Hydrogen Sulfide-induced Barley Resilience to Drought and Salinity through Protein Persulfidation

Plant Physiology and Biochemistry 221 (2025) 109644 Available online 11 February 2025 0981-9428/© 2025 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Hydrogen sulfide-induced barley resilience to drought and salinity through protein persulfidation Reyes Carrillo , Inmaculada Moreno , Luis C. Romero , Angeles Aroca ** , Cecilia Gotor * Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Sevilla, Spain ARTICLE INFO Keywords: Amino acids ABA Abiotic stress Hydrogen sulfide Na+/K+ratio Persulfidation ROS ABSTRACT Barley (Hordeum vulgare) is a widely cultivated cereal crops, and its production is increasingly threatened by environmental stresses such as drought and salinity. Hydrogen sulfide is established as a signaling molecule that promotes tolerance to plant stress throught persulfidation, a post-translational modification of cysteine residues in proteins. The purpose of this study is to explore the impact of NaHS (sulfide donor) pretreatment on barley plants in enhancing tolerance to drought and salinity stresses, and determine if persulfidation is involved. In pretreated-plants, phenotypical traits and pigment contents showed an improvement in the survival of the plants under stress conditions. Quantification of stress-markers such as anthocyanin, proline, and reactive oxygen species also showed significant decreased contents in pretreated compared to untreated plants. In addition, the accumulation of amino acids under drought stress was significantly reduced when plants were pretreated with NaHS. Similarly, the increase of ABA content as a typical drought response was reduced in the pretreated plants. When plants are exposed to salt stress, the Na + /K + ratio was maintained low in NaHS-pretreated plants, by increasing K + levels. The sulfide ameliorative effect to salt was also observed during germination in previously NaHS-soaked seeds. Our findings suggest that sulfide pretreatment prepares barely plants to better deal with drought and salinity. Moreover, persulfidation was analyzed under all conditions, exhibiting enhanced levels under stress when plants were pretreated with NaHS. Our findings indicate that sulfide pretreatment induces a previous state in barley to respond more efficiently to stress and propose persulfidation is the underlying mechanism. 1. Introduction Drought stands as the most destructive environmental limitation, inflicting significantly higher yield losses than any other abiotic stressor, covering more than 20% of agricultural areas worldwide (Rojas, 2020). Moreover, projections indicate an escalation in drought frequency and intensity within currently arid regions owing to climatic change. Additionally, soil salinity is another critical abiotic stress that has become a major concern, hindering agricultural production and posing risks to environmental health and economic stability (Hassani et al., 2021). Both abiotic stresses, salinity and drought, substantially influence the food supply’s safety worldwide (Ortas et al., 2021). Barley (Hordeum vulgare L.), one of the most widely cultivated cereal crops, plays a crucial role in global food security and agricultural economies. However, barley production is increasingly threatened by environmental stresses such as drought and salinity (Bindereif et al., 2021). These abiotic stresses can lead to significant reductions in crop yield and quality, which pose a challenge to sustainable agriculture. Drought and salinity stresses induce oxidative stress in plants, characterized by the overproduction of reactive oxygen species (ROS) that can damage cellular structures, proteins, and nucleic acids (Cruz de Carvalho, 2008). To survive and thrive under these adverse conditions, barley plants have evolved a variety of physiological and biochemical strategies. These include the synthesis of osmoprotectants, the activation of antioxidant defense systems, and the modulation of stress-responsive genes (Abdelrady et al., 2024). Despite these natural defense mechanisms, the severity of drought and salinity stresses often exceeds the plant’s intrinsic tolerance levels, necessitating the Abbreviations: ABA, abscisic acid; ICP-OES, inductively coupled plasma optical emission spectroscopy; PBS, phosphate-buffered saline; PEG, polyethylene glycol; PTM, post-translational modification; ROS, reactive oxygen species; UPLC-MS/MS, ultra performance liquid chromatography tandem mass spectroscopy. