Moderate salinity enhances the antioxidative response in the halophyte Hordeum maritimum L. under potassium deficiency

Environmental and Experimental Botany 69 (2010) 129–136

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Moderate salinity enhances the antioxidative response in the halophyte Hordeum maritimum L. under potassium deficiency Chokri Hafsi a,∗ , María C. Romero-Puertas b , Dharmendra K. Gupta b , Luis A. del Río b , Luisa M. Sandalio b , Chedly Abdelly a a b

Laboratoire d’Adaptation des Plantes aux Stress Abiotiques, Centre de Biotechnologie à la Technopole de Borj Cédria, BP 901, Hammam-Lif 2050, Tunisia Departamento de Bioquímica, Biología celular y Molecular de Plantas, Estación Experimental del Zaidín, CSIC, Apartado 419, E-18080 Granada, Spain

a r t i c l e

i n f o

Article history: Received 31 August 2009 Received in revised form 18 March 2010 Accepted 13 April 2010 Keywords: Antioxidants Hordeum maritimum L. Potassium deficiency Reactive oxygen species Salinity

a b s t r a c t Changes in the leaf antioxidant metabolism upon exposure to salinity and potassium deficiency were investigated in the annual halophyte Hordeum maritimum L. Plants were hydroponically grown either with a complete nutrient solution containing 3 mM K+ without (+K/−NaCl) or with 100 mM NaCl (+K/+NaCl), or in K+ -free medium containing 100 mM NaCl (−K/+NaCl). Malondialdehyde (MDA), carbonyl group (C O), and hydrogen peroxide (H2 O2 ) contents as well as antioxidant enzyme activities [superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), guaiacol peroxidase (GPX, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (GR, EC 1.6.4.2)] and non-enzymatic antioxidant contents (ascorbate and glutathione) were determined. Plants exposed to salinity, either alone or in combination with K+ deprivation, showed enhanced lipid peroxidation along with higher antioxidative response. This tendency was generally more marked in −K/+NaCl plants as compared to +K/+NaCl plants. H2 O2 concentration was negatively correlated with the plant antioxidative capacity, either enzymatic or non-enzymatic. As a whole, these data suggest that the enhancement of the antioxidative response is of crucial significance for H. maritimum plants growing under salinity and potassium deficiency. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Salinity is one of the major environmental factors restricting plant growth and productivity especially in arid and semi-arid areas. The deleterious effects of salinity on plant growth are associated with (i) osmotic stress, (ii) nutrient deficiencies, (iii) specific ion toxicities, or (iv) a combination of these factors (Ashraf, 1994). Salinity is known to inhibit photosynthesis in a number of plant species as a consequence of stomatal closure, thereby limiting CO2 diffusion into chloroplasts (Hernández et al., 1999; Centritto et al., 2003; Degl’Innocenti et al., 2009). When utilization of light energy absorbed during CO2 fixation is restricted by salinity, overreduction of photosynthetic electron transport chain may occur, resulting in an accumulation of reactive oxygen species (ROS), such as singlet oxygen (1 O2 ), hydrogen peroxide (H2 O2 ), superoxide anion (O2 •− ), and hydroxyl radical (• OH) (Hoshida et al., 2000). In chloroplast, ROS can be generated by direct transfer of excitation energy from chlorophyll to produce single oxygen, or by univalent oxygen reduction of photosystem I (I), in the Mehler

∗ Corresponding author. Tel.: +216 71 430 855; fax: +216 71 430 934. E-mail address: [email protected] (C. Hafsi). 0098-8472/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2010.04.008

reaction (Asada, 1999). As soon as the carbon fixation inside chloroplasts decreases, there also is a lower NADP+ availability to accept electrons from PSI, thus initiating O2 reduction resulting in ROS formation (Sudhakar et al., 2001). Additionally, photorespiration, another consequence of the low chloroplastic CO2 /O2 ratio, tends to enhance the cellular level of ROS such as hydrogen peroxide (Hernández et al., 2000). In addition to chloroplasts, ROS are generated in other cell compartments including cytosol, mitochondria, peroxisomes, and apoplastic space (Bowler and Fluhr, 2000; Mittler, 2002; del Río et al., 2006). Restriction in K+ uptake, as a consequence of Na+ /K+ competition for entry into plant cells is among the major effects caused by salinity (Rubio et al., 1995; Hafsi et al., 2007). A higher K+ efflux may also occur because of NaCl-induced plasma membrane depolarization, as suggested by membrane potential measurements and patchclamp studies on barley and Arabidopsis thaliana roots (Shabala et al., 2003). In addition, the over-production of ROS caused by salinity usually leads to lipid peroxidation and induces K+ leak from cells by activating K+ efflux channels (Demidchik et al., 2003; Cuin and Shabala, 2007). Therefore, plant cell ability to maintain an appropriate K+ /Na+ ratio is one of the key features of plant salt tolerance (Cuin et al., 2003; Tester and Davenport, 2003). Because salt stress impairs K+ uptake in plants, Cakmak (2005) suggested that

