Different antioxidant defense responses to salt stress

Environmental and Experimental Botany 77 (2012) 63–76

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Different antioxidant defense responses to salt stress during germination and vegetative stages of endemic halophyte Gypsophila oblanceolata Bark. Askim Hediye Sekmen, Ismail Turkan ∗ , Zehra Ozgecan Tanyolac, Ceyda Ozfidan, Ahmet Dinc Department of Biology, Faculty of Science, Ege University, Bornova, Izmir 35100, Turkey

a r t i c l e

i n f o

Article history: Received 19 September 2011 Received in revised form 25 October 2011 Accepted 27 October 2011 Keywords: Antioxidant enzymes Germination Gypsophila oblanceolata Ion accumulation Salt stress Vegetative growth

a b s t r a c t Salinity is a major limiting factor to agricultural productivity. To ensure future productivity of the agricultural regions and achieve a selection of genetically transformed salt-tolerant plants, there is a need to select and characterize salt-tolerant plants. Gypsophila oblanceolata Bark. is a Turkish endemic and endangered halophyte occurring in salt marshes/hydromorphic soils. The aim of this experiment was to determine the responses of G. oblanceolata to salt stress during germination and vegetative growth. Therefore, effects of salinity (0, 50, 100, 150, 300 mM NaCl) on germination and changes in the activities of antioxidant enzymes/isoenzymes (SOD, CAT and POX) during germination under stress and recovery after stress were determined. Moreover, during vegetative growth (60 d old plants), changes in physiological parameters, ion concentrations, proline (Pro) content, lipid peroxidation (MDA), H2 O2 content, NADPH oxidase activity and, antioxidant enzyme/isoenzyme system (superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductase (GR)) were also investigated. Salt stress decreased both the germination percentage and rate. Few seeds germinated at 100 mM NaCl. Exposure to high concentrations of NaCl did not permanently inhibit germination. Salinity (50 mM NaCl) caused a decrease in activities of SOD, CAT and POX during germination. However, after stress, the activities of all enzymes were increased in recovered-plants. During vegetative growth, increased activities of SOD, CAT and APX in 50 and 100 mM NaCl treated-plants may help to avoid oxidative damage in G. oblanceolata. However, at higher doses, induced stimulation of the CAT, POX and GR was not sufficient to cope with the enhanced ROS production and MDA level. From the results obtained in present study, it can be suggested that G. oblanceolata is a moderately salt-tolerant species. Differential responses of antioxidant enzymes to salt stress during germination and vegetative growth suggested different antioxidant metabolism in G. oblanceolata. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Salinity is one of the major environmental constraints on plants growth and productivity. Desertification and salinization are rapidly increasing on global scale and currently affect more than 10% of arable land, which results in a decline of the average yields of major crops greater than 50% (FAO, 2008). Increasing salinity leads to a reduction and/or delay in germination of plants and death of seeds before germination (Song et al., 2005). Moreover, salt stress affects a wide variety of physiological and metabolic processes in plants in their vegetative stages leading to growth reduction (Di Baccio et al., 2003). The initial growth reduction is due to osmotic effect of salt outside roots and subsequent growth reduction is due to ionic stress which is caused by the inability to prevent salt from

∗ Corresponding author. Tel.: +90 232 311 24 43; fax: +90 232 311 17 38. E-mail address: [email protected] (I. Turkan). 0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2011.10.012

reaching toxic levels in transpiring leaves (Munns, 2005). Plants growing naturally on saline soils have evolved various mechanisms to cope with negative effects of ionic stress (Flowers et al., 1977; Munns, 2002; Parida et al., 2004; Turkan and Demiral, 2009). These mechanisms rely on controlled uptake, exclusion, compartmentalization and increased extrusion of salts (Flowers and Colmer, 2008). In addition to these primary effects, salinity also leads to oxidative stress through an increase in reactive oxygen species (ROS), such as superoxide anion (O2 •− ), hydrogen peroxide (H2 O2 ) and hydroxyl radicals (OH• ) (Zheng et al., 2009). It is now widely accepted that these cytotoxic ROS are responsible for various stress-induced damages to macromolecules and ultimately to cellular structure (Asada, 1999; Sairam and Tyagi, 2004; Amor et al., 2006). To cope with oxidative damage initiated by ROS formed under salt stress, plants possess a complex antioxidant defense system including non-enzymatic antioxidants such as ascorbic acid, glutathione, tocopherols and carotenoids as well as antioxidative enzymes such as superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), peroxidase (POX; EC 1.11.1.7) and enzymes

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A.H. Sekmen et al. / Environmental and Experimental Botany 77 (2012) 63–76

of the so-called ascorbate–glutathione cycle: ascorbate peroxidase (APX; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2) (Lee et al., 2001). Recently, a correlation between the antioxidant capacity and salt tolerance has been found in different halophytic plant species, including Centaurea tuzgoluensis (Yıldıztugay et al., 2011), Plantago maritima (Sekmen et al., 2007) and Cakila maritima (Amor et al., 2006). To ensure future productivity of the agricultural regions, we need to (i) select and characterize salt-tolerant plants and (ii) identify the mechanisms of effects of their antioxidative responses. Gypsophila oblanceolata Bark. (Caryophyllaceae) is a perennial halophyte. This species occurs very abundantly in salt marshes/hydromorphic soils of Salt Lake (Konya, Aksaray and Nigde) and Sultansazlı˘gı in Central Anatolian part of Turkey (Hamzaoglu and Aksoy, 2009). Its natural habitat is characterized by fluctuating salinity due to changes in water regime. Besides its obvious interest as a naturally salt-tolerant plant, this species has also potential economic importance at detergent, pharmaceutical and food industries (as decolorant and stickiness improver). Indeed, G. oblanceolata rhizome and roots are rich in saponin which have antimicrobial and antifungal properties, along with its antiinflammatory and immune-stimulating properties. Moreover, this species is a Turkish endemic and endangered halophyte, which is included in a threatened category (Ekim et al., 2000). “The Red Book of Turkish Plants”, adopting the IUCN red list categories, listed this species as “vulnerable” to extinction (Ekim et al., 2000). The responses of G. oblanceolata are of particular interests as it is sensitive to salt stress throughout the germination, but can withstand NaCl concentrations up to 300 mM NaCl at the vegetative stage. However, there is no study enlightening the seed germination requirements of this species. How much salinity this species can withstand during germination? Can its germinability recover following alleviation of high salinity stress? How this species responds to salinity stress during vegetative stage? Which extent of salt stress this species can withstand to? Is there any role of increase in the activities of their antioxidant enzymes on salt tolerance of this species? To answer these questions, G. oblanceolata was selected as a research material in the present study. With this aim, the responses to salt stress of G. oblanceolata were characterized in two distinctive stages of plant development; germination and vegetative growth. Therefore, two experiments were conducted. In experiment 1, the effects of salinity on seed germination (final germination percentage, rate of germination) and recovery responses of G. oblanceolata were determined. Moreover, changes in the activities of antioxidant enzymes/isoenzymes (SOD, CAT and POX) during germination under salt stress and recovery after stress were also identified. In experiment 2, the effects of salt stress during vegetative growth on relative growth rate (RGR), leaf relative water content (RWC), leaf osmotic potential ( ), chlorophyll fluorescence (Fv /Fm ), ion concentrations (Na+ , K+ and Ca2+ ), proline (Pro) content, the activities of antioxidant enzyme/isoenzyme (SOD, CAT, POX, APX and GR), NADPH oxidase (NOX) activity, lipid peroxidation and H2 O2 content of salt-treated G. oblanceolata plants (60 d) during 14 day were investigated.

2. Materials and methods 2.1. Plant material Seeds of G. oblanceolata were collected from the salty steppe habitat in west of Tersakan Lake near Salt Lake (Yıldıztugay 2485, 38◦ .32 .933 N, 33◦ .08 .967 E). Seeds were separated from the inflorescence and stored at 4 ◦ C. G. oblanceolata seeds were sterilized with 5% sodium hypochlorite for 10 min and were washed

