Thylakoid membrane responses to moderately high leaf temperature in Pima cotton

Photosynthesis is inhibited by high temperatures that plants are likely to experience under natural conditions. Both increased thylakoid membrane ionic conductance and ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) deactivation have been suggested as the primary cause. The moderately heat‐tolerant crop Pima S‐6 cotton (Gossypium barbadense) was used to examine heat stress‐induced inhibition of photosynthesis. Previous field‐work indicated that moderate heat stress (T = 35–45 °C) is associated with very rapid leaf temperature changes. Therefore, a system was devised for rapidly heating intact, attached leaves to mimic natural field heat‐stress conditions and monitored Rubisco activation, carbon‐cycle metabolites, thylakoid ionic conductance, and photosystem I activity. As a proxy for NADPH and stromal redox status the activation state of NADP‐malate dehydrogenase (NADP‐MDH) was measured. In dark‐adapted cotton leaves, heating caused an increase in thylakoid permeability at temperatures as low as 36 °C. The increased permeability did not cause a decline in adenosine 5′‐triphosphate (ATP) levels during steady‐state or transient heating. Rapid heating caused a transient decline in ribulose 1,5‐bisphosphate without a decrease in Rubisco activation. Sustained heating caused a decline in Rubisco activation and also oxidized the stroma as judged by NADP‐MDH activation and this is hypothesized to result from increased cyclic photophosphorylation, explaining the maintenance of ATP content in the face of increased thylakoid membrane ion leakiness.     

Electron transport is the functional limitation of photosynthesis in field-grown Pima cotton plants at high temperature

Restrictions to photosynthesis can limit plant growth at high temperature in a variety of ways. In addition to increasing photorespiration, moderately high temperatures (35–42 °C) can cause direct injury to the photosynthetic apparatus. Both carbon metabolism and thylakoid reactions have been suggested as the primary site of injury at these temperatures. In the present study this issue was addressed by first characterizing leaf temperature dynamics in Pima cotton (Gossypium barbadense) grown under irrigation in the US desert south‐west. It was found that cotton leaves repeatedly reached temperatures above 40 °C and could fluctuate as much as 8 or 10 °C in a matter of seconds. Laboratory studies revealed a maximum photosynthetic rate at 30–33 °C that declined by 22% at 45 °C. The majority of the inhibition persisted upon return to 30 °C. The mechanism of this limitation was assessed by measuring the response of photosynthesis to CO₂ in the laboratory. The first time a cotton leaf (grown at 30 °C) was exposed to 45 °C, photosynthetic electron transport was stimulated (at high CO₂) because of an increased flux through the photorespiratory pathway. However, upon cooling back to 30 °C, photosynthetic electron transport was inhibited and fell substantially below the level measured before the heat treatment. In the field, the response of assimilation (A) to various internal levels of CO₂ (C_i) revealed that photosynthesis was limited by ribulose‐1,5‐bisphosphate (RuBP) regeneration at normal levels of CO₂ (presumably because of limitations in thylakoid reactions needed to support RuBP regeneration). There was no evidence of a ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) limitation at air levels of CO₂ and at no point on any of 30 A–C_i curves measured on leaves at temperatures from 28 to 39 °C was RuBP regeneration capacity measured to be in substantial excess of the capacity of Rubisco to use RuBP. It is therefore concluded that photosynthesis in field‐grown Pima cotton leaves is functionally limited by photosynthetic electron transport and RuBP regeneration capacity, not Rubisco activity.     

