叶绿体翻译延伸因子Tu对镁离子螯合酶活性调控及其机制研究

Chlorophyll, a key pigment in chloroplast, plays an indispensable role in maintaining the photosynthesis. The chlorophyll biosynthesis is one of the most important pathways in chloroplast and its first and rate-limiting step is catalyzed by Mg chelatase. Mg chelatase is a heterotrimeric enzyme consisting of three subunits: CHLH, CHLI and CHLD subunits. Each of the subunits is necessary for Mg chelatase activity (Luo et al., 2013) and modulated by regulatory proteins. So far, a regulatory protein, GUN4 (Genome uncoupled 4) has been reported to regulate the CHLH subunit. And Our previous result showed that the CHLI subunit is regulated by chloroplast thioredoxins (Luo et al., 2012). However, as to our knowledge, not any regulatory proteins of CHLD subunit have been identified. Our present study has identified a putative CHLD-interacting protein chloroplast elongating factor Tu (chloroplast EF-Tu) by means of screening a yeast two hybrid cDNA library using CHLD subunit as bait. After performing five different methods of protein-protein interaction assays, we validated the in vivo and in vitro interactions between CHLD and chloroplast EF-Tu. In addition, we also found that chloroplast EF-Tu was sufficient for renaturation of CHLD in vitro. Recently, Chloroplast EF-Tu has been report to protect the chloroplast proteins against heat stress. Taking all together, the present study intend to analyze the regulatory effect of chloroplast EF-Tu on Mg chelatase activity and explore its mechanism under normal condition and heat stress. The results of this study will not only open up new ideas of the research on the regulation of chlorophyll synthesis pathway but also provide the theoretical basis for increasing crop yield by enhanced photosynthetic efficiency.

镁离子螯合酶是叶绿素合成途径的第一个酶，也是限速酶。它是由H、I和D三个亚基组成的异源三聚体，各亚基对其活性必不可少，且受调控蛋白调节。我们之前结果显示I亚基在叶绿体内受硫氧还蛋白调控；H亚基则被报道受GUN4蛋白调控，但D亚基的调控蛋白仍不清楚。本项目前期通过酵母双杂交筛选到D亚基的互作候选蛋白叶绿体翻译延伸因子Tu（叶绿体EF-Tu），通过不同方法验证了它们的互作，并发现它在体外能够帮助D亚基复性，其基因沉默豌豆植株呈现黄化表型，这些结果暗示叶绿体EF-Tu可能是镁离子螯合酶活性的调控蛋白。最近研究表明叶绿体EF-Tu在高温逆境下能够保护叶绿体蛋白。因此本项目拟分别在常温和高温逆境下探讨叶绿体EF-Tu对镁离子螯合酶活性的调控作用及其具体机制，其结果将不仅为叶绿素合成途径的研究提供新思路，而且为利用遗传改良手段提高作物光合效率提供理论依据。

叶绿素作为光合作用至关重要的色素分子，其合成代谢调控一直是研究热点。镁离子螯合酶是叶绿素合成途径的限速酶，研究其活性调控机制对揭示叶绿素合成调控途径具有重要意义。本课题以前期通过酵母双杂交技术筛选到的镁离子螯合酶D亚基互作蛋白叶绿体翻译延伸因子Tu（EF-Tu）为切入点，在通过体内和体外实验验证了它们互作的前提下，进一步研究EF-Tu对镁离子螯合酶的调控作用及其机制。结果显示：1）EF-Tu在体外对D亚基具有伴侣蛋白的功能，能帮助D亚基从包涵体中复性成有活性的可溶性蛋白；2）利用病毒介导的基因沉默技术在豌豆植株中沉默EF-Tu基因后，沉默植株出现黄化表型、且黄化叶片中镁离子螯合酶活性下降、叶绿体超微结构异常、镁离子螯合各亚基以及叶绿素合成途径相关基因的基因表达和蛋白含量发生不同程度改变、5-氨基乙酰丙酸合成速率下降，但四吡咯代谢中间产物原卟啉IX和镁原卟啉IX的含量未有显著变化；3）沉默植株黄化叶片中D亚基在叶绿体中不能正确定位以及形成同源聚合体；4）高温逆境能降低镁离子螯合酶活性，而叶绿体EF-Tu在体外和体内对镁离子螯合酶三个亚基和调控蛋白GUN4具有明显热保护作用。综上所述，叶绿体EF-Tu作为镁离子螯合酶D亚基的互作蛋白，对D亚基在叶绿体中的定位以及同源聚合体的形成并对镁离子螯合酶各亚基具有热保护作用，从而调控镁离子螯合酶活性。以上部分结果已发表在Plant cell Tiss Organ Cult、遗传以及植物生理学报等国内外知名期刊，部分内容处于论文撰写、投稿中，有望于年内发表。后续将进一步研究EF-Tu对镁离子螯合酶热保护的具体机制。这些工作不仅为叶绿素合成途径的研究提供了新思路，而且为利用遗传改良手段提高作物光合效率和高温逆境耐受能力提供了理论依据。