EFFECT OF PRETREATMENT WITH HYDROGEN SULFIDE DONOR

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Biologia 69/8: 1001—1009, 2014 Section Botany DOI: 10.2478/s11756-014-0396-2

Effect of pretreatment with hydrogen sulfide donor sodium hydrosulfide on heat tolerance in relation to antioxidant system in maize (Zea mays) seedlings Zhong-Guang Li*, Xiao-Yun Yi & Yu-Ting Li School of Life Sciences, Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Key Laboratory of Biomass Energy and Environmental Biotechnology, Yunnan Province, Yunnan Normal University, Kunming 650092, P.R. China; e-mail: zhongguang [email protected]

Abstract: Hydrogen sulfide (H2 S) is a signal molecule that is involved in plant growth, development and the acquisition of stress tolerance including heat tolerance, but the mechanism of H2 S-induced heat tolerance is not completely clear. In present study, the effect of sodium hydrosulfide (NaHS), a H2 S donor, treatment on heat tolerance of maize seedlings in relation to antioxidant system was investigated. The results showed that NaHS treatment improved survival percentage of maize seedlings under heat stress in a concentration-dependent manner, indicating that H2 S treatment could improve heat tolerance of maize seedlings. To further study mechanism of NaHS-induced heat tolerance, catalase (CAT), guaiacol peroxidase (GPX), superoxide dismutase (SOD), glutathione reductase (GR) and ascorbate peroxidase (APX) activities, and glutathione (GSH) and ascorbic acid (AsA) contents in maize seedlings were determined. The results showed that NaHS treatment increased the activities of CAT, GPX, SOD and GR, and GSH and AsA contents as well as the ratio of reduced antioxidants to total antioxidants [AsA/(AsA+DHA) and GSH/(GSH +GSSG)] in maize seedlings under normal culture conditions compared with the control. Under heat stress, antioxidant enzymes activities, antioxidants contents and the ratio of the reduced antioxidants to total antioxidants in control and treated seedlings all decreased, but NaHS-treated seedlings maintained higher antioxidant enzymes activities and antioxidants levels as well as the ratio of reduced antioxidants to total antioxidants. All of above-mentioned results suggested that NaHS treatment could improve heat tolerance of maize seedlings, and the acquisition of this heat tolerance may be relation to enhanced antioxidant system activity. Key words: antioxidants; antioxidant enzymes; heat stress; heat tolerance; hydrogen sulfide; maize seedlings Abbreviations: ANOVA, analysis of variance; APX, ascorbate peroxidase; AsA, ascorbic acid; CAT, catalase; CO, carbon monoxide; DTT, dithiothreitol; DHA, dehydroascorbate; FW, fresh weight; GR, glutathione reductase; GSH, glutathione; GSSG, oxidized glutathione; GST, glutathione S-transferase; MDA, malondialdehyde; NO, nitric oxide; NBT, nitroblue tetrazolium; GPX, guaiacol peroxidase; ROS, reactive oxygen species; SOD, superoxide dismutase; TBA, 2-thiobarbituric acid; TCA, trichloroacetic acid.

Introduction Land plants, being sessile organisms, are constantly subjected to various kinds of abiotic and biotic stresses like extremes in temperature, drought, high salinity, and mechanical stress. Among these stress factors, high temperature is a major abiotic factor affects metabolism, growth, development, reproduction, yield, and even survival of plants including crop plants like maize (Zea mays L.) (Li & Gong 2011; Saidi et al. 2011; Mittler et al. 2012; Wu et al. 2012; Grover et al. 2013; Piterková et al. 2013). In crop plants, maize not only is a new model plant, but also the third most important food grain crop after wheat and rice, and heat stress is the principal cause of maize failure worldwide, global warming accentuates this problem (Leip-

ner & Stamp 2009; Strable & Scanlon 2009). Numerous studies have clearly illustrated high temperature stress commonly leads to oxidative stress as result of peroxidation of membrane lipids, degradation of proteins, inactivation of enzymes, DNA damage, etc., (Saidi et al. 2011; Mittler et al. 2012; Wu et al. 2012; Grover et al. 2013; Piterková et al. 2013; Tari et al. 2013). The degree of oxidative stress is determined by the level of reactive oxygen species (ROS) such as superoxide radi. cal (O2− ), hydrogen peroxide (H2 O2 ), hydroxyl radical . − (OH ) (Saidi et al. 2011; Mittler et al. 2012; Wu et al. 2012; Piterková et al. 2013). During the process of evolution, plants have developed the antioxidant system, including antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), guaiacol peroxidase (GPX) and ascor-

