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Hydrogen sulfide and signaling in plants John T. Hancock1,4, Miroslav Lisjak2, Tihana Teklic2, Ian D. Wilson1 and Matthew Whiteman3 1 Faculty of Health and Life Sciences, University of the West of England, Bristol, UK 2 Department of Agroecology, Faculty of agriculture, University of Josip Juraj Strossmayer, Osijek, Croatia 3 Peninsula Medical School, University of Exeter, Exeter, UK 4: to whom correspondence should be addressed: Faculty of Health and Life Sciences University of the West of England Coldharbour Lane Bristol BS16 1QY, UK [email protected] Key words: plants; hydrogen sulfide; signaling; nitric oxide; GYY4137; NaSH. 1 Abstract Several relatively reactive compounds exist which are considered to play major roles in plant cell signaling. These include reactive oxygen species such as hydrogen peroxide and nitric oxide (NO). Until recently hydrogen sulfide (H2S) has commonly been thought of as a phytotoxin, but a growing body of evidence now points to the fact that H2S may also have a signaling role and that it should be ranked as an important signal alongside NO and ROS. At high concentrations H2S will inhibit enzymes such as cytochrome oxidase. However, at lower concentrations it may act in a more positive manner. A renewed interest in the role of H2S in biological systems has been evidenced in the results of research investigating cell signaling in both animals and plants. The growth and development of plants may be affected, for example, during the promotion of adventitious root formation. Stomatal closure has also been shown to be altered by H2S and it has been reported to be involved in the tolerance of plants to metals such as aluminium and copper. The treatment of plant cells with H2S affects cysteine and glutathione metabolism and there is a growing body of evidence to suggest that the presence of H2S may impact on oxidative stress metabolism and nitric oxide signaling. New H2S donor molecules are appearing in the literature, such as GYY4137, and with such new tools the true extent of the role of H2S in the control of plant signalling will no doubt be unravelled in the future. 2 Review Methodology This review was written after searches for papers in the following databases: Pubmed., Science Direct, Google Scholar, Cab Abstracts using the keyword searches with both UK English and American English Spelling, such as hydrogen sulphide & hydrogen sulfide, signalling & signaling. In addition we used the references from within the articles obtained to check for additional relevant material. Introduction It is now well established that many reactive chemicals are involved in the control of cellular events both in animals and in plants. Such chemicals include reactive oxygen species (ROS) and reactive nitrogen species (RNS). Among these the ROS most studied are hydrogen peroxide and superoxide anions while the RNS investigated include nitric oxide (NO) and peroxynitrite [1, 2]. However, although considered as a signal in animals [3, 4] hydrogen sulfide (H2S) should also to be counted in this group of small, sometimes gaseous compounds which are used by plants to control their physiological and biochemical activities. In animals H2S has been dubbed the “third gasotransmitter” [5], the others being NO and carbon monoxide (CO) and several research groups are now focussing on H2S and its role as a signal in plants. 3 However, for molecules to be truly counted as signaling components there are several criteria which must be adhered to and as such ROS and RNS have been judged. Signaling molecules need to be produced when required and to move to a site of action where they must be perceived and subsequently induce cellular responses. Such compounds also need to be readily removed when no longer required and are often seen to interact with a variety of other signal molecules so that they readily partake in signaling cross-talk. Thus, for hydrogen sulfide to be truly counted as a reactive species signal similar judgements need to be made. This article will review the generation and perception of and responses of plants to H2S and so consider whether it can be classed as a plant cell signaling molecule. The generation of hydrogen sulfide H2S is a colorless, flammable gas and therefore might not be considered to be ideal as a signaling molecule. Certainly, these properties make it difficult to study. That said, in biological systems H2S can be measured, one approach being based on the formation of methylene blue from sulfide and N,N-dimethyl-p-phenylenediamine in the presence of Fe3+ and its spectrophotometric detection at 675 nm [6]. This and similar assays can be applied to detect H2S in plants and their environments. Plants may be externally exposed to H2S either atmospherically or by its presence in the rhizosphere. Certainly, anoxic soil [7], such as that found in marshlands, can generate H2S and as such the roots of plants in these areas will be so exposed. Other sulfurous compounds will also be present in such soils. SO42- reducing bacteria such as Desulfovibriao, found in waterlogged 4 soils and marshes, can generate sulfite [8] and sulfite levels in marine soils have been reported to be higher than 1mM [9]. The aerial parts of plants may often be exposed to atmospheric H2S. H2S is commonly emitted from many sources such as waste treatment installations [10], agricultural industries [11] and from geothermal power plants [12]. It has also been found to be at surprisingly high concentrations in some urban environments with car catalytic converters being suggested as a potential source [13]. However, the fact that plants respond to such exogenous sources of H2S does not necessarily indicate that it has a signaling role. Often, in such contexts, the responses of the plants are those associated with the aberrant biochemistry of an organism exposed to toxic levels of this compound. After all, it is for its phytotoxic effects at high levels that H2S has become well known [14, 15]. To truly be a cell signaling molecule, H2S must be generated by plants and indeed endogenous production of H2S can be observed. Using a sulfurspecific flame photometric detector, Wilson et al. [16] showed that, among other plants, cucumber, squash, pumpkin, soybean and cotton were able to produce volatile sulfur compounds including H2S. Rates of emission were variable but were up to 10 nmol min-1 for leaves of about 50 cm2. This ability was described as being light-dependent and when roots were supplied with sulfate and the plants illuminated the emissions lasted for several hours. Furthermore, if either the leaves were fed sulfate through their petioles or if the roots of the plants were mechanically damaged the rate of the H2S emission was significantly increased. Sekiya et al. [17] also measured H2S emissions from cucumber (Cucumis sativus) leaf discs given sulfate in the light, emitting at a rate of 50–100 pmol min-1 cm-2. Again, using cucumber they 5 also reported that young leaves emit much more H2S than mature leaves [18]. The release of H2S from plants has also been confirmed by Rennenberg [19] who found that pumpkin leaves emitted H2S if supplied with sulfate, sulfite, cysteine or SO2. Different metabolic pathways were described to account for the use of the different sulfur sources to produce the H2S in each case. In animal cells the production of H2S results from the action of two enzymes involved in the metabolism of cysteine, cystathionine gamma-lyase and cystathionine beta synthase [5, 20]. In plants it appears that the enzymes responsible are desulfhydrases. A plastid located cysteine desulfhydrases has been reported in Arabidopsis [21] while others report the presence of a similar enzyme in the mitochondria [22]. The levels of such enzymes are not static and their activity has been shown to increase after, for example, pathogen challenge [23]. Therefore, there appear to be inducible and regulated enzymes capable of making H2S and thus, a mechanism for its generation when required, one criterion that must be met if it is to be considered as a cellular signal. Of course H2S acting as a signal may not come from the plant per se. Animals and bacteria have been shown to generate H2S. For example microorganisms which are invading plants, such as pathogenic bacteria, may be able to release H2S [24] which will then affect the activities of the plant. Bacterial release of H2S having profound effects on animals has been reported, such as the promotion of IL-8 production from epithelial cells [25] and therefore such interactions would not be unlikely in plants too. 6 The effects of hydrogen sulfide on plants Hydrogen sulfide is a reactive and toxic compound and in animals is well known to be lethal in high doses, causing inhibition of cytochrome oxidase in the mitochondria [for example 26]. It was found that cytochrome oxidase in olfactory epithelium was decreased with H2S at a concentration of 30 ppm or above. It is, therefore, not surprising that over a number of years it has been established that hydrogen sulfide (H2S) may also have similar effects on plants and that it has generally been thought to be a phytotoxin. For example, 35 years ago it was described as inhibiting oxygen release from various cultivars of young rice seedlings [27]. H2S was used at concentrations of 0.2 to 10 μg mL-1. It was also noted that in some cultivars of rice nutrient uptake was also reduced, while in other cultivars it was increased. Phosphorous uptake was also inhibited in this plant species. Thompson and Kats [28] continuously fumigated various species of plants with H2S. In Medicago, grape, lettuce, sugar beet, pine and fir 3000 parts per billion (ppb) H2S caused lesions on the leaves, defoliation and reduced growth of the plants. However, H2S treatment has not always been shown to be deleterious. Interestingly, lower levels of fumigation, 100 ppb, caused a significant increase in the growth of Medicago, lettuce and sugar beet [28]. While working with beet it was also noted that there was less fungal attack following H2S treatment, suggesting that the H2S may inhibit the growth of the fungi. However, H2S was observed to reduce the sugar content of the roots of the beet plants. An increase in either the tolerance to or the protection against some plant stresses has also been found to be mediated by H2S. In a similar 7 manner to the above [19], Hällgren and Fredriksson [29] showed that when pine (Pinus silvestris L.) needles were subjected to low concentrations of SO2 emissions of H2S could be measured that resulted in an increase in the tolerance of the plants to the SO2. The emissions were light dependent, lasted for a considerable time after the SO2 was removed and it was suggested that sulfur metabolism in the chloroplasts was responsible. A similar example of H2S-induced SO2 tolerance was seen with young Cucurbitaceae leaves [17]. Takemoto et al. [30] detected increased emission of H2S and thiol accumulation in duckweed (Lemnaceae) under high irradiance and hypothesized that this was important for sulfite tolerance. Much of the work on H2S and its effect on plants was carried out many years ago. However, more recently there has been a renewed interest. In 2004 Bloem et al. [23] showed that fungal infection, particularly with Pyrenopeziza brassicae caused an increase in the activity of an enzyme capable of generating H2S, and thus, a greater potential for H2S release from the plant being infected. More recently Zhang et al. [31] showed that the H2S donor NaSH could alleviate the osmotic-induced decrease in chlorophyll concentration in sweet potato. Furthermore, spraying the plants with NaSH induced increases in the activities of the antioxidant enzymes superoxide dismutase, catalase and ascorbate peroxidase and decreases in the concentration of hydrogen peroxide and the activity of lipoxygenase, suggesting that H2S has a role in protecting plants against oxidative stress. Other plant responses linked to H2S include freezing tolerance [32], a process which also has links to changes in oxidative stress metabolism. Similar H2S mediated stress tolerance has also been reported in animals [20]. 8 Aluminium is known to inhibit seed germination and pre-treatment with NaSH has been shown to alleviate this in a concentration-dependent manner with an optimum at 0.3 nmol L-1 [33]. Following NaSH treatment, endogenous H2S was seen to increase and again the levels of enzymes involved in oxidative stress were altered. There was a decrease in the activity of lipoxygenase, but an increase in the activities of catalase, superoxide dismutase, ascorbate peroxidase and guaiacol peroxidase. Clearly, there appears to be a link between the presence of H2S and the oxidative stress responses of plant cells and similarly. NaSH has also been shown to alleviate the copper inhibition of germination in wheat [34]. In this case, NaSH caused an increase in superoxide dismutase and catalase activities, decreased lipoxygenase activity, left the activity of ascorbate peroxidase unchanged, increased esterase and amylase activities and caused a reduction in hydrogen peroxide and malondialdehyde levels. Further work in which wheat seeds were pre-treated with NaSH for 12 h showed that H2S preferentially affected the activity of endosperm β-amylase and that the synthesis and activity of α-amylase remained unaffected [35]. However, overall such studies highlight the probable interaction of H2S and ROS metabolism. Intracellular responses to hydrogen sulfide It has been suggested that H2S is a signaling molecule in animals [3-5, 36-38] and that it is likely, therefore, that the same is true for plants [34, 35]. However, to be a signal H2S needs to be perceived by plant cells and there needs to be a response. 9 Intracellular responses to H2S have been studied and it has been found that there are a range of effects if plant cells are so treated. For example, on the fumigation of spinach with H2S (250 ppb: 380 g m-3) it was found that glutathione levels increased [39], which fits well with the notion put forward by Zhang et al. [31, 33] that H2S may impinge on oxidative stress responses. It was estimated that approximately 40% of the H2S was converted to glutathione in the leaves. On cessation of the fumigation glutathione levels once again fell, with the levels being comparable to control levels after 48 h of no H2S treatment. Changes in glutathione will have profound effects on the intracellular redox poise of the cells. The redox environment of proteins in cells is crucially important for the correct functioning of many enzymes and was discussed eloquently by Buettner and Schafer [40], and especially many proteins that are involved in signalling [41]. It has been remarked that H2S may indeed modulate intracellular redox status as H2S in an aqueous solution is a weak reducing agent [42]. It was also pointed out that there was much to investigate in this area too and that the exact impact of H2S requires further investigation. However, at the same time that glutathione was being altered photosynthetic carbon fixation and photosynthetic electron transport were reported to be insensitive to the presence of H2S [37]. Fumigation of poplar (Populus tremula X Populus alba) cuttings with H2S showed that a significant amount of the H2S was incorporated into organic sulfur compounds. When H2S was taken up by the leaves they had increased levels of cysteine as well as increased levels of -glutamylcysteine synthetase. As in the spinach, 10 glutathione was also increased in poplar leaves, roots, xylem sap and phloem exudate [43]. In another study adenosine 5’-phosphosulfate reductase (APR) was found to be highly inhibited by the short term exposure of shoots of Brassica oleracea L. (curly kale) to atmospheric H2S at 0.2-0.8 uL L-1 [44], although roots of these plants were not affected. Other enzymes assayed were ATPsulfurylase (ATPS), serine acetyltransferase (SAT) and Oacetylserine(thiol)lyase (OAS-TL), but none of these appeared to be affected by the H2S treatment. However, in the shoots of these plants the thiol content increased. This included increases in the level of cysteine, with thiols increasing 3 fold after 5 days exposure to H2S. Thiols were also a focus of a study by Riemenschneider et al. [45]. Using the model plant Arabidopsis exposures of up to 48 h to atmospheric H2S caused significant increases in both cysteine and glutathione levels. Further work in this paper reported on the levels of encoding messenger RNAs and levels and activities of enzymes involved in cysteine metabolism. The activity of 3-mercaptopyruvate sulfurtransferase was only slightly higher after the longest exposure to H2S while the activities of other enzymes which could either remove H2S, O-acetyl-l-serine(thiol)lyase, or generate H2S, Lcysteine desulfhydrase, were not significantly affected by the H2S treatment. Low levels of H2S (up to 0.5 microL L-1) induced an increase in the levels of the encoding messenger RNAs for and higher protein levels of such enzymes, but high concentrations (0.75 microL L-1) of H2S were seen to have the opposite effect. 