumulating activase 40% of WT (R100) exhibited similar inhibitions in the Pn and Rubisco activation state. Despite better membrane integrity than WT, plants having less polyunsaturation of thylakoid lipids (fad7/8 double mutant) failed to maintain greater Pn than the WT. Plants expressing the 46 kDa isoform only (rwt46) exhibited the most inhibition, but plants expressing a 46 kDa isoform incapable of redox regulation (C411A) were similar to the WT. The null mutant (rca) exhibited a continuous decline in Pn. As measured by fluorescence, electron transport activity decreased concomitantly with Pn but PSII was not damaged. Following a quick recovery to 25 from 40 degrees C, whereas most lines recovered 90% Pn, the rwt46 and rca lines recovered only to 59 and <10%, respectively. As measured by NADP-malate dehydrogenase activation, after an initial increase at 30 degrees C, stromal oxidation in the WT and rwt46 plants did not increase further as Pn decreased. These results provide additional insight into the role of Rubisco activation and activase in the reversible heat inhibition of Pn.     

The effects of Rubisco activase on C4 photosynthesis and metabolism at high temperature

The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. This enzyme facilitates the release of sugar phosphate inhibitors from Rubisco catalytic sites thereby influencing carbamylation. T(1) progeny of transgenic Flaveria bidentis (a C(4) dicot) containing genetically reduced levels of Rubisco activase were used to explore the role of the enzyme in C(4) photosynthesis at high temperature. A range of T(1) progeny was screened at 25 degrees C and 40 degrees C for Rubisco activase content, photosynthetic rate, Rubisco carbamylation, and photosynthetic metabolite pools. The small isoform of F. bidentis activase was expressed and purified from E. coli and used to quantify leaf activase content. In wild-type F. bidentis, the activase monomer content was 10.6+/-0.8 micromol m(-2) (447+/-36 mg m(-2)) compared to a Rubisco site content of 14.2+/-0.8 micromol m(-2). CO(2) assimilation rates and Rubisco carbamylation declined at both 25 degrees C and 40 degrees C when the Rubisco activase content dropped below 3 mumol m(-2) (125 mg m(-2)), with the status of Rubisco carbamylation at an activase content greater than this threshold value being 44+/-5% at 40 degrees C compared to 81+/-2% at 25 degrees C. When the CO(2) assimilation rate was reduced, ribulose-1,5-bisphosphate and aspartate pools increased whereas 3-phosphoglycerate and phosphoenol pyruvate levels decreased, demonstrating an interconnectivity of the C(3) and C(4) metabolites pools. It is concluded that during short-term treatment at 40 degrees C, Rubisco activase content is not the only factor modulating Rubisco carbamylation during C(4) photosynthesis.     

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.     

Inhibition of photosynthesis by heat stress: the activation state of Rubisco as a limiting factor in photosynthesis

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. 30–42°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.     

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.     

The temperature response of CO2 assimilation, photochemical activities and Rubisco activation in Camelina sativa, a potential bioenergy crop with limited capacity for acclimation to heat stress

The temperature optimum of photosynthesis coincides with the average daytime temperature in a species’ native environment. Moderate heat stress occurs when temperatures exceed the optimum, inhibiting photosynthesis and decreasing productivity. In the present study, the temperature response of photosynthesis and the potential for heat acclimation was evaluated for Camelina sativa, a bioenergy crop. The temperature optimum of net CO₂ assimilation rate (A) under atmospheric conditions was 30–32?°C and was only slightly higher under non-photorespiratory conditions. The activation state of Rubisco was closely correlated with A at supra-optimal temperatures, exhibiting a parallel decrease with increasing leaf temperature. At both control and elevated temperatures, the modeled response of A to intercellular CO₂ concentration was consistent with Rubisco limiting A at ambient CO₂. Rubisco activation and photochemical activities were affected by moderate heat stress at lower temperatures in camelina than in the warm-adapted species cotton and tobacco. Growth under conditions that imposed a daily interval of moderate heat stress caused a 63?% reduction in camelina seed yield. Levels of cpn60 protein were elevated under the higher growth temperature, but acclimation of photosynthesis was minimal. Inactivation of Rubisco in camelina at temperatures above 35?°C was consistent with the temperature response of Rubisco activase activity and indicated that Rubisco activase was a prime target of inhibition by moderate heat stress in camelina. That photosynthesis exhibited no acclimation to moderate heat stress will likely impact the development of camelina and other cool season Brassicaceae as sources of bioenergy in a warmer world.     

Activase region on chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase. Nonconservative substitution in the large subunit alters species specificity of protein interaction

In the active form of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC ), a carbamate at lysine 201 binds Mg(2+), which then interacts with the carboxylation transition state. Rubisco activase facilitates this spontaneous carbamylation/metal-binding process by removing phosphorylated inhibitors from the Rubisco active site. Activase from Solanaceae plants (e.g. tobacco) fails to activate Rubisco from non-Solanaceae plants (e.g. spinach and Chlamydomonas reinhardtii), and non-Solanaceae activase fails to activate Solanaceae Rubisco. Directed mutagenesis and chloroplast transformation previously showed that a proline 89 to arginine substitution on the surface of the large subunit of Chlamydomonas Rubisco switched its specificity from non-Solanaceae to Solanaceae activase activation. To define the size and function of this putative activase binding region, substitutions were created at positions flanking residue 89. As in the past, these substitutions changed the identities of Chlamydomonas residues to those of tobacco. Whereas an aspartate 86 to arginine substitution had little effect, aspartate 94 to lysine Rubisco was only partially activated by spinach activase but now fully activated by tobacco activase. In an attempt to eliminate the activase/Rubisco interaction, proline 89 was changed to alanine, which is not present in either non-Solanaceae or Solanaceae Rubisco. This substitution also caused reversal of activase specificity, indicating that amino acid identity alone does not determine the specificity of the interaction.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> <Emphasis Type="Italic">thaliana</Emphasis> expressing a thermostable chimeric Rubisco activase exhibits enhanced growth and higher rates of photosynthesis at moderately high temperatures

