A dominant‐negative Arabidopsis cation exchanger 1 (<scp>CAX1</scp>): N‐terminal autoinhibition and membrane topology

Calcium (Ca²⁺) is essential for plant growth and cellular homeostasis, with cation exchangers (CAXs) regulating Ca²⁺ transport into plant vacuoles. In Arabidopsis, multiple CAXs feature a common structural arrangement, comprising an N-terminal autoinhibitory domain followed by two pseudosymmetrical modules. Mutations in CAX1 enhance stress tolerance, notably tolerance to anoxia (a condition marked by oxygen depletion), crucial for flood resilience. Here we engineered a dominant-negative CAX1 variant, named ½N-CAX1, incorporating the autoinhibitory domain and the N-terminal pseudosymmetrical module, which, when expressed in wild-type Arabidopsis plants, phenocopied the anoxia tolerance of cax1. Physiological evaluations, yeast assays, and calcium imaging demonstrated that wild-type plants expressing ½N-CAX1 have phenotypes consistent with inhibition of CAX1, which is likely through direct interaction of ½N-CAX1 with CAX1. Eliminating segments within the N-terminal pseudosymmetrical module, as well as incorporating modules from other plant CAXs and expressing these variants into wild-type plants, failed to produce anoxia tolerance. This underscores the requirement for both the CAX1 autoinhibitory domain and the intact pseudosymmetrical module to produce the dominant-negative phenotype. Our study elucidates the interaction of this ½N-CAX1 variant with CAX1 and its impact on anoxia tolerance, offering insights into further approaches for engineering plant stress tolerance.