Introduction

Under high salinity conditions, the growth and survival of plants depends on adaptations to both low water potentials and high sodium concentrations. High salinity in the external solution of plant cells imposes ion-specific stresses resulting from Na+ and Cl− concentrations and ion inbalance (Serrano et al. 1999). It also inflicts hyper osmotic shock on plants, as the chemical activity of water decreases causing the loss of cell turgor. Therefore, survival and growth depends on adaptation to re-establish homeostasis (Zhu 2001). Both a salt-induced reduction in chloroplast stromal volume and the generation of reactive oxygen species (ROS) can also play an important role in decreasing plant photosynthetic and respiratory capacities, thus inhibiting growth (Price and Hendry 1991). Plants are equipped with an array of non-enzymatic scavengers and antioxidant enzymes that act in concert to alleviate cellular damage under oxidative stress (Foyer and Noctor 2000). Superoxide dismutase (SOD) reacts with the superoxide radical to produce H2O2 (Scandalios 1993) which is scavenged by peroxidases, especially ascorbate peroxidase (APX) and catalase (CAT). CAT has been found predominantly in leaf peroxisomes where it basically functions to remove H2O2 formed in the photorespiration or in β-oxidation of fatty acids in the glyoxysomes (Dat et al. 2000). However, CAT activity is also found in the roots (Corpas et al. 1999; Shalata et al. 2001). APX which uses ascorbic acid (AA) as a reductant in the first step of the ascorbate glutathione cycle is the most important plant peroxidase in H2O2 detoxification (Foyer and Halliwell 1976). In addition, ROS are scavenged non-enzymatically by hydrophilic antioxidants, such as ascorbate (AA) and glutathione (GSH), found mainly in photosynthetic tissues, however these antioxidants have been detected in the roots of bean (Cuypers et al. 2000) and tomato (Shalata et al. 2001).

Increasing evidence indicates that changes in the concentrations of antioxidant molecules and enzyme activity are among the first signs of tolerance and/or adaptation to stress (Di Baccio et al. 2004). Various abiotic stresses, such as heavy metals (Rucinska et al. 1999), hypoxia (Biemelt et al. 2000), chilling (Queiroz et al. 1998) and salinity (Lee et al. 2001; Khan et al. 2002) have been shown to differently alter leaf and root antioxidant system activity. If NaCl, for example, is responsible for the modulation of these antioxidants, then different concentrations of NaCl stress should be expected to produce different degrees of effect.

Several studies have demonstrated that salt-tolerant species increase their antioxidant enzyme activities and antioxidant contents in response to salt stress, while salt-sensitive species failed to do so (Lopez et al. 1996; Meneguzzo et al. 1999; Shalata and Tal 1998). Recent studies show that even under control conditions, salt tolerant plants display a substantially higher abundance of major antioxidant components and expression of antioxidant enzyme activities than sensitive plants (Taji et al. 2004; M’rah et al. 2006).

In Tunisia, Crithmum maritimum L. (sea fennel) thrives in coastal bluff communities where the accumulation of salt spray on leaves is considerable (Orcut and Nilsen 2000). This species has the potential to grow in salt-affected areas (Shay 1990). Zarrouk et al. (2004) reported clearly that the quality of extracted oil from C. maritimum seeds is similar to that of Olive and Canola. This observation supports reports on the potential of this halophyte to act as a new source of edible oil (Weber et al. 2007). Leaves of C. maritimum contain a high concentration of ascorbic acid and traditionally have protected sailors from scurvy (Shay 1990).

Crithmum maritimum is a Na+-accumulator halophyte (Ben Hamed et al. 2004; Ben Amor et al. 2005) and is reported to have efficient antioxidant enzyme systems which are crucial in the tolerance to salt accumulation in its leaves (Ben Amor et al. 2005, 2006). In the present study, we focus on the oxidative stress symptoms and compare enzymatic and non-enzymatic antioxidant activities in the roots and leaves of control and salt treated plants.

Materials and methods

Plant material and growth conditions

Seeds of C. maritimum were collected from the shores of Korbous, about 40 km in the south east of Tunis (bioclimatic semi arid stage). Seeds were sown in pots (two seeds per pot), filled with inert sand and irrigated with distilled water until germination. At the early development stages, seedlings were watered daily with Hewitt nutrient solution (pH 7.3, EC 2.7 mS cm−1) (Hewitt 1966). About 2-month-old seedlings were divided into three blocks, which were treated with solutions of 0, 100, and 300 mM NaCl, respectively. Salt concentrations were increased daily by 50 mM NaCl, until reaching the required concentration. Plants were irrigated with their corresponding solutions every 2 days. Salt-treated plants were irrigated with distilled water between successive salt irrigations in order to eliminate excess in salt accumulated in the sand. Plant culture took place in a greenhouse at 25, 70% relative humidity (RH) during the day (16 h) and 15°C, 90% RH at night (8 h).

