Jasmonate‐responsive MdMYC2/MdMED25 complex regulates malic acid accumulation in apples through the miR858– <i>MdMYB73</i> module

Malic acid is a crucial determinant of apple (Malus domestica) fruit quality, influencing acidity and flavor. While transcriptional regulation of malic acid metabolism is well-studied, post-transcriptional control and the role of jasmonate (JA) remain largely unexplored. We identify a novel regulatory pathway involving JA signaling, a microRNA (miRNA), and vacuolar transport regulators that control malic acid accumulation in apple fruit. We show that mdm-miR858, which increases during fruit maturation, directly targets and cleaves MdMYB73 transcripts. MdMYB73 is a known positive regulator of vacuolar H⁺-pumping and malate transport, activating genes like MdVHA-A, MdVHP, and MdALMT9. Overexpression of mdm-miR858 suppressed MdMYB73, thereby reducing MdVHA-A, MdVHP, and MdALMT9 expression and malic acid content in apple calli, fruits, and GL-3 plantlets, while silencing mdm-miR858 had opposite effects. Crucially, the JA-responsive transcription factor MdMYC2, the expression of which increases during fruit maturation, directly binds the mdm-miR858 promoter and activates its expression. Furthermore, the Mediator complex subunit MdMED25 interacts with MdMYC2, enhancing this activation. Manipulating MdMYC2 or MdMED25 expression altered mdm-miR858 levels, MdMYB73 expression, and malic acid accumulation, mirroring exogenous methyl jasmonate (MeJA) treatment effects. A miR858-resistant MdMYB73 variant confirmed the miRNA-target interaction's specificity and functional significance. Our findings reveal a novel JA-MdMYC2/MdMED25-miR858-MdMYB73 regulatory cascade controlling malic acid accumulation in apple, providing a mechanistic link between hormonal signaling and post-transcriptional regulation of fruit acidity. This discovery offers new targets for manipulating fruit quality.