Cloning and functional analysis of chloroplast division gene NtFtsZ2-l in Nicotiana tabacum

FtsZ protein plays an important role in the division of chloroplasts. With the finding and functional analysis of higher plant FtsZ proteins, people have deepened the understanding in the molecular mechanism of chloroplast division. Multiple ftsZ genes are diversified into two families in higher plants, ftsZ1 and ftsZ2 . On the basis of the research on ftsZl family, we analyzed the function of NtFtsZ2-l gene in Nicotiana tabacum . Microscopic analysis of the sense and antisense NtFtsZ2-l transgenic tobacco plants revealed that the chloroplasts were abnormal in size and also in number when compared with wild-type tobacco chloroplasts. Our investigations confirmed that the NtFtsZ2-l gene is involved in plant chloroplast division.     

Cloning of two plastid division ftsZ genes from Nicotiana tabacum and their expression in E.coli

Two cDNAs of plastid division gene NtFtsZ1-1 and NtFtsZ1-2 are isolated from Nicotiana tabacum by RT-PCR and rapid amplification cDNA ends (RACE) method. Analysis of the deduced amino acid sequences encoded by NtFtsZ1-1 and NtFtsZ1-2 indicate that these two proteins possess the typical conservative motifs and GTP binding sites existing in all FtsZ proteins. The existence of putative plastid transit peptide in their N-terminal suggests that there are at least two transit-peptide containing FtsZ proteins in higher plants. Phylogenetic analysis based on amino acid sequences of FtsZ proteins also supports this interference. These two NtFtsZ genes demonstrate a similar expression pattern during the plant development, detected by Northern blot. Expression of NtFtsZ1-1 and NtFtsZ1-2 in E.coli interrupts the normal division process of host cells. These results suggest the diverse functions of FtsZ proteins in higher plants.     

The essential bacterial cell-division protein FtsZ is a GTPase.

Cytokinesis defines the last stage in the division cycle, in which cell constriction leads to the formation of daughter cells. The biochemical mechanisms responsible for this process are poorly understood. In bacteria, the ftsZ gene product, FtsZ, is required for cell division, playing a prominent role in cytokinesis. The cellular concentration of FtsZ regulates the frequency of division and genetic studies have indicated that it is the target of several endogenous division inhibitors. At the time of onset of septal invagination, the FtsZ protein is recruited from the cytoplasm to the division site, where it assembles into a ring that remains associated with the leading edge of the invaginating septum until septation is completed. Here we report that FtsZ specifically binds and hydrolyses GTP. The reaction can be dissociated into a GTP-dependent activation stage that is markedly affected by the concentration of FtsZ, and a hydrolysis stage in which GTP is hydrolysed to GDP. The results indicate that GTP binding and hydrolysis are important in enabling FtsZ to support bacterial cytokinesis, either by facilitating the assembly of the FtsZ ring and/or by catalysing an essential step in the cytokinetic process itself.     

Escherichia coli cell-division gene ftsZ encodes a novel GTP-binding protein.

Escherichia coli divides by forming a septum across the middle of the cell. The biochemical mechanism underlying this process is unknown. Genetic evidence suggests that of all the fts (filamentation temperature sensitive) genes involved in E. coli cell division, ftsZ plays a central role at the earliest known step of septation. Here we show that FtsZ protein binds GTP in vitro using unusual sequence elements. In contrast, such binding to the product of the conditional-lethal ftsZ84 allele is impaired. Purified FtsZ displays a Mg(2+)-dependent GTPase activity which is markedly reduced in the FtsZ84 protein. FtsZ copurifies with near stoichiometric amounts of noncovalently-bound GDP, implying the presence of a GTPase cycle in vivo, similar to that known for signal-transducing GTP-binding proteins. We also show that a small fraction of FtsZ exists as a distinct membrane-associated species that binds GTP. The membrane association of FtsZ and the known ability of GTPases to act as molecular switches implicate FtsZ in a GTP-activated signal transduction pathway that may regulate the start of septation in E. coli.     

FtsZ ring structure associated with division in Escherichia coli

Genes for cell division have been identified in Escherichia coli by the isolation of conditional lethal mutations that block cell division, but do not affect DNA replication or segregation. Of these genes, ftsZ is of great interest as it acts earliest in the division pathway, is essential, its level dictates the frequency of division, and it is thought to be the target of two cell-division inhibitors, SulA, produced in response to DNA damage, and MinCD, which prevents division at old sites. Here we have used immunoelectronmicroscopy to localize the FtsZ protein to the division site. The results suggest that FtsZ self-assembles into a ring structure at the future division site and may function as a cytoskeletal element. The formation of this ring may be the point at which division is regulated.     

