yellow-in-the-dark Mutants of Chlamydomonas Lack the CHLL Subunit of Light-Independent Protochlorophyllide Reductase

Correspondence to: Michael P. Timko, mpt9g{at}virginia.edu (E-mail), 804-982-5626 (fax)

Light-independent protochlorophyllide reduction leading to chlorophyll formation in the dark requires both chloroplast and nuclear gene expression in Chlamydomonas reinhardtii. Mutations in any one of the plastid (chlL, chlN, and chlB) or nuclear (y-1 to y-10) genes required for this process result in the phenotype of the yellow-in-the-dark or y mutants. Analysis of the chlL, chlN, and chlB transcript levels in both light- and dark-grown wild-type and y mutant cells showed that the y mutations have no effect on the transcription of these plastid genes. Protein gel blot analysis showed that the CHLN and CHLB proteins are present in similar amounts in light- and dark-grown wild-type cells, whereas CHLL is present only in wild-type cells grown in the dark or at light intensities 15 µmol m^-2 sec^-1. Analysis of chlL transcript distribution on polysome profiles and rates of protein turnover in chloramphenicol-treated cells suggested that CHLL formation is most probably blocked at translation initiation or elongation. Furthermore, treatment of cells with metabolic inhibitors and uncouplers of photosynthetic electron transport showed that regulation of CHLL formation is linked to the physiologic status of the chloroplast. Similar to wild-type cells, y mutants contain nearly identical amounts of CHLN and CHLB when grown in either light or darkness. However, no CHLL is present in any of the y mutants except y-7, which contains an immunoreactive CHLL smaller than the expected size. Our findings indicate that CHLL translation is negatively photoregulated by the energy state or redox potential within the chloroplast in wild-type cells and that nuclear y genes are required for synthesis or accumulation of the CHLL protein.

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