亚适温弱光对黄瓜幼苗光合酶活性和基因表达的影响

以‘津优3号’为试材,研究亚适温弱光(18℃/12℃,100 μmol·m-2·s-1)下黄瓜幼苗叶片核酮糖-1,5-二磷酸羧化/加氧酶(Rubisco)、果糖-1,6-二磷酸酶(FBPase)、甘油醛-3-磷酸脱氢酶(GAPDH)、果糖-1,6-二磷酸醛缩酶(FBA)、转酮醇酶(TK) mRNA表达量及活性的变化.结果表明:亚适温弱光处理的单株叶面积和干物质量均明显减小.处理初期,Rubisco大亚基(rbcL)、小亚基(rbcS)、FBPase、GAPDH、FBA及TK的基因表达量大幅度下降,多数酶活性明显减弱(TK变化不明显),光合速率(Pn)快速降低;处理3d后,亚适温弱光处理的rbcL、rbcS基因表达量和Rubisco初始活性持续下降,但下降幅度明显减小,Rubisco总活性及FBPase、GAPDH、FBA和TK基因表达与活性均呈上升趋势,Pn同步回升;处理时间超过6d时,Rubisco和FBPase基因表达与活性趋于平稳,其他酶和Pn呈下降趋势.可见,亚适温弱光下黄瓜光合酶基因表达量和活性的降低是Pn降低的重要原因,光合机构对亚适温弱光的适应与光合酶的活化机制有关.     

钙对亚适温弱光下黄瓜幼苗Ca2+分布及叶绿素荧光参数的影响

以'津优3号'黄瓜幼苗为试验材料,采用焦锑酸钙沉淀的电镜细胞化学方法,研究了CaCl2预处理对亚适温(昼/夜18℃/12℃)弱光(100 μmol·m-2·s-1)下黄瓜幼叶细胞中Ca2+分布、Ca2+-ATP酶活性及叶绿素荧光参数的影响.结果显示:正常温光条件(CK)下,黄瓜幼叶细胞Ca2+主要存在于液泡和液泡膜上,细胞质中含量较低;经亚适温和弱光处理7 d后,叶片细胞质和细胞膜中形成较大的钙沉淀颗粒,液泡中的Ca2+颗粒聚集成团,膜组织边缘模糊,Ca2+-ATPase活性降低;胁迫前用CaCl2预处理的细胞质中Ca2+颗粒略有增加,且分布较均匀,膜组织完整,Ca2+-ATPase活性与CK差异不显著;而经LaCl3、EGTA和CPZ预处理的Ca2+多呈大颗粒状聚积在细胞质、液泡膜或细胞壁上,Ca2+-ATPase活性大幅度下降.在7 d亚适温弱光处理后,各处理黄瓜叶片的Fv/Fm变化不大,而ΦPSⅡ、qP和ETR显著降低;与水预处理相比,叶片ΦPSⅡ、qP和ETR在CaCl2处理下显著增加,而在EGTA和CPZ处理下显著减小,LaCl3处理的无显著变化.研究表明,亚适温弱光处理能打破黄瓜幼苗细胞内的Ca2+平衡,使其膜组织受到一定程度破坏;CaCl2可维持胞内较高的Ca2+-ATP酶活性,保持Ca2+平衡,保护细胞膜组织结构完整,并参与了光合作用光能捕获和光合效率的调控,能有效减轻亚适温弱光对黄瓜幼苗光合作用的不良影响.     

