高等植物叶绿体和线粒体免疫亲近性的研究

以火箭免疫电泳分析表明：大豆叶绿体抗体与大豆线粒体有免疫交叉反应,同时大豆线粒体抗体与大豆叶绿体也有免疫交叉反应,但是大豆线粒体的抗体与鼠肝线粒体之间无免疫交叉反应。这说明高等植物线粒体对叶绿体比之对动物线粒体在免疫特性上有更大的亲近性;亦即高等植物线粒体和高等植物的叶绿体有更大的同源性。经火箭免疫电泳,交叉免疫电泳和线状免疫电泳进一步分析表明：菠菜偶联因子抗体和大豆线粒体,大豆叶绿体间,大豆线粒     

植物细胞核DNA,叶绿体DNA和线粒体DNA的比较

植物一般有细胞核,叶绿体和线粒体三套遗传体系,本文结合近年来植物分子生物学研究的最新进展,系统比较了细胞核ＤＮＡ,叶绿体ＤＮＡ和线粒体ＤＮＡ在组织结构,遗传方式,基因表达（转录,翻译,ＲＮＡ加工）等方面的差异。     

高等植物叶绿体和线粒体免疫亲近性的研究

以火箭免疫电泳分析表明:大豆叶绿体抗体与大豆线粒体有免疫交叉反应,同时大豆线粒体抗体与大豆叶绿体也有免疫交叉反应,但是大豆线粒体的抗体与鼠肝线粒体之间无免疫交叉反应。这说明高等植物线粒体对叶绿体比之对动物线粒体在免疫特性上有更大的亲近性,亦即高等植物线粒体和高等植物的叶绿体有更大的同源性。经火箭免疫电泳、交叉免疫电泳和线状免疫电泳进一步分析表明:菠菜偶联因子抗体(AbCF_1)和大豆线粒体、大豆叶绿体间,大豆线粒体抗体与CF_1和大豆叶绿体之间,以及大豆叶绿体的抗体(AbC)与CF_1和大豆线粒体间有免疫交叉反应,说明两种换能器之间有免疫亲近性,并分别与CF_1存在免疫亲近性。这揭示两种换能器免疫亲近性的表现是由于存在共同物质基础所致,这内在共同物质基础是偶联因子。这个结果有力地支持高等植物叶绿体和线粒体在结构和功能上以及发生上存在同源性的观点,在理论上也为两种换能器的起源和演化上存在同源性提供了一些依据。     

高山植物叶绿体与线粒体位置相关性研究

利用透射电镜对生长于青藏高原东北部达坂山(海拔3900m)的4种高山植物叶肉细胞进行了超微结构观察。首次在蒲公英(Taraxacum mongolicum)和乳白香青(Anaphalis lacten)的叶肉细胞中发现了叶绿体“吞噬”线粒体的现象。在所研究的4种高山植物中,线粒体的数量均较多,线粒体在细胞中的分布表现出不均一,且常分布在叶绿体附近,二者靠的很紧,常常可以观察到5～6个线粒体将叶绿体包围起来的现象。研究表明,高山植物叶肉细胞中叶绿体和线粒体在位置上的这种变化是对逆境的一种适应,是青藏高原特殊生态条件长期胁迫的结果。     

植物叶绿体和线粒体的超微弱发光

介绍叶绿体和线粒体的超弱发光基础,以及各自的发光特点与影响因素.     

高等植物叶绿体基因工程

叶绿体基因工程作为一项新技术具有一系列传统核基因工程所不具备的优点,在基础性及应用性研究中极具吸引力,已经成功应用于了解质体基因组,调控植物代谢系统,农作物抗旱、抗虫、抗病、抗除草剂及以植物为生物反应器生产抗体、疫苗等方面的研究.本文主要介绍叶绿体基因工程的原理、操作体系及其在高等植物中的应用.     

