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Michael E Salvucci - One of the best experts on this subject based on the ideXlab platform.
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Activation of interspecies-hybrid RuBisCO enzymes to assess different models for the RuBisCO-RuBisCO activase interaction.
Photosynthesis research, 2013Co-Authors: Rebekka M Wachter, Michael E Salvucci, A Elizabete Carmo-silva, Csengele Barta, Todor Genkov, Robert J SpreitzerAbstract:Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is prone to inactivation from non-productive binding of sugar-phosphates. Reactivation of RuBisCO requires conformational remodeling by a specific chaperone, RuBisCO activase. RuBisCO activase from tobacco and other plants in the family Solanaceae is an inefficient activator of RuBisCO from non-Solanaceae plants and from the green alga Chlamydomonas reinhardtii. To determine if the RuBisCO small subunit plays a role in the interaction with RuBisCO activase, a hybrid RuBisCO (SSNT) composed of tobacco small subunits and Chlamydomonas large subunits was constructed. The SSNT hybrid, like other hybrid RuBisCOs containing plant small subunits, supported photoautotrophic growth in Chlamydomonas, but growth in air was much slower than for cells containing wild-type RuBisCO. The kinetic properties of the SSNT hybrid RuBisCO were similar to the wild-type enzyme, indicating that the poor growth in air was probably caused by disruption of pyrenoid formation and the consequent impairment of the CO2concentrating mechanism. Recombinant RuBisCO activase from Arabidopsis activated the SSNT hybrid RuBisCO and hybrid RuBisCOs containing spinach and Arabidopsis small subunits at rates similar to the rates with wild-type RuBisCO. However, none of the hybrid RuBisCOs was activated by tobacco RuBisCO activase. That replacement of Chlamydomonas small subunits with plant small subunits does not affect the species-specific interaction between RuBisCO and RuBisCO activase suggests that the association is not dominated by the small subunits that surround the RuBisCO central solvent channel. Therefore, the geometry of a side-on binding mode is more consistent with the data than a top-on or ring-stacking binding mode.
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RuBisCO activity and regulation as targets for crop improvement
Journal of experimental botany, 2012Co-Authors: Martin A. J. Parry, Michael E Salvucci, A Elizabete Carmo-silva, P. John Andralojc, Joanna C. Scales, Hernan Alonso, Spencer M. WhitneyAbstract: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 CO2 and NH3 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 CO2 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.
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RuBisCO activase activity assays.
Methods in molecular biology (Clifton N.J.), 2010Co-Authors: Csengele Barta, A Elizabete Carmo-silva, Michael E SalvucciAbstract:Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO) activase functions as a mechano-chemical motor protein using the energy from ATP hydrolysis to contort the structure of its target protein, RuBisCO. This action modulates the activation state of RuBisCO by removing tightly-bound inhibitory sugar-phosphates from RuBisCO's catalytic sites, thereby restoring the sites to catalytic competence. This chapter reports methods developed for assaying the two activities of RuBisCO activase: ATP hydrolysis and RuBisCO activation.
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Two Residues of RuBisCO Activase Involved in Recognition of the RuBisCO Substrate
The Journal of biological chemistry, 2005Co-Authors: Michael E Salvucci, Archie R. PortisAbstract:Abstract 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.
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RuBisCO activase constrains the photosynthetic potential of leaves at high temperature and co2
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Steven J Craftsbrandner, Michael E SalvucciAbstract: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 carboxylase/oxygenase (RuBisCO) activation in cotton and tobacco leaves and compared the responses to those of the isolated enzymes. Depending on the CO2 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 CO2 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 CO2. The ability of activase to maintain or promote RuBisCO activation in vitro also decreased with temperature but was not affected by CO2. Increasing the activase/RuBisCO 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 CO2 was not caused by a faster rate of deactivation, but by reduced activase activity possibly in response to unfavorable ATP/ADP ratios. When adjustments were made for changes in activation state, the kinetic properties of RuBisCO predicted the response of Pn at high temperature and CO2.
Amane Makino - One of the best experts on this subject based on the ideXlab platform.
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Overexpression of both RuBisCO and RuBisCO activase rescues rice photosynthesis and biomass under heat stress.
