MsERF4-like1/2 transcription factors positively regulate anthocyanin biosynthesis in Malus spectabilis

Crabapple is a horticulturally valuable ornamental plant renowned for its diverse flower and leaf colors, with its aesthetic and economic value closely linked to anthocyanin production. While anthocyanin-mediated pigmentation has been widely studied in fruits and flowers, the regulatory networks governing leaf coloration remain less understood. In this study, we employed Malus spectabilis, which displays natural red/green leaf color variation, as a model to investigate the molecular mechanisms of leaf anthocyanin biosynthesis. We found that the expression of two Arabidopsis ERF4 homologs, MsERF4-like1 and MsERF4-like2, was significantly higher in red leaves compared to green leaves, suggesting coordinated upregulation with key anthocyanin structural genes. Overexpression of MsERF4-like1 and MsERF4-like2 in apple fruit peel enhanced anthocyanin accumulation compared to the empty vector control. Crucially, yeast one-hybrid and dual-luciferase reporter assays demonstrated that both MsERF4-like1 and MsERF4-like2 directly bind the promoter of the anthocyanin structural gene MsCHI, thereby activating its transcription and promoting anthocyanin accumulation. Collectively, these findings reveal that MsERF4-like1 and MsERF4-like2 directly regulate anthocyanin biosynthetic genes to control leaf coloration in M. spectabilis, offering potential targets for genetic improvement of foliage color in ornamental plants.