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H2S signaling in plants and applications in agriculture

Graphical abstract Summary of the main physiological or adverse environmental situations in higher plants where the hydrogen sulfide (H2S) participates.


Introduction
The description of the gasotransmitter hydrogen sulfide (H 2 S), with its toxic impact on the metabolism of animal and plant cells, changed drastically when this molecule was shown to be endogenously generated in cells. However, its signaling capacity has particularly fascinated researchers in many fields of investigation [1][2][3][4][5]. A number of studies in the field of plants began to show that H 2 S is directly or indirectly involved in a wide range of physiological processes including seed germination [6], root organogenesis [7,8], photosynthesis [9], stomatal movement [10][11][12][13], fruit ripening [14,15], as well as senescence in leaves, flowers and fruits [16,17]. H 2 S has also been shown to be involved in the mechanism of response to adverse biotic and abiotic environmental conditions [18,19]. Research has shown a significant correlation between the functions of H 2 S and nitric oxide (NO), another simple molecule, whose metabolisms appear regulate each other [4]. Fig. 1 summarizes the principal functions of H 2 S in higher plants. The main aim of this review is to provide a broad overview of the major role played by H 2 S in higher plants, with particular attention paid to the beneficial effects of its biotechnological application in crop plants, especially under adverse stressful conditions.

Plant biochemistry of H 2 S: An overview
The study of H 2 S as a signaling molecule has focused on its capacity to interact with thiol (-SH) groups present in protein cysteine residues through the post-translational modification (PTM) persulfidation [4,20]. It is important to point out the major regulatory role played by protein thiol groups involved in multiple interactions which can activate or inhibit the function of the target proteins [21,22]. H 2 S competes with other molecules, such as nitric oxide (NO), glutathione (GSH), cyanide and fatty acids, which generate the PTMs S-nitrosation [4,23], S-glutathionylation [24,25], S-cyanylation [26] and S-acylation [27][28][29], respectively. Fig. 2 shows a simple model of these PTMs involving protein thiol groups. However, fewer studies have explored the potential protein targets of persulfidation, previously known as S-sulfhydration, and how this PTM affects up-regulates and down-regulates these proteins.
Information garnered from initial plant proteomic analyses focusing on the model plant Arabidopsis thaliana [30,31] and that obtained from animal cells [32,33], as well as complementary studies, have facilitated the evaluation of the in vitro effect of H 2 S on a specific plant protein using different H 2 S donors [15,34,35]. Table 1 shows a list of plant proteins, which have been observed to undergo persulfidation, and how their protein function is modulated [36,37]. In some cases, a specific purified protein can behave differently under in vitro conditions depending on whether the H 2 S donor is applied to the whole plant, added to the nutrient solution or growth media or sprayed on the aerial part of the plant. This is due to the complex action of H 2 S characterized by its functional interaction/competition in whole cells with other molecules including nitric oxide (NO) [4], melatonin [38] and phytohormones such as ethylene, auxin and abscisic acid [39,40].
Although the precise mechanisms involved remain unknown, H 2 S has been shown to regulate gene expression [41,42]. Exogenous applications of H 2 S to grapevine (Vitis vinifera L.) plants trigger gene expression involved in the synthesis of secondary metabolites as well as various defensive compounds which boosts plant development and abiotic resistance [43]. In addition, microarray analysis of differentially expressed genes of tomato plants supplemented with NaHS has shown that 5349 genes were up-regulated, while 5536 were down-regulated [44].
However, any precise biochemistry of endogenous H 2 S in plant cells, as well as how and where H 2 S is produced and its metabolic interactions with other molecules, is still in its infancy. In higher plant systems, several enzymes involved in cysteine metabolism present in subcellular compartments (the cytosol, chloroplasts, mitochondria and peroxisomes) are available for the production of H 2 S [35,45,46]. These enzymes include L-cysteine desulfhydrase (L-DES), L-cysteine desulfhydrase 1 (DES1), previously known as Cys synthase-like (CS-LIKE), and cysteine synthase (CS) in the cytosol; D-cysteine desulfhydrase (D-DES) and cyano alanine synthase (CAS) in mitochondria; and sulfite reductase (SiR) in the chloroplast [3,[46][47][48]. However, given its highly lipophilic nature, the H 2 S molecule can spread with ease throughout the lipid bilayer of cell membranes [49]. New promising data also show how activities, such as cysteine desulfhydrases, in some of these enzymes are up-regulated under red light and down-regulated by blue and white light [50].

