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Plant, Cell and Environment (2013) 36, 1564–1572

doi: 10.1111/pce.12092

Hydrogen sulphide may be a novel downstream signal molecule in nitric oxide-induced heat tolerance of maize (Zea mays L.) seedlings ZHONG-GUANG LI, SHI-ZHONG YANG, WEI-BIAO LONG, GUO-XIAN YANG & ZHEN-ZHEN SHEN

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 Normal University, Kunming 650092, Yunnan Province, China

ABSTRACT Nitric oxide (NO) is a second messenger with multifunction that is involved in plant growth, development and the acquisition of stress tolerance. In recent years, hydrogen sulphide (H2S) has been found to have similar functions, but crosstalk between NO and H2S in the acquisition of heat tolerance is not clear. In this study, pretreatment with the NO donor sodium nitroprusside (SNP) improved the survival percentage of maize seedlings and alleviated an increase in electrolyte leakage and a decrease in tissue vitality as well as accumulation of malondialdehyde, indicating that pretreatment with SNP improved the heat tolerance of maize seedlings. In addition, pretreatment with SNP enhanced the activity of L-cystine desulfhydrase, which, in turn, induced accumulation of endogenous H2S, while application of H2S donors, NaHS and GYY4137, increased endogenous H2S content, followed by mitigating increase in electrolyte leakage and enhanced survival percentage of seedlings under heat stress. Interestingly, SNP-induced heat tolerance was enhanced by application of NaHS and GYY4137, but was eliminated by inhibitors of H2S synthesis DL-propargylglycine, aminooxyacetic acid, potassium pyruvate and hydroxylamine, and the H2S scavenger hypotaurine. All of the above-mentioned results suggest that SNP pretreatment could improve heat tolerance, and H2S may be a downstream signal molecule in NO-induced heat tolerance of maize seedlings. Key-word: heat stress. Abbreviations: AOA, aminooxyacetic acid; anova, analysis of variance; L-DES, L-cystine desulfhydrase; FW, fresh weight; GYY4137, morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate; HA, hydroxylamine; HT, hypotaurine; MDA, malondialdehyde; NO, nitric oxide; PAG, DLpropargylglycine; PP, potassium pyruvate; SNP, sodium nitroprusside; TTC, triphenyl tetrazolium chloride.

INTRODUCTION Because of the sessile characteristic of land plants, they are constantly exposed to various abiotic and biotic stresses such Correspondence: Z.-G. Li. e-mail: [email protected] 1564

as extreme temperatures, drought, high salinity and mechanical stress. Among these stresses, high temperature is a central environmental factor with a far-reaching impact on metabolism, growth, development, reproduction and yield of plants (Königshofer, Tromballa & Loppert 2008; Li & Gong 2011; Saidi, Finka & Goloubinoff 2011; Mittler, Finka & Goloubinoff 2012; Wu et al. 2012). Maize is 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 (Leipner & Stamp 2009). Many studies have illustrated that high temperatures lead to an increase in fluidity of membrane lipids and loss of membrane integrity, protein denaturation and aggregation, inhibition of protein synthesis and acceleration of protein degradation, inactivation of enzymes in chloroplast and mitochondria, oxidative stress and osmotic stress (Wahid et al. 2007; Hanumappa & Nguyen 2010; Saidi et al. 2011; Mittler et al. 2012; Wu et al. 2012). Based on these negative effects, plants are forced to invest valuable resources in modifying their metabolism to prevent damage caused by heat, in a process generally referred to as heat acclimation or heat tolerance, corresponding adaptive mechanism including repair and reestablishment of biomembrane, stress protein synthesis, enhancement in antioxidant capacity, osmotic adjustment and so on (Wahid et al. 2007; Königshofer et al. 2008; Saidi et al. 2011; Mittler et al. 2012; Wu et al. 2012). Nitric oxide (NO), an ubiquitous gaseous molecule, has gained an increasing attention of researchers as a secondary messenger in plant cells (Blokhina & Fagerstedt 2010; Moreau et al. 2010; Siddiqui, Al-Whaibi & Basalah 2011; Hancock et al. 2011a; Fan, Du & Guo 2013; García-Mata & Lamattina 2013; Gill et al. 2013). It is now largely accepted that NO is a ‘jack-of-all-trades’ molecule, regulating plant cell responses under physiological and pathological conditions throughout the entire plant life cycle. In addition to that, NO controls plant responses to a wide range of abiotic factors such as extreme temperatures, high salinity, hypoxic conditions, mechanical and oxidative injuries, heavy metal accumulation, treatment with herbicides, osmotic stresses, light, gravity and UV-B (Santa-Cruz et al. 2010; Baudouin 2011; Siddiqui et al. 2011; Fan et al. 2013; García-Mata & Lamattina 2013; Gill et al. 2013). Pretreatment with sodium nitroprusside (SNP), an NO donor, could improve the heat tolerance of rice seedlings (Uchida et al. 2002) and callus of reed (Song © 2013 John Wiley & Sons Ltd

