1. OST1 is Limiting in ABA Responses of Arabidopsis Guard Cells Biswa R. Acharya 1, Byeong Wook Jeon 1, Wei Zhang 1, 2* and Sarah M. Assmann 1, * *Corresponding author (WZ): Tel: (86)-531-88364332 e-mail: weizhang@sdu.edu.cn *Corresponding author (SMA): Tel.: (814)863-9579 FAX: (814) 360-1089 e-mail: sma3@psu.edusma3@psu.edu Supporting Information

2. Fig. S1. Identification of C-terminal FLAG peptide-tagged transgenic OST1 overexpressing Arabidopsis thaliana lines. Expression status of indicated transgenic lines was determined by RT-PCR analysis. Forward primer specific to OST1 (N-terminus region) and reverse primer specific to the region encoding for C-terminal FLAG peptide-tag of OST1-FLAG were used. Actin gene specific primers were used as internal control. Empty vector transgenic plants (EV) and Ler were used as negative controls Actin Ler OV1OV2 OV3OV4OV5 OV6OV7OV8OV9 EV1EV3 OST1-FLAG

3. 0 1 µM10 µM50 µM closure Aperture (µm) 0 1 µM 10 µM 50 µM Aperture (µm) opening a b Fig. S2. ABA inhibition of light induced stomatal opening and ABA promotion of stomatal closure in Arabidopsis thaliana wild type (Ler) and empty vector lines (EV1 and EV3). Data are means ± SE of three independent replicates. In each replicate, > 130 stomata were measured for each genotype per treatment. The means of the stomatal apertures of EV lines are not significantly different from those of wild type (Ler), as assessed by two- sample t-test (P > 0.05). (a) ABA inhibition of stomatal opening in wild type and EV control lines. (b) ABA promotion of stomatal closure in wild type and EV control lines.

4. Aperture (µm) Time (h) a b

5. Fig S3. Time courses of stomatal aperture changes induced by 10 µM ABA in Arabidopsis thaliana wild type (Ler), two empty vector control lines (EV1 and EV3), two OST1 overexpressing lines (OV1 and OV4) and two ost1 mutants (ost1-1 and ost1-2). For opening experiments, the intact leaves of each genotypes were floated on opening solution and kept in darkness for 2.5 h. After 2.5 h, leaves were illuminated with white light at 450 µmol m -2 sec -1. Two leaves each were taken at indicated time points for measurement of stomatal aperture. For closure experiments, the intact leaves of each genotypes were floated on closing solution and illuminated for 2.5 h to induce stomatal opening. After 2.5 h illumination, leaves were treated with 10 µM ABA. Two leaves each were taken at indicated time points for measurement of stomatal aperture. Data are means ± SE of three independent replicates. In each replicate, > 180 stomata were measured for each genotype per treatment. (a) ABA inhibition of light-induced stomatal opening (b) ABA promotion of stomatal closure. At 0.5 h (↑), the means of the stomatal apertures of OV1 and OV4 are statistically different (two-sample t-test) from those of wild-type (Ler) (P 0.05).

6. a b Fig. S4. ABA inhibition of whole cell K + in currents in guard cells of Arabidopsis thaliana wild type (Ler) and empty vector lines (EV1 and EV3). (a) Typical whole-cell recordings of K + currents with or without 50 µM ABA treatment. (b) I/V curves (mean ± SE) of time-activated whole-cell K + currents indicated in (a). Numbers of guard cells analyzed were: Ler (8), Ler + ABA (7), EV1 (7), EV1 + ABA (6), EV3 (6), EV3 + ABA (7).

