The DNA Demethylase CaDML2 Functions as a Transcriptional Activator in the Control of Pepper Fruit Ripening

Fruit ripening is controlled by intertwined hormonal, transcriptional, and epigenetic networks. Although DNA methylation dynamics modulate ripening in several species, their regulatory effects on non-climacteric pepper (Capsicum annuum) fruit ripening and carotenoid-based secondary metabolism remain poorly understood. Here, pepper fruit ripening was accompanied by global CHH hypermethylation and non‑CHH (CG/CHG) hypomethylation. CHH methylation was associated with the activation of a non‑canonical, RDR6/DCL2‑dependent RNA-directed DNA methylation (RdDM) pathway, whereas non‑CHH demethylation was correlated with the strong activation of the DNA demethylase DEMETER-like 2 (CaDML2). CaDML2 not only functions as a DNA demethylase but also directly activates the transcription of downstream target genes, including RIN, DIVARICATA1, capsanthin/capsorubin synthase (CCS), and the abscisic acid (ABA) biosynthesis-related gene NCED1. Moreover, CaDML2-induced demethylation of cytosines within the promoter regions of these target genes leads to increased transcription, thereby promoting ABA accumulation and enhancing carotenoid synthesis. Silencing of CaDML2 delayed pepper fruit ripening, whereas its heterologous expression restored ripening in tomato sldml2 mutants. These results reveal a dual epigenetic module in which CaDML2‑mediated DNA demethylation and transcriptional activation refine programmes for ABA production and carotenoid metabolism, clarifying the epigenetic basis of non‑climacteric fruit ripening and identifying CaDML2‑centred pathways as targets for manipulating pepper fruit ripening and quality traits.