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1002 bate peroxidase (APX), as well as low-molecular weight water-soluble antioxidants ascorbic acid (AsA) and glutathione (GSH), to maintain ROS homeostasis in plant cells (Foyer & Noctor 2009, 2011; Jaleel et al. 2009; Ahmad et al. 2010; Szarka et al. 2012; Garg & Kaur 2013). Therefore, the balance between ROS and antioxidant system plays crucial role in the acquisition of heat stress in plants (Wahid et al. 2007; Ahmad et al. 2010; Saidi et al. 2011; Mittler et al. 2012; Szarka et al. 2012; Wu et al. 2012). However, the effect of hydrogen sulfide treatment on heat tolerance of maize seedlings in relation to antioxidant system is not fully understood. Hydrogen sulfide (H2 S) has recently been considered as a third endogenous gaseous transmitter in animal systems after nitric oxide (NO) and carbon monoxide (CO), and it participates in multiple physiological processes (Li et al. 2011; Wang 2012; Hancock & Whiteman 2014). In plant systems, hydrogen sulfide has been found to play multiple physiological roles in plant growth, development and the acquisition of stress tolerance like heat tolerance (Lisjak et al. 2010, 2013; Li 2013). The novel functions of hydrogen sulfide have been uncovered in seed germination (Zhang et al. 2010a; Li et al. 2012a), root organogenesis (Zhang et al. 2009a) and stomata movement (Lisjak et al. 2010; Liu et al. 2011; Jin et al. 2013), as well as in alleviation in osmotic stress (Zhang et al. 2009b), salt stress (Wang et al. 2012), oxidative stress (Shan et al. 2011, Zhang et al. 2011), heavy metal stress (Zhang et al. 2008, 2010b; Chen et al. 2013) and pathogen infection (Bloem et al. 2011). Our previous results also showed that pretreatment with sodium hydrosulfide (NaHS), a hydrogen sulfide donor, could improve resistance of tobacco cells (Li et al. 2012b) and maize seedlings (Li et al. 2013a,b, 2014) to high temperature, but its mechanism is not fully clear. In this study, effect of NaHS pretreatment on heat tolerance of maize seedlings in relation to antioxidant system was investigated. The objective was to further expound possible mechanisms of NaHS-induced heat tolerance in maize seedlings. Material and methods Plant material and sodium hydrosulfide treatment Commercial variety of maize (Zea mays L., Huidan No. 4) was used in the present experiments. The seeds were sterilized in 0.1% HgCl2 for 10 min, and pre-soaked in distilled water for 12 h for imbibition. The soaked seeds were sowed on six layers of wetted filter papers in trays (24 cm×16 cm, approximately 300 seeds per tray) with covers and germinated at 26 ◦C in the dark for 2.5 d. After germination, the seedlings with unanimous growth were irrigated with 100 mL of 0 (control), 0.1, 0.3, 0.5, 0.7, 0.9, 1.5, 2.0 and 3.0 mM sodium hydrosulfide (NaHS) (the pH of the solution was adjusted to 6.0 with 1 M HCl), a hydrogen sulfide donor (Lisjak et al. 2013; Wang 2012; García-Mata & Lamattina 2013) for 6 h (pretreatment with NaHS had no significant effect on the growth of seedlings). Afterward, NaHS-treated seedlings in trays with covers were exposed to high temperature at 47 ◦C in the dark (70% RH) for 15 h for heat stress. At the end of heat stress, seedlings were transferred to a climate chamber with 26 ◦C, 100 µmol m−2 s−1 and 12 h pho-

