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Dual Role of Hydrogen Peroxide in Arabidopsis Guard Cells in Response to Sulfur Dioxide

Dual Role of Hydrogen Peroxide in Arabidopsis Guard Cells in Response to Sulfur Dioxide Dual Role of Hydrogen Peroxide in Arabidopsis Guard Cells in Response to Sulfur Dioxide <meta name="citation_title" content="Dual Role of Hydrogen Peroxide in Arabidopsis Guard Cells in Response to Sulfur Dioxide"/> <meta name="dc.title" content="Dual Role of Hydrogen Peroxide in Arabidopsis Guard Cells in Response to Sulfur Dioxide"/> div.banner_title_bkg div.trangle { border-color: #376240 transparent transparent transparent; opacity:0.8; /*new styles start*/ -ms-filter:"progid:DXImageTransform.Microsoft.Alpha(Opacity=80)" ;filter: alpha(opacity=80); /*new styles end*/ } div.banner_title_bkg_if div.trangle { border-color: transparent transparent #376240 transparent ; opacity:0.8; /*new styles start*/ -ms-filter:"progid:DXImageTransform.Microsoft.Alpha(Opacity=80)" ;filter: alpha(opacity=80); /*new styles end*/ } div.banner_title_bkg div.trangle { width: 221px; } #banner { background-image: url('http://images.hindawi.com/journals/atox/atox.banner.jpg'); background-position: 50% 0;} Hindawi Publishing Corporation Home Journals About Us Advances in Toxicology About this Journal Submit a Manuscript Table of Contents Journal Menu About this Journal · Abstracting and Indexing · Aims and Scope · Article Processing Charges · Author Guidelines · Bibliographic Information · Contact Information · Editorial Board · Editorial Workflow · Free eTOC Alerts · Publication Ethics · Recently Accepted Articles · Reviewers Acknowledgment · Submit a Manuscript · Subscription Information · Table of Contents Open Special Issues · Special Issue Guidelines Abstract Full-Text PDF Full-Text HTML Full-Text ePUB Linked References How to Cite this Article Advances in Toxicology Volume 2014 (2014), Article ID 407368, 9 pages http://dx.doi.org/10.1155/2014/407368 Research Article Dual Role of Hydrogen Peroxide in Arabidopsis Guard Cells in Response to Sulfur Dioxide Huilan Yi , 1 Xin Liu , 1,2 Min Yi , 1,3 and Gang Chen 2 1 School of Life Science, Shanxi University, Taiyuan 030006, China 2 Shanxi Provincial Guoxin Energy Development Group Co., LTD., Taiyuan 030006, China 3 Department of Statistics, University of Missouri-Columbia, Columbia, MO 65211, USA Received 28 April 2014; Accepted 8 September 2014; Published 30 September 2014 Academic Editor: Mugimane Manjanatha Copyright © 2014 Huilan Yi et al. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Sulfur dioxide (SO 2 ) is a major air pollutant and has significant impacts on plant physiology. Plant can adapt to SO 2 stress by controlling stomatal movement, gene expression, and metabolic changes. Here we show clear evidences that SO 2 -triggered hydrogen peroxide (H 2 O 2 ) production mediated stomatal closure and cell death in Arabidopsis leaves. High levels of SO 2 caused irreversible stomatal closure and decline in guard cell viability, but low levels of SO 2 caused reversible stomatal closure. Exogenous antioxidants ascorbic acid (AsA) and catalase (CAT) or Ca 2+ antagonists EGTA and LaCl 3 blocked SO 2 -induced stomatal closure and decline in viability. AsA and CAT also blocked SO 2 -induced H 2 O 2 and elevation. However, EGTA and LaCl 3 inhibited SO 2 -induced increase but did not suppress SO 2 -induced H 2 O 2 elevation. These results indicate that H 2 O 2 elevation triggered stomatal closure and cell death via signaling in SO 2 -stimulated Arabidopsis guard cells. NADPH oxidase inhibitor DPI blocked SO 2 -induced cell death but not the stomatal closure triggered by low levels of SO 2 , indicating that NADPH oxidase-dependent H 2 O 2 production plays critical role in SO 2 toxicity but is not necessary for SO 2 -induced stomatal closure. Our results suggest that H 2 O 2 production and accumulation in SO 2 -stimulated plants trigger plant adaptation and toxicity via reactive oxygen species mediating Ca 2+ signaling. 1. Introduction Sulfur dioxide (SO 2 ) is a harmful gas that is emitted largely from burning coal, high-sulfur oil, and fuels. During the past few decades, the concentration of SO 2 in the atmosphere has increased in many areas of the world, especially in the developing countries. High levels of SO 2 can injure many plant species and varieties, resulting in photosynthesis decline, growth inhibition, and even death [ 1 – 4 ]. Sulfur dioxide enters plants mainly through the open stomata [ 5 ]. Once it enters the leaf, SO 2 is hydrated to form and . The toxicity of SO 2 is derived from molecular species sulfite ( ) and bisulfite ( ) generated after SO 2 is dissolved in cellular fluid [ 6 ]. Sulfite oxidation, which is the detoxification reaction of sulfite to sulfate ( ), leads to the formation of reactive oxygen species (ROS) in plant cells [ 7 , 8 ]. The production and accumulation of ROS are one of the key events in plant response to SO 2 [ 9 – 12 ]. ROS have been proposed as central components of plant response to both biotic and abiotic stresses. Under such conditions, ROS may play two very different roles: exacerbating damage or signaling the activation of defense responses [ 13 , 14 ]. It has been reported that ROS act as signaling molecules mediating a variety of physiological responses, including stomatal movement and gene expression [ 15 – 17 ], although they can attack biomolecules such as nucleic acids, proteins, and lipids leading to cell damage and death [ 16 , 18 , 19 ]. Results of previous studies have shown that plants can adapt to SO 2 stress by controlling stomatal movement and gene transcription [ 11 , 12 , 20 ]. Stomatal closure could protect the leaves against further entry of the environmental SO 2 , while differential gene expression could regulate the metabolic routes of plant cells providing long-term adaptation to environmental stress. However, up to now, it is not clear if the ROS production is closely associated with the initial physiological mechanisms responsible for plant responses to SO 2 stress. ROS overproduction can trigger plant adaptation or cell damage during abiotic stress. However, there has been little overlap between these two fields of research. In the present study, guard cells of A. thaliana leaves, which are a well-developed model cell system for studying the signal transduction in plant cells [ 21 – 23 ], were employed to investigate the cellular mechanism of plant response to SO 2 stress. To the best of our knowledge, this is the first report of H 2 O 2 mediating both adaptation response (stomatal movement) and cytotoxicity (cell death) in plant response to SO 2 stress. Our results show that H 2 O 2 elevation triggered both stomatal closure and cell viability loss via Ca 2+ signaling in SO 2 -treated plants. 