Antioxidant Enzyme Activities as a Tool to Discriminate Ecotypes of Crithmum maritimum L. Differing in Their Capacity to Withstand Salinity

Salt stress causes a range of adverse effects in plants, mainly ionic disorders, osmotic stress and nutritional imbalance. A common feature of these effects is the production of reactive oxygen species (ROS) (Ashraf and Foolad, 2007). Thus, salt stress causes stomatal closure, which reduces the CO2/O2 ratio inside leaf tissues and inhibits CO2 fixation (Hernandez et al., 1999). As a consequence, an over reduction of the photosynthetic electron transport chain occurs, which causes the generation of ROS such as singlet oxygen (1O2), superoxide anion (O2.−), hydrogen peroxide (H2O2), and hydroxyl radical (OH). It is now widely accepted that ROS are responsible for various stress-induced damages to macromolecules and ultimately to cellular structure (Mittler, 2000; Ashraf, 2009). Plants are equipped with a set of non-enzymatic scavengers and of antioxidant enzymes that act in concert to alleviate cellular damage under oxidative stress conditions (Foyer and Noctor, 2000). Superoxide dismutase (SOD) reacts with the superoxide radical at almost diffusion-limited rates to produce H2O2 (Scandalios, 1993). H2O2 is scavenged by peroxidases, especially ascorbate peroxidase (APX), and catalase (CAT). CAT has been found predominantly in leaf peroxisomes where it functions chiefly to remove H2O2 formed in photorespiration or in β-oxidation of fatty acids in the glyoxysomes (Dat et al., 2000). APX, which uses ascorbic acid as a reductant in the first step of the ascorbateglutathione cycle is the most important plant peroxidase involved in H2O2 detoxification (Foyer and Halliwell, 1976; Noctor and Foyer, 1998). ROS are also scavenged nonenzymatically by hydrophilic antioxidants, such as ascorbate and glutathione (GSH), most of them being found in photosynthetic tissues. In the last two decades, a number of reviews have concentrated much on the role of various antioxidant enzymes and metabolites in plant salt tolerance (Jitesh et al., 2006; Ashraf, 2009). Due to considerable variations in the mechanisms of defense against ROS among plant species, it is difficult to generalize the involvement of this phenomenon in salt tolerance