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K+ deficiency at the cellular level might be a contributory factor to salt-induced oxidative stress. ROS are highly reactive and can damage lipids, proteins, nucleic acids, and photosynthetic components (Alscher et al., 1997; Meloni et al., 2003; Implay, 2003). In plant cells, ROS detoxification is controlled by enzymatic and non-enzymatic defence systems. Nonenzymatic compounds include lipid-soluble antioxidants (such as ␣-tocopherol and ␤-carotene) and water-soluble reductants (such as ascorbate and glutathione) (Kim et al., 2001). ROS-scavenging by antioxidative enzymes is achieved through series of complex reactions including O2 •− dismutation to H2 O2 by superoxide dismutase (SOD) (Asada, 1999; Meloni et al., 2003) and H2 O2 detoxification by numerous enzymes like catalase (Corpas et al., 1999; Lin and Kao, 2000) and peroxidases (Dionisio-Sese and Tobita, 1998). APX, MDHAR, DHAR, and GR are part of an effective enzymatic ROS-scavenging system, called ascorbate–glutathione or Foyer–Halliwell–Asada pathway that catalyzes H2 O2 conversion into water (Foyer and Halliwell, 1976; Asada, 1999). A strong antioxidant response mechanism is of crucial significance for plants coping with low K+ availability and salinity (Jithesh et al., 2006). The annual facultative halophyte Hordeum maritimum, potentially useful for forage production in saline zones, can survive up to 300 mM NaCl under potassium deficiency conditions (Hafsi et al., 2007). Improving our knowledge with respect to the agronomic, physiological, biochemical, and molecular responses of this species to the interactive effects of salinity and potassium deficiency might contribute to the development of salt-tolerant barley cultivars. Since data concerning the effect of salinity and potassium shortage on the plant antioxidative capacity are scarce, the main question of this work was: how can the salt-induced antioxidative response of H. maritimum be modulated by potassium status? 2. Materials and methods 2.1. Plant material and growth conditions H. maritimum L. seeds were collected from Soliman sabkha (saline area, 20 km south of Tunis). Seeds were disinfected for 2 min with 1% NaClO, abundantly rinsed with distilled water, and germinated (at 20 ◦ C) in Petri dishes on filter paper moistened with distilled water. Three days after sowing, seedlings were transferred into plastic pots (24 plants/pot) and irrigated with 5 L of modified Hewitt’s nutrient solution (1966). The nutrient solution contained the following macronutrients (mM): 1.5 MgSO4 , 7H2 O, 3.5 Ca (NO3 )2 , 4H2 O, 5.4 NaNO3 , 2 NH4 H2 PO4 , and 3 KCl. The micronutrients (ppm) were: Mn (0.5), Cu (0.04), Zn (0.05), B (0.5), Mo (0.02) (Arnon and Hoagland, 1940) and Fe (3) as Fe–tartaric acid complex. Nutrient solutions were continuously aerated and renewed twice a week. After a pretreatment period of 28 days, plants were divided into three lots. The following treatments: 3 mM K–0 mM NaCl (+K/−NaCl), 3 mM K–100 mM NaCl (+K/+NaCl), and 0 mM K–100 mM NaCl (−K/+NaCl) were used. The culture was made in a growth chamber with day/night temperatures of 25 ◦ C/18 ◦ C, a 16-h photoperiod, a photon flux density of 400 ␮mol m−2 s−1 , and a relative humidity of 70–75%. After 14 days of treatment, leaves were collected for the determination of the different parameters of oxidative stress (MDA, carbonyl groups, and H2 O2 contents), the activities of antioxidative enzymes, and the accumulation of ascorbate and glutathione. 2.2. Lipid peroxidation Lipid peroxidation was assessed by monitoring malondialdehyde (MDA) formation using the thiobarbituric acid method

