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. 2014 May 19;165(3):1315–1327. doi: 10.1104/pp.114.239723

Cytochrome P450 93G1 Is a Flavone Synthase II That Channels Flavanones to the Biosynthesis of Tricin O-Linked Conjugates in Rice1,[C],[W]

Pui Ying Lam 1,2, Fu-Yuan Zhu 1,2, Wai Lung Chan 1,2, Hongjia Liu 1,2, Clive Lo 1,2,*
PMCID: PMC4081339  PMID: 24843076

The rice cytochrome P450 enzyme CYP93G1 is a phylogenetically unique flavone synthase II that converts flavanones directly to flavones, a key branch point reaction leading to the production of tricin O-linked glycosides and flavanolignans.

Abstract

Flavones are a major class of flavonoids with a wide range of physiological functions in plants. They are constitutively accumulated as C-glycosides and O-linked conjugates in vegetative tissues of grasses. It has long been presumed that the two structural modifications of flavones occur through independent metabolic routes. Previously, we reported that cytochrome P450 93G2 (CYP93G2) functions as a flavanone 2-hydroxylase (F2H) that provides 2-hydroxyflavanones for C-glycosylation in rice (Oryza sativa). Flavone C-glycosides are subsequently formed by dehydratase activity on 2-hydroxyflavanone C-glycosides. On the other hand, O-linked modifications were proposed to proceed after the flavone nucleus is generated. In this study, we demonstrate that CYP93G1, the closest homolog of CYP93G2 in rice, is a bona fide flavone synthase II (FNSII) that catalyzes the direct conversion of flavanones to flavones. In recombinant enzyme assays, CYP93G1 desaturated naringenin and eriodictyol to apigenin and luteolin, respectively. Consistently, transgenic expression of CYP93G1 in Arabidopsis (Arabidopsis thaliana) resulted in the accumulation of different flavone O-glycosides, which are not naturally present in cruciferous plants. Metabolite analysis of a rice CYP93G1 insertion mutant further demonstrated the preferential depletion of tricin O-linked flavanolignans and glycosides. By contrast, redirection of metabolic flow to the biosynthesis of flavone C-glycosides was observed. Our findings established that CYP93G1 is a key branch point enzyme channeling flavanones to the biosynthesis of tricin O-linked conjugates in rice. Functional diversification of F2H and FNSII in the cytochrome P450 CYP93G subfamily may represent a lineage-specific event leading to the prevalent cooccurrence of flavone C- and O-linked derivatives in grasses today.

Flavonoids are a large class of secondary metabolites widespread in vascular plants and some bryophytes. Currently, more than 9,000 flavonoid structures have been reported (Yun et al., 2008), and different classes are assigned based on the oxidation state in the middle C-ring (Schijlen et al., 2004). Among them, flavones are found extensively in land plants but are absent in almost all members of the Cruciferae (Martens and Mithöfer, 2005; Zhou and Ibrahim, 2010). In plants, flavones play important physiological roles, including UV protection (Schmitz-Hoerner and Weissenböck, 2003), interactions with other organisms (Peters et al., 1986; Kong et al., 2007), regulation of auxin transport (Mathesius et al., 1998), and copigmentation in flowers (Goto and Kondo, 1991). On the other hand, flavones as dietary constituents or supplements have become increasingly popular due to their health-beneficial effects, such as cancer prevention (Bontempo et al., 2007; Cai et al., 2007; Liu et al., 2007), antioxidation (Park et al., 2007), antiviral activities (Yarmolinsky et al., 2012), reduction of risks of cardiovascular diseases (Baek et al., 2009), and suppression of cholesterol levels (Dharmarajan and Arumugam, 2012).

The biosynthesis of flavones begins with flavanones, which are the precursors for all the major flavonoid classes. In dicots, two completely different flavone synthase (FNS) enzymes, FNSI and FNSII, have long been described for the conversion of flavones from flavanones. FNSI is a soluble dioxygenase (DOX) that requires Fe2+ as cofactor and 2-oxoglutarate as a cosubstrate and is mainly confined to the Apiaceae (Martens and Mithöfer, 2005). Molecular and phylogenetic analysis of parsley (Petroselinum crispum) FNSI suggested that it evolved from the functional diversification of a paralog of flavanone 3-hydroxylase (F3H), which is also a DOX accepting flavanone substrates (Gebhardt et al., 2007). In other dicot species, flavone formation is primarily catalyzed by cytochrome P450 enzymes belonging to the CYP93B subfamily. While most dicot FNSII recombinant enzymes oxidize flavanones to flavones directly, CYP93B members from two legume species were demonstrated to show flavanone 2-hydroxylase (F2H) activities in vitro (Akashi et al., 1998; Zhang et al., 2007). In licorice (Glycyrrhiza echinata), CYP93B1 is believed to generate 2-hydroxyflavanones as a common precursor for licodione and flavone formation (Akashi et al., 1998).

