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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- O -glycosyltransferase (UF3GT), is one of the common enzymes which could catalyze the glycosylation of both flavonol and anthocyanidin aglycons simultaneously  in vitro . However, whether and how  UF3GT  paralogous genes function diversely at the biochemical and transcriptional levels are largely unknown. Recently,  Fh3GT1  was identified to be a member of  UF3GT s in  Freesia hybrida . 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  Freesia , three closest candidate  UFGT  genes— Fh3GT2 ,  Fh3GT3 , and  Fh3GT4 —were mined from the  Freesia  transcriptomic database and isolated from the flowers of the widely distributed  Freesia  cultivar, Red River ® . Based on bioinformatic analysis and enzymatic assays,  Fh3GT2  turned out to be another  bona fide  glycosyltransferase gene. Biochemical analysis further proved that Fh3GT2 preferentially glucosylated kaempferol while  Fh3GT1  controlled the glucosylation of quercetin and anthocyanidins. In addition, transfection assays demonstrated that  Fh3GT2  could be mainly activated by the flavonol regulator FhMYBF1 or the anthocyanin regulator FhPAP1, whereas  Fh3GT1  could only be activated by FhPAP1. These findings suggested that  Fh3GT s might have functionally diverged in flavonoid biosynthesis at both the biochemical and transcriptional levels.

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