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. 2025 Jun 12;66(7):1076–1085. doi: 10.1093/pcp/pcaf042

Sulfur dioxide-induced guard cell death and stomatal closure are attenuated in nitrate/proton antiporter AtCLCa mutants

Lia Ooi 1,2,*, Takakazu Matsuura 3, Izumi C Mori 4,*
PMCID: PMC12344094  PMID: 40478634

Abstract

Guard cells surrounding the stomata play a crucial role in regulating the entrance of hazardous gases such as SO2 into leaves. Stomatal closure could be a plant response to mitigate SO2 damage, although the mechanism for SO2-induced closure remains controversial. Proposed mediators for SO2-induced stomatal closure include phytohormones, reactive oxygen species, gasotransmitters, and cytosolic acidification. In this study, we investigated the mechanism of stomatal closure in Arabidopsis in response to SO2. Despite an increment in auxin and jasmonates after SO2 exposure, the addition of auxin did not cause stomatal closure and jasmonate-insensitive mutants exhibited SO2-induced stomatal closure suggesting auxin and jasmonates are not mediators leading to the closure. In addition, supplementation of scavenging reagents for reactive oxygen species and gasotransmitters did not inhibit SO2-induced closure. Instead, we found that cytosolic acidification is a credible mechanism for SO2-induced stomatal closure in Arabidopsis. CLCa mutants coding H+/nitrate antiporter, involved in cytosolic pH homeostasis, showed less sensitive stomatal phenotype against SO2. These results suggest that cytosolic pH homeostasis plays a tenable role in SO2 response in guard cells.

Keywords: airborne pollutants, cytosolic acidification, stomatal closure, sulfur dioxide

Introduction

Stomatal pores facilitate gas exchange within the epidermis in vascular plants. These pores are composed of a pair of guard cells. The turgor regulation of guard cells drives stomatal movements in response to environmental changes (Hetherrington and Woodward 2003). Stomata play a crucial role not only in the absorption of carbon dioxide for photosynthesis and the dissipation of water from leaves but also in serving as entry points for air pollutants. Ozone, nitrogen dioxide, and sulfur dioxide (SO2) can invade leaves through stomatal pores, causing damage to foliage at elevated levels.

Stomatal closure plays a role in defense to air pollutants. An earlier study demonstrated that induction of artificial stomatal closure made plants resistant to SO2 fumigation (Kondo and Sugahara 1978). In the same study, it was reported that there was a significant variability in SO2 sensitivity among plant species, which showed a positive correlation with abscisic acid (ABA) content in the leaves. In a field study conducted in a heavily air-polluted area, it was observed that higher levels of SO2 pollution were associated with reduced stomatal conductance in tropical trees (Rao and Dubey 1990). These findings suggest that stomatal closure is crucial for mitigating the impact of hazardous gases in plants (Majernik and Mansfield 1970, Taylor 1978).

It has been noted that the response of stomata to SO2 is complex. Leaf age, stomatal position, and sulfur availability within leaves can lead to different response of stomata to SO2, even within the same plant (Olszyk and Tibbitts 1981a, 1981b, Mozhgani et al. 2024). A study conducted on Helianthus annuus (common sunflower) demonstrated that when SO2 damage is mild, it can be reversed. For instance, stomata which close upon SO2 exposure can reopen after a few hours post-exposure. However, when the damage is severe, it became irreversible (Omasa et al. 1985). This suggests a correlation between the extent of damage and the degree of stomatal opening. Irreversible leaf injury is characterized by cell collapse and leaf wilting. The mechanisms responsible for these effects in response to SO2 exposure remained to be revealed. Furthermore, the signaling mechanisms involved in SO2 response in guard cells remained poorly understood.

Several reports have highlighted the potential role of ABA in regulating the process of SO2-induced stomatal closure. External application of ABA has been shown to induce stomatal closure, resulting in increased resistance to SO2 (Kondo and Sugahara 1978). Additionally, ABA levels in Glycine max (soybean) leaves were found to increase in response to SO2 fumigation in concentration- and time-dependent manners (Gupta et al. 1991). An analysis of SO2 resistance in Coleus scutellarioides cultivars revealed a correlation between higher ABA levels, reduced transpiration rates, and fewer SO2-induced lesions (Krizek et al. 2001). While the involvement of numerous phytohormones in regulating stomatal aperture has gained recognition (Murata et al. 2015), the potential roles of plant hormones in SO2-induced stomatal closure have not been thoroughly explored, apart from ABA.

Lipid and protein oxidation are also proposed as mechanisms for SO2 toxicity in plants. A study reported that the occurrence of hydrogen peroxide and singlet oxygen molecules in strawberry leaves following SO2 exposure leads to plant damages (Muneer et al. 2014). Numerous reports have demonstrated that superoxide detoxification by superoxide dismutase is a critical step in enhancing SO2 resistance in various plant species (Shimazaki et al. 1980, Tanaka and Sugahara 1980, Rao and Dubey 1990, Madamanchi and Alscher 1991, Madamanchi et al. 1994). A deficiency in ascorbic acid has also been linked to increased sensitivity to SO2 (Conklin et al. 1996). It can be hypothesized that oxidative damage may also serve as a potential trigger for stomatal closure, similar as observed in ozone exposure (Vainonen and Kangasjarvi 2015).

