The regulatory module MsSPX4/MsPHL11–MsWRKY91 mediates low-phosphorus-induced anthocyanin synthesis in Malus spectabilis leaves

Phosphorus (P) is a macronutrient necessary for plant growth and development. Phosphorus deficiency usually causes changes in plant secondary metabolites, mainly including anthocyanin synthesis. However, the regulatory mechanism of anthocyanin synthesis in Malus spectabilis leaves under low-phosphorus conditions remains unclear. In this study, anthocyanin accumulation in M. spectabilis leaves gradually increased as phosphorus application decreased, reaching a maximum anthocyanin concentration at 300 µM phosphorus. Through transcriptome sequencing, MsSPX4 was identified as a key regulatory factor for anthocyanin synthesis under low-phosphorus conditions. The protein complex MsSPX4-MsPHL11 was confirmed through Y2H, BiFC, and LCI assays, and its function was validated through transient and stable transformation experiments in Malus spp. Under conditions of sufficient Pi, MsSPX4 inhibited MsPHL11 activity by interacting with MsPHL11, whereas phosphorus starvation disrupted the MsSPX4/MsPHL11 complex, alleviating the inhibitory effect on MsPHL11. MsPHL11 bound to the P1BS element on the MsWRKY91 promoter to activate MsWRKY91 expression. Furthermore, MsWRKY91 activated MsF3'H expression by binding to the W-box element on the MsF3'H promoter, thereby positively regulating anthocyanin biosynthesis. Overexpression of MsPHL11 promoted the expression of genes related to phosphorus metabolism and transport, thereby increasing M. 'Gala' low-phosphorus resistance. By contrast, MsSPX4-stabilized transgenic M. 'Gala' lines exhibited lower phosphorus resistance. Our results elucidate the mechanism by which the MsSPX4/MsPHL11-MsWRKY91 module regulates anthocyanin synthesis in M. spectabilis leaves under low-phosphorus conditions. These findings provide a reference for further exploration of the molecular mechanism of anthocyanin synthesis in plants under low-phosphorus conditions.