Hydrogen sulfide induced by hydrogen peroxide mediates darkness-induced stomatal closure in Arabidopsis thaliana

Abstract

Background

Hydrogen sulfide, a novel gasotransmitter, has a similar function with carbon monoxide (CO) and nitric oxide (NO). Early research is devoted to its toxicity studies, while neglecting its physiological functions in plants and animals. Nowadays, H₂S is reported as a new type of gas signal molecule, which regulates many physiological processes of animals and plants. For example, in animals, H₂S is involved in brain development, inflammation, neuronal antioxidant protection, regulation of heart and nervous system, diastolic cardiovascular and digestive tract smooth muscle [1-3]. Compared with the research degree in animals, the understanding of the function of H₂S in plant growth and development is far from enough. However, with the increasing attention of researchers, there are also many new discoveries about the physiological role of H₂S in plants. H₂S not only promotes the growth and development of plants, for example, increasing the seed germination rate, promoting the root development, enhancing the photosynthesis, prolonging the florescence and delaying senescence, but also enhances the resistance of plant to abiotic stresses [4-9]. In addition, it is also found that H₂S is an important signaling molecule regulating stomatal movement [10]. Furthermore, H₂S interacts with abscisic acid (ABA) and participates in regulating stomatal movement in Arabidopsis thaliana, which located upstream of ATP-binding cassette (ABC) transporter regulating stomatal closure by ABA [6]. H₂S also participates in stomatal closure by ethylene, and NO acts upstream of it [11].

Hydrogen peroxide (H₂O₂), another kind of signal molecule in plant cells, is one of the main reactive oxygen species (ROS) produced in many metabolism processes with relatively stable molecular properties [12]. H₂O₂ is generated by enzymatic sources including cell wall peroxidases in plant cells, NADPH oxidases (NOX), or polyamine oxidases [13-15]. A large number of studies have shown that plant tissue can resist all kinds of abiotic or biological stresses via the production of H₂O₂, including extreme temperature, ABA, ultraviolet-b radiation (UV-B), darkness, ethylene and bacterial invasion etc [16-21]. Moreover, H₂O₂ also mediates different physiological processes in plants, and resistance to adversity, defense response to pathogenic bacteria, gene expression, stomatal movement, and programmed cell death, all of which have important regulatory roles [16, 22-25].

Both H₂O₂ and H₂S have been reported to mediate stomatal closure by darkness, and H₂S functions downstream of H₂O₂ during the process in Vicia faba [14, 26]. However, it is unclear that whether H₂S participates in stomatal closure by darkness in A. thaliana, the interaction between H₂S and H₂O₂ and their enzymatic pathways in the process needs to be elucidated. To address these questions, A. thaliana genotypes ( Atl-cdes，Atd-cdes, AtrbohD, AtrbohF, AtrbohD/F mutants and wild-type) were adopted to investigate the significance and interactions between H₂S and H₂O₂ in stomatal closure by darkness.

Results

Involvement of H₂S in stomatal closure by darkness

Influences of darkness on stomatal aperture in wild-type, Atl-cdes and Atd-cdes

To analyze whether H₂S mediates stomatal closure by darkness, the influences of H₂S synthesis inhibitors AOA, NH₂OH, C₃H₃KO₃+NH_3, H₂S scavenger HT and the producer of L-/D-cysteine desulfhydrase (L-/D-CDes) on stomatal aperture of wild-type (Col-0), as well as the influence of darkness on stomatal aperture of Atl-cdes and Atd-cdes which are T-DNA insertion lines were detected. Moreover, it is found that the presence of HT, AOA, NH₂OH, and C₃H₃KO₃+NH₃ inhibited darkness-induced stomatal closure. The stomatal closure was not induced when NH₂OH, AOA, HT, and C₃H₃KO₃+NH₃ were applied in light (As shown in Fig. 1a). Actually, Atl-cdes and Atd-cdes stomatal aperture could not be changed by darkness (Fig. 1b). From the results, we found that L-/D-CDes generated H₂S might participate into stomatal closure by darkness in A. thaliana.

