Integrated Metabolomic and Transcriptomic Analyses Reveal the Coumarin Biosynthesis Pathway and Key Regulatory Genes...

Coumarins in the pericarp of Zanthoxylum contribute to the characteristic numbing-aromatic flavor and are associated with diverse bioactivities. To characterize coumarin divergence between two Zanthoxylum materials, mature pericarps of Dahongpao Z. bungeanum (red Sichuan pepper) and Z. planispinum var. dingtanensis (green Sichuan pepper) were analyzed by widely targeted UPLC-ESI-MS/MS metabolomics integrated with transcriptome sequencing. This approach enabled joint profiling of metabolites and transcripts to identify genes associated with material-specific coumarin accumulation. Across the two materials, 583 metabolites were detected, with flavonoids, phenolic acids, and alkaloids as the predominant classes. Among these, 24 coumarins were identified, and most showed significantly higher abundance in green Sichuan pepper than in red Sichuan pepper. Pathway enrichment analysis indicated that differentially accumulated coumarins were mainly associated with the phenylpropanoid biosynthesis pathway, consistent with coordinated metabolic and transcriptional regulation. The integration of metabolite abundance with gene expression patterns identified 56 candidate genes strongly correlated with scopoletin and scopolin accumulation. To evaluate functional relevance, CCoAOMT, COMT, and F6H were cloned and transiently overexpressed in Nicotiana benthamiana. Transient expression assays showed that overexpression of each gene increased scopoletin and scopolin, supporting their involvement in coumarin biosynthesis. Collectively, these results clarify molecular determinants of coumarin variation between the two materials and highlight candidate genes for quality improvement and metabolic engineering.