Hu et al. (2014) reported that nitric oxide (NO) scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), and hydrogen sulfide (H2S) scavenger, hypotaurine, were able to block SO2-induced stomatal closure in Ipomoea batatas. This study implicates that SO2 signaling mechanism in guard cells is mediated by two gasotransmitters, NO and H2S. In Hemerocallis fulva, mitigation of SO2-induced guard cell death by treatments with catalase and ascorbate, suggesting the role of ROS in this process (Wei et al. 2013). This study also reported the NO scavenger, cPTIO and Ca2+ channel blocker, LaCl3, decreased cell death rate. The generality of the involvement of these gasotransmitters in SO2-induced stomatal closure in other plant species remains to be evaluated.

Open Stomata 1 (OST1) is a SnRK2-type serine/threonine kinase. It plays a critical role in induction of stomatal closure in response to myriad environmental and endogenous stimuli, such as methyl jasmonate (MeJA), microbe-associated molecular pattern, and ozone (Vahisalu et al. 2010, Ye et al. 2015, Yin et al. 2016), as well as ABA (Mustilli et al. 2002). However, our previous study (Ooi et al. 2019) demonstrated that the loss-of-function mutations in OST1 did not affect SO2 response of stomata. This suggests that the mechanism for SO2-induced stomatal closure is distinct from that for those stimuli mediated with OST1 kinase.

NADPH oxidases play critical roles in reactive oxygen species (ROS) signaling in plants in addition to the roles in ROS burst during defense response (Fluhr 2009). Respiratory burst oxidase homologs, RbohD and RbohF are reported to participate in ABA- and methyl jasmonate-induced stomatal closure (Kwak et al. 2003, Suhita et al. 2004). Our previous report showed that SO2-induced stomatal closure was not impaired in the rbohd rbohf double mutant of Arabidopsis (Ooi et al. 2019). This result is one of the negative pieces of evidence for the involvement of oxidative signals in SO2-induced stomatal closure.

Cell collapse is a characteristic of SO2 damage in plant cells. In our previous study, we showed that cell death of guard cells is likely associated with stomatal closure (Ooi et al. 2019). This cell death is not related to chromosome fragmentation, suggesting it may be a form of necrotic cell death rather than apoptotic cell death. That study postulated that the mechanism behind the collapse of guard cells induced by SO2 involves cytosolic acidification, although further assessment is needed to confirm this hypothesis. CLORIDE CHANNEL A (CLCa) gene in Arabidopsis codes a proton-nitrate antiport protein localized in vacuolar membrane (De Angeli et al. 2006). This antiporter is not only involved in nitrate accumulation, but also involved in pH homeostasis of guard cells (Demes et al. 2020). It is a good candidate to confirm our hypothesis.

Here, we investigated the involvements of phytohormones, ROS, gasotransmitters, and cytosolic acidification in SO2-induced stomatal closure. To examine the involvement of plant hormones in SO2-induced stomatal closure, we took advantage of simultaneous multiple hormone analysis using liquid chromatography–mass spectrometry (LC–MS) in SO2-exposed Arabidopsis leaves. Genetic mutants related to the hormones demonstrated changes in the contents were analyzed to address the involvement of phytohormones in the process. The involvement of productions of ROS in SO2-induced stomatal closure was assessed using two ROS scavengers, N-acetylcysteine (NAC) and 1,2-dihydroxybenzene-3,5- disulfonic acid (tiron). We also examined the effects of the NO scavenger, cPTIO, and the H2S scavenger, hypotaurine to investigate the contribution of NO and H2S in SO2-induced stomatal closure in Arabidopsis. Furthermore, taking advantage of the cytosolic pH homeostasis-impaired phenotype of clca mutants, we tested the hypothesis of cytosolic acidification as a critical mechanism responsible for guard cell death induced by SO2 exposure.

Materials and Methods

Plant materials and growth conditions

Arabidopsis thaliana wild types (ecotypes Col and WS), coronatine insensitive 1 (coi1) mutant (Munemasa et al. 2007), and chloride channel-a mutants, clca2 and clca3 (Wege et al. 2014, Mozhgani et al. 2024) were grown in pots filled with Vermiculite GS (Nittai Co. Ltd., Osaka) and seedling soil (SK Agri, Kiryu, Japan) in a 4:3 ratio, in a growth chamber (Biotron LPH 200, NK System, Osaka) with 16 h-light/8 h-dark photoperiod regime at 135 µmol/m2/s–1, 23 ± 0.5°C, and 65–80% relative humidity.

Chemicals

All chemicals used in this study were of the special grade or the highest quality grade procured from Nacalai Testque, Inc. (Kyoto, Japan), Fujifilm Wako Pure Chemical Corporation (Osaka, Japan), or Kanto Chemical Co., Inc. (Tokyo, Japan), unless stated otherwise.

Phytohormone quantification

The contents of nine phytohormones, GA1, GA4, SA, JA, JA-Ile, IAA, tZ, iP, and ABA, in excised leaves treated with 180 min aqueous SO2 solution were quantified by LC–MS as previously described (Cho et al. 2022).

