Identification and analysis of Chlamydomonas mutants with defects in chlorophyll synthesis

莱茵衣藻突变株能耐受叶绿素合成过程中的遗传损伤，只要积累的叶绿素前体不会产生光氧化胁迫。因此，叶绿素合成基因的敲除突变体可以在黑暗中生长在额外的碳源上并进行分析，而相应的植物突变体将是胚胎致死的。我们继续筛选一系列具有棕色、黄色和黄绿色色素沉着的黑色生长的有趣的光敏突变体，这些突变体都是由不同的积累的叶绿素前体指定的，这些前体使这些突变体变得光敏，并使它们无法严格地光自养生长。主要目的是详细分析三个缺失GUN4或HEM4基因或显示镁原卟啉单甲酯积聚的突变体，表明环化反应受损。将在这些突变体中研究四吡咯生物合成的基因表达和酶活性，从而预测衣藻影响四吡咯生物合成和光合作用的转录和翻译后调节机制，这可能与以前分析的维管束植物不同。其他研究包括评估(I)LHC在光强增加时的表达，以响应受损的叶绿素生物合成；(Ii)作为有效底物通道和避免依赖四吡咯的光氧化胁迫的先决条件的酶和镁分支因子之间的蛋白质相互作用。

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.