Hydrogen Sulfide Signaling in Plants

Abstract

Significance:

Hydrogen sulfide (H₂S) is a multitasking potent regulator that facilitates plant growth, development, and responses to environmental stimuli.

Recent Advances:

The important beneficial effects of H₂S in various aspects of plant physiology aroused the interest of this chemical for agriculture. Protein cysteine persulfidation has been recognized as the main reduction–oxidation (redox) regulatory mechanism of H₂S signaling. An increasing number of studies, including large-scale proteomic analyses and functional characterizations, have revealed that H₂S-mediated persulfidations directly regulate protein functions, altering downstream signaling in plants. To date, the importance of H₂S-mediated persulfidation in several abscisic acid signaling-controlling key proteins has been assessed as well as their role in stomatal movements, largely contributing to the understanding of the plant H₂S-regulatory mechanism.

Critical Issues:

The molecular mechanisms of the H₂S sensing and transduction in plants remain elusive. The correlations of H₂S-mediated persulfidation with other oxidative post-translational modifications of cysteines are still to be explored.

Future Directions:

Implementation of advanced detection approaches for the spatiotemporal monitoring of H₂S levels in cells and the current proteomic profiling strategies for the identification and quantification of the cysteine site-specific persulfidation will provide insight into the H₂S signaling in plants. Antioxid. Redox Signal. 39, 40–58.

Introduction

Sulfur is the 10th most abundant chemical element in the universe and is essential for all living organisms (Räisänen, 2005). It occupies a unique position in the reduction–oxidation (redox) biology due to its availability to reach many distinct oxidation states, ranging from −2 to +6 (Fig. 1). As the planet got oxidized, sulfate (+6) became the most abundant inorganic form of sulfur on earth. Hydrogen sulfide (H₂S), the most reduced inorganic form of sulfur (−2) (Fig. 1), is a colorless, but flammable, gas, with a smell of rotten eggs that is naturally released from volcanic emissions or other geothermal activities and from decaying plant and animal proteins.

FIG. 1.

Sulfur oxidation states in some biologically relevant compounds. The oxidation states of sulfur in different organic and inorganic compounds range from −2 to +6. R refers to the remainder of the molecule and symbolizes the variable side chain in protein structures.

As H₂S is a weak acid with the dissociation constants pK_a1 of 6.9 (Pomeroy, 1941) and pK_a2 of between 12 and 17 (Ellis and Golding, 1959; Meyer et al., 1983), it can be dissociated into hydrosulfide anion (HS^¯) and sulfide anion (S^2¯) in aqueous solutions. H₂S usually stands for all species, including H₂S, HS^¯, and S^2¯ (Paulsen and Carroll, 2013). Nevertheless, in solutions at an approximately physiological pH of 7.4, H₂S releases negligible amounts of S^2¯ and exists primarily as HS^¯ (Hughes et al., 2009).

Given the basic chemical properties of H₂S and HS^¯ with the lowest oxidation state of −2, they both can only be oxidized. Whereas H₂S is a gasotransmitter that can diffuse freely across cellular membranes, HS^¯ needs specific ion channels to move between different subcellular organelles or cells (Kabil and Banerjee, 2010). Accordingly, the H₂S and HS^¯ might regulate cellular functions differently. H₂S, HS^¯, and S^2¯, together with various chemically reactive forms of cysteine thiols (see in section H₂S Signaling via Protein Persulfidation) and other sulfur-containing compounds that either reduce or oxidize biomolecules, can be classified as reactive sulfur species (RSS).

H₂S has been implicated in the origin of life (Filipovic et al., 2018; Olson and Straub, 2016). Life began 3.8 billion years ago (bya) in an anoxic and ferrous ion (Fe²⁺)-rich ocean. Cyanobacteria, the first photosynthetic oxygen-generating organisms, are believed to have then evolved and contributed to the Great Oxygenation Event ∼2.5 bya (Demoulin et al., 2019; Fournier et al., 2021; Planavsky et al., 2014). Along with the slightly increased oxygen level, sulfur oxidized to sulfate, which was further reduced to sulfide by ubiquitous Fe²⁺ present in the ocean, greatly increasing the H₂S level and, consequently, leading to an anoxic and sulfidic ocean (Cortese-Krott et al., 2017).

The first eukaryotes appeared and adapted under this condition using H₂S as their major energy source (Olson and Straub, 2016). Green algae, one of the earliest photosynthetic eukaryotes of the Plantae kingdom that contained primary chloroplasts, derived from endosymbiosis with cyanobacteria, are assumed to have evolved as early as 1.0 bya (Tang et al., 2020). The combined activity of cyanobacteria and algae tremendously increased the level of ambient oxygen ∼0.6 bya, sequentially, land plants have evolved.

The antioxidant enzymes, such as superoxide dismutase (Cannio et al., 2000; Miller, 2012), catalase (Zamocky et al., 2008), glutathione (GSH) peroxidase (Margis et al., 2008), peroxiredoxins (Dietz, 2011; Knoops et al., 2007), thioredoxins (TRXs) (Balsera and Buchanan, 2019), and glutaredoxins (GRXs) (Alves et al., 2009), were already present in an anoxic and high H₂S environment as early as 2.0 bya (Cortese-Krott et al., 2017). Therefore, these redox regulation systems most probably evolved primarily to use H₂S as energy source or to deal with RSS, which was later amended to regulate reactive oxygen species (ROS) (Cortese-Krott et al., 2017; Olson and Straub, 2016).

Along with the evolution and increasing complexity of organisms, the ancient mechanisms of H₂S metabolism and regulation had to be adapted. For example, the sulfate transport system in chloroplasts has been suggested to undergo several adaptions, spanning the evolution of green algae, liverworts, and flowering plants (Kopriva et al., 2015; Mendoza-Cózatl et al., 2005; Takahashi et al., 2012). In brief, H₂S metabolism as well as regulatory mechanisms in living organisms has evolved as a result of environmental adaptation, while the fundamental principles might remain conserved (Yamasaki and Cohen, 2016).

Besides its implied vital role in evolution, H₂S has been recognized as a potent signaling molecule in the regulation of critical cellular processes (Wang, 2014; Wang, 2002). Increasing evidence has shown that H₂S is not only involved in plant growth, development, reproduction processes (Baudouin et al., 2016; Chen et al., 2011; Ma et al., 2021), and promotion of nodulation in the rhizobium–legume symbiosis (Zou et al., 2019), but also facilitates tolerance to various environmental stresses, such as drought (Jin et al., 2011; Shen et al., 2013), salinity (Christou et al., 2013; Li et al., 2014a), heavy metals (Fu et al., 2019; Zhang et al., 2020b), and extreme temperatures (Du et al., 2021; Li et al., 2012).

Furthermore, H₂S has been reported to improve the quality maintenance during postharvest storage of fruit (Ge et al., 2017; Hu et al., 2012), vegetables (Li et al., 2014b), and flowers (Zhang et al., 2011). Given the important beneficial effects of H₂S in multiple aspects of plant physiology, exogenous H₂S seems to be a promising biotechnological strategy with a great agronomic interest.

