Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) catalyzes the first step in net photosynthetic CO2 assimilation and photorespiratory carbon oxidation. The enzyme is notoriously inefficient as a catalyst for the carboxylation of RuBP and is subject to competitive inhibition by O2, inactivation by loss of carbamylation, and dead-end inhibition by RuBP. These inadequacies make Rubisco rate limiting for photosynthesis and an obvious target for increasing agricultural productivity. Resolution of X-ray crystal structures and detailed analysis of divergent, mutant, and hybrid enzymes have increased our insight into the structure/function relationships of Rubisco. The interactions and associations relatively far from the Rubisco active site, including regulatory interactions with Rubisco activase, may present new approaches and strategies for understanding and ultimately improving this complex enzyme.

Net photosynthesis (Pn) is inhibited by moderate heat stress. To elucidate the mechanism of inhibition, we examined the effects of temperature on gas exchange and ribulose 1,5-bisphosphate carboxylase͞oxygenase (Rubisco) activation in cotton and tobacco leaves and compared the responses to those of the isolated enzymes. Depending on the CO 2 concentration, Pn decreased when temperatures exceeded 35-40°C. This response was inconsistent with the response predicted from the properties of fully activated Rubisco. Rubisco deactivated in leaves when temperature was increased and also in response to high CO 2 or low O2. The decrease in Rubisco activation occurred when leaf temperatures exceeded 35°C, whereas the activities of isolated activase and Rubisco were highest at 42°C and >50°C, respectively. In the absence of activase, isolated Rubisco deactivated under catalytic conditions and the rate of deactivation increased with temperature but not with CO 2. The ability of activase to maintain or promote Rubisco activation in vitro also decreased with temperature but was not affected by CO 2. Increasing the activase͞Rubisco ratio reduced Rubisco deactivation at higher temperatures. The results indicate that, as temperature increases, the rate of Rubisco deactivation exceeds the capacity of activase to promote activation. The decrease in Rubisco activation that occurred in leaves at high CO 2 was not caused by a faster rate of deactivation, but by reduced activase activity possibly in response to unfavorable ATP͞ADP ratios. When adjustments were made for changes in activation state, the kinetic properties of Rubisco predicted the response of Pn at high temperature and CO 2.C urrent models of global climate predict a gradual increase in the atmospheric concentrations of greenhouse gases over the next century and associated increases in global temperature (1). The resulting effects on net photosynthesis will have significant ecological and agricultural consequences. For temperate C3 plants, the rate of photosynthesis under current atmospheric conditions exhibits a broad temperature optimum, with the maximum rate occurring near the daytime temperature under which the plants are grown (2). The broad temperature optimum for photosynthesis has been attributed to differential changes in the solubility of CO 2 and O 2 with temperature (3, 4) and to specific changes in the kinetic properties of ribulose 1,5-bisphosphate carboxylase͞oxygenase (Rubisco) (4-6). Increasing the CO 2 concentration above ambient generally increases the temperature optimum for photosynthesis by several degrees (2, 4).Models of photosynthesis have been developed to explain the response of photosynthesis to environmental conditions (7,8). These models make a clear distinction between limitations attributable to Rubisco and those attributable to factors that limit the rate of ribulose-1,5-bisphosphate (RuBP) regeneration, but they usually do not include changes in the activation state of Rubisco among the factors that influence the rate of photosynthesis. Th...

Although the catalytic activity of Rubisco increases with temperature, the low affinity of the enzyme for CO(2) 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 degrees C. Inhibition of photosynthesis by moderate heat stress (i.e. 30-42 degrees 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 CO(2) indicate that electron transport activity is not limiting at temperatures that inhibit CO(2) fixation. Instead, recent evidence shows that inhibition of net photosynthesis correlates with a decrease in the activation state of Rubisco in both C(3) and C(4) 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.

Past increases in yield potential of wheat have largely resulted from improvements in harvest index rather than increased biomass. Further large increases in harvest index are unlikely, but an opportunity exists for increasing productive biomass and harvestable grain. Photosynthetic capacity and efficiency are bottlenecks to raising productivity and there is strong evidence that increasing photosynthesis will increase crop yields provided that other constraints do not become limiting. Even small increases in the rate of net photosynthesis can translate into large increases in biomass and hence yield, since carbon assimilation is integrated over the entire growing season and crop canopy. This review discusses the strategies to increase photosynthesis that are being proposed by the wheat yield consortium in order to increase wheat yields. These include: selection for photosynthetic capacity and efficiency, increasing ear photosynthesis, optimizing canopy photosynthesis, introducing chloroplast CO(2) pumps, increasing RuBP regeneration, improving the thermal stability of Rubisco activase, and replacing wheat Rubisco with that from other species with different kinetic properties.

