In the developing and under developed world, about 2.5 billion people survive on wheat as an element source of energy ( http://faostat.fao.org ). In many arid and semi-arid regions in the world, wheat production yield is limited by the availability of water resources, the use of poor water quality and also when soil drainage is poor. Salinity is one of the major abiotic stress issues worldwide affecting plant cultivation and productivity, and its adverse impacts are getting more serious problem in regions where saline water is used for irrigation ( Türkan & Demiral, 2009 ). The sodium exclusion from the leaves is the major mechanism that confers salt tolerance in wheat ( Gorham et al. , 1990 ; Husain et al. , 2003 ). In general, the hexaploid bread wheat ( Triticum aestivum : AABBDD) has a better capacity to exclude Na ⁺ than durum wheat ( Triticum durum : AABB), which is linked to the Kna1 locus on chromosome 4D ( Dvorák et al. , 1994 ). High salinity causes a primary effect like hyperosmotic stress and ion disequilibrium producing secondary effects, such as ion toxicity, oxidative stress, hormonal imbalances and nutrient disturbances ( Ashraf, 2009 ; Türkan & Demiral, 2009 ). In plant cells, salinity induces the production of reactive oxygen species (ROS) such as superoxide radical (O ₂ ^{), hydrogen peroxide (H ₂ O ₂ ), and hydroxyl radical (OH - ). ROS are continuously generated during normal metabolic processes in the mitochondria, peroxisomes and cytoplasm. But, at high level they are highly cytotoxic and can react with vital biomolecules and consequently causing peroxidation, protein denaturation and DNA mutation ( Choudhury et al. , 2013 ; Hossain et al. , 2015 ).}

In plant cells, a complex defense system is implicated to maintain the redox homeostasis, which comprise of enzymatic and non-enzymatic components. In plants, many genes encode for different ROS-detoxifying or ROS-producing enzymes forming well organized ROS gene web ( Mittler et al. , 2004 ). Several enzymes are involved in the detoxification of ROS like superoxide dismutase (SOD; EC 1.15.1.1), ascorbate peroxidase (APX; EC 1.11.1.11), catalase (CAT; E.C 1.11.1.6), glutathione reductase (GR; EC 1.6.4.2) and glutathione peroxidase (GPX; EC 1.11.1.9). SOD is the first enzyme implicated in the removal of ROS, and it catalyzes the conversion of O ₂ ^{to H ₂ O ₂ , and it occurs ubiquitously in every plant cells ( Breusegem et al. , 1999 ; Ashraf, 2009 ). H ₂ O ₂ is a toxic ROS and has deleterious effects in plant tissue. It is scavenged by CAT and different classes of peroxidase (POD) ( Mhamdi et al. , 2010 ). Other enzymes that are very important in the ROS scavenging system and function in the ascorbate-glutathione cycle are glutathione reductase (GR), monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) and dehydroascorbate reductase (DHAR; EC 1.8.5.1) ( Foyer & Noctor, 2011 ). Several genes encoding for plant antioxidant enzymes have been cloned, characterized, and used in the construction of transgenic lines. Transgenic plants overexpressing SOD ( Bowler et al. , 1991 ; Van Camp et al. , 1996 ; Breusegem et al. , 1999 ; Alscher et al. , 2002 ; Feki et al. , 2016 ), APX ( Wang et al. , 1999 ), GR ( Foyer et al. , 1995 ), GPX ( Roxas et al. , 1997 , 2000 ) and CAT ( Feki et al. , 2015 ) showed tolerance to various abiotic stresses.}

It has been demonstrated that NaCl tolerance is closely correlated with the antioxidant capacity in numerous plants, such as bread wheat ( Sairam et al. , 2002 , 2005 ; Mandhania et al. , 2006 ; Choudhury et al. , 2013 ; Hossain et al. , 2015 ). However, it has been showed that the increase of the activity of some antioxidant enzymes is concomitantly with salt sensitivity. For example, in the leaves of the salt-sensitive rice plants, NaCl stress preferentially enhanced the activities of some antioxidant enzymes like SOD and APX ( Lee et al. , 2001 ). In addition, it has been reported that the responses of plant antioxidant enzymes to salinity show varying activity patterns according to the species and analyzed tissues ( Gossett et al. , 1994 ).