Potential biotechnological applications of exogenously applied H 2 S
Although further basic research on H 2 S is required, sufficient experimental data show that the exogenous application of H 2 S to different plant species at different stages of development can  palliate damage caused by abiotic stress and enhance physiological features such as seed germination, root development and postharvest preservation of vegetables [4,51,52]. However, an empirical evaluation of how H 2 S is to be applied and appropriate dosages is also required. Up to now, exogenous applications have been carried out using chemicals capable of delivering H 2 S. In animal research on biomedical applications, different families of chemicals, with the capacity to slowly release H 2 S into cells, have been developed. This has led to the development of water-soluble molecules such as (p-methoxyphenyl)morpholino-phosphinodithioic acid (GYY4137) and a family of cysteine-activated H 2 S donors (5a, 8l, and 8o) [53]. Few plant studies have used these chemicals [54] which are comparatively more expensive to produce than standard chemicals such as sodium hydrosulfide (NaHS) and inorganic sodium polysulfides (Na 2 S n ) such as Na 2 S 2 , Na 2 S 3 , and Na 2 S 4 . Thus, in aqueous solutions, delivery of H 2 S by these polysulfides depends on medium pH and the corresponding pKa [55]. In plant research, the cheaper NaHS is exogenously added to hydroponic solutions and in vitro growth media or is sprayed directly on plants. NaHS, which is a short-lived donor and does not mimic the slow continuous process of H 2 S generation in vivo, is used in a wide range of concentrations. The chemical dialkyldithiophosphate, which is capable of slowly releasing H 2 S [56], has recently been demonstrated to increase corn plant weight by up to 39% after 4.5 weeks of treatment. Other compounds, which are capable of releasing NO combined with H 2 S, are being used in antiinflammatory pharmaceutical treatments [57].

H 2 S and abiotic stress
Many adverse external conditions are well known to negatively affect plant growth, development and productivity [58]. To palliate these effects, plants have developed various strategies which differ according to the type of stress and plant species involved. In many cases, these stresses are associated with unregulated overproduction of reactivate oxygen and nitrogen species (ROS/RNS) which can trigger nitro-oxidative stress [59] characterized by an increase in key parameters such as lipid peroxidation, protein tyrosine nitration and oxidative damage to proteins and nucleic acids. Table 2 shows different examples of the beneficial effects of the exogenous application of H 2 S through the use of different donors on a wide range of agronomically important plants affected by stresses such as heavy metals (cadmium, aluminum, chromium, copper, iron, zinc), metalloids (arsenic), salinity, drought, as well as high and low temperatures . Apart from certain specific responses, in most cases, the application of exogenous H 2 S appears to cause an increase in the different components of antioxidant systems, such as catalase, superoxide dismutase (SOD) isozymes, as well as enzymatic and non-enzymatic components of the ascorbate-glutathione cycle, which enables H 2 O 2 levels and lipid peroxidation content to be reduced.

H 2 S in fruit ripening and post-harvest damage to fresh produce
Information available on endogenous H 2 S metabolism in fruits and vegetables is highly limited. Recently, endogenous H 2 S content in non-climacteric sweet pepper (Capsicum annumm L) fruits was reported to increase during the transition from green immature to red ripe [15]. However, the number of studies focusing on the economic impact of biotechnological applications of H 2 S on fruit ripening and post-harvest storage, which prevent the loss of fresh produce caused by fungi, bacteria, viruses and low temperatures used to store fruits and vegetables, has increased over the last ten years. Given that all these factors are usually associated with oxidative stress, many studies have shown that the exogenous application of H 2 S could have a beneficial effect on the shelf life of a diverse range of fruits, vegetables and flowers [14,16,38,85,86]. Table 3 provides representative examples of the exogenous application of H 2 S to fruits and vegetables [87][88][89][90][91][92][93][94] which enables their quality to be maintained. Another common effect observed following exogenous treatment with H 2 S is an increase in antioxidant systems which prevent ROS overproduction and consequently oxidative damage.

