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。     

水稻转绿型白化突变系W25转绿过程中Rubisco、Rubisco活化酶活性与光合速率的变化

水稻 (OryzasativaL .)转绿型白化突变系W2 5在转绿过程中叶绿素、可溶性蛋白质和Rubisco含量的动态变化过程表明 ,白化突变体内叶绿素、可溶性蛋白质和Rubisco含量极低 ,随着转绿过程各组分含量迅速提高 ,转绿至第 30天时超过野生种 2 177s;Rubisco初始活力与Rubisco活化酶含量呈极显著正相关。Rubisco活化酶基因表达的研究结果表明 ,突变体的Rubisco活化酶表达高于野生种 2 177s。在转绿过程中 ,Rubisco活化酶含量的提高要先于Rubisco和光合速率     

UV-B辐射引起的绿豆幼苗叶片Rubisco量降低与H2O2含量升高有关

为了探讨UV-B辐射引起的Rubisco含量降低的可能机制,研究了两个绿豆品种(秦豆-20和中绿-1)幼苗在UV-B辐射下叶片Rubisco含量、蛋白水解酶活性和H2O2含量的变化.结果表明:UV-B辐射显著加速了两个绿豆品种幼苗叶片H2O2含量和蛋白水解酶活性上升,使Rubiscco含量下降.秦豆-20品种在UV-B辐射下H2O2含量和蛋白水解酶活性的上升程度明显大于中绿-1,相应其Rubisco含量的下降程度也大于中绿-1.抗坏血酸处理能明显降低UV-B辐射下两品种幼苗叶片H2O2含量,同时明显抑制蛋白水解酶活性的上升及Rubisco含量的下降.结果说明UV-B辐射诱导Rubisco含量的降低可能通过提高H2O2水平从而加强蛋白水解酶系统的活化而加速了Rubisco的降解.     

水稻温敏型突变体叶片间断失绿的超微结构

在短时降温诱导下,水稻温敏型突变体１１０３ｓ（Ｏｒｙｚａ　ｓａｔｉｖａ　ｓｓｐ．ｉｎｄｉｃａ）植株间断失绿性状表达（临界温度２３．１℃）过程中,叶绿体含量的增减与叶色变化相符。电镜观察发现,性状表达时叶片间断失绿区叶绿体内部结构发生退化,呈现基粒垛叠片层数的异常减少,或基粒消失仅剩基粒残迹,有的甚至整个叶绿体为高电子密度的囊泡状结构。但在同一叶片的绿区,叶绿体仅表现基粒片层数减少、排列不规则,嗜锇小球聚集。在叶片失绿区的复绿过程中,叶绿体的这些变化又可逆转,内部结构重建,最后整个叶绿体结构基本恢复正常。水稻温敏突变体１１０３ｓ叶片间断失绿性状表达过程,实质上是一个由温度调控的叶绿体结构退化与修复的可逆过程。     

—个新的水稻斑马叶突变（zebraleaf1）对叶绿体发育的影响（简报）

通过γ射线诱变．在水稻粳稻栽培品种9522中得到一个斑马叶突变体zebraleaf1。为了研究zl1的功能．我们对突变体进行了形态学和细胞学的分析．同时也对此基因突变以后对叶绿体发育和光合作用的影响作了评价。突变体叶片上绿色和枯白色条纹相间．叶绿素含量显著的下降。电镜显示叶绿体类囊体的排列被打乱．变得杂乱无章。这表明。Zl1突变体在叶绿体发育过程中出现障碍。zl1基因的突变使得净光合速率显著的下降。参与光合作用的一些关键蛋白．比如核酮糖1,5-二磷酸羧化酶／加氧酶（Rubisco）、Rubisco活化酶、D1蛋白、CF1β亚基的表达量也显著的下调。但是．zl1突变体对外界环境非常敏感．有时会没有表型。     

