四吡咯代谢中间产物在植物质体向细胞核信号转导中的作用

在植物细胞内,除了顺向的信号转导通路,即核基因控制着质体基因的转录和翻译之外,还存在着逆向的信号转导通路,即质体的代谢状况作为一种信号去调控核基因的表达。过去对这条逆向的信号转导通路,亦称质体因子,研究得非常少。近几年来,随着对基因组解偶联突变体的深入研究,人们对这条通路的认识大大加深了。现着重介绍质体中的四吡咯代谢中间产物参与信号的产生,以及质体向细胞质搬运这些中间产物启动了对编码质体蛋白的核基因的表达调控。     

Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme

We affinity-purified the tobacco plastid-encoded plastid RNA polymerase (PEP) complex by the alpha subunit containing a C-terminal 12 x histidine tag using heparin and Ni(2+) chromatography. The composition of the complex was determined by mass spectrometry after separating the proteins of the >900 kDa complex in blue native and SDS polyacrylamide gels. The purified PEP contained the core alpha, beta, beta', beta" subunits and five major associated proteins of unknown function, but lacked sigma factors required for promoter recognition. The holoenzyme efficiently recognized a plastid psbA promoter when it was reconstituted from the purified PEP and recombinant plastid sigma factors. Reconstitution of a plastid holoenzyme with individual sigma factors will facilitate identification of sigma factor-specific promoter elements.     

DCL is a plant-specific protein required for plastid ribosomal RNA processing and embryo development

The defective chloroplast and leaf-mutable (dcl-m) mutation of tomato blocks chloroplast differentiation in leaf mesophyll cells and a signaling system that appears to be required for morphogenesis of palisade cells during leaf growth. To dissect the function of DCL, mutants with stable dcl alleles (dcl-s) were generated and examined for their phenotype. DCL/dcl-s plant produce dcl-s/dcl-s seeds with embryos arrested at the globular stage of development. The levels of several chloroplast- and nuclear-encoded proteins are strongly reduced in dcl-m mutant leaf sectors without significant changes in their corresponding mRNAs. The 4.5S rRNA fails to be processed efficiently, however, suggesting that DCL has a direct or indirect function in rRNA processing or correct ribosome assembly. Accordingly, chloroplasts in dcl-m sectors are impaired in polysome assembly, which can explain the reduced accumulation of chloroplast-encoded proteins. These results suggest that DCL is required for chloroplast rRNA processing, and emphasize the importance of plastid function during embryogenesis.     

植物叶绿体分裂的分子机制

叶绿体是植物细胞内一种重要的细胞器．它不仅是光合作用的场所,还是其它多种中间代谢的场所．叶绿体起源于蓝细菌,与其原核祖先类似,通过二分裂方式进行增殖．最近的研究表明,叶绿体的分裂装置包含原核起源和真核起源的蛋白质,它们在叶绿体的内膜内侧和外膜外侧协同作用以完成叶绿体的分裂．在过去十几年里,包括丝状温度敏感蛋白Z（FtsZ）、Min系统蛋白、质体分裂蛋白（PDV）和ARC蛋白等在内的多个叶绿体分裂相关组分被分离鉴定．本文简要介绍了叶绿体分裂装置各成员的发现、叶绿体被膜的收缩和叶绿体分裂位点的选择机制．另外,植物发育过程中叶绿体分裂可能受到细胞的控制,但目前对细胞如何调控叶绿体分裂知之甚少．本文对该领域的最新研究进展也进行了综述．     

Phylogeny of flowering plants by the chloroplast genome sequences: in search of a “lucky gene”

One of the most complicated remaining problems of molecular-phylogenetic analysis is choosing an appropriate genome region. In an ideal case, such a region should have two specific properties: (i) results of analysis using this region should be similar to the results of multigene analysis using the maximal number of regions; (ii) this region should be arranged compactly and be significantly shorter than the multigene set. The second condition is necessary to facilitate sequencing and extension of taxons under analysis, the number of which is also crucial for molecular phylogenetic analysis. Such regions have been revealed for some groups of animals and have been designated as "lucky genes". We have carried out a computational experiment on analysis of 41 complete chloroplast genomes of flowering plants aimed at searching for a "lucky gene" for reconstruction of their phylogeny. It is shown that the phylogenetic tree inferred from a combination of translated nucleotide sequences of genes encoding subunits of plastid RNA polymerase is closest to the tree constructed using all protein coding sites of the chloroplast genome. The only node for which a contradiction is observed is unstable according to the different type analyses. For all the other genes or their combinations, the coincidence is significantly worse. The RNA polymerase genes are compactly arranged in the genome and are fourfold shorter than the total length of protein coding genes used for phylogenetic analysis. The combination of all necessary features makes this group of genes main candidates for the role of "lucky gene" in studying phylogeny of flowering plants.     

Chloroplast Genetic Engineering: Recent Advances and Future Perspectives

Chloroplast genetic engineering offers a number of unique advantages, including a high-level of transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects, and undesirable foreign DNA. Thus far, over forty transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer important agronomic traits, as well as express industrially valuable biomaterials and therapeutic proteins. The hyperexpression of recombinant proteins within plastid engineered systems offers a cost effective solution for using plants as bioreactors. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Oral delivery of vaccine antigens against cholera, tetanus, anthrax, plague, and canine parvovirus are made possible because of the high expression levels and antibiotic-free selection systems available in plastid transformation systems. Plastid genetic engineering also has become a powerful tool for basic research in plastid biogenesis and function. This approach has helped to unveil a wealth of information about plastid DNA replication origins, intron maturases, translation elements and proteolysis, import of proteins and several other processes. Although many successful examples of plastid engineering have set a foundation for various future applications, this technology has not been extended to many of the major crops. Highly efficient plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors in soybean, carrot, and cotton. Transgenic carrots were able to withstand salt concentrations that only halophytes could tolerate; more than twice the effectiveness of other engineering attempts. Recent advances in plastid engineering provide an efficient platform for the production of therapeutic proteins, vaccines, and biomaterials using an environmentally friendly approach. This review takes an in-depth look into the state of the art in plastid engineering and offers directions for further research and development.     

The presence and synthesis of the NADPH-protochlorophyllide oxidoreductase in barley leaves with a high temperature-induced deficiency of plastid ribosomes

High-temperature-induced deficiency of plastid ribosomes in barley plants (Hordeum vulgare L.) was used as a system for studying the role of the cytoplasm in the synthesis of the NADPH-protochlorophyllide oxidoreductase. The enzyme is present in 33° C-grown plants. The failure of high-temperature-grown plants to accumulate chlorophyll during illumination is not caused by the absence of the protochlorophyllide-reducing enzyme. The synthesis of the NADPH-protochlorophyllide oxidoreductase was studied by feeding [³⁵S]methionine to the seedling and by following the incorporation of the radioactively labeled amino acid into plastid proteins. The NADPH-protochlorophyllide oxidoreductase was labeled in high-temperature-grown barley plants to the same extent as in control plants grown at 25° C. It is concluded that the 36,000-M_r polypeptide of the NADPH-protochlorophyllide oxidoreductase is synthesized outside the plastid on cytoplasmic 80S ribosomes.