Proc Natl Acad Sci U S A. 2008 April 1; 105(13): 5271–5276

Intact Plant Transpiration Measurements. Plants were individually grown on compost in plastic containers in a growth chamber (21°C, 70% relative humidity, 8-h/16-h light/dark, 300 μE·m−2·s−1). Each container was closed by a screw cap to avoid evaporation from the compost surface. After sowing, each plant grew through a hole pierced in the middle of the cap. This experimental device ensured that water loss (decrease in weight) could be ascribed to leaf transpiration. Periodic addition of water into the container maintained the compost water content close to 75% (wt/wt). The transpirational water loss was expressed on leaf area basis, estimated from daily plant photographs using OPTIMAS 6.1 software. A controlled automated growth chamber [Phenopsis robot (28)] was used to impose variable micrometeorological conditions (light and VPD; the containers were weighed every 15 min). To study the effect of Na+ on transpiration, the plants were hydroponically grown in half-strength Hoagland solution using the same device (plastic containers) as described above for plants grown on compost. Na+ was added to the nutrient solution as a chloride salt.

Proc Natl Acad Sci U S A. 2008 April 1; 105(13): 5271–5276

Reverse Transcription Experiments. Total RNA was extracted from Arabidopsis leaves by using TRIzol reagent (Invitrogen). After conversion to first-strand cDNA, KAT2 and EF1α were amplified by PCR from the same amounts of cDNA by using the following couples of primers: KAT2-3000 (5′-gcgtcttagacgagttagctcgc-3′) and KAT2-3930 (5′-ccgtgaaataggtagacgttctgaacgattggg-3′) or EF1α-350 (5′-ccaccactggtggttttgaggctggtatc-3′) and EF1α-900 (5′-cattgaacccaacgttgtcacctggaag-3′).

Proc Natl Acad Sci U S A. 2008 April 1; 105(13): 5271–5276

Generation of the domneg-1 and kincless Mutants. Site-directed mutagenesis was performed on the KAT2 cDNA to replace the selectivity filter GlyTyrGlyAsp motif (hallmark of K+-selective channels) by ArgArgGlyAsp. The mutated cDNA, named domneg, was cloned downstream of the KAT2 promoter region (2.258 kb upstream from the initiation codon) into a binary vector (pBIB-HYGRO), and the resulting plasmid was introduced into Agrobacterium tumefaciens GV3010 (pMP90) strain as described (10). WT plants (Wassilevskija ecotype) and kat2-1 mutant plants were transformed by using the floral dip method (27). Selection on hygromycin allowed us to identify transformed lines and to obtain a fixed transgenic domneg-1 or kincless line, respectively.

Proc Natl Acad Sci U S A. 2008 April 1; 105(13): 5271–5276

Isolation of the T-DNA-Tagged Mutant kat2-1 Disrupted in the KAT2 Gene. The kat2-1 knockout line was obtained by PCR screening of 40,000 Arabidopsis thaliana T-DNA insertion mutants [Wassilevskija ecotype; library constructed by Institut National de la Recherche Agronomique, Versailles (27)]. A Southern blot with a probe targeting the T-DNA right border revealed a single insertion locus. The exact position of the T-DNA insertion was determined by sequencing the T-DNA flanking sequences. Plants homozygous for the disruption were selected by PCR in the F3 progeny.

Proc Natl Acad Sci U S A. 2008 April 1; 105(13): 5271–5276

Fig. 5. Disruption of inward K+ channel activity affects stomatal circadian rhythm and renders stomatal opening sensitive to the duration of the preceding photoperiods. (A and B) Effect on stomatal circadian rhythm. (A) Continuous recording of the transpiration rate in a single WT or kincless plant during 7 days. Black and white boxes under the curves indicate dark and light periods, respectively. Two photoperiods were suppressed, on days 4 and 5. (B) Enlargement from A (dotted boxed regions) highlighting stomatal preopening in darkness in the WT plant. (C and D) Sensitivity to the duration of the preceding photoperiods. (C) Transpiration rates recorded in 5-week-old WT or kincless plants exposed to an 8-h photoperiod since sowing. (D) Transpiration rates recorded in the same plants after exposure to a 3-h photoperiod during 2 days. Data are means ± SE; n = 15 plants per genotype. Proc Natl Acad Sci U S A. 2008 April 1; 105(13): 5271–5276

PLoS ONE. 2008; 3(6): e2491.

