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Supporting Text

Stomatal aperture was determined in epidermal strips prepared from the abaxial side of leaves. The epidermal strips were preincubated in a Petri dish containing 10 mM Mes-NaOH (pH 6.1), 5 mM KCl, and 0.1 mM CaCl₂ with or without 10 mM tautomycin for 2.5 h in the dark. Then, the epidermal strips were illuminated with red light (150 mmol·m^-2·s^-1) from the bottom with or without blue light (10 mmol·m^-2·s^-1) from the top for 2.5 h. For fusicoccin-induced stomatal opening, the epidermal peels were kept in darkness for 2.5 h, then 10 mM fusicoccin was added and stomatal apertures were measured microscopically (Eclipse TS100; Nikon, Tokyo, Japan) after another 2.5 h in the dark. For measurements of half stomatal apertures in transformed guard cells, epidermal strips were incubated as described above, and the guard cells that expressed the sGFP fusion protein were photographed.

Full-length inhibitor-2, VfPP1c-1, VfPP1c-3, VfPP1c-1-H137N, and VfPP1c-3-H121N were subcloned into CaMV35SW-sGFP(S65T)-nos3' (1). Individual constructs were introduced into Vicia leaves by particle bombardment using gold microcarriers (particle size 1.0 mm). Bombardments were carried out under the following conditions: vacuum, 25 in Hg; target distance, 6 cm; and He pressure, 1350 psi. After 12-16 h, epidermal strips were excised from the abaxial side of leaves and used for the stomatal opening assay or microscopic observation. Fluorescent images were obtained with a confocal laser-scanning fluorescent microscopy (Digital Eclipse C1; Nikon) with excitation at 488 nm and the emission signal recovered at 515-530 nm for sGFP, and with excitation at 543 nm and the emission signal recovered between 578-632 nm for DsRed.

A full-length VfPP1c-1 and VfPP1c-1-H137N were subcloned into pGEX-2T (Pharmacia Biotech, Tokyo, Japan) and transformed into Escherichia coli strain BL21. The transformants were grown overnight at 30°C in LB medium supplemented with 50 mg/ml ampicillin and 0.1 mM CoCl₂ according to ref. 2. The culture was inoculated into fresh medium and grown at 20°C until the absorbance at 600 nm reached a value of about 0.5. Isopropyl-b-d-thiogalactopyranoside (IPTG) was then added to a final concentration of 0.3 mM, and cultures were further grown overnight. Cells were harvested and resuspended in 20 mM Tris-HCl (pH 7.4), 140 mM NaCl, 1 mM CoCl₂, 2 mM phenylmethanesulfonyl fluoride (PMSF), and 200 mM leupeptin. Cells were disrupted by sonication, Triton X-100 was added to a final concentration of 1% (wt/vol), and the mixture was incubated for 30 min at room temperature. The suspension was centrifuged at 12,000 ´g for 10 min at 4°C, the supernatant liquid was mixed with Glutathione-Sepharose 4B (GS4B; Pharmacia Biotech) and incubated with gentle shaking for 1 h at 4°C. The GS4B was washed three times and thrombin (30 units; Pharmacia Biotech) was added. The suspension was further incubated at room temperature for 16 h and then centrifuged at 10,000 ´g for 1 min; the supernatant was used for the assay.

Immunodetections of Vfphots and the plasma membrane H⁺-ATPase were performed according to the previous methods (3) with slight modification. Guard cell protoplasts (400 mg of protein per ml) were preincubated for 30 min under the background red light (600 mmol·m^-2·s^-1) at 24°C, and then were exposed to 10 mM tautomycin for 2 h. Afterward, a pulse of blue light (100 mmol·m^-2·s^-1) was applied.

For the detection of Vfphots, the reaction was terminated at 1 min after the starting of blue light pulse by adding trichloroacetic acid to the final concentration of 10% (wt/vol) to the guard cell suspension (70 mg of protein). The sample was kept on ice for 15 min, followed by centrifugation for 10 min at 10,000 ´ g. The resulting pellet was washed three times with 50 mM Tris·HCl (pH 8.0), and resuspended in solubilizing medium [10 mM Tris·HCl (pH 8.0), 15% (wt/vol) Suc, 1% (wt/vol) SDS, 1 mM EDTA, 2.5% (wt/vol) 2-mercaptoehanol, and 0.02% (wt/vol) Coomassie brilliant blue]. After boiling at 95°C for 3 min, the sample was subjected to SDS/PAGE.

For the detection of the H⁺-ATPase, the reaction was terminated at 3.5 min after the starting of blue light pulse by adding an equal volume of the medium containing 100 mM Mops-KOH (pH 7.5), 5 mM EDTA, 200 mM NaCl, 1 mM PMSF, 20 mM leupeptin, 4 mM DTT, 20 mM NaF, 1 mM ammonium molybdate, 100 nM calyculin A, and 2% (wt/vol) Trinton X-100 to the guard cell suspensions (50 mg of protein), followed by centrifugation for 1 min at 10,000 ´ g. The resulting supernatant was mixed with protein A-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 4°C on a rotating apparatus to remove nonspecifically bound proteins. After spin down beads, the supernatant was mixed with anti-H⁺-ATPase antibody conjugated to protein A-agarose with dimethyl pimelimidate dihydrochloride (DMP). After incubation for 12 h with gentle mixing at 4°C, the sample was centrifuged for 1 min at 10,000 ´ g, and the pellet was washed three times with Tris-based saline [20 mM Tris·HCl (pH 7.4) and 140 mM NaCl]. The pellet was resuspended in solubilizing medium and subjected to SDS/PAGE.

After separation of proteins by SDS/PAGE gel, individual proteins were transferred to nitrocellulose membrane at 1 mA·cm^-2 in transfer buffer [48 mM Tris, 39 mM Gly, and 20% (vol/vol) methanol] by electroblotting (Transblot, Bio-Rad, Hercules, CA). The resulting membranes were treated with Qentix Western Blot Signal Enhancer Kit (Pierce, Rockford, IL) according to the manufacturer's protocol. Afterward, the membranes were incubated with blocking buffer for 1 h [0.05% (wt/vol) Tween 20, 5% (wt/vol) nonfat dry milk, 20 mM Tris·HCl (pH 7.4), and 140 mM NaCl] and then reacted with polyclonal antibodies at a dilution of 1:2000 for Vfphots and 1:3000 for the H⁺-ATPase in blocking buffer, respectively, at room temperature for 12 h. The membrane was then rinsed three times for 5 min each in T-TBS containing 0.05% (wt/vol) Tween 20, 20 mM Tris·HCl (pH 7.4), and 140 mM NaCl and reacted with a goat anti-rabbit IgG secondary antibody conjugated to alkaline phosphatase (Bio-Rad Laboratories) at a dilution of 1:3,000 in blocking buffer at room temperature for 2 h. Development of the alkaline phosphatase reaction was performed by the addition of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazorium.

1. Niwa, Y., Hirano, T., Yoshimoto, K., Shimizu, M. & Kobayashi, H. (1999) Plant J. 18, 455-463.

2. Watanabe, T., da Cruz e Silva, E.F., Huang, H.-B., Starkova, N., Kwon, Y.-G., Horiuchi, A., Greengard, P. & Nairn, A.C. (2003) Methods Enzymol. 366, 321-338.

3. Kinoshita, T., Emi, T., Tominaga, M., Shigenaga, A., Doi, M. & Shimazaki, K. (2003) Plant Physiol. 133, 1453-1463.