In monocots, the enzymology of flavone biosynthesis remained largely elusive until recent years. Previously, we demonstrated the expression of genes encoding chalcone synthase and chalcone isomerase in nonpigmented rice (Oryza sativa) seedlings (Shih et al., 2008), suggesting that flavonoids other than anthocyanins are synthesized. In fact, metabolite analysis revealed the accumulation of both C- and O-glycosides of flavones in vegetative tissues of rice (Shih et al., 2008). Glycosylation is a common structural modification in flavonoids, affecting their solubility, stability, and activities (Zhou and Ibrahim, 2010). Subsequently, a rice C-glucosyltransferase (OsCGT) was shown to catalyze UDP-Glc-dependent C-glucosylation of 2-hydroxyflavanone substrates (Brazier-Hicks et al., 2009). In addition, dehydratase activities that preferentially converted 2-hydroxyflavanone C-glucosides to flavone C-glucosides were demonstrated in both rice and wheat (Triticum aestivum) extracts (Brazier-Hicks et al., 2009). These results strongly suggested that enzyme activities that generate 2-hydroxyflavones are necessary for channeling flavanones to flavone C-glycoside formation in cereals. In fact, we have filled this remaining gap in the biosynthetic route for flavone C-glycosides with the characterization of CYP93G2, a rice P450 enzyme that is a bona fide F2H (Du et al., 2010; Fig. 1). Yeast (Saccharomyces cerevisiae) microsomes expressing CYP93G2 convert naringenin and eriodictyol to the respective 2-hydroxyflavanones. Consistently, 2-hydroxyflavanone O-glycosides are synthesized by transgenic Arabidopsis (Arabidopsis thaliana) plants overexpressing CYP93G2. We further confirmed that CYP93G2 and OsCGT function together along the same metabolic pathway in planta, resulting in the production of 2-hydroxyflavanone C-glycosides in transgenic Arabidopsis. Recently, similar biochemical steps were established in maize (Zea mays) for the biosynthesis of the insecticidal maysin, which is also a flavone C-glycoside (Morohashi et al., 2012; Falcone Ferreyra et al., 2013). For example, CYP93G5 is a functional F2H under the genetic control of P1, which encodes an R2R3-MYB transcription factor in maize (Morohashi et al., 2012). Coexpression of CYP93G5 and a maize UDP-dependent glycosyltransferase (UGT708A6) in yeast cells resulted in the production of flavone C-glycosides (Falcone Ferreyra et al., 2013). Interestingly, UGT708A6 was further shown to be a bifunctional enzyme leading to the production of both C- and O-glycosylated flavonoids.

Figure 1.

Figure 1.

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Biosynthesis pathways for flavone-derived metabolites in rice. Previously, we showed that CYP93G2 functions as an F2H that converts flavanones to 2-hydroxyflavanones. Subsequent C-glycosyltransferase (OsCGT) and dehydratase activities resulted in the production of flavone C-glycosides. Formation of flavone O-linked conjugates is believed to proceed through a separate metabolic route. FNSI or FNSII may generate the flavone nucleus directly, followed by different O-linked modifications.

In rice F2H mutant seedlings, substantial amounts of tricin-derived metabolites were detected despite their deficiency in the accumulation of flavone C-glycosides (Du et al., 2010). Tricin is a 3′,5′-dimethoxylated flavone that is almost exclusively present as O-glycosides in grasses (Harborne and Hall, 1964). Apparently, a completely different enzyme system is utilized for the formation of flavone O-glycosides in monocots, at least in the case of rice. However, whether an FNSI or FNSII is involved in generating the flavone nucleus for O-linked modifications in rice remains an intriguing question (Fig. 1). In this study, recombinant enzyme activity assays and transgenic Arabidopsis analysis established CYP93G1 as a functional FNSII enzyme that converts flavanones to flavones directly. Metabolite profiling investigations further revealed the depletion of tricin O-conjugated metabolites in a CYP93G1 transfer DNA (T-DNA) insertion rice mutant. At the same time, metabolic flux through the flavone C-glycoside pathway is enhanced. Collectively, our work provides conclusive evidence that CYP93G1 catalyzes an essential step along the biosynthesis pathway for tricin O-linked conjugates in rice.

RESULTS

Putative FNSII Enzymes in Rice

Previously, we demonstrated that the P450 enzyme CYP93G2 is an F2H required for flavone C-glycoside formation in rice (Du et al., 2010). As FNSII and F2H are related sequences in dicots, a BLASTP search of the rice proteome (http://rice.plantbiology.msu.edu/) for sequences with closest homology to CYP93G2 was performed to identify potential FNSII candidates. Two sequences, Os04G01140 and Os06G4107, were retrieved with 60% and 48% identity to CYP93G2, respectively. Both of them are cytochrome P450 proteins containing diagnostic signatures of a Pro hinge region, an oxygen-binding pocket, and a heme-binding motif. Previously, Os04G01140 and Os06G4107 were designated as P450 enzymes CYP93G1 and CYP93F1, respectively (http://drnelson.uthsc.edu/rice.html). CYP93G1 and CYP93F1 show 40% to 46% identity to known FNSII enzymes from Gerbera spp. hybrids (CYP93B2; Martens and Forkmann, 1999), Antirrhinum majus (CYP93B3; Akashi et al., 1999), Torenia hybrida (CYP93B4; Akashi et al., 1999), and Perilla frutescens (CYP93B6; Kitada et al., 2001). To infer the phylogeny of our rice sequences within the CYP93 family, phylogenetic analysis was performed together with members of CYP93A and CYP93B as well as the most closely related proteins to CYP93F1, CYP93G1, and CYP93G2 based on homology searches against different cereal proteomes (http://www.phytozome.net). As shown in Figure 2A, CYP93G1 and CYP93G2 each forms its own clade with its related cereal sequences. CYP93G1 is clustered with sorghum (Sorghum bicolor) and maize cognate proteins with unknown functions, while CYP93G2 is mainly clustered with sorghum and maize F2H enzymes (CYP93G3 and CYP93G5). Similarly, CYP93F1 is clustered with their close cereal homologs and sister to the CYP93G clade. Overall, the CYP93G and CYP93F subfamilies constitute an independent group diverged from the CYP93B subfamily that includes dicot FNSII and F2H enzymes. CYP93B is sister to the CYP93A subfamily containing several soybean (Glycine max) uncharacterized proteins.

Figure 2.

Figure 2.

