Exogenous nitric oxide protect cucumber roots against oxidative stress induced by salt stress

Exogenous nitric oxide protect cucumber roots against oxidative stress induced by salt stress

Plant Physiology and Biochemistry 45 (2007) 542e550 www.elsevier.com/locate/plaphy Research article Exogenous nitric oxide protect cucumber roots ag...

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Plant Physiology and Biochemistry 45 (2007) 542e550 www.elsevier.com/locate/plaphy

Research article

Exogenous nitric oxide protect cucumber roots against oxidative stress induced by salt stress Qinghua Shi, Fei Ding, Xiufeng Wang*, Min Wei State Key Laboratory of Crop Biology, College of Horticulture Science and Engineering, Shandong Agricultural University, Taian 271018, PR China Received 19 August 2006; accepted 23 May 2007 Available online 27 May 2007

Abstract Mitochondria are subcellular organelles with an essentially oxidative type of metabolism. The production of reactive oxygen species (ROS) in it increases under stress conditions and causes oxidative damage. In the present study, effects of exogenous sodium nitroprusside (SNP), a nitric oxide (NO) donor, on both the ROS metabolism in mitochondria and functions of plasma membrane (PM) and tonoplast were studied in cucumber seedlings treated with 100 mM NaCl. NaCl treatment induced significant accumulation of H2O2 and led to serious lipid peroxidation in cucumber mitochondria, and the application of 50 mM SNP stimulated ROS-scavenging enzymes and reduced accumulation of H2O2 in mitochondria of cucumber roots induced by NaCl. As a result, lipid peroxidation of mitochondria decreased. Further investigation showed that application of SNP alleviated the inhibition of Hþ-ATPase and Hþ-PPase in PM and/or tonoplast by NaCl. While application of sodium ferrocyanide (an analog of SNP that does not release NO) did not show the effect of SNP, furthermore, the effects of SNP were reverted by addition of hemoglobin (a NO scavenger). Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Cucumber; Salt stress; Nitric oxide; Oxidative stress; Mitochondria; Plasma membrane; Tonoplast

1. Introduction In many areas of the world, salinity has been seriously imposed on crops and forms the main constraint for agriculture [43]. Several physiological and biochemical processes are affected by salinity in plants. When plants are exposed to NaCl, cellular ion homeostasis may be impaired. Under salinity conditions, tolerant plants typically maintain low sodium (Naþ) in the cytosol of cells [18]. Such mechanisms involve Naþ compartmentalization into vacuoles and/or extrusion to the external medium and these processes appear to be

Abbreviations: APX, ascorbate peroxidase (EC 1.11.1.11); CAT, catalase (EC 1.11.1.6); DHAR, dehydroascorbate reductase (EC 1.8.5.1); EDTA, ethylene diamine tetra-acetic acid; GR, glutathione reductase (EC 1.6.4.2); GPX, guaiacol peroxidase (EC 1.11.1.7); NBT, nitroblue tetrazolium; ROS, reactive oxygen species; SNP, sodium nitroprusside; SOD, superoxide dismutase (EC 1.15.1.1); TBARS, thiobarbituric acid reactive substance. * Corresponding author. Tel./fax: þ86 538 8242456. E-mail address: [email protected] (X. Wang). 0981-9428/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2007.05.005

mediated by the transport systems in PMs and tonoplasts [47,51].The control of Naþ movement across the PM and tonoplast to maintain a low Naþ concentration in the cytosol is a key factor to the cell adaptation to salt stress [47,51]. To support the assumption described above, proton pumps such as plasma membrane Hþ-ATPase as well as tonoplast HþATPase and Hþ-PPase must be investigated in order to identify their role in the plant tolerance to salt stress. Another common biochemical change occurring when plants are subjected to salt stress is the accumulation of ROS, which unbalances the cellular redox in favor of oxidized forms, thereby creating oxidative stress that can damage DNA, inactivate enzymes and cause lipid peroxidation [41,52,64]. Many cell compartments produce ROS and mitochondria are important source of ROS due to respiratory electron transport, especially under stress conditions [4,55]. To control the level of ROS, plants have evolved the antioxidant defense system comprising of enzymes such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR),

