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. 2012 Nov 15;161(1):521–534. doi: 10.1104/pp.112.208421

One Divinyl Reductase Reduces the 8-Vinyl Groups in Various Intermediates of Chlorophyll Biosynthesis in a Given Higher Plant Species, But the Isozyme Differs between Species1,[W],[OA]

Pingrong Wang 1, Chunmei Wan 1, Zhengjun Xu 1, Pingyu Wang 1, Wenming Wang 1, Changhui Sun 1, Xiaozhi Ma 1, Yunhua Xiao 1, Jianqing Zhu 1, Xiaoling Gao 1, Xiaojian Deng 1,*
PMCID: PMC3532282  PMID: 23154534

Abstract

Divinyl reductase (DVR) converts 8-vinyl groups on various chlorophyll intermediates to ethyl groups, which is indispensable for chlorophyll biosynthesis. To date, five DVR activities have been detected, but adequate evidence of enzymatic assays using purified or recombinant DVR proteins has not been demonstrated, and it is unclear whether one or multiple enzymes catalyze these activities. In this study, we systematically carried out enzymatic assays using four recombinant DVR proteins and five divinyl substrates and then investigated the in vivo accumulation of various chlorophyll intermediates in rice (Oryza sativa), maize (Zea mays), and cucumber (Cucumis sativus). The results demonstrated that both rice and maize DVR proteins can convert all of the five divinyl substrates to corresponding monovinyl compounds, while both cucumber and Arabidopsis (Arabidopsis thaliana) DVR proteins can convert three of them. Meanwhile, the OsDVR (Os03g22780)-inactivated 824ys mutant of rice exclusively accumulated divinyl chlorophylls in its various organs during different developmental stages. Collectively, we conclude that a single DVR with broad substrate specificity is responsible for reducing the 8-vinyl groups of various chlorophyll intermediates in higher plants, but DVR proteins from different species have diverse and differing substrate preferences, although they are homologous.

Chlorophyll (Chl) molecules universally exist in photosynthetic organisms. As the main component of the photosynthetic pigments, Chl molecules perform essential processes of absorbing light and transferring the light energy in the reaction center of the photosystems (Fromme et al., 2003). Based on the number of vinyl side chains, Chls are classified into two groups, 3,8-divinyl (DV)-Chl and 3-monovinyl (MV)-Chl. The DV-Chl molecule contains two vinyl groups at positions 3 and 8 of the tetrapyrrole macrocycle, whereas the MV-Chl molecule contains a vinyl group at position 3 and an ethyl group at position 8 of the macrocycle. Almost all of the oxygenic photosynthetic organisms contain MV-Chls, with the exceptions of some marine picophytoplankton species that contain only DV-Chls as their primary photosynthetic pigments (Chisholm et al., 1992; Goericke and Repeta, 1992; Porra, 1997).

The classical single-branched Chl biosynthetic pathway proposed by Granick (1950) and modified by Jones (1963) assumed the rapid reduction of the 8-vinyl group of DV-protochlorophyllide (Pchlide) catalyzed by a putative 8-vinyl reductase. Ellsworth and Aronoff (1969) found evidence for both MV and DV forms of several Chl biosynthetic intermediates between magnesium-protoporphyrin IX monomethyl ester (MPE) and Pchlide in Chlorella spp. mutants. Belanger and Rebeiz (1979, 1980) reported that the Pchlide pool of etiolated higher plants contains both MV- and DV-Pchlide. Afterward, following the further detection of MV- and DV-tetrapyrrole intermediates and their biosynthetic interconversion in tissues and extracts of different plants (Belanger and Rebeiz, 1982; Duggan and Rebeiz, 1982; Tripathy and Rebeiz, 1986, 1988; Parham and Rebeiz, 1992, 1995; Kim and Rebeiz, 1996), a multibranched Chl biosynthetic heterogeneity was proposed (Rebeiz et al., 1983, 1986, 1999; Whyte and Griffiths, 1993; Kolossov and Rebeiz, 2010).

Biosynthetic heterogeneity refers to the biosynthesis of a particular metabolite by an organelle, tissue, or organism via multiple biosynthetic routes. Varieties of reports lead to the assumption that Chl biosynthetic heterogeneity originates mainly in parallel DV- and MV-Chl biosynthetic routes. These routes are 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). DV-MPE could be converted to MV-MPE in crude homogenates from etiolated wheat (Triticum aestivum) seedlings (Ellsworth and Hsing, 1974). Exogenous DV-Pchlide could be partially converted to MV-Pchlide in barley (Hordeum vulgare) plastids (Tripathy and Rebeiz, 1988). 8-Vinyl chlorophyllide (Chlide) a reductases in etioplast membranes isolated from etiolated cucumber (Cucumis sativus) cotyledons and barley and maize (Zea mays) leaves were found to be very active in the conversion of exogenous DV-Chlide a to MV-Chlide a (Parham and Rebeiz, 1992, 1995). Kim and Rebeiz (1996) suggested that Chl biosynthetic heterogeneity in higher plants may originate at the level of DV magnesium-protoporphyrin IX (Mg-Proto) and would be mediated by the activity of a putative 8-vinyl Mg-Proto reductase in barley etiochloroplasts and plastid membranes. However, since these reports did not use purified or recombinant enzyme, it is not clear whether the reductions of the 8-vinyl groups of various Chl intermediates are catalyzed by one enzyme of broad specificity or by multiple enzymes of narrow specificity, which actually has become one of the focus issues in Chl biosynthesis.

Nagata et al. (2005) and Nakanishi et al. (2005) independently identified the AT5G18660 gene of Arabidopsis (Arabidopsis thaliana) as an 8-vinyl reductase, namely, divinyl reductase (DVR). Chew and Bryant (2007) identified the DVR BciA (CT1063) gene of the green sulfur bacterium Chlorobium tepidum, which is homologous to AT5G18660. An enzymatic assay using a recombinant Arabidopsis DVR (AtDVR) on five DV substrates revealed that the major substrate of AtDVR is DV-Chlide a, while the other four DV substrates could not be converted to corresponding MV compounds (Nagata et al., 2007). Nevertheless, a recombinant BciA is able to reduce the 8-vinyl group of DV-Pchlide to generate MV-Pchlide (Chew and Bryant, 2007). Recently, we identified the rice (Oryza sativa) DVR encoded by Os03g22780 that has sequence similarity with the Arabidopsis DVR gene AT5G18660. We also confirmed that the recombinant rice DVR (OsDVR) is able to not only convert DV-Chlide a to MV-Chlide a but also to convert DV-Chl a to MV-Chl a (Wang et al., 2010). Thus, it is possible that the reductions of the 8-vinyl groups of various Chl biosynthetic intermediates are catalyzed by one enzyme of broad specificity.

