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.     

CsRCA超表达对高温胁迫下黄瓜幼苗光合作用的影响

以CsRCA超表达黄瓜株系T1-7、T1-2和野生型‘08-1’为试材,在三叶一心时用光照培养箱模拟高温环境[40 ℃,光量子通量密度(PFD) 600 μmol·m^-2·s^-1],研究了CsRCA超表达对高温胁迫下黄瓜幼苗光合作用的调控机理.结果表明: CsRCA超表达可显著提高转基因黄瓜幼苗核酮糖-1,5-二磷酸羧化/加氧酶(Rubisco)大、小亚基的mRNA 表达量,Rubisco和Rubisco活化酶(RCA)活性亦显著高于野生型植株.高温胁迫2 h后,超表达和野生型黄瓜幼苗的光合速率(P_n)、以吸收光能为基础的光化学性能指数(PI_ABS)、Rubisco活性和RCA活性及其mRNA表达量均显著降低.经JIP-test分析发现,高温胁迫导致叶绿素荧光快速诱导动力学曲线中K点明显上升, 而捕获的激子将电子传递到电子传递链中Q_A下游的其他电子受体的概率(Ψ_o)和用于电子传递的产额(φ_E0)均显著下降,说明PSⅡ放氧复合体(OEC)和Q_A之后的电子传递链在高温下受到抑制,但是超表达植株的变化幅度要小于野生型植株.可见CsRCA超表达可以通过提高Rubisco、RCA和PSⅡ活性,缓解高温对黄瓜幼苗光合作用的影响,增强其对高温的适应性.     

Photosynthetic characteristics and tolerance to photo-oxidation of transgenic rice expressing C4 photosynthesis enzymes

The photosynthetic characteristics of four transgenic rice lines over-expressing rice NADP-malic enzyme (ME), and maize phosphoenolpyruvate carboxylase (PC), pyruvate,orthophosphate dikinase (PK), and PC+PK (CK) were investigated using outdoor-grown plants. Relative to untransformed wild-type (WT) rice, PC transgenic rice exhibited high PC activity (25-fold increase) and enhanced activity of carbonic anhydrase (more than two-fold increase), while the activity of ribulose-bisphosphate carboxylase/oxygenase (Rubisco) and its kinetic property were not significantly altered. The PC transgenic plants also showed a higher light intensity for saturation of photosynthesis, higher photosynthetic CO₂ uptake rate and carboxylation efficiency, and slightly reduced CO₂ compensation point. In addition, chlorophyll a fluorescence analysis indicates that PC transgenic plants are more tolerant to photo-oxidative stress, due to a higher capacity to quench excess light energy via photochemical and non-photochemical means. Furthermore, PC and CK transgenic rice produced 22–24% more grains than WT plants. Taken together, these results suggest that expression of maize C₄ photosynthesis enzymes in rice, a C₃ plant, can improve its photosynthetic capacity with enhanced tolerance to photo-oxidation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Responses of Rubisco and Rubisco activase in cucumber seedlings to low temperature and weak light

Using 'Jinyou 3' cucumber seedlings as test materials, this paper studied their photosynthetic rate (P(n)), Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase (RCA) activities, and gene expression of Rubisco and RCA under optimal temperature and weak light (WL: 25 degrees C/18 degrees C, 100 micromol x m(-2) x s(-1)), suboptimal temperature and weak light (ST+WL: 18 degrees C/12 degrees C, 100 micromol x m(-2) x s(-1)), and low temperature and weak light (LT+WL: 10 degress C/5 degrees C, 100 micromol x m(-2) x s(-1)). Comparing with the control (25 degrees C/18 degrees C, 400 micromol x m(-2) x s(-1)), treatments WL, ST+WL, and LT+WL all led to a remarkable decrease in leaf area and dry matter mass. At initial stage, the P(n), Rubisco activity, rbcL and rbcS expression, RCA activity, and CsRCA expression in the three treatments declined by a big margin; 5-7 days later, these parameters tended to be less changed in treatment WL, ascended slowly in treatment ST+WL, and decreased continuously in treatment LT+WL. These results suggested that the photosynthetic apparatus of test cucumber seedlings could gradually adapt to weak light or suboptimal temperature and weak light. The Rubisco and RCA activities and the gene expression of Rubisco and RCA showed the similar responses to low temperature and weak light as the P(n), suggesting that the decline in Rubisco and RCA activities and gene expression in cucumber seedlings under low temperature and weak light could be the important reason leading to the decrease of P(n).     