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glutathione reductase (GR) and non-enzymatic constituents such as ascorbate and glutathione, which are responsible for scavenging excessively accumulated ROS in plants under stress conditions [24]. Accordingly, the regulation of these antioxidant constituents by an exogenous substance might mediate the plant tolerance to salt stress. Nitric oxide (NO) is a small, highly diffusible gas and a ubiquitous bioactive molecule. Its chemical properties make NO a versatile signal molecule that functions through interactions with cellular targets via either redox or additive chemistry [27]. In recent years there has been increasing evidence that NO is involved many key physiological processes of plants, such as germination [6], mitochondrial functionality [65], gravitopism [22] and floral regulation [20]. On the other hand, NO can mediate plant regulators and ROS metabolism, and many experiments have shown it is involved in signal transduction [12,35,45,53] and responses to biotic and abiotic stresses [28,35,61,63]. Under an ordinary physiological condition, SOD rapidly converts O2 to H2O2 and an oxygen molecule. However, a large amount of NO may combine with O2 to form peroxynitrite (ONOO), which has been reported to damage lipids, proteins and nucleic acids [33,60]. Nevertheless, O2 and H2O2 are more toxic than NO and ONOO; therefore, NO may protect cells from destruction [59]. In accordance, NO has been suggested to have dual roles, either toxic or protective, depending on its environments [5,6]. In previous studies, it has been reported that exogenous NO stimulated the germination of NaCl-treated Suaeda salsa seeds [31], stimulated the expression of PM Hþ-ATPase in reed under salt stress [63] and dramatically promoted the germination of wheat seeds under osmotic stress by improving antioxidant capacity [62]. Based on the above observations, the objectives of the present experiment were to investigate whether NO is involved in regulation of ROS metabolism in cucumber root mitochondria and ATPase, PPase activities in plasma membrane and/or tonoplast, and therefore to elucidate the physiological mechanism of exogenous NO increasing cucumber plants tolerance to salt stress. 





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100 mM NaCl þ 50 mM SNP; N þ F, 100 mM NaCl þ 50 mM sodium ferrocyanide; N þ S þ H, 100 mM NaCl þ 50 mM SNP þ 0.1% (w/v) hemoglobin) and was arranged in a randomized, complete block design with three replicates, giving a total of 12 containers. The light period was 12e14 h, and the air temperature was 20e33  C and 16e25  C day and night, respectively. After 4 or 8 days, fresh roots were taken for the isolation of mitochondria, plasma membrane and tonoplast vesicles. 2.2. Plant growth measurement After 8 days of treatment, the plants were harvested, divided into shoots and roots, dried at 70  C for a period of 4 days and weighed. 2.3. Isolation of mitochondria Mitochondria were isolated according to the method of Leaver et al. [29]. The young root tissue (10 g) was homogenized in grinding buffer (25 mM MOPS; pH7.8; 8 mM Cyshydrochloride; 0.1% BSA [w/v]; 1 mM EGTA) in a chilled mortar and pestle at 4  C. Mitochondria were subsequently purified by two rounds of differential centrifugation. Further purification of mitochondria was done using sucrose density gradient centrifugation. The mitochondria were recollected from the sucrose gradient, pelleted, and suspended in resuspending buffer (250 mM sucrose; 10 mM TricineeKOH; pH 7.2; 1 mM MgCl2; and 1 mM KH2PO4). The mitochondria were lysed by a freezeethaw cycle, and the lysate was used for the spectrophotometric analysis of enzyme activities. 2.4. H2O2 content assay H2O2 content was determined according to Patterson et al. [49]. 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 H2O2.

