Exogenous nitric oxide improves seed germination in wheat against mitochondrial oxidative damage induced by high salinity

Exogenous nitric oxide improves seed germination in wheat against mitochondrial oxidative damage induced by high salinity

Environmental and Experimental Botany 67 (2009) 222–227 Contents lists available at ScienceDirect Environmental and Experimental Botany journal home...

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Environmental and Experimental Botany 67 (2009) 222–227

Contents lists available at ScienceDirect

Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot

Exogenous nitric oxide improves seed germination in wheat against mitochondrial oxidative damage induced by high salinity Chunfang Zheng a,c , Dong Jiang a,∗ , Fulai Liu b , Tingbo Dai a , Weicheng Liu c , Qi Jing a , Weixing Cao a a Key Laboratory of Crop Physiology and Ecology in Southern China, Ministry of Agriculture/Hi-Tech Key Laboratory of Information Agriculture of Jiangsu Province, Nanjing Agricultural University, PR China b Department of Agriculture and Ecology, Faculty of Life Sciences, University of Copenhagen, Højbakkegaard Allé 13, DK-2630 Taastrup, Denmark c Zhejiang Mariculture Research Institute, Wenzhou 325000, PR China

a r t i c l e

i n f o

Article history: Received 21 January 2009 Received in revised form 9 May 2009 Accepted 13 May 2009 Keywords: Antioxidative enzymes Ion balance Seed respiration Sodium nitroprusside (SNP) Starch degradation

a b s t r a c t Effects of exogenous nitric oxide (NO) on starch degradation, oxidation in mitochondria and K+ /Na+ accumulation during seed germination of wheat were investigated under a high salinity level. Seeds of winter wheat (Triticum aestivum L., cv. Huaimai 17) were pre-soaked with 0 mM or 0.1 mM of sodium nitroprusside (SNP, as nitric oxide donor) for 20 h just before germination under 300 mM NaCl. At 300 mM NaCl, exogenous NO increased germination rate and weights of coleoptile and radicle, but decreased seed weight. Exogenous NO also enhanced seed respiration rate and ATP synthesis. In addition, seed starch content decreased while soluble sugar content increased by exogenous NO pre-treatment, which was in accordance with the improved amylase activities in the germinating seeds. Exogenous NO increased the activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6); whereas decreased the contents of malondialdehyde (MDA) and hydrogen peroxide (H2 O2 ), and superoxide anions (O2 •− ) release rate in the mitochondria. Exogenous NO also decreased Na+ concentration while increased K+ concentration in the seeds thereby maintained a balance between K+ and Na+ during germination under salt stress. It is concluded that exogenous NO treatment on wheat seeds may be a good option to improve seed germination and crop establishment under saline conditions. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Nearly half of the irrigated land and 20% of the world’s cultivated land are currently affected by salinity (Zhu, 2001). Salinity greatly affects seed germination (Misra and Dwivedi, 2004), and consequently induces a reduction in germination rate and a delay in the initiation of the germination and seedling establishment (Almansouri et al., 2001). Thus, it is worthwhile to study the physiological mechanisms of poor seed germination caused by salt stress and to develop suitable measures to alleviate the negative effects of salinity on seed germination thereby crop establishment on saline soils. The effects of salt stress on plant physiology have been well documented. Salt stress can cause ion toxicity, osmotic stress and reactive oxygen species (ROS) stress (Mittler, 2002), leading to gradual peroxidation of lipid and antioxidant enzyme inactivation (Tanou et al., 2009). In plant cells, mitochondria are major

∗ Corresponding author at: College of Agriculture, Nanjing Agricultural University, No. 1 Weigang Road, Nanjing 210095, Jiangsu Province, PR China. Fax: +86 25 8439 6575. E-mail address: [email protected] (D. Jiang). 0098-8472/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2009.05.002

