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1. Proteomic Insights into Trichome Responses to Elevated Elemental Stress in Cation Exchanger (CAX) Mutants

   https://doi.org/10.1093/pcp/pcae097  

   Guo, Qi; Sarkar, Shayan; Punshon, Tracy; Tappero, Ryan; Barkla, Bronwyn_J; Hirschi, Kendal_D (September 2024, Plant And Cell Physiology)

   Abstract Research on elemental distribution in plants is crucial for understanding nutrient uptake, environmental adaptation and optimizing agricultural practices for sustainable food production. Plant trichomes, with their self-contained structures and easy accessibility, offer a robust model system for investigating elemental repartitioning. Transport proteins, such as the four functional cation exchangers (CAXs) in Arabidopsis, are low-affinity, high-capacity transporters primarily located on the vacuole. Mutants in these transporters have been partially characterized, one of the phenotypes of the CAX1 mutant being altered with tolerance to low-oxygen conditions. A simple visual screen demonstrated trichome density and morphology in cax1, and quadruple CAX (cax1-4: qKO) mutants remained unaltered. Here, we used synchrotron X-ray fluorescence (SXRF) to show that trichomes in CAX-deficient lines accumulated high levels of chlorine, potassium, calcium and manganese. Proteomic analysis on isolated Arabidopsis trichomes showed changes in protein abundance in response to changes in element accumulation. The CAX mutants showed an increased abundance of plasma membrane ATPase and vacuolar H-pumping proteins, and proteins associated with water movement and endocytosis, while also showing changes in proteins associated with the regulation of plasmodesmata. These findings advance our understanding of the integration of CAX transport with elemental homeostasis within trichomes and shed light on how plants modulate protein abundance under conditions of altered elemental levels. 

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2. The vacuolar H+/Ca transporter CAX1 participates in submergence and anoxia stress responses

   https://doi.org/10.1093/plphys/kiac375  

   Yang, Jian; Mathew, Iny Elizebeth; Rhein, Hormat; Barker, Richard; Guo, Qi; Brunello, Luca; Loreti, Elena; Barkla, Bronwyn J.; Gilroy, Simon; Perata, Pierdomenico; et al (August 2022, Plant Physiology)

   Abstract A plant’s oxygen supply can vary from normal (normoxia) to total depletion (anoxia). Tolerance to anoxia is relevant to wetland species, rice (Oryza sativa) cultivation, and submergence tolerance of crops. Decoding and transmitting calcium (Ca) signals may be an important component to anoxia tolerance; however, the contribution of intracellular Ca transporters to this process is poorly understood. Four functional cation/proton exchangers (CAX1–4) in Arabidopsis (Arabidopsis thaliana) help regulate Ca homeostasis around the vacuole. Our results demonstrate that cax1 mutants are more tolerant to both anoxic conditions and submergence. Using phenotypic measurements, RNA-sequencing, and proteomic approaches, we identified cax1-mediated anoxia changes that phenocopy changes present in anoxia-tolerant crops: altered metabolic processes, diminished reactive oxygen species production post anoxia, and altered hormone signaling. Comparing wild-type and cax1 expressing genetically encoded Ca indicators demonstrated altered cytosolic Ca signals in cax1 during reoxygenation. Anoxia-induced Ca signals around the plant vacuole are involved in the control of numerous signaling events related to adaptation to low oxygen stress. This work suggests that cax1 anoxia response pathway could be engineered to circumvent the adverse effects of flooding that impair production agriculture. 

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3. A dominant‐negative Arabidopsis cation exchanger 1 ( CAX1 ): N‐terminal autoinhibition and membrane topology

   https://doi.org/10.1111/tpj.16966  

   Sarkar, Shayan; Rhein, Hormat_Shadgou; Pittman, Jon_K; Hirschi, Kendal_D (August 2024, The Plant Journal)

   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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4. The vacuolar Ca ²⁺ transporter CATION EXCHANGER 2 regulates cytosolic calcium homeostasis, hypoxic signaling, and response to flooding in Arabidopsis thaliana

   https://doi.org/10.1111/nph.19274  

   Bakshi, Arkadipta; Choi, Won‐Gyu; Kim, Su‐Hwa; Gilroy, Simon (September 2023, New Phytologist)

   Summary Flooding represents a major threat to global agricultural productivity and food security, but plants are capable of deploying a suite of adaptive responses that can lead to short‐ or longer‐term survival to this stress. One cellular pathway thought to help coordinate these responses is via flooding‐triggered Ca²⁺signaling.We have mined publicly available transcriptomic data from Arabidopsis subjected to flooding or low oxygen stress to identify rapidly upregulated, Ca²⁺‐related transcripts. We then focused on transporters likely to modulate Ca²⁺signals. Candidates emerging from this analysis includedAUTOINHIBITED Ca²⁺ATPASE 1andCATION EXCHANGER 2. We therefore assayed mutants in these genes for flooding sensitivity at levels from growth to patterns of gene expression and the kinetics of flooding‐related Ca²⁺changes.Knockout mutants inCAX2especially showed enhanced survival to soil waterlogging coupled with suppressed induction of many marker genes for hypoxic response and constitutive activation of others.CAX2mutants also generated larger and more sustained Ca²⁺signals in response to both flooding and hypoxic challenges.CAX2 is a Ca²⁺transporter located on the tonoplast, and so these results are consistent with an important role for vacuolar Ca²⁺transport in the signaling systems that trigger flooding response. 

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5. CAX control: multiple roles of vacuolar cation/H ⁺ exchangers in metal tolerance, mineral nutrition and environmental signalling

   https://doi.org/10.1111/plb.13698  

   Pittman, J K; Hirschi, K D (October 2024, Plant Biology)

   Abstract Plant vacuolar transporters, particularly CAX (Cation/H⁺Exchangers) responsible for Ca²⁺/H⁺exchange on the vacuole tonoplast, play a central role in governing cellular pH, ion balance, nutrient storage, metal accumulation, and stress responses. Furthermore, CAX variants have been employed to enhance the calcium content of crops, contributing to biofortification efforts. Recent research has uncovered the broader significance of these transporters in plant signal transduction and element partitioning. The use of genetically encoded Ca²⁺sensors has begun to highlight the crucial role of CAX isoforms in generating cytosolic Ca²⁺signals, underscoring their function as pivotal hubs in diverse environmental and developmental signalling networks. Interestingly, it has been observed that the loss of CAX function can be advantageous in specific stress conditions, both for biotic and abiotic stressors. Determining the optimal timing and approach for modulating the expression of CAX is a critical concern. In the future, strategically manipulating the temporal loss of CAX function in agriculturally important crops holds promise to bolster plant immunity, enhance cold tolerance, and fortify resilience against one of agriculture's most significant challenges, namely flooding. 

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