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (A. Aroca), [email protected] (C. Gotor). Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy https://doi.org/10.1016/j.plaphy.2025.109644 Received 21 October 2024; Received in revised form 15 January 2025; Accepted 10 February 2025 Plant Physiology and Biochemistry 221 (2025) 109644 2 development of additional protective strategies. Recent studies have identified hydrogen sulfide (H₂S) as a promising molecule for enhancing plant stress tolerance (Aroca et al. 2018,2021). H 2 S has been demonstrated to play a crucial role in numerous biological functions within plants, including diverse developmental and physiological processes, as well as stress responses aimed at managing challenges such as heavy metal toxicity, salinity, and heat stress, among others (Zhang et al. 2015,2021;Zhao et al., 2018). Despite its traditionally known toxicity at high concentrations, H₂S has gained recognition as an important signaling molecule involved in various plant physiological processes. Recent research highlights the potential of H₂S as a signaling molecule in plants that can mitigate the adverse effects of drought and salinity. The key protective mechanism of H₂S is through persulfidation (Aroca et al., 2015), a post-translational modification (PTM) of cysteine residues in proteins. This PTM can enhance protein function, stability, and interaction, thereby improving the plant’s stress response. Persulfidation helps to regulate the activity of antioxidant enzymes (Aroca et al., 2015), ion channels, and transcription factors (Aroca et al. 2017, 2021;Zhang et al., 2024), which are critical for maintaining cellular homeostasis under stress conditions. By modulating these proteins, H₂S can improve the plant’s ability to cope with drought and salinity stresses, reducing oxidative damage and enhancing overall stress tolerance. In fact, research in different organism systems has proved that after sulfide treatment, persulfidation levels experienced a notable increase, consequently safeguarding cysteines from overoxidation (Garcia-Calderon et al., 2023;Jurado-Flores et al. 2023a,2023b;Zivanovic et al., 2019). Therefore, H₂S has been proposed as an effective cellular mechanism of protein protection against irreversible oxidation imposed by abiotic stresses in plants. Nevertheless, no previous studies are available about the influence of H 2 S-induced persulfidation and the outcome of H 2 S pretreatment in the comparison of drought and salinity stress tolerance in barley. In this context, this study explores the role of hydrogen sulfide in enhancing barley tolerance to drought and salinity stresses, and the mechanism of persulfidation involved. By elucidating the molecular and physiological pathways influenced by H₂S, we aim to provide insights into how this gaseous molecule can be harnessed to improve crop resilience and productivity under challenging environmental conditions caused by climate change. Understanding these mechanisms not only contributes to the fundamental knowledge of plant stress biology, but also offers practical applications for agricultural management and crop improvement strategies. 2. Material and methods 2.1. Plant material, growth conditions and treatments Barley (Hordeum vulgare, Scarlet cultivar, Spain) kindly provided by Dr. Gonz´ alez-Andr´ es, University of Le´ on, was used in this study. The seeds were sterilized and germinated in distilled water for 2–3 days at 25 ◦C, and were sown in soil. Barley plants grown in soil and irrigated with water for 15 days were divided into two batches, one batch irrigated with water and the other with 50 μ M NaHS for 7 additional days. Each batch was then divided into two new batches, one was water irrigated and the other was subjected to 20% PEG 6000 (w/v) for 6 days (for the drought stress study), or alternatively one irrigated with water and the other irrigated with 300 mM NaCl for 15 days (for the salinity stress study). For germination experiments, barley seeds were sterilized and divided into two batches, one was kept in distilled water and the other in 50 μ M NaHS, for 18 h at 4 ◦C in the dark. Subsequently, each batch was divided into two