according to Buege and Aust (1978). Leaf samples were grounded in a cold mortar with 50 mM Tris–HCl (pH 7.5) buffer containing 0.2 mM EDTA and 0.2% (v/v) Triton X-100. The homogenate was centrifuged at 16,700 × g for 30 min. To 200 ␮l of aliquote of supernatant was added 1000 ␮l of 0.375% (w/v) thiobarbituric acid containing 15% (w/v) trichloroacetic acid and 0.01% butylated hydroxytoluene. The mixture was heated at 100 ◦ C for 15 min and then quickly cooled in ice bath. After centrifugation at 5500 × g for 5 min, the absorbance of the supernatant was read at 535 and 600 nm. The MDA content was calculated from the difference in absorbance at 535 and 600 nm using a molar extinction coefficient of 155 mM−1 cm−1 . 2.3. Hydrogen peroxide Hydrogen peroxide was determined using a PeroXOquantTM Quantitative Peroxide Assay Kits (Pierce product). This method detects peroxide based on oxidation of ferrous to ferric ion in the presence of xylenol orange. Leaf samples were grounded in 25 mM H2 SO4 as buffer. The homogenate was centrifuged at 13,000 × g for 30 min at 4 ◦ C. To 20 ␮l of aliquote of supernatant was added 200 ␮l of a mixture containing reagent A (composition: 25 mM ammonium ferrous (II) sulphate, 2.5 M H2 SO4 ) and reagent B (composition: 100 mM sorbitol, 125 ␮M xylenol orange in water) in a microplate. After 20 min incubation at room temperature, the absorbance at 595 nm was measured. A standard curve covering the range of 1–32 ␮M H2 O2 was used. 2.4. Protein oxidation Protein oxidation was estimated by determining carbonyl groups using the dinitrophenyl hydrazine (DNPH) method (Romero-Puertas et al., 2002). 0.5 mg protein samples were incubated for 20 min with 0.55% (v/v) Triton X-100 and 10% (w/v) streptomycin sulphate to remove the nucleic acids. After centrifuging at 5500 × g for 10 min, supernatants (200 ␮l) were mixed with 300 ␮l of 10 mM DNPH in 2 M HCl. The blank was incubated in 2 M HCl. After 1 h incubation at room temperature, proteins were precipitated with 10% (w/v) trichloroacetic acid (TCA) and the pellets were washed three times with 500 ␮l of ethanol/ethylacetate (1/1). The pellets were finally dissolved in 6 M guanidine hydrochloride in 20 mM potassium phosphate buffer (pH 2.3) and the absorbance at 370 nm was measured. Protein recovery was estimated for each sample by measuring the absorbance at 280 nm. Carbonyl content was calculated using a molar absorbance coefficient for aliphatic hydrazones of 22,000 M−1 cm−1 (Levine et al., 1994). 2.5. Enzyme extractions and assays All operations were carried out at 4 ◦ C. Frozen leaf tissues (1000 ␮l/500 mg fresh weight) were ground in a mortar with liquid nitrogen in 50 mM Tris–HCl buffer (pH 7.5) containing 0.1 mM EDTA, 0.2% (v/v) Triton X-100, 2 mM DTT, and 1 mM PMSF. For APX extraction, 2 mM ascorbate was added and EDTA was omitted. The homogenate was centrifuged at 16,700 × g for 30 min and the supernatants were collected and used for different enzyme assays. SOD isoenzymes were individualized by native-PAGE on 12% acrylamide gels and were localized by photochemical method (Beauchamp and Fridovich, 1971). The developed gel was scanned on a Bio-Rad imaging densitometer system and densitogram was recorded using Scion Image software. CAT activity was measured spectrophotometrically according to Aebi (1984) by monitoring the disappearance of H2 O2 at 240 nm and 25 ◦ C for 2 min (ε = 39.58 M−1 cm−1 ). The reaction mixture (1 ml) contained 50 mM potassium phosphate buffer (pH 7.0), to which 10.6 mM H2 O2 was added. The reaction was initiated by