He-Ne激光和增强UV-B辐射对小麦幼叶叶绿素荧光和Rubisco活化酶的影响

选用小麦‘ML7113’品种为材料,人工模拟He-Ne激光(5mJ·s-1·mm-2)、增强UV-B(10.8kJ·m-2·d-1)辐射及两者复合辐照进行处理,利用叶绿素荧光仪、考马斯亮蓝G-250染色法和PCR技术研究7d龄小麦幼苗叶绿素荧光特性、Rubisco活化酶含量、基因表达量及其基因序列的变化。结果表明:(1)与对照组相比,增强UV-B辐射后,小麦幼苗叶绿素荧光特性减弱,Rubisco活化酶含量及其基因表达量均下降;而低剂量的He-Ne激光辐照后能够在一定程度上修复经UV-B辐射后对小麦幼苗叶绿素荧光特性所造成的损伤,且使Rubisco活化酶含量及其基因表达量上升。(2)与对照组相比,经He-Ne激光和增强UV-B辐射以及两者复合辐照处理后基因序列均出现两个相同的点突变,但并未造成氨基酸序列的变化。研究认为,低剂量He-Ne激光辐照能够在一定程度上修复受UV-B辐射小麦幼苗叶绿素荧光活性、Rubisco活化酶含量及其基因表达量的降低;He-Ne激光和增强UV-B辐射对小麦幼苗Rubisco活化酶活性的影响可能发生在其转录水平,从而使小麦光合能力发生相应的变化。     

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.     

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。     

低温弱光对辣椒幼苗抗氧化酶活性与质膜透性的影响

以辣椒CapsicumannuumL.幼苗为材料,研究了辣椒幼苗叶片中活性氧清除系统对低温弱光的响应.结果表明:随着低温弱光胁迫程度和时间的增加,辣椒幼苗叶片中POD活性提高,但SOD和CAT活性下降,MDA含量增加,细胞膜透性增大;就温度而言,临界低温15℃/8℃比偏低温19℃/12℃对植株的影响更为显著;在偏低温19℃/12℃下,弱光90μmol·m-2·s-1使POD活性上升更大,而SOD和CAT活性的下降、MDA含量的增加和细胞膜透性的增大更小,而在临界低温15℃/8℃下则相反;陇椒2号耐低温弱光能力强,各项指标均优于七寸红.     

低温弱光对茄子幼苗光合特性的影响

以4～5叶的 二苠 茄幼苗为试材,研究了其在低温弱光 10℃/5℃昼/夜,光强60、120μmol·m-2·s-1 胁迫7d并恢复7d后的光合特性变化.结果表明,低温弱光胁迫后茄子幼苗的净光合速率、气孔导度和叶绿素含量显著降低;光补偿点、光饱和点、光饱和时的Pn、表观量子产额降低;CO2补偿点升高,CO2饱和点、CO2饱和时的Pn、光合能力、CO2羧化效率降低;以低温下较强光照时 120μmol·m-2·s-1 的变化幅度较大;恢复7d后各项指标仍然不能恢复到对照水平.试验条件已对茄子幼苗叶片光合机构的结构和活性造成了不可恢复的伤害.     

持续低温弱光对黄瓜叶片气体交换、叶绿素荧光猝灭和吸收光能分配的影响

持续常温弱光(25℃/18℃,l00umol m-2 s-1)、低温弱光(12℃/12℃,100 umol m-2 s-1和7℃/7℃,l00μmolm-2s-1)均导致黄瓜生长减慢或停滞、叶绿素含量、气孔导度和净光合速率、光合电子传递速率下降以及胞间CO2浓度上升.常温弱光和12℃弱光处理对光系统II的最大光化学效率Fv/Fm无显著影响,而7℃弱光处理导致Fv/Fm的可逆性下降.常温弱光和7℃、12℃弱光处理均导致了光化学反应速率的降低以及天线热耗散和反应中心过剩能量的增加.在胁迫后,12℃弱光0比7℃弱光更有利于植株光合功能的恢复.     

