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 39,59-dimethoxyflavone nucleus is formed before O-linked conjugations. Previously, flavonoid 39,59-hydroxylases belonging to the CYP75A subfamily were believed to generate tricetin from apigenin for 39,59-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 39-methoxylated flavone). CYP75B4 functions as a bona fide flavonoid 39-hydroxylase by restoring the accumulation of 39-hydroxylated flavonoids in Arabidopsis (Arabidopsis thaliana) transparent testa7 mutants and catalyzing in vitro 39-hydroxylation of different flavonoids. In addition, overexpression of both CYP75B4 and CYP93G1 (a flavone synthase II) in Arabidopsis resulted in tricin accumulation. Specific 59-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 39-hydroxylase sequences. Recruitment of chrysoeriol-specific 59-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.

Silencing CHALCONE SYNTHASE in Maize Impedes the Incorporation of Tricin into Lignin and Increases Lignin Content

“…Additionally, we found luteolin and chrysoeriol derivatives as differential flavones. Our data thus support the recent finding that chrysoeriol, instead of tricetin, is the intermediate in tricin biosynthesis in grasses (Lam et al, 2015).…”

“…The incorporation of tricin into dicot lignins that normally do not produce tricin, or its overproduction in grasses through genetic engineering, may be a promising strategy to test the direct influence of tricin levels in the lignin on lignocellulose recalcitrance. The production of tricin in species that normally do not produce this metabolite has already been achieved in the dicot Arabidopsis (Lam et al, 2015), but its incorporation into lignins and the effect on saccharification have not yet been investigated.…”

“…This enzyme converts p-coumaroyl-CoA into naringenin chalcone, which is then isomerized by chalcone isomerase (CHI) to the flavanone naringenin, a common precursor for several flavonoid classes (Shih et al, 2008;Dixon and Pasinetti, 2010). The biochemical route from naringenin toward tricin in grasses has only recently been fully resolved (Lam et al, 2014(Lam et al, , 2015. In rice (Oryza sativa), naringenin is desaturated by flavone synthase to form apigenin, after which an extra hydroxyl group is added by flavonoid 39-hydroxylase to produce luteolin.…”

“…PMT has been shown in rice (Withers et al, 2012), brachypodium (Petrik et al, 2014), and maize (Marita et al, 2014). The flavone biosynthesis pathway is shown according to Zhou et al (2008), Corté s-Cruz et al (2003, and Lam et al (2014Lam et al ( , 2015. The enzymes involved in the metabolic route via F2H toward apigenin and luteolin conjugates has been described in rice, wheat, and buckwheat (Fagopyrum esculentum;Brazier-Hicks et al, 2009;Du et al, 2010).…”

“…This hydroxyl group is then methylated by flavonoid O-methyltransferase (FOMT) to form chrysoeriol. Another hydroxyl group is added to the same ring by flavonoid 59-hydroxylase, and the resulting selgin is then again methylated by FOMT to form tricin (Lam et al, 2015). In maize, two genes have been described to encode CHS: Colorless2 (C2) and Whitepollen1 (Whp1; Coe et al, 1981;Franken et al, 1991).…”