H2S mediates NO-induced heat tolerance et al. 2006). NO acts as a second messenger downstream of hormones in many signalling cascades involving Ca2+, cGMP, MAPK, CDPK and TFs, among other cellular regulators (Astier et al. 2010; Saidi et al. 2011; Hancock, Neill & Wilson 2011b; Ma et al. 2012; García-Mata & Lamattina 2013). Hydrogen sulphide (H2S), a small flammable colourless gas with a characteristic odour of rotten eggs, has long been known as a phytotoxin (Zhang et al. 2010a,b; Chen et al. 2011; Li, Rose & Moore 2011; García-Mata & Lamattina 2013; Jin et al. 2013; Lisjak et al. 2013). For example, exposure to 3000 parts per billion (ppb) in air H2S caused lesions on leaves, defoliation and reduced growth of the plants (Nakamura et al. 2009). However, in animal systems, H2S has recently been identified as a third endogenous gaseous transmitter after NO and CO; it plays multiple physiological roles (Li et al. 2011). In plant systems, the positive effect of H2S is being emerged in seed germination (Zhang et al. 2010b; Li, Gong & Liu 2012a), organogenesis (Zhang et al. 2009a), stomata opening (Lisjak et al. 2010; Liu et al. 2011; Jin et al. 2013), osmotic stress (Zhang et al. 2009b), salt stress (Wang et al. 2012), oxidative stress (Shan et al. 2011; Zhang et al. 2011) and heavy metal stress (Zhang et al. 2008, 2010a; Chen et al. 2013). Our previous results also showed that treatment with sodium hydrosulphide (NaHS), an H2S donor, can improve resistance of tobacco cells (Li et al. 2012b) and maize seedlings (Li, Ding & Du 2013) to high temperature and the acquisition of heat tolerance involved in calcium messenger system and osmolyte proline. More recently, it was reported that ethylene-induced stomatal closure requires H2S, and the Arabidopsis mutant Atl-cdes does not close the stomata in response to ethylene or NO, suggesting that ethylene and NO act upstream of H2S (García-Mata & Lamattina 2013). On the basis of these studies, we hypothesized that interaction between NO and H2S may exist in the acquisition of heat tolerance of plants. However, in maize seedlings, crosstalk between NO and H2S in the acquisition of thermotolerance is poorly known. In this study, the effect of SNP, an NO donor, treatment on heat tolerance and involvement of H2S in maize seedlings was investigated; the results indicated that SNP treatment could improve heat tolerance, and hydrogen sulphide may be a downstream signal molecule in NOinduced heat tolerance of maize seedlings.

MATERIALS AND METHODS Plant material and sodium nitroprusside treatment A commercial variety of maize (Zea mays L., Yedan no. 51) was used in the present experiments. The seeds were sterilized in 0.1% HgCl2 for 10 min and pre-soaked in distilled water 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 germinated for 2.5 d, the seedlings with unanimous growth were treated with 0 (control), 50, 100, 150, 200, 300 and 400 mm SNP for 6 h

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(pretreatment with SNP had no significant effect on the growth of seedlings). Afterwards, treated seedlings were exposed to high temperature at 48 °C for 18 h for heat stress. At the end of heat stress, seedlings were transferred to a climate chamber with 26 °C and 30 mmol m-2 s-1 as well as 12 h photoperiod for a week for recovery and 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. 2013).

