Origin and evolution of steroidal saponin biosynthesis and its regulation by the DcWRKY33-DcWRKY11 module in Dioscorea composita

Abstract

Although steroidal saponins from Dioscorea species have substantial economic value and versatile utilization potential, the evolutionary origin and regulatory mechanisms underlying their biosynthesis remain poorly understood. Here, we conducted a comparative genomic analysis across representative plant lineages ranging from algae to angiosperms to examine the copy number variation and syntenic organization of steroidal saponin biosynthetic genes. Our results reveal that ancestral sterol biosynthetic components were already present in bryophytes. However, synteny analysis indicates that stable genomic integration of these genes was established no earlier than in pteridophytes. By integrating transcriptome–metabolome association analyses with branch-site selection tests, we identified strong positive selection acting on DcWRKY33 within the Dioscorea lineage, suggesting adaptive evolution and highlighting the importance of regulatory innovation in steroidal saponin diversification. Functional analyses demonstrated that DcWRKY33 functions as a positive regulator of steroidal saponin biosynthesis: transient overexpression upregulates the expression of structural genes and increases saponin accumulation, whereas gene silencing produced opposite effects. Mechanistically, dual-luciferase and yeast one-hybrid assays indicated that DcWRKY33 activates DcHDR and DcCYP51, while indirectly regulating DcHMGR through modulation of DcWRKY11, thereby orchestrating biosynthesis across both the MEP and MVA pathways. Promoter analysis revealed W-box motifs partially absence in non-saponin-producing Dioscorea, and heterologous expression in tobacco confirmed lineage-specific regulatory capacity despite conserved cis-elements. Collectively, our study links pathway evolution with regulatory innovation and establishes a mechanistic framework for understanding the evolution and transcriptional control of steroidal saponin biosynthesis in D. composita, providing insights for metabolic improvement of medicinal and horticultural yam species.