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 ...

SUMMARYThe air pollutant ozone can be used as a tool to unravel in planta processes induced by reactive oxygen species (ROS). Here, we have utilized ozone to study ROS-dependent stomatal signaling. We show that the ozonetriggered rapid transient decrease (RTD) in stomatal conductance coincided with a burst of ROS in guard cells. RTD was present in 11 different Arabidopsis ecotypes, suggesting that it is a genetically robust response. To study which signaling components or ion channels were involved in RTD, we tested 44 mutants deficient in various aspects of stomatal function. This revealed that the SLAC1 protein, essential for guard cell plasma membrane S-type anion channel function, and the protein kinase OST1 were required for the ROS-induced fast stomatal closure. We showed a physical interaction between OST1 and SLAC1, and provide evidence that SLAC1 is phosphorylated by OST1. Phosphoproteomic experiments indicated that OST1 phosphorylated multiple amino acids in the N terminus of SLAC1. Using TILLING we identified three new slac1 alleles where predicted phosphosites were mutated. The lack of RTD in two of them, slac1-7 (S120F) and slac1-8 (S146F), suggested that these serine residues were important for the activation of SLAC1. Mass-spectrometry analysis combined with site-directed mutagenesis and phosphorylation assays, however, showed that only S120 was a specific phosphorylation site for OST1. The absence of the RTD in the dominant-negative mutants abi1-1 and abi2-1 also suggested a regulatory role for the protein phosphatases ABI1 and ABI2 in the ROS-induced activation of the S-type anion channel.

Transpiration and ozone uptake rates were measured simultaneously in sunflower leaves at different stomatal openings and various ozone concentrations. Ozone uptake rates were proportional to the ozone concentration up to 1500 nanoliters per liter. The leaf gas phase diffusion resistance (stomatal plus boundary layer) to water vapor was calculated and converted to the resistance to ozone multiplying it by the theoretical ratio of diffusion coefficients for water vapor and ozone in air (1.67). The ozone concentration in intercellular air spaces calculated from the ozone uptake rate and diffusion resistance to ozone scattered around zero. The ozone concentration in intercellular air spaces was measured directly by supplying ozone to the leaf from one side and measuring the equilibrium concentration above the other side, and it was found to be zero. The total leaf resistance to ozone was proportional to the gas phase resistance to water vapor with a coefficient of 1.68. It is concluded that ozone enters the leaf by diffusion through the stomata, and is rapidly decomposed in cell walls and plasmalemma. diffusion resistance of the whole gaseous pathway from cell surfaces to ambient air:where E2 is the transpiration rate (minus cuticular transpiration); rg, the diffusion resistance in the leaf gaseous phase to water vapor; wi, the water vapor concentration at evaporating cell surfaces; and wa, that in the ambient air. CO2 is a heavier gas (M = 44) than water vapor (M = 18); therefore, CO2 moves more slowly than water vapor through the same diffusion pathway and at the same concentration difference. The ratio of the diffusion coefficients of H20 and CO2 in the leaf gaseous pathway was measured to be 1.62 (9).We could not find a value of the diffusion constant for 03 in air, DZ, in the literature. However, diffusion constants for various gas mixtures may be calculated using the molecular parameters of component gases (1) 0.43 X (T)00) X Abbreviations: E, transpiration rate; wa, wi, water vapor concentration in ambient air (a) and on evaporating cell surfaces (i); r8, rg, leaf gas phase diffusion resistance to water vapor (w) and to ozone (z); M, molecular weight; DW, Dz, diffusion constant for water vapor (w) and for ozone (z); T, temperature, Tk, critical temperature; Vk, critical volume; P, atmospheric pressure; Za, zi, ozone concentration in ambient air (a) and in the leaf intercellular air space (i); Q, ozone uptake rate; v, gas flow rate; S, leaf area; gz, total leaf conductance for ozone; rz, total leaf resistance to ozone; gge ggz, leaf gas phase diffusion conductance for water vapor (w) and for ozone (z).

To follow stomatal responses to ozone (O3) in different Arabidopsis lines, we constructed a rapid‐response O3 exposure/gas‐exchange measurement device consisting of eight through‐flow whole‐rosette cuvettes. To separate rosette from roots and growth substrate, plant is grown through an agar‐filled hole in a polished glass plate fixed on the pot. Following insertion of the plant, the plate forms air‐tight bottom surface of the cuvette; thus the rosette is enclosed without touching it during any phase of the insertion of the plant to the cuvette. The device allows monitoring rapid responses in the stomatal function. For example, an acute exposure to 150 ppb O3 decreased stomatal conductance to 60–70% of its initial value within 9–12 min. Thereafter, the conductance regained its preexposure value within further 30–40 min in spite of the continuing O3 exposure. The transient decrease was absent in the abscisic acid‐insensitive mutant abi2 defective in a class 2C protein phosphatase. This provides an in vivo confirmation that the early transient decrease in stomatal conductance is not a result of physical damage by the reactive oxygen species (ROS) formed from O3 breakdown but reflects the biological action of ROS, transduced through a signalling cascade. Thus, the apparatus will be helpful in specifying complex molecular and genetic interactions in rapid responses in guard cells in vivo.

To estimate protection of the plasmalemma against ozone by the cell apoplast, the decomposition network of ozone in the mesophyll cell wall is analysed in consideration of data on published bimolecular reaction rate constants and concentrations of the reactants involved. The effect of dimerization of ascorbate free radicals (AFR) on the stoichiometric ratio of ozone reduction by ascorbate is quantified over the range of cell wall acidity, pH = 5.0‐6.5. As the disproportionation of AFR decreases sharply towards higher pH, the flow of AFR through dimerization is low over the pH range 5.5‐6.5, allowing abstraction of the second electron from AFR and formation of dehydroascorbate with a nearly 1:1 stoichiometric ratio in relation to ozone. The direct reaction between ozone and ascorbate (AA) in cell walls 0.3‐0.5 µm thick and at an AA concentration of 0.5 mM is able to detoxify 50‐70% of the O3 that impinges on the wall surface. Generation of singlet oxygen and the hydroxyl radical, which are more reactive to AA than O3, decreases markedly the O3 flow to the plasmalemma. The question is raised whether cell wall alkalinization under ozone may hasten the decomposition of the pollutant due to the more rapid generation of hydroxyl radical by phenolic compounds.