Atmospheric H2S exposure does not affect stomatal aperture in maize

Main conclusion Stomatal aperture in maize is not affected by exposure to a subtoxic concentration of atmospheric H2S. At least in maize, H2S, thus, is not a gaseous signal molecule that controls stomatal aperture. Abstract Sulfur is an indispensable element for the physiological functioning of plants with hydrogen sulfide (H2S) potentially acting as gasotransmitter in the regulation of stomatal aperture. It is often assumed that H2S is metabolized into cysteine to stimulate stomatal closure. To study the significance of H2S for the regulation of stomatal closure, maize was exposed to a subtoxic atmospheric H2S level in the presence or absence of a sulfate supply to the root. Similar to other plants, maize could use H2S as a sulfur source for growth. Whereas sulfate-deprived plants had a lower biomass than sulfate-sufficient plants, exposure to H2S alleviated this growth reduction. Shoot sulfate, glutathione, and cysteine levels were significantly higher in H2S-fumigated plants compared to non-fumigated plants. Nevertheless, this was not associated with changes in the leaf area, stomatal density, stomatal resistance, and transpiration rate of plants, meaning that H2S exposure did not affect the transpiration rate per stoma. Hence, it did not affect stomatal aperture, indicating that, at least in maize, H2S is not a gaseous signal molecule controlling this aperture. Electronic supplementary material The online version of this article (10.1007/s00425-020-03463-6) contains supplementary material, which is available to authorized users.