Growth activity

The relative growth rate (RGR) was calculated according to Hunt (1990).

$$ {\text{RGR (day}}^{{ - {\text{1}}}} {\text{)}} = {\text{M/}}\underline{{\text{M}}} \Updelta t $$

where Δ is the difference between the values at the final and initial harvests, t the salt treatment duration (days), and M the natural logarithmic mean of M, the whole plant dry weight (g):

$$ \underline{{\text{M}}} = \Updelta {\text{M/}} \Updelta \ln {\text{M}} $$

H2O2 determination

The H2O2 concentration was measured colorimetrically (Chaparzadeh et al. 2004). H2O2 was extracted by homogenizing leaf and root materials with phosphate buffer (50 mM, pH 6.8) including 1 mM hydroxylamine. The homogenate was centrifuged at 6,000g for 25 min and 1 ml of the supernatant was added to 2 ml 0.1% titanium chloride in 20% (v/v) H2SO4. After centrifugation at 6,000g for 15 min, the absorbance of the supernatant was recorded at 410 nm. H2O2 concentration was calculated using the extinction coefficient 0.25 mM−1 cm−1. H2O2 concentration was expressed on the basis of fresh weight (FW).

Lipid peroxidation

The extent of lipid peroxidation (LP) was estimated by determining the concentration of malondialdehyde (MDA) (Draper and Hadley 1990). Leaf and root material was homogenized in 0.1% (w/v) TCA solution. The homogenate was centrifuged at 15,000g for 10 min and 1 ml of the supernatant obtained was added to 4 ml 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. Samples were centrifuged at 10,000g 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

The leaf and root samples (0.2 g) were placed in test tubes containing 10 ml of double distilled water. The leaves were cut into discs of uniform size (5 mm length). The tubes were incubated in a water bath at 32°C for 2 h and the initial electrical conductivity of the medium (EC1) measured. The samples were autoclaved at 121°C for 20 min to release all the electrolytes, cooled to 25°C and the final electrical conductivity (EC2) measured (Dionisio-Sese and Tobita 1998). The electrolyte leakage (EL) was calculated by using the formula: EL = (EC1/ EC2) × 100.

Enzyme extractions

All of the following operations were performed at 4°C. The fresh leaf and root samples (0.5 g) were rapidly extracted in a pre-chilled mortar with 10% (w/w) PVP (polyvinylpyrrolidone) in 50 mM K-phosphate buffer (pH 8), containing 0.1 mM EDTA (ethylenediaminetetraacetic acid), 1 mM DTT (dithiothreitol), and 0.5 mM PMSF (phenyl-methyl-sulphonyl-fluoride). For APX activity, 20 mM ascorbate was added to the extraction medium to maintain the enzyme active during extraction. The homogenate was centrifuged at 12,000g for 30 min. For APX, the supernatant was dialysed for 2 h against the same buffer used for the homogenization containing 5 mM sodium ascorbate. Three replicates per treatment were used. The supernatants were collected and their protein concentrations were determined according to Bradford (1976), using bovine serum albumin as a standard.

Enzyme assays

The total 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 reaction colour was measured spectrophotometrically at 560 nm. The control reaction mixture had no enzyme extract, while blanks had the same complete reaction mixture, but were kept in the dark. The volume of sample causing 50% inhibition of colour development was taken as one unit of SOD activity and the activity was expressed as units per mg of protein.

The total CAT (EC 1.11.1.6) activity was measured spectrophotometrically according to the method of Luck (1965), by monitoring the decline in absorbance at 240 nm as H2O2 was consumed, against a plant extract-free blank. The 3 ml reaction mixture contained 66 mM sodium phosphate buffer (pH 7.0), to which 30% (w/v) H2O2 was added (the optical density was around 0.5 at 240 nm 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. The time Δt required for a decrease in the optical density of from 0.45 to 0.4 was used for CAT activity calculations. CAT activity was expressed as unit per mg of protein. A unit is the amount of an enzyme which liberates half the peroxide oxygen from a hydrogen peroxide solution of any concentration in 100 s at 25°C.