Z.-G. Li et al. toperiod for a week for recovery, as well as irrigated with 1/2 Hoagland solution daily. Survival percentage (%) was counted after recovery, and the seedlings that could regrow and become green during recovery were considered to have survived (Li et al. 2013a, b). Determination of antioxidant enzyme activities During the course of NaHS treatment and heat stress, antioxidant enzymes CAT, GPX, SOD, GR and APX in maize seedlings were extracted and measured according to our methods described previously (Li et al. 2013c). In brief, maize seedlings (0.5 g) were ground in a mortar with a pestle in 5 mL of extraction buffer contained 50 mM Tris-HCl (pH 7.0), 0.1 mM EDTA, 1 mM AsA, 1 mM dithiothreitol (DTT) and 5 mM MgCl2 . The homogenates were centrifuged at 10,000 g for 15 min at 4 ◦C. The supernatants were used for assays of antioxidant enzymes. Detailed measurement protocols were described as follow, respectively. CAT (EC1.11.1.6) activity was determined by measuring the decrease in the absorbance of H2 O2 at 240 nm. The 3 ml reaction mixture consisted of 50 mM Tris-HCl (pH 7.0), 0.1 mM EDTA and 0.1 mL enzyme extract. The reaction was initiated by adding 12.5 mM H2 O2 (final concentration). CAT activity was computed using the extinction coefficient of 0.04 mM−1 cm−1 at 240 and expressed as µmol H2 O2 g−1 dry weight (DW) min−1 . GPX (EC1.11.1.7) activity was estimated by measuring the increase in absorbance at 470 nm due to guaiacol oxidation. The reaction mixture contained 50 mM TrisHCl (pH 7.0), 10 mM guaiacol and 5 mM H2 O2 . The reaction was initiated by adding 0.1 mL enzyme extract. GPX activity was counted using the extinction coefficient of 26.6 mM−1 cm−1 at 470 nm and expressed as µmol g−1 DW min−1 . SOD (EC1.11.1.6) activity was determined by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT). The 3 mL reaction mixture contained 50 mM Tris-HCl (pH 7.8), 13.37 mM methionine, 0.1 mM NBT, 0.1 mM riboflavin, 0.1 mM EDTA and 0.1 mL enzyme extract. One unit of enzyme activity was defined as the amount of the enzyme bringing about 50% inhibition of the photochemical reduction of NBT and the activity of SOD was expressed as U g−1 DW. GR (EC1.6.4.2) was assayed by monitoring the increase in absorbance at 340 nm. The reaction mixture contained 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5 mM MgCl2 , 0.2 mM NADPH and 0.1 mL enzyme extract, and distilled water to make up a volume of 1 mL. Reaction was initiated by adding 0.5 mM GSSG (oxidized glutathione, final concentration). GR activity was calculated using the extinction coefficient of 6.2 mM−1 cm−1 at 340 and expressed as µmol g−1 DW min−1 . APX (EC1.11.1.1) activity was measured by monitoring the rate of AsA oxidation at 290 nm. The assay mixture contained 50 mM Tris-HCl (pH 7.0), 0.1 mM H2 O2 , 0.1 mM EDTA and 0.1 mL enzyme extract. The reaction was initiated by adding 0.5 mM AsA (final concentration). APX was detected according to the reduction value of the absorbance at 290 nm per unit time, and APX activity was counted using the extinction coefficient of 2.8 mM−1 cm−1 at 290 and expressed as µmol g−1 DW min−1 . Measurement of water-soluble antioxidant content In addition to antioxidant enzyme activities, during the process of NaHS treatment and heat stress, antioxidants GSH and AsA in maize seedlings were extracted and measured as our procedures described previously (Li et al. 2013c). Unauthenticated Download Date | 9/13/18 9:12 PM

