Integrated Transcriptomic and Proteomic Analysis Provides Insights into Sepal Doubling in Ornamental Pomegranate

Sepal doubling in Punica granatum (pomegranate), characterized by calyx splitting and bilayered structure formation, confers distinctive ornamental value but its developmental mechanisms remain poorly understood. Elucidating the molecular mechanisms of sepal doubling and screening for associated genes and proteins in pomegranate are of theoretical and practical significance. Herein, transcriptomic and proteomic analyses were performed on sepals from single, double, and proliferated flowers. Overall, 23,630 genes and 5803 proteins were identified, with 5634 quantified at both levels. Pairwise comparisons revealed stronger correlations between differentially expressed genes (DEGs) and differentially abundant proteins (DAPs) than between the total gene and protein sets. Because SeSi and SeDo/SePr were obtained from different cultivars, SeSi comparisons are partly confounded by genotype, whereas SeDo vs. SePr is the least cultivar-affected comparison. Cell wall-related terms were detected in the transcriptomic, proteomic and integrated datasets, alongside dominant photosynthesis-related terms in the transcriptomic comparisons. Eleven cell wall metabolism-related DEGs and three cell wall metabolism-related DAPs were identified, including ten concordant DEG–DAP pairs. These candidates included both concordant DEG–DAP pairs and those supported at a single omics level. Among them, cellulose synthase A (CESA), extensin-2 (EXT2), endo-1,4-β-D-glucanase (EG), and xyloglucan endotransglucosylase/hydrolase 16 (XTH16) were associated with bilayered calyx formation, whereas pectinesterase (PE), alpha-galactosidase (α-Gal), xylan α-glucuronyltransferase (α-GLU), and polygalacturonase (PG) were associated with calyx splitting. Two protein-modifying enzymes, serine/threonine-protein kinase (STPK) and serine carboxypeptidase (SCP), were associated with calyx splitting. Additionally, two transcription factors, basic helix–loop–helix (bHLH) and cold-shock domain protein (CSD), were associated with sepal doubling. These findings provide a molecular basis for further studies of floral development and type improvement in pomegranate.