Effects of H₂S modulators on darkness-induced L-/D-CDes activity and H₂S content in wild-type

To confirm the metabolic pathways participated in H₂S systhesis, L- and D-CDes activities and H₂S content in wild-type  were determined. In fact, the content of H₂S in darkness was greatly higher than that in light, while HT, AOA, NH₂OH, and C₃H₃KO₃+NH₃ could reduce darkness-induced H₂S production (Fig. 2a). Similarly, there was a remarkable increase in activity by darkness, and L- and D-CDes activities were reduced in the presence of HT, AOA, NH₂OH, and C₃H₃KO₃+NH₃ in darkness (Fig. 2b and 2c). However, no effect was observed when HT, AOA, NH₂OH, and C₃H₃KO₃+NH₃ were applied in light (Fig. 2a-c). These results suggested that darkness-induced H₂S biosynthesis might function via L- and/or D-CDes in A. thaliana.

Relationship between H₂O₂ and H₂S in stomatal closure by darkness

Effects of H₂O₂ modulators on L-/D-CDes activity, and H₂S content in darkness

For the sake of analyzing the relationship between H₂O₂ and H₂S during stomatal closure by darkness, H₂S content and L-and D-CDes activities in leaves of wild-type plants were examined with application of H₂O₂ synthesis inhibitors SHAM, DPI, and H₂O₂ scavenger ASA, CAT in darkness [27-29]. Moreover, the treatment with SHAM, ASA, DPI, and CAT in darkness not only decreased H₂S content, but also reduced L- and D-CDes activities in wild-type (Fig. 3a-c). We proposed that both NADPH oxidase-derived and peroxidase-derived H₂O₂ might be a novel upstream component of H₂S signaling cascade during stomatal closure by darkness in A. thaliana.

Influences of darkness on L-/D-CDes activity and H₂S content in AtrbohD, AtrbohF, and AtrbohD/F

To further investigate the interaction between H₂O₂ and H₂S, L- and D-CDes activities, as well as H₂S content, were detected in the leaves of AtrbohD, AtrbohF, and AtrbohD/F. H₂S content in leaves of wild-type and AtrbohD in darkness was apparently higher than that in the light, and L-and D-CDes activities of leaves was significantly enhanced as well (Fig. 4 a-c). However, darkness couldn’t induce any increase in H₂S content and L-and D-CDes activities in AtrbohF and AtrbohD/F (Fig. 4 a-c). The results further suggested that H₂S acted downstream of H₂O₂ in darkness-regulated stomatal closure in A. thaliana.

Influences of H₂S modulators on the guard cells H₂O₂ levels by darkness in wild-type

For the sake of further validating the relationship between H₂O₂ and H₂S, the fluorescence of H₂O₂ in guard cells after using HT, NH₂OH, AOA, and N₃H₃KO₃+NH₃ in darkness was examined by H₂DCF-DA, which was previously used to measure the production of H₂O₂ in stomatal closure by darkness by Allan and Fluhr (1997) [30]. A great increase was caused by darkness in H₂O₂ levels of guard cells (Fig. 5b). However, treatment with HT, NH₂OH, AOA, and N₃H₃KO₃+NH₃ in darkness had no clear effects on H₂O₂ levels (Fig. 5c-f). Therefore, a fact was consolidated by these results, H₂S functioned downstream of H₂O₂ in stomatal closure by darkness in A. thaliana.

Influences of darkness on H₂O₂ production in Atl-cdes and Atd-cdes

To confirm that H₂S mediated darkness-induced stomatal closure as a downstream factor of H₂O₂, H₂O₂ production of Atl-cdes and Atd-cdes was detected. In darkness, H₂O₂ fluorescence in Atl-cdes and Atd-cdes guard cells were significantly stronger than that in light (Fig. 6c-f) without significant difference from the wild-type (Fig. 6a, b). Additionally, the above data further demonstrated that H₂S, as a downstream factor of H₂O_2, mediated darkness-induced stomatal closure in A. thaliana.

Discussion

Stomata are important structures to exchange gases and water in higher plants, which respond to different environmental factors by controlling their aperture. The mechanism of regulating stomatal movement is extremely complex. Other than the classical theory of cell turgor pressure, the stomatal closure is also affected by cytoplasmic calcium concentration changes, pH, protein phosphorylation, K⁺ and anion channel regulation [31]. During the continuous exploration of stomatal movement mechanism, some essential regulators of signal transduction emerged gradually, such as NO, H₂O₂, H₂S and so on.