温敏失绿突变体水稻1103s在失绿过程中全叶蛋白的变化

从全叶蛋白比较入手,研究了温敏失绿突变体水稻（ＯｒｙｚａｓａｔｉｖａＬ．）１１０３ｓ失绿前、后的变化,并结合该突变体在不同温度处理和遗传背景下叶片全蛋白的变化特征分析了变温诱导与失绿的关系。结果表明,诱导后１１０３ｓ的叶片上,失绿部分的组织中没有出现冷胁迫的迹象,其Ｒｕｂｉｓｃｏ的大、小亚基表现正常。一个５１ｋＤ（ＰＩ＝４．５）的特异蛋白Ｐ１在失绿部分的组织中消失,而在叶片绿色部分的组织中检测到了Ｐ１蛋白,不过量有所减少,说明在失绿叶片上突变基因的表达存在组织差异性。Ｐ１蛋白在常温下生长的１１０３ｓ叶片中为一大量蛋白,此蛋白在“８９０２ｓ”、“窄叶青８号”等品种中均可检测到;并且持续低温处理的１１０３ｓ植株和１１０３ｓ×８９０２ｓ杂交一代的叶片中Ｐ１蛋白的表达未受影响。由此推测,Ｐ１蛋白是水稻叶片中的一个与叶绿素的代谢过程密切相关的重要功能蛋白。在１１０３ｓ中,Ｐ１蛋白的变化不是温度诱导的直接后果,而是受突变所导致的温敏过程调控的下游变化。     

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.     

日本桃叶珊瑚的冷驯化及抗寒机制研究

冷驯化能大大提高植物的抗寒性。研究测定了引进日本桃叶珊瑚(Auccuba japonica Thunb.cv.Variegata)苗木在不同越冬条件下的抗寒性指标及在低温锻炼前后的半致死温度,并通过双向电泳分离与抗寒性有关的特异蛋白。结果表明,日本桃叶珊瑚能安全地越冬(最低气温为-18℃),日本桃叶珊瑚叶片抗冻的细胞结构特征在于其叶肉细胞排列疏松,间隙大,有利于防止细胞内结冰;经过4℃冷驯化的植株低温半致死温度为-21℃,而未经冷驯化的植株低温半致死温度为-7．5℃;双向电泳分析发现至少有3个常温下存在而低温下消失的蛋白多肽,分子量分别为A15kD、B14kD、C56kD、D26kD。依据分子量和等电点初步分析,A与组蛋白H2A相似,B与组蛋白H2B相似;另B与Rubisco小亚基分子量相近,C与Rubisco大亚基分子量相近,前一种情况可能与有关抗寒基因的表达有关,后一种情况可能与低温下的光合作用减弱有关。     

家蚕雌性附腺及其Ng突变体蛋白质组双向电泳图谱分析

分别对家蚕(Bombyx mori．L)正常及Ng突变体雌蛾件附腺分泌部组织的蛋白质进行提取,并采用双向凝胶电泳和计算机辅助分析方法,对提取的蛋白质混合物进行分离和比较分析。用银染的方法,平均每张电泳图谱可以分离约700个蛋白质点,其中大部分的蛋白质点分布在pH4～8范围内,在分子量上主要集中在30～70kD区域：比较分析发现,有4种蛋白只在正常性附腺组织中特异表达,而有2种蛋白只在Ng突变体的组织中特异表达。另外约有29种蛋白在正常性附腺分泌部组织中的表达水平明显高于Ng突变,而约有15种蛋白在Ng突变体的分泌部组织中表达水平较高。这些差异蛋白质可能与Ng突变的形成和导致这种突变体的性附腺不能正常分泌粘性蛋白的性状有关。     

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.     

