Characterization of the anthocyanin-related <i>flavanone 3-hydroxylase</i> (<i>F3H</i>) genes and functional analysis of <i>VcF3H2</i> in blueberry

Flavanone 3-hydroxylase (F3H) is a key enzyme involved in plant flavonoid/anthocyanin biosynthesis pathways. In this study, we identified eight candidate anthocyanin-related <italic>F3H</italic> genes from blueberry (<italic>Vaccinium corymbosum</italic>), and characterized their sequences and expression patterns. These anthocyanin-related VcF3Hs are conserved in sequences and their encoding genes express highly in blueberry fruits at late ripening stages. By using apple and blueberry fruits transient overexpression analysis, the function of <italic>VcF3H2</italic> was studied. Its overexpression significantly improved the anthocyanin accumulation and upregulated the expression levels of anthocyanin biosynthesis-related structural genes in fruit peels of both apple and blueberry. Numerous binding sites for MYB and bHLH transcription factors (TFs) were identified in promoters of <italic>VcF3Hs</italic>, especially <italic>VcF3H1</italic>~<italic>4</italic>. Consistently, our yeast one-hybrid and dual-luciferase assay results showed that both the anthocyanin-related VcMYB-1 and VcAN1 could bind to and activate the promoter of <italic>VcF3H2</italic>, indicating that its roles in anthocyanins biosynthesis were regulated by anthocyanin-related TFs. Our study characterized the anthocyanin-related <italic>F3Hs</italic> in blueberry and demonstrated the role of the 'VcMYB-1/VcAN1-VcF3H2' module in regulating blueberry anthocyanin biosynthesis.