Phenotypic and RNA-seq Profiles Identified Key Pathways and Genes Involved in Gleditsioside Biosynthesis in Gleditsia sinensis Lam.

Gleditsia sinensis Lam. (G. sinensis) is a widely known medicinal plant, and its primary bioactive compound is gleditsioside. So far, the significant economic and medicinal value of gleditsioside has been widely recognized. However, the transcriptional regulation governing the biosynthesis of gleditsioside during G. sinensis pod development remains unclear. In this investigation, we observed that gleditsioside levels increased in the pods of G. sinensis from June to November, and we performed a transcriptome analysis to explore the phenomenon. A total of 703 and 162 differentially expressed unigenes (DEGs) were identified in the terpenoid backbone and triterpenoid biosynthesis pathways, respectively. In total, 99 unigenes encoding 17 enzymes, such as ENIN, cytochrome P450 (CYP93E1), and UDP-glucosyltransferase, were identified in the gleditsioside biosynthesis pathway. Moreover, DEGs encoding crucial enzymes, such as HMGCR and AGBH, might determine gleditsioside synthesis during G. sinensis pod development. Interestingly, the gleditsioside synthesis pathway extended to ten metabolic pathways, including the sterol biosynthesis pathway and the brassinolide biosynthesis pathway, among other pathways involved in various hormonal regulations. These pathways shared the same precursor substances (IPP and DMAPP). In addition, weighted gene correlation network analysis (WGCNA) revealed that CL5845.Contig1 (HMGCR) and CL8823.Contig2 (LUP4) might be involved in the gleditsioside biosynthesis. Furthermore, transient transformation validation experiments demonstrated overexpression of CL5845.Contig1 (HMGCR), CL8823.Contig2 (LUP4), and CL11248.Contig4 (CYP93E1) significantly enhanced gleditsioside biosynthesis. Overall, our findings provide important genetic resources for future functional research and new insights into the basic mechanism of saponin biosynthesis.