MdPHYB2-MdPIF1 Module Regulates Transcription Factors to Mediate Light-Dependent ALA-Induced Apple Anthocyanin Biosynthesis.

Light is a primary environmental signal that induces plant anthocyanin accumulation. The plant growth regulator 5-aminolevulinic acid (ALA) has also been proven to effectively promote anthocyanin accumulation in apple. However, the underlying regulatory mechanisms remain unknown. In this study, apple phytochrome B2 (MdPHYB2) was identified as the key phytochrome involved in the transcriptional response to ALA under light conditions. Overexpressing (OE)-MdPHYB2 promoted anthocyanin accumulation, while RNA interfering (RNAi)-MdPHYB2 suppressed it. Library screening and a series of validation tests proved that apple phytochrome-interacting factor 1 (MdPIF1) is an interactive protein of MdPHYB2, which can interact with each other only under light conditions. OE-MdPIF1 inhibited anthocyanin accumulation, while RNAi-MdPIF1 promoted it. MdPIF1 can directly bind to E-box motifs of the promoters of MdWRKY71, MdMADS1, and MdNAC33 to repress all gene expression, but not to the promoters of MdPAL, MdCHS, MdF3H, and MdUFGT, the structural genes of anthocyanin biosynthesis. Combined with the previous findings, we propose that MdPIF1 is a primary repressor, negatively transcriptionally regulating the expression of MdWRKY71, MdMADS1 (responsible for the non-MBW route of anthocyanin biosynthesis) and MdNAC33 (responsible for the MBW route). ALA upregulated MdPHYB2 but depressed MdPIF1 expression under light conditions, then cytoplasmic MdPHYB2 was shifted into the nucleus, interacting with MdPIF1 and antagonizing the binding with the promoters of downstream genes, alleviating the gene repression and activating anthocyanin biosynthesis. These findings establish an MdPHYB2-MdPIF1 module as a central regulatory hub that coordinates light perception and ALA signalling to promote apple anthocyanin biosynthesis.