低温弱光胁迫及恢复对切花菊光合作用和叶绿素荧光参数的影响

以切花菊品种‘神马’为试材,在偏低温弱光(16℃/12℃,PFD100μmol.m-2.s-1)和临界低温弱光(12℃/8℃,PFD60μmol.m-2.s-1)下分别胁迫11d,然后转入正常条件(22℃/18℃,PFD450μmol.m-2.s-1)恢复11d,研究不同低温弱光强度及恢复对菊花光合作用和叶绿素荧光参数的影响.结果表明:低温弱光导致菊花叶片的净光合速率(Pn)和气孔限制值(Ls)下降,而胞间CO2浓度(Ci)上升.偏低温弱光胁迫下菊花叶片暗适应下最大光化学效率(Fv/Fm)和初始荧光(Fo)无明显变化,但光适应下最大光化学效率(Fv′/Fm′)在处理前期略有下降,后期则有所回升;而临界低温弱光处理的Fo明显升高,Fv/Fm和Fv′/Fm′显著降低.PSⅡ光合电子传递量子效率(ΦPSⅡ)、光化学猝灭系数(qP)和表观光合电子传递速率(ETR)均随着低温弱光胁迫程度的增加和时间的延长而降低;偏低温弱光处理植株在解除胁迫后能迅速恢复到对照水平,而临界低温弱光处理植株回升速度较慢;同时,低温弱光胁迫下吸收光强用于分配光化学反应部分(Prate)的比例减少,而天线热耗散(Drate)和反应中心的能量耗散(Ex)比例上升,但天线热耗散为过剩光能的主要分配途径.     

低温弱光下外源褪黑素对黄瓜幼苗生长及抗氧化系统的影响

以黄瓜品种‘津优4号’为材料,利用人工气候箱进行低温弱光处理(昼/夜,18℃/10℃),研究外源褪黑素(MT)对低温胁迫下黄瓜幼苗生长和抗氧化系统等生理指标的影响。结果显示:低温弱光下,与对照相比,外源褪黑素处理显著提高了黄瓜幼苗株高、茎粗、植株鲜重和干重,叶绿素和根系活力分别显著提高了13.3%和18.8%,抗氧化物质GSH及ASA的含量分别显著增加了39.3%和24.7%,保护酶SOD、POD、CAT、APX活性均显著升高;同时,外源褪黑素处理还显著提高了黄瓜叶片质膜及液泡膜H+-ATP酶的活性,从而使黄瓜幼苗叶片MDA含量和电解质渗漏率分别显著降低了28.7%和29.7%。研究表明,低温弱光下外源褪黑素可通过提高黄瓜幼苗保护酶活性、抗氧化物质的含量、细胞膜ATP酶活性等来降低质膜过氧化水平,保持细胞膜的完整性和功能,从而增强黄瓜幼苗对低温弱光的适应性,维持其正常生长,并以200μmol·L-1褪黑素处理效果较好。     

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.     

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.     

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.     

Light Activation of Rubisco by Rubisco Activase and Thylakoid Membranes

A reconstituted system comprising ribulose bisphosphate carboxylase/oxygenase(rubisco), rubisco activase, washed thylakoid membranes, andATP was used to demonstrate a light-dependent stimulation ofrubisco activation. ATP, ribulose bisphosphate, H⁺, and Mg²⁺concentrations are normally light-dependent variables in thechloroplast but were maintained at pre-determined levels. Resultsindicated that rubisco activase and washed thylakoid membranesare sufficient to catalyze light stimulation of rubisco activationwith the reconstituted system, and that rubisco activase isrequired for this light stimulation. The washed thylakoid membranesdid not exhibit rubisco activase activity, nor was rubisco activaseprotein detected immunologically. Light-dependent activationof rubisco in the reconstituted system was similar in whole-chainand PS I electron transport reactions, and saturated at approximately100 µmol photons m^–2 s^–1. ¹ Present address: Department of Biological Sciences, LouisianaTech University, Ruston, LA 71272, U.S.A.     

