Functional Differentiation of Duplicated Flavonoid 3-O-Glycosyltransferases in the Flavonol and Anthocyanin Biosynthesis of Freesia hybrida

Abstract

Flavonols and anthocyanins are two widely distributed groups of flavonoids that occurred apart during plant evolution and biosynthesized by shared specific enzymes involved in flavonoid metabolism. UDP-glucose, flavonoid 3-<i>O</i>-glycosyltransferase (UF3GT), is one of the common enzymes which could catalyze the glycosylation of both flavonol and anthocyanidin aglycons simultaneously <i>in vitro</i>. However, whether and how <i>UF3GT</i> paralogous genes function diversely at the biochemical and transcriptional levels are largely unknown. Recently, <i>Fh3GT1</i> was identified to be a member of <i>UF3GT</i>s in <i>Freesia hybrida</i>. However, its expression patterns and enzymatic characteristics could not coincide well with flavonol accumulation. In an attempt to characterize other flavonoids, especially flavonol glycosyltransferase genes in <i>Freesia</i>, three closest candidate <i>UFGT</i> genes-<i>Fh3GT2</i>, <i>Fh3GT3</i>, and <i>Fh3GT4</i>-were mined from the <i>Freesia</i> transcriptomic database and isolated from the flowers of the widely distributed <i>Freesia</i> cultivar, Red River^®. Based on bioinformatic analysis and enzymatic assays, <i>Fh3GT2</i> turned out to be another <i>bona fide</i> glycosyltransferase gene. Biochemical analysis further proved that Fh3GT2 preferentially glucosylated kaempferol while <i>Fh3GT1</i> controlled the glucosylation of quercetin and anthocyanidins. In addition, transfection assays demonstrated that <i>Fh3GT2</i> could be mainly activated by the flavonol regulator FhMYBF1 or the anthocyanin regulator FhPAP1, whereas <i>Fh3GT1</i> could only be activated by FhPAP1. These findings suggested that <i>Fh3GT</i>s might have functionally diverged in flavonoid biosynthesis at both the biochemical and transcriptional levels.