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	Fig. 1. AtALMT12 is expressed in guard cells. (A) The expression of AtALMT12 (At4g17970), SLAC1 (At1g12480), KAT1 (At5g46240), CBP (At4g33050), actin (At5g09810) and β-tubulin genes (AT1g75780, AT5g62690, AT5g62700 and AT5g44340) in guard cells (GC) and mesophyll cells (MC) was detected by RT–PCR. Note that three amplification products are detected for AtALMT12 (249 bp, black arrowhead; 335 bp, red arrowhead; 412 bp, blue arrowhead). (B) Comparison of the expression level of AtALMT12 among plant organs. (C) Expression of AtALMT12 determined by real-time PCR using primer set #2 (see Fig. 2A). Plants (Columbia) were grown in hydroponic medium and RNA was isolated from whole seedlings. Relative expression levels were normalized against the values of the EF1α transcript (At5g60390). Bars show the mean ± SEM (n = 3). (D–G) GUS reporter expression in seedling (D), leaf (E), guard cell (F) and root (G). GUS is driven by the region 3,157 bp upstream of the start codon. Bars = 5 mm (D), 1 mm (E), 20 μm (F) and 500 μm (G).
	Fig. 2. Knockdown mutation of AtALMT12 impairs stomatal responses and has a wilty phenotype. (A) Schematic of the AtALMT12 locus (At4g17970) showing T-DNA insertion sites and primer locations (arrowheads) for RT–PCR analysis. (B) RT–PCR analysis using primer set #1 (or β-tubulin primers). (C) One-week-old wild-type (WT) and atalmt12-1 plants were subjected to water withholding for a further 2 weeks. Photographs of representative plants from three independent replicates were taken from the side and top. (D) Dark-induced stomatal closure (n = 4 experiments). (E) Calcium-induced stomatal closure (n = 4 experiments). (F) ABA-induced stomatal closure (n = 10 experiments for 0, 1 and 10 μM ABA; n = 4 experiments for 50 μM ABA). For D–F, 20 stomata were measured for each genotype in each experiment, and symbols or bars plot the mean ± SEM. (G) Light-induced stomatal opening in the atalmt12-1 mutant (n = 5 experiments). (H) Water loss of detached leaves (n = 3). Symbols plot the mean ± SEM. (I) Stomatal density. Plants were grown under short days (8L:16D) for 3 months or long days (16L:8D) for 3 weeks. Data are presented as the mean ± SD (n = 5 leaves). Differences from WT values were significant at *P < 0.05 and **P < 0.01, respectively.
	Fig. 3. Complementation of the ABA-insensitive phenotype of atalmt12-1 with genomic AtALMT12 sequences. Plants were treated with or without 1 μM ABA and stomatal closure was assayed. Complementation of atalmt12-1 by the native promoter (NP)-driven genomic sequence of AtALMT12 with or without GFP fusion (NP::genome, NP::genome–GFP; n = 5–6 independent experiments), and the NP-driven coding sequence (NP::ORF, n = 8 independent experiments).
	Fig. 4. Localization of AtALMT12. The AtALMT12 coding sequence–GFP (ORF::GFP) construct under the control of the 35S promoter was transiently expressed in onion epidermal cells (A–O) and V. faba guard cells (P–V). As control, GFP alone was expressed in onion cells (A–C) or V. faba cells (P–R). Expression of GFP::AtALMT12 (N-terminal fusion, D–F) or AtALMT12::GFP (C-terminal fusion, G–I) shows similar localizations in onion cells. GFP fluorescence surrounds the nucleus, suggesting that AtALMT12 localized to both endomembranes and plasma membrane (J–L). Plasmolysis of AtALMT12::GFP-expressing cells with 1 M mannitol shows that the Hechtian strands attaching the plasma membrane to the cell wall are labeled, confirming that the protein is localized on the plasma membrane. Co-expression of AtALMT12::GFP with Cyt b5::mRFP as an ER marker (S–V). Fluorescence from AtALMT12::GFP co-localized with Cyt b5::mRFP suggests that AtALMT12 is targeted to the ER. Photographs show GFP fluorescence images (A, D, G, J, M, P, S), mRFP images (T), transmitted light images (C, I, L, O, R, V) and merged images (B, E, H, K, N, Q, U).
	Fig. 5. Electrophysiological properties of AtALMT12 expressed in Xenopus oocyte plasma membranes. (A) Typical traces of anion currents across the plasma membrane in oocytes expressing AtALMT12 (ORF) (left) and water-injected controls (right). The dotted line indicates zero current level (±0 nA). (B) Mean current–voltage relationships in AtALMT12 (ORF)-expressing oocytes recorded with a range of extracelluar NaCl concentrations [96 mM (n = 23), 48 mM (n = 12), 24 mM (n = 11), 0 mM (n = 11) and 96 mM TEA-Cl (n = 11)]. (C) Current–voltage relationships for AtALMT12-expressing (ORF: open symbols) and water-injected control (water: filled symbols) oocytes recorded with various anions (n = 4 for each solution). (D) Current–voltage relationships for oocytes expressing the splicing variant SV1 (n = 10) and SV2 (n = 12) and water-injected controls (n = 10). All symbols plot the mean ± SEM.
	Fig. 6. S-type and R-type anion currents in A. thaliana guard cell protoplasts. (A, B) Whole-cell S-type anion currents in response to a high extracellular calcium concentration (40 mM CaCl2 in the bath solution). (A) Representative traces of calcium-activated S-type anion currents. (B) Current–voltage relationships of the wild type (n = 6), atalmt12-1 (n = 6) and cpk6-1 (n = 3). (C–E) Whole-cell S-type anion currents with 10 μM (C, D) or 50 μM (E) ABA in the bath solution. (C) Representative traces of S-type anion currents. (D) Current–voltage relationships of the wild type (n = 5) and atalmt12-1 (n = 7). (E) Current–voltage relationship of the wild type and atalmt12-1 (n = 2). (F) Representative traces of R-type anion currents in the wild-type and atalmt12-1. (G) Average peak R-type anion channel current in the wild type (n = 4) and atalmt12-1 (n = 3). All data are the mean ± SEM. Significant differences (Student’s t-test) were not observed between the wild type and atalmt12-1 in both the S-type anion current (E; P = 0.587 at −115 mV) and the R-type anion current (F; P = 0.348 at negative peak values).

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