SOD1 is an essential H2S detoxifying enzyme.

Although hydrogen sulfide (H2S) is an endogenous signaling molecule with antioxidant properties, it is also cytotoxic by potently inhibiting cytochrome c oxidase and mitochondrial respiration. Paradoxically, the primary route of H2S detoxification is thought to occur inside the mitochondrial matrix via a series of relatively slow enzymatic reactions that are unlikely to compete with its rapid inhibition of cytochrome c oxidase. Therefore, alternative or complementary cellular mechanisms of H2S detoxification are predicted to exist. Here, superoxide dismutase [Cu-Zn] (SOD1) is shown to be an efficient H2S oxidase that has an essential role in limiting cytotoxicity from endogenous and exogenous sulfide. Decreased SOD1 expression resulted in increased sensitivity to H2S toxicity in yeast and human cells, while increased SOD1 expression enhanced tolerance to H2S. SOD1 rapidly converted H2S to sulfate under conditions of limiting sulfide; however, when sulfide was in molar excess, SOD1 catalyzed the formation of per- and polysulfides, which induce cellular thiol oxidation. Furthermore, in SOD1-deficient cells, elevated levels of reactive oxygen species catalyzed sulfide oxidation to per- and polysulfides. These data reveal that a fundamental function of SOD1 is to regulate H2S and related reactive sulfur species.

Reactive sulfur species disaggregate the SQSTM1/p62-based aggresome-like induced structures via the HSP70 induction and prevent parthanatos.

Reactive sulfur species (RSS) have emerged as key regulators of protein quality control. However, the mechanisms by which RSS contribute to cellular processes are not fully understood. In this study, we identified a novel function of RSS in preventing parthanatos, a nonapoptotic form of cell death that is induced by poly (ADP-ribose) polymerase-1 and mediated by the aggresome-like induced structures (ALIS) composed of SQSTM1/p62. We found that sodium tetrasulfide (Na2S4), a donor of RSS, strongly suppressed oxidative stress-dependent ALIS formation and subsequent parthanatos. On the other hand, the inhibitors of the RSS-producing enzymes, such as 3-mercaptopyruvate sulfurtransferase and cystathionine γ-lyase, clearly enhanced ALIS formation and parthanatos. Interestingly, we found that Na2S4 activated heat shock factor 1 by promoting its dissociation from heat shock protein 90, leading to accelerated transcription of HSP70. Considering that the genetic deletion of HSP70 allowed the enhanced ALIS formation, these findings suggest that RSS prevent parthanatos by specifically suppressing ALIS formation through induction of HSP70. Taken together, our results demonstrate a novel mechanism by which RSS prevent cell death, as well as a novel physiological role of RSS in contributing to protein quality control through HSP70 induction, which may lead to better understanding of the bioactivity of RSS.

Analysis and characterization of sulfane sulfur.

In the late 1970s, sulfane sulfur was defined as sulfur atoms covalently bound only to sulfur atoms. However, this definition was not generally accepted, as it was slightly vague and difficult to comprehend. Thus, in the early 1990s, it was defined as "bound sulfur," which easily converts to hydrogen sulfide upon reduction with a thiol-reducing agent. H2S-related bound sulfur species include persulfides (R-SSH), polysulfides (H2Sn, n ≥ 2 or R-S(S)nS-R, n ≥ 1), and protein-bound elemental sulfur (S0). Many of the biological effects currently associated with H2S may be attributed to persulfides and polysulfides. In the 20th century, quantitative determination of "sulfane sulfur" was conventionally performed using a reaction called cyanolysis. Several methods have been developed over the past 30 years. Current methods used for the detection of H2S and polysulfides include colorimetric assays for methylene blue formation, sulfide ion-selective or polarographic electrodes, gas chromatography with flame photometric or sulfur chemiluminescence detection, high-performance liquid chromatography analysis with fluorescent derivatization of sulfides, liquid chromatography with tandem mass spectrometry, the biotin switch technique, and the use of sulfide or polysulfide-sensitive fluorescent probes. In this review, we discuss the methods reported to date for measuring sulfane sulfur and the results obtained using these methods.

Sodium thiosulfate: A donor or carrier signaling molecule for hydrogen sulfide?

