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The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles

Abstract

Nitrate, the major nitrogen source for most plants, is widely used as a fertilizer and as a result has become a predominant freshwater pollutant. Plants need nitrate for growth and store most of it in the central vacuole1. Some members of the chloride channel (CLC) protein family, such as the torpedo-fish ClC-0 and mammalian ClC-1, are anion channels2,3, whereas the bacterial ClC-ec1 and mammalian ClC-4 and ClC-5 have recently been characterized as Cl-/H+ exchangers with unknown cellular functions4,5,6. Plant members of the CLC family are proposed to be anion channels7,8 involved in nitrate homeostasis9; however, direct evidence for anion transport mediated by a plant CLC is still lacking. Here we show that Arabidopsis thaliana CLCa (AtCLCa) is localized to an intracellular membrane, the tonoplast of the plant vacuole, which is amenable to electrophysiological studies, and we provide direct evidence for its anion transport ability. We demonstrate that AtCLCa is able to accumulate specifically nitrate in the vacuole and behaves as a NO3-/H+ exchanger. For the first time, to our knowledge, the transport activity of a plant CLC is revealed, the antiporter mechanism of a CLC protein is investigated in a native membrane system, and this property is directly connected with its physiological role.

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Figure 1: AtCLCa resides in the tonoplast.
Figure 2: Whole-vacuole NO 3 - currents are abolished in vacuoles from clca knockout mutants.
Figure 3: AtCLCa mediates specific NO 3 - transport into the vacuole.
Figure 4: AtCLCa functions as a 2NO 3 - /1H + antiporter.

References

  1. Martinoia, E. & Wiemken, A. Vacuoles as storage compartments for nitrate in barley leaves. Nature 289, 292–294 (1981)

    ADS  CAS  Article  Google Scholar 

  2. Miller, C. & White, M. M. A voltage-dependent chloride conductance channel from Torpedo electroplax membrane. Ann. NY Acad. Sci. 341, 534–551 (1980)

    ADS  CAS  Article  Google Scholar 

  3. Steinmeyer, K., Ortland, C. & Jentsch, T. J. Primary structure and functional expression of a developmentally regulated skeletal muscle chloride channel. Nature 354, 301–304 (1991)

    ADS  CAS  Article  Google Scholar 

  4. Accardi, A. & Miller, C. Secondary active transport mediated by a prokaryotic homologue of ClC Cl- channels. Nature 427, 803–807 (2004)

    ADS  CAS  Article  Google Scholar 

  5. Picollo, A. & Pusch, M. Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5. Nature 436, 420–423 (2005)

    ADS  CAS  Article  Google Scholar 

  6. Scheel, O., Zdebik, A. A., Lourdel, S. & Jentsch, T. J. Voltage-dependent electrogenic chloride/proton exchange by endosomal CLC proteins. Nature 436, 424–427 (2005)

    ADS  CAS  Article  Google Scholar 

  7. Lurin, C., Geelen, D., Barbier-Brygoo, H., Guern, J. & Maurel, C. Cloning and functional expression of a plant voltage-dependent chloride channel. Plant Cell 8, 701–711 (1996)

    CAS  Article  Google Scholar 

  8. Hechenberger, M. et al. A family of putative chloride channels from Arabidopsis and functional complementation of a yeast strain with a CLC gene disruption. J. Biol. Chem. 271, 33632–33638 (1996)

    CAS  Article  Google Scholar 

  9. Geelen, D. et al. Disruption of putative anion channel gene AtCLC-a in Arabidopsis suggests a role in the regulation of nitrate content. Plant J. 21, 259–267 (2000)

    CAS  Article  Google Scholar 

  10. Loudet, O., Chaillou, S., Krapp, A. & Daniel-Vedel, F. Quantitative trait loci analysis of water and anion content in interaction with nitrogen availability in Arabidopsis thaliana. Genetics 163, 711–722 (2003)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Harada, H., Kuromori, T., Hirayama, T., Shinozaki, K. & Leigh, R. A. Quantitative trait loci analysis of nitrate storage in Arabidopsis leading to an investigation of the contribution of the anion channel gene, AtCLC-c, to variation in nitrate levels. J. Exp. Bot. 55, 2005–2014 (2004)

    CAS  Article  Google Scholar 

  12. Lurin, C. et al. CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers. Biochem. J. 348, 291–295 (2000)

    CAS  Article  Google Scholar 

  13. Teardo, E. et al. Localisation of a putative ClC chloride channel in spinach chloroplasts. FEBS Lett. 579, 4991–4996 (2005)

    CAS  Article  Google Scholar 

  14. Bertl, A. et al. Electrical measurements on endomembranes. Science 258, 873–874 (1992)

    ADS  CAS  Article  Google Scholar 

  15. Cerana, R., Giromini, L. & Colombo, R. Malate-regulated channels permeable to anions in vacuoles of Arabidopsis thaliana. Aust. J. Plant Physiol. 22, 115–121 (1995)

