Heat sensitivity of Rubisco,Rubisco activase and Rubisco binding protein in higher plants

During the past few years the investigations concerning Rubisco and the changes of its activity and properties at elevated temperature were reconsidered with special reference to the important role of Rubisco activase and Rubisco binding protein. The major changes in Rubisco, Rubisco activase and Rubisco binding protein reported recently are presented in this review. New information on these proteins, including their changes under heat stress conditions, is discussed together with open questions.     

Alterations in Photosystem II electron transport as revealed by thermoluminescence of Cu-poisoned chloroplasts

The Rubisco activase amino acid sequences of spinach and tobacco are 79% identical, yet the tobacco protein does not facilitate the activation of the uncarbamylated, ribulose bisphosphate bound form of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) and vice versa. In contrast, combinations of the spinach Rubisco activase with Rubisco from non-Solanaceae species and combinations of tobacco Rubisco activase with Rubisco from other Solanaceae species are almost as effective as the analogous combination. To examine the basis of the preference of an activase protein for either Solanaceae or non-Solanaceae Rubisco, several recombinant chimeric proteins were obtained by combining regions from the cDNAs of spinach and tobacco activase and expression in Escherichia coli. The chimeric proteins were analyzed for ATP hydrolysis and ability to activate spinach and tobacco Rubisco. Comparisons of Rubisco preference with composition of the various activase chimeras indicate that the major determinants of Rubisco preference seem to be localized in the carboxyl-terminal region.     

植物Rubisco活性中心的模拟分析

通过对与不同配基结合的植物Rubisco复合物结构的重叠比较分析 ,发现Rubisco的活性差异是由其中一段Loop6环序列所造成的 ;金属离子与活性中心的结合会造成活性中心巨大的构象变化 .进一步用SwissPDBViewer软件模拟不同配基的植物Rubisco活性中心与此Loop环的氢键相互作用 .结果表明 ,有 3个Lys残基Lys2 0 1、Lys334、Lys175与Rubisco是否处于活性状态密切相关 ,这些残基的结构变化对分子设计可能有重要的参考价值     

Rubisco活化酶的研究进展

Rubisco活化酶是近年中发现的一种可以调节Rubisco活性的酶 ,它能使Rubisco在植株体内条件下达到最大活化程度。Rubisco活化酶不仅具有活化Rubisco的活性 ,而且具有ATP水解酶活性。在ATP水解过程中 ,Rubisco活化酶促使各种磷酸糖抑制物从Rubisco上解离下来 ,恢复Rubisco活性。Rubisco活化酶的发现与研究使许多Rubisco体内活化中的疑难问题得到了阐明。本文还介绍了Rubisco活化酶的分子特性、酶作用机制以及环境因素对它活性影响等方面的最新研究进展。     

大麦和玉米叶片叶绿体中Rubisco及其活化酶的免疫金标记定位

运用免疫金标记电镜技术研究了禾本科C3植物大麦(Hordeum vulgare L.)和C4植物玉米(Zea mays L.)叶片中Rubisoo及其活化酶(RCA)的细胞定位,结果表明:两种植物叶片解剖结构及叶绿体超微结构差别明显.在大麦叶细胞中,只有一种叶肉细胞叶绿体,Rubisoo和RCA主要分布于叶绿体的间质中.在玉米叶细胞中,存在着维管束鞘细胞和叶肉细胞两种类型叶绿体,Rubisco主要分布于鞘细胞叶绿体的基质中,但在叶肉细胞叶绿体中亦有少量特异性标记;RCA在鞘细胞叶绿体和叶肉细胞叶绿体的基质中都有分布.两种植物叶绿体结构及光合作用关键酶定位的不同,体现了C3植物和C4植物在光合器结构与功能上的差异.     

水稻Rubisco和RCA的日变化及其细胞定位

采用免疫胶体金标记电镜技术对水稻(0ryza sativa subsp.indica cv.浙农952)叶片中的Rubisco及其活化酶(RCA)进行细胞器定位和定量,同时用免疫扩散法进行叶片含量分析,研究了这两种酶含量及活力的日变化.结果表明Rubisco主要分布于叶绿体,RCA分布于叶绿体和线粒体中;光合速率(Pn)、Rubisco初始活力和RCA活力与光合日变化密切相关;在光照最强的13时,出现光合"午休",叶绿体中Rubisco的密度有一定程度降低,而全叶的总Rubisco保持稳定,Rubisco初始活力也有明显的"午休",这意味着体内Rubisco的活力除受RCA调节外,可能还与叶绿体中Rubisco的分布有关.RCA活力变化与叶绿体中RCA含量变化较为一致,表明RCA在叶绿体中的分布对调节其本身活力和Rubisco活性有重要作用.     