Overexpression of transketolase gene promotes chilling tolerance by increasing the activities of photosynthetic enzymes,alleviating oxidative damage and stabilizing cell structure in Cucumis sativus L.

Despite being a key enzyme of Cavin cycle, transketolase (TK) is believed to be related to abiotic resistance in higher plants. However, how TK affects chilling tolerance still remains largely unknown. Here, we describe the effect of overexpression of the Cucumis sativa TK gene (CsTK) on growth, photosynthesis, ROS metabolism and cell ultrastructure under chilling stress. Low temperature led to a decrease of the photosynthetic rate (Pn), the stomatal conductance (Gs), the actual photochemical efficiency (ΦPSII) and the sucrose content, whereas there was an increase of the intercellular CO₂ concentration (Ci) and MDA content. These changes were alleviated in the CsTK plants after 5 days of chilling stress, however, inhibition of CsTK showed the opposite results. Furthermore, transgenic plants with overexpression of CsTK showed higher increase in leaf area and dry matter, higher activity of the enzymes and higher increase in the contents of metabolism substance involved in Calvin cycle and reactive oxygen scavenging system as well as lower ^?OH and H₂O₂ content, superoxide anion production rate compared with the control cucumber plants under chilling stress. At the end of the chilling stress, compared to wild‐type (WT) which exhibited dramatically destroyed cell ultrastructure, expanded chloroplast, broken cell and chloroplast membranes as well as the disappeared grana lamella, the CsTK sense plants showed a more complete cell ultrastructure, whereas, the damage of the cell ultrastructure was aggravated in CsTK antisense plants. Taken together, these results imply that CsTK promoted chilling tolerance in cucumber plants mainly through increasing the capacity to assimilate carbon, alleviating oxidative damage and stabilizing cell structure.     

Increased Rubisco content in maize mitigates chilling stress and speeds recovery

Many C₄ plants, including maize, perform poorly under chilling conditions. This phenomenon has been linked in part to decreased Rubisco abundance at lower temperatures. An exception to this is chilling‐tolerant Miscanthus, which is able to maintain Rubisco protein content under such conditions. The goal of this study was to investigate whether increasing Rubisco content in maize could improve performance during or following chilling stress. Here, we demonstrate that transgenic lines overexpressing Rubisco large and small subunits and the Rubisco assembly factor RAF1 (RAF1‐LSSS), which have increased Rubisco content and growth under control conditions, maintain increased Rubisco content and growth during chilling stress. RAF1‐LSSS plants exhibited 12% higher CO₂ assimilation relative to nontransgenic controls under control growth conditions, and a 17% differential after 2 weeks of chilling stress, although assimilation rates of all genotypes were ~50% lower in chilling conditions. Chlorophyll fluorescence measurements showed RAF1‐LSSS and WT plants had similar rates of photochemical quenching during chilling, suggesting Rubisco may not be the primary limiting factor that leads to poor performance in maize under chilling conditions. In contrast, RAF1‐LSSS had improved photochemical quenching before and after chilling stress, suggesting that increased Rubisco may help plants recover faster from chilling conditions. Relatively increased leaf area, dry weight and plant height observed before chilling in RAF1‐LSSS were also maintained during chilling. Together, these results demonstrate that an increase in Rubisco content allows maize plants to better cope with chilling stress and also improves their subsequent recovery, yet additional modifications are required to engineer chilling tolerance in maize.     

