Stage-Specific Metabolic and Proteomic Dynamics Underlying Ripening and Quality Formation in Tropical Evergreen Pomegranate

Abstract

Pomegranate fruit development involves coordinated physiological, metabolic, and proteomic changes shaping coloration and quality. Fruit expansion and peel/aril pigmentation were most active between 90 and 130 days, when soluble solids increased and organic acids fluctuated, producing the highest sugar-acid ratio. Widely targeted metabolomics detected 1,815 metabolites, including 945 differentially accumulated metabolites (DAMs; 90d_vs_15d) and 170 DAMs (130d_vs_90d), mainly phenolic acids, flavonoids, terpenoids, and alkaloids. Early DAMs were enriched in nucleotide and phenylalanine metabolism, whereas late DAMs were linked to anthocyanin biosynthesis and hormone signaling. Cyanidin- and pelargonidin-derived flavonoids paralleled fruit coloration. Proteomics quantified 9,585 proteins, identifying 4,031 early and 329 late differentially abundant proteins (DAPs), with dynamic regulation of phenylpropanoid, flavonoid, carbohydrate metabolism, glycolysis, gluconeogenesis, and TCA pathways. WGCNA revealed stage-specific modules associated with phenylpropanoid biosynthesis, carbohydrate metabolism, and amino acid pathways. These integrated results provide a temporal multi-omics framework linking metabolite accumulation, sugar-acid ratio, and pathway regulation to pomegranate ripening and quality formation, highlighting how stage-specific metabolic and proteomic shifts underpin the development of color, flavor, and overall fruit quality during fruit maturation.