In this report, we extended our studies to four DVR proteins and five DV substrates. First, ZmDVR and CsDVR genes were isolated from maize and cucumber genomes, respectively, using a homology-based cloning approach. Second, enzymatic assays were systematically carried out using recombinant OsDVR, ZmDVR, CsDVR, and AtDVR as representative DVR proteins and using DV-Chl a, DV-Chlide a, DV-Pchlide a, DV-MPE, and DV-Mg-Proto as DV substrates. Third, we examined the in vivo accumulations of various Chl intermediates in rice, maize, and cucumber. Finally, we systematically investigated the in vivo accumulations of Chl and its various intermediates in the OsDVR (Os03g22780)-inactivated 824ys mutant of rice (Wang et al., 2010). The results strongly suggested that a single DVR protein with broad substrate specificity is responsible for reducing the 8-vinyl groups of various intermediate molecules of Chl biosynthesis in higher plants, but DVR proteins from different species could have diverse and differing substrate preferences even though they are homologous.

RESULTS

Cloning of Maize and Cucumber DVR Genes

So far, two DVR genes (AtDVR and OsDVR) have been characterized from Arabidopsis and rice, the model plants for dicots and monocots, respectively, and their translated amino acid sequences share 66% similarity (Nagata et al., 2005; Nakanishi et al., 2005; Wang et al., 2010). In this study, we further isolated the ZmDVR and CsDVR genes from maize and cucumber, the representatives for monocots and dicots, respectively, using a homology-based cloning approach.

BLAST search in the genome database revealed that ZmDVR (PCB2, Zm124787) is a single-copy gene with an open reading frame (ORF) of 1,206 bp encoding a 401-amino acid protein with a molecular mass of approximately 43 kD. Similarly, CsDVR (Csa000053) is also a single-copy gene, consisting of a 1,260-bp ORF encoding a protein of 419 amino acids with a molecular mass of approximately 46 kD. Similar to OsDVR and AtDVR, the ZmDVR and CsDVR proteins also contain apparent chloroplast-targeting sequences of 54 and 71 amino acids at their N termini, respectively (http://www.cbs.dtu.dk/services/TargetP/; Supplemental Fig. S1).

Multiple amino acid sequence alignment indicated that ZmDVR has higher sequence similarity with OsDVR than with AtDVR, while CsDVR has higher sequence similarity with AtDVR than with OsDVR. The ZmDVR protein is 82% and 62% identical to the OsDVR and AtDVR proteins, respectively, and the CsDVR protein is 62% and 71% identical to the OsDVR and AtDVR proteins, respectively. In addition, the CsDVR protein also has 68% sequence similarity with the ZmDVR protein (Supplemental Fig. S1).

Enzymatic Assays Using Four Recombinant DVR Proteins and Five DV Substrates

It has been implied that heterogeneity of DVR activities could exist in various species (Tripathy and Rebeiz, 1988; Parham and Rebeiz, 1995; Chew and Bryant, 2007; Nagata et al., 2007; Wang et al., 2010). In this study, to further probe the substrate specificity of this enzyme, we carried out a series of enzymatic assays using recombinant OsDVR, ZmDVR, CsDVR, and AtDVR as representative DVR proteins and using DV-Chl a, DV-Chlide a, DV-Pchlide a, DV-MPE, and DV-Mg-Proto as DV substrates.

First, we investigated the effects of temperature and pH on DVR activities. The recombinant DVR proteins were expressed in Escherichia coli, and the cell extracts were incubated with each of five substrates in the presence of NADPH. Incubation time was set to 1 min, 10 min, or 5 h according to DVR activity of every substrate/enzyme combination, temperature was set from 10°C to 40°C at pH 7.0, and pH values were 6.0 to 8.0 at 30°C (for DV-Chlide a and DV-Chl a) or 20°C (for DV-Pchlide a, DV-MPE, and DV-Mg-Proto). The results were as follows. (1) OsDVR was able to convert all of five substrates to corresponding MV compounds, and CsDVR was able to convert DV-Chlide a, DV-Pchlide a, and DV-MPE, but not DV-Chl a and DV-Mg-Proto, to corresponding MV compounds (Fig. 1). (2) The optimum temperatures and pH values for reductive activities were similar among the different DVRs tested when the activities were detectable on a substrate but could be different for different substrates. More specifically, 25°C and pH 7.0 were the optimum conditions for DV-Chlide a (Fig. 1, A1 and B1), 25°C and pH 6.5 were optimal for DV-Chl a (Fig. 1, A2 and B2), and 20°C and pH 6.5 were optimal for DV-MPE (Fig. 1, A4 and B4) and DV-Mg-Proto (Fig. 1, A5 and B5). As for DV-Pchlide a, the optimum pH value was 7.0 (Fig. 1B3) and the optimum temperature was 20°C or 10°C, because the difference of DVR activities between the two temperature conditions was subtle (Fig. 1A3).

Figure 1.

Figure 1.

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Effects of temperature (A) and pH (B) on DVR activities. Each of five substrates (DV-Chlide a, DV-Chl a, DV-Pchlide a, DV-MPE, and DV-Mg-Proto) was incubated with recombinant DVR proteins (OsDVR, CsDVR, and AtDVR) expressed in E. coli. Incubation time in every substrate/enzyme combination was set as follows: DV-Chlide a/OsDVR, 1 min; DV-Chlide a/CsDVR and AtDVR, DV-Chl a/OsDVR and CsDVR, 10 min; DV-Pchlide a, DV-MPE, and DV-Mg-Proto/OsDVR and CsDVR, 5 h. Subsequently, the pigments extracted from reaction mixtures were subjected to HPLC. Each data point is the mean of three replicates, and error bars represent sd. A1 to A5, DVR activities for DV-Chlide a, DV-Chl a, DV-Pchlide a, DV-MPE, and DV-Mg-Proto were measured at various temperatures (10°C–40°C) at pH 7.0. B1 to B5, DVR activities for DV-Chlide a, DV-Chl a, DV-Pchlide a, DV-MPE, and DV-Mg-Proto were measured at various pH values (6.0–8.0) at 30°C (for DV-Chlide a and DV-Chl a) or 20°C (for DV-Pchlide a, DV-MPE, and DV-Mg-Proto).

Subsequently, we determined catalytic activities of DVR proteins. Each substrate was incubated with four recombinant DVR proteins under the optimum temperature and pH conditions (Fig. 1) of this substrate. Incubation time was set to 20 s to 10 h based on DVR activity for the conversion of a substrate. The results were as follows.

(1) Reductive activities of DVRs on different substrates were significantly different. For example, reaction velocities of OsDVR for conversion of DV-Chlide a and DV-Chl a in the first 1 min of incubation amounted to 8.02 and 5.21 nmol mg−1 protein min−1, respectively, while those for DV-Pchlide a and DV-MPE in the first 1 h of incubation were only 2.95 and 1.51 nmol mg−1 protein h−1, respectively (Fig. 2, B1–B4). The slowest reaction velocity was observed for DV-Mg-Proto (Fig. 2B5). Catalytic activity of OsDVR on DV-Chlide a was about 50% higher than that on DV-Chl a and about 160- and 320-fold higher than that on DV-Pchlide a and DV-MPE, respectively, indicating that OsDVR has quite different catalytic activities on different substrates.

Figure 2.

Figure 2.

