Auxin regulates anthocyanin biosynthesis through the Aux/IAA–ARF signaling pathway in apple

Auxin signaling, which is crucial for normal plant growth and development, mainly depends on ARF-Aux/IAA interactions. However, little is known regarding the regulatory effects of auxin signaling on anthocyanin metabolism in apple (Malus domestica). We investigated the functions of MdARF13, which contains a repression domain and is localized to the nucleus. This protein was observed to interact with the Aux/IAA repressor, MdIAA121, through its Cterminal dimerization domain. Protein degradation experiments proved that MdIAA121 is an unstable protein that is degraded by the 26S proteasome. Additionally, MdIAA121 stability is affected by the application of exogenous auxin. Furthermore, the overexpression of MdIAA121 and MdARF13 in transgenic red-fleshed apple calli weakened the inhibitory effect of MdARF13 on anthocyanin biosynthesis. These results indicate that the degradation of MdIAA121 induced by auxin treatment can release MdARF13, which acts as a negative regulator of the anthocyanin metabolic pathway. Additionally, yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays confirmed that MdMYB10 interacts with MdARF13. A subsequent electrophoretic mobility shift assay and yeast one-hybrid assay demonstrated that MdARF13 directly binds to the promoter of MdDFR, which is an anthocyanin pathway structural gene. Interestingly, chromatin immunoprecipitation-quantitative real-time PCR results indicated that the overexpression of MdIAA121 clearly inhibits the recruitment of MdARF13 to the MdDFR promoter. Our findings further characterized the mechanism underlying the regulation of anthocyanin biosynthesis via Aux/IAA-ARF signaling.