As the third gasotransmitter, H₂S has become a new star in the transduction process of plant signal. It has been indicated that exogenous H₂S released by NaHS caused stomatal opening [32, 33]. However, the other results indicated that H₂S induced the stomatal closure in diferent plants [10]. It has been shown that H₂S mediates ABA, ethylene and darkness-regulated stomatal movement [6, 11, 26, 34], and that cytosolic alkalization, NO, CO, H₂O₂ sphingosine-1-phosphate (S1P), and intracellular calcium mediate stomatal movement by darkness [17, 18, 35-39]. However, the mechanism of H₂S in stomatal movement by darkness is unclear. Our results suggested that stomatal closure by darkness was significantly inhibited by H₂S scavenger HT, the inhibitors of H₂S biosynthesis NH₂OH and AOA, and the products of L- and/or D-cysteine desulfhydrase C₃H₃KO₃+NH₃ in wild-type A. thaliana (Fig. 1a). Darkness could induce stomatal closure in wild-type without influence on stomatal aperture of Atl-cdes and Atd-cdes (Fig. 1b). Additionally, HT, AOA, NH₂OH, and C₃H₃KO₃+NH₃ not only reduced the production of darkness-induced H₂S (Fig. 2a), but also diminished L-CDes activity (Fig. 2b) as well as D-CDes activity in darkness (Fig. 2c). From these results, we could find that H₂S was needed for darkness-regulated stomatal closure in A. thaliana, and L-/D-CDes was one of the key enzymes for its synthesis, which was consistent with the results of Ma et al. (2018) in V. faba [26].

Hydrogen peroxide (H₂O₂) is another important signal molecule involved in the signal transduction of plant cells. As the second messenger in plants, H₂O₂ participates in many physiological processes including stomatal movement. McAinsh et al. (1996) proved for the first time that H₂O₂ had an effect on stomatal movement in plants, and exogenous H₂O₂ could promote calcium level increase, leading to stomatal closure [40]. For the past few years, deep research on the function of H₂O₂ has been carried out in stomatal movement. It has been proved that guard cells accumulate H₂O₂ to promote stomatal closure under the stimulation of drought, salt, high concentration of CO₂, ethylene, ABA, UV-B, brassinosteroid, as well as darkness [16, 18, 19, 30, 41-46]. Data from Desikan et al. (2004) indicated that stomatal closure was related to H₂O₂ accumulation in guard cells of pea, and the large gp91phox subunit (rboh genes) encoding NOX was appeared in guard cells of pea , which probably participates in ABA responses and darkness [17]. It has been reported that soaking with H₂O₂ could not only increase L-CDes activity, promote H₂S production, but also improve the germination rate of Jatropha curcas seeds [47]. Additionally, Li and He (2015) showed that H₂O₂ acted downstream of H₂S in seed germination of Vigna radiata [48]. Wang et al. (2015) indicated that H₂O₂ was involved in exogenous H₂S-induced stomatal closure [49]. In fact, H₂O₂ has been reported to function upstream of H₂S in darkness and salt stress-induced stomatal closure in V. faba [26, 50]. It has been proved that H₂O₂ mediates darkness-induced stomatal closure [17, 18, 26]. However, the interaction between H₂O₂ and H₂S was still unclear in darkness-induced stomatal closure in A. thaliana. The data displayed here suggested that H₂O₂ modulators ASA, CAT, DPI and SHAM could significantly inhibit the production of darkness-induced H₂S and L-/D-CDes activity enhancement in A.thaliana wild-type leaves (Fig. 3), H₂S scavenger HT and its synthesis inhibitors AOA, NH₂OH and C₃H₃KO₃+NH₃ had no significant effects on H₂O₂ levels of wild-type guard cells in darkness (Fig. 5). Compared with light treatment, there was no significant effect of darkness on H₂S content and L-/D-CDes activity in AtrbohF and AtrbohD/F leaves (Fig. 4), but could increase H₂O₂ levels in Atl-cdes and Atd-cdes (Fig. 6). These data indicated that NADPH oxidase-derived and peroxidase-derived H₂O₂ could act upstream of L-/D-CDes-generated H₂S in stomatal closure by darkness in A. thaliana, as the same as the results of Ma et al. (2018) in V. faba [26].

