Hydrogen Sulfide-Mediated Activation of O-Acetylserine (Thiol) Lyase and l/d-Cysteine Desulfhydrase Enhance Dehydration Tolerance in Eruca sativa Mill

Hydrogen sulfide (H2S) has emerged as an important signaling molecule and plays a significant role during different environmental stresses in plants. The present work was carried out to explore the potential role of H2S in reversal of dehydration stress-inhibited O-acetylserine (thiol) lyase (OAS-TL), l-cysteine desulfhydrase (LCD), and d-cysteine desulfhydrase (DCD) response in arugula (Eruca sativa Mill.) plants. Dehydration-stressed plants exhibited reduced water status and increased levels of hydrogen peroxide (H2O2) and superoxide (O2•−) content that increased membrane permeability and lipid peroxidation, and caused a reduction in chlorophyll content. However, H2S donor sodium hydrosulfide (NaHS), at the rate of 2 mM, substantially reduced oxidative stress (lower H2O2 and O2•−) by upregulating activities of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase) and increasing accumulation of osmolytes viz. proline and glycine betaine (GB). All these, together, resulted in reduced membrane permeability, lipid peroxidation, water loss, and improved hydration level of plants. The beneficial role of H2S in the tolerance of plants to dehydration stress was traced with H2S-mediated activation of carbonic anhydrase activity and enzyme involved in the biosynthesis of cysteine (Cys), such as OAS-TL. H2S-treated plants showed maximum Cys content. The exogenous application of H2S also induced the activity of LCD and DCD enzymes that assisted the plants to synthesize more H2S from accumulated Cys. Therefore, an adequate concentration of H2S was maintained, that improved the efficiency of plants to mitigate dehydration stress-induced alterations. The central role of H2S in the reversal of dehydration stress-induced damage was evident with the use of the H2S scavenger, hypotaurine.


Introduction
Climate change is the main factor triggering various environmental stresses, affecting agriculture productivity in almost all the areas of the world. Being sessile in nature, plants are always exposed Dehydration stress-induced generation of ROS was measured in terms of H2O2 and O2 •− content. Perusal of the data shows that dehydration-stressed plants generated 25.9% and 38.8% more H2O2 and O2 •− content, respectively, as compared with the control ( Figure 2C,D). However, H2S showed an inhibitory effect on the generation of these ROS. Application of H2S to stressed seedlings caused a reduction of 16.4% and 20.7% in H2O2 and O2 •− content, respectively, compared with stressed samples. Dehydration-stressed seedlings showed an increase of 63.2% in electrolyte leakage as compared with the control ( Figure 1A), whereas application of H 2 S to stressed seedlings reduced electrolyte leakage by 31.4% as compared with dehydration-stressed plants. On the contrary, an addition of HT countered the impact of H 2 S and, again, caused a significant increase in electrolyte leakage to a level higher than the dehydration-suffered seedlings ( Figure 1A).
Dehydration-stressed seedlings exhibited 44.0% higher value for TBARS as compared with the control ( Figure 1C). However, H 2 S reduced the level of TBARS by 24.8% in stressed plants in comparison with dehydration-stressed seedlings not treated with H 2 S ( Figure 1C). On the contrary, the H 2 S scavenger, HT, mutated the stress, alleviating the impact of H 2 S and further caused a significant increase in TBARS, reaching the value statistically close to the value recorded from dehydration-stressed seedlings ( Figure 1C).

Hydrogen Peroxide (H 2 O 2 ) and Superoxide (O 2 •− ) Content
The visual effect of dehydration stress and H 2 S on the production of ROS in roots was tested by assessing in situ formation of H 2 O 2 and O 2 •− in roots using a DCF-DA and DHE fluorescence probe, respectively. Roots of the seedlings exposed to DS exhibited sharper green (with DCF-DA probe) and red fluorescence (with DHE probe) signal than the control plants. However, a lower fluorescence signal was observed in the dehydration-suffered roots treated with H 2 S donor, NaHS, as compared with the stressed plants not treated with NaHS, whereas fluorescence signals again sharpened in the roots of stressed plants that received H 2 S scavenger HT along with NaHS ( Figure 2A,B)