Photosynthetic performance at low temperature of Bouteloua gracilis Lag., a high-altitude C4 grass from the Rocky Mountains, USA

The mechanisms controlling the photosynthetic performance of C₄ plants at low temperature were investigated using ecotypes of Bouteloua gracilis Lag. from high (3000 m) and low (1500 m) elevation sites in the Rocky Mountains of Colorado. Plants were grown in controlled‐environment cabinets at a photon flux density of 700 μ mol m^?2 s^?1 and day/night temperatures of 26/16 °C or 14/7 °C. The thermal response of the net CO₂ assimilation rate (A) was evaluated using leaf gas‐exchange analysis and activity assays of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco), phosphoenolpyruvate carboxylase (PEPCase) and pyruvate,orthophosphate dikinase (PPDK). In both ecotypes, a reduction in measurement temperature caused the CO₂‐saturated rate of photosynthesis to decline to a greater degree than the initial slope of A versus the intercellular CO₂ response, thereby reducing the photosynthetic CO₂ saturation point. As a consequence, A in normal air was CO₂‐saturated at sub‐optimal temperatures. Ecotypic variation was low when grown at 26/16 °C, with the major difference between the ecotypes being that the low‐elevation plants had higher A; however, the ecotypes responded differently when grown at cool temperature. At temperatures below the thermal optimum, A in high‐elevation plants grown at 14/7 °C was enhanced relative to plants grown at 26/16 °C, while A in low‐elevation plants grown at 14/7 °C was reduced compared to 26/16 °C‐grown plants. Photoinhibition at low growth temperature was minor in both ecotypes as indicated by small reductions in dark‐adapted F_v/F_m. In both ecotypes, the activity of Rubisco was equivalent to A below 17 °C but well in excess of A above 25 °C. Activities of PEPCase and PPDK responded to temperature in a similar proportion relative to Rubisco, and showed no evidence for dissociation that would cause them to become principal limitations at low temperature. Because of the similar temperature response of Rubisco and A, we propose that Rubisco is a major limitation on C₄ photosynthesis in B. gracilis below 17 °C. Based on these results and for theoretical reasons associated with how C₄ plants use Rubisco, we further suggest that Rubisco capacity may be a widespread limitation upon C₄ photosynthesis at low temperature.     

Modulation of Rubisco activity in leaves of <Emphasis Type="Italic">Prosopis juliflora</Emphasis> in response to tropical conditions in north India

The success of P. juliflora, an evergreen woody species has been largely attributed to temperature acclimation and stomatal control of photosynthesis under wide range of environmental conditions prevalent in India. We studied the contribution of the enzyme ribulose-1,5 bisphosphate carboxylase/oxygenase (Rubisco) in diurnal and seasonal photosynthesis changes in P. juliflora. The changes observed in photosynthesis under natural conditions could be effected by the growth temperatures, which ranged from 10–30 °C in winter to 30–47 °C in summer. However, the Total Rubisco activity displayed a constant diurnal pattern and showed a maximum at 1200 in all seasons namely spring, summer, monsoon and winter irrespective of the changes in temperature. The Total Rubisco activity from two cohorts of leaves produced in spring and monsoon appeared to be down-regulated differentially at low PPFD during the evening. The in vivo and in vitro measurements of carboxylation efficiency of Rubisco showed wide variation during the day and were correlated with the photosynthesis rate. The light activation of Rubisco showed the acclimation to moderately high temperatures in different seasons except in summer. The exceptionally high temperatures (>45 °C) in summer, though not affecting Total activity, severely inhibited the light activation of Rubisco and also modulated the recovery process for the activation of Rubisco. Our studies suggest that the modulation of Rubisco driven by Rubisco activase and not Rubisco per se was crucial for the diurnal regulation of photosynthesis. NBRI Publication No.: 528     

A novel method for determination of the 15N isotopic composition of Rubisco in wheat plants exposed to elevated atmospheric carbon dioxide

Although ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) is mostly known as a key enzyme involved in CO₂ assimilation during the Calvin cycle, comparatively little is known about its role as a pool of nitrogen storage in leaves. For this purpose, we developed a protocol to purify Rubisco that enables later analysis of its ¹⁵N isotope composition (δ¹⁵N) at the natural abundance and ¹⁵N‐labeled plants. In order to test the utility of this protocol, durum wheat (Triticum durum var. Sula) exposed to an elevated CO₂ concentration (700 vs 400 µmol mol^?1) was labeled with K¹⁵NO₃ (enriched at 2 atom %) during the ear development period. The developed protocol proves to be selective, simple, cost effective and reproducible. The study reveals that ¹⁵N labeling was different in total organic matter, total soluble protein and the Rubisco fraction. The obtained data suggest that photosynthetic acclimation in wheat is caused by Rubisco depletion. This depletion may be linked to preferential nitrogen remobilization from Rubisco toward grain filling.     

