Effect of salt stress on physiological and morphological parameters of rapeseed cultivars.
Abiotic stresses such as salt excess (NaCl) and drought are among factors most limiting to plant productivity [2,6,7]. High salinity in soil or irrigation water is a common environmental problem affecting plant growth and productivity by provoking osmotic stress and ion toxicity together with induction of oxidative stress. There is an increasing body of evidence which suggests that together with osmotic adjustment and ion compartmentalization, an efficient antioxidant system is also important in combating salinity stress. Results have indicated that salinity affects growth and development of plants through oxidative, osmotic and ionic stresses. Because of accumulated salts in soil under salt stress condition plant wilts apparently while soil salts such as Na+ and [Cl.sup.-] disrupt normal growth and development of plant [9, 25, 38]. Salt tolerance of wheat cultivars have a direct relationship with [Na.sup.+]/[K.sup.+] ratio so that the ratio increased with the increase of salinity level but less increase is observed in tolerant cultivars, they concluded that [Na.sup.+]/[K.sup.+] ratio can be a measure of salt stress tolerance [10, 26].
One of the biochemical changes occurring when plants are subjected to biotic or abiotic stresses is the production of reactive oxygen species (ROS) . ROS are highly reactive and in the absence of any protective mechanism they can seriously disrupt normal metabolism through oxidative damage to lipids, protein and nucleic acids. In order to avoid the production of these reactive molecules plants have evolved an effective scavenging system involving antioxidant molecules like carotenoids, ascorbate, glutathione and tocopherols as well as antioxidant enzymes such as super oxide dismutase, catalase and glutathione reductase. Malondialdehyde (MDA) as the decomposition product of polyunsaturated fatty acids of biomembranes, showed greater accumulation under salt stress [23, 25]. Cell membrane stability has been widely used to differentiate stress tolerant and susceptible cultivars of some crops and and in some cases higher membrane stability and prolin content could be correlated with abiotic stress tolerance [27, 28].
The aim of this study was to evaluate the effects of salt stress on the activity of antioxidative enzymes; the lipid membrane peroxidation; the prolin content and the [Na.sup.+] and [K.sup.+] content in four rape seed cultivars, in order to better understand their differences on salt stress tolerance.
Material and methods
A research was carried out to evolution effect of salt stress on physiological and morphological parameters of four rapeseed cultivars, Slm04, Opera, Zarfam and Modena in University of Tehran and Islamic Azad University (Shoushtar Branch) in 2010. Salt stress treatments were applied using salt solutions with EC values of 0.6 (control), 4 and 8 ds/[m.sup.-1]. These solutions were called S0, S1 and S2 respectively. Required amount of each solid salt for preparing one liter salt solution was calculated through the following formula first :
TDS (mg/lit) = EC x 640
Where: TDS= total soluble solid salt amount (mg/lit)
EC= given electro conductivity value (ds/m)
Then EC value of each solution was read by means of EC meter and reached the desirable EC with addition of solid salt or distilled water. 10 Seeds of each cultivar were sown in germination boxes filled with perlite. The germination boxes were placed greenhouse where temperature ranged between 22[degrees]C and 25[degrees]C for a period of 3 weeks. The boxes irrigated daily with three different Hoagland solutions by the use of NaCl. Electrical conductivities (EC) at 25[degrees]C of the three salinity levels were 0.6(control), 4 and 8dS [m.sup.-1], respectively. After 21 days, plants were harvested for morphological, physiological and biochemical determinations. The layout of the experiment was a Factorial complete block Design. There were four replicates in each treatment group.
For enzyme assays and estimation of lipid peroxidation, frozen leaf samples were ground to a fine powder with liquid nitrogen and extracted with ice-cold 50 nM phosphate buffer (pH 7.0). The extracts were centrifuged at 4[degrees]C for 30 min at 20000g and the resulting supernatants; hereafter referred to as crude extracts, was collected and used for protein content assay and enzyme activities. Protein content was determined according to Bradford  with bovine serum albumin as the standard. Peroxidase activity was determined using the guaiacol oxidation method  in a 3 ml reaction mixture containing 10 mM phosphate buffer (pH 6.4), 8 mM guaiacol, 100-200 ml enzyme extract and 2.75 mM H2O2. The increase in absorbance was recorded at 470 nm within 30 s (linear phase) after H2O2 was added. CAT extraction was performed in a 50 mM Tris- HCl buffer. The enzyme activity was assayed by measuring the reduction of H2O2 at 240 nm and 25[degrees]C as described by Dionisio-Sese et al. .
