Sulfur dioxide (SO2) is a major air pollutant and has significant impacts on plant physiology. Plant can adapt to SO2 stress by controlling stomatal movement, gene expression, and metabolic changes. Here we show clear evidences that SO2-triggered hydrogen peroxide (H2O2) production mediated stomatal closure and cell death in Arabidopsis leaves. High levels of SO2 caused irreversible stomatal closure and decline in guard cell viability, but low levels of SO2 caused reversible stomatal closure. Exogenous antioxidants ascorbic acid (AsA) and catalase (CAT) or Ca2+ antagonists EGTA and LaCl3 blocked SO2-induced stomatal closure and decline in viability. AsA and CAT also blocked SO2-induced H2O2 and elevation. However, EGTA and LaCl3 inhibited SO2-induced increase but did not suppress SO2-induced H2O2 elevation. These results indicate that H2O2 elevation triggered stomatal closure and cell death via signaling in SO2-stimulated Arabidopsis guard cells. NADPH oxidase inhibitor DPI blocked SO2-induced cell death but not the stomatal closure triggered by low levels of SO2, indicating that NADPH oxidase-dependent H2O2 production plays critical role in SO2 toxicity but is not necessary for SO2-induced stomatal closure. Our results suggest that H2O2 production and accumulation in SO2-stimulated plants trigger plant adaptation and toxicity via reactive oxygen species mediating Ca2+ signaling.

1. Introduction

Sulfur dioxide (SO2) is a harmful gas that is emitted largely from burning coal, high-sulfur oil, and fuels. During the past few decades, the concentration of SO2 in the atmosphere has increased in many areas of the world, especially in the developing countries. High levels of SO2 can injure many plant species and varieties, resulting in photosynthesis decline, growth inhibition, and even death [14]. Sulfur dioxide enters plants mainly through the open stomata [5]. Once it enters the leaf, SO2 is hydrated to form and . The toxicity of SO2 is derived from molecular species sulfite () and bisulfite () generated after SO2 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 SO2 [912].

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 [1517], 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 SO2 stress by controlling stomatal movement and gene transcription [11, 12, 20]. Stomatal closure could protect the leaves against further entry of the environmental SO2, 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 SO2 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 [2123], were employed to investigate the cellular mechanism of plant response to SO2 stress. To the best of our knowledge, this is the first report of H2O2 mediating both adaptation response (stomatal movement) and cytotoxicity (cell death) in plant response to SO2 stress. Our results show that H2O2 elevation triggered both stomatal closure and cell viability loss via Ca2+ signaling in SO2-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 μmoL 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 μmoL 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 SO2 hydrates (a mixture of sodium sulfite and sodium bisulfite, 3 : 1 mM/mM, prepared freshly before use) under continuous illumination of 180 μmoL 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 SO2 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 Ca2+ antagonists LaCl3 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 SO2 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 μM 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 μM 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 H2O2 level for each treatment.

2.5. Measurement of Intracellular Ca2+

The concentration of intracellular Ca2+ () 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. SO2-Evoked H2O2 Production Is Involved in the Regulation of Stomatal Movement

As shown in Figure 1, SO2 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 SO2 hydrates at concentrations of 10 to 500 μM. However, stomatal closure evoked by low SO2 concentrations (below 200 μM) could be reversed completely after SO2 removal; otherwise the decline of stomatal aperture in high SO2 concentration (500 μM) group could be partly reversed.

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 μM SO2 hydrates and continued to decline at a slower rate in the remaining 90 minutes of SO2 exposure (Figure 1(c)).

SO2-induced stomatal closure was associated with an elevated H2O2 level in Arabidopsis guard cells. Exposure to SO2 hydrates not only caused smaller stomatal apertures (Figure 1) but also evoked increased H2O2 level (1.2- to 1.7-fold) in Arabidopsis guard cells (Figure 2). When H2O2 elevation was blocked by exogenous H2O2 scavengers CAT or AsA, SO2-induced stomatal closure was efficiently reversed (Figure 2). These results indicate that H2O2 production in SO2-stimulated guard cells mediates stomatal movement upon SO2 stress.