Electrolyte leakage, tissue vitality and malondialdehyde (MDA) content assays To study further the effect of SNP pretreatment on electrolyte leakage, tissue vitality and lipid peroxidation, the 2.5day-old seedlings were subjected to heat stress at 48 °C for 18 h; after being treated with different concentrations of SNP, electrolyte leakage of roots, tissue vitality and MDA (indicator of lipid peroxidation) content in coleoptiles were assayed according to our previous methods (Li et al. 2012b, 2013). One centimetre roots in seedlings treated with different concentrations of SNP were cut off and the electrolyte leakage from the roots was measured with a conductometer. Similarly, the vitality of maize coleoptiles and roots was measured using triphenyltetrazolium chloride (TTC): 0.2 g of sample was cultured in 0.6% TTC solution at 27 °C for 15 h, then the TTC solution was drained off and the coleoptile samples were homogenized in 95% (v/v) ethanol. The crude homogenate was heated in an 80 °C water bath for 10 min for the extraction of formazan, then 95% ethanol was added to the homogenate to a final volume of 25 mL and the mixture was centrifuged at 10 000 g for 10 min. The absorbance of the supernatant at 485 nm was determined spectrophotometrically. In addition, the level of lipid peroxidation in coleoptiles and roots of maize seedlings under heat stress was measured in terms of MDA content, which was determined by thiobarbituric acid reaction. Electrolyte leakage, tissue vitality and MDA content were expressed as %, A485 and nmol g-1 FW (fresh weight), respectively.

Determination of activity of L-cysteine desulfhydrase L-cysteine desulfhydrase (L-DES, E.C. 4.4.1.1.) is considered to be a key enzyme in hydrogen sulphide biosynthesis in plants (Wang 2012; García-Mata & Lamattina 2013; Lisjak et al. 2013). The 2.5-day-old seedlings were treated with 150 mm SNP, and then the activity of L-DES was determined as previously described in the methods of Li et al. (2012a). Coleoptiles or roots (5 g) were ground with a mortar and pestle in liquid nitrogen and the soluble proteins were extracted by adding 5 mL of 20 mm Tris–HCl (pH 8.0). After centrifugation, the protein content of the supernatant was adjusted to 100 mg mL-1 to obtain equal amounts of protein in each assay sample. L-cysteine desulfhydrase activity was determined by the release of H2S from L-cysteine in the presence of dithiothreitol (DTT).

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The assay contained in a total volume of 1 mL: 0.8 mm L-cysteine, 2.5 mm DTT, 100 mm Tris–HCl (pH 9.0) and 10 mg protein solution. The reaction was initiated by the addition of L-cysteine; after incubation for 15 min at 37 °C, the reaction was terminated by adding 100 mL of 30 mm FeCl3 dissolved in 1.2 m HCl and 100 mL of 20 mm N,Ndimethyl-p-phenylenediamine dihydrochloride dissolved in 7.2 m HCl. The amount of H2S was determined colorimetrically at 667 nm after incubation for 15 min at room temperature. Blanks were prepared by the same procedures and known concentrations of Na2S were used in a standard curve, and the activity of L-cysteine desulfhydrase was expressed as nmol g-1 FW min-1.

Measurement of H2S content In order to understand the effect of SNP treatment on endogenous H2S content, H2S in coleoptiles and roots of seedlings treated with 150 mm SNP was measured by the formation of methylene blue from dimethyl-p-phenylenediamine in H2SO4, according to our previous method (Li et al. 2012a, 2013). Coleoptiles or roots (5 g) were ground and extracted in 10 mL of phosphate-buffered saline (pH 6.8, 50 mm) containing 0.1 mm EDTA and 0.2 mm ascorbic acid. The homogenate was mixed in a test tube containing 100 mm phosphate-buffered saline (pH 7.4), 10 mm L-cysteine and 2 mm phosphopyridoxal at room temperature, and the released H2S was absorbed in a zinc acetate trap, a small glass tube containing zinc acetate, fixed at the bottom of the test tube. After 30 min of reaction, 0.3 mL of 5 mm dimethyl-pphenylenediamine dissolved in 3.5 mm H2SO4 was added to the trap, followed by injection of 0.3 mL of 50 mm ferric ammonium sulphate in 100 mm H2SO4. The amount of H2S in the zinc acetate trap was determined colorimetrically at 667 nm after incubation for 15 min at room temperature. A calibration curve was made according to the above methods, and H2S content in maize seedlings was expressed as nmol g-1 FW.

