Plant Physiol. 2007 February; 143(2): 1068–1077.

Oil Treatment of PoresThe cuvette allows micromanipulation on the lower leaf surface by micropipettes inserted through small holes in the cuvette wall. Micropipettes were drawn from 1.5-mm borosilicate glass capillaries with a pipette puller (L/M-3P-A; List). Tips were ground to a diameter of approximately 5 μm. Mineral oil (M8662; Sigma-Aldrich) was applied to pores by approaching the oil-filled pipette to a pore and gently pressurizing it manually by pressing a rubber ball. In each experiment, six to 20 pores with a spacing of at least 2 mm were selected. Either the observed pore or the observed pore plus the adjacent pores was sealed with oil. In an additional experiment, which was only used for the model parameter estimation, only the adjacent pores were sealed (leaving the observed central pore free). In all experiments, a control sample of the same size as the treatment sample was observed to detect and correct for interday variability, which, however, was found to be small.

Plant Physiol. 2007 February; 143(2): 1068–1077.

Plant Culture and Experimental SetupExperiments were performed on attached leaves of potted Sambucus nigra plants of approximately 50 to 80 cm in size. Plants were drawn from cuttings and cultivated in 40-cm pots in a climatic chamber at a PPFD of 220 μmol m−2 s−1 (16-h light/8-h dark) and a temperature of 20°C. Plants were amply supplied with water and nutrients. Stomatal movements were observed on mature leaves in a gas-exchange chamber designed for simultaneous measurement of CO2-water gas exchange and microscopic observation of stomatal movements under controlled light, humidity, temperature, and CO2 conditions (Kaiser and Kappen, 2001). Temperature, leaf-to-air mole fraction of water vapor (ΔW) and [CO2] in the cuvette were set to 20°C to 22°C, 2 mmol mol−1, and approximately 360 μmol mol−1, respectively. Irradiance (500 μmol m−2 s−1, 16-h light/8-h dark) was provided by a fiber-optic illuminator (Kaltlicht-Fiberleuchte FL-400 with Spezial Fiberoptik 400-F; Walz). After mounting the leaf in the gas-exchange cuvette, the plant was allowed to adjust to measuring conditions for at least 24 h. Leaves were fixed with the adaxial side to a Perspex plate with double-sided transparent adhesive tape (Tesa 56661–2; Tesa) to allow micromanipulation. Subsequently, the plate was mounted inside the cuvette, which allows observation of the lower leaf surface with a long-distance microscope lens (50×) led through the bottom of the gas-exchange cuvette (Kaiser and Grams, 2006). The microscope (Axiovert 25CFL; Zeiss) is mounted on a motorized translation stage, which allows repositioning of samples of selected stomata. Digital images of stomata were recorded with a video camera, digitized, and stored for subsequent measurement of aperture with custom image analysis software. The aperture of oil-treated pores can no longer be measured due to refraction of the oil. In these experiments, the area of the guard cell pair between the anticlinal walls was measured. This measure is linearly related to aperture when pores are open (Kaiser and Kappen, 2001) and can be taken as a surrogate measure of stomatal opening. Pore area or guard cell pair area were converted to circularity (c = width × 100/length) to allow comparison between differently sized stomata. The humidity control by a bypass compensation system was used to perform quick changes in air humidity from ΔW = 2 to approximately 18 mmol mol−1 by switching the humidity of the incoming air to lower humidity. Within approximately 90 s, ΔW arrived at its new steady state (Fig. 3). One hour before increasing ΔW, [CO2] was reduced to approximately 60 to 70 μmol mol−1, which is approximately the CO2 compensation point for C3 plants (von Caemmerer and Farquhar, 1982). This avoids intercellular [CO2] gradients due to locally suppressed CO2 diffusion into the mesophyll. Stomatal responses to a reduction in air humidity were observed before and after the treatment with oil or adhesive foil on subsequent days, always beginning at the same time 4 h after illumination was switched on. Stomatal apertures were observed from at least 30 min before to 1.5 h after reduction of air humidity. Images were taken every 3 to 4 min if apertures changed fast, otherwise at longer intervals of up to 10 min.