Inhibition of photosynthesis by high temperature in oak (Quercus pubescens L.) leaves grown under natural conditions closely correlates with a reversible heat-dependent reduction of the activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase

Inhibition of the net photosynthetic CO₂ assimilation rate (P_n) by high temperature was examined in oak (Quercus pubescens L.) leaves grown under natural conditions. Combined measurements of gas exchange and chlorophyll (Chl) a fluorescence were employed to differentiate between inhibition originating from heat effects on components of the thylakoid membranes and that resulting from effects on photosynthetic carbon metabolism. Regardless of whether temperature was increased rapidly or gradually, P_n decreased with increasing leaf temperature and was more than 90% reduced at 45 °C as compared to 25 °C. Inhibition of P_n by heat stress did not result from reduced stomatal conductance (g_s), as heat‐induced reduction of g_s was accompanied by an increase of the intercellular CO₂ concentration (C_i). Chl a fluorescence measurements revealed that between 25 and 45 °C heat‐dependent alterations of thylakoid‐associated processes contributed only marginally, if at all, to the inhibition of P_n by heat stress, with photosystem II being remarkably well protected against thermal inactivation. The activation state of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) decreased from about 90% at 25 °C to less than 30% at 45 °C. Heat stress did not affect Rubisco per se, since full activity could be restored by incubation with CO₂ and Mg²⁺. Western‐blot analysis of leaf extracts disclosed the presence of two Rubisco activase polypeptides, but heat stress did not alter the profile of the activase bands. Inhibition of P_n at high leaf temperature could be markedly reduced by artificially increasing C_i. A high C_i also stimulated photosynthetic electron transport and resulted in reduced non‐photochemical fluorescence quenching. Recovery experiments showed that heat‐dependent inhibition of P_n was largely, if not fully, reversible. The present results demonstrate that in Q. pubescens leaves the thylakoid membranes in general and photosynthetic electron transport in particular were well protected against heat‐induced perturbations and that inhibition of P_n by high temperature closely correlated with a reversible heat‐dependent reduction of the Rubisco activation state.     

Inadequacy of Rubisco initial and total activities to account for observed rates of photosynthetic carbon dioxide assimilation by Scenedesmus ecornis

Both initial and total activity of ribulose-1,5, bisphosphate carboxylase/oxygenase (Rubisco) measured for the green alga Scenedesmus ecornis are affected by the experimental procedure and they are not sufficiently high to account for the rates of ¹⁴C fixation by photosynthesis. The very low β-carboxylase activities detected (less than 3% of the Rubisco total activity) cannot explain the difference in CO₂ fixation. Attempts to obtain possible optimal conditions (pH, duration of activation with Mg²⁺ and HCO^- ₃, absence of proteases, linearity of ¹⁴C fixation with time) did not lead to increased activity yields. The substrate ribulose-1,5-bisphosphate was found to decrease the initial activity at concentrations higher than 25 μM for algae harvested by centrifugation and having thus experienced several minutes of darkness. Deactivation seems to be primarily responsible for this loss of activity. Furthermore, initial and total activities decrease when the delay before freezing increases, suggesting accumulation of an inhibitor from the light-dark transition metabolism during the first minutes of harvesting.     