7. Fig. S5. Arabidopsis thaliana KAT1 does not interact with AtrbohD. Protein-protein interaction between KAT1 and AtrbohD was performed by BiFC assays in epidermal cells of Nicotiana benthamiana. KAT1-Venus N was co-expressed with Venus C -AtrbohD (by cotransformation method). A representative confocal image does not show fluorescence from BiFC, which indicates that KAT1 does not interact with AtrbohD. Similar results were obtained in more than three replicates. (BiFC: BiFC signal; Chl: chlorophyll autofluorescence) BiFCChl BiFCChl

8. ab Fig. S6. ABA activation of whole cell anion currents in guard cells of Arabidopsis thaliana wild type (Ler) and empty vector lines (EV1 and EV3). (a) Typical whole-cell recordings of anion currents (Ler, EV1 and EV3) with or without 50 µM ABA treatment. (b) I/V curves (mean ± SE) of time-activated anion currents as indicated in (a). Numbers of guard cells analyzed were: Ler (7), Ler + ABA (8), EV1 (6), EV1 + ABA (7), EV3 (7), EV3 + ABA (8).

9. ab Fig. S7. ABA activation of guard cell anion currents is impaired in Arabidopsis thaliana ost1 mutants under weak pH buffered pipette solution condition. (a) Typical whole-cell anion currents in wild type and ost1 mutant guard cells with or without 50 µM ABA. Time and current scales as shown in top left panel apply to all traces. (b) I/V curves (mean ± SE) of time-activated anion currents as recorded in (A). Numbers of guard cells analyzed were: Ler (7), Ler + ABA (9), ost1-1 (7), ost1-1 + ABA (9), ost1-2 (9), ost1-2 + ABA (10).

10. Fig. S8. Arabidopsis thaliana SLAC1 does not interact with AtrbohF. Protein-protein interaction between SLAC1 and AtrbohF was performed by BiFC assay in epidermal cells of Nicotiana benthamiana. SLAC1-Venus N was coexpressed with Venus C -AtrbohF (by cotransformation method). A representative confocal image does not show fluorescence from BiFC, which indicates that SLAC1 does not interact with AtrbohF. Similar negative results were obtained in more than three replicates. (BiFC: BiFC signal; Chl: chlorophyll autofluorescence) BiFCChl BiFC

11. Fig. S9. ABA activation of whole cell Ba 2+ currents through Ca 2+ -permeable channels in guard cells of Arabidopsis thaliana wild type (Ler) and empty vector lines (EV1 and EV3). (a-c) Typical whole-cell recordings of Ba 2+ currents (Ler, EV1 and EV3) with or without 50 µM ABA treatment. (d) I/V curves (mean ± SE) of time-activated Ba 2+ currents as indicated in (A-C). Numbers of guard cells analyzed were: Ler (9), Ler + ABA (9), EV1 (8), EV1 + ABA (7), EV3 (9), EV3 + ABA (7). a b c d

12. Fig. S10. ABA-induced ROS production in guard cells of Arabidopsis thaliana wild type (Ler) and empty vector lines (EV1 and EV3). (a) Representative images of ROS production in response to 50 µM ABA in indicated genotypes. (b) Kinetics of ROS production in guard cells after treatment with 50 µM ABA (final concentration) or with ethanol (solvent control). Results are the averages ± SE (n > 6) from at least 3 independent experiments; Ler + ABA (n=10), EV1 + ABA (n=11), EV3 + ABA (n=12), Ler (n=13), EV1 (n=12), and EV3 (n=10). EV3EV1Ler 0 10 min 30 min a Fluorescence (% of initial) Time (min) Ler Ler + ABA EV1 EV1 + ABA EV3 EV3 + ABA 100 120 140 160 05101520 2530 b

13. Fig. S11. Arabidopsis thaliana KAT1 does not interact with AtrbohF. Protein-protein interaction between KAT1 and AtrbohF was performed by BiFC assay in epidermal cells of Nicotiana benthamiana. KAT1-Venus N coexpressed with Venus C -AtrbohF (by cotransformation method). A representative confocal image does not show fluorescence from BiFC, which indicates that KAT1 does not interact with AtrbohF. Similar negative results were obtained in more than three replicates. (BiFC: BiFC signal; Chl: chlorophyll autofluorescence) BiFCChl

