Measures of stomatal size, density, and index in Kl-4, Ga-0, Chi-1, and Col-0.

<p>(A) Guard cell lengths, which were used as a measure of stomatal size. (B) Stomatal densities. (C) Stomatal index. Data presented are means ± SE (<i>n</i> = 3). No significant differences were observed between the four ecotypes.</p

Stomatal conductance in <i>Arabidopsis</i> ecotypes that have demonstrated a low CO₂ responsiveness.

<p>The plants were exposed to 0 ppm CO₂ for 2 h and then transferred to 1,000 ppm CO₂ for 1.5 h at 40% RH. Relative conductance levels (Relative <i>g</i>_<i>s</i>) were calculated as (<i>g</i>_<i>s</i> at 1,000 ppm CO₂)/(<i>g</i>_<i>s</i> at 0 ppm CO₂); large values represent small responses. Data presented are means ± SE (<i>n</i> = 3). The commonly used model ecotype Col-0 is highlighted in red and three particularly unresponsive ecotypes that were selected for further experiments are shown in yellow.</p

Relative sensitivities to environmental factors among Kl-4, Ga-0, Chi-1, and Col-0.

<p>The diagram summarizes the relative sensitivities of Kl-4, Ga-0, Chi-1, and the reference ecotype Col-0 to CO₂, light, humidity, and ABA. Kl-4 and Col-0 show similar levels of sensitivity to changes in RH. Chi-1 and Ga-0 show similar levels of sensitivity to ABA.</p

Natural Variation in Stomatal Responses to Environmental Changes among <i>Arabidopsis thaliana</i> Ecotypes

<div><p>Stomata are small pores surrounded by guard cells that regulate gas exchange between plants and the atmosphere. Guard cells integrate multiple environmental signals and control the aperture width to ensure appropriate stomatal function for plant survival. Leaf temperature can be used as an indirect indicator of stomatal conductance to environmental signals. In this study, leaf thermal imaging of 374 <i>Arabidopsis</i> ecotypes was performed to assess their stomatal responses to changes in environmental CO2 concentrations. We identified three ecotypes, Köln (Kl-4), Gabelstein (Ga-0), and Chisdra (Chi-1), that have particularly low responsiveness to changes in CO2 concentrations. We next investigated stomatal responses to other environmental signals in these selected ecotypes, with Col-0 as the reference. The stomatal responses to light were also reduced in the three selected ecotypes when compared with Col-0. In contrast, their stomatal responses to changes in humidity were similar to those of Col-0. Of note, the responses to abscisic acid, a plant hormone involved in the adaptation of plants to reduced water availability, were not entirely consistent with the responses to humidity. This study demonstrates that the stomatal responses to CO2 and light share closely associated signaling mechanisms that are not generally correlated with humidity signaling pathways in these ecotypes. The results might reflect differences between ecotypes in intrinsic response mechanisms to environmental signals.</p></div

The phenotype of Kl-4, Ga-0, and Chi-1, which exhibit low CO₂ responsiveness.

<p>(A) Thermal imaging of the three selected ecotypes Kl-4, Ga-0, Chi-1, and the commonly used ecotype Col-0. Plants were subjected to 0 ppm CO₂ for 2 h and then 1,000 ppm CO₂ for 1 h at 40% RH. The subtractive image on the right shows that the largest temperature changes were exhibited by Col-0. (B) Time courses of stomatal conductance (<i>g</i>_<i>s</i>) in response to changes in CO₂ concentration in Kl-4, Ga-0, Chi-1, and Col-0. Col-0 is more responsive to changes in CO₂ concentration than Kl-4, Ga-0, Chi-1. (C) Sizes of stomatal apertures at low and high CO₂ concentrations. Plants were subjected to 0 ppm CO₂ for 2 h and then transferred to 700 ppm CO₂ for 1 h at 40% RH with 150 μmol m^-2 s^-1 photosynthetically active radiation. (D) The relative changes in stomatal aperture (relative stomatal aperture) were calculated as (stomatal aperture in 0 ppm CO₂)/(stomatal aperture in 700 ppm CO₂). Large values represent small responses. Data presented are means ± SE (<i>n</i> = 60) of five independent experiments. Significant differences from Col-0 at <i>p</i> < 0.05 (Student’s t test) are indicated by asterisks.</p

An S-Type Anion Channel SLAC1 Is Involved in Cryptogein-Induced Ion Fluxes and Modulates Hypersensitive Responses in Tobacco BY-2 Cells