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Higher activities of antioxidant enzymes (SOD, POX, and CAT) were recorded in stressed seedlings as compared with the control plants ( Figure 3C,D). Furthermore, an additional increase of 17.5%, 15.4%, and 13.5% was recorded in SOD, POX, and CAT, respectively, in dehydration-stressed seedlings supplemented with H2S donor NaHS. On the other hand, the inclusion of HT to NaHStreated stressed plants (HT + NaHS + DS) suppressed the activities of SOD, POX, and CAT by 23.0%, 17.6%, and 16.2%, respectively, compared with dehydration-stressed plants ( Figure 3C,D).

Activity of O-Acetylserine (Thiol) Lyase (OAS-TL) Enzyme and Cysteine (Cys) Content
Perusal of the data shows that DS enhanced the activity of Cys-synthesizing enzyme OAS-TL, as well as Cys content by 20.0% and 25.6%, respectively, when compared with the control ( Figure   Figure 2. Fluorescence microscopy imaging of reactive oxygen species (ROS):  Figure 2C,D).

Proline (Pro) and Glycine Betaine (GB) Content
In the present study, dehydration-stressed plants accumulated 37.3% and 24.4% more Pro and GB content, respectively, than the control plants ( Figure 3A,B). Moreover, application of H 2 S further elevated Pro and GB content by 16.9% and 31.1%, respectively, as compared with dehydration-stressed plants, whereas the presence of HT in the incubation medium (HT + NaHS + DS) reversed the effect of H 2 S on these osmolytes and, as a result, Pro and GB content dropped to 16.9% and 12.4%, respectively, as compared with the stressed plants ( Figure 3A,B). 6 enhanced these parameters to the highest level. The treatment of NaHS + DS enhanced OAS-TL activity and Cys content by 20.7% and 22.9%, respectively, compared with the stressed plants. On the contrary, the inclusion of H2S scavenger HT (HT + NaHS + DS) slowed down the activity of OAS-TL by 27.9%, which was 20.7% higher when HT was not present (NaHS + DS) in the incubation medium ( Figure 2E,F). A similar inhibitory effect of HT was also noticed on Cys content, which exhibited a 31.9% lower value than the stressed seedlings ( Figure 3F).

Activities of Antioxidant Enzymes
Higher activities of antioxidant enzymes (SOD, POX, and CAT) were recorded in stressed seedlings as compared with the control plants ( Figure 3C,D). Furthermore, an additional increase of 17.5%, 15.4%, and 13.5% was recorded in SOD, POX, and CAT, respectively, in dehydration-stressed seedlings supplemented with H 2 S donor NaHS. On the other hand, the inclusion of HT to NaHS-treated stressed plants (HT + NaHS + DS) suppressed the activities of SOD, POX, and CAT by 23.0%, 17.6%, and 16.2%, respectively, compared with dehydration-stressed plants ( Figure 3C,D).

Activity of O-Acetylserine (Thiol) Lyase (OAS-TL) Enzyme and Cysteine (Cys) Content
Perusal of the data shows that DS enhanced the activity of Cys-synthesizing enzyme OAS-TL, as well as Cys content by 20.0% and 25.6%, respectively, when compared with the control (Figure 3E,F). In addition, treatment of dehydration-stressed plants with NaHS (NaHS + DS) further enhanced these parameters to the highest level. The treatment of NaHS + DS enhanced OAS-TL activity and Cys content by 20.7% and 22.9%, respectively, compared with the stressed plants. On the contrary, the inclusion of H 2 S scavenger HT (HT + NaHS + DS) slowed down the activity of OAS-TL by 27.9%, which was 20.7% higher when HT was not present (NaHS + DS) in the incubation medium ( Figure 2E,F). A similar inhibitory effect of HT was also noticed on Cys content, which exhibited a 31.9% lower value than the stressed seedlings ( Figure 3F).