Lipid peroxidation and Electrolyte leakage:
Lipid peroxidation was determined by measuring the amount of malondialdehyde (MDA) formation using the thiobarbituric acid method described by Stewart and Bewley. The crude extract preparation was mixed with the same volume of a 0.5% (w: v) thiobarbituric acid solution containing 20% (w: v) trichloroacetic acid. The mixture was heated at 95[degrees]C for 30 min and the reaction was stopped by quickly placing in an ice-bath. The cooled mixture was centrifuged at 10000g for 10 min, and the absorbance of the supernatant at 532 and 600 nm was read. After subtracting the nonspecific absorbance at 600 nm, the MDA concentration was determined by its extinction coefficient of 155 m[M.sup.-1] [cm.sup.-1]. 
To determine electrolyte leakage, 100 mg fresh leaf samples were cut into 5 mm length and placed in test tubes containing 10 ml distilled deionized water. The tubes were covered with plastic caps and placed in a water bath maintained at the constant temperature of 32[degrees]C. After 2 h the initial electrical conductivity of the medium (EC1) was measured using an electrical conductivity meter. The samples were autoclaved
afterwards at 120[degrees]C for 20 min to completely kill the tissues and release all electrolytes. Samples were then cooled to 25[degrees]C and the final electrical conductivity (EC2) was measured. The electrolyte leakage (EL) was expressed following the formula EL=EC1/EC2x 100 .
Proline determination was carried out according to the method of Bates et al. .
Sodium and Potassium determination:
For the determination of sodium and Potassium in the leaf, 10 mg dried material was cut into 1 cm length, placed in test tubes containing 20 ml distilled deionized water, and heated in a boiling water bath for 1 h. The tubes were then autoclaved at 120[degrees]C for 20 min and cooled. The sodium content in 15 time's diluted extract was determined by atomic absorption spectrophotometry.
All data were subjected to ANOVA test and means were compared by the Duncan's. Comparisons with P values B/0.05 were considered significantly different.
Results and discussion
Effect of Salinity on Growth;
Seedling Fresh and dry weight, shoot length and root length of four rapeseed cultivars subjected to 3week salinity treatments are shown in Table1. Cultivars which are considered as salt-tolerant, that is, Zarfam and Modena, showed higher Shoot length, fresh and dry weight at 4 dS [m.sup.-1] salinity level compared to the non-salt-treated plants. The salt sensitive cultivars, Slm04 and Opera, on the other hand, did not show this growth stimulation at moderate salinity level. At 8 dS [m.sup.-1] salinity level Zarfam and Modena cultivars showed higher root length, shoot length, fresh and dry weight than Opera and Slm04. In all cultivars decrease of shoot length in compared to root length was higher.
Effect of salinity on catalase and peroxidase activities
Fig. 1 and Fig.2 showed the effect of increasing level of NaCl salinity on catalase and peroxidase activities of the four rapeseed cultivars after 3 week exposure to salinity. The Results showed that salt stress decrease catalase activity in four rapeseeds cultivars but this decrease was slightly in Zarfam and Modena than other cultivars. In fact decrease of catalase in Zarfam and Modena was very slightly in compared to other cultivars (we can say catalase activity unchanged in salt tolerance rapeseeds cultivars). The salt-tolerant cultivars, Modena and Zarfam showed a increase in peroxidase activity at high salinity level, whereas the salt-sensitive cultivars, Slm04 and Opera, did not show any increase in peroxidase activity at all. In Slm04 Cultivar peroxidase activity unchang under salinity condition and in Opera Cultivar peroxidase activity decrease in compared to non salinity condition.