Introduction
Sulfur is an essential macronutrient for plants, which plants usually acquire as sulfate via the root (Hawkesford and De Kok 2006). After its uptake, sulfate is reduced via several intermediates to sulfide, which is subsequently incorporated in cysteine via the reaction of sulfide with O-acetylserine (OAS), catalyzed by the enzyme O-acetylserine(thiol)lyase (OAS-TL; Hawkesford and De Kok 2006). Cysteine functions as the precursor and reduced sulfur donor for the synthesis of other organic compounds.
It is often assumed that sulfur-containing metabolites might modulate physiological processes in plants. Hydrogen sulfide (H 2 S) might act as endogenous gasotransmitter that affects plant development and stress tolerance (Sirko and Gotor 2007;Calderwood and Kopriva 2014;Maniou et al. 2014;Hancock 2018). Moreover, H 2 S might control the aperture of stomata (Lisjak et al. 2010(Lisjak et al. , 2011Scuffi et al. 2014;Honda et al. 2015;Li et al. 2016;Aroca et al. 2018;Zhang et al. 2019). It is assumed that H 2 S is metabolized into cysteine to stimulate the synthesis of abscisic acid (ABA), which is the canonical trigger for stomatal closure (Batool et al. 2018;Rajab et al. 2019).
The physiological significance of H 2 S for stomatal closure should, however, be questioned. Research with thale cress (Arabidopsis thaliana), maize (Zea mays), cabbage (Brassica olerecea), pumpkin (Curcubita pepo), spruce (Picea abies), and spinach (Spinacea oleracea) showed that exposure to atmospheric H 2 S did not affect transpiration rates, measured at the whole plant level, at various concentrations and under all exposure periods applied (which ranged from minutes to days; De Kok et al. 1989; Van der Stuiver and De Kok 2001;Tausz et al. 1998).
63 Page 2 of 9 Accordingly, there are at least two caveats pertaining studies that reported impacts of H 2 S on stomatal dynamics. First, uncontrolled, potentially very high, levels of H 2 S have been used (e.g., Scuffi et al. 2014;Zhang et al. 2019). Sodium hydrosulfide (NaHS) has been used as H 2 S donor and it was added to nutrient or tissue incubation solutions at pH < 7.0. However, if NaHS is used at this pH range, HS − is rapidly converted to gaseous H 2 S (HS − + H + ⇄ H 2 S; pKa = 7.0; Lee et al. 2011). Since H 2 S is rather poorly soluble in water (the Henry's law solubility constant for H 2 S is 0.086 M atm −1 at 25 °C), it is quickly released into the atmosphere, where it may transiently reach phytotoxic (growth-inhibiting) levels (Lee et al. 2011;Riahi and Rowley 2014). H 2 S may bind to metallo-groups in enzymes and other proteins (Beauchamp et al. 1984;Maas and De Kok 1988). Reported impacts of H 2 S on stomatal aperture could possibly be the consequence of such toxicity, instead of being specifically related to H 2 S functioning as gasotransmitter. One should further bear in mind that especially thale cress, which functioned as model plant, is rather susceptible to atmospheric H 2 S (Van der Birke et al. 2015).
Secondly, in some studies (e.g., Zhang et al. 2019), mutants with a modified H 2 S homeostasis were used. Genetic manipulation of H 2 S homeostasis may not only alter tissue H 2 S content, but also the contents of other metabolites. These associated changes in metabolite contents may impact stomatal aperture. Hence, perceived impacts on stomatal aperture in mutants cannot directly be ascribed to the modification in H 2 S homeostasis (viz., genotypic variation cannot directly be translated to phenotypic variation; Piersma and Van Gils 2011;Noble 2013;Noble et al. 2014).
The application of controlled, subtoxic (non-growthinhibiting) levels of atmospheric H 2 S to non-mutant plants can provide a physiologically realistic view of the role of H 2 S in stomatal regulation. Plants absorb atmospheric H 2 S via stomata, since the leaf's cuticle is hardly permeable for gases (Ausma and De Kok 2019). At the pH of leaf cells (i.e., ~ 5-6.4) absorbed H 2 S remains largely undissociated, causing it to easily pass cellular and subcellular membranes (Lee et al. 2011;Riahi and Rowley 2014). Foliar H 2 S levels increase significantly upon H 2 S fumigation (Ausma and De Kok 2019). For instance, exposure of thale cress to 0.5 and 1.0 µl l −1 H 2 S enhanced leaf H 2 S levels by approximately twofold and threefold, respectively (Birke et al. 2015). Since H 2 S is rapidly and with high affinity metabolized in cysteine, H 2 S fumigation also strongly enhanced foliar cysteine content and that of the tripeptide glutathione (De Kok et al. 1997;Birke et al. 2015;Ausma et al. 2017;Ausma and De Kok 2019). Thus, fumigation with low H 2 S levels may profoundly alter tissue sulfur status, without affecting plant growth (Ausma and De Kok 2019).
Plants may switch from using sulfate to using H 2 S as sulfur source: H 2 S absorbance by the foliage may partially downregulate the uptake and subsequent metabolism of sulfate (Buchner et al. 2004;De Kok et al. 1997). Plants may even grow with atmospheric H 2 S as the only sulfur source (viz., in the absence of a root sulfate supply; De Kok et al. 1997;Koralewska et al. 2007Koralewska et al. , 2008. Whereas sulfate deprivation may reduce plant growth rate as well as endogenous cysteine and glutathione levels, fumigation with a sufficiently high H 2 S level may fully alleviate these reductions. Here, we study the importance of H 2 S as gaseous signal molecule for the regulation of stomatal aperture in maize (Zea mays). Initially, we determined the H 2 S level that is subtoxic for maize, though sufficiently high to fully cover the plant's sulfur demand for growth (viz., the H 2 S concentration at which H 2 S-fumigated plants have a similar biomass as non-fumigated sulfate-sufficient plants). We then exposed plants for several days to this atmospheric H 2 S level in the presence or absence of a root sulfate supply. We measured plant growth, sulfur status, stomatal density, stomatal resistance, and transpiration rates. We conclude that, at least in maize, H 2 S is not a gaseous signal molecule that controls stomatal opening.