2. Materials and Methods 2.1. Plant Material Preparation Plants of Arabidopsis thaliana (L.) ecotype Columbia (Col-0) were grown in soil (Klasmann-Deilmann) in temperature-controlled growth rooms at 22°C with an average light intensity of 240 μ moL m −1 s −1 , a 16 h photoperiod per day, and 60% relative humidity. Young fully expanded leaves were harvested from 4-week-old Arabidopsis plants. The abaxial epidermes were peeled from the underside of each leaf and cut into small pieces. The isolated epidermal strips were immediately floated in 10 mM 2-(N-morpholino)ethanesulfonic acid (MES; Bio Basic Inc.) buffer (10 mM MES-Tris, pH 7.0, and 50 mM KCl). 2.2. Determination of Stomatal Aperture The isolated epidermal strips were incubated in 10 mM MES buffer for 2 h, for stomata-opening under continuous light of 180 μ moL m −2 s −1 at 22°C or for stomata-closuring in the dark, and then incubated for 2 h in MES buffer containing a certain amount of SO 2 hydrates (a mixture of sodium sulfite and sodium bisulfite, 3 : 1 mM/mM, prepared freshly before use) under continuous illumination of 180 μ moL m −2 s −1 at 22°C. Control samples were treated under the same conditions with 10 mM MES buffer. The isolated epidermal strips were treated with a mixture of SO 2 hydrates and a certain amount of antagonists which include NADPH oxidase inhibitor diphenylene iodonium (DPI, Sigma), antioxidants catalase (CAT, Sigma), and ascorbic acid (AsA, Sigma) and Ca 2+ antagonists LaCl 3 and ethylene glycol tetraacetic acid (EGTA, Sigma) to examine the protective effects. After 2 h of chemical exposure, the epidermal strips were mounted on a microscopy slide, moistened with 10 mM MES buffer, and covered with a slip. Stomatal aperture was measured by using a digital microscope camera system (DP72, Olympus) and an attached DP2-BSW software. At least three leaves and 300 stomata per leaf were measured in each treatment and all of the experiments were independently repeated at least three times. For the recovery groups, after 2 h of chemical exposure, the isolated strips were resuspended in MES buffer for 2 h under continuous light followed by stomatal aperture measurement. For time-course experiment, the stomatal apertures were examined every five minutes after isolated strips were incubated in SO 2 hydrates. 2.3. Determination of Cell Viability Cell viability was assessed by using the method of double staining with fluorescein diacetate (FDA; Bio Basic Inc.) and propidium iodide (PI; Sigma). After 2 h of chemical exposure, the epidermal strips were simultaneously stained with 0.1 mg·L −1 FDA and 10 μ M PI for visualizing cell viability. At least three leaves and 300 guard cells per leaf were observed in each treatment and all of the experiments were independently repeated three times. 2.4. Measurement of Reactive Oxygen Species Reactive oxygen species in guard cells of epidermal peels were detected using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime) according to the methods described by Yi et al. [ 24 ]. After 2 h of exposure to the chemicals, the epidermal strips were incubated in 20 μ M DCFH-DA for 30 min in the dark. The mean value of fluorescence intensity, resulting from 600 guard cells in three independent experiments, represents the intracellular H 2 O 2 level for each treatment. 2.5. Measurement of Intracellular Ca 2+ The concentration of intracellular Ca 2+ ( ) was measured using fluo-3 acetomethoxyester (Fluo-3 AM; Beyotime) as described in our previous report [ 24 ]. The average fluorescence intensity of Fluo-3 AM obtained from three different leaves and 200 guard cells represents the level for each treatment. All experiments were independently repeated three times. 2.6. Statistical Analysis All values of mean and standard deviation (SD) were obtained from three independent experiments. Analysis of variance (ANOVA) and Dunnett’s -test were used to determine the significant differences among the control and a series of treatment groups. 3. Results 3.1. SO 2 -Evoked H 2 O 2 Production Is Involved in the Regulation of Stomatal Movement As shown in Figure 1 , SO 2 hydrates promote stomatal closure and inhibit light-promoted stomatal opening in Arabidopsis leaves. The width of stomatal aperture decreased in a concentration-dependent manner and showed a significant decrease after the isolated strips were exposed for 2 h to SO 2 hydrates at concentrations of 10 to 500 μ M. However, stomatal closure evoked by low SO 2 concentrations (below 200 μ M) could be reversed completely after SO 2 removal; otherwise the decline of stomatal aperture in high SO 2 concentration (500 μ M) group could be partly reversed. Figure 1: Promotion of stomatal closure by SO 2 and its reversibility upon SO 2 removal. Stomata of Arabidopsis leaf epidermis were allowed to open in light [(a) and (c)] or to close in the dark (b) for 2 h and then were incubated for 2 h in SO 2 hydrates. b, A significant difference at from control. c, A significant difference at between SO 2 treatment and its recovery group. Time course analysis of stomatal movement showed that the diameter of the stomatal aperture decreased markedly within the first 30 minutes of exposure to 10 μ M SO 2 hydrates and continued to decline at a slower rate in the remaining 90 minutes of SO 2 exposure (Figure 1(c) ). SO 2 -induced stomatal closure was associated with an elevated H 2 O 2 level in Arabidopsis guard cells. Exposure to SO 2 hydrates not only caused smaller stomatal apertures (Figure 1 ) but also evoked increased H 2 O 2 level (1.2- to 1.7-fold) in Arabidopsis guard cells (Figure 2 ). When H 2 O 2 elevation was blocked by exogenous H 2 O 2 scavengers CAT or AsA, SO 2 -induced stomatal closure was efficiently reversed (Figure 2 ). These results indicate that H 2 O 2 production in SO 2 -stimulated guard cells mediates stomatal movement upon SO 2 stress. Figure 2: Effects of antioxidants and calcium antagonists on SO 2 -induced stomatal closure and H 2 O 2 elevation in Arabidopsis guard cells. Different superscript letters indicate significant differences ( ). The same letters indicate no significant difference. The green fluorescence (DCFH-DA) of guard cells indicates H 2 O 2 content. 