Implication of H 2 S in rhizobium-legume symbiosis
In agriculture and natural ecosystems, a major source of nitrogen-fixation is throughout the nodule formation during the plant-rhizobia interaction [95]. As happened with the NO that was seen to be involved in the interaction rhizobium-legume symbiosis [96][97][98], H 2 S seems to be also involved in different ways in this process. A recent report indicates that exogenous H 2 S promotes plant growth, nodulation and nitrogenase activity in the functional symbiosis between rhizobium (Sinorhizobium fredii) and soybean (Glycine max) plants [99]. Furthermore, the synergy between H 2 S and rhizobia allowed the increase of soybean nitrogen contents by the regulation of related enzymes at different levels (activity, protein, and gene expression) as well as senescence-associated genes which were also regulated [100]. Moreover, new data obtained during the Mesorhizobium-Lotus Promote ABA signaling. Promote ABA-induced stomatal closure [12] Respiratory burst oxidase homolog protein D (RBOHD) Generation of superoxide radical Activity up-regulated [13] symbiosis indicate that this interaction is regulated by the crosstalk among H 2 S with other signaling molecules including NO and ROS [101].
Conclusions and future perspectives H 2 S, which is part of the plant sulfur metabolism, is a new signal molecule whose regulatory function acts through redox interactions, especially the protein post-translational modification persulfidation. The application of exogenous H 2 S, involving a signaling mechanism, causes an increase in different components of the antioxidant system at both the gene and protein level. Nevertheless, the precise biochemical and molecular mechanisms involved in these processes need to be further investigated in future research. However, the exogenous application of H 2 S undoubtedly has a beneficial effect on different plant species, especially those of considerable agronomic interest under adverse environmental conditions. Therefore, the use of H 2 S alone or combined with other molecules, such as nitric oxide, melatonin, thiourea, silicon, chitosan and calcium, which appear to beneficially affect crop plants, needs to be explored in light of climate change [102][103][104][105][106][107][108]. Thus, additional research is necessary in order to decipher the unknowns of H 2 S and its interaction with the metabolism of ROS and RNS under physiological and stressful conditions [109], as well as to establish biotechnological strategies to combat these stresses, Table 2 Main effects of the exogenous application of H 2 S to plants exposed to diverse environmental stresses. ABA, abscisic acid. APX, ascorbate peroxidase. AsA, ascorbate. CAT, catalase. GR, glutathione reductase. GSH, reduced glutathione. GSNOR, S-nitrosoglutathione reductase. HT, high temperature. MDA, malondialdehyde. POD, peroxidase. NaHS, sodium hydrosulfide. PIP, plasma membrane intrinsic proteins. PM, plama membrane. SOD, superoxide dismutase. Increases GSNOR activity and reduce HT-induced damage to the photosynthetic system [83] NaHS (100) or GYY4137 (10)

Arabidopsis thaliana
Enhances seed germination rate under HT.Increases gene expression of ABI5 (ABA-INSENSITIVE 5). [84] which are responsible for major losses in plant yield and crop productivity.

Compliance with Ethics requirements
This article does not contain any studies with human or animal subjects.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 1 mM NaHS Alleviates postharvest senescence of grape and maintain high fruit quality [89] Banana (Musa acuminata, AAA group) 1 mM NaHS Alleviates fruit softening. Antagonizes ethylene effects [14] Tomato (Solanum lycopersicum L.) 'Micro Tom' 0.9 mM NaHS Postpones ripening and senescence of postharvest tomato fruits by antagonizing the effects of ethylene [90] Hawthorn (Crataegus oxyacantha) fruit 1.5 mM NaHS Confers tolerance to chilling. Triggers H 2 S accumulation, increase antioxidant enzyme activities of and promote phenolics accumulation [91] Avocado (Persea americana Mill, cv. 'Hass') 200 mMNaHS Protects against frost and day high light [92] Kiwifruit (Actinidia chinensis) 2 0 mM H 2 S Delays ripening and senescence. Inhibits ethylene production. Increases antioxidant activities. Regulates the cell wall degrading enzyme gene [42] Daylily (Hemerocallis fulva) 4 mMNaHS Delays senescence of postharvest daylily flowers. Increases antioxidant capacity to maintain the redox balance [93] Tomato (Solanum lycopersicum L.). 1 M NaHS Inhibits ethylene-induced petiole abscission [94]