## 3 Discussion  
Transcription factors play a crucial role in regulating diosgenin biosynthesis. Previous studies have suggested that members of the WRKY, MYB, bZIP, bHLH, NAC, HD-ZIP, and AP2/ERF families may be involved in this process [8; 11; 20; 21]. In the present study, we identified DZHDZ32 as a key HD-ZIP transcription factor that regulates the biosynthesis and accumulation of diosgenin. Phylogenetic analysis classified DZHDZ32 as a member of the HD-ZIP I subfamily, the largest among the four HD-ZIP subfamilies. HD-ZIP I transcription factors are known to respond to a wide range of abiotic stresses, including drought, salinity, cold, exposure to toxic metals/metalloids, and light [28; 48; 49; 50; 51; 52; 53; 54]. They are also responsive to plant hormones such as ABA (abscisic acid), MeJA, and GA (gibberellin) [28; 55; 56; 57]. In addition to their roles in stress signaling, HD-ZIP I factors influence metabolite accumulation, fruit abscission, and plant morphogenesis and structural development [34; 38; 53; 58]. These diverse functions underscore the significance of HD-ZIP I transcription factors in plant growth, development, and adaptation. Our study demonstrates the potential of DZHDZ32 to enhance diosgenin production, as evidenced by its elevated expression and strong correlation with diosgenin accumulation. This was further validated by the generation of _DZHDZ32_ overexpression transgenic lines. Notably, genetic transformation in _D. zingiberensis_ has historically been challenging due to the lack of a well-established transformation system, which has limited functional genomic studies in this species. The transformation method developed in this study significantly reduces the time required to obtain transgenic lines, enabling the generation of complete transgenic plantlets in approximately four months. Moreover, the improved transformation efficiency facilitates more efficient and comprehensive investigations into gene function in _D. zingiberensis_. The _DZHDZ32_OE lines showed no obvious phenotypic differences compared to WT plants. However, diosgenin content increased by 41.68% and 68.07% in the leaves of the two transgenic lines, respectively. Given that diosgenin is a valuable secondary metabolite and a precursor for various steroidal drugs, this increase is of considerable significance. Concurrently, we observed upregulation of key biosynthetic genes, including _ACAT_, _MVD_, _GPPS1_, and _CYP72A12-4_, suggesting that DZHDZ32 positively regulates diosgenin synthesis through the modulation of these genes. Subcellular localization experiments confirmed that DZHDZ32 is localized to the nucleus. Interestingly, DZHDZ32 protein expression was detectable as early as 33 hpi in _D. zingiberensis_, earlier than the typical 36-48 h observed for other proteins. Since diosgenin biosynthetic genes are nuclear-encoded, the early nuclear localization of DZHDZ32 supports its regulatory role in this pathway. The binding specificity of HD-ZIP I transcription factors can vary across plant species, reflecting the structural diversity of these proteins. The most common binding motif is CAAT(A/T)ATTG, as observed in _A. thaliana_ (e.g., ATHB1). However, other variants have been identified. For example, the HD-ZIP I factor VFHB21 from _Vernicia fordii_ (tung tree) binds to multiple motifs, including CAATAATTG, GAATG, TAATTATA, TAATAATTA, AAAATTATA, and TAATTATG [34]. A general consensus sequence for HD-ZIP I binding is considered to be NATTNATTN, with a central A or T nucleotide and complementary sequences at both ends enhancing binding affinity. These variations reflect the adaptability of HD-ZIP I proteins in recognizing diverse promoter elements and regulating a wide array of biological processes. In our study, Y1H and dual-LUC assays confirmed that DZHDZ32 binds to the upstream promoter regions of diosgenin biosynthetic genes. Furthermore, _DZHDZ32_ overexpression led to the upregulation of these genes in transgenic lines compared to WT plants.