Integrated analysis of metabolomics and transcriptomics reveals potential transcription factors regulating anthocyanin synthesis in pomegranate fruit

• 3 cyanidins, 3 delphinidin, 2 pelargonidin and 2 procyanidins predominate. • The expression of F3H, F3’H, DFR, DFR-like, ANS is higher in ‘Acid’ pomegranate. • TFs such as AP2/ERF, WRKY and MYB show differences among different varieties. • WGCNA identified 19/14 TFs respectively linked to peel/aril anthocyanin. • PgMYB61-like and PgMYB4-like positively regulate anthocyanin accumulation. Although pomegranate fruit is rich in anthocyanins, the profiles of specific anthocyanins and their regulatory transcription factors (TFs) in the peel and aril tissues remain incompletely characterized. In this study, We identified 103 anthocyanidins across three pomegranate cultivars. Notably, Cyanidin-3,5-O-diglucoside, Cyanidin-3-O-glucoside, and Cyanidin-3-gentiobioside were abundant in both peels and arils of ‘Six-month red’ and ‘Acid’, whereas Delphinidin-3,5-O-diglucoside, Delphinidin-3-O-galactoside, and Delphinidin-3-O-glucoside exhibited preferential accumulation in the aril of these cultivars. Pelargonidin derivatives showed cultivar-specific distribution: Pelargonidin-3,5-O-diglucoside was most abundant in the peel of ‘Acid’, whereas Pelargonidin-3-O-glucoside preferentially accumulated in the peel of both ‘Six-month red’ and ‘Acid’. Procyanidin B1 and B3 showed pronounced peel-specific accumulation in all cultivars examined. Transcriptome analysis revealed that differentially expressed genes (DEGs) were significantly enriched in the phenylpropanoid biosynthesis (ko00940) and flavonoid biosynthesis (ko00941) pathways. Further sub-pathway analysis identified 36 structural genes involved in anthocyanin biosynthesis ( F3H, F3’H, DFR, ANS, ANR ) showing elevated expression in the arils of the ‘Six-month red’ and ‘Acid’. Weighted gene co-expression network analysis (WGCNA) identified that 19 TFs (predominantly AP2/ERF, WRKY and MYB) and 14 TFs (mainly AP2/ERF, NAC and bZIP) closely associated with the anthocyanin accumulation in the peel and aril, respectively. In addition, suppression of the PgMYB61-lik e and PgMYB4-like expression promoted anthocyanin accumulation in the peel and aril, respectively. This study characterizes the distinct anthocyanin profiles in the peel and aril of three pomegranate cultivars and identifies potential TFs influencing anthocyanin biosynthesis through integrated transcriptomic and metabolomic analyses. These findings provide new targets for future studies on pomegranate anthocyanin regulation.