Transcriptome and DNA methylome analyses of the effects of postharvest 5-azacytidine treatment on pigment and ascorbic acid metabolism in Rosa roxburghii fruit

DNA methylation is a key epigenetic regulator of gene expression, yet its role in pigment and ascorbic acid (AsA) metabolism in Rosa roxburghii remains unclear. This study examined the effects of 5-azacytidine (5-azaC), a DNA methyltransferase inhibitor, on postharvest fruit quality after cold storage. 5-azaC treatment reduced decay, delayed the onset of color change, preserved overall fruit brightness, maintained firmness, and suppressed respiration during cold storage. It inhibited chlorophyll a degradation and enhanced zeaxanthin, lutein, α-carotene, phytoene, and violaxanthin levels. 5-azaC enhanced AsA content, accompanied by increased activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione reductase (GR), ascorbate oxidase (AO), and ascorbate peroxidase (APX). Integrated RNA-seq and WGBS analysis showed that 5-azaC induced carotenoid biosynthesis genes ( RrBCH, RrVDE, RrNSY, RrDXS and RrZE P), with RrVDE hypomethylated. RrMDHAR, RrDHAR, RrAO1, RrGR, RrGALUR, RrGalDH and RrIMPL2 were upregulated by 5-azaC, with reduced DNA methylation levels observed in RrAO1, RrGalDH and RrIMPL2 . Transcription factors RrAP2, RrMYB21, RrMYB44 and RrPosF21-like were induced by 5-azaC, with RrMYB21 hypomethylated. DNA demethylases ( RrROS1 and RrROS1-like ) were upregulated, while methyltransferases ( RrCMT-like, RrMET, RrMDR2 and RrDMR2-like ) were downregulated. Silencing RrAP2, RrMYB21, RrMYB44, RrPosF21-like and RrROS1-like reduced AsA content and downregulated AsA metabolism-related genes in R. roxburghii fruit . These findings provide preliminary evidence that 5-azaC may regulate pigment and AsA metabolism via epigenetic mechanisms during cold storage.