2. Materials and methods 2.5. Lipid peroxidation assay 2.1. Plant materials Cucumber (Cucumis sativus L. cv. Jinchun 5) seeds were germinated on moist filter paper in the dark at 28  for 2 days, then germinated seedlings were transferred to the growth chamber filled with vermiculite and grown in a greenhouse for 8 days. Then they were transplanted into 5 l black plastic containers containing aerated full nutrient solution: 4 mM Ca(NO3)2; 4 mM KNO3; 2.5 mM KH2PO4; 2 mM MgSO4; 29.6 mM H3BO3; 10 mM MnSO4; 50 mM Fe-EDTA; 1.0 mM ZnSO4; 0.05 mM H2MoO4; 0.95 mM CuSO4; with three seedlings per container. The pH was maintained close to 6.5 by adding H2SO4 or KOH. After 9 days of pre-culture, the treatments were started. The experimental design consisted of a control (no added SNP and NaCl, indicated by CK) and five treatments (S, 50 mM SNP; N, 100 mM NaCl; N þ S,

Lipid peroxidation was determined as the amount of thiobarbituric acid reactive substances (TBARS) [54]. 2.6. Antioxidant enzyme assays SOD activity was assayed by measuring its ability to inhibit the photochemical reduction of nitro blue tetrazolium (NBT) [50]. One unit of the enzyme activity was defined as the amount of enzyme required to result in a 50% inhibition of the rate of nitro blue tetrazolium reduction at 560 nm. CAT activity was measured as the decline in absorbance at 240 nm due to the decline of extinction of H2O2. The reaction mixture containing 25 mM sodium phosphate buffer (pH 7.0), 10 mM H2O2 and 0.1 ml enzyme extract. The reaction was started by adding H2O2 [10].

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GPX activity was measured by the increase in absorbance at 470 nm due to guaiacol oxidation [46]. The reaction mixture contained: 25 mM guaiacol; 10 mM H2O2; and 0.1 ml enzyme extract. The reaction was stared by adding H2O2. APX activity was measured according to Nakano and Asada [44]. The assay depends on the decrease in absorbance at 290 nm as ascorbate is oxidized. The 2 ml reaction mixture contained: 25 mM sodium phosphate buffer (pH 7.0); 0.5 mM ascorbate; 0.1 mM H2O2; 0.1 mM EDTA; and 0.1 ml enzyme extract. The reaction was started by adding H2O2. The assay of DHAR activity was carried out by measuring the increase in absorbance at 265 nm due to ASC formation [44]. The reaction mixture contained: 25 mM sodium phosphate buffer (pH 7.0); 2.5 mM reduced glutathione (GSH); 0.4 mM dehydroascorbate (DHA); and 0.1 ml enzyme extract. GR activity was measured according to Foyer and Halliwell [14], which depends on the rate of decrease in the absorbance of NADPH at 340 nm. The reaction mixture consisted of: 25 mM HEPES buffer (pH 7.0); 0.5 mM oxidized glutathione (GSSG); 0.12 mM NADPH; and 0.2 mM EDTA. 2.7. Isolation of plasma membrane and tonoplast vesicles For the preparation of the membrane fraction enriched in plasma membrane and tonoplast vesicles, the method was modified from Ballesteros [1]. Excised roots were homogenized (1/2, w/v) with a mortar and pestle in a cold grinding medium containing: 60 mM HEPESeTris (pH 7.2); 300 mM sucrose; 0.5% (w/v) BSA: 5 mM EDTA; 0.5 mM EGTA; 2 mM DTT; 1 mM PMSF; and 5% (w/v) PVPP. All steps were carried out at 4  C. The homogenate was filtered through four layers of cheesecloth and centrifuged at 10,000  g for 20 min. The supernatant was layered on top of a step gradient consisting of 6, 9 and 6 ml of 45, 33 and 15% (w/w) sucrose, respectively, and then centrifuged for 2.5 h at 80,000  g. Each in gradient buffer contained: 20 mM HEPESeTris (pH 7.2); 5 mM EDTA; 0.5 mM EGTA; 2 mM DTT; and 0.1 mM PMSF. The tonoplast-enriched fraction was collected at the 15/33% sucrose interface, and was diluted two-fold in the gradient buffer. The plasma membrane-enriched fraction was collected at the 33/45% sucrose interface, and was diluted four-fold in the gradient buffer. Each fraction was centrifuged for 1 h at 100 000  g. The resulting pellet was resuspended in a medium containing: 20 mM HEPESeTris (pH 7.2); 3 mM MgCl2; 0.5 mM EGTA; 300 mM sucrose; and 0.1 mM PMSF and was stored at 70  C for further use. 2.8. Determination of Hþ-ATPase in PMs Hþ-ATPase activity in PMs was determined by measuring the release of inorganic phosphate from ATP according to a modified method of Briskin et al. [9]. The 0.5 ml assay medium contained: 250 mM HEPESeTris (pH7.5); 25 mM ATP (Na salt); 3 mM sodium molybdate; 1 mM sodium azide; 1 mM EDTA; and 0.02% Triton X-100 (v/v). The reaction was started by adding 50 ml PM vesicles. After incubation for 30 min at 37  C, the reaction was stopped by adding

