Molecular Mechanism of AtCAX3-Induced BPA Tolerance in Plants

Cation/H⁺ Exchangers (CAXs) are involved in the sequestration of cations such as Ca²⁺, Mn²⁺, Li⁺, Zn²⁺, and Cd²⁺ from the cytosol into the vacuole using proton gradients. Our previous studies demonstrated the involvement of AtCAX3 in Cd²⁺ tolerance. In this study, we investigated whether AtCAX3 is also involved in tolerance to the endocrine disruptor Bisphenol A (BPA). Furthermore, to examine the role of the autoinhibitory domain of AtCAX3 in BPA tolerance, the empty vector (pYES5), AtCAX3, Δ36-AtCAX3 (deletion of the N-terminal regulatory region; NRR), and Δ90-AtCAX3 (deletion of both the NRR and regulatory-dependent region; RDR) were overexpressed in Saccharomyces cerevisiae and the atcax3 knockout mutant of Arabidopsis thaliana. In Arabidopsis, BPA tolerance, Ca²⁺ levels, and antioxidant enzyme activities were highest in Δ90-AtCAX3-overexpressing plants, followed by Δ36-AtCAX3, AtCAX3, Col-0, and atcax3. Conversely, ROS levels followed the opposite pattern. Interestingly, the expression levels of Ca²⁺ importer genes (AtCNGC10 and AtMCA1) were increased, whereas those of Ca²⁺ exporter genes (AtACA10) were decreased in the order of Δ90-AtCAX3 > Δ36-AtCAX3 > AtCAX3 > Col-0 > atcax3. These results suggest that elevated Ca²⁺ levels may contribute to ROS reduction. Taken together, deletion of the autoinhibitory domain of AtCAX3 enhances BPA tolerance by reducing BPA-induced ROS accumulation through increased Ca²⁺ accumulation and activation of antioxidant defense systems. Furthermore, enhanced Ca²⁺ homeostasis may interfere with the positive feedback loop between BPA-induced ROS generation and Ca²⁺ spikes. These findings can be applied to develop BPA phytoremediators and BPA-resilient crops.