Introduction

Salinity is a major stress limiting food crops and is one of the most brutal environmental stresses that hamper crop productivity worldwide (Flowers 2004; Munns and Tester 2008). Salt stress exerts its undesirable effects through osmotic inhibition, ionic toxicity and also by disturbing the uptake and translocation of nutritional ions (Misra and Dwivedi 2004). These effects can disturb the physiological and biochemical functions of the plant cell, leading to cell death (Xiong and Zhu 2002).

During salt stress, accumulation of osmolytes and proteins occurs to combat adverse effects and to enhance tolerance (Sakamoto and Murata 2002). Reactive oxygen species (ROS) such as O2−, H2O2 and OH− are also produced throughout plant development and in response to biotic and abiotic stresses. High ROS concentrations damage membrane lipids, proteins, chlorophyll, and nucleic acids. They also trigger genetically programmed cell death. To maintain the balance between ROS production and scavenging, plants regulate the activity of antioxidant enzymes, including superoxide dismutases, ascorbate peroxidases (APXs), glutathione reductase, catalase (CAT) and guaiacol peroxidase (POX) (Yan et al. 2003).

Auxin hormone is involved in plant adaptation to biotic and abiotic stresses. The plant hormone auxin activates many early response genes that are thought to be responsible for diverse aspects of plant growth and development (Yang et al. 2009). Protein kinases constitute one of the major classes of signal transducers involved in mediating a cell’s response to external stimuli. They are involved in many aspects of cellular regulation and 1–3% of functional eukaryotic genes are predicted to encode protein kinases (Stone and Walker 1995). MAPK cascades have also been implicated in plant signal transduction including response to wounding, pathogens, abiotic stresses, plant hormones, and elicitors (Pedley and Martin 2005).

Occurrence of both salinity and drought stresses is being increased due to global environmental changes. Both stresses trigger cellular dehydration leading to reduction of cytosolic and vacuolar volume. Moreover, Fan et al. (2015) proposed multi stress tolerance is regulated by genes with pleiotropic effects. Therefore, developing drought-tolerant and salt-tolerant crops will be sustainable and economical solution.

Brassica is an important source of vegetable oil and holds the third position among the oilseed crops in the world (Ashraf and McNeilly 2004). This crop is mainly grown in arid and semiarid areas of the world and is the most affected by drought and salinity compared to other major food crops. In total, drought- and salt-affected soils cover areas of 60 and 10.5 million km2, respectively and salinization and drought are the major challenges for world food supplies due to loss of farmable lands (Zhang et al. 2014). Therefore, development of varieties with optimum yield levels under abiotic stresses is an urgent need.

This study aimed to investigate biochemical and molecular aspects of salinity effect on canola seedlings differing in drought tolerance. Hence, several biochemical parameters as well as the expression of four genes involved in signal transduction pathway including Auxin responsive protein, Protein kinase, MAPK3 and MAPK4 were investigated in Licord (drought sensitive cultivar) and SLM046 (drought tolerant cultivar) of Brassica napus.

Materials and methods

Plant material and treatment

Seeds of Brassica napus namely, Licord (drought sensitive, ST index = 0.56) and SLM046 (drought tolerant, ST index = 1.06) (Shirani Rad et al. 2010) were obtained from the Seed and Plant Improvement Research Institute, Karaj, Iran. These genotypes were selected based on the following parameters: plant height, number of branch plant−1, number of silique plant−1, number of grain silique−1, 1000 grain weight, grain yield, and oil yield. Seeds were surface sterilized with 70% ethanol for 2 min, then with 20% sodium hypochlorite solution for 10 min and washed four times with deionized water thoroughly. Seeds were germinated on plates with half-strength Murashige and Skoog (MS) medium (pH 5.8) containing 0.8% agar under a 16 h light/8 h dark cycle at 25 °C for 10 days (Murashige and Skoog 1962). Ten-day-old seedlings were transferred into MS medium containing 12 dSm−1 NaCl for 0, 3, 6, 12 and 24 h, respectively. Plant materials were harvested and wrapped by aluminum foil and stored at − 20 °C before enzymatic analysis. Treated Plant were also immediately frozen in liquid nitrogen and stored at − 80 °C for further molecular analysis.