thoroughly with deionized water. Preliminary studies using various light (24 h dark, 24 h light and 16 h light/8 h dark) and temperatures regimes (5 ◦ C, 15 ◦ C, 24 ◦ C and 30 ◦ C) have shown that optimum conditions for germination of G. oblanceolata seeds occurred at 24 ◦ C and 16 h light/8 h dark, with a germination percentage approaching to 90%. In this study, two experiments were conducted. Salt stress imposed during the germination stage in experiment 1 and during vegetative stage in experiment 2. All germination tests were conducted in petri dishes containing three layers of filter paper, to which 9 ml of treatment solution was added. Petri dishes were sealed with parafilm to prevent evaporation. 2.2. Experimental design 2.2.1. Experiment 1: effects of salinity on germination The effects of 0, 50, 100, 150 and 300 mM NaCl on germination of seeds were tested at 24 ◦ C in the 16 h light/8 h dark regime. For each treatment, three replicates of 50 seeds were used. Every 2 d, distilled water equal to the mean loss of water from dishes was added to each petri dish. During 10 days, germinated seeds were counted every 2 days and removed from the dishes. And, the final percentage germination was recorded 10 d after incubation. Seeds were considered to have germinated when radicle had emerged and elongated by at least 2 mm. Two characteristics of germination were determined: final germination percentage and germination rate. Final germination percentage was recorded 10 d after incubation. by using a modified Timson The germination rate was estimated index of germination velocity = G/t, where “G” is percentage of seed germination at 2 d intervals and “t” is total germination period (10 d) (Khan and Ungar, 1997). To determine whether germinability could be recovered following alleviation of high salinity stress, un-germinated seeds were transferred from the NaCl treatments to distilled water, recovery germination was also recorded at 2 day intervals for 10 days. Moreover, recovery percentage, rate of recovery and days required for 50% recovery of germination were determined. The final recovery percentage was determined by following formula (Khan et al., 2000; Sekmen et al., 2004): a−b × 100 c−b where “a” is the total number of seeds germinated after being transferred to distilled water, “b” is the total number of seed germination in saline solution and “c” is the total number of seeds. The rate of recovery germination was estimated by using a modified Timson index of recovery germination velocity = G/t where “G” is percentage of recovery germination after stress at 2 d intervals and “t” is total recovery period (10 d). Plants were harvested on the 10 d of germination (under salt stress) and recovery (after stress) and then, stored at −80 ◦ C until analyses of antioxidant enzyme/isoenzyme activity. 2.2.2. Experiment 2: effects of salinity on vegetative growth In this experiment, seeds were germinated at 24 ◦ C and 16 h light/8 h dark for 10 days. After germination, seedlings were transferred into the pots filled with perlite. Seedlings were grown for two months (60 d) under controlled conditions (16 h light/8 h dark at 25 ◦ C, relative humidity of 70%, and photosynthetic photon flux density of (PAR) 350 ␮M m−2 s−1 ). They were sub-irrigated every other day with half strength Hoagland’s solution (Hoagland and Arnon, 1950). For stress treatments, on day 60 of normal growth, a salt stress treatment was initiated by giving Hoagland’s solution containing 0, 50, 100, 150 and 300 mM NaCl. Plants were harvested


on the 0 and 14th days of salt treatment and then, stored at −80 ◦ C until further analyses. In experiment 2, the changes in relative growth rate (RGR), leaf relative water content (RWC), leaf osmotic potential ( ), chlorophyll fluorescence (Fv /Fm ), ion concentrations (Na+ , K+ and Ca2+ ), proline (Pro) content, lipid peroxidation and H2 O2 content of salt-treated 60 d-old plants during 14 day were investigated. Moreover, the activities of antioxidant enzymes/isoenzymes during vegetative growth of G. oblanceolata under salt stress were also determined.

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concentration of MDA was calculated using an extinction coefficient of 155 mM−1 cm−1 . 2.9. Determination of hydrogen peroxide (H2 O2 ) accumulation

In experiment 2, 6 random plants for each group were used for the growth analyses. They were separated to shoot and root fractions on the 0 and 14 d of stress treatment. Shoot length, fresh weights (FW) and dry weights (DW) – after the samples were dried at 70 ◦ C for 72 h – of seedlings were measured to calculate the relative growth rate (RGR) of seedlings according to using the formula given by Hunt et al. (2002).

Determination of H2 O2 content was performed according to Liu et al. (2010). Samples used in detection of H2 O2 content were stored in liquid nitrogen immediately after harvesting until analyses. 0.5 g of samples were grounded with liquid nitrogen and were homogenized in 3 ml of cold acetone (−20 ◦ C) and centrifuged at 3000 × g at 4 ◦ C for 10 min. 1 ml of the supernatant was mixed with 0.1 ml of titanium reagent (prepared in concentrated hydrochloric acid containing 20% (v/v) titanium tetrachloride) then 0.2 ml of ammonium hydroxide was added to precipitate the titanium-peroxide complex. Reaction mixture was centrifuged at 16000 × g at 4 ◦ C for 10 min, and the pellet was washed with cold acetone (−20 ◦ C). The pellet was dissolved in 2 ml of 1 M H2 SO4 . The absorbance of the solution was measured at 410 nm against water blank. H2 O2 concentrations were calculated using a standard curve prepared with known concentrations of H2 O2 .

2.4. Leaf osmotic potential (

2.10. Proline level

2.3. Growth analysis

)

Leaf osmotic potential was measured on the 0 and 14 d of treatment by Vapro Vapor pressure Osmometer 5520. The data were collected from six sample leaves per replicate. These results were converted to MPa according to Santa-Cruz et al. (2002) by multiplying with coefficient of 2.408 × 10−3 . 2.5. Leaf relative water content (RWC) After harvest on 0 d and 14 d, six leaf disks were obtained from G. oblanceolata plants and their fresh weight (FW) was determined. The leaf discs were floated on de-ionised water for 6 h under low irradiance and then the turgid tissue was then quickly blotted dry prior to determining turgid weight (TW). Dry weight (DW) was determined after oven drying at 70 ◦ C for 72 h, the time point at which a constant weight was reached. The relative water content (RWC) was calculated by the following formula (Smart and Bingham, 1974): RWC (%) =

FW − DW TW − DW

× 100

Determination of free Pro content was done according to Bates et al. (1973). Gypsophila leaves (0.5 g) were homogenized in 3% sulphosalycylic acid and homogenate was filtered through filter paper. After addition of acid ninhydrin and glacial acetic acid, resulting mixture was heated at 100 ◦ C for 1 h in water bath. Reaction was then stopped using ice bath. The mixture was extracted with toluene, and the absorbance of fraction with toluene aspired from liquid phase was read at 520 nm. Pro concentration was determined using calibration curve as mmol proline g−1 FW. 2.11. NADPH oxidase (NOX) activity NOX (EC 1.6.3. 1) activity was measured according to Jiang and Zhang (2002). The assay medium contained 50 mM Tris–HCl buffer, pH 7.5, 0.5 mM XTT, 100 ␮M NADPH·Na4 and 20 ␮g of protein. After the addition of NADPH, XTT reduction was followed at 470 nm. The corrections of background production were determined in the presence of 50 U SOD. Activity was calculated using the extinction coefficient, 2.16 × 104 M−1 cm−1 . One unit of NOX was defined as 1 nmol ml−1 XTT oxidized min−1 .

2.6. Chlorophyll fluorescence (Fv /Fm )

2.12. Enzyme extractions and assays

After harvest on 0 d and 14 d of stress treatment, chlorophyll fluorescence parameters (PSII maximum efficiency, Fv /Fm ) of leaves of six plants for each group were measured by Plant Efficiency Analyzer of Hansatech, UK. This parameter provided an estimate of the maximum photochemical efficiency of the photosystem II (PSII).

Responses of antioxidant enzyme systems to salt stress during germination (in 10 d plants), recovery germination (10 d of recovery) after stress and vegetative stages (in 60 d plants) in G. oblanceolata were compared. All assays were performed at 4 ◦ C. For protein and enzyme extractions, 0.5 g of samples were grounded to fine powder by liquid nitrogen and then homogenized in 1.25 ml of 50 mM Tris–HCl, pH 7.8, containing 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.2% (w/v) Triton X-100, 1 mM phenylmethylsulphonyl fluoride (PMSF), and 2 mM dithiothreitol (DTT). For APX activity determination, 5 mM ascorbate was added into homogenization buffer and PVP (2%, w/v) was used instead of DTT. Samples were centrifuged at 14,000 × g for 30 min, and supernatants were used for the determination of protein content and enzyme activities. Total soluble protein contents of the enzyme extracts were determined according to Bradford (1976) using bovine serum albumin as a standard. All spectrophotometric analyses were conducted on a Shimadzu (UV 1600) spectrophotometer. CAT (EC 1.11.1.6) activity was estimated according Bergmeyer (1970), which measures the initial rate of disappearance of H2 O2

2.7. Ion concentrations Samples were finely ground, and 0.5 g plant material was digested with concentrated nitric acid (HNO3 ) in a microwave system (CEM, Mars 5). The Na+ , K+ and Ca2+ in extracts were analyzed by ICP-AES (Varian-Vista) (Nyomora et al., 1997). 2.8. Lipid peroxidation The level of lipid peroxidation was determined in terms of malondialdehyde (MDA) content according to the method of Madhava Rao and Sresty (2000). MDA concentration was calculated from the absorbance at 532 nm and measurements were corrected for nonspecific turbidity by subtracting the absorbance at 600 nm. The