sites for the generation of reactive oxygen species (ROS) in nonphotosynthetic cells (Møller, 2001; Fleury et al., 2002). Electrons leaked from the electron transport chains can react with O2 during normal aerobic metabolism to produce ROS such as superoxide (O2 •− ), hydrogen peroxide (H2 O2 ), especially when plants are subjected to biotic or abiotic stresses (Dixit et al., 2002; Mittova et al., 2004). In plants, the antioxidant enzymes are important components in scavenging ROS (Ashraf, 2009). ROS scavenging enzymes in plant mitochondria have been identified as Mn-superoxide dismutase (Mn-SOD) (Streller et al., 1994), peroxidases (POD) ˇ (Hadˇzi-Taˇskovic´ Sukalovi c´ et al., 2007), catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) (Shi et al., 2007). A large body of evidence has demonstrated that the antioxidant systems play important roles in protecting plants against oxidative damage induced by salt stress. It has been reported that salt stress caused significant decreases in growth and the relative water content of cucumber root, which are associated with increases of free radical production and membrane damage resulting from decrease in the activity of antioxidant enzymes (Duan et al., 2008). NaCl treatment also causes suppression of growth of purslane seedlings due to a decreased antioxidant enzyme activity (Yazici et al., 2007). Therefore, enhancing the activity of antioxidant enzymes

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in plants organs is necessary for improving plant’s tolerance to salt stress. Nitric oxide (NO) is an important signaling molecule exerting various physiological functions in plants. For instance, NO is reported to mediate the plant’s responses to both biotic and abiotic stresses (Crawford and Guo, 2005; Delledonne, 2005); and NO plays a crucial role in regulating plant growth and development, including germination, flowering, fruit ripening and organ senescence (Arasimowicz and Floryszak-Wieczorek, 2007). NO treatment has shown to increase dry matter accumulation of maize seedlings under salt stress (Zhang et al., 2006), and to improve activities of SOD and CAT in root of Lupinus luteus under heavy metal and salin´ z, ´ 2003). Moreover, NO is believed ity stresses (Kopyra and Gwózd to be involved in two respiratory electron transport pathways in mitochondria (Yamasaki et al., 2001; Zottini et al., 2002), where it mediates the modulation of ROS and enhances antioxidant defense system in plants subjected to various abiotic stresses, such as heat (Uchida et al., 2002), iron deficiency (Sun et al., 2007), and salt and heavy metals (Singh et al., 2008). Recent evidence in cucumber reveals that exogenous NO treatment protects plant cells from oxidation damage by promoting the transformation of O2 •− to H2 O2 and O2 , and by enhancing the H2 O2 -scavenging enzymes activities and consequently reducing H2 O2 accumulation in root mitochondria under salt stress (Shi et al., 2007). However, until now little is known about the influences of exogenous NO treatment on oxidative damage and antioxidant enzyme activities in mitochondria in wheat seeds during germination under salt stress. Therefore, the objective of the present study was to investigate the effect of exogenous NO treatment on seed germination, antioxidant enzyme activity and lipid peroxidation in mitochondria in wheat seeds under a severe salt stress. The results may help to elucidate the regulatory effects of exogenous NO on ROS metabolism in seeds mitochondria and the physiological roles of NO in anti-salt stress during seed germination in wheat. 2. Materials and methods 2.1. Experimental design Seeds of winter wheat (Triticum aestivum L.) cultivar of Huaimai 17 were surface sterilized with 2.5% sodium hypochlorite for 10 min and washed seven times with sterile distilled water. Sodium nitroprusside (SNP) was used as NO donor. Just before germination, two treatments were set by soaking the seeds in distilled water as control (SNP0) and 0.1 mM of SNP solution (SNP0.1) for 20 h, respectively. The soaked 50 seeds were put on a filter paper in petri dishes with a diameter of 9 cm, and 7 ml of 300 mM NaCl was then added into each dish. The dishes were incubated at 25 ± 1 ◦ C for 5 days in an illumination incubator. Seeds were sampled at days 1, 2, 3, 4 and 5 after treatment for bio-chemical and physiological measurements, and another batch was sampled at day 7 after treatment for determinations of dry weight and seed germination rate. 2.2. Dry weight assays Coleoptiles, radicles and the remnant seeds (mainly endosperms and pericarps) were isolated and dried in oven at 105 ◦ C for 2 h, and then maintained at 80 ◦ C for 3 days to determine their dry weights. 2.3. Amylase enzyme extraction and assays Amylase enzymes were extracted and estimated according to the procedure of Kishorekumar et al. (2007) and Tárrago and Nicolás (1976). One gram of germinating seeds was ground and homogenized in a pre-chilled mortar and pestle with 10 ml ice-cold