Numerous studies have uncovered the positive physiological impact of H₂S on plants (Corpas, 2019; Corpas and Palma, 2020; Zhang et al., 2021), but how plants sense and transduce H₂S signals remains elusive. H₂S can modify proteins through post-translational modification (PTM), a process named persulfidation. Persulfidation on critical proteins plays an important role in activating downstream signaling as demonstrated by both proteomic analyses and functional characterizations (Aroca et al., 2021a, Aroca et al., 2021b, Aroca et al., 2017b, Aroca et al., 2015; Fu et al., 2020a; Laureano-Marín et al., 2020; Zivanovic et al., 2019).

Recently, H₂S-induced protein persulfidation has been found to regulate stomatal movements in the abscisic acid (ABA) signaling pathway (Chen et al., 2020; Shen et al., 2020; Zhou et al., 2021), contributing to the understanding of the molecular mechanism of H₂S signaling in plants.

In this review, we provide a wide perspective of H₂S in plants, including H₂S biosynthesis, exogenous application, endogenous detection methods, and the mechanisms and identification strategies for protein persulfidation. We further give insights into the molecular mechanisms of persulfidation in the regulation of ABA-mediated stomatal closure by highlighting several recent functional studies.

H₂S Biosynthesis in Plants

Plants generate H₂S endogenously through several biosynthesis pathways in different subcellular organelles (Fig. 2). The major H₂S source is associated with the photosynthetic sulfate assimilation pathway in chloroplasts. Plants take up sulfate from the environment through sulfate transporters (Takahashi et al., 2011), a protein class with high sulfate affinity that facilitates sulfate trafficking across membranes, into the chloroplasts, where H₂S is mainly generated through sulfur metabolism.

FIG. 2.

H₂S biosynthesis in plant cells. H₂S is produced in different subcellular organelles in the plant cells via various enzymes. The assimilated sulfate in plant cells is transported to the chloroplasts, where the sulfate is reduced first to APS, then to sulfite that is subsequently reduced to sulfide (H₂S) via SiR. Sulfide can be further catalyzed by OAS-TL in the presence of OAS to generate cysteine. In chloroplasts, this reaction is catalyzed by OASB, while the same reaction is catalyzed by OASA1 in the cytosol and by OASC in mitochondria. In the cytosol, l-cysteine and d-cysteine are degraded by l-CDes and d-CDes, respectively, to produce H₂S, pyruvate, and NH₃. Thus far, DES1 is the best H₂S-producing enzyme. NifS has been suggested to catalyze cysteine to H₂S in chloroplasts and mitochondria. In mitochondria, CAS catalyzes the reaction of cyanide and l-cysteine to produce H₂S and β-cyanoalanine. APS, adenosine 5′-phosphosulfate; CAS, β-cyanoalanine synthase; d-CDes, d-cysteine desulfhydrase; DES1, l-cysteine desulfhydrase 1; H₂S, hydrogen sulfide; l-CDes, l-cysteine desulfhydrase; NH₃, ammonia; NifS, nitrogenase Fe–S cluster; OAS, O-acetylserine; OAS-TL, O-acetylserine (thiol)lyase; SiR, sulfite reductase.

Sulfate is reduced by ATP sulfurylase to form the adenosine 5′-phosphosulfate (APS) intermediate that is further reduced to sulfite by the APS reductase. H₂S is then produced from sulfite in the reaction catalyzed by sulfite reductase (Takahashi et al., 2011) (Fig. 2). H₂S reacts with O-acetylserine (OAS), generating cysteine via catalyzation by OAS (thiol)lyase (OAS-TL) (Fig. 2). Based on an in vitro activity assay, OAS-TL has been suggested to catalyze the reverse reaction to break down cysteine into H₂S and OAS (Burandt et al., 2001). However, negligible amounts of H₂S were formed when compared with the cysteine production, indicating that the OAS-TL reaction is a net H₂S-consuming reaction (Bloem et al., 2004).

Moreover, the production of endogenous H₂S in planta by OAS-TL remains unclear. In plants, the main OAS-TLs responsible for cysteine synthesis are the cytosolic OAS-TL A1, the chloroplastic OAS-TL B, and the mitochondrial OAS-TL C (Fig. 2). Recently, the H₂S level was found to be higher in an oas-tl a1 mutant than in wild-type Arabidopsis thaliana plants, confirming the major biological function of OAS-TL in cysteine biosynthesis rather than in H₂S generation (Li et al., 2018).

Cysteine desulfhydrase (CDes) was the first studied H₂S-producing enzyme (Harrington and Smith, 1980; Tishel and Mazelis, 1966). Because of its ubiquitous activity in various physiological processes of different plant species (Zhao et al., 2020), CDes is considered to be the most critical enzymatic source of H₂S in plants. There are two types of CDes: l-cysteine desulfhydrase (l-CDes) that degrades l-cysteine and d-cysteine desulfhydrase (d-CDes) that uses d-cysteine as substrate. In the cytosol, l/d-cysteine is catalyzed by l/d-CDes to produce H₂S, ammonia (NH₃), and pyruvate (Fig. 2).

l-CDes 1 (DES1), an OAS-TL homolog located in the cytosol, had originally been thought to have a function similar to that of OAS-TL A1, until its CDes activity had been proven (Álvarez et al., 2010). The Arabidopsis DES1 gene is ubiquitously expressed at all developmental stages in plants (Laureano-Marín et al., 2014), and its function has been extensively investigated in the context of H₂S signaling in ABA-mediated stomatal movement (see below).

Moreover, the nitrogenase Fe–S cluster (NifS), localized both in chloroplasts and in mitochondria, is a putative H₂S-producing enzyme due to its L-CDes-like activity (Pilon-Smits et al., 2002; Van Hoewyk et al., 2008). In addition, the mitochondrial β-cyanoalanine synthase (CAS) detoxifies cyanide that appeared in the cells to β-cyanoalanine in the presence of cysteine, along with the production of H₂S (Hatzfeld et al., 2000) (Fig. 2).

In mammals, 3-mercaptopyruvate sulfurtransferase (MST) that belongs to the sulfurtransferase (STR) family is one of the most important H₂S-producing enzymes, but information about MST in plants is scarce. Recently, two Arabidopsis MSTs, the mitochondrial STR1 and the cytosolic STR2, were characterized by means of an in vitro activity assay, revealing their H₂S-producing capability in the presence of reducing systems, such as TRXs and GRXs (Moseler et al., 2021). Nevertheless, the in planta contribution to H₂S biosynthesis from STR1 and STR2 awaits to be further investigated.

Exogenous H₂S Application in Plants

Currently, investigation of the physiological roles of H₂S is mainly based on studies applying exogenous H₂S donors. To date, various H₂S donors have been developed (Powell et al., 2018; Yang et al., 2022) and have been widely used in plant studies (Corpas, 2019; Corpas and Palma, 2020; Liu et al., 2021). Here, we provide a short discussion and an update on these donors.

Sulfide salts, such as sodium hydrosulfide (NaHS) and sodium sulfide (Na₂S), are inorganic compounds that release H₂S by hydrolysis. To date, NaHS is the most popular and widely applied H₂S donor in various plant species (Corpas, 2019; Corpas and Palma, 2020; Liu et al., 2021). The use of NaHS as H₂S donor has greatly improved our understanding of the biological function of H₂S, but the NaHS shortcoming is that it does not mimic the biological effects of the physiological H₂S generation.

