Identification and Characterization of MYB-bHLH-WD40 Regulatory Complex Members Controlling Anthocyanidin Biosynthesis in Blueberry Fruits Development

Anthocyanins is the main representative of flavonoids in blueberry fruits. The anthocyanins biosynthetic pathway has been extensively studied in numerous model plants and fruit crops at biochemical, genetic, and molecular levels. However, the mechanisms by which the MYB transcription factor/basic helix-loop-helix (bHLH) domain protein/WD-repeat (MYB-bHLH-WD40) complexes regulate anthocyanin biosynthesis in blueberry is still limited. In the present study, we identified 11 MYB, 7 bHLH, and 6 WD40 genes in blueberry fruits, using amino acid sequences of homologous MYB-bHLH-WD40 complexes in Arabidopsis, apple, grape, and strawberry. To understand these mechanisms, the expression patterns of MYB-bHLH-WD40 genes were examined and validated using differentially expressed gene (DEG) analysis and quantitative real-time reverse transcription PCR (qRT-PCR), respectively. The expression patterns of MYB-bHLH-WD40 genes positively correlated with anthocyanin accumulation and color development in blueberry fruits. Consistent with the effects of other transcriptional regulators, the VcMYBL1::GFP, VcbHLH1::GFP, and VcWDL2::GFP fusion proteins were only observed in the nucleus. The protein-protein interactions (PPIs) and bimolecular fluorescence complementation (BiFC) assay suggested a possible link between VcbHLHL1 and VcMYBL1. Finally, a model was proposed and discussed for how the expression of the MYB-bHLH-WD40 complexes can promote anthocyanin biosynthesis in blueberry fruits. To our knowledge, this study was the first to evaluate MYB-bHLH-WD40 complexes in blueberry fruits, and it provides a foundation to dissect the function of the mechanism.