3.2. SO 2 -Evoked H 2 O 2 Elevation Triggered Cell Death To investigate the role of H 2 O 2 in irreversible stomatal closure in response to SO 2 , we detect the viability of Arabidopsis guard cells by double staining with fluorescein diacetate (FDA) and propidium iodide (PI). The results showed that SO 2 induced guard cell death in a concentration-dependent manner (Figure 3 ). Cell death reached 24% in 6 mM SO 2 hydrates treatment group, but no obvious cell death could be observed in 10 to 200 μ M SO 2 hydrates treatment groups. The H 2 O 2 level of guard cells exposed to 2 to 6 mM SO 2 hydrates showed a statistically significant increase (1.90- to 2.30-fold), which is higher than those exposed to 10 to 500 μ M SO 2 hydrates. Moreover, exposure to SO 2 hydrates simultaneously with 200 U·mL −1 CAT or 0.1 mM AsA, SO 2 -induced cell death was efficiently blocked, associated with a significant decrease in H 2 O 2 level of guard cells (Figure 3 ). These results clearly demonstrate that an elevated H 2 O 2 level can trigger guard cell death leading to stomatal dysfunction and irreversible stomatal closure in SO 2 -treated Arabidopsis leaves. Figure 3: Sulfur dioxide caused viability decline associated with H 2 O 2 elevation in Arabidopsis guard cells. Different superscript letters indicate significant differences ( ). The same letters indicate no significant difference. To further confirm the role of H 2 O 2 in SO 2 toxicity, we investigate the protective effects of CAT on cell viability in SO 2 -treated samples. The results showed that application of 1000 U·mL −1 CAT could completely block the cell death evoked by 2 mM and lower concentrations of SO 2 hydrates, but the cytotoxicity evoked by 6 mM SO 2 hydrates was only partly blocked (Figure 4 ). These results clearly demonstrate that H 2 O 2 production, which may work together with other molecules, is enough to trigger SO 2 toxicity. Figure 4: Hydrogen peroxide scavenger and NADPH oxidase inhibitor DPI blocked SO 2 -induced cell death, but DPI cannot block SO 2 -induced stomatal closure. Different superscript letters indicate significant difference at . The same letters indicate no significant difference. 3.3. H 2 O 2 Action Is Dependent on Its Concentrations and Spatial Generation Patterns As shown above, SO 2 can cause stomatal closing and decline in cell viability, which is dependent on SO 2 concentrations and H 2 O 2 level. However, there was no clear dividing line between a safe level and a toxic level. In order to understand what constitutes a safe level, an inhibitor of NADPH oxidase was used to detect its inhibitory effects. The results show that application of 20 μ M NADPH oxidase inhibitor DPI for 2 h markedly blocked cell death evoked by 2 mM SO 2 hydrates. But 20 μ M DPI did not inhibit stomatal closing evoked by 10 μ M SO 2 hydrates (Figure 4 ). These findings indicate that SO 2 -triggered stomatal closing can be driven by NADPH oxidase-dependent and -independent H 2 O 2 generation, but NADPH oxidase-dependent H 2 O 2 generation is involved in SO 2 -caused cytotoxicity. 3.4. H 2 O 2 Acts Upstream of Ca 2+ Signaling in Response to SO 2 Stress SO 2 exposure enhanced the fluorescence intensity of Fluo-3 AM (Ca 2+ indicator) in Arabidopsis guard cells. The relative fluorescence intensity of Fluo-3 AM, resulting from more than 600 guard cells per treatment group in three independent experiments, increased obviously in guard cells exposed to 10 μ M to 6 mM SO 2 hydrates for 2 h but decreased markedly when isolated strips were treated simultaneously with SO 2 hydrates and 0.1 mM calcium channel blocker LaCl 3 (Figure 5 ). These results indicate that SO 2 exposure evokes an elevation of level, and Ca 2+ influx through Ca 2+ channels in the plasma membrane results in elevation. Figure 5: Effects of antioxidants and Ca 2+ antagonists on SO 2 -induced Ca 2+ elevation and cell death in Arabidopsis guard cells. Different superscript letters indicate significant differences ( ). The same letters indicate no significant difference. As shown in Figures 2 and 5 , the addition of LaCl 3 and calcium chelator EGTA to SO 2 hydrates blocked elevation, stomatal closure, and cell death evoked by SO 2 . Stomatal closure and cell death occurred with increased in SO 2 -treated samples, but both of them were significantly suppressed by 0.1 mM LaCl 3 or EGTA. These results indicate that elevated level is an important stimulus driving stomatal movement and cytotoxicity. To understand the pathways to plant responses, we study the signaling pathways in Arabidopsis guard cells. As shown in Figures 2 , 3 , and 5 , both antioxidants AsA and CAT and Ca 2+ antagonists LaCl 3 and EGTA can suppress SO 2 -induced stomatal closure and cell death, but application of H 2 O 2 scavenger CAT (200 U·mL −1 ) decreased elevation evoked by SO 2 , whereas application of Ca 2+ channel inhibitor LaCl 3 (0.1 mM) did not affect H 2 O 2 elevation evoked by SO 2 . These results confirm the involvement of Ca 2+ downstream of H 2 O 2 production, indicating that H 2 O 2 triggers stomatal movement and cell death via Ca 2+ signaling in plant response to SO 2 stress. 