烟草叶绿体基因组和线粒体基因组SSR位点分析

利用生物信息学方法对烟草叶绿体和线粒体基因组数据中的SSR信息进行了分析.结果表明,在叶绿体和线粒体基因组中分别获得186和578个SSR位点,SSR间的平均距离分别为838 bp和745 bp.在SSR的分布区域上,绝大多数SSR位点分布在UTR(尤其是5’UTR)区域;在SSR重复碱基类型上,主要集中在二、三碱基重复,二者占总SSR位点的90％以上,其中三碱基重复类型丰度最高.利用全部657对SSR引物在供试的10份烟草材料中进行扩增,发现所有引物均能获得目的片段,但在普通烟草内品种间并未检测到多态性,而在烟草种间有26对叶绿体基因组SSR引物和178对线粒体基因组SSR引物扩增出多态性条带,表明来源于烟草叶绿体基因组和线粒体基因组的SSR标记适合用于烟草种间进化、分类、遗传多样性等方面研究.     

高等植物叶绿体基因组转化的应用

叶绿体基因组转化技术由于其独特的优越性,现已成为植物基因工程的研究热点。本文简单介绍了叶绿体基因组转化技术的原理和方法;并重点综述了该技术在基础研究和实践中的应用。这些应用主要包括利用叶绿体基因组转化技术进行Rubisco的组装,叶绿体基因结构、转录、翻译和RNA编辑等研究;利用叶绿体作为生物反应器生产人生长激素、霍乱毒素抗体、聚羟基丁酸脂和生物弹性蛋白等;获得抗虫、抗病、抗除草剂和耐旱的转基因植物;以及降低转基因植物的外源基因扩散等。     

Dual Targeting to Mitochondria and Chloroplasts： Characterization of Thr-tRNA Synthetase Targeting Peptide

There is a group of proteins that are encoded by a single gene, expressed as a single precursor protein and dually targeted to both mitochondria and chloroplasts using an ambiguous targeting peptide. Sequence analysis of 43 dual targeted proteins in comparison with 385 mitochondrial proteins and 567 chloroplast proteins ofArabidopsis thaliana revealed an overall significant increase in phenylalanines, leucines, and serines and a decrease in acidic amino acids and glycine in dual targeting peptides （dTPs）. The N-terminal portion of dTPs has significantly more serines than mTPs. The number of arginines is similar to those in mTPs, but almost twice as high as those in cTPs. We have investigated targeting determinants of the dual targeting peptide of Thr-tRNA synthetase （ThrRS-dTP） studying organellar import of N- and C-terminal deletion constructs of ThrRS-dTP coupled to GFR These results show that the 23 amino acid long N-terminal portion of ThrRS-dTP is crucial but not sufficient for the organellar import. The C-terminal deletions revealed that the shortest peptide that was capable of conferring dual targeting was 60 amino acids long. We have purified the ThrRS- dTP（2-60） to homogeneity after its expression as a fusion construct with GST followed by CNBr cleavage and ion exchange chromatography. The purified ThrRS-dTP（2-60） inhibited import of pF1β into mitochondria and of pSSU into chloroplasts at μM concentrations showing that dual and organelle-specific proteins use the same organellar import pathways. Furthermore, the CD spectra of ThrRS-dTP（2-60） indicated that the peptide has the propensity for forming α-helical structure in membrane mimetic environments; however, the membrane charge was not important for the amount of induced helical structure. This is the first study in which a dual targeting peptide has been purified and investigated by biochemical and biophysical means.     

Import Determinants of Organelle-Specific and Dual Targeting Peptides of Mitochondria and Chloroplasts in Arabidopsis thaliana

Most of the mitochondrial and chloroplastic proteins are synthesized in the cytosol as precursor proteins carrying an N-terminal targeting peptide （TP） directing them specifically to a correct organelle. However, there is a group of proteins that are dually targeted to mitochondria and chloroplasts using an ambiguous N-terminal dual targeting peptide （dTP）. Here, we have investigated pattern properties of import determinants of organelle-specific TPs and dTPs combining mathematical multivariate data analysis （MVDA） with in vitro organellar import studies. We have used large datasets of mitochondrial and chloroplastic proteins found in organellar proteomes as well as manually selected data sets of experimentally confirmed organelle-specific TPs and dTPs from Arabidopsis thaliana. Two classes of organelle-specific TPs could be distinguished by MVDA and potential patterns or periodicity in the amino acid sequence contributing to the separation were revealed, dTPs were found to have intermediate sequence features between the organelle-specific TPs. Interestingly, introducing positively charged residues to the dTPs showed clustering towards the mitochondrial TPs in silico and resulted in inhibition of chloroplast, but not mitochondrial import in in vitro organellar import studies. These findings suggest that positive charges in the N-terminal region of TPs may function as an ＇avoidance signal＇ for the chloroplast import.     