1 ml of termination reagent (4.2 g (NH4)6Mo7O24$4H2O, which was obtained by dissolving 28.6 ml H2SO4 and 10 g SDS in 1 l distilled water). Fifty microliters of 10% ascorbate were added and color allowed to develop for 40 min at room temperature before the absorbance at 660 nm was recorded. The Hþ-ATPase hydrolysis activity in PMs was calculated from the amount of inorganic phosphate released in the absence and presence of 250 mM Na3VO4. 2.9. Determination of Hþ-ATPase and Hþ-PPase activity in tonoplast The activities of Hþ-ATPase and Hþ-PPase in tonoplast were determined by a modified method according to Lin and Morales [32]. Activities of tonoplast Hþ-ATPase were measured in a 0.5 ml reaction mixture containing: 30 mM HEPESeTris (pH7.5); 3 mM MgSO4; 100 mM KCl; 0.5 mM NaN3; 0.1 mM Na3VO4; 0.1 mM (NH4)4MoO4; 0.02% Triton X-100 (v/v); and 3 mM ATPNa2. The reaction was started by adding 50 ml tonoplast vesicles. After incubation for 30 min at 37 deg;C, the reaction was terminated and the color developed as in the PM-ATPase assay. The ATPase hydrolysis activity in tonoplast was calculated from the amount of inorganic phosphate released in the absence and presence of 100 mM KNO3. The PPase activity in the tonoplast was performed by analogy in the presence of 3 mM Na-pyrophosphate instead of ATPNa2, and determined as the difference of activity in the absence and presence of 100 mM KCl. 2.10. Protein content assay Protein content was measured using bovine serum as standard according to the method of Bradford [8]. 2.11. Statistics Values presented were means  one standard deviation (SD) of three replicates. Statistical analyses were performed by analysis of variance (ANOVA) using SAS software (SAS Institute, Cary, NC). Differences between treatments were separated by the least significant difference (LSD) test at a 0.05 probability level. 3. Results 3.1. Plant growth As shown in Fig. 1, the dry weight of both shoot and root of cucumber plants was significantly decreased by NaCl stress (P < 0.05), and the inhibition was significantly alleviated by exogenous NO treatment(P < 0.05). Under non-salt stress conditions, NO treatment slightly increased the dry weight of both shoots and roots. The alleviating salt stress of exogenous NO was blocked by hemoglobin (an NO scavenger), moreover, sodium ferrocyanide, an analog of SNP that does not release NO, had no obvious effect on alleviation of salt stress to cucumber plants.

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Fig. 1. Effects of SNP (sodium nitroprusside, a nitric oxide donor) supply on shoot dry weight (A) and root dry weight (B) of 8 day treatment cucumber plants grown in nutrient solutions without or with 100 mM NaCl. Data are means  SD of three replicates. CK, control; S, 50 mM SNP treatment; N, 100 mM NaCl treatment; N þ S, 100 mM NaCl þ 50 mM SNP treatment; N þ F, 100 mM NaCl þ 50 mM sodium ferrocyanide (an analog of SNP that does not release NO) treatment; N þ S þ H, 100 mM NaCl þ 50 mM SNP þ 0.1 (w/v) hemoglobin (a NO scavenger) treatment.