In vivo temperature response functions of parameters required to model RuBP-limited photosynthesis

The leaf model of C₃ photosynthesis of Farquhar, von Caemmerer & Berry (Planta 149, 78–90, 1980) provides the basis for scaling carbon exchange from leaf to canopy and Earth‐System models, and is widely used to project biosphere responses to global change. This scaling requires using the leaf model over a wider temperature range than that for which the model was originally parameterized. The leaf model assumes that photosynthetic CO₂ uptake within a leaf is either limited by the rate of ribulose‐1,5‐bisphosphate (RuBP) regeneration or the activity of RuBP carboxylase‐oxygenase (Rubisco). Previously we reported a re‐parameterization of the temperature responses of Rubisco activity that proved robust when applied to a range of species. Herein this is extended to re‐parameterizing the response of RuBP‐limited photosynthesis to temperature. RuBP‐limited photosynthesis is assumed to depend on the whole chain electron transport rate, which is described as a three‐parameter non‐rectangular hyperbolic function of photon flux. Herein these three parameters are determined from simultaneous measurement of chlorophyll fluorescence and CO₂ exchange of tobacco leaves, at temperatures from 10 to 40 °C. All varied significantly with temperature and were modified further with variation in growth temperature from 15 to 35 °C. These parameters closely predicted the response of RuBP‐limited photosynthesis to temperature measured in both lemon and poplar and showed a significant improvement over predictions based on earlier parameterizations. We provide the necessary equations for use of the model of Farquhar et al. (1980) with our newly derived temperature functions for predicting both Rubisco‐ and RuBP‐limited photosynthesis.     

Decrease in leaf sucrose synthesis leads to increased leaf starch turnover and decreased RuBP regeneration-limited photosynthesis but not Rubisco-limited photosynthesis in Arabidopsis null mutants of SPSA1

We investigated the individual effect of null mutations of each of the four sucrose‐phosphate synthase (SPS) genes in Arabidopsis (SPSA1, SPSA2, SPSB and SPSC) on photosynthesis and carbon partitioning. Null mutants spsa1 and spsc led to decreases in maximum SPS activity in leaves by 80 and 13%, respectively, whereas null mutants spsa2 and spsb had no significant effect. Consistently, isoform‐specific antibodies detected only the SPSA1 and SPSC proteins in leaf extracts. Leaf photosynthesis at ambient [CO₂] was not different among the genotypes but was 20% lower in spsa1 mutants when measured under saturating [CO₂] levels. Carbon partitioning at ambient [CO₂] was altered only in the spsa1 null mutant. Cold treatment of plants (4 °C for 96 h) increased leaf soluble sugars and starch and increased the leaf content of SPSA1 and SPSC proteins twofold to threefold, and of the four null mutants, only spsa1 reduced leaf non‐structural carbohydrate accumulation in response to cold treatment. It is concluded that SPSA1 plays a major role in photosynthetic sucrose synthesis in Arabidopsis leaves, and decreases in leaf SPS activity lead to increased starch synthesis and starch turnover and decreased Ribulose 1,5‐bisphosphate regeneration‐limited photosynthesis but not ribulose 1·5‐bisphosphate carboxylase/oxygenase (Rubisco)‐limited photosynthesis, indicating a limitation of triose‐phosphate utilization (TPU).     

Effects of growth and measurement light intensities on temperature dependence of CO2 assimilation rate in tobacco leaves