Introduction
Salt stress causes a range of adverse effects in plants, mainly ionic disorders, osmotic stress and nutritional imbalance.A common feature of these effects is the production of reactive oxygen species (ROS) (Ashraf and Foolad, 2007).Thus, salt stress causes stomatal closure, which reduces the CO 2 /O 2 ratio inside leaf tissues and inhibits CO 2 fixation (Hernández et al., 1999).As a consequence, an over reduction of the photosynthetic electron transport chain occurs, which causes the generation of ROS such as singlet oxygen ( 1 O 2 ), superoxide anion (O 2 .−), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (•OH).It is now widely accepted that ROS are responsible for various stress-induced damages to macromolecules and ultimately to cellular structure (Mittler, 2000;Ashraf, 2009).Plants are equipped with a set of non-enzymatic scavengers and of antioxidant enzymes that act in concert to alleviate cellular damage under oxidative stress conditions (Foyer and Noctor, 2000).Superoxide dismutase (SOD) reacts with the superoxide radical at almost diffusion-limited rates to produce H 2 O 2 (Scandalios, 1993).H 2 O 2 is scavenged by peroxidases, especially ascorbate peroxidase (APX), and catalase (CAT).CAT has been found predominantly in leaf peroxisomes where it functions chiefly to remove H 2 O 2 formed in photorespiration or in β-oxidation of fatty acids in the glyoxysomes (Dat et al., 2000).APX, which uses ascorbic acid as a reductant in the first step of the ascorbateglutathione cycle is the most important plant peroxidase involved in H 2 O 2 detoxification (Foyer and Halliwell, 1976;Noctor and Foyer, 1998).ROS are also scavenged nonenzymatically by hydrophilic antioxidants, such as ascorbate and glutathione (GSH), most of them being found in photosynthetic tissues.In the last two decades, a number of reviews have concentrated much on the role of various antioxidant enzymes and metabolites in plant salt tolerance (Jitesh et al., 2006;Ashraf, 2009).Due to considerable variations in the mechanisms of defense against ROS among plant species, it is difficult to generalize the involvement of this phenomenon in salt tolerance (Ashraf and Harris, 2004).A number of experimental approaches have been adopted to understand the link between salt tolerance and antioxidant activities.Several studies compared crop species, genera and cultivars differing in salt tolerance, and showed considerable variations in the production of antioxidant in response to salt stress.Moreover, increased levels of antioxidant enzymes and/or metabolites have been correlated to the degree of salt tolerance in a number of plant species including wheat, rice, maize, cotton, tomato and potato (Ashraf, 2009).However, such relationship could not be found in other plants such as cowpea (Cavalcanti et al., 2004), Arabidopsis (Katsuhara et al., 2005) and strawberry (Turhan et al., 2008).Recently, many authors addressed investigations on naturally salt tolerant plants (or halophytes) in which the mechanisms of salt tolerance are fully developed and functional to make them productive under stress.Jithesh et al. (2006) reviewed the regulation of antioxidant enzymes under salt stress in halophytes, especially those of mangroves.They concluded that antioxidant enzymes protect halophytes from deleterious ROS production during salt stress.The antioxidant response in halophytes, like in glycophytes, varies among species and genera.Most studies reported an induction in the activities of antioxidant enzymes and an accumulation of antioxidant metabolites, in response to salinity (Cherian et al., 1999;Takemura et al., 2000;Parida et al., 2004).Moreover, some halophytes like Thelungiella halophila exhibited high levels of SOD expression even in unstressed conditions (Taji et al., 2004).Thus, the SOD activities in mangrove Rhizophora stylosa were more than 40 times higher than those of the glycophyte pea.High concentration of ascorbate was also observed in the leaves of that plant.This robust antioxidant activity is considered to be effective in protecting mangroves from excess irradiance (which generates ROS) under natural conditions (Cheeseman et al., 1997).Higher antioxidant defences in leaves compared to those of roots accounted for the survival of the halophyte Crithmum maritimum L. under high salinity conditions (Ben Hamed et al., 2007).In the same species, Ben Amor et al. (2005) showed an increased total SOD activity in shoot tissues in presence of 50 mM NaCl stress while SOD activity in root tissues decreased upon salt treatment (up to 200 mM).These findings showed that the activities of antioxidant enzymes under salt stress may vary depending on the tissues.It may also depend on the conditions of treatments and on the species from which ecotypes were collected.In the present work, we examined the effects of salt stress on two ecotypes of Crithmum maritimum grown under the same controlled conditions.The effects of salt stress were investigated on growth, lipid peroxidation, electrolyte leakage, hydrogen peroxide levels and antioxidative enzyme activity.The differential response of that system might contribute to better understand the mechanism of salinity resistance and, in turn, might lead to the development of elite ecotypes able to withstand high salinity levels in the soils.

Plant material and growth conditions
Seeds of two Crithmum maritimum (Apiaceae) ecotypes were collected from coastal sites near Tabarka (North-West of Tunisia, humid bioclimatic stage) and Korbous (North-East of Tunisia, semi-arid bioclimatic stage).Seeds were sown in pots (4 seeds per pot) filled with 3 kg of inert sand and irrigated with distilled water until germination.Then, seedlings were daily watered with Hewitt solution (Ben Hamed et al., 2007).Thirty four day-old seedlings were partitioned into 3 lots, which were treated with 0, 100, and 300 mM NaCl, respectively (the salt concentrations were daily stepwise increased with 50 mM NaCl).The experiment was conducted in a growth room under controlled conditions : 15-25°C temperature and 70-90% relative humidity, 8-16 h night-day photoperiod, and 440 µmol m -2 s -1 photosynthetic active radiations (PAR).Plants were harvested at the beginning of salt treatment (initial harvest) and after 50 days.For plant growth, plants were separated into leaves, stems and roots.Samples from fully expanded leaves were liquid N 2 frozen and stored at -80 °C for enzyme activity analysis.

Plant growth
Plant shoot and root dry weights (DW) were measured after 96 h at 80°C.The relative growth rate (RGR) was also calculated.

RGR (day
Where ∆ is the difference between the values at the final and initial harvests, t the salt treatment duration (days), and M the logarithmic mean of M, the whole plant dry weight (g): was extracted by homogenizing plant leaves with phosphate buffer (50 mM, pH 6.8) including 1 mM hydroxylamine.The homogenate was centrifuged at 6000 g for 25 min.The H 2 O 2 concentration in the supernatant was then measured colorimetrically (Chaparzadeh et al., 2004).Aliquot of the extract was mixed with 0.1% titanium chloride in 20% (v/v) H 2 SO 4 and the mixture was centrifuged at 6000 g for 15 min.The intensity of yellow color of the supernatant was measured at 410 nm.H 2 O 2 concentration was calculated using the extinction coefficient 0.25 mM -1 cm -1 , and expressed on the basis of fresh weight (FW).

