CaNAC2 orchestrates Capsicum annuum fruit ripening by coordinated carotenoid and ABA metabolism under epigenetic control.

Fruit ripening in non-climacteric species such as pepper (Capsicum annuum L.) is governed by a complex interplay of hormonal, transcriptional, and epigenetic factors. Here, we identified the NAC transcription factor CaNAC2 as an important regulator orchestrating pepper fruit ripening. CaNAC2 directly activated CaCCS, a key gene in capsanthin and capsorubin biosynthesis, and repressed CaCYP707A2, which encodes an ABA catabolic enzyme. Virus-induced gene silencing and overexpression assays confirmed that CaNAC2 promotes carotenoid accumulation and ABA biosynthesis, thereby accelerating ripening. Notably, CaNAC2 expression was activated by the ABA-responsive transcription factor CaABF3, forming a positive feedback loop (CaNAC2-CaCYP707A2-ABA-CaABF3) that sustained ABA accumulation in late ripening stages. Integrated DAP-seq and RNA-seq analyses revealed that CaNAC2 binds to and modulates the expression of multiple genes associated with carotenoid and ABA pathways. Moreover, DNA methylation levels at the promoters of CaNAC2, CaCYP707A2, and CaABF3 declined prior to ripening initiation, and 5-azacytidine treatment enhanced CaNAC2 expression and promoted ripening. While methylation-sensitive EMSAs suggested that cytosine methylation does not directly block transcription factor binding, elevated ABA levels suppressed DNA methyltransferase (CaCMT2) and induced demethylase (CaDML2) expression, reinforcing DNA hypomethylation. Together, these findings uncover CaNAC2 as a key integrator of hormonal and epigenetic signals and provide mechanistic insight into the coordinated transcriptional network driving pepper fruit ripening.