Biochemical and Molecular Characterization of a Flavonoid 3-O-glycosyltransferase Responsible for Anthocyanins and Flavonols Biosynthesis in Freesia hybrida.

Wei Sun,Xiang Gao,Yueqing Li,Fengzhan Gao,Xingxue Liu,Lingjie Liang,Xiangyu Meng,Li Wang,Shucai Wang

doi:10.3389/fpls.2016.00410

Wei Sun, Xiang Gao + Show 7 more

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https://doi.org/10.3389/fpls.2016.00410

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Abstract

The glycosylation of flavonoids increases their solubility and stability in plants. Flowers accumulate anthocyanidin and flavonol glycosides which are synthesized by UDP-sugar flavonoid glycosyltransferases (UFGTs). In our previous study, a cDNA clone (Fh3GT1) encoding UFGT was isolated from Freesia hybrida, which was preliminarily proved to be invovled in cyanidin 3-O-glucoside biosynthesis. Here, a variety of anthocyanin and flavonol glycosides were detected in flowers and other tissues of F. hybrida, implying the versatile roles of Fh3GT1 in flavonoids biosynthesis. To further unravel its multi-functional roles, integrative analysis between gene expression and metabolites was investigated. The results showed expression of Fh3GT1 was positively related to the accumulation of anthocyanins and flavonol glycosides, suggesting its potential roles in the biosynthesis of both flavonoid glycosides. Subsequently, biochemical analysis results revealed that a broad range of flavonoid substrates including flavonoid not naturally occurred in F. hybrida could be recognized by the recombinant Fh3GT1. Both UDP-glucose and UDP-galactose could be used as sugar donors by recombinant Fh3GT1, although UDP-galactose was transferred with relatively low activity. Furthermore, regiospecificity analysis demonstrated that Fh3GT1 was able to glycosylate delphinidin at the 3-, 4-′, and 7- positions in a sugar-dependent manner. And the introduction of Fh3GT1 into Arabidopsis UGT78D2 mutant successfully restored the anthocyanins and flavonols phenotypes caused by lost-of-function of the 3GT, indicating that Fh3GT1 functions as a flavonoid 3-O-glucosyltransferase in vivo. In summary, these results demonstrate that Fh3GT1 is a flavonoid 3-O-glycosyltransferase using UDP-glucose as the preferred sugar donor and may involve in flavonoid glycosylation in F. hybrida.

Highlights

Flavonoids, the important polyphenolic secondary metabolites, are widely distributed in plant species and possess diverse biological functions including pollination, pigmentation, auxin transport inhibition, UV light protection and other defense mechanism (Brenda, 2001; Koes et al, 2005; Charles et al, 2010)
In order to examine the subcellular distribution of Fh3GT1, transgenic Arabidopsis plants expressing this enzyme fused to the C terminus of GFP were produced, and the results showed that this protein seemed to be localized in both the nuclei and the cytosol (Supplementary Figure S1)
These results demonstrated that only one molecule glucose/galactose had been transferred to the substrates

Summary

Introduction

Flavonoids, the important polyphenolic secondary metabolites, are widely distributed in plant species and possess diverse biological functions including pollination, pigmentation, auxin transport inhibition, UV light protection and other defense mechanism (Brenda, 2001; Koes et al, 2005; Charles et al, 2010). Colorless co-pigments, affect the brightness and brilliance of colors and have vital roles in pollen germination (Ferreyra et al, 2012; Zhao and Tao, 2015). Anthocyanins and flavonols play an important role in human health and have potential medicinal uses, as the intake of them can protect against cardiovascular disease, cancer, and many other diseases (Petroni et al, 2014; Santos-Buelga et al, 2014)

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