Sodium thiosulfate has been used for decades in the treatment of calciphylaxis and cyanide detoxification, and has recently shown initial therapeutic promise in critical diseases such as neuronal ischemia, diabetes mellitus, heart failure and acute lung injury. However, the precise mechanism of sodium thiosulfate remains incompletely defined and sometimes contradictory. Although sodium thiosulfate has been widely accepted as a donor of hydrogen sulfide (H2S), emerging findings suggest that it is the executive signaling molecule for H2S and that its effects may not be dependent on H2S. This article presents an overview of the current understanding of sodium thiosulfate, including its synthesis, biological characteristics, and clinical applications of sodium thiosulfate, as well as the underlying mechanisms in vivo. We also discussed the interplay of sodium thiosulfate and H2S. Our review highlights sodium thiosulfate as a key player in sulfide signaling with the broad clinical potential for the future.

Experimental Insights into the Formation, Reactivity, and Crosstalk of Thionitrite (SNO-) and Perthionitrite (SSNO-).

Hydrogen sulfide (H2S) and nitric oxide (NO) are important gaseous biological signaling molecules that are involved in complex cellular pathways. A number of physiological processes require both H2S and NO, which has led to the proposal that different H2S/NO• crosstalk species, including thionitrite (SNO-) and perthionitrite (SSNO-), are responsible for this observed codependence. Despite the importance of these S/N hybrid species, the reported properties and characterization, as well as the fundamental pathways of formation and subsequent reactivity, remain poorly understood. Herein we report new experimental insights into the fundamental reaction chemistry of pathways to form SNO- and SSNO-, including mechanisms for proton-mediated interconversion. In addition, we demonstrate new modes of reactivity with other sulfur-containing potential crosstalk species, including carbonyl sulfide (COS).

An Expanded Palette of Fluorescent COS/H2S-Releasing Donors for H2S Delivery, Detection, and In Vivo Application.

Hydrogen sulfide (H2S) is an important reactive sulfur species that is involved in many biological functions, and H2S imbalances have been indicated as a potential biomarker for various diseases. Different H2S donors have been developed to deliver H2S directly to biological systems, but few reports include donors with optical responses that allow for tracking of H2S release. Moreover, donor systems that use the same chemistry to deliver H2S across a palette of fluorescent responses remain lacking. Here we report five thiol-activated fluorescence turn-on COS/H2S donors that utilize blue, yellow, orange, red, and near infrared-emitting dyes functionalized with an H2S-releasing sulfenyl thiocarbonate scaffold. Upon treatment with thiols, each donor provides a fluorescence turn-on response (3-310-fold) and high H2S release efficiencies (>60 %). Using combined electrode and fluorescence experiments, we directly correlate the measured H2S release with the fluorescence response. All donors are biocompatible and release H2S in live cell environments. In addition, we demonstrate that the NIR donor allows for imaging H2S release in live rats via subcutaneous injection of the donor loaded into an alginate gel, which to the best of our knowledge is the first in vivo tracking of H2S release from a fluorogenic donor in non-transparent organisms.

Regulation of the redox metabolome and thiol proteome by hydrogen sulfide.

Overproduction of reactive oxygen species and compromised antioxidant defenses perturb intracellular redox homeostasis and is associated with a myriad of human diseases as well as with the natural process of aging. Hydrogen sulfide (H2S), which is biosynthesized by organisms ranging from bacteria to man, influences a broad range of physiological functions. A highly touted molecular mechanism by which H2S exerts its cellular effects is via post-translational modification of the thiol redox proteome, converting cysteine thiols to persulfides, in a process referred to as protein persulfidation. The physiological relevance of this modification in the context of specific signal transmission pathways remains to be rigorously established, while a general protective role for protein persulfidation against hyper-oxidation of the cysteine proteome is better supported. A second mechanism by which H2S modulates redox homeostasis is via remodeling the redox metabolome, targeting the electron transfer chain and perturbing the major redox nodes i.e. CoQ/CoQH2, NAD+/NADH and FAD/FADH2. The metabolic changes that result from H2S-induced redox changes fan out from the mitochondrion to other compartments. In this review, we discuss recent developments in elucidating the roles of H2S and its oxidation products on redox homeostasis and its role in protecting the thiol proteome.

Sulfur-Containing Persistent Free Radicals and Reactive Species on Photoaged Microplastics: Identification and the Formation Mechanism.