Plant cell & environment, 2021Co-Authors: Kazuma Sakoda, Ichiro Terashima, Hiroshi Fukayama, Amane Makino, Yuji Suzuki, Eri Kondo, Wataru YamoriAbstract:Global warming threatens food security by decreasing crop yields through damage to photosynthetic systems, especially RuBisCO activation. We examined whether co-overexpression of RuBisCO and RuBisCO activase improves the photosynthetic and growth performance of rice under high temperatures. We grew three rice lines-the wild-type (WT), a RuBisCO activase-overexpressing line (oxRCA), and a RuBisCO- and RuBisCO activase-co-overexpressing line (oxRCA-RBCS)-and analyzed photosynthesis and biomass at 25 and 40°C. Compared with the WT, the RuBisCO activase content was 153% higher in oxRCA and 138% higher in oxRCA-RBCS, and the RuBisCO content was 27% lower in oxRCA and similar in oxRCA-RBCS. The CO2 assimilation rate (A) of WT was lower at 40°C than at 25°C, attributable to RuBisCO deactivation by heat. On the other hand, that of oxRCA and oxRCA-RBCS was maintained at 40°C, resulting in higher A than WT. Notably, the dry weight of oxRCA-RBCS was 26% higher than that of WT at 40°C. These results show that increasing the RuBisCO activase content without the reduction of RuBisCO content could improve yield and sustainability in rice at high temperature. This article is protected by copyright. All rights reserved.
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Co-overproducing RuBisCO and RuBisCO activase enhances photosynthesis in the optimal temperature range in rice.
Plant physiology, 2021Co-Authors: Mao Suganami, Wataru Yamori, Yuji Suzuki, Youshi Tazoe, Amane MakinoAbstract:RuBisCO limits C3 photosynthesis under some conditions and is therefore a potential target for improving photosynthetic efficiency. The overproduction of RuBisCO is often accompanied by a decline in RuBisCO activation, and the protein ratio of RuBisCO activase (RCA) to RuBisCO (RCA/RuBisCO) greatly decreases in RuBisCO-overproducing plants (RBCS-ox). Here, we produced transgenic rice (Oryza sativa) plants co-overproducing both RuBisCO and RCA (RBCS-RCA-ox). RuBisCO content in RBCS-RCA-ox plants increased by 23%-44%, and RCA/RuBisCO levels were similar or higher than those of wild-type plants. However, although the activation state of RuBisCO in RBCS-RCA-ox plants was enhanced, the rates of CO2 assimilation at 25°C in RBCS-RCA-ox plants did not differ from that of wild-type plants. Alternatively, at a moderately high temperature (optimal range of 32°C-36°C), the rates of CO2 assimilation in RBCS-ox and RBCS-RCA-ox plants were higher than in wild-type plants under conditions equal to or lower than current atmospheric CO2 levels. The activation state of RuBisCO in RBCS-RCA-ox remained higher than that of RBCS-ox plants, and activated RuBisCO content in RCA overproducing, RBCS-ox, RBCS-RCA-ox, and wild-type plants was highly correlated with the initial slope of CO2 assimilation against intercellular CO2 pressures (A:Ci) at 36°C. Thus, a simultaneous increase in RuBisCO and RCA contents leads to enhanced photosynthesis within the optimal temperature range.
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Effects of Overproduction of RuBisCO Activase on RuBisCO Content in Transgenic Rice Grown at Different N Levels.
International journal of molecular sciences, 2020Co-Authors: Mao Suganami, Yuji Suzuki, Eri Kondo, Shinji Nishida, So Konno, Amane MakinoAbstract:It has been reported that overproduction of RuBisCO activase (RCA) in rice (Oryza sativa L.) decreased RuBisCO content, resulting in declining photosynthesis. We examined the effects of RCA levels on RuBisCO content using transgenic rice with overexpressed or suppressed RCA under the control of different promoters of the RCA and RuBisCO small subunit (RBCS) genes. All plants were grown hydroponically with different N concentrations (0.5, 2.0 and 8.0 mM-N). In RCA overproduced plants with > 2-fold RCA content (RCA-HI lines), a 10%-20% decrease in RuBisCO content was observed at 0.5 and 2.0 mM-N. In contrast, at 8.0 mM-N, RuBisCO content did not change in RCA-HI lines. Conversely, in plants with 50%-60% increased RCA content (RCA-MI lines), RuBisCO levels remained unchanged, regardless of N concentration. Such effects on RuBisCO content were independent of the promoter that was used. In plants with RCA suppression to < 10% of the wild-type RCA content, RuBisCO levels were increased at 0.5 mM-N, but were unchanged at 2.0 and 8.0 mM-N. Thus, the effects of the changes in RCA levels on RuBisCO content depended on N supply. Moreover, RCA overproduction was feasible without a decrease in RuBisCO content, depending on the degree of RCA production.