(P < 0.05), and the effect of the exogenous NO was removed by addition of hemoglobin. Application of sodium ferrocyanide did not affect TBARS level in salt-treated plants. Under normal conditions application of NO had no significant effects on TBARS (Fig. 2B).

3.2. H2O2 concentrations and lipid peroxidation Compared to control, salt stress significantly increased H2O2 accumulation in root mitochondria (P < 0.05), especially 8 days after treatment. Application of exogenous NO dramatically decreased accumulation of H2O2 in cucumber root mitochondria under salt stress (P < 0.05), especially 8 days after treatment, and the effect of exogenous NO was also blocked by addition of hemoglobin. Addition of sodium ferrocyanide had no significant effect on accumulation of H2O2 under salt stress. While under normal conditions, NO treatment slightly increased H2O2 levels in cucumber root mitochondria (Fig. 2A). Lipid peroxidation was estimated by TBARS concentration. Similar to H2O2 change, salt stress significantly increased TBARS concentration in cucumber root mitochondria (P < 0.05) both 4 and 8 days after treatments, and application of NO significantly reduced the accumulation of TBARS

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Under normal conditions, application of exogenous NO did not greatly change SOD activity in cucumber root mitochondria. Salt stress significantly inhibited SOD activity, especially 8 days after treatment (P < 0.05), and application of NO greatly decreased the inhibition (P < 0.05), while the addition of hemoglobin removed the effect of exogenous NO, and application of sodium ferrocyanide had not change the SOD activity in salt-treated plants (Fig. 3A). Salt stress had different effects on CAT activity. On the 4th day after treatment, salt stress greatly induced CAT activity

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Fig. 2. Effects of SNP (sodium nitroprusside: a nitric oxide donor) supply on contents of H2O2 (A) and TBARS (B) in mitochondria of 4 and 8 day treatment cucumber root grown in nutrient solutions without or with 100 mM NaCl. Data are means  SD of three replicates. CK, control; S, 50 mM SNP treatment; N, 100 mM NaCl treatment; N þ S, 100 mM NaCl þ 50 mM SNP treatment; N þ F, 100 mM NaCl þ 50 mM sodium ferrocyanide (an analog of SNP that does not release NO) treatment; N þ S þ H, 100 mM NaCl þ 50 mM SNP þ 0.1 (w/v) hemoglobin (a NO scavenger) treatment.

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Fig. 3. Effects of SNP (sodium nitroprusside, a nitric oxide donor) supply on activities of SOD (A), CAT (B), GPX (C), APX (D), DHAR (E) and GR (F) in mitochondria of 4 and 8 day treatment cucumber root grown in nutrient solutions without or with 100 mM NaCl. Data are means  SD of three replicates. CK, control; S, 50 mM SNP treatment; N, 100 mM NaCl treatment; N þ S, 100 mM NaCl þ 50 mM SNP treatment; N þ F, 100 mM NaCl þ 50 mM sodium ferrocyanide (an analog of SNP that does not release NO) treatment; N þ S þ H, 100 mM NaCl þ 50 mM SNP þ 0.1 (w/v) hemoglobin (a NO scavenger) treatment.

(P < 0.05), and higher CAT activity was observed in SNP þ NaCl treatment. On the 8th day after treatment, salt stress significantly inhibited CAT activity (P < 0.05), and application of NO still kept CAT relatively high activity under salt stress (P < 0.05). Addition of hemoglobin or sodium ferrocyanide did not greatly affect the change of CAT activity.