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at 240 nm. The reaction mixture contained 50 mM Na–phosphate buffer (pH 7.0) with 0.1 mM EDTA and 3% H2 O2 . The decrease in the absorption was followed for 3 min and 1 mmol H2 O2 ml−1 min−1 was defined as 1 unit of CAT. POX (EC1.11.1.7) activity was based on the method described by Herzog and Fahimi (1973). The reaction mixture contained 3,3 -diaminobenzidine-tetra hydrochloride dihydrate solution containing 0.1% (w/v) gelatine and 150 mM Na–phosphate–citrate buffer (pH 4.4) and 0.6% H2 O2 . The increase in the absorbance at 465 nm was followed for 3 min. A unit of POX activity was defined as mmol H2 O2 decomposed ml−1 min−1 . APX (EC 1.11.1.11) activity was measured according to Nakano and Asada (1981). The assay depends on the decrease in absorbance at 290 nm as ascorbate was oxidized. The reaction mixture contained 50 mM K–phosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.1 mM EDTA Na2 , 0.1 mM H2 O2 and 0.1 ml of enzyme extract in a final assay volume of 1 ml. The concentration of oxidized ascorbate was calculated by using extinction coefficient of 2.8 mM−1 cm−1 . One unit of APX was defined as 1 mmol ml−1 ascorbate oxidized min−1 . GR (EC 1.6.4.2) activity was measured according to Foyer and Halliwell (1976). The assay medium contained 25 mM Na–phosphate buffer (pH 7.8), 0.5 mM GSSG, and 0.12 mM NADPH. Na4 and 0.1 ml enzyme extract in a final assay volume of 1 ml. NADPH oxidation was followed at 340 nm. Activity was calculated using the extinction coefficient of NADPH (6.2 mM−1 cm−1 ). One unit of GR was defined as 1 mmol ml−1 GSSG reduced min−1 . The specific enzyme activity for all enzymes was expressed as in unit mg−1 protein. 2.13. Identification of isoenzymes Samples containing equal amounts of protein were subjected to non-denaturing polyacrylamide gel electrophoresis (PAGE) as described by Laemmli (1970) except that sodium dodecyl sulphate was omitted. For the separation of SOD isoenzymes, 4.5% stacking and 12.5% separating gels under constant current (120 mA) at 4 ◦ C were used. SOD activity was detected by photochemical staining with riboflavin and NBT as described by Beauchamp and Fridovich (1971). The unit activity of each SOD isoenzyme was calculated by running a SOD standard from bovine liver (Sigma Chemical Co.). The different types of SOD were differentiated by incubating gels in inhibitors of SOD before staining, such as 2 mM KCN to inhibit Cu/Zn-SOD activity and 3 mM H2 O2 to inhibit Cu/Zn-SOD and FeSOD activities as described by Vitória et al. (2001) (Mn-SOD activity is resistant to both inhibitor treatments). CAT isoforms were detected according to Woodbury et al. (1971). The electrophoretic separation was performed on nondenaturating polyacrylamide mini gels using 10% separating gel under constant current (30 mA). The gels were incubated in 0.01%

H2 O2 for 5 min. After incubation gels were washed with distilled water twice and incubated for 20 min in staining solution containing 1% FeCl3 and 1% K3 Fe(CN6 ). For identification of the different CAT isozymes 3-amino-1,2,4-triazole (3-AT) was added to the H2 O2 solution for 2.5 min in a final concentration of 10 mM. POX isoforms were detected according to Seevers et al. (1971). The electrophoretic separation was performed on nondenaturating polyacrylamide mini gels using 10% separating gel under constant current (30 mA). The gels were loaded with 20 ␮g protein. The gels were incubated for 30 min at 25 ◦ C in 200 mM Na–acetate buffer (pH 5.0) containing 1.3 mM benzidine and 3% hydrogen peroxide. The gels were then stored in 7% acetic acid. GR isoforms were detected using 7.5% native PAGE according to Hou et al. (2004). After electrophoresis of the samples containing 20 ␮g protein, GR isoforms were detected by incubating the gels in a solution containing 10 mM Tris–HCl (pH 7.9), 4 mM GSSG, 1.5 mM NADPH·Na4 and 2 mM DTNB (5,5 -dithiobis (2-nitro-benzoic acid) for 20 min. After a brief rinse with 50 mM Tris–HCl buffer (pH 7.9), GR activity was negatively stained by 1.2 mM MTT (thiazolyl blue tetrazolium bromide), 1 mM DPIP (2,6-dichloroindophenol) and 1.6 mM PMS (N-methylphenazonium methyl sulphate) for 5–10 min at room temperature. Gels stained for SOD, CAT, POX and GR activities were photographed with Vilber Lourmat gel imaging system and then analyzed with Bio-Profil Bio-1D Windows Application V11.9 software package (Vilber Lourmat, Marne la Vallée, France). In densitometric analyses of CAT, POX and GR activities, activities of control plants were taken as 100% and % of control values for each treatment are shown. The values are average of data from three independent gels ± SE. Within each isoenzyme, means with the same letter are not significantly different at p < 0.05 by Tukey. 3. Results 3.1. Effects of salinity on seed germination The germination responses of G. oblanceolata seeds to salinity are shown in Fig. 1A. Seeds of G. oblanceolata germinated rapidly in distilled water during the initial 2 d. Percentage of germination was 30% on the first day, and 58% on the second day. The highest germination percentage was found in distilled water, followed by 50 mM NaCl. When seeds were germinated on filter papers moistened with solutions of NaCl (50, 100, 150 and 300 mM), germination decreased with increasing salt concentrations (Fig. 1A). In 50 mM NaCl, germination percentage declined from 90% to 51%. While few seeds germinated in 100 mM NaCl, germination was not observed over 100 mM NaCl (at 150 and 300 mM NaCl). Ungerminated seeds under salt stress showed recovery after transferred to dIH2 O (Fig. 2A). Percentage of recovery germination

Fig. 1. Percentage of germination (A) and rate of germination (B) of G. oblanceolata seeds under salt stress (0 (control), 50, 100, 150 and 300 mM NaCl).


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Fig. 2. Percentage of recovery germination (A), rate of recovery germination (B) and time required to attain 50% recovery of germination (C) of G. oblanceolata seeds under salt stress (0 (control), 50, 100, 150 and 300 mM NaCl).

of G. oblanceolata seeds was enhanced with an increase in NaCl concentrations. The highest increase was observed in 300 mM NaCl treated-seeds. While the germination rate of G. oblanceolata seeds decreased (Fig. 1B), the rate of recovery germination increased with an increasing salinity (Fig. 2B). The decrease in germination rate was started at 4 d and 2 d of germination under 50 and 100 mM NaCl stress, respectively. On the other hand, after transfer from NaCl to dI-H2 O, the rate of recovery was minimal at 50 mM NaCl, it was maximum at 300 mM NaCl. G. oblanceolata seeds showed 50% recovery at 3 d after transfer from 50 mM, 100 mM and 150 mM NaCl to dI-H2 O (Fig. 2C). However, at 300 mM NaCl, their 50% recovery occurred just 2 d after transfer, indicating a faster recovery at 300 mM NaCl as compared to other salt concentrations.

NaCl, the decrease in SOD activity coincided with the decrease in the intensities of these isoenzymes. Moreover, the highest activity was observed at Fe-SOD isoenzyme under salt stress. After stress, in all recovered plants, Fe-SOD activity was gradually increased, as

3.2. The changes in the activities of antioxidant enzymes/isoenzymes during germination under salt stress and recovery after stress The total activities of all antioxidant enzymes (SOD, CAT and POX) were decreased after 10 d of germination under salt stress (Fig. 3). SOD, CAT and POX activities were decreased by 15%, 13.38% and 10%, respectively, as compared to control groups. On the other hand, SOD activity was increased up to control levels under recoveries of 50, 100 and 150 mM stress conditions and also 300 mM NaCl treated-recovery group showed 23.05% increased SOD activity as compared to 50 mM treated-recovered group (Fig. 3A). CAT activities were significantly increased approximately 2-fold under recovery of stress conditions (Fig. 3B). POX activity of 50 mM NaCl treated-recovered groups was decreased 29.66% as compared to 50 mM stressed group. However, POX activities of 100, 150 and 300 mM NaCl treated-recovered groups increased gradually, 103.7%, 137.41%, and 216.6%, respectively. As shown in Fig. 4A, SOD isoenzymes were identified as one Mn-SOD (not inhibited by KCN or H2 O2 ), one Fe-SOD (not inhibited by KCN, but inhibited by H2 O2 ). These isoenzymes were named in the order of their migration distance. Under control conditions, the intensities of Mn-SOD were higher than that of Fe-SOD. At 50 mM

Fig. 3. Effects of 10 d salt stress (S) (50 mM NaCl) and 10 d recovery (R) from stress (50, 100, 150 and 300 mM NaCl) on the activities of SOD (A), CAT (B) and POX (C) of G. oblanceolata during germination. Data represents the average of two experiments with three replicates. Vertical bars indicate ±SE and values sharing a common letter are not significantly different at p < 0.05.

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Fig. 4. Effects of 10 d salt stress (50 mM NaCl) (S) and 10 d recovery from stress (R) on the activity staining and % change of SOD (A), CAT (B) and POX (C) isoenzymes of G. oblanceolata during germination. Samples applied to the gels contained 20 ␮g protein for SOD and POX, 15 ␮g for CAT. Lanes: C: control; S50: germinated plants (10 d) under 50 mM NaCl stress; R50, R100, R150, R300: recovered plants (10 d) after stress (50, 100, 150, 300 mM NaCl). The different letters are significantly different (p < 0.05).

compared to salt-treated plants (50 mM NaCl). Similarly, the activities of Mn-SOD were also increased in recovered-plants after 50, 100, and 300 NaCl stress. However, after 150 mM NaCl, it was observed a decrease (15.5%) in Fe-SOD activity of recoveredplants. As shown in Fig. 4B, only one CAT isoenzyme (CAT1) was determined in all treatment groups during germination period. The intensity of this isoenzyme was decreased by 13.44% after 10 d of germination under salt stress. However, the activity of same isoenzyme was increased in all recovered-plants after stress, as compared to germinated-plants under 50 mM NaCl stress. Moreover, the highest increase (92.1%) in CAT1 isoenzyme was observed in recovered-plants after 150 mM NaCl stress. As shown in Fig. 4C, G. oblanceolata had three POX isoenzymes (POX1, POX3 and POX5) after 10 d of germination under stress, while four POX isoenzymes (POX1, POX2, POX3, POX5) were determined under recovery of this group. If we examine the seeds that non germinated after 50, 100, 150 and 300 mM stresses at the sight of recovery, POX1 and POX3 isoenzymes were observed in all treatment groups. Recovered plants after 50 and 100 mM NaCl stress had the same four POX isoenzymes. Moreover, POX6 was detected only at 150 and 300 mM stress recovery plants. After 10 d of germination under stress, while intensities of POX3 and POX5 isoenzymes were decreased, POX1 isoenzyme was increased by 29.49% as compared to non-treated control group. If we look closely to POX1 isoenzyme activity, after recovery of 50 mM stress, it decreased 34.15%. But after 100, 150 and 300 mM stress it showed increasing activity reached to 130%. After recovery of 50 mM stress, POX5 activity was decreased by 76.42%, as compared to its non treated control group. But after recovery of 100 mM stress it increased sharply. Furthermore, POX6 took an important role for recovery of severe stress (150 and 300 mM NaCl) treated groups while POX4 had been observed only under 300 mM stress recovered plants.