Lipid peroxidation
The extent of lipid peroxidation was estimated by determining the concentration of malondialdehyde (MDA) (Hagege et al., 1990).Leaf materials were homogenized in 0.1% (w/v) TCA solution.The homogenate was centrifuged at 15000 g for 10 min and 1 ml of the supernatant obtained was added to 4 ml of 0.5% (w/v) TBA in 20% (w/v) TCA.The mixture was incubated at 90°C for 30 min, and the reaction was stopped by placing the reaction tubes in an ice water bath.Then, the samples were centrifuged at 10000 g for 5 min, and the absorbance of the supernatant was read at 532 nm.The value for non-specific absorption at 600 nm was subtracted.The concentration of MDA was calculated from the extinction coefficient 155 mM -1 cm -1 .

Electrolyte leakage
Fresh leaf samples were cut into discs of uniform size (5 mm diameter) and placed in test tubes containing 10 ml of double distilled water.The tubes were incubated in a water bath at 32°C for 2 h and the initial electrical conductivity of the medium (EC1) was measured.Samples were heated at 100°C for 20 min to release all electrolytes, cooled to 25°C and the final electrical conductivity (EC2) was measured (Sairam et al., 2002).The electrolyte leakage (EL), expressed in % of total electrolytes, was calculated by using the formula: EL= (EC1/ EC2) x 100.
For ascorbate peroxidase extraction, 20 mM sodium ascorbate was added to the medium to maintain the enzyme active during the extraction procedure.The homogenate was centrifuged at 12000 g for 30 min.and the supernatant was dialysed for 2 h against the extraction buffer containing only 5 mM sodium ascorbate.Three replicates per treatment were used.The supernatants were collected and protein concentration was determined according to Bradford (1976), using bovine serum albumin as a standard.

Enzyme assays
Total superoxide dismutase (SOD, EC 1.11.1.5)activity was assayed according to Scebba et al. (1999).Increasing volumes (5, 10, 20, and 40 µl) of leaf and root crude extracts were added to the reaction mixture at a final volume of 3 ml.The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM L-methionine, 2 µM riboflavin and 75 µM NBT (nitroblue tetrazolium).The reaction was started by exposing the mixture to cool white fluorescent light for 15 min.The blue colour developed was measured spectrophotometrically at 560 nm.The volume of sample causing 50% inhibition of colour development (compared to the blank where sample was replaced by extraction buffer) contained one unit of SOD activity and the specific activity of plant extracts was expressed as units per mg of protein.
Total catalase (CAT, EC 1.11.1.6)activity was measured spectrophotometrically according to the method of Lück (1965), by monitoring the decline in absorbance at 240 nm due to H 2 O 2 consumption.The 3 ml reaction mixture contained 66 mM sodium phosphate buffer (pH 7.0) and 30% H 2 O 2 (the absorbance should be about 0.5 at 240 nm and with a 1 cm light path).The reaction was initiated by adding an appropriate dilution of the shoot or root crude extract to this solution.CAT activity was expressed as units per mg of protein, one unit being the amount of enzyme which liberates half the peroxide in 100 s at 25°C (Lück, 1965).Total peroxidase (POD, EC 1.11.1.7)activity was determined spectrophotometrically by measuring the oxidation of o-dianisidine (3,3'-dimethoxybenzidine) at 460 nm (Ranieri et al., 2000).The reaction mixture contained 20 mM phosphate buffer (pH 5.0), 1 mM dianisidine, 3 mM H 2 O 2 and 50 µl of extract.POD specific activity was expressed as units (µmol of dianisidine oxidized per minute) per mg of protein.Total ascorbate peroxidase (APX, EC 1.11.1.11)activity was measured spectrophotometrically according to Nakano and Asada (1981) by following the decline in absorbance at 290 nm due to ascorbate oxidation.The oxidation rate of ascorbate was estimated between 1 and 60 s after starting the reaction with the addition of H 2 O 2. The 1 ml reaction mixture contained 50 mM HEPES-NaOH (pH 7.6), 0.22 mM ascorbate, 1 mM H 2 O 2, and an aliquot of enzyme extract.Corrections were made for the low, non-enzymatic oxidation of ascorbate by H 2 O 2 and for the oxidation of ascorbate in the absence of H 2 O 2 .APX activity was expressed as units (µmol of ascorbate oxidized per minute) per mg of protein.
Total glutathione reductase (GR, EC 1.6.4.2) activity was determined by following the rate of GSSG-dependent oxidation of NADPH, through the decrease in the absorbance at 340 nm (Di Baccio et al., 2004).The assay mixture (1 ml final volume) was composed of 0.4 M potassium phosphate buffer (pH 7.5), 0.4 mM Na 2 EDTA, 5.0 mM GSSG and 100 µl of crude extract.The reaction was initiated by the addition of 2.0 mM NADPH.Corrections were made for the background absorbance at 340 nm, without NADPH.Activity was expressed as units (µmol of NADPH oxidized per minute) per mg of protein.