4. Discussion Plant could adapt to environmental challenges through various means. Our recent findings showed that SO 2 fumigation caused an increased ROS production accompanied with differential gene expression and stomatal closure in Arabidopsis plants [ 12 , 20 ]. The results of the present study show the evidences that H 2 O 2 acts as an important signaling molecule triggering stomatal movement and cell death in plant response to SO 2 . SO 2 -caused cell viability decline could interfere with the normal function of stomata to further affect plant physiology under environmental stress. However, the occurrence of SO 2 -caused guard cell death was associated with decreased stomatal aperture, suggesting the existence of a complex signaling network in plant responses to environmental stress. Environmental challenges including biotic and abiotic stresses could induce ROS production in plant cells [ 25 ]. It has been documented that ROS play an important role in signal transduction of stomatal movement regulation and gene expression activation in plant response to environmental stresses [ 26 – 29 ]. ROS, which can be used as rapid long-distance autopropagating signals that are transferred throughout the plant in response to different environmental conditions, are widely considered to be an important player in guard cell signaling [ 26 , 30 ]. The data presented above indicate a requirement for H 2 O 2 production in plant response to SO 2 stress. First, SO 2 induces stomatal closure and cell death associated with an increased intracellular H 2 O 2 level (Figures 2 and 3 ). Second, two types of H 2 O 2 scavengers CAT and AsA could block SO 2 -evoked stomatal closure and cell death; in particular, the effects of SO 2 at low concentrations could be completely reversed by CAT (Figures 2 and 4 ). Third, H 2 O 2 scavenger blocks the SO 2 -induced increase in intracellular Ca 2+ required for stomatal closure and toxicity (Figure 5 ). Our study, validating the strong positive correlation between intracellular H 2 O 2 level and stomatal closure/cell death, demonstrates a key role of H 2 O 2 as trigger of stomatal closure and/or guard cell death in response to SO 2 . Sulfur dioxide inhibited light-promoted stomatal opening and promoted stomatal closure leading to the smaller size of Arabidopsis stomata. High concentrations of SO 2 also caused viability loss of Arabidopsis guard cells. The time course experiments show that H 2 O 2 level of guard cells increased gradually during SO 2 exposure (date not shown); therefore, an increased H 2 O 2 level in SO 2 -stimulated Arabidopsis cells might trigger stomatal closure firstly and then cell viability loss through ROS-mediated cell death pathway as shown in V. faba cells [ 24 ] or through the gradual accumulation of free radical damage to biomolecules during SO 2 exposure. There are two primary sources of H 2 O 2 in guard cells: chloroplasts and plasma membrane-associated NADPH oxidase [ 22 , 31 , 32 ]. The results of the time course experiments with single cell assays using the fluorescent probe DCFH-DA showed that H 2 O 2 generation was dependent on SO 2 concentration and that the increase in fluorescence intensity of chloroplasts occurred significantly earlier than within the other regions of guard cells (date not shown), demonstrating an enhanced H 2 O 2 production in chloroplasts of guard cells. NADPH oxidase in plasma membrane contributes to O 2 · generation and ROS elevation in plant responses to abiotic stresses [ 33 ]. Application of DPI, which is widely accepted as a relatively specific direct inhibitor of NADPH oxidase, markedly blocked SO 2 -induced cell death but cannot suppress stomatal closure evoked by low concentrations of SO 2 . These findings indicate that H 2 O 2 -mediated stomatal closure in Arabidopsis leaves exposed to low concentrations of SO 2 is not dependent on the activity of plasma membrane NADPH oxidase, but cell death evoked by high concentrations of SO 2 is NADPH oxidase-dependent. SO 2 exposure caused stomatal closure and guard cell death associated with elevation, whereas application of either Ca 2+ chelator EGTA or Ca 2+ channel inhibitor LaCl 3 blocking SO 2 -evoked elevation, stomatal closure and cell death evoked by SO 2 were effectively blocked. These results demonstrate that a channel-mediated Ca 2+ influx across the plasma membrane contributes to the elevation of and subsequent stomatal closure and cell death in SO 2 -stimulated guard cells. However, it is not clear how Ca 2+ signaling recognizes the different situations of cells to mediate appropriate processes such as stomatal closure and cell death. It has been found that H 2 O 2 could activate plasma membrane Ca 2+ channels leading to increase in plant cells [ 34 – 36 ]. Therefore, H 2 O 2 activation of plasma membrane Ca 2+ channels may be a central step in SO 2 -induced stomatal closure and/or cell death. The results of our present study also showed that application of antioxidant CAT and AsA significantly decreased SO 2 -evoked elevation, but application of Ca 2+ channel blocker LaCl 3 did not affect SO 2 -evoked H 2 O 2 increase, indicating that H 2 O 2 acts upstream of Ca 2+ signaling in SO 2 -induced stomatal closure and/or cell death. These observations were consistent with other previous reports that a increase was linked to H 2 O 2 production and was involved in ROS-mediated stomatal closure/cell death [ 37 – 39 ]. Therefore, H 2 O 2 elevation and subsequent activation of Ca 2+ channels are events occurring in SO 2 -induced stomatal closure/cell death. Ca 2+ influx from extracellular region results in increase, and then elevation mediates subsequent stomatal closure/cell death. These results suggested that H 2 O 2 mediates SO 2 -induced stomatal movement/cytotoxicity by targeting Ca 2+ channels in the plasma membrane. Briefly, environmental SO 2 has a remarkable effect on the size of the stomatal aperture. Exposure to SO 2 induced the overproduction of H 2 O 2 in guard cells, as shown in other plant cells exposed to environmental challenges [ 40 , 41 ]. Elevated H 2 O 2 acts in conjunction with other factors to activate stomatal movement and cell death under SO 2 stress. Guard cells are a well-developed model system for characterizing early signal transduction mechanisms in plants. In this study, the dual role of H 2 O 2 in plant cells in response to air pollutant was clearly displayed in Arabidopsis guard cells, which additionally broaden the role of stomatal guard cells in cytotoxicity study. Our results suggest that guard cells are a valuable model system for the study of cytotoxicity in plant cells. 5. Conclusion Sulfur dioxide exposure caused an elevated H 2 O 2 level in Arabidopsis guard cells. H 2 O 2 elevation triggered by SO 2 mediated both stomatal closure and cell death via Ca 2+ signaling. Intracellular Ca 2+ increase is necessary for stomatal closure and cell death in Arabidopsis guard cells in response to SO 2 . H 2 O 2 production by NADPH oxidase plays a critical role in SO 2 toxicity; however NADPH oxidase activation is sufficient but not necessary for SO 2 -triggered stomatal closure. 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Dual Role of Hydrogen Peroxide in Arabidopsis Guard Cells in Response to Sulfur Dioxide

