An ABA-MabZIP60 Module Links DNA Methylation to Anthocyanin Biosynthesis during Mulberry Fruit Ripening

Abstract Mulberry fruit ripening involves complex regulatory mechanisms influenced by epigenetic modifications, yet the role of DNA methylation in this process remains poorly understood. Through integrated transcriptome and methylome analysis of mulberry fruits at different developmental stages, 9528 differentially expressed genes (DEGs) and 1797 differentially methylated genes (DMGs) were identified. Among these, 621 genes exhibited concurrent changes in both methylation and expression levels throughout fruit ripening. Notably, a prevailing genome-wide hypomethylation trend was observed. Intriguingly, the MabZIP60 gene showed increased expression accompanied by promoter hypermethylation during ripening, and this response was further enhanced by abscisic acid (ABA). MabZIP60 interacts with two unfolded protein response (UPR) components: luminal-binding protein 5 (BiP5) and subtilisin-like protease SBT1.4. ABA triggers proteolytic cleavage, resulting in the release of MabZIP60 from the membrane-bound complex and its subsequent translocation into the nucleus. Within the nucleus, MabZIP60 directly binds to the promoters of CHS, ANS, and LAR, activating their transcription to enhance anthocyanin production, and ABA treatment further enhances the MabZIP60-mediated transcriptional activation. Our findings reveal a novel regulatory module in which ABA recruits DNA methylation as an activating switch to control a central transcription factor network, thereby integrating hormonal signaling, epigenetic modification, and metabolic regulation to orchestrate ripening in mulberry and other horticultural crops.