3.3. The changes in the physiological parameters during vegetative growth of G. oblanceolata under salt stress 3.3.1. Relative growth rate (RGR) 50 mM and 100 mM NaCl had no effect on RGR of G. oblanceolata seedling on 14 d of salt treatment (Table 1). However, RGR showed 40% and 41% decrease at 150 and 300 mM NaCl, respectively, in comparison to control group. RWC did not change at 50 mM NaCl (Table 1). However, other salt concentrations caused decrease in RWC. This effect was slight at 100 and 150 mM NaCl-treated plants and strong (14.01%) in 300 mM NaCl-treated plants. decreased with an increasing in the salt concentration (Table 1). Whereas smaller changes were observed in the leaves of 50 mM NaCl-treated plants (from −1.051 MPa to −1.237 MPa), at 300 mM NaCl, was declined from −1.051 MPa (before stress) to −2.161 MPa (after stress). No significant impact of stress treatment on chlorophyll fluorescence was found in present study (Table 1). 3.3.2. Ion concentrations NaCl treatments increased the concentrations of Na+ in the leaves, as compared to control groups (Fig. 5A). The highest enhancement was observed in 300 mM NaCl-treated leaves. Na+ concentrations in the leaves ranged from 112.95 ppm to 346.35 ppm at 50 mM and 300 mM NaCl at 14 d, respectively. Salt treatment caused a significant decrease in K+ and Ca2+ concentrations in the leaves of G. oblanceolata (Fig. 5A). However, at 300 mM NaCl, contents of these ions were close to control group. On the other hand, under normal and stress conditions (except for 300 mM NaCl), G. oblanceolata had higher leaf K+ and Ca2+ concentrations than those of Na+ . K+ /Na+ ratio was decreased with increasing NaCl. The highest decrease in K+ /Na+ was observed by 83.85% in the leaves of 300 mM NaCl-treated

Table 1 Effects of salt stress on relative growth rate (RGR; mg mg−1 d−1 ), osmotic potential (MPa), relative water content (RWC; %) and Fv /Fm (maximum quantum yield of PSII) of G. oblanceolata. The different letters are significantly different (p < 0.05) values. 0: control; 50: 50 mM NaCl; 100: 100 mM NaCl; 150: 150 mM NaCl; 300: 300 mM NaCl. NaCl concentration (mM) 0 RGR (mg mg−1 d−1 ) RWC (%) Osmotic potential (MPa) Fv /Fm

0.190 84.78 −1.051 0.841

50 ± ± ± ±

0.007a 1.766a 0.099a 0.0124a

0.189 81.51 −1.237 0.839

100 ± ± ± ±

0.06a 1.158a 0.010b 0.0125a

0.182 79.22 −1.474 0.824

150 ± ± ± ±

0.028a 0.494b 0.087c 0.0106a

0.112 79.42 −1.762 0.842

300 ± ± ± ±

0.006b 1.473b 0.014d 0.0192a

0.114 70.77 −2.161 0.848

± ± ± ±

0.002b 1.452c 0.018e 0.048a


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Fig. 5. Effect of salt treatment on ion content (Na+ , K+ and Ca2+ ) (A) and K+ /(K+ + Na+ ) ratio (B) in the leaves of G. oblanceolata (60 d) on day 14. The different letters are significantly different (p < 0.05).

plants. The selective accumulation of K+ over Na+ was estimated by the ratio of K+ /(K+ + Na+ ) (Fig. 5B). Although salt treatment caused a significant decrease in K+ concentrations, plant exhibited a strong selectivity for K+ uptake, as shown by Fig. 5A and B. 3.4. The changes in the biochemical mechanism during vegetative growth of G. oblanceolata under salt stress H2 O2 content was increased by salt stress in all treatment groups, as compared to control group (Fig. 6A). The highest increase (57.95%) was observed at 300 mM NaCl-treated plants. Moreover, there were no significant changes in H2 O2 content of 50, 100 and 150 mM NaCl-treated plants. The level of salt-induced oxidative damage was determined by monitoring the differences in lipid peroxidation (Fig. 6B). Salt stress caused a significant increase in MDA content of all treatment

groups. G. oblanceolata showed significant increase by 9.3%, 20.4%, 56.68% and 72.87% at 50 mM, 100 mM, 150 mM and 300 mM, respectively, as compared to its control group. Moreover, there were no significant changes between lipid peroxidation levels of 50 mM and 100 mM NaCl-treated seedlings. Free Pro level of the leaves showed an enhancement with an increase in salt concentration (Fig. 6C). The highest Pro content was recorded (18.20 fold) in 300 mM NaCl-treated plants. Moreover, Pro levels were increased by approximately 2.5 fold at all other salt concentrations. To measure the effects of salt stress on ROS production during vegetative growth of G. oblanceolata, NADPH oxidase (NOX) activity was measured (Fig. 7). Salt stress caused a significant increase in NOX activity. NOX activity was induced by 3.37, 3.65 and 3.86 fold by 50 mM, 100 mM and 150 mM NaCl, respectively, as compared to control groups. However, a stronger induction of NOX was observed in 300 mM NaCl-treated plants by 4.86 fold, as compared to its control groups. 3.5. The changes in the activities of antioxidant enzymes/isoenzymes during vegetative growth of G. oblanceolata under salt stress Salt stress-induced changes in the isoenzyme pattern of SOD at vegetative growth of G. oblanceolata were detected by Native-PAGE electrophoresis. As shown in Fig. 8A, SOD isoenzymes were identified as five Mn-SOD, two Fe-SOD and one Cu/Zn-SOD. In Fig. 8B, one band includes both Fe-SOD2 as the major component and MnSOD5 as the minor component. At 50 mM and 100 mM NaCl, while total SOD activity was increased, total Mn-SOD and Fe-SOD2 activities were also increased. However, under the same conditions, Fe-SOD1 isoenzyme was not determined. At 150 mM NaCl, while the decrease in SOD activity coincided with decreases in Mn-SOD5 and Fe-SOD2, Cu/Zn-SOD was not determined. On the other hand,

Fig. 6. The effects of different salt concentrations (0 (control), 50, 100, 150 and 300 mM NaCl) on H2 O2 (␮mol g−1 FW) (A), MDA (nmol g−1 FW) (B) and proline (nmol g−1 FW) contents (C) of G. oblanceolata during vegetative growth. Data represents the average of two experiments with three replicates. Vertical bars indicate ±SE and values sharing a common letter are not significantly different at p < 0.05.

Fig. 7. The effects of different salt concentrations (0 (control), 50, 100, 150 and 300 mM NaCl) on NOX (units mg−1 protein) activity of G. oblanceolata during vegetative stage. Data represents the average of two experiments with three replicates. Vertical bars indicate ±SE and values sharing a common letter are not significantly different at p < 0.05.

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Fig. 8. (A) Activity staining and % change of SOD isoenzymes in the crude extracts of leaves of G. oblanceolata exposed to 0, 50, 100, 150 and 300 mM NaCl on vegetative stage. Samples applied to the gels contained 75 ␮g protein. Lanes SOD std.: 0.5 unit SOD; 0: Control; 50: 50 mM NaCl-treated groups; 100: 100 mM NaCl-treated groups; 150: 150 mM NaCl-treated groups; 300: 300 mM NaCl-treated groups. The different letters represent significantly different values (p < 0.05). (B) Zymograms and identification of SOD isoenzymes in leaves of G. oblanceolata exposed to 150 mM NaCl on vegetative stage. Staining for activity was performed in the presence of 2 mM KCN which inhibits Cu/Zn-SOD, or in the presence of 3 mM H2 O2 which inhibits both Cu/Zn- and Fe-SOD.

total SOD activity was increased in 300 mM NaCl-treated plants, as compared to 150 mM NaCl-treated plants, and reached the control level. Under same conditions, while intensities of Mn-SOD3, 4, 5 and Fe-SOD2 showed concomitant increase with total SOD activity, as compared to 150 mM NaCl-treated plants. 50 mM and 100 mM NaCl treatments significantly increased SOD activity (10.01% and 76.95% of control at 50 and 100 mM NaCl, respectively). On the other hand, SOD activity slightly decreased by 13.58% at 150 mM NaCl, but at 300 mM NaCl, its activity reached to control level (Fig. 9A). Salt stress caused a significant increase in CAT activity of all treatment groups (Fig. 9B). The highest (fold 2.90) and lowest (fold 1.69) increases CAT activities between stress-treated groups were recorded in 100 mM and 300 mM NaCl-treated groups, respectively, as compared to control group. Only one isoenzyme (CAT1) of CAT was identified (Fig. 10A). The number of CAT isoenzymes in G. oblanceolata did not change under stress. Intensity of CAT1 was increased by salt stress, as compared to control group. The intensities of CAT1 were increased by 69.46%, 102%, 93.66% and 70.61% at 50 mM, 100 mM, 150 mM and 300 mM NaCl, respectively.

