Integrated metabolome and transcriptome analysis reveals volatile aroma compounds and their regulatory mechanisms in chili pepper fruits

Aroma is a key determinant of flavor quality and consumer preference in chili peppers, mainly derived from volatile organic compounds (VOCs). Understanding the chemical and genetic basis of the unique aroma in geographical indication (GI) varieties is crucial for quality control and breeding, but remains poorly characterized. In this study, we employed an integrated metabolomic-transcriptomic approach to investigate aroma dynamics in C. annuum 'Sichuan Erjingtiao' chili peppers across four stages: mature green (MG), breaker (Br), mature red (MR), and dried mature red (DMR). Through headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS), we identified 602 VOCs. Terpenoids were predominant during the MG stage, while the contents of aldehydes and esters increased significantly during the MR and DMR stages, respectively. Relative odor activity value (rOAV) analysis identified 96 key aroma compounds. Specifically, α/β-ionone was the marker for the MG stage and (E,E)-2,4-hexadienal was characteristic of the MR and DMR stages. Transcriptomic sequencing revealed 8,830 differentially expressed genes (DEGs), and co-expression network analysis identified a WRKY/bZIP-FAD/LOX regulatory module regulating aroma formation in 'Sichuan Erjingtiao' chili peppers. Expression of the FAD gene CA07g02040 and the LOX gene CA08g12080 was strongly correlated with (E,E)-2,4-hexadienal accumulation. Cis -acting element analysis indicated that CA07g02040 and CA08g12080 are potentially regulated by WRKY and bZIP transcription factors via conserved W-box/G-box/TGACG motifs, thereby modulating (E,E)-2,4-hexadienal biosynthesis. This study comprehensively analyzed aroma metabolism in 'Sichuan Erjingtiao' peppers and identified key regulatory genes, laying the groundwork for flavor-oriented genetic enhancement and molecular breeding in chili peppers.