Under normal conditions, CAT activity was slightly promoted by NO treatment (Fig. 3B). Similar to the change of CAT activity, salt stress significantly promoted cucumber root mitochondria GPX activity on the 4th day of treatment and significantly inhibited GPX activity on the 8th day of treatment. Under salt stress, application

of NO did not increase GPX activity under salt stress on the 4th day of treatment but significantly enhanced GPX activity on the 8th day of treatment. Application of hemoglobin and sodium ferrocyanide did not significantly influence GPX activity on the 4th day of salt stress, and on the 8th day of treatment, hemoglobin slightly increased GPX activity in salt treatment, while still being much lower than the treatment of salt with application of exogenous NO. Under normal conditions, application of NO significantly increased GPX activity on both the 4th and 8th day of treatment (Fig. 3C). On the 4th day of treatment, APX activity in cucumber root mitochondria was significantly induced by salt stress (P < 0.05), and application of NO had no obvious effect on APX activity. On the contrary, on the 8th day of treatment, salt stress considerably inhibited APX activity, and the inhibition was significantly alleviated by application of NO (P < 0.05). Application of both hemoglobin and sodium ferrocyanide did not greatly change APX activity in the salt treatment (Fig. 3D). DHAR activity in cucumber root mitochondria was significantly induced on the 4th day of treatment and inhibited on the 8th day of treatment by salt stress. Exogenous NO significantly increased DHAR activity under both normal conditions and salt stress during the treatment period. DHAR activity did not show statistically significant differences between salt treatment, salt treatment with addition of sodium ferrocyanide and salt treatment with addition of both SNP and hemoglobin (Fig. 3E). On the 4th day of treatment, there was no significant difference in GR activity between control and salt-stressed plants. Application of NO significantly increased GR activity under normal conditions and salt stress (P < 0.05). On the 8th day of treatment, GR activity was greatly induced by salt stress (P < 0.05), and application of NO slightly increased GR activity but was not statistically significant. Under salt stress, the treatment with both sodium ferrocyanide and SNP with addition of hemoglobin did not greatly change GR activity (Fig. 3F). 3.4. Hþ-ATPase activity in PMs

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On the 8th day of treatment, H -ATPase activity in cucumber root plasma membrane was assayed. As shown in Fig. 4, under normal conditions, application of NO did not significantly change Hþ-ATPase activity in cucumber root plasma membrane. Salt stress significantly inhibited Hþ-ATPase activity, and application of NO dramatically decreased the inhibition. Application of sodium ferrocyanide and SNP with addition of hemoglobin had no significant effects on Hþ-ATPase activity in PMs under salt stress (Fig. 4). 3.5. Hþ-ATPase and Hþ-PPase activity in tonoplast Cucumber root tonoplast Hþ-ATPase and Hþ-PPase activity was determined on the 8th day of treatment. Their activities showed the similar change tendency, salt stress markedly inhibited activities tonoplast Hþ-ATPase and Hþ-PPase, exogenous NO significantly alleviated the inhibition. The treatment

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Fig 4. Effects of SNP (sodium nitroprusside: a nitric oxide donor) supply on activity of Hþ-ATPase in plasma membrane of 8 day treatment cucumber root grown in nutrient solutions without or with 100 mM NaCl. Data are means  SD of three replicates. CK, control; S, 50 mM SNP treatment; N, 100 mM NaCl treatment; N þ S, 100 mM NaCl þ 50 mM SNP treatment; N þ F, 100 mM NaCl þ 50 mM sodium ferrocyanide (an analog of SNP that does not release NO) treatment; N þ S þ H, 100 mM NaCl þ 50 mM SNP þ 0.1 (w/v) hemoglobin (a NO scavenger) treatment.