Hydrogen sulphide donor treatment and heat tolerance NaHS and morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (GYY4137) were commonly used as donors for H2S. They release H2S when dissolved in water; NaHS giving a relatively short burst of H2S, while GYY4137 giving a longer more prolonged exposure to H2S (Wang 2012; García-Mata & Lamattina 2013; Lisjak et al. 2013). To study the effect of NaHS and GYY4137 pretreatment on heat tolerance and H2S content of maize seedlings, the 2.5-day-old seedlings were transferred to aqueous solution of 0 (control), 100, 500, 1000 and 1500 mm NaHS or GYY4137 (the pH of the solution was adjusted to 6.0 with 1 m HCl) for 6 h (pretreatment with NaHS or GYY4137 had no significant effect on the growth of seedlings), and the survival percentage, electrolyte leakage of roots and content of endogenous H2S in coleoptiles and roots were measured as in the above-mentioned method.

Inhibitors of H2S biosynthesis and its scavenger treatment and heat tolerance DL-propargylglycine (PAG), aminooxyacetic acid (AOA), potassium pyruvate (PP) and hydroxylamine (HA) are commonly used as inhibitors of H2S biosynthesis in plants, while hypotaurine (HT) is a scavenger of H2S (Liu et al. 2011; Li et al. 2012a; Lisjak et al. 2013). To study the effect of this chemical pretreatment on SNP-induced heat tolerance of maize seedlings, the 2.5-day-old seedlings were transferred to aqueous solution of 0 mm SNP (-S), 150 mm SNP (+S), 150 mm SNP + 500 mm NaHS (S + SH), 150 mm SNP + 500 mm GYY4137 (S + GY), 150 mm SNP + 300 mm PAG (S + PG), 150 mm SNP + 300 mm AOA (S + AA), 150 mm SNP + 300 mm PP (S + PP), 150 mm SNP + 300 mm HA (S + HA), 150 mm SNP + 300 mm HT (S + HT) for 6 h (chemical treatments had no significant effect on the growth of seedlings), respectively, and the survival percentage of seedlings were measured as in the above-mentioned method.

Statistical analysis All experiments were repeated at least three times and two replications in each time. The results were processed statistically using one-way analysis of variance (anova). The figures were drawn by SigmaPlot 10.0 (Systat Software Inc., London, UK); error bars represent standard error, and each data in the figure represents the mean ⫾ SE of at least three experiments.

RESULTS Effect of SNP treatment on heat tolerance in maize seedlings The 2.5-day-old maize seedlings treated with SNP were subjected to heat stress at 48 °C for 18 h. As shown in Fig. 1, SNP pretreatments increased the survival percentage of maize seedlings and alleviated the increase in electrolyte leakage that occurred under high temperature stress; in particular, the treatment with 150 mm SNP showed a very significant difference compared with the control without SNP treatment among the five concentrations (P < 0.01). Therefore, the concentration of 150 mm SNP was used in further experiments. In addition to measuring the survival percentage and electrolyte leakage, the effect of SNP on heat tolerance of maize seedlings was further investigated. Tissue vitality (triphenyl tetrazolium chloride reduction) and MDA content were measured in coleoptiles and roots after the 2.5-day-old seedlings were treated with different concentrations of SNP and exposed to heat stress at 48 °C for 18 h; the results showed that pretreatment with SNP alleviated the reduction in tissue vitality (Fig. 2a) and accumulation of MDA (Fig. 2b) in coleoptiles and roots of maize seedlings under heat stress at 48 °C for 18 h. The treatment with 150 mm SNP showed the most significant difference compared with the control among the five concentrations (P < 0.01), similar to the change in survival percentage (Fig. 1), but higher concentration of SNP (ⱖ300 mm) reduced the survival percentage (Fig. 1) and tissue vitality, as well as accelerated MDA accumulation