Plant Physiol. 2007 February; 143(2): 1068–1077.

Plant water loss is tightly balanced with water uptake to maintain beneficial water status. The most important control on water transport is the change of stomatal aperture, which governs water diffusion from the leaf interior to the atmosphere, as well as the opposite flow of carbon dioxide (CO2) into the photosynthesizing mesophyll. To balance transpiration and photosynthesis, guard cells may sense and integrate many environmental as well as physiological signals related to photosynthesis, the transpirational demand of the atmosphere, and the plant's current hydraulic status (Buckley, 2005; Roelfsema and Hedrich, 2005). Atmospheric humidity is one of the key environmental signals that stomata need to sense to adjust water loss. The sensing mechanisms involved in the stomatal humidity response are nonetheless still not identified.

Plant Cell. 2008 January; 20(1): 75–87

The Arabidopsis Small G Protein ROP2 Is Activated by Light in Guard Cells and Inhibits Light-Induced Stomatal Opening

Plant Physiol. 2007 February; 143(2): 745–758

Germination and Root Elongation Tests For germination tests, seeds harvested from the same batches were cold pretreated and then grown on modified Murashige and Skoog medium supplemented with Glc, Suc, NaCl, ABA, glufosinate ammonium, or fluridone at various concentrations listed in the “Results.” The medium was autoclaved and cooled to 50°C to 60°C prior to the addition of filter-sterilized ABA, glufosinate ammonium, or fluridone. For root elongation experiments, cold-pretreated seeds from different genotypes were first grown on agar plates with 1% Suc for 4 or 5 d, then uniform seedlings of similar size and primary root length were transferred to appropriate fresh medium and grown vertically for another 6 or 7 d.

Plant Physiol. 2007 November; 145(3): 853–862.

Figure 2. Localization of suberin and ABA in tumors. A, Four-week-old tumor, induced by A. tumefaciens (strain C58) at an inflorescence stalk of Arabidopsis. The arrow indicates the section plane of the cross sections shown in B to I. B to D, ABA immunolocalization in tumor cross sections using a primary mouse hybridoma monoclonal antibody against ABA and a secondary antibody labeled with the green Alexa 488 chromophore. Images were taken with a confocal laser scanning microscope and show overlays of images taken in a confocal and differential interference contrast mode. B, Fluorescence signals were strongest around vascular tissue (vascular, vs) of the tumor/host inflorescence stalk interface. C, ABA immunofluorescence at cellular resolution in thin cytoplasmic layers of cells near the xylem vessels (xy-ve). D, Control cross section of a tumor, treated with 1% (w/v) rabbit serum and the secondary Alexa 488 antibody conjugate in the absence of the primary antibody against ABA, revealed no significant fluorescence signal. E to G, Images from bright-field microscopy. H and I, UV illuminated (488 nm) cross sections. E, Cross section of a tumor (tu) with a disrupted epidermis attached to an inflorescence stalk (stalk). The reddish color of the outer tumor cell layers marks Sudan-III-stained cells indicating suberin. Note that the stalk is covered by an intact epidermis, containing a cuticle (arrows). F, Closeup of outer cell layers from the tumor, attached to the host inflorescence stalk as shown in E with suberized cell walls (red) and of outer cell layers (G) from the host inflorescence stalk shown in E with a cuticle (red). H, Strong autofluorescence indicates aromatic compounds of lignified xylem vessels and outer cell layers of the tumor. I, Closeup of outer cell layers from the border between the tumor and inflorescence stalk shown in H. Autofluorescence marks cell walls of cells at the tumor surface but not at the surface of the stalk (H, white arrows). Bars: A, 5 mm; B, 100 μm; C, D, F, G, and I, 50 μm; E and H, 200 μm. Plant Physiol. 2007 November; 145(3): 853–862.