The elemental composition may affect the persistent free radical (PFR) and reactive species (RS) formation associated with photoaging microplastics; however, a relevant study is still lacking. This study systematically investigated the formation, evolution, and types of PFRs and RS on sulfur-containing microplastics (S-MPs) under simulated sunlight. Electron paramagnetic resonance detection and power saturation curve analysis isolated three different PFRs on each photoaging poly(phenylene sulfide) (PPS) and polysulfone (PSF). Combining the results of characterization and density functional theory calculation, these observed PFRs on the irradiated S-MPs were classified as oxygen-centered radicals with an adjacent S atom (namely, thio-oxygen radicals), oxygen-centered and sulfur-centered radicals, where the thio-oxygen radicals on PPS were benzenethiol-like radicals, and oxygen-centered radicals and sulfur-centered radicals on PSF that were identified as benzenesulfonic-like radicals and phenyl sulfonyl-like radicals, respectively. Moreover, potential precursor molecule fragments of PFRs on the photoaging S-MPs, including p-toluenesulfinic acid and benzenesulfonic acid, were detected by pyrolysis-gas chromatography/mass spectrometry and liquid chromatography-mass spectrometry. Interestingly, reactive sulfur species (SO3•-) was also observed on irradiated S-MPs in addition to reactive oxygen species, which was mainly derived from the reaction of •OH and sulfonyl radicals. These results have implications for assessing the potential risks of atmospheric S-MPs.

Echinochrome Prevents Sulfide Catabolism-Associated Chronic Heart Failure after Myocardial Infarction in Mice.

Abnormal sulfide catabolism, especially the accumulation of hydrogen sulfide (H2S) during hypoxic or inflammatory stresses, is a major cause of redox imbalance-associated cardiac dysfunction. Polyhydroxynaphtoquinone echinochrome A (Ech-A), a natural pigment of marine origin found in the shells and needles of many species of sea urchins, is a potent antioxidant and inhibits acute myocardial ferroptosis after ischemia/reperfusion, but the chronic effect of Ech-A on heart failure is unknown. Reactive sulfur species (RSS), which include catenated sulfur atoms, have been revealed as true biomolecules with high redox reactivity required for intracellular energy metabolism and signal transduction. Here, we report that continuous intraperitoneal administration of Ech-A (2.0 mg/kg/day) prevents RSS catabolism-associated chronic heart failure after myocardial infarction (MI) in mice. Ech-A prevented left ventricular (LV) systolic dysfunction and structural remodeling after MI. Fluorescence imaging revealed that intracellular RSS level was reduced after MI, while H2S/HS- level was increased in LV myocardium, which was attenuated by Ech-A. This result indicates that Ech-A suppresses RSS catabolism to H2S/HS- in LV myocardium after MI. In addition, Ech-A reduced oxidative stress formation by MI. Ech-A suppressed RSS catabolism caused by hypoxia in neonatal rat cardiomyocytes and human iPS cell-derived cardiomyocytes. Ech-A also suppressed RSS catabolism caused by lipopolysaccharide stimulation in macrophages. Thus, Ech-A has the potential to improve chronic heart failure after MI, in part by preventing sulfide catabolism.

Redox and Nucleophilic Reactions of Naphthoquinones with Small Thiols and Their Effects on Oxidization of H2S to Inorganic and Organic Hydropolysulfides and Thiosulfate.

Naphthoquinone (1,4-NQ) and its derivatives (NQs, juglone, plumbagin, 2-methoxy-1,4-NQ, and menadione) have a variety of therapeutic applications, many of which are attributed to redox cycling and the production of reactive oxygen species (ROS). We previously demonstrated that NQs also oxidize hydrogen sulfide (H2S) to reactive sulfur species (RSS), potentially conveying identical benefits. Here we use RSS-specific fluorophores, mass spectroscopy, EPR and UV-Vis spectrometry, and oxygen-sensitive optodes to examine the effects of thiols and thiol-NQ adducts on H2S-NQ reactions. In the presence of glutathione (GSH) and cysteine (Cys), 1,4-NQ oxidizes H2S to both inorganic and organic hydroper-/hydropolysulfides (R2Sn, R=H, Cys, GSH; n = 2-4) and organic sulfoxides (GSnOH, n = 1, 2). These reactions reduce NQs and consume oxygen via a semiquinone intermediate. NQs are also reduced as they form adducts with GSH, Cys, protein thiols, and amines. Thiol, but not amine, adducts may increase or decrease H2S oxidation in reactions that are both NQ- and thiol-specific. Amine adducts also inhibit the formation of thiol adducts. These results suggest that NQs may react with endogenous thiols, including GSH, Cys, and protein Cys, and that these adducts may affect both thiol reactions as well as RSS production from H2S.