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with the elongation of treatment time; 4 and 6 h of treatment reached very significant difference (P < 0.01; Fig. 3a). In addition, L-DES activity in roots was slightly higher than that of coleoptiles from beginning to end (Fig. 3a). For change in H2S content, as shown in Fig. 3b, the pretreatment with SNP gradually increased the content of H2S in coleoptiles and roots compared with the control, and reached very significant difference level at 4 and 6 h of treatment (P < 0.01; Fig. 3b), consisting the change in L-DES activity (Fig. 3a). Furthermore, H2S content in roots was higher than that of coleoptiles throughout, similar to L-DES activity (Fig. 3a).

Effect of H2S donor pretreatment on heat tolerance and endogenous H2S content of maize seedlings The 2.5-day-old seedlings were treated with different concentrations of NaHS and GYY4137 (Fig. 4). Pretreatment with

Figure 1. Effects of sodium nitroprusside (SNP) pretreatment on the survival percentage (a) and electrolyte leakage (b) of maize seedlings under heat stress at 48 °C for 18 h. The 2.5-day-old seedlings of maize were subjected to heat stress at 48 °C for 18 h after being treated with 0 (control), 50, 100, 150, 200, 300 and 400 mm SNP for 6 h, respectively, and the survival percentage of maize seedlings and electrolyte leakage of roots were determined. Error bars represent standard error and each data in figure represents the mean ⫾ SE of three experiments, and asterisk and double asterisks indicate significant difference (P < 0.05) and very significant difference (P < 0.01) from the control without SNP treatment, respectively.

under heat stress (Fig. 2). Higher concentration of SNP may have toxic effects on seedlings of maize under heat stress, coinciding with one of the characteristics of signal molecules such as Ca2+ and H2O2. All of the above-mentioned results illustrated that pretreatment with SNP could improve the heat tolerance of maize seedlings in a concentration-dependent manner. Figure 2. Effects of sodium nitroprusside (SNP) pretreatment on

Effect of SNP on L-DES activity and H2S content in maize seedlings As mentioned earlier, L-DES is the key enzyme in H2S biosynthesis in plants (Wang 2012; García-Mata & Lamattina 2013; Lisjak et al. 2013).After the 2.5-day-old maize seedlings were treated with 150 mm SNP, L-DES activity and H2S content in coleoptiles and roots were measured every 2 h. Pretreatment with SNP increased the activity of L-DES in maize coleoptiles and roots, and this improvement increased

tissue vitality (a) and malondialdehyde (MDA) content (b) in coleoptiles and roots of maize seedlings under heat stress at 48 °C for 18 h. The 2.5-day-old seedlings of maize were subjected to heat stress at 48 °C for 18 h after being treated with 0 (control), 50, 100, 150, 200, 300 and 400 mm SNP for 6 h, respectively, and the tissue vitality and MDA content in coleoptiles and roots of maize seedlings were determined. Error bars represent standard error and each data in figure represents the mean ⫾ SE of three experiments, and asterisk and double asterisks indicate significant difference (P < 0.05) and very significant difference (P < 0.01) from the control without SNP treatment, respectively.

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Z-G. Li et al. survival percentage and electrolyte leakage, pretreatment with 500 mm NaHS or GYY4137 rapidly improved accumulation of endogenous H2S in coleoptiles and roots; 2, 4 and 6 h of treatment reached very significant difference level (P < 0.01; Fig. 5), and H2S accumulation by NaHS treatment was faster than that of GYY4137, as well as this accumulation of endogenous H2S was quicker than that of induction by SNP (Fig. 3b). In addition, H2S accumulation in roots by NaHS was higher than that of coleoptiles from beginning to end (Fig. 5), similar to induction by SNP (Fig. 3b).