Lipid peroxidation assay

Lipid peroxidation in leaf tissue was determined by measuring malondialdehyde (MDA), a major thiobarbituric acid reactive species (TBARS) and product of lipid peroxidation (Heath and Packer 1968). The shoot and root tissue (1 g) was ground in 2.5 ml of trichloroacetic acid (0.1%, w/v). The homogenate was centrifuged at 15,000×g for 10 min at 4 °C. An equal volume of supernatant and 0.5% thiobarbituric acid (TBA) were added to 20% TCA. Samples were heated at 96 °C for 30 min and cooled in ice for 5 min. Absorbances were read at 532 and 600 nm (E = 155 mM−1 cm−1).

Total protein measurement

The protein content of B. napus varieties was measured quantitatively by using the method as given by Lowry et al. (1951). The intensity of blue color was measured at 600 nm. The amount of protein was determined from the standard curve prepared using BSA as the standard.

Soluble sugar measurement

To determine the soluble Sugar Phenol–sulfuric method was used. Dry shoot and root samples (100 mg) were placed into test tube. Then 10 ml of 70% ethanol was added and placed at 5 °C to release soluble sugars for 1 week. After 1 week, the samples were centrifuged for 20 min and then 1 ml of the clear solution removed. Then, 1 ml of phenol and 3 ml sulfuric acid added. The tubes were left for 1 h had to be stabilized color and appearance instead of extracts, 1 ml distill water was used for control. Absorbance were read at 580 nm using a standard curve of soluble sugars, sugar levels in shoots and roots were determined in control and treatments. Finally, the amount of soluble sugars was calculated based on mg g−1 dry wt. (Kochert 1978).

Preparation of extracts

Shoot and root samples (0.5 g) were homogenized in a mortar and pestle with 3 ml ice-cold extraction buffer (0.05 M Tris–HCL buffer, pH 7.5, 3 mM MgCl2, 1 mM EDTA) with the addition of 2 mM ascorbate in the case of APX assay. The homogenate was centrifuged at 5000g for 30 min at 4 °C, and then the supernatant was filtered through Whatman No. 10 filter paper. The supernatant fraction was used as a crude extract for the assay of enzyme activity. All operations were carried out at 4 °C.

Peroxidase (POX) assay

Activity of guaiacol peroxidase was measured by following the change of absorption at 420 nm due to guaiacol oxidation. The activity was assayed for 2 min in a reaction solution containing 2.5 ml of 50 mM potassium phosphate buffer (pH 7.0), 1 ml guaiacol 1%, 1 ml H2O2 1% and 0.3 ml of enzyme extract (Gueta-Dahan et al. 1997). The enzyme activity was calculated using the extinction coefficient (E = 26.6 mM−1 cm−1).

Ascorbate peroxidase (APX) assay

Ascorbate peroxidase activity was determined by monitoring a decrease in absorbance at 290 nm due to ascorbate oxidation (Nakano and Asada 1981). The assay mixture contained 2.5 ml of 50 mM potassium phosphate buffer (pH 7.0) 0.1 mM EDTA, 1 mM ascorbate sodium, 1.2 mM H2O2 1% and 0.1 ml of enzyme extract. The activity was calculated using the extinction coefficient (E = 2.8 mM−1 cm−1).

Catalase (CAT) assay

Catalase activity was determined by monitoring the disappearance of H2O2 at 240 nm according to the method of Aebi (1984). The reaction mixture contained 2.5 ml of 50 mM K-phosphate buffer (pH 7.0), 1 mM EDTA, 0.2 ml H2O2 1% and 0.3 ml enzyme extract (E = 39.4 mM−1 cm−1).