Yi, Huilan; Liu, Xin; Yi, Min; Chen, Gang
Advances in Toxicology , Volume 2014 (2014) – Sep 30, 2014
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Dual Role of Hydrogen Peroxide in Arabidopsis Guard Cells in Response to Sulfur Dioxide <meta name="citation_title" content="Dual Role of Hydrogen Peroxide in Arabidopsis Guard Cells in Response to Sulfur Dioxide"/> <meta name="dc.title" content="Dual Role of Hydrogen Peroxide in Arabidopsis Guard Cells in Response to Sulfur Dioxide"/> div.banner_title_bkg div.trangle { border-color: #376240 transparent transparent transparent; opacity:0.8; /*new styles start*/ -ms-filter:"progid:DXImageTransform.Microsoft.Alpha(Opacity=80)" ;filter: alpha(opacity=80); /*new styles end*/ } div.banner_title_bkg_if div.trangle { border-color: transparent transparent #376240 transparent ; opacity:0.8; /*new styles start*/ -ms-filter:"progid:DXImageTransform.Microsoft.Alpha(Opacity=80)" ;filter: alpha(opacity=80); /*new styles end*/ } div.banner_title_bkg div.trangle { width: 221px; } #banner { background-image: url('http://images.hindawi.com/journals/atox/atox.banner.jpg'); background-position: 50% 0;} Hindawi Publishing Corporation Home Journals About Us Advances in Toxicology About this Journal Submit a Manuscript Table of Contents Journal Menu About this Journal · Abstracting and Indexing · Aims and Scope · Article Processing Charges · Author Guidelines · Bibliographic Information · Contact Information · Editorial Board · Editorial Workflow · Free eTOC Alerts · Publication Ethics · Recently Accepted Articles · Reviewers Acknowledgment · Submit a Manuscript · Subscription Information · Table of Contents Open Special Issues · Special Issue Guidelines Abstract Full-Text PDF Full-Text HTML Full-Text ePUB Linked References How to Cite this Article Advances in Toxicology Volume 2014 (2014), Article ID 407368, 9 pages http://dx.doi.org/10.1155/2014/407368 Research Article Dual Role of Hydrogen Peroxide in Arabidopsis Guard Cells in Response to Sulfur Dioxide Huilan Yi , 1 Xin Liu , 1,2 Min Yi , 1,3 and Gang Chen 2 1 School of Life Science, Shanxi University, Taiyuan 030006, China 2 Shanxi Provincial Guoxin Energy Development Group Co., LTD., Taiyuan 030006, China 3 Department of Statistics, University of Missouri-Columbia, Columbia, MO 65211, USA Received 28 April 2014; Accepted 8 September 2014; Published 30 September 2014 Academic Editor: Mugimane Manjanatha Copyright © 2014 Huilan Yi et al. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Sulfur dioxide (SO 2 ) is a major air pollutant and has significant impacts on plant physiology. Plant can adapt to SO 2 stress by controlling stomatal movement, gene expression, and metabolic changes. Here we show clear evidences that SO 2 -triggered hydrogen peroxide (H 2 O 2 ) production mediated stomatal closure and cell death in Arabidopsis leaves. High levels of SO 2 caused irreversible stomatal closure and decline in guard cell viability, but low levels of SO 2 caused reversible stomatal closure. Exogenous antioxidants ascorbic acid (AsA) and catalase (CAT) or Ca 2+ antagonists EGTA and LaCl 3 blocked SO 2 -induced stomatal closure and decline in viability. AsA and CAT also blocked SO 2 -induced H 2 O 2 and elevation. However, EGTA and LaCl 3 inhibited SO 2 -induced increase but did not suppress SO 2 -induced H 2 O 2 elevation. These results indicate that H 2 O 2 elevation triggered stomatal closure and cell death via signaling in SO 2 -stimulated Arabidopsis guard cells. NADPH oxidase inhibitor DPI blocked SO 2 -induced cell death but not the stomatal closure triggered by low levels of SO 2 , indicating that NADPH oxidase-dependent H 2 O 2 production plays critical role in SO 2 toxicity but is not necessary for SO 2 -induced stomatal closure. Our results suggest that H 2 O 2 production and accumulation in SO 2 -stimulated plants trigger plant adaptation and toxicity via reactive oxygen species mediating Ca 2+ signaling. 1. Introduction Sulfur dioxide (SO 2 ) is a harmful gas that is emitted largely from burning coal, high-sulfur oil, and fuels. During the past few decades, the concentration of SO 2 in the atmosphere has increased in many areas of the world, especially in the developing countries. High levels of SO 2 can injure many plant species and varieties, resulting in photosynthesis decline, growth inhibition, and even death [ 1 – 4 ]. Sulfur dioxide enters plants mainly through the open stomata [ 5 ]. Once it enters the leaf, SO 2 is hydrated to form and . The toxicity of SO 2 is derived from molecular species sulfite ( ) and bisulfite ( ) generated after SO 2 is dissolved in cellular fluid [ 6 ]. Sulfite oxidation, which is the detoxification reaction of sulfite to sulfate ( ), leads to the formation of reactive oxygen species (ROS) in plant cells [ 7 , 8 ]. The production and accumulation of ROS are one of the key events in plant response to SO 2 [ 9 – 12 ]. ROS have been proposed as central components of plant response to both biotic and abiotic stresses. Under such conditions, ROS may play two very different roles: exacerbating damage or signaling the activation of defense responses [ 13 , 14 ]. It has been reported that ROS act as signaling molecules mediating a variety of physiological responses, including stomatal movement and gene expression [ 15 – 17 ], although they can attack biomolecules such as nucleic acids, proteins, and lipids leading to cell damage and death [ 16 , 18 , 19 ]. Results of previous studies have shown that plants can adapt to SO 2 stress by controlling stomatal movement and gene transcription [ 11 , 12 , 20 ]. Stomatal closure could protect the leaves against further entry of the environmental SO 2 , while differential gene expression could regulate the metabolic routes of plant cells providing long-term adaptation to environmental stress. However, up to now, it is not clear if the ROS production is closely associated with the initial physiological mechanisms responsible for plant responses to SO 2 stress. ROS overproduction can trigger plant adaptation or cell damage during abiotic stress. However, there has been little overlap between these two fields of research. In the present study, guard cells of A. thaliana leaves, which are a well-developed model cell system for studying the signal transduction in plant cells [ 21 – 23 ], were employed to investigate the cellular mechanism of plant response to SO 2 stress. To the best of our knowledge, this is the first report of H 2 O 2 mediating both adaptation response (stomatal movement) and cytotoxicity (cell death) in plant response to SO 2 stress. Our results show that H 2 O 2 elevation triggered both stomatal closure and cell viability loss via Ca 2+ signaling in SO 2 -treated plants. 