3.2. SO2-Evoked H2O2 Elevation Triggered Cell Death

To investigate the role of H2O2 in irreversible stomatal closure in response to SO2, we detect the viability of Arabidopsis guard cells by double staining with fluorescein diacetate (FDA) and propidium iodide (PI). The results showed that SO2 induced guard cell death in a concentration-dependent manner (Figure 3). Cell death reached 24% in 6 mM SO2 hydrates treatment group, but no obvious cell death could be observed in 10 to 200 μM SO2 hydrates treatment groups. The H2O2 level of guard cells exposed to 2 to 6 mM SO2 hydrates showed a statistically significant increase (1.90- to 2.30-fold), which is higher than those exposed to 10 to 500 μM SO2 hydrates. Moreover, exposure to SO2 hydrates simultaneously with 200 U·mL−1 CAT or 0.1 mM AsA, SO2-induced cell death was efficiently blocked, associated with a significant decrease in H2O2 level of guard cells (Figure 3). These results clearly demonstrate that an elevated H2O2 level can trigger guard cell death leading to stomatal dysfunction and irreversible stomatal closure in SO2-treated Arabidopsis leaves.

To further confirm the role of H2O2 in SO2 toxicity, we investigate the protective effects of CAT on cell viability in SO2-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 SO2 hydrates, but the cytotoxicity evoked by 6 mM SO2 hydrates was only partly blocked (Figure 4). These results clearly demonstrate that H2O2 production, which may work together with other molecules, is enough to trigger SO2 toxicity.

3.3. H2O2 Action Is Dependent on Its Concentrations and Spatial Generation Patterns

As shown above, SO2 can cause stomatal closing and decline in cell viability, which is dependent on SO2 concentrations and H2O2 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 μM NADPH oxidase inhibitor DPI for 2 h markedly blocked cell death evoked by 2 mM SO2 hydrates. But 20 μM DPI did not inhibit stomatal closing evoked by 10 μM SO2 hydrates (Figure 4). These findings indicate that SO2-triggered stomatal closing can be driven by NADPH oxidase-dependent and -independent H2O2 generation, but NADPH oxidase-dependent H2O2 generation is involved in SO2-caused cytotoxicity.

3.4. H2O2 Acts Upstream of Ca2+ Signaling in Response to SO2 Stress

SO2 exposure enhanced the fluorescence intensity of Fluo-3 AM (Ca2+ 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 μM to 6 mM SO2 hydrates for 2 h but decreased markedly when isolated strips were treated simultaneously with SO2 hydrates and 0.1 mM calcium channel blocker LaCl3 (Figure 5). These results indicate that SO2 exposure evokes an elevation of level, and Ca2+ influx through Ca2+ channels in the plasma membrane results in elevation.

As shown in Figures 2 and 5, the addition of LaCl3 and calcium chelator EGTA to SO2 hydrates blocked elevation, stomatal closure, and cell death evoked by SO2. Stomatal closure and cell death occurred with increased in SO2-treated samples, but both of them were significantly suppressed by 0.1 mM LaCl3 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 Ca2+ antagonists LaCl3 and EGTA can suppress SO2-induced stomatal closure and cell death, but application of H2O2 scavenger CAT (200 U·mL−1) decreased elevation evoked by SO2, whereas application of Ca2+ channel inhibitor LaCl3 (0.1 mM) did not affect H2O2 elevation evoked by SO2. These results confirm the involvement of Ca2+ downstream of H2O2 production, indicating that H2O2 triggers stomatal movement and cell death via Ca2+ signaling in plant response to SO2 stress.

4. Discussion

Plant could adapt to environmental challenges through various means. Our recent findings showed that SO2 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 H2O2 acts as an important signaling molecule triggering stomatal movement and cell death in plant response to SO2. SO2-caused cell viability decline could interfere with the normal function of stomata to further affect plant physiology under environmental stress. However, the occurrence of SO2-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 [2629]. 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 H2O2 production in plant response to SO2 stress. First, SO2 induces stomatal closure and cell death associated with an increased intracellular H2O2 level (Figures 2 and 3). Second, two types of H2O2 scavengers CAT and AsA could block SO2-evoked stomatal closure and cell death; in particular, the effects of SO2 at low concentrations could be completely reversed by CAT (Figures 2 and 4). Third, H2O2 scavenger blocks the SO2-induced increase in intracellular Ca2+ required for stomatal closure and toxicity (Figure 5). Our study, validating the strong positive correlation between intracellular H2O2 level and stomatal closure/cell death, demonstrates a key role of H2O2 as trigger of stomatal closure and/or guard cell death in response to SO2.

