Molecular Characterisation of Flavanone O-methylation in Eucalyptus

Flavonoids are ubiquitous polyphenolic compounds in plants, long recognised for their health-promoting properties in humans. Methylated flavonoids have received increasing attention due to the potential of methylation to enhance medicinal efficacy. Recently, Eucalyptus species with high levels of the O-methylated flavanone pinostrobin have been identified. Pinostrobin has potential commercial value due to its numerous pharmacological and functional food benefits. Little is known about the identity or mode of action of the enzymes involved in methylating flavanones. This study aimed to identify and characterise the methyltransferase(s) involved in the regiospecific methylation of pinostrobin in Eucalyptus and thereby add to our limited understanding of flavanone biosynthesis in plants. RNA-seq analysis of leaf tips enabled the isolation of a gene encoding a flavanone 7-O-methyltransferase (EnOMT1) in Eucalyptus. Biochemical characterisation of its in vitro activity revealed a range of substrates upon which EnOMT1 acts in a regiospecific manner. Comparison to a homologous sequence from a Eucalyptus species lacking O-methylated flavonoids identified critical catalytic amino acid residues within EnOMT1 responsible for its activity. This detailed molecular characterisation identified a methyltransferase responsible for chemical ornamentation of the core flavanone structure of pinocembrin and helps shed light on the mechanism of flavanone biosynthesis in Eucalyptus.