Stomatal closure assay

Measurement of stomatal aperture width was conducted essentially as previously described (Ooi et al. 2019). In brief, excised rosette leaves of 4- to 6 week-old plants were pre-incubated for 2 h floated on stomatal opening solution containing 5 mM KCl, 50 µM CaCl2, and 10 mM 2-(N-morpholino)ethanesulfonic acid-tris(hydroxymethyl)aminomethane (pH 5.7) under white fluorescent tube illumination at 120 µmol/m2/s, followed by a 3-h treatment of aqueous SO2 added in the stomatal opening solution, unless stated otherwise. Concentrations of H2SO3 in the solution were estimated as previously described (Ooi et al. 2019). After H2SO3 treatment, epidermal fragments were released from SO2-treated leaves by blending using a Waring Bar Blender (BB-900, Waring Products Inc., Torrington, CT, USA). Stomatal aperture width was measured under a microscope (BA300, Shimadzu Rika Corporation, Kyoto, Japan). Concentrations of the protonated forms of formic acid and maleic acid were estimated from their pKa1 values and pH of buffering solution (Supplemental Table S1).

Treatments with H2S, NO, and ROS scavengers

Excised rosette leaves were pre-incubated for 2 h, under white fluorescent tube illumination at 120 µmol/m2/s, in stomata opening solution supplemented with the H2S scavenger, hypotaurine (Merck KGaA, Darmstadt, Germany), the NO scavenger, cPTIO (Dojindo Laboratories, Kumamoto, Japan), the ROS scavengers, tiron (Tokyo Chemical Industry Co. Ltd., Tokyo, Japan) or NAC before they were exposed to H2SO3 for 2 h. Measurement of stomatal aperture width was conducted as described earlier.

Guard cell viability test

The viability of guard cells was assessed as previously described (Ooi et al. 2019). In brief, released epidermal fragments from SO2-treated excised rosette leaves were double-stained with 50 ng/ml of carboxyfluorescein diacetate (CFDA, Molecular Probes, Eugene, OR, USA) for 20 min and 2 ng/µl of propidium iodide (PI, Life technologies, Eugene, OR, USA) for 10 min in the stomatal opening solution. Stained epidermal fragments were thoroughly rinsed with distilled water and observed under a fluorescence microscope (Biozero BZ-X700, Keyence Corporation, Osaka, Japan) with BZ-X filter GFP for CFDA (excitation wavelength: 470/40 nm, emission wavelength: 525/50 nm, dichroic mirror wavelength: 495 nm) and BZ-X filter TRITC for PI (excitation wavelength: 545/25 nm, emission wavelength: 605/70 nm, dichroic mirror wavelength: 565 nm, Keyence Corporation). CFDA-positive and PI-positive cells were counted as viable cells and dead cells, respectively.

Results

Potential involvement of phytohormones in SO2-induced stomatal closure

In our previous report, we identified that when SO2 diffused into water, three chemical species H2SO3, HSO3−, and SO32− are formed; H2SO3 is the sole SO2-derived species which is responsible for SO2-induced stomatal closure (Ooi et al. 2019). An earlier study in various crop plants postulated that SO2-induced stomatal closure was mediated by ABA (Kondo and Sugahara 1978), while this statement requires further clarification. To investigate the roles of plant hormones in the process of SO2-induced stomatal closure, we quantified the contents of nine plant hormones in excised leaves exposed to H2SO3 for a period of 3 h, by LC–MS. We chose two H2SO3 concentrations (1.1 µM and 1.2 mM) for SO2 exposure here, since these concentrations resulted in distinct response in stomatal behavior: a slight widening (1.1 µM) and substantial closure of stomatal aperture (1.2 mM), respectively (Fig. 1A).

Figure 1.

Figure 1.

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Stomatal closure induction and hormone contents in H2SO3-treated leaves. (a) Time course of stomatal aperture width in a period of 180 min incubation in H2SO3; Contents of (b) jasmonic acid (JA), (c) jasmonoyl-isoleucine (JA-Ile), (d) indoleacetic acid (IAA) and (e) abscisic acid (ABA) in H2SO3-treated leaves. Mature rosette leaves of wild-type plants were incubated in stomata opening buffer containing 0 (control), 1.1 μM, and 1.2 mM H2SO3 for 180 min under white light radiation. Error bars represent SD. Some error bars are too small to be seen. Lower case letters indicate significant differences at 0.05 via one-way ANOVA followed by Tukey’s honestly significant post hoc test at each exposure time.

