Rice Research Institute , Sichuan Agricultural University, Chengdu 611130, China.
Plant Physiol. 2010 Jul;153(3):994-1003. doi: 10.1104/pp.110.158477. Epub 2010 May 18.
3,8-Divinyl (proto)chlorophyll(ide) a 8-vinyl reductase (DVR) catalyzes the reduction of 8-vinyl group on the tetrapyrrole to an ethyl group, which is indispensable for monovinyl chlorophyll (Chl) synthesis. So far, three 8-vinyl reductase genes (DVR, bciA, and slr1923) have been characterized from Arabidopsis (Arabidopsis thaliana), Chlorobium tepidum, and Synechocystis sp. PCC6803. However, no 8-vinyl reductase gene has yet been identified in monocotyledonous plants. In this study, we isolated a spontaneous mutant, 824ys, in rice (Oryza sativa). The mutant exhibited a yellow-green leaf phenotype, reduced Chl level, arrested chloroplast development, and retarded growth rate. The phenotype of the 824ys mutant was caused by a recessive mutation in a nuclear gene on the short arm of rice chromosome 3. Map-based cloning of this mutant resulted in the identification of a gene (Os03g22780) showing sequence similarity with the Arabidopsis DVR gene (AT5G18660). In the 824ys mutant, nine nucleotides were deleted at residues 952 to 960 in the open reading frame, resulting in a deletion of three amino acid residues in the encoded product. High-performance liquid chromatography analysis of Chls indicated the mutant accumulates only divinyl Chl a and b. A recombinant protein encoded by Os03g22780 was expressed in Escherichia coli and found to catalyze the conversion of divinyl chlorophyll(ide) a to monovinyl chlorophyll(ide) a. Therefore, it has been confirmed that Os03g22780, renamed as OsDVR, encodes a functional DVR in rice. Based upon these results, we succeeded to identify an 8-vinyl reductase gene in monocotyledonous plants and, more importantly, confirmed the DVR activity to convert divinyl Chl a to monovinyl Chl a.
3,8-二乙烯基(原)叶绿素(ide)a 8-乙烯基还原酶(DVR)催化四吡咯上 8-乙烯基基团还原为乙基,这对于单乙烯基叶绿素(Chl)合成是必不可少的。迄今为止,已从拟南芥(Arabidopsis thaliana)、绿菌(Chlorobium tepidum)和集胞藻 6803(Synechocystis sp. PCC6803)中鉴定出三个 8-乙烯基还原酶基因(DVR、bciA 和 slr1923)。然而,单子叶植物中尚未鉴定出 8-乙烯基还原酶基因。在这项研究中,我们从水稻(Oryza sativa)中分离出一个自发突变体 824ys。该突变体表现出黄-绿叶片表型,叶绿素水平降低,叶绿体发育受阻,生长速度减慢。824ys 突变体的表型是由水稻 3 号染色体短臂上一个核基因的隐性突变引起的。该突变体的图位克隆导致鉴定出一个与拟南芥 DVR 基因(AT5G18660)具有序列相似性的基因(Os03g22780)。在 824ys 突变体中,开放阅读框 952 到 960 位的九个核苷酸缺失,导致编码产物中三个氨基酸残基缺失。叶绿素的高效液相色谱分析表明,突变体仅积累二乙烯基叶绿素 a 和 b。在大肠杆菌中表达由 Os03g22780 编码的重组蛋白被发现能够催化二乙烯基叶绿素(ide)a 向单乙烯基叶绿素(ide)a 的转化。因此,已证实 Os03g22780,更名为 OsDVR,在水稻中编码一种功能性 DVR。基于这些结果,我们成功地在单子叶植物中鉴定出 8-乙烯基还原酶基因,更重要的是,证实了 DVR 活性可以将二乙烯基叶绿素 a 转化为单乙烯基叶绿素 a。