Activities of LCD and DCD Enzymes and H 2 S Content
It is evident from Figure 4A,B that DS induced the activities of LCD and DCD enzymes, and H 2 S content. Dehydration-stressed samples showed 30.0% and 10.3% higher activities of LCD and DCD enzymes, respectively, compared with the control. Likewise, an increase of 19.7% was also recorded in H 2 S content of dehydration-stressed plants. Additionally, exogenous application of H 2 S to dehydration-stressed seedlings (NaHS + DS) further elevated the activities of LCD and DCD enzymes and H 2 S content by 24.3%, 41.9%, and 28.7%, respectively, compared with the stressed plants ( Figure 3A,B). Nevertheless, the addition of HT to H 2 S-supplemented dehydration-stressed plants (HT + NaHS + DS) suppressed the LCD and DCD activities, and H 2 S content. HT scavenged 79.8% H 2 S in H 2 S-supplemented stressed seedlings (NaHS + DS) as compared with dehydration-stressed seedlings ( Figure 4B). It is worth mentioning here that impact of HT on H 2 S was more prominent than on the activities of LCD and DCD enzymes.
It is evident from Figure 4A,B that DS induced the activities of LCD and DCD enzymes, and H2S content. Dehydration-stressed samples showed 30.0% and 10.3% higher activities of LCD and DCD enzymes, respectively, compared with the control. Likewise, an increase of 19.7% was also recorded in H2S content of dehydration-stressed plants. Additionally, exogenous application of H2S to dehydration-stressed seedlings (NaHS + DS) further elevated the activities of LCD and DCD enzymes and H2S content by 24.3%, 41.9%, and 28.7%, respectively, compared with the stressed plants ( Figure  3A,B). Nevertheless, the addition of HT to H2S-supplemented dehydration-stressed plants (HT + NaHS + DS) suppressed the LCD and DCD activities, and H2S content. HT scavenged 79.8% H2S in H2S-supplemented stressed seedlings (NaHS + DS) as compared with dehydration-stressed seedlings ( Figure 4B). It is worth mentioning here that impact of HT on H2S was more prominent than on the activities of LCD and DCD enzymes.

Photosynthetic Pigments and Carbonic Anhydrase (CA) Activity
As evident from previous studies, DS causes destruction of photosynthetic pigments. A similar trend was also recorded in the present investigation. Dehydration-suffered plants showed a 25.5%, 46.9%, and 32.9% decrease in Chl a, Chl b, and total Chl content, respectively, as compared with the control (Table 1). However, stressed plants showed an increase of 40.1% in Chl a/b ratio, as compared with control. Incubation of dehydration-stressed plants with H2S improved Chl a, Chl b, and total Chl content by 29.2%, 66.7%, and 39.4%, respectively, but H2S decreased Chl a/b ratio by 22.7% compared with the stressed seedlings. Plants exposed to DS exhibited a 12.8% increase in carotenoid content, as compared with the control. Furthermore, dehydration-stressed plants treated with H2S exhibited a further increase of 15.2% in total carotenoid content as compared with the stressed plants that did not receive H2S treatment (Table 1). Conversely, the presence of HT, along with exogenous H2S in incubation medium, created stress-like conditions, as reflected by the reduced values of all these parameters, except Chl a/b ratio, which showed an increase when compared with dehydration-

Photosynthetic Pigments and Carbonic Anhydrase (CA) Activity
As evident from previous studies, DS causes destruction of photosynthetic pigments. A similar trend was also recorded in the present investigation. Dehydration-suffered plants showed a 25.5%, 46.9%, and 32.9% decrease in Chl a, Chl b, and total Chl content, respectively, as compared with the control (Table 1). However, stressed plants showed an increase of 40.1% in Chl a/b ratio, as compared with control. Incubation of dehydration-stressed plants with H 2 S improved Chl a, Chl b, and total Chl content by 29.2%, 66.7%, and 39.4%, respectively, but H 2 S decreased Chl a/b ratio by 22.7% compared with the stressed seedlings. Plants exposed to DS exhibited a 12.8% increase in carotenoid content, as compared with the control. Furthermore, dehydration-stressed plants treated with H 2 S exhibited a further increase of 15.2% in total carotenoid content as compared with the stressed plants that did not receive H 2 S treatment (Table 1). Conversely, the presence of HT, along with exogenous H 2 S in incubation medium, created stress-like conditions, as reflected by the reduced values of all these parameters, except Chl a/b ratio, which showed an increase when compared with dehydration-stressed plants without H 2 S (Table 1). Table 1. Effect of hydrogen sulfide and dehydration stress on chlorophyll content, total carotenoids, and carbonic anhydrase activity in arugula.

