小麦Rubisco 活化酶基因的克隆和表达特性

Rubisco活化酶是广泛存在于光合生物中调节Rubisco活性的酶, 我们利用PCR技术, 从小麦(Triticum aestivum)叶片cDNA文库中克隆得到Rubisco活化酶基因cDNA片段, 该片段长度为850 bp, 编码201个氨基酸。Northern blot表明, 小麦叶片在暗诱导衰老的条件下, 叶片中活化酶基因表达水平逐渐下降; 同时, 小麦叶片的光合特性、叶绿素含量和Rubisco活性呈现下降趋势。这些结果表明, 衰老时小麦叶片Rubisco活化酶基因表达水平下降与光合速率下降密切相关。     

小麦Rubisco活化酶基因的克隆和表达特性

Rubisco活化酶是广泛存在于光合生物中调节Rubisco活性的酶,我们利用PCR技术,从小麦(Triticum aestivum)叶片cDNA文库中克隆得到Rubisco活化酶基因cDNA片段,该片段长度为850 bp,编码201个氨基酸.Northern blot表明,小麦叶片在暗诱导衰老的条件下,叶片中活化酶基因表达水平逐渐下降;同时,小麦叶片的光合特性、叶绿素含量和Rubisco活性呈现下降趋势.这些结果表明,衰老时小麦叶片Rubisco活化酶基因表达水平下降与光合速率下降密切相关.     

Regulation of Rubisco activase and its interaction with Rubisco

The large, alpha-isoform of Rubisco activase confers redox regulation of the ATP/ADP response of the ATP hydrolysis and Rubisco activation activities of the multimeric activase holoenzyme complex. The alpha-isoform has a C-terminal extension that contains the redox-sensitive cysteine residues and is characterized by a high content of acidic residues. Cross-linking and site-directed mutagenesis studies of the C-terminal extension that have provided new insights into the mechanism of redox regulation are reviewed. Also reviewed are new details about the interaction between activase and Rubisco and the likely mechanism of 'activation' that resulted from mutagenesis in a 'Sensor 2' domain of activase that AAA(+) proteins often use for substrate recognition. Two activase residues in this domain were identified that are involved in Rubisco recognition. The results directly complement earlier studies that identified critical residues for activase recognition in the large subunit of Rubisco.     

光和糖对水稻Rubisco活化酶基因表达的影响

水稻黄化苗在光照2h内其Rubisco。活化酶的mRNA和蛋白量明显增加,然后维持在相对稳定的水平。光对水稻Rubisco活化酶的基因表达的诱导作用主要在转录水平上。Rubisco活化酶主要在绿叶中表达,这与Rubisco基因表达的器官特异性完全一致。用等渗葡萄糖喂养成熟的水稻叶片1h,促使水稻Rubisco大、小亚基和Rubisco活化酶可翻译mRNA含量下降。同样蔗糖对Rubisco小亚基和Rubisco活化酶的表达也有抑制,其作用弱于葡萄糖。     

Evidence for contribution of autophagy to Rubisco degradation during leaf senescence in Arabidopsis thaliana

During leaf senescence, Rubisco is gradually degraded and its components are recycled within the plant. Although Rubisco can be mobilized to the vacuole by autophagy via specific autophagic bodies, the importance of this process in Rubisco degradation has not been shown directly. Here, we monitored Rubisco autophagy during leaf senescence by fusing synthetic green fluorescent protein (sGFP) or monomeric red fluorescent protein (mRFP) with Rubisco in Arabidopsis (Arabidopsis thaliana). When attached leaves were individually exposed to darkness to promote their senescence, the fluorescence of Rubisco‐sGFP was observed in the vacuolar lumen as well as chloroplasts. In addition, release of free‐sGFP due to the processing of Rubisco‐sGFP was observed in the vacuole of individually darkened leaves. This vacuolar transfer and processing of Rubisco‐sGFP was not observed in autophagy‐deficient atg5 mutants. Unlike sGFP, mRFP was resistant to proteolysis in the leaf vacuole of light‐grown plants. The vacuolar transfer and processing of Rubisco‐mRFP was observed at an early stage of natural leaf senescence and was also obvious in leaves naturally covered by other leaves. These results indicate that autophagy contributes substantially to Rubisco degradation during natural leaf senescence as well as dark‐promoted senescence.     

Structure and functional annotation of hypothetical proteins having putative Rubisco activase function from Vitis vinifera

Rubisco is a very large, complex and one of the most abundant proteins in the world and comprises up to 50% of all soluble protein in plants. The activity of Rubisco, the enzyme that catalyzes CO₂ assimilation in photosynthesis, is regulated by Rubisco activase (Rca). In the present study, we searched for hypothetical protein of Vitis vinifera which has putative Rubisco activase function. The Arabidopsis and tobacco Rubisco activase protein sequences were used as seed sequences to search against Vitis vinifera in UniprotKB database. The selected hypothetical proteins of Vitis vinifera were subjected to sequence, structural and functional annotation. Subcellular localization predictions suggested it to be cytoplasmic protein. Homology modelling was used to define the three-dimensional (3D) structure of selected hypothetical proteins of Vitis vinifera. Template search revealed that all the hypothetical proteins share more than 80% sequence identity with structure of green-type Rubisco activase from tobacco, indicating proteins are evolutionary conserved. The homology modelling was generated using SWISS-MODEL. Several quality assessment and validation parameters computed indicated that homology models are reliable. Further, functional annotation through PFAM, CATH, SUPERFAMILY, CDART suggested that selected hypothetical proteins of Vitis vinifera contain ATPase family associated with various cellular activities (AAA) and belong to the AAA+ super family of ring-shaped P-loop containing nucleoside triphosphate hydrolases. This study will lead to research in the optimization of the functionality of Rubisco which has large implication in the improvement of plant productivity and resource use efficiency.     