new batches and the seeds were placed in Petri dishes containing water or 300 mM NaCl and kept for 7 days in the dark at 20–22 ◦C. 2.2. Determination of chlorophyll and carotenoid contents Chlorophyll and carotenoids were determined spectrophotometrically (Jana Braniˇ sa et al., 2014). Plant material previously collected and frozen at −80 ◦C, was ground in liquid nitrogen and 50 mg of powder was transferred to a 1.5 mL microtube. Subsequently, we added 1 mL of 100% (v/v) methanol, and kept it for 24 h on a rotary shaker, at 4 ◦C under dark conditions. Then, 1/10 dilutions of the samples were made and the absorbance was measured at 666, 653 and 470 nm. Concentration was calculated using the following equations: Chlorophyll a( μ g mg −1 FW) =[(15.65 ×A 666 )–(7.34 ×A 653 )] / mg FW. Chlorophyll b( μ g mg −1 FW) =[(27.05 ×A 653 )–(11.21 ×A 666 )] / mg FW. Carotenoids (units mg −1 FW) =[(1000 ×A 470 –2.86 ×chlorophyll a– 129.2 ×chlorophyll b) / 221)] / mg FW. 2.3. Determination of anthocyanin and proline contents Total anthocyanin content of leaves was extracted using propanol/ HCl/water (18/1/81, v/v/v), kept in a boiling-water bath for 3 min, and for 24 h on a rotary shaker at 4 ◦C under dark conditions. The mixture was then centrifuged at 10,000 rpm for 10 min. The absorbance of the supernatant was measured at 535 nm and 650 nm. Anthocyanin content was determined using the following formula: A 535 =A 535 –2.2 ×A 650 and referred to the fresh weight, as described (Jurado-Flores et al., 2023a). Total proline content of leaves was determined following previous method (Carillo et al., 2011). The plant tissue was extracted using a cold mixture of ethanol/water (40/60, v/v) and kept it for 24 h on a rotary shaker, at 4 ◦C under dark conditions. Then, the mixture was centrifuged at 15,000 rpm for 5 min and 50 μ L of supernatants was mixed with 100 μ L of reaction mix: [1% ninhydrin (w/v) in 60% acetic acid (v/v) and 20% ethanol (v/v)]. The mixture was kept in a boiling-water bath for 20 min, protected from light. After cooling at room temperature, 100 μ L of the mixture were transferred to a microplate well. The absorbance was measured at 520 nm. To determine the proline content, a calibration line of proline solution ranging from 0.4 to 1 mM was used, and referred to fresh weight. 2.4. Amino acid and abscisic acid determination by UPLC-MS/MS For free amino acid determination, approximately 100 mg of frozen plant tissue was homogenized in liquid nitrogen. The metabolites were extracted from each aliquot in 400 μ L of 0.1 N HCL and 0.1% formic acid (v/v) with shaking for 30 min at 4 ◦C. Samples were centrifuged 15 min, 15,000 rpm at 4 ◦C. Samples were immediately subjected to UPLC‒MS/ MS as described previously (Jurado-Flores et al., 2023a). For abscisic acid determination, approximately 50 mg of frozen plant tissue was homogenized in 1.5-mL Eppendorf tubes for 2 min at maximum speed with a Retsch ball mill 153 (MM400; Retsch). The metabolites were extracted from each aliquot in 0.8 mL of solvent mixture containing methyl-tert-butyl-ether/methanol (MTBE/MeOH, 3/ 1, v/v) cooled to −20 ◦C, with shaking for 30 min at 4 ◦C. The samples were sonicated in an ice-cooled bath for 15 min. An aliquot of 0.4 mL of acidified water (0.1% HCl) was added and thoroughly vortexed for 1 min. After that, the samples were kept on an orbital shaker for an additional 30 min at 4 ◦C. The samples were centrifuged at 15,000 rpm for 10 min at 4 ◦C. A fixed volume of the upper supernatant was transferred to a fresh tube and dried down using a Speed-Vac concentrator at room temperature. The dried pellets were resuspended in 100 μ L of water/methanol (50/50, v/v) solution, and the resuspended samples R. Carrillo et al. Plant Physiology and Biochemistry 221 (2025) 109644 3 were immediately subjected to UPLC‒MS/MS hormonal analysis as described (Zhang et al., 2024). External abscisic acid standard solutions were used for quantitation. 