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Phylogenetic and expression analyses of CYP93G1 and CYP93F1. A, Phylogenetic relationships of CYP93G1, CYP93F1, known FNSII and F2H enzymes, and their homologous sequences. The unrooted phylogenetic tree was constructed by the maximum likelihood method using MEGA6. Bootstrapping with 1,000 replications was performed to show the confidence level of the branching. The scale bar represents 0.1 substitutions per site. B, Expression analysis of CYP93G1 and CYP93F1 in rice. RT-PCR experiments were performed on cDNA prepared from 2-week-old rice seedlings using gene-specific primers. A rice actin gene (OsActin) was used as a positive gene expression control. WT, Wild type.

Reverse transcription (RT)-PCR experiments with gene-specific primers were performed to analyze the expression of CYP93G1 and CYP93F1 in 2-week-old rice wild-type seedlings that were previously reported to accumulate substantial amounts of both O- and C-glycosides of flavones (Shin et al., 2006; Du et al., 2010). As shown in Figure 2B, the expression of CYP93G1 was detected in leaf tissues. However, no RT-PCR products were detected for CYP93F1. Therefore, CYP93G1 is more likely to play a role in flavone accumulation in rice seedlings.

FNSII in Vitro Enzyme Activity Assays

To investigate the enzymatic functions of CYP93G1 and CYP93F1, the coding region of each gene was expressed in the yeast Saccharomyces cerevisiae. Microsomes were prepared from recombinant yeast cells and incubated with flavanone substrates in the presence of NADPH for FNSII enzyme assays. Reaction products were then detected by HPLC-tandem mass spectrometry (MS/MS) analysis. CYP93G1-expressing microsomes were found to catalyze the conversion of naringenin to apigenin, which was identified by its retention time and MS/MS fragmentation pattern in comparison with an authentic standard (Fig. 3, A and B). Similarly, eriodictyol was desaturated to luteolin following the microsome and NADPH incubation (Fig. 3, C and D). On the other hand, no activities were detected when yeast-expressed CYP93F1 or microsome-free samples were assayed against naringenin or eriodictyol (data not shown), indicating that CYP93F1 does not have FNSII activities. The maximum FNSII activities with naringenin as a substrate were recorded at pH 8 and 30°C for CYP93G1. Determination of kinetic parameters for CYP93G1 revealed that the Km and Vmax for naringenin were 3.2 μm and 18.5 fkat mg−1, respectively, and the corresponding values for eriodictyol were 1.5 μm and 14.6 fkat mg−1 (Fig. 3F). The relatively larger Vmax/Km ratio for eriodictyol suggested that it is slightly more preferred than naringenin as an in vitro substrate for CYP93G1.

Figure 3.

Figure 3.

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HPLC-MS/MS analysis of the recombinant CYP93G1 enzyme assays using flavanone substrates. A to D, A single peak at m/z 271 (A) or m/z 289 (C) was detected in the reaction mixture containing yeast microsomes expressing CYP93G1, NADPH, and naringenin or eriodictyol as a substrate, respectively. The dotted lines represent chromatographs of microsome-free controls. MS/MS spectra for m/z 271 (B) and m/z 289 (D) were consistent with the fragmentation patterns of apigenin and luteolin standards, respectively (Supplemental Fig. S1). E, Direct conversion of flavanones to flavone by CYP93G1, which functions as an FNSII. F, Kinetic parameters of recombinant CYP93G1. Km and Vmax values were determined by the Lineweaver-Burk plot. All assays were performed using yeast microsome extract from the same preparation. [See online article for color version of this figure.]

Transgenic Analysis of Putative FNSI and FNSII Genes in the Arabidopsis transparent testa6 Mutant

To understand its metabolic roles in planta, the coding sequence of CYP93G1 was placed under the control of the cauliflower mosaic virus 35S promoter and transformed into the Arabidopsis transparent testa6 (tt6) mutant. Competition for flavanone substrates with endogenous enzymes is minimized in the transgenic plants because of the lack of F3H activity (tt6 mutation). Transgene expression was confirmed by RT-PCR (data not shown), and five independent transformant lines were selected for metabolite investigations. Transgenic seeds were germinated on medium devoid of nitrogen sources. Under such conditions, the endogenous flavonoid pathway in Arabidopsis is highly active in providing potential substrates for the engineered enzyme. Ten-day-old T1 seedlings were harvested for methanol extraction, followed by acid hydrolysis, which releases flavonoid aglycones from O-linked conjugates. HPLC-MS/MS analysis detected the presence of apigenin, luteolin, and chrysoeriol in all of the CYP93G1 transgenic plant samples (Fig. 4A). None of these flavone metabolites are synthesized by cruciferous plants, including Arabidopsis (Martens and Mithöfer, 2005). Recently, two rice genes (Os10g39140 and Os03g03034) encoding DOX enzymes were reported to show FNSI activities in vitro (Kim et al., 2008; Lee et al., 2008b). In this study, we also generated transgenic Arabidopsis tt6 plants overexpressing either rice gene for metabolite analysis. However, no flavones were detected in acid-hydrolyzed methanol extracts prepared from five different transformant lines for each of the DOX transgenes (data not shown).

Figure 4.

Figure 4.

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HPLC-MS/MS metabolite analysis of Arabidopsis tt6 plants overexpressing CYP93G1. A, Apigenin, luteolin, and chrysoeriol were detected in the acid-hydrolyzed methanol extract prepared from transgenic plants but not in tt6 plants. Their identities were confirmed by retention time and fragmentation patterns in comparison with authentic standards. XIC, Extracted ion chromatogram. B, Precursor ions of m/z 271 were scanned following HPLC separation. Five distinct peaks corresponding to different apigenin O-glycosides were detected in the transgenic plant samples. C, MS/MS spectrum of a major peak (M2; m/z 579) indicating the loss of hexose (162 D) and Rha (146 D) units. D, MS3 analysis of the m/z 271 daughter ion in C, producing a fragmentation pattern consistent with that for an apigenin standard (Supplemental Fig. S1). HPLC-MS/MS PIS data for the detection of different apigenin, luteolin, and chrysoeriol O-glycosides are shown in Supplemental Figures S2 to S4.