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Catalytic activities of four recombinant DVR proteins. Each of five substrates (DV-Chlide a, DV-Chl a, DV-Pchlide a, DV-MPE, and DV-Mg-Proto) was incubated with each of four recombinant DVR proteins (OsDVR, ZmDVR, CsDVR, and AtDVR) expressed in E. coli, and the pigments extracted from reaction mixtures were subjected to HPLC. Each data point is the mean of three replicates, and error bars represent sd. A1 to A5, Conversion rates of DV-Chlide a, DV-Chl a, DV-Pchlide a, DV-MPE, and DV-Mg-Proto to their corresponding MV products during various incubation times (reaction time) under the optimum temperature and pH conditions depicted in Figure 1. B1 to B5, Reaction velocities of DVR proteins for conversion of DV-Chlide a, DV-Chl a, DV-Pchlide a, DV-MPE, and DV-Mg-Proto in the first 1 min or 1 h of incubation under the optimum temperature and pH conditions, which were calculated as described in “Materials and Methods.”

(2) Although having no obvious target band in SDS-PAGE (Supplemental Fig. S2), the bacterial lysate expressing recombinant ZmDVR protein in E. coli had DVR activities very similar to that expressing OsDVR protein under the same experimental conditions (including adding the same volume of crude lysate in an enzymatic reaction), which suggested that, like OsDVR, ZmDVR was able to convert all five DV-Chl intermediates into corresponding MV compounds (Figs. 2–5).

Figure 5.

Figure 5.

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Enzymatic assays of four recombinant DVR proteins using DV-MPE (A) and DV-Mg-Proto (B) as substrates. Each substrate was incubated for 10 h with OsDVR, ZmDVR, CsDVR, and AtDVR proteins expressed in E. coli, and the pigments extracted from reaction mixtures were subjected to HPLC. The left column shows the chromatograms that were detected at 410 nm by HPLC, and the right column shows the spectrum of each peak. A1 and B1, DV-MPE and DV-Mg-Proto standards purchased from Frontier Scientific. A2 and B2, Products synthesized after DV-MPE and DV-Mg-Proto were incubated with E. coli lysates expressing the empty vector, which were used as negative controls. A3 to A6, Products synthesized after DV-MPE was incubated with E. coli lysates expressing OsDVR, ZmDVR, CsDVR, and AtDVR, respectively. B3 to B6, Products synthesized after DV-Mg-Proto was incubated with E. coli lysates expressing OsDVR, ZmDVR, CsDVR, and AtDVR, respectively.

(3) CsDVR and AtDVR were able to efficiently convert DV-Chlide a to MV-Chlide a with reaction velocities of 3.62 and 2.92 nmol mg−1 protein min−1, respectively, in the first 1 min of incubation (Fig. 2B1). In addition, the CsDVR and AtDVR proteins were also able to convert DV-Pchlide a and DV-MPE into MV-Pchlide a and MV-MPE, respectively. Although the reaction velocities were extremely slow, small amounts of the two substrates were robustly detected to be converted to the corresponding MV products after 10 h of incubation (Figs. 2, A3 and A4, 4, A6 and A7, and 5, A5 and A6). However, both CsDVR and AtDVR were incapable of converting DV-Chl a and DV-Mg-Proto to corresponding MV compounds (Figs. 2, A2 and A5, 3, B6 and B7, and 5, B5 and B6), even though the incubation time of DV-Chl a was prolonged to 2 h (Supplemental Fig. S3) or the substrate concentration of DV-Mg-Proto was increased from 2 to 15 nmol in the enzyme reaction.

Figure 4.

Figure 4.

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Enzymatic assay of four recombinant DVR proteins using DV-Pchlide a as a substrate. The substrate was incubated for 10 h with OsDVR, ZmDVR, CsDVR, and AtDVR proteins expressed in E. coli, and the pigments extracted from reaction mixtures were subjected to HPLC. The left column shows the chromatograms that were detected at 440 nm by HPLC, and the right column shows the spectrum of each peak. A1 and A2, DV- and MV-Pchlide a prepared from the 6-d-old etiolated seedlings of the rice 824ys mutant and its wild type. A3, Product synthesized after incubation with E. coli lysates expressing the empty vector, which was used as a negative control. A4 to A7, Products synthesized after incubation with E. coli lysates expressing OsDVR, ZmDVR, CsDVR, and AtDVR, respectively.

Figure 3.

Figure 3.

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Enzymatic assays of four recombinant DVR proteins using DV-Chlide a (A) and DV-Chl a (B) as substrates. Each substrate was incubated for 10 min with OsDVR, ZmDVR, CsDVR, and AtDVR expressed in E. coli, and the pigments extracted from reaction mixtures were subjected to HPLC. The left column shows the chromatograms that were detected at 440 nm for Chlide a or at 660 nm for Chl a, and the right column shows the spectrum of each peak. A1 and A2, DV- and MV-Chlide a prepared from DV- and MV-Chl a by the enzymatic reaction of chlorophyllase isolated from garland chrysanthemum leaves, respectively. B1 and B2, DV- and MV-Chl a prepared from the rice 824ys mutant and its wild type, respectively. A3 and B3, Products synthesized after DV-Chlide a and DV-Chl a were incubated with E. coli lysates expressing the empty vector, which were used as negative controls. A4 to A7, Products synthesized after DV-Chlide a was incubated with E. coli lysates expressing OsDVR, ZmDVR, CsDVR, and AtDVR, respectively. B4 to B7, Products synthesized after DV-Chl a was incubated with E. coli lysates expressing OsDVR, ZmDVR, CsDVR, and AtDVR, respectively. A0, MV-Chlide a resulting from incubation only with an acetone powder of garland chrysanthemum leaves, suggesting that a small amount of MV-Chl a still remained in the acetone powder after being washed repeatedly; the minor peak (peak 2) of A1 and A3 was exactly the same as the MV-Chlide a converted from residue MV-Chl a in the acetone powder.

(4) OsDVR and ZmDVR had significantly higher DVR activities than CsDVR and AtDVR on the same substrate. For example, the conversions of DV-Chlide a to MV-Chlide a by OsDVR and ZmDVR were obviously faster than that by CsDVR and AtDVR (Fig. 2, A1 and B1).

These data confirmed that the OsDVR, ZmDVR, CsDVR, and AtDVR proteins all have broad substrate specificity, although with significant differences in DVR activities.