Temperature is one of the most important factors controlling growth, development, and reproduction in plants. The rate of photosynthesis declines at moderately high temperatures in plants and particularly in temperate species like Arabidopsis thaliana. This can be attributed to a reduced ability of Rubisco activase to achieve optimum activation of Rubisco, leading to reduced Rubisco activity. In order to overcome this problem, we transformed the Arabidopsis rca mutant with a more thermostable, chimeric activase where a Rubisco recognition domain in the more thermostable tobacco activase was replaced with that from Arabidopsis. Transgenic lines expressing this activase showed higher rates of photosynthesis than the wild type after a short exposure to higher temperatures and they also recovered better, when they were returned to the normal temperature. Moreover, under extended exposure to moderately elevated temperature, the transgenic lines had higher biomass and seed yield when compared with the wild type plants.     

Rubisco activase is a key regulator of non-steady-state photosynthesis at any leaf temperature and, to a lesser extent, of steady-state photosynthesis at high temperature

The role of Rubisco activase in steady-state and non-steady-state photosynthesis was analyzed in wild-type (Oryza sativa) and transgenic rice that expressed different amounts of Rubisco activase. Below 25°C, the Rubisco activation state and steady-state photosynthesis were only affected when Rubisco activase was reduced by more than 70%. However, at 40°C, smaller reductions in Rubisco activase content were linked to a reduced Rubisco activation state and steady-state photosynthesis. As a result, overexpression of maize Rubisco activase in rice did not lead to an increase of the Rubisco activation state, nor to an increase in photosynthetic rate below 25°C, but had a small stimulatory effect at 40°C. On the other hand, the rate at which photosynthesis approached the steady state following an increase in light intensity was rapid in Rubisco activase-overexpressing plants, intermediate in the wild-type, and slowest in antisense plants at any leaf temperature. In Rubisco activase-overexpressing plants, Rubisco activation state at low light was maintained at higher levels than in the wild-type. Thus, rapid regulation by Rubisco activase following an increase in light intensity and/or maintenance of a high Rubisco activation state at low light would result in a rapid increase in Rubisco activation state and photosynthetic rate following an increase in light intensity. It is concluded that Rubisco activase plays an important role in the regulation of non-steady-state photosynthesis at any leaf temperature and, to a lesser extent, of steady-state photosynthesis at high temperature.     

Regulation of Rubisco activation in antisense plants of tobacco containing reduced levels of Rubisco activase

Following an increase in photon flux density (PFD), ribulose bisphosphate carboxylase/oxygenase (Rubisco) undergoes a slow activation which substantially limits the rate of photosynthesis. This activation process is mediated in part by Rubisco activase. Antisense DNA plants of tobacco were used to quantify the degree to which activase limits Rubisco activation. Reductions in leaf activase content caused proportional reductions in the rate of Rubisco activation following a PFD increase from 110 to 1200 micromol m(-2) sec(-1). This was the case for activase levels up to and slightly beyond normal wild-type activase levels. Activase therefore has a flux control coefficient of unity with respect to the Rubisco activation flux. Such a high control coefficient has rarely been measured for any metabolic system, and this is the highest control coefficient measured for an important photosynthetic flux. In contrast, the rate of Rubisco inactivation in leaves following a drop in PFD of 1200 to 110 micromol m(-2) sec(-1) was unchanged by a 60% reduction in activase levels. Despite the high degree of control that activase exerts over the rate of activation, and thus non-steady-state photosynthesis, it was shown that steady-state photosynthesis was largely unaffected by activase concentration until it was reduced below approximately 15% of the wild-type level. The significance of these results and their implications for published models of Rubisco activation are discussed.     

Inhibition and Acclimation of Photosynthesis to Heat Stress Is Closely Correlated with Activation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

Increasing the leaf temperature of intact cotton (Gossypium hirsutum L.) and wheat (Triticum aestivum L.) plants caused a progressive decline in the light-saturated CO₂-exchange rate (CER). CER was more sensitive to increased leaf temperature in wheat than in cotton, and both species demonstrated photosynthetic acclimation when leaf temperature was increased gradually. Inhibition of CER was not a consequence of stomatal closure, as indicated by a positive relationship between leaf temperature and transpiration. The activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which is regulated by Rubisco activase, was closely correlated with temperature-induced changes in CER. Nonphotochemical chlorophyll fluorescence quenching increased with leaf temperature in a manner consistent with inhibited CER and Rubisco activation. Both nonphotochemical fluorescence quenching and Rubisco activation were more sensitive to heat stress than the maximum quantum yield of photochemistry of photosystem II. Heat stress led to decreased 3-phosphoglyceric acid content and increased ribulose-1,5-bisphosphate content, which is indicative of inhibited metabolite flow through Rubisco. We conclude that heat stress inhibited CER primarily by decreasing the activation state of Rubisco via inhibition of Rubisco activase. Although Rubisco activation was more closely correlated with CER than the maximum quantum yield of photochemistry of photosystem II, both processes could be acclimated to heat stress by gradually increasing the leaf temperature.