Cloning, characterization and functional analysis of a flavonol synthase from Vaccinium corymbosum

Key message VcFLS from Vaccinium corymbosum promoted myricetin biosynthesis in Arabidopsis thaliana and VcFLS expression was induced by salicylic acid. Flavonoids are polyphenols with important functions in pigmentation, UV filtration, and symbiotic nitrogen fixation. Flavonols are a class of flavonoids that are produced by the desaturation of dihydroflavanols in a reaction that is catalyzed by flavonol synthase (FLS). In the study reported here, we cloned the full-length cDNA of FLS (designated as VcFLS ) from Vaccinium corymbosum (blueberry) using rapid amplification of cDNA ends (RACE). The cDNA contained a 1005-bp open reading frame that encoded a 334-amino acid protein. Phylogenetic analysis showed that VcFLS was closely related to FaFLS, a flavonol synthase that catalyzed the formation of kaempferol and had little effect on the formation of quercetin. Quantitative RT-PCR analysis demonstrated that VcFLS was expressed in all of the tissues tested, with particularly high expression in the petals and young leaves (both green and red). The flavanols myricetin and quercetin also occurred in all of these tested tissues, with the highest levels detected in mature leaves. The expression of VcFLS was not consistent with the accumulation of quercetin and myricetin in different tissues, nor were the expressions of VcFLS , VcPAL , VcCHS , VcF3H , and VcF3′5′H consistent with the accumulation of the quercetin during fruit development. However, the change in the trend of VcCHS and VcF3H expression was similar with myricetin accumulation during fruit development. Expression profiling analysis revealed that VcFLS expression was induced by salicylic acid, a phytohormone involved in plant defense against pathogens, and was suppressed by gibberellic acid, a phytohormone involved in various aspects of plant development. Heterologous expression of VcFLS in Arabidopsis thaliana increased the content of myricetin, but did not affect quercetin content. Thus, we conclude that VcFLS is a key enzyme in the flavonol biosynthetic pathway and would appear to be involved in the plant defense response.