Mechanisms For Transcriptional Regulation Of Anthocyanin Biosynthesis By MYB-bHLH-WD40in Chinese Bayberry

Chinese bayberry (Myrica rubra Sieb. et. Zucc.) is an economically important subtropical fruit crop belonging to the Myricaceae family and native to southern China and other Asian countries. Anthocyanins are the principal color compounds in Chinese bayberry and play an essential role in the fruit quality. The biosynthesis of anthocyanin is mainly regulated by MYB-bHLH-WD40transciption factor complex. Based on the previous results, the MrMYB1which regulates the anthocyanin biosynthesis in Chinese bayberry had been isolated and verified, thus this paper carried out the identification of bHLH and WD40and focused on the mechanisms for the anthocyanin biosynthesis regulation by MYB-bHLH-WD40and lightness in Chinese bayberry. The main results are as follows:1. The isolation and verification of bHLH member(s) related to anthocyanin biosynthesis in Chinese bayberry. MrbHLH1and MrbHLH2were isolated from117bHLH members contained in RNA-Seq database based on their homology to known plant bHLHs involved in anthocyanin biosynthesis regulation. Both of them expressed constitutively in different developmental stages of fruits, different cultivars and tissues, however, only the expression of MrbHLH1showed positive correlation with the anthocyanin accumulation during the fruit development. Moreover, the coexpression of MrbHLHl and MrMYB1in tobacco transient assays could stimulate the activity of AtDFR promoter and regulate obvious anthocyanin biosynthesis. In contrast, MrbHLH2has no function on the regulation of anthocyanin accumulation.2. The verification of WD40related to anthocyanin biosynthesis in Chinese bayberry. By searching and assembling the sequences available in the RNA-Seq database of Chinese bayberry, sixty WD40members were obtained and two of them, designated as MrWD40-1and MrWD40-2respectively, had been isolated as the putative WD40members regulating anthocyanin biosynthesis. However, positive correlation was observed between the anthocyanin accumulation and the expression patterns of MrWD40-1but 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-MrWD40-1induced anthocyanin accumulation earlier and stronger than with binary expression of MrMYB1-MrbHLH1in the absence of MrWD40-1, while MrWD40-2could not improve the anthocyanin accumulation even with MrMYB1and MrbHLH1.3. The mechanisms for the anthocyanin biosynthesis regulation by MYB-bHLH-WD40in Chinese bayberry. Firstly, the function of MrMYBl-MrbHLHl complex on the regulation of anthocyanin biosynthesis is much stronger than the sole member. The results were verified with transient assays in bayberry fruits. Furthermore, MrMYB1-MrbHLH1stimulated the anthocyanin accumulation in the whole flower and leaves of the transformed tobacco lines, while that restricted to petals and anthers of35S::MrMYB1lines, and only in petals of35S::MrbHLH1lines. Moreover, lightness is the essential factor in the regulation of biosynthetic genes by MrMYB1-MrbHLH1complex. Compared with the strong anthocyanin accumulation in tobacco leaves which over-expressing MrMYB1-MrbHLH1in the normal light condition, bagging treatment could significantly inhibite the biosynthesis of anthocyanin. Secondly, the MBW complex regulated the biosynthetic genes selectively. The significant up-regulations had been obsevered in the promoters of MrCHI, MrF3’H, MrDFRl, MrANS and MrUFGT, with the over-expression of MBW members. Moreover, light played an irreplaceable role in the regulation progress by MrMYB1-MrbHLH1. Last but not important, these three members interacted physically with each other during the regulation of anthocyanin biosynthesis in Chinese bayberry. Based on the results of Y2H, the interaction existed both between MrWD40-1-BD and MrMYB1-AD and between MrWD40-1-BD and MrbHLHl-AD. Moreover, MrbHLH1-BD physically interacted with MrMYB1-AD as well.