Effects of growth light intensity on the temperature dependence of CO₂ assimilation rate were studied in tobacco (Nicotiana tabacum) because growth light intensity alters nitrogen allocation between photosynthetic components. Leaf nitrogen, ribulose 1·5‐bisphosphate carboxylase/oxygenase (Rubisco) and cytochrome f (cyt f) contents increased with increasing growth light intensity, but the cyt f/Rubisco ratio was unaltered. Mesophyll conductance to CO₂ diffusion (g_m) measured with carbon isotope discrimination increased with growth light intensity but not with measuring light intensity. The responses of CO₂ assimilation rate to chloroplast CO₂ concentration (C_c) at different light intensities and temperatures were used to estimate the maximum carboxylation rate of Rubisco (V_cmax) and the chloroplast electron transport rate (J). Maximum electron transport rates were linearly related to cyt f content at any given temperature (e.g. 115 and 179 µmol electrons mol^?1 cyt f s^?1 at 25 and 40 °C, respectively). The chloroplast CO₂ concentration (C_trans) at which the transition from RuBP carboxylation to RuBP regeneration limitation occurred increased with leaf temperature and was independent of growth light intensity, consistent with the constant ratio of cyt f/Rubisco. In tobacco, CO₂ assimilation rate at 380 µmol mol^?1 CO₂ concentration and high light was limited by RuBP carboxylation above 32 °C and by RuBP regeneration below 32 °C.     

A reversible decrease in ribulose 1,5‐bisphosphate carboxylase/oxygenase carboxylation activity caused by the aggregation of the enzyme's large subunit is triggered in response to the exposure of moderate irradiance‐grown plants to low irradiance

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) is highly regulated in response to fluctuations in the environment, including changes in irradiance. However, no complex data are available on Rubisco regulatory mechanisms triggered in plants which are submitted to moderate–low irradiance shift. Therefore, we investigated in a comprehensive way the changes at the level of amount of Rubisco protein, its structural organization and carboxylase activity of the holoenzyme as triggered by exposure of moderate irradiance‐grown Arabidopsis thaliana plants to low irradiance conditions. An exposure of moderate irradiance‐grown plants to low irradiance for a single photoperiod caused the exclusion of a certain pool of Rubisco under altered conditions owing to oxidative modifications resulting in the formation of protein aggregates involving Rubisco large subunit (LS). As a result, both initial and total Rubisco carboxylase activities were reduced, whereas Rubisco activation state remained largely unchanged. The results of the determination of reactive oxygen species indicated that a moderate/low irradiance transition had stimulated ¹O₂ accumulation and we strongly suggest that Rubisco oxidative modifications leading to formation of aggregates encompassing Rubisco‐LS were triggered by ¹O₂. When moderate irradiance regime was resumed, the majority of Rubisco‐LS containing aggregates tended to be resolubilized, and this allowed Rubisco carboxylation activities to be largely recovered, without changes in the activation state of the enzyme. In the longer term, these results allow us to better understand a complexity of Rubisco regulatory mechanisms activated in response to abiotic stresses and during recovery from the stresses.     

Light-dependent modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in the genus Phaseolus

Modulation of the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in low light and darkness was measured in A) 25 genotypes from the four cultivated species of Phaseolus (P. vulgaris, P. acutifolius, P. lunatus and P. coccineus), B) 8 non-cultivated Phaseolus species, and C) the related species Macroptileum atropurpureum. The activity ratio of Rubisco (the ratio of initial and total Rubisco activities, which reflects Rubisco carbamylation), and the molar activity of fully-activated Rubisco (which primarily reflects the inhibition of Rubisco activity by carboxyarabinitol 1-phosphate, CA1P) were assayed in leaves from the cultivated species sampled at midday in full sunlight, in low light at dusk (60 to 100 mol photons m^-2s^-1), and after at least 4 h in darkness. Dark inhibition of Rubisco molar activity was compared in both cultivated and non-cultivated species. In all cultivated genotypes, a significant reduction of the activity ratio of Rubisco was measured in leaves sampled at low light; however, the molar activity of fully activated Rubisco was not greatly reduced in these low light samples. In darkened leaves, molar activities substantially declined in most Phaseolus species with 11 of 13 exhibiting greater than 60% reduction. In P. vulgaris, the reduction of molar activity was extensive (greater than 69%) in all genotypes studied, which included wild progenitors as well as ancient and advanced cultivars. These results indicate that at low light late in the day, modulation of Rubisco activity is primarily through changes in carbamylation state, with CA1P playing a more limited role. By contrast in the dark, binding of CA1P dominates the modulation of Rubisco activity in Phaseolus in a pattern that appears to be conserved within a species, but can vary significantly between species within a genus. The degree of CA1P inhibition in Phaseolus was associated with phylogenetic affinities within the genus, as the species with extensive dark-inhibition of Rubisco activity tended to be more closely related to each other than to species with reduced inhibition of Rubisco activity.Abbreviations CA1P carboxyarabinitol 1-phosphate - CABP carboxyarabinitol bisphosphate - PFD photon flux density between 400 and 700 nm - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase     

