A dominant-negative arabidopsis cation exchanger 1(CAX 1): N-terminal autoinhibition and membrane topology

Authors: SARKAR, SHAYAN - Children'S Nutrition Research Center (CNRC); RHEIN, HORMAT SHADGOU - Children'S Nutrition Research Center (CNRC); PITTMAN, JON - University Of Manchester; HIRSCHI, KENDAL - Children'S Nutrition Research Center (CNRC)

Submitted to: The Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/25/2024
Publication Date: 8/23/2024
Citation: Sarkar, S., Rhein, H., Pittman, J.K., Hirschi, K.D. 2024. A dominant-negative arabidopsis cation exchanger 1(CAX 1): N-terminal autoinhibition and membrane topology. The Plant Journal. 119:2982-2999. https://doi.org/10.1111/tpj.16966.
DOI: https://doi.org/10.1111/tpj.16966

Interpretive Summary: Plants need calcium to grow and survive stress, especially during floods when oxygen is low. In this study, scientists made a modified version of a plant protein that affects how calcium is used inside cells. When added to normal plants, this protein helped them survive low-oxygen stress. The helpful version had to include two key parts of the original protein to work. By using this modified version in crop plants, we can enhance their ability to survive flooding and other challenging conditions, which is crucial for food security.

Technical Abstract: 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.