Ultrastructural Observation on Chloroplast of the Thermo sensitive Green yellow Banded Leaf Mutant in Rice (Oryza sativa ssp.indica)

The green-yellow bands interrupted discolorated transverse bands on the leaves of the thermo-sensitive rice line l103s (Oryza sativa ssp. indica ) was induced by cold shock and the change of leaf color coincided with the change of chlorophyll contents. Uhrastmctural observation revealed that the chloroplast slructure in the yellow part degenerated, i.e., the number of stocks in granum abnormally decreased, thylakoid system was disintegrated and replaced by few single thylakoid, and then the whole structure of some chloroplast became vesicle-hke structure with high electronic density. Nonetheless, in the green zone of the green-yellow banded leaf, there were slight decrease in the number granum thylakoids, disordered grana arrangement in the chloroplast, and aggregation of osmiophile globule. However, it was found that these structural alternations were reversable in the process of recovery of the yellow part of the leaves into green, and the chloroplast structure renewed normally. It might be concluded that the character of green-yellow bands induced by cold shock was consequent upon a reverse process of degeneration and regeneration.     

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.     

Mobilization of rubisco and stroma-localized fluorescent proteins of chloroplasts to the vacuole by an ATG gene-dependent autophagic process

During senescence and at times of stress, plants can mobilize needed nitrogen from chloroplasts in leaves to other organs. Much of the total leaf nitrogen is allocated to the most abundant plant protein, Rubisco. While bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have visualized the fate of Rubisco, stroma-targeted green fluorescent protein (GFP) and DsRed, and GFP-labeled Rubisco in order to investigate the involvement of autophagy in the mobilization of stromal proteins to the vacuole. Using immunoelectron microscopy, we previously demonstrated that Rubisco is released from the chloroplast into Rubisco-containing bodies (RCBs) in naturally senescent leaves. When leaves of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing stroma-targeted fluorescent proteins were incubated with concanamycin A to inhibit vacuolar H(+)-ATPase activity, spherical bodies exhibiting GFP or DsRed fluorescence without chlorophyll fluorescence were observed in the vacuolar lumen. Double-labeled immunoelectron microscopy with anti-Rubisco and anti-GFP antibodies confirmed that the fluorescent bodies correspond to RCBs. RCBs could also be visualized using GFP-labeled Rubisco directly. RCBs were not observed in leaves of a T-DNA insertion mutant in ATG5, one of the essential genes for autophagy. Stroma-targeted DsRed and GFP-ATG8 fusion proteins were observed together in autophagic bodies in the vacuole. We conclude that Rubisco and stroma-targeted fluorescent proteins can be mobilized to the vacuole through an ATG gene-dependent autophagic process without prior chloroplast destruction.     

'Senescence-associated vacuoles' are involved in the degradation of chloroplast proteins in tobacco leaves

Massive degradation of photosynthetic proteins is the hallmark of leaf senescence; however the mechanism involved in chloroplast protein breakdown is not completely understood. As small 'senescence-associated vacuoles' (SAVs) with intense proteolytic activity accumulate in senescing leaves of soybean and Arabidopsis, the main goal of this work was to determine whether SAVs are involved in the degradation of chloroplastic components. SAVs with protease activity were readily detected through confocal microscopy of naturally senescing leaves of tobacco (Nicotiana tabacum L.). In detached leaves incubated in darkness, acceleration of the chloroplast degradation rate by ethylene treatment correlated with a twofold increase in the number of SAVs per cell, compared to untreated leaves. In a tobacco line expressing GFP targeted to plastids, GFP was re-located to SAVs in senescing leaves. SAVs were isolated by sucrose density gradient centrifugation. Isolated SAVs contained chloroplast-targeted GFP and the chloroplast stromal proteins Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and glutamine synthetase, but lacked the thylakoid proteins D1 and light-harvesting complex II of the photosystem II reaction center and photosystem II antenna, respectively. In SAVs incubated at 30 degrees C, there was a steady decrease in Rubisco levels, which was completely abolished by addition of protease inhibitors. These results indicate that SAVs are involved in degradation of the soluble photosynthetic proteins of the chloroplast stroma during senescence of leaves.     

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.