Nuclear Factors Regulating Light-Independent Chlorophyll Formation in Chlamydomonas

调节衣藻中不依赖于光的叶绿素形成的核因子

• 批准号: 9818037
• 负责人: Michael Timko
• 金额: $ 20万
• 依托单位: University of Virginia Main Campus
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9818037&HistoricalAwards=false
• 关键词: Nuclear; Factors; Regulating; Light; Independent

During evolution two distinct mechanisms were established for the reduction of protochlorophyllide (pchlide) to chlorophyllide (chlide), a key step in the chlorophyll biosynthesis pathway. One mechanism, catalyzed by the enzyme NADPH: protochlorophylide oxidoreductase (POR), is completely dependent upon light for its activity. Light-dependent POR activity is present in cyanobacteria, green algae, most non-vascular and vascular plants, and is the only mechanism used for chlorophyll formation in angiosperms. The second mechanism, present in anoxygenic photosynthetic bacteria as well as cyanobacteria, non-vascular plants, ferns and gymnosperms, is capable of reducing pchlide to chlide in a light-independent manner. Organisms containing this pchlide reduction mechanism are all capable of chlorophyll formation in the dark. While a significant amount of information is now available on the regulation of POR biosynthesis and activity, little is known about the enzyme mediating light-independent pchlide reduction (designated LIPOR), the factors that regulate its formation, and its requirements for catalytic function. In this proposal the factors involved in regulating the biosynthesis and activity of the polypeptide subunits that comprise the LIPOR activity in the green alga, Chlamydomonas reinhardtii will be elucidated. Previous studies have shown that the products of three chloroplast genes (designated chIL, caIN, and chIB) and at least seven independent nuclear loci (designated y for yellow-in-the-dark) are required in this process. Mutations in either the plastid-or nuclear-encoded genes result in cells with identical "yellow-in-the-dark" phenotypes, reflecting a loss of chlorophyll formation and the accumulation of the biosynthetic precursor pchlide in dark-grown cells. Although direct biochemical evidence is still lacking, the products of the plastid-encoded chIL, chIN, and chIB genes are thought to constitute the subunits of an oligomeric LIPOR complex. No structural information is available for any y gene and no specific function has yet to be ascribed to their products. However, it is thought that these genes are involved in either regulating the transcription or posttranscriptional activities of the three plastid-encoded genes. The overall goal of this research is to characterize the various y genes and their encoded products and determine their role(s) in the process of light-independent chlorophyll biosynthesis. Within this broad framework it is proposed(1) to define the effect(s) of the various y mutations on the transcription and postransciptional activities of the plastid-encoded chIL, chIN, and chIB genes. In particular, we would like to know if any of the known y mutations specifically affect the expression of a particular chl gene or subset of chl genes, or if they have an indirect or general effect on plastid function or development. (2) If specific alterations of chl gene expression associated with various y mutations are observed, an attempt to define the level at which this regulation occurs (e.g., transcription, splicing, translation initiation, turnover, etc) and the structural determinants within the chl gene or its product that mediate these activities will be made. (3) Experiments using a combination of molecular and genetic approaches to isolate one or more y loci for which a defined regulatory effect can be attributed will be initiated. The structure of the gene(s) at the particular locus and the role their encoded product(s) play in the regulation of LIPOR synthesis, assembly, or catalytic function will be determined.Our studies should provide significant new information on the biochemical and genetic factors that regulate this critical biosynthetic process and provide new insights into how nuclear and organellar compartments coordinate their genetic and biosynthetic activities. Since both the light-dependent and light independent mechanisms for pchlide reduction operate in the Chlamydomonas, these studies should also contribute to our eventual understanding of how these two enzymatic processes are integrated and coordinated in their operation

在进化过程中，原叶绿内酯（pchlide）还原为叶绿内酯（chlide）建立了两种不同的机制，这是叶绿素生物合成途径的关键步骤。一种由NADPH酶催化的机制：原叶绿素氧化还原酶（POR），其活性完全依赖于光。光依赖性POR活性存在于蓝藻、绿藻、大多数非维管和维管植物中，是被子植物中叶绿素形成的唯一机制。第二种机制存在于无氧光合细菌以及蓝藻、非维管植物、蕨类植物和裸子植物中，能够以不依赖光的方式将pchlide还原为chlide。含有这种叶绿体还原机制的生物都能在黑暗中形成叶绿素。虽然现在有大量关于POR生物合成和活性调控的信息，但对介导不依赖光的pclide还原（称为LIPOR）的酶，调节其形成的因素及其催化功能的要求知之甚少。本文将对绿藻莱茵衣藻（Chlamydomonas reinhardtii）中组成LIPOR活性的多肽亚基的生物合成和活性的调控因素进行阐述。先前的研究表明，在这个过程中需要三个叶绿体基因（指定为chIL， caIN和chIB）和至少7个独立的核位点（暗黄色指定为y）的产物。无论是质体基因还是核编码基因的突变都会导致细胞具有相同的“黑暗中变黄”表型，这反映了叶绿素形成的丧失和黑暗生长细胞中生物合成前体叶绿体的积累。虽然直接的生物化学证据仍然缺乏，但质体编码的chIL， chIN和chIB基因的产物被认为构成低聚LIPOR复合物的亚基。没有任何y基因的结构信息，也没有任何特定的功能被归因于它们的产物。然而，人们认为这些基因参与调控三种质体编码基因的转录或转录后活性。本研究的总体目标是表征各种y基因及其编码产物，并确定它们在不依赖光的叶绿素生物合成过程中的作用。在这个广泛的框架内，我们提出(1)定义各种y突变对质体编码的chIL、chIN和chIB基因的转录和转录后活性的影响。特别是，我们想知道是否有任何已知的y突变特异性地影响特定chl基因或chl基因子集的表达，或者它们是否对质体功能或发育有间接或一般的影响。(2)如果观察到与各种y突变相关的chl基因表达的特定改变，则尝试定义这种调节发生的水平（例如，转录，剪接，翻译起始，翻转等）以及chl基因或其产物中介导这些活动的结构决定因素。(3)将启动结合分子和遗传方法分离一个或多个y基因座的实验，这些基因座可以归因于明确的调节作用。特定位点的基因结构及其编码产物在LIPOR合成、组装或催化功能的调节中所起的作用将被确定。我们的研究将为调控这一关键生物合成过程的生化和遗传因素提供重要的新信息，并为细胞核和细胞器室如何协调其遗传和生物合成活动提供新的见解。由于衣藻中氯代物还原的光依赖性和光非依赖性机制都在起作用，这些研究也应该有助于我们最终理解这两种酶的过程是如何在其操作中整合和协调的