To further elucidate whether CYP93G1 functions as FNSII or F2H in planta, HPLC-MS/MS precursor ion scan (PIS) analyses were performed to detect the presence of flavone or 2-hydroxyflavanone O-glycosylated derivatives using nonhydrolyzed samples, since acid treatment is known to convert 2-hydroxyflavanones to flavones (Akashi et al., 1998; Zhang et al., 2007). PIS of protonated 2-hydroxynarigenin or 2-hydroxyeriodictyol ion did not generate any distinct peaks in the LC-MS/MS profiles of all the CYP93G1 transgenic plant samples (data not shown). Such conditions were previously shown to reveal the accumulation of 2-hydroxyflavanone O-glycosides in CYP93G2 (F2H)-overexpressing Arabidopsis plants (Du et al., 2010). On the other hand, PIS of protonated apigenin, luteolin, and chrysoeriol ions revealed the accumulation of different flavone O-glycosides in the transgenic seedlings. For example, the PIS of mass-to-charge ratio (m/z) 271, which is consistent with an [apigenin + H]+ ion, resulted in the detection of several distinct peaks in the LC-MS/MS chromatogram (Fig. 4B). The most prominent peak, M2 (m/z 579), generated the diagnostic m/z 271 ion after MS/MS fragmentation (Fig. 4C). The successive losses of 162 and 146 D are indicative of the presence of O-linked hexose and Rha, respectively, in the parent compound. Multistage tandem MS (MS3) analysis of the m/z 271 daughter further confirmed its identity as an [apigenin + H]+ ion (Fig. 4D). Similarly, the other PIS peaks detected in the transgenic plant sample were identified as O-hexoside (M3; m/z 433), O-rhamnoside (M5; m/z 417), di-O,O-hexosides (M1; m/z 595), and di-O,O-rhamnosides (M4; m/z 563) of apigenin (Table I; Supplemental Fig. S2). Furthermore, different O-glycosides of luteolin and chrysoeriol were identified in the CYP93G1-expressing Arabidopsis plants following PIS, MS/MS, and MS3 analyses (Table I; Supplemental Figs. S3 and S4).

Table I. List of flavone O-glycosides accumulated in Arabidopsis CYP93G1 overexpression plants.

Metabolites were identified in nonhydrolyzed extracts by LC-MS/MS PIS analysis. The identities of the protonated flavone ions (in boldface) were confirmed by MS3 analysis.

Peaks Precursor Ion Retention Time MS/MS Ions Compound Assignment
m/z min m/z
M3 433 19.5 271 Apigenin O-hexoside
M5 417 23.9 271 Apigenin O-rhamnoside
M2 579 17.2 417, 271 Apigenin O-hexosyl rhamnoside
M4 563 22.4 417, 271 Apigenin di-O,O-rhamnosides
M1 595 14.7 433, 271 Apigenin di-O,O-hexosides
M7 449 17.8 287 Luteolin O-hexoside
M8, M9 595 19.0, 19.7 449, 287 Luteolin O-hexosyl rhamnoside
M6 611 14.8 449, 287 Luteolin di-O,O-hexoside
M15 447 23.7 301 Chrysoeriol O-rhamnoside
M12 549 18.2 463, 301 Chrysoeriol O-malonyl hexoside
M11, M13, M14 609 17.8, 19.3, 19.9 463, 301 Chrysoeriol O-hexosyl rhamnoside
M10 625 15.1 463, 301 Chrysoeriol di-O,O-hexosides
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To explore whether CYP93G1 may provide flavone substrates for C-glycosylation, Arabidopsis plants coexpressing CYP93G1 and OsCGT were generated for metabolite analysis. However, no flavone C-glycosides were detected in the double transgenic lines following LC-MS/MS analysis (data not shown). Previously, we demonstrated that coexpression of CYP93G2 and OsCGT led to the accumulation of 2-hydroxyflavanone C-hexosides in transgenic Arabidopsis (Du et al., 2010). This study further suggested that OsCGT does not accept flavone substrates for C-glycosylation in planta.

Profiling of Flavone Metabolites in Rice T-DNA Insertion Mutants

To evaluate the contribution of CYP93G1 toward the biosynthesis of different flavone derivatives in rice, an insertion line was identified at the RiceGE database (http://signal.salk.edu/cgi-bin/RiceGE), and seeds were acquired for detailed metabolite characterizations. The mutant harbors a T-DNA insertion in the second exon of Os04g01140 (Fig. 5A). Homozygous (HM) and wild-type sibling lines were isolated from seedlings derived from a hemizygous insertion parent. Using gene-specific primers flanking the insertion site, RT-PCR experiments demonstrated that the intact CYP93G1 transcript was not present in the HM seedlings (Fig. 5B), confirming the insertional mutation at the expression level. On the other hand, the two DOX genes (Os10g39140 and Os03g03034) and CYP93G2 were found to be expressed normally in the CYP93G1 HM mutant seedlings (Fig. 5B).

Figure 5.

Figure 5.

