The transcriptional repressor MdMYB21 negatively regulates anthocyanin and malic acid accumulation in red-fleshed...

Red-fleshed apples are characterized by high nutritional value due to their abundant anthocyanin content. However, their commercial development is often constrained by a decline in fresh-eating quality caused by excessive acidity. The molecular mechanism underlying the coordinated accumulation of anthocyanins and malic acid in apple flesh remains largely unclear. In this study, transcriptome analysis of an F₁ hybrid population derived from a cross of Malus sieversii 'CSR6R6' and Malus domestica cv. 'Royal Gala' identified an R2R3-MYB transcription factor, MdMYB21, which was further confirmed as a key negative regulator of both metabolic pathways. Functional assays revealed that overexpressing MdMYB21 significantly inhibited the accumulation of anthocyanins and malic acid in apple calli and fruit tissues. Mechanistic investigations using chromatin immunoprecipitation sequencing (ChIP-Seq), electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter assays demonstrated that MdMYB21 directly binds to promoters of core genes involved in anthocyanin synthesis and transport-including the glycosyltransferase gene MdAGT1 and glutathione S-transferase genes MdGSTU15, MdGSTU16, and MdGSTU22-and acts as a transcriptional repressor that inhibits their expression. These findings uncover a novel mechanism by which MdMYB21 coordinately regulates color and flavor metabolism in apple, providing an important genetic target for breeding new red-fleshed apple varieties with a desirable balance of visual and sensory quality.