The contents of the major active gibberellins (gibberellinA1 [GA1], gibberellin A4 [GA4]), free salicylic acid (SA), and major active cytokinins (trans-zeatin [tZ], N6-isopentenyl adenine [iP]) in the leaves did not show significant changes by either 1.1 µM or 1.2 mM H2SO3 exposure (Supplementary Fig. S1). Therefore, the potential involvement of these five natural plant hormones in stomatal response to SO2 was inferred as less probable. Contents of jasmonates (jasmonic acid [JA], jasmonoyl-isoleucine [JA-Ile]) changed upon H2SO3 treatments (Fig. 1B and C). In the first 30 min, JA and JA-Ile contents decreased from the initial value in the control and 1.1 µM H2SO3 treatments. This may be attributed to the recovery from injury by leaves excision, which transiently increases JA and JA-Ile contents. For 1.2 mM H2SO3 treatment, JA and JA-Ile contents elevated significantly at 30 min and the elevation persisted for at least 180 min. At 1.1 µM H2SO3, the increase in JA contents was not observed, while JA-Ile contents increase slightly at 180 min. Auxin (indole-3-acetic acid [IAA]) contents were not significantly different at 1.1 µM H2SO3 compared to the control (Fig. 1D). Nevertheless, 1.2 mM-H2SO3 treatment caused a significant increase in IAA contents at 30 min and decreased to a lower level below the original level, at 180 min. The contents of ABA transiently increased after a 60 min treatment with 1.2 mM H2SO3, while it was not statistically significant (Fig. 1E).

In accordance with our hormone quantification results, we explored the potential participation of jasmonates and ABA in SO2-induced stomatal closure utilizing hormone-insensitive mutants. We also examined the involvement of IAA in stomatal closure induction utilizing exogenous application of IAA (Fig. 2). H2SO3 treatment of the JA-insensitive mutant, coronatine-insensitive 1 (coi1), showed no significant difference from the wild type. Reportedly, stomata of coi1 did not close by the application of meJA (Fig. 2A). The lower concentration (1.1 µM) of H2SO3 slightly increased the stomatal aperture width and the higher concentration (1.2 mM) induced substantial stomatal closure both in the wild type and coi1 similarly (Fig. 2B). These results suggest that jasmonates do not play a critical role in SO2-induced stomatal closure.

Figure 2.

Figure 2.

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Induction of stomatal closure by meJA, IAA and ABA in wild type (Col), and MeJA- and ABA-insensitive mutants (coi1 and sextuple). (a) MeJA-induced stomatal closure in Col and the jasmonate-insensitive coronatine-insensitive 1 (coi1) mutant. Four biological replicates consisted of 80 stomata. (b) Stomatal closure induction by H2SO3 in Col and coi1. Four biological replicates consisted of 80 stomata. (c) Stomatal movement of Col against 2 h of 0–100 µM IAA incubation. 0 IAA indicates 2 h treatment with 0.1% ethanol as the solvent control; eight biological replicates consisted of 160 stomata. (d) Stomatal movement of Col against 0, 15, 30, and 60 min of 10 µM IAA incubation. Four biological replicates consisted of 80 stomata. (e) Stomatal closure induction by pulse treatment of 10 µM IAA. Pre-incubated leaves of Col were treated with 30 min of 10 µM IAA in stomata opening buffer, rinsed with distilled water, followed by 150 min of incubation without IAA before the measurement of stomatal aperture width; three biological replicates consisted of 60 stomata. Insert panel illustrates the overview of experimental procedure. (f) ABA-induced stomatal closure in Col and sextuple mutant of the ABA receptor genes (sextuple). Three biological replicates consisted of 60 stomata. (g) Stomatal closure induction by H2SO3 in Col and sextuple; three biological replicates consisted of 60 stomata. Error bars indicate standard error of the mean. Dunnett’s tests (α = 0.05) performed on (c) and (d), and Student’s t-test (P > .05) conducted for data in (b) and (e) found no significant differences as compared to the respective controls.

The effect of exogenous IAA was examined to assess the role of IAA in SO2-induced stomatal closure (Fig. 2C-E). Pre-opened stomata were exposed to a wide range of IAA concentrations for 2 h. The aperture width of stomata treated with 1, 10 and 100 µM IAA were similar in size (P > .05, Fig. 2C). Considering the dynamic as observed in the hormone quantification data, of which bidirectional time-dependent shifts in IAA levels following SO2 treatment were observed (Fig. 1D), we conducted a time-course analysis of stomatal aperture after the application of IAA. Time-course experiment with 10 µM IAA showed a constant stomatal aperture width over 1 h (Fig. 2D). Furthermore, we artificially create a transient IAA elevation in the first 30 min (pulse exposure) to mimic the observed changes in IAA contents after SO2 treatment, as in Fig. 1D. Sizes of stomatal aperture between solvent control and 10 µM IAA pulse exposure were not significantly different (Fig. 2E). We did not find a clue for the involvement of IAA in SO2-induced stomatal closure from this attempt. Lastly, we examined the effect of H2SO3 on the sextuple ABA receptor mutant, sextuple. While the mutant showed strong insensitivity to ABA application (Fig. 2F), stomatal aperture responded similar to H2SO3, as compared to the wild type (Fig. 2G). This suggests that the canonical ABA signaling mediated by the six major PYR1/PYLs/RCARs ABA receptors (Gonzalez-Guzman et al. 2012, Merilo et al. 2013) does not participate in SO2-induced stomatal closure. This again reject the hypothesis that ABA is involved in regulating SO2-induced stomatal closure, at least in Arabidopsis.