Effect of N source on concentration of Rubisco in Eucalyptus diversicolor, as measured by capillary electrophoresis

Proteins in plant tissues have been extensively characterised by conventional methods such as liquid chromatography and polyacrylamide gel electrophoresis – methods that are tedious and time‐consuming. Capillary electrophoresis is potentially a more simple and cost‐effective method (with respect to time and consumables) but needs substantial development, especially for native plants which are frequently poor in protein and rich in interfering substances (oils, tannins, phenols). We report here the development of capillary electrophoresis (CE) for the separation of SDS‐protein complexes (by molecular mass) and their quantification in plant tissues. In leaf extracts, two peaks dominated the electropherograms, these peaks had migration times corresponding to the small and large subunits of Rubisco (ribulose‐1,5‐bisphosphate carboxylase/oxygenase; EC 4.1.1.39) and co‐migrated with added purified Rubisco. Linearity of peak area, reproducibility of migration time and peak areas for the small and large subunit were excellent, suggesting Rubisco could be quantified with a high degree of accuracy. We determined how the concentration (0.5 or 4 mM) and form of N applied (nitrate versus ammonium) affects partitioning of N to Rubisco in seedlings of Eucalyptus diversicolor. Analysis of extracts from leaves of Eucalyptus diversicolor was only possible after precipitation of proteins with trichloroacetic acid (TCA). Precipitation with TCA was highly reproducible and recovery of added Rubisco through procedures of extraction, precipitation and analysis were close to 100% for both subunits. An 8‐fold difference in the concentration of N applied did not affect total N, the concentration of Rubisco or the fraction of N present as Rubisco. The similarity of total N may well reflect faster rates of growth in those plants receiving 4 mM N, and a subsequent ‘dilution’ of tissue N. The N source did not affect total N, the concentration of Rubisco or the fraction of N present as Rubisco. Despite similar Rubisco concentrations, the total concentration of soluble proteins was greater in ammonium‐grown plants.     

From chaperonins to Rubisco assembly and metabolic repair

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) mediates the fixation of atmospheric CO₂ in photosynthesis by catalyzing the carboxylation of the 5‐carbon sugar ribulose‐1,5‐bisphosphate (RuBP). Despite its pivotal role, Rubisco is an inefficient enzyme and thus has been a key target for bioengineering. However, efforts to increase crop yields by Rubisco engineering remain unsuccessful, due in part to the complex machinery of molecular chaperones required for Rubisco biogenesis and metabolic repair. While the large subunit of Rubisco generally requires the chaperonin system for folding, the evolution of the hexadecameric Rubisco from its dimeric precursor resulted in the dependence on an array of additional factors required for assembly. Moreover, Rubisco function can be inhibited by a range of sugar‐phosphate ligands. Metabolic repair of Rubisco depends on remodeling by the ATP‐dependent Rubisco activase and hydrolysis of inhibitors by specific phosphatases. This review highlights our work toward understanding the structure and mechanism of these auxiliary machineries.     