一种新型叶绿体表达系统的构建

针对目前的叶绿体转基因技术存在载体构建步骤繁琐耗时的问题,建立了一种新型的不需自带启动子的叶绿体表达系统,选择模式植物烟草叶绿体中的假基因作为外源基因的插入位点.通过基因枪转化法获得4株独立的叶绿体转化植株.此新技术可实现叶绿体载体构建的时间、技术成本最小化,从而促进转基因叶绿体的应用.     

Phototropin-related NPL1 controls chloroplast relocation induced by blue light

In photosynthetic cells, chloroplasts migrate towards illuminated sites to optimize photosynthesis and move away from excessively illuminated areas to protect the photosynthetic machinery. Although this movement of chloroplasts in response to light has been known for over a century, the photoreceptor mediating this process has not been identified. The Arabidopsis gene NPL1 (ref. 2) is a paralogue of the NPH1 gene, which encodes phototropin, a photoreceptor for phototropic bending. Here we show that NPL1 is required for chloroplast relocation induced by blue light. A loss-of-function npl1 mutant showed no chloroplast avoidance response in strong blue light, whereas the accumulation of chloroplasts in weak light was normal. These results indicate that NPL1 may function as a photoreceptor mediating chloroplast relocation.     

转SOD基因烟草的光合特性初探

 以转基因Fe-SOD,Mn-SOD高表达、转基因Mn-SOD反义低表达烟草及非转基因品系为供试材料,比较CO₂光强对烟草光合作用和蒸腾作用的影响及不同叶位叶片的光合差异.在相同条件下,Mn-SOD高表达烟草CO₂补偿点最高(105μmol·mol^-1),Mn-SOD低表达烟草最低(78μmol·mol^-1),CO₂浓度对非转基因烟草蒸腾作用的影响比对其他三个转基因品系的影响要强.Mn-SOD低表达烟草的光补偿点最高(45μmol·m^-2·s^-1),而Mn-SOD高表达烟草最低(25μmol·m^-2·s^-1),并且强光对非转基因烟草的光合作用抑制大,转基因品系对强光具有较强的耐受力.同一叶位叶片,转基因烟草的光合速率、蒸腾速率更强.但转基因SOD高表达烟草在整体上并没有表现出明显的光合作用优势.     

Responses of ferns to red light are mediated by an unconventional photoreceptor

Efficient photosynthesis is essential for plant survival. To optimize photosynthesis, plants have developed several photoresponses. Stems bend towards a light source (phototropism), chloroplasts move to a place of appropriate light intensity (chloroplast photorelocation) and stomata open to absorb carbon dioxide. These responses are mediated by the blue-light receptors phototropin 1 (phot1) and phototropin 2 (phot2) in Arabidopsis (refs 1-5). In some ferns, phototropism and chloroplast photorelocation are controlled by red light as well as blue light. However, until now, the photoreceptor mediating these red-light responses has not been identified. The fern Adiantum capillus-veneris has an unconventional photoreceptor, phytochrome 3 (phy3), which is a chimaera of the red/far-red light receptor phytochrome and phototropin. We identify here a function of phy3 for red-light-induced phototropism and for red-light-induced chloroplast photorelocation, by using mutational analysis and complementation. Because phy3 greatly enhances the sensitivity to white light in orienting leaves and chloroplasts, and PHY3 homologues exist among various fern species, this chimaeric photoreceptor may have had a central role in the divergence and proliferation of fern species under low-light canopy conditions.     

Single gene circles in dinoflagellate chloroplast genomes.

Photosynthetic dinoflagellates are important aquatic primary producers and notorious causes of toxic 'red tides'. Typical dinoflagellate chloroplasts differ from all other plastids in having a combination of three envelope membranes and peridinin-chlorophyll a/c light-harvesting pigments. Despite evidence of a dinoflagellete satellite DNA containing chloroplast genes, previous attempts to obtain chloroplast gene sequences have been uniformly unsuccessful. Here we show that the dinoflagellate chloroplast DNA genome structure is unique. Complete sequences of chloroplast ribosomal RNA genes and seven chloroplast protein genes from the dinoflagellate Heterocapsa triquetra reveal that each is located alone on a separate minicircular chromosome: 'one gene-one circle'. The genes are the most divergent known from chloroplast genomes. Each circle has an unusual tripartite non-coding region (putative replicon origin), which is highly conserved among the nine circles through extensive gene conversion, but is very divergent between species. Several other dinoflagellate species have minicircular chloroplast genes, indicating that this type of genomic organization may have evolved in ancestral peridinean dinoflagellates. Phylogenetic analysis indicates that dinoflagellate chloroplasts are related to chromistan and red algal chloroplasts and supports their origin by secondary symbiogenesis.     

