Introduction

Salt stress is regarded as a major environmental stress and a substantial constraint to crop production (Mahajan and Tuteja 2005). About half of world's irrigated lands and nearly 20% of the world's cultivated area are affacted by salinity (Zhu 2001). Salt stress is an ever-present threat to crop yield, especially in countries where irrigation is an essential aid to agricultuer (Flower 2004). Salinity alters various biochemical and physiological responses in plant, and thus affects almost all plant processes including photosynthesis, growth and development (Iqbal et al. 2006). In plant, salt stress leads to the imbalance of production and escavenging the reactive oxgen species (ROS) as peroxidizing agents such as hydrogen peroxide (H₂O₂), superoxide anions (O₂^· ‾) and hydroxyl radical (OH^·) at many sites (Mittler 2002). ROS can seriously disturb normal metabolism through oxidative damage to membrane lipids, proteins, pigments and nucleic acids (Misra and Gupta 2006). There are several known sources for ROS that include the leakage of electrons to O₂ from electron transport chains in the chloroplast and mitochondria of the plant cell (Dat et al. 2000). Oxidative damage in the most plant tissues is lessened by a concerted action of both enzymatic and non-enzymatic antioxidant metabolisms (Hasegawa et al. 2000). Some investigators have shown that plants with high levels of antioxidants are more resistant to damage by ROS and better adapted to tolerate abiotic stress conditions (Prasad et al. 1999; Koca et al. 2007; Javadian et al. 2010; Zamani et al. 2011). The increase in antioxidant enzymes, such as superoxide desmutase (SOD, EC: 1.15.1.1), catalase (CAT, EC: 1.11.1.6) and peroxidase (POD, EC: 1.11.1.17), is closely related to salt tolerance of many plants (Lee et al. 2001; Tseng et al. 2007; Gao et al. 2008).

SOD is a major scavenger of O₂^·‾ and its enzymatic action result in the formation of H₂O₂ and molecular O₂ (Tuna et al. 2008). The CAT and POD enzymes are scavengers of H₂O₂. A more efficient antioxidant system may correlate with tolerance to salt stress(Lin and Kao 2000). POD is involved in various processes, including lignification, auxin metabolism, salt tolerance and heavy metal stress in higher plants (Passardi et al. 2005). POD has often served as a parameter of metabolism activity in growth alteration and environmental stress conditions (Gao et al. 2008). Different affinities of important antioxidant enzymes including APX and CAT (µM and mM, respectively) for H₂O₂ suggest that they belong to the two different classes of H₂O₂-scavenging enzymes: CAT might be responsible for removal of the excess ROS during stress, whereas APX might be responsible for the fine modulation of ROS for signaling (Mittler 2002). Under environmental stress circumstances, increased antioxidant enzyme system lead to the decrease of ROS in plants (Abd El-baky et al. 2003). Salt stress causes increase in superoxide radical levels and an upregulation of antioxidant defense system (Vital et al. 2008). Many reports showed intimate relationship between enhanced constitutive antioxidant enzyme activities and increased resistance to environmental stresses (Vranova et al. 2002; Bor et al. 2003).

Oilseed rape is the second largest oilseed crop in the world (Raymer 2002) which is growing in many parts of Iran. Canola has high oil levels for agronomical benefits (Omidi et al. 2008). This study was aimed to assess the effect of salt stress on the activity of major antioxidant enzymes (SOD, POD, CAT) in the seedlings’ roots and shoots of three (salt tolerant, semi tolerant and susceptible) winter canola cultivars.

Materials and Methods

Plant growth conditions and experimental treatments

Seeds of three winter canola (Brassica napus L.) cultivars, Colvert (salt tolerant), Symbol (semi tolerant to salt) and Agat (salt susceptible), were obtained from the Seed and plant Improvement Institute (SPII), Karaj, Iran. The seeds were surface sterilized with 50% (v/v) sudium hypochlorite solution for five minutes followed by three times rinsing with distilled water. They were then germinated in petry dishes between two moisted layers of Watmann paper for 5 d and the seedlings were grown in aerated Hoagland^ۥs solution in an environmentally controlled growth chamber (Department of Plant Biology, Faculty of Biological Sciences, Tarbiat Modares University, Iran) with 16 h photoperiod at 22 ± 1ºC. Salt treatments were achieved as 0 (Control), 50, 100 and 150 mM NaCl for 24 h (Ashraf et al. 2001; Sergeeva et al. 2005; Ahmadi and Ardakani 2006; Mokhamed et al. 2006). After the treating period, roots and shoots were collected separately. Aliquotes were kept at -80 ºC to be used for enzyme assay. The remained samples were used for measuring fresh and dry weights.

