Steroidal saponins are an important type of plant-specific metabolite that are essential for plants’ responses to biotic and abiotic stresses. Because of their extensive pharmacological activities, steroidal saponins are also important industrial raw materials for the production of steroidal drugs. In recent years, more and more studies have explored the biosynthesis of steroidal saponins in plants, but most of them only focused on the biosynthesis of their molecular skeleton, diosgenin, and their subsequent glycosylation modification mechanism needs to be further studied. In addition, the biosynthetic regulation mechanism of steroidal saponins, their distribution pattern, and their molecular evolution in plants remain unclear. In this review, we summarized and discussed recent studies on the biosynthesis, molecular regulation, and function of steroidal saponins. Finally, we also reviewed the distribution and molecular evolution of steroidal saponins in plants. The elucidation of the biosynthesis, regulation, and molecular evolutionary mechanisms of steroidal saponins is crucial to provide new insights and references for studying their distribution, diversity, and evolutionary history in plants. Furthermore, a deeper understanding of steroidal saponin biosynthesis will contribute to their industrial production and pharmacological applications.

Diosgenin, a crucial precursor for steroidal drug production, has poorly understood regulatory pathways. Diosgenin is the primary active component of Dioscorea zingiberensis. Notably, D. zingiberensis also possesses the highest diosgenin content among Dioscorea species, reaching up to 16.15% of dry weight. This study identified DZHDZ32 as a potential regulator of diosgenin biosynthesis in D. zingiberensis through transient overexpression. To validate its function, we developed an optimized genetic transformation method for D. zingiberensis and generated two DZHDZ32-overexpressing lines. The DZHDZ32 transcription factor belongs to the HD-ZIP I subfamily and is localized to the nucleus. Notably, overexpression of DZHDZ32 resulted in a significant increase in its transcript levels in leaves (264.59- and 666.93-fold), leading to elevated levels of diosgenin and its biosynthetic intermediates, including cholesterol and β-sitosterol. Specifically, diosgenin content increased by 41.68% and 68.07%, cholesterol by 10.29% and 16.03%, and β-sitosterol by 12.33% and 19.49% in leaves compared to wild-type plants. Yeast one-hybrid and dual-luciferase assays demonstrated that DZHDZ32 directly binds to the promoters of ACAT and GPPS1, consistent with the significant upregulation of ACAT and GPPS1 expression (3.69- and 4.87-fold and 4.75- and 6.53-fold, respectively) in the overexpressing lines. This study established an optimized genetic transformation method for D. zingiberensis and identified DZHDZ32 as a key regulator of diosgenin biosynthesis. The discovery of DZHDZ32 has significant implications for enhancing diosgenin production and advancing steroidal drug development.