干旱胁迫对3种不同光合类型荒漠植物叶绿体和线粒体超微结构的影响

以荒漠木本C_3植物天山猪毛菜、C_3-C_4中间型植物松叶猪毛菜、C_4植物木本猪毛菜为研究对象,采用盆栽控水试验,设置正常供水和轻度、中度和重度干旱处理(土壤含水量分别为田间持水量的80%、60%、45%和35%),研究不同程度干旱胁迫对3种不同光合类型荒漠植物叶片超微结构的影响。结果表明:(1)正常水分条件下,叶肉细胞中各细胞器结构完整。(2)轻度干旱胁迫下,3种植物叶片超微结构未受损伤,无明显变化。(3)中度干旱胁迫下,天山猪毛菜和松叶猪毛菜叶肉细胞壁界限不清晰,类囊体片层扩张且排列不紧密,不同之处在于,天山猪毛菜线粒体最先出现降解,内含物流失,而松叶猪毛菜线粒体外膜轮廓变形,嵴减少;木本猪毛菜线粒体无明显变化,叶绿体轻微扩张。(4)重度干旱胁迫下,天山猪毛菜和松叶猪毛菜叶绿体受损且结构混乱,线粒体出现降解;木本猪毛菜叶绿体出现膨胀,线粒体外膜轮廓模糊,嵴减少且结构模糊不清楚。研究认为,不同程度干旱胁迫下木本猪毛菜叶绿体和线粒体的受损程度都最低;干旱胁迫下天山猪毛菜和松叶猪毛菜叶绿体的受损程度大致相似;松叶猪毛菜和木本猪毛菜线粒体对干旱胁迫的耐受力要比叶绿体强。     

植物叶绿体发育及调控研究进展

植物的光合作用几乎是所有生物生存和发展的物质基础。叶绿体是绿色植物进行光合作用的重要细胞器。尽管叶绿体发育及调控一直受到人们的关注,但其装备及调控的分子机制尚不完全清楚。该文对叶绿体装备过程、叶绿体发育调控及质体-细胞核反向信号的研究进展进行概述,以使人们从整体上认识叶绿体发育及调控机制。     

干旱胁迫对5种植物叶绿体和线粒体超微结构的影响

应用温室盆栽方法,研究了土壤干旱胁迫对麻栎（Quercus acutissima Carruth）、黄檀（Dalbergia hupeana Hance）、黄连木(Pistacia chinensis)、湿地松（Pinus elliottii）、朴树（Celtis sinesis Pers）5个树种叶肉细胞超微构的影响。结果表明：正常水分条件下,叶肉细胞中各细胞器结构完整。轻度干旱胁迫下,湿地松的叶片超微结构未受损伤。麻栎线粒体无明显变化,叶绿体有扩张现象。黄连木与黄檀线粒体外膜有降解现象,叶绿体膨胀。朴树线粒体与叶绿体受损明显。重度胁迫下,湿地松和麻栎的线粒体内部出现降解,叶绿体受损。黄连木与黄檀出现质壁分离,叶绿体与线粒体受到严重损伤。朴树细胞内部受损最严重。可将5个树种分为3种不同的抗旱等级：湿地松与麻栎抗旱性较强,黄连木与黄檀抗旱性中等,朴树抗旱性较弱。     

哺乳动物核移植中线粒体命运

线粒体是哺乳动物细胞中一种重要的产能、供能细胞器,与生长、发育、衰老和凋亡等多种细胞事件以及多种疾病有关.哺乳动物核移植中,供体细胞和受体卵胞质两种来源的线粒体在重构胚胎发育进程中的变化一直是科学家们研究的热点.对哺乳动物同种胚胎细胞核移植、同种体细胞核移植、异种核移植研究中线粒体的变化进行了综述.     