适于双向电泳分析的银杏叶绿体蛋白质提取方法

【目的】获得银杏叶绿体蛋白质的提取方法,在蛋白质水平探讨银杏光合作用对环境的响应机制。【方法】建立一种适合银杏叶绿体蛋白双向电泳分离的实验体系,采用Tris-平衡酚抽提法对银杏叶绿体蛋白质进行提取,并与三氯乙酸(TCA)-丙酮沉降法进行对比分析。【结果】Percoll密度梯度离心法适用于银杏叶绿体的提取,提取的叶绿体平均完整率在85%左右。单向电泳结果显示,与TCA-丙酮沉降法相比,在用Tris-平衡酚抽提法分离的叶绿体蛋白质泳道中,低分子质量蛋白质条带更多、更清晰。进一步的双向电泳结果表明,用Tris-平衡酚抽提法对银杏叶绿体蛋白质进行提取,蛋白质产量更高,图谱清晰,所分离的蛋白点更多,形态更好,条纹影响相对较小。【结论】Tris-平衡酚抽提法可有效地提取高质量的叶绿体蛋白质并进行双向电泳,可用于银杏叶绿体的蛋白质组学分析。     

大麦叶绿体分裂的研究

报道了大麦叶绿体分裂的全过程。发现叶绿体是在长轴的中部或近中部产生缢痕、逐渐紧缩并断裂为两个子叶绿体的。并对遗传性黄化大麦叶绿体的结构特征、叶绿体分裂机制、同步性及子叶绿体的大小等进行讨论。     

Synthesis of medium-chain-length-polyhydroxyalkanoates in tobacco via chloroplast genetic engineering

Medium-chain-length-polyhydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters containing monomers ranging from 6 to 14 carbons in length. The key enzymes of their biosynthesis are PHA-polymerase (product of phaC gene) and 3-hydroxyacyl-acyl carrier protein-CoA transferase (product of phaG gene). With aadA (aminoglycoside 3‘-adenylyltransferase) gene as screening marker, two chloroplast transformation vectors of pTC2 harboring phaC2 gene only and pTGC harboring both phaC and phaG genes were constructed and introduced into tobacco chloroplast genome through particle bombardment. PCR and Southern blot analysis confirmed the insertion of the introduced genes into chloroplast genome. The content of mcl-PHAs accumulated in transgenic plants was analyzed by gas chromatography, mcl-PHAs accumulated up to 4.8 mg/g dry weight (dw) in transgenic line $4-3; their monomers were 3-hydroxyoctanoate and 3-hydroxydecanoate. Accumulation of mcl-PHAs polymers in the tobacco chloroplast was also observed by transmission electron microscopy. To our knowledge, this is the first report on the synthesis of mclPHAs in tobacco via chloroplast genetic engineering.     

Cloning of plastid division gene GlFtsZ from Gentiana lutea and its expression during petal development

A full-length cDNA of GlFtsZ was isolated by screening the cDNA library of Gentiana lutea. Analysis of the deduced amino acid sequence encoded by GlFtsZ indicated that GlFtsZ protein possesses the typical conservative motifs existed in all FtsZ proteins. The existence of putative plastid transit peptide in its N-terminus suggested that GlFtsZ might function inside of plastids. With the deve- lopmental process of petals of Gentiana lutea, the expression of plastid division gene GlFtsZ declined gradually, whereas the expression of carotenoids biosynthesis gene Zds increased obviously; meanwhile, in contrast to the increment of carotenoids, the content of chlorophyll in petals decreased sharply. The chloroplasts turned into chromoplasts, and the color of petals also turned from green to golden. All of these results suggested that the expression of GlFtsZ is accompanied with the development and differentiation of plastids.     

