Integrating metabolomic and transcriptomic analyses to identify candidate genes and transcriptional regulators associated with triterpenoid saponin accumulation in developing Momordica charantia fruits

Abstract

Momordica charantia is an economically and pharmacologically important vegetable, with its medicinal value largely attributed to its diverse triterpenoid saponins. Although the decline of total saponin content during fruit development is well-documented, the precise metabolic profiles and the underlying molecular regulatory mechanisms governing individual saponin accumulation remain poorly understood. We performed integrated metabolomic and transcriptomic analyses on fruits of the Momordica charantia cultivar ‘Ruyu No. 45’ (‘RY45’) at 10 and 15 days post-anthesis (DPA). Metabolites were profiled using ultra-performance liquid chromatography-tandem mass spectrometry, and transcriptomes were analyzed via RNA sequencing. Weighted gene co-expression network analysis and Pearson correlation analysis were applied to identify transcription factors and structural genes (cytochrome P450 monooxygenases and UDP-glycosyltransferases) potentially involved in saponin biosynthesis. We identified 473 terpenoid metabolites, of which 56 triterpenoid saponins were tentatively identified (Metabolomics Standards Initiative Level 2), comprising cucurbitane-type (44.6%), oleanane-type (53.6%), and ursane-type (1.8%). Total saponin content decreased significantly from 10 DPA to 15 DPA, with 27 individual triterpenoid saponins showing significant downregulation. Transcriptome analysis revealed 3,291 differentially expressed genes, including 50 candidate genes potentially involved in triterpenoid saponin biosynthesis. Candidate structural genes ( McCYP72A219 , McCYP86A7 , McCYP704C1 , McUGT73C6 , McUGT84B1 and McβAS ) were markedly downregulated at 15 DPA and positively correlated with multiple saponin contents. Additionally, transcription factors McbHLH93–1 and McNAC90–1 were identified as co-expressed with candidate structural genes. Transient overexpression of McCYP72A219 was associated with increased total saponin content, providing preliminary functional clues for its potential role in saponin accumulation. This study provides a systematic overview of the metabolic decline of triterpenoid saponins in bitter gourd at 15 DPA compared to 10 DPA, identifies a suite of candidate structural genes and transcription factors and constructs a preliminary co-expression regulatory network as an exploratory analysis. These findings offer new insights into the transcriptional regulation of triterpenoid saponin accumulation in bitter gourd and lay a molecular foundation for future metabolic engineering and quality improvement. However, the proposed regulatory relationships are primarily correlative and require further genetic and biochemical validation.