Comparison of Reactive Sites in 2(1H)-Quinolone Derivatives for the Detection of Biologically Important Sulfur Compounds.

Novel fluorescent probes based on 2(1H)-quinolone skeleton containing a malonate group (Q1-Q3) were synthesized and proposed for biothiols detection. Their chemical reactivity toward thiols was compared to the reactivity of derivative having a dicyanovinyl group (Q4) as a reactive site. The detailed photophysical properties of these compounds were assessed through the determination of absorption and fluorescence spectra, fluorescence quantum yield, and fluorescence lifetime. In the presence of biothiols, an increase in the fluorescence intensity of compounds Q1-Q3 and a hypsochromic shift in their emission bands were observed. In contrast, the compound with the dicyanovinyl group (Q4) in the presence of biothiols and cyanide ion showed the quenching of fluorescence, while a fluorescence "turn on" effect was observed toward reactive sulfur species.

Synergistic Activation of Nitrite and Thiocarbonyl Compounds Affords NO and Sulfane Sulfur via (Per)thionitrite (SNO- /SSNO- ).

(Per)thionitrite (SNO- /SSNO- ) intermediates play vital roles in modulating nitric oxide (NO) and hydrogen sulfide (H2 S) dependent bio-signalling processes. Whilst the previous preparations of such intermediates involved reactive H2 S/HS- or sulfane sulfur (S0 ) species, the present report reveals that relatively stable thiocarbonyl compounds (such as carbon disulfide (CS2 ), thiocarbamate, thioacetic acid, and thioacetate) react with nitrite anion to yield SNO- /SSNO- . For instance, the reaction of CS2 and nitrite anion (NO2 - ) under ambient condition affords CO2 and SNO- /SSNO- . A detailed investigation involving UV/Vis, FTIR, HRMS, and multinuclear NMR studies confirm the formation of SNO- /SSNO- , which are proposed to form through an initial nucleophilic attack by nitrite anion followed by a transnitrosation step. Notably, reactions of CS2 and nitrite in the presence of thiol RSH show the formation of organic polysulfides R-Sn -R, thereby illustrating that the thiocarbonyls are capable of influencing the pool of bioavailable sulfane sulfurs. Furthermore, the availability of both NO2 - and thiocarbonyl motifs in the biological context hints at their synergistic metal-free activations leading to the generation of NO gas and various reactive sulfur species via SNO- /SSNO- .

Signaling by reactive molecules and antioxidants in legume nodules.

Legume nodules are symbiotic structures formed as a result of the interaction with rhizobia. Nodules fix atmospheric nitrogen into ammonia that is assimilated by the plant and this process requires strict metabolic regulation and signaling. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved as signal molecules at all stages of symbiosis, from rhizobial infection to nodule senescence. Also, reactive sulfur species (RSS) are emerging as important signals for an efficient symbiosis. Homeostasis of reactive molecules is mainly accomplished by antioxidant enzymes and metabolites and is essential to allow redox signaling while preventing oxidative damage. Here, we examine the metabolic pathways of reactive molecules and antioxidants with an emphasis on their functions in signaling and protection of symbiosis. In addition to providing an update of recent findings while paying tribute to original studies, we identify several key questions. These include the need of new methodologies to detect and quantify ROS, RNS, and RSS, avoiding potential artifacts due to their short lifetimes and tissue manipulation; the regulation of redox-active proteins by post-translational modification; the production and exchange of reactive molecules in plastids, peroxisomes, nuclei, and bacteroids; and the unknown but expected crosstalk between ROS, RNS, and RSS in nodules.

Peroxynitrite-Promoted Persulfide Prodrugs with Protective Potential against Paracetamol Poisoning.