2.5. Determination of cysteine content Total Cys content was quantified by reverse-phase HPLC after derivatization with monobromobimane as described previously (Jurado-Flores et al., 2023a). Approximately 100 mg of frozen plant tissue were homogenized in liquid nitrogen and Cys extracted in 400 μ L of 0.1 N HCl and 1 mM EDTA. Samples were centrifuged 15 min, 15,000 rpm at 4 ◦C and an aliquot of 100 μ L was mixed with 200 μ L 200 mM CHES (pH 9.3) and incubated 30 min at 4 ◦C. Then, the derivatization reaction was performed by adding 20 μ L of 15 mM monobromobimane, incubating for 60 min, and stopped by adding 5 μ L 20% formic acid (v/v). The mixture was centrifuged at 12,000 rpm for 10 min and immediately subjected to reverse-phase HPLC. 2.6. Determination of hydrogen peroxide content Hydrogen peroxide (H 2 O 2 ) was quantified using the Amplex redbased method (Thermo Fisher Scientific), as described previously (Garcia-Calderon et al., 2023). Briefly, 50 mg of barley leaves was ground to a fine powder in liquid nitrogen, and H 2 O 2 was extracted with 50 mM sodium phosphate (pH 7.4), centrifuged and the supernatant used for H 2 O 2 detection. A 100 μ L of reaction buffer containing 25 μ M Amplex Red, 10 U mL −1 horseradish peroxidase (HRP) and 5 μ L of supernatant in 50 mM PBS (pH 7.4) was incubated for 30 min and changes in fluorescence caused by the formation of oxidized Amplex-red (Ex/Em: 560 nm/590 nm) were recorded. Standard calibration curves were obtained with known H 2 O 2 concentrations. 2.7. Determination of Na + and K + by ICP-OES The determination of these elements was performed as previously described (Mostofa et al., 2015). Before analyzing the plant samples, the calibration curve was represented using different concentrations (from 1 ppm to 100 ppm) of each element. Approximately, 1g of fresh plant was dried for 6 d at 65 ◦C. Subsequently, 15 mL of a solution of 1 M HNO 3 and 10 mM MgCl 2 was added, and kept for 7 d at room temperature for digestion. The digested samples were then measured in triplicate by inductively coupled plasma-optical emission spectrometry (ICP-OES) using an iCAP 7200 ICP-OES Duo (ThermoFisher Scientific) at the Atomic Emission Spectrometry Service of the ICMS (Institute of Materials Science of Sevilla). 2.8. In-gel persulfidation detection Protein persulfidation patterns in plant tissue were analyzed with the dimedone switch method (Aroca et al., 2022). In detail, 150 mg of plant leaf material was ground in liquid nitrogen with 200 μ L of cold PBS lysis buffer [1 ×PBS pH 7.4, 1 mM EDTA, 2% SDS (w/v)] supplemented with 1×protease inhibitor (Pierce™, Thermo Scientific). Then, samples were incubated with 5 mM 4-chloro-7-nitrobenzofurazan (Cl-NBF) at 37 ◦C for 30 min, protected from light. Methanol/chloroform precipitation was performed to eliminate excess Cl-NBF, and the protein pellets obtained were washed with cold methanol, dried, and redissolved in 1 × PBS with 2% (w/v) SDS supplemented with 1 ×protease inhibitor. Proteins were incubated with 25 μ M DAz-2/Cy-5 preclick mix at 37 ◦C for 30 min. Following incubation, methanol/chloroform precipitation was performed, and the pellets were washed with methanol as described above. Protein labeling was analyzed using SDS-PAGE. After SDS PAGE, gels were fixed for 30 min in 12.5% methanol (v/v) and 4% acetic acid (v/v) protected from light. The gel was imaged at 640 nm for the Cy5 signal and 488 nm for the NBF-Cl signal. The persulfidation levels were quantified by measuring the Cy5/Cl-NBF fluorescence signal ratio. 2.9. Statistical analysis All data are the means of at least three independent experiments. The data were subjected to one-way analysis of variance (ANOVA), and different letters indicate significant differences between treatments at p >0.05, according to Dunnett’s multiple comparisons test using the software package for statistical analysis GraphPad Prism 9.5.0. 3. Results 3.1. Impact of hydrogen sulfide pretreatment on barley plants subjected to different stress conditions Significant evidence of the protective role of H 2 S in plants has already been generated under stress conditions (Aroca et al., 2021; Gotor et al., 2019), but a comparative study in the same plants subjected to different stress conditions is still lacking. To investigate this, barley was selected as a crop species and the performance of NaHS-pretreated plants was determined when they were exposed to drought by PEG treatment or alternatively to salinity stress by NaCl treatment. Our stablished experimental system consisted of 15-day-old barley plants grown in soil that were divided into two batches, one was watered, and the other was treated with 50 μ M NaHS for 7 additional days. Then, each batch was divided into two new batches, one