Divinyl Chlorophyll(ide) a Can Be Converted to Monovinyl Chlorophyll(ide) a by a Divinyl Reductase in Rice

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.Chlorophyll (Chl) is the main component of the photosynthetic pigments. Chl molecules universally exist in photosynthetic organisms and perform essential processes of harvesting light energy in the antenna systems and by driving electron transfer in the reaction centers (Fromme et al., 2003). In higher plants, there are two Chl species, Chl a and Chl b. The photosynthetic reaction centers contain only Chl a, and the peripheral light-harvesting antenna complexes contain Chl a and Chl b (Grossman et al., 1995). Chl a is synthesized from glutamyl-tRNA, and Chl b is synthesized from Chl a at the last step of Chl biosynthesis (Beale, 1999). So far, genes for all 15 steps in the Chl biosynthetic pathway have been identified in higher plants, at least in angiosperms represented by Arabidopsis (Arabidopsis thaliana; Beale, 2005; Nagata et al., 2005). Analysis of the complete genome of Arabidopsis showed that it has 15 enzymes encoded by 27 genes for Chl biosynthesis from glutamyl-tRNA to Chl b (Nagata et al., 2005). However, only six genes encoding three enzymes involved in Chl biosynthesis have been identified in rice (Oryza sativa). Magnesium chelatase comprises three subunits (ChlH, ChlD, and ChlI) and catalyzes the insertion of Mg²⁺ into protoporphyrin IX, the last common intermediate precursor in both Chl and heme biosyntheses. Jung et al. (2003) characterized OsCHLH gene for the OsChlH subunit of magnesium chelatase, and Zhang et al. (2006) cloned Chl1 and Chl9 genes encoding the OsChlD and OsChlI subunits of magnesium chelatase. Chl synthase catalyzes esterification of chlorophyllide (Chlide), resulting in the formation of Chl a. Wu et al. (2007) identified the YGL1 gene encoding the Chl synthase. Chl b is synthesized from Chl a by Chl a oxygenase; Lee et al. (2005) identified OsCAO1 and OsCAO2 genes for Chl a oxygenase.According to the number of vinyl side chains, Chls of oxygenic photosynthetic organisms are classified into two groups: 3,8-divinyl Chl (DV-Chl) and 3-vinyl Chl (monovinyl Chl MV-Chl]). Almost all of the oxygenic photosynthetic organisms contain MV-Chls, regardless of the variation in their indigenous environments (Porra, 1997). The exceptions are species of Prochlorococcus marinus, marine picophytoplanktons that contain DV-Chls as their photosynthetic pigments (Chisholm et al., 1992).Chl biosynthetic heterogeneity is assumed to originate mainly in parallel DV- and MV-Chl biosynthetic routes interconnected by 8-vinyl reductases that convert DV-tetrapyrroles to MV-tetrapyrroles by conversion of the vinyl group at position 8 of ring B to the ethyl group (Parham and Rebeiz, 1995; Rebeiz et al., 2003). Most of Chls carry an ethyl group or, less frequently, a vinyl group. For example, Chl a and b occur as the MV-derivatives in green plants, but Chl precursors sometimes accumulate as DV-intermediates, and the ratio between the two forms can vary depending on the species, tissue, and growth conditions (Shioi and Takamiya, 1992; Kim and Rebeiz, 1996). So far, five 8-vinyl reductase activities have been detected at the levels of DV Mg-protoporphyrin IX (Kim and Rebeiz, 1996), Mg-protomonomethyl ester (Kolossov et al., 2006), protochlorophyllide (Pchlide) a (Tripathy and Rebeiz, 1988), Chlide a (Kolossov and Rebeiz, 2001; Nagata et al., 2005), and Chl a (Adra and Rebeiz, 1998). What is not clear at this stage is whether the various 8-vinyl reductase activities are catalyzed by one enzyme of broad specificity or by a family of enzymes of narrow specificity encoded by one gene or multiple genes, as is the case for NADPH Pchlide oxidoreductases (Rebeiz et al., 2003). The issue could be settled by purification of the various putative reductases and comparison of their properties.Nagata et al. (2005) and Nakanishi et al. (2005) independently identified the AT5G18660 gene of Arabidopsis as a divinyl reductase (DVR) that has sequence similarity to isoflavone reductase. Chew and Bryant (2007) demonstrated that BciA (CT1063), which is an ortholog of the Arabidopsis gene, encodes a DVR of the green sulfur bacterium Chlorobium tepidum TLS. They also considered that BchJ, which had been reported to be a vinyl reductase (Suzuki and Bauer, 1995), is not the enzyme, but it may play an important role in substrate channeling and/or regulation of bacteriochlorophyll biosynthesis. Islam et al. (2008) and Ito et al. (2008) independently identified a novel 8-vinyl reductase gene (Slr1923) in DVR-less cyanobacterium Synechocystis sp. PCC6803. However, no DVR gene has yet been identified in monocotyledonous plants.In this study, we isolated a spontaneous mutant, 824ys, from indica rice cv 824B. The mutant exhibited a yellow-green leaf phenotype throughout the growth stage, reduced level of Chls, arrested development of chloroplasts, and retarded growth rate. Map-based cloning of the mutant resulted in the identification of the OsDVR gene, showing sequence similarity to the DVR gene of Arabidopsis. In the 824ys mutant, nine nucleotides were deleted at residues 952 to 960 in the open reading frame (ORF), resulting in three amino acid deletion in the encoded protein. HPLC analysis of Chls indicated the mutant accumulates only DV-Chls. Enzymatic analysis demonstrated that the recombinant protein expressed in Escherichia coli is able to catalyze the conversion of DV-Chl(ide) a to MV-Chl(ide) a. Therefore, this study has confirmed that the OsDVR gene encodes a functional DVR in rice.