PRELIMINARY INVESTIGATIONS INTO THE STRUCTURE AND ACTIVITY OF RIBULOSE BISPHOSPHATE CARBOXYLASE FROM TWO PHOTOSYNTHETIC DINOFLAGELLATES1

Ribulose bisphosphate carboxylase / oxygenase (Rubisco) from the dinoflagellates Symbiodinium sp. Freudenthal and Amphidinium carterae Hulburt rapidly loses activity following cell lysis. Evidence presented indicates that this is not due to proteolysis. Using the tight binding inhibitor [¹⁴C] carboxyarabinitol bisphosphate as a marker, the Rubisco large subunit (LSu) from Symbiodinium sp. was purified. The subunit molecular weight was 56 kDa, while non-denaturing polyacrylamide gel electrophoresis indicated that the purified protein had a molecular weight significantly less than that expected of the intact hexadecameric protein. No trace of the small subunit was apparent. The initial loss of carboxylase activity following cell lysis may be due to instability of the quaternary structure of the enzyme. Antibodies prepared to the purified LSU cross-reacted with LSus from other dinoflagellates but not with the LSus of higher plants, diatoms, and other chromophytic algae. This suggests that the LSu of at least some dinoflagellates is antigenically different from that of other eukaryotes.     

Mutation design of a thermophilic Rubisco based on three‐dimensional structure enhances its activity at ambient temperature

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) plays a central role in carbon dioxide fixation on our planet. Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk‐Rubisco) shows approximately twenty times the activity of spinach Rubisco at high temperature, but only one‐eighth the activity at ambient temperature. We have tried to improve the activity of Tk‐Rubisco at ambient temperature, and have successfully constructed several mutants which showed higher activities than the wild‐type enzyme both in vitro and in vivo. Here, we designed new Tk‐Rubisco mutants based on its three‐dimensional structure and a sequence comparison of thermophilic and mesophilic plant Rubiscos. Four mutations were introduced to generate new mutants based on this strategy, and one of the four mutants, T289D, showed significantly improved activity compared to that of the wild‐type enzyme. The crystal structure of the Tk‐Rubisco T289D mutant suggested that the increase in activity was due to mechanisms distinct from those involved in the improvement in activity of Tk‐Rubisco SP8, a mutant protein previously reported to show the highest activity at ambient temperature. Combining the mutations of T289D and SP8 successfully generated a mutant protein (SP8‐T289D) with the highest activity to date both in vitro and in vivo. The improvement was particularly pronounced for the in vivo activity of SP8‐T289D when introduced into the mesophilic, photosynthetic bacterium Rhodopseudomonas palustris, which resulted in a strain with nearly two‐fold higher specific growth rates compared to that of a strain harboring the wild‐type enzyme at ambient temperature. Proteins 2016; 84:1339–1346. © 2016 Wiley Periodicals, Inc.     

Effect of heat stress on the inhibition and recovery of the ribulose-1,5-bisphosphate carboxylase/oxygenase activation state

Experiments were conducted to determine the relative contributions of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) activation state vis-à-vis Rubisco activase and metabolite levels to the inhibition of cotton (Gossypium hirsutum L.) photosynthesis by heat stress. Exposure of leaf tissue in the light to temperatures of 40 or 45 °C decreased the activation state of Rubisco to levels that were 65 or 10%, respectively, of the 28 °C control. Ribulose-1,5-bisphosphate (RuBP) levels increased in heat-stressed leaves, whereas the 3-phosphoglyceric acid pool was depleted. Heat stress did not affect Rubisco per se, as full activity could be restored by incubation with CO₂ and Mg²⁺. Inhibition and recovery of Rubisco activation state and carbon dioxide exchange rate (CER) were closely related under moderate heat stress (up to 42.5 °C). Moderate heat stress had negligible effect on Fv/Fm, the maximal quantum yield of photosystem II. In contrast, severe heat stress (45 °C) caused significant and irreversible damage to Rubisco activation, CER, and Fv/Fm. The rate of Rubisco activation after alleviating moderate heat stress was comparable to that of controls, indicating rapid reversibility of the process. However, moderate heat stress decreased both the rate and final extent of CER activation during dark-to-light transition. Treatment of cotton leaves with methyl viologen or an oxygen-enriched atmosphere reduced the effect of heat stress on Rubisco inactivation. Both treatments also reduced tissue RuBP levels, indicating that the amount of RuBP present during heat stress may influence the degree of Rubisco inactivation. Under both photorespiratory and non-photorespiratory conditions, the inhibition of the CER during heat stress could be completely reversed by increasing the internal partial pressure of CO₂ (Ci). However, the inhibition of the CER by nigericin, a K⁺ ionophore, was not reversible when the Ci was increased at ambient or high temperature. Our results indicate that inhibition of photosynthesis by moderate heat stress is not caused by inhibition of the capacity for RuBP regeneration. We conclude that heat stress inhibits Rubisco activation via a rapid and direct effect on Rubisco activase, possibly by perturbing Rubisco activase subunit interactions with each other or with Rubisco. Received: 25 February 2000 / Accepted: 13 May 2000     