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distilled water at 4 ◦ C. The extract was centrifuged at 15,000 × g for 30 min at 4 ◦ C. The supernatant was collected for estimating ␣- and ␤-amylase activities. Activity of ␣-amylase was assayed after inactivating ␤-amylase activity under higher temperature. Five ml of enzyme extract and 3 ml of 3 mM CaCl2 were mixed and incubated at 70 ◦ C for 5 min. The reaction mixture (2 ml) contained 0.1 mM citrate buffer (pH 5.0), 2% soluble starch solution and 0.7 ml of hot enzyme extract. The reaction was incubated at 30 ◦ C for 5 min and stopped by adding 2 ml colour reagent (the colour reagent was obtained by dissolving 1 g 3 ,5-dinitrosalicylic acid in 20 ml of 2 M NaOH and 30 g potassium sodium tartrate in 100 ml distilled water). The mixture was heated at 50 ◦ C for 5 min, and the final volume of the solution was made up to 10 ml with distilled water. Activity of ␣-amylase was then determined spectrophotometrically (UV-2401, Shimadzu Corp., Japan) at 540 nm. Activity of ␤-amylase was estimated after inactivating ␣amylase at a low pH of 3.4 with 0.1 M EDTA. Two ml of reaction solution contained 0.1 mM citrate buffer (pH 3.4), 2% soluble starch and 0.7 ml of EDTA treated enzyme extract. The reaction was allowed for 5 min after the addition of starch at 30 ◦ C. After reaction, 2 ml of colour reagent was added to stop the reaction. The activity was then assayed following the same method of ␣-amylase activity analysis as described above. Soluble protein content in seeds was determined according to Bradford (1976) using bovine serum albumin (BSA) as standard. Both ␣- and ␤-amylase activities were expressed as unit mg−1 protein, and one unit is equivalent to release of one milligram maltose from starch per minute by the enzyme. 2.4. Measurement of seed respiration rate A close gas collecting system was used to measure the CO2 production via respiration during seed germination. Germinating seeds were sealed in an internally ventilated chamber with a volume of 200 cm3 . The chamber was coupled with a GXH-3010F IRGA (infra-red gas analyzer, Huayun Instrument Research Institute Co., Beijing, China). The changes in CO2 concentration in the chamber was logged in 3 min just after the CO2 concentration increased linearly. The respiration rate of seed was then calculated according to the slope of CO2 increase in the chamber. 2.5. Determination of ATP content in the seeds One g germinating seeds were finely sliced and were put into 5 ml acetone, and then kept in boiling water bath for 5 min until the acetone fully evaporated. Three ml of 20 mM Tris–HCl buffer at pH 7.6 were added to the sample and heated in a boiled water bath for 10 min, and then immediately cooled down in an ice bath. The extraction was centrifuged at 3000 × g for 10 min, and the supernatant was collected. Bioluminescence produced by adding the ATP extract was then measured with the ATP Bioluminescent Assay Kit (luciferin-luciferase reagent, the Detect Technical Institute, Shanghai, China) using a SHG-D Bioluminescence and Chemiluminescence Meter (The Detect Technical Institute, Shanghai, China). 2.6. Starch content and soluble sugar assays One gram of germinating seeds powder was heated with 10 ml 0.33 mM HCl at 100 ◦ C for 10 min to extract starch. 0.5 ml of 30% (w/v) ZnSO4 was added to the extraction to deposit protein. 0.5 ml 30% (w/v) K3 [Fe(CN)6 ] was then added to the extraction and mixed by shaking, and the mixture was adjusted to a volume of 20 ml. After fully shaken and filtrated, starch was determined as the amount of glucose by an Automatic Recording Polarimeter (WZZ-2B, Shanghai,

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China) using the polarimetric analysis at 20–25 ◦ C according to Xie et al. (2003). Dried germinating seeds sample powder (0.1 g) was extracted twice with 80% (v/v) ethanol at 80 ◦ C. Soluble sugar was measured using the anthrone method (Fales, 1951).

pension, and heated at 95 ◦ C for 25 min and then centrifuged at 3000 × g for 5 min for clarification. MDA content was measured at 532 nm and corrected by subtracting the absorbance at 600 nm for non-specific turbidity. 2.10. Statistical analysis

2.7. K+ and Na+ content assays About 0.1 g dried germinating seeds sample powder was completely digested with 5 ml 98% H2 SO4 at 350 ◦ C by supplemented with a few drops of H2 O2 (30%, v/v). K+ and Na+ contents were then determined using an atomic absorption spectrometer (Beijing Purkinje General Instrument Co., Ltd., China).