subjected to water irrigation, and the second one to stress conditions, which were drought by irrigating with 20% PEG (w/v) for 6 days, or salinity by irrigating with 300 mM NaCl for 15 days. Therefore, at the end, we had water-irrigated plants (control), NaHS-treated plants (NaHS), nontreated-plants irrigated with PEG (drought), nontreated plants under salinity (NaCl), NaHS-treated plants subjected to PEG treatment (NaHS +drought) and NaHS-treated plants subjected to salt (NaHS +NaCl). The typical wilting and browning phenotypes of plants under drought were observed that matched a significant decrease in fresh weight and a lower reduction in shoot length compared to control plants. When plants were pretreated with NaHS, they partially recovered from both phenotypic parameters under drought (Fig. 1A). When plants were subjected to NaCl stress, a decrease in fresh weight was also observed, although it was lower than the effect caused by drought. However, pretreatment with NaHS also had a significant positive effect recovering partially the non-stressed phenotype (Fig. 1B). 3.2. Biochemical characterization of the effect of NaHS pretreatment on barley plants under stress Further analysis of the impact of NaHS pretreatment was performed by quantifying different biochemical parameters in plant samples. We observed a strong reduction in both chlorophylls (a and b) together with carotenoids content in plants after drought compared to control conditions, which was improved in NaHS-pretreated plants under drought, without reaching the values observed under control and NaHS conditions. Similar results were obtained when salt stress conditions were studied (Fig. 2). In addition, different biochemical parameters that are well-known markers in plants harmed by stress conditions, such as anthocyanin and proline, were quantified. Under drought, the content of both compounds was higher than in the control plants, showing a very strong increase in proline content, which was indicative of the plants being affected by stress. Whereas the level of both markers significantly decreased in NaHS-pretreated plants under drought, reaching the same level as plants grown under control conditions. When salt stress was examined, we also observed an increase in both stress markers, although in the case of proline, the increase was significantly lower than that observed under drought conditions. Similarly, NaHS-pretreated plants showed a reduction in proline content when were subjected to salt stress compared to the untreated plants, however, no change was observed in the anthocyanin content (Fig. 3). These findings demonstrate that pretreatment with sulfide causes an improvement in the survival of barley R. Carrillo et al. Plant Physiology and Biochemistry 221 (2025) 109644 4 Fig. 1. Effect of NaHS pretreatment on the phenotype of barley plants under stress conditions. Phenotypes and quantification of fresh weight and shoot height of barley plants treated as indicated. A) Drought and B) saline stress. Data are means ±SD. Same letters indicate no statistically significant differences. P <0.05. R. Carrillo et al. Plant Physiology and Biochemistry 221 (2025) 109644 5 plants under stress conditions, being more efficient under drought. The high content of proline found under drought might not be due exclusively to the accumulation of this amino acid, because the method used for proline quantification was a non-specific chemical method, thoroughly used in several publications (Carillo et al., 2011). In order to determine the steady-state level of individual amino acids, we quantified their accumulation by UPLC-MS/MS in plant extracts under our experimental conditions. Cysteine content was also determined by reverse-phase HPLC after derivatization with monobromobimane. We found indeed a strong accumulation of proline under drought stress, that was significantly reduced when plants were NaHS-pretreated, but in addition, we clearly observed the accumulation of other amino acids. The most significant accumulation under drought was observed for tryptophan, which was substantially reduced when NaHS pretreatment was imposed (Fig. 4). However, other amino acids, such as glutamine, methionine, cysteine, threonine, phenylalanine, valine, isoleucine, leucine, and tyrosine, also accumulated under drought stress and decreased in