Plant Physiol. 2006 January; 140(1): 302–310

RNA Isolation, RT-PCR, and Real-Time PCRTotal RNA was prepared from plants by using the RNeasy plant minikit (Qiagen). Two micrograms of RNA was used as a template for first-strand DNA synthesis using the SuperScript II first-strand synthesis system for RT (Invitrogen). PCR amplification was performed using Taq DNA polymerase (Invitrogen). Real-time quantitative PCR was run on an ABI 7500 real-time PCR system (Applied Biosystems) according to the manufacturer's recommendations. Real-time quantitative PCR reaction contained 25 μL 2× SYBR Premix Ex Taq (TaKaRa), 2 μL primer mix, 1 μL 50× ROX Reference Dye II, 4 μL cDNA, and 18 μL deionized water to make a total volume of 50 μL. After setting the amplification conditions, experiments were repeated twice. The primers used were as follows: ABA2 (At1g52340), 5′-ctcgctttggctcatttgc-3′ and 5′-ccgtcagttccaccccttt-3′; NCED3 (At3g14440), 5′-ccggtggtttacgacaagaa-3′, and 5′-cccaagcgttccagagatg-3′; and Actin2 (At3g18780), 5′-gctgagagattcagactgccca-3′ and 5′-cacagttttcgcgatccagac-3′. For relative quantification the method of Pfaffl (2001) was used to determine the relative expression ratio. This determines the expression of the target gene relative to reference gene (ACTIN2) in a test sample compared with an untreated Col sample.

Plant Physiol. 2006 January; 140(1): 302–310

Hormonal and Sugar Treatment The effects of ABA on seed germination were studied by determining the germination rates of 70 to 100 seeds pretreated with deionized water or 100 μm fluridone and planted in triplicate on medium containing ABA (mixed isomers; Sigma). The effects of Glc, Suc, mannitol, and sorbitol were studied in a similar manner. Sterilized seeds were stratified at 4°C for 48 h and sown on plates containing different concentrations or types of sugars (d-Glc, Suc, d-mannitol, and d-sorbitol) in triplicate. For direct comparisons of germination rates, each plate was subdivided and all seed lines were planted on the same plate.

Plant Physiol. 2007 November; 145(3): 853–862.

Plant Material and Tumor Induction Plant cultivation and tumor induction were performed as previously described (Deeken et al., 2006). Wild-type Arabidopsis (Arabidopsis thaliana; ‘Wassilewskija’, ‘WS-2’; Col-0 Heynh.; Landsberg erecta) and mutant plants (aba3-1, abi1-1, abi2-1, abi3-1, abi4-1, abi1-1R4, and abi1-1R5) were inoculated with the nopaline-utilizing Agrobacterium tumefaciens strain C58noc (nopalin catabolism construction; no. 584; Max-Planck-Institute for Plant Breeding Research).

Plant Physiol. 2007 February; 143(2): 745–758

RT-PCRTotal RNA was extracted from wild type, aba2, and ABA2 overexpression lines using TRIzol reagent (Invitrogen). Six micrograms of total RNA of each genotype with 1 μg oligo(dT) primer (Invitrogen) were heated at 70°C for 5 min and then chilled on ice immediately. RNA was then subjected to RT with reverse-transcriptase Avian myeloblastosis virus (Roche) at 42°C for 1 h according to the manufacturer's protocol. Synthesized cDNA was used as a template for PCR.

Plant Physiol. 2007 February; 143(2): 745–758

GUS Activity AssayCold-pretreated transgenic seeds harboring the ABA2GUS transgene were grown on agar plates or in soil for different time periods, depending on experiments, and then subjected to various stresses (cold, drought, or salinity). The treated plants were harvested and ground with extraction buffer (50 mm NaHPO4 [pH 7.0], 10 mm β-mercaptoethanol, 10 mm Na2EDTA, 0.1% sodium lauryl sarcosine, 0.1% Triton X-100). After centrifugation at 13K rpm for 10 min at 4°C, the supernatants were removed for further GUS assay, essentially according to the protocol of Jefferson et al. (1987), using a DyNA Quant 200 fluorometer (Amersham-Pharmacia Biotech).