Antisense inhibition of Rubisco activase increases Rubisco content and alters the proportion of Rubisco activase in stroma and thylakoids in chloroplasts of rice leaves

BACKGROUND AND AIMS: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA) is a nuclear-encoded chloroplast protein that modifies the conformation of Rubisco, releases inhibitors from active sites, and increases enzymatic activity. It appears to have other functions, e.g. in gibberellin signalling and as a molecular chaperone, which are related to its distribution within the chloroplast. The aim of this research was to resolve uncertainty about the localization of RCA, and to determine whether the distributions of Rubisco and RCA were altered when RCA content was reduced. The monocotyledon, Oryza sativa was used as a model species. METHODS: Gas exchange and Rubisco were measured, and the sub-cellular locations of Rubisco and RCA were determined using immunogold-labelling electron microscopy, in wild-type and antisense rca rice plants. KEY RESULTS: In antisense rca plants, net photosynthetic rate and the initial Rubisco activity decreased much less than RCA content. Immunocytolocalization showed that Rubisco in wild-type and antisense plants was localized in the stroma of chloroplasts. However, the amount of Rubisco in the antisense rca plants was greater than in the wild-type plants. RCA was detected in both the chloroplast stroma and in the thylakoid membranes of wild-type plants. The percentage of RCA labelling in the thylakoid membrane was shown to be substantially decreased, while the fraction in the stroma was increased, by the antisense rca treatment. CONCLUSIONS: From the changes in RCA distribution and alterations in Rubisco activity, RCA in the stroma of the chloroplast probably contributes to the activation of Rubisco, and RCA in thylakoids compensates for the reduction of RCA in the stroma, allowing steady-state photosynthesis to be maintained when RCA is depleted. RCA may also have a second role in protecting membranes against environmental stresses as a chaperone.     

Effect of Nd3+ ion on carboxylation activity of ribulose-1,5-bisphosphate carboxylase/oxygenase of spinach

Neodymium (Nd), as a member of rare earth elements, proved to enhance the photosynthesis rate and organic substance accumulation of spinach through the increase in carboxylation activity of Rubisco. Although the oxygenase activity of spinach Rubisco was slightly changed with the Nd(3+) treatment, the specific factor of Rubisco was greatly increased. It was partially due to the promotion of Rubisco activase (R-A) activity but mainly to the formation of Rubisco-Rubisco activase super-complex, a heavier molecular mass protein (about 1200kD) comprising both Rubisco and Rubisco activase. This super-complex was found during the extraction procedure of Rubisco by the gel electrophoresis and Western-blot studies. The formation of Rubisco-R-A super-complex suggested that the secondary structure of the protein purified from the Nd(3+)-treated spinach was different from that of the control. Extended X-ray absorption fine structure study of the 'Rubisco' purified from the Nd(3+)-treated spinach revealed that Nd was bound with four oxygen atoms and two sulfur atoms of amino acid residues at the Nd-O and Nd-S bond lengths of 2.46 and 2.89A, respectively.     

Proteolytic activities and ribulose 1,5 bisphosphate carboxlyase degradation in leaves of soybean (Glycine max L. Merril) with different nitrogen status

Changes in soluble proteins and Rubisco (E.C.4.1.1.39) contents were examined in leaves of nitrogen-deprived and nitrogen-sufficient soybeans. Rubisco content was very responsive to nitrogen stress, and this protein appeared to be the largest source of mobilizable nitrogen in the senescent leaf. Loss of soluble proteins and Rubisco was associated with a decrease in the activities of several proteolytic enzymes measured using artificial substrates: carboxypeptidase, aminopeptidase and haemoglobinase.The in vitro activity of enzyme(s) which can degrade Rubisco was investigated using endogenous Rubisco and in vitro radiolabelled Rubisco as substrates. Highest endopeptidic cleavage of endogenous Rubisco occured at pH 4; the enzyme responsible for this breakdown appeared to be a sulfhydryl-dependent proteinase. In contrast, [¹⁴C] Rubisco was attacked preferentially at pH 9, by a peptide hydrolase sensitive to EDTA. No increase in Rubisco-degrading activities was detected in nitrogen-deficient soybean leaves compared to control plant leaves.Abbreviations EDTA Ethylenediaminetetraacetate - LS Large Subunit of Rubisco - NEM N-ethylmaleimide - pCMB Parachloromercuribenzoate - PMSF Phenylmethylsulfonyl-fluoride - Rubisco Ribulose 1,5 Bisphosphate Carboxylase/Oxygenase     