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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
Plant Journal, 2012Co-Authors: Wataru Yamori, Chisato Masumoto, Hiroshi Fukayama, Amane MakinoAbstract: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.
Wataru Yamori - One of the best experts on this subject based on the ideXlab platform.
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Overexpression of both RuBisCO and RuBisCO activase rescues rice photosynthesis and biomass under heat stress.
Plant cell & environment, 2021Co-Authors: Kazuma Sakoda, Ichiro Terashima, Hiroshi Fukayama, Amane Makino, Yuji Suzuki, Eri Kondo, Wataru YamoriAbstract:Global warming threatens food security by decreasing crop yields through damage to photosynthetic systems, especially RuBisCO activation. We examined whether co-overexpression of RuBisCO and RuBisCO activase improves the photosynthetic and growth performance of rice under high temperatures. We grew three rice lines-the wild-type (WT), a RuBisCO activase-overexpressing line (oxRCA), and a RuBisCO- and RuBisCO activase-co-overexpressing line (oxRCA-RBCS)-and analyzed photosynthesis and biomass at 25 and 40°C. Compared with the WT, the RuBisCO activase content was 153% higher in oxRCA and 138% higher in oxRCA-RBCS, and the RuBisCO content was 27% lower in oxRCA and similar in oxRCA-RBCS. The CO2 assimilation rate (A) of WT was lower at 40°C than at 25°C, attributable to RuBisCO deactivation by heat. On the other hand, that of oxRCA and oxRCA-RBCS was maintained at 40°C, resulting in higher A than WT. Notably, the dry weight of oxRCA-RBCS was 26% higher than that of WT at 40°C. These results show that increasing the RuBisCO activase content without the reduction of RuBisCO content could improve yield and sustainability in rice at high temperature. This article is protected by copyright. All rights reserved.
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Co-overproducing RuBisCO and RuBisCO activase enhances photosynthesis in the optimal temperature range in rice.
Plant physiology, 2021Co-Authors: Mao Suganami, Wataru Yamori, Yuji Suzuki, Youshi Tazoe, Amane MakinoAbstract:RuBisCO limits C3 photosynthesis under some conditions and is therefore a potential target for improving photosynthetic efficiency. The overproduction of RuBisCO is often accompanied by a decline in RuBisCO activation, and the protein ratio of RuBisCO activase (RCA) to RuBisCO (RCA/RuBisCO) greatly decreases in RuBisCO-overproducing plants (RBCS-ox). Here, we produced transgenic rice (Oryza sativa) plants co-overproducing both RuBisCO and RCA (RBCS-RCA-ox). RuBisCO content in RBCS-RCA-ox plants increased by 23%-44%, and RCA/RuBisCO levels were similar or higher than those of wild-type plants. However, although the activation state of RuBisCO in RBCS-RCA-ox plants was enhanced, the rates of CO2 assimilation at 25°C in RBCS-RCA-ox plants did not differ from that of wild-type plants. Alternatively, at a moderately high temperature (optimal range of 32°C-36°C), the rates of CO2 assimilation in RBCS-ox and RBCS-RCA-ox plants were higher than in wild-type plants under conditions equal to or lower than current atmospheric CO2 levels. The activation state of RuBisCO in RBCS-RCA-ox remained higher than that of RBCS-ox plants, and activated RuBisCO content in RCA overproducing, RBCS-ox, RBCS-RCA-ox, and wild-type plants was highly correlated with the initial slope of CO2 assimilation against intercellular CO2 pressures (A:Ci) at 36°C. Thus, a simultaneous increase in RuBisCO and RCA contents leads to enhanced photosynthesis within the optimal temperature range.
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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
Plant Journal, 2012Co-Authors: Wataru Yamori, Chisato Masumoto, Hiroshi Fukayama, Amane MakinoAbstract: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.