Treatments Parameters
Chl a (mg g −1 FW) Results showed that DS upregulated carbonic anhydrase (CA) activity and caused an increase of 16.3% compared with the control plants (Table 1). In addition, stressed plants, when treated with NaHS (NaHS + DS), exhibited a further increase of 15.5% in CA activity than the dehydration-stressed plants that did not receive H 2 S. On the other hand, addition of HT (HT + NaHS + DS) invalidated the effect of H 2 S and caused a reduction in the activity of CA (Table 1).

Discussion
It is well established that DS has multiple effects on plants. Reduction in water status is the hallmark of DS. Therefore, keeping an eye on the water status of plants would be amongst one of the strategies to save crop plants from DS. To test the effect of dehydration on water status, we measured LRWC ( Figure 1A). Plants under DS showed lower values for LRWC and an enhanced rate of water loss over the time. Consistent DS leads to the creation of hyperosmotic stress, which affects water relations and causes loss of turgor [34] that reduces water uptake capacity of plants, as reflected by lower LRWC ( Figure 1A). To evaluate the water-retaining capacity of plants, we performed a water loss assay. Dehydration-stressed seedlings showed a parallel increase in the rate of water loss from 1 to 6 h of estimation ( Figure 1B). Plants counter osmotic stress through osmotic adjustment, which is normally accomplished by the accumulation of osmolytes, such as Pro and GB, that maintain normal water status in plants under abiotic stress [35,36]. Enhanced levels of Pro and GB were recorded in the same dehydration-stressed plants in which lower LRWC and a higher rate of water loss was noticed. This indicated that enhanced levels of Pro and GB were not sufficient to maintain the normal hydration level of the plants. However, the presence of H 2 S in the growth medium further enhanced Pro and GB content ( Figure 3A,B) to a level that vigorously countered dehydration-induced osmotic stress, as reflected by enhanced LRWC and a lower rate of water loss in stressed plants ( Figure 1A,B). An increment in Pro and GB content by H 2 S can be explained on the basis of the role of H 2 S in enhancing the activities of key enzymes responsible for Pro and GB synthesis [37,38]. Furthermore, H 2 S has been shown to increase the expression levels of abscisic acid (ABA) biosynthesis and reactivation genes [39], leading to stomatal closure [40]. However, H 2 S inhibits ethylene synthesis through suppressing the activity of 1-aminocyclopropane-1-carboxylic acid oxidase [41]. An increase in ABA and decrease in ethylene has been reported to be associated with a decrease in transpiration rate and leaf water potential [42], which improved the water-retaining capacity of plants, as witnessed by improved LRWC and reduced rate of water loss ( Figure 1A,B). Alleviation of DS and an increase in relative water content due to H 2 S treatment was also reported by García-Mata and Lamattina [43], and Chen et al. [44]. On the contrary, H 2 S scavenger HT mutated the effect of H 2 S that resulted in suppressed accumulation of Pro and GB content ( Figure 3A,B) and, in turn, lower LRWC ( Figure 1A), and a higher rate of water loss ( Figure 1B) was recorded.
Continued drought results in severe adverse effects as it creates oxidative stress through overproduction of ROS. Excessive generation of ROS causes peroxidation of membrane lipids and damage to cellular compartments, proteins, and RNA and DNA molecules [6,45]. The present study also shows higher levels of ROS (H 2 O 2 and O 2 •− content) ( It is well known that Cys is synthesized by incorporation of reduced sulfur to O-acetylserine in a reaction catalyzed by the enzyme OAS-TL ( Figure 5). Cys serves as a sulfur donor for synthesis of S-containing compounds [49], and as a precursor of various antioxidants and defense compounds [50], and induces tolerance to various stresses [51]. Results show that DS enhanced the activity of OAS-TL, that resulted in enhancement of Cys content ( Figure 3E,F). As discussed in the preceding paragraph, DS also enhanced H 2 O 2 and O 2 •− content, electrolyte leakage, and TBARS. It shows that seedlings tried to counter DS by increasing Cys synthesis, but an increase in Cys was not enough to counter available ROS. Nevertheless, application of NaHS to stressed seedlings further enhanced OAS-TL activity and Cys content coupled with a decrease in water loss, ROS, electrolyte leakage, and TBARS, and an increase in LRWC ( Figure 1A-C and Figure 2A-D). On the other hand, treatment with HT reversed the effect of NaHS, that resulted in enhanced levels of water loss, ROS, electrolyte leakage, and TBARS, and reduced LRWC. Thus, it is clear from the results that exogenous application of NaHS enhances Cys-synthesizing enzyme and Cys content, that directly or indirectly gives protection against stress-induced constraints. It is evident from the results shown in Figure 4A,B that DS significantly enhanced the activities of H 2 S-synthesizing enzymes LCD and DCD, and H 2 S content. The enzymes LCD and DCD synthesize H 2 S from L-Cys and D-Cys, respectively ( Figure 5) [52,53]. Therefore, an endogenous level of H 2 S directly depends on the availability of Cys. In the present study, DS enhanced OAS-TL, LCD, and DCD activities, and Cys and H 2 S content, but seedlings failed to counter imposed DS. Moreover, treatment with H 2 S further enhanced these parameters. Enhanced levels of H 2 S proved effective in protecting plants against DS via enhancing the accumulation of osmolytes and activities of antioxidant enzymes, as witnessed by improved hydration level and reduced water loss, electrolyte leakage, and TBARS [25,54]. On the other hand, the addition of HT to the incubation medium scavenged