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Analysis of the rice CYP93G1 T-DNA insertion mutant. A, CYP93G1 (Os04g01140) gene structure and insertion site of the T-DNA (not drawn to scale; the coding region is shaded). B, RT-PCR analysis of HM mutant and wild-type (WT) seedlings (two samples each). Note the absence of CYP93G1 RT-PCR product in the HM samples when primers CL2632 and CL2633 were used. The expression of the DOX genes (Os10g39140 and Os03g03034) and CYP93G2 (F2H) was not affected. C, HPLC-MS/MS chromatograms of tricin and naringenin in acid-hydrolyzed methanol extracts of wild-type and HM plants. D, Relative levels of different flavone C-glycosides in the acid-hydrolyzed extracts of wild-type and HM plants. a, Apigenin C-hexoside; b, luteolin C-hexoside; c, chrysoeriol C-hexoside; d, apigenin C-hexoside C-pentoside; e, luteolin C-hexoside C-pentoside; f, chrysoeriol C-hexoside C-pentoside. The quantity of each flavone C-glycoside is expressed in peak area (as determined by HPLC-MS/MS) per unit fresh weight. Error bars represent sd (n = 5; **P < 0.01, ***P < 0.001 by Student’s t test).

Tissues from CYP93G1 wild-type and HM 3-week-old seedlings (five lines each) were extracted in methanol, acid hydrolyzed, and analyzed by LC-MS/MS. As shown in Figure 5C, the CYP93G1 gene insertion resulted in the absence of tricin detection in the HM plant extracts. However, these mutant seedlings showed significant increases (2- to 60-fold) in the accumulation of different flavone (apigenin, luteolin, and chrysoeriol) C-glycosides (Fig. 5D). Moreover, the flavanone naringenin was detected in the HM extracts but not in the wild-type samples (Fig. 5C). On the other hand, other common flavanones, including eriodictyol and dihydrotricetin, were not detected in both samples. We also obtained rice T-DNA mutant lines for the two DOX genes and analyzed their metabolite profiles accordingly. However, the accumulation of tricin and isovitexin (apigenin 6C-glucoside) derivatives was not affected in HM mutant seedlings of either DOX gene (Supplemental Fig. S5). Taken together, the above results demonstrated that CYP93G1 is essential for the biosynthesis of tricin-derived metabolites and that its disruption also results in enhanced metabolic flux through the flavone C-glycoside biosynthesis pathway.

To further investigate the types of tricin-derived metabolites whose accumulation is affected in the CYP93G1 HM seedlings, PIS experiments for m/z 331, which is consistent with a protonated tricin ion, were performed using wild-type and HM nonhydrolyzed extracts. LC-MS/MS chromatograms revealed the presence of eight distinct peaks (M16–M23) that were detected in the wild-type samples but not the HM samples (Fig. 6A). In all cases, the m/z 331 daughter ion of these precursor ions was confirmed to be [tricin + H+]+ by MS3 analysis (Fig. 6D). Three of the peaks were identified as tricin O-glycosides, including an O-hexoside (M17; m/z 493), an O-hexoside O-rhamnoside (M16; m/z 639), and an O-sinapoylhexoside (M20; m/z 699). Based on published MS/MS spectra (Yang et al., 2013), the other prominent precursor ions of [tricin + H]+ were identified as several flavanolignans, including tricin O-guaiacylglyceryl ether (M21 and M22; m/z 527) and its O-hexoside (M18 and M19; m/z 689) as well as tricin O-(guaiacyl-O-methyl-glyceryl) ether (M23; m/z 541; Table II; Supplemental Fig. S6). Their MS/MS spectra showed the diagnostic loss of the O-guaiacylglyceryl (−196 D) or O-guiaycl-O-methyl-glyceryl (−210 D) moieties (Fig. 6, B and C). Results from our metabolite profiling experiments further indicated that CYP93G1 is essential for the accumulation of different tricin O-conjugated derivatives in rice seedlings.

Figure 6.

Figure 6.

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HPLC-MS/MS analysis of tricin-derived metabolites in rice seedlings. A, Precursor ions of m/z 331 were scanned following HPLC separation. Eight distinct peaks corresponding to different tricin O-linked conjugates were detected in the wild type but not the CYP93G1 HM samples. B, MS/MS spectrum of a major precursor ion (M22; m/z 527) indicating the loss of a guaiacylglyceryl ether unit (196 D). C, MS/MS spectrum of a major peak (M23; m/z 541) indicating the loss of a guaiacylmethylglyceryl ether unit (210 D). D, Representative MS3 spectrum of the m/z 331 daughter ion in B and C, producing a fragmentation pattern consistent with that for a tricin standard (Supplemental Figure S1).

Table II. List of tricin O-linked conjugates depleted in the CYP93G1 mutant line.

Metabolites were identified in non-acid-hydrolyzed extracts by LC-MS/MS PIS analysis. The identities of the protonated tricin ions (in boldface) were confirmed by MS3 analysis.

Peaks Precursor Ion Retention Time MS/MS Ions Compound Assignment
m/z min m/z
M16 639 18.8 493, 331 Tricin O-hexosyl rhamnoside
M17 493 19.3 331 Tricin O-hexoside
M18, M19 689 20.6, 22.2 527, 331 Tricin O-guaiacylglyceryl ether hexoside
M20 699 20.6 331 Tricin O-sinapoyl hexoside
M21, M22 527 24.3, 25.0 331 Tricin O-guaiacylglyceryl ether
M23 541 28.0 331 Tricin O-guaiacylmethylglyceryl ether
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DISCUSSION