Effect of salinity on lipid peroxidation, Proline content and electrolyte leakage
The effect of increasing of salinity stress on MDA formation in the leaves of the four rapeseed cultivars after 3 week salinity treatment is shown in Table 2. With increasing level of salinity stress, the MDA content increased in the four cultivars, but this increase was higher in Opera and Slm04 than other cultivars. On the other hand, in Zarfam and Modena did not exhibit strongly increase in lipid peroxidation with a 3 week exposure to salinity stress. The amount of electrolyte leakage from the leaves of the four rapeseed cultivars subjected to increasing level of salinity stress is shown in Table2. Increasing of salinity stress increased the amount of electrolyte leakage from the leaves of Opera and Slm04 but this increase was slowly in Zarfam and Modena. The amount of proline from the leaves of the four rapeseed cultivars subjected to increasing level of salinity stress is shown in Table2. Salinity significantly increase proline content in all rapeseed cultivars but this increase was not significantly in Slm04 and Opera.
Effect of salinity on [Na.sup.+] and [K.sup.+] uptake:
There was a marked varietals difference in the accumulation of [Na.sup.+] and [K.sup.+] in rapeseed leaves in response to salinity. with increasing Stalinization, in all cultivars [Na.sup.+] was increase, but in Zarfam and Slm04 this increase were slowly than other two cultivars. With increasing Stalinization, [K.sup.+] was decrease in all cultivars, but this decrease was slowly in Zarfam Cultivar. Data showed that in Zarfam, [K.sup.+] was higher than other cultivars and Higher [K.sup.+]: [Na.sup.+] ratio showed in Zarfam than other cultivars (Table3).
Morphologically, the most typical symptom of saline injury to a plant is retarded growth due to inhibition of cell elongation . In this study Salinity decrease dry and fresh weight of rapeseed seedling but this decrease was lower in salt tolerance cultivars (Table 1). Researchers reported that accumulation of salts and ions in plant growth environment causes osmotic and drought stress leading to decrease of water absorption by plant tissues. Decrease of tissue water content results in reduction of cellular growth and development. Therefore, restriction of water absorption and its consequences for cellular growth and development is one of the most important causes of decreased growth of stem and root [2,9, 12, 16, 22]. In supporting of our observation, Hernandez et al.  reported a dose-dependent reduction in the growth of pea plants subjected to NaCl stress. Similarly, in rice leaves, under higher saline conditions, relative growth rate was decreased in salt sensitive cultivar whereas salt tolerant cultivars exhibited no significant change [13, 28]. Data of this study showed salt stress decrease shoot and root length but this decrease was higher in shoot, especially in salt sensitive cultivars. Bandeoglu et al. showed salt stress decrease root and shoot growth of lentil seedling but decrease of shoot growth was higher than root growth.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
Results of the analysis of [Na.sup.+] and [K.sup.+] percentage of leaves of rapeseed cultivars differing in salt tolerance agree with the view that there is an inverse relationship between shoot [Na.sup.+] and [K.sup.+] concentration and salt tolerance [36,38]. The salt-tolerant cultivars Modena and Zarfam did not exhibit a significant increase in [Na.sup.+] accumulation in the leaves even at high salinity level whereas the salt-sensitive cultivars Slm04 and Opera showed pronounced accumulation. Our results showed that salt tolerant cultivars have higher [K.sup.+] concentration and [K.sup.+]: [Na.sup.+] than salt sensitive cultivars. On the other hand, this study showed composite correlation between increase of [K.sup.+] and decrease of Na+ with growth of rapeseed seedling. Ashraf and McNilley . showed that salt stress increase of Na+ and decrease of K+ in shoot of rapeseed and salt tolerance rapeseed have higher [K.sup.+]:[Na.sup.+] ratio than salt sensitive cultivars . Chen et al.  studied soybean, wheat, maize and cotton and suggested that Na+ concentration increased with the increase in salinity level in all of these plants. Root Na+ content of cotton which is a salt stress tolerant plant was more than soybean root indicating preservation of [Na.sup.+] in cotton root and lack of Na+ transportation to shoot. Data of this study showed negative correlation between [Na.sup.+] concentrations in shoot of rapeseeds cultivars with MAD concentration and electrolyte leakage and positive correlation between [K.sup.+] concentration and these parameters.