Statistical analysis
Analysis of variance (ANOVA) using AV1W MSUSTAT program with orthogonal contrasts and mean comparison procedures was performed to detect significant differences between treatments.Mean separation procedures were carried out using the multiple range tests with Student's least significant difference (LSD) (P ≤ 0.05).

Plant growth parameters
Growth of both ecotypes was estimated through dry mass production and RGR.The response of Tabarka plants to salinity was different to that of Korbous ecotype: whereas growth decreased under low (100 mM) and significantly under high (300 mM NaCl) salinity in Korbous plants (Fig. 1A and B), it increased by 40% under 100 mM NaCl in Tabarka plants.
In the following sections, we will try to check if the different physiological behavior of the ecotypes of C. maritimum observed mainly under low salinity (100 mM NaCl) is concomitant with different or similar antioxidant responses.

Oxidative stress parameters
Lipid peroxidation, as estimated by the MDA content, electrolyte leakage and H 2 O 2 content are considered as the most useful indicators to detect oxidative disturbances in plants.In Korbous ecotype, MDA level (Fig. 2A), H 2 O 2 concentration (Fig. 2B) and electrolyte leakage (Fig. 2C) increased in the leaves of salt treated plants.Conversely, all these parameters were higher in control plants of Tabarka ecotype.

Activity of antioxidant enzymes
Superoxide dismutase (SOD) is the first enzyme that eliminates superoxide radicals in plant cells.Total SOD activity in Korbous plants was unaffected by NaCl exposure but it increased in the leaves of salt-treated Tabarka ecotype (Fig. 3A).Individual activities of SOD isoforms in response to NaCl was examined after polyacrylamide gel electrophoresis (Fig. 3  B).The gel showed that plants from Korbous subjected to salt maintained steady activities of Mn and Fe SOD isoforms.Conversely, each isoform was stimulated by NaCl treatment in Tabarka ecotype and a new isoform (type Cu-Zn) was induced.Cu Zn, Mn and Fe SOD isoforms were identified in the presence of specific inhibitors (Figure 3 C).SOD reaction on O 2 -. results in the production of H 2 O 2, which needs to be controlled.The enzyme that are well known to play major roles in the detoxification of H 2 O 2 are catalase (CAT), dianisidine (POD) and ascorbate peroxidases (APX) and Glutathione reductase (GR).In the absence of salt, the CAT activity in Tabarka plant was significantly higher than that of Korbous (Fig. 4A).The CAT activity did not change in plants from Korbous treated by 100 mM, while it increased in salt treated plants from Tabarka.POD activities increased under salinity conditions compared to control plants in both ecotypes (Fig. 4B).Also, we noticed higher control POD activities in Korbous ecotype.The activity of APX was significantly higher in Tabarka, both in the absence and presence of NaCl (Fig. 4C).Salt treatment did not affect enzyme activities in the two ecotypes.Under control conditions, the activity of GR was 50% higher in Korbous than in Tabarka plants (Fig. 4D).Salt-treated plants from Korbous exhibited a slight (-10%) decrease in GR activity, while a strong increase (+80%) in GR activity was observed in Tabarka ecotype.

