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
