The MrWD40-1 Gene of Chinese Bayberry (Myrica rubra) Interacts with MYB and bHLH to Enhance Anthocyanin Accumulation

Anthocyanins are important for fruits as they contribute not only to fruit color but also to human health. Anthocyanin biosynthesis is transcriptionally regulated by the MYB-bHLH-WD40 transcription complex. For Chinese bayberry (Myrica rubra), the MYB and bHLH regulating anthocyanin accumulation, named as MrMYB1 and MrbHLH1, respectively, have been isolated previously. In this study, by searching and assembling the sequences available in the RNA-Seq database of Chinese bayberry, 60 WD40 members were obtained. Through phylogenetic analysis of these members with those related to anthocyanin biosynthesis regulation in other plants, unigenes 803 and 11128, designated as MrWD40-1 and MrWD40-2, respectively, have been isolated as the putative WD40 members regulating anthocyanin biosynthesis. However, positive correlation was observed between the anthocyanin accumulation and the expression patterns of MrWD40-1 but not MrWD40-2, both during fruit development, and in different tissues or cultivars of Chinese bayberry. Tobacco transient assays indicated that the ternary expression of MrMYB1-MrbHLH1-MrWD40-1 induced anthocyanin accumulation earlier and stronger than with binary expression of MrMYB1-MrbHLH1 in the absence of MrWD40-1. Compared with the enhancement effect on anthocyanin biosynthesis of MrWD40-1, MrWD40-2 could not improve the anthocyanin accumulation even with MrMYB1 and MrbHLH1, although the highly conserved four WD repeat motifs were also present in MrWD40-2. Moreover, it was observed that MrWD40-1 physically interacted with both MrMYB1 and MrbHLH1 according to yeast two-hybrid analysis. These results indicated that MrWD40-1, but not MrWD40-2, is the member regulating anthocyanin biosynthesis in Chinese bayberry through the formation of a ternary complex with MrMYB1 and MrbHLH1.