SLAC1 is required for plant guard cell S-type anion channel function in stomatal signalling

Abstract

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 fixation1 and climate change2. The aperture of stomatal pores is regulated by the transport of osmotically active ions and metabolites across guard cell membranes3,4. Despite the vital role of guard cells in controlling plant water loss3,4, ozone sensitivity1,2 and CO2 supply2,5,6,7, 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 stimuli3,4,8. 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 CO2, abscisic acid, ozone, light/dark transitions, humidity change, calcium ions, hydrogen peroxide and nitric oxide. Mutations in SLAC1 impair slow (S-type) anion channel currents that are activated by cytosolic Ca2+ and abscisic acid, but do not affect rapid (R-type) anion channel currents or Ca2+ channel function. A low homology of SLAC1 to bacterial and fungal organic acid transport proteins, and the permeability of S-type anion channels to malate9 suggest a vital role for SLAC1 in the function of S-type anion channels.

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Figure 1: Membrane protein SLAC1 controls leaf ozone and water-loss responses.
Figure 2: Mutations in SLAC1 impair stomatal responses to changes in environment.
Figure 3: Impaired stomatal responses to ABA, H2O2, NO and Ca2+ in slac1.
Figure 4: Ca 2+ and ABA activations of S-type anion channels are impaired in slac1 guard cells.

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Primary accessions

ArrayExpress

Data deposits

The primary microarray data reported has been deposited with the ArrayExpress database under accession number E-MEXP-1388.

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Acknowledgements

We thank M. Uuskallio and I. Puzõrjova for technical help. This research was supported by the Academy of Finland Centre of Excellence programme and Helsinki University Environmental Research Centre (to J.K.), by Estonian Science Foundation and University of Tartu start-up grants (to H.K.), by NIH, NSF and, in part, DOE grants (to J.I.S.), and a Leverhulme Trust Early Career Fellowship (to R.D.)

Author Contributions T.V., H.K. and Y.-F.W. contributed equally to this work. J.K. and H.K. designed the experiments in Figs 1 and 2. A.L., H.K. and T.V. identified the SLAC1 gene. T.V. and M.B. performed the expression, complementation and subcellular localization analyses in Fig. 1 and Supplementary Fig. 5. H.K. and H.M. performed experiments in Fig. 2. H.K. performed experiments in Supplementary Figs 1 and 2. R.D. designed and performed experiments in Fig. 3b, c and Supplementary Fig. 6b. J.I.S. and J.K. designed experiments in Figs 3a and d, and 4, and Supplementary Figs 6a, 7, 8 and 9. W.-Y.C. and G.V. performed experiments in 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.

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Correspondence to Jaakko Kangasjärvi.

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Supplementary Information

The file contains Supplementary Figures 1-9 with Legends and Legend to Supplementary Movie. (MOV 12881 kb)

Supplementary Movie 1

The file contains Supplementary Movie 1. The movie shows confocal images converted to a rotating 3D image showing the relative localization of SLAC1:GFP to the nucleus stained with DAPI. (PDF 1419 kb)

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Vahisalu, T., Kollist, H., Wang, YF. et al. SLAC1 is required for plant guard cell S-type anion channel function in stomatal signalling. Nature 452, 487–491 (2008). https://doi.org/10.1038/nature06608

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