H 2 S to a considerable limit, and suppressed the activities of LCD and DCD enzymes ( Figure 4A,B). Photosynthetic pigments, the key light harvesting devices of plants, are responsible for the conversion of light energy to usable chemical energy for all the creatures of planet Earth. Plants under DS exhibited reduced levels of the studied photosynthetic pigments viz. Chl a,b, and total Chl content with the exception of Chl a/b ratio and carotenoid content which showed an increase compared with control plants (Table 1). A decrease in Chl content by DS has already been reported by several scientists [25,34,55,56]. A decline in Chl content was probably the result of an inhibitory effect of DS on Chl biosynthetic intermediates and decrease in Chl biosynthesis enzymes [57]. Moreover, DSinduced overproduction of ROS might have caused photo-oxidative damage to Chl, as shown by reduced Chl a,b and total Chl content ( Table 1). It is interesting to note, here, that dehydration-stressed plants show increased Chl a/b ratio than the non-stressed control plants. This indicates that Chl b is more susceptible to dehydration-induced damage than Chl a, as shown by higher decrease in Chl b content (46.9%) than Chl a (25.5%) [58]. However, treatment of plants with H2S donor NaHS overcame the effect of DS and reduced the level of ROS through enhancing the activity of antioxidant enzymes ( Figure 3C,D). Reduction in ROS gave protection to cellular membranes that resulted in the suppression of electrolyte leakage and TBARS ( Figure 1A,C). As a result, H2S created a condition that could be suitable for the biosynthesis of Chl and resulted in improved Chl content (Table 1) [59][60][61]. Besides antioxidant enzymes in plants, carotenoids act as first line of defense in chloroplast against O2 toxicity, especially singlet oxygen ( 1 O2). Dehydration stress induced carotenoids, and a further increase was also noticed in H2S-treated stressed plants that might have prohibited oxidative stress in the chloroplast, leading to the normal synthesis of Chl even under stressful conditions. On the contrary, the addition of H2S scavenger, HT, validated the role of H2S by reducing Chl and carotenoid content (Table 1).
Carbonic anhydrase (CA) is a chloroplast-localized enzyme that catalyzes the reversible hydration of CO2, and maintains its continuous supply to Rubisco, a key enzyme responsible for the Photosynthetic pigments, the key light harvesting devices of plants, are responsible for the conversion of light energy to usable chemical energy for all the creatures of planet Earth. Plants under DS exhibited reduced levels of the studied photosynthetic pigments viz. Chl a,b, and total Chl content with the exception of Chl a/b ratio and carotenoid content which showed an increase compared with control plants (Table 1). A decrease in Chl content by DS has already been reported by several scientists [25,34,55,56]. A decline in Chl content was probably the result of an inhibitory effect of DS on Chl biosynthetic intermediates and decrease in Chl biosynthesis enzymes [57]. Moreover, DS-induced overproduction of ROS might have caused photo-oxidative damage to Chl, as shown by reduced Chl a,b and total Chl content ( Table 1). It is interesting to note, here, that dehydration-stressed plants show increased Chl a/b ratio than the non-stressed control plants. This indicates that Chl b is more susceptible to dehydration-induced damage than Chl a, as shown by higher decrease in Chl b content (46.9%) than Chl a (25.5%) [58]. However, treatment of plants with H 2 S donor NaHS overcame the effect of DS and reduced the level of ROS through enhancing the activity of antioxidant enzymes ( Figure 3C,D). Reduction in ROS gave protection to cellular membranes that resulted in the suppression of electrolyte leakage and TBARS ( Figure 1A,C). As a result, H 2 S created a condition that could be suitable for the biosynthesis of Chl and resulted in improved Chl content (Table 1) [59][60][61]. Besides antioxidant enzymes in plants, carotenoids act as first line of defense in chloroplast against O 2 toxicity, especially singlet oxygen ( 1 O 2 ). Dehydration stress induced carotenoids, and a further increase was also noticed in H 2 S-treated stressed plants that might have prohibited oxidative stress in the chloroplast, leading to the normal synthesis of Chl even under stressful conditions. On the contrary, the addition of H 2 S scavenger, HT, validated the role of H 2 S by reducing Chl and carotenoid content (Table 1).
Carbonic anhydrase (CA) is a chloroplast-localized enzyme that catalyzes the reversible hydration of CO 2 , and maintains its continuous supply to Rubisco, a key enzyme responsible for the fixation of CO 2 . In addition to its direct role in photosynthesis, expression of CA is related with environmental stresses, and CA activity is upregulated under stresses [62]. An increase in CA activity was recorded in dehydration-stressed plants; moreover, treatment with NaHS further enhanced CA activity in non-stressed, as well as stressed, plants (Table 1). Increased activity of CA controls ROS levels during DS, and helps the cells to become more resistant to cytotoxic concentrations of H 2 O 2 [63,64]. Therefore, it can be explained that H 2 S-induced reduction in ROS was due to enhanced levels of SOD and carotenoids, that converted oxygen radicals to H 2 O 2 and, further, the level of H 2 O 2 was regulated by the joint action of CA, POX, and CAT. The effect of reduced concentrations of ROS is also witnessed by enhanced LRWC and reduced water loss, electrolyte leakage, and TBARS in treated plants ( Figure 1A-E). This is the first report regarding the response recorded from the effect of H 2 S in the reduction of ROS generation by the enhanced activity of CA under DS.