The experiment was a completely randomized block design. All data were subjected to one-way ANOVA using the SPSS10.0 software package (SPSS Chicago, IL, USA). Difference at P < 0.05 was considered significant between treatments. 3. Results

2.8. Preparation of mitochondria

3.1. Effect of exogenous NO on seed germination rate

Mitochondria were isolated and further purification according to the methods of Pomeroy (1974) and Braidot et al. (1998). Around 15 g seeds were weighted and then homogenized in 50 ml of refrigerated grinding medium containing 500 mM sucrose, 1 mM EDTA, 67 mM KH2 PO4 and 0.75% (w/v) bovine serum albumin at pH 7.2. The homogenate was filtered through two layers of gauze and centrifuged at 2000 × g for 5 min. The supernatant was further centrifuged at 28,000 × g for 4 min. The crude mitochondrial fraction was then suspended in 1 ml medium containing: 20 mM 3-(N-Morpholino) propanesulfonic acid (MOPS)–KOH (7.2), 0.3 M mannitol, 1 mM EDTA, 0.1% (w/v) BSA, and purified on a discontinuous gradient formed by three layers of 45%, 21% and 13.5% (v/v) percoll in 20 mM MOPS-KOH (7.2), 0.5 M sucrose and 0.2% (w/v) BSA. The gradient was centrifuged at 20,000 × g for 40 min and the mitochondria were collected at the 21%/45% interface. Mitochondria were washed from Percoll in 50 ml re-suspending medium and centrifuged at 28,000 × g for 5 min. Purified mitochondria were resuspended in 0.6 ml of 20 mM HEPES–Tris, 0.4 M sucrose and 0.1% BSA (7.5). All the above operations were performed at 0–4 ◦ C.

After day 7 of treatment, the germination rate of cultivar was significantly enhanced by exogenous NO under salt stress (P < 0.05; Fig. 1). In addition, exogenous NO treatment apparently stimulated growth of the coleoptile and radicle, and led to a significant increase in their dry weights under salt stress (P < 0.05). Consequently, dry weight of the seeds decreased significantly (P < 0.05) under NO treatment as compared to the non-NO treatment (SNP0) under salt stress.

Seed starch content decreased, whereas total soluble sugar content increased along with germination progress in wheat (Fig. 2). Under salt stress, exogenous NO treatment further decreased seed starch content from 3 days of treatment (P < 0.05). In contrast, exogenous NO treatment significantly increased the total soluble sugar content in germinating seeds (P < 0.05). At day 5 after treatment, soluble sugar content under the SNP0.1 treatment was about 23.4% higher than that in the control in Huaimai 17.

2.9. Analysis of antioxidant enzyme and O2 •− production rate and H2 O2 content in the mitochondria

3.3. Effect of exogenous NO treatment on seed ˛- and ˇ-amylase activities

Activities of superoxide dismutase (SOD), catalase, and peroxidase (POD) were measured by adopting the method of Tan et al. (2008). SOD activity was assayed on the basis of its ability to inhibit the photochemical reduction of nitro blue tetrazolium (NBT). One unit of SOD activity was defined as the amount of enzyme that caused 50% inhibition of the rate of the reaction in the absence of enzyme. Total SOD activity was expressed as unit mg−1 protein. CAT activity was estimated by the residual H2 O2 in the reaction solution using 10 mM permanganate titration to pink. The reaction was started by adding 1 ml of 50 ␮M H2 O2 , and stopped the enzyme activity by adding 2 ml of 10% H2 SO4 . POD activity was measured as the change rate of absorbance of the reaction solution at 470 nm. O2 •− production rate was determined according to Sui et al. (2007). 80 ␮l mitochondria supernatant, 1.7 ml phosphate (pH 7.8) and 0.1 ml 10 mM hydroxylammonium chloride were incubated at 25 ◦ C for 20 min, then added to 1 ml 17 mM P-aminobenzene sulfonic acid and 1 ml 7 mM ␣-naphthylamine, and the mixture was incubated at 25 ◦ C for 20 min. The O2 •− generation was determined as the absorbance at 530 nm. H2 O2 content was measured according to the method of Moloi and Westhuizen (2006). The assay was based on the absorbance change of the titanium peroxide complex at 415 nm. Absorbance values were quantified using standard curve generated from known concentrations of H2 O2 . Malondialdehyde (MDA) in the mitochondria was estimated by the method of Du and Bramlage (1992). 20% trichloroacetic acid containing 0.5% 2-thiobarturic acid was added to mitochondria sus-

