Divinyl chlorophyll(ide) a can be converted to monovinyl chlorophyll(ide) a by a divinyl reductase in rice

Abstract

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.

Figure 1.

Plant phenotype of the rice 824ys mutant. A, Four-week-old plants of wild type (left) and 824ys mutant (right). B, Thirteen-week-old plants of wild type (left) and 824ys mutant (right). Bars = 5 cm.

Figure 2.

Fine mapping of the 824ys locus. The map was constructed based on the publicly available sequence of rice chromosome 3. Two InDel markers (PR809 and PR826) were developed during this study, while three SSR markers (RM5748, RM1319, and RM6676) were obtained from the public database. A, The 824ys locus was mapped to a region between SSR markers RM1319 and RM6676 on the short arm of rice chromosome 3 (Chr.3) with 214 recessive individuals. B, Fine mapping of the 824ys locus between InDel markers PR809 and PR826 from a segregating population of 848 recessive individuals. C, Two bacterial artificial chromosome contigs (AC134231 and AC135257) covered the 824ys locus, which was narrowed down to a 142-kb DNA region between PR809 and PR826.

Figure 3.

A phylogenetic tree representing alignment of OsDVR protein and its homologs. The rooted neighbor-joining tree using percentage identities was constructed based on a multiple sequence alignment generated with the program DNAMAN. Accession numbers for the respective protein sequences are as follows: rice (OsDVR, Os03g22780, ADE43128); sorghum (XP_002465274); maize (PCB2, NP_001148282); Arabidopsis (DVR, AT5G18660, NP_197367); Synechococcus sp. WH 8102 (SYNW0963, NP_897056); Chlamydomonas reinhardtii (XP_001690168); C. tepidum TLS (BciA [CT1063], NP_661954); and Rhodobacter sphaeroides (ZP_00006667). Scale represents percentage substitution per site.

Figure 4.

HPLC analysis of Chls. The elution profiles of wild type (A) and 824ys mutant (B) were detected at 440 nm. Peak 1, Chl b; peak 2, Chl a; peak 1′, Chl b-like pigment; peak 2′, Chl a-like pigment. The absorption spectra of the peaks 2 and 2′ (C) and the peaks 1 and 1′ (D) in acetone were compared, respectively.

Figure 5.

Enzymatic assay of OsDVR. DV-Chl a (A), -Chl b (B), -Chlide a (C), and -Chlide b (D) were incubated with OsDVR expressed in E. coli, and the pigments extracted from reaction mixtures were subjected to HPLC as described in “Materials and Methods,” respectively. The left column is the chromatogram that was detected at 440 nm by HPLC and the right column depicts the spectrum of each peak. A1 and B1 stand for MV-Chl a and b prepared from the wild-type MV-Chls, respectively. A2 and B2 represent DV-Chl a and b prepared from the 824ys mutant DV-Chls, respectively. C1, D1, C2, and D2 stand for MV-Chlide a, MV-Chlide b, DV-Chlide a, and DV-Chlide b prepared from MV-Chl a, MV-Chl b, DV-Chl a, and DV-Chl b by the enzymatic reaction of chlorophyllase isolated from G. chrysanthemum leaves, respectively. A3, B3, C3, and D3 represent products synthesized after incubation with E. coli lysates expressing the empty vector, which were used as negative controls, respectively. A4, B4, C4, and D4 stand for products synthesized after incubation with E. coli lysates expressing Osdvr, which were also used as negative controls, respectively. A5, B5, C5, and D5 represent products synthesized after incubation with E. coli lysates expressing OsDVR, respectively. Each pigment, DV-Chl a (A), -Chl b (B), -Chlide a (C), and -Chlide b (D), was used as a substrate for enzymatic reaction of OsDVR. C0 (blank control) represents MV-Chlide a resulted from incubation only with an acetone powder of G. chrysanthemum leaves, suggesting a small amount of MV-Chl a still remained in the acetone powder after being washed repeatedly, and the minor peak (peak 1) of C2, C3, and C4 was exactly the MV-Chlide a converted from residue MV-Chl a in the acetone powder.

Figure 6.

Activity of DVR. DVR activities described previously (A) and proposed in this study (B). In current Chl biosynthetic pathway, there are the two DVR activities that have been confirmed (A), i.e. DV-Chlide a is converted to MV-Chlide a by the Arabidopsis DVR (Nagata et al., 2007) and DV-Pchlide a is converted to MV-Pchlide a by the green sulfur bacterium DVR (BciA; Chew and Bryant, 2007). Here we succeeded to demonstrate the third DVR activity, i.e. DV-Chl a is converted to MV-Chl a by OsDVR (B).CHLG, Chl synthase; POR, Pchlide oxidoreductase.