Antioxidant system involved in H2 S-induced thermotolerance Briefly, seedlings (0.5 g) were ground in a mortar with a pestle in 3 mL of 5 % (v/v) sulfosalicylic acid. The homogenates were centrifuged at 10,000 g for 15 min at 4 ◦C. The supernatants were used for assays of glutathione and ascorbic acid. Reduced glutathione GSH and oxidized glutathione GSSG were determined by the 5,5-dithiobis-(2-nitrobenzoic acid)-GR recycling procedure, the increase in absorbance of the reaction mixtures were measured at 412 nm. Ascorbic acid was determined using a method based on the reduction of ferric ion to ferrous ion with reduced ascorbic acid in acid solution and then the formation of the red chelate between ferrous ion and 2,2’-dipyridyl, which absorbs at 525 nm. Oxidized ascorbic acid DHA was measured according to this methods after being reduced to reduced ascorbic acid by adding DTT. The contents of GSH, GSSG, AsA and DHA, as well as the ratio of reduced antioxidants to total antioxidants [AsA/(AsA+DHA) and GSH/(GSH+GSSG)] were expressed as nmol g−1 DW, µmol g−1 DW and %, respectively. Hydrogen peroxide and malondialdehyde (MDA) contents assays To further study the effect of NaHS pretreatment and heat stress on hydrogen peroxide content and lipid peroxidation. During the course of NaHS pretreatment and heat stress, measurement of hydrogen peroxide and malondialdehyde (MDA) (indicator of peroxidation of membrane lipids) contents in maize seedlings were performed as described by Chirstou et al. (2013) with modifications. Seedling material (5 g) was ground in 5 mL of 0.1% (w/v) trichloroacetic acid (TCA) with a mortar and pestle on ice. The homogenate was centrifuged at 15,000 g for 15 min at 4 ◦C and 3 ml of the supernatant was added to 0.1 mL of 0.1% (w/v) titanium sulfate dissolved in 20% (v/v) H2 SO4 . The absorbance of assay mixture was read at 410 nm, the content of H2 O2 was calculated based on a standard curve of known concentrations of H2 O2 and expressed as µmol g−1 DW. In addition, the level of lipid peroxidation in maize seedlings was measured in terms of MDA content determined by thiobarbituric acid reaction. Maize seedlings (0.5 g) were homogenized in 0.5 mL of 0.1% (w/v) trichloroacetic acid (TCA) and centrifuged at 10,000 g for 10 min at 4 ◦C. Supernatant was mixed with 1.5 mL of 20% (w/v) TCA containing 0.5% (w/v) 2-thiobarbituric acid (TBA). The mixtures were heated at 95 ◦C for 30 min and then quickly cooled in an ice bath. The mixtures were centrifuged at 10,000 g for 5 min at 4 ◦C and their absorbance was measured at 532 nm. The value of non-specific absorption at 600 nm was subtracted from the 532 nm reading. The MDA content was calculated using the extinction coefficient of 155 mM−1 cm−1 at 532 nm and expressed as nmol g−1 DW. Statistics analysis The experiment was set up according to a completely randomized design with at least tree replications. The data were processed statistically using software package SPSS version 21.0 (SPSS, Chicago, USA) and the comparison of averages of each treatment was based on the analysis of variance (oneway ANOVA) according to Duncan’s multiple range test at a significance level of 5% (P < 0.05). Figures were drawn by SigmaPlot 12.5 (Systat Software Inc., London, UK), error bars represent standard error and each data in figure represents the mean ± SE of at least three independent experiments, different letters indicate significant differences (P < 0.05).

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Fig. 1. Effect of sodium hydrosulfide (NaHS) treatment on survival percentage of maize seedlings under heat stress at 47 ◦C. 2.5-day-old seedlings of maize were irrigated with 0 (control), 0.1, 0.3, 0.5, 0.7, 0.9, 1.5, 2.0 and 3.0 mM NaHS for 6 h (approximately 300 seedlings were investigated each treatment), and then the control and NaHS-treated seedlings were exposed to heat stress at 47 ◦C for 15 h. Survival percentage (%) of the seedlings was counted after recovery under normal growth conditions for a week. Error bars represent standard error and each data in the figures represents the mean ± SE of at least three independent experiments, different letters indicate significant differences (P < 0.05) according to Duncan’s multiple test.