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Effect of RuBisCO activase deficiency on the temperature response of CO2 assimilation rate and RuBisCO activation state: insights from transgenic tobacco with reduced amounts of RuBisCO activase.
Plant physiology, 2009Co-Authors: Wataru Yamori, Susanne Von CaemmererAbstract:The activation of RuBisCO in vivo requires the presence of the regulatory protein RuBisCO activase. To elucidate its role in maintaining CO(2) assimilation rate at high temperature, we examined the temperature response of CO(2) assimilation rate at 380 microL L(-1) CO(2) concentration (A(380)) and RuBisCO activation state in wild-type and transgenic tobacco (Nicotiana tabacum) with reduced RuBisCO activase content grown at either 20 degrees C or 30 degrees C. Analyses of gas exchange and chlorophyll fluorescence showed that in the wild type, A(380) was limited by ribulose 1,5-bisphosphate regeneration at lower temperatures, whereas at higher temperatures, A(380) was limited by ribulose 1,5-bisphosphate carboxylation irrespective of growth temperatures. Growth temperature induced modest differences in RuBisCO activation state that declined with measuring temperature, from mean values of 76% at 15 degrees C to 63% at 40 degrees C in wild-type plants. At measuring temperatures of 25 degrees C and below, an 80% reduction in RuBisCO activase content was required before RuBisCO activation state was decreased. Above 35 degrees C, RuBisCO activation state decreased slightly with more modest decreases in RuBisCO activase content, but the extent of the reductions in RuBisCO activation state were small, such that a 55% reduction in RuBisCO activase content did not alter the temperature sensitivity of RuBisCO activation and had no effect on in vivo catalytic turnover rates of RuBisCO. There was a strong correlation between RuBisCO activase content and RuBisCO activation state once RuBisCO activase content was less that 20% of wild type at all measuring temperatures. We conclude that reduction in RuBisCO activase content does not lead to an increase in the temperature sensitivity of RuBisCO activation state in tobacco.
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Effects of RuBisCO kinetics and RuBisCO activation state on the temperature dependence of the photosynthetic rate in spinach leaves from contrasting growth temperatures
Plant cell & environment, 2006Co-Authors: Wataru Yamori, Kensaku Suzuki, Ko Noguchi, Masato Nakai, Ichiro TerashimaAbstract:Recently, several studies reported that the optimum temperature for the initial slope [IS(Ci)] of the light-saturated photosynthetic rate (A) versus intercellular CO2 concentration (Ci) curve changed, depending on the growth temperature. However, few studies compare IS(Ci) with ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) properties. Here, we assessed RuBisCO activation state and in vitro RuBisCO kinetics, the main determinants of IS(Ci), in spinach leaves grown at 30/25 [high temperature (HT)] and 15/10 °C [low temperature (LT)]. We measured RuBisCO activation state and A at a CO2 concentration of 360 µL L−1 (A360) at various temperatures. In both HT and LT leaves, the RuBisCO activation state decreased with increasing temperatures above the optimum temperatures for A360, while the activation state remained high at lower temperatures. To compare RuBisCO characteristics, temperature dependences of the maximum rate of ribulose 1,5-bisphosphate (RuBP) carboxylation (Vcmax), specificity factor (Sc/o) and thermal stability were examined. We also examined Vcmax and thermal stability in the leaves that were transferred from HT to LT conditions and were subsequently kept under LT conditions for 2 weeks (HL). RuBisCO purified from HT, LT and HL leaves are called HT, LT and HL RuBisCO, respectively. Thermal stabilities of LT and HL RuBisCO were similar and lower than that of HT RuBisCO. Both Vcmax and Sc/o in LT RuBisCO were higher than those of HT RuBisCO at low temperatures, while these were lower at high temperatures. Vcmax in HL RuBisCO were similar to those of LT RuBisCO at low temperatures, and to those of HT RuBisCO at high temperatures. The predicted photosynthetic rates, taking account of the RuBisCO kinetics and the RuBisCO activation state, agreed well with A360 in both HT and LT leaves. This study suggests that photosynthetic performance is largely determined by the RuBisCO kinetics at low temperature and by RuBisCO Kinetics and the RuBisCO activation state at high temperature.