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adding 10 ␮l of the leaf crude extract to this solution for 2 min at 25 ◦ C. GPX activity was determined according to Quessada and Macheix (1984) by monitoring the increase in absorbance at 470 nm and 25 ◦ C for 2 min (ε = 26.6 mM−1 cm−1 ). The reaction mixture (1 ml) consisted of 50 mM potassium phosphate buffer (pH 6.1), 6.25 mM guaiacol, and 25 ␮M H2 O2 . The reaction was started by the addition of 10 ␮l of the enzyme extract. APX activity was assayed as described by Jiménez et al. (1997) by monitoring the oxidation of ascorbate at 240 nm for 2 min at 25 ◦ C (ε = 2.8 mM−1 cm−1 ). The reaction mixture (1 ml) consisted of 50 mM HEPES–NaOH buffer (pH 7.6) containing 0.2 mM ascorbate, 0.3 mM H2 O2 , and 100 ␮l of supernatant. The reaction was started by adding H2 O2 . Corrections were made for the non-enzymatic oxidation of ascorbate by H2 O2 and for the oxidation of ascorbate in the absence of H2 O2 . APX was measured in the presence and absence of the specific inhibitor 4-chloromercuribenzoic acid (0.5 mM). GR activity was measured according to Edwards et al. (1990) by monitoring the rate of NADPH oxidation as the decrease in absorbance at 340 nm for 2 min at 25 ◦ C (ε = 6.22 mM−1 cm−1 ). The reaction mixture (1 ml) consisted of 100 mM HEPES–NaOH (pH 7.8) contained 1 mM EDTA, 3 mM MgCl2 , 0.5 mM GSSG, and 100 ␮l of enzyme extract. The reaction was initiated by the addition of 0.2 mM NADPH. MDHAR activity was determined according to Jiménez et al. (1997) by monitoring the decrease in absorbance at 340 nm and 25 ◦ C for 2 min because of the oxidation of NADH. The assay mixture (977 ␮l) consisted of 50 mM Tris–HCl (pH 7.8) containing 0.2 mM NADH, 1 mM ASC, 0.5 U of ascorbate oxidase, which generated saturating concentrations of MDHA, and 25 ␮l of enzyme extract. The reaction was started by adding NADH. DHAR activity was measured according to Dalton et al. (1993) by monitoring ASC formation via DHA reduction at 265 nm and 25 ◦ C for 1 min. The reaction mixture (1 ml) consisted of 50 mM potassium phosphate buffer (pH 6.5) containing 0.1 mM EDTA, 4 mM DHA, 50 mM GSH, and 30 ␮l of enzyme extract. The reaction was started by adding DHA. The reaction rate was corrected for the nonenzymatic reduction of DHA by GSH. A factor of 0.98, accounting for the small contribution of the absorbance by GSSG, was used. 2.6. Extraction of ascorbate and glutathione Leaf samples were grounded in a cold mortar with liquid nitrogen and homogenized with 25 mM sulphuric acid (800 ␮l/400 mg fresh weight). The homogenate was centrifuged at 16,700 × g for 30 min at 4 ◦ C, and the supernatant was collected for the analysis of ascorbate and glutathione. 2.7. Quantification of reduced (ASC) and oxidized (DHA) ascorbate The assay is based on the reduction of Fe3+ to Fe2+ by ascorbic acid (Kampfenkel et al., 1995). Fe2+ forms a red complex with bipyridyl that absorbs at 550 nm. DHA was reduced to ASC by pre-incubating the sample with dithiothreitol (DTT). After DTT excess was removed with N-ethylmaleimide (NEM), total ascorbate (ASC + DHA) content was measured. DHA content was then estimated from the difference between total ascorbate and the ASC. A standard curve covering the range of 2.5–60 ␮mol ASC was used.

