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Phosphorylation of the vacuolar anion exchanger AtCLCa is required for the stomatal response to abscisic acid
One Exchanger to Open and Close the Pores
Turgor pressure in the guard cells of plant leaves controls pores called stomata, which enable gas exchange and photosynthesis when open and limit water loss when closed. Light opens these pores, whereas the hormone abscisic acid (ABA) keeps them closed. Ions and water imported into or released from the vacuole control the turgor pressure of guard cells. Wege et al. found that Arabidopsis plants lacking the vacuolar anion/proton exchanger AtCLCa did not effectively open or close their stomata. In light, AtCLCa mediated the uptake of anions into the vacuole to aid in opening the pores, and ABA stimulated AtCLCa phosphorylation and enhanced its efflux activity, which helped to close the pores. Thus, a single exchanger can move ions bidirectionally depending on the plant’s photosynthetic and water conservation needs.
Abstract
Eukaryotic anion/proton exchangers of the CLC (chloride channel) family mediate anion fluxes across intracellular membranes. The Arabidopsis thaliana anion/proton exchanger AtCLCa is involved in vacuolar accumulation of nitrate. We investigated the role of AtCLCa in leaf guard cells, a specialized plant epidermal cell that controls gas exchange and water loss through pores called stomata. We showed that AtCLCa not only fulfilled the expected role of accumulating anions in the vacuole during stomatal opening but also mediated anion release during stomatal closure in response to the stress hormone abscisic acid (ABA). We found that this dual role resulted from a phosphorylation-dependent change in the activity of AtCLCa. The protein kinase OST1 (also known as SnRK2.6) is a key signaling player and central regulator in guard cells in response to ABA. Phosphorylation of Thr38 in the amino-terminal cytoplasmic domain of AtCLCa by OST1 increased the outward anion fluxes across the vacuolar membrane, which are essential for stomatal closure. We provide evidence that bidirectional activities of an intracellular CLC exchanger are physiologically relevant and that phosphorylation regulates the transport mode of this exchanger.
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Supplementary Material
Summary
Fig. S1. The ABA response of stomata aperture is similar in the presence of nitrate instead of chloride.
Fig. S2. Purity and activity of recombinant OST1 and OST1-G33R.
Fig. S3. Characterization of AtCLCa1–86 phosphorylation by mass spectrophotometry.
Fig. S4. Activity of recombinant OST1 in patch-clamp buffer and effect of OST1 on current families evoked in clca-2 knockout mutant.
Fig. S5. Abundance of AtCLCa mRNA in the different genotypes used in the study.
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Published In
Science Signaling
Volume 7 | Issue 333
July 2014
July 2014
Copyright
Copyright © 2014, American Association for the Advancement of Science.
Submission history
Received: 30 January 2014
Accepted: 18 June 2014
Acknowledgments
We thank L. Sago and M. Argentini (CNRS-FRC3115 and IFR115, Centre de Recherche de Gif, Imagif, Gif sur Yvette, France) for their help with the mass spectrometry analyses, M.-N. Soler (CNRS-FRC3115 and IFR87, Centre de Recherche de Gif, Imagif, Gif sur Yvette, France) for her help with the confocal microscopy analyses, M. Allot (CNRS, ISV, Gif sur Yvette, France) for her technical support with GUS staining, and E. Diatloff for critical reading. The cDNA of AtSK2.3 was provided by M. Boudsocq (INRA-UEVE, URGV, Evry, France). Funding: This research was financially supported by the EU Marie Curie Research Training Network “VaTEP” (MRTN-CT-2006-035833), by the Agence Nationale de la Recherche (ANR-Nitrapool ANR-08-BLAN-0008-02), the University of Aix-Marseille II (MENRT 26591-2007), and the French-Italian CNRS-CNR bilateral cooperation program (EDC21599). Author contributions: S.W., L.K., and N.L. performed stomata aperture measurements. A.D.A. performed dehydration measurements. S.W. performed BiFC, confocal microscopy, and peptide assays. S.W. and M.-J.D. produced recombinant proteins and performed kinase assays. M.-J.D. performed Western blots. A.D.A. performed electrophysiology. S.W. performed protoplast transformation. D.C. performed mass spectroscopy. A.D.A., S.T., S.W., S.M., and S.F. designed the research and wrote the manuscript. S.F. supervised all experiments. H.B.-B., F.G., N.L., and E.M. helped design experiments, analyzed the data, and contributed to the writing of the manuscript. Competing interests: The authors declare that they have no competing interests.
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