SO2-induced stomatal closure is not mediated by hydrogen sulfide and nitric oxide in Arabidopsis

It was reported that gasotransmitters such as H2S and NO mediate SO2-induced stomatal closure in Ipomoea batatas (Hu et al. 2014). Here, we investigated the involvement of these gasotransmitters in Arabidopsis using scavenging reagents, hypotaurine, and cPTIO, which abolish H2S and NO generation, respectively. In the absence of H2SO3, 100 µM hypotaurine and 200 µM cPTIO did not alter stomatal aperture width in Arabidopsis (Fig. 3A). Stomata stayed open when treated with 1.1 µM H2SO3, in all conditions, with and without the presence of scavengers. Exposure of leaves to 1.2 mM H2SO3 caused significant stomatal closure in all conditions, even in the presence of hypotaurine and cPTIO. These scavengers did not inhibit 1.2 mM H2SO3-induced stomatal closure (Fig. 3A), unlike reported in I. batatas (Hu et al. 2014). This suggests that SO2-induced signaling mechanism in guard cells which involved H2S and NO, is distinct between I. batatas and A. thaliana.

Figure 3.

Figure 3.

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Effect of Hypotaurine (HT), 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide sodium salt (cPTIO), N-acetyl-l-cysteine (NAC) and tiron (1,2-Dihydroxybenzene-3,5-disulfonic acid) on stomatal aperture width in the presence of H2SO3. Pre-opened stomata were incubated in the experimental solution containing H2SO3 added with either 100 µM HT or 200 µM cPTIO (a), and 1 mM NAC or 5 mM tiron (b), for 2 h. Four biological replicates per bar consisted of 80 stomata. All scavengers (HT, cPTIO, NAC, and Tiron) used are dissolved in purified water. ‘Solvent control’ represents H2SO3 treatments without the presence of scavengers. Error bars indicate SE. Results from scavenger treatments showed non-significant differences from the solvent control by Student’s t-test, for treatments with the same H2SO3 concentration.

Reactive oxygen species do not play a critical role in SO2-induced stomatal closure in Arabidopsis

Earlier studies have suggested that ROS formation occurs after SO2 exposure, and the activation of antioxidant systems is associated with resistance mechanism against SO2 (Asada and Kiso 1973, Rao and Dubey 1990, Madamanchi and Alscher 1991). To investigate the involvement of antioxidant systems in stomatal response of Arabidopsis against SO2, the effects of two ROS scavengers, NAC and tiron on SO2-induced stomatal closure were examined (Fig. 3B). Given that ROS signaling positively mediates SO2-induced stomatal closure, the application of ROS scavengers would inhibit stomatal closure. In the absence of H2SO3, neither NAC nor Tiron apparently affect the stomatal aperture width. In the presence of 1.1 µM and 1.2 mM H2SO3, no significant difference was observed among the scavenger treatments in any case. This indicates that ROS formation does not play a role in SO2-induced stomatal closure, at least in Arabidopsis. On the other hand, it might be one of the mechanisms resulting from SO2-induced injury.

Role of CLCa proton/nitrate antiporter in guard cell resistance against SO2

In our previous study, we proposed that a potential mechanism of SO2-induced cell death in guard cells is cytosolic acidification (Ooi et al. 2019). To further elucidate this hypothesis, we conducted stomatal assays in chloride channel a (clca) mutants. CLCa transporter protein is well-known for its crucial role in facilitating proton-coupled anion transport across both plasma membrane and vacuolar membrane. However, emerging evidence suggests that CLCa also play a role in maintaining cytosolic pH homeostasis (Demes et al. 2020).

Using clca mutants (clca2 on Wassilewskija [WS] genetic background and clca3 on Columbia-0 [Col] genetic background), we explored the relationship between cytosolic pH homeostasis and the response of stomata to H2SO3 (Fig. 4). Stomatal aperture of wild types was narrowed by increasing concentrations of H2SO3 (Fig. 4A and B). When treated with 0.3 mM H2SO3, the aperture width became 2.04 ± 0.21 µm (WS) and 1.79 ± 0.68 µm (Col). Different from the wild types, aperture width of the clca mutants stayed wide at 2.69 ± 0.16 µm (clca2) and 2.93 ± 0.22 µm (clca3) (Fig. 4A and B). In the previous study, it was reported that stomatal closure of Arabidopsis after H2SO3 exposure consisted of two directional responses: slight widening at modest H2SO3 concentrations and tightly closed at higher H2SO3 concentrations, which bring about two separable stomatal populations (Ooi et al. 2019). That study implies that tightly closed stomatal population was of dead guard cells and the open-wide stomatal population was of viable guard cells. Here, we took a histogram analysis to investigate the stomatal populations of wild types and clca mutants (Fig. 4C and D). The histograms showed two phasic distributions of stomatal aperture widths. It was apparent that the frequency of wider populations was greater in the clca mutants than the wild types, suggesting more viable guard cells were sustained in clca mutants after a 3-h of 0.3 mM H2SO3 treatment. Based on our previous and current results, we infer that guard cells of clca mutants exhibit higher resistance to SO2 than the wild types. We then examined the viability of SO2-treated guard cells in both clca mutants and the wild types (Fig. 4E-H).

