Cytochrome P450 93G1 Is a Flavone Synthase II That Channels Flavanones to the Biosynthesis of Tricin O-Linked Conjugates in Rice    

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.

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

“…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).…”

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

“…FNSIs are soluble Fe 2+ /oxoglutaratedependent dioxygenases restricted to Apiaceae, while FNSIIs are cytochrome P450 enzymes (CYP93B subfamily) identified in other flavone-accumulating dicots. Recently, we established CYP93G1 and CYP93G2 as branch point enzymes channeling flavanones to the formation of tricin O-linked conjugates (glycosides and flavanolignans) and flavone (apigenin, luteolin, and chrysoeriol) C-glycosides, respectively, in rice (Du et al, 2010;Lam et al, 2014). CYP93G1 functions as an FNSII, which generates flavone aglycones for different O-linked modifications (Lam et al, 2014).…”

“…Recently, we established CYP93G1 and CYP93G2 as branch point enzymes channeling flavanones to the formation of tricin O-linked conjugates (glycosides and flavanolignans) and flavone (apigenin, luteolin, and chrysoeriol) C-glycosides, respectively, in rice (Du et al, 2010;Lam et al, 2014). CYP93G1 functions as an FNSII, which generates flavone aglycones for different O-linked modifications (Lam et al, 2014). On the other hand, CYP93G2 is a flavanone 2-hydroxlase, which produces 2-hydroxyflavanones for immediate C-glycosylation, followed by the formation of the flavone nucleus (Du et al, 2010).…”

Functional expression of cytochrome P450 in <i>Escherichia coli</i>: An approach to functional analysis of uncharacterized enzymes for flavonoid biosynthesis

“…On the other hand, although no cDNA or gene for I2′H has been identified from soybean, the specificity of its I2′H to 4′-hydroxy substrates is expected because of predominant accumulation of 4′-hydroxy type pterocarpan phytoalexins in soybean. In contrast with leguminous isoflavonoids, flavones are widely distributed in higher plants and are produced by the CYPs flavanone 2-hydroxylase (F2H) or flavone synthase II (FNS II) (Figure 1), which have been identified separately in multiple plant species (Akashi et al 1999a(Akashi et al , 1999bDu et al 2010aDu et al , 2010bFliegmann et al 2010;Kitada et al 2001;Lam et al 2014;Martens and Forkmann 1999;Nakatsuka et al 2005;Zhang et al 2007).…”