Indeed, NaHS hydrolyzes immediately in aqueous solutions, and instantaneously releases large amounts of H₂S, HS⁻, and S²⁻ species, a process very different from the endogenously slow and continuous H₂S enzymatic production. Therefore, chemicals with a slow H₂S-releasing rate are required.

The morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (GYY4137) is such a slow releasing H₂S compound that had initially been synthesized and evaluated with a vasodilatory and antihypertensive activity (Li et al., 2008). Due to the commercial availability and application feasibility, GYY4137 is the most widely used H₂S donor, besides sulfide salts in the mammalian field (Powell et al., 2018). In plants, GYY4137 often applied in parallel with NaHS that has similar effects. For example, similarly to NaHS, GYY4137 application could induce stomatal closure in Arabidopsis (García-Mata and Lamattina, 2010; Honda et al., 2015) and Nicotiana tabacum (tobacco) (Papanatsiou et al., 2015), and could improve growth of Pisum sativum (pea), Lactuca sativa (lettuce), and Raphanus sativa (radish) (Carter et al., 2018).

Recent environmentally friendly slow H₂S-releasing chemicals, dialkyldithiophosphates and disulfidedithiophosphates, have been shown to improve the growth of Zea mays (maize) plants, hinting at potential applicability in agriculture (Brown et al., 2021; Carter et al., 2019). Another novel H₂S donor is a class of nitric oxide (NO)–hydrogen sulfide-releasing hybrid (NOSH) compounds that release NO and H₂S simultaneously, which was designed for its extreme effectiveness in growth inhibition of human cancer cell lines (Kodela et al., 2012). NOSH and its aspirin hybrid (NOSH-aspirin) that also releases acetylsalicylic acid have been shown to improve drought tolerance of Medicago sativa (alfalfa) (Antoniou et al., 2020), suggesting that NOSH might be a promising plant priming agent against environmental stresses.

H₂S has been reported to act as electron donor for respiration, and to contribute to ATP production in mitochondria of prokaryotes (Sakurai et al., 2010) and in a variety of species, such as California killifish (Fundulus parvipinnis) (Bagarinao and Vetter, 1990), marine mussel (Geukensia demissa) (Doeller et al., 2001; Doeller et al., 1999; Parrino et al., 2000), sandworm (Arenicola marina) (Vökel and Grieshaber, 1997), chicken (Gallus gallus) (Yong and Searcy, 2001), and human (Homo sapiens) (Goubern et al., 2007).

A mitochondria-specific H₂S donor (10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl)phenoxy)decyl), triphenylphosphonium bromide (AP39) (Le Trionnaire et al., 2014), can be used as a useful tool to specifically investigate the biological function of H₂S in mitochondria. AP39 was initially used in murine microvascular endothelial cells, had an antioxidant and cytoprotective impact under oxidative stress conditions (Szczesny et al., 2014), and later improved the mitochondrial function in the nematode Caenorhabditis elegans (Fox et al., 2021). H₂S is certainly tightly linked to the mitochondrial electron transport chain (ETC) activity in the above-mentioned species. However, the knowledge of this aspect in plants remains modest.

Recently, AP39 was applied in Arabidopsis to investigate stomata movement (Pantaleno et al., 2023). In addition to the induction of stomatal closure, AP39 could modulate mitochondrial ETC activity and redox homeostasis of guard cells, providing the first piece of evidence that H₂S modulates mitochondrial energetics in plants.

In vivo Detection of H₂S in Plants

The important physiological functions of H₂S have attracted attention in plant research, compelling the development of detection techniques in living cells, tissues, and different organisms, but the direct detection of endogenous H₂S in plants remains a challenge. Traditional detection methods for H₂S, such as colorimetric assays (Siegel, 1965), gas chromatography (Hannestad et al., 1989), high-performance liquid chromatography (Shen et al., 2011), polarographic H₂S sensor (Doeller et al., 2005), and ion-selective electrode (Li et al., 2000), typically require sample destruction and are limited to in vitro detection.

Fluorescent probes are emerging as tools for the noninvasive study of reactive species in situ in different biological systems, because of their high cell permeability and specificity. Different fluorescent approaches, including chemical and genetically encoded probes, have been used for H₂S detection (Chen et al.,2013b, Chen et al., 2012; Lin et al., 2015; Liu et al., 2011a; Youssef et al., 2019). Despite the considerably fewer reports in plants than in the mammalian field, fluorescent probes are considered effective and noninvasive tools for the real-time detection and imaging of H₂S in plants.

As the methods for H₂S detection have been extensively reviewed (Filipovic et al., 2018; Kong et al., 2022; Lin et al., 2015; Luo et al., 2022; Zeng et al., 2021; Zhao et al., 2020), we will discuss the fluorescent probes recently used to discover endogenous H₂S in different plant species and organisms. In general, the various strategies can be categorized into three groups based on the reaction types, namely azide/nitro/nitroso reduction, copper sulfide precipitation, and nucleophilic reaction-based methods (Luo et al., 2022). The fluorescent probes used in plant studies are mainly based on azide reduction and nucleophilic reaction (Fig. 3).

FIG. 3.

H₂S detection with fluorescent probes in plants. Various fluorescent probes have been used to visualize and determine the H₂S level in different plant species. In the chemical structures, the red star highlights the reaction moiety for H₂S, and the yellow color marks the fluorophore group. AzMC is an azide (N₃)-based chemical probe, used to determine the H₂S level in guard cells induced by ABA in wild-type Arabidopsis (accession Columbia-0 [Col-0]), but not in des1 mutant plants, whereas the H₂S donor NaHS induced H₂S production in the guard cells of both genotypes (Zhang et al., 2020a). Another azide-based probe, SiND-ANPA-N₃, had been tested in inner-layer epidermal tissues of onion (Fu et al., 2020b). A nucleophilic reaction-based chemical probe, WSP-1, containing a pyridyl disulfide moiety as H₂S reaction site, has been used in tomato roots to determine H₂S induction upon NO donor and scavenger treatments, and in the root of turnip for the observation of H₂S production repression upon selenium treatment (Chen et al., 2014; Li et al., 2014c). Two sulfane sulfur probes, SSP4 and SSNIP, contain thiophenol moiety. The SSP4 probe has been used to reveal the H₂S production during symbiosis in the nodules of Lotus japonicus (Fukudome et al., 2020). SSNIP is a recent NIR probe used in Arabidopsis roots (Jiang et al., 2019). The most recent fluorescent probe applied in plants is the NIR-based probe, HBTP-H₂S, showing an increased H₂S level upon Al³⁺ and flooding stresses (Wang et al., 2021). ABA, abscisic acid; Al³⁺, aluminum ion; AzMC, 7-azido-4-methylcoumarin; BF, bright field; HBTP-H₂S, 2-(2-hydroxyphenyl) benzothiazole-based H₂S probe; NaHS, sodium hydrosulfide; NIR, near-infrared; NO, nitric oxide; SiND-ANPA-N_3, silicon nanodots-4-azido-N-alanine-1,8-naphthalimide; SSNIP, sulfane sulfur near-infrared probe; SSP4, sulfane sulfur probe 4; WSP-1, Washington State probe-1.

