Stomatal pores, formed by two surrounding guard cells in the epidermis of plant leaves, allow influx of atmospheric carbon dioxide in exchange for transpirational water loss. Stomata also restrict the entry of ozone-an important air pollutant that has an increasingly negative impact on crop yields, and thus global carbon fixation 1 and climate change 2 . The aperture of stomatal pores is regulated by the transport of osmotically active ions and metabolites across guard cell membranes 3,4 . Despite the vital role of guard cells in controlling plant water loss 3,4 , ozone sensitivity 1,2 and CO 2 supply 2,5-7 , the genes encoding some of the main regulators of stomatal movements remain unknown. It has been proposed that guard cell anion channels function as important regulators of stomatal closure and are essential in mediating stomatal responses to physiological and stress stimuli 3,4,8 . However, the genes encoding membrane proteins that mediate guard cell anion efflux have not yet been identified. Here we report the mapping and characterization of an ozone-sensitive Arabidopsis thaliana mutant, slac1. We show that SLAC1 (SLOW ANION CHANNEL-ASSOCIATED 1) is preferentially expressed in guard cells and encodes a distant homologue of fungal and bacterial dicarboxylate/malic acid transport proteins. The plasma membrane protein SLAC1 is essential for stomatal closure in response to CO 2 , ©2008 Nature Publishing GroupCorrespondence and requests for materials should be addressed to J.K. (jaakko.kangasjarvi@helsinki.fi). † Present address: Division of Biology, Imperial College London, London SW7 2AZ, UK. * These authors contributed equally to this work Fig. 3d and Supplementary Fig. 6a. N.N. performed experiments in Fig. 3a and Supplementary Fig. 7. Y.-F.W. performed experiments in Fig. 4 and Supplementary Figs 8 and 9. J.K. and J.I.S. wrote the paper. All the authors discussed the results, and commented on and edited the manuscript.The primary microarray data reported has been deposited with the ArrayExpress database under accession number E-MEXP-1388.Reprints and permissions information is available at www.nature.com/reprints. 8,11 by mediating anion efflux and causing membrane depolarization, which controls K + efflux through K + channels. So far, none of the candidates for plant anion channels -the plant homologues to the animal CLC chloride channels -has been localized to the plasma membrane 10 , and the first plant CLC channel that was functionally characterized encodes a central vacuolar proton/nitrate exchanger 12 , rather than an anion channel. Thus, despite their proposed importance in several physiological and stress responses in plants 8,10,11 , the molecular identity of the guard cell plasma membrane proteins that mediate anion channel activity has remained unknown. NIH Public AccessIn a mutant screen for O 3 sensitivity, a series of Arabidopsis ethyl methanesulphonate (EMS) mutants called radical-induced cell death (rcd) was identified 13,14 . One of them, a recessive mutant originally referred ...

T-DNA disruption mutations in the AtHKT1 gene have previously been shown to suppress the salt sensitivity of the sos3 mutant. However, both sos3 and athkt1 single mutants show sodium (Na+) hypersensitivity. In the present study we further analyzed the underlying mechanisms for these non-additive and counteracting Na+ sensitivities by characterizing athkt1-1 sos3 and athkt1-2 sos3 double mutant plants. Unexpectedly, mature double mutant plants grown in soil clearly showed an increased Na+ hypersensitivity compared with wild-type plants when plants were subjected to salinity stress. The salt sensitive phenotype of athkt1 sos3 double mutant plants was similar to that of athkt1 plants, which showed chlorosis in leaves and stems. The Na+ content in xylem sap samples of soil-grown athkt1 sos3 double and athkt1 single mutant plants showed dramatic Na+ overaccumulation in response to salinity stress. Salinity stress analyses using basic minimal nutrient medium and Murashige-Skoog (MS) medium revealed that athkt1 sos3 double mutant plants show a more athkt1 single mutant-like phenotype in the presence of 3 mM external Ca2+, but show a more sos3 single mutant-like phenotype in the presence of 1 mM external Ca2+. Taken together multiple analyses demonstrate that the external Ca2+ concentration strongly impacts the Na+ stress response of athkt1 sos3 double mutants. Furthermore, the presented findings show that SOS3 and AtHKT1 are physiologically distinct major determinants of salinity resistance such that sos3 more strongly causes Na+ overaccumulation in roots, whereas athkt1 causes an increase in Na+ levels in the xylem sap and shoots and a concomitant Na+ reduction in roots.