Constitutive accumulation of zeaxanthin in tomato alleviates salt stress-induced photoinhibition and photooxidation

Zeaxanthin (Z) has a role in the dissipation of excess excitation energy by participating in non‐photochemical quenching (NPQ) and is essential in protecting the chloroplast from photooxidative damage. To investigate the physiological effects and functional mechanism of constitutive accumulation of Z in the tomato at salt stress‐induced photoinhibition and photooxidation, antisense‐mediated suppression of zeaxanthin epoxidase transgenic plants and the wild‐type (WT) tomato were used. The ratio of Z/(V + A + Z) and (Z + 0.5A)/(V + A + Z) in antisense transgenic plants were maintained at a higher level than in WT plants under salt stress, but the value of NPQ in WT and transgenic plants was not significantly different under salt stress. However, the maximal photochemical efficiency of PSII (Fv/Fm) and the net photosynthetic rate (Pn) in transgenic plants decreased more slowly under salt stress. Furthermore, transgenic plants showed lower level of hydrogen peroxide (H₂O₂), superoxide anion radical (O₂^??) and ion leakage, lower malondialdehyde content. Compared with WT, the content of D1 protein decreased slightly in transgenic plants under salt stress. Our results suggested that the constitutive accumulation of Z in transgenic tomatoes can alleviate salt stress‐induced photoinhibition because of the antioxidant role of Z in the scavenging quenching of singlet oxygen and/or free radicals in the lipid phase of the membrane.     

Antisense-mediated suppression of tomato zeaxanthin epoxidase alleviates photoinhibition of PSII and PSI during chilling stress under low irradiance

A tomato (Lycopersicon esculentum Mill.) zeaxanthin epoxidase gene (LeZE) was isolated and antisense transgenic tomato plants were produced. Northern, southern, and western blot analyses demonstrated that antisense LeZE was transferred into the tomato genome and the expression of LeZE was inhibited. The ratio of (A+Z)/(V+A+Z) in antisense transgenic plants was maintained at a higher level than in the wild type (WT) plants under high light and chilling stress with low irradiance. The value of non-photochemical quenching (NPQ) in WT and transgenic plants was not affected during the stresses. The oxidizable P700 and the maximal photochemical efficiency of PSII (F_v/F_m) in transgenic plants decreased more slowly at chilling temperature under low irradiance. These results suggested that suppression of LeZE caused zeaxanthin accumulation, which was helpful in alleviating photoinhibition of PSI and PSII in tomato plants under chilling stress.     

黄瓜幼苗光合作用对高温胁迫的响应与适应

以‘津优35号’黄瓜幼苗为试材,研究高温(HT: 42 ℃/32 ℃)和亚高温(SHT: 35 ℃/25 ℃)胁迫对黄瓜幼苗光合作用及生长量的影响.结果表明: 高温、亚高温明显抑制幼苗生长.随着胁迫时间的延长,黄瓜幼苗叶片的光合速率(P_n)逐渐降低,胞间CO₂浓度(C_i)趋于升高,气孔导度(g_s)、蒸腾速率(T_r)、光呼吸速率(P_r)和暗呼吸速率(D_r)先上升后下降,高温、亚高温引起P_n降低的主要原因是非气孔限制.高温、亚高温可使黄瓜幼苗叶片的暗下光系统Ⅱ最大光化学效率(F_v/F_m)、光下实际光化学效率(Φ_PSII)、光化学猝灭系数(q_P)和电子传递效率(ETR)显著降低,初始荧光(F_o)和非化学猝灭系数(NPQ)逐渐升高.随着胁迫时间的延长,HT处理的RuBP羧化酶(RuBPCase)和Rubisco活化酶(RCA)活性及其mRNA表达量逐渐降低,而SHT处理的胁迫初期变化不大,3 d后趋于降低;HT和SHT处理的景天庚酮糖-1,7-二磷酸酶(SBPase)和果糖-1,6-二磷酸醛缩酶(FBA)活性与mRNA表达均呈先升高后降低趋势.可见,适宜光强下短时亚高温处理黄瓜幼苗不会产生明显光抑制,高温胁迫会对其PSⅡ反应中心造成严重损伤;光合酶受高温胁迫诱导,但其诱导效应与温度升高幅度和高温持续时间有关.     

