The C-glycosylation of flavonoids in cereals

Abstract

Flavonoids normally accumulate in plants as O-glycosylated derivatives, but several species, including major cereal crops, predominantly synthesize flavone-C-glycosides, which are stable to hydrolysis and are biologically active both in planta and as dietary components. An enzyme (OsCGT) catalyzing the UDP-glucose-dependent C-glucosylation of 2-hydroxyflavanone precursors of flavonoids has been identified and cloned from rice (Oryza sativa ssp. indica), with a similar protein characterized in wheat (Triticum aestivum L.). OsCGT is a 49-kDa family 1 glycosyltransferase related to known O-glucosyltransferases. The recombinant enzyme C-glucosylated 2-hydroxyflavanones but had negligible O-glucosyltransferase activity with flavonoid acceptors. Enzyme chemistry studies suggested that OsCGT preferentially C-glucosylated the dibenzoylmethane tautomers formed in equilibrium with 2-hydroxyflavanones. The resulting 2-hydroxyflavanone-C-glucosides were unstable and spontaneously dehydrated in vitro to yield a mixture of 6C- and 8C-glucosyl derivatives of the respective flavones. In contrast, in planta, only the respective 6C-glucosides accumulated. Consistent with this selectivity in glycosylation product, a dehydratase activity that preferentially converted 2-hydroxyflavanone-C-glucosides to the corresponding flavone-6C-glucosides was identified in both rice and wheat. Our results demonstrate that cereal crops synthesize C-glucosylated flavones through the concerted action of a CGT and dehydratase acting on activated 2-hydroxyflavanones, as an alternative means of generating flavonoid metabolites.

FIGURE 1.

Glycosylflavone biosynthesis in plants. A, FNS converts flavanones (compound 1) to flavones (compound 2), which are then conjugated by an OGT. B, in cereals, the flavanone also undergoes conversion to the 2-hydroxyflavanone (compound 4a), which exists in equilibrium with its open-chain form (compound 4b), the latter apparently being acted on by the C-glucosyltransferase (CGT) to produce 2-hydroxyflavanone C-glucosides (compound 5, a–c). These are then dehydrated to yield the flavone-6C-glucoside (compound 6) and flavone-8C-glucoside (compound 7). C and D, the hydroxylation (C) and C-glycosylation (D) of the flavonoids referred to under “Results.”

FIGURE 2.

Flavonoids in 7 day old rice foliage. A, HPLC profile of UV absorbing metabolites extracted with methanol from rice shoots. mAU, milliabsorbance units. B, identification of the flavone structure and C-glycosylation (C-gly) pattern of key rice metabolites. O-gly, O-glycosylation; ara, arabinose; glc, glucose.

FIGURE 3.

Sequential purification of CGT activity from rice. CGT activity (■-■-■-■) was monitored along with elution of UV absorbing proteins (····) on phenyl-Sepharose (A), mono Q (B), and Superdex 200 columns (C). AU, absorbance units. D, analysis of polypeptides corresponding to the active fraction from the gel filtration chromatography (C) as determined by SDS-PAGE. Proteins designated by arrows were subjected to MALDI-TOF-MS sequencing (supplemental Fig. S2).

FIGURE 4.

CGT activities of pure recombinant OsCGT toward flavonoids and related compounds. A and B, respective specific activities were determined (A), and where possible, kinetic data were determined (B). n.a., not available; Bn, benzyl. pkat, picokatals.

FIGURE 5.

Analysis of reaction products derived from the C-glucosylation of 2,5,7-trihydroxyflavanone. A, determination by HPLC, with the initial product (peak 5) spontaneously dehydrating to give peaks 6 and 7. B and C, the fragmentation of peak 5 (B) and mass ions associated with peaks 6 and 7 (C) are shown. D, peak 5 was then incubated with either an enzyme preparation from rice cultures (------) or boiled protein (····).