Figure 4.

Figure 4.

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Stomatal closure and cell death induction of H2SO3 in wild-types and clca mutants. (a and b) Stomatal closure induction of H2SO3 in wild types (Col and WS); clca2 and clca3 mutants. Four biological replicates consisted of 80 stomata. Asterisks indicate significant differences (α = 0.05) by Student’s t-test. Error bars represent standard error of the mean. Some error bars are too small to be seen. (c and d) Distribution of stomatal aperture width in leaves treated with 3 h of 0.3 mM H2SO3. Bars represent the frequency of aperture width of wild types and clca mutants. Eighty stomata were measured for each line. (E–H) Guard cell viability of H2SO3-exposed wild-types and clca mutants. Four independent experiments consisted of 150–200 guard cells were observed for each experiment. Error bars indicate standard error of the mean. Some of the error bars are too small to be seen. Asterisks represent significant different via one-way ANOVA followed by Dunnett’s test (α = 0.05).

In accordance with our previous report, increasing concentrations of H2SO3 induced cell death of guard cells in wild types above 10–6 m (Fig. 4E and G, Ooi et al. 2019). In clca mutants, the H2SO3 concentrations that killed ∼100% of guard cells were higher compared to the corresponding wild types (Fig. 4F and H). This result is in a good agreement with the stomatal aperture width results (Fig. 4A-D), if we consider that observed stomatal closure induced by H2SO3 is associated with cell death. Furthermore, the results demonstrated in Fig. 4, collectively, suggest that sensitivity of Arabidopsis stomata to SO2 is affected by cytosolic pH homeostasis.

Given that cytosolic acidification act as the key role in stomatal closure in the exposure to H2SO3, other weak acids would also induce stomatal closure. To test that hypothesis, we assessed the effects of formic acid and maleic acid on stomatal closure in wildtypes and clca mutants (Supplemental Fig. S2). Maleic acid at 1.2 mM induced stomatal closure in Col and WS. This was partially attenuated in clca mutants. On the other hand, formic acid did not induce the closure evidently. This may be due to the difference in pKa1 of these chemicals, pKa1 of formic acid, maleic acid, and sulfurous acid are 3.75, 1.90 and 1.89, respectively. The range of pKa1 around 1.9 may efficiently acidify the guard cell cytosol, favorably kill the cells. Notably, the effect of maleic acid was attenuated in clca mutants (Supplemental Fig. S2), suggesting that pH homeostasis in the mutants was associated with the closure phenotype as observed for H2SO3 (Fig. 4). Unlike in the Col background, formic acid modestly induced stomatal closure in WS and clca2. The degree of the closure seemed comparable between WS and clca2. The WS background might be more sensitive to dicarboxylic acid than the Col background, and this sensitivity is not mediated via cytosolic pH. This can explain the remaining sensitivity of clca2 mutant to 1.2 mM maleic acid. Our results suggest that weak acids with pKa1 around 1.9 can induce stomatal closure in Arabidopsis.

Higher SO2 tolerance of clca stomata rendered whole-leaf phenotype to be more SO2-sensitive

Considering that stomatal closure is a stress avoidance response against hazardous gases, keeping stomatal aperture to stay widely opened can lead to a greater invasion of SO2 into leaf tissues, thus resulting to higher susceptibility of leaves to SO2. H2SO3-treated clca mutant leaves appeared paler than each corresponding wild type (Fig. 5A), indicating higher sensitivities of clca mutant leaves to H2SO3 as compared to wild types. There was no apparent difference in chlorophyll contents between WS and clca2 (Fig. 5B). However, chlorophyll contents in clca3 after a 3-h exposure to H2SO3 were found to be lesser than Col (Fig. 5C). There could be a minor difference in whole-leaf phenotype between wild types and clca mutants due to the difference in stomatal sensitivity against SO2. The clca mutants have stronger guard cell resistance against SO2 allowing them to stay alive and their stomata to stay open for longer time, resulting in paler leaves from larger amount of SO2 entrance into the leaves. Nevertheless, the discrepancy in chlorophyll content is not significant in WS and clca2. There may be other factors for whole-leaf SO2 sensitivity beside prolonged guard cell resistance gained from cytosolic pH homeostasis.

Figure 5.

Figure 5.

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Effects of H2SO3 on the appearance of whole rosettes of wild types and clca mutants. (a) Representative images of excised rosette leaves after a 3 hr of H2SO3 exposure. (b) Chlorophyll content in H2SO3-treated leaves, n = 3. Asterisks indicate significant differences (α = 0.05) by Student’s t-test. Error bars represent standard error of the mean. Some error bars are too small to be seen.

