Identification of genes for chlorophyll biosynthesis and its roles in stay green phenotype and cell death

近年来，叶绿素代谢与其他细胞过程（包括叶片衰老、细胞程序性死亡和质体信号传导）之间的联系也取得了重大进展。本研究的目的是完成叶绿素代谢途径，并确定参与该途径的所有基因。叶绿素代谢在各种生物中得到了广泛的研究，几乎所有的叶绿素生物合成基因都在高等植物中被鉴定出来。然而，光合生物产生单叶绿素或二叶绿素所必需的8-乙烯基还原酶（DVR） 3,8 -二乙烯基原叶绿素内酯（DVR）的基因尚未确定。在本研究中，我们通过乙基甲烷磺酸诱变分离了一个积累二乙烯基叶绿素而不是单乙烯基叶绿素的拟南芥突变体。对该突变体的图谱克隆鉴定出一个与异黄酮还原酶基因序列相似的基因（At5g18660）。与野生型基因的转化补充了突变型表型。在大肠杆菌中表达了一个由At5g18660编码的重组蛋白，发现该蛋白可催化二酰基叶绿素内酯转化为单酰基叶绿素内酯；从而表明该基因编码功能性DVR。DVR由拟南芥基因组中的一个单拷贝基因编码。随着DVR基因的鉴定，高等植物叶绿素合成所需的所有基因最终都被鉴定出来。对拟南芥全基因组的分析表明，拟南芥含有从3 glutamyl-tRNAglu到叶绿素b的27个基因编码的15个叶绿素合成酶。

Recently, significant progress has also been made toward elucidating the linkages between chlorophyll metabolism and other cellular processes including leaf senescence, programmed cell death, and plastid signaling. The purpose of the present research is to complete chlorophyll metabolic pathway and to identify all the genes involved in the pathway. Chlorophyll metabolism has been extensively studied with various organisms and almost all of the chlorophyll biosynthetic genes have been identified in higher plants. However, only the gene for 3, 8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), which is indispensable for the photosynthetic organisms to produce monovinylchlorophyll or dinvinyl chlorophyll, has not been identified yet. In this study, we isolated an Arabidopsis mutant that accumulated divinyl chlorophyll instead of monovinyl chlorophyll by ethyl-methane sulfonate mutagenesis. Map-based cloning ofthis mutant resulted in the identification of a gene (At5g18660) that shows sequence similarity with isoflavon reductase genes. The mutant phenotype was complemented by the transformation with the wild-type gene. A recombinant protein encoded by At5g18660 was expressed in E.coli and found to catalyze the conversion of divinyl chlorophyllide to monovinyl chlorophyllide ; thereby indicating that the gene encodes a functional DVR. DVR is encoded by a single-copy gene in the Arabidopsis genome. With the identification of DVR, finally all genes required for chlorophyll biosynthesis have been identified in higher plants. Analysis of the complete genome of Arabidopsis showed that it has 15 enzymes encoded by 27 genes for chlorophyll biosynthesis from 3 glutamyl-tRNAglu to chlorophyll b.