The total 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 as ascorbate was oxidized (Σ = 2.8 mM−1 cm−1). The oxidation rate of ascorbate was estimated between 1 and 60 s after starting the reaction with the addition of H2O2. The 1 ml reaction mixture contained 50 mM HEPES-NaOH (pH 7.6), 0.22 mM ascorbate, 1 mM H2O2, and an enzyme sample. The control reaction mixture was prepared without the enzyme extract. Corrections were made for the low, non-enzymatic oxidation of ascorbate by H2O2 and for the oxidation of ascorbate in the absence of H2O2. The activity was expressed as units (μmol of oxidized ascorbate per min) per mg of protein.

The total GR (EC 1.6.4.2) activity was determined by following the rate of NADPH oxidation as measured by the decrease in the absorbance at 340 nm against a extract-free blank. The assay mixture (1.0 ml final volume) was composed of 0.4 mol/l potassium phosphate buffer, pH 7.5, 0.4 mmol l−1 Na2EDTA, 5.0 mmol l−1 GSSG, 2.0 mmol l−1 NADPH, and 100 μl of crude extract. The reaction was initiated by NADPH addition and the GR activity was determined by monitoring GSSG-dependent oxidation of NADPH at 340 nm and 30°C (Di Baccio et al 2004). Corrections were made for the background absorbance at 340 nm, without NADPH. Activity was expressed as units (μmol of oxidized NADPH per min) per mg of protein.

Ascorbate determination

The aliquots of the fresh leaf and root tissues (0.5 g) were homogenized in ice-cold 6% (w/v) TCA, using a cold mortar and pestle. Total and reduced AA were determined in the supernatant after centrifugation at 15,000g for 10 min at 4°C. The assay is based on the reduction of Fe3+ to Fe2+ by AA in acid solution and the spectrophotometric detection of Fe2+ complexed with 2,2-dipyridyl (which absorbs at 525 nm). Total ascorbate was determined through a reduction of DHA to AA by 10 mM DTT. Excess DTT was removed with N-ethylmaleimide (NEM) 4% (w/v). A standard curve covering the range of 10–50 μmol AA was used (Kampfenkel et al. 1995).

Glutathione determination

The aliquots of the fresh leaf and root tissue (0.5 g) were homogenized in ice-cold 5% (w/v) TCA, using a cold mortar and pestle and centrifuged at 15,000g for 10 min at 4°C. Total (GSG + GSSG) and oxidized (GSSG) glutathione were determined in the supernatant by the 5,5-dithiobis-nitrobenzoic acid (DTNB)-GR recycling procedure as reported in Sgherri and Navari-Izzo (1995). Changes in the absorbance of the reaction mixture were measured at 412 nm at 25°C. The total glutathione concentration was calculated from a standard curve in which GSH equivalents (1–10 nmol) were plotted against the rate of change at 412 nm. GSSG was determined after GSH had been removed by 2-vinylpyridine derivatization. GSH was determined by subtracting GSSG, as GSH equivalents, from the total glutathione concentration.

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).

Results

Biomass and growth of leaves and roots under NaCl conditions

Leaf biomass (FW and DW) and growth (RGR) were unaffected by 100 mM NaCl. However at 300 mM, they significantly decreased compared to the controls. Biomass and growth of roots were significantly reduced by 100 and 300 mM NaCl (Table 1). The decrease in RGR at 300 mM NaCl was more significant in roots than in leaves.

Table 1 Biomass and RGR in control and NaCl treated leaves and roots
Full size table

Oxidative stress in control and salt treated roots and leaves

H2O2 concentrations were higher in leaves than in roots (Table 2). They significantly increased with salt concentration. On the contrary, lipid peroxidation (LP) levels, as estimated from MDA concentrations, were significantly higher in roots than in leaves either under control or saline conditions (Table 2). The electrolyte leakage (EL) increased in leaves and roots with increasing salinity (Table 2).

Table 2 Measures of oxidative stress (hydrogen peroxide concentration, lipid peroxidation and electrolyte leakage) in control and NaCl treated leaves and roots
Full size table

Leaf and root antioxidant enzyme activities

Leaf CAT, APX and GR activities were significantly lower than the control in plants treated with 300 mM NaCl, but they were not affected by 100 mM NaCl. Only SOD activity was reduced in the latter treatment (Fig. 1). In roots, SOD and APX responded to NaCl. Root SOD activity was significantly decreased in response to 300 mM NaCl only, and root APX activity was significantly higher in plants treated with 100 and 300 mM compared to controls. The other activities in roots were insensitive to salt (Fig. 1).