Plant Culture and Treatments
Healthy and uniform seeds of arugula (Eruca sativa Mill.) were surface-sterilized with sodium hypochlorite (1%), followed by repeated washing with double distilled water (DDW). The seeds were sown 2 cm deep in plastic pots (20 cm diameter and 20 cm height) containing a mixture of soil/vermiculite (1:1). The pots were kept under natural illuminated conditions with an average day/night temperature of 28/10 ± 3 • C, and were well-watered (100% field capacity). After one week, the seedlings were exposed to 2 mM sodium hydrosulfide (NaHS), 1 mM hypotaurine (HT), and dehydration stress (DS). Dehydration stress was imposed by maintaining the water level in soil at 30% through withholding the water supply. During the dehydration period, soil relative water content was monitored daily, and DS was continued up to 10 days when the desired level of soil relative water content (30%) was attained. Plants (control) which were not exposed to dehydration were irrigated with DDW throughout the period of dehydration. The treatments were given as (i) DDW: Control, (ii) Dehydration stress (DS), (iii) 2 mM NaHS + DDW: (NaHS), (iv) 2 mM NaHS + DS: (NaHS + DS) and (v) 1 mM HT + 2 mM NaHS + DS: (HT + NaHS + DS). NaHS was used as H 2 S donor, while HT was used as H 2 S scavenger. The design of the experiment was simple-randomized, with three replicates per treatment. After 10 days of treatments, the seedlings were uprooted carefully, and the response of seedlings to the treatments was further evaluated.