Enzymes assay

For the extraction of SOD enzyme, roots and shoots (0.2 g of fresh weight) were homogenized in HEPES-KOH buffer (pH 7.3), containing 0.1 mM EDTA. The homogenate was centrifuged at 13000 × g for 15 min at 4 ºC and the supernatant was used for assaying the activity of SOD enzyme. This activity was assayed by monitoring its ability to inhibit the photochemical reduction of Nitro Blue Tetrazolium (NBT; Giannopolitis and Ries 1977). One unit of SOD is defined as the amount of enzyme required to result in 50% inhibition of the rate of NBT reduction at the 560 nm absorbance and was recorded by using spectrophtometre GBC Cintra 6 (Australia). Reaction mixture (3 ml) contained HEPES-KOH buffer (pH 7.3), 0.1 mM EDTA, 50 mM Na₂CO₃ (pH 10.2), 12 mM L-methionine, 75 mM NBT, 1 μM riboflavine and 100 μl of crude enzyme. Roots and shoots tissues (0.2 g) were homogenized in 50 mM sodium phosphate buffer (pH 7.0), centrifuged at 13000 × g for 15 min at 4 ºC and the supernatant was used for assaying the activities of POD and CAT enzymes (Chance and Maehley 1955). The POD activity was assayed by adding tissue extract (100 μl) to the reaction mixture, containing 13 mM guaicol, 5 mM H₂O₂ and 50 mM sudium phoshate buffer. Changes in the absorbance at 470 nm were read every 15 s (Chance and Maehley 1955).

Data analysis

The data were first tested for normality and then analyzed, using two-factorial balanced analysis of variance (ANOVA) on the basis of completely randomized design with three replications, using Minitab Statistical Software (Minitab Inc., State College, PA, USA; Ryan and Joiner 2001). Cultivars and salt treatments were considered as factors with three and four levels, respectively. Means were compared by the LSD method using MSTATC Statistical Software.

Results and Discussion

According to their capacity for growth on high salt medium, plants are classified as glycophytes or halophytes. Most glycophytes can not tolerate salt-stress (Sairam and Tyagi 2004). Results of ANOVA showed that for both roots and shoots, the main effects were highly significant (P<0.001) for all five parameters studied (Table 1). The interactions were also significant in shoots and roots except for root's dry weight and SOD activity and shoot's catalase activity (Table 1). Based on mean comparisons, high level of salinity caused more reduction in root and shoot fresh and dry weight in all canola cultivars (Figures 1 and 2) Colvert, the salt tolerant cultivar, showed smaller decrease than Symbol and Agat. This finding was in agreement with that reported by Ashraf and Ali (2008) in canola (cvs. Cyclon, Dunkeld, CON-III and Rainbow). Salt stress caused more reduction in fresh and dry weights in Cyclon (sensitive) than those in Dunkeld (tolerant). CON-III and Rainbow had intermediate shoot and root weights under salt stress. Similar findings were reported by other workers (Ashraf et al. 2001; Qasim et al 2003; Jamil et al. 2005).

Table 1. Analysis of variance for physiological parameters in roots (a) and shoots

 (b) of three winter canola cultivars

^*, ^**, ^*** Significant at 0.05, 0.01 and 0.001 probability levels, respectively

^ns Not significant at 0.05 probability level

Figure 1. Fresh weight (g) of a) root and b) shoot of three winter canola cultivars

at four salt stress treatments

With the increase in salt concentration, SOD activity was enhanced in roots and shoots of all cultivars. However,  Colvert had the highest SOD activity at all conditions (Figures 3a and 3b). In the stress conditions, SOD constitutes the first line of defence against ROS (Alscher et al. 2002). Since SOD activity was high, ROS, in particular superoxide radical, scavenging was done properly and therefore, damage to membranes and oxidative stress decreased, leading to the increase of tolerance to oxidative stress (Esfandiari et al. 2007). Khosravinejad et al. (2008) reported that under saline stress, activities of protective enzymes (APX, CAT, GPX and SOD) were increased in roots and shoots of barley cultivars. In the present work, the higher POD activity in roots, was detected for Symbol and Colvert cultivars (Figure 4a) and to a lesser extent in Agat. POD activity was increased with increasing salt concentrations in all studied cultivars. Colvert, the salt tolerant cultivar, had the highest POD activity in the shoots followed by Symbol  and Agat cultivars  (Figure 4b).

Figure 2. Dry weight (g) of a) root and b) shoot of three winter canola cultivars at four salt stress treatments

A significant increase in CAT activity was  detected in the roots. Except for Colvert, the highest CAT activity in other cultivars were observed at 150 mM NaCl (Figure 5a). CAT activity of roots in the Symbol was very similar to Colvert. In shoots, the highest CAT activity was recognized in Colvert, the salt tolerant cultivar (Figure 5b) followed by Symbol and Agat, the semi tolerant and sensitive canola cultivars, respectively. Uunder salt stress, antioxidant enzymes activity were increased in roots and shoots, but this increase was more in shoots than in roots. Lin and Kao (2000) reported that the activity of antioxidant enzymes (SOD, POD, CAT) increased at 200 mM NaCl salt stress in the rice leaves. In other work, Ashraf and Ali (2008) reported that salt stress (150 mM NaCl) increased the activity of antioxidant enzymes in diploid Brassica species. They noticed that SOD, POD and CAT activities of  tolerant cultivars were higher than sensitive cultivars. Our data were in agreement with those reported by the above-mentioned workers. With respect to asseyed parameters, Colvert was more tolerant to salt stress than the other two cultivars. In conclusion, our results showed that enhanced activites of antioxidant enzymes could be related to salt stress tolerance in the canola cultivars under study.