Discussion
The two ecotypes of Crithmum maritimum exhibited strongly divergent responses to salinity.Tabarka ecotype showed an halotolerant behavior, as shown by stimulation of leaf biomass production and leaf expansion under 100 mM NaCl.Conversely, leaf growth and expansion of Korbous ecotype were significantly reduced under salt treatment, even at low NaCl concentration.These results showed an intraspecific variability in the response to salt stress of C. maritimum, a perennial Apiaceae, which is considered as a promising crop species for oil production (Zarrouk et al., 2004).The same conclusion was drawn for another oilseed halophyte, Cakile maritima (Ben Amor et al., 2006;Ksouri et al., 2007;Megdiche et al., 2009).Salt stress is usually accompanied by the formation of various reactive oxygen species (ROS).These are toxic and may result in a variety of metabolic disturbances including photosynthetic component damages, protein and enzyme inactivation, and membrane permeabilization because of lipid peroxidation (Price and Hendry, 1991;Mittler, 2002;Meloni et al., 2003).MDA level, H 2 O 2 concentration and electrolyte leakage are considered as reliable indicators of the oxidative stress resulting from several abiotic constraints (Shulavev and Oliver, 2006).Our results showed variability of these indicators in leaves of the two ecotypes exposed to salt stress.The significant increases in MDA and H 2 O 2 concentration and electrolyte leakage in Korbous leaves at 100 mM NaCl, compared to the lower MDA and H 2 O 2 concentrations and electrolyte leakage in Tabarka leaves under the same conditions suggest that the latter is better protected against oxidative damage under salt stress.Another explanation would be that unlike Korbous, Tabarka leaves escape from the applied stress.For instance, the two accessions could differ in their capacity to compartmentalize salt in their leaf cells, or to maintain the leaf water equilibrium.This point deserves further investigation.
The maintaining of leaf growth in Tabarka plants might be the result of the presence of more efficient antioxidative systems.The activities of antioxidant enzymes are determinant in the functioning of these systems.Under NaCl stress, the up-regulation of antioxidative enzymes characterized the response of the more tolerant ecotype.These enzymes include SOD and POD.SOD plays a significant role in protecting living cells against the toxicity of active O 2 species due to their capacity to scavenge superoxide (Scandalios, 1993).The effects of salinity stress on SOD activity has been reported in a number of plant species, lines and varieties.Thus, considerable variations in the activity of SOD in response to salinity appeared at the inter-specific or intra-specific level (Ashraf, 2009).In the present study, the activity of this enzyme increased in the leaves of the more tolerant ecotype (Tabarka), in the presence of NaCl, but no significant change in SOD activity was detected in the more sensitive ecotype (Korbous).Such a differential response to salt stress was reported between cultivars of potato (Rahnama and Ebrahimzadeh, 2005), rice (Dionisio-Sese and Tobita 1998), millet (Srenivasulu et al., 2000), wheat (Sairam et al., 2002), tomato (Shalata and Tal, 1998), strawberry (Turhan and Gulen 2008), cotton (Gossett et al.,1994) and beet (Bor et al., 2003).In plants, scavenging of superoxides occurs by differential regulation of SOD isoforms (Wang et al., 2004).Studies on regulation of individual isoforms of SOD are therefore important because their role during salt stress could be elucidated (Jithesh et al., 2006).However, all the previous reports reflect the effect of NaCl stress on total SOD activity.Most of the studies in halophytes have revealed increased activities of Mn SOD and Fe SOD isoforms localized in mitochondria and chloroplasts, respectively (Parida et al., 2004;Wang et al., 2004).This is not surprising because the immediate targets of salt stress are the chloroplasts and mitochondria.Superoxide radicals and other ROS are formed in the chloroplasts during photosynthesis, and could be translocated in mitochondria during oxygen reduction.However, superoxide radicals could also leak towards the cytosol, resulting in the induction of the cytosolic Cu-Zn SOD.Accordingly, our study revealed an increased activity of Cu-Zn SOD isoform in Tabarka ecotype in the presence de NaCl and suggested that superoxides were mainly over-produced in cytosol under salinity.Similar results were reported for the halophyte Bruguiera gymnorrhiza in presence of NaCl, mannitol and abscissic acid (Takemura et al., 2000).H 2 O 2 , which is toxic and produced by the activity of SOD to prevent cellular damage, must be eliminated by conversion to H 2 O in subsequent reactions.CAT and APX regulate H 2 O 2 level in plants (Mittler, 2002).Our data showed that CAT activities in leaves of both ecotypes decreased at 100 mM NaCl while APX activity did not change.Contrarily to our results, Shalata and Tal (1998), Bor et al. (2003), Vaidyanathan et al. (2003) and Sekmen et al. (2007) observed salt-induced activities of CAT and APX in salt-tolerant tomato, sugar beet, rice and plantain, respectively.It could be that, in our case, little H 2 O 2 is formed in the leaves of salt-treated C. maritimum, so that CAT and APX activations are not required to detoxify H 2 O 2 .Another explanation could be the involvement of POD in H 2 O 2 elimination since POD are known to participate in H 2 O 2 scavenging.Salt tolerant plants often exhibit a stimulation of POD activities under salt stress (Bor et al., 2003, Meloni et al., 2003;Ben Amor et al., 2006).Here, we obtained the same results with Crithmum maritimum plantlets.PODs are therefore probably the most important enzymes in the detoxification of H 2 O 2 in that species.However, our results show that POD activity is constitutively higher in the more sensitive ecotype.Some studies reported that high levels of POD activity in condition of saline stress are correlated with a more reduced growth of plants and it appeared that POD activity is involved in inhibiting the growth of aerial parts, rather than in protecting tissues against the accumulation of H 2 O 2 (Lin and Kao, 2000).Such growth reduction has been attributed to the hemicellulose ferruloylation and the insolubilization of hydroxyproline rich glycoprotein (HPRG), those reactions being catalyzed by POD (Dionisio-Sese and Tobita, 1998).A similar role of POD been proposed in cowpea plants subjected to NaCl (200 mM), where induction of POD activity in leaf tissues could be involved in stopping growth through the activation of tissue lignification (Cavalcanti et al., 2004).GR, the last enzyme of ascorbate-glutathione cycle, catalyzes NADPH-dependent reduction of oxidized glutathione.GR is important for plant protection against oxidative stress (Foyer and Halliwell, 1976).Although 100 mM NaCl treatment remarkably enhanced GR activity of Tabarka ecotype, slightly decreased that of Korbous ecotype.Because decreased GR activity enhances plant sensitivity to environmental stress (Aono et al., 1995), the salt induced decrease in GR activity found in leaves of Korbous plants may explain the impairment of leaf growth and loss of membrane integrity.In the same way, oxidative stress in Tabarka appears to be prevented by the strong activity of SOD, POD and the ascorbate-glutathione cycle, as shown by the increased GR activities under 100 mM NaCl stress.The induction of GR by NaCl could increase the NADP / NADPH ratio, promoting the availability of NADP to accept electrons from the chain of electron transfer and limiting ROS levels in chloroplasts (Ben Amor et al., 2006).