Sulfur dioxide inhibited light-promoted stomatal opening and promoted stomatal closure leading to the smaller size of Arabidopsis stomata. High concentrations of SO2 also caused viability loss of Arabidopsis guard cells. The time course experiments show that H2O2 level of guard cells increased gradually during SO2 exposure (date not shown); therefore, an increased H2O2 level in SO2-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 SO2 exposure.

There are two primary sources of H2O2 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 H2O2 generation was dependent on SO2 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 H2O2 production in chloroplasts of guard cells. NADPH oxidase in plasma membrane contributes to O2· 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 SO2-induced cell death but cannot suppress stomatal closure evoked by low concentrations of SO2. These findings indicate that H2O2-mediated stomatal closure in Arabidopsis leaves exposed to low concentrations of SO2 is not dependent on the activity of plasma membrane NADPH oxidase, but cell death evoked by high concentrations of SO2 is NADPH oxidase-dependent.

SO2 exposure caused stomatal closure and guard cell death associated with elevation, whereas application of either Ca2+ chelator EGTA or Ca2+ channel inhibitor LaCl3 blocking SO2-evoked elevation, stomatal closure and cell death evoked by SO2 were effectively blocked. These results demonstrate that a channel-mediated Ca2+ influx across the plasma membrane contributes to the elevation of and subsequent stomatal closure and cell death in SO2-stimulated guard cells. However, it is not clear how Ca2+ signaling recognizes the different situations of cells to mediate appropriate processes such as stomatal closure and cell death.

It has been found that H2O2 could activate plasma membrane Ca2+ channels leading to increase in plant cells [3436]. Therefore, H2O2 activation of plasma membrane Ca2+ channels may be a central step in SO2-induced stomatal closure and/or cell death. The results of our present study also showed that application of antioxidant CAT and AsA significantly decreased SO2-evoked elevation, but application of Ca2+ channel blocker LaCl3 did not affect SO2-evoked H2O2 increase, indicating that H2O2 acts upstream of Ca2+ signaling in SO2-induced stomatal closure and/or cell death. These observations were consistent with other previous reports that a increase was linked to H2O2 production and was involved in ROS-mediated stomatal closure/cell death [3739]. Therefore, H2O2 elevation and subsequent activation of Ca2+ channels are events occurring in SO2-induced stomatal closure/cell death. Ca2+ influx from extracellular region results in increase, and then elevation mediates subsequent stomatal closure/cell death. These results suggested that H2O2 mediates SO2-induced stomatal movement/cytotoxicity by targeting Ca2+ channels in the plasma membrane.

Briefly, environmental SO2 has a remarkable effect on the size of the stomatal aperture. Exposure to SO2 induced the overproduction of H2O2 in guard cells, as shown in other plant cells exposed to environmental challenges [40, 41]. Elevated H2O2 acts in conjunction with other factors to activate stomatal movement and cell death under SO2 stress. Guard cells are a well-developed model system for characterizing early signal transduction mechanisms in plants. In this study, the dual role of H2O2 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 H2O2 level in Arabidopsis guard cells. H2O2 elevation triggered by SO2 mediated both stomatal closure and cell death via Ca2+ signaling. Intracellular Ca2+ increase is necessary for stomatal closure and cell death in Arabidopsis guard cells in response to SO2. H2O2 production by NADPH oxidase plays a critical role in SO2 toxicity; however NADPH oxidase activation is sufficient but not necessary for SO2-triggered stomatal closure. Both stomatal closing and stomatal opening inhibition evoked by SO2 lead to a decline in stomatal aperture, protecting the leaf against further entry of the pollutant but also curtailing photosynthesis. Cell death evoked by high concentrations of SO2 indicated the cytotoxicity of SO2, but might provide appropriate protection by reducing ROS production in Arabidopsis plants.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.


This study was supported by the National Natural Science Foundation of China (Grant nos. 30870454, 30470318, and 31371868), Research Fund for the Doctoral Program of Higher Education of China (Grant no. 20070108007), and Shanxi Scholarship Council of China (Grant no. 2009022).