Identification and Analysis of Chlamydomonas Mutants With Defects in Chlorophyll Synthesis

Mutants of Chlamydomonas reinhardtii tolerate genetic lesions in chlorophyll synthesis as long as accumulating chlorophyll precursors cannot generate photooxidative stress. Therefore, knock-out mutants for genes in chlorophyll synthesis can grow in darkness on an additional carbon source and be analyzed, while corresponding plant mutants would be embryo-lethal. We continue to screen a collection of dark-grown interesting photosensitive mutants with brown, yellow and yellow-green pigmentation, which are all specified by diverse accumulating chlorophyll precursors that photosensitize these mutants and disable them to grow strictly photoautotrophically. The main objective is the detailed analysis of three mutants with either deleted GUN4 or HEM4 genes or displaying an accumulation of Mg protoporphyrin monomethylester indicating an impaired cyclase reaction. The expression of genes as well as enzyme activities of tetrapyrrole biosynthesis will be studied in these mutants allowing a prediction of transcriptional and posttranslational regulatory mechanisms of Chlamydomonas affecting regulation of tetrapyrrole biosynthesis and photosynthesis, which might be different from those of previously analyzed vascular plants. Other studies include assessments of (i) LHC expression during increasing light intensities in response to impaired chlorophyll biosynthesis and (ii) protein interactions among enzymes and factors of the Mg branch as a prerequisite for efficient substrate channeling and avoidance of tetrapyrrole-dependent photooxidative stress.