In Vivo Accumulation of Chl Intermediates in Rice, Maize, and Cucumber

Pchlide is the end product of the Chl biosynthetic pathway in etiolated seedlings because the reduction of Pchlide to Chlide absolutely requires light in angiosperms (Griffiths, 1975, 1978; Masuda and Takamiya, 2004; Tanaka and Tanaka, 2007). Thus, the accumulation of MV-Pchlide a can reflect the conversion efficacy of DV-Pchlide a and/or DV-MPE and/or DV-Mg-Proto to corresponding MV compounds in vivo. To test if our observations of the different conversion velocities of different DVRs on DV-Chl intermediates in vitro are true in vivo, we measured the accumulation of Chl intermediates in rice, maize, and cucumber. First, we measured the accumulation of MV-Pchlide a. After being grown in the dark for 6 d, the etiolated rice and maize seedlings almost entirely accumulated MV-Pchlide a (Fig. 6, A and B). Conversely, the etiolated cucumber cotyledons accumulated only about 68% of MV-Pchlide a, and nearly one-third of DV-Pchlide a remained to be converted (Fig. 6C). These data suggested that rice, maize, and cucumber can convert DV-Pchlide a and/or DV-MPE and/or DV-Mg-Proto to corresponding MV compounds in vivo, although the conversion efficacy in the three plants could be different. Next, we examined the reaccumulation of Pchlide. Etiolated cucumber cotyledons and rice and maize seedlings were illuminated for 14 h and then returned to the dark for 0 min, 10 min, 2 h, and 10 h. As shown in Figure 7, at the beginning of the dark period (0 min), rice, maize, and cucumber only accumulated a very small amount of Pchlide a, and their Pchlide a pools consisted almost entirely of DV-Pchlide a (Fig. 7, A1–A3). After 10 min of dark incubation, Pchlide a pools consisted of about 26% MV-Pchlide a in rice and maize (Fig. 7, B1 and B2). After 2 h of dark incubation, the amount of MV-Pchlide a in rice and maize accumulated higher than that of DV-Pchlide a (Fig. 7, C1 and C2). By the end of 10 h of dark incubation, the Pchlide pools consisted almost completely of MV-Pchlide a in the etiolated rice and maize seedlings (Fig. 7, D1 and D2). Contrary to these observation, MV-Pchlide a was still undetectable in cucumber after 2 h of dark incubation (Fig. 7, B3 and C3). However, by the end of 10 h of dark incubation, a considerable amount (about 40%) of MV-Pchlide a was detected in the etiolated cucumber cotyledons (Fig. 7D3). These results once again indicated that DV-Pchlide a and/or DV-MPE and/or DV-Mg-Proto can be converted to corresponding MV compounds in rice, maize, and cucumber in vivo, but the conversion velocities in rice and maize are much faster than that in cucumber. These observations were consistent with our in vitro data that showed the efficient conversions of DV-Pchlide a and/or DV-MPE and/or DV-Mg-Proto to corresponding MV compounds by DVRs from rice and maize, while they were extremely slow by DVR from cucumber.

Figure 6.

Figure 6.

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Accumulation of Pchlide in 6-d-old etiolated seedlings. Wild-type rice (A), maize (B), cucumber (C), and rice 824ys mutant (D) were grown in the dark at 28°C for 6 d, and Pchlide a was extracted and subjected to HPLC. The left column shows the chromatograms that were detected at 440 nm by HPLC, and the right column shows the spectrum of each peak. Peak 1, DV-Pchlide a; peak 2, MV-Pchlide a.

Figure 7.

Figure 7.

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Reaccumulation of Pchlide during different periods in the dark. Rice, maize, and cucumber were grown in the dark at 28°C for 6 d. Subsequently, the etiolated seedlings were exposed to 14 h of continuous illumination (50–70 μmol m−2 s−1) and then returned to the dark for 0 min (A), 10 min (B), 2 h (C), and 10 h (D). Pigments were extracted and then detected at 440 nm by HPLC. Peak 1, DV-Pchlide a; peak 2, MV-Pchlide a. The absorption spectra of peaks 1 and 2 are as in Figure 6.

In addition, we investigated the accumulation of protoporphyrin IX, Mg-Proto, and MPE. Picolinic acid (PA) was shown to induce the accumulation of these intermediates in plants (Mayasich et al., 1990; Nandihalli and Rebeiz, 1991; Kim and Rebeiz, 1996). By growing rice, maize, and cucumber in the dark at 28°C for 5 d, we incubated the etiolated cucumber cotyledons and rice and maize leaves in 30 mm PA and 40 mm 5-aminolevulinic acid (ALA) in darkness at 28°C for 14 h. Then we measured the accumulation of tetrapyrroles. As shown in Figure 8, a certain amount of MV-MPE was detected in etiolated rice, maize, and cucumber (Fig. 8, A–C, peak 3′), suggesting that DV-MPE and/or DV-Mg-Proto can be converted to corresponding MV compounds in these plants in vivo. Moreover, a certain amount of MV-Mg-Proto was also detected in the etiolated rice (Fig. 8A, peak 1′), confirming that DV-Mg-Proto can be converted to MV-Mg-Proto in rice in vivo.

Figure 8.

Figure 8.

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Accumulation of tetrapyrroles in the etiolated cucumber cotyledons and rice and maize leaves treated with ALA and PA. Wild-type rice (A), maize (B), cucumber (C), and rice 824ys mutant (D) were grown in the dark at 28°C for 5 d. Subsequently, the etiolated cucumber cotyledons and rice and maize leaves were incubated with 40 mm ALA and 30 mm PA in darkness at 28°C for 14 h. Then, tetrapyrroles were extracted and subjected to HPLC. The left column shows the chromatograms that were detected at 410 nm by HPLC, and the right column shows the spectrum of each peak.

In Vivo Accumulation of Chl and Its Intermediates in Rice 824ys Mutant

In the previous study, we demonstrated that the rice 824ys mutant accumulated only DV-Chls and had no MV-Chls, because it had a defective DVR protein encoded by a mutant gene of Os03g22780 (Wang et al., 2010). However, at that time, we examined Chl compositions of the mutant only using leaf blades of 4-week-old plants (at the five-leaf stage). We could not exclude the possible accumulation of MV-Chls generated by DVRs other than OsDVR in the mutant if we did not check various tissues during various developmental stages (Islam et al., 2008; Ito et al., 2008). To this end, we systematically investigated Chl compositions in leaf blade, leaf sheath, uppermost internode, panicle rachis, glume, and caryopsis of the 824ys mutant at seedling, tillering, flowering, filling, and maturation stages. As shown in Figure 9, contrary to the wild-type rice that contained MV-Chl a and MV-Chl b, the 824ys mutant exclusively accumulated DV-Chls; indeed, it did not accumulate any MV-Chls in any tissues during any developmental stages tested, including rapidly greening tissues and senescent tissues. In addition, we examined Pchlide accumulation of the etiolated 824ys mutant seedlings grown in the dark for 6 d, showing that this mutant exclusively accumulated DV-Pchlide a in the dark (Fig. 6D). Moreover, we investigated the accumulation of protoporphyrin IX, Mg-Proto, and MPE in the etiolated 824ys mutant leaves treated with PA and ALA, showing that this mutant accumulated only DV intermediates (Fig. 8D). These observations demonstrated that the 824ys mutant cannot convert any DV intermediates of Chls to corresponding MV compounds in vivo, strongly suggesting that the OsDVR encoded by Os03g22780 is the only protein that is responsible for reducing the 8-vinyl groups of various intermediates of Chl biosynthesis in rice.

Figure 9.

Figure 9.

