VcHY5-VcbHLH004 module regulates anthocyanin synthesis in response to light in blueberry.

Light is an essential factor for the anthocyanin accumulation in blueberry. However,the molecular mechanism of light on blueberry anthocyanin synthesis remains unclear.In this study, we discovered a VcHY5-VcbHLH004 molecular module that is responsive to light and regulates anthocyanin biosynthesis in bluebery.This study revealed that blueberry VcbHLH004 and VcHY5 positively regulate anthocyanin biosynthesis in response to light. Specifically, light exposure induced VcbHLH004 and VcHY5 expression and anthocyanin accumulation in blueberry calli. VcbHLH004-OE and VcHY5-OE calli exhibited enhanced red coloration, whereas VcbHLH004-RNA-interference and VcHY5-RNA-interferencecalli experienced a reduction in anthocyanin accumulation.We also found that VcHY5 interacts with VcbHLH004 and identified the specific domains responsible for this interaction. The VcHY5-VcbHLH004 complex enhances VcbHLH004's binding to the promoter of its target genes and increases the protein abundance of VcbHLH004. This interaction upregulates the transcription of VcbHLH004's downstream anthocyanin-associated genes, VcF3'5'H4 and VcDFR, whose promoters contain G-box motifs.This interaction upregulates the transcription of VcbHLH004's downstream anthocyanin-associated genes,VcF3'5'H4 and VcDFR. Furthermore, transcriptome and metabolic data revealed significantly positive correlations between VcbHLH004, VcHY5, and VcF3'5'H4 genes and flavonoids (especially anthocyanins), confirming the reliability of our results. Our findings demonstrated that the transcriptional regulatory mechanism by which the VcHY5-VcbHLH004 module induces anthocyanin accumulation.