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2-nitrobenzene). GSSG was reduced to GSH by the action of glutathione reductase and NADPH. GSSG was assayed from the sample after the removal of GSH by 2-vinylpyridine and triethanolamine derivatizations. The rate of DTNB reduction was recorded at 412 nm for 2 min. The contents were calculated using a standard curve. The GSH content was calculated from the difference between the total glutathione and GSSG. 2.9. Protein determination Proteins were determined by the method of Bradford (1976) using bovine serum albumin (BSA) as standards. 2.10. Statistical analysis All data were subjected to one-way ANOVA test, and means were compared using Duncan’s multiple-range test at 5% level of significance by means of SPSS.10.0 Windows (1996). 3. Results 3.1. Oxidative stress evaluation MDA content of leaves increased by about 98% and 68% at 100 mM NaCl, either alone or in combination with potassium deficiency, respectively (Table 1). Contents of carbonyl groups remained unchanged (Table 1). 100 mM NaCl salinity had no significant effect on H2 O2 contents. Nevertheless, the interactive effects of salinity and potassium deprivation induced a significant reduction in H2 O2 content by 56% in comparison with the control (Table 1). 3.2. Leaf antioxidant enzyme activities Two different isoenzymes of SOD could be detected using native-PAGE and SOD staining (Fig. 1). SOD activity was increased by salinity and to a higher extent by the combined treatment (salinity-potassium deprivation) as compared to the control. CAT activity was also enhanced by 36% and 178% by salt alone or in combination with potassium deficiency, respectively, as compared to the control (Fig. 2A). While salinity alone had no significant effect on GPX activity as compared to the control, the recorded value of this parameter was 34% higher in plants submitted to the interactive effects of salinity and potassium deficiency (Fig. 2B). APX activity was significantly stimulated (+16%) in plants subjected to salinity as compared to the control. Yet, both treatments applied simultaneously (−K/+NaCl) increased APX activity by 74% in comparison with the control (Fig. 3A). GR activity was enhanced by all the treatments applied, the highest increase being observed −K/+NaCl-treated plants (+271% in comparison with the control) (Fig. 3B). MDHAR activity increased by 155% and 124% in +K/+NaCl and −K/+NaCl plants, respectively (Fig. 4A). Finally, DHAR activity was significantly increased (+62% and +52%) by salinity and the combined effects of salinity and K+ starvation, respectively (Fig. 4B).

Table 1 Changes in malondialdehyde (MDA, nmol g−1 FW), carbonyl groups (nmol C O mg−1 protein), and hydrogen peroxide (H2 O2 , ␮M g−1 FW) contents in H. maritimum grown in a nutrient solution containing different concentrations (mM) of K+ and NaCl: 3 and 0 (+K/−NaCl), 3 and 100 (+K/+NaCl), and 0 and 100 (−K/+NaCl), respectively. Data are the mean of three replicates ± SE. Means followed by the same letters are not significantly different at 5% according to the Duncan’s multiple-range test.

2.8. Quantification of reduced (GSH) and oxidized (GSSG) glutathione

Treatments

The contents of GSH and GSSG were determined as described by Griffith (1980) based on the oxidation of GSH by DTNB (5,5 -dithio-bis-nitrobenzoic acid) to give GSSG and TNB (5-thio-

+K/−NaCl +K/+NaCl −K/+NaCl

Parameters MDA

C O

H2 O2

1.89 (0.24) a 3.74 (0.27) b 3.18 (0.02) b

13.37 (1.33) a 13.38 (1.50) a 13.49 (0.70) a

7.12 (0.53) c 6.07 (0.43) bc 3.11 (0.58) a

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Fig. 1. Activity staining of SOD isozymes after native-PAGE and densitograms of H. maritimum leaves grown in a nutrient solution containing different concentrations (mM) of K+ and NaCl: 3 and 0 (+K/−NaCl), 3 and 100 (+K/+NaCl), and 0 and 100 (−K/+NaCl), respectively. Samples applied to the gels contained 30 ␮g of protein. Areas under the peak are expressed in arbitrary units.

Fig. 2. Changes in CAT (A) and GPX (B) activities in H. maritimum grown in a nutrient solution containing different concentrations (mM) of K+ and NaCl: 3 and 0 (+K/−NaCl), 3 and 100 (+K/+NaCl), and 0 and 100 (−K/+NaCl), respectively. Data are the mean of three replicates ± SE. Means followed by the same letters are not significantly different at 5% according to the Duncan’s multiple-range test.

3.3. Ascorbate and glutathione Total ascorbate content increased by 31% as compared to the control in plants submitted to the combined effects of salinity and K+ deprivation, whereas salinity treatment alone had no effect. The same trend was observed with respect to reduced ascorbate

(Table 2). Oxidized ascorbate content was only slightly reduced by salinity relative to the control. Yet, the decline was more marked (−63%) in plants subjected to the interactive effects of the two constraints. Salinity, either alone or in combination with potassium deficiency caused respectively 1.6- and 5.6-fold increases in the ASC/DHA ratio respectively (Table 2). All treatments had no signif-

Fig. 3. Changes in APX (A), and GR (B) activities in H. maritimum grown in a nutrient solution containing different concentrations (mM) of K+ and NaCl: 3 and 0 (+K/−NaCl), 3 and 100 (+K/+NaCl), and 0 and 100 (−K/+NaCl), respectively. Data are the mean of three replicates ± SE. Means followed by the same letters are not significantly different at 5% according to the Duncan’s multiple-range test.