7-Azido-4-methylcoumarin

The azide reduction-based chemical probe, 7-azido-4-methylcoumarin (AzMC), is commercially available. The strong electron-withdrawing ability of the azide group of AzMC quenches its fluorescence, whereas H₂S reduces the azide to amine, thus turning on the fluorescence (Kong et al., 2022). Initially, AzMC has been used in photoaffinity labeling of the substrate-binding site of the human phenol sulfotransferase (Chen et al., 1999), and later for H₂S detection in living cells and cardiac tissues (Chen et al., 2013a). In plants, AzMC was first applied to measure the H₂S level in tomato (Solanum lycopersicum) guard cells in response to ethylene (ET) signal transduction upon osmotic stress (Jia et al., 2018).

Recently, this probe has been utilized to determine H₂S levels in Arabidopsis guard cells from wild-type and des1 mutant plants, which are deficient in cytosolic H₂S generation due to the lack of DES1, in response to ABA-induced—stomatal closure (Shen et al., 2020; Zhang et al., 2020a). The ABA-induced H₂S accumulation in guard cells of wild-type plants was abolished in the des1 mutant plants, whereas the H₂S donor NaHS could clearly induce H₂S in plants of both genotypes (Fig. 3), indicating that DES1 is responsible for the sensitivity of ABA-induced stomatal closure (Zhang et al., 2020a).

Silicon nanodots-4-azido-N-alanine-1,8-naphthalimide

Another azide reduction-based chemical probe is the silicon nanodots-4-azido-N-alanine-1,8-naphthalimide (SiND-ANPA-N₃ ). This probe contains three moieties: the two-photon fluorophore dye N-alanine-1,8-naphthalimide, the azido adduct responsible for reduction-activated fluorescence, and the attached silicon nanodot (SiND) that increases water solubility and cell permeability. This probe has been tested in the inner-layer epidermal tissues of onion (Allium cepa), and evaluated as a potential probe for H₂S detection in aqueous media and living cells (Fu et al., 2020b) (Fig. 3).

Washington state probe-1

The Washington state probe-1 (WSP-1) is a nucleophilic reaction-based chemical probe (Liu et al., 2011a) that contains a pyridyl disulfide moiety and a fluorophore group. H₂S reacts with the pyridyl disulfide and generates a persulfide (–SSH) intermediate that undergoes a spontaneous intramolecular cyclization to release the fluorophore. By using WSP-1 in tomato roots, an increased level of H₂S was detected upon an exogenous NO donor treatment, which was inhibited by applying the NO scavenger cPTIO (Li et al., 2014c) (Fig. 3). Until now, WSP-1 was utilized for endogenous H₂S detection in the roots of turnip (Brassica rapa), and the H₂S level decreased upon selenium treatment (Chen et al., 2014) (Fig. 3).

Sulfane sulfur probe 4

The nucleophilic reaction-based sulfane sulfur probe, sulfane sulfur probe 4 (SSP4), has been developed based on the original design of the SSP1 and SSP2 probes (Chen et al., 2013b). Sulfane sulfur reacts with the nucleophilic thiols in the nonfluorescent SSP4 to form a persulfide intermediate that reacts with electrophilic ester groups, leading to spontaneous intramolecular cyclization and release of the fluorophore.

Recently, SSP4 has been used for endogenous H₂S detection during the root nodule symbiosis in the legume Lotus japonicas, and an increased level of H₂S has been observed during nodulation (Fukudome et al., 2020) (Fig. 3). SSP4 is commercially available, and has a relatively high selectivity and sensitivity to sulfane sulfurs. However, sulfane sulfur probes are not specific for H₂S, because they also react with persulfides and polysulfides.

Sulfane sulfur near-infrared probe

Another sulfane sulfur probe that shares a thiophenol moiety and an ester linker with the SSP4 probe is attached to a near-infrared (NIR) fluorophore, designated sulfane sulfur NIR probe (SSNIP). The SSNIP probe has been tested in Arabidopsis roots (Fig. 3) during different growth stages (Jiang et al., 2019).

2-(2-Hydroxyphenyl) benzothiazole-based H₂S probe

The 2-(2-hydroxyphenyl) benzothiazole-based H₂S probe, HBTP-H₂S, is a recent NIR fluorescent probe and contains a dinitrophenyl (DNP) ether that undergoes thiolysis under nucleophilic attacks by H₂S, releasing the fluorophore. This NIR fluorescent probe has been applied for in situ bioimaging of endogenous H₂S in rice (Oryza sativa) roots, and revealed an increased level of H₂S under aluminum ion (Al³⁺) and flooding stresses (Wang et al., 2021) (Fig. 3).

Genetically encoded H₂S sensors

Since 2012, reaction-based genetically encoded fluorescent H₂S sensors have been studied, and a probe based on p-azidophenylalanine (pAzF) was originally developed and tested by expressing the cpGFP-Tyr66pAzF in Hela cells (Chen et al., 2012). This pAzF-based genetic probe has been optimized by modification with pAzF of the chromophore of a circularly permutated, superfolder green fluorescent protein (cpsGFP) to derive the cpsGFP-pAzF, which subsequently served as a Förster resonance energy transfer acceptor to an enhanced blue fluorescent protein EBFP2 (Youssef et al., 2019).

Thus far, the H₂S genetic probe has not been utilized in plant research, whereas other genetic sensors, such as HyPer or the roGFP series for hydrogen peroxide (H₂O₂) sensing, and SoNar or Peredox for NADH/NAD⁺ sensing (Müller-Schüssele et al., 2021), have been extensively used for monitoring the redox states in living plant cells. The development or application of genetic probes for real-time H₂S monitoring in plant research would be greatly beneficial for understanding the H₂S-regulatory mechanisms in plants.

H₂S Signaling via Protein Persulfidation

Protein persulfidation, also called S-sulfhydration, as a type of oxidative PTM of cysteines, has been increasingly recognized as the main redox mechanism directly regulating diverse biological processes in H₂S signaling, such as protein function, structure, and subcellular localization. Protein cysteine thiols (–SH) are very susceptible to H₂O₂ and can undergo different oxidative PTMs. Initially, thiol reacts with H₂O₂ to form sulfenic acid (–SOH) (Allison, 1976) that is intrinsically unstable and an intermediary en route to other PTMs.

In the presence of H₂O₂ excess, –SOH forms the relatively more stable sulfinic acid (–SO₂H) and sulfonic acid (–SO₃H) (Cremlyn, 1996), which are generally considered to cause protein overoxidation (Fig. 4). The –SO₃H formation is irreversible, whereas –SO₂H can be reduced via an ATP-dependent reaction by sulfiredoxin of certain proteins (Akter et al., 2018; Biteau et al., 2003).

FIG. 4.