Thylakoid Protein Phosphorylation in Higher Plant Chloroplasts Optimizes Electron Transfer under Fluctuating Light

Several proteins of photosystem II (PSII) and its light-harvesting antenna (LHCII) are reversibly phosphorylated according to light quantity and quality. Nevertheless, the interdependence of protein phosphorylation, nonphotochemical quenching, and efficiency of electron transfer in the thylakoid membrane has remained elusive. These questions were addressed by investigating in parallel the wild type and the stn7, stn8, and stn7 stn8 kinase mutants of Arabidopsis (Arabidopsis thaliana), using the stn7 npq4, npq4, npq1, and pgr5 mutants as controls. Phosphorylation of PSII-LHCII proteins is strongly and dynamically regulated according to white light intensity. Yet, the changes in phosphorylation do not notably modify the relative excitation energy distribution between PSII and PSI, as typically occurs when phosphorylation is induced by “state 2” light that selectively excites PSII and induces the phosphorylation of both the PSII core and LHCII proteins. On the contrary, under low-light conditions, when excitation energy transfer from LHCII to reaction centers is efficient, the STN7-dependent LHCII protein phosphorylation guarantees a balanced distribution of excitation energy to both photosystems. The importance of this regulation diminishes at high light upon induction of thermal dissipation of excitation energy. Lack of the STN7 kinase, and thus the capacity for equal distribution of excitation energy to PSII and PSI, causes relative overexcitation of PSII under low light but not under high light, leading to disturbed maintenance of fluent electron flow under fluctuating light intensities. The physiological relevance of the STN7-dependent regulation is evidenced by severely stunted phenotypes of the stn7 and stn7 stn8 mutants under strongly fluctuating light conditions.Several proteins of PSII and its light-harvesting antenna (LHCII) are reversibly phosphorylated by the STN7 and STN8 kinase-dependent pathways according to the intensity and quality of light (Bellafiore et al., 2005; Bonardi et al., 2005). The best-known phosphorylation-dependent phenomenon in the thylakoid membrane is the state transition: a regulatory mechanism that modulates the light-harvesting capacity between PSII and PSI. According to the traditional view, “state 1” prevails when plants are exposed to far-red light (state 1 light), which selectively excites PSI. Alternatively, thylakoids are in “state 2” when plants are exposed to blue or red light (state 2 light), favoring PSII excitation. In state 1, the yield of fluorescence from PSII is higher in comparison with state 2 (for review, see Allen and Forsberg, 2001). State transitions are dependent on the phosphorylation of LHCII proteins (Bellafiore et al., 2005) and their association with PSI proteins, particularly PSI-H (Lunde et al., 2000). Under state 2 light, both the PSII core and LHCII proteins are strongly phosphorylated, whereas the state 1 light induces dephosphorylation of both the PSII core and LHCII phosphoproteins (Piippo et al., 2006; Tikkanen et al., 2006). In nature, however, such extreme changes in light quality rarely occur. The intensity of light, on the contrary, fluctuates frequently in all natural habitats occupied by photosynthetic organisms, thus constantly modulating the extent of thylakoid protein phosphorylation in a highly dynamic manner (Tikkanen et al., 2008a).The regulation of PSII-LHCII protein phosphorylation by the quantity of light is much more complex than the regulatory circuits induced by the state 1 and state 2 lights. Whereas changes in light quality induce a concurrent increase or decrease in the phosphorylation levels of both the PSII core (D1, D2, and CP43) and LHCII (Lhcb1 and Lhcb2) proteins, the changes in white light intensity may influence the kinetics of PSII core and LHCII protein phosphorylation in higher plant chloroplasts even in opposite directions (Tikkanen et al., 2008a). Indeed, it is well documented that low light (LL; i.e. lower than that generally experienced during growth) induces strong phosphorylation of LHCII but relatively weak phosphorylation of the PSII core proteins. Exposure of plants to high light (HL) intensities, on the contrary, promotes the phosphorylation of PSII core proteins but inhibits the activity of the LHCII kinase, leading to dephosphorylation of LHCII proteins (Rintamäki et al., 2000; Hou et al., 2003).Thylakoid protein phosphorylation induces dynamic migrations of PSII-LHCII proteins along the thylakoid membrane (Bassi et al., 1988; Iwai et al., 2008) and modulation of thylakoid ultrastructure (Chuartzman et al., 2008). According to the traditional state transition theory, the phosphorylation of LHCII proteins decreases the antenna size of PSII and increases that of PSI, which is reflected as a quenched fluorescence emission from PSII. Alternatively, subsequent dephosphorylation of LHCII increases the antenna size of PSII and decreases that of PSI, which in turn is seen as increased PSII fluorescence (Bennett et al., 1980; Allen et al., 1981; Allen and Forsberg, 2001). This view was recently challenged based on studies with thylakoid membrane fractions, revealing that modulations in the relative distribution of excitation energy between PSII and PSI by LHCII phosphorylation specifically occur in the areas of grana margins, where both PSII and PSI function under the same antenna system, and the energy distribution between the photosystems is regulated via a more subtle mechanism than just the robust migration of phosphorylated LHCII (Tikkanen et al., 2008b). It has also been reported that most of the PSI reaction centers are located in the grana margins in a close vicinity to PSII-LHCII-rich grana thylakoids (Kaftan et al., 2002), providing a perfect framework for the regulation of excitation energy distribution from LHCII to both PSII and PSI.When considering the natural light conditions, the HL intensities are the only known light conditions that in higher plant chloroplasts specifically dephosphorylate only the LHCII proteins but not the PSII core proteins. However, such light conditions do not lead to enhanced function of PSII. Instead, the HL conditions strongly down-regulate the function of PSII via nonphotochemical quenching of excitation energy (NPQ) and PSII photoinhibition (for review, see Niyogi, 1999). On the other hand, after dark acclimation of leaves and relaxation of NPQ, PSII functions much more efficiently when plants/leaves are transferred to LL despite strong phosphorylation of LHCII, as compared with the low phosphorylation state of LHCII upon transfer to HL conditions.The delicate regulation of thylakoid protein phosphorylation in higher plant chloroplasts according to prevailing light intensity is difficult to integrate with the traditional theory of state transitions (i.e. the regulation of the absorption cross-section of PSII and PSI by reversible phosphorylation of LHCII). Moreover, besides LHCII proteins, reversible phosphorylation of the PSII core proteins may also play a role in dynamic light acclimation of plants. Recently, we demonstrated that the PSII core protein phosphorylation is a prerequisite for controlled turnover of the PSII reaction center protein D1 upon photodamage (Tikkanen et al., 2008a). This, however, does not exclude the possibility that the strict regulation of PSII core protein phosphorylation is also connected to the regulation of light harvesting and photosynthetic electron transfer. Moreover, the interactions between PSII and LHCII protein phosphorylation, nonphotochemical quenching, and cyclic electron flow around PSI in the regulation of photosynthetic electron transfer reactions remain poorly understood. To gain a deeper insight into such regulatory networks, we explored the effect of strongly fluctuating white light on chlorophyll (chl) fluorescence in Arabidopsis (Arabidopsis thaliana) mutants differentially deficient in PSII-LHCII protein phosphorylation and/or the regulatory systems of NPQ.     