NaHS-preteated plants. These results indicate that the accumulation of proline as a major compatible osmolyte under drought conditions go together with the readjustment of amino acid metabolism, as well as, that NaHS pretreatment regulates these alterations. Both water deficit and salinity are stress conditions that alter cell homeostasis, producing reactive oxygen species (ROS) accumulation and subsequent oxidative damage (Miller et al., 2010). In accordance, Fig. 2. Effect of NaHS pretreatments on pigment contents under different stresses. Chlorophyll aand b, and carotenoids content of barley plants treated as indicated. A) Drought and B) saline stress. Data are means ±SD. Same letters indicate no statistically significant differences. P <0.05. R. Carrillo et al. Plant Physiology and Biochemistry 221 (2025) 109644 6 H 2 O 2 levels were quantified in barley plants under the different treatments. A significant production of H 2 O 2 was determined when plants were subjected to any of these stress conditions, that was reduced when plants were treated with NaHS prior to the stress (Fig. 5). Thus, the results are indicative of the role of sulfide alleviating the oxidative stress of the plants. 3.3. Effect of NaHS pretreatment on stress-specific plant responses Although plants share common responses to cope with different stress conditions, some are more specific to one condition than to the other. Various environmental stresses, primarily dehydration, trigger the production of the phytohormone abscisic acid (ABA) to activate the ABA-signaling pathway and induce tolerance to stress (Cutler et al., 2010). The interrelation between sulfide and ABA has been widely evidenced and in particular, the action of sulfide in ABA-dependent stomatal closure (Aroca et al., 2021). Therefore, the level of ABA was quantified in the plant samples under drought conditions and a strong induction of ABA was determined in plants subjected to PEG treatment compared to plants under control or sulfide-irrigated conditions. Significantly, when plants were pretreated with NaHS, a reduction in the level of ABA was observed under drought conditions (Fig. 6). These Fig. 3. Effect of NaHS pretreatment on biochemical stress marker contents of barley plants under different stresses. Anthocyanin and proline contents of barley plants treated as indicated. A) Drought and B) saline stress. Data are means ±SD. Same letters indicate no statistically significant differences. P <0.05. R. Carrillo et al. Plant Physiology and Biochemistry 221 (2025) 109644 7 Fig. 4. Effect of NaHS pretreatment on content of free amino acids under drough stress in barley plants. Plants were treated as indicated and the levels of individual amino acid were determined by UPLC-MS/MS. Data are means ±SD. Same letters indicate no statistically significant differences. P <0.05. R. Carrillo et al. Plant Physiology and Biochemistry 221 (2025) 109644 8 findings further suggest that sulfide pretreatment prepares plants to better deal with drought. A key adaptive response to cope with salt stress is the maintenance of the balance of cellular ions, which is disrupted by the accumulation of toxic Na + that displaces essential ions such as K + (Park et al., 2016). Accordingly, a low Na + /K + ratio is indicative of improved plant tolerance to salt stress. In our experimental system, we measured the Na + and K + contents and determined very low levels of Na + and significant high levels of K + in plants that were grown under control conditions or after irrigation with NaHS. Upon salinity, the levels of both Na + and K + ions were equally in untreated plants, however, when plants were NaHS-pretreated, the K + content significantly increased to a level similar to that of water-irrigated plants, while Na + content remained similar to that of the untreated plants. Consequently, the Na + /K + ratio was reduced by NaHS pretreatment (Fig. 7), suggesting an improvement of plant tolerance to salinity by sulfide action. 