Results Effect of NaHS treatment on heat tolerance in maize seedlings 2.5-day-old maize seedlings treated with different concentrations of NaHS were subjected to heat stress at 47 ◦C for 15 h. As shown in Fig. 1, NaHS pretreatments improved survival percentage of maize seedlings under heat stress, and this improvement enhanced with the increasing concentration of NaHS, reaching the maximum value at 0.7 mM, approximately increased by 40% compared with the control. On the contrary, compared with the control, pretreatment with higher concentration of donors (1.5 and 2.0 mM NaHS) had no significant effects on survival percentage of maize seedlings under heat stress, but 3.0 mM NaHS weakened seedlings heat tolerance (Fig. 1). Therefore, the concentration of 0.7 mM NaHS was used in further experiments. These results implied that NaHS treatment could enhance heat tolerance of maize seedlings in a concentration-dependent manner. Effect of NaHS treatment and heat stress on antioxidant enzyme activities of maize seedlings In order to further understand the possible mechanisms of NaHS-induced heat tolerance, during the process of NaHS treatment and heat stress, the activities of the antioxidant enzymes CAT, GPX, SOD, GR and APX in maize seedlings were determined. NaHS treatment increased the activities of CAT, GPX, SOD and GR under normal culture conditions (Fig. 2a–d), and the increase in CAT and GPX activities were faster than those of SOD and GR (Fig. 2a–b). In addition, increased enzymes activities enhanced with the extension of NaHS treatment time, CAT, GPX, SOD and GR activities reached the maximum value at 6 h of NaHS treatUnauthenticated Download Date | 9/13/18 9:12 PM

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Fig. 2.Effect of NaHS treatment and heat stress on CAT (a), GPX (b), SOD (c), GR (d) and APX (e) activities in maize seedlings. 2.5-day-old seedlings of maize were irrigated with 0 (control) or 0.7 mM NaHS for 6 h, and then the control and NaHS-treated seedlings were exposed to heat stress at 47 ◦C for 15 h. During the process of NaHS treatment and heat stress, the activities of CAT, GPX, SOD, GR and APX in maize seedlings were measured. Error bars represent standard error and each data in the figures represents the mean ± SE of at least three independent experiments, different letters indicate significant differences (P < 0.05) according to Duncan’s multiple test.

ment, approximately increasing by 45%, 40%, 29% and 11% compared with the control, respectively (Fig. 2a– d). In contrast, pretreatment with NaHS lowered APX activity with the elongation of NaHS treatment time, 6 h of treatment reached significant difference, approximately decreasing by 11% (Fig. 2e). Under heat stress, all of antioxidant enzymes activities in control and treated seedlings declined with the prolongation of heat stress time, but NaHS-treated seedlings maintained higher antioxidant enzymes activities compared with the control (Fig. 2a–d). These results displayed that antioxidant enzymes CAT, GPX, SOD and GR play crucial role in NaHS-induced heat tolerance in maize seedlings.

Effect of NaHS treatment and heat stress on antioxidants GSH and AsA contents of maize seedlings In addition, during the process of NaHS treatment and heat stress, the level of the antioxidants GSH and AsA in maize seedlings was measured. During the process of NaHS treatment, GSH and AsA contents as well as the ratio of reduced antioxidants to total antioxidants [AsA/(AsA+DHA) and GSH/(GSH+GSSG)] in maize seedlings increased with the extension of NaHS treatment time, and the accumulation of GSH and increase in GSH/(GSH+GSSG) ratio were faster than those of AsA (Fig. 3a, c; Fig. 4a, c). The contents of GSH and AsA, as well as the ratios of GSH/(GSH+GSSG) and AsA/(AsA+DHA) reached the maximum value at 6 h Unauthenticated Download Date | 9/13/18 9:12 PM