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°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°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°C. The activation state of phosphoenolpyruvate carboxylase decreased marginally at leaf temperatures above 40°C, and the activity of pyruvate phosphate dikinase was insensitive to temperature up to 45°C. The activation state of Rubisco decreased at temperatures exceeding 32.5°C, with nearly complete inactivation at 45°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°C.It has long been recognized that C4 plant species have a higher temperature optimum for photosynthesis than C3 plants due to the operation of a CO 2 -concentrating system that inhibits Rubisco oxygenase activity (Berry and Bjö rkman, 1980;Edwards and Walker, 1983). In C3 plants, inhibition of net photosynthesis (Pn) at moderately high temperatures has usually been ascribed to an increase in the ratio of Rubisco oxygenase:Rubisco carboxylase activities. As temperature increases, the ratio of dissolved O 2 /CO 2 and the specificity of Rubisco for O 2 increase, thus favoring oxygenase activity (Monson et al., 1982; Jordan and Ogren, 1984; Sage and Sharkey, 1987) and resulting in inhibition of Pn. As a consequence, when C3 plants are exposed to high CO 2 and/or low O 2 , i.e. conditions that reduce oxygenase activity, the temperature optimum for Pn is increased (Berry and Bjö rkman, 1980;Edwards and Walker, 1983).For C3 and C4 plants, the temperature range for optimum Pn is broad, and at temperatures above this range, Pn decreases (Edwards and Walker, 1983). Temperature-induced decreases in Pn in C3 species are closely associated with inactivation of Rubisco (Law and Crafts-Brandner, 1999), and when the activation state of Rubisco and g...

Inhibition of net photosynthesis (Pn) by moderate heat stress has been attributed to an inability of Rubisco activase to maintain Rubisco in an active form. To examine this proposal, the temperature response of Pn, Rubisco activation, chlorophyll fluorescence, and the activities of Rubisco and Rubisco activase were examined in species from contrasting environments. The temperature optimum of Rubisco activation was 108C higher in the creosote bush (Larrea tridentata) compared with the Antarctic hairgrass (Deschampsia antarctica), resembling the temperature response of Pn. Pn increased markedly with increasing internal CO 2 concentration in Antarctic hairgrass and creosote bush plants subjected to moderate heat stress even under nonphotorespiratory conditions. Nonphotochemical quenching of chlorophyll fluorescence, the effective quantum yield of photochemical energy conversion (DF/F m 9) and the maximum yield of PSII (F v /F m ) were more sensitive to temperature in Antarctic hairgrass and two other species endemic to cold regions (i.e. Lysipomia pumila and spinach [Spinacea oleracea]) compared with creosote bush and three species (i.e. jojoba [Simmondsia chinensis], tobacco [Nicotiana tabacum], and cotton [Gossypium hirsutum]) from warm regions. The temperature response of activity and the rate of catalytic inactivation of Rubisco from creosote bush and Antarctic hairgrass were similar, whereas the optimum for ATP hydrolysis and Rubisco activation by recombinant creosote bush, cotton, and tobacco activase was 88C to 108C higher than for Antarctic hairgrass and spinach activase. These results support a role for activase in limiting photosynthesis at high temperature.

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.

Rubisco (ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase) enables net carbon fixation through the carboxylation of RuBP. However, some characteristics of Rubisco make it surprisingly inefficient and compromise photosynthetic productivity. For example, Rubisco catalyses a wasteful reaction with oxygen that leads to the release of previously fixed CO(2) and NH(3) and the consumption of energy during photorespiration. Furthermore, Rubisco is slow and large amounts are needed to support adequate photosynthetic rates. Consequently, Rubisco has been studied intensively as a prime target for manipulations to 'supercharge' photosynthesis and improve both productivity and resource use efficiency. The catalytic properties of Rubiscos from diverse sources vary considerably, suggesting that changes in turnover rate, affinity, or specificity for CO(2) can be introduced to improve Rubisco performance in specific crops and environments. While attempts to manipulate plant Rubisco by nuclear transformation have had limited success, modifying its catalysis by targeted changes to its catalytic large subunit via chloroplast transformation have been much more successful. However, this technique is still in need of development for most major food crops including maize, wheat, and rice. Other bioengineering approaches for improving Rubisco performance include improving the activity of its ancillary protein, Rubisco activase, in addition to modulating the synthesis and degradation of Rubisco's inhibitory sugar phosphate ligands. As the rate-limiting step in carbon assimilation, even modest improvements in the overall performance of Rubisco pose a viable pathway for obtaining significant gains in plant yield, particularly under stressful environmental conditions.