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

Abstract

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 'Halloween'. Our study employed 'Halloween' leaves for experimental analysis, incorporating 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. Phylogenetic analyses suggest AaMYB4 as a transcription factor suppressing flavonoid biosynthesis. Comparative analysis of protein sequences revealed that AaMYB4 functions as an R2R3-MYB transcription factor, characterized by a typical repressive EAR motif. AaMYB4 was cloned into Arabidopsis by inflorescence infestation. The WT and stably overexpressing AaMYB4 T2-generation Arabidopsis were subsequently grown under stress conditions including nitrogen deficiency, high light exposure, 6% sucrose, and abscisic acid (ABA) treatment. The results indicated that the anthocyanin content was significantly reduced in AaMYB4 overexpressing Arabidopsis compared with the WT under the four treatments described above, and the structural genes for anthocyanin synthesis were down-regulated in the AaMYB4 transgenic Arabidopsis. Moreover, the expression levels of the positively regulated MYB and bHLH transcription factors involved in anthocyanin synthesis, specifically AtPAP1 and AtTT8, exhibited a significant downregulation in Arabidopsis. Yeast two-hybrid assays revealed no interaction between AaMYB4 and AaTT8, and the AaMYB4 protein itself can interact. This research confirms AaMYB4's role 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.