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Fig. 4. Changes in MDHAR (A) and DHAR (B) activities in H. maritimum grown in a nutrient solution containing different concentrations (mM) of K+ and NaCl: 3 and 0 (+K/−NaCl), 3 and 100 (+K/+NaCl), and 0 and 100 (−K/+NaCl), respectively. Data are the mean of three replicates ± SE. Means followed by the same letters are not significantly different at 5% according to the Duncan’s multiple-range test.

Table 2 Changes in ascorbate (reduced (ASC) and oxidized (DHA) ascorbate) and glutathione (reduced (GSH) and oxidized (GSSG) glutathione) contents in H. maritimum grown in a nutrient solution containing different concentrations (mM) of K+ and NaCl: 3 and 0 (+K/−NaCl), 3 and 100 (+K/+NaCl), and 0 and 100 (−K/+NaCl), respectively. Data are the mean of three replicates ± SE. Means followed by the same letters are not significantly different at 5% according to the Duncan’s multiple-range test. Treatments +K/+NaCl

−K/+NaCl

Ascorbate content ASC + DHA 43.05 (1.46) a ASC 26.38 (0.71) a DHA 16.67 (1.42) c ASC/DHA 1.59 (0.15) a

41.88 (0.70) a 29.69 (2.88) a 12.19 (2.19) bc 2.50 (0.73) b

56.32 (0.43) b 50.17 (2.39) b 6.16 (1.63) a 8.96 (1.67) c

Glutathione content GSH + GSSG 13.13 (1.18) a GSH 9.32 (0.79) a GSSG 3.80 (0.84) a GSH/GSSG 2.52 (0.61) ab

14.24 (0.89) a 8.30 (0.45) a 5.94 (0.53) a 1.40 (0.09) a

13.68 (1.09) a 9.72 (1.31) a 3.96 (0.27) a 2.47 (0.48) ab

+K/−NaCl

icant effect on the total glutathione (GSH + GSSG), GSSG, and GSH contents and the GSH/GSSG ratio (Table 2). 4. Discussion The present study was conducted to evaluate the participation of antioxidants in tolerance of H. maritimum to salinity alone or in combination with potassium deficiency. Because of salt stress impairs K+ uptake of plants, it has been suggested that K deficiency might be a contributor factor to salt-induced oxidative stress and related cell damage. Due to impairment in: (i) stomata regulation, (ii) conversion of light energy into chemical energy, and (iii) phloem transport of photoassimilates from leaves into sink organs, photosynthetic CO2 fixation is limited (Cakmak, 2005). In a recent study and under the same conditions (Degl’Innocenti et al., 2009), we showed that potassium deprivation increased the negative effects of salinity on several parameters (CO2 net assimilation rate, stomatal conductance, transpiration rate, and electron transport rate) of H. maritimum photosynthetic activity. Disturbances in potassium status and increased sodium and chloride contents were also observed. Such unfavourable conditions could trigger metabolic disturbances in photosystems I and II, leading to an overproduction of ROS, and may cause oxidative damage to the biomolecules such as lipids, proteins, and nucleic acids as suggested by Kanazawa et al. (2000).

In the present study, two biomarkers of oxidative damage to macromolecules (the extent of lipid peroxidation and the carbonyl group of proteins) were considered. Our results showed that MDA content was enhanced either by 100 mM NaCl or by the interactive effects of the two treatments (salinity and K+ deprivation), indicating that membrane polyunsaturated fatty acids underwent peroxidation. Lipid peroxidation could be a result of the light-dependent formation of singlet oxygen during stress conditions (Boo and Jung, 1999). The slight and non-significant decrease in MDA content in −K/+NaCl treatment as compared to +K/+NaCl treatment could be explained by the significant increase in reduced ascorbate (ASC) pool, which is known to play an important role in reducing membrane damage during environmental stresses (Shalata and Neumann, 2001; Moradi and Ismail, 2007). Nevertheless, protein structure, assessed through leaf carbonyl group content was unaffected by the two applied treatments (Table 1). The effect depends on species, organ, stress intensity, treatment duration, and stress nature. Similar results were reported by Rodríguez-Serrano et al. (2006) in roots of pea (Pisum sativum) exposed to cadmium stress. However, Sandalio et al. (2001) observed an increase in both lipid peroxidation and carbonyl group content in leaves of pea plants grown with cadmium. Several environmental stresses enhance H2 O2 production in different compartments of plant cells by enzymatic and nonenzymatic processes (Asada, 1999). In the present study, no significant effect of NaCl on H2 O2 production was registered. However, a reduction in H2 O2 content occurred in response to the interactive effects of salinity and potassium deficiency (Table 1). According to Jithesh et al. (2006), a strong antioxidant response is important under conditions where physiological adaptations to low K+ are not sufficient at the cell level to cope with salinity stress. This observation is significant with halophytes. Our results agree with this suggestion, since the increase in the plant antioxidative response (both enzymatic and non-enzymatic) generally observed following salt exposure under potassium-deficient conditions was higher in comparison with plants subjected to salinity alone. The higher antioxidant enzyme activities and the accumulation of the non-enzymatic antioxidants were correlated with stability or decrease in H2 O2 concentration in H. maritimum leaves, indicating involvement of these antioxidant systems in alleviating oxidative stress and consequently protecting cells from oxidative damage. The decrease in CO2 fixation as a result of stress conditions may result in over reduction of components of photosynthetic electron transport system leading to increased ROS production (Hoshida et

