## 2 Result
### qRT-PCR Analysis of KrCaCAs Response to Heavy Metal Stress  
To further investigate the expression patterns of the _RrCaCA_ genes in _R. roxburghii_ under various heavy metal stresses, qRT-PCR analyses were conducted. The results indicated that _RrCAX1a_ and _RrCAX1b_ genes exhibited significant upregulation under Ca\({}^{2+}\) treatment, with _RrCAX1a_ reaching 14 times the control level and _CAX1b_ about 8 times. Both genes showed reduced expression under Na\({}^{+}\), Mg\({}^{2+}\), and Mn\({}^{2+}\) treatments. Similarly, _CAX3a_ displayed a notable increase in expression under the Ca\({}^{2+}\) treatment, approximately 8 times the control level, but decreased significantly under the Mn\({}^{2+}\) treatment. _RrCCX4_ and _RrCCX1 Ia_ also demonstrated the highest expressions under the Mg\({}^{2+}\) and Ca\({}^{2+}\) treatments, respectively, with _RrCCX1 Ia_ exceeding 20 times the control level. _RrMHX1_ and _RrMHX2_ showed the highest expressions under the Ca\({}^{2+}\) treatment. In contrast, _RrNCL1_ and _RrNCL2_ exhibited minimal expression changes across all conditions (Figure 7). These findings underscore the significant role of the _RrCaCA_ gene family in responding to calcium stress, highlighting their potential importance in heavy metal stress adaptation. Figure 6: Heatmap showing predicted cis-acting elements in promoter regions of _RrCaCA_. The bar chart on the left visualizes promoter elements, with colors representing functional categories as indicated in the legend. The heatmap on the right shows the frequency of specific cis-regulatory motifs in the promoters of the analyzed genes, with numbers indicating motif occurrences. Subcellular Localization of _RrcAX1a_ and Its Heterologous Expression Enhanced Tobacco Resistance to Calcium Stress  
To further verify the function of _RrcAX1a_, known for its high responsiveness to Ca\({}^{2+}\) stress, we analyzed its subcellular localization. The coding sequence of _RrcAX1_ was fused with green fluorescent protein (GFP). Transient expression in tobacco leaves revealed distinct green fluorescence on the cell membrane, confirming that _RrcAX1a_ is localized to the cell membrane (Figure 8a). In _A. thaliana_, membrane-localized transporters such as _AtCAX1/3_ are phosphorylated and activated by Ca\({}^{2+}\) sensor kinase modules to facilitate Ca\({}^{2+}\) efflux and maintain cytoplasmic homeostasis [31]. The localization of _RrcAX1a_ to the  
Figure 7: Expression profiles of _RrcACA_ under metal stress conditions. The qRT-PCR results normalized with the 2\({}^{-\Delta\Delta\text{CT}}\) Ct method using GADPH as an internal reference. The experiment was performed three times with three biological replicates. Error bars represent mean \(\pm\) SD. CK refers to the untreated _R. rxuburghii_, while the other treatments include _R. rxuburghii_ subjected to 200 mM NaCl, 50 mM MgCl\({}_{2}\), 50 mM MnCl\({}_{2}\), and 100 mM CaCl\({}_{2}\). Different lowercase letters indicate significant differences, as determined by one-way ANOVA followed by [PERSON]’s post hoc test (\(p<0.05\)). Groups with different letters are significantly different from each other, with the letter assignments based on the results of pairwise comparisons. cell membrane suggests its potential involvement in similar calcium transport mechanisms under Ca\({}^{2+}\) stress conditions. Figure 8: Subcellular localization of \(RrCAX1a\) and its role in enhancing calcium stress resistance in tobacco. (**a**) Subcellular localization of \(RrCAX1a\) showing its membrane-bound localization; CK represents control group transformed with empty pSuper1300-GFP vector. RrCAX1a-GFP indicates experimental group transformed with RrCAX1a-GFP fusion protein, demonstrating clear GFP signals localized to plasma membrane. GFP signals (**left**), bright-field images (**middle**), and merged images (**right**) are shown. Scale bar = 30 μm. (**b**) Overexpression of \(RrCAX1a\) in tobacco enhances resistance to calcium stress; CK represents transgenic tobacco plants transformed with pBI121 empty vector.