RESUMEN
In red-fleshed kiwifruit, anthocyanin pigmentation is a crucial commercial trait. The MYB-bHLH-WD40 (MBW) complex and other transcription factors regulate its accumulation. Herein, a new SEP gene, AcMADS68, was identified as a regulatory candidate for anthocyanin biosynthesis in the kiwifruit by transcriptome data and bioinformatic analyses. AcMADS68 alone could not induce the accumulation of anthocyanin both in Actinidia arguta fruit and tobacco leaves. However, in combination with AcMYBF110, AcMYB123, and AcbHLH1, AcMADS68 co-overexpression increased anthocyanin biosynthesis, whereas its silencing reduced anthocyanin accumulation. The results of the dual-luciferase reporter, firefly luciferase complementation, yeast two-hybrid and co-immunoprecipitation assays showed that AcMADS68 could interact with both AcMYBF110 and AcMYB123 but not with AcbHLH1, thereby co-regulating anthocyanin biosynthesis by promoting the activation of the target genes, including AcANS, AcF3GT1, and AcGST1. Moreover, AcMADS68 also could activate the promoter of AcbHLH1 surported by dual-luciferase reporter and yeast one-hybrid assays, thereby further amplifying the regulation signals from the MBW complex, thus resulting in enhanced anthocyanin accumulation in the kiwifruit. These findings may facilitate better elucidation of various regulatory mechanisms underlying anthocyanin accumulation and contribute to the quality enhancement of red-fleshed kiwifruit.
RESUMEN
The anthocyanin synthetic pathway is regulated centrally by an MYB-bHLH-WD40 (MBW) complex. Anthocyanin pigmentation is an important fruit quality trait in red-fleshed kiwifruit; however, the underlying regulatory mechanisms involving the MBW complex are not well understood. In this study, one R2R3MYB (AcMYBF110 expressed in fruit characteristically), one bHLH (AcbHLH1), two upstream regulators of AcbHLH1 (AcbHLH4 and AcbHLH5), and one WDR (AcWDR1) are characterized as being involved in the regulation of anthocyanin synthesis in kiwifruit. AcMYBF110 plays an important role in the regulation of anthocyanin accumulation by specifically activating the promoters of several anthocyanin pathway genes including AcCHS, AcF3'H, AcANS, AcUFGT3a, AcUFGT6b, and AcGST1. Coexpression of AcbHLH1, AcbHLH4, or AcbHLH5 together with AcMYBF110 induces much greater anthocyanin accumulation in both tobacco leaves and in Actinidia arguta fruit compared with AcMYBF110 alone. Moreover, this activation is further enhanced by adding AcWDR1. We found that both AcMYBF110 and AcWDR1 interact with all three AcbHLH factors, while AcMYBF110 also interacts with AcWDR1 to form three different MBW complexes that have different regulatory roles in anthocyanin accumulation of kiwifruit. The AcMYBF110-AcbHLH1-AcWDR1 complex directly targets the promoters of anthocyanin synthetic genes. Other features of the regulatory pathways identified include promotion of AcMYBF110, AcbHLH1,and AcWDR1 activities by this MBW complex, providing for both reinforcement and feedback regulation, whereas the AcMYBF110-AcbHLH4/5-AcWDR1 complex is indirectly involved in the regulation of anthocyanin synthesis by activating the promoters of AcbHLH1 and AcWDR1 to amplify the regulation signals of the first MBW complex.
