Isolation and functional characterization of two 5-O-glucosyltransferases related to anthocyanin biosynthesis from Freesia hybrida

Glycosylation modification fulfils an important role in increasing the stability and solubility of anthocyanin in plants. In our paper, two genes encoding anthocyanin 5-O-glucosyltransferase, designated as Fh5GT1 and Fh5GT2, were isolated from flowers of Freesia hybrida and then functionally characterized. Sequence alignment revealed that the coding regions of Fh5GTs were highly homologous with 5GTs from other plant species and had typical conserved amino acid residues that are crucial for 5GT enzyme activity. Subsequently, subcellular location analysis showed that these two proteins were both localized in the nucleus as well as cytoplasm. Fh5GTs transcripts were detected in various tissues of F. hybrida and were highest expressed in petals. Throughout the development of flowers, Fh5GT1 and Fh5GT2 showed significantly higher transcript levels at the last stage when petals were fully pigmented, and this expression patterns corresponded to the accumulation of anthocyanin in F. hybrida. Additionally, the in vivo function of Fh5GT1 and Fh5GT2 was investigated by their overexpression in Arabidopsis mutant and tobacco plants. Transgenic Arabidopsis seedlings expressing Fh5GTs regained red color pigmentation of the cotyledon and hypocotyl as well as anthocyanin 5-O-glycosides, indicating that Fh5GT1 and Fh5GT2 encoded functional 5GT protein for anthocyanin biosynthesis. When comparing with the wild-type plants, transgenic tobacco overexpressing Fh5GT1 and Fh5GT2 also showed enhanced red color pigmentation. Overall, our results not only demonstrate Fh5GT1 and Fh5GT2 may perform crucial roles in the glycosylation of anthocyanin in F. hybrida but also will aid attempts to manipulate anthocyanin biosynthesis in other plants.