Comparative Analysis of Ca2+/Cation Antiporter Gene Family in Rosa roxburghii and Enhanced Calcium Stress Tolerance via Heterologous Expression of RrCAX1a in Tobacco

<i>Rosa roxburghii</i>, a calciphilic species native to the mountainous regions of Southwest China, is renowned for its high vitamin C and bioactive components, making it valuable for culinary and medicinal uses. This species exhibits remarkable tolerance to the high-calcium conditions typical of karst terrains. However, the underlying mechanisms of this calcium resilience remain unclear. The Ca²⁺/cation antiporter (CaCA) superfamily plays a vital role in the transport of Ca²⁺ and other cations and is crucial for plant tolerance to metal stress. However, the roles and evolutionary significance of the CaCA superfamily members in <i>R. roxburghii</i> remain poorly understood. This study identified 22 CaCA superfamily genes in <i>R. roxburghii</i>, categorized into four subfamilies. The gene structures of these <i>RrCaCAs</i> show considerable conservation across related species. Selection pressure analysis revealed that all <i>RrCaCAs</i> are subject to purifying selection. The promoter regions of these genes contain numerous hormone-responsive and stress-related elements. qRT-PCR analyses demonstrated that H⁺/cation exchanger (CAX) <i>RrCAX1a</i> and <i>RrCAX3a</i> were highly responsive to Ca²⁺ stress, cation/Ca²⁺ exchanger (CCX) <i>RrCCX4</i> to Mg²⁺ stress, and <i>RrCCX11a</i> to Na⁺ stress. Subcellular localization indicated that <i>RrCAX1a</i> is localized to the plant cell membrane, and its stable transformation in tobacco confirmed its ability to confer enhanced resistance to heavy Ca²⁺ stresses, highlighting its crucial role in the high-calcium tolerance mechanisms of <i>R. roxburghii</i>. This research establishes a foundation for further molecular-level functional analyses of the adaptation mechanisms of <i>R. roxburghii</i> to high-calcium environments.