The sequential activities of chalcone synthase and chalcone isomerase direct the flow of carbon from the phenylpropanoid pathway to the production of flavanones from which different classes of flavonoids are derived. Due to the absence of F3H gene expression, dihydroflavonols and the downstream 3-hydroxylated flavonoids, such as flavonols and anthocyanins, are not accumulated in vegetative tissues of rice (Shih et al., 2008). Instead, flavone-derived metabolites are the predominant flavonoids synthesized in rice seedlings (Shih et al., 2008; Du et al., 2010). The formation of flavones from flavanones, which involves C2-C3 desaturation in the C-ring, can be catalyzed by either FNSI or FNSII. These two mechanistically different enzymes are apparently consequences of independent recruitment by taxonomically diverse plant species. Interestingly, two recombinant rice DOX enzymes encoded by Os10g39140 and Os03g03034 were previously reported to convert naringenin to apigenin in vitro (Kim et al., 2008; Lee et al., 2008b). However, we found that their transgenic expression did not lead to flavone accumulation in Arabidopsis, suggesting that they may not function as FNSI in planta. Furthermore, since the flavone metabolite profiles were essentially unaffected in their T-DNA HM mutants (Supplemental Fig. S5), the two DOX enzymes are not likely to play crucial roles in the biosynthesis of flavones in rice. In fact, both genes were expressed normally in the CYP93G1 HM mutant seedlings, which are depleted in tricin-derived metabolites (Fig. 5). The in vitro FNSI activities suggest the possibility of substrate promiscuity in DOX enzymes.

On the other hand, FNSII activities of the rice cytochrome P450 enzyme CYP93G1 were established by both recombinant enzyme assays and transgenic Arabidopsis analysis in this study. The in vitro FNSII activities of CYP93G1 were also described recently (Brazier-Hicks and Edwards, 2013). Consistently, our CYP93G1-overexpressing seedlings accumulate O-glycosides of apigenin, luteolin, and chrysoeriol, which are not naturally present in Arabidopsis. Apparently, the different flavones generated by CYP93G1 were modified by endogenous flavonoid O-glycosyltransferases. It is not clear whether the 3′-O-methylation in chrysoeriol occurs before or after flavone formation. Interestingly, two Arabidopsis O-methyltransferases, AtOMT1 and CCoAOMT7, were both reported to show catalytic activity toward luteolin in vitro (Muzac et al., 2000; Wils et al., 2013). Hence, it is possible that luteolin is methylated to chrysoeriol by AtOMT1 or CCoAOMT7 in our CYP93G1 transgenic Arabidopsis plants. In licorice and Medicago truncatula, flavones are converted from 2-hydroxyflavanones generated by CYP93B P450 enzymes that function as F2H (Akashi et al., 1998; Zhang et al., 2007). However, this alterative catalytic mechanism for CYP93G1 can be excluded due to the absence of the accumulation of 2-hydroxyflavanone derivatives in our transgenic lines. Taken together, CYP93G1 is a bona fide FNSII that catalyzes the direct transformation of flavanones to flavones (Fig. 3E).

In dicots, FNSII enzymes are widespread in a number of families while F2H enzymes are only described in leguminous plants, and both FNSII and F2H belong to the CYP93B subfamily. As members of the CYP93G subfamily, the rice F2H and FNSII enzymes are apparently derived from an origin independent from their dicot counterparts. This is in contrast to another flavonoid P450 enzyme, flavonoid 3′-hydroxylase, which belongs to the CYP75B subfamily, consisting of both dicot and monocot members. On the other hand, the coexistence of F2H and FNSII activities may not be unique in rice. Phylogeny analysis revealed the presence of a potential FNSII sequence (CYP93B16) in M. truncatula (Fliegmann et al., 2010), which contains two F2H enzymes (CYP93B10 and CYP93B11) involved in root nodulation (Zhang et al., 2007). In grasses, the exclusive occurrence of apigenin and luteolin as C-glycosides and tricin as O-glycosides was documented several decades ago (Harborne and Hall, 1964). However, the hypothesis of two separate metabolic pathways involved in their biosynthesis was only recently supported by molecular genetics evidence. In rice, CYP93G2 converts flavanones to 2-hydroxyflavanones, which are substrates for C-glycosylation by OsCGT (Brazier-Hicks et al., 2009; Du et al., 2010). The same metabolic steps (F2H and CGT) were also established in maize for the biosynthesis of maysin (Morohashi et al., 2012; Falcone Ferreyra et al., 2013). In this study, we further establish that CYP93G1 channels flavanones toward the biosynthesis of different tricin O-linked conjugates in rice seedlings. Disruption of CYP93G1 in a T-DNA mutant resulted in the depletion of tricin O-glycosides and O-linked flavanolignans (Fig. 6). By contrast, elevated levels of different flavone C-glycosides were observed (Fig. 5), indicating a redirection of metabolic flow to 2-hydroxyflavanones when the entry point to the flavone O-linked conjugate biosynthetic route is blocked. In fact, both CYP93G1 and CYP93G2 are competing for flavanone substrates, converting them to flavones or 2-hydroxyflavanones, respectively. The competing nature of these two pathways was also described previously. Following treatment with cloquintocet mexyl (an herbicide safener), there was a depletion of flavone C-glycoside content but preferential accumulation of tricin in wheat seedlings (Cummins et al., 2006). Interestingly, naringenin was detected in the acid-hydrolyzed samples of the CYP93G1 mutant but not wild-type seedlings (Fig. 5C). It is possible that the increased flavanone availability for CYP93G2 had exceeded the capacity of the flavone C-glycoside biosynthesis pathway, thus building up an internal pool of naringenin normally not present in wild-type rice plants.