POX activity was reduced only at 50 mM NaCl-treated plants (Fig. 9C). Although 100 and 150 mM NaCl did not change POX activity, 300 mM NaCl caused an increase by 32.75% in POX activity of G. oblanceolata seedlings. Three isoenzymes of POX were identified (Fig. 10B). Intensity of POX1 was increased by increasing salt concentration. While the highest increase in POX1 intensity was observed at 300 mM NaCl, the highest decrease in the POX2 and POX3 were observed in 100 mM NaCl. However, 50 mM, 150 mM and 300 mM NaCl increased POX3 intensity. Salt stress (except for 300 mM NaCl) induced APX activity (Fig. 9D). This effect was stronger (30.60%) at 50 mM NaCl-treated plants than the others. However, 300 mM NaCl reduced APX activity by 15.61%. 50 mM NaCl caused a slight decrease in GR activity (Fig. 9E). However, other NaCl concentrations (100 mM, 150 mM and 300 mM) induced GR activity of G. oblanceolata. The highest GR activity was observed at 150 mM NaCl-treated plants. Two isoenzyme of GR was determined (Fig. 10C). GR1 and GR2 isoenzymes were observed in all treatment groups. The intensity of GR1


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Fig. 9. Total SOD (A), CAT (B), POX (C), APX (D) and GR (E) activities in the crude extracts of leaves of G. oblanceolata exposed to 0, 50, 100, 150 and 300 mM NaCl on vegetative stage. Data represents the average of two experiments with three replicates. Vertical bars indicate ±SE and values sharing a common letter are not significantly different at p < 0.05.

isoenzyme was increased with an increasing salinity. GR2 was observed as a faint band at control groups, but its intensity was increased by salt stress. Activities of GR2 were induced by 50.76%, 96.79% and 59.93% at 100, 150 and 300 mM NaCl.

4. Discussion 4.1. Effects of salinity on seed germination Previous studies with halophytes indicated that they generally germinate in spring, rather than in summer, because high levels of salt in soil composition caused by high evaporation causes seeds to remain dormant in a saline environment (Keiffer and Ungar, 1997; Song et al., 2005). Also, seeds of halophytes tend to be less salt tolerant than growing plants (Meyer and Poljakoff-Mayber, 1963; Ungar, 1995, 1996). In its natural habitats, seeds of G. oblanceolata mature at the end of the autumn. Seed germination starts after winter rains, when salt concentration is decreased in soil. Similarly, in present study, we observed that seeds of G. oblanceolata cannot germinate over 100 mM NaCl. However, when ungerminated seeds under salt stress are transferred to dIH2 O for recovery, germination can be observed. Seed survival under hypersaline conditions rather than germinability has been used as criteria for salt tolerance. Moreover, survival skills of halophyte seeds like observed in G. oblanceolata under hypersaline conditions gives a selective advantage to halophytes and distinguishes them from most of glycophytes (Ungar, 1991, 1996; Sekmen et al., 2004).

4.2. The changes in the activities of antioxidant enzymes/isoenzymes during germination under salt stress and recovery after stress ROS, which occurs during germination especially under various abiotic stresses such as high salinity, inevitably lead to oxidative stress and cellular damage, thus resulting in inhibition of seed germination and even seed deterioration (Bailly, 2004). Most studies proposed that one of the effective mechanism of defense in plants against oxidative damage resulted from salt stress is ROS detoxification by enzymatic (SOD, CAT, POX, GR and APX) antioxidants (Verslues et al., 2006; Khan and Panda, 2008). It is relevant now that, a higher constitutive (inherent response) and induced antioxidant defense is related to an increased resistance to salt stress (Meneguzzo et al., 1999; Sreenivasulu et al., 2000; Mittova et al., 2003). In present study, we observed a significant difference between the changes in activities of SOD and CAT enzymes of G. oblanceolata during germination and vegetative growth under salt stress. While the activities of CAT and SOD during germination were higher as compared to vegetative stage, there was no difference in POX activity between these stages. According to these results, the higher tolerance to salt stress during vegetative growth might be associated with significant changes in composition of SOD and POX isozymes. Our results demonstrated that salt stress significantly decreased the activities of SOD, CAT and POX in G. oblanceolata during germination. However, the activities of all enzymes were increased during recovery germination after stress.

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and cv. begunbitchi). On the contrary, in present study, both CAT and POX activities were decreased during germination under salt stress. Decreased CAT activity in stressed plants might have promoted H2 O2 accumulation, which could result in a Haber–Weiss reaction to form hydroxyl radicals which are known to damage biological systems (Gill and Tuteja, 2010). However, after recovery from salt stress, CAT and POX activities were increased in the poststress recovered plants. These results indicated that post-stress recovered plants had higher capacity for the decomposition of H2 O2 than germinated plants under salt stress. 4.3. The changes in the physiological parameters during vegetative growth of G. oblanceolata under salt stress

Fig. 10. Activity staining and % change of CAT (A), POX (B), GR (C) isoenzymes in the crude extract of leaves of G. oblanceolata exposed to 0, 50, 100, 150 and 300 mM NaCl on vegetative stage. Samples applied to the gels contained 20 ␮g protein for GR and 15 ␮g protein for CAT and POX enzymes. The abbreviations on the gel are the same as explained in Fig. 8. The different letters represent significantly different values (p < 0.05).

Superoxide dismutase (SOD) catalyzing the dismutation of O2 • − to H2 O2 constitutes the first line of cellular defense against oxidative stress (Alscher et al., 2002; Mittler et al., 2004). In present study, it was obvious that NaCl stress caused reduction in the SOD activity of G. oblanceolata during germination as also demonstrated by Cavalcanti et al. (2007) and Khan and Panda (2008) working with cowpea and rice, respectively. This reduction in SOD activity could diminish the ability of the seedlings to scavenge O2 •− radicals favoring an accumulation of ROS, which could cause membrane damage. On the other hand, this decrease in SOD activity coincided with decreases in the activities of Mn-SOD1 and Fe-SOD1, as compared to control groups. However, SOD activity in recovered plants reached to control levels. Moreover, Mn-SOD1 and Fe-SOD1 showed concomitant increase with total enzyme activity in recovered plants, compared to salt-treated plants. CAT has significant roles in plant defense system against oxidative stress. CAT is important in the removal of H2 O2 generated in peroxisomes by photorespiration, purine catabolism and ␤oxidation of lipids (Gill and Tuteja, 2010). Beside CAT, POXs are other important H2 O2 detoxifying enzymes. Sharma and Dubey (2005) reported a decrease in CAT activity in rice seedlings under salt and drought stress, respectively. Moreover, as reported by Khan and Panda (2008), salt stress decreased total activity of CAT, but after recovery, CAT activity increased in rice cultivars (cv. lunishree

Some halophytes such as Suaeda fruticosa (Khan et al., 2000), Suaeda maritima (Moghaieb et al., 2004), Atriplex portulocoides (Redondo-Gomez et al., 2007) and Salicornia europea (Aghaleh et al., 2009) show optimal growth in saline conditions. On the other hand, other halophytes such as Crithmum maritimum (Amor et al., 2005), P. maritima (Sekmen et al., 2007) and C. tuzgoluensis (Yıldıztugay et al., 2011) grow optimally in the absence of salt (Flowers and Colmer, 2008). Similarly, also in present study, while RGR of G. oblanceolata was not stimulated by 50 and 100 mM NaCl, 150 mM NaCl caused an inhibition (40%) in its growth activity, without exhibiting any further toxicity symptoms on the leaves. However, interestingly, this inhibition was not stronger in 300 mM NaCltreated plants. Reduction rate in RGR of these plants was same with that of 150 mM NaCl-treated plants. 150 mM and 300 mM NaCl might inhibit plant growth due to osmotic and ionic effects of salinity, as evident from decreased RWC, and increased ion i.e. Na+ content in the leaves (Munns and Tester, 2008; Turkan and Demiral, 2009). On the other hand, previous studies showed that RGR degree of glycophytes was decreased by 50 mM and 100 mM NaCl as reported in rice by Lutts et al. (1996) and in sorghum by de Lacerda et al. (2005). These results suggested that G. oblanceolata showed much higher tolerance to salinity as compared to other glycophytes. Moreover, this species can withstand moderate doses of NaCl in the medium. RWC was decreased by salt stress throughout the experiment, as previously observed in other halophytes (Amor et al., 2005; Tounekti et al., 2011), but this effect was more pronounced in 300 mM NaCl-treated plants. On the other hand, seedlings treated with 50 mM, 100 mM and 150 mM NaCl were able to maintain RWC values above 79%, indicating that G. oblanceolata have the ability to sustain their water content under moderate stress (50–100–150 mM NaCl). This improvement in RWC might be result of osmotic adjustment because of increased Pro level, one of the compatible solutes, which accumulates in large quantities in response to environmental stresses (Hsu et al., 2003; Claussen, 2005). Moreover, our results demonstrated that 300 mM NaCltreated plants showed the lowest osmotic potential as well as highest Na+ and proline accumulation while RWC values were at 70.77%. These results suggest that Pro accumulation might have helped to maintain suitable leaf turgor under salt stress, as has been also shown in other species such as olive (Gucci et al., 1997) and rosemary (Tounekti et al., 2011). Similarly, studies with C. tuzgoluensis and rosemary indicated that these salt-adapted species had higher accumulation of free proline compared with non-stressed plants (Yıldıztugay et al., 2011; Tounekti et al., 2011). Pro, which is one of the compatible solutes, accumulates in large quantities in response to environmental stresses (Hsu et al., 2003; Kishor et al., 2005). Pro have a major role as an osmolyte in osmotic adjustment (Voetberg and Sharp, 1991). In present study, proline concentration increased 18.20 fold its control level in 300 mM NaCl treated stressed plants but, proline content was only increased by 2.93 fold in 150 mM NaCl treatment. This difference in induction