Antioxidant system involved in H2 S-induced thermotolerance

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Fig. 3. Effect of NaHS treatment and heat stress on GSH (a) and GSSG (b) contents, and GSH/(GSH+GSSG) ratio (c) in maize seedlings. 2.5-day-old seedlings of maize were irrigated with 0 (control) or 0.7 mM NaHS for 6 h, and then the control and NaHS-treated seedlings were exposed to heat stress at 47 ◦C for 15 h. During the process of NaHS treatment and heat stress, the contents of GSH and GSSG as well as GSH/(GSH+GSSG) ratio in maize seedlings were measured. Error bars represent standard error and each data in the figures represents the mean ± SE of at least three independent experiments, different letters indicate significant differences (P < 0.05) according to Duncan’s multiple test.

Fig. 4. Effect of NaHS treatment and heat stress on AsA (a) and DHA (b) contents, and AsA/(AsA+DHA) ratio (c) in maize seedlings. 2.5-day-old seedlings of maize were irrigated with 0 (control) or 0.7 mM NaHS for 6 h, and then the control and NaHS-treated seedlings were exposed to heat stress at 47 ◦C for 15 h. During the process of NaHS treatment and heat stress, the contents of AsA and DHA as well as AsA/(AsA+DHA) ratio in maize seedlings were measured. Error bars represent standard error and each data in the figures represents the mean ± SE of at least three independent experiments, different letters indicate significant differences (P < 0.05) according to Duncan’s multiple test.

of NaHS treatment, approximately increasing by 84%, 13%, 14% and 7% compared with the control, respectively (Fig. 3a, c; Fig. 4a, c), similar to the trend of antioxidant enzymes activities (Fig. 2), but the change in contents of DHA and GSSG was not significantly difference (Fig. 3b; Fig 4b). In addition, during the

course of heat stress, antioxidants contents and the ratio of reduced antioxidants to total antioxidants in control and treated seedlings all declined, but NaHStreated seedlings maintained higher antioxidants contents and the ratio compared with the control (Fig. 3a, c; Fig. 4a, c), analogous to the change of antioxidant Unauthenticated Download Date | 9/13/18 9:12 PM

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the elongation of heat stress time, but NaHS-treated seedlings maintained lower level compared with the control (Fig. 5a, b), indicating that NaHS treatment could alleviate peroxidation of membrane lipids by reducing accumulation of H2 O2 as a result of enhanced antioxidant system activity. Discussion

Fig. 5. Effect of NaHS treatment and heat stress on H2 O2 (a) and malondialdehyde (MDA) (b) contents in maize seedlings. 2.5-dayold seedlings of maize were irrigated with 0 (control) or 0.7 mM NaHS for 6 h, and then the control and NaHS-treated seedlings were exposed to heat stress at 47 ◦C for 15 h. During the process of NaHS treatment and heat stress, the contents of H2 O2 and MDA in maize seedlings were measured. Error bars represent standard error and each data in the figures represents the mean ± SE of at least three independent experiments, different letters indicate significant differences (P < 0.05) according to Duncan’s multiple test.

enzymes activities (Fig. 2). In contrast, oxidized glutathione GSSG and oxidized ascorbic acid DHA contents increased with duration of heat stress, but NaHStreated seedlings maintained relatively lower level compared with the control (Fig. 3b; Fig. 4b). All of the above-mentioned results suggested that the acquisition of heat tolerance induced by NaHS could be relative to antioxidants GSH and AsA in maize seedlings. Effect of NaHS treatment and heat stress on H2 O2 and MDA contents of maize seedlings To further explore effect of NaHS treatment on endogenous H2 O2 and MDA contents in maize seedlings, during the process of NaHS treatment and heat stress, H2 O2 and MDA contents in maize seedlings were determined. The data showed that pretreatment with NaHS was not significant impact on the change in endogenous H2 O2 and MDA contents in maize seedlings under normal culture conditions (Fig. 5a, b). Under heat stress conditions, H2 O2 and MDA contents increased with