Updated Progress on Group II Intron Splicing Factors in Plant Chloroplasts

Group II introns are large catalytic RNAs (ribozymes) in the bacteria and organelle genomes of several lower eukaryotes. Many critical photosynthesis-related genes in the plant chloroplast genome also contain group II introns, and their splicing is critical for chloroplast biogenesis and photosynthesis processes. The structure of chloroplast group II introns was altered during evolution, resulting in the loss of intron self-splicing. Therefore, the assistance of protein factors was required for their splicing processes. As an increasing number of studies focus on the mechanism of chloroplast intron splicing; many new nuclear-encoded splicing factors that are involved in the chloroplast intron splicing process have been reported. This report reviewed the research progress of the updated splicing factors found to be involved in the splicing of chloroplast group II introns. We discuss the main problems that remain in this research field and suggest future research directions.     

Peptide Chain Initiation in Bacteria,Mitochondria and Chloroplasts

Abstract

Ribosomes prepared from the bacterium Bacillus subtilis, from mitochondria of Neurospora crassa and from chloroplasts of Euglena gracilis carry out the ApUpG-dependent reactions for peptide chain initiation demonstrated for Escherichia coli ribosomes. Initiation factors functionally similar to those from E. coli appear to be present on the above reported ribosomal preparations. The activity of ribosomes washed with high concentrations of ammonium chloride may be restored by adding initiation factors prepared from E. coli.