<Emphasis Type="Italic">ftsZ</Emphasis> gene and plastid division

As the important cellular organelles in plants, plas-tids comprise one of the primary features that distinguish plant cells from those of other eukaryotes. Seen from the origin, plastids derive from endosymbiotic photosynthetic bacteria. Subsequently, plastids have evolved to become essential components for plant cell function. Besides the important role of chloroplasts in photosynthesis, some water-soluble proteins that involved in biosynthesis of starch, fatty acids, amino acids, nucleic aci…     

Homologous plant and bacterial proteins chaperone oligomeric protein assembly

An abundant chloroplast protein is implicated in the assembly of the oligomeric enzyme ribulose bisphosphate carboxylase-oxygenase, which catalyses photosynthetic CO2-fixation in higher plants. The product of the Escherichia coli groEL gene is essential for cell viability and is required for the assembly of bacteriophage capsids. Sequencing of the groEL gene and the complementary cDNA encoding the chloroplast protein has revealed that these proteins are evolutionary homologues which we term 'chaperonins'. Chaperonins comprise a class of molecular chaperones that are found in chloroplasts, mitochondria and prokaryotes. Assisted post-translational assembly of oligomeric protein structures is emerging as a general cellular phenomenon.     

拟南芥中一个镍离子转运蛋白的亚细胞定位

离子的跨膜转运是细胞获取养分的重要环节，亦是植物在组织和器官水平上进行养分吸收运移的基础．在植物中镍（Ni）元素主要以Ni^2＋的形式存在，并通过Ni^2＋转运蛋白将其跨膜转运至相应的组织器官，参与氢酶和脲酶的合成．生物信息学分析表明，拟南芥中一个Ni^2＋转运蛋白AT2G16800含有叶绿体定位信息．克隆该基因5’端编码转运肽的272bp片段，与绿色荧光蛋白（GFP）基因融合后，在拟南芥中高效表达，对其进行了亚细胞定位的研究．转基因植株通过共聚焦扫描显微镜的观察，发现GFP荧光信号只存在于叶绿体中，该结果表明A他G16800为叶绿体蛋白．     

Cyclin D control of growth rate in plants

The mechanisms by which plants modulate their growth rate in response to environmental and developmental conditions are unknown, but are presumed to involve specialized regions called meristems where cell division is concentrated. The possible role of cell division in influencing meristem activity and overall plant growth rate is controversial, with a prevailing view that cell division is secondary to higher order meristem controls. Here we show that a reduction in the length of the cell-cycle G1 phase and faster cell cycling occur when the rate of cell division in transgenic tobacco plants is increased by the plant D-type cyclin CycD2 (ref. 8). The plants have normal cell and meristem sizes, but elevated overall growth rates, an increased rate of leaf initiation and accelerated development in all stages from seedling to maturity. We conclude that cell division is a principal determinant of meristem activity and overall growth rate, and propose that modulation of plant growth rate is achieved through regulation of G1.     

Transfer of a eubacteria-type cell division site-determining factor CrldfnD 8ene to the nucleus from the chloroplast genome in Chlamydomonas reinhardtii

MinD is a ubiquitous ATPase that plays a crucial role in selection of the division site in eubacteria, chloroplasts, and probably Archaea. In four green algae, MesosUgma viride, Nephroselmis olivacea, Chlorella vulgaris and Prototheca wickerhamii, MinD homologues are encoded in the plastid genome. However, in Arabidopsis, MinD is a nucleus-encoded, chloroplast-targeted protein involved in chloroplast division, which suggests that MinD has been transferred to the nucleus in higher land plants. Yet the lateral gene transfer （LGT） of MinD from plastid to nucleus during plastid evolution remains poorly understood. Here, we identified a nucleus-encoded MinD homologue from unicellular green alga Chlamydomonas reinhardtii, a basal species in the green plant lineage. Overexpression of CrMinD in wild type E. coil inhibited cell division and resulted in the filamentous cell formation, clearly demonstrated the conservation of the MinD protein during the evolution of photosynthetic eukaryotes. The transient expression of CrMinD-egfp confirmed the role of CrMinD protein in the regulation of plastid division. Searching all the published plastid genomic sequences of land plants, no MinD homologues were found, which suggests that the transfer of MinD from plastid to nucleus might have occurred before the evolution of land plants.     

Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast

The use of plants as bioreactors for the expression oftherapeutic proteins has developed into a new field in biotechnology research[1]. Plant systems provide simple genetic manipulation, low production costs, and low risk of contamination by animal viruse…