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

Xanthomonas campestris pv campestris (Xcc) is a bacterial intravascular phytopathogen that is the causal agent of the black rot of crucifers. It has a broad host range that includes a majority of members of the Brassicaceae family. Whereas Xcc is widely considered to use hydathodes and wounds as preferential ways of entry into the leaf, it can also penetrate this organ through stomata (Buell, 2002). In Arabidopsis, Xcc 8004 can enter the leaf through both hydathodes and stomata, and the preferred route of entry depends both on the particular Arabidopsis ecotype and on environmental conditions (Hugouvieux et al., 1998). Because at least under some conditions and in certain ecotypes Xcc can enter Arabidopsis leaves through stomata, it is likely that Xcc possesses some mechanism to overcome stomatal defense.

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

Little is known about the signaling events downstream of PAMPs in guard cells leading eventually to promotion of stomatal closure. 一般用于introduction的后面，接下来阐述自己的观点和研究

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

Figure 8. Proposed model of the mode of inhibition of stomatal closure by Xcc. The DSF (diamonds), which mediates cell-to-cell communication, interacts with the cell surface receptor RpfC, which in turn triggers signaling that leads to transcription of genes required for the synthesis of the stomatal inhibitory compound (triangles). This molecule diffuses to the extracellular space and gets contact with stomatal guard cells, where it blocks stomatal closure induced by ABA or PAMPs by interfering with some signaling component within a putative signaling pathway branch including H2O2 and MPK3. MPK3 is absolutely required for bacterial or PAMP-induced promotion of closure pathway, as shown by the failure of MPK3 antisense plants to close stomata in response to these stimuli. For ABA, instead, other pathways, including ABA-induced cytosolic pH increase, would compensate for the absence of MPK3. The failure of the stomatal inhibitor to interfere with promotion of closure by ABA in MPK3 antisense plants could be explained by compensatory adjustments within the guard-cell-signaling network, indicated by thick arrows, that would make up for the absence of MPK3. WT, Wild type; LRR, Leu-rich repeat receptor. Plant Physiol. 2009 February; 149(2): 1017–1027.

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

Foliar bacterial phytopathogens initially colonize the leaf surface as epiphytes, but subsequently become endophytes as the infection progresses. Because bacteria cannot directly penetrate the leaf epidermis, endophytic colonization occurs through natural openings, such as hydathodes and stomata, or through accidental wounds. Stomata are small pores located in the leaf surface that allow plants to exchange gases with the environment. Stomatal apertures are finely regulated in response to hormones and environmental factors such as light intensity, air humidity, and CO2 concentration, which allow the plant to maximize CO2 intake required for photosynthesis, while minimizing water loss. Several internal and external stimuli, such as the hormone abscisic acid (ABA), low humidity, or a high concentration of CO2, can bring about stomatal closure through a reduction in turgor of the two guard cells that constitute the stomatal pore. This is achieved at least in part through the efflux of osmotically active ions from these cells (Schroeder et al., 2001; Pandey et al., 2007).

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

Pathogen-induced stomatal closure is part of the plant innate immune response. Phytopathogens using stomata as a way of entry into the leaf must avoid the stomatal response of the host.

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

Xanthomonas campestris Overcomes Arabidopsis Stomatal Innate Immunity through a DSF Cell-to-Cell Signal-Regulated Virulence Factor

Plant Physiol. 2008 June; 147(2): 922–930

Preparation of Epidermal Fragments and Determination of Stomatal Aperture Adiantum plants were kept in the dark for 16 h before the preparation of epidermal fragments to induce stomatal closing. Ten to 15 leaves were harvested and blended three times in cold water using a Waring blender for 30 s at full speed. The blender contents were filtered through a 200-μm nylon mesh, and the retained samples were washed with 5 mm MES, 50 mm KCl, and 0.1 mm CaCl2, pH 6.5. The resultant epidermal fragments were floated in the same solution in petri dishes and kept in the dark for 1 h. The epidermal fragments were irradiated for 2 h, then 1% (w/v) fluorescein diacetate in acetone was added at 0.1 μg mL−1 to determine the viability of the guard cells. Micrographs of over 50 viable stomata were obtained using a Nikon fluorescence microscope equipped with a CCD camera. The stomatal aperture surrounded by viable guard cells was defined as the width to length ratio of the stomatal pores.

Plant Physiol. 2008 June; 147(2): 922–930

Gas Exchange MeasurementsPhotosynthetic CO2 fixation and stomatal conductance were measured using a gas exchange system (LI-6400; LI-COR) equipped with an Arabidopsis (Arabidopsis thaliana) chamber (LI-COR) as previously described (Doi et al., 2004). Red light was provided from a tungsten lamp (MHF-G150LR; Moritex) by passing the light through a red cutoff filter (2-61; Corning) or from an LED Lamp Drive unit (SS-001; Shimatec) equipped with a red LED (WBS-Φ-30-16R, λ = 656 ± 18 nm). Blue light was provided by a metal halide lamp (LS-M250; Moritex) by passing the light through a blue filter (5-60; Corning) or an LED Lamp Drive unit equipped with a blue LED (WBS-Φ-30-16B, λ = 467 ± 19 nm). Green light was provided from a tungsten lamp by passing the light through a green interference filter (λ = 500 ± 26 nm; Optical Coatings Japan). Far-red light was provided from an LED Lamp Drive unit equipped with a far-red LED (WBS 720; λ = 723 ± 12 nm). PFD was measured using an LI-2500 light meter with an LI190SA quantum sensor (LI-COR) and an SKR 110 sensor (Skye Instruments). To administer DCMU to Adiantum leaves, we cut off the leaf with its petiole and placed the petiole in water under darkness. The next morning, the leaf was transferred to 0.05% ethanol solution that contained 50 μm DCMU and was kept under fluorescent light at 30 μmol m−2 s−1 for 1 h. Then, the Adiantum leaf was returned to the darkness for 3 h and used for the measurement.