Conclusions

In this study, we explored the interaction between H₂O₂ and H₂S in stomatal closure by darkness. Our results indicated that darkness induced H₂O₂ synthesis via promoting the activity of NADPH oxidase and peroxidase, and further led to the production of L-/D-CDes-derived H₂S and stomatal closure in A. thaliana. Additionally, studies showed that L-/D-CDes-generated H₂S functioned downstream of H₂O₂ in A. thaliana. In fact, our work not only enriches the signal transduction network to regulate the stomatal movement induced by darkness, but also provides experimental evidences in plant physiology, cell biology and genetics for the interaction between H₂O₂ and H₂S in stomatal movement.

Methods

Chemicals

The molecular probe H₂DCF-DA was bought from Biotium (Hayward, CA, USA), while 2-(N-morpholino) ethanesulfonic acid (MES), salicylhydroxamic acid (SHAM), potassium pyruvate (C₃H₃KO₃), aminooxy acetic acid (AOA), hypotaurine (HT), hydroxylamine (NH₂OH), catalase (CAT), ammonia (NH₃), diphenylene iodonium (DPI), D-cysteine, dimethyl sulfoxide (DMSO), ascorbic acid (ASA), L-cysteine, dithiothreitol (DTT) and N, N-dimethyl-p-phenylenediamine dihydrochloride were acquired from Sigma-Aldrich (Located in St Louis, MO, USA). Unless stated otherwise, the other chemicals were purchased from various Chinese suppliers with highest analytical grade.

Plant materials

A. thaliana ecotype Columbia (Col-0) was applied throughout this study. Seeds of L-/D-cysteine desulfhydras deletion mutants of AtL-CDes T-DNA insertion line (N541918, designated Atl-cdes), AtD-CDes T-DNA insertion line (CS853264, designated Atd-cdes), NADPH oxidase gene single mutant line (N9555, designated AtrbohD and N9557, designated AtrbohF), and homozygous transposon insertion double mutant line (N9558, designated AtrbohD/F ) were provided by Nottingham Arabidopsis Stock Centre (NASC, Nottingham, UK). The mutant Atd-cdes, Atl-cdes and AtrbohF, AtrbohD, AtrbohD/F has been respectively identified by PCR and RT-PCR [51-53].Wild-type and mutants seeds of A. thaliana. were surface-sterilized and sown on sterilized vermiculite. Seedlings were stratified in darkness for 2-4d at 4 °C. After growing 4 euphylla, they were transferred in a controlled-environment chamber with a humidity of 80%, 16-h light/8-h dark cycle, and day/night temperature cycle of 22°C/18°C with a photon flux density of 100 μmol·m⁻²s⁻¹ PAR generated by cool white fluorescent tubes (Philips, New York, NY, USA). Fully expanded leaves were harvested at 4-6 weeks for immediate use.

Stomatal bioassays

Stomatal bioassay was performed as described by McAinsh et al. (1996) with minor modifications [40]. The epidermal strips newly prepared were treated with MES-KCl buffer (10 mM MES, 50 mM KCl, 100 μM CaCl₂, pH 6.15) alone or containing various compounds or inhibitors in light (100 μmol·m⁻²·s⁻¹) or darkness. And then the stomatal apertures were recorded by an optical microscope and eyepiece graticule previously calibrated with a stage micrometer. In each treatment, 30 randomly-selected apertures were scored per replicating and the treatment was repeated three times at least. The data provided are the mean ± s.e. of 90 measurements.

Measurement of H₂S emission

Measurement of H₂S emission was determined by the formation of methylene blue, which was performed as described by Sekiya et al. (1982) and Hou et al. (2013) with slight modifications [34, 54]. Fully expanded leaves were utilized to measure H₂S emission. Firstly, the leaves were treated with MES-KCl buffer alone or containing various scavengers or synthesis inhibitors in light (100 μmol·m^{− 2}·s^{− 1}) or darkness for 3h, and then 0.1 g of them was taken for grinding by adding 0.9 mL 20 mM Tris-HCl (pH 8.0) buffer. After the centrifugation, the supernatant and a trap with 1% of zinc acetate were put into a test tube, and then the tube was quickly sealed with a Parafilm at the same time. Then 100 μL 20 mM N,N-dimethyl-p-phenylenediamine dihydrochloride dissolved in 7.2 M HCl and 100 μL 30 mM FeCl₃ dissolved in 1.2 M HCl were added into the trap after the absorption of H₂S for 30 min at 37 °C. Finally, the absorbance was measured at 670 nm. In additional, a calibration curve was also drawn with known concentrations of Na₂S solution. Each treatment was repeated three times, and all the data presented are the mean ± s.e.