Regulation of Rubisco activity in vivo

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is not able to achieve and maintain adequate CO₂ and Mg²⁺ activation under physiological conditions. Higher plants and green algae contain Rubisco activase, a soluble protein which not only facilitates Rubisco activation in situ but also regulates enzyme activity in response to irradiance and other factors. Regulation of Rubisco activity by modulation of activation state coordinates the rate of CO₂ fixation with the rate of substrate regeneration. This regulation may be required to ensure that the levels of photosynthetic metabolites in the chloroplast are optimal for photosynthesis under a variety of environrmental conditions. Some plant species also appear to regulate Rubisco activity by synthesizing 2-carboxyarabinitol 1-phosphate, an inhibitor of Rubisco in the dark. This inhibitor may function primarily as a regulator of metabolite binding in the dark rather than as a modulator of Rubisco activity in the light.     

水稻Rubisco和RCA的日变化及其细胞定位

采用免疫胶体金标记电镜技术对水稻(Oryza satova subsp.indica cv.浙农952)叶片中的Rubisco及其活化酶(RCA)进行细胞器定位和定量,同时用免疫扩散法进行叶片含量分析,研究了这两种酶含量及活力的日变化。结果表明Rubisco主要分布于叶绿体,RCA分布于叶绿体和线粒体中;光合速率(Pn)、Rubisco初始活力和RCA活力与光合日变化密切相关;在光照最强的13时,出现光合“午休”,叶绿体中Rubisco的密度有一定程度降低,而全叶的总Rubisco保持稳定,Rubisco初始活力也有明显的“午休”,这意味着体内Rubisco的活力除受RCA调节外,可能还与叶绿体中Rubisco的分布有关。RCA活力变化与叶绿体中RCA含量变化较为一致,表明RCA在叶绿体中的分布对调节其本身活力和Rubisco活性有重要作用。     

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.     

Binding of a Transition State Analog to Newly Synthesized Rubisco

Radioactive amino acids, when added to isolated pea chloroplasts or chloroplast extracts engaged in protein synthesis, are incorporated into Rubisco large subunits that co-migrate with native Rubisco during nondenaturing electrophoresis. We have added the transition state analog 2′-carboxyarabinitol bisphosphate (CABP) to chloroplast extracts after in organello or in vitro incorporation of radioactive amino acids into Rubisco large subunits. Upon addition of CABP the radioactive bands co-migrating with native Rubisco undergo a readily detected shift in electrophoretic mobility just as the native enzyme, thus demonstrating the ability of the newly assembled molecules to interact with this transition state analog.     

羊草光合作用相关基因的克隆与分析

在构建了羊草叶片cDNA文库的基础上,利用M13载体通用引物筛选其亚文库,挑选阳性克隆进行测序,将测序结果在NCBI基因库中进行比对,得到一个Rubisco大亚基基因全长序列和Rubisco小亚基基因部分序列,并对其核苷酸及其编码的氨基酸序列进行分析。结果显示,Rubisco大亚基基因长度为1 796 bp,与禾本科大麦、小麦、野雀麦、粗山羊草、旱麦草、异形花草、黑麦等的核苷酸序列同源性达98%以上;羊草的Rubisco小亚基基因部分序列含有一个开放阅读框,其长度为186 bp,编码61个氨基酸,与禾本科的小麦、大麦、燕麦、黑麦以及扁穗雀麦Rubisco小亚基基因氨基酸序列的同源性分别为93%、93%、91%、91%、92%。羊草Rubisco基因的克隆与分析有利于进一步研究其光合作用效率。     

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.     

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

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.     

A role for differential Rubisco activase isoform expression in C4 bioenergy grasses at high temperature

Rubisco activase (Rca) facilitates the release of sugar‐phosphate inhibitors at Rubisco catalytic sites during CO₂ fixation. Most plant species express two Rca isoforms, the larger Rca‐α and the shorter Rca‐β, either by alternative splicing from a single gene or expression from separate genes. The mechanism of Rubisco activation by Rca isoforms has been intensively studied in C₃ plants. However, the functional role of Rca in C₄ plants where Rubisco and Rca are located in a much higher [CO₂] compartment is less clear. In this study, we selected four C₄ bioenergy grasses and the model C₄ grass setaria (Setaria viridis) to investigate the role of Rca in C₄ photosynthesis. All five C₄ grass species contained two Rca genes, one encoding Rca‐α and the other Rca‐β, which were positioned closely together in the genomes. A variety of abiotic stress‐related motifs were identified in the Rca‐α promoter of each grass, and while the Rca‐β gene was constantly highly expressed at ambient temperature, Rca‐α isoforms were expressed only at high temperature but never surpassed 30% of Rca‐β content. The pattern of Rca‐α induction on transition to high temperature and reduction on return to ambient temperature was the same in all five C₄ grasses. In sorghum (Sorghum bicolor), sugarcane (Saccharum officinarum), and setaria, the induction rate of Rca‐α was similar to the recovery rate of photosynthesis and Rubisco activation at high temperature. This association between Rca‐α isoform expression and maintenance of Rubisco activation at high temperature suggests that Rca‐α has a functional thermo‐protective role in carbon fixation in C₄ grasses by sustaining Rubisco activation at high temperature.     