Hydrogen sulfide is emerging as a potential messenger molecule that is involved in modulation of many physiological processes and the acquisition of stress tolerance in plants (Hancock et al. 2011; Li et al. 2011; Lisjak et al. 2013; Li 2013; Hancock & Whiteman 2014). In cucumber (Cucumis sativus) seedlings, root elongation was significantly inhibited by exposure of cucumber seedlings to solutions containing 5 mM boron (B), but this inhibitory effect was substantially alleviated by treatment with NaHS, a hydrogen sulfide donor (Wang et al. 2010). In addition, under chromium (Cr) stress, the percentage of germination of wheat seeds decreased, but this decrease could be alleviated by pretreatment with NaHS in a dose-dependent manner (Zhang et al. 2010a). Exposure of alfalfa (Medicago sativa) to 100 mM NaCl, seed germination and thereafter seedling growth were inhibited, but this negative effects could significantly be attenuated by NaHS at 100 µM (Wang et al. 2012). Zhang et al. (2009b) reported that osmotic stress mimicked by PEG-6000 decreased dramatically chlorophyll in seedling leaves of sweetpotato (Ipomoea batata), and the osmotic-induced decrease in chlorophyll concentration was alleviated by spraying NaHS in a dose-dependent manner, similar to the reports of Zhang et al. (2010b) in wheat seedlings. Also, In bermudagrass, exogenous application of NaHS improved salt (150 and 300 mM NaCl), osmotic (15% and 30% PEG6000) and cold (4 ◦C) tolerances, which were evidenced by decreased electrolyte leakage and increased survival rate under stress conditions (Shi et al. 2013). Similarly, in strawberry, hydroponic pretreatment of roots with NaHS at 100 µM for 48 h could induce long-lasting priming effects and tolerance to subsequent exposure to 100 mM NaCl or 10% (w/v) PEG6000 for 7 d (Christou et al. 2013). Additionally, Fu et al. (2013) found that pretreatment with sodium hydrosulfide improved the survival rate of grape seedlings under chilling stress at 4 ◦C. In contrast, hypotaurine (a H2 S scavenger) treatment displayed contrary effect under the chilling temperature, suggesting that H2 S might be directly involved in the cold signal transduction pathway of grape. Our previous results found that NaHS pretreatment markedly improved germination percentage of seeds and survival percentage of seedlings of maize (Li et al. 2013a) and wheat (Wu et al. 2013) under drought and heat stress, and alleviated an increase in electrolyte leakage of roots, a decrease in tissue vitality and accumulation of MDA in seedlings. Similarly, pretreatment with the NO donor sodium nitroprusside (SNP) improved heat tolerance of maize seedlings, and the acquisition of this heat tolerance was involved in Unauthenticated Download Date | 9/13/18 9:12 PM