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al., 2000; Moradi and Ismail, 2007) which may modulate the antioxidative response. In the present study, SOD, which catalyzes the conversion of superoxide radical to molecular oxygen and H2 O2 (Asada, 1999; Meloni et al., 2003), activity increased in particular under salinity with K+ limited supply (Fig. 1) suggesting a better superoxide radical scavenging ability. Based on previous works (Mascher et al., 2005; Pérez-López et al., 2009), the two isoforms can be identified as Cu/Zn SOD 1 and 2, the Cu/Zn SOD 2 being the most abundant. No induction of new isoforms was observed, suggesting that enhanced SOD activity was due to increased activation of already synthesized SOD isozymes. Wang et al. (2004) reported that Cu/Zn SOD 1 isoform activity decreased and that of Cu/Zn SOD 2 isoform remained unaltered in response to NaCl and in presence of K+ ions. In absence of K+ , activities of both Cu/Zn SOD isoenzymes were unaffected under NaCl treatment. On the other hand, Mn SOD and Fe SOD 1 specific activities increased in absence of K+ ions during NaCl treatment. H2 O2 which is toxic must be scavenged by its conversion into H2 O in subsequent reactions (Sekmen et al., 2007). In our experimental conditions, GPX did not show statistically significant changes by NaCl addition alone. On the other hand, under K+ limitation, salinity significantly increased GPX activity (Fig. 2B). Azevedo Neto et al. (2006) and Ben Amor et al. (2006) observed increased peroxidase activity respectively in maize and Cakile maritima as subjected to salt stress. Nevertheless, peroxidase activity was lower in marigold and purslane plants subjected to NaCl (Chaparzadeh et al., 2004; Yazici et al., 2007). Besides their role in H2 O2 scavenging from chloroplasts and the cytosol, peroxidases are involved in numerous physiological functions including oxidation of toxic compounds, biosynthesis of cell walls (lignin and suberin), growth and development processes, etc. (Jbir et al., 2001; Dicko et al., 2006). CAT is a key antioxidant enzyme that decomposes H2 O2 producing O2 and H2 O (Corpas et al., 1999; Lin and Kao, 2000). Our results showed increased CAT activity, especially in −K/+NaCl plants (Fig. 2A). Increases in CAT activity have been mentioned by Pérez-López et al. (2009) and Yang et al. (2009) after salt exposure. However, a decrease of CAT activity has been reported in certain plant species exposed to numerous environmental stresses like salinity (Parida et al., 2004; Ben Amor et al., 2006), drought (Zhang and Kirkham, 1994), and heavy metals (Rodríguez-Serrano et al., 2006; Romero-Puertas et al., 2007). Ascorbate–glutathione pathway, another important antioxidant mechanism involved in H2 O2 elimination, is composed of three interdependent redox couples: ASC/DHA, GSH/GSSG, and NADPH/NADP, and the enzymes APX, MDHAR, DHAR, and GR (Noctor and Foyer, 1998). APX, that eliminates peroxides by converting ASC into DHA, is one of the most important enzymes in detoxifying plant cells from H2 O2 (Foyer et al., 1994; Foyer, 1996). In our study, an increase in APX activity was observed especially under −K/+NaCl conditions suggesting its involvement in the elimination of H2 O2 (Fig. 3A). A salt-dependent increase of APX was reported in Bruguiera parviflora (Parida et al., 2004), Suaeda salsa (Cai-Hong et al., 2005), and barley (Pérez-López et al., 2009), while the opposite effect was observed in sunflower plants (Baccio et al., 2004). In addition to antioxidative enzymes, non-enzymatic antioxidants also play a key role in detoxifying free radicals in plants during salt stress (Jithesh et al., 2006). Lower ascorbate contents have been reported in diverse species exposed to a range of abiotic stresses (Hernández et al., 2000; Ben Amor et al., 2006; RodríguezSerrano et al., 2006). In this work, no significant effect of salinity was observed on both total ascorbate and reduced ascorbate contents (Table 2). However, an increase was observed in response to salt exposure in absence of K+ . Such an increase in ascorbate content suggests that ascorabate synthesis may be higher than ascorbate catabolism. In our case, regeneration of ASC is likely to

