A Novel Ca2+-Dependent O -Methyltransferase Involved in Flavonoid Methylation in Tartary Buckwheat

Methoxyflavonoids have attracted increasing attention because of their diverse biological activities and improved physicochemical properties. Although methylation is pivotal to methoxyflavonoid biosynthesis, the O-methyltransferases (OMTs) responsible for this process in Tartary buckwheat remain poorly characterized. In this study, the Tartary buckwheat cultivar "Y324" was used to systematically analyze the accumulation patterns and spatial distribution of methoxyflavonoids during achene development. Most methoxyflavonoids accumulated progressively during achene development, and they were specifically enriched in the hull. Guided by these metabolite profiles, FtOMT19, a Ca2+-dependent member of the true CCoAOMT subfamily, was identified. Integrating in vitro enzymatic assays with in vivo hairy root overexpression experiments showed that FtOMT19 displays broad substrate promiscuity, efficiently catalyzing the methylation of various flavonoids bearing ortho-dihydroxyl moieties. The enzyme showed a strong preference for the flavonol quercetin, and it can efficiently methylate the 6-, 7-, 8-, 3'-, 4'-, and 5'-hydroxyl positions. Notably, its in vivo role was supported by the increased accumulation of multiple methoxyflavonoids in FtOMT19-overexpressing hairy roots. Mechanistic studies combining molecular docking and site-directed mutagenesis further revealed that the catalytic activity of FtOMT19 depends on the Ca2+-stabilized active center formed by key residues D167, K170 and N194. Meanwhile, D193 contributes to partial activity maintenance, which orchestrates a general base catalytic mechanism. Collectively, this study expands the functional understanding of the true CCoAOMT subfamily and confirms that FtOMT19 is a highly efficient, multifunctional O-methyltransferase that can participate in the biosynthesis of methoxyflavonoids in Tartary buckwheat, providing useful mechanistic insights and genetic resources for further studies of methoxyflavonoid biosynthesis.