Co-overproducing Rubisco and Rubisco activase enhances photosynthesis in the optimal temperature range in rice

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.

A simultaneous increase in Rubisco and RCA contents in transgenic rice leads to an enhancement of photosynthesis at moderately high temperatures within the optimal temperature range.     

Overexpression of chloroplastic monodehydroascorbate reductase enhanced tolerance to temperature and methyl viologen‐mediated oxidative stresses

A tomato (Lycopersicon esculentum Mill.) monodehydroascorbate reductase gene (LeMDAR) was isolated. The LeMDAR–green fluorescence protein (GFP) fusion protein was targeted to chloroplast in Arabidopsis mesophyll protoplast. RNA and protein gel blot analyses confirmed that the sense‐ and antisense‐ LeMDAR were integrated into the tomato genome. The MDAR activities and the levels of reduced ascorbate (AsA) were markedly increased in sense transgenic lines and decreased in antisense transgenic lines compared with wild‐type (WT) plants. Under low and high temperature stresses, the sense transgenic plants showed lower level of hydrogen peroxide (H₂O₂), lower thiobarbituric acid reactive substance (TBARS) content, higher net photosynthetic rate (P_n), higher maximal photochemical efficiency of PSII (F_v/F_m) and fresh weight compared with WT plants. The oxidizable P700 decreased more obviously in WT and antisense plants than that in sense plants at chilling temperature under low irradiance. Furthermore, the sense transgenic plants exhibited significantly lower H₂O₂ level, higher ascorbate peroxidase (APX) activity, greater P_n and F_v/F_m under methyl viologen (MV)‐mediated oxidative stresses. These results indicated that overexpression of chloroplastic MDAR played an important role in alleviating photoinhibition of PSI and PSII and enhancing the tolerance to various abiotic stresses by elevating AsA level.     

Brassinosteroids promote photosynthesis and growth by enhancing activation of Rubisco and expression of photosynthetic genes in <Emphasis Type="Italic">Cucumis sativus</Emphasis>

Brassinosteroids (BRs) are a new group of plant growth substances that promote plant growth and productivity. We showed in this study that improved growth of cucumber (Cucumis sativus) plants after treatment with 24-epibrassinolide (EBR), an active BR, was associated with increased CO₂ assimilation and quantum yield of PSII (Φ_PSII). Treatment of brassinazole (Brz), a specific inhibitor for BR biosynthesis, reduced plant growth and at the same time decreased CO₂ assimilation and Φ_PSII. Thus, the growth-promoting activity of BRs can be, at least partly, attributed to enhanced plant photosynthesis. To understand how BRs enhance photosynthesis, we have analyzed the effects of EBR and Brz on a number of photosynthetic parameters and their affecting factors, including the contents and activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Northern and Western blotting demonstrated that EBR upregulated, while Brz downregulated, the expressions of rbcL, rbcS and other photosynthetic genes. In addition, EBR had a positive effect on the activation of Rubisco based on increased maximum Rubisco carboxylation rates (V _c,max), total Rubisco activity and, to a greater extent, initial Rubisco activity. The accumulation patterns of Rubisco activase (RCA) based on immunogold-labeling experiments suggested a role of RCA in BR-regulated activation state of Rubisco. Enhanced expression of genes encoding other Calvin cycle genes after EBR treatment may also play a positive role in RuBP regeneration (J _max), thereby increasing maximum carboxylation rate of Rubisco (V _c,max). Thus, BRs promote photosynthesis and growth by positively regulating synthesis and activation of a variety of photosynthetic enzymes including Rubisco in cucumber.     

