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
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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.
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