The CsMYB123 and CsbHLH111 are involved in drought stress-induced anthocyanin biosynthesis in Chaenomeles speciosa.

Drought stress has been demonstrated to enhance the biosynthesis of anthocyanins in the leaves, resulting in an increased aesthetic appeal. However, the molecular mechanisms underlying drought-induced anthocyanin biosynthesis in Chaenomeles speciosa remain unclear. In this study, the metabolites of C. speciosa leaves were analyzed, and it was found that the content of cyanidin-3-O-rutinoside increased significantly under drought stress. The differentially expressed genes CsMYB123 and CsbHLH111 were isolated by transcriptomics data analysis and gene cloning, and gene overexpression and VIGS experiments verified that both play important roles in anthocyanin biosynthesis. Subsequently, Y1H and Dual-luciferase reporter assay showed that CsMYB123 binds to the promoters of anthocyanin biosynthesis-related structural genes (such as CsCHI, CsF3H, and CsANS), while CsbHLH111 was shown to bind to the promoter of CsCHI, positively regulating its activity. Furthermore, BIFC and Y2H assays unveiled potential protein-protein interactions between CsMYB123 and CsbHLH111 at the cell nucleus. Collectively, these results shed light on the critical roles played by CsMYB123 and CsbHLH111 in anthocyanin biosynthesis, thus providing a valuable insight into understanding the molecular mechanisms of how the MYB and bHLH genes regulate anthocyanin biosynthesis in the process of leaf coloration in C. speciosa.