Nuclear Factors Regulating Light-Independent Chlorophyll Formation in Chlamydomonas

在进化过程中，建立了两种不同的机制，用于将原叶绿素（pchlide）还原为叶绿素（chlide），这是叶绿素生物合成途径中的关键步骤。 一种机制，由酶NADPH催化：原叶绿素氧化还原酶（POR），是完全依赖于其活动的光。 光依赖性POR活性存在于蓝细菌、绿色藻类、大多数非维管和维管植物中，并且是被子植物中用于叶绿素形成的唯一机制。 第二种机制存在于不产氧光合细菌以及蓝藻、非维管植物、蕨类植物和裸子植物中，能够以不依赖于光的方式将pchlide还原为chlide。 含有这种叶绿素还原机制的生物都能够在黑暗中形成叶绿素。 虽然现在有大量关于POR生物合成和活性调节的信息，但对介导光非依赖性pchlide还原的酶（指定为LIPOR），调节其形成的因子及其催化功能的要求知之甚少。 在这个提议中，将阐明参与调节绿色衣藻（Chlamydiumreinhardtii）中包含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，在这一过程中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位点（命名为y，表示黑暗中的黄色）的产物。 质体或核编码基因的突变导致细胞具有相同的“黑暗中变黄”表型，反映了叶绿素形成的损失和生物合成前体叶绿素在黑暗生长细胞中的积累。 虽然直接的生物化学证据仍然缺乏，质体编码的chIL，chIN和chIB基因的产品被认为是构成一个寡聚体LIPOR复合物的亚基。 没有任何y基因的结构信息，也没有特定的功能被归因于它们的产物。 然而，据认为，这些基因参与调节三个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，并确定它们在光非依赖性叶绿素生物合成过程中的作用。 在这个广泛的框架内，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基因表达的特异性改变，则试图确定这种调节发生的水平（例如，转录、剪接、翻译起始、转换等），并产生CH1基因或其产物内介导这些活性的结构决定簇。 (3)将启动使用分子和遗传方法的组合来分离一个或多个y基因座的实验，所述y基因座可以归因于确定的调节效应。 在特定位点的基因的结构（S）和作用，其编码的产品（S）发挥的调节LIPOR的合成，装配，或催化功能将被determined.Our研究应提供重要的新信息的生化和遗传因素，调节这一关键的生物合成过程，并提供新的见解如何核和细胞器隔间协调其遗传和生物合成活动。 由于光依赖性和光非依赖性机制都在衣原体中起作用，这些研究也有助于我们最终理解这两种酶过程是如何整合和协调的

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