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

最近，也取得了重大进展，阐明叶绿素代谢和其他细胞过程，包括叶片衰老，程序性细胞死亡，质体信号之间的联系。本研究的目的是完善叶绿素代谢途径，并对参与该途径的所有基因进行鉴定。叶绿素代谢已被广泛研究与各种生物和几乎所有的叶绿素生物合成基因已被确定在高等植物。然而，只有3，8-二乙烯基原叶绿素a 8-乙烯基还原酶（DVR）的基因尚未被鉴定，DVR是光合生物产生单乙烯基叶绿素或二乙烯基叶绿素所必需的。在这项研究中，我们分离出一个拟南芥突变体，积累二乙烯基叶绿素，而不是单乙烯基叶绿素的乙基甲烷磺酸诱变。基于图位克隆的突变体导致在鉴定的基因（At 5g 18660），显示序列相似性与葡萄糖醛酸还原酶基因。突变体表型通过用野生型基因转化来补充。在大肠杆菌中表达了由At 5g 18660编码的重组蛋白，发现其催化二乙烯基叶绿素转化为单乙烯基叶绿素;从而表明该基因编码功能性DVR。DVR由拟南芥基因组中的单拷贝基因编码。随着DVR的鉴定，最终在高等植物中鉴定了所有叶绿素生物合成所需的基因。对拟南芥全基因组的分析表明，拟南芥有15个酶，由27个基因编码，从3个谷氨酰-tRNAglu到叶绿素B。

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.