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Chl compositions of various organs of the rice 824ys mutant during various developmental stages. Chls were extracted and subjected to HPLC. The elution profiles of wild-type (A) and 824ys mutant (B) rice were detected at 660 nm. Absorption spectra of peaks 1 and 1′ (C) and peaks 2 and 2′ (D) in acetone were compared. Peak 1, MV-Chl b; peak 2, MV-Chl a; peak 1′, DV-Chl b; peak 2′, DV-Chl a. A1 and A2, Seedlings of wild-type rice at the one-leaf stage (heterotrophic period) and the five-leaf stage, respectively. B1 and B2, Seedlings of 824ys mutant rice at the one-leaf stage (heterotrophic period) and the five-leaf stage, respectively. B3 and B4 Leaf blade and sheath of 824ys mutant rice during tillering stage, respectively. B5 to B8 Leaf blade, leaf sheath, uppermost internode (including panicle rachis), and glume of 824ys mutant rice during flowering stage, respectively. B9 to B13, Leaf blade, leaf sheath, uppermost internode (including panicle rachis), glume, and caryopsis of 824ys mutant rice during grain-filling stage, respectively. B14 to B18, Leaf blade, leaf sheath, uppermost internode (including panicle rachis), glume, and caryopsis of 824ys mutant rice during maturation stage, respectively.

Collectively, the in vivo accumulation of Chl intermediates in rice, maize, and cucumber were consistent with the results of the above in vitro enzymatic assays. These data provide robust supporting evidence that a single DVR protein with broad substrate specificity is responsible for reducing the 8-vinyl groups of various intermediate molecules of Chl biosynthesis in higher plants, but homologous DVR proteins from different species could have quite different reductive activities.

DISCUSSION

A Single DVR Protein Has Broad Substrate Specificity

So far, five DVR activities have been detected at the levels of DV-Mg-Proto (Kim and Rebeiz, 1996), DV-MPE (Ellsworth and Hsing, 1974; Kim and Rebeiz, 1996), DV-Pchlide a (Tripathy and Rebeiz, 1988), DV-Chlide a (Parham and Rebeiz, 1992, 1995; Kolossov and Rebeiz, 2001), and DV-Chl a (Adra and Rebeiz, 1998). However, it is unclear whether these DVR activities are catalyzed by one enzyme with broad substrate specificity or by multiple enzymes with narrow specificity. Nagata et al. (2007) reported that AtDVR is able to convert DV-Chlide a to MV-Chlide a but unable to convert DV-Pchlide a, DV-Chlide b, DV-Chl a, and DV-Chl b to corresponding MV compounds. Nevertheless, Chew and Bryant (2007) demonstrated that a recombinant DVR protein (BciA) of the green sulfur bacterium C. tepidum successfully reduced the 8-vinyl group of DV-Pchlide a in vitro. In a previous report, we confirmed that OsDVR is able to convert both DV-Chlide a and DV-Chl a to MV-Chlide a and MV-Chl a, respectively (Wang et al., 2010). In this study, we found that OsDVR and ZmDVR have broader substrate specificity than CsDVR and AtDVR. Both OsDVR and ZmDVR proteins can convert the five Chl biosynthetic intermediates, DV-Chl a, DV-Chlide a, DV-Pchlide a, DV-MPE, and DV-Mg-Proto, into corresponding MV compounds (Figs. 1–5). Both CsDVR and AtDVR proteins can convert DV-Chlide a, DV-Pchlide a, and DV-MPE, but not DV-Chl a and DV-Mg-Proto, into corresponding MV compounds (Figs. 1–5). These data indicate that a single DVR protein has broad substrate specificity with different range.

Homologous DVR Proteins from Different Species Have Diverse and Differing Substrate Preferences

DVR proteins derived from different plant species have quite different reductive activities on the same or different substrates. DV-Pchlide could be partially converted to MV-Pchlide in barley plastids but not in cucumber plastids (Tripathy and Rebeiz, 1988). The velocity of converting DV-Chlide a in etioplast membranes from etiolated cucumber cotyledons and barley and maize leaves was 50- to 300-fold higher than that of converting DV-Pchlide a in barley etioplasts (Tripathy and Rebeiz, 1988; Parham and Rebeiz, 1995). Our data demonstrate that OsDVR and ZmDVR have much higher catalyzing activities than CsDVR and AtDVR in the conversion of DV-Pchlide a and DV-MPE to the corresponding MV compounds (Figs. 2, 4, and 5A). These observations reasonably explain why the relative amounts of MV-Pchlide a in the Pchlide pools in some plant species such as rice, maize, and wheat are significantly higher than those in other plant species such as cucumber and mustard (Brassica juncea) in the dark for 6 d or 10 h (Figs. 6 and 7; Carey and Rebeiz, 1985). In addition, the same DVR protein also has quite different reductive activities on different substrates. Both OsDVR and ZmDVR have significantly higher efficacy in converting DV-Chlide a and DV-Chl a to MV-Chlide a and MV-Chl a, respectively, than that in converting DV-Pchlide a, DV-MPE, and DV-Mg-Proto to corresponding MV compounds (Figs. 2–5). Both CsDVR and AtDVR also have substantially higher efficiency to reduce DV-Chlide a into MV-Chlide a than that to reduce DV-Pchlide a and DV-MPE into corresponding MV compounds (Figs. 2, 3A, 4, and 5A).

A Single DVR Protein Is Responsible for Reducing the 8-Vinyl Groups in Higher Plants

Chl biosynthetic heterogeneity is assumed to originate mainly in parallel DV-Chl and MV-Chl biosynthetic routes interconnected by 8-vinyl reductases (Parham and Rebeiz, 1995; Rebeiz et al., 2003). In past decades, 8-vinyl Mg-Proto, MPE, Pchlide a, Chlide a, and Chl a reductases were all proposed as a kind of 8-vinyl reductase (Ellsworth and Hsing, 1974; Tripathy and Rebeiz, 1988; Parham and Rebeiz, 1992, 1995; Kim and Rebeiz, 1996; Adra and Rebeiz, 1998; Kolossov and Rebeiz, 2010), but none of these enzymes had been successfully purified until 2001, when the enzyme was solubilized and partially purified from etiolated barley leaves (Kolossov and Rebeiz, 2001). In recent years, AtDVR (AT5G18660) and OsDVR (Os03g22780) genes were isolated from Arabidopsis and rice, respectively, and identified as homologs encoding an 8-vinyl reductase (Nagata et al., 2005, 2007; Wang et al., 2010). However, it has not been fully clear whether one or multiple enzymes are responsible for the reduction of the 8-vinyl groups of various Chl intermediates. A maize mutant accumulated only DV-Chls (Bazzaz, 1981; Bazzaz and Brereton, 1982; Bazzaz et al., 1982), which implied that a single gene product might be responsible for the reduction of the 8-vinyl group of Chl intermediates, although the possibility that the gene encodes a regulator for the divinyl reduction could not be excluded. The AtDVR (AT5G18660)-inactivated pcb2 mutant of Arabidopsis also exclusively accumulated DV-Chls in 16-d-old whole plants, which suggested that Arabidopsis could contain only one functional DVR (Beale, 2005; Nakanishi et al., 2005), but other possibilities were also proposed (Islam et al., 2008; Ito et al., 2008). Previously, we reported that the OsDVR (Os03g22780)-inactivated 824ys mutant of rice accumulated only DV-Chls and no MV-Chls in leaf blade of 4-week-old plants (Wang et al., 2010). Here, we further demonstrated that the 824ys mutant is unable to convert the 8-vinyl group of any Chl intermediates to the ethyl group in vivo (Figs. 6D, 8D, and 9B). Moreover, the enzymatic assays clearly demonstrated that a single DVR protein has broad substrate specificity, although homologous DVR proteins from different species could have quite different DVR activities (Figs. 1–5). In addition, the DVR proteins used in this study all are encoded by a single-copy gene, and no other homologous genes were found in the rice, maize, cucumber, and Arabidopsis genomes. Collectively, we are confident that a single DVR protein is responsible for reducing the 8-vinyl groups of various intermediate molecules of Chl biosynthesis in higher plants, at least in angiosperms represented by rice and Arabidopsis.