The newly emerging persulfide prodrugs provide additional options for the profound study of persulfide, a fascinating molecule expected to intervene in biological functions and even diseases. Peroxynitrite is often the culprit in pathological processes characterized by oxidative stress, while the persulfide prodrug responsive to it is still pending. To enrich the family of redox-activated prodrugs, we designed prodrugs with a 2-oxo-2-phenylacetamide trigger, which achieved the release of persulfide via 1, 6-N, S-relay. The degradation of prodrugs and the formation of persulfides were confirmed to be peroxynitrite-responsible by the qualitative and quantitative studies based on LC-MS/MS methods and a spectrophotometry-based tag-switch strategy. Furthermore, these prodrugs showed potent peroxynitrite scavenging activity, cellular therapeutic potential against paracetamol poisoning in HepG2 and oxidative stress in H9c2, as well as desirable in vitro metabolic properties.

Distinct Pharmacological Profiles of Monosulfide and Trisulfide in an Experimental Model of Intracerebral Hemorrhage in Mice.

Hydrogen sulfide and polysulfides are increasingly recognized as bioactive signaling molecules to produce various actions and regulate (patho)physiological processes. Here we examined the effects of sodium sulfide (Na2S) and sodium trisulfide (Na2S3) on an experimental model of intracerebral hemorrhage (ICH) in mice. Na2S or Na2S3 (25 µmol/kg, intraperitoneally (i.p.)) was administered 30 min before ICH induction by intrastriatal injection of collagenase. We found that Na2S significantly ameliorated sensorimotor functions of mice after ICH. Histopathological examinations revealed that Na2S inhibited neuron loss in the striatum, prevented axon degeneration in the internal capsule, and ameliorated axonal transport dysfunction in the striatum and the cerebral cortex where the edge of hematoma was located. Although Na2S did not suppress accumulation of activated microglia/macrophages in the peri-hematoma region, it suppressed ICH-induced upregulation of inflammatory mediators such as C-X-C motif ligand 2. On the other hand, Na2S3 did not ameliorate ICH-induced sensorimotor dysfunction. Although the effect of Na2S3 on several parameters such as axon degeneration and axonal transport dysfunction was comparable to that of Na2S, Na2S3 did not significantly inhibit neuron loss and upregulation of inflammatory mediators. These results suggest that the regulation of multiple pathological events is involved in the effect of Na2S leading to amelioration of neurological symptoms associated with ICH.

Naphthoquinones Oxidize H2S to Polysulfides and Thiosulfate, Implications for Therapeutic Applications.

1,4-Napththoquinones (NQs) are clinically relevant therapeutics that affect cell function through production of reactive oxygen species (ROS) and formation of adducts with regulatory protein thiols. Reactive sulfur species (RSS) are chemically and biologically similar to ROS and here we examine RSS production by NQ oxidation of hydrogen sulfide (H2S) using RSS-specific fluorophores, liquid chromatography-mass spectrometry, UV-Vis absorption spectrometry, oxygen-sensitive optodes, thiosulfate-specific nanoparticles, HPLC-monobromobimane derivatization, and ion chromatographic assays. We show that NQs, catalytically oxidize H2S to per- and polysulfides (H2Sn, n = 2−6), thiosulfate, sulfite and sulfate in reactions that consume oxygen and are accelerated by superoxide dismutase (SOD) and inhibited by catalase. The approximate efficacy of NQs (in decreasing order) is, 1,4-NQ ≈ juglone ≈ plumbagin > 2-methoxy-1,4-NQ ≈ menadione >> phylloquinone ≈ anthraquinone ≈ menaquinone ≈ lawsone. We propose that the most probable reactions are an initial two-electron oxidation of H2S to S0 and reduction of NQ to NQH2. S0 may react with H2S or elongate H2Sn in variety of reactions. Reoxidation of NQH2 likely involves a semiquinone radical (NQ·−) intermediate via several mechanisms involving oxygen and comproportionation to produce NQ and superoxide. Dismutation of the latter forms hydrogen peroxide which then further oxidizes RSS to sulfoxides. These findings provide the chemical background for novel sulfur-based approaches to naphthoquinone-directed therapies.

Thionitrite (SNO- ) and Perthionitrite (SSNO- ) are Simple Synthons for Nitrosylated Iron Sulfur Clusters.