Discussion

A potential role of ABA in mediating SO2-induced stomatal closure was proposed with experiments in several plant species (Kondo and Sugahara 1978, Gupta et al. 1991, Krizek et al. 2001). It is well known that not only ABA but also other plant hormones are involved in regulating stomatal closure (Murata et al. 2015). In this study, we found that contents of jasmonates and IAA in Arabidopsis leaves changed upon H2SO3 exposure (Fig. 1B-D). ABA contents also changed modestly (Fig. 1E). Based on these findings, we assessed stomatal phenotype in JA- and ABA-insensitive mutants (Fig. 2A, B, F and G). In addition, the response of stomata to exogenous application of IAA was also assessed (Fig. 2C-E). IAA-impaired mutants were not used since IAA-insensitive mutants show dwarfism that would hamper stomatal assay (Leyser et al. 1993, Prigge et al. 2020). The results in this study showed no sign of involvement of plant hormones in H2SO3-induced stomatal closure in Arabidopsis. We employed several Arabidopsis genotypes as study materials here using hormone-insensitive mutants available. Exploitation of mutants exhibiting hormone insensitivity in other plant species is not as easy as Arabidopsis. Meanwhile, Raphanus sativus, a Brassicaceae species was shown to be more sensitive to SO2 as compared to Arachis hypogaea (Fabaceae) and Solanum lycopersicum (Solanaceae) (Kondo and Sugahara 1978). Since Arabidopsis is a Brassicaceae species, it may exhibit SO2 sensitivity like R. sativus. Considering that there could be a potential variation in SO2 sensitivity among different species, a distinct phytohormone contribution to SO2 response may be found in other species.

It was proposed that ROS production is involved in SO2 toxicity (Li and Yi 2012). It is well acknowledged that ROS production plays a role in stomatal closure (Pham and Desikan 2009). Therefore, we hypothesized that ROS production following SO2 exposure takes part in SO2-induced stomatal closure. A pharmacological study showed that gasotransmitters H2S and NO are involved in SO2-induced stomatal closure in I. batatas and H. fulva (Wei et al. 2013, Hu et al. 2014). In ABA signaling in guard cells, ROS, H2S, and NO are postulated to coordinately function in activating Ca2+ channels (Honda et al. 2015). Thus, it is tempting to speculate that ROS production and successive interaction with H2S and NO participate in SO2-induced stomatal closure. In this study, evidence for the involvement of ROS in SO2-induced stomatal closure was not obtained based on the result from coincubation of SO2-treated leaves with ROS scavengers (Fig. 3B). In addition, the presence of scavengers of NO and H2S did not inhibit SO2-induced stomatal closure (Fig. 3A). Our previous study showed that SO2-induced stomatal closure was not impaired in the NADPH oxidase double knockout mutant, rbohD/F (Ooi et al. 2019). These findings suggest that the coordinate action of ROS, H2S, and NO does not occur in the process of SO2-induced stomatal closure in Arabidopsis. However, this does not reject the possibility that the involvement of ROS, H2S, and NO in SO2 response might be diverse across plant species.

We have anticipated that acidification of guard cell cytosol and successive cell deaths are the events contributing to the mechanism for SO2-induced stomatal closure. In the previous study, we showed that the toxicity of aqueous solution of SO2 was owing to H2SO3 but not HSO3− or SO32− (Ooi et al. 2019). Given that the acidification of guard cell is critical for SO2-induced stomatal closure, a mutant associated with proton regulation/pH homeostasis would affect SO2 sensitivity of stomata. CLCa is a tonoplast-localized proton/nitrate antiporter and plays myriads of roles in plant cells. It is involved in nitrate uptake, nitrogen distribution among organic and inorganic compounds, osmotic regulation, stomatal response to abscisic acid, and pH homeostasis (Geelen et al. 2000, De Angeli et al. 2006, Wege et al. 2014, Hodin et al. 2023). In this study, we showed that the guard cells of clca mutants were more resistant to H2SO3 toxicity, and stomata of clca mutants stayed wide open with larger aperture widths as compared to the wild types upon SO2 exposure (Fig. 4). CLCa is shown to participate in pH homeostasis in Arabidopsis guard cells (Demes et al. 2020), most likely via regulating the balance of cytosolic proton and vacuolar nitrate. It is conceivable that stabilized pH homeostasis in clca would rescue the cell from H2SO3-induced cell death. Alternatively, it is also conceivable that enormous acidification in the cytosol by H2SO3 would result in a sudden exchange in nitrate between the vacuole and the cytosol, consequently malfunctioning cellular function via an abrupt change in ion strength and/or membrane potential in guard cells. This change in nitrate concentration may not occur in clca mutants since the proton/nitrate antiporter is not functional. Although the fine mechanism for the resistance of clca mutant guard cells to SO2 remained to be further clarified, our study clearly showed the SO2 toxicity can be mitigated by disrupting CLCa function in guard cells. Given that the biochemical role of CLCa as proton/nitrate antiporter (De Angeli et al. 2006), it is plausible that SO2 toxicity on guard cells and cytosolic pH are tightly related. It should be noted that some other proton-coupled transporters may also be involved in SO2 sensitivity through cytosolic pH homeostasis beyond CLCa. To further demonstrate that cell acidification plays a role in cell death and stomatal closure, we investigated the effects of weak acids other than sulfurous acid on stomatal aperture. Maleic acid (pKa1 = 1.9) strongly induced stomatal closure, suggesting that another weak acid capable of sufficiently acidifying the cytoplasm can trigger stomatal closure. In contrast, formic acid (pKa1 = 3.75) did not significantly promote stomatal closure. Since the pKa1 of sulfurous acid is approximately 1.9, we hypothesized that weak acids within a specific pKa range can acidify the cytoplasm and ultimately lead to guard cell death. This model is consistent with the working hypothesis derived from experiments using the clca mutants. It is supported by experiments with the clca mutants, where the effect of maleic acid was attenuated, aligning with our working hypothesis.

