Auxin Response Factor 4 Regulates the ABA Catabolism Gene <i>CYP707A2</i> to Reduce the Accumulation of ABA and Inhibit Anthocyanin Synthesis in <i>Malus</i> “<i>Pinkspire</i>” Fruits

Anthocyanins are natural pigments that have attracted significant attention due to their broad-spectrum antioxidant, antimutagenic, and health-promoting properties. During fruit ripening, endogenous auxin levels decrease, while abscisic acid (ABA) and anthocyanin contents increase. However, the interaction between endogenous auxin and ABA and the molecular mechanisms regulating anthocyanin biosynthesis remain unclear. Here, we report that the auxin response factor MpARF4 plays a key role in anthocyanin synthesis in <i>Malus</i> "<i>Pinkspire</i>" peel by regulating the catabolism of ABA. Transient overexpression assays and RT-qPCR demonstrated that <i>MpARF4</i> overexpression significantly reduced ABA content in peel and inhibited anthocyanin synthesis. Virus-induced gene silencing experiments confirmed that silencing <i>MpARF4</i> markedly promoted ABA accumulation and anthocyanin biosynthesis, indicating that <i>MpARF4</i> negatively regulates both ABA and anthocyanin accumulation. The results of yeast single hybridization, electrophoretic mobility shift assays, and dual fluorescein tests showed that MpARF4 activates the expression of the ABA catabolism gene <i>MpCYP707A2</i> to reduce the level of ABA. The decreased level of ABA led to the inhibition of the expression of anthocyanin synthesis regulatory factors <i>MpMYB10</i> and <i>MpDFR</i>, thereby affecting the biosynthesis of anthocyanin. Our research has revealed the mechanism by which auxin response factor regulate anthocyanin synthesis through controlling the ABA catabolism.