莱茵衣藻四吡咯分子胆色素通过镁螯合酶调控蛋白GUN4调控叶绿素合成的机制研究

光合生物通过四吡咯途径产生叶绿素和血红素，尚不清楚血红素分支是否能够直接调控叶绿素的合成。衣藻中血红素代谢产物胆色素与催化叶绿素合成第一步反应的镁螯合酶(MgCh)调控蛋白GUN4互作，增强MgCh体外活性50倍以上。本项目多角度解析胆色素通过GUN4调控叶绿素合成的分子机制：鉴定胆色素与GUN4在体内的互作及胆色素与四吡咯分子竞争结合GUN4；构建胆色素缺陷及gun4双突变体，通过添加胆色素及遗传互补、免疫沉淀等方法鉴定胆色素-GUN4对MgCh活性、叶绿体中分布及蛋白稳定性的影响；分析胆色素-GUN4对四吡咯前体分子ALA合成的反馈调控作用；鉴定GUN4在黑暗/光照转变过程中介导胆色素从叶绿体到细胞质的转运及胆色素信号调控活性氧代谢相关基因表达的功能。本项目的实施将丰富对叶绿素合成调控机制的认识，有望揭示在藻类及植物等光合生物中可能普遍存在的这样一条血红素分支直接调控叶绿素合成的途径。

Chlorophyll and heme are derived from tetrapyrrole biosynthetic pathway in photosynthetic organisms, yet it is still unclear whether or not heme branch can directly regulate chlorophyll biosynthesis. The magnesium chelatase (MgCh) catalyzes the first step of chlorophyll biosynthetic pathway. We observed that Chlamydomonas heme catabolite bilin could interact with the MgCh regulatory protein GUN4, and therefore enhanced the MgCh activity more than 50 folds in vitro. This project is aimed to investigate the molecular mechanism of chlorophyll biosynthetic regulation by bilin and GUN4 from many aspects: characterizing the interaction between bilin and GUN4 in vivo and competitive binding to GUN4 among bilin and other tetrapyrrole intermediates; construction of double mutant containing both bilin biosynthetic deficiency and gun4 mutation, identification of the impact on MgCh activity, subchloroplastic distribution and protein stability by bilin-GUN4 by employing various approaches including in vitro feeding of bilin, genetic complementation and immunoprecipitation, etc; analysis of bilin-GUN4-mediated feedback regulation of biosynthesis of tetrapyrrole precursor ALA (5-Aminolevulinic acid); identification of the role of GUN4 in mediating bilin transport from chloroplast to cytosol and in regulating expression of reactive oxygen species (ROS) metabolism-related genes by the bilin retrograde signaling during dark to light transition in Chlamydomonas reinhardtii. The implementation of this project will enrich our understanding about the mechanism of chlorophyll biosynthetic regulation, and is expected to reveal a widely present regulatory pathway in photosynthetic species including algae and plants in that heme branch can directly regulate chlorophyll biosynthesis.