Completion of Tricin Biosynthesis Pathway in Rice: Cytochrome P450 75B4 Is a Unique Chrysoeriol 5'-Hydroxylase

Abstract

Flavones are ubiquitously accumulated in land plants, but their biosynthesis in monocots remained largely elusive until recent years. Recently, we demonstrated that the rice (Oryza sativa) cytochrome P450 enzymes CYP93G1 and CYP93G2 channel flavanones en route to flavone O-linked conjugates and C-glycosides, respectively. In tricin, the 3',5'-dimethoxyflavone nucleus is formed before O-linked conjugations. Previously, flavonoid 3',5'-hydroxylases belonging to the CYP75A subfamily were believed to generate tricetin from apigenin for 3',5'-O-methylation to form tricin. However, we report here that CYP75B4 a unique flavonoid B-ring hydroxylase indispensable for tricin formation in rice. A CYP75B4 knockout mutant is tricin deficient, with unusual accumulation of chrysoeriol (a 3'-methoxylated flavone). CYP75B4 functions as a bona fide flavonoid 3'-hydroxylase by restoring the accumulation of 3'-hydroxylated flavonoids in Arabidopsis (Arabidopsis thaliana) transparent testa7 mutants and catalyzing in vitro 3'-hydroxylation of different flavonoids. In addition, overexpression of both CYP75B4 and CYP93G1 (a flavone synthase II) in Arabidopsis resulted in tricin accumulation. Specific 5'-hydroxylation of chrysoeriol to selgin by CYP75B4 was further demonstrated in vitro. The reaction steps leading to tricin biosynthesis are then reconstructed as naringenin → apigenin → luteolin → chrysoeriol → selgin → tricin. Hence, chrysoeriol, instead of tricetin, is an intermediate in tricin biosynthesis. CYP75B4 homologous sequences are highly conserved in Poaceae, and they are phylogenetically distinct from the canonical CYP75B flavonoid 3'-hydroxylase sequences. Recruitment of chrysoeriol-specific 5'-hydroxylase activity by an ancestral CYP75B sequence may represent a key event leading to the prevalence of tricin-derived metabolites in grasses and other monocots today.

Figure 1.

Tricin biosynthesis pathway. Tricin occurs exclusively as O-linked conjugates in grasses, and the flavone nucleus is formed before O-linked modifications. Naringenin is desaturated to apigenin by CYP93G1, which functions as an FNSII. Previously, tricetin was proposed to be an intermediate in the pathway (illustrated in light color and dotted arrows). In this study, chrysoeriol is established as an intermediate during the biotransformation of apigenin to tricin. Hence, apigenin is converted to luteolin by an F3′H (CYP75B3 or CYP75B4) and then to chrysoeriol by an OMT (e.g. ROMT9). CYP75B4 functions as a chrysoeriol 5′-hydroxylase, which generates selgin, the immediate precursor of tricin.

Figure 2.

Analysis of the rice CYP75B4 T-DNA insertion mutant. A, Gene structure of CYP75B4 (Os10g16974). The T-DNA is inserted in the first exon of the gene in the mutant. B, RT-PCR gene expression analysis of HM and wild-type (WT) seedlings. In the HM samples, the CYP75B4 RT-PCR product was absent when primers CL1758 and CL1759 were used. The expression of CYP75B3, CYP75A11, and ROMT9 was not affected. C, Relative quantities of apigenin, luteolin, chrysoeriol, and tricin detected in acid-hydrolyzed HM and wild-type extracts. Error bars represent sd (n = 5; **, P < 0.01; and ***, P < 0.001 by Student’s t test).

Figure 3.

Transgenic analysis of CYP75B4 in the Arabidopsis mutants. A, CYP75B4 expression restored the ability of tt7 plants to accumulate proanthocyanin in seed coats and purple anthocyanin when grown on a medium devoid of nitrogen. B, CYP75B4 expression restored the accumulation of 3′-modified flavonols in tt7 plants. C, CYP75B4 plus CYP93G1 expression resulted in the accumulation of 5′-modified flavones in the tt6/tt7 double mutant. LC-MS chromatograms are representatives of at least three independent experiments. Metabolites in acid-hydrolyzed extracts were identified by retention time and MS/MS spectra in comparison with authentic standards. Selgin identification is described in Figure 4 and Supplemental Figure S4. WT, Wild type; cps, counts per second. Bar = 1 mm.

Figure 4.

LC-MS analysis of in vitro enzyme assays. A, A major peak at m/z 317 was detected in the CYP75B4 reaction containing chrysoeriol as a substrate. The same peak was identified when tricetin was incubated with ROMT9, and the expected product was selgin. MS/MS spectra for the two products were identical (Supplemental Fig. S4). B, Addition of ROMT9 to the chrysoeriol plus CYP75B4 reaction further converted selgin to tricin. The same product was also detected in the tricetin plus ROMT9 reaction after prolonged incubation. C, No selgin was detected when chrysoeriol was incubated with CYP75A11 or CYP75B3. CYP75A1 (petunia F3′5′H) was used as a positive enzyme control. D, Enzymatic reaction steps for A and B. LC-MS chromatograms are representatives of at least three independent experiments. cps, Counts per second.

Figure 5.

Phylogenetic analysis of CYP75A and CYP75B sequences. The unrooted tree was constructed by maximum-likelihood method using MEGA6. Bootstrapping with 1,000 replications was performed. Canonical F3′5′H and F3′H enzymes belong to CYP75A and CYP75B subfamilies, respectively. Functionally characterized F3′H and F3′5′H are annotated. Acquisition of 5′-hydroxylase activity by CYP75B members occurred independently in Poaceae and Asteraceae. Bar = 0.1 substitution per site.