An Excel tool for deriving key photosynthetic parameters from combined gas exchange and chlorophyll fluorescence: theory and practice

Combined photosynthetic gas exchange and modulated fluorometres are widely used to evaluate physiological characteristics associated with phenotypic and genotypic variation, whether in response to genetic manipulation or resource limitation in natural vegetation or crops. After describing relatively simple experimental procedures, we present the theoretical background to the derivation of photosynthetic parameters, and provide a freely available Excel‐based fitting tool (EFT) that will be of use to specialists and non‐specialists alike. We use data acquired in concurrent variable fluorescence–gas exchange experiments, where A/C_i and light–response curves have been measured under ambient and low oxygen. From these data, the EFT derives light respiration, initial PSII (photosystem II) photochemical yield, initial quantum yield for CO₂ fixation, fraction of incident light harvested by PSII, initial quantum yield for electron transport, electron transport rate, rate of photorespiration, stomatal limitation, Rubisco (ribulose 1·5‐bisphosphate carboxylase/oxygenase) rate of carboxylation and oxygenation, Rubisco specificity factor, mesophyll conductance to CO₂ diffusion, light and CO₂ compensation point, Rubisco apparent Michaelis–Menten constant, and Rubisco CO₂‐saturated carboxylation rate. As an example, a complete analysis of gas exchange data on tobacco plants is provided. We also discuss potential measurement problems and pitfalls, and suggest how such empirical data could subsequently be used to parameterize predictive photosynthetic models.     

Study on salt tolerance with YHem1 transgenic canola (Brassica napus)

5‐Aminolevulinic acid (5‐ALA) has been suggested for improving plant salt tolerance via exogenous application. In this study, we used a transgenic canola (Brassica napus), which contained a constituted gene YHem1 and biosynthesized more 5‐ALA, to study salt stress responses. In a long‐term pot experiment, the transgenic plants produced higher yield under 200 mmol L^?1 NaCl treatment than the wild type (WT). In a short‐term experiment, the YHem1 transformation accelerated endogenous 5‐ALA metabolism, leading to more chlorophyll accumulation, higher diurnal photosynthetic rates and upregulated expression of the gene encoding Rubisco small subunit. Furthermore, the activities of antioxidant enzymes, including superoxide dismutase, guaiacol peroxidase, catalase and ascorbate peroxidase, were significantly higher in the transgenic plants than the WT, while the levels of O₂·^? and malondialdehyde were lower than the latter. Additionally, the Na⁺ content was higher in the transgenic leaves than that in the WT under salinity, but K⁺ and Cl^? were significantly lower. The levels of N, P, Cu, and S in the transgenic plants were also significantly lower than those in the WT, but the Fe content was significantly improved. As the leaf Fe content was decreased by salinity, it was suggested that the stronger salt tolerance of the transgenic plants was related to the higher Fe acquisition. Lastly, YHem1 transformation improved the leaf proline content, but salinity decreased rather than increased it. The content of free amino acids and soluble sugars was similarly decreased as salinity increased, but it was higher in the transgenic plants than that in the WT.     

Transgenic tomato plants overexpressing chloroplastic monodehydroascorbate reductase are resistant to salt- and PEG-induced osmotic stress

RNA gel hybridization showed that the expression of monodehydroascorbate reductase (MDHAR) in the wild type (WT) tomato was decreased firstly and then increased under salt- and polyethylene glycol (PEG)-induced osmotic stress, and the maximum level was observed after treatment for 12 h. WT, sense transgenic and antisense transgenic tomato plants were used to analyze the antioxidative ability to cope with osmotic stresses. After salt stress, the fresh mass (FM) and height of sense transgenic lines were greater than those of antisense lines and WT plants. Under salt and PEG treatments, sense transgenic plants showed a lower level of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), a higher net photosynthetic rate (P _N), and the maximal photochemical efficiency of PSII (F_v/F_m) compared with WT and antisense transgenic plants. Moreover, sense lines maintained higher ascorbate peroxidase (APX) activity than WT and antisense plants under salt- and PEG-induced osmotic stress. These results indicate that chloroplastic MDHAR plays an important role in alleviating photoinhibition of PSII by elevating ascorbate (AsA) level under salt- and PEG-induced osmotic stress.     