Effects of light and regulators on senescence-related changes in soluble proteins in detached oat (Avena sativa L.) leaves

The rate of senescence and the two-dimensional pattern of soluble proteins from detached oat leaves senescing in either darkness or light were analyzed, and compared to those of leaves in which senescence was delayed by application of the cytokinin benzyladenine or enhanced through the action of abscisic acid.Senescence of detached leaves in light did not differ significantly from senescence in attached leaves on intact plants. In darkness, protein was lost at a higher rate than in light, but several individual proteins showed relative increases. Notably, proteins previously characterized as high-molecular-weight proteins and senescence-associated proteins (Klerk et al., 1992) increased. Changes observed during incubation in light or darkness appeared to be related to this condition rather than the rate or progress of senescence. Cytokinins delayed and abscisic acid accelerated the changes in protein pattern compared to water. Beside changes previously identified in leaves senescing on the plant, detached leaves show alterations that reflect their condition of incubation rather than their developmental progress.Abbreviations 2D-PAG two-dimensional polyacrylamide gel electrophoresis - ABA abscisic acid - BA N⁶-benzyladenine - BSA bovine serum albumin - EDTA ethylenediamine tetraacetic acid - IEF isoelectric focusing - Rubisco ribulosebisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate - Tris tris (hydroxymethyl) aminomethane     

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

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

Importance of Rubisco activase in maize productivity based on mass selection procedure

The original maize (Zea mays L.) var. Zacatecas 58 (Z0) and five composites of cycles 5, 10, 15, 20, and 23 of stratified mass selection (Z5, Z10, Z15, Z20, and Z23) for improved productivity applied to the original variety were used as the model system. Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) (Rubisco) activity and the rates of net photosynthesis in the leaves above the ear were compared in all the composites during the grain filling period. Leaves were sampled weekly from anthesis to physiological maturity. Results showed a significant and gradual increase in Rubisco activity for the improved populations. In vivo photosynthesis measured by IRGA during the same period also showed increased levels associated with increases of grain yield. The highest Rubisco activity, photosynthetic rate and grain yield were found in the Z23 population. Western blot analysis for Rubisco protein did not show significant differences either during the filling period or between populations. The same analysis, however, for Rubisco activase protein showed increasing contents in the improved populations. These data confirm and complement previous findings, indicating that the stratified visual mass selection procedure applied to maize plants is associated with leaf content of Rubisco activase protein. The possible regulatory role of Rubisco activase during grain filling is discussed.     

Determination of Apparent K(m) Values for Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Using the Spectrophotometric Assay of Rubisco Activity

The spectrophotometric assay for ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) was used to determine the rate of increase in Rubisco activity over time in the presence or absence of Rubisco activase. Polynomial approximations to the raw data were used to smooth out minor fluctuations in the spectrophotometer readings, and Rubisco activase activity was expressed as nanomoles of activated Rubisco per minute. This assay was used to examine the effects of CO₂ and the inactive-Rubisco:ribulose 1,5-bisphosphate complex (ER) on the activase-catalyzed activation reaction. Double-reciprocal plots of activase activity and ER at several concentrations of CO₂ were consistent with two-substrate Michaelis-Menton kinetics, and the apparent K_m (CO₂) and K_m(ER) were determined to be 53 and 2.7 micromolar, respectively. These data do not prove that ER and CO₂ are substrates for the reaction catalyzed by activase, but they may be important to our understanding of the activation process in vivo. The implications of these data and their relation to previously published data on the effects of ER and CO₂ on activase are discussed.     

Rubisco, Rubisco activase, and global climate change

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

Subunit interactions of Rubisco activase: polyethylene glycol promotes self-association, stimulates ATPase and activation activities, and enhances interactions with Rubisco.