The non-enzymatic antioxidant system includes ascorbic acid (AsA), glutathione (GSH), a-tocopherols (vitamin E), flavonoids, anthocyanines, polyphenolic compounds and carotenoids ( Schafer et al. , 2002 ; Suzuki et al. , 2012 ). The most abundant soluble antioxidants in plants are AsA and GSH, which play a key role in plant defense against oxidative stress ( Foyer & Noctor, 2011 ; Venkatesh & Park, 2014 ). a-tocopherol, an abundant vitamin E compound, is a lipid soluble antioxidant found in chloroplasts where it counteracts lipid peroxidation through scavenging of lipid peroxyl radicals and detoxifies singlet oxygen and hydroxyl radicals ( Munné-Bosch, 2005 ). Oxidative stress activates the expression of genes responsible for the synthesis of tocopherols in higher plants, generating its accumulation in plants ( Shao et al. , 2007 ; Wu et al. , 2007 ).

In a previous work, the physiological analysis of the two Tunisian durum wheat genotypes showed differential tolerance to salinity ( Brini et al. , 2009 ). However, data concerning the effects of salinity on ROS production and antioxidant defense system in these two durum wheat cultivars are still lacking. In this work, we aimed to provide an insight view to antioxidant enzyme system in two Tunisian durum wheat treated with different NaCl stress conditions, in order to understand the mechanisms relevant in salt tolerance.

The extent of lipid peroxidation was estimated by determining the amount of MDA in the leaves by the method of Ben Amor et al. (2005) . Fresh shoots (0.15 g) were homogenized in 1.5 mL of 0.1% (w/v) trichloroacetic acid (TCA) solution. The homogenate was centrifuged at 15,000 х g for 30 min. To 0.5 mL aliquot of the supernatant, 1.0 mL of 0.5% (w/v) thiobarbituric acid (TBA) in 20% (w/v) TCA solution was added. The mixture was heated at 90°C for 30 min, and then cooled on ice. The MDA equivalent was calculated by measuring absorbance at 532 and 600 nm in reference to a MDA standard curve.

In the present study, MDA content in the shoots of the two Tunisian durum wheat cultivars correlated with growth inhibition produced by NaCl stress. Moreover, MDA content increased significantly in cv. Mahmoudi shoots as compared to the other cultivar with the increase of NaCl concentration in the medium (Fig. 2).

The increase in the salt level of the medium caused a consistent increase in H ₂ O ₂ in these wheat cultivars. Accumulation of H ₂ O ₂ was superior in salt sensitive cultivar. Indeed, in the comparison with the non-treated plants, the presence of high NaCl concentration in the medium (200 mM NaCl) caused about three-fold increase on H ₂ O ₂ content in cv. Mahmoudi. However, this increase was lower in cv. Om Rabia3 (Fig. 2).

Under standard conditions, a weak NBT staining was observed in the different part of these two wheat cultivars. After salt stress and in contrast to the cv. Om Rabia3, an intense NBT staining was observed on cv. Mahmoudi leaves and roots, indicating an increase in the amount of O ₂ ^{(Fig. 3).}