S/N crosstalk species derived from the interconnected reactivity of H2 S and NO facilitate the transport of reactive sulfur and nitrogen species in signaling, transport, and regulatory processes. We report here that simple Fe2+ ions, such as those that are bioavailable in the labile iron pool (LIP), react with thionitrite (SNO- ) and perthionitrite (SSNO- ) to yield the dinitrosyl iron complex [Fe(NO)2 (S5 )]- . In the reaction of FeCl2 with SNO- we were able to isolate the unstable intermediate hydrosulfido mononitrosyl iron complex [FeCl2 (NO)(SH)]- , which was characterized by X-ray crystallography. We also show that [Fe(NO)2 (S5 )]- is a simple synthon for nitrosylated Fe-S clusters via its reduction with PPh3 to yield Roussin's Red Salt ([Fe2 S2 (NO)4 ]2- ), which highlights the role of S/N crosstalk species in the assembly of fundamental Fe-S motifs.

Intramolecular Thiol- and Selenol-Assisted Delivery of Hydrogen Sulfide.

Arylthioamides have been frequently employed to assess the chemical biology and pharmacology of hydrogen sulfide (H2 S). From this class of donors, however, extremely low H2 S releasing efficiencies have been reported and proper mechanistic studies have been omitted. Consequently, millimolar concentrations of arylthioamides are required to liberate just trace amounts of H2 S, and via an unidentified mechanistic pathway, which obfuscates the interpretation of any biological activity that stems from their use. Herein, we report that H2 S release from this valuable class of donors can be markedly enhanced through intramolecular nucleophilic assistance. Specifically, we demonstrate that both disulfide- and diselenide-linked thioamides are responsive to biologically relevant concentrations of glutathione and release two molar equivalents of H2 S via an intramolecular cyclization that significantly augments their rate and efficiency of sulfide delivery in both buffer and live human cells.

H2S and reactive sulfur signaling at the host-bacterial pathogen interface.

Bacterial pathogens that cause invasive disease in the vertebrate host must adapt to host efforts to cripple their viability. Major host insults are reactive oxygen and reactive nitrogen species as well as cellular stress induced by antibiotics. Hydrogen sulfide (H2S) is emerging as an important player in cytoprotection against these stressors, which may well be attributed to downstream more oxidized sulfur species termed reactive sulfur species (RSS). In this review, we summarize recent work that suggests that H2S/RSS impacts bacterial survival in infected cells and animals. We discuss the mechanisms of biogenesis and clearance of RSS in the context of a bacterial H2S/RSS homeostasis model and the bacterial transcriptional regulatory proteins that act as "sensors" of cellular RSS that maintain H2S/RSS homeostasis. In addition, we cover fluorescence imaging- and MS-based approaches used to detect and quantify RSS in bacterial cells. Last, we discuss proteome persulfidation (S-sulfuration) as a potential mediator of H2S/RSS signaling in bacteria in the context of the writer-reader-eraser paradigm, and progress toward ascribing regulatory significance to this widespread post-translational modification.

Adenosine 5' phosphosulfate reductase and sulfite oxidase regulate sulfite-induced water loss in Arabidopsis.

Chloroplast-localized adenosine-5'-phosphosulphate reductase (APR) generates sulfite and plays a pivotal role in reduction of sulfate to cysteine. The peroxisome-localized sulfite oxidase (SO) oxidizes excess sulfite to sulfate. Arabidopsis wild type, SO RNA-interference (SO Ri) and SO overexpression (SO OE) transgenic lines infiltrated with sulfite showed increased water loss in SO Ri plants, and smaller stomatal apertures in SO OE plants compared with wild-type plants. Sulfite application also limited sulfate and abscisic acid-induced stomatal closure in wild type and SO Ri. The increases in APR activity in response to sulfite infiltration into wild type and SO Ri leaves resulted in an increase in endogenous sulfite, indicating that APR has an important role in sulfite-induced increases in stomatal aperture. Sulfite-induced H2O2 generation by NADPH oxidase led to enhanced APR expression and sulfite production. Suppression of APR by inhibiting NADPH oxidase and glutathione reductase2 (GR2), or mutation in APR2 or GR2, resulted in a decrease in sulfite production and stomatal apertures. The importance of APR and SO and the significance of sulfite concentrations in water loss were further demonstrated during rapid, harsh drought stress in root-detached wild-type, gr2 and SO transgenic plants. Our results demonstrate the role of SO in sulfite homeostasis in relation to water consumption in well-watered plants.