    CAS  Google Scholar 

  16. Hafke, J. B., Hafke, Y., Smith, J. A., Luttge, U. & Thiel, G. Vacuolar malate uptake is mediated by an anion-selective inward rectifier. Plant J. 35, 116–128 (2003)

    CAS  Article  Google Scholar 

  17. Hurth, A. et al. Impaired pH homeostasis in Arabidopsis lacking the vacuolar dicarboxylate transporter and analysis of carboxylic acid transport across the tonoplast. Plant Physiol. 137, 901–910 (2005)

    CAS  Article  Google Scholar 

  18. Pei, Z., Ward, J., Harper, J. F. & Schroeder, J. A novel chloride channel in Vicia faba guard cell vacuoles activated by the serine/threonine kinase, CDPK. EMBO J. 15, 6564–6574 (1996)

    CAS  Article  Google Scholar 

  19. Accardi, A., Kolmakova-Partensky, L., Williams, C. & Miller, C. Ionic currents mediated by a prokaryotic homologue of CLC Cl- channels. J. Gen. Physiol. 123, 109–119 (2004)

    CAS  Article  Google Scholar 

  20. Gross, E. & Hopfer, U. Activity and stoichiometry of Na+:HCO3- cotransport in immortalized renal proximal tubule cells. J. Membr. Biol. 152, 245–252 (1996)

    CAS  Article  Google Scholar 

  21. Accardi, A. et al. Separate ion pathways in a Cl-/H+ exchanger. J. Gen. Physiol. 126, 563–570 (2005)

    CAS  Article  Google Scholar 

  22. Cookson, S. J., Williams, L. E. & Miller, A. J. Light–dark changes in cytosolic nitrate pools depend on nitrate reductase activity in Arabidopsis leaf cells. Plant Physiol. 138, 1097–1105 (2005)

    CAS  Article  Google Scholar 

  23. Miller, C. ClC chloride channels viewed through a transporter lens. Nature 440, 484–489 (2006)

    ADS  CAS  Article  Google Scholar 

  24. Thomine, S., Lelievre, F., Debarbieux, E., Schroeder, J. I. & Barbier-Brygoo, H. AtNRAMP3, a multispecific vacuolar metal transporter involved in plant responses to iron deficiency. Plant J. 34, 685–695 (2003)

    CAS  Article  Google Scholar 

  25. Song, W. Y. et al. Engineering tolerance and accumulation of lead and cadmium in transgenic plants. Nature Biotechnol. 21, 914–919 (2003)

    CAS  Article  Google Scholar 

  26. Sarafian, V., Kim, Y., Poole, R. J. & Rea, P. A. Molecular cloning and sequence of cDNA encoding the pyrophosphate-energized vacuolar membrane proton pump of Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 89, 1775–1779 (1992)

    ADS  CAS  Article  Google Scholar 

  27. Neher, E. Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol. 207, 123–131 (1992)

    CAS  Article  Google Scholar 

  28. Lange, N. & Dean, J. A. in Lange's Handbook of Chemistry (ed. Dean, J. A.) 5-3–5-7 (McGraw-Hill, New York, 1973)

    Google Scholar 

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Acknowledgements

We thank C. Lurin and C. Maurel for help at the initial stage of the project. We are grateful to S. Bolte for offering the confocal microscopy facilities of the Cell Biology Platform of IFR 87 “La Plante et son Environnement”. We would like to thank K. Czempinski for providing the pA7-GFP plasmid; P. Rea for anti-pyrophosphatase IgG; V. Lanquar for help in vacuole purification; and J. Scholz-Starke for reading the manuscript. This project was supported by the Centre National de la Recherche Scientifique. A.DeA. and D.M. were funded by the European Research Training Network NICIP.

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Correspondence to H. Barbier-Brygoo.

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Supplementary information

Supplementary Figure 1

Malate currents: clca-2 knock-out mutant vacuoles show wild-type malate currents. (PDF 106 kb)

Supplementary Figure 2

AtCLCa current in different cytosolic NO3- concentrations. I-V curves of AtCLCa current in different cytosolic NO3- concentrations normalised to capacitance. (PDF 63 kb)

Supplementary Figure 3

Conserved regions of CLC proteins. Sequence alignment of conserved regions of different CLC proteins. (PDF 135 kb)

Supplementary Notes

Cytosolic solutions used for selectivity experiments in Figure 3c and d. Equation used for deriving the H+/NO3- transport. Numerical calculations. (PDF 70 kb)

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De Angeli, A., Monachello, D., Ephritikhine, G. et al. The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles. Nature 442, 939–942 (2006). https://doi.org/10.1038/nature05013

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