Researchers suggested that [K.sup.+] concentration observed in salt stress tolerant plants were more than that of susceptible cultivars led to decreased [Na.sup.+] toxicity. Increased Na+ content led to decrease in seed germination level and seedling fresh weight in such plants [10, 11,32, 34, 35, 38, 40, 41,42,43]. Morant et al.  working on Triticale cultivars suggested that [K.sup.+]:[Na.sup.+] ratio decreased with the increase in salt stress level in growth environment in all of investigated cultivars, however, more increase value was observed among salt stress susceptible plants and reported decreased [K.sup.+] absorption in presence of NaCl as the cause of this observation.
Proline is one of the most important osmoprotectant in plants. Under salt stress most plant species exhibit a remarkable increase in their proline content [4, 12, 16,17]. In our experiments we also observed a similar behavior in the seedling of rapeseeds. Supporting findings come from other plants [35, 3] where salt stress resulted in extensive proline accumulation. In support of our observations, recently in rice roots exposed to NaCl stress, a uniform accumulation of proline was shown to be related with increasing NaCl concentrations [15, 21, 39].
The extent of damage to the membrane was monitored by measuring the amount of MDA produced when polyunsaturated fatty acids in the membrane undergo peroxidation. Membrane structure and properties, this enhanced free radical formation and lipid peroxidation under salt stress in salt-sensitive cultivars may have also brought about an increase in membrane permeability or loss of membrane integrity, as evidenced by the increase in solute leakage (Table 2). Salt stress-induced electrolyte leakage has also been previously observed in foxital millet .
An increase in MDA contents upon salt stress has been reported in different plant species [13, 16, 29, 34]. This increase was shown to be related to the amount of stress and well correlated with lipid membrane damage. Our results also demonstrated a marked increase in MDA content in leaves of rapeseeds seedlings but this increase were higher in salt sensitive cultivars. Valentovic et al.  showed salinity increase MAD content in corn salt sensitive cultivar but in tolerant cultivar, MAD unchanched. Mansour  reported that application of proline prior to salt stress protected plasma membranes of onion cells form stress mediated oxidative damage. Therefore, a higher percentage of increase in proline content and a lower extent of increase in MDA levels of seedling tissues as observed in our study was most probably the possible explanation of a reduced membrane damage of seedling tissues under salinity stress. Data of this study showed positive correlation between low of electrolyte leakage and MDA content with seedling dry weight (data don't show).
Various researchers dealing with plants [30, 39,] have also reported increase in antioxidant enzyme like as peroxidase (POD), super oxide dismutaz (SOD)and catalase activity in salt-tolerance cultivars under salt stress. In tolerant plant species, POD activity was found to be higher, enabling plants to protect themselves against the oxidative stress whereas such activity was not observed in sensitive plants . In the present study, the POD activity significantly increased in Zarfam and Modena but remained unchanged in Slm04 and decrease in Opera. On the other hand, The salt-induced enhancement of POD activity in salt tolerant cultivars indicated that it had a higher capacity for the scavenge ROS. Data of this study showed positive correlation between peroxidase activity and low of MAD. Study of Bandeoglu et al. on Lentil showed under salt stress increase of peroxidase enzyme decrease effect of salt stress on growth of lentil seedling but activity of catalase decrease under salt stress in lentil. Meloni et al.  showed under salinity, antioxidant enzyme like as peroxidase increase in salt tolerant cotton variety but in salt sensitive variety peroxidase activity was lower than salt tolerance variety. Increase in peroxidase in salt tolerant variety led to increase of photosynthesis of cotton in compared to salt sensitive variety. Conversely, as observed in this study, in potato rice . Catalase activity did not change under salt stress. Neto et al.  showed salt stress reduced catalase activity of corn sensitive cultivar but did not effect on catalase activity of corn resistance cultivar. They suggested that catalase was sensitive antioxidant enzyme in compared to other enzymes such as peroxidase and super oxide desmutase under stress condition.
