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Abstract Multiple Arabidopsis H+/Cation exchangers (CAXs) participate in high‐capacity transport into the vacuole. Previous studies have analysed single and double mutants that marginally reduced transport; however, assessing phenotypes caused by transport loss has proven enigmatic. Here, we generated quadruple mutants (cax1‐4: qKO) that exhibited growth inhibition, an 85% reduction in tonoplast‐localised H+/Ca transport, and enhanced tolerance to anoxic conditions compared to CAX1 mutants. Leveraging inductively coupled plasma mass spectrometry (ICP‐MS) and synchrotron X‐ray fluorescence (SXRF), we demonstrate CAX transporters work together to regulate leaf elemental content: ICP‐MS analysis showed that the elemental concentrations in leaves strongly correlated with the number of CAX mutations; SXRF imaging showed changes in element partitioning not present in single CAX mutants and qKO had a 40% reduction in calcium (Ca) abundance. Reduced endogenous Ca may promote anoxia tolerance; wild‐type plants grown in Ca‐limited conditions were anoxia tolerant. Sequential reduction of CAXs increased mRNA expression and protein abundance changes associated with reactive oxygen species and stress signalling pathways. Multiple CAXs participate in postanoxia recovery as their concerted removal heightened changes in postanoxia Ca signalling. This work showcases the integrated and diverse function of H+/Cation transporters and demonstrates the ability to improve anoxia tolerance through diminishing endogenous Ca levels.
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A dominant‐negative Arabidopsis cation exchanger 1 ( CAX1 ): N‐terminal autoinhibition and membrane topology
SUMMARY 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 ofcax1. 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.
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- Award ID(s):
- 2042513
- PAR ID:
- 10535494
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- The Plant Journal
- Volume:
- 119
- Issue:
- 6
- ISSN:
- 0960-7412
- Format(s):
- Medium: X Size: p. 2982-2999
- Size(s):
- p. 2982-2999
- Sponsoring Org:
- National Science Foundation
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