2. Materials and Methods 2.1. Plant Material Preparation Plants of Arabidopsis thaliana (L.) ecotype Columbia (Col-0) were grown in soil (Klasmann-Deilmann) in temperature-controlled growth rooms at 22°C with an average light intensity of 240 μ moL m −1 s −1 , a 16 h photoperiod per day, and 60% relative humidity. Young fully expanded leaves were harvested from 4-week-old Arabidopsis plants. The abaxial epidermes were peeled from the underside of each leaf and cut into small pieces. The isolated epidermal strips were immediately floated in 10 mM 2-(N-morpholino)ethanesulfonic acid (MES; Bio Basic Inc.) buffer (10 mM MES-Tris, pH 7.0, and 50 mM KCl). 2.2. Determination of Stomatal Aperture The isolated epidermal strips were incubated in 10 mM MES buffer for 2 h, for stomata-opening under continuous light of 180 μ moL m −2 s −1 at 22°C or for stomata-closuring in the dark, and then incubated for 2 h in MES buffer containing a certain amount of SO 2 hydrates (a mixture of sodium sulfite and sodium bisulfite, 3 : 1 mM/mM, prepared freshly before use) under continuous illumination of 180 μ moL m −2 s −1 at 22°C. Control samples were treated under the same conditions with 10 mM MES buffer. The isolated epidermal strips were treated with a mixture of SO 2 hydrates and a certain amount of antagonists which include NADPH oxidase inhibitor diphenylene iodonium (DPI, Sigma), antioxidants catalase (CAT, Sigma), and ascorbic acid (AsA, Sigma) and Ca 2+ antagonists LaCl 3 and ethylene glycol tetraacetic acid (EGTA, Sigma) to examine the protective effects. After 2 h of chemical exposure, the epidermal strips were mounted on a microscopy slide, moistened with 10 mM MES buffer, and covered with a slip. Stomatal aperture was measured by using a digital microscope camera system (DP72, Olympus) and an attached DP2-BSW software. At least three leaves and 300 stomata per leaf were measured in each treatment and all of the experiments were independently repeated at least three times. For the recovery groups, after 2 h of chemical exposure, the isolated strips were resuspended in MES buffer for 2 h under continuous light followed by stomatal aperture measurement. For time-course experiment, the stomatal apertures were examined every five minutes after isolated strips were incubated in SO 2 hydrates. 2.3. Determination of Cell Viability Cell viability was assessed by using the method of double staining with fluorescein diacetate (FDA; Bio Basic Inc.) and propidium iodide (PI; Sigma). After 2 h of chemical exposure, the epidermal strips were simultaneously stained with 0.1 mg·L −1 FDA and 10 μ M PI for visualizing cell viability. At least three leaves and 300 guard cells per leaf were observed in each treatment and all of the experiments were independently repeated three times. 2.4. Measurement of Reactive Oxygen Species Reactive oxygen species in guard cells of epidermal peels were detected using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; Beyotime) according to the methods described by Yi et al. [ 24 ]. After 2 h of exposure to the chemicals, the epidermal strips were incubated in 20 μ M DCFH-DA for 30 min in the dark. The mean value of fluorescence intensity, resulting from 600 guard cells in three independent experiments, represents the intracellular H 2 O 2 level for each treatment. 2.5. Measurement of Intracellular Ca 2+ The concentration of intracellular Ca 2+ ( ) was measured using fluo-3 acetomethoxyester (Fluo-3 AM; Beyotime) as described in our previous report [ 24 ]. The average fluorescence intensity of Fluo-3 AM obtained from three different leaves and 200 guard cells represents the level for each treatment. All experiments were independently repeated three times. 2.6. Statistical Analysis All values of mean and standard deviation (SD) were obtained from three independent experiments. Analysis of variance (ANOVA) and Dunnett’s -test were used to determine the significant differences among the control and a series of treatment groups. 3. Results 3.1. SO 2 -Evoked H 2 O 2 Production Is Involved in the Regulation of Stomatal Movement As shown in Figure 1 , SO 2 hydrates promote stomatal closure and inhibit light-promoted stomatal opening in Arabidopsis leaves. The width of stomatal aperture decreased in a concentration-dependent manner and showed a significant decrease after the isolated strips were exposed for 2 h to SO 2 hydrates at concentrations of 10 to 500 μ M. However, stomatal closure evoked by low SO 2 concentrations (below 200 μ M) could be reversed completely after SO 2 removal; otherwise the decline of stomatal aperture in high SO 2 concentration (500 μ M) group could be partly reversed. Figure 1: Promotion of stomatal closure by SO 2 and its reversibility upon SO 2 removal. Stomata of Arabidopsis leaf epidermis were allowed to open in light [(a) and (c)] or to close in the dark (b) for 2 h and then were incubated for 2 h in SO 2 hydrates. b, A significant difference at from control. c, A significant difference at between SO 2 treatment and its recovery group. Time course analysis of stomatal movement showed that the diameter of the stomatal aperture decreased markedly within the first 30 minutes of exposure to 10 μ M SO 2 hydrates and continued to decline at a slower rate in the remaining 90 minutes of SO 2 exposure (Figure 1(c) ). SO 2 -induced stomatal closure was associated with an elevated H 2 O 2 level in Arabidopsis guard cells. Exposure to SO 2 hydrates not only caused smaller stomatal apertures (Figure 1 ) but also evoked increased H 2 O 2 level (1.2- to 1.7-fold) in Arabidopsis guard cells (Figure 2 ). When H 2 O 2 elevation was blocked by exogenous H 2 O 2 scavengers CAT or AsA, SO 2 -induced stomatal closure was efficiently reversed (Figure 2 ). These results indicate that H 2 O 2 production in SO 2 -stimulated guard cells mediates stomatal movement upon SO 2 stress. Figure 2: Effects of antioxidants and calcium antagonists on SO 2 -induced stomatal closure and H 2 O 2 elevation in Arabidopsis guard cells. Different superscript letters indicate significant differences ( ). The same letters indicate no significant difference. The green fluorescence (DCFH-DA) of guard cells indicates H 2 O 2 content. 