Flavonoid O-Methyltransferases in Eucalyptus—Biosynthesis of Alpinetin via a Methylated Chalcone Precursor

Methylated flavonoids are abundant phytochemicals in Eucalyptus and are of interest because methylation can alter flavonoid diversity, bioactivity, and stability. However, the enzymes responsible for flavonoid methylation in eucalypts remain largely uncharacterised. We used comparative leaf transcriptomics of two species with contrasting flavanone profiles, together with protein-structure-guided candidate selection, to identify prospective O-methyltransferases involved in methylated flavonoid biosynthesis. Five candidate methyltransferases from E. eugenioides were cloned, heterologously expressed, and assayed against a panel of flavonoids and a chalcone precursor. The enzymes showed distinct substrate preferences and regioselectivities. EeOMT1 acted as a broad 7-O-methyltransferase, whereas EeOMT3–EeOMT5 preferentially methylated B- and C-ring hydroxyl groups, with differing capacities for sequential methylations at different sites. EeOMT2 was of particular interest because it methylated pinocembrin chalcone to alpinetin chalcone more efficiently than it converted the flavanone pinocembrin to alpinetin. Expression–metabolite analyses across E. eugenioides genotypes were consistent with roles for EeOMT2 and EeOMT1 in the in planta accumulation of 5-O- and 7-O-methylated flavanones, respectively. These findings support a revised model in which alpinetin biosynthesis proceeds, at least in part, through methylation of a chalcone precursor before flavanone formation. This work provides a foundation for elucidating flavonoid methylation pathways in plants and for engineering the production of tailored methylated flavonoids.