be through efficient operation of ASC–GSH cycle, as indicated by the increase in the activities of APX, MDHAR, DHAR, and GR. The latter, that catalyzes NADPH-dependent reduction of GSSG, is an important enzyme in protecting many plants from oxidative stress (Foyer et al., 1991). Enhancement in foliar GR activity could increase NADP+ /NADPH ratio, thereby ensuring the ability of NADP+ to accept electrons from photosynthetic electron transport chain and minimising ROS accumulation in chloroplasts (Gómez et al., 2004). The parallel enhancement of MDHAR (Fig. 4A) and DHAR (Fig. 4B) activities suggests that both pathways are involved in ASC regeneration. Hernández et al. (2000) documented ASC regeneration by DHAR in stressed pea plants. In contrast, Chaparzadeh et al. (2004) observed a reduction in MDHAR and DHAR activities, suggesting that ASC was regenerated through a non-enzymatic pathway. The decrease in MDHAR and DHAR activities in the −K/+NaCl treatment as compared to plants subjected to +K/+NaCl treatment could be due to the decrease in DHA pool and/or feedback inhibition due to increased ASC content in −K/+NaCl treatment. The change in the ASC/DHA ratio, an important indicator of the cell redox status, is one of the first signs of oxidative stress (Meneguzzo et al., 1999). An increase in the ASC to DHA ratio was observed particularly in plants grown under salinity and potassium deprivation (Table 2). This indicates that a considerable amount of total ascorbate was still in the reduced form, a favourable condition for maintaining APX activity, as APX could be irreversibly damaged when ASC concentration falls (Asada, 1999; Miyagawa et al., 2000). Besides its participation in H2 O2 detoxification via ASC–GSH cycle, ascorbate can directly scavenge superoxide, hydroxyl radicals, and singlet oxygen. It can also serve as an enzyme cofactor, for example for violaxanthin de-epoxidase in xanthophyll cycle (Fedoroff, 2006). As for ascorbate, glutathione protects cells against oxidative stress. In addition to its involvement in the re-reduction of ascorbate in ASC–GSH cycle (Dalton et al., 1993; Noctor et al., 1998), glutathione reacts also non-enzymatically with singlet oxy´ gen, superoxide, and hydroxyl radical (Kuzniak and Sklodowska, 2001). In the present study, no significant changes in total glutathione, GSH, and GSSG contents and in GSH to GSSG ratio were registered (Table 2), indicating that glutathione metabolism was unaffected although ASC–GSH cycle was functioning. The previously described improvement in antioxidative response either enzymatic or non-enzymatic suggest that the excess energy generated in H. maritimum following the limitation in CO2 photoassimilation (Degl’Innocenti et al., 2009) under the same conditions was effectively dissipated through upregulation of the reactive oxygen detoxifying system. 5. Conclusion This study showed an upregulation of the antioxidative system to detoxify ROS produced in leaves of H. maritimum plants growing under moderate salinity. Potassium deficiency modulated the plant antioxidative response, as indicated by the enhancement of several antioxidative enzymes and the accumulation of non-enzymatic antioxidants, especially ascorbate. Acknowledgements This work was supported by grant BIO2008-04067 from the Ministry of Science and Innovation, and Junta de Andalucía (Research Group BIO-0192), Spain and Tunisian Ministry of higher Education, Scientific Research and technology (LR02CB02). We wish to thank Mrs Elena Sanchez for technical assistance. We thank also Dr. Ahmed Debez and Dr. Mokded Rabhi for English revision of the manuscript.

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