TfWRKY40 positively regulates diosgenin biosynthesis in Trigonella foenum-graecum L.

Diosgenin is a bioactive steroidal natural product extraced from plants and serves as an important precursor for the industrial production of steroidal hormone drugs. Despite its pharmacological significance, the biosynthetic and regulatory mechanisms underlying diosgenin production in the medicinal plant <i>T. foenum-graecum</i> remain poorly understood. In this study, we identified <i>Tf</i>WRKY40, a WRKY transcription factor from <i>T. foenum-graecum</i>, whose expression strongly correlates with diosgenin accumulation. Using RNA interference and overexpression strategies combined with transcriptomic analysis and targeted metabolite quantification, we demonstrated that silencing of <i>TfWRKY40</i> led to a 67.60% reduction in diosgenin content, which was accompanied by downregulation of key biosynthetic genes or transcript variants including <i>ACAT1</i>, <i>HMGR1</i>, <i>PMK1</i>, <i>MVD</i>, <i>FPS</i>, <i>SQE2</i>, <i>CAS1</i>, <i>SMO3-1</i>, <i>SMO3-2</i>, <i>8,7-SI</i>, <i>SMO4-3</i>, <i>CYP90B50</i>, and <i>CYP82J17</i> in the transgenic hairy roots. Conversely, overexpression of <i>TfWRKY40</i> resulted in a 59.25% increase in diosgenin levels, along with upregulation of these biosynthetic genes or transcript variants. Taken together, these findings suggest that <i>TfWRKY40</i> acts as a positive regulator of diosgenin biosynthesis in <i>T. foenum-graecum</i>, likely by activating the transcription of critical pathway genes, particularly <i>CAS1</i>, <i>HMGR1</i>, and <i>CYP90B50</i>. This work highlights <i>TfWRKY40</i> as a promising target for metabolic engineering strategies aimed at enhancing diosgenin production and facilitating the development of diosgenin-derived steroidal therapeutics.