14. KAT1-CubKAT2-Cub OST1-Nub G __ Nub G -OST1__ Nub wt -OST1 (positive control) +++++ (Nub G Vector) (Negative Control) __ Table S1. Interaction analyses between Arabidopsis thaliana OST1 and K + channel proteins, KAT1 and KAT2, in yeast (mbSUS assay). Protein-protein interaction analysis between Nub-fusion proteins of OST1 (OST1-Nub G and Nub G -OST1) and Cub-fusion protein of K + channel proteins. Growth phenotypes of yeast on interaction-selective media (minimal media containing 150 µM methionine). -: No yeast growth (no interaction); ++: Moderate yeast growth (moderate positive interaction); +++: Heavy yeast growth (heavy positive interaction). Note that Cub fusions are always on the C- terminus of the protein, while two different Nub G constructs: C-terminal (X-Nub G ) and N- terminal (Nub G -X) are used.

15. Video S1. ROS imaging in a guard cell pair of Arabidopsis thaliana wild type (Ler) in response to 50 μM ABA. For all videos, reactive oxygen species production in Arabidopsis guard cells was monitored using the dye 2′,7′-dichlorofluorescin diacetate (H 2 DCF-DA, Invitrogen, USA) (Murata et al., 2001) and analyzed by image J software (NIH, Bethesda, MD, USA) as earlier (Zhang et al., 2011). Leaves from Arabidopsis plants (indicated genotype) were incubated for 3 h in 30 mM KCl and 10 mM MES-KOH, pH 6.5 under white light (150 µmol m -2 sec -1 ). After incubation, leaves were cut into slices (less than 3 mm 2 ). Sliced leaves were then incubated with H 2 DCF- DA at a final concentration of 30 µM for 20 min under dark. After 20 min, excess dye was removed by three washes with distilled water. Leaf slices were then mounted on to an incubation chamber with medical adhesive (Hollister, Libertyville, IL). Samples were then incubated for the indicated time with 50 µM ABA or with an equal volume of ethanol added as a solvent control. Guard cells were then observed with a laser scanning confocal microscope (LSM 510 Meta; Carl Zeiss, Thornwoood, NY, USA) using a C-Apochromat 40×/1.2 W corr water immersion objective. H 2 DCF-DA was excited by the 488-nm line of the argon laser at 4.9 A tube current and 99% attenuation (equal to 1% laser intensity). The fluorescence of H 2 DCF was detected by a bandpass emission filter (500-550 nm). Time-lapse imaging of guard cell pairs was performed at 30 sec interval for 30 min. Movie files were generated using Image J software.

16. Video S2. ROS imaging in a guard cell pair of Arabidopsis thaliana ost1-1 mutant in response to 50 μM ABA. Video S3. ROS imaging in a guard cell pair of Arabidopsis thaliana ost1-2 mutant in response to 50 μM ABA. Video S4. ROS imaging in a guard cell pair of Arabidopsis thaliana empty vector line EV1 in response to 50 μM ABA. Video S5. ROS imaging in a guard cell pair of Arabidopsis thaliana empty vector line EV3 in response to 50 μM ABA. Video S6. ROS imaging in a guard cell pair of Arabidopsis thaliana OST1 overexpressing line OV1 in response to 50 μM ABA. Video S7. ROS imaging in a guard cell pair of Arabidopsis thaliana OST1 overexpressing line OV4 in response to 50 μM ABA. Video S8. ROS imaging in a guard cell pair of Arabidopsis thaliana wild type (Ler) in response to ethanol. Video S9. ROS imaging in a guard cell pair of Arabidopsis thaliana ost1-1 mutant in response to ethanol. Video S10. ROS imaging in a guard cell pair of Arabidopsis thaliana empty vector line EV1 in response to ethanol. Video S11. ROS imaging in a guard cell pair of Arabidopsis thaliana OST1 overexpressing line OV1 in response to ethanol.