of Pro accumulation between 150 and 300 mM NaCl stress might be related to difference of water potential of these NaCl solutions and the severity of osmotic stress caused by them. Theoretically, water potentials of these solutions can be calculated as −0.704 and −1.391 MPa for 150 and 300 mM, respectively (Lang, 1967). Girousse et al. (1996) and Handa et al. (1986) also found a similar threshold value and sharp increase in Pro concentration of alfalfa and tomato cell culture under low water potential condition, respectively. Salinity caused an increase in Na+ content and a decrease in ratio of K+ /Na+ , Ca2+ and K+ contents. In this case, many halophytic plants accumulate inorganic ions to concentrations equal to or greater than that of the surrounding root solution to uptake water from medium (Bradley and Morris, 1991; Aghaleh et al., 2009). In G. oblanceolata, although salinity (except for 300 mM NaCl) caused an increase in Na+ content or a decrease in K+ /Na+ ratio, K+ and Ca2+ content, this plant had higher leaf K+ and Ca2+ concentrations than those of Na+ . At 300 mM NaCl, although Na+ content in plants increases to 5.7 times that of the control, this was not lethal for G. oblanceolata. However, reduction in its growth was observed due to decreased K+ /Na+ and K+ /(K+ + Na+ ). All data showed that this species have a capacity to regulate K+ transport during salt stress. Fv /Fm , one of the chlorophyll fluorescence parameters, is related to photochemical efficiency of PSII value. The photosynthetic efficiency of PSII is known to decrease by environmental stress conditions such as salt stress (Flanagan and Jefferies, 1988; Force et al., 2003). Therefore, Fv /Fm can be used as an indicator of the stress damage (Uzilday et al., 2011). In the present study, no significant effect of salt stress in G. oblanceolata was observed. Moreover, Fv /Fm remained above 0.8 in G. oblanceolata, indicating optimal functioning of PSII. Parallel to our findings, Yıldıztugay et al. (2011) and Uzilday et al. (2011) also found that photosynthetic efficiency of PSII in C. tuzgolunensis (moderate-halophyte) and Cleome gynandra under salt and drought stress are protected. 4.4. The changes in the biochemical mechanism during vegetative growth of G. oblanceolata under salt stress H2 O2 production during drought stress may act as an oxidative stress agent and a local or systemic signal which activates various antioxidant enzymes (Demiral et al., 2011). In present study, salt stress caused a rapid accumulation of H2 O2 during stress treatment, but higher in 300 mM NaCl-treated plants. This result was confirmed by Sairam et al. (2002) who found a strong increase in H2 O2 in wheat under salt stress. This increase in H2 O2 content in 50 mM and 100 mM NaCl-treated plants can be connected with (i) enhancement of activities of SOD isoenzymes which dismutates O2 •− that has been produced in the mitochondrial respiratory electron transport chain or during chloroplastic photosynthetic electron transport, (ii) H2 O2 produced during increased photorespiratory flux (Uzilday et al., 2011). On the other hand, despite the decreased or unchanged SOD activity in 150 and 300 mM NaCltreated plants, H2 O2 production was increased. In this case, these results might indicate an increase in activity of other potential sources of H2 O2 (except SOD), including cell wall peroxidases, apoplastic oxalate oxidases, and amine oxidases (Parida et al., 2004). Lipid peroxidation is widely used as marker of stress-induced damage (Hernandez and Almansa, 2002). In this study, the level of lipid peroxidation was determined by measuring MDA content, which is a product of lipid peroxidation. Extent of peroxidation of membrane lipids has often been as mean to assess the severity of the salt induced oxidative stress and the degree of plant sensitivity (Perez-Lopez et al., 2009). Our results demonstrate that MDA accumulation increased in 150 and 300 mM NaCl-treated plants. However, in lower NaCl concentrations (50 and 100 mM NaCl),

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MDA accumulation was higher only by 10% and 20% compared to control group, respectively. Therefore, our results showed that G. oblanceolata can withstand moderate doses of salt stress (up to 100 mM NaCl) while higher doses of NaCl led to oxidative stress as indicated by MDA accumulation. The highest MDA accumulation was observed at 300 mM NaCl-treated plants, coincident with the unchanged SOD, increased CAT, POX and GR activity and increased accumulation of Pro. NADPH oxidases are membrane bound O2 •− producing enzymes, which mediate the regulation of biological processes such as growth, cell cycle, programmed cell death, hormone signaling, stress responses, and development (Mittler et al., 2004; Foyer and Noctor, 2005). Many studies have shown that salt stress increased NOX activity (Jiang and Zhang, 2002; Duan et al., 2009). In agreement with these studies, we also observed a strong increase in NOX activity of G. oblanceolata under stress. Acclimation to stress might be achieved by this increasing pattern before it gets more severe. 4.5. The changes in the activities of antioxidant enzymes/isoenzymes during vegetative growth of G. oblanceolata under salt stress Stimulation of the antioxidant enzyme activities such as SOD, CAT and APX in 50 and 100 mM NaCl-treated plants might result from an induction of synthesis of these enzymes via the production of O2 •− and H2 O2 (Jung, 2004). Hence, as compared to 150 mM and 300 mM NaCl-treated plants, these inductions in 50 and 100 mM NaCl-treated plants caused a decrease in the level of ROS under salt stress. This was indicated in a lower MDA content in 50 and 100 mM NaCl-treated plants under same conditions. In contrast, 150 mM and 300 mM NaCl-treated plants exhibited a greater extent of MDA due to unchanged or decreased SOD activity, lack of induction of CAT, POX, APX and GR, or excess H2 O2 content. Increased SOD activity is implicated in combating oxidative stress caused due to biotic and abiotic stress and have a critical role in the survival of plants under environmental stress (Gill and Tuteja, 2010). Significant increase in SOD activity has been observed in plants such as sugarbeet under salt stress (Bor et al., 2003). In present study, increased SOD activity especially under 100 mM NaCl seems to be essential for salt tolerance during vegetative growth. These results indicated that this increase in SOD activity might reflect enhanced O2 •− production. On the other hand, we found a decrease in G. oblanceolata under 150 mM NaCl, as compared to control groups. And, this decrease negatively affected scavenging efficiency of G. oblanceolata against salt stress inducedROS formation as evidenced by the increased MDA level. Different SOD isoenzymes in G. oblanceolata exhibited different intensities under salt stress. As compared to control group, under 50 and 100 mM NaCl, intensities of all SOD isoenzymes (except Cu/Zn-SOD at 50 mM and Fe-SOD1 at 50 and 100 mM) were increased. Similar to our findings, Slesak and Miszalski (2003) and Seckin et al. (2010) also found significant increase in both Cu/ZnSOD and Mn-SOD activities in the Mesembryanthemum crystallinum and Hordeum marinum under salt stress. Although total SOD activity was decreased by 150–300 mM NaCl stress, new Mn-SOD isoenzymes were determined under same conditions. Therefore, these results suggested that the mitochondrial compartment might be important in scavenging O2 •− in the stressed plants of G. oblanceolata due to the higher intensity of Mn-SOD isoenzymes. And, this may be general adaptive defense of G. oblanceolata to environmental stresses. On the other hand, although Fe-SOD is known as minor enzyme in the chloroplast (Hernandez et al., 2000), in present study, we have found that Fe-SOD2 is the major isoenzyme in the band including Mn-SOD5 and Fe-SOD2 (Fig. 8B). However, as it can be seen in Fig. 8A, role of Fe-SOD1 in control plants was very low (6%) as compared to other isoenzymes (MnSODs, FeSOD2

