Abstract
To investigate the molecular mechanisms underlying ascorbic acid (AsA) accumulation in pepper fruits and to identify key genes involved in its biosynthesis, we performed integrated metabolomic and transcriptomic analyses on two pepper cultivars-T41 (sweet pepper) and 22-5 (chili pepper)-at three developmental stages, including young fruit, green ripe, and color change stages. The results show that AsA content in both cultivars was significantly higher at the green ripe stage than the young fruit stage, with T41 exhibiting significantly higher AsA levels than 22-5 at both the young fruit and green ripe stages. Transcriptomic analysis identified a total of 24,433 differentially expressed genes (DEGs). KEGG pathway enrichment analysis revealed that genes associated with AsA biosynthesis were mainly enriched in the "ascorbate and aldarate metabolism" pathway. Follow-up validation confirmed APX3 as the most likely candidate gene responsible for the difference in AsA content between the two pepper fruit types, with its expression pattern negatively correlating with AsA accumulation. This study unveils the molecular regulatory mechanisms underlying AsA biosynthesis and provides a theoretical foundation for breeding pepper cultivars with elevated AsA levels. This study provides valuable insights into the molecular regulation of AsA biosynthesis and lays a theoretical foundation for breeding high-AsA pepper varieties.