Protein extraction, cell fractionation, SDS-PAGE and western blottingFor cell fractionation 100 mg tissue of transiently transformed tobacco leafs were homogenised in liquid nitrogen and the homogenate was extracted in 2 ml homogenization buffer (25 mM MOPS, 0.1 mM MgCl2, 8 mM L-cysteine, 2.5 mM EDTA, 2× protease inhibitor mix (Roche), 250 mM sucrose; pH 7.8). The crude extract was cleared from debris by centrifugation (4000xg, 40 min, 4°C). The microsomal fraction was separated from the soluble fraction by ultracentrifugation (100,000xg, 30 min, 4°C). The pellet was washed three times in homogenization buffer supplemented with 0.05% Triton X-100 and resuspended in 50 μl SDS-PAGE sample buffer. The soluble fraction was mixed with SDS-PAGE sample (ratio: 21 v/v). For SDS-PAGE 18 μl of the soluble fraction and 10 μl of microsomal fraction were loaded. Western blot analysis and immunodetection were performed according to [61] using anti-GFP antibody (Roche, Switzerland) to detect GFP-AHK5, BRI1-GFP, ERS1-GFP and ARR4-GFP. An anti-mouse-AP conjugate (BioRad, UK) was used as secondary antibody.

Proc Natl Acad Sci U S A. 2008 April 1; 105(13): 5271–5276

Fig. 3. Inward K+ channel activity underlies stomatal responsiveness to changes in light, VPD, and CO2 conditions. (A) Rate of light-induced stomatal opening. The consequences of the absence of GCKin activity on leaf transpiration were then investigated by using a setup allowing to continuously monitor transpirational water loss in an intact plant (10). (Left) Typical recordings of transpiration in a WT or kincless plant during a whole nycthemeral period (8-h/16-h day/night). (Right) Time constants describing the increase in transpiration rate induced by light (derived by fitting the kinetics with monoexponential functions). Data are means ± SE; n = 4. For each of the four plants tested per genotype, the individual time constant integrated values derived from the recording of transpiration rate during at least five successive photoperiods. (B) Responsiveness to a decrease in VPD. An automated growth chamber [Phenopsis robot (28)] was used to impose rapid changes in VPD. (Left) WT or kincless plant transpiration (bottom) during a climatic scenario comprising (top) a dark-light transition (open arrow) 2 h before a sudden decrease in VPD. Data are means ± SE; n = 60. (Right) Kinetics of the increase in transpiration rate due to stomatal aperture readjustment during the first half hour (boxed region in Left) after the change in VPD. (C) Responsiveness to CO2 availability. (Left) Epidermal peel bioassays. Epidermal strips were peeled at the end of the dark period and incubated in the dark in 30 mM K+. Stomatal opening was induced by bubbling CO2-free air for 3 h. Stomatal aperture was measured just before (control values, two bars on the left) and after (two bars on the right) this 3-h treatment. (Right) In planta stomatal conductance measurement in WT and kincless leaves. Time courses of stomatal conductance in response to changes in CO2 concentration under dark (Upper) or light (Lower) conditions. Dark and light periods are indicated by black and white boxes under the graphs. The changes in CO2 concentration in the air flow are indicated (expressed in ppm) in the gray boxes. The decrease in CO2 concentration in the air flow under dark conditions mimics depletion of internal CO2 driven by photosynthetic activity (independent of light signal and photosynthesis). Proc Natl Acad Sci U S A. 2008 April 1; 105(13): 5271–5276.

Proc Natl Acad Sci U S A. 2008 April 1; 105(13): 5271–5276

Plant adaptation to fluctuating environment and biomass production are strongly dependent on guard cell potassium channels

Plant Physiol. 2009 February; 149(2): 1017–1027

Bacterial Growth Assay Plant inoculations and bacterial growth assays were performed as previously described (Tornero and Dangl, 2001; Yun et al., 2006). Briefly, 3-week-old plants were infected with bacterial suspensions (108 cfu/mL except where indicated) resuspended in 10 mm MgCl2, and Silwet L-77 (200 μL/L). The aerial part of plants growing in pots were submerged upside down in the bacterial solution for 30 s and then covered with a transparent lid. Two hours after inoculation, the lid was removed and the collected sample was considered as zero time point; samples were taken every 24 h over 4 d.

Plant Physiol. 2009 February; 149(2): 1017–1027

Assays of Bacterial Migration For the assays of bacterial migration across epidermal peels, X. campestris strains were transformed with the plasmid pRU1319, which expresses the green fluorescent protein (GFPuv; Allaway et al., 2001). Bacteria were cultured and processed as described above. Peels were floated on 10:10 buffer containing bacteria under the light. When extracts were used, resuspended bacteria were preincubated for 30 min with extracts. After 3 h, peels were briefly washed in 10:10 to avoid carryover of bacteria from the incubation medium and observed in a confocal laser-scanning microscope (Carl Zeiss LSM510-Axiovert 100 m, 488-nm excitation with argon laser line, and 505-nm long-pass emission).

Plant Physiol. 2009 February; 149(2): 1017–1027

Preparation of Extracts of Bacterial Culture Supernatants One hundred-milliliter cultures of Xanthomonas campestris strains were grown overnight in peptone, yeast, and malt extract medium. Bacteria were centrifuged for 30 min at 6,000g. The supernatant was transferred to a new centrifuge bottle and centrifuged for 90 min at 20,000g. The supernatant was transferred to 50-mL Falcon tubes and was extracted with one-third volume of ethyl acetate. Phases were separated by centrifugation for 15 min at 13,000g. The organic phase was evaporated using a Speedvac concentrator and was resuspended in 500 μL of water. For stomatal bioassays, 6 μL of extracts were used for every milliliter of incubation buffer.

Plant Physiol. 2009 February; 149(2): 1017–1027

Bacterial Strains Xcc strains 8004 (wild type; Daniels et al., 1984), 8523 (rpfFTn5lac; Tang et al., 1991), and 8557 (rpfCpUIRM504; Slater et al., 2000) were grown in peptone, yeast, and malt extract medium (Cadmus et al., 1976). Escherichia coli DH5α were grown at 37°C in Luria-Bertani medium (Sambrook et al., 1989). Pst DC3000 and mutant derivatives were cultured at 28°C in Luria-Bertani medium supplemented with appropriate antibiotics. All strains were grown overnight with the appropriate antibiotics. Bacteria were collected by centrifugation and resuspended in 10:10 buffer. The final bacterial concentration for stomatal bioassays was 108 cfu/mL for all strains. In coincubation experiments, 108 cfu/mL of each strain were added to the incubation buffer.

Plant Physiol. 2009 February; 149(2): 1017–1027

Stomatal Aperture Bioassays For all experiments, epidermal peels from the two or three youngest fully expanded leaves from 3- to 4-week-old, unbolted Arabidopsis plants were used. Unless otherwise stated, bioassays were performed in Col-0 cultivar. To measure promotion of stomatal closure, epidermal peels were floated in 10:10 buffer under light (under the same conditions as used previously for plant growth) for at least 2 h. Then ABA, LPS, and bacterial suspensions were added to the incubation medium, and peels were further incubated as indicated. For the inhibition of opening experiments, peels were floated in the dark in 10:0 buffer (10 mm MES-KOH, pH 6.15) for 2 h to promote stomatal closure. Peels were then transferred to 10:10 buffer containing ABA for a further 2 h and were subsequently placed on a microscope slide, where apertures of 40 stomata from each experiment were measured in a Carl Zeiss microscope (400×) with the aid of an eyepiece micrometer. Data are presented as the average from 80 to 120 aperture measurements per treatment, collected from two or three independent experiments. For V. faba stomatal bioassays, peels were obtained from mature leaves of 2- to 3-week-old plants. Assays were performed as described for Arabidopsis, except that CO2-free 10:10 buffer was used.

Plant Physiol. 2009 February; 149(2): 1017–1027

Plant Material Arabidopsis (Arabidopsis thaliana L. Heynh.) ecotype Col-0, Ler, and MPK3 guard cell-specific antisense line (Gudesblat et al., 2007) seeds were surface sterilized in 10% (v/v) commercial bleach with 0.01% (v/v) Tween 20 for 10 min and rinsed four times in sterile water. Seeds were plated in petri dishes containing one-half-strength Murashige and Skoog medium with 1% (w/v) Suc and 0.6% (w/v) agar. Seeds on plates were maintained in the dark for 2 to 3 d at 4°C to break dormancy before being transferred to a growth room at 22°C to 23°C, with a 12-h light photoperiod under light intensity of 90 μE m−2 s−1. After 5 or 6 d, seedlings were transferred to pots containing a mixture of vermiculite, peat, and perlite (1:1:1) and fertilized every 2 d. Vicia faba plants were grown in soil inside a growth room at 22°C to 23°C, with a 16-h light photoperiod.