In conclusion, this study showed that the difference of antioxidant enzyme activities and ion content in the four cultivars could be described to the difference in mechanisms underlying salt stress injury and subsequent tolerance to salinity. Notably Zarfam and Modena cultivars, which exhibited higher salt tolerance, had also higher antioxidant enzyme activity and K+:Na+ ratio than Modena and Opera. Data obtained of this study indicated that the relative NaCl stress tolerance of Zarfam and Modena may be due to a lower rate of peroxidation of its lipids and a higher constitutive activity of antioxidant enzymes. These results confirm that the scavenging system forms the primary defense line in protecting the plant tissue against ROS in rapeseed. MDA is produced when polyunsaturated fatty acids in the membrane undergo peroxidation. The results reported here show that the degree of accumulation of MDA was higher in Opera and Slm04 than in Zarfam and Modena, indicating a high rate of lipid peroxidation in Opera and Slm04 due to salt stress. The lesser degree of membrane damage (as indicated by low MDA content and electrolyte leakage) and the higher activity of peroxidase and catalase observed in NaCl treated plants of Zarfam and Modena indicated that these rapeseed cultivars had a higher capacity for the tolerant salinity in comparison with sensitive cultivars.
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Department of Agronomy, Islamic Azad University, Shoushtar Branch, Shoushtar, Iran
Rozbeh Farhoudi, Department of Agronomy, Islamic Azad University, Shoushtar Branch, Shoushtar, Iran E-mail: firstname.lastname@example.org email@example.com
Table1: Effect of salt stress on seedling growth of four rapeseed cultivars Shoot Root Salt level Cultivar length(cm) length(cm) S0 Slm04 6.75 a 11.3 a Opera 6.50 a 11.1 a Modena 6.60 a 11.2 a Zarfam 6.90 a 11.3 a S1 Slm04 5.10 c 8.10 c Opera 5.10 c 8.30 c Modena 6.20 b 10.1 b Zarfam 6.30 b 10.2 b S2 Slm04 3.70 d 6.20 d Opera 3.70 d 6.30 d Modena 5.20 c 9.20 b Zarfam 5.30 c 9.10 b Seedling Seedling Salt level Cultivar FW(mg) * DW(mg) * S0 Slm04 230.8 a 24.2 a Opera 228.0 a 22.4 a Modena 226.7 a 23.7 a Zarfam 228.7 a 23.8 a S1 Slm04 165.2 c 18.2 c Opera 161.2 c 18.9 c Modena 180.3 b 20.2 b Zarfam 181.1 b 20.1 b S2 Slm04 90.3 e 14.3 e Opera 92.4 e 14.7 d Modena 134.7 d 16.7 d Zarfam 137.3 d 17.0 d * FW: Fresh Weight ** DW: Dry Weight Means followed by the same letter(s) are not significantly different at P = 0.01 according to Duncan test Table 2: Effect of salt stress on some physiological trait of four rapeseed cultivars Proline MAD Electrolyte Salt level Cultivar (mg/grfw) (nmol/grfw) leakage(%) S0 Slm04 22.7 a 4.7 a 8.30 a Opera 21.8 a 4.9 a 9.10 a Modena 20.9 a 5.1 a 8.20 a Zarfam 22.6 a 4.4 a 8.70 a S1 Slm04 23.4 a 8.2 b 15.3 b Opera 21.6 a 9.0 c 16.1 b Modena 32.9 b 6.1 b 9.80 a Zarfam 31.8 b 5.8 a 10.10 a S2 Slm04 27.2 a 12.1 d 38.80 c Opera 22.7 a 11.4 d 37.0 c Modena 50.23 c 6.6 c 14.10 b Zarfam 51.0 c 6.3 c 15.00 b Means followed by the same letter(s) are not significantly different at P = 0.01 according to Duncan test Table 3: Effect of salt stress on shoot [Na.sup.+] and [K.sup.+] percentage of rapeseed cultivars [K.sup.+]/ [Na.sup.+] [K.sup.+] [Na.sup.+] Salt level Cultivar % % ratio S0 Slm04 3.6 c 10.6 a 2.9 a Opera 3.2 c 10.9 a 3.2 a Modena 3.4 c 10.5 a 3.1 a Zarfam 3.4 c 10.7 a 3.1 a S1 Slm04 4.4 b 8.4 c 1.9 c Opera 4.4 b 8.2 c 1.8 c Modena 4.1 b 9.1 b 2.2 b Zarfam 3.8 c 9.4 b 2.4 b S2 Slm04 5.9 a 6.1 d 1.3 e Opera 5.7 a 6.3 d 1.1 e Modena 4.9 b 8.1 c 1.6 d Zarfam 4.6 b 8.2 c 1.9 c Means followed by the same letter(s) are not significantly different at P = 0.01 according to Duncan test