RNA isolation

At the end of each treatment, various parts of the seedlings were frozen in liquid nitrogen for RNA isolation. Approximately 100 mg of leaf tissue was sampled and placed into 1.5 ml eppendorf tube and immediately transferred into liquid nitrogen. Plant materials were ground to powder by mortar and pestle. Total RNA was extracted according to the method described for Trizol.

RT-PCR reaction

First-strand cDNA was synthesized in a 12 μl reaction system containing 1 μl oligo dT and 2 μl total RNA and 9 μl DEPS water at 65 °C for 5 min followed by addition 2 μl dNTP, 1 μl Reverse transcriptase, 1 μl RNase inhibitor and 4 μl reaction buffer at 42 °C for 1 h and 70 °C for 5 min. RT-PCR reaction was performed according to the manufacturer’s instructions, with gene-specific primers (Table 1). PCRs were conducted in 25 μl volumes containing 12.5 μl master mix, 1 μl cDNA, 0.75 µM of each of the primers and 10 μl H2O. The reactions were initiated by 95 °C for 3 min, followed by 28–30 cycle of: 95 °C 25 s, 58–62 °C 20 s, 72 °C 25 s and a final extension at 72 °C for 7 min. The intensity of PCR amplified bands was visualized under UV and measured using gel documentation system (Gel Logic 212 Pro Imaging System, Carestream, USA).

Table 1 List of primers used in RT-PCR study
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Statistical analysis

Biochemical and molecular data were presented as mean-standard deviation (SD) of three biological replicates (each replicate contained 30 seedlings). Statistical analysis was performed using one-way-analysis-of variance (ANOVA). One-way ANOVA analysis compared each cultivar at different time points under same NaCl concentration. This was followed by Tukey’s post hoc multiple comparison test using SPSS (version 16.0) for data statistics of each cultivar at different time points and lower case letters (P < 0.05) were considered as statistically significant.

Results and discussion

The highest MDA concentration was observed in shoots of Licord (1.23 µmol g−1 fr.wt.) after 12 h while the maximum level of MDA recorded in SLM046 (1.24 µmol g−1 fr.wt.) after 6 h. The MDA level was decreased in both lines over time (Fig. 1A). In roots, the highest MDA content was exhibited after 6 h (2.25 µmol g−1 fr.wt.) in Licord compared with control. However, in SLM046 cultivar peaked after 24 h (2.30 µmol g−1 fr.wt.) (Figure 1B). Exposure to 12 dSm−1 NaCl imposed reduction in MDA level of shoots in SLM046 seedlings after 24 h, while inducing its content in roots. The MDA is produced when polyunsaturated fatty acids in the membrane undergo peroxidation and can be used as indicator of stress-induced damages at the cellular level (Masood et al. 2006). Variations in MDA contents were reported in rice (Tijen and Ismail 2005) and cotton (Meloni et al. 2003) cultivars in relation to salt tolerance. Our research found significant increase of MDA content in Brassica napus L. under salt stress in initial hours after treatment. The MDA accumulation was higher in roots than shoots indicating a higher degree of lipid peroxidation in root tissues due to salt stress. However, SLM046 was able to manage lipid peroxidation of membranes in shoot tissues (76% of control) better than Licord (as same as control) after 24 h exposure to salt.

Fig. 1
figure 1

Effect of 12 dSm−1 NaCl on MDA content (µmol mg−1 fr.wt.) in A shoots and B roots of Licord and SLM046 canola plants. All the values followed by the same letter in each column are not statistically different at the P < 0.05 probability level in comprise of each line with control

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Total protein content increased in both cultivars with time. The highest level of protein was recorded in Licord after 6 h (0.117 µg g−1 dry wt.) and 24 h (0.116 µg g−1 dry wt.). In SLM046 cultivar, the highest protein content was recorded after 3 h (0.117 µg g−1 dry wt.) and 24 h (0.105 µg g−1 dry wt.) treatments (Fig. 2A). Both cultivars showed significant decline in protein level after 12 h (0.076 and 0.083 µg g−1 dry wt.) which reached the protein concentration to almost control level. A high content of soluble proteins has been observed in salt tolerant cultivars of barley, sunflower, finger millet, and rice (Ashraf and Harris 2004). The increase in protein content has also been recorded in tomato (Lycopersicon esculentum L.) clover (Medicago citrna L.), barley (Hordeum vulgare L.), and Vigna mungo L. in response to sodium chloride treatment (Abdul Qados 2011). In our experiment, initial increase in protein content also suggests adaptive responses to salinity, conferring tolerance. But due to salt stress the protein content reduced at later stages and again increased in both cultivars.