Estimation of Leaf Relative Water Content (LRWC), Electrolyte Leakage, and Rate of Water Loss
After 10 days of dehydration treatment, the fresh weight (FW) of the leaves was measured. To determine turgid weight (TW), the leaves were immersed in DDW inside the covered Petri dish. After 4 h, the water from leaf surface was wiped with blotting paper, and TW was recorded. Finally, the leaf samples were oven-dried at 80 • C for 24 h, and then the dry weight (DW) was determined. Leaf relative water content was estimated according to Yamasaki and Dillenburg [65], using the following formula: LRWC (%) = (FW−DW)/(TW−DW) × 100 The effect of DS and H 2 S on membrane permeability was assessed by measuring electrolyte leakage by the method of Lutts et al. [66]. After 10 days of treatment, electrical conductivity of the leaves incubated with DDW for 24 h (EC1), and leaves autoclaved at 120 • C for 20 min (EC2), were measured. The electrolyte leakage (%) was calculated as ((EC1/EC2) × 100).
To determine the rate of water loss, treated leaves were placed on a filter paper at room temperate under white fluorescent light. These leaves were weighed periodically at an interval of 1 h for up to 6 h. The rate of water loss was expressed as a percentage of the control. Hydrogen peroxide (H 2 O 2 ) in the leaves was determined using 10 M potassium phosphate buffer and 1 M potassium iodide [68]. The absorbance of the samples was read at 390 nm. The H 2 O 2 content was measured by comparing with a standard curve, and was expressed as µmol g −1 leaf FW.

Detection of Hydrogen
Superoxide (O 2 •− ) content was determined by adopting the method of Elstner and Heupel [69] with some modifications. The absorbance was measured at 530 nm. The content of O 2 •− was calculated by comparing with a standard curve.

Analysis of Lipid Peroxidation
Peroxidation of lipids was tested by measuring the thiobarbituric acid reactive substances (TBARS), as described by Cakmak and Horst [70]. The absorbance of the supernatant was measured at 532 nm and 600 nm. The values were corrected for non-specific turbidity by subtracting the absorbance. TBARS content was expressed as nmol g −1 DW.

Determination of Proline (Pro) and Glycine Betaine (GB) Content
Proline (Pro) content was determined based on the reaction of proline with acid ninhydrin [71]. The mixture was extracted with toluene, and the free toluene was quantified at 528 nm using L-proline as standard.
Glycine betaine (GB) content was measured by the method of Grieve and Grattan [72]. Using aqueous extracts of dry-ground material after reaction with KI 2 -I 2 , GB concentration was estimated at 365 nm.

Assay of Antioxidant Enzymes
A crude enzyme extract was prepared prior to determination of activities of antioxidant enzyme. Seedlings were homogenized with three volumes (w/v) of an ice-cold extraction buffer (1 mM MgCl 2 , 1.5% (w/w) polyvinylpyrrolidone, 1 mM EDTA, and 50 mM Tris-HCl, pH 7.8). The supernatant was collected after centrifugation of homogenate at 15,000×g at 4 • C for 20 min. The supernatant was stored at −20 • C, and was used as the crude extract for the assay of activities of superoxide dismutase (SOD), peroxidase (POX), and catalase (CAT). Superoxide dismutase (SOD; EC 1.15.1.1) activity was determined by estimating the enzymes's capacity of inhibiting the photochemical reduction of nitroblue tetrazolium [73]. The absorbance of the solution was measured at 560 nm.
Catalase (CAT; EC 1.11.1.6) activity was measured according to Cakmak and Marschner [75]. The reaction mixture was comprised of 0.1 mL enzyme extract, 10 mM H 2 O 2 , and 25 mM sodium phosphate buffer (pH 7.0). A decrease in absorbance due to the decline of extinction of H 2 O 2 was recorded at 240 nm.