Plant material and growth conditions
Seeds of maize (Zea mays; cultivar number 669; Van Der Wal; Hoogeveen; The Netherlands) were germinated between moistened filter paper in the dark at 23 °C. After 3 days, the seedlings were put on 15 l boxes containing aerated tap water, which were placed in a climate-controlled room. Air temperature was 23 °C (± 1 °C), relative humidity was 60-70%, and the photoperiod was 16 h at a photon fluency rate of 300 ± 20 µmol m -2 s -1 (within the 400-700 nm range) at plant height, supplied by Philips GreenPower LED (deep red/white 120) production modules.
Plants were fumigated either with 0, 0.5, 1.0, or 1.5 µl l −1 H 2 S. Pressurized H 2 S diluted with N 2 (1.0 ml l −1 ) was injected into the incoming air stream and the concentration in the cabinet was adjusted to the desired level using electronic mass flow controllers (ASM; Bilthoven; The Netherlands). H 2 S levels in the cabinets were monitored by an SO 2 analyzer (model 9850) equipped with a H 2 S converter (model 8770; Monitor Labs; Measurements Controls Corporation; Englewood; CO; USA). Sealing of the lid of the boxes and plant sets prevented absorption of H 2 S by the nutrient solutions.
In the first experiment, plants were harvested after 10 days of exposure. In the second experiment after 7 days of exposure per treatment, sets of 4 plants were weighted (viz., total biomass was determined). Subsequently, each plant set was transferred to a separate vessel containing 1.1 l of a similar 50% Hoagland nutrient solution as the set was grown on before ( Supplementary Fig. S1). Vessels with plant sets were placed in the stainless-steel cabinets described above (with similar H 2 S levels) and plants were grown for an additional 3 days before harvest.

Growth analyses
Plant harvesting took place 3 h after the onset of the light period. To remove ions and other particles attached to the root, plants were placed with their roots in ice-cold de-mineralized water (3 × 20 s). Thereafter, the root and shoot were separated and weighted. In the second experiment, the shoot was additionally separated in leaf blades and the whorl of leaf sheaths (viz., the seedlings did not yet possess a true stem, since all leaves emerged from the shoot base). Moreover, the total leaf blade area (abaxial plus adaxial) of the plants was determined by drawing the outlines of all leaf blades on graph paper.

Stomatal resistance
On the harvest day, stomatal resistance was analyzed at the abaxial and adaxial side of nascent leaf blades using a portable leaf porometer (AP4 Leaf Porometer; Delta-T-Devices Ltd.; Cambridge; UK). Measurements were performed 2-3 h after the onset of the light period.

Plant sulfur status
In whole shoots (leaf blades plus sheaths) and roots, which were stored at − 20 °C after harvest, sulfate levels were determined via high-performance liquid chromatography (HPLC) following Maas et al. (1986). Additionally, watersoluble non-protein thiols were extracted from freshly harvested shoots and roots. The total water-soluble non-protein thiol and cysteine content were determined colorimetrically according to De Kok et al. (1988).

Stomatal density
For the determination of stomatal density, silicone impression paste was prepared by 1:1 mixing of catalyst and base material (Provil Novo Light; Kulzer GmbH; Hanau; Germany). Subsequently, freshly harvested nascent leaf blades were gently pressed in the paste with either their abaxial or adaxial side. Once the paste had solidified, the leaf blades were removed and the mould was filled with transparent nail polish, as described by Kraaij and van der Kooi (2020). The positive (nail polish) replica was next examined under an Olympus CX-41 microscope and photographed using a Euromex CMEX 5000 camera with ImageFocus v3.0 software. From the obtained photographs, stomatal density (number of stomata per leaf area) was determined. Importantly, during trial experiments, also leaf sheaths were examined, but these did not hold stomata.