Hiroshi Fukayama - One of the best experts on this subject based on the ideXlab platform.
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Overexpression of both RuBisCO and RuBisCO activase rescues rice photosynthesis and biomass under heat stress.
Plant cell & environment, 2021Co-Authors: Kazuma Sakoda, Ichiro Terashima, Hiroshi Fukayama, Amane Makino, Yuji Suzuki, Eri Kondo, Wataru YamoriAbstract:Global warming threatens food security by decreasing crop yields through damage to photosynthetic systems, especially RuBisCO activation. We examined whether co-overexpression of RuBisCO and RuBisCO activase improves the photosynthetic and growth performance of rice under high temperatures. We grew three rice lines-the wild-type (WT), a RuBisCO activase-overexpressing line (oxRCA), and a RuBisCO- and RuBisCO activase-co-overexpressing line (oxRCA-RBCS)-and analyzed photosynthesis and biomass at 25 and 40°C. Compared with the WT, the RuBisCO activase content was 153% higher in oxRCA and 138% higher in oxRCA-RBCS, and the RuBisCO content was 27% lower in oxRCA and similar in oxRCA-RBCS. The CO2 assimilation rate (A) of WT was lower at 40°C than at 25°C, attributable to RuBisCO deactivation by heat. On the other hand, that of oxRCA and oxRCA-RBCS was maintained at 40°C, resulting in higher A than WT. Notably, the dry weight of oxRCA-RBCS was 26% higher than that of WT at 40°C. These results show that increasing the RuBisCO activase content without the reduction of RuBisCO content could improve yield and sustainability in rice at high temperature. This article is protected by copyright. All rights reserved.
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Expression level of RuBisCO activase negatively correlates with RuBisCO content in transgenic rice.
Photosynthesis research, 2018Co-Authors: Hiroshi Fukayama, Chiaki Ueguchi, Tomoko Hatanaka, Akina Mizumoto, Jun Katsunuma, Ryutaro Morita, Daisuke Sasayama, Tetsushi AzumaAbstract:The relationship between ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and RuBisCO activase (Rca) levels was studied using transgenic rice overexpressing maize Rca (OX-mRca) and knockdown transgenic rice expressing antisense Rca (KD-Rca). The ratio of RuBisCO to total soluble protein was lower in OX-mRca, whereas it was higher in KD-Rca than in WT, indicating that Rca expression was negatively correlated with RuBisCO content. The expressions of other Calvin-Benson-Bassham cycle enzymes such as sedoheptulose-1,7-bisphosphatase and phosphoribulokinase analyzed by immunoblotting did not show such a negative correlation with Rca, suggesting that the effect of Rca on protein expression may be specific for RuBisCO. Although RuBisCO content was decreased in OX-mRca, the transcript levels of the RuBisCO large subunit (OsRbcL) and the RuBisCO small subunit mostly increased in OX-mRca as well as in KD-Rca. Additionally, polysome loading of OsRbcL was slightly higher in OX-mRca than it was in WT, suggesting that the OsRbcL translation activity was likely stimulated by overexpression of Rca. 35S-methionine labeling experiments demonstrated that there was no significant difference in the stability of newly synthesized RuBisCO among genotypes. However, 35S-methionine-labeled RuBisCO was marginally decreased in OX-mRca and increased in KD-Rca compared to the WT. These results suggest that Rca negatively affects the RuBisCO content, possibly in the synthesis step.
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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
Plant Journal, 2012Co-Authors: Wataru Yamori, Chisato Masumoto, Hiroshi Fukayama, Amane MakinoAbstract: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.