<div><p>Pharmacological evidence suggests that anion channel-mediated plasma membrane anion effluxes are crucial in early defense signaling to induce immune responses and hypersensitive cell death in plants. However, their molecular bases and regulation remain largely unknown. We overexpressed Arabidopsis <i>SLAC1</i>, an S-type anion channel involved in stomatal closure, in cultured tobacco BY-2 cells and analyzed the effect on cryptogein-induced defense responses including fluxes of Cl⁻ and other ions, production of reactive oxygen species (ROS), gene expression and hypersensitive responses. The SLAC1-GFP fusion protein was localized at the plasma membrane in BY-2 cells. Overexpression of <i>SLAC1</i> enhanced cryptogein-induced Cl⁻ efflux and extracellular alkalinization as well as rapid/transient and slow/prolonged phases of NADPH oxidase-mediated ROS production, which was suppressed by an anion channel inhibitor, DIDS. The overexpressor also showed enhanced sensitivity to cryptogein to induce downstream immune responses, including the induction of defense marker genes and the hypersensitive cell death. These results suggest that SLAC1 expressed in BY-2 cells mediates cryptogein-induced plasma membrane Cl⁻ efflux to positively modulate the elicitor-triggered activation of other ion fluxes, ROS as well as a wide range of defense signaling pathways. These findings shed light on the possible involvement of the SLAC/SLAH family anion channels in cryptogein signaling to trigger the plasma membrane ion channel cascade in the plant defense signal transduction network.</p></div

Effect of <i>SLAC1</i>-overexpression on cryptogein-induced defense gene expressions.

<p>(A and B) Cryptogein-induced the expression of <i>HIN1</i> in a dose-dependent manner (A) and <i>Hsr203j</i> (B) in <i>SLAC1</i>-overexpressing cells. The amount of each mRNA was calculated from the threshold point located in the log-linear range of the RT-PCR. The relative level of each gene in the control cells at time 0. Total RNA was isolated from BY-2 cells harvested 5 h after the addition of cryptogein at various concentrations. Data are the mean ± SE of three independent experiments. * <i>p</i><0.05, ** <i>p</i><0.005, significantly different from the control line.</p

Effect of <i>SLAC1</i>-overexpression on cryptogein-induced ion fluxes.

<p>(A) Time course of cryptogein-induced [Cl^–]_ext changes in BY-2 cells. BY-2 cells were treated with cryptogein (0.25 µM). (B) Effect of DIDS on cryptogein-induced [Cl^–]_ext changes. DIDS (100 µM) was added to BY-2 cells 15 min prior to the elicitor (0.25 µM) treatment. DMSO was used as a control. (C) Time course of cryptogein-induced [pH]_ext changes. BY-2 cells were treated with cryptogein (1 µM). ** <i>p</i><0.005, *** <i>p</i><0.001, significantly different from the control line. (D) Effect of DIDS on cryptogein-induced [pH]_ext changes. DIDS (50 µM) was added to BY-2 cells 15 min prior to the elicitor treatment. DMSO was used as a control. Data are the mean ± SE of three independent experiments. * <i>p</i><0.05, significantly different from the control line.</p

Intracellular localization of the SLAC1 protein in tobacco BY-2 cells.

<p>Confocal fluorescence images (<b>a</b>-<b>f</b>) of BY-2 protoplast expressing SLAC1-GFP (<b>a</b>–<b>c</b>) or GFP (<b>d–f</b>) stained with the fluorescent styryl membrane probe FM4-64. Fluorescence of GFP (<b>a</b> and <b>d</b>) and FM4-64 (<b>b</b> and <b>e</b>). Scale bar: 10 µm. FM4-64 was kept as a 17 mM stock solution in sterile water, and used at a final concentration of 4.25 µM. Tobacco BY-2 protoplasts were treated with FM4-64 for 10 min and washed twice with the wash buffer at room temperature to label the PM.</p

Involvement of SLAC1 in the regulation of cryptogein-induced biphasic ROS production mediated by NADPH oxidases.

<p>(A) Cryptogein (0.25 µM)-induced rapid transient ROS production within 15 min in BY-2 cells. Data is representative of three experiments (B) The effects of inhibitors on cryptogein-induced ROS production within 15 min. To quantify the effects of inhibitors on ROS production, the peak intensity of luminol chemiluminescence was compared with the control. DIDS or an NADPH oxidase inhibitor, DPI, was added to BY-2 cells 15 min prior to the elicitor treatment. DMSO was used as a control. Data are the mean ± SE of three independent experiments. ** <i>p</i><0.005, significantly different from the control line. (C) Effect of <i>SLAC1</i>-overexpression on cryptogein-induced prolonged ROS production. BY-2 cells were treated with cryptogein (0.25 µM). Data are the mean ± SE of three independent experiments. * <i>p</i><0.05, ** <i>p</i><0.005, *** <i>p</i><0.001, significantly different from the control line. (D) Quantitative expression levels of <i>NtRbohD</i> mRNAs in <i>SLAC1</i>-overexpressing cells by real-time quantitative PCR. Total RNA was isolated from BY-2 cells harvested 5 h after the addition of cryptogein at various concentrations. The amount of each mRNA was calculated from the threshold point located in the log-linear range of the RT-PCR. The relative level of each gene in the control cells at time 0. Data are the mean ± SE of four independent experiments. * <i>p</i><0.005, ** <i>p</i><0.005, significantly different from the control line.</p