Transpiration rate
The transpiration rate of plants, expressed on a whole plant fresh weight basis, was calculated over the 3-day period that plants were grown on the vessels as follows: where I t represents the transpiration rate, I u the water uptake rate, and I g the amount of water required for plant growth (all expressed as g H 2 O g −1 FW plant day −1 ). Furthermore, P represents the whole plant's fresh weight, S the shoot's fresh weight, R the root's fresh weight, and I m the total solution weight in the vessels, with the subscripts 1 and 2 denoting the parameters' value at the start and at the end of the 3-day exposure period, respectively. Moreover, whereas the factor 3 in the formulas refers to the 3-day duration of the experiment, the factor 8.95 refers to the average difference in solution weight of 4 vessels, which did not hold a plant set, between the start and end of the 3-day exposure period, respectively (standard deviation of this measurement was 0.61). Finally, the factors 0.9 and 0.95 represent the fraction (1) of a maize shoot and root consisting of water, respectively (Ausma et al. 2017). It deserves mentioning that during the 3-day exposure period, the proportion of biomass allocated to the different plant organs was not affected.

Statistics
Statistical analyses were performed in GraphPad Prism (version 8.4.1; GraphPad Software; San Diego; CA; USA). Treatment means were compared using a two-way analysis of variance (ANOVA) with a Tukey's HSD test as post hoc test at the P ≤ 0.05 level.