Overview of cysteine thiols undergoing different oxidative post-translational modifications. Protein cysteine –SH reacts with H₂O₂ to form –SOH that can be further oxidized by H₂O₂ to –SO₂H and –SO₃H, both considered protein overoxidations. Via an ATP-dependent reaction –SO₂H can be reduced by SRX, whereas –SO₃H is an irreversible modification. The –SOH protein can react with –SH or with GSH to form –SS– or –SSG, respectively. The proteins –SS– and –SSG can be reduced by redoxins, including TRXs and GRXs. The protein cysteine –SH reacts with NO to form –SNO that can also be reduced by redoxins. H₂S reacts with –SOH and –SS– to form –SSH, which has been suggested to be formed also by the reaction of H₂S with –SNO or –SSG. The protein –SSH can be oxidized by H₂O₂ to form –SSOH, –SSO₂H, and–SSO₃H and –SSH, and its oxidative derivatives can be reduced by redoxins. GRX, glutaredoxin; GSH, glutathione; H₂O_2, hydrogen peroxide;–SH, thiol; –SNO, S-nitrosothiol;–SO₂H, sulfinic acid; –SO₃H, sulfonic acid; –SOH; sulfenic acid; SRX, sulfiredoxin; –SS–, intra/intermolecular disulfides; –SSG, glutathione adduct; –SSH, persulfide; –SSO₂H, perthiosulfinic acid; –SSO₃H, perthiosulfonic acid; –SSOH, perthiosulfenic acid; TRX, thioredoxin.

Alternatively, –SOH can react with proximal –SHs from proteins and with GSH, forming intra/intermolecular disulfide (–SS–) (Nagy and Winterbourn, 2010; Turell et al., 2021) and S-glutathione adduct (–SSG) (Turell et al., 2008), respectively (Fig. 4). Disulfides and GSH adducts can be enzymatically reduced to –SH by thiol reductases, so-called redoxins, such as TRXs and GRXs (Huang et al., 2018; Willems et al., 2021). Besides H₂O₂, reactive nitrogen species, which mainly refer to NO, trigger the formation of S-nitrosothiols (–SNO) (Hess et al., 2005) that can also be reduced by redoxins.

H₂S reacts with oxidized, not reduced, thiols, –SOH, and disulfides specifically, to form persulfides (–SSH) (Cuevasanta et al., 2015). The kinetics of the H₂S reactions with low-molecular weight albumin disulfides and –SOH have been determined. The rate constant of H₂S with –SOH for the formation of persulfides is 270 M ⁻¹s⁻¹, significantly higher than that of disulfides (0.6 M ⁻¹s⁻¹), implying that the formation of protein persulfides might mainly occur through reaction of H₂S with –SOH. However, extremely high reaction rates of protein disulfides with H₂S have been detected in some special cases, such as human sulfide quinone oxidoreductase (Cuevasanta et al., 2017).

Furthermore, protein disulfides are relatively more stable than the labile –SOH in the environment; hence, the generation of protein persulfides via the H₂S reaction with disulfides cannot be excluded. Persulfides might be formed by reaction of H₂S with –SNO or –SSG (Francoleon et al., 2011; Iciek et al., 2015; Mishanina et al., 2015), but this reaction mainly remains hypothetical and needs further investigation.

Protein persulfides can be oxidized by H₂O₂ to form perthiosulfenic acid (–SSOH), perthiosulfinic acid (–SSO₂H), and perthiosulfonic acid (–SSO₃H). In contrast to the hardly reversible –SO₂H and irreversible –SO₃H formed upon H₂O₂ overoxidation, the corresponding –SSH and its oxidative derivatives can be reduced by redoxins (Dóka et al., 2020; Filipovic, 2015; Ju et al., 2016; Wedmann et al., 2016) (Fig. 4).

Protein Persulfidation Detection in Plants

In the past decade, a variety of detection methods for efficient persulfidation labeling and identification have been developed to investigate the H₂S signaling executed via persulfidation. Protein persulfidation can be directly detected by means of mass spectrometry (MS), because of the mass increase of 31.972 Da by the addition of one sulfur atom, but is difficult to distinguish from other modification, such as –SO₂H due to the addition of two oxygen atoms (mass increase of 31.99 Da). Thus, specific labeling with chemical probes is required for persulfidation detection.

Initially, a modified biotin switch method had been applied in human cells, in which methyl methanethiosulfonate (MMTS) was believed to specifically block –SH, whereafter –SSH was targeted and enriched with N-[6-(biotinamido) hexyl]-3′-(2′-pyridyldithio)propionamide (biotin-HPDP) (Mustafa et al., 2009). In plants, this method was first utilized in Arabidopsis leaf extracts, and 106 persulfided proteins were identified (Aroca et al., 2015). However, because the reactivity toward MMTS of SSH was higher than that of thiols (Pan and Carroll, 2013), this method was questioned and should be used with caution.

The most challenging aspect of persulfidation detection is the discriminative labeling from thiols, because of their similar reactivity to commonly used reagents through alkylation, such as maleimide, N-ethylmaleimide (NEM), and iodoacetamide (IAM) (Pan and Carroll, 2013). Due to their greater nucleophilicity, persulfides react even much faster than thiols to thiol-labeling reagents (Cuevasanta et al., 2015). Nevertheless, because alkylation of thiols yields thioethers, whereas persulfides generate disulfides; many thiol label-based detection approaches have been exploited based on this characteristic.

At first, a fluorescent probe, designated red maleimide, had been used to study the persulfidation of the p65 subunit of mice NF-κB (Sen et al., 2012). Both −SH and −SSH are labeled with red maleimide, followed by dithiothreitol (DTT) reduction, so that only the labeled −SSH, namely the R−S−S−maleimide-red adducts, are reduced (Fig. 5A). The protein samples are subsequently separated by gel electrophoresis and detected by in-gel fluorescence. The loss of fluorescence can be calculated for evaluation of the persulfidation level. This method revealed the persulfidation of recombinant human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein at Cys150, which enhances its catalytic activity (Gao et al., 2015).

FIG. 5.

Persulfidation detection approaches. (A) Red maleimide and MalP methods. Both –SH and –SSH are initially labeled, but only the labeled –SSH can be reduced by DTT. In the red maleimide method, –SSH is detected by SDS-PAGE and displays fluorescence loss. As the Mal-P labeling results in a molecular mass increase of 1.95 kDa, –SSH is discovered as a decreased mobility shift in SDS-PAGE. (B) BTA method for proteomic analysis. Both –SH and –SSH are labeled with maleimide-biotin and enriched by streptavidin magnetic beads. The enriched –SSH proteins are eluted by DTT reduction, digested with trypsin, and subjected to MS analysis. (C) Tag-switch and dimedone-switch methods. In the tag-switch method, MSBT is used to block both –SH and –SSH, the latter forming –SS–MSBT that further reacts with CN-biotin to be biotinylated, which is further enriched by streptavidin AP, digested by trypsin, and subjected to MS analysis. Alternatively, CN-BOT and CN-Cy3 are used instead of CN-biotin to visualize –SSH in situ by confocal microscopy and in-gel fluorescent detection, respectively. In the dimedone-switch assay, NBF-Cl reacts with –SSH, –SH, and –SOH, whereafter DCP-Bio1 or DCP-N₃ is added to selectively switch with the –SS–NBF adducts. When DCP-Bio1 is utilized, the –SSH proteins are subsequently enriched with streptavidin AP and identified by MS. When DCP-N₃ and the Cy5-alkyne click mix is applied, the –SSH is detected by in-gel fluorescent assay or with confocal microscopy. (D) Direct labeling method at low pH for proteomic analysis. Both –SH and –SSH are labeled by IPM at pH 5.0, resulting in proteins with S-IPM and SS-IPM adducts that are further digested by trypsin. The digested peptides are biotinylated by a click reaction with Az-UV-biotin reagents, enriched by streptavidin AP, cleaved by UV light to remove the biotin moiety, and subjected to MS analysis. AP, affinity purification; Az-UV-biotin, UV cleavable biotin-azide; BTA, biotin thiol assay; CN-biotin, cyanoacetate biotin; CN-BOT, cyanoacetate with the fluorescent BODIPY moiety; CN-Cy3, cyanoacetate with Cy3-dye; DCP-Bio1, dimedone-based biotin-conjugated analog; DCP-N₃, dimedone-based azide-conjugated analog; DTT, dithiothreitol; IPM, N-propynyliodoacetamide; MalP, maleimide peptide; MS, mass spectrometry MSBT, methylsulfonyl benzothiazole; NBF-Cl, 4-chloro-7-nitrobenzofurazan; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; –SS–MSBT, MSBT disulfide adducts; UV, ultraviolet.