Nonetheless, two DVRs, BciA (CT1063) and BciB (CvrA, Slr1923), have been identified in photosynthetic bacteria and are sufficient to independently produce 8-vinyl reductase activity (Chew and Bryant, 2007; Islam et al., 2008; Ito et al., 2008; Liu and Bryant, 2011). Multiple amino acid sequence alignment indicated that BciA is a homolog of both OsDVR (Os03g22780) and AtDVR (At5G18660), whereas BciB has no homology to both OsDVR and AtDVR. Interestingly, homologs of the BciB gene also exist in the rice (Os04g25400) and Arabidopsis (At1G04620) genomes, but the At1G04620 gene product was recently characterized as a 7-hydroxymethyl Chl a reductase that converts 7-hydroxymethyl Chl a to Chl a (Meguro et al., 2011).

Chl Biosynthetic Pathways Include Multibranched Routes That Resulted from a Single DVR Protein in Higher Plants

A multibranched Chl biosynthetic pathway was proposed due to the detection of MV and DV tetrapyrrole intermediates and their biosynthetic interconversion in extracts of different plant tissues (Rebeiz et al., 1983, 1986, 1999; Whyte and Griffiths, 1993; Kolossov and Rebeiz, 2010). Nagata et al. (2007) suggested that the major route for Chl synthesis includes DV-Chlide a reduction in Arabidopsis, but they also observed that DV-Pchlide a can be converted to MV-Pchlide a in vivo, although the conversion of DV-Pchlide a to MV-Pchlide a is much slower than that of DV-Chlide a to MV-Chlide a, which actually indicated the existence of a multibranched pathway in Arabidopsis. In this paper, three or two MV-Chl intermediates were simultaneously detected in etiolated rice, maize, and cucumber after ALA and PA treatment (Fig. 8, peaks 1′, 2′, and 3′), also suggesting that multibranched pathways do exist. In addition, the enzymatic assays demonstrated that both OsDVR and ZmDVR can convert five DV substrates to corresponding MV compounds, and both CsDVR and AtDVR can convert three of them (Figs. 1–5), which match the branch points of the multipathways converting DV-Chl to MV-Chl intermediates. Meanwhile, our data indicated that a single DVR is responsible for reducing 8-vinyl groups of various Chl intermediates. These results strongly suggested that Chl biosynthetic pathways include multibranched routes that resulted from a single DVR in higher plants.

On the basis of MV- or DV-Pchlide accumulation during the dark and light phases of the photoperiod, green plants have been classified into three different greening groups: dark divinyl-light divinyl (DDV-LDV), dark monovinyl-light divinyl (DMV-LDV), and dark monovinyl-light monovinyl (DMV-LMV) plants (Carey et al., 1985; Carey and Rebeiz, 1985; Shioi and Takamiya, 1992; Ioannides et al., 1994; Mageed et al., 1997). DDV-LDV plants, such as cucumber and Arabidopsis, accumulate mainly DV-Pchlide a at night and in daytime. DMV-LMV plants, such as johnsongrass (Sorghum halepense), accumulate mainly MV-Pchlide a at night and in daytime. DMV-LDV plants, such as rice, maize, and wheat, accumulate MV-Pchlide a at night, but in daytime they accumulate mainly DV-Pchlide a (Ioannides et al., 1994). The three greening groups actually result from the above multibranched routes of Chl biosynthesis. Our results demonstrated that homologous DVR proteins from different species could have quite different DVR activities on the same or different substrates (Figs. 1–7), which reasonably explains differences in DV- and MV-Pchlide accumulation of the three different greening groups.

In summary, we here propose that the multibranched Chl biosynthetic pathways resulted from a single DVR protein in higher plants (Fig. 10), which have the following characteristics. (1) Chl biosynthetic pathways include multibranched routes. (2) The multibranched routes result from a single DVR with broad substrate specificity. (3) DVR proteins derived from different species could have diverse and differing substrate preferences even though they are homologous, and the same DVR protein could also have quite different DVR activities on different substrates. (4) The origin of the multibranched routes could not be the same in various species. The routes may originate at the level of DV-MPE in some species such as cucumber and Arabidopsis or at the level of DV-Mg-Proto in other species such as rice and maize. (5) The conversion efficiency of various DV substrates to corresponding MV compounds could be very different. For example, the conversion of DV-Pchlide a and DV-MPE is generally much slower than that of DV-Chlide a. (6) The contributions of various routes could be very different in the overall synthesis of Chl. The major route(s) could include the reduction of the 8-vinyl groups of only DV-Chlide a in some species such as cucumber and Arabidopsis or that of both DV-Chlide a and DV-Chl a in other species such as rice and maize.

Figure 10.

Figure 10.

Open in a new tab

Multibranched Chl biosynthetic pathways resulting from a single DVR protein in higher plants. Arrows joining the DV and MV branches indicate the reactions catalyzed by a single DVR. The thickness of arrow lines represents relative DVR activities based on the results of this study. Virtual boxes indicate the enzymatic reactions mainly occurring in the dark. Proto, Protoporphyrin IX. A, The pathway may operate mainly in DDV-LDV plants represented by cucumber and Arabidopsis. B, The pathway may operate mainly in DMV-LDV plants represented by rice and maize.

In the dark, the Chl synthesis pathway leads only to the formation of Pchlide in angiosperms. Once a critical level of Pchlide has been reached, ALA synthesis slows down (Griffiths, 1975, 1978; Masuda and Takamiya, 2004). Our data demonstrated that the conversions of DV-Pchlide a, DV-MPE, and DV-Mg-Proto to corresponding MV compounds were much slower than that of DV-Chlide a and DV-Chl a (Figs. 1–7), which could be a kind of adaptation to the diurnal variation of Chl biosynthesis. On the other hand, ALA-induced tetrapyrrole accumulation in green plants could cause extensive photodynamic damage to some plant species, while other plant species remained unaffected. The nonsusceptible plant species maintained higher relative levels of MV-Pchlide than the susceptible ones under the subdued light levels (Carey and Rebeiz, 1985). So the conversion of DV-Pchlide to MV-Pchlide may be considered as one means of eliminating the excess DV-Pchlide (Tripathy and Rebeiz, 1988). Therefore, the multibranched Chl biosynthetic pathways could be physiologically important.