Rubisco和RCA在青菜叶绿体中的分布及病毒侵染对其细胞定位的影响

运用免疫金标电镜术观察了青菜叶细胞中光合作用关键酶Rubisco和Rubisco活化酶(RCA)的细胞化学定位,结果显示Rubisco和RCA免疫金颗粒主要分布于薄壁组织叶绿体的间质中,在基粒片层上很少,表皮的气孔保卫细胞和维管束薄壁细胞叶绿体内也有分布,在细胞质及线粒体等细胞器中无特异性分布。同时比较观察了感染芜菁花叶病毒(TuMV)的青菜叶绿体Rubisco和RCA免疫金标记结果,发现病组织中结构尚完整的叶绿体Rubisco和RCA标记率略有下降,而结构严重破坏的叶绿体中两种酶标记率分别仅为正常叶绿体的58.44%和64.67%,表明病毒侵染可导致Rubisco和RCA含量下降,影响寄主植物的光合作用。     

水稻温敏失绿突变体的Rubisco活化酶活性、蛋白质与氨基酸组分的变化

在水稻温敏失绿突变性状表达过程中,对其Rubsico 含量、Rubsico 活化酶活性,全叶蛋白及游离氨基酸组分变化进行测定。结果表明:突变体的Rubisco 结构和含量与野生型一样,保持相对稳定;而其Rubisco 活化酶活性则随一个分子量为56.2kD(PI=4.5)的特异蛋白质的存在与消失发生明显改变。当突变性状表达时,分子量为56.2kD(PT=4.5)的特异蛋白消失,其Rubisco 活化酶活性下降;当叶片失绿区域复绿时,56.2kD(PI=4.5)特异蛋白出现,则Rubisco 活化酶活性上升。这一密切地相关关系表明,突变体的Rubisco 活化酶活性变化在光合作用过程中,除与自身结构和含量有关外,还与叶片中这一特异蛋白的存在密切相关,它可能是Rubisco 活化酶活性的调节蛋白。这种调节具体表现在氨基酸代谢上,是对上游氨基酸的阻遏调控,从而使叶绿体的结构物质合成受阻,最终导致类囊体膜的退化。     

Regulation of photosynthesis in developing leaves of soybean chlorophyll-deficient mutants

In this report we examine the factors that regulate photosynthesis during leaf ontogeny in y3y3 and Y11y11, two chlorophyll-deficient mutants of soybean. Photosynthetic rates were similar during wild type and Y11y11 leaf development, but the senescence decline in photosynthesis was accelerated in y3y3. Photosynthetic rates fell more rapidly than chlorophyll concentrations during senescence in wild type leaves, indicating that light harvesting is not strongly limiting for photosynthesis during this phase of leaf development. Chlorophyll concentrations in Y11y11, though significantly lower than normal, were able to support normal photosynthetic rates throughout leaf ontogeny. Chlorophyll a/b ratios were constant during leaf development in the wild type, but in the mutants they progressively increased (y3y3) or decreased (Y11y11). In all three sets of plants, photosynthetic rates were directly proportional to Rubisco contents and activities, suggesting that Rubisco plays a dominant role in regulating photosynthesis throughout leaf ontogeny in these plants. The expression of some photosynthetic proteins, such as Rubisco activase, was coordinately regulated with that of Rubisco in all three genotypes, i.e. an early increase, coincident with leaf expansion, followed by a senescence decline in the fully-expanded leaf. On the other hand, the light harvesting chlorophyll a/b-binding proteins of PS II (the CAB proteins), while they showed a profile similar to that of Rubisco in the wild type and y3y3, progressively increased in amount during Y11y11 leaf development. We conclude that Y11y11 may be defective in the accumulation of a component required for LHC II assembly or function, while y3y3 has more global effects and may be a regulatory factor that controls the duration of senescence.