## 2 Results
### Subcellular Localization of DZHDZ32  
DZHDZ32 encodes a transcription factor that is typically localized in the nucleus [34; 46]. To verify the subcellular localization of DZHDZ32, its amino acid sequence was analyzed using three different online prediction tools, all of which consistently predicted nuclear localization (Supplementary Materials Table S2). To experimentally confirm this prediction in _D. zingiherensis_, the _DZHDZ32_ coding sequence was fused to EGFP (enhanced green fluorescent protein) and transiently expressed in young leaves. The empty vector PQG110-EGFP served as a control (Figure 5a). Figure 5: Subcellular localization of the DZHDZ32 protein in mesophyll cells of _D. zingiherensis_. (**a**) Confocal microscopy images of leaf mesophyll cells at 48 hpi with _Agrobacteriumium_ carrying the empty vector PQG110 (355: EGFP); this sample serves as the negative control. (**b**) Leaves expressing the DZHDZ32-EGFP fusion protein are shown at 30 hpi, (**c**) at 33 hpi, (**d**) at 36 hpi, and (**e**) at 48 hpi. The red arrow indicates the fluorescence signal of DZHDZ32-EGFP. Three biological replicates were performed in this study. DAPI staining was used to mark nuclear localization. Bar = 5 μm  
Confocal microscopy analysis confirmed that DZHDZ32 is localized in the nucleus (Figure 5b-d). Notably, DZHDZ32 exhibited an early expression pattern, with strong nuclear fluorescence observed as early as 30 hpi (hours post-infiltration) (Figure 5b). Fluorescence intensity peaked at 33 hpi (Figure 5c), then gradually declined, with a noticeable reduction by 36 hpi (Figure 5d). By 48 hpi, the nuclear fluorescence signal of the DZHDZ32-EGFP fusion protein was markedly weaker compared to the control (Figure 5e). Correlation Analysis Expression of DZHDZ32 with Genes Involved in the Diosgenin Biosynthetic Pathway  
Given that diosgenin is predominantly synthesized in leaves and that _DZHDZ32_ exhibits its highest expression in leaf tissues [1], along with the observation that diosgenin content is elevated to varying degrees in both _D. zingiberensis_ overexpression lines, these findings suggest a potential involvement of _DZHDZ32_ in the diosgenin biosynthetic pathway. To identify specific points of interaction, we analyzed the correlation between _DZHDZ32_ expression and genes involved in diosgenin biosynthesis using transcriptome data (accession number CRA007729). Under drought stress conditions, the expression of _DZHDZ32_ exhibited strong positive correlations (Spearman correlation coefficient \(\geq\) 0.8) with the expression levels of several key biosynthetic genes, including _ACAT_ (_acetyl-CoA acetyltransferase gene_), _HMGR_ (_3-hydroxy-3-methylglutaryl-CoA reductase gene_), _MVD_ (_diphosphomevolonate decarboxylase gene_), _CMK_ (_4-diphosphocytidyl-2-C-methyl-D-erythnit kinase gene_), _CAS_ (_cycloartenol synthase gene_), _SMT1_ (_steroid C-24 methyltransferase gene_), and cytochrome P450 genes _CYP94D14_ and _CYP94A11_. Similarly, diosgenin content was strongly correlated with _GPPS1_ (_geranyl-geranyl pyrophosphate synthase gene_), _CAS_, _SMT2_, and _CYP51_ (_steroid C-14 demethylase gene_) (Figure 6a). Following MeJA treatment, the expression of _DZHDZ32_ also displayed strong positive correlations with the expression levels of _ACAT_, _PMK_ (_phosphomevolonate kinase gene_), _MVD_, _DXS_ (_1-deoxy-D-xylulose-5-phosphate synthase 1 gene_), _CMK_, _SMT1_, _CYP72A12-4_, and _CYP94A11_ (Figure 6b). Notably, the correlation coefficient between _DZHDZ32_ expression and diosgenin content under MeJA treatment was 1.0, indicating a perfect positive correlation. To determine whether these strongly correlated genes serve as functional links between _DZHDZ32_ and the diosgenin biosynthetic pathway, we examined their expression levels in _DZHDZ32_OES lines compared with WT plants. As shown in Figure 6c, several genes were significantly upregulated or downregulated in the transgenic lines.