Natural selection has often produced multiple (bio)chemical and physical ways of conveying the same message. It is also possible that, via natural selection, Chl biosynthetic heterogeneity has imparted an evolutionary advantage to higher plants. Ioannides et al. (1994) proposed that the DDV-LDV greening group is evolutionarily ancestral, because so far all representative primitive plant species, including algae, bryophytes, ferns, and gymnosperms, fall into this greening group. The DMV-LMV greening group comprises a small number of plant species, and evolutionary studies suggested that it is derived (Ioannides et al., 1994). The DMV-LDV greening group comprises by far the largest number of plant species surveyed to date, and plant species of major agronomic importance belong to this group, which was proposed to be evolutionarily intermediate (Ioannides et al., 1994). Accordingly, our results demonstrated that DVR proteins of DMV-LDV rice and maize have broader substrate specificity and higher catalyzing activities (especially for DV-Pchlide a and DV-MPE) than those of DDV-LDV cucumber and Arabidopsis (Figs. 1–7), suggesting that the heterogeneity of DVR activity could be a species-dependent phenomenon with evolutionary significance.

MATERIALS AND METHODS

Plant Materials

Rice (Oryza sativa; including the 824ys mutant and its wild-type 824B), maize (Zea mays), and cucumber (Cucumis sativus) were planted under natural conditions in April to August in Wenjiang District (latitude 30°42′N, longitude 103°50′E, altitude 539.3 m), Chengdu City, Sichuan Province (Wang et al., 2010). Arabidopsis (Arabidopsis thaliana) was grown at 23°C with a 14-h-light/10-h-dark photoperiod. Etiolated seedlings of rice, maize, cucumber, and wheat (Triticum aestivum) were grown in the dark at 28°C for 6 d.

Sequence Analysis

The full-length DNA and protein sequences of OsDVR, ZmDVR, and AtDVR genes were retrieved from GenBank (http://www.ncbi.nlm.nih.gov). The full-length DNA and protein sequences of CsDVR were retrieved from http://cucumber.genomics.org.cn/page/cucumber/index.jsp. The four genes were isolated from rice, maize, cucumber, and Arabidopsis genomic DNA by PCR amplification. The chloroplast signal peptide was predicted with TargetP (http://www.cbs.dtu.dk/services/TargetP; Emanuelsson et al., 2000). The amino acid alignment was conducted using ClustalX (Thompson et al., 1997).

Pigment Standards

Chl a and b standards were purchased from Sigma. DV-MPE, DV-Mg-Proto, and DV protoporphyrin IX were purchased from Frontier Scientific.

Analysis of Chl Composition

Chls used for Chl composition analysis were extracted from the 824ys mutant and its wild-type 824B with 100% acetone. The extract was centrifuged at 12,857g (Eppendorf 5804R; 10,000 rpm) for 15 min, and the supernatants were subjected to HPLC analysis on a C18 column (4.6 mm i.d. × 150 mm long, 5 µm; Agilent) and eluted with solvent (methanol:acetonitrile:acetone, 1:3:1) at a flow rate of 1.0 mL min−1 at 40°C (Nakanishi et al., 2005). Elution profiles were monitored by measuring A660, and Chl a and b standards were used as a control.

Preparation of Chl a and Chlide a

DV/MV-Chl a were prepared from the 824ys mutant and its wild-type 824B of rice, respectively. Chls were extracted from fresh rice leaf tissue with 100% acetone, the extract was centrifuged at 12,857g (5804R; Eppendorf) for 15 min, and the supernatants were dried under N2 gas and redissolved with 100% acetone. DV/MV-Chl a were separated by HPLC on a C18 column (4.6 mm i.d. × 150 mm long, 5 μm; Agilent) and eluted with solvent (methanol:acetonitrile:acetone, 1:3:1) at a flow rate of 1.0 mL min−1 at 40°C (as described by Nakanishi et al. [2005]). Elution profiles were monitored by measuring A660.

Chlorophyllase (an acetone powder of garland chrysanthemum [Chrysanthemum coronarium] leaves) was prepared according to the method of Ito et al. (1994). A total of 200 μg of purified DV/MV-Chl a was dissolved in 4 mL of a solution containing 25 mm Tris-HCl (pH 7.5) and 40% acetone and then incubated with 200 mg of an acetone powder of garland chrysanthemum leaves for 2 h at 28°C (Holden,1961; Ito et al., 1996). After incubation of the mixture in the dark, DV/MV-Chlide a were purified as described by Ito et al. (1993). The DV/MV-Chlide a were analyzed by HPLC on a C8 column (4.6 mm i.d. × 150 mm long, 3.5 µm; Agilent) according to the method of Zapata et al. (2000). Elution profiles were monitored by measuring A440.

Extraction of Pchlide a

DV-Pchlide a was prepared from 6-d-old etiolated seedlings of the rice 824ys mutant, and MV-Pchlide a was prepared from that of wild-type rice 824B. MV-Pchlide a prepared from 6-d-old etiolated seedlings of wheat was used as a control (Carey and Rebeiz, 1985).

Etiolated cucumber cotyledons and rice, maize, and wheat seedlings were homogenized in acetone containing 0.1 m NH4OH under a green safelight. Tris-HCl (pH 7.6) was added to the acetone solution to a final concentration of 200 mm in order to protect against modification of Pchlide. Then, an equal volume of hexane was applied to the acetone solution to remove Chls and carotenoid. Finally, Pchlide in the acetone solution was transferred to diethylether and dried under N2 gas (Nagata et al., 2007). The Pchlide was analyzed by HPLC on a C8 column (4.6 mm i.d. × 150 mm long, 3.5 µm; Waters) according to the method of Zapata et al. (2000). Elution profiles were monitored by measuring A440.

Induction of the Accumulation of MPE, Mg-Proto, and Protoporphyrin IX in Vivo

Five-day-old etiolated cucumber cotyledons and rice and maize leaves (about 0.2 g fresh weight) were cut out and incubated in a deep petri dish (8.5 cm i.d. × 1.5 cm deep) in 10 mL of solution containing 40 mm ALA and 30 mm PA. The incubation was carried out in darkness at 28°C for 14 h (Kim and Rebeiz, 1996). Subsequently, the incubated tissues were homogenized in acetone:0.1 n NH4OH (9:1, v/v) under a green safelight. The homogenate was centrifuged at 7,197g (Eppendorf 5430R; 7,830 rpm) at 1°C for 30 min, and the resulting supernatant was stored at −80°C until tetrapyrrole extraction. Then, Chl and other fully esterified tetrapyrroles were transferred from acetone to hexane by extraction with an equal volume of hexane, followed by a second extraction with one-third volume of hexane (Kim and Rebeiz, 1996). The remaining hexane-extracted acetone residue was analyzed by HPLC on a C8 column (4.6 mm i.d. × 150 mm long, 3.5 µm; Waters) according to the method of Zapata et al. (2000). Elution profiles were monitored by measuring A410.