11 Alcohol dehydrogenase activity in plants cells was found to be inhibited by H2S [14] in a study which used from 0.5 mM up to 4 mM. This was accompanied by a decrease in the levels of total adenine nucleotides in the root and also decreased nitrogen uptake and leaf growth. Alcohol dehydrogenase is found to be an enzyme which is sensitive to both ROS [46] and NO [47], again suggesting an interplay between H2S, ROS and NO metabolism. H2S was found to promote adventitious roots formation. The H2S donor NaSH caused an increase in endogenous H2S, NO and indole acetic acid (IAA) in shoot tips of sweet potato seedlings suggesting that H2S is acting upstream of both IAA and NO in signalling pathways [48]. Although the transpiration rates of several species of plants including maize, pumpkins and spinach were unaffected by short-term exposure to atmospheric H2S, recent work [49] found that H2S caused stomatal opening in Arabidopsis [50]. Using Arabidopsis thaliana as a model system, H2Smediated opening was seen in plants treated with either NaSH or with the H2S donor, GYY4137 [51-53]. If leaves were not pre-opened in the light, the effects of both NaSH and GYY4137 were seen to be more pronounced. Furthermore, when endogenous nitric oxide was measured using a NO sensitive probe (DAF2-DA: [54]) in conjunction with confocal microscopy, it appeared that NO levels were lower in guard cells after treatment with either NaSH or GYY4137, suggesting that H2S interferers with NO signalling, perhaps through a scavenging role. However, this is not consistent with the findings of Zhang et al. [48] who found that a H2S donor increased NO accumulation in roots and suggested that H2S was upstream of NO in a 12 signaling pathway. Furthermore, Lamattina and Garcia-Mata [55] found that both H2S donors NaSH and GY4137 caused stomatal closure in Vicia faba, Arabidopsis thaliana and Impatiens walleriana. The ABC transport inhibitor glibenclamide impaired the effect, as did the inhibitor propargylglycine which effects cystathione lyase and L-Cys desulfhydrase, enzymes which may be responsible for H2S synthesis. They suggested that H2S partakes in the ABA signaling pathway in guard cells. Therefore, there is conflicting evidence as to the effect of H2S on guard cells. However, such contrary evidence has been seen with other signals in stomatal regulation. Tanaka et al. [56] reported that ethylene inhibited ABA-induced stomatal closure and some considered that ethylene mediated auxin-induced opening of stomata [57], but others subsequently showed that ethylene caused stomatal closure [58]. Many environmental factors clearly impinge on the exact responses recorded in such experiments and future work performed under standardised conditions will no doubt reveal how compounds like H2S are truly acting. The suggestion that there is an interaction between H2S and NO is not new [20]. It has been shown in animal systems that H2S inhibits nitric oxide synthase (NOS) isoforms, probably through an interaction between H2S and the co-factor tetrahydrobiopterin (BH4) [59]. However, there is considerable debate about the presence of NOS-like enzymes in plants [see 60] so this is unlikely to be the mode of action for H2S here. Alternatively, it has been suggested that NO and H2S react together to form novel nitrosothiols/nitrothiol-like species which themselves may have cellular effects [61]. 13 However a note of caution should be added here. Several studies [for example 50 and 55] used H2S donors such as NaSH and GY4137. Concentrations of these are given in papers but the exact release of H2S into solution and to which the cells are exposed is not known or indeed measured. Therefore such studies are hard to compare to others when the role of H2S as a signal or simply as a phytotoxin are being discussed. If H2S is indeed acting as a signal, then it would need to be removed when no longer required. Tobacco plants transformed with a gene for Oacetylserine lyase were more resistant to H2S treatment, suggesting that this enzyme was used in its removal and detoxification [62, 63]. Conclusion For hydrogen sulfide to be considered a signalling molecule in plants it needs to have a way of being generated as and when needed, to be perceived and to elicit a response. It has been shown that desulfhydrases are able to make H2S, that H2S can be measured both intracellularly and extracellularly and that many physiological and biochemical responses occur as a result of the presence of H2S. It appears to be implicated in the transduction pathways of several other cellular signals including ABA, auxin and NO. 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