Fig. 1
figure 1

Activities of SOD (A), CAT (B), APX (C) and GR (D) in the leaves and the roots of C. maritimum under control and saline conditions. Results are the means of five replicates of five plants each. Means followed by different letters are significantly different at P ≤ 0.05 according to student’s LSD test

Full size image

Ascorbic acid (AA) and glutathione (GSH) concentrations under salinity conditions

Under control conditions, the concentration of AA was approximately 20-fold higher in leaves than in roots (Table 3). In leaves, the concentration of AA was significantly lower in plants treated with 100 and 300 mM NaCl than in control plants (Table 3). In roots, the concentration of AA in plants treated with 300 mM NaCl was 38 % lower than the control. The concentrations of GSH in salt-treated leaves and roots were not significantly different from the controls (Table 3). In both organs, AA and GSH were predominating in the total pool in ascorbic acid (AA + DHA) and glutathione (GSH + GSSG), either in control or saline conditions (Table 3).

Table 3 Ascorbic acid (AA) and glutathione (GSH) concentrations in control and NaCl treated leaves and roots
Full size table

Discussion

Differences in the leaf and root responses to NaCl

Moderate salinity (100 mM NaCl) reduced biomass or RGR of the roots only, whereas high salinity resulted in a significant reduction in both the leaves and the roots. This agrees with the assumption that roots, being the first part of the plant to encounter soil salinity, are more markedly affected by saline conditions than are the leaves (Di Baccio et al. 2004; Ballesteros et al. 1997).

An increase in H2O2 and MDA concentration upon salt stress has been reported in different plant species (Dionisio-Sese and Tobita 1998; Lee et al. 2001, Sudhakar et al. 2001; Sairam and Srivastava 2002; Bandeoglu et al. 2004) and this has been shown to be related to stress levels and well correlated with lipid membrane damage. In our study, lipid membrane damage was only observed in the roots under high salinity condition, whereas leaves were less affected by the NaCl-induced oxidative stress than the roots. Mansour (1998) reported that the reduced membrane damage of onion leaf tissues under salinity stress could be explained by a reduction in the concentrations of H2O2 and MDA in leaf tissues. Lower lipid peroxidation under salt stress has also been reported in rice leaves (Dionisio-Sese and Tobita 1998), tomato (Shalata et al. 2001), sugar beet (Bor et al. 2003) and cotton (Meloni et al. 2003).

The origin of oxidative stress under salinity conditions is well documented in leaves where the inhibition of Calvin cycle results in over-reduction of oxygen and formation of superoxides (Grassmann et al. 2002). In roots, little is known about the origin of oxidative stress, although salt stress-related impairment of mitochondrial function is likely to be involved (Hamilton and Heckathorn 2001). Recent studies have revealed that salt-induced oxidative stress occurs in root mitochondria in a wild salt-tolerant tomato species as indicated by increased concentrations of H2O2 and MDA (Mittova et al. 2004). It is now widely accepted that superoxide radicals produced during respiration, in response to salt stress, are the main precursors of mitochondrial H2O2 and make an important contribution to the oxidative load experienced by the cell (Møller 2001). Root mitochondrial ROS production is increased as a result of high salinity (Gómez et al. 1999) as constraints are imposed on electron transport through mitochondrial complexes I and II (Hamilton and Heckathorn 2001).

Antioxidant responses of roots and leaves under control conditions

Recent studies have shown that even under control conditions salt tolerant plants display a substantially higher abundance of major antioxidant components along with an increased induction of antioxidant enzyme activity and gene expression compared to sensitive plants (Taji et al. 2004; M’rah et al. 2006). Taji et al. (2004) showed that the SOD gene induced by salt in Arabidopsis was overexpressed in unstressed conditions in Thellungiella halophila. In the same study, they also reported that Thellungiella was more tolerant not only to high salinity but also to oxidative stress. The specific SOD activity (120 U) measured in the control leaves of C. maritimum (Fig. 1) was 13 to 120-fold higher than the activities reported for wheat (Meneguzzo et al. 1999), pea (Hernandez et al. 1999) and cotton (Gossett et al. 1994), respectively. It was similar to SOD activity measured in salt tolerant species such as Najas gramenia (Rout and Shaw 2001), Suaeda salsa (Qiu Fang et al. 2005) and Mesembryanthemum crystallinum (Slesak et al. 2003). An elevated SOD activity could be considered as an advantage which allows the plants to resist potential oxidative damage without the requirement of further induction of SOD activity. These results are in agreement with those obtained by Stepien and Klobus (2005) where a positive correlation between salt tolerance of C4 plants, and high constitutive SOD activity was found. Like SOD, leaf CAT activity exceeded root activity under both control and stress conditions. CAT is crucial in removing photorespiratory H2O2 generated in C3 plants, under salinity conditions (Cavalcanti et al. 2004). Indeed, several experimental observations have conclusively demonstrated the necessity of sufficient CAT activity to cope with the abundant H2O2 production that accompanies the high photorespiratory flux (Foyer and Noctor 2003).