Two Rubisco activase isoforms may play different roles in photosynthetic heat acclimation in the rice plant

Studies on some plant species have shown that increasing the growth temperature gradually or pretreating with high temperature can lead to obvious photosynthetic acclimation to high temperature. To test whether this acclimation arises from heat adaptation of ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) activation mediated by Rubisco activase (RCA), gene expression of RCA large isoform (RCA_L) and RCA small isoform (RCA_S) in rice was determined using a 4‐day heat stress treatment [40/30°C (day/night)] followed by a 3‐day recovery under control conditions [30/22°C (day/night)]. The heat stress significantly induced the expression of RCA_L as determined by both mRNA and protein levels. Correlative analysis indicated that RCA_S protein content was extremely significantly related to Rubisco initial activity and net photosynthetic rate (Pn) under both heat stress and normal conditions. Immunoblot analysis of the Rubisco–RCA complex revealed that the ratio of RCA_L to Rubisco increased markedly in heat‐acclimated rice leaves. Furthermore, transgenic rice plants expressing enhanced amounts of RCA_L exhibited higher thermotolerance in Pn and Rubisco initial activity and grew better at high temperature than wild‐type (WT) plants and transgenic rice plants expressing enhanced amounts of RCA_S. Under normal conditions, the transgenic rice plants expressing enhanced amounts of RCA_S showed higher Pn and produced more biomass than transgenic rice plants expressing enhanced amounts of RCA_L and wild‐type plants. Together, these suggest that the heat‐induced RCA_L may play an important role in photosynthetic acclimation to moderate heat stress in vivo, while RCA_S plays a major role in maintaining Rubisco initial activity under normal conditions.     

Reversible inhibition of CO2 fixation by ribulose 1,5‐bisphosphate carboxylase/oxygenase through the synergic effect of arsenite and a monothiol

The activity of the photosynthetic carbon‐fixing enzyme, ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco), is partially inhibited by arsenite in the millimolar concentration range. However, micromolar arsenite can fully inhibit Rubisco in the presence of a potentiating monothiol such as cysteine, cysteamine, 2‐mercaptoethanol or N‐acetylcysteine, but not glutathione. Arsenite reacts specifically with the vicinal Cys172‐Cys192 from the large subunit of Rubisco and with the monothiol to establish a ternary complex, which is suggested to be a trithioarsenical. The stability of the complex is strongly dependent on the nature of the monothiol. Enzyme activity is fully recovered through the disassembly of the complex after eliminating arsenite and/or the thiol from the medium. The synergic combination of arsenite and a monothiol acts also in vivo stopping carbon dioxide fixation in illuminated cultures of Chlamydomonas reinhardtii. Again, this effect may be reverted by washing the cells. However, in vivo inhibition does not result from the blocking of Rubisco since mutant strains carrying Rubiscos with Cys172 and/or Cys192 substitutions (which are insensitive to arsenite in vitro) are also arrested. This suggests the existence of a specific sensor controlling carbon fixation that is even more sensitive than Rubisco to the arsenite–thiol synergism.