Nuclear Factors Regulating Light-Independent Chlorophyll Formation in Chlamydomonas

在进化过程中，原叶绿素(Pchlide)被还原为叶绿素(Chlide)的两个不同的机制被建立起来，这是叶绿素生物合成途径中的关键步骤。一种机制是由NADPH酶催化的：原叶绿素氧化还原酶(POR)，其活性完全依赖于光。光依赖的POR活性存在于蓝藻、绿藻、大多数非维管植物和维管束植物中，是被子植物形成叶绿素的唯一机制。第二种机制存在于产氧光合细菌以及蓝藻、非维管植物、蕨类和裸子植物中，能够以不依赖于光的方式将pchlide还原为chlide。含有这种pchlide还原机制的生物都能在黑暗中形成叶绿素。虽然目前关于POR生物合成和活性的调控信息很多，但对介导光非依赖性pchlide还原的酶(简称LIPOR)、调节其形成的因素及其对催化功能的要求知之甚少。在这项建议中，将阐明参与调节组成LIPOR活性的多肽亚基的生物合成和活性的因素。以前的研究表明，这一过程需要三个叶绿体基因(命名为Chil、Cain和ChiB)和至少七个独立的核基因(y表示暗黄色)的产物。无论是叶绿体编码基因还是核编码基因的突变都会导致细胞具有相同的“暗黄”表型，这反映了在暗生长的细胞中叶绿素形成的丧失和生物合成前体pchlide的积累。尽管目前还缺乏直接的生化证据，但叶绿体编码的Chil、Chin和ChiB基因的产物被认为构成了一个寡聚体LIPOR复合体的亚单位。目前还没有任何y基因的结构信息，也没有特定的功能被归因于他们的产品。然而，人们认为这些基因参与调节这三个叶绿体编码基因的转录或转录后活性。这项研究的总体目标是确定各种y基因及其编码产物的特征，并确定它们在与光无关的叶绿素生物合成过程中的作用(S)。在这个广泛的框架内，建议(1)定义各种y突变对叶绿体编码的Chil、Chin和ChiB基因转录和转录后活性的影响(S)。特别是，我们想知道是否有任何已知的y突变专门影响特定的chl基因或chl基因的子集的表达，或者它们是否对叶绿体的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基因表达的特定变化，将试图定义这种调控发生的水平(例如，转录、剪接、翻译起始、转换等)以及chl基因或其产物中调节这些活性的结构决定因素。(3)将启动使用分子和遗传方法相结合的实验，以分离一个或多个可归因于特定调控效应的y基因座。该基因(S)在特定基因座上的结构及其编码产物(S)在调节LIPOR合成、组装或催化功能中所起的作用将被确定。我们的研究将提供关于调控这一关键生物合成过程的生化和遗传因素的重要新信息，并为核和细胞器间如何协调其遗传和生物合成活动提供新的见解。由于光依赖和光非依赖的pchlide还原机制都在衣藻中运行，这些研究也应该有助于我们最终理解这两个酶过程是如何在它们的操作中整合和协调的

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