REGULATION OF CHLOROPHYLL-APOPROTEIN SYNTHESIS

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The long range goal of this research is to define biochemical mechanisms which regulate the synthesis and assembly of chlorophyll and chlorophyll- apoproteins. The first specific aim is to elucidate how D1 synthesis is regulated by chlorophyll alpha and a nuclear gene which is disrupted in the barley mutant vir115. D1 is a chlorophyll-apoprotein which, together with D2, binds pheophytin, chlorophyll and quinones. This heterodimer forms the reaction center of Photosystem II, a large protein complex which mediates oxygen evolution and plastoquinone reduction in chloroplasts. D1 does not accumulate in dark-grown plants which lack chlorophyll. However, mRNA encoding this protein is associated with polysomes and translation intermediates can be detected in pulse-labeled etioplasts. D1 accumulates when plants are illuminated and chlorophyll is synthesized. This data suggests that chlorophyll binding will be analyzed by carrying steps in D1 translation, translocation into membranes and chlorophyll binding will be analyzed by carrying out several specific objectives; (1) Early steps in D1 translation will be analyzed using toeprint assays to identify ribosome binding sites, (2) The nature of membrane bound D1 translation intermediates will be investigated using pulse-chase assays, determining if the intermediates are associated with polysomes and by determining the distribution of ribosomes on psbA mRNA found in membrane polysomes, (3) Translocation of D1 into thylakoid membranes will be studied and the coordination of translocation and chlorophyll-binding investigated, (4) Proteolysis of D1 will be examined and the role of a nuclear encoded protein in D1 synthesis identified, (5) Knowledge gained through the study of D1 will be extended to the analysis of three additional chl-apoproteins (CP47, P700 chl-apoproteins). In addition, the mechanisms by which blue light regulates the psbD-psbC RNA population will be studied; 1. The influence of transcription and RNA stability on the light-induced accumulation of two psbD-psbC RNAs will be quantitated, 2. Factors which stimulate psbD-psbC transcription will be identified and 3. Blue light inducible nuclear genes which encode the plastid proteins regulating psbD- psbC expression will be isolated.