Salinization of cropland in the Mediterranean region is a major limitation to crop yields. The ability to limit the accumulation of Na ⁺ in leaves may be an important mechanism in salt tolerance because the excessive accumulation of Na ⁺ causes the premature senescence of leaves ( Pardo, 2010 ). In a previous work, it has been demonstrated that the response to NaCl treatment is different between the two Tunisian wheat cultivars (Om Rabia3 and Mahmoudi) at short and long-term. In this study, these two Tunisian wheat genotypes were grown for seven days and then subjected to NaCl treatment for short period (3 days). Significant differences of shoots and roots DW were detected between the non-treated and the stressed plants. Moreover, the salt-tolerant cultivar (Om Rabia3) presented higher roots and shoots DW than the salt-sensitive cultivar (Mahmoudi) under high NaCl concentration (Fig. 1). Similar to our findings, dry weight was less affected in salt tolerant sesame, sugar beet and moderately salt tolerant cotton ( Greenway & Munns, 1980 ; Koca et al. , 2007 ).

Salt stress induces oxidative damage in many plants like pea, rice and tomato ( Gómez et al. , 1999 ; Hernández et al. , 2001 ; Uchida et al. , 2002 ; Mittova et al. , 2003 ). The MDA content is considered as an indicator of the level of lipid peroxidation. Many studies showed that abiotic stresses lead to lipid peroxidation and consequently produce an accumulation of the MDA ( Chaoui et al. , 1997 ; Mandhania et al. , 2006 ; Distelbarth et al. , 2012 ). In agreement with these observations, our results showed that salinity induced oxidative stress in these two Tunisian durum wheat leaves, manifested by an accumulation of O ₂ ^{and an increase in MDA and H ₂ O ₂ contents (Figs. 2, 3). Nevertheless, this increase was different between these two cultivars. Indeed, under NaCl treatment the level of lipid peroxidation and H ₂ O ₂ content were higher in the cv. Mahmoudi than in the other cultivar. These data suggest that the cv. Om Rabia3 was better protected against oxidative damage under NaCl stress. Parallel to our results, low MDA content is important in terms of salt tolerance as represented in different studies. Salt tolerant barley ( Liang et al. , 2003 ), tobacco ( Ruiz et al. , 2005 ) and wheat cultivars ( Sairam et al. , 2002 , 2005 ; Mandhania et al. , 2006 ) also showed low level of lipid peroxidation which is important sign of higher oxidative damage limiting capacity under salinity.}

AsA is the major antioxidant in the plant cell. As the generation of ROS is increased under stress conditions, AsA is believed to contribute actively in enhancing tolerance to various environmental stresses ( Noctor & Foyer, 1998 ). It is clear that high level of endogenous AsA is essential effectively to maintain the antioxidant system that protects plants from oxidative damage due to abiotic and biotic stresses ( Shigeoka et al. , 2002 ). Salt induces a decrease in AsA content in wheat at the vegetative stage ( Sairam et al. , 2005 ) and at the reproductive stage ( Athar et al. , 2008 ). In agreement with this observation, our study showed that salinity induces a decrease in AsA in the two durum wheat cultivars particularly in the cv. Mahmoudi (Fig. 4). This larger AsA decrease was also found in salt sensitive pea cultivar ( Hernández et al. , 2001 ).