Later, another thiol label-based probe, a maleimide compound containing a peptide arm, designated maleimide peptide (MalP, 1.95 kDa), was used to study the persulfidation of the iron–sulfur cluster machinery in mammalian proteins (Parent et al., 2015). As with the red maleimide fluorescent probe, both −SH and −SSH are labeled with MalP, and only the labeled −SSHs are further reduced by the subsequent DTT incubation and release the succinimide-peptide moiety (the product of the maleimide reaction with a sulfhydryl). As a result, the mobility shift in the protein migration can be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 5A).

The two aforementioned approaches offered many advantages, such as relative application simplicity and radiometric read-outs (−SSH vs. −SH), giving extra quantification information. Nevertheless, these methods are restricted to biochemical characterizations and are rather not applicable to large-scale MS-based proteomic analyses.

A MS-coupled thiol labeling-based proteomic approach, termed biotin thiol assay (BTA), had initially been utilized for mapping protein persulfidation in mammalian cells by means of maleimide-biotin (Cuevasanta et al., 2015), maleimide-PEG2-biotin (Gao et al., 2015), or iodoacetyl-PEG2-biotin (Dóka et al., 2019; Dóka et al., 2016). In this method, −SH and −SSH are first labeled with biotin-tagged alkylating reagents that are sequentially enriched on an avidin column, whereafter the labeled −SSHs are selectively eluted by reduction via DTT or tris(2-carboxyethyl)phosphine (Fig. 5B).

By labeling the eluted persulfide-derived thiols by isotope-labeled (D5, heavy) or normal (H5, light) maleimide, a quantitative analysis under different conditions could be achieved (Gao et al., 2015). Recently, a BTA combined with the iodoacetyl isobaric tandem mass tag system allowed the quantitative cysteine site-specific identification of persulfidation (Gao et al., 2020).

In addition, a “tag-switch” method was developed for persulfidation detection (Zhang et al., 2014). Here, methylsulfonyl benzothiazole (MSBT) was used to block both −SH and −SSH, whereafter solely MSBT disulfide adducts (–SS–MSBT) could react with cyanoacetate biotin (CN-biotin), hence designated “tag switch” (Fig. 5C). This method was further improved by attaching cyanoacetate with the fluorescent BODIPY moiety (CN-BOT) or the Cy3 dye (CN-Cy3), so that persulfidation could be visualized in situ by fluorescence confocal microscopy or in-gel fluorescent detection from the cell lysates (Kouroussis et al., 2019; Wedmann et al., 2016).

In plants, the “tag-switch” method was applied in wild-type and des1 mutant Arabidopsis plants (Aroca et al., 2017a). In total, 2015 and 2130 persulfidated proteins were identified in the wild-type and des1 plants, respectively, suggesting that a large fraction of the Arabidopsis proteome undergoes persulfidation even under nonstressed conditions (Aroca et al., 2017a). Recently, the application of the “tag-switch” method on Arabidopsis root tissue identified 5214 –SSH proteins (Jurado-Flores et al., 2021).

A variant of this tag-switch method for the –SSH identification is called the “dimedone-switch” assay (Zivanovic et al., 2019) (Fig. 5C). In this assay, –SSH, –SH, and –SOH first react with 4-chloro-7-nitrobenzofurazan (NBF-Cl), whereafter a dimedone-based probe, such as a biotin-conjugated analog dimedone-based biotin-conjugated analog (DCP-Bio1) (Poole et al., 2007) or azide-conjugated analog DCP-N₃, selectively switches with –SS–NBF disulfides. The biotin-tagged persulfides are subsequently enriched, and identified by MS and by in-gel fluorescence or confocal microscopy when DCP-Bio1 and DCP-N₃ and Cy5-alkyne click mix are used, respectively (Zivanovic et al., 2019). This dimedone-switch method recently applied in Arabidopsis revealed persulfidation of Cys103 of the autophagy-related protein 18a, thereby activating its phospholipid-binding activity in a reversible manner and, hence, regulating autophagy under endoplasmic reticulum stress (Aroca et al., 2021a).

Currently, a direct persulfidation detection method was developed for proteomic analysis by labeling –SSH at pH 5.0 by means of an alkyne-linked IAM, N-propynyliodoacetamide (IPM) (Fu et al., 2020a) (Fig. 5D). Given the lower pK_a of persulfide (4.3) than that of thiol (8.29), persulfides maintain a relatively high reactivity at pH 5.0 when compared with protonated thiols. Hence, efficient labeling of –SSH can be achieved by labeling proteome extracts with IPM at a low pH, resulting in disulfide adducts (SS-IPM), in addition to thioether adducts (S-IPM) due to the unavoidable –SH labeling.

IPM-labeled peptides are further biotinylated by reaction with UV cleavable biotin-azide (Az-UV-biotin) reagents through a click reaction, the addition of biotin facilitating peptide enrichment. Two types of probe-modified peptides, including the disulfide forms derived from –SSH and the thioether forms derived from –SH, can be analyzed by MS (Fu et al., 2020a).

H₂S-Mediated Persulfidation in ABA-Regulated Stomatal Movement

Stomata are pores on the epidermis of plant leaves surrounded by a pair of guard cells. Stomatal movement regulates gas and water exchange between the plants and the environment, and is important for plant growth, development, and response to environmental stimuli (Kim et al., 2010). That H₂S induced stomatal closure and participated in ABA signaling was first evidenced by application of NaHS in epidermal strips of Vicia faba (broad bean), A. thaliana, and Impatiens walleriana (impatiens) (García-Mata and Lamattina, 2010). In contrast, H₂S was reported to cause stomatal opening in Arabidopsis (Lisjak et al., 2010) and pepper (Capsicum annuum) (Lisjak et al., 2011).

The reason of the opposite effects of H₂S on stomatal movement remains inconclusive. However, an increasing number of studies subsequently revealed that H₂S is a key regulator of stomatal closure triggered by different environmental stresses, such as drought (Jin et al., 2017), cold (Du et al., 2019), and mediated by phytohormones, such as ABA that accumulates under drought stress (Jin et al., 2013), ET (Hou et al., 2016; Liu et al., 2011b), salicylic acid (SA) (He et al., 2020), and jasmonic acid (JA) (Deng et al., 2020) (Fig. 6).

FIG. 6.