L-/D-cysteine desulfhydrase activity measurements

H₂S was determined to further study the activity of L-/D-cysteine desulfhydrase (L-/D-CDes), which was released from L-/D-cysteine within a certain period of time [34, 55]. The assay contained in the total volume of 1mL includes 100 μL 0.8 mM D-/L-cysteine, 400 μL 100 mM Tris-HCl, 400 μL 2.5 mM DTT, and 100 μL supernatant. Then 100 μL 20 mM N,N-dimethyl-p-phenylenediamine dihydrochloride dissolved in 7.2 M HCl and 100 μL 30 mM FeCl₃ dissolved in 1.2 M HCl were added into the trap after reaction for 30 min at 37 °C. And the rate of H₂S released was presented by the determination of absorbance at 670 nm. Besides, the activity of L-CDes and D-CDes was also confirmed by the same method, but the pH of Tris-HCl buffer used previously was 8, and the latter was 9. Each treatment was repeated three times, and the data presented were the mean ± s.e.

Measurement of endogenous H₂O₂

H₂O₂ levels were measured with 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA) by the method of Allan and Fluhr (1997) with minor modifications [30]. In order to research the influence of H₂S scavenger and synthesis inhibitors on darkness-induced H₂O₂ production in guard cells, the epidermal strips were incubated in MES-KCl buffer alone in light or MES-KCl buffer alone or containing ASA, CAT, DPI, and SHAM in darkness for 3 h, and then immediately loaded with 50 μM H₂DCF-DA in Tris-KCl buffer (10 mM Tris, 50 mM KCl, pH 7.2) for 10 min in darkness. To study the effects of darkness on H₂O₂ levels in guard cells of Atl-cdes and Atd-cdes mutnts, the epidermal strips were incubated in MES-KCl buffer alone in light or MES-KCl buffer alone in darkness for 3h, and then immediately loaded with 50 μM H₂DCF-DA in Tris-KCl buffer for 10 min in darkness. After that, excess dye was washed off with fresh Tris-KCl loading buffer in darkness, and the epidermal strips were immediately examined by TCS SP5 laser-scanning confocal microscopy (Leica Lasertechnik Gmbh, Heidelberg, Germany) with following settings: excitation 488 nm, emission 530 nm, power 10%, zoom about 4, normal scanning speed, and frame 512×512 pixels. Leica image software and Photoshop 7.0 (Adobe, San Jose, CA, USA) were used to analyze and process the images acquired. Each treatment was repeated at least three times. The depicted confocal images represent similar results from three replications.

Statistical analyses

The statistical importance of treatments was checked by one-way ANOVA as well as Duncan’s multiple range test. The data was considered to be statistically important when P-values were below 0.05. All the figures were plotted by Origin6.1 (Microcal Software, Nothampton, MA, USA) and processed with Photoshop 7.0 (Adobe, San Jose, CA, USA).

Abbreviations

ABA - abscisic acid

ABC - ATP-binding cassette

AOA - aminooxy acetic acid

ASA - ascorbic acid

A.thaliana - Arabidopsis thaliana

CAT - catalase

CO - carbon monoxide

DPI - diphenylene iodonium

DTT - N,N-dimethyl-p-phenylenediamine dihydrochloride and dithiothreitol

H₂DCF-DA - 2′,7′-dichlorodihydrofluorescein diacetate

H₂O_{2 -} hydrogen peroxide

H₂S - hydrogen sulfide

HT - hypotaurine

D-/L-CDes - D-/L-cysteine desulfhydrase

NaHS - sodium hydrosulfide

N₃H₃KO_3 - potassium pyruvate

NH_3 - ammonia

NH₂OH - hydroxylamine

NO - nitric oxide

NOX - NADPH oxidase

ROS - reactive oxygen species

SHAM - salicylhydroxamic acid

UV-B - ultraviolet-b radiation

V. faba - Vicia faba

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by grants from the Innovative Training Program for College Students in Shanxi, China (No.2019229). The funding bodies had no role in the experimental design, data analysis, decision to publish, or preparation of the manuscript.

Authors' contributions

YM designed the experiments and wrote the manuscript. LS and JN performed the experiments. YM, LS and JN analyzed the data. The final manuscript was reviewed and approved by all authors.

Acknowledgements

Not applicable.

Authors' Information

College of Life Sciences, Shanxi Normal University, Linfen 041004, People's Republic of China

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