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and Cu/Zn-SOD). Moreover, this band cannot be detected in salt stress groups. These results showed that contrary to Fe-SOD2, Fe-SOD1 does not take a role against salt-induced oxidative stress in vegetative stage of G. oblanceolata. Also, in germinated plants (10 d) under salt stress, Fe-SOD plays a major role. CAT enzyme eliminate H2 O2 in mitochondria and microbodies (Shigeoka et al., 2002), while APX, a primary enzyme of the ascorbate–glutathione cycle, suppresses the accumulation of H2 O2 in chloroplast, cytosol, peroxisomes and apoplast (Diaz-Vivancos et al., 2006; Liu et al., 2010). The activities of these enzymes were increased in salt stressed-plants such as Catharanthus roseus (Jaleel et al., 2007). Similarly, a strong implication of CAT and APX in the scavenging of stress-induced H2 O2 was reported by Amor et al. (2005). Bian and Jiang (2009) observed an increase in both CAT and APX activity of Kentucky bluegrass under salt stress. Similarly, in present study, although CAT and APX have different affinities for H2 O2 , in G. oblanceolata, increasing of CAT and APX activity under moderate salinity (50–100 mM NaCl) may facilitate leaf cells to scavenge H2 O2 in an efficient way. However, increased CAT and APX activity could not prevent the increase in the H2 O2 accumulation under high salinity level, as shown in Fig. 6A. Therefore, in 50 and 100 mM NaCl treated-plants, reduced MDA content seems to be essentially due to more efficient scavenging of H2 O2 by CAT and APX that correlated with improved salt tolerance. The CAT isoenzymes have been studied extensively in higher plants (Polidoros and Scandalios, 1999) such as two isoenzymes in Hordeum vulgare and C. tuzgoluensis (Azevedo et al., 1998; Yıldıztugay et al., 2011), three isoenzymes in maize (Scandalios, 1990). Regarding the isoenzymatic pattern of CAT, only one isoenzyme was detected in G. oblanceolata. Gill and Tuteja (2010) reported that the activity of POX varies considerably depending upon plant species and stress condition. Its activity increased in NaCl-exposed plants of Phaseolus mungo (Dash and Panda, 2001) and Oryza sativa (Satoh et al., 2009). In present study, the alteration of POX activity under salt stress was not basically in the same pattern with those of CAT and APX. POX activity was increased only in 300 mM NaCl-treated plants. However, this increase could not prevent increase in the H2 O2 accumulation. These results indicate that POX enzyme does not take a crucial part in defense mechanisms of G. oblanceolata against oxidative stress as also reported in Calendula officinalis (Chaparzadeh et al., 2004). In fact, for most plants, the POX consists of several isoforms, but the regulation of these isoforms has not been well clarified (Dalton et al., 1998; Jebara et al., 2005). In present study, three POX bands were identified in G. oblanceolata which were differentially affected by salt stress. 150 and 300 mM NaCl enhanced the intensities of POX1 and POX3 isoenzymes. From these results it seems that salt stress enhanced POX activity by increasing the activation of already synthesized POX enzyme isoforms as also reported in wheat by Seckin et al. (2009). Beside APX, activity of GR enzyme, the last enzyme of the ascorbate–glutathione cycle, catalyzes NADPH-dependent reduction of oxidized glutathione (Foyer et al., 1991). It was already shown that GR have a protective role in defense of different plant species against abiotic stress-induced oxidative damage including salinity (Shalata and Tal, 1998; Demiral and Turkan, 2005; Chalapathi Rao and Reddy, 2008). Parallel to these results, we also observed an increase in GR activity (except for 50 mM NaCl). Therefore, 50 and 100 mM NaCl-induced oxidative stress in G. oblanceolata appears to be prevented more efficiently by the activity of the ascorbate–glutathione cycle due to increased APX and GR activities under salt stress. However, at high salinity level (300 mM NaCl), while GR was increased, APX was decreased. Therefore, under same conditions, oxidative stress cannot be prevented due to decreased activity of the ascorbate–glutathione cycle. Kocsy et al. (2001) reported that stress increase GR activity

by the appearance of new GR isoenzymes. However, in present study, salt stress enhanced GR activity by increasing the activation of already synthesized GR enzyme isoforms (GR1 and GR2). 5. Conclusion (i) Optimal germination percentage of G. oblanceolata occurred at 25 ◦ C and 16 h light/8 h dark, (ii) salt stress decreased both the germination percentage and germination rate, (iii) the activities of antioxidant enzymes (SOD, CAT and POX) were decreased during germination under salt stress. However, their activities were increased during recovery germination after stress, (iv) G. oblanceolata is a moderately salt-tolerant species on vegetative stage, (v) G. oblanceolata display a general adaptive defense against 50 mM and 100 mM NaCl stress that involves increased SOD, CAT and APX. Nevertheless, MDA content was the highest in 150 mM and 300 mM NaCl-treated plants, indicating that the generation of endogenous H2 O2 exceeds the capacity of the cellular antioxidant defense system to eliminate H2 O2 , (vi) differential responses of antioxidant enzymes to salt stress during germination and vegetative growth suggested different antioxidant metabolism in G. oblanceolata, (vii) the higher tolerance to salt stress during vegetative growth might be associated with significant changes in composition of SOD and POX isozymes, (viii) beside of induced-antioxidant enzyme activities and changes in composition of isoenzymes, salt tolerance of G. oblanceolata would be partially based on to higher proline accumulation. Acknowledgements This work was supported by Ege University Research Foundation [2010-FEN-067]. The authors wish to thank Dr. Evren Yıldıztugay for providing G. oblanceolata seeds and his technical assistance on ICP-AES analyses. References Aghaleh, M., Niknam, V., Ebrahimzadeh, H., Razavi, K., 2009. Salt stress effects on growth, pigments, proteins and lipid peroxidation in Salicornia persica and S. europaea. Biol. Plantarum 53, 243–248. Alscher, R.G., Erturk, N., Heath, L.S., 2002. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J. Exp. Bot. 53, 1331–1341. Amor, N.B., Hamed, K.B., Debez, A., Grignon, C., Abdelly, C., 2005. Physiological and antioxidant responses of the perennial halophyte Crithmum maritimum to salinity. Plant Sci. 168, 889–899. Amor, N.B., Jimenez, A., Megdiche, W., Lundqvist, M., Sevilla, F., Abdelly, C., 2006. Response of antioxidant systems to NaCl stress in the halophyte Cakile maritima. Physiol. Plant. 126, 446–457. Asada, K., 1999. The water–water cycle in chloroplasts: scavenging of active oxygen’s and dissipation of excess photons. Annu. Rev. Plant Phys. 50, 601–639. Azevedo, R.A., Alas, R.M., Smith, R.J., Lea, P.J., 1998. Response of antioxidant enzymes to transfer from elevated carbon dioxide to air and ozone fumigation, in the leaves and roots of wild-type and a catalase-deficient mutant of barley. Physiol. Plant. 104, 280–292. Bailly, C., 2004. Active oxygen species and antioxidants in seed biology. Seed Sci. Res. 14, 93–107. Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for water-stress studies. Plant Soil 39, 205–207. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287. Bergmeyer, N., 1970. Methoden der enzymatischen Analyse, vol. 1. Akademie Verlag, Berlin, pp. 636–647. Bian, S., Jiang, Y., 2009. Reactive oxygen species, antioxidant enzyme activities and gene expression patterns in leaves and roots of Kentucky bluegrass in response to drought stress and recovery. Sci. Hortic. 120, 264–270. Bor, M., Ozdemir, F., Turkan, I., 2003. The effect of salt stress on lipid peroxidation and antioxidants in leaves of sugar beet Beta vulgaris L. and wild beet Beta maritima L. Plant Sci. 164, 77–84. Bradford, M.M., 1976. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of the protein–dye binding. Anal. Biochem. 72, 248–254. Bradley, P.M., Morris, J.T., 1991. relative importance of ion exclusion, secretion and accumulation in Spartina alterniflora Loisel. J. Exp. Bot. 42, 1525–1532.

