The ABA signaling responsive gene VcCRF9 positively regulates anthocyanin synthesis in blueberry (Vaccinium corymbosum)

Anthocyanin content is a core nutritional indicator for evaluating blueberry quality, and anthocyanin accumulation in blueberry fruit largely dictates its economic value. In fruit trees, abscisic acid (ABA) promotes anthocyanin accumulation via the AP2/ERF family of transcription factors. However, the molecular mechanism behind ABA-regulated anthocyanin biosynthesis has not been fully elucidated in blueberry fruits. Using transcriptomics analysis, we identified an AP2/ERF transcription factor, termed VcCRF9, that responds to ABA signaling. Homologous transformation assays demonstrated that VcCRF9 significantly promoted anthocyanin biosynthesis in blueberry leaves, fruits, and adventitious shoots. Previous studies have shown that VcMYB1 is an important regulatory factor promoting anthocyanin synthesis in blueberry fruits. Quantitative real-time PCR indicated that VcCRF9 overexpression led to significant upregulation of structural genes related to anthocyanin synthesis. Using a combination of yeast one-hybridization (Y1H), firefly luciferase complementation (LUC), and electrophoretic mobility shift assays (EMSA), we found that VcCRF9 interacts with VcMYB1 at the DNA-protein level. Furthermore, the interactions between VcCRF9 and structural genes related to anthocyanin synthesis were also examined. VcCRF9 was found to directly bind to the G-box motif in the promoter region of the anthocyanin synthase gene VcANS and activated its expression, resulting in anthocyanin accumulation. These findings not only provide a foundation for modifying blueberry fruit quality through exogenous ABA application but also deepen our understanding of the molecular regulatory network of anthocyanin biosynthesis in blueberry.