Molecular cloning and functional analysis of a flavanone 3-hydroxylase gene from blueberry

Vaccinium corymbosum (blueberry) is touted as a superfood with numerous health benefits due to its high levels of flavonoids. Flavanone 3-hydroxylase (F3H) is a key regulatory enzyme of the flavonoid pathway. In this study, we cloned the full-length cDNA of F3H (designated VcF3H) from young blueberry leaves using rapid amplification of cDNA ends (RACE). The cDNA contained a 1080-bp open reading frame that encoded a 359-amino acid protein. The deduced VcF3H protein showed high similarities to other plant F3Hs. Conserved amino acid motifs required for ferrous iron binding (HXD) and 2-oxoglutarate binding (RXS) were identified in VcF3H, VcFLS (flavonol synthase), and VcANS (anthocyanidin synthase). Quantitative RT-PCR analysis demonstrated that VcF3H was expressed in all tissues tested, with particularly high expression in young leaves, fruits (pink and blue), and stems. Anthocyanins accumulated mainly in fruits, whereas flavonols were found mainly in leaves and stems. Furthermore, the expression pattern of VcF3H was similar to that of VcCHS, VcDFR, and VcANS in various tissues. Heterologous expression of VcF3H in Arabidopsis thaliana increased the anthocyanin content in leaves, but did not affect the flavonol content. Thus, VcF3H seems to be involved in anthocyanin synthesis in the flavonoid biosynthetic pathway when ectopically expressed in Arabidopsis.