An integrated analysis with transcriptomics and metabolomics highlights VdMYB56 regulation anthocyanin biosynthesis in rabbiteye blueberry (Vaccinium ashei)

Blueberry (Vaccinium ashei) is highly characterized by its nutritional value, with an extremely high anthocyanin content, and rabbiteye blueberry is widely grown across China. However, molecular regulatory mechanisms underlying the high anthocyanin accumulation during the fruit development and colouration of rabbiteye blueberry fruit, have not yet been fully clarified so far. The fruit anthocyanin content of rabbiteye blueberry in the karstic area of Guizhou Province, China, is much higher compared to that in other regions, and the fruit colour is highly affected by anthocyanin accumulation. Currently, the untargeted metabolomics and HPLC assays have been carried out using rabbiteye blueberry fruit at various stages, and it was investigated that cyanidin (Cy) and pelargonidin (Pg) reached their peaks at the red fruit (RF) stage, whereas delphinidin (Dp), petudinin (Pt), malvidin (Mv), and peonidin (Pn) got their ceilings at the mature fruit (MF) stage. Transcriptome and co-expression network analyses showed that 27 differentially expressed genes (DEGs) were associated with anthocyanin content, among which VdMYB56, belonging to the R2R3-MYB family, was markedly up-regulated during the development and colouration of fruit, and was significantly higher in the skin than in the pulp. Furthermore, VdMYB56-overexpressing tomato fruits demonstrated a substantial elevation in anthocyanin content on the 35th day after flowering (DAF). It was worth noting that VdMYB56 could directly bind to the promoter of Vd3GT to enhance its expression, thereby further strengthening the anthocyanin accumulation. Meantime, multiple assays verified that VdMYB69, an R2R3-MYB member, might interact with VdMYB56, leading to the promotion of VdMYB56 expression. Conclusively, the VdMYB56-VdMYB69 module is a positive regulator of anthocyanin biosynthesis in rabbiteye blueberry, which may provide new insights into high-anthocyanin breeding, particularly for the southern karstic regions.