The effect of polyethylene glycol (PEG) on the enzymatic and physical properties of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase was examined. In the presence of PEG, Rubisco activase exhibited higher ATPase and Rubisco activating activities, concomitant with increased apparent affinity for ATP and Rubisco. Specific ATPase activity, which was dependent on Rubisco activase concentration, was also higher in the presence of Ficoll, polyvinylpyrrolidone, and bovine serum albumin. The ability of Rubisco activase to facilitate dissociation of the tight-binding inhibitor 2-carboxyarabinitol 1-phosphate from carbamylated Rubisco was also enhanced in the presence of PEG. Mixing experiments with Rubisco activase from two different sources showed that tobacco Rubisco activase, which exhibited little activation of spinach Rubisco by itself, was inhibitory when included with spinach Rubisco activase. Polyethylene glycol improved the ability of tobacco and a mixture of tobacco plus spinach Rubisco activase to activate spinach Rubisco. Estimates based on rate zonal sedimentation and gel-filtration chromatography indicated that the apparent molecular mass of Rubisco activase was two- to fourfold higher in the presence of PEG. The increase in apparent molecular mass was consistent with the propensity of solvent-excluding reagents like PEG to promote self-association of proteins. Likewise, the change in enzymatic properties of Rubisco activase in the presence of PEG and the dependence of specific activity on protein concentration resembled changes that often accompany self-association. For Rubisco activase, high concentrations of protein in the chloroplast stroma would provide an environment conducive to self-association and cause expression of properties that would enhance its ability to function efficiently in vivo.     

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.     

Rubiscolytics: fate of Rubisco after its enzymatic function in a cell is terminated

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the predominant protein in photosynthesizing plant parts and the most abundant protein on earth. Amino acids deriving from its net degradation during senescence are transported to sinks (e.g. developing leaves, fruits). Rubisco catabolism is not controlled only by the overall sink demand. An accumulation of carbohydrates may also accelerate senescence and Rubisco degradation under certain conditions. Amino acids produced by proteolysis are rapidly redistributed in plants with proper source-sink relationships. In leaves of wheat plants with reduced sink capacity (e.g. sink removal, phloem interruption by steam girdling at the leaf base), Rubisco is degraded and free amino acids accumulate. They may be washed out in the rain during late senescence. In leaves of depodded soybeans, Rubisco is degraded and amino acids can be reutilized in these leaves for the synthesis of special vacuolar proteins in the paraveinal mesophyll (vegetative storage proteins). Nitrogen deriving from Rubisco degradation in older (senescing) leaves of annual crops is integrated to some extent again in newly synthesized Rubisco in younger leaves or photosynthesizing tissues of fruits. Finally, a high percentage of this nitrogen is accumulated in protein bodies (storage proteins). At the subcellular level, Rubisco can be degraded in intact chloroplasts. Reactive oxygen species may directly cleave the large subunit or modify it to become more susceptible to proteolysis. A metalloendopeptidase may play an important role in Rubisco degradation within intact chloroplasts. Additionally, the involvement of vacuolar endopeptidase(s) in Rubisco catabolism (at least under certain conditions) was postulated by various laboratories.     

Antisense RNA inhibition of Rubisco activase expression

Ribulose bisphosphate carboxylase (Rubisco) activase catalyzes the activation of Rubisco in vivo. Activase antisense DNA mutants of tobacco have been generated to explore the control that activase exerts on the photosynthetic process. These mutants have up to 90% reductions in activase protein levels as a consequence of an inhibition of activase mRNA accumulation. It is shown that photosynthesis, measured as the rate of CO₂ exchange (CER), is modestly decreased in plants exposed to high irradiances. The decreases in CER in the transgenic plants are accompanied by corresponding decreases in Rubisco activation, indicating that activase has a direct effect on photosynthetic rates in the antisense plants by influencing the activation state of Rubisco. It is concluded that in high light conditions, control of photosynthesis is largely shared between Rubisco and activase. Plant growth is also impaired in mutant plants that have severe reductions in activase. The inhibition of activase in the antisense plants does not have an impact on the accumulation of Rubisco large subunit or small subunit mRNAs or proteins. This indicates that the concerted expression of the genes for activase (Rca) and Rubisco (rbcL and rbcS) in response to light, developmental factors and circadian controls is not due to feedback regulation of rbcL or rbcS by the amount of activase protein.     

Mechanism for deactivation of Rubisco under moderate heat stress

Photosynthesis is particularly sensitive to direct inhibition by heat stress. This inhibition is closely associated with the inactivation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). To develop a more complete understanding of the mechanism of inactivation of Rubisco under moderate heat stress, various aspects of the process were examined both in vivo and in vitro. Experiments with isolated Rubisco revealed that the rate of synthesis of the catalytic misfire product, xylulose-1,5-bisphosphate, increased with temperature. Activated Rubisco, produced by reaction with activase at a control temperature of 25°C or by incubation with high CO₂, deactivated when the temperature of the reaction exceeded temperatures that were equivalent to the optimum for activase adenosine triphosphatase (ATPase) activity. Measurements of the activation state of Rubisco in cotton and tobacco leaves showed that Rubisco inactivated within 7 s of imposing a heat stress. Thus, elevated temperature had an opposite effect on the two processes that ultimately determine the activation state of Rubisco, decreasing activase activity but stimulating the catalytic misfire reaction that inactivates Rubisco. These data support a mechanism for the inactivation of Rubisco at high temperature involving an inability of activase to overcome the inherently faster rates of Rubisco inactivation. That the net effect of elevated temperatures on Rubisco activation is similar both in vivo and under controlled conditions in vitro argues for a direct effect of temperature on the activation of Rubisco by activase and against the proposal that the deactivation of Rubisco under moderate heat stress is a secondary consequence of perturbations in the thylakoid membrane.