It is generally believed that O-linked modifications of flavonoids occur at a terminal step after the aglycone is produced (Iwashina, 2000). In fact, early radiotracer experiments in Lemnaceae plants demonstrated that 14C-labeled flavones were incorporated into O-glycosylated and O-methylated flavones but not flavone C-glycosides (Wallace et al., 1969). The formation of tricin (3′,5′-O-dimethoxylated) requires the activities of flavonoid 3′,5′-hydroxylase (F3′5′H) to generate hydroxyl groups in the B ring for the O-methylation reactions (Fig. 7). Genes encoding the cytochrome P450 F3′5′H enzymes have received enormous attention because of their applications in engineering purple/blue pigmentation in ornamental plants. The 3′- and 5′-hydroxylation of dihydroflavonols is an important step that determines whether delphinidin derivatives are formed (Nishihara and Nakatsuka, 2011; Tanaka and Brugliera, 2013). For tricin formation, it was previously proposed that the 3′,5′-hydroxylation occurs before flavone formation in wheat (Cummins et al., 2006). However, as naringenin but not eriodictyol or dihydrotricetin accumulates in our T-DNA seedlings, it is likely to be the preferred flavanone substrate for CYP93G1 in planta (Fig. 7). On the other hand, there is evidence suggesting that the 3′,5′-O-methylation occurs after flavone formation. For example, tricetin was sequentially O-methylated in vitro to selgin (3′-O-methylated) and then to tricin by recombinant rice OMTs (ROMT9, ROMT15, and ROMT17; Kim et al., 2006; Lee et al., 2008a) and wheat OMT (TaOMT2; Zhou et al., 2006). However, the precise contribution of these different OMTs in the formation of tricin in planta remains to be elucidated.

Figure 7.

Figure 7.

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Proposed biosynthesis pathway for tricin O-linked conjugates in rice. CYP93G1 is likely to function as an FNSII converting naringenin to apigenin. Tricin 7-O-glucoside, tricin O-guaiacylglyceryl ether (R = H), and tricin O-guaiacylmethylglyceryl ether (R = CH3) were previously detected in rice tissues.

Tricin occurs naturally as O-conjugated derivatives in plants. The most common form is tricin 7-O-glucopyranoside, which is found in taxonomically diverse species including sugarcane (Saccharum officinarum; Mabry et al., 1984), Phoenix hanceana (Lin et al., 2009), Sasa borealis (Park et al., 2007), Lygodium japonicum (Zhang et al., 2008a), and Ficus stenophylla (Zhang et al., 2008b). Our metabolite profiling of rice seedlings showed that tricin is mostly present as O-linked flavanolignans and their O-glycosides, which were previously identified in several rice varieties (Jeong et al., 2011, 2014; Yang et al., 2013) and other grass species (Bouaziz et al., 2002; Wenzig et al., 2005). The different tricin-type flavanolignans were reported to exhibit a range of health-beneficial properties, such as antioxidation, antiinflammation, cardiovascular protection, and induction of tumor cell apoptosis (Chang et al., 2010; Jeong et al., 2011; Mohanlal et al., 2011, 2013). However, the biosynthesis of flavanolignans in plants is not well understood. The final reaction step is likely to proceed through the oxidative coupling of flavonoid and coniferyl alcohol catalyzed by a peroxidase (Begum et al., 2010). Our work strongly supports that CYP93G1 catalyzes an essential step in the generation of tricin nucleus for O-linked flavanolignan and glycoside formation in rice. Complete understanding of the biosynthetic route and its regulation will facilitate future attempts at targeted engineering of tricin-type flavanolignans as nutraceuticals in the edible tissue of rice (i.e. endosperm), which mainly consists of starch and protein but is deficient in phytochemicals (Zhou and Ibrahim, 2010). Using several combinations of rice and nonrice flavonoid structural genes under the control of tissue-specific promoters, different classes of flavonoids were recently successfully engineered in rice endosperm, embryo, and aleurone layer (Ogo et al., 2013). In particular, tricin was detected in the transgenic lines by introducing a composite cassette comprising rice Phe ammonia lyase, chalcone synthase, and OMT genes as well as parsley FNSI, soybean FNSII, and Viola spp. F3′5′H genes.

In summary, this study and our previous work (Du et al., 2010) firmly established CYP93G1 and CYP93G2 as key branch-point enzymes in rice controlling the metabolic flux from flavanones to the biosynthesis of tricin O-linked conjugates (glycosides and flavonolignins) and flavone (apigenin, luteolin, and chrysoeriol) C-glycosides, respectively (Fig. 1). Flavones are ubiquitous in land plants with a high degree of structural diversity, which is likely to be contributed by different lineage-specific metabolic pathways. The CYP93G subfamily constitutes a unique group of P450 proteins present in monocots, or at least in cereals. Functional diversification of FNSII and F2H activities from an ancestral CYP93G sequence may represent a key process giving rise to the cooccurrence of the two biosynthetically distinct groups of flavones in many grass species today.

MATERIALS AND METHODS

Yeast Expression of Cytochrome P450 Proteins and Enzyme Assays

The coding sequences of CYP93G1 and CYP93F1 were amplified from the rice (Oryza sativa subsp. japonica) full-length complementary DNA (cDNA) clones AK100972 and AK287657 (National Institute of Agrobiological Science of Japan), respectively, using the high-fidelity Pfx DNA polymerase (Invitrogen). They were ligated directly to the yeast expression vector pYES2.1/V5-His-TOPO (Invitrogen). Clones confirmed by DNA sequencing were transformed into Saccharomyces cerevisiae INVSc1 cells according to the manufacturer’s instructions. Yeast growth and preparation of microsome extracts were performed essentially as described (Du et al., 2010). Expression of the rice genes in the yeast cells was confirmed by RT-PCR. Typical FNSII enzyme assays were carried out at 30°C for 30 min in 100 mm potassium phosphate buffer (pH 8.0) containing 2 mm l-glutathione, 5 mm NADPH, 100 µm substrate, and 200 µg of microsome protein (CYP93F1 or CYP93G1). Protein concentrations were determined by the Bradford assay (Bio-Rad). For kinetic analysis, CYP93G1 was tested against naringenin or eriodictyol in a concentration series of 0.5, 1, 2, 5, 10, 20, and 50 μm using the same batch of microsomal preparation. All the reactions were terminated by extraction twice in ethyl acetate, vacuum dried, and redissolved in HPLC-grade methanol for LC-MS/MS analysis.

