, it was shown that the application of NO donor, SNP reduces harmful effects of salinity  and heavy metals on plants . However, nitric oxide itself is a reactive nitrogen species, and studies have shown that its effect on different cells is either protective or toxic, depending on its concentration and on the position of action . Furthermore, NO as an antioxidant agent can scavenge ROS, also as a signaling molecule leading to alterations of antioxidative gene expression and thus protects plant cells from oxidative damage . NO one can be generated non-enzymatically, by the chemical breakdown of NO donor molecules, such as sodium nitroprusside (SNP), Snitroso-N-acetylpenicillamine, S-nitrosoglutathione (GSNO) and 3-morpholinosydnomine.
2. Materials and Methods
2.1. Plant Growth and Treatment
Seeds of soybean (Glycine max L. cv. Union × Elf) were surface-sterilized with 0.5% (v/v) sodium hypochlorite for 5 min and then washed thoroughly several times with sterile distilled water. Sterilized G. max seeds were placed between two layers of filter paper in petri dishes containing water and kept at 25˚C. After germination, 7- day-old seedlings were cultivated in a hydroponics system in a growth chamber (Hoagland solution, a 14-h light, day/night temperatures of 25˚C/20˚C, photon flux density 300 μM·m–2·s–1 at the leaf level and 70% relative humidity).
Before application of NaCl salinity and nitric oxide donor, seedlings were pretreated with SA (0 and 100 µM) for a week. Sodium chloride (0 and 100 mM) without or with SNP (0 and 100 µM) was added to Hoagland solution during the third week of seedling growth. SNP (Merck, Germany) was used as a donor of NO. The Hoagland solution was daily renewed and pH was adjusted close to 6.5 . The control plants have only been treated with sterile distilled water.
After pre-treatment with SA and then treatment with SNP and NaCl, the plants were harvested and the roots and shoots were separated and washed with deionized distilled water. For the estimation of Na+, K+ and Ca2+ contents, the plants were dried at 70˚C for 48 h. For assay of enzyme activities, determination of flavonoids and anthocyanins concentration and also protein content. Fresh plant materials were frozen in liquid nitrogen and stored at –70˚C until use.
2.2. Analysis of Na+, K+ and Ca2+ Contents
The dried roots and shoots were wet-ashed in HNO3 and HCl solution (5:1; v/v). The Na+ and K+ contents of plant tissue were assayed by flame emission spectrophotometry (Model 410, Sherwood Company) and Ca2+ concentration was also measured by atomic absorption spectrophotometry (Spectra A-200, Varian Australia). The contents of these cations expressed as [mg·g–1 (d.m)].
2.3. Germination Percentage
Germination studies were conducted in incubators with a 14-h photoperiod (Gallekan Sanyong Model with cool white fluorescent lamps, 25 mM·photons·m–2·s–1 photosynthetically active radiation). Twelve treatments, including three levels of NaCl (0 and 100 mM), two levels of SA (0 and 100 µM) and two levels of SNP (0 and 100 µM) were used in this experiment. When no seeds germinated in a treatment, it was considered that germination had completed for that treatment. Finally, the seeds were checked every 24 h for 3 d. The germination index was estimated using the following equation .
In this formula, is the number of germinated seeds and N is the number of used seeds in each petri dish.
2.4. Flavonoids and Anthocyanins Concentration
Flavonoids were extracted from 0.1 g of freshly collected leaves by 10 ml acetic ethanol. Concentration of flavonoids was determined according to the formula described by Krizek et al.  and read at 300 nm.
In this formula, C is the content of flavonoids in V ml solution, ABS is the absorbance at 300 nm and Σ is the correction coefficient. Anthocyanins were extracted from leaf (100 mg fresh weight) by 10 ml acetic methanol. Concentrations of anthocyanins were determined according to the method of Wagner  and read at 300 nm. The amount of flavonoids and anthocyanins were expressed as [mg·g–1 (f.m.)].
2.5. Assay of Enzyme Activities
Frozen leaf samples (0.5 g) were used for protein extraction according to the method of Bradford . Samples were ground in 5 ml of 50 mM phosphate buffer (pH 7.5) using pre-chilled mortar and pestle. The phosphate buffer contained 1 mM EDTA, 1 mM PMSF and 1% PVP-40. Then the extract was centrifuged at 4˚C at 15,000 × g for 30 min. The supernatant (protein extract) was used for measurement of protein content, the activity of SOD and PPO activity.