3.4. Effect of NaHS pretreatment on the persulfidation level The best mechanism studied by which sulfide plays its role as a signaling molecule in plants is the posttranslational modification of protein cysteines named persulfidation, which consists of the formation of cysteine persulfides (Aroca et al., 2021;Gotor et al., 2019). To analyze whether persulfidation is responsible for the better plant performance observed in NaHS-pretreated plants under stress, in gel-persulfidation detection was performed. We determined a slight increase in the persulfidation level when plants were irrigated with NaHS compared to the plants watered. If these NaHS-irrigated plants were subjected to drought stress, the level of persulfidation increased significantly (Fig. 8A). Likewise, we observed a significant induced level of persulfidation in plants under salt stress when they were previously treated with NaHS (Fig. 8B). This enhanced persulfidation level was accompanied by an improved tolerance to drought and salinity as previously observed, suggesting that protective role of sulfide pretreatment under stress could be related to an induced persulfidation level. 3.5. NaHS pretreatment improves barley seed germination under salt stress Different studies have shown a protective role for sulfide in seed germination of different plant species under certain abiotic stresses, mainly hevay metal stress (Zhang et al. 2008,2010). To extend the study to other abiotic condition, we investigated the effect of NaHS pretreatement on the ability of barley seeds to germinate under salt stress conditions. Barley seed germination was strongly affected by salt stress, as was observed at first sight; however, seeds preteated with NaHS showed a clear improvement in germination rate (Fig. 9A). To quantify the effect of salt stress and the role of sulfide in germination, different variables were studied, such as the percentage of germinated seeds, the length of sprouts and roots and seed vigour. Seeds germinated under salt stress showed a strong reduction in all of these variables, demonstrating the negative effect of salt on germination. However, the effect of NaHS in pretreated seeds under salinity was ameliorated, mainly observed in the percentage of germination and seed vigour (Fig. 9B). Fig. 5. Effect of NaHS pretreatment on hydrogen peroxide content of barley plants under different stresses. H₂O₂, content of barley plants treated as indicated. A) Drought and B) saline stress. Data are means ±SD. Same letters indicate no statistically significant differences. P <0.05. Fig. 6. Effect of NaHS pretreatment on ABA content of barley plants under drought stress. Plants were treated as indicated and ABA content was measured. Data are means ±SD. Same letters indicate no statistically significant differences. P <0.05. R. Carrillo et al. Plant Physiology and Biochemistry 221 (2025) 109644 9 4. Discussion Substantial studies have already demonstrated the role of hydrogen sulfide as inducer of plant resistance, mainly by sulfide exogenous application combined to the stress condition (Aroca et al., 2021;Corpas, 2019;Li et al., 2016;Zhang et al., 2021). Protein persulfidation has been demonstrated as the molecular mechanism involved in sulfide signaling (Gotor et al., 2019;Huang and Xie, 2023), and several proteomic analyses have shown an inventory of persulfidated proteins. This list has been mainly obtained from Arabidopsis either under non-stress or stress conditions in the absence of sulfide treatment (Aroca et al. 2015,2017; Garcia-Calderon et al., 2023;Jurado-Flores et al. 2021,2023a,2023b; Laureano-Marin et al., 2020). As well, a catalog of persulfidated proteins has been described in some crops such as rice (Zhang et al., 2024), bean (Matamoros et al., 2024) and pepper (Mu˜ noz-Vargas et al., 2024), also in the absence of sulfide treatment. Therefore, the endogenous sulfide is responsible of the persulfidation level in these conditions and plants, however, the effect of the exogenous sulfide pretreatment on the persulfidation level either in the absence of stress or before imposing the stress is unknown. This study explores the effect of pretreatment with NaHS as a sulfide donor on the plant performance of a crop species as barley under physiological growth conditions, as well as on plant tolerance to drought-associated osmotic stress and salinity. In general, no significant Fig. 7. Effect of NaHS pretreatment on the ratio Na + /K + of barley plants under salt stress. Plants were treated as indicated, and A) Na + and K + contents were determined by ICP and B) Ratio Na + /K + was calculated. Data are means ±SD. Same letters indicate no statistically significant differences. P <0.05. R. Carrillo et al.