Results and discussion
To test the relevance of H 2 S for the regulation of stomatal aperture, maize seedlings were grown with atmospheric H 2 S in the presence or absence of sulfate in the root environment.
We first assessed what H 2 S level is subtoxic for maize, albeit sufficiently high to fully cover the plant's sulfur demand for growth. Sulfur-deficiency symptoms manifested after 10 days of sulfur deprivation (Table 1). The biomass of sulfate-deprived seedlings was on average 36% lower than that of sulfate-sufficient seedlings, which could be ascribed to both a lower root (33%) and shoot (37%) biomass (Table 1).
H 2 S fumigation can alleviate sulfur-deficiency symptoms. If maize was H 2 S fumigated in the absence of a sulfate supply, the plants did not develop any sulfur-deficiency symptoms (Table 1). The biomass of sulfate-deprived plants that were fumigated with 0.5 or 1.0 µl l −1 H 2 S was comparable to that of sulfate-sufficient, non-fumigated plants (Table 1), meaning that, analogous to the many plant species tested previously (Ausma et al. 2017;Ausma and De Kok 2019), maize can use H 2 S as a sulfur source. The results further demonstrate that maize is rather insusceptible for the potential phytotoxicity of H 2 S. Only exposure to 1.5 µl l −1 H 2 S negatively affected plant growth (Table 1). Generally, monocots are highly H 2 S tolerant (Stulen et al. 1990(Stulen et al. , 2000. In monocots, the shoot's meristem is sheltered by the whorl of leaves. Therefore, H 2 S can hardly penetrate the meristem, which may explain why grasses are relatively H 2 S insusceptible (Stulen et al. 1990(Stulen et al. , 2000. Tissue H 2 S, cysteine, and glutathione levels may be more profoundly affected at higher H 2 S levels (Birke et al. 2012;Ausma and De Kok 2019). Thus, in a second experiment, plants were fumigated with 1.0 µl l −1 H 2 S instead of 0.5 µl l −1 H 2 S. Similar to our previous observations (Table 1), sulfate-deprived plants had a lower biomass than sulfate-sufficient plants, owing to a lower root (34%) and leaf sheath biomass (22%; Table 2). Leaf blade biomass was comparable between sulfate-sufficient and sulfate-deprived plants (Table 2).
The biomass of plants that were fumigated with 1.0 µl l −1 H 2 S was comparable to that of sulfate-sufficient nonfumigated plants (Table 2). Thiol levels were higher in H 2 S-fumigated plants compared to non-fumigated plants (Fig. 1). Under sulfate-sufficient conditions, shoot total water-soluble non-protein thiol and cysteine levels were 1.4-and 2.0-fold higher in fumigated plants compared to non-fumigated plants, respectively (Fig. 1). Moreover, under sulfate-deprived conditions, fumigated plants had a 5.0-fold higher shoot total water-soluble non-protein thiol level, a 1.9-fold higher root water-soluble non-protein thiol level, and a 3.0-fold higher root cysteine level compared to non-fumigated plants (Fig. 1). Shoot cysteine levels in sulfate-deprived fumigated plants were even 1.5-fold higher compared to sulfate-sufficient non-fumigated plants (Fig. 1). Apparently, absorbed H 2 S was metabolized with high affinity into cysteine and subsequently into glutathione.
H 2 S-fumigated plants additionally had a higher shoot sulfate content compared to non-fumigated plants (Fig. 1). Whereas sulfate-sufficient fumigated plants had a 1.5-fold Fig. 1 The content of sulfate, total water-soluble non-protein thiols, and cysteine in maize as affected by H 2 S fumigation and sulfate deprivation. For experimental details, see the legend of Table 2. Data, representing 3 measurements with 4 plants in each, are presented as boxes with a 5-95 percentile and whiskers. Different letters indicate significant differences between treatments (P ≤ 0.05; two-way ANOVA; Tukey's HSD test as a post hoc test) higher shoot sulfate content compared to sulfate-sufficient non-fumigated plants, sulfate-deprived fumigated plants had a 5.0-fold higher shoot sulfate content compared to sulfatedeprived non-fumigated plants (Fig. 1). The higher sulfate content in fumigated plants might be related to the oxidation of absorbed H 2 S and/or the degradation of excessively accumulated organic compounds (Ausma and De Kok 2019). However, it may also be due to H 2 S absorbance only partially downregulating root sulfate uptake (Ausma and De Kok 2019). Further research should elucidate the source of the accumulated sulfate.
Exposure of maize to 1.0 µl l −1 H 2 S did not affect the total leaf blade area and stomatal density at the abaxial and adaxial side of nascent leaves (Figs. 2 and 3). There were approximately 75 stomata mm −2 at the adaxial leaf side and 50 at the abaxial leaf side (Fig. 3). Similar densities were reported previously (e.g., Zheng et al. 2013). Based on these observations, it is concluded that H 2 S fumigation does not affect the total number of stomata per plant.
Based on these observations, it is also concluded that it is unlikely that H 2 S regulates the formation of aerenchyma in maize leaves. Aerenchyma can be formed via programmed cell death (PCD) events and H 2 S is hypothesized to be a signal molecule stimulating PCD (Maniou et al. 2014). However, H 2 S fumigation did neither alter leaf biomass nor leaf area (Figs. 2 and 3). It did thus not affect the specific leaf weight, which implies H 2 S did not induce aerenchyma formation in the foliage. In accordance with this result, previously, it was shown that exposure of maize to atmospheric H 2 S did not trigger the aerenchyma formation in roots (Ausma et al. 2017).  Table 2. Data, representing 4 measurements with 4 plants in each, are presented as boxes with a 5-95 percentile and whiskers. Different letters indicate significant differences between treatments (P ≤ 0.05; two-way ANOVA; Tukey's HSD test as a post hoc test) Fig. 3 Stomatal density at the abaxial and adaxial side of leaf blades of maize as affected by H 2 S fumigation and sulfate deprivation. For experimental details, see the legend of Table 2. Data, representing 4 measurements with 2 plants in each, are presented as boxes with a 5-95 percentile and whiskers. Different letters indicate significant differences between treatments (P ≤ 0.05; two-way ANOVA; Tukey's HSD test as a post hoc test)  Table 2. Data, representing 4 measurements with 4 plants in each, are presented as boxes with a 5-95 percentile and whiskers. Different letters indicate significant differences between treatments (P ≤ 0.05; two-way ANOVA; Tukey's HSD test as a post hoc test)  Table 2. Data, representing 18 measurements on different plants, are presented as boxes with a 5-95 percentile and whiskers. Different letters indicate significant differences between treatments (P ≤ 0.05; two-way ANOVA; Tukey's HSD test as a post hoc test)