Simplified scheme of H₂S-triggered stomatal closure in ABA-regulated stomatal closure under drought stress. Under drought or cold stress, ABA or other phytohormones, such as ET, SA, and JA, accumulate in the cells and regulate stomatal movement via H₂S signaling. ABA induces the expression of DES1, increasing the H₂S production. DES1-catalyzed H₂S signals activate downstream H₂O₂ and NO signaling to trigger stomatal closure. ET, ethylene; JA, jasmonic acid; SA, salicylic acid.

To date, the H₂S signaling function has been best characterized by the ABA-regulated stomatal movement. ABA has been generally recognized as eliciting the DES1 expression in guard cells, which increases the endogenous level of H₂S, because DES1-mediated H₂S production is required for downstream NO signaling (Scuffi et al., 2014) and respiratory burst oxidase homolog (RBOH)-dependent H₂O₂ signaling (Scuffi et al., 2018) to activate stomatal closure (Fig. 6).

Besides DES1 and RBOH (Shen et al., 2020), abscisic acid insensitive 4 (ABI4) (Zhou et al., 2020) and SNF1-related protein kinase2.6 (SnRK2.6), also known as Open stomata 1 (OST1) (Chen et al., 2020), have been found as key proteins involved in H₂S signaling in ABA-regulated stomatal movement (see below). In addition, by means of pharmacological and genetic approaches, phospholipase D and mitogen-activated protein kinase 4 were shown to participate in H₂S-mediated guard cell signaling (Scuffi et al., 2018) and to be an important downstream signal of H₂S in stomatal movement in response to drought stress, respectively (Du et al., 2019).

Persulfidation on several key proteins in plants has been characterized, such as the critical antioxidant enzyme ascorbate peroxidase 1 (Aroca et al., 2015), the moonlighting cytosolic GAPDH protein (Aroca et al., 2017b), the autophagy-related protein 4 (Laureano-Marín et al., 2020) and 18 (Aroca et al., 2021a). For a recent review, see Aroca et al. (2021b). Here, we focus on the most recent proteins, that is, DES1, respiratory burst oxidase homolog D (RBOHD), OST1, and ABI4, which are involved in H₂S signaling in ABA-regulated stomatal closure (Fig. 7).

FIG. 7.

H₂S-triggered persulfidation in ABA-regulated guard cell movement. When ABA is present, it binds PYR/PYL/RCAR to form a receptor complex that interacts with and inhibits the catalytic activity of PP2C, activating SnRK2.6. SnRK2.6 undergoes H₂S-triggered persulfidation, thereby promoting its activity and interaction with ABF2. SnRK2 phosphorylates downstream the transcription factor RAV1, hampering the expression of ABI4, whereas DES1-mediated H₂S production induces ABI4 persulfidation that enhances its transactivation activity on MAPKKK18, thereby initiating a MAPK cascade. DES1-mediated H₂S triggers its autopersulfidation, leading to a H₂S burst. The H₂S accumulation induces RBOHD persulfidation, thereby increasing its activity and leading to ROS overproduction. The ROS accumulation increases the Ca²⁺ influx, contributing to stomatal closure in response to ABA. ABF2, ABA response element-binding factor 2; ABI4, abscisic acid insensitive 4; Ca²⁺, calcium cation; MAPK, mitogen-activated protein kinase; MAPKKK18, mitogen-activated protein kinase kinase kinase 18; PP2C, protein phosphatase type 2C; PYR/PYL/RCAR, pyrabactin resistance/PYR-like/regulatory component of ABA receptor; RAV1, ABI3/VP1-like 1; RBOHD, respiratory burst oxidase homolog D; ROS, reactive oxygen species; SnRK2.6, SNF1-related protein kinase2.6.

The ABA level in guard cells under normal conditions remains low but increases upon stress stimuli. When ABA is absent, the protein phosphatase type 2C (PP2C) binds to the SnRK2.6 kinase domain and inhibits the kinase activity by dephosphorylation. In the presence of ABA, it binds to the ABA receptor pyrabactin resistance/PYR-like/regulatory component of ABA receptor (PYR/PYL/RCAR) to form a complex that binds to PP2C and inhibits the catalytic activity of PP2C (Fig. 7), thereby activating SnRK2.6 because of its dissociation from PP2C and autophosphorylation (Soon et al., 2012). The activated SnRK2.6 then transmits the ABA signal by phosphorylating downstream factors, such as RBOH, ultimately inducing rapid ROS production and cellular calcium cation (Ca²⁺) influx and activating specific ion channels that trigger stomatal closure (Fig. 7).

ABA induces the DES1 expression transcriptionally through an unclear mechanism, thereby inducing the H₂S production in guard cells (Scuffi et al., 2014). The ABA-induced H₂S production results in the persulfidation of SnRK2.6 at Cys131 and Cys137, which are adjacent to the catalytic loop of the kinase and the pivotal phosphorylation site Ser175 (Chen et al., 2020) (Fig. 7).

Persulfidation of SnRK2.6 promotes its activity and interacts with the transcription factor ABA response element-binding factor 2 (ABF2) that enhances the cytosolic Ca²⁺ signaling. Persulfidation of SnRK2.6 at Cys131 and Cys137 alters its protein structure, hence bringing the Ser175 residue closer to the phosphate-acceptor Asp140 and improving its activity (Chen et al., 2021). In contrast, NO negatively regulates ABA signaling in guard cells by inhibiting SnRK2.6 through formation of -SNO at Cys137 (Wang et al., 2015).

SnRK2 phosphorylates the downstream transcription factor related to ABI3/VP1-like 1 (RAV1) to inhibit the expression of ABI4 (Feng et al., 2014). ABI4 has been considered as a versatile activator or repressor of its downstream target genes (Wind et al., 2013). The DES1-mediated H₂S production has been reported to induce the persulfidation of ABI4 at Cys250, which is essential for the ABI4 function in the regulation of plant responses to ABA (Zhou et al., 2021). The persulfidation of ABI4 at Cys250 enhances its transactivation activity on mitogen-activated protein kinase kinase kinase 18 (MAPKKK18), thereby activating a mitogen-activated protein kinase (MAPK) cascade (Fig. 7). Furthermore, transactivation of DES1 could be mediated by ABI4 persulfidation, hinting at the existence of a regulatory loop.

In addition, we recently demonstrated that H₂S-mediated persulfidation is involved in ABA signaling in guard cells by directly regulating the activity of both ROS and H₂S-producing enzymes (Shen et al., 2020). Upon ABA treatment, DES1-mediated H₂S triggers its autopersulfidation at Cys44 and Cys205, leading to a burst of H₂S in guard cells. The accumulation of H₂S further induces the persulfidation of RBOHD Cys825 and Cys890, enhancing the RBOHD activity and leading to ROS overproduction (Fig. 7).

NO was demonstrated to negatively regulate Arabidopsis RBOHD by forming -SNO at Cys 890 during the hypersensitive response in plant immunity (Yun et al., 2011). Nevertheless, NO-mediated modifications on RBOHD still need to be investigated regarding the impact on the ABA signaling in guard cells. Here, the interplays between H₂S, ROS, and NO involved in the regulatory ABA signaling mechanism in guard cells occur most probably through redox PTMs of key proteins.

