MdBBX47, a B-box transcription factor directly and indirectly regulates ALA-induced anthocyanin accumulation in apple

The red pigmentation of apple (Malus × domestica Borkh.) fruit peel arises from the accumulation of anthocyanins. Anthocyanins, a pivotal determinant of fruit coloration, not only define commercial appeal but also confer nutritional and antioxidant benefits. 5-Aminolevulinic acid (ALA), a naturally occurring plant growth substance, has been shown to significantly intensify fruit redness by stimulating anthocyanin biosynthesis. Nevertheless, the underlying molecular mechanism remains largely unclear. In this investigation, we identified and functionally characterized MdBBX47, an ALA-responsive transcription factor whose transcriptional activation exhibits strong correlation with anthocyanin accumulation across apple fruits, leaves, and callus tissues, implicating it is a key regulatory node in the anthocyanin biosynthesis cascade. Subcellular localization assays confirmed that MdBBX47 is localized within the nucleus. Overexpression of MdBBX47 substantially elevated the anthocyanin content, whereas RNA interference-mediated silencing markedly attenuated ALA-induced pigmentation, underscoring its positive regulatory function. Yeast one-hybrid (Y1H), luciferase (LUC), GUS, and electrophoretic mobility shift assays (EMSA) revealed that MdBBX47 directly binds to the promoters of MdCHS and MdUFGT to activate their expressions. Additionally, MdBBX47 can bind to the promoter of MdMYB110a, indirectly mediating anthocyanin accumulation. Collectively, these findings elucidate how MdBBX47 serves as a novel regulatory component involved in ALA-induced apple anthocyanin accumulation.