## 1 Introduction  
_D. zingiberensis_ is a species belonging to the genus _Dioscorea_ (family _Dioscorea_) [1, 2, 3]. Its rhizces are particularly rich in diosgenin, with concentrations reaching up to 16.15% of dry weight [1, 4, 5]. This high diosgenin content makes _D. zingiberensis_ not only a valuable medicinal resource but also a critical raw material for steroidal drug production with significant industrial relevance [6, 7, 8, 9]. In addition to its industrial importance, this species also demonstrates notable medicinal properties [10]. Traditionally, it has been used in the treatment of rheumatism and arthritis and exhibits anti-inflammatory and analgesic effects [1, 11, 10, 12]. Furthermore, it shows potential for managing cardiovascular diseases and regulating endocrine function [3, 10, 11]. Diosgenin is a type of steroidal sapogenin and an aglycone component of steroidal saponins [13]. Steroidal sapogenins are a class of compounds characterized by their steroid nucleus structure. Diosgenin, for instance, is a spirostane-type sapogenin. Diosgenin was first isolated from the tubers of _Dioscorea_tokoro_[14], and in 1943, it was identified as a key precursor in the synthesis of steroidal hormone drugs [15]. Due to its cost-effective biosynthetic pathway, diosgenin remains an ideal starting material for steroid hormone production [16]. Despite its importance, the regulatory mechanisms underlying diosgenin biosynthesis remain largely unexplored. Some studies have shown that low concentrations of copper ions, ethylene, and jasmonic acid can enhance diosgenin accumulation [6; 11; 17; 18; 19]. The transcription factor DCWRKY11, which includes a WD (WRKY domain) in _Dioscorea composita_ and is associated with the MeJA (methyl jasmonate) signaling pathway, has been reported to promote the expression of genes involved in steroidal saponin biosynthesis--the storage form of diosgenin [11]. Other transcription factors, including those from the WRKY, MYB (v-myb avian myeloblastosis viral oncogene homolog), bZIP (basic region-leucine zipper), bHLH (basic helix-loop-helix), NAC (NAM, ATAF, and CUC), HD-ZIP (homeodomain-leucine zipper), and AP2/ERF (APETALA2/ethylene responsive factor) families, may also play roles in diosgenin biosynthesis [8; 11; 20; 21]. However, genetic transformation remains a significant challenge in _D. zingiherensis_, severely limiting molecular studies. Few studies have employed transgenic approaches in _D. zingiherensis_ or closely related species, and investigations into transcriptional regulation remain scarce [22; 23; 24]. HD-ZIP transcription factors are widely distributed in plants and play vital roles in growth, development, and responses to environmental stressors [25; 26; 27; 28]. However, their involvement in plant secondary metabolism, particularly in the biosynthesis of steroidal compounds, is not well understood. For example, in _Artemisia annua_, the HD-ZIP transcription factor AAHD1 regulates artemisinin accumulation [29]; in _Arabidopsis thaliana_, ATHB8 positively regulates brassinosterol biosynthesis by modulating the expression of _CPD_ (_constitutive photomoprogenic dwarf_), a key gene in the brassinosterol pathway [30]. HD-ZIP transcription factors are also implicated in the regulation of steroidal alkaloid biosynthesis in _Veratrum mengtzeanum_[31]. HAT1, an HD-ZIP transcription factor in _A. thaliana_, can suppress the expression of the BR biosynthesis gene _BR6 OX2_ (_brassinosterol-6-oxidase2_), thereby reducing BR content [32]. Currently, there are only speculative studies that lack experimental validation reporting that HD-ZIP transcription factors regulate the biosynthesis of diosgenin. In this study, we use diosgenin as a model compound to investigate the regulatory role of HD-ZIP transcription factors. HD-ZIP proteins are divided into four subfamilies (I-IV) based on DNA-binding specificity [33; 34], sequence similarity, conserved motifs, and physiological functions [35].