Coordinate Expression of Rubisco Activase and Rubisco during Barley Leaf Cell Development

We have utilized the cellular differentiation gradient and photomorphogenic responses of the first leaf of 7-day-old barley (Hordeum vulgare L.) to examine the accumulation of mRNA and protein encoded by the ribulose-1,5-biphosphate carboxylase holoenzyme (rubisco) activase gene (rca). Previous studies have revealed a pattern of coordinate expression of rubisco subunit polypeptides during development. We compared the expression of rubisco polypeptides and mRNAs with those encoded by rca. The mRNAs encoding both rubisco activase and rubisco are expressed exclusively in leaf tissue of 7-day-old barley seedlings; mRNAs and polypeptides of rca accumulate progressively from the leaf base in a pattern that is qualitatively similar to that of rubisco subunit mRNAs and polypeptides. The parallel pattern of rca protein and mRNA accumulation indicate that a primary control of rca gene expression in this system lies at the level of mRNA production. Light-induced expression of rca in etiolated barley follows a different pattern from that of the acropetal barley leaf gradient, however. Etiolated, 7-day-old barley seedlings contain levels of rca mRNA near the limit of detection in Northern blot hybridization assays. White light induces a 50- to 100-fold accumulation of rca mRNA, which is detectable within 30 min after the onset of illumination. In contrast, steady state levels of mRNAs encoding the small rubisco subunit are affected little by light, and mRNAs encoding the large subunit accumulate about 5-fold in response to illumination. While rca mRNA levels are low in etiolated barley leaves, levels of the protein are approximately 50 to 75% of those found in fully green leaves.     

Heat Denaturation Profiles of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) and Rubisco Activase and the Inability of Rubisco Activase to Restore Activity of Heat-Denatured Rubisco

We compared the heat-denaturation profiles of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase and further examined the ability of Rubisco activase to restore the activity of heat-denatured Rubisco originally reported (E. Sanchez de Jimenez, L. Medrano, and E. Martinez-Barajas [1995] Biochemistry 34: 2826-2831). Rubisco was heat-treated in both the carbamylated and uncarbamylated forms and in the presence and absence of 10 mM dithiothreitol (DTT). Both forms were highly resistant to heat denaturation and further protection was gained in the presence of DTT. A 50% loss in total activity occurred after 1 h at 57.5 and 55.2[deg]C for uncarbamylated Rubisco and at 60.2 and 59.6[deg]C for carbamylated Rubisco, in each case with and without DTT, respectively. In contrast, Rubisco activase lost 50% activity after only 5 min at 33[deg]C and the loss in activity was not affected by the presence of Rubisco. When Rubisco, heat-denatured to various extents, was incubated at room temperature with Rubisco activase or bovine serum albumin as a control, Rubisco activase did not have a significant specific ability to restore Rubisco activity. We conclude that Rubisco activase alone does not have the ability to restore the activity of heat-denatured Rubisco and is unlikely to protect or restore Rubisco activity from heat denaturation in vivo because it is more heat-labile than Rubisco.     

Catalysis and regulation in Rubisco

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the incorporation of inorganic CO(2) into the organic molecules of life. Rubisco is extremely inefficient as a catalyst and its carboxylase activity is compromised by numerous side-reactions including oxygenation of its sugar phosphate substrate by atmospheric O(2). The reduction in the catalytic efficiency as a result of these processes has implications for crop yield, nitrogen and water usage, and for the global carbon cycle. Several aspects of Rubisco including its complex biosynthesis and multi-step catalytic reaction are subject to tight control involving light, cellular metabolites, and molecular chaperones. Numerous high-resolution crystal structures of different forms of Rubisco are now available, including structures of mutant enzymes. These provide a molecular framework for the understanding of these processes at the molecular level.     

Structure and function of Rubisco

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating CO(2) into the biosphere. At the same time Rubisco is an extremely inefficient catalyst and its carboxylase activity is compromised by an opposing oxygenase activity involving atmospheric O(2). The shortcomings of Rubisco have implications for crop yield, nitrogen and water usage, and for the global carbon cycle. Numerous high-resolution crystal structures of different forms of Rubisco are now available, including structures of mutant enzymes. This review uses the information provided in these structures in a structure-based sequence alignment and discusses Rubisco function in the context of structural variations at all levels--amino acid sequence, fold, tertiary and quaternary structure--with an evolutionary perspective and an emphasis on the structural features of the enzyme that may determine its function as a carboxylase.     

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).