3.2. Effect of exogenous NO on starch and soluble sugar contents

In the first ␣-amylase activity of wheat decreased day 2 after treatment, and slightly increased thereafter till day 5 of the treatment under salt stress (Fig. 3). Compared to the control, exogenous NO significantly enhanced ␣-amylase activity in wheat (P < 0.05).

Fig. 1. Effect of exogenesis nitric oxide on germination rate, dry weights of seed, radicle and coleoptile after day 7 of germination under salt stress in wheat. Data are means + SD (n = 3). Different small letters in each cultivar indicate significant difference between treatments at P < 0.05.

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Fig. 2. Effect of exogenesis nitric oxide on changes in contents of starch and soluble sugar in wheat seeds during 5 days germination under salt stress. Data are means + SD (n = 3). Different small letters at each day indicate significant difference between treatments at P < 0.05.

Fig. 3. Effect of exogenesis nitric oxide on changes in activities of ␣- and ␤-amylase in wheat seeds during 5 days germination under salt stress. Data are means + SD (n = 3). Different small letters at each day indicate significant difference between treatments at P < 0.05.

␤-Amylase activity showed a reverse pattern to ␣-amylase activity and it decreased after day 3 of treatment in wheat (Fig. 3). However, exogenous NO significantly alleviated the inhibition of ␤-amylase activity in wheat seed subject to salt stress (P < 0.05). 3.4. Effect of exogenous NO treatment on seed respiration rate and ATP content At first, seed respiration rate decreased sharply at day 2 after treatment and increased thereafter in wheat (Fig. 4). Compared to the control, exogenous NO treatment significantly increased the respiration rate in wheat (P < 0.05). A similar pattern of changes in ATP content in the germinating seed in response to exogenous NO was observed in wheat under salt stress (Fig. 4). It was found that exogenous NO treatment significantly increased ATP content in seeds (P < 0.05). Compared to the control, ATP content in NO pretreated seeds at days 2, 3, 4 and 5 after treatment increased 34.6%, 30.7%, 27.8% and 20.9%, respectively.

Fig. 5. Effect of exogenesis nitric oxide on changes in contents of MDA and H2 O2 , and O2 •− release rate and activities of SOD, POD and CAT in mitochondria of wheat seeds during 5 days germination under salt stress. Data are means + SD (n = 3). Different small letters at each day indicate significant difference between treatments at P < 0.05.

3.5. Effect of exogenous NO treatment on the rate of O2 •− generation, H2 O2 and MDA concentrations and the activities of antioxidant enzymes in the seed mitochondria Contents of MDA and H2 O2 , and release rate of O2 •− in wheat seed mitochondria increased during germination under salt stress (Fig. 5). Exogenous NO treatment significantly alleviated the saltinduced accumulation of MDA and H2 O2 , and decreased the release rate of O2 •− of the mitochondria in the germinating wheat seeds (P < 0.05). Exogenous NO treatment significantly increased SOD and CAT activities in the mitochondria during germination under salt stress (P < 0.05; Fig. 5). The inducible effect of exogenous NO on the activity of POD in wheat was not significant, except for at the first day after treatment when exogenous NO treatment significantly increased POD activity than the control (P < 0.05). 3.6. Effect of exogenous NO treatment on the concentrations of Na+ and K+ in the germinating wheat seeds Seed Na+ concentration increased, whereas seed K+ concentration decreased after the onset of treatments (Fig. 6). Exogenous NO treatment decreased Na+ concentration, but increased that of K+ under salinity in wheat. At day 5 after treatment, K+ concentration under the SNP0.1 treatment increased by 23.0%, and Na+ concentration decreased by 14.7%, as compared with the control. 4. Discussion

Fig. 4. Effect of exogenesis nitric oxide on changes in respiration rate and ATP content in wheat seeds during 5 days germination under salt stress. Data are means + SD (n = 3). Different small letters at each day indicate significant difference between treatments at P < 0.05.

