An HD-ZIP I Transcription Factor DZHDZ32 Upregulates Diosgenin Biosynthesis in .

Diosgenin, a crucial precursor for steroidal drug production, has poorly understood regulatory pathways. Diosgenin is the primary active component of . Notably, also possesses the highest diosgenin content among species, reaching up to 16.15% of dry weight. This study identified DZHDZ32 as a potential regulator of diosgenin biosynthesis in through transient overexpression. To validate its function, we developed an optimized genetic transformation method for and generated two -overexpressing lines. The DZHDZ32 transcription factor belongs to the HD-ZIP I subfamily and is localized to the nucleus. Notably, overexpression of 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 and , consistent with the significant upregulation of and 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 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.