Low-temperature photosynthetic performance of a C4 grass and a co-occurring C3 grass native to high latitudes

The photosynthetic performance of C₄ plants is generally inferior to that of C₃ species at low temperatures, but the reasons for this are unclear. The present study investigated the hypothesis that the capacity of Rubisco, which largely reflects Rubisco content, limits C₄ photosynthesis at suboptimal temperatures. Photosynthetic gas exchange, chlorophyll a fluorescence, and the in vitro activity of Rubisco between 5 and 35 °C were measured to examine the nature of the low‐temperature photosynthetic performance of the co‐occurring high latitude grasses, Muhlenbergia glomerata (C₄) and Calamogrostis canadensis (C₃). Plants were grown under cool (14/10 °C) and warm (26/22 °C) temperature regimes to examine whether acclimation to cool temperature alters patterns of photosynthetic limitation. Low‐temperature acclimation reduced photosynthetic rates in both species. The catalytic site concentration of Rubisco was approximately 5.0 and 20 µmol m^?2 in M. glomerata and C. canadensis, respectively, regardless of growth temperature. In both species, in vivo electron transport rates below the thermal optimum exceeded what was necessary to support photosynthesis. In warm‐grown C. canadensis, the photosynthesis rate below 15 °C was unaffected by a 90% reduction in O₂ content, indicating photosynthetic capacity was limited by the capacity of P_i‐regeneration. By contrast, the rate of photosynthesis in C. canadensis plants grown at the cooler temperatures was stimulated 20–30% by O₂ reduction, indicating the P_i‐regeneration limitation was removed during low‐temperature acclimation. In M. glomerata, in vitro Rubisco activity and gross CO₂ assimilation rate were equivalent below 25 °C, indicating that the capacity of the enzyme is a major rate limiting step during C₄ photosynthesis at cool temperatures.     

Phosphorylation pattern of Rubisco activase in Arabidopsis leaves

Rubisco activase (RCA) is an ancillary photosynthetic protein essential for Rubisco activity. Some data suggest that post‐translational modifications (such as reduction of disulphide bridges) are involved in the regulation of RCA activity. However, despite the key role of protein phosphorylation in general metabolic regulation, RCA phosphorylation has not been well characterised. We took advantage of phosphoproteomics and gas exchange analyses with instant sampling adapted to Arabidopsis rosettes to examine the occurrence and variations of phosphopeptides associated with RCA in different photosynthetic contexts (CO₂ mole fraction, light and dark). We detected two phosphopeptides from RCA corresponding to residues Thr 78 and Ser 172, and show that the former is considerably more phosphorylated in the dark than in the light, while the latter show no light/dark pattern. The CO₂ mole fraction did not influence phosphorylation of either residue. Phosphorylation thus appears to be a potential mechanism associated with RCA dark inactivation, when Rubisco‐catalysed carboxylation is arrested. Since Thr 78 and Ser 172 are located in the N and Walker domains of the protein, respectively, the involvement of phosphorylation in protein–protein interaction and catalysis is likely.     

The relationship between CO2 assimilation, photosynthetic electron transport and water–water cycle in chill-exposed cucumber leaves under low light and subsequent recovery