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functional incorporation of sorghum small subunit increases the catalytic turnover rate of RuBisCO in transgenic rice
Plant Physiology, 2011Co-Authors: Chie Ishikawa, Tomoko Hatanaka, Shuji Misoo, Chikahiro Miyake, Hiroshi FukayamaAbstract:RuBisCO limits photosynthetic CO2 fixation because of its low catalytic turnover rate (kcat) and competing oxygenase reaction. Previous attempts to improve the catalytic efficiency of RuBisCO by genetic engineering have gained little progress. Here we demonstrate that the introduction of the small subunit (RbcS) of high kcat RuBisCO from the C4 plant sorghum (Sorghum bicolor) significantly enhances kcat of RuBisCO in transgenic rice (Oryza sativa). Three independent transgenic lines expressed sorghum RbcS at a high level, accounting for 30%, 44%, and 79% of the total RbcS. RuBisCO was likely present as a chimera of sorghum and rice RbcS, and showed 1.32- to 1.50-fold higher kcat than in nontransgenic rice. RuBisCO from transgenic lines showed a higher Km for CO2 and slightly lower specificity for CO2 than nontransgenic controls. These results suggest that RuBisCO in rice transformed with sorghum RbcS partially acquires the catalytic properties of sorghum RuBisCO. RuBisCO content in transgenic lines was significantly increased over wild-type levels but RuBisCO activation was slightly decreased. The expression of sorghum RbcS did not affect CO2 assimilation rates under a range of CO2 partial pressures. The Jmax/Vcmax ratio was significantly lower in transgenic line compared to the nontransgenic plants. These observations suggest that the capacity of electron transport is not sufficient to support the increased RuBisCO capacity in transgenic rice. Although the photosynthetic rate was not enhanced, the strategy presented here opens the way to engineering RuBisCO for improvement of photosynthesis and productivity in the future.
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SDS-dependent proteases induced by ABA and its relation to RuBisCO and RuBisCO activase contents in rice leaves.
Plant physiology and biochemistry : PPB, 2010Co-Authors: Hiroshi Fukayama, Rie Abe, Naotsugu UchidaAbstract:Protease activities and its relation to the contents of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and RuBisCO activase were investigated in detached leaves of rice (Oryza sativa L.) floated on the solutions containing abscisic acid (ABA) or benzyladenine (BA). RuBisCO and RuBisCO activase contents were decreased during the time course and the decreases were enhanced by ABA and suppressed by BA. The decrease in RuBisCO activase was faster than that in RuBisCO. SDS-dependent protease activities at 50-70 kDa (rice SDS-dependent protease: RSP) analyzed by the gelatin containing PAGE were significantly enhanced by ABA. RSPs were also increased in attached leaves during senescence. RSPs had the pH optimum of 5.5, suggesting that RSPs are vacuolar protease. Both decrease in RuBisCO and RuBisCO activase contents and increase in RSPs activities were suppressed by cycloheximide. These findings indicate that the activities of RSPs are well correlated with the decrease in these protein contents. Immunoblotting analysis showed that RuBisCO in the leaf extracts was completely degraded by 5h at pH 5.5 with SDS where it was optimal condition for RSPs. However, the degradation of RuBisCO did not proceed at pH 7.5 without SDS where it is near physiological condition for stromal proteins. RuBisCO activase was degraded at similar rate under both conditions. These results suggest that RSPs can functions in a senescence related degradation system of chloroplast protein in rice leaves. RuBisCO activase would be more susceptible to proteolysis than RuBisCO under physiological condition and this could affect the contents of these proteins in leaves.
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Phylogenetic and evolutionary relationships of RuBisCO and the RuBisCO-like proteins and the functional lessons provided by diverse molecular forms.
Philosophical transactions of the Royal Society of London. Series B Biological sciences, 2008Co-Authors: F. Robert Tabita, Thomas E. Hanson, Sriram Satagopan, Brian Witte, Nathanial KreelAbstract:Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO) catalyses the key reaction by which inorganic carbon may be assimilated into organic carbon. Phylogenetic analyses indicate that there are three classes of bona fide RuBisCO proteins, forms I, II and III, which all catalyse the same reactions. In addition, there exists another form of RuBisCO, form IV, which does not catalyse RuBP carboxylation or oxygenation. Form IV is actually a homologue of RuBisCO and is called the RuBisCO-like protein (RLP). Both RuBisCO and RLP appear to have evolved from an ancestor protein in a methanogenic archaeon, and comprehensive analyses indicate that the different forms (I, II, III and IV) contain various subgroups, with individual sequences derived from representatives of all three kingdoms of life. The diversity of RuBisCO molecules, many of which function in distinct milieus, has provided convenient model systems to study the ways in which the active site of this protein has evolved to accommodate necessary molecular adaptations. Such studies have proven useful to help provide a framework for understanding the molecular basis for many important aspects of RuBisCO catalysis, including the elucidation of factors or functional groups that impinge on RuBisCO carbon dioxide/oxygen substrate discrimination.