Fig. 2
figure 2

Effect of 12 dSm−1 NaCl on A protein and B sugar contents (µg g−1dry wt.) in shoots of Licord and SLM046 canola plants. All the values followed by the same letter in each column are not statistically different at the P < 0.05 probability level in comprise of each line with control

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Carbohydrates such as sugars and starch are accumulated under salt stress (Parida et al. 2002) and their key roles in stress mitigation are osmoprotection, osmotic adjustment, carbon storage, and radical scavenging activity. Increase in reducing sugars (glucose, fructose), sucrose, and fructans have been reported in a number of plants under salt stress (Singh et al. 2000; Parida et al. 2002). In this study, soluble sugar level increased in both cultivars with the highest amount observed at 12 h (1.01 µg g−1 dry wt.) and 24 h (1.00 µg g−1 dry wt.) treatments in Licord (Fig. 2B). In SLM046, sugar content was at highest level at 6 h (0.94 µg g−1 dry wt.), 12 h (0.93 µgg−1 dry wt.) and 24 h (0.94 µg g−1 dry wt.) exposures. Both cultivars responded similarly to salt stress during the first 24 h of salinity exposure.

The antioxidant enzymes and metabolites increase under various environmental stresses (Gueta-Dahan et al. 1997; Hernandez et al. 1995). Moreover, higher activity has been reported in tolerant cultivars than the susceptible ones (Hernandez et al. 2000; Sreenivasulu et al. 2000) suggesting that higher antioxidant enzymes activity have a role in imparting tolerance to these cultivars against environmental stresses. According to the analysis, the POX enzyme activity increased over time in shoots of both cultivars (Fig. 3A) with 1.74 fold induction in Licord and 3.14 fold in SML046 at 12 h exposure. This observation suggests that the SLM046 (drought-tolerant cultivar) possesses a better ROS scavenging ability. In Licord roots, the highest activity of the enzyme was observed after 12 h (2.48 fold of control) which returned to control level (Fig. 3B). But in SLM046 cultivar, POX activity reached to its maximum level at 3 h (1.63 fold of control) and 6 h (1.55 fold of control). This result suggests quicker response of SLM046 (drought-tolerant cultivar) in ROS scavenging activity than Licord. There was steep decrease in POX activity at 24 h treatment in both cultivars which was not significant for Licord compared to control. Induction of POX activity was much more in roots of Licord compared to SLM046. On the contrary, NaCl exposed shoots of SLM046 showed higher POX activity compared to Licord. Meneguzzo et al. (1999) reported an increase in antioxidant enzyme activities in shoots of durum wheat while the roots responded differently with reduction. The mechanism by which salinity affects the antioxidant responses is not yet clear. The increase in expression of POX could be part of the oilseed rape’s response to the oxidative damage caused by the high level of salt. POX induction has also been shown in wild type and transgenic Arabidopsis seedlings as well as Citrus sinensis under salt stress (Borsani et al. 2001). Comparing two cultivars, the POX activity increased more rapidly in SLM046 (drought tolerant) shoot at initial hours of salinity stress indicating stronger response of this cultivar. Concerning roots, less activity fluctuation was observed for SLM046 as well.