3.2. SO 2 -Evoked H 2 O 2 Elevation Triggered Cell Death To investigate the role of H 2 O 2 in irreversible stomatal closure in response to SO 2 , we detect the viability of Arabidopsis guard cells by double staining with fluorescein diacetate (FDA) and propidium iodide (PI). The results showed that SO 2 induced guard cell death in a concentration-dependent manner (Figure 3 ). Cell death reached 24% in 6 mM SO 2 hydrates treatment group, but no obvious cell death could be observed in 10 to 200 μ M SO 2 hydrates treatment groups. The H 2 O 2 level of guard cells exposed to 2 to 6 mM SO 2 hydrates showed a statistically significant increase (1.90- to 2.30-fold), which is higher than those exposed to 10 to 500 μ M SO 2 hydrates. Moreover, exposure to SO 2 hydrates simultaneously with 200 U·mL −1 CAT or 0.1 mM AsA, SO 2 -induced cell death was efficiently blocked, associated with a significant decrease in H 2 O 2 level of guard cells (Figure 3 ). These results clearly demonstrate that an elevated H 2 O 2 level can trigger guard cell death leading to stomatal dysfunction and irreversible stomatal closure in SO 2 -treated Arabidopsis leaves. Figure 3: Sulfur dioxide caused viability decline associated with H 2 O 2 elevation in Arabidopsis guard cells. Different superscript letters indicate significant differences ( ). The same letters indicate no significant difference. To further confirm the role of H 2 O 2 in SO 2 toxicity, we investigate the protective effects of CAT on cell viability in SO 2 -treated samples. The results showed that application of 1000 U·mL −1 CAT could completely block the cell death evoked by 2 mM and lower concentrations of SO 2 hydrates, but the cytotoxicity evoked by 6 mM SO 2 hydrates was only partly blocked (Figure 4 ). These results clearly demonstrate that H 2 O 2 production, which may work together with other molecules, is enough to trigger SO 2 toxicity. Figure 4: Hydrogen peroxide scavenger and NADPH oxidase inhibitor DPI blocked SO 2 -induced cell death, but DPI cannot block SO 2 -induced stomatal closure. Different superscript letters indicate significant difference at . The same letters indicate no significant difference. 3.3. H 2 O 2 Action Is Dependent on Its Concentrations and Spatial Generation Patterns As shown above, SO 2 can cause stomatal closing and decline in cell viability, which is dependent on SO 2 concentrations and H 2 O 2 level. However, there was no clear dividing line between a safe level and a toxic level. In order to understand what constitutes a safe level, an inhibitor of NADPH oxidase was used to detect its inhibitory effects. The results show that application of 20 μ M NADPH oxidase inhibitor DPI for 2 h markedly blocked cell death evoked by 2 mM SO 2 hydrates. But 20 μ M DPI did not inhibit stomatal closing evoked by 10 μ M SO 2 hydrates (Figure 4 ). These findings indicate that SO 2 -triggered stomatal closing can be driven by NADPH oxidase-dependent and -independent H 2 O 2 generation, but NADPH oxidase-dependent H 2 O 2 generation is involved in SO 2 -caused cytotoxicity. 3.4. H 2 O 2 Acts Upstream of Ca 2+ Signaling in Response to SO 2 Stress SO 2 exposure enhanced the fluorescence intensity of Fluo-3 AM (Ca 2+ indicator) in Arabidopsis guard cells. The relative fluorescence intensity of Fluo-3 AM, resulting from more than 600 guard cells per treatment group in three independent experiments, increased obviously in guard cells exposed to 10 μ M to 6 mM SO 2 hydrates for 2 h but decreased markedly when isolated strips were treated simultaneously with SO 2 hydrates and 0.1 mM calcium channel blocker LaCl 3 (Figure 5 ). These results indicate that SO 2 exposure evokes an elevation of level, and Ca 2+ influx through Ca 2+ channels in the plasma membrane results in elevation. Figure 5: Effects of antioxidants and Ca 2+ antagonists on SO 2 -induced Ca 2+ elevation and cell death in Arabidopsis guard cells. Different superscript letters indicate significant differences ( ). The same letters indicate no significant difference. As shown in Figures 2 and 5 , the addition of LaCl 3 and calcium chelator EGTA to SO 2 hydrates blocked elevation, stomatal closure, and cell death evoked by SO 2 . Stomatal closure and cell death occurred with increased in SO 2 -treated samples, but both of them were significantly suppressed by 0.1 mM LaCl 3 or EGTA. These results indicate that elevated level is an important stimulus driving stomatal movement and cytotoxicity. To understand the pathways to plant responses, we study the signaling pathways in Arabidopsis guard cells. As shown in Figures 2 , 3 , and 5 , both antioxidants AsA and CAT and Ca 2+ antagonists LaCl 3 and EGTA can suppress SO 2 -induced stomatal closure and cell death, but application of H 2 O 2 scavenger CAT (200 U·mL −1 ) decreased elevation evoked by SO 2 , whereas application of Ca 2+ channel inhibitor LaCl 3 (0.1 mM) did not affect H 2 O 2 elevation evoked by SO 2 . These results confirm the involvement of Ca 2+ downstream of H 2 O 2 production, indicating that H 2 O 2 triggers stomatal movement and cell death via Ca 2+ signaling in plant response to SO 2 stress. 4. Discussion Plant could adapt to environmental challenges through various means. Our recent findings showed that SO 2 fumigation caused an increased ROS production accompanied with differential gene expression and stomatal closure in Arabidopsis plants [ 12 , 20 ]. The results of the present study show the evidences that H 2 O 2 acts as an important signaling molecule triggering stomatal movement and cell death in plant response to SO 2 . SO 2 -caused cell viability decline could interfere with the normal function of stomata to further affect plant physiology under environmental stress. However, the occurrence of SO 2 -caused guard cell death was associated with decreased stomatal aperture, suggesting the existence of a complex signaling network in plant responses to environmental stress. Environmental challenges including biotic and abiotic stresses could induce ROS production in plant cells [ 25 ]. It has been documented that ROS play an important role in