Cloning and functional identification of anthocyanin synthesis-regulating transcription factor AaMYB4 in Aeonium arboreum Webb & Berthel ‘Halloween’

Succulents are widely appreciated for indoor decoration, primarily due to their vibrant leaf colors. However, the underlying mechanisms of leaf color development in succulents remain largely unexplored. In this study, we isolated AaMYB4, an MYB transcription factor that represses anthocyanin synthesis, from an ornamental succulent, Aeonium arboreum Webb & Berthel ‘Halloween’. Our study incorporated gene cloning, bioinformatics, functional validation of transgenes, and yeast two-hybrid assays to investigate AaMYB4’s role. The finding revealed that the full-length Open Reading Frame (ORF) of AaMYB4 spans 825 base pairs, encoding 274 amino acids. Bioinformatics analyses suggested that AaMYB4 functions as an R2R3-MYB transcription factor suppressing flavonoid biosynthesis, characterized by a typical repressive EAR motif. Functional validation of transgenes for AaMYB4 indicated that the anthocyanin content was significantly reduced in AaMYB4 overexpressing Arabidopsis compared with the WT under nitrogen deficiency, high light exposure, 6% sucrose, and abscisic acid (ABA) treatment, and the structural genes for anthocyanin synthesis were down-regulated in the AaMYB4 transgenic Arabidopsis. Furthermore, in terms of transcription factors in the AaMYB4 transgenic Arabidopsis, the expression levels of AtPAP1 and AtTT8 exhibited a significant downregulation. Yeast two-hybrid assays revealed no interaction between AaMYB4 and AaTT8, and the AaMYB4 protein can self-interact. This research confirmed the role of AaMYB4 in inhibiting anthocyanin synthesis in ‘Halloween’ leaves, enriching our understanding of the molecular basis of leaf color formation in succulents. Additionally, it offers valuable genetic insights for developing new ‘Halloween’ colorful leaf varieties.