Determination of OAS-TL Enzyme Activity and Cys Content
The activity of O-acetylserine (thiol) lyase (OAS-TL; EC 2.5.1.47) and cysteine content was determined according to Gaitonde [76], as explained by Riemenschneider et al. [77] with a few modifications. OASTL activity and Cys content were determined in soluble protein extract. The assay mixture (1 mL) for OAS-TL activity contained 50 mL enzyme extract, 100 mM Tris-HCl (pH 7.5), 5 mM Na 2 S, 33.4 mM dithiotreitol, and 5 mM O-acetyl serine (OAS) [78]. The Cys concentration (n mol Cys min −1 mg −1 protein) was determined by adding Na 2 S to the reaction mixture, and incubated for 30 min at 37 • C, followed by the addition of 1 mL acid ninhydrin reagent [76]. The Cys content was estimated using pure Cys as standard. The absorbance was measured at 560 nm, and the result was expressed as n mol g −1 DW.

Measurement of LCD and DCD Enzyme Activities and H 2 S Content
The activities of L-cysteine desulfhydrase (LCD; EC 4.4.1.1) and D-cysteine desulfhydrase (DCD; EC 4.4.1.15) were determined as described by Bloem et al. [79] and Riemenschneider et al. [80], respectively. LCD activity in enzyme extract (extracted in Tris-HCl buffer, pH 9.0) was measured by the release of H 2 S from L-Cys. The absorbance was read at 670 nm. DCD activity was determined by the same method, except that D-Cys was used instead of L-Cys, and the pH of the Tris-HCl buffer was 8.0 instead of 9.0. The standard curve of known concentrations of Na 2 S were prepared for the quantification of H 2 S.
Concentration of H 2 S in fresh tissues was determined according to Nashef et al. [81]. The assay mixture contained 20 mL of 20 mM 5,5'-dithiobis(2-nitrobenzoic acid) and 1880 µL extraction buffer. The assay mixture was incubated at room temperature for 2 min, and the absorbance was read at 412 nm. The values were compared with the standard curve of Na 2 S solutions of different concentrations.

Estimation of Photosynthetic Pigments and Carbonic Anhydrase (CA) Activity
The method of Lichtenthaler and Buschmann [82] was used for the estimation of chlorophyll (Chl) and total carotenoid content. The pigment solution of fresh tissue was prepared by grinding the samples with 100% acetone. The optical density of the pigment solution was recorded at 662, 645, and 470 nm to determine Chl a, Chl b, and total carotenoid content, respectively, using a spectrophotometer.

Statistical Analysis
Data were presented as the means ± SE of three independent replicates. The data were analyzed statistically with SPSS-17 statistical software (SPSS Inc., Chicago, IL, USA). Means were statistically compared by Duncan's multiple range test (DMRT) at p < 0.05% level.

Conclusions
Dehydration stress enhanced the accumulation of osmolytes and activities of antioxidant enzymes. Concurrently, DS also caused excessive generation of ROS and decrease in hydration level of plants. Therefore, dehydration-induced enhancement of osmolytes and antioxidant enzymes was not capable of providing enough protection against the detrimental effects of DS. However, application of H 2 S donor NaHS induced Cys content via enhancing the activity of OAS-TL enzyme. Exogenous application of H 2 S also induced the activities of LCD and DCD enzymes that synthesized more H 2 S from accumulated Cys, and enhanced the concentration of endogenous H 2 S. An enhanced level of H 2 S accelerated the synthesis of osmolytes and activities of antioxidant enzymes and CA, that enabled the plants to counter the damaging effects of DS. A constructive effect of H 2 S on dehydration-induced impairment was also witnessed by reduced H 2 O 2 and O 2 •− content, electrolyte leakage, and TBARS, resulting in an improved hydration level, enhanced concentration of photosynthetic pigments, and upregulated CA activity, a key enzyme of photosynthesis ( Figure 5). Consequently, it can be concluded that H 2 S alleviated DS by enhancing Cys synthesis and endogenous H 2 S accumulation, that induced osmolyte accumulation and an antioxidant system which enabled the plants to perform normally even under adverse conditions of dehydration ( Figure 5).