Generation of Transgenic Arabidopsis Plants and Metabolite Extraction

To generate Arabidopsis (Arabidopsis thaliana) plants overexpressing CYP93G1 or the DOX genes, their coding sequences were each cloned into the binary vector pCAMBIA1300 (CAMBIA) under the control of the cauliflower mosaic virus 35S promoter and the nopaline 3′ terminator. Each construct was transformed into Arabidopsis tt6 mutants (SALK_113904) by Agrobacterium tumefaciens strain GV3101 using the floral dip method (Clough and Bent, 1998). The transformants were selected on Murashige and Skoog (Sigma) plates (Murashige and Skoog, 1962) containing 3% (w/v) Suc and 25 µg mL−1 hygromycin, followed by transfer to soil for further growth. PCR genotyping and RT-PCR were performed to confirm the presence and expression of transgenes, respectively. Seeds from five independent transformant lines for each construct were germinated on Murashige and Skoog plates containing 3% (w/v) Suc but without nitrogen sources. They were then placed in the dark at 4°C for 2 d, followed by incubation in a tissue culture room at 23°C (16 h of light and 8 h of dark). Ten day-old plants (0.5 g) were harvested and ground to powder in liquid nitrogen. HPLC-grade methanol (500 µL) was used for metabolite extraction. For acid hydrolysis, an equal volume of 2 n HCl was added to the samples, followed by incubation at 90°C for 1 h. Transgenic Arabidopsis plants coexpressing CYP93G1 and OsCGT were generated by crossing between the CYP93G1- and OsCGT-overexpressing lines (Du et al., 2010).

Characterization of Rice T-DNA Insertion Mutants

Rice wild-type and T-DNA insertion mutant seeds were obtained from the Crop Biotech Institute of Kyung Hee University. Mutants of DOX-encoding genes Os10g39140 (accession 3A-07540; cv Dongjin) and Os03g03034 (accession 3A-17437; cv Dongjin) as well as CYP93G1 (accession K-00244; cv Kitaake) were used in this study. Seeds were incubated in 1% (v/v) nitric acid in darkness at room temperature for 24 h, after which they were transferred to distilled water in darkness at 37°C for 2 d. The germinated seeds were then placed in rice growth medium (Yoshida et al., 1976) and incubated in a growth chamber (12 h of light at 28°C and 12 h of dark at 22°C). Genomic PCR genotyping was performed to identify wild-type and HM seedlings derived from heterozygous insertion parents. Three-week-old seedlings were used for metabolite extraction in methanol as described above.

RNA Extraction and RT-PCR Experiments

Plant tissues were harvested and ground in liquid nitrogen for total RNA extraction using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. After DNase I (Invitrogen) treatment, RNA samples (4 µg) were reverse transcribed by reverse transcriptase (M-MLV RT RNase [H-] point mutant; Promega) using an oligo(dT) primer. PCR analysis of the cDNA samples was then performed using different gene-specific primers. The thermal cycling program was set as follows: preincubation (94°C for 10 min); 30 cycles of denaturation (94°C for 30 s), annealing (55°C for 30 s), and elongation (72°C for 1 min); and final extension (72°C for 10 min).

Primers for Molecular Biology Experiments

Primers for vector construction as well as the molecular analysis of transgenic Arabidopsis and rice mutants are listed in Supplemental Table S1.

LC-MS/MS Analysis of Enzyme Reaction Products and Plant Metabolites

Filtered samples (10 µL) of enzyme assays and plant extracts were separated on a Nucleosil 100-5 C18 column (5 μm, 150 × 2 mm; Agilent Technologies) connected to the HP1100 series HPLC system (Agilent Technologies). For the analysis of enzyme reaction products, a solvent system of 0.5% (v/v) formic acid/water (A) and 0.5% (v/v) formic acid/methanol (B) with a linear gradient of 15% to 90% B over 15 min was used. For the analysis of nonhydrolyzed plant extracts, a solvent system of methanol (A) and 10 mm ammonium acetate (pH 5.6; B) with a linear gradient of 10% to 60% B over 18 min was used. For the analysis of acid-hydrolyzed plant extracts, the mobile phase and separation conditions were essentially as described (Du et al., 2010). In all cases, the HPLC flow rate was maintained at 0.2 mL min−1 and the elution was analyzed with an AP3200-QTRAP mass spectrometer (AB SCIEX). Precursor ion scan, product ion scan, and MS3 spectra were obtained and acquired as described previously (Lo et al., 2007; Shih et al., 2008). Quantification, data analysis, and peak integration were conducted using the Analyst 1.5.2 software (AB SCIEX).

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AK100972 (CYP93G1) and AK287657 (CYP93F1).

Supplemental Data

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

Supplementary Material

Supplemental Data
supp_165_3_1315__index.html (1.2KB, html)

Glossary

T-DNA

transfer DNA

RT

reverse transcription

MS/MS

tandem mass spectrometry

LC

liquid chromatography

PIS

precursor ion scan

m/z

mass-to-charge ratio

HM

near-isogenic homozygous

cDNA

complementary DNA

Footnotes

1

This work was supported by the Research Grants Council of the Hong Kong Special Administrative Region, China (grant no. HKU7736/11M).

[C]

Some figures in this article are displayed in color online but in black and white in the print edition.

[W]

The online version of this article contains Web-only data.

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