Exogenous NO has a strong stimulating effect on seed germination under stress or no-stress conditions (Beligni and Lamattina, 2000). For instance, NO induces a rapid increase in ␤-amylase activity in germinating wheat seeds (Zhang et al., 2005), and greatly promotes germination process by enhancing the activities of amylase in wheat seeds under copper stress (Hu et al., 2007).

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seed Na+ concentration during germination, whereas increased K+ concentration (especially at the 4 and 5 days of germination), led to a higher K+ /Na+ ratio in germinating wheat seeds under salt stress (Fig. 6). These results indicated that exogenesis NO may help maintain the ion balance between K+ and Na+ in germinating wheat seeds, which might have also contributed to the improved germination rate under salt condition. Acknowledgments

+

+

Fig. 6. Effect of exogenesis nitric oxide on changes in contents of Na and K in wheat seeds during 5 days germination under salt stress. Data are means + SD (n = 3). Different small letters at each day indicate significant difference between treatments at P < 0.05.

In agreement with these, we observed that exogenous NO treatment significantly stimulated seed germination under severe salt stress in wheat (Fig. 1). Germination rate, weights of coleoptiles and radicels were also significantly increased due to exogenous NO treatment. During seed germination, activities of both ␣-amylase and ␤-amylase were significantly enhanced by the exogenous NO treatment (Fig. 3). Thus, exogenous NO facilitated the conversion of starch into sugars (Fig. 2), which benefited seed germination of wheat under salt stress. In the plant mitochondria, electron transfer along the respiration chain is coupled to the formation of ATP (Maxwell et al., 1999; Affourtit et al., 2001), and the redundant electron leads to the formation of ROS if the ATP synthesis was blocked (Petrussa et al., 2008). Mittova et al. (2003) reported that salt stress increased the contents of H2 O2 and MDA in the tomato mitochondria, and which was probably due to a salt-induced increase in the rate of O2 •− production (Møller, 2001). In the present study, we observed that exogenous NO treatment significantly decreased the MDA and H2 O2 contents and the O2 •− production rate in the wheat seed mitochondria during germination under salt stress (Fig. 5). A similar anti-oxidative stress function of exogenous NO was also observed in salt-stressed barley plants (Li et al., 2008). Therefore, exogenous NO treatment could be an effective practice to protect plants/germinating seeds against oxidative damage caused by salt stress. It is well known that the antioxidant enzymes such as SOD, CAT and POD play a significant role in scavenging ROS in saltstressed plants (Tseng et al., 2007; Ashraf and Ali, 2008; Tuna et al., 2008; Ashraf, 2009). Earlier studies have demonstrated that exogenous NO protects leaves against oxidative damage in reed under heat (Song et al., 2006) and in wheat under drought (Tian and Lei, 2006), ascribes mainly to the increased activities of SOD, CAT and POD. In accordance with these findings, we observed that activities of both SOD and CAT in the seed mitochondria were significantly increased by exogenous NO treatment in the present study (Fig. 5), and which might have contributed to the alleviated oxidative stress in the mitochondria of germinating wheat seeds and thereby improved germinating rate under salt stress. In addition, exogenous NO treatment significantly increased seed respiration rate during germination under salt stress (Fig. 4), and this was coincided with the increased ATP content in the seeds. Thus, it is suggested that exogenous NO may promote ATP synthesis and enhance seed activity (respiration rate), and both have a positive effect on seed germination under salt stress. Moreover, some studies have shown that salt stress may cause imbalance of the inner cellular ions and thus resulting in ion toxicity and osmotic stress in plant cells (Chung et al., 2006; Raza et al., 2007; Athar et al., 2008). Therefore, to enhance Na+ exclusion and K+ absorption thereby maintaining optimum K+ /Na+ ratio is crucial for salt tolerance in higher plants (Zhu, 2001; Liu et al., 2008). In the present study, we found that exogenous NO treatment decreased

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