Fig. 3
figure 3

Effect of 12 dSm−1 NaCl on peroxidase activity (µmol mg−1 fr.wt. min−1) in A shoots and B roots of Licord and SLM046 plants. All the values followed by the same letter in each column are not statistically different at the P < 0.05 probability level in comprise of each line with control

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To be able to endure oxidative damage under conditions such as salinity, plants must possess efficient antioxidant system in the form of enzymes such as APX (Smirnoff 1995). According to the results, the salinity increased shoot APX enzyme activity at the early 3 h of exposure in both cultivars compared to their controls. The pattern of enzyme activity in shoots of both cultivars showed a similar trend (Fig. 4A). However, reduction of activity was less in Licord (62.5%) than SLM046 (76%) compared to control. In roots, highest enzyme activity was assayed in 6 h treated seedlings in Licord cultivar. The enzyme activity showed increase in time dependent manner in roots of SLM046 cultivar (Fig. 4B) indicating better scavenging activity for this cultivar. High levels of intercellular H2O2 induces cytosolic APX activity under salt stress (Lee et al. 2001), and APX activity may have an important role in the mechanism of salt tolerance in plants (Meneguzzo et al.1999). Higher APX activity has also been reported in wild salt tolerant tomato and radish plants (Lopez et al. 1996). Our presented data supports higher APX activity in shoots than roots. Moreover, roots of SLM046 showed 8.8 fold higher APX activity compared to Licord (2.75 fold) after 24 h salinity exposure, compared to their controls. This observation implies better enduring oxidative damage by this cultivar.

Fig. 4
figure 4

Effect of 12 dSm−1 NaCl on ascorbate peroxidase activity (µmol mg−1 fr.wt. min−1) in A shoots and B roots of Licord and SLM046 plants. All the values followed by the same letter in each column are not statistically different at the P < 0.05 probability level in comprise of each line with control

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The CAT activity was significantly increased after 6 h treatment compared to control in shoots of both cultivars (Fig. 5A). However, the SLM046 shoot could boost the enzyme activity to higher level (2.85 fold) than Licord (1.45 fold) compared to their control (0 h). Similar to our findings, increase in CAT activity was found in maize (Azevedo et al. 2006) and Sesamum indicum (Koca et al. 2007) under salt exposure. Subsequently, this induction was followed by decrease of activity which reached to its lowest level at 24 h treatment. However, the extent of reduction was higher in Licord (81%) than SLM046 (47.6%). This result shows better response of SLM046 cultivar to elevated level of salt stress at the early hours of exposure. Interestingly, CAT enzymatic activity was about fourfold higher in roots of SLM046 than Licord with threefold induction in Licord and 84% reduction in SLM046 after 24 h (Fig. 5B).

Fig. 5
figure 5

Effect of 12 dSm−1 NaCl on catalase activity (µmol mg−1 fr.wt. min−1) in A shoots and B roots of Licord and SLM046 plants. All the values followed by the same letter in each column are not statistically different at the P < 0.05 probability level in comprise of each line with control

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Effect of salinity on gene expression

To investigate the expression profile of Auxin responsive protein gene under high salt treatment, its transcript level was analyzed using RT-PCR approach. Compared to expression at 0 h, Auxin responsive protein displayed higher expression at 3 h time point in both cultivars (Figs. 6A, 7A). This observation indicates up-regulation of the gene in response to salinity in the early hours of NaCl treatment. However, the transcription was down regulated at 12 and 24 h time points in both cultivars. Several plant hormones such as auxin, abscisic acid, ethylene, acetic acid and acetylsalicylic acid are involved in response to the abiotic and biotic stresses. In accordance with our finding, Liang et al. (2010) demonstrated the highest level of Auxin responsive protein transcript accumulation after 3 h treatment in Zhongyou 821 cultivar under drought stress induced by mannitol. The gene showed less alteration of expression at mRNA level in SLM046 cultivar compared to Licord indicating more tolerance facing adverse condition.