In Arabidopsis cells exposed to oxidative stress, some gene showed changes in expression levels. These genes encoded for proteins with antioxidant functions or were associated with defense responses or other stresses ( Desikan et al. , 2001 ). For example, there are many reports on the changes in the activity of various SOD isoenzymes and corresponding mRNA under osmotic stresses ( Zhu & Scandalios, 1994 ). Our results showed that higher NaCl concentration produces an elevate expression of CAT, MnSOD and APX genes (Fig. 5). Interestingly, the higher expression of these three genes was observed in the cv. Om Rabia3. APX gene is induced by various stress conditions suggesting his crucial role in many stress tolerance like drought and heat shock ( Mittler & Zilinskas, 1992 ). Concerning the antioxidative enzymes activities, NaCl stress caused up-regulation of the activities in these wheat cultivars. Comparing the enzymatic activity in the two Tunisian durum wheat cultivars, the higher activities were obtained in the salt tolerant cultivar (Om Rabia3) than in the sensitive one (cv. Mahmoudi). This result suggested that the cv. Om Rabia3 presents higher capacity for scavenging ROS than the cv. Mahmoudi. Thus, the decrease in the content of H ₂ O ₂ in cv. Om Rabia3 is the result of SOD reaction which is accompanied by an increased enzymatic capacity to decompose it. This was particularly clear in cv. Om Rabia3, in which a greater and parallel increase in SOD, CAT, POD, and APX activities occurred under low or high salinity (Fig. 6). The correlation between salinity tolerance and the increase of SOD activity has been demonstrated in many works ( Hernández et al. , 2001 ; Sreenivasulu et al. , 2000 ; Ben Amor et al. , 2005 ; Mandhania et al. , 2006 ; Koca et al. , 2007 ; Ellouzi et al. , 2011 ). Increased SOD activity in the cv. Om Rabia3 at higher NaCl level probably coped with injuring effects of O ₂ ^{. This suggestion was confirmed by the low amount of O ₂ in the different part of this cultivar. APX utilizes AsA as its specific electron donor to reduce H ₂ O ₂ to water with the generation of monodehydroascorbate ( Shigeoka et al. , 2002 ), and plays a crucial role in the management of ROS during stress ( Ahmad et al. , 2008 ). It has been reported previously that salinity increases APX activity in many salt tolerant cultivars ( Hernández et al. , 2001 ; Sairam et al. , 2005 ; Koca et al. , 2007 ). The susceptibility of the cv. Mahmoudi to salinity could be due to the inhibition of APX activity at higher salinity level. This inhibition was also observed previously in two bread wheat cultivars HD 2009 and HD 2687 ( Sairam et al. , 2005 ). Concerning POD, our results showed that salt stress produces an enhancement of POD activity in the two durum wheat cultivars. Nevertheless, POD activity remained unchanged in the salt sensitive cv. Mahmoudi treated with high NaCl concentration. Interestingly, the increase of POD activity in cv. Om Rabia3 is concomitant with the enhanced intensity of POD2 isoform, which probably produces more activity in this cultivar than in the cv. Mahmoudi. CAT is the main scavenger of strong oxidant H ₂ O ₂ in peroxisomes and it converts H ₂ O ₂ to water and molecular oxygen ( Willekens et al. , 1995 ). Higher activities of CAT and APX decrease H ₂ O ₂ level in cell and increase the stability of membranes. In both cultivars, CAT activity and salt stress effect increased in parallel. In the presence of high salinity level (200 mM NaCl), higher CAT activity was observed in the cv. Om Rabia3 resulting a better cope with reactive oxygen species. Similar to our results, higher CAT activity was found in many salt tolerant cultivars such as sesame, maize and wheat ( Azevedo Neto et al. , 2006 ; Koca et al. , 2007 ). It is worth to note that the enzyme POD increased more its activity in the tolerant cultivar in response to salt stress. Consequently, the low amount of H ₂ O ₂ in the cv. Om Rabia3 under high salt stress condition could be attributed to an increased detoxification capacity mediated by the POD2 isoform activity (Fig. 7). Taken together, it seems that the salt tolerance phenotype of the cv. Om Rabia3 could be due to a low production of ROS or to its better ability to counteracting ROS than the salt sensitive cultivar.}

In this study, we show that the two Tunisian durum wheat cultivars respond differently to salt stress. Salt leads to oxidative stress and it is an abiotic elicitor of antioxidative defenses. The better development of the cv. Om Rabia3 plant results from the greater efficiency of the antioxidative response under NaCl stress conditions. In fact, in this cultivar the antioxydant enzyme system is enhanced more efficiently compared to the salt sensitive cultivar with higher capacity to accumulate ascorbate. Consequently, under saline conditions, the growth inhibition of this cultivar is retarded with low lipid damage. Thus, it is important to understand the genetics of detoxification of ROS in order to enhance crop salt tolerance.