| Description: | We have isolated a mutant, conditional chlorina (cch) that disassembles its light harvesting complex (LHC) under moderate light. The biochemical activities that underly this disassembly process have been proposed to consist of proteases that specifically degrade LHC proteins and enzymes that convert Chl b into Chl a. We propose using cch in a microarray analysis to try to define genes that are essential for LHC disassembly. When grown at low light (60 micromol photons m-2 sec-1), cch has a near normal phenotype, but when transferred to moderate light (150 micromol photons m-2 sec-1), cch appears yellow-green. The molecular basis of the cch mutant is a missense mutation (P642L) in the H subunit of Mg-chelatase, the first committed step in Chl versus heme biosynthesis. Demands on Chl synthesis increase after transfer to moderate light, and the mutant Mg-chelatase cannot synthesize Chl at an adequate rate. We hypothesize that during acclimation, the Chl that had been bound to the LHCs is recycled so that it can be used to build new photosystems in the growing cell. As Chl b can only be used for LHCs, it is likely that Chl b is reconverted into Chl a. It is unlikely that the Chl is detoxified, and then sent to the vacuole as in senescence, because the plant needs Chl. Rather, the initial steps of senescence are occuring (LHC disassembly), but the latter are not (removal of the phytol tail and the Mg ion, and tetrapyrrole ring cleavage). A rapid way to find genes involved in LHC disassembly would be a microarray experiment. We would look for mRNA species that are induced when cch plants acclimate to moderate light. These genes must be specific for LHC disassembly, and not genes whose expression is increased during faster growth rates. We propose to use two mRNA samples, WT and cch, both isolated from plants that had been in moderate light for two days. Genes whose expression is increased by higher growth rates should be found in both samples, and therefore will not be selected. Genes involved in the disassembly of the LHC and in Chl redistribution should only be expressed in cch, and therefore should be selected. The suite of genes selected by the microarray process will be analyzed using BLAST and ChloroP searches, and genes that lack chloroplast leaders will be excluded. Candidate genes that have chloroplast leaders will be overexpressed in WT and in cch, and their effects on Chl levels will be analyzed. |
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