of both sodium ferrocyanide and SNP with addition of hemoglobin did not significantly influenced their activities under salt stress (Fig. 5A,B). 4. Discussion Increased H2O2 and lipid peroxidation has been obtained in mitochondria of pea leaves, tomato leaves and roots under salt stress [17,39,40]. Results of this experiment also revealed that NaCl treatment induced oxidative stress in cucumber root mitochondria as indicated by the increased accumulation of H2O2 and TBARS. The increased H2O2 concentration of root mitochondria in the salt-treated plants probably resulted from a salt-induced increase in the rate of O2 production, considered to be the main precursor of mitochondrial H2O2 [42]. Zottini et al. [65] has reported previously that NO can affect plant mitochondrial functionality and this experiment showed that oxidative stress of cucumber root mitochondria induced by salt was effectively alleviated by application of SNP, while the application of sodium ferrocyanide and the application of SNP with hemoglobin did not alleviate the oxidative stress induced by salt stress. Because SNP is a NO donor, while sodium ferrocyanide is an analog of SNP that does not release NO and hemoglobin is a NO scavenger, therefore, by comparing these results, we can conclude that SNP alleviating the oxidative stress by salt stress was attributed to its releasing NO. In animals, NO has been reported to block the oxidative damage associated with NO, including modulating antioxidant enzyme activity to exert protective functions [7]. Also, Uchida et al. [56] reported that NO could increase rice tolerance to salt and heat stress by inducing SOD activities. 

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induction might play an important role in cucumber root mitochondria tolerance to salt stress. DHAR activity was increased on the 4th day of treatment and was not unaffected on the 8th day of treatment under salt stress. Some of the observed results were consistent with the results of Lechno et al. [30] and Zhu et al. [64], and some of the observed results were not consistent with them. The difference indicates that the influence of salt stress on the antioxidant enzymes are very complex and related to the plant treatment time, plant tissues, plant species and genotypes, and the different influence of salt stress on the ROS-scavenging enzyme activity has been observed with treatment time in the present experiment. In many studies, it was found that the function of NO alleviation of oxidative stress was attributed to induction of various ROS-scavenging enzyme activity [11,26,56]. Cheng et al. [11] concluded that the inhibition of polyethylene glycol (PEG)- and dehydration (DH)-enhanced senescence of rice leaves by NO is most likely mediated through an increase in SOD activity and a decrease in lipid peroxidation. In the present study, application of SNP significantly decreased the inhibited SOD activity by salt stress, which suggested that application of NO could promote the conversation from O2 into H2O2 and O2, which is an important step in protecting the cell. If the produced H2O2 cannot be scavenged efficiently, it can interact with O2 to form highly reactive hydroxyl radicals ($OH) that are thought to be primarily responsible for oxygen toxicity in the cell. Therefore, the efficient scavenging of H2O2 is very important for normal metabolism of plant. In the experiment, application of NO greatly induced the H2O2-scavenging enzymes CAT, APX and GPX under salt stress. Similar results have been observed in the experiment of NO stimulating Lupinus luteus seed germination under heavy metal and salt stress [26] and the experiment of NO increasing rice tolerance to salt and heat stress [56]. APX reduction of H2O2 requires ascorbate as substrate, and dehydroascorbate conversion back to ascorbate needs glutathione as substrate, therefore, higher activities of DHAR and GR are very important for keeping higher substrate concentration for APX. In the experiment, application of NO promoted DHAR and GR activities under salt stress, which is important for the efficient H2O2-scavenging by APX in cucumber mitochondria. Plasma membrane Hþ-ATPase is a P-type proton pump in plant. The transmembrane electrochemical gradient generated by the enzyme is the primary force for ions’ cross-membrane transports [38]. Effects of salt stress on plasma membrane HþATPase are well documented. In tomato and sunflower roots, the plasma membrane bound Hþ-ATPase activity was inhibited by salt stress [2,19], while the contrary, i.e. an induction by salt stress, in tomato was observed [25]. In the present experiment, salt stress significantly inhibited plasma membrane Hþ-ATPase in cucumber roots; this might be attributed to oxidative stress induced by salt treatment, because the optical activity of plasma membrane Hþ-ATPase needs a specific lipid environment, while lipid content and composition of the plasma membrane also depends on the generation of free radicals [16,52], such as hydroperoxides of polyunsaturated fatty acid, the process undergoes a variety of reactions 

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Fig. 5. Effects of SNP (sodium nitroprusside, a nitric oxide donor) supply on activities of Hþ-ATPase and Hþ-PPase in tonoplast of 8 day treatment cucumber root grown in nutrient solutions without or with 100 mM NaCl. Data are means  SD of three replicates. CK, control; S, 50 mM SNP treatment; N, 100 mM NaCl treatment; N þ S, 100 mM NaCl þ 50 mM SNP treatment; N þ F, 100 mM NaCl þ 50 mM sodium ferrocyanide (an analog of SNP that does not release NO) treatment; N þ S þ H, 100 mM NaCl þ 50 mM SNP þ 0.1 (w/v) hemoglobin (a NO scavenger) treatment.