Effect of inhibitors of H2S biosynthesis and its scavenger on SNP-induced heat tolerance in maize seedlings Pretreatment with SNP improved the activity of L-DES (Fig. 3a) and brought about increase in accumulation of H2S

Figure 3. Effects of sodium nitroprusside (SNP) pretreatment on L-cystine desulfhydrase (L-DES) activity (a) and endogenous H2S content (b) in coleoptiles and roots of maize seedlings under normal culture conditions. The 2.5-day-old seedlings of maize were treated with 150 mm SNP for 6 h, and the L-DES activity and endogenous H2S content in both coleoptiles and roots were determined every 2 h. Error bars represent standard error and each data in figure represents the mean ⫾ SE of three experiments, and asterisk and double asterisks indicate significant difference (P < 0.05) and very significant difference (P < 0.01) from the control without sodium hydrosulphide (NaHS) treatment, respectively.

NaHS and GYY4137 increased the survival percentage of seedlings and alleviated the electrolyte leakage of roots under heat stress at 48 °C. Treatment with 500 mm NaHS or GYY4137 showed the most significant difference compared with the control without donor treatment among the four concentrations (P < 0.01). Therefore, the concentration of 500 mm NaHS was used in further experiments. On the contrary, higher concentration of donors (ⱖ1000 mm NaHS or >1000 mm GYY4137) may have toxic effects on seedlings under heat stress, that is, no significant effect on survival percentage and electrolyte leakage compared with the control, similar to SNP treatment (Fig. 1). These results implied that NaHS or GYY4137 treatment could enhance heat tolerance of maize seedlings in a concentrationdependent manner. H2S is a highly lipophilic molecule, which easily penetrates the lipid bilayer of the cell membranes (Wang 2012; García-Mata & Lamattina 2013). In this study, in addition to

Figure 4. Effects of sodium hydrosulphide (NaHS) and morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (GYY4137) pretreatment on the survival percentage (a) and electrolyte leakage (b) of maize seedlings under heat stress at 48 °C for 18 h. The 2.5-day-old maize seedlings were subjected to heat stress at 48 °C for 18 h after being treated with 0, 100, 500, 1000 and 1500 mm NaHS or GYY4137 for 6 h, and the survival percentage of maize seedlings and electrolyte leakage of roots were measured. Error bars represent standard error and each data in figure represents the mean ⫾ SE of three experiments, and asterisk and double asterisks indicate significant difference (P < 0.05) and very significant difference (P < 0.01) from the control without NaHS treatment, respectively.

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DISCUSSION Because NO is a second messenger with properties such as being a free radical, having small size, no charge, short-lived and highly diffusible across biological membranes, it has many roles in plant growth, development and regulation of remarkable spectrum of plant cellular mechanisms (Gupta et al. 2011; Siddiqui et al. 2011; Wendehenne & Hancock 2011; Fan et al. 2013; García-Mata & Lamattina 2013; Gill et al. 2013). In many studies, researchers tried to find out the role of NO in the alleviation of abiotic stress including salt, drought, heavy metal stress, UV-B and extreme temperatures (Siddiqui et al. 2011; Wendehenne & Hancock 2011; Fan et al. 2013; García-Mata & Lamattina 2013; Gill et al. 2013). Pretreatment with 100 mm SNP significantly improved the growth of cucumber seedlings under NaCl stress for 8 d, as indicated by increased plant height, stem thickness, fresh weight and increased dry matter accumulation, by regulating the content and proportions of the different types of free polyamines (Fan et al. 2013). Exogenous SNP increased the activities of superoxide dismutase and catalase, maintained higher relative water content, lowered water loss in the leaves of wheat seedlings exposed to polyethylene glycol (PEG), termed as drought stress, alleviated the oxidative damage, accelerated protein synthesis and enhanced photosynthesis rate. Interestingly, such effects of SNP were reversed by the addition of c-PTIO, a specific NO scavenger

Figure 5. Effects of sodium hydrosulphide (NaHS) (a) and morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate (GYY4137) (b) pretreatment on endogenous H2S content in coleoptiles and roots of maize seedlings. The 2.5-day-old maize seedlings were treated with 500 mm NaHS or GYY4137 for 6 h, and the endogenous H2S content in both coleoptiles and roots of maize seedlings were measured every 2 h. Error bars represent standard error and each data in figure represents the mean ⫾ SE of three experiments, and double asterisks indicate very significant difference (P < 0.01) from the control without NaHS treatment.

