mdm-miR395 targets MdAPS1(ATP sulfurylase 1) to regulate sulfur homeostasis and anthocyanin accumulation in Malus

Although miR395 is well established as a key regulator of sulfur (S) metabolism, its potential role in integrating light signaling with S homeostasis to coordinate secondary metabolite biosynthesis remains underexplored. Here, we identify a regulatory module comprising mdm-miR395, its target MdAPS1, and the transcription factor MdWRKY26, which integrates light and S signaling to promote anthocyanin and ascorbic acid (AsA) accumulation in Malus. Subcellular localization analysis revealed dual localization of MdAPS1 in the cell membrane and mitochondrial, suggesting its role in sensing extracellular S concentrations and modulating assimilation efficiency. Transient suppression of miR395 in apple fruit peel enhanced light-induced anthocyanin accumulation, whereas miR395 overexpression produced opposite effects, underscoring its regulatory role in S homeostasis. Genetic manipulation demonstrated that MdAPS1 positively regulates reduced glutathione (GSH) levels, with overexpression increasing anthocyanin and AsA accumulation. Notably, MdWRKY26 was identified as a novel positive regulator of both GSH and anthocyanin accumulation. Exogenous GSH application significantly enhanced pigmentation in apple fruits and plantlets. Our findings reveal that light signals modulate S homeostasis through the miR395-MdAPS1 and MdWRKY26, and establish a GSH-mediated regulatory node linking light signals to anthocyanin and AsA accumulation in Malus, providing a basis for optimizing S nutrition to improve apple fruit coloration.