Fig. 6
figure 6

Relative mRNA expression of Auxin responsive protein, Protein kinase, MPK3 and MAPK4 in A Licord and B SLM046 cultivars of rapeseed (Brassica napus L.). Ten day old seedlings were treated with 12 dSm−1 NaCl for 0, 3, 6, 12 and 24 h

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Fig. 7
figure 7

Relative mRNA expression of A Auxin responsive protein, B Protein kinase, C MPK3 and D MPK4 in Licord and SLM046 cultivars of rapeseed (Brassica napus L.). Ten day old seedlings were treated with 12 dSm−1 NaCl for 0,3, 6, 12 and 24 h. Values were compared with Actin2 gene and experiments carried out with three replications. All the values followed by the same letter in each column are not statistically different at the P < 0.05 probability level with control

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According to our semi quantitative RT-PCR results, Protein kinase gene expression significantly decreased after 6 h following slight induction after 12 h. Exposure of 10 day old SLM046 canola seedlings to high salinity, displayed significant increase at 12 h time point which followed by gradual reduction (Figs. 6B, 7B). Gene expression in both cultivars decreased overtime, indicating salinity adaptation. The highest level of protein kinase mRNA expression has also been reported after 6 h NaCl treatment in 7 day old Zhongyou 821 cultivar following decline (Liang et al. 2010). Our RT-PCR data shows threefold higher transcript level of the gene in SLM046 (drought tolerant) cultivar compared to Licord at 0 h. Protein kinases are involved in many aspects of cellular regulation (Stone and Walker 1995). The higher level of its mRNA expression in SLM046 during experiment indicates more adaptability of the cultivar to imposed stress.

Mitogen-activated protein kinase (MAPK) cascades play an important role in protein phosphorylation of signal transduction events (Rodriguez et al. 2010). They are key enzymes that mediate adaptive responses to various abiotic and biotic stresses, including pathogen challenge and elicitor induced immunity (MAPK 2002). Pharmacological studies suggest that MAPK cascade has an important role in the antioxidant defense with rapid increase in the level of reactive oxygen species (ROS) (Wang et al. 2009). In order to determine the kinetics of BnMPK3 and BnMPK4 expression in B. napus, a time course study was performed through semi-quantitative RT-PCR. Both cultivars responded to 12 dSm−1 NaCl by down-regulating of BnMPK3 after 3 h exposure. But the down-response of SLM046 came back at 6 h after treatment and up regulated after 12 h (Figs. 6C, 7C). Opposite to our finding, salinity has increased BnMPK3 gene expression in 10 day old seedlings of Huyou 15 cultivar in the early hours of treatment (Yu et al. 2005).

RT-PCR results for BnMPK4 gene showed significant up regulation after 6 h under high salt treatment following significant decrease after 12 h (Figs. 6D, 7D). The AtMPK4 plant is one of the major MAPK which is involved in the regulation of plant defense. Environmental stresses such as low temperature, low humidity, high salinity, exposure and injury could activate BnMPK4 (Wang et al. 2009). Although, two cultivars did not show any significant difference in MPK expression after 12 and 24 h exposure of seedlings to salt, but SLM046 declined the gene transcript level at earlier times to lower its signaling action. However, the transcript level of BnMPK4 always appeared in higher level in SLM046 (drought tolerant) during our experimental period which implies the higher tolerance of this cultivar facing salt stress. Since, overexpression of OsMAPK5 in rice transgenic plants could increase tolerance while its suppression has led to hypersensitivity to salt (Xiong and Yang 2003).

Conclusion

The data generated in this study will facilitate to understand mechanisms of salt and drought tolerance in B. napus. Since, most of Brassica crops do not perform well under drought and salt stresses due to sensitivity to both stresses. The biochemical and molecular data presented significant differences between antioxidant defense enzymes activity and gene expression between two cultivars when exposed to salinity. Based on our results, the SLM046 responded better to salinity condition than Licord cultivar. Identification of tolerant cultivars to both stresses will be beneficial in breeding programs of Brassica for areas threatened by salt and drought. Drought and salt stresses have many common features such as dehydration of cell and osmotic imbalance. Hence, more investigation on mechanisms of drought and salt stresses might lead to generalized tolerance mechanism in this plant. Studying the roles of stress-inducible genes in detail will further clarify the molecular mechanism of response to high-salinity and drought stresses, and also provide us the basis of effective genetic engineering strategies for improving stress tolerance in B. napus.