signal transduction of stomatal movement regulation and gene expression activation in plant response to environmental stresses [ 26 – 29 ]. ROS, which can be used as rapid long-distance autopropagating signals that are transferred throughout the plant in response to different environmental conditions, are widely considered to be an important player in guard cell signaling [ 26 , 30 ]. The data presented above indicate a requirement for H 2 O 2 production in plant response to SO 2 stress. First, SO 2 induces stomatal closure and cell death associated with an increased intracellular H 2 O 2 level (Figures 2 and 3 ). Second, two types of H 2 O 2 scavengers CAT and AsA could block SO 2 -evoked stomatal closure and cell death; in particular, the effects of SO 2 at low concentrations could be completely reversed by CAT (Figures 2 and 4 ). Third, H 2 O 2 scavenger blocks the SO 2 -induced increase in intracellular Ca 2+ required for stomatal closure and toxicity (Figure 5 ). Our study, validating the strong positive correlation between intracellular H 2 O 2 level and stomatal closure/cell death, demonstrates a key role of H 2 O 2 as trigger of stomatal closure and/or guard cell death in response to SO 2 . Sulfur dioxide inhibited light-promoted stomatal opening and promoted stomatal closure leading to the smaller size of Arabidopsis stomata. High concentrations of SO 2 also caused viability loss of Arabidopsis guard cells. The time course experiments show that H 2 O 2 level of guard cells increased gradually during SO 2 exposure (date not shown); therefore, an increased H 2 O 2 level in SO 2 -stimulated Arabidopsis cells might trigger stomatal closure firstly and then cell viability loss through ROS-mediated cell death pathway as shown in V. faba cells [ 24 ] or through the gradual accumulation of free radical damage to biomolecules during SO 2 exposure. There are two primary sources of H 2 O 2 in guard cells: chloroplasts and plasma membrane-associated NADPH oxidase [ 22 , 31 , 32 ]. The results of the time course experiments with single cell assays using the fluorescent probe DCFH-DA showed that H 2 O 2 generation was dependent on SO 2 concentration and that the increase in fluorescence intensity of chloroplasts occurred significantly earlier than within the other regions of guard cells (date not shown), demonstrating an enhanced H 2 O 2 production in chloroplasts of guard cells. NADPH oxidase in plasma membrane contributes to O 2 · generation and ROS elevation in plant responses to abiotic stresses [ 33 ]. Application of DPI, which is widely accepted as a relatively specific direct inhibitor of NADPH oxidase, markedly blocked SO 2 -induced cell death but cannot suppress stomatal closure evoked by low concentrations of SO 2 . These findings indicate that H 2 O 2 -mediated stomatal closure in Arabidopsis leaves exposed to low concentrations of SO 2 is not dependent on the activity of plasma membrane NADPH oxidase, but cell death evoked by high concentrations of SO 2 is NADPH oxidase-dependent. SO 2 exposure caused stomatal closure and guard cell death associated with elevation, whereas application of either Ca 2+ chelator EGTA or Ca 2+ channel inhibitor LaCl 3 blocking SO 2 -evoked elevation, stomatal closure and cell death evoked by SO 2 were effectively blocked. These results demonstrate that a channel-mediated Ca 2+ influx across the plasma membrane contributes to the elevation of and subsequent stomatal closure and cell death in SO 2 -stimulated guard cells. However, it is not clear how Ca 2+ signaling recognizes the different situations of cells to mediate appropriate processes such as stomatal closure and cell death. It has been found that H 2 O 2 could activate plasma membrane Ca 2+ channels leading to increase in plant cells [ 34 – 36 ]. Therefore, H 2 O 2 activation of plasma membrane Ca 2+ channels may be a central step in SO 2 -induced stomatal closure and/or cell death. The results of our present study also showed that application of antioxidant CAT and AsA significantly decreased SO 2 -evoked elevation, but application of Ca 2+ channel blocker LaCl 3 did not affect SO 2 -evoked H 2 O 2 increase, indicating that H 2 O 2 acts upstream of Ca 2+ signaling in SO 2 -induced stomatal closure and/or cell death. These observations were consistent with other previous reports that a increase was linked to H 2 O 2 production and was involved in ROS-mediated stomatal closure/cell death [ 37 – 39 ]. Therefore, H 2 O 2 elevation and subsequent activation of Ca 2+ channels are events occurring in SO 2 -induced stomatal closure/cell death. Ca 2+ influx from extracellular region results in increase, and then elevation mediates subsequent stomatal closure/cell death. These results suggested that H 2 O 2 mediates SO 2 -induced stomatal movement/cytotoxicity by targeting Ca 2+ channels in the plasma membrane. Briefly, environmental SO 2 has a remarkable effect on the size of the stomatal aperture. Exposure to SO 2 induced the overproduction of H 2 O 2 in guard cells, as shown in other plant cells exposed to environmental challenges [ 40 , 41 ]. Elevated H 2 O 2 acts in conjunction with other factors to activate stomatal movement and cell death under SO 2 stress. Guard cells are a well-developed model system for characterizing early signal transduction mechanisms in plants. In this study, the dual role of H 2 O 2 in plant cells in response to air pollutant was clearly displayed in Arabidopsis guard cells, which additionally broaden the role of stomatal guard cells in cytotoxicity study. Our results suggest that guard cells are a valuable model system for the study of cytotoxicity in plant cells. 5. Conclusion Sulfur dioxide exposure caused an elevated H 2 O 2 level in Arabidopsis guard cells. H 2 O 2 elevation triggered by SO 2 mediated both stomatal closure and cell death via Ca 2+ signaling. Intracellular Ca 2+ increase is necessary for stomatal closure and cell death in Arabidopsis guard cells in response to SO 2 . H 2 O 2 production by NADPH oxidase plays a critical role in SO 2 toxicity; however NADPH oxidase activation is sufficient but not necessary for SO 2 -triggered stomatal closure. 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Advances in Toxicology – Hindawi Publishing Corporation

Published: Sep 30, 2014

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