Gueta-Dahan et al. (1997) indicated that APX seems to be a key enzyme in determining the salt tolerance in citrus, as its constitutive activity was much higher in a salt tolerant cultivar. Our results demonstrated that the constitutive activity of APX was significantly greater in leaves than in the roots. The converse was reported in wheat (Meneguzzo et al. 1999) and lentil (Bandeoglu et al. 2004) where root tissues markedly exhibited higher APX activity compared to the shoot.

Antioxidant responses of roots and leaves at moderate and high salinity

Leaves and roots exhibited different responses to 100 mM NaCl with respect to the activities of SOD and APX. SOD activity decreased significantly in leaves at 100 mM NaCl compared to the control, but not in roots. APX activity increased significantly in roots in response to 100 mM and was insensitive to this concentration in leaves. The activities of CAT and GR in the roots of plants treated with 100 mM NaCl were not different from those in control roots.

Both increase as well as decrease in the activity of SOD has been reported in plants in response to salinity stress (Dionisio-Sese and Tobita 1998; Shalata et al. 2001; Jitesh et al. 2006). It appears that the activity of SOD under salinity varies depending upon plant species, organ analyzed as well as the level of salinity (Chaparzadeh et al 2004). Decreased leaf SOD activity at 100 mM NaCl indicates that in leaves this enzyme is not crucial detoxification of ROS, and non enzymatic routes for conversion of O −·2 to H2O2 via antioxidants such as GSH and AA may compensate for reduced activity of SOD. This view is supported from our results on GSH and AA concentrations under salt stress. At low and even high salinity, leaves exhibited higher concentrations of GSH and AA than roots. Increases in CAT and peroxidase activity is suggested to be an adaptive trait to overcome tissue damage of the metabolism by reducing toxic concentrations of H2O2 (Agarwal and Pandey 2004). In this study, the salt induced enhancement of APX (but not of CAT) in roots may suggest a more effective scavenging mechanism of APX than CAT in removing H2O2 and imparting tolerance against NaCl stress.

Several studies have implied that H2O2 can act as a signal of biotic and abiotic stress (Noctor and Foyer 1998). However, high concentrations of H2O2 directly damage organelles and accelerate Haber Weiss reactions, resulting in the formation of hydroxyl radicals (OH·) that can cause lipid peroxidation (Mittler 2002). Apart from causing peroxidation of membrane lipids, high concentrations of H2O2 cause a net reduction in photosynthesis (Vaidyanathan et al. 2003). Moreover, by channeling most of the photoreductants for the detoxification of H2O2 produced under stress and by shifting carbon photoassimilates to the production of defense and repair metabolites, plant growth under high salinity may undergo a significant reduction as shown here. In the roots of C. maritimum, The accumulation of H2O2 at 300 mM NaCl is suggested to be due to NaCl enhanced SOD and NaCl-deactivated CAT activities. Similarly, Elkahoui et al. (2005) reported that in salt sensitive cells of Catharanthus roseus, the strong induction in SOD activity was not accompanied by an increase in CAT activity. Clearly, the salt-induced stimulation of the APX activity was not sufficient to cope with the enhanced SOD activity and the reduced ability to scavenge H2O2 by CAT, probably leading to an increased generation of OH·. This is likely linked to the more drastic decrease in the root growth and the higher lipid peroxidation found in 300 mM NaCl-treated roots.

Conclusion

The present study shows an antioxidative response of C. maritimum to moderate and severe NaCl stress. The different activity patterns of antioxidant enzymes indicated that the leaves and the roots reacted differently to NaCl. The stimulated SOD and APX activities and the decreased CAT activities in the roots of the plants treated with 300 mM can be considered as response regulation under severe saline conditions. Besides, this study shows that the mechanisms which protect the plant against the salt-generated oxidative stress are more efficient in leaves than in roots. An important element of these mechanisms seems to be the higher constitutive antioxidant defense in leaves, as recently described for the model halophyte Thellungiella halophila (Taji et al. 2004; M’rah et al. 2006).