As key elements in the defense mechanisms, varying reactions of ROS-scavenging enzymes in plants have been observed under salt stress. For example, it has been reported that ROS-scavenging enzymes increased under saline conditions in the case of salt-tolerant cotton [36], shoot cultures of rice [13], cucumber [30], wheat shoot [37] and pea [21], but decreased in wheat roots [37], or were unaffected as in the case of SOD in cucumber [30]. In the present experiment, activities of CAT, APX and GPX in cucumber root mitochondria were greatly induced on the 4th day of treatment, but were significantly inhibited on the 8th day of treatment, and SOD activity was inhibited during the whole treatment-period by NaCl stress, while GR activity was increased during the whole treatment-period by NaCl stress, the result implies that GR

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including the generation of free radicals which provoke changes in membrane properties, ultimately resulting in the dysfunction of the lipid bilayer and membrane deterioration [16]. Application of NO significantly stimulated PM Hþ-ATPase in cucumber roots, which might be attributed to optical lipid environment and be one mechanism of NO increasing cucumber tolerance to salt stress. In the calluses of reed, Zhao et al. [63] found that NO serves as a signal for inducing salt resistance by increasing the K to Na ratio, which is also dependent on the increased PM Hþ-ATPase. Sequestration of Naþ in the vacuole is a possible survival strategy of plants under salt stress by maintaining a higher Kþ/Naþ ratio in cytoplasm [18,58]. Naþ transport from the cytoplasm into the vacuole via the tonoplast Naþ/Hþ antiporter [3,15] is dependent on the activity of V-Hþ-ATPase and V-Hþ-PPase, which establishes an electrochemical Hþ-gradient across the tonoplast that energizes the transport of Naþ against the concentration gradient. In the present study, V-Hþ-ATPase and V-Hþ-PPase activities of tonoplast in cucumber roots were greatly inhibited under salt stress, but were significantly stimulated by the application of NO. The results indicate that less severe tonoplast damage occurred in NO-treated roots of cucumber under salt stress, and this might also be ascribed to the alleviation of ROS under salt stress by NO-enhanced activities of antioxidant enzymes. It is well known that the degree of lipid peroxidation is closely related to the accumulation of ROS, and the lipid process is one important factor exerting an effect on ATPase under changing environmental conditions [57]. As a result of lipid peroxidation induced by salt stress, ATPase and PPase proteins were disturbed [48]. Therefore, exogenous NO decreasing lipid peroxidation should be considered as one cause maintaining higher ATPase activity. It has been reported that many genes are induced by NO. In Arabidopsis, endogenous NO mediated UV-B induction of CHS gene expression, which is important in conferring UV-B protection [34]. Furthermore, some antioxidant genes including APX, CAT are induced by NO in Arabidopsis suspension cells [23]. In the present study, exogenous NO greatly elevated activities of antioxidant enzymes, and alleviated oxidative stress to cucumber mitochondria induced by salt stress. Thus, the protective role of NO may be attributed to its mediating the expression of genes encoding these ROS-scavenging enzymes under salt stress. To conclude, lower lipid peroxidation might result in better functioning of the plasma membrane and tonoplast, and reduce the salt stress to cucumber plants as indicated by plant growth. Based on the results, during the growth of vegetable plants in saline soil, application of exogenous NO can be a method to decrease salt stress to plants. However, the application dose of NO donor needs further investigation according to different plant species and different growth stages. Acknowledgements This work was supported by the Young Scientist Innovation Science Foundation of Shandong Agricultural University (No. 23407).

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