Conclusion
In the present study, plant growth and leaf antioxidant activities were analyzed in two Tunisian ecotypes of the halophyte Crithmum maritimum (Tabarka and Korbous, sampled from humid and semi-arid bioclimatic stages, respectively) under controlled salt stress.Both ecotypes responded differently to low salt treatment.Korbous ecotype behaved like a facultative halophyte, with growth decreasing as NaCl levels rised in the medium.Conversely, biomass production in Tabarka ecotype was stimulated by 100 mM NaCl.Our results showed also that intraspecific variability in C. maritimum salt tolerance coincides with differential antioxidant responses.As a whole, our findings suggest the capacity of a plant ecotype or species to induce antioxidative enzymes in response to salt may greatly contribute to its ability to sustain growth.

Fig. 1 .
Fig. 1.Effect of NaCl on plant dry weight (A) and relative growth rate (B) of Crithmum maritimum ecotypes.The results are the means of 10 replicates.Means followed by different letters are significantly different at P ≤ 0.05 according to student's LSD test.

Fig. 2 .
Fig.2.Effect of NaCl on malondialdehyde levels (A), hydrogen peroxide (H 2 O 2 ) concentration (B), and % of electrolyte leakage (C) in leaves of Crithmum maritimum ecotypes.The results are the means of 5 replicates.Means followed by different letters are significantly different at P ≤ 0.05 according to student's LSD test.

Fig. 3 .
Fig. 3. Effect of NaCl on the total activity of superoxide dismutases (A) and in gel activities of SOD isoforms (B).Differentiation of CuZn, Mn and Fe SOD in the crude extract of leaves of salt-treated plants of C. maritimum (ecotype Tabarka) (C).The results in (A) are the means of 5 replicates.Means followed by different letters are significantly different at P ≤ 0.05 according to student's LSD test.