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Function, Structure, and Evolution of the RuBisCO-Like Proteins and Their RuBisCO Homologs
Microbiology and molecular biology reviews : MMBR, 2007Co-Authors: F. Robert Tabita, Thomas E. Hanson, Sriram Satagopan, Jaya Singh, Sum ChanAbstract:Summary: About 30 years have now passed since it was discovered that microbes synthesize RuBisCO molecules that differ from the typical plant paradigm. RuBisCOs of forms I, II, and III catalyze CO2 fixation reactions, albeit for potentially different physiological purposes, while the RuBisCO-like protein (RLP) (form IV RuBisCO) has evolved, thus far at least, to catalyze reactions that are important for sulfur metabolism. RuBisCO is the major global CO2 fixation catalyst, and RLP is a somewhat related protein, exemplified by the fact that some of the latter proteins, along with RuBisCO, catalyze similar enolization reactions as a part of their respective catalytic mechanisms. RLP in some organisms catalyzes a key reaction of a methionine salvage pathway, while in green sulfur bacteria, RLP plays a role in oxidative thiosulfate metabolism. In many organisms, the function of RLP is unknown. Indeed, there now appear to be at least six different clades of RLP molecules found in nature. Consideration of the many RuBisCO (forms I, II, and III) and RLP (form IV) sequences in the database has subsequently led to a coherent picture of how these proteins may have evolved, with a form III RuBisCO arising from the Methanomicrobia as the most likely ultimate source of all RuBisCO and RLP lineages. In addition, structure-function analyses of RLP and RuBisCO have provided information as to how the active sites of these proteins have evolved for their specific functions.
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a ribulose 1 5 bisphosphate carboxylase oxygenase RuBisCO like protein from chlorobium tepidum that is involved with sulfur metabolism and the response to oxidative stress
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Thomas E. Hanson, F R TabitaAbstract:A gene encoding a product with substantial similarity to ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was identified in the preliminary genome sequence of the green sulfur bacterium Chlorobium tepidum. A highly similar gene was subsequently isolated and sequenced from Chlorobium limicola f.sp. thiosulfatophilum strain Tassajara. Analysis of these amino acid sequences indicated that they lacked several conserved RuBisCO active site residues. The Chlorobium RuBisCO-like proteins are most closely related to deduced sequences in Bacillus subtilis and Archaeoglobus fulgidus, which also lack some typical RuBisCO active site residues. When the C. tepidum gene encoding the RuBisCO-like protein was disrupted, the resulting mutant strain displayed a pleiotropic phenotype with defects in photopigment content, photoautotrophic growth and carbon fixation rates, and sulfur metabolism. Most important, the mutant strain showed substantially enhanced accumulation of two oxidative stress proteins. These results indicated that the C. tepidum RuBisCO-like protein might be involved in oxidative stress responses and/or sulfur metabolism. This protein might be an evolutional link to bona fide RuBisCO and could serve as an important tool to analyze how the RuBisCO active site developed.
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A ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)-like protein from Chlorobium tepidum that is involved with sulfur metabolism and the response to oxidative stress
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Thomas E. HansonAbstract:A gene encoding a product with substantial similarity to ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was identified in the preliminary genome sequence of the green sulfur bacterium Chlorobium tepidum. A highly similar gene was subsequently isolated and sequenced from Chlorobium limicola f.sp. thiosulfatophilum strain Tassajara. Analysis of these amino acid sequences indicated that they lacked several conserved RuBisCO active site residues. The Chlorobium RuBisCO-like proteins are most closely related to deduced sequences in Bacillus subtilis and Archaeoglobus fulgidus, which also lack some typical RuBisCO active site residues. When the C. tepidum gene encoding the RuBisCO-like protein was disrupted, the resulting mutant strain displayed a pleiotropic phenotype with defects in photopigment content, photoautotrophic growth and carbon fixation rates, and sulfur metabolism. Most important, the mutant strain showed substantially enhanced accumulation of two oxidative stress proteins. These results indicated that the C. tepidum RuBisCO-like protein might be involved in oxidative stress responses and/or sulfur metabolism. This protein might be an evolutional link to bona fide RuBisCO and could serve as an important tool to analyze how the RuBisCO active site developed.