The effects of chilling under low light (9/7 °C, 100 µmol m^?2 s^?1) on the photosynthetic and antioxidant capacities and subsequent recovery were examined in two (one tolerant and one sensitive) cucumber genotypes. Chilling resulted in an irreversible inhibition of net CO₂ assimilation and growth for the sensitive genotype, which was accompanied by decreases in the maximum velocity of RuBP carboxylation by Rubisco (V_cmax), the capacity for ribulose‐1,5‐bisphosphate regeneration (J_max), Rubisco content and activity, and the quantum efficiency of photosystem II, in the absence of any stomatal limitation of CO₂ supply or inorganic phosphate limitation. In contrast, CO₂ assimilation for the tolerant genotype fully recovered after chill. The chill‐induced decrease in the proportion of electron flux for photosynthetic carbon reduction was mostly compensated by an O₂‐dependent alternative electron flux driven by the water–water cycle, especially in the sensitive genotype. Compared with the tolerant genotype, the sensitive genotype after chill showed reduced capacity for scavenging reactive oxygen species and increased accumulation of reactive oxygen species. The balance between O₂‐dependent alternative electron flux and the capacity for scavenging reactive oxygen species in response to chill plays a major role in determining the tolerance of cucumber leaves to this stress factor. It is concluded that the water–water cycle operates at high rates when CO₂ assimilation is restricted in cucumber leaves subjected to chill and low light conditions.     

Temperature response of photosynthesis in transgenic rice transformed with 'sense' or 'antisense' rbcS

The responses of chlorophyll fluorescence, gas exchange rate and Rubisco activation state to temperature were examined in transgenic rice plants with 130 and 35% of the wild-type (WT) Rubisco content by transformation with rbcS cDNA in sense and antisense orientations, respectively. Although the optimal temperatures of PSII quantum efficiency and CO(2) assimilation were found to be between 25 and 32 degrees C, the maximal activation state of Rubisco was found to be between 16 and 20 degrees C in all genotypes. The Rubisco flux control coefficient was also the highest between 16 and 20 degrees C in the WT and antisense lines [>0.88 at an intercellular CO(2) pressure (Ci) of 28 Pa]. Gross photosynthesis at Ci = 28 Pa per Rubisco content in the WT between 12 and 20 degrees C was close to that of the antisense lines where high Rubisco control is present. Thus, Rubisco activity most strongly limited photosynthesis at cool temperatures. These results indicated that a selective enhancement of Rubisco content can enhance photosynthesis at cool temperatures, but in the sense line with enhanced Rubisco content Pi regeneration limitation occurred. Above 20 degrees C, the Rubisco flux control coefficient declined. This decline was associated with a decline in Rubisco activation. The activation state of Rubisco measured at each temperature decreased with increasing Rubisco content, and the slope of activation to Rubisco content was independent of temperature. We discuss the possibility that the decline in Rubisco activation at intermediate and high temperatures is part of a regulated response to a limitation in other photosynthetic processes.     

Response of xanthophyll cycle and chloroplastic antioxidant enzymes to chilling stress in tomato over-expressing glycerol-3-phosphate acyltransferase gene

Over-expression of chloroplastic glycerol-3-phosphate acyltransferase gene (LeGPAT) increased unsaturated fatty acid contents in phosphatidylglycerol (PG) of thylakoid membrane in tomato. The effect of this increase on the xanthophyll cycle and chloroplast antioxidant enzymes was examined by comparing wild type (WT) tomato with the transgenic (TG) lines at chilling temperature (4 °C) under low irradiance (100 μmol m⁻² s⁻¹). Net photosynthetic rate and the maximal photochemical efficiency of photosystem (PS) 2 (F_v/F_m) in TG plants decreased more slowly during chilling stress and F_v/F_m recovered faster than that in WT plants under optimal conditions. The oxidizable P700 in both WT and TG plants decreased during chilling stress under low irradiance, but recovered faster in TG plants than in the WT ones. During chilling stress, non-photochemical quenching (NPQ) and the de-epoxidized ratio of xanthophyll cycle in WT plants were lower than those of TG tomatoes. The higher activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in TG plants resulted in the reduction of O₂ ^−· and H₂O₂ contents during chilling stress. Hence the increase in content of unsaturated fatty acids in PG by the over-expression of LeGPAT could alleviate photoinhibition of PS2 and PS1 by improving the de-epoxidized ratio of xanthophyll cycle and activities of SOD and APX in chloroplast.