Enzymatic Activity Assays

Full-length OsDVR, ZmDVR, CsDVR, and AtDVR genomic DNAs were amplified by PCR from rice, maize, cucumber, and Arabidopsis genomic DNAs using primers OS (forward, 5′-CAGGATCCATGGCTGCCCTCCTCCTCT-3′; reverse, 5′-GAAGAATTCCGAGGCCTAGAAGATGGT-3′), ZM (forward, 5′-CCGGATCCATGGCGACCATCCTCCTATC-3′; reverse, 5′-ATGGAATTCGCCTAGAAGATGGTCTGCTC-3′), CS (forward, 5′-GAGGATCCATGTCCATTTGCTCCACCGTT-3′; reverse, 5′-ACCGAGCTCAAAAAACGCTCTGTTCACC-3′), and AT (forward, 5′-ATGGATCCATGTCACTTTGCTCTTCCTTCAACG-3′; reverse, 5′-CGGAATTCCTAGAAGAACTGTTCACCGAGTTC-3′), respectively. The primers incorporated a BamHI site at the N-terminal end and a EcoRI (or SacI) site at the C-terminal end of these ORFs. PCR products were inserted into the pMD18-T vector (TaKaRa) and sequenced to obtain the correct clones, pMD-OsDVR, pMD-ZmDVR, pMD-CsDVR, and pMD-AtDVR, respectively. The pMD-OsDVR, pMD-ZmDVR, pMD-CsDVR, and pMD-AtDVR plasmids were then digested by BamHI and EcoRI (or SacI) enzymes; cloned into the corresponding site of the bacterial expression vector pET30a(+) (Novagen) to generate pET30-OsDVR, pET30-ZmDVR, pET30-CsDVR, and pET30-AtDVR, respectively; sequenced to confirm OsDVR, ZmDVR, CsDVR, and AtDVR sequences, respectively; and finally introduced into Escherichia coli BL21 for protein expression. The cells transformed with an empty vector were used as a negative control.

Protein expression and recombinant enzyme activity assays were performed according to the methods described by Nagata et al. (2005, 2007) and Wang et al. (2010) with slight modifications. A culture of E. coli strain BL21 containing pET30-OsDVR, pET30-ZmDVR, pET30-CsDVR, pET30-AtDVR, or pET30 plasmid alone was grown overnight in 2 mL of Luria-Bertani broth containing 50 μg mL−1 kanamycin. Overnight cultures were subsequently used to inoculate 50 mL of Luria-Bertani broth containing 50 μg mL−1 kanamycin. To maximize protein production, isopropylthio-β-galactoside was added to the culture at a final concentration of 0.5 mm after 30 min at 30°C. After 7 h of incubation, the culture was harvested and resuspended with 2.5 mL of solution containing 6.7 μg mL−1 lysozyme and 3.3 μg mL−1 DNaseI in 50 mm Tris-HCl, pH 8.0. The culture lysate was stored at −20°C until use. The culture lysate corresponding to 200 μg of DVR proteins (or 700 μL of the lysate expressing ZmDVR) was used as the source of the enzyme for every experiment. DVR activity was determined in 700 μL of incubation buffer (40 mm citric acid, 80 mm K2HPO4, 0.5 mm NADPH, and 20% acetone, adjusted to the pH as indicated in “Results” with KOH at room temperature). The incubation buffer was preincubated with the culture lysate for 5 min at various temperatures as indicated in “Results” before initiation of the reaction. The reaction was started by adding 2 nmol of a substrate that was dissolved in acetone and incubated for the length of time indicated in “Results.” The reaction was stopped by adding 700 μL of acetone. The pigments were transferred from acetone to diethylether and dried under N2 gas. These pigments were dissolved in a small volume of acetone and analyzed by HPLC on a C8 column (4.6 mm i.d. × 150 mm long, 3.5 µm; Agilent) for Chlide a, on a C8 column (4.6 mm i.d. × 150 mm long, 3.5 µm; Waters) for Pchlide a, MPE, and Mg-Proto according to the above-mentioned methods of Zapata et al. (2000), and on a C18 column (4.6 mm i.d. × 150 mm long, 5 µm; Agilent) for Chl a according to the above-mentioned methods of Nakanishi et al. (2005). Elution profiles were monitored by measuring A440 for Chlide a and Pchlide a, A410 for MPE and Mg-Proto, and A660 for Chl a.

Concentrations (μg μL−1) of the DVR proteins expressed in E. coli were determined by SDS-PAGE using Protein Mr Marker (SM0431; Fermentas) and the Easy Protein Quantitative Kit (DQ101-01; TransGen Biotech; Supplemental Fig. S2). The pigment concentrations (mol L−1) were quantified with a spectrophotometer according to the methods described by Rebeiz (2002) and Nagata et al. (2007). The percentage of MV intermediate was determined by the proportion of peak area of the MV intermediate to total peak area of the MV intermediate and its corresponding DV intermediate in the same elution curve of HPLC as described by Shioi and Beale (1987) and Shioi and Takamiya (1992).

Reaction velocities were calculated as follows:

graphic file with name PP_208421E01_LW.jpg

where ν = reaction velocity (nmol mg−1 protein min−1 for DV-Chlide a and DV-Chl a as substrates or nmol mg−1 protein h−1 for DV-Pchlide a, DV-MPE, and DV-Mg-Proto as substrates); S = amount (nmol) of a substrate, which here was equal to 2 nmol in every enzymatic reaction; C = conversion percentage of a substrate during a period of time when there was a linear increase in product with time, which was represented by percentage of MV product (nmol) to total products (nmol) of the corresponding DV and MV tetrapyrroles in the first 1 min of incubation for DV-Chlide a and DV-Chl a as substrates, or in the first 1 h of incubation for DV-Pchlide a, DV-MPE, and DV-Mg-Proto as substrates (Fig. 2A); and E = amount (mg) of DVR protein, which here was equal to 0.2 mg (200 μg) of target protein in every enzymatic reaction.

As an exception, the lysate expressing ZmDVR had no obvious target band in SDS-PAGE (Supplemental Fig. S2), but the DVR activity of 700 μL of lysate expressing ZmDVR was very similar to that of the lysate corresponding to 200 μg of OsDVR protein in an enzymatic reaction under the same experimental conditions. So reaction velocity (ν) of ZmDVR was presented in nmol 3.5 mL−1 lysate min−1 for DV-Chlide a and DV-Chl a as substrates or nmol 3.5 mL−1 lysate h−1 for DV-Pchlide a, DV-MPE, and DV-Mg-Proto as substrates. Accordingly, E = volume (3.5 mL) of the lysate expressing ZmDVR, which here was equal to one-fifth of 3.5 mL in a reaction.

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: OsDVR, Os03g22780, ADE43128 in rice; ZmDVR, Zm124787, AFR53112 in maize; CsDVR, Csa000053, AFR53113 in cucumber; AtDVR, At5G18660, NP_197367 in Arabidopsis.

Supplemental Data

The following materials are available in the online version of this article.

Glossary

Chl

chlorophyll

DV

3,8-divinyl

MV

3-monovinyl

Pchlide

protochlorophyllide

MPE

magnesium-protoporphyrin IX monomethyl ester

Chlide

chlorophyllide

Mg-Proto

magnesium-protoporphyrin IX

DVR

divinyl reductase

ORF

open reading frame

PA

picolinic acid

ALA

5-aminolevulinic acid

DDV-LDV

dark divinyl-light divinyl

DMV-LDV

dark monovinyl-light divinyl

DMV-LMV

dark monovinyl-light monovinyl

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