Stomata serve as the gateway for SO2 entry into leaves, thus linking whole-leaf SO2 sensitivity with its stomatal phenotype. In Columbia ecotype background, clca3 mutant demonstrated increased sensitivity to SO2 in terms of chlorosis (Fig. 5). This increased sensitivity may be attributed to prolonged opening of stomata at higher levels of SO2 due to higher resistance of guard cells of clca3. It is worth noting that whole leaf becomes more sensitive when guard cells exhibit greater resistance to SO2. Stomata close due to cell death of guard cells in response to SO2. Consequently, whole leaves initially appear more resistant in Col wild type in a 3-h SO2 exposure, although this may not be conductive to long-term survival. To enhance resistance to SO2 exposure, it is crucial for both mesophyll and guard cells to develop resistance to SO2. Additionally, it is important to recognize that the leaves of WS ecotype displayed greater resistance to SO2-induced chlorosis as compared to those of the Col ecotype (Fig. 5), indicating a variation in SO2 sensitivity even within a single species.

In this study, we observed that clca mutant in Col ecotype exhibits gained resistance to SO2 only in guard cells but not in whole leaf (Figs. 4 and 5). Nonetheless, it is conceivable that the manipulation of CLC-related genes in fruits, flowers, and mesophyll tissues could potentially lead to improved SO2 resistance in crops. We explored the mechanisms behind SO2-induced stomatal closure in Arabidopsis. The participation of ABA in this process was not substantiated, with observations from hormone profiling and SO2 treatment in a sextuple ABA receptor knockout mutant. Involvement of jasmonates and IAA were also denied via investigation employing a JA-insensitive mutant and exogenous IAA treatment (Fig. 2). Pharmacological evidence using scavengers did not support the involvement of ROS, H2S, and NO in the process of SO2-induced stomatal closure (Fig. 3). Our findings indicate the crucial role of the proton-nitrate antiporter, CLCa, in resisting SO2 toxicity. Reverse genetic study using clca mutants demonstrated lower sensitivity of guard cells against SO2, suggesting that acidification of guard cell cytosol is the primary mechanism leading to guard cell death and subsequent stomatal closure.

Supplementary Material

pcaf042_Supp
pcaf042_supp.zip (305.7KB, zip)

Acknowledgments

The authors thank Dr. Shintaro Munemasa for providing coi1 mutant seeds, Dr. Sophie Filleur for providing clca2 and clca3 mutant seeds, and Mr. Mahdi Mozhgani for his assistance in double-checking a part of experimental results.

Contributor Information

Lia Ooi, Institute of Plant Science and Resources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama 710-0046, Japan; Plant & Microbial Research Unit, Research, Technology & Value Creation Division, Nagase Viita Co., Ltd., 675-1 Fujisaki, Naka-ku, Okayama 702-8006, Japan.

Takakazu Matsuura, Institute of Plant Science and Resources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama 710-0046, Japan.

Izumi C Mori, Institute of Plant Science and Resources, Okayama University, 2-20-1 Chuo, Kurashiki, Okayama 710-0046, Japan.

Supplementary Data

Supplementary Data is available at PCP online.

Author Contributions

O.L. and I.C.M. conceived the research. O.L. performed the experiments. O.L., T.M. and I.C.M. performed LC-MS analysis. O.L. and I.C.M. wrote the manuscript.

Conflict of interest

None declared.

Funding

This work was supported by Japan Society for the Promotion of Science (grant number 18F18391, 18H02169, 23K05025), Research Grant for Encouragement of Students FY2017, Graduate School of Environmental and Life Science, Okayama University.

Data Availability

Sequence data used in this article can be found in the Arabidopsis Information Resource database (https://www.arabidopsis.org/) under the following accession numbers: CLCa (At5g40890), COI1 (At2g39940), PYR1 (At4g17870), PYL1 (At5g46790), PYL2 (At2g26040), PYL4 (At2g38310), PYL5 (At5g05440), and PYL8 (At5g53160). The authors confirm that the data supporting the findings of this study are available within the article and its online supplementary materials. Additional data related to this article may be shared on reasonable request to the corresponding authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

pcaf042_Supp
pcaf042_supp.zip (305.7KB, zip)

Data Availability Statement

Sequence data used in this article can be found in the Arabidopsis Information Resource database (https://www.arabidopsis.org/) under the following accession numbers: CLCa (At5g40890), COI1 (At2g39940), PYR1 (At4g17870), PYL1 (At5g46790), PYL2 (At2g26040), PYL4 (At2g38310), PYL5 (At5g05440), and PYL8 (At5g53160). The authors confirm that the data supporting the findings of this study are available within the article and its online supplementary materials. Additional data related to this article may be shared on reasonable request to the corresponding authors.

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