A.H. Sekmen et al. / Environmental and Experimental Botany 77 (2012) 63–76 Cavalcanti, F.R., Lima, J.P.M.S., Ferreira-Silva, S.L., Viegas, R.A., Silveira, J.A.G., 2007. Roots and leaves display contrasting oxidative response during salt stress and recovery in cowpea. J. Plant Physiol. 164, 591–600. Chalapathi Rao, A.S.V., Reddy, A.R., 2008. Glutathione reductase: a putative redox regulatory system in plant cells. In: Khan, N.A., Singh, S., Umar, S. (Eds.), Sulfur Assimilation and Abiotic Stress in Plants. Springer, pp. 111–147. Chaparzadeh, N., D’amico, M.L., Khavari-Nejad, R.A., Izzo, R., Navari-Izzo, F., 2004. Antioxidative responses of Calendula officinalis under salinity conditions. Plant Physiol. Biochem. 42, 695–701. Claussen, W., 2005. Proline as a measure of stress in tomato plants. Plant Sci. 168, 241–248. Dalton, D.A., Joyner, S.l., Becana, M., Iturbe-Ormaetxe, I., Chatfield, J.M., 1998. Antioxidant defences in the peripheral cell layers of legume root nodules. Plant Physiol. 116, 37–43. Dash, M., Panda, S.K., 2001. Salt stress induced changes in growth and enzyme activities in germinating Phaseolus mungo seeds. Biol. Plantarum 44, 587–589. de Lacerda, C.F., Cambraia, J., Oliva, M.A., Ruiz, H.A., 2005. Changes in growth and in solute concentrations in sorghum leaves and roots during salt stress recovery. Environ. Exp. Bot. 54, 69–76. Demiral, T., Turkan, I., 2005. Comparative lipid peroxidation, antioxidant defense systems and proline content in roots of two rice cultivars differing in salt tolerance. Environ. Exp. Bot. 53, 247–257. Demiral, T., Turkan, I., Sekmen, A.H., 2011. Signaling strategies during drought and salinity, recent news. In: Turkan, I. (Ed.), Plant Responses to Drought and Salinity Stress: Developments in a Post-Genomic Era. Academic Press Elsevier, pp. 293–317. Di Baccio, D., Tognetti, R., Sebastiani, L., Vitagliano, C., 2003. Responses of Populus deltoides × Populus nigra (Populus × euramericana) clone I-214 to high zinc concentrations. New Phytol. 159, 443–452. Diaz-Vivancos, Rubio, M., Mesonero, V., Periago, P.M., Ros Barcelo, A., MartinezGomez, P., Hernandez, J.A., 2006. The apoplastic antioxidant system in Prunus: response to long-term plum pox virus infection. J. Exp. Bot. 57, 3813–3824. Duan, Z., Bai, L., Zhao, Z.G., Zhang, G.P., Cheng, F.M., Jiang, L.X., Chen, K.M., 2009. Drought stimulated activity of plasma membrane nicotineamide adenine dinucleotide phosphate oxidase and its catalytic properties in rice. J. Integr. Plant Biol. 51, 1104–1115. Ekim, T., Koyunce, M., Vural, H., Duman, H., Aytac, Z., Adiguzel, N., 2000. The red book of Turkish plants, Turkish Association for the Conservation of Nature TTKD and Van Yuzuncu Yil Unv. Ankara, 245. Flanagan, L.B., Jefferies, R.L., 1988. Stomatal limitation of photosynthesis and reduced growth of the halophyte, Plantago maritima L. at high salinity. Plant Cell Environ. 11, 239–245. Flowers, T.J., Colmer, T.D., 2008. Salinity tolerance in halophytes. New Phytol. 179, 945–963. Flowers, T.J., Troke, P.F., Yeo, A.R., 1977. The mechanism of salt tolerance in halophytes. Annu. Rev. Plant Physiol. 28, 89–121. Food and Agriculture Organization (FAO), 2008. Land and Plant Nutrition Management Service. Available from: http://www.fao.org/ag/ag/agll/spush. Force, L., Critchley, C., van Rensen, J.S., 2003. New fluorescence parameters for monitoring photosynthesis in plants. Photosynth. Res. 78, 17–33. Foyer, C., Lelandais, M., Galap, C., Kunert, K.J., 1991. Effects of elevated cytosolic glutathione reductase activity on the cellular glutathione pool and photosynthesis in leaves under normal and stress conditions. Plant Physiol. 97, 863–872. Foyer, C.H., Halliwell, B., 1976. The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 133, 21–25. Foyer, C.H., Noctor, G., 2005. Oxidant and antioxidant signalling in plants: a reevaluation of the concept of oxidative stress in a physiological context. Plant Cell Environ. 28, 1056–1071. Gill, S.S., Tuteja, N., 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 48, 909–930. Girousse, C., Bournoville, R., Bonnemain, J.L., 1996. Water deficit-induced changes in concentrations in proline and some other amino acids in the phloem sap of alfalfa. Plant Physiol. 111, 109–113. Gucci, R., Lombardini, L., Tttini, M., 1997. Analysis of leaf water relations in leaves of two olive (Olea europea) cultivars differing in tolerance to salinity. Tree Physiol. 17, 13–21. Hamzaoglu, E., Aksoy, A., 2009. A phytosociological study on the halophytic communities of sultansazligi (Inner Anatolia-Turkey). Phytosociological studies on the halophytic communities of central Anatolia. Ekoloji 18 (71), 1–14. Handa, S., Handa, A.K., Hasegawa, P.M., Bressan, R.A., 1986. Proline accumulation and the adaptation of cultured plant cells to water stress. Plant Physiol. 80, 938–945. Hernandez, J.A., Almansa, M.S., 2002. Short-term effects of salt stress on antioxidant systems and leaf water relations of pea leaves. Physiol. Plant. 115, 251–257. Hernandez, J.A., Jimenez, A., Mullineaux, P., Sevilla, F., 2000. Tolerance pf pea (Pisum sativum L.) to long term salt stress is associated with induction of antioxidant defences. Plant Cell Environ. 23, 853–862. Herzog, V., Fahimi, H., 1973. Determination of the activity of peroxidase. Anal. Biochem. 55, 554–562. Hoagland, D.R., Arnon, D.I., 1950. The water culture method for growing plants without soil. Calif. AES Bull. 347, 1–32. Hou, D., Fujii, M., Terahara, N., Yoshimoto, M., 2004. Molecular mechanisms behind the chemo preventive effects of anthocyanidins. J. Biomed. Biotechnol. 5, 321–325. Hsu, S.Y., Hsu, Y.T., Kao, C.H., 2003. The effect of polyethylene glycol on proline accumulation in rice leaves. Biol. Plantarum 46, 73–78.

75

Hunt, R., Causton, D.R., Shipley, B., Askew, A.P., 2002. A modern tool for classical plant growth analysis. Ann. Bot. 90, 485–488. Jaleel, C.A., Gopi, R., Manivannan, P., Panneerselvam, R., 2007. Responses of antioxidant defense system of Catharanthus roseus (L.) G. Don. to paclobutrazol treatment under salinity. Acta Physiol. Plant. 29, 205–209. Jebara, S., Jebara, M., Limam, F., Aouani, M.E., 2005. Changes in ascorbate peroxidase, catalase, guaiacol peroxidase and superoxide dismutase activities in common bean (Phaseolus vulgaris) nodules under salt stress. J. Plant Physiol. 162, 929– 936. Jiang, M., Zhang, J., 2002. Involvement of plasma membrane NADPH oxidase in abscicic acid- and water stress-induced antioxidant defense in leaves of maize seedlings. Planta 215, 1022–1030. Jung, S., 2004. Variation in antioxidant metabolism of young and mature leaves of Arabidopsis thaliana subjected to drought. Plant Sci. 166, 459–466. Keiffer, C.M., Ungar, I.A., 1997. The effect of extended exposure to hypersaline conditions on the germination of five inland halophyte species. Am. J. Bot. 84, 104–111. Khan, M.A., Ungar, I.A., 1997. Effects of thermoperiod on recovery of seed germination of halophytes in saline conditions. Am. J. Bot. 84, 279–283. Khan, M.A., Ungar, I.A., Showalter, A.M., 2000. The effect of salinity on the growth, water status, and ion content of a leaf succulent perennial halophyte, Suaeda fruticosa (L.) Forssk. J. Arid Environ. 45, 73–84. Khan, M.H., Panda, S.K., 2008. Alterations in root lipid peroxidation and antioxidative responses in two rice cultivars under NaCl-salinity stress. Acta Physiol. Plant. 30, 81–89. Kishor, P.B.K., Sangam, S., Amrutha, R.N., Sri Laxmi, P., Naidu, K.R., Rao, K.R.S.S., Rao, S., Reddy, K.J., Theriappan, P., Sreenivasulu, N., 2005. Regulation of proline biosynthesis, degradation, uptake and transport in higher plants: its implications in plant growth and abiotic stress tolerance. Curr. Sci. 88, 424–438. Kocsy, G., Galiba, G., Brunold, C., 2001. Role of glutathione in adaptation and signalling during chilling and cold acclimation in plants. Physiol. Plant. 113, 158–164. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lang, A.R.G., 1967. Osmotic coefficients and water potentials of sodium chloride solutions from 0 to 40 ◦ C. Aust. J. Chem. 20, 2017–2023. Lee, D.H., Kim, Y.S., Lee, C.B., 2001. The inductive responses of the antioxidant enzymes by salt stress in the rice (Oryza sativa L.). Plant Physiol. 158, 735– 747. Liu, Z.J., Guo, Y.K., Bai, J.G., 2010. Exogenous hydrogen peroxide changes antioxidant enzyme activity and protects ultra structure in leaves of two cucumber ecotypes under osmotic stress. J. Plant Growth Regul. 29, 171–183. Lutts, S., Kinet, J.M., Bouharmont, J., 1996. Effects of salt stress on growth, mineral nutrition and proline accumulation in relation to osmotic adjustment in rice (Oryza sativa L.) cultivars differing in salinity resistance. Plant Growth Regul. 19, 207–218. Madhava Rao, K.V., Sresty, T.V.S., 2000. Antioxidative parameters in the seedlings of pigeon pea (Cajanus cajan L. Millspaugh) in response to Zn and Ni stresses. Plant Sci. 157, 113–128. Meneguzzo, S., Navari-Izzo, F., Izzo, R., 1999. Antioxidative responses of shoots and roots of wheat to increasing NaCl concentrations. J. Plant Physiol. 155, 274–280. Meyer, A.M., Poljakoff-Mayber, A., 1963. The Germination of Seeds. MacMillan Company, New York, 236 pp. Mittler, R., Vanderauwera, S., Gollery, M., van Breusegem, F., 2004. Reactive oxygen gene network of plants. Trends Plant Sci. 9, 490–498. Mittova, V., Tal, M., Volokita, M., Guy, M., 2003. Up-regulation of the leaf mitochondrial and peroxisomal antioxidative systems in response to salt-induced oxidative stress in the wild salt-tolerant tomato species Lycopersicon pennellii. Plant Cell Environ. 26, 845–856. Moghaieb, R.E.A., Saneoka, H., