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

Figure 1 Subcellular localisation of AHK5 in plant cells. (A) Confocal images of Arabidopsis protoplasts and tobacco (Nicotiana benthamiana) leaf cells transiently transformed with a construct expressing P35S:GFP-AHK5 cDNA. Left panel, GFP fluorescence; right panel, bright field image. The bars represent 10 μm. (B) Western blot showing the expression of full-length GFP-AHK5 in transiently transformed tobacco leaf cells using anti-GFP antibody. Lane 1, protein standard; lane 2, extracts from cells transformed with a P35S-GFP-AHK5 construct; lane 3, extracts from cells transformed with the empty vector. (C) Cell fractionation of transiently transformed tobacco leaf cells expressing either GFP-AHK5, the microsomal marker BRI-GFP, the ER marker ERS1-GFP or the soluble marker ARR4-GFP.Two days after the infiltration of the Agrobacteria the leaf tissue was harvested and total protein extracted. The microsomal fraction (M) and the soluble fraction (S) were separated by ultracentrifugation. Equal cell equivalents were loaded per lane. (D) Fluorescence intensity images (upper panel) and the corresponding intensity profiles (lower diagram) of the indicated plasmalemma-cell wall section (blue bar in the magnification) of two adjacent, transiently transformed tobacco leaf cells co-expressing GFP-AHK5 (green dots) and the plasma membrane marker pm-rk-CD3-1007 (red dots). The red line represents the mono-peak Gauss fit of RFP fluorescence and the green line the multi-peak Gauss fit of GFP fluorescence (green). The single fits which compose the multi-peak Gauss fit of GFP, are shown in black. PLoS ONE. 2008; 3(6): e2491.

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

Figure 2 Expression pattern of AHK5 and characterisation of the T-DNA insertion sites in ahk5-1 and ahk5-3. (A) Steady-state levels of AHK5 transcript in different tissues and developmental stages of Arabidopsis detected by semi-quantitative RT-PCR using different cycle numbers. For the detection of AHK5, up to 40 cycles of PCR were performed using primers 3 and 10 (left panel) or primers 8 and 9 and 40 cycles of PCR (right panel). GC, cDNA from guard cell-enriched non-treated leaf sample; GC+H, cDNA from guard cell-enriched leaf samples treated with 0.5 mM H2O2 for 2 h; WL, cDNA from whole-leaf sample. ACTIN and EF1 were used as controls. Primer numbers indicated in panel C. (B) Expression of a PAHK5-GFP-AHK5genomic construct in transiently transformed tobacco leaf cells. Agrobacteria carrying the PAHK5-GFP-AHK5 construct were infiltrated into the abaxial side of the leaf and the GFP fluorescence analysed by CLSM 2 days later. Top panels = GFP images, lower panels = GFP image overlaying bright field image. The scale bar represents 20 μm. Images shown from repeat experiments. (C) AHK5 gene structure, position of primers used for genomic and RT-PCR and T-DNA insertions in ahk5-1 (Col-0) and ahk5-3 (Ws4). (D) Analysis of AHK5 expression in seedlings of wild type, ahk5-1 and ahk5-3 mutant, P35S-AHK5 complemented and AHK5 over-expressing plants using the indicated primer pairs (see Supplementary data for sequences). PLoS ONE. 2008; 3(6): e2491.

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

As demonstrated by the wild type behaviour of both ahk5 mutants to ABA, the AHK5-dependent signalling pathway does not contribute to the ABA response pathway in guard cells.

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

Growth and maintenance of plants Wild type and mutant seeds of Arabidopsis thaliana ecotype Columbia (Col-0) and Wassilewskijia (Ws4) were sown on Levington's F2 compost and grown under a 16 h photoperiod (100–150 μE m−2 s−1), 22°C and 65% relative humidity in controlled environment growth chambers (Sanyo Gallenkamp, UK). atrbohD/F seeds were obtained from J Jones (Sainsbury Laboratory, Norwich, UK).]. Details of the T-DNA insertion lines in AHK5 are as follows: ahk5-1 mutant seeds (SAIL 50_H11) were originally obtained from Syngenta (SAIL collection, now available at ABRC/NASC), and the ahk5-3 seeds (FLAG_271G11) were obtained from the INRA/FLAG-FST collection at Versailles [29], [30].

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

Stomatal bioassaysStomatal assays were performed on leaves essentially as described in [15]. Leaves were floated for 2.5 h under continuous illumination (100–150 μE m−2 s−1) in Mes/KCl buffer (5 mM KCl/10 mM Mes/50 μM CaCl2, pH 6.15). Once the stomata were fully open, leaves were treated with various compounds for a further 2.5 h. The leaves were subsequently homogenised individually in a Waring blender for 30 s and the epidermal fragments collected on a 100 μm nylon mesh (SpectraMesh, BDH-Merck, UK). Stomatal apertures from epidermal fragments were then measured using a calibrated light microscope attached to an imaging system (Leica QWin software, Leica, UK). Flg22 and elf-26 peptides were a kind gift from J Mansfield (Imperial College London). Elicitors were added to the incubation buffer at 2.5 h and stomatal apertures measured after a further 3 h. For bacterial experiments, Pseudomonas syringae pv DC3000 or P. syringae hrpA− mutant were grown overnight in LB media and overnight cultures centrifuged, resuspended in 10 mM MgCl2 at an OD600=0.2 (equivalent to 2×108 cfu/ml). Silwet (0.002% v/v) was added to cultures or MgCl2 to act as a wetting agent. Bacteria were gently coated onto the abaxial side of leaves on intact plants (controls were MgCl2 with Silwet alone). Plants were left in the growth chambers with a covered lid (to increase humidity) for 3 h, inoculated leaves subsequently detached and stomatal apertures measured.

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

Measurement of H2O2 and NO using confocal/fluorescent microscopy Epidermal peels from mature leaves, prepared as described above, were incubated in Mes/KCl buffer for 2–3 h. Following this, the fragments were loaded by incubation in 50 μM of the H2O2-sensitive fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Molecular Probes, Leiden, The Netherlands) for 10 min. After washing in fresh buffer for a further 20 min, the fragments were challenged with various compounds as indicated in the figure legends. For dark treatments the peels were incubated in darkness for 30 min and microscopy performed. Confocal laser scanning microscopy was used to visualise fluorescence, using an excitation wavelength of 488 nm and an emission wavelength of 515–560 nm (Nikon PCM2000, Nikon Europe B.V. Badhoewvedorp, The Netherlands). Images were acquired and analysed using Scion Image software (Scion Corp., USA) to measure the relative fluorescence intensities in the cells following various treatments. For the data in Figure 7D, fluorescent microscopy (Zeiss Axioskop2, Zeiss, UK) was used with filter set 10 (excitation filter BP 450–490 nm, beam splitter FT 510 nm and emission filter BP 515–565 nm). Images were acquired and analysed using Image J software (NIH, USA). Data represent fluorescence intensities expressed as average fluorescence or as a percent of the control values, from several guard cells analysed in different experiments. For NO fluorescence, epidermal peels were loaded with 10 μM of the NO-sensitive dye diaminofluorescein diacetate (DAF2-DA, Calbiochem, UK) using exactly the same dye loading procedure, and images acquired using confocal microscopy as described above.

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

Transient transformation of tobacco leaf cells and Arabidopsis protoplasts, GFP and RFP analyses The p19 protein from tomato bushy stunt virus cloned in pBIN61 [60] was used to suppress gene silencing in tobacco (Nicotiana benthamiana). All plasmids were transformed in Agrobacterium tumefaciens strain GV3101 pMP90, which was grown in YEB medium to OD600 1.0 and prior to infiltration resuspended in AS medium (10 mM MgCl2, 150 μM acetosyringone and 10 mM MES pH 5.7) to OD600=0.8. The Agrobacterium strains containing the GFP or p19 construct were mixed in a 11 relationship and co-infiltrated into leaves of 4-week-old tobacco plants as described in [60]. The abaxial epidermis of infiltrated tobacco leaves was assayed for fluorescence by CLSM (confocal laser-scanning microscopy) 2 to 3 days post infiltration according to [61]. Arabidopsis protoplasts were transformed using PEG mediated transformation procedure and assayed for fluorescence by CLSM after 20 h [61]. CLSM was performed using a Leica TCS SP2 confocal microscope (Leica Microsystems, Germany). These CLSM images were obtained using the Leica Confocal Software and the HCX PL APO 63×/1.2 W CORR water-immersion objective. For recording the RFP and GFP intensity profiles a homemade confocal laser-scanning microscope, based on a Zeiss Axiovert was used [62], [63]. The microscope was equipped with an avalanche photodiode (APD, SPCM-AQR-14, Perkin Elmer, USA) as a spectrally integrating detector. A pulsed 473 nm diode laser (Picoquant LDH-P-C470) operating at a repetition rate of 10 MHz served as excitation source. Fluorescence intensity images were obtained by raster scanning the sample and detecting emission intensity for every spot on the sampled area. The setup was equipped with a 480 nm long pass filter (Semrock Razor Edge LP02-473RU-25) to block back-scattered excitation light, a 500 nm bandpass filter (Semrock BrightLine BL500/24) to detect GFP-fluorescence and a 590 nm bandpass filter (Semrock FF01-590/20-25) to detect RFP-fluorescence in front of the APD. The processing of the obtained fluorescence intensity images was accomplished with the WSxM software [64].

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.