(Fig. 3b), which, in turn, enhanced the survival percentage of maize seedlings (Fig. 1). To study further the effect of inhibitors of H2S and its scavenger on SNP-induced heat tolerance in maize seedlings, the 2.5-day-old seedlings were treated with the combination of 150 mm SNP and NaHS, GYY4137, PAG, AOA, PP, HA or HT for 6 h, and then were subjected to heat stress at 48 °C for 18 h. The results showed that pretreatment with 150 mm SNP improved the survival percentage of seedlings (Figs 1 & 6), and this improvement was enhanced by application of 500 mm NaHS or GYY4137, respectively, but was eliminated by inhibitors of H2S synthesis PAG, AOA, PP and HA, as well as H2S scavenger HT. In addition, inhibitors of H2S and its scavenger treatment alone had no significant effect on the survival percentage of seedlings under heat stress compared with the control (data not shown).

Figure 6. Effects of inhibitors of H2S synthesis DL-propargylglycine (PAG), aminooxyacetic acid (AOA) and potassium pyruvate (PP) as well as H2S scavenger hypotaurine (HT) on sodium nitroprusside (SNP)-induced heat tolerance of maize seedlings under heat stress at 48 °C for 18 h. The 2.5-day-old maize seedlings were exposed to heat stress at 48 °C for 18 h after being treated with 0 mm SNP (-S), 150 mm SNP (+S), 150 mm SNP + 500 mm NaHS (S + SH), 150 mm SNP + 500 mm GYY4137 (S + GY), 150 mm SNP + 300 mm PAG (S + PG), 150 mm SNP + 300 mm AOA (S + AA), 150 mm SNP + 300 mm PP (S + PP), 150 mm SNP + 300 mm HA (S + HA) and 150 mm SNP + 300 mm HT (S + HT), and the survival percentage of maize seedlings were determined. Error bars represent standard error and each data in figure represents the mean ⫾ SE of three experiments, and double asterisks indicate very significant difference (P < 0.01) from the control without NaHS treatment.

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(Tan et al. 2008). In rice seedlings, 100 mm SNP treatment decreased Cd accumulation in both cell walls and soluble fraction of leaves, while it increased Cd accumulation in the cell wall of rice roots clearly, which alleviated Cd toxicity by increasing pectin and hemicellulose content in the cell wall of roots (Xiong et al. 2009). In addition, Uchida et al. (2002) found that pretreatment with SNP increased the reactive oxygen species (ROS)-scavenging enzyme activities and induced expression of transcripts for oxidative stress-related gene encoding sucrose-phosphate synthase, d-pyrroline-5carboxylate synthase, and small heat shock protein 26 in rice seedlings, which, in turn, improved the survival of more green leaf tissue and resulted in higher quantum yield for photosynthesis II than in controls without SNP treatment under salinity and heat stress. Song et al. (2006) also reported that exogenous SNP elevated the activities of antioxidant enzymes superoxide dismutase, ascorbic acid peroxidase, catalase and peroxidase in callus tissue; it also alleviated the electrolyte leakage, growth suppression and cell viability decrease in callus of reed under heat stress. In the present study, SNP pretreatments could increase the survival percentage of maize seedlings and alleviate the increase in electrolyte leakage (Fig. 1) in roots and decrease in tissue vitality as well as MDA accumulation in coleoptiles and roots (Fig. 2). These results suggest that NO can effectively protect plants from abiotic stress including heat stress, and it plays an important role in the tolerance of plants to abiotic stress by acting as a signal molecule. H2S, similar to NO and CO, participates in plant systems in controlling growth and development, and in modulating many adaptive responses to different biotic and abiotic stresses (Mancardi et al. 2009; Zhang et al. 2010a,b; Chen et al. 2011; Li et al. 2011; Wang 2012; Lisjak et al. 2013). The results of García-Mata & Lamattina (2010) showed that NaHS or GYY 4137 treatment increased the relative water content and protected plants against drought stress. Zhang et al. (2008, 2010a, b, 2011) found that NaHS treatment could enhance the activities of amylase, esterase, catalase and ascorbate peroxidase in wheat seeds, and significantly reduce MDA and H2O2 accumulation, which, in turn, improved germination percentage of wheat seeds under normal conditions or PEG, Cu2+, Cr and Al3+ stress, respectively. In cucumber seedlings, NaHS alleviated the inhibition of root elongation, expression of genes encoding PME (CsPME) and increase in the activity of pectin methylesterase (PME) by boron stress (Wang et al. 2009). Interestingly, in Caenorhabditis elegans, Miller & Roth (2007) found that H2S treatment can increase thermotolerance and lifespan. Our previous results also found that NaHS pretreatment could improve heat tolerance in tobacco suspension cultured cells (Li et al. 2012b) and maize seedlings (Li et al. 2013), and the acquisition of this heat tolerance involved in calcium messenger system and proline accumulation. These investigations indicated that H2S may be involved in the acquisition of multiple abiotic stress tolerance like heat stress in plants. Based on the above studies, the data implied that crosstalk between NO and H2S in plants exists in the acquisition of abiotic tolerance like heat stress and other physiological

