Integrated Transcriptomic and Metabolomic Analysis Reveals the Metabolic Basis and Regulatory Networks of Triterpenoid Biosynthesis in Ziziphus jujuba Mill. cv. ‘Junzao’ Fruits at Different Harvest Times

Triterpenoids are the primary bioactive constituents responsible for the medicinal efficacy of Ziziphus jujuba Mill. cv. ‘Junzao’ (Junzao) fruits, and there are significant differences in their functional composition depending on the harvest period. However, the metabolic characteristics and dynamic accumulation patterns of triterpenoids during fruit development remain poorly understood. This study represented the first systematic integration of transcriptomic and metabolomic analysis combined with weighted gene co-expression network analysis (WGCNA) to elucidate the biosynthetic pathways of triterpenoids during the harvest times of Junzao fruits, and provide a scientific basis for the future development of functional foods. Total triterpenoid content exhibited a stage-specific accumulation pattern, peaking at the YG (young fruit) stage of fruit development, declining sharply by 37% at the PD (expansion) stage, rebounding at the BS (white-ripe) stage, and gradually decreasing through the CS (crispy-ripe) stage and WS (full-ripe) stages. A total of 347 terpenoid differentially accumulated metabolites (DAMs) and 18,925 differentially expressed genes (DEGs) were identified, among which 224 triterpenoids were predominant. A total of 347 terpenoid differentially accumulated metabolites (DAMs) and 18,925 differentially expressed genes (DEGs) were identified, among which 224 triterpenoids were predominant. WGCNA identified six key modules (salmon, midnightblue, black, blue, yellow, and brown modules) strongly correlated with the accumulation of methyl oleanolate, 3-oxopomolic acid, hederagenin, and other triterpenoids. Furthermore, integrated correlation analysis revealed that cytochrome P450 family genes, particularly CYP716, CYP72A and CYP88, were likely the hub genes governing triterpenoid biosynthesis and RT-qPCR validation of eight key genes confirmed the transcriptome expression trends. These findings provide a comprehensive framework for understanding triterpenoid biosynthesis and offer theoretical foundations for optimizing harvest timing and advancing metabolic engineering in Junzao fruits.