Hydrogen sulfide induces Ca²⁺ signal in guard cells by regulating reactive oxygen species accumulation

ABSTRACT

Hydrogen sulfide (H₂S) is an important gas signal molecule, but little is known about its signal mechanism. Ca²⁺ is an important second messenger in plant cells, and its fluctuation in the cytoplasm causes downstream physiological responses. Our previous study found that H₂S can induce the accumulation of hydrogen peroxide (H₂O₂). We also found that reactive oxygen species (ROS) can further induce the Ca²⁺ influx in guard cells by noninvasive micro-teat technology (NMT). This study confirmed that the accumulation of reactive oxygen species to induce Ca²⁺ signal in guard cells, resulting in stomatal closure. Thus, revealing a novel mechanism of H₂S promoting stomatal closure.

KEYWORDS:

• H₂S
• persulfidation
• Ca²⁺ influx
• ROS

H₂S is an important gas signal molecule. In plants, there are mainly three kinds of gas signal molecules: Nitric oxide (NO), Carbon monoxide (CO) and H₂S.¹ H₂S is involved in regulating stomatal movement, promoting root development, affecting cytoskeleton, alleviating drought and metal ions.^2–6 Although many studies have been done on the physiological effects of H₂S, little is known about its detailed mechanism. At present, based on the chemical properties of H₂S, two regulating mechanisms have been proposed. First, H₂S can redox with ROS, H₂O₂ and peroxynitrite to reduce cellular oxidation level;¹ Second, H₂S can mediate post-translational modification on the cysteine residue of the protein, namely persulfidation.⁷

Ca²⁺ is an important second messenger in plant cells and plays a key role in regulating plant growth and development and responding to stress.⁸ The most important calcium ion pool in plant cells is vacuoles, and calcium ions also exist in chloroplasts, mitochondria and endoplasmic reticulum. There are voltage-gated channels on the vacuole membrane. When cells are stimulated, calcium ions in the calcium pool flow into the cytoplasm and stimulate downstream signal molecules.⁹ As an important signal molecule in cells, calcium ions have fingerprint characteristics, and their fluctuation is an important mechanism for maintaining physiological and biochemical functions in organisms. The volatility of calcium ions is highly consistent at the spatial and practical levels, which is characterized by different frequencies, periods or amplitudes.¹⁰  Calmodulin is a receptor protein in calcium signal transduction, and its main function is to regulate the expression of downstream proteins and the physiological process of guard cells.⁸

Our previous report showed that H₂S could induce ROS accumulation in Arabidopsis thaliana roots.¹¹ In this study, H₂O₂ was visualized using the specific H₂O₂ fluorescent probe dichlorofluorescein diacetate (H2DCF-DA) according to the method described by Maffei et al.¹² We found that H₂S could induce the accumulation of H₂O₂ in guard cells of leaf in Arabidopsis thaliana (Figure 1a,b). Recent studies have also shown that H₂S activates NADPH oxidase RBOHD and RBOHF through S-thiosulfhydrylation, to induce H₂O₂ accumulation.¹⁴ Reactive oxygen species can further induce the appearance of Ca²⁺ signal.⁸ In this study, net stomatal fluxes of Ca²⁺ were measured with a noninvasive micro-test technology (NMT), according to methods described by Chen et al.¹³ and Yan et al.¹⁵ The epidermal strips were peeled from the rosette leaves of 5-week-old seed. A 100 mM NaHS or or 10 mM H₂O₂ was added instantly, after 3–5 min of detecting the basic Ca²⁺ flux. Data were recorded for 10 min. As shown in Figure 1c, the application of 100 μM NaHS and 10 mM H₂O₂ caused a significant Ca²⁺ influx in guard (approximately 63.6 ± 11 and 32.3 ± 7 pmol cm⁻² s⁻¹, respectively). Therefore, we believe that H₂S can promote the accumulation of ROS and induce the emergence of Ca²⁺ signal in plant guard cells.

Figure 1. Assay of H₂O₂ content and net Ca²⁺ fluxes in Arabidopsis thaliana guard cells. (a) The effect of NaHS on H₂O₂ content in guard cells. 100 μM NaHS were used for various treatments for 20 min. Bar = 10 μm. (b) Quantification of H₂O₂ H₂DCF-DA fluorescence density for (a). The average of the fluorescence of each guard cell was calculated. The data are the mean values ± SE (n = 10). (c) The effect of NaHS and H₂O₂ on Ca²⁺ fluxes in guard cells. The Ca²⁺ influxes in guard cells were measured, and 100 mM NaHS or 10 mM H₂O₂ was used. The negative value indicates influx. The data are the mean values ± SE (n = 3). (d) H₂S induces Ca²⁺ signal in guard cells by regulating protein persulfidation and reactive oxygen species accumulation.^13,14 Within each set of experiments, bars with different letters are significantly different at the P < .05 level (Duncan’s multiple range tests)

In our recent report, it has been proved that H₂S can also activate ABA-SnRK2.6 signal pathway to induce the emergence of Ca²⁺ signal in guard cells.¹³ In persulfidation-site mutant lines, persulfidation of SnRK2.6 resulted in a decrease of Ca²⁺ influx induced by ABA and H₂S (Figure 1d). H₂S also can activate NADPH oxidase RBOHD and RBOHF¹⁴ (Figure 1d). Both H₂S and H₂O₂ induce the stomatal closure, in which H₂S activates NADPH oxidase RBOH and promotes the accumulation of H₂O₂.^13,14 H₂S does not induce Ca²⁺ influx in ost1-3, suggesting that H₂S induces Ca²⁺ influx through persulfidation of SnRK2.6. The increase of Ca²⁺ leads to stomatal closure by inhibiting inward K⁺ channels and activating the outward anion channels.⁸ As we know K⁺ and anion result in a dramatic decrease in osmotic pressure. As a result, a large amount of water flux from guard cells following stomatal closure. However, the increase of Ca²⁺ content results in partial or complete loss of stomatal closure in persulfidation-site mutant lines. And drought intolerance occurred in persulfidation-site mutants, especially in ost1-3/2.6 ^{C131SC137S-GFP} double-site mutant lines.¹³

In conclusion, H₂S can induce Ca²⁺ signal in guard cells by regulating the persulfidation of SnRK2.6 protein kinase and the accumulation of reactive oxygen species¹³ (Figure 1d). This study revealed a novel mechanism that H₂S promotes stomatal closure.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [No. 31770648, 31400246], was partially supported by the Key Research and Development Program of Shaanxi [2020NY-190], Natural Science Foundation of Shaanxi Province [2018JM3017].