Summary and Perspectives

In the past decades, numerous studies revealed the multitasking capacity of H₂S that is involved in many physiological and pathological processes in mammals (Dilek et al., 2020; Kimura et al., 2012; Murphy et al., 2019; Shatalin et al., 2011) and growth, development, and response to environmental stimuli in plants (Baudouin et al., 2016; Chen et al., 2011; Jin et al., 2017; Jin et al., 2013; Li et al., 2014a; Zou et al., 2019). Nevertheless, our current knowledge on the molecular mechanisms by which H₂S executes its signaling function remains limited.

The emerging studies focusing on H₂S-mediated persulfidations in plants, especially the recently reported cases showing the key function of persulfidation in ABA-regulated stomatal movements, have greatly contributed to the understanding of the H₂S regulatory mechanism in plants. Although several aspects regarding H₂S signaling in plants still await to be assessed, we believe that they will be solved in the near future by the application of advanced techniques, such as quantitative proteomics, real-time imagining, and structural biology.

Intra/interdisulfide formation and S-glutathionylation are known to be the major redox mechanisms that protect –SH from overoxidation, with GSH being the most important low molecular weight antioxidant in the cells. Recently, the reaction rate of SOH with H₂S has been reported to be 2700 M ⁻¹s⁻¹ (Cuevasanta et al., 2015), which is much faster than that of –SH (21.6 M ⁻¹s⁻¹) or that of GSH (2.9 M ⁻¹s⁻¹) for the formation of disulfides and -SSG, respectively (Turell et al., 2008). In addition, increased persulfidation has been observed as a response to H₂O₂ stress in mammalian cells (Wedmann et al., 2016), indicating that the persulfidation chemistry involves the preferential addition of H₂S to a –SOH protein.

Furthermore, H₂S might play an important role as an antioxidant through persulfidation in protecting thiols from irreversible overoxidations, such as –SO₃H. To compare the antioxidant role of H₂S-mediated persulfidation with that of other PTMs, such as GSH-mediated S-glutathionylation, several features need to be taken into account, including the cellular concentration of H₂S in the microenvironment.

The concentrations of H₂O₂ and GSH in plant cells have been well documented (Cheeseman, 2006; Hasanuzzaman et al., 2017; Smirnoff and Arnaud, 2019), and measurements at a spatiotemporal resolution of H₂O₂ and GSH have been improved by means of genetically encoded sensors (Niemeyer et al., 2021; Nietzel et al., 2019; Ugalde et al., 2022; Ugalde et al., 2021a; Ugalde et al., 2021b).

Unfortunately, the information on the cellular H₂S content is scarce. Although the available H₂S fluorescent probes provide useful tools to detect H₂S production in situ, the noninvasive application of chemical fluorescent probes in planta for real-time H₂S measurements remains a challenge because plant tissues are rather rigid. In our opinion, the implementation of genetic sensors for spatiotemporal monitoring of H₂S levels in cells would greatly advance our understanding of H₂S signaling in plants.

Besides GSH, ascorbate, another abundant antioxidant in plant cells, occurs in all subcellular compartments with particularly high levels in the chloroplasts (Smirnoff and Wheeler, 2000). Ascorbate–GSH cycle has been recognized as one of the major redox regulation pathways to detoxify H₂O₂ in plant cell (Foyer and Noctor, 2011). Although ascorbate was thought to selectively reduce –SNO formation and had been used extensively for the –SNO protein identification in proteomic studies (Willems et al., 2021), it has been found to also reduce –SOH of several proteins, including 1-Cys peroxiredoxin (Monteiro et al., 2007) and the thiol-specific antioxidant enzyme 2 (Anschau et al., 2020) from Saccharomyces cerevisiae and papain from Mus musculus (Zito et al., 2012). Therefore, concern about the selectivity of ascorbate toward –SNO in terms of proteomic analysis is increasing.

More importantly, these findings hint at an alternative pathway of thiol redox regulation via ascorbate. As H₂S is known to react with –SOH to form –SSH, ascorbate might presumably affect persulfidation of certain proteins indirectly via reduction of –SOH. Nevertheless, the direct correlation between ascorbate and H₂S-mediated persulfidation remains elusive.

In addition to ascorbate itself, ascorbate peroxidase (APX), the enzyme catalyzing the conversion of H₂O₂ into H₂O in the presence of ascorbate, undergoes several types of thiol-based PTMs. The enzymatic activity of cytosolic APX1 in Arabidopsis is enhanced by NO-triggered –SNO formation (Begara-Morales et al., 2014; Yang et al., 2015) or H₂S-induced –SSH formation (Aroca et al., 2015) at Cys32. Large-scale proteomic studies revealed that tAPX and sAPX from chloroplast were also sensitive to thiol modifications (Huang et al., 2019; Wei et al., 2020). However, which are the exact types of thiol-based PTMs and how they regulate the biological function of chloroplastic APXs still await to be explored by functional analysis.

To better understand the function of H₂S-mediated persulfidation, it is crucial to find out the occupancy of –SSH and its correlation with other thiol-based PTMs. Such a correlation was evidenced by a comparative analysis based on proteomics data between –SSH and –SOH or –SH events in mammalian studies (Fu et al., 2020a; Zivanovic et al., 2019) and between –SSH, –SOH, and –SNO in Arabidopsis (Aroca et al., 2021b; Zhang et al., 2021; Zhou et al., 2020).

Likewise many thiol-based PTMs, –SSH sites have been mapped at the whole-proteome level in mammals (Fu et al., 2020a), but remain uncharted territory in plants. Adoption of advanced proteomic profiling strategies for identification and quantification of the complete repertoire of persulfidation-undergoing Cys sites in plants will prove a promising future endeavor.

Footnotes

Acknowledgment

The authors thank Martine De Cock for her kind support on the article preparation.

Authors' Contributions

J.H. conceived and wrote the article, Y.X. revised and submitted the article.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by the Jiangsu Natural Science Foundation for Distinguished Young Scholars (No. BK20220084 to Y.X.), the Fundamental Research Funds for the Central Universities (No. XUEKEN2022002 to Y.X.), and the Research Foundation-Flanders FWO Senior Postdoctoral fellowship (No. 1227020N to J.H.).

Abbreviations Used

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Hydrogen Sulfide Signaling in Plants

Abstract

Significance:

Recent Advances:

Critical Issues:

Future Directions:

Introduction

H2S Biosynthesis in Plants

Exogenous H2S Application in Plants

In vivo Detection of H2S in Plants

7-Azido-4-methylcoumarin

Silicon nanodots-4-azido-N-alanine-1,8-naphthalimide

Washington state probe-1

Sulfane sulfur probe 4

Sulfane sulfur near-infrared probe

2-(2-Hydroxyphenyl) benzothiazole-based H2S probe

Genetically encoded H2S sensors

H2S Signaling via Protein Persulfidation

Protein Persulfidation Detection in Plants

H2S-Mediated Persulfidation in ABA-Regulated Stomatal Movement

Summary and Perspectives

Footnotes

Acknowledgment

Authors' Contributions

Author Disclosure Statement

Funding Information

Abbreviations Used

References

H₂S Biosynthesis in Plants

Exogenous H₂S Application in Plants

In vivo Detection of H₂S in Plants

2-(2-Hydroxyphenyl) benzothiazole-based H₂S probe

Genetically encoded H₂S sensors

H₂S Signaling via Protein Persulfidation

H₂S-Mediated Persulfidation in ABA-Regulated Stomatal Movement