Antioxidant system involved in H2 S-induced thermotolerance the accumulation of endogenous H2 S by enhancing the activity of L-cystine desulfhydrase, a key enzyme in H2 S biosynthesis in plants, indicating H2 S may be a downstream signal molecule in NO-induced heat tolerance of maize seedlings. In present study, pretreatment with NaHS enhanced survival percentage of 2.5-dayold maize seedlings in a concentration-dependent manner (Fig. 1), consistent with the results of Zhang et al. (2009b, 2010a) in wheat and sweetpotato under Cr and osmotic stress. All of above-mentioned studies indicated that H2 S may be relative to the acquisition of stress tolerance including heat tolerance in plants, but their mechanisms are not fully clear. Cellular redox homeostasis is considered to be an “integrator” of information from metabolism and the environment controlling plant growth and acclimation responses (Foyer & Noctor 2009; Jaleel et al. 2009; Scheibe & Dietz 2012). In cucumber seedlings, total and isozymatic activities and corresponding transcripts of SOD, CAT, GPX and APX were activated differentially by NaHS, thus resulting in the alleviation of oxidative damage induced by boron (Wang et al. 2010, 2012). Pretreatment with exogenous NaHS enhanced the activities of SOD, CAT, APX and GPX in wheat (Triticum aestivum L.) seedlings, reduced the Cr-induced increase in over-production of MDA and H2 O2 , followed by alleviated decrease in the germination rate of wheat seeds under Cr stress in a dose-dependent manner (Zhang et al. 2010a). Again, application of NaHS increased the activities of APX, GR, dehydroascorbate reductase (DHAR) and γ-glutamylcysteine synthetase (γGCS), a key enzyme of GSH biosynthesis, as well as the contents of AsA, GSH, total ascorbate and total glutathione under water stress, which in turn decreased the MDA accumulation induced by water deficiency in wheat compared to control without NaHS treatment (Shan et al. 2011). Further experiments showed that NaHS treatment significantly increased CAT and APX activities, reduced lipoxygenase activity, MDA and H2 O2 accumulation in wheat seeds under osmotic stress (Zhang et al. 2010b). In addition, root pretreatment with NaHS resulted in gene expression of key antioxidant enzymes APX, CAT, SOD, GR as well as ascorbate and glutathione biosynthesis glutamylcysteine synthetase (GCS), l-galactose dehydrogenase (GDH) and glutathione synthetase (GS), reduced oxidative and nitrosative stress in strawberry plants, manifesting via lower levels of synthesis of NO and H2 O2 in leaves, and maintained high ascorbate and glutathione redox states, which in turn improved the resistance of plants to subsequent salt and non-ionic osmotic stresses (Christou et al. 2013). Similarly, NaHS treatment alleviated the reactive oxygen species (ROS) burst and cell damage induced by salt, osmotic and cold stress, via modulating metabolisms of several antioxidant enzymes CAT, POD and GR, and non-enzymatic glutathione antioxidant pool and redox state (Shi et al. 2013). In present experiment, pretreatment with NaHS increased CAT, GPX, SOD and GR activities (Fig. 2a– d), but reduced the activity of APX (Fig. 2e), these

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positive and inhibitory effects reached the maximum value at 6 h of treatment, indicating synergistic effect among antioxidant enzymes. In addition, exogenous application of NaHS also improved the contents of GSH and AsA contents, as well as the ratio of the reduced antioxidants to total antioxidants [AsA/(AsA+DHA) and GSH/(GSH+GSSG)] (Fig. 3a, c; Fig. 4a, c) under normal culture conditions. Enhancement of abovementioned antioxidant system activity may be achieved through upregulation of gene expression of antioxidant enzymes, antioxidant biosynthetic enzymes, and heat shock proteins (Christou et al. 2014). Under heat stress conditions, treated seedlings maintained higher antioxidant enzymes activities and antioxidants levels as well as the ratio of the reduced antioxidants to total antioxidants from beginning to end compared with the control (Figs 2, 3, 4), which in turn alleviated the accumulation of H2 O2 and MDA (Fig. 5). These investigations showed that hydrogen sulfide involved in the acquisition of various stress tolerance including heat tolerance, and antioxidant system plays a very important role in hydrogen sulfide-induced stress tolerance in plants. In summary, it is clearly shown that NaHS treatment could improve survival percentage of maize seedlings in a concentration-dependent manner under heat stress. In addition, NaHS treatment also could increase activities of CAT, GPX, SOD and GR, and levels of antioxidants AsA and GSH, as well as the ratio of reduced antioxidants to total antioxidants [AsA/(AsA+DHA) and GSH/(GSH+GSSG)] under normal culture conditions. Under heat stress, NaHS-treated seedlings maintained higher antioxidant enzyme activities and antioxidant levels, as well as the ratio of reduced antioxidants to total antioxidants from beginning to end compared with control. These results indicated that NaHS pretreatment could improve heat tolerance of maize seedlings, and the acquisition of heat tolerance induced by NaHS may be relative to enhanced antioxidant system activity. However, signal crosstalk among Ca2+ , H2 O2 , NO, H2 S, and plant hormones in the acquisition of heat tolerance in plants needs to be further illustrated in future study.

Acknowledgements This work was supported by the National Natural Science Foundation of China (31360057). We appreciate the editors and reviewers for their exceptionally helpful comments about the manuscript.

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