Plant Physiol. 2008 June; 147(2): 922–930.

Figure 4. Dependencies of PFD for stomatal conductance increase and photosynthetic CO2 fixation. Solid and dotted lines represent the increase in stomatal conductance and photosynthetic CO2 fixation, respectively. Red light was irradiated onto the upper (black circles) or lower side (white circles) of the leaf. Plant Physiol. 2008 June; 147(2): 922–930.

Plant Physiol. 2008 June; 147(2): 922–930.

Figure 1. Stomatal responses to light in intact leaves of A. capillus-veneris. A, Stomatal conductance, photosynthetic rate, and Ci in response to red (left) and blue (right) light. Each light at an intensity of 600 μmol m−2 s−1 was applied to the upper surface of a leaf as indicated by the upward-pointing arrowhead, and turned off as indicated by the downward-pointing arrowhead. B, PFD response curves for stomatal opening. For each type of light, opening rates were measured at five to seven PFDs with separate leaves of the same plant. Circles, Blue light; squares, red light; triangles, green light. Black, gray, and white symbols represent the separate experiments that were conducted on the different days, respectively. Solid, broken, and dotted lines represent the regression curves for blue, red, and green lights, respectively. Plant Physiol. 2008 June; 147(2): 922–930.

Plant Physiol. 2008 June; 147(2): 922–930.

Adiantum capillus-veneris belongs to the Leptosporangiopsida, which are newly diversified fern species that grow in shadow beneath angiosperms (Schneider et al., 2004), and usually makes its habitat beneath the canopy, where the ambient light is weak and rich in far-red. The stomata are found only on the lower surface of leaves and lack subsidiary cells, and their guard cells contain densely arranged chloroplasts. Recently, we reported that the stomata of the fern A. capillus-veneris lacked a blue light-specific opening response but did open in response to red light (Doi et al., 2006). A lack of blue light-specific response has commonly been seen in other species of Leptosporangiopsida. We also showed that phy3 (neochrome 1; Suetsugu et al., 2005), a chimeric protein of phototropin and phytochrome, did not work as a photoreceptor for red light-induced stomatal opening in Adiantum. Therefore, the red light-induced stomatal opening in Adiantum could be explained by the photosynthetic activity and/or by the response via phytochrome. Here, we investigated the stomatal responses of Adiantum to light and CO2 in the dark. We demonstrated that the guard cells are insensitive to CO2 and that the guard cell chloroplasts are responsible for stomatal opening in response to light in this plant species.

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

Fig. 1. Absence of inward K+ channel activity in guard cells of the kincless mutant. (A) Representative macroscopic current traces recorded with the patch-clamp technique in guard cell protoplasts from WT (Upper Left), kat2-1 (knockout mutant disrupted in the KAT2 gene) (Upper Right), domneg-1 (expressing a dominant negative kat2 construct in WT background) (Lower Right), or kincless (expressing the same domneg construct in the kat2-1 background) (Lower Left) plants. In all recordings, the holding potential was −100 mV; voltage steps were applied to potentials ranging between −100 and +120 mV in 20-mV increments (top traces for each genotype) or between −100 and −260 mV in increments of −20 mV (bottom traces for each genotype). (B) Comparison of current–voltage relationships from the different genotypes. Total current (I tot, sampled at time marked by symbols over the recordings in A) is plotted against membrane potential. Data are means ± SE (with the number of repeats in brackets). 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

Stomatal Aperture Measurements. Epidermal peels were prepared from abaxial epidermis as described (10) and incubated in 30 or 10 mM KCl and 10 mM Mes-iminodiacetic acid (pH 6.5) at 20°C, unless otherwise noted. To standardize the initial state, the epidermal strips were kept in the incubation solution for 30 min in darkness. Then, they were exposed to treatments inducing stomatal aperture: white light (300 μE·m−2·s−1, 30 mM KCl), blue light (30 μE·m−2·s−1, 30 mM KCl), red light (80 μE·m−2·s−1, 30 mM KCl), fusicoccin (10 μM, 10 mM KCl), or CO2-free air (30 mM KCl). Effects of Cs+ or Na+ on stomatal behavior were investigated either in 30 mM CsNO3 and 10 mM KCl, using fusicoccin (10 μM) to trigger stomatal opening, or in 20 mM NaCl and 10 mM KCl, using white light (300 μE·m−2·s−1) to trigger stomatal opening. Stomatal apertures were measured (pore width; at least 40 measurements per epidermis for each experimental point) with an optical microscope (Optiphot; Nikon) fitted with a camera lucida and a digitizing table (Houston Instruments) linked to a personal computer.

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

Stomatal Conductance. Measurements of stomatal conductance were performed in intact leaves on intact plants using a Li-COR 6400 infrared gas analyzer-based gas exchange system (Li-COR). Leaves were kept at 75% ± 2% relative humidity and 22°C.

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

Transpiration and CO2 Assimilation in Individual Whole Plants. Plants were hydroponically grown in a growth chamber (22°C, 65% relative humidity, 8-h/16-h light/dark, 300 μE·m−2·s−1) for 4 weeks before being transferred (a single plant per experiment) to an experimental chamber allowing continuous gas exchange measurements (as described in ref. 10).