processes, but evidence is insufficient. During the process of stomatal movement, it was reported that ethylene treatment increased transcripts and activity of L-DES, a key enzyme in H2S biosynthesis in plants, which, in turn, induced H2S generation in the leaves of Arabidopsis, followed by stomatal closure, while AOA, PP and HA, inhibitors of H2S synthesis, were found to block ethylene-induced stomatal closure, suggesting that H2S and NO are involved in the signal transduction pathway of ethylene-induced stomatal closure, and H2S may represent a novel downstream indicator of NO during ethylene-induced stomatal movement (Liu et al. 2011). In the present work, 150 mm SNP treatment clearly improved the activity of L-DES in maize seedlings (Fig. 3a), which, in turn, increased the content of H2S (Fig. 3b). In addition, pretreatment with 500 mm NaHS and GYY4137 could also rapidly improve accumulation of endogenous H2S in maize seedlings (Fig. 5), followed by an increase in the survival percentage of seedlings and alleviation of electrolyte leakage of roots under heat stress at 48 °C (Fig. 4), respectively, while SNPinduced heat tolerance was enhanced by application of NaHS or GYY4127, and eliminated by PAG, AOA, PP, HA and HT, respectively (Fig. 6). These results illustrated that both NO and H2S are involved in the acquisition of heat tolerance in maize seedlings, and H2S may be a novel downstream signal molecule in NO-induced heat tolerance. In conclusion, pretreatment with NO donor SNP significantly improved the activity of L-DES and endogenous H2S accumulation, which, in turn, alleviated the increase in electrolyte leakage and decrease in tissue vitality as well as accumulation of MDA, and it finally enhanced the survival percentage of maize seedlings. In addition, exogenously applied NaHS or GYY4137 also elevated the endogenous H2S level, induced the acquisition of heat tolerance in maize seedlings, while this heat tolerance was enhanced by application of NaHS or GYY4137, but was eliminated by inhibitors of H2S synthesis PAG, AOA, PP and HA as well as H2S scavenger HT. All of the above-mentioned results indicated that SNP treatment could improve heat tolerance, and H2S may be a downstream signal molecule in NO-induced heat tolerance of maize seedlings. However, the perception of heat, signal transduction and the acquisition of heat tolerance in plants is a complex event, involving many second messengers such as Ca2+, H2O2, NO, H2S and plant hormones, even crosstalk among these signal molecules, and in all aspects of physiological, biochemical and molecular mechanisms, which needs to be further illustrated in future study.

ACKNOWLEDGMENTS This research was supported by the Natural Science Foundation of Yunnan Province of China (2010ZC066) and the Education Department Foundation of Yunnan Province of China (09Y0145). We appreciate the reviewers and editors for their exceptionally helpful comments about the manuscript.

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Received 31 December 2012; accepted for publication 25 February 2013

© 2013 John Wiley & Sons Ltd, Plant, Cell and Environment, 36, 1564–1572