Plant Physiol. 2007 February; 143(2): 745–758

NaCl and Dehydration TreatmentsWild type, aba2, and ABA2 overexpression seeds with cold pretreatment were grown on agar plates with various NaCl concentrations for 18 or 28 d. The ratios of bleached to total plants were counted to define tolerance to salinity. Seeds with cold pretreatment were also grown in soil for 3 weeks; then plants were subjected to watering with solutions containing four gradually increased concentrations of NaCl (50, 100, 150, and 200 mm), each for 4 d for a total of 16 d (Shi et al., 2003). For dehydration, seeds with cold pretreatment were grown in soil for 21 d and then plants were excised. The aerial parts of tissues were placed on plastic weighing boats and kept in an electronic dry box (model-DX-76; Taiwan Dry Tech Corp.) with approximately 40% relative humidity. The fresh weights of tissues were measured at 0-, 1.5-, and 3-h time points.

Plant Physiol. 2007 February; 143(2): 745–758

Preparation of Low-Water-Potential Medium Plates One-half-strength modified Murashige and Skoog medium (pH 5.7) with 0.7% Phyto agar (Duchefa Biochemie B.V.) was autoclaved. The sterilized medium was cooled to 50°C to 60°C and then aliquoted into petri dishes (100- × 20-mm depth), 20 mL each, for solidification. PEG-infused plates were made by dissolving PEG-8000 (Sigma) powder into one-half-strength Murashige and Skoog solution (pH 5.7) with the above-mentioned components, except phyto agar, and then filter sterilized; this PEG solution was then overlaid on agar-solidified medium at a ratio of 3:2 (v/v) and equilibrated overnight (≥12 h). The excess PEG solution was then removed. The procedure essentially followed the protocol of Verslues and Bray (2004).

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

Guard Cell Protoplast Isolation and Electrophysiological Recordings. Plants were grown on compost for 5 weeks in a greenhouse. Guard cell protoplasts were enzymatically isolated, and patch-clamp experiments were carried out as described (10). The pipette solution contained 100 mM K-glutamate, 2 mM MgATP, 5 mM EGTA, 1 mM CaCl2 (50 nM free Ca2+), 0.5 mM MgCl2, 300 mM d-mannitol, and 20 mM Hepes-KOH (pH 7.25). The bath solution contained 20 mM CaCl2, 2 mM MgCl2, 100 mM K-glutamate, 225 mM d-mannitol, and 10 mM Mes-HCl (pH 5.5). Liquid junction potentials at the pipette–bath interface were measured and corrected.

Plant Physiol. 2007 February; 143(2): 745–758

Transgene Constructs and Transgenic Plant Isolation For overexpression, ABA2 (GIN1) full-length cDNA was amplified by RT-PCR and cloned into pGEM-T Easy vector (Promega), followed by subcloning into the binary vector (pSMAB704) driven by a constitutive 35S promoter (35SABA2). For tissue-specific expression and GUS assay, the ABA2 promoter, approximately 2.9 kb upstream of the ATG start codon, was amplified by PCR and fused to a GUS coding region to generate ABA2GUS in the pSMAB704 binary vector. Transgene constructs were confirmed by sequencing and subsequently transformed into wild type or the aba2 mutant (T0) by use of the floral-dip method (Clough and Bent, 1998). T1 seeds with cold pretreatment were screened on 1% Suc agar plates containing 25 mg/L herbicide, glufosinate ammonium. At least 10 homozygous lines resistant to herbicide were obtained at the T3 or T4 generation. Three homozygous lines were randomly chosen for further study.