2.5.1. Lipoxygenase (LOX, EC 188.8.131.52)
Activity was estimated according to the method of Doderer and coworkers . For measurement of LOX activity, the substrate solution was prepared by adding 35 μL linoleic acid to 5 ml distilled water containing 50 μL Tween-20. After adjusting the pH to 6.5, 0.1 M phosphate buffer (pH 6.5) was added to make a total volume of 100 ml. LOX activity was determined by adding 50 μL of enzyme extracts to 2.95 ml substrate. The solution absorbance was recorded at 234 nm and the activity was calculated using the extinction coefficient (25 mM−1·cm−1) and expressed as units (1 μmol of substrate oxidized per minute) per mg protein.
2.5.2. Superoxide Dismutase (SOD, EC 184.108.40.206)
Superoxide Dismutase (SOD, EC 220.127.116.11) activity was measured using the method of Giannopolitis and Reis . The reaction solution (3 mL) contained 75 μM NBT, 1.3 μM riboflavin, 13 mM methionine, 0.1 mM EDTA, 50 mM phosphate buffer (pH 7.8), and 100 μL of the enzyme extracts. The absorbance of the irradiated solution was read at 560 nm using a spectrophotometer. One unit of SOD activity was defined as the amount of enzymes that inhibited photoreduction of 50% ρ-nitro blue tetrazolium chloride (NBT).
2.5.3. Polyphenol Oxidase (PPO, EC 18.104.22.168)
Polyphenol Oxidase (PPO, EC 22.214.171.124) activity was determined according to the method of Raymond et al.  at 430 nm and expressed as [∆A430 mg–1 (protein) min–1].
2.5.4. Phenylalanine Ammonia Lyase (PAL, 126.96.36.199)
To measure the activity of this enzyme, 0.1 g fresh root and leaves of soybean were homogenised with β-mercaptoethanol (15 mM), Tris-HCl (pH 8.2, 100 mM). The formation of trans-cinnamic acid was monitored at 290 nm. PAL activity is defined as the amount of enzymes forming 1 nmol of trans-cinnamic acid from the substrate phenylalanine and expressed as µmol cinamic acid mg−1 protein .
All spectrophotometric analyses were conducted at 25˚C with a UV-vis spectrophotometer (Model UV-2101 PC, Shimadzu, Japan).
2.6. Statistical Analysis
Data for germination, cation content, flavonoids and anthocyanins concentration; and the activities of SOD, LOX, PPO and PAL were analysed using ANOVA (completely randomized) to determine if significant differences were present among means. A Duncan's multiple range tests were carried out to determine if significant (P < 0.05) differences occurred between individual treatments (SPSS- 16).
3.1. Na+, K+ and Ca2+ Contents
Application of NaCl (100 mM) to G. max increased the Na+ concentration of shoot and root as compared with control plants, while reduced the K+ content in the same conditions (Table 1). Accumulation of Na+ in the root of soybean seedlings is more than of shoot. The results indicated that Na+/K+ ratio of root and shoot was enhanced 4.1 and 5.2 times more than of control in the presence of 100 mM NaCl, respectively. As it is shown in the table, the application of SA, NO donor and SA + SNP decreased the Na+ content of shoot under salt stress, but they had no effect on the Na+ content of root. In the presence of salt
Table 1. Effects of SA, SNP, and SA + SNP supply on Na and K contents, and Na/K ratio of root and shoot in soybean plants under saline and non-saline conditions.
stress, treatment with SA + SNP could reduce the shoot Na+ content more than of SA and NO donor alone. As indicated in table, treatment of seedlings with SA, SNP and SA + SNP pronouncedly increased the K+ concentration in soybean root and shoot subjected to 100 mM NaCl. The ratio of the shoot Na+/K+ decreased by SA, NO donor and especially SA + SNP in the presence of salinity.
The content of shoot and root Ca2+ was significantly decreased in the presence of NaCl salinity (Figure 1). The data have been supported that using SA with SNP enhanced the content of the root and shoot Ca2+ of soybean seedlings in the medium containing of NaCl, while the application of SA or NO could not be effective.
3.2. Germination Percentage
Figure 2(a) demonstrates the inhibitory effects of NaCl on the germination of soybean seeds. In this experiment, germination of seeds at 100 mM NaCl was decreased by 25%, respectively, as compared with the control in the absence of NaCl. The role of SA, NO donor and their interaction in controlling the germination of G. max was studied seeds under salinity. Application of SA or NO donor could significantly enhance germination under NaCl stress. Also, the combination of SA with SNP elevated it in the presence of 100 mM NaCl (Figure 2(a)).
Figure 1. Effects of SA, SNP, and SA + SNP on the amount of root Ca+2 (a) and shoot Ca+2 (b) in soybean under saline and non-saline conditions.
Figure 2. Effects of SA, SNP, and SA + SNP on germination (a) the contents of flavonoids and anthocyanins (b, c) in soybean under saline and non-saline conditions.
3.3. Flavonoids and Anthocyanins Concentration
Salinity induced by NaCl decreased the contents of flavonoids (Figure 2(b)). In this experiment, treatment with SA + NO in the absent medium of NaCl and in the presence of 100 mM NaCl increased concentration of flavonoids markedly.
Measurement of G. max anthocyanins content under salinity conditions indicated that salinity reduced the contents of these pigments. As shown in Figure 2(c), the effect of salinity in decreasing anthocyanins was obvious. The application of NO donor or SA and their combination elevated the content of anthocyanins in the present of 100 mM NaCl.
3.4. Enzyme Activities
About the role of enzymes, in this research the activity of LOX, SOD, PPO and PAL enzymes was measured in G. max leaves under various treatments (Figure 3). As shown in Figure 3(a), treatment with 100 mM NaCl caused a significant increase in the activity of LOX. The activity of this enzyme was decreased by NO donor and SA + SNP exposed to 100 mM NaCl. The activity of SOD enzyme was considerably declined in response salinity stress as compared with control (Figure 3(b)). Application of SA in the present of salinity and the interaction of SA with SNP under saline and non-saline conditions enhanced SOD activity significantly.
Figure 3(c) demonstrates that salt stress pronouncedly decreased the activity of polyphenol oxidase, while the exogenous application of SA, SNP and SA + SNP in the absent salinity and in the presence of NaCl stress markedly increased the activity of this enzyme. Therefore, the addition of SA, SNP and SA + SNP was significantly enhanced PPO activity under non-saline conditions in comparison to control plants. The results have been data in Figure 3(d) showed that in G. max, the activity of PAL enzyme decreased in the presence of 100 mM NaCl. In the presence of exogenous SA and/or NO donor SNP, the activity of PAL considerably increased subjected to 100 mM NaCl. The application of SA with SNP could significantly increase PAL activity under salinity.
The results indicated that the content of Na+ was increased in the root and shoot of G. max under salinity (Table 1), while K+ and Ca2+ was decreased. However, excessive soluble ions such as sodium and chloride are harmful to most plants, including all major crops . It has been reported that Na+ is an inhibitory ion for cytosolic processes . There is a direct relationship between the contents of Na+ in root and shoot with applied concentration of NaCl. The results showed that increase in the Na+ content is correlated to decrease in K+ uptake. The Na+ uptake might be mediated by K+ channel and non-selective cation channels . K+ is a necessary ion to translate mRNA to protein thought coupling ribosome subunits. To investigate the effects of exogenous SA and NO donor on Na+, K+ and Ca2+ uptake, were measured the contents of Na+, K+ and Ca2+ in G. max. The results showed that treatment with SA, SNP and especially the combination of SA and SNP significantly decreased the
Figure 3. Effects of SA, SNP, and SA + SNP on the activity of LOX (a), SOD (b), PPO (c) and PAL (d) in soybean under saline and non-saline conditions.
Na+ content of shoot under NaCl salinity, but application of SA and/or NO donor had no significant effect. In contrast, externally application of SA, SNP and SA + SNP in the same condition enhanced the contents of K+ in the root of soybean plants. Moreover, treatment with SA + SNP could increase the contents of Ca2+ in the root and shoot of soybean plants under 100 mM NaCl. Enhancing effects of 100 µM SA and/or 100 µM SNP in the K+ and Ca2+ uptake reduced the Na+ Content and the Na+/K+ ratio significantly. Treatment with exogenous SA and NO donor maintained a lower ratio of Na+/K+ in NaClstressed plants. The results suggested that NO might confer salt tolerance in G. max by preventing both oxidative membrane damage and translocation of Na+ from the root to shoot that coincident with the obtained results by Guo and coworkers . It has been reported that Arabidopsis mutant Atnoa1 with an impaired in vivo NO synthase (NOS) activity and a reduced endogenous NO level was more sensitive to NaCl stress in comparison with wild type. Atnoa1 mutants displayed a greater Na+/K+ ratio in shoot than wild type when exposed to NaCl, but SNP treatment attenuated this elevation of Na+/K+ ratio . Also, It is reported that NO enhanced salt tolerance in maize seedlings, through increasing K+ accumulation in root, leaves and sheathes, while decreased Na+ accumulation . Furthermore, treatment with SA could decrease the Na+ uptake, while increased the uptake of K+ and Ca2+ in Catha edulis . Ca2+ is widely accepted as an important second messenger in the intracellular signal transduction from the cell surface to internal compartments of the cell . Ca2+ alleviates Na+ toxicity through different mechanisms, including the control of Na+/K+ selective accumulation and others that are now being elucidated .
Experiment showed that exogenous application of SA, SNP and SA + SNP increased NaCl-induced reduction in the germination of soybean seeds. It was suggested presowing seed treatment with SA leads to an activation of germination and seedling growth . There is a growing body of evidence that NO plays a role in seed dormancy and germination, but its mechanism of action at the cellular level is unknown yet. NO scavengers have also been used to implicate NO as the effective component when dormancy is broken by a range of N-containing compounds, including NO donors. For example, cPTIO prevented germination of Arabidopsis seeds that would otherwise be induced by SNP .
Leaf flavonoids and anthocyanins act as the scavenger of free radicals which protect cells against oxidative stress induced by the damaging effects of reactive oxygen species . Morever, flavonoids are able to chelate iron atom and prevent Fenton reaction . Exposure to NaCl salinity, treatment with SA, SNP and SA + SNP could enhance the content of flavonoids and anthocyanins in leaves to confer resistance of soybean seedlings to salinity. The results suggested that the role of SA + SNP in increasing these pigments was the more effective than of treatment with SA and NO donor SNP alone. The results have been suggested that the application of SA and SNP can affect the biosynthesis pathways of these pigments and counteract NaCl-induced stress .
It has been reported that stimulation of LOX activity under stress conditions reflects higher lipolytic activity in membranes and oxidation of membrane-bound fatty acids by causing propagation of lipid peroxidation . It has been showed that LOX activity was increased in the presence of 100 mM NaCl. In addition, an inhibiting effect of LOX activity was found in soybean plants treated with SNP and SA + SNP under salt stress. These results provide evidence that SA and especially NO donor may reduce LOX activity to against lipid peroxidation and membrane damage produced by NaCl salinity. It has been suggested that NO-activated antioxidative systems protected membrane lipids against peroxidation by interacting with lipid peroxyl radicals or by blocking LOX activity .
Salt stress often leads to increased production of ROS in plants, including H2O2, hydroxyl radicals (OH·) and superoxide anions () . The superoxide dismutase (SOD) is one of the most important enzymes of the antioxidant defense system in the cells of all organisms. It has been observed a progressive increase in SOD activity to SA + SNP in the absence of salinity and by SA and SA + SNP under 100 mM NaCl. In addition, the enhanced activities of SOD due to NO donor application might have been one of the factors contributing to improved growth in soybean plants under saline conditions. Further results proved that interaction of SA with NO donor significantly enhanced the activities of SOD and catalase (CAT), both of which separately contributed to the delay of and H2O2 accumulation in wheat leaves under salt stress [2,37]. Therefore, it has been conclude that the protective effect of SA and NO in stressed soybean roots may be partly due to the stimulation of SOD activity and/or direct scavenging of the superoxide anion. It has been suggested SA-induced SOD activation in wheat plants was consistent with the results obtained with other plants such as maize, cucumber and rice .
It is reported that treatment of cowpea genotypes with SA results in the induction of PAL activity and quantitative change in polyphenoloxidase (PPO) isozyme patterns and also increase in the level of endogenous SA . They as well showed the increase in activity of PPO can be attributed to minimize production of ROS. The application of SA, NO donor and the combination SA with SNP in this research could enhance the activity of PPO under salt and non-saline stress. NO-derived induction for phenylalanine ammonia lyase (PAL) gene was shown in tobacco and soybean . Nitric oxide also affects the expression of numerous plant genes, including those that encode PAL and pathogenesis related 1 (PR1). Because the accumulation of PAL has been detected in tobacco cell suspensions that were treated with a membrane-permeable analogue of cGMP, and taking into account that the induction of PAL in tobacco suspension cells by NO can be suppressed by several inhibitors of guanylate cyclase. Moreover, It is thought the activation of some of NO-dependent defense genes be mediated by cGMP . PAL is known to play a key role in the synthesis of phenolic substances necessary for lignification . It is shown that SA increased the activity of PAL in the roots of wheat seedlings. Therefore, the involvement of SA in the acceleration of lignification of cell walls which in turn might have contributed to an increase in salt resistance of wheat plants was suggested .
Salt stress often leads to increased production of ROS in plants, while application of SA, NO donor and especially the crosstalk between SA with SNP reduce the damaging effects of salinity.
We are grateful to the coworkers of the laboratory of lahijan, science and research branch, Islamic Azad University, for their support.
FW: Fresh weight;
GP: Germination percentage;
NOS: Nitric oxide synthase;
PAL: Phenylalanine ammonia lyase;
PPO: Polyphenol oxidase;
SA: Salicylic acid;
SNP: Sodium nitroprusside;
SOD: Superoxide dismutase.