Expression and regulation of fruit colour in pepper by CaNAC78-like

• CaNAC78-like was identified as a nucleus-localized NAC transcription factor highly expressed in pepper fruits and flowers. • Silencing of CaNAC78-like delayed fruit coloring and reduced carotenoid content and related gene expression. • Overexpression of CaNAC78-like accelerated fruit ripening and enhanced carotenoid accumulation. • CaNAC78-like directly binds to the promoter of CaPSY1 , a key gene in carotenoid biosynthesis. • This study reveals a novel regulatory mechanism of fruit coloration mediated by NAC transcription factors in pepper. Capsanthin, as the most important component of carotenoids, is uniquely present in pepper fruit, and NAC transcription factors play important roles in carotenoids metabolism, but there was little study about the relationship between NAC and pepper fruit color. This study investigates the role of the CaNAC78-like (NM_001324670.1) in fruit color of pigment pepper, which can provide theoretical guidance for the breeding of higher-color-value pepper varieties. We analysed its sequence and expression pattern, and investigated its function in pepper fruit via VIGS silencing and overexpression techniques. Direct downstream target genes were identified using yeast one-hybrid and dual luciferase reporter assays, while carotenoid composition was analysed by high-performance liquid chromatography. Results indicate that CaNAC78-like localises to the cell nucleus. Silencing CaNAC78-like delayed fruit colouration, reduced total carotenoid content, and downregulated carotenoid synthesis genes (CaPSY1, CaLCYB, CaZEP, and CaCCS). Conversely, its overexpression accelerated ripening and enhanced carotenoid deposition. Yeast one-hybrid and dual luciferase assays confirmed that CaNAC78-like directly binds to and activates the promoter of the carotenoid biosynthesis gene CaPSY1 (NM_001324967.1). LC-MS analysis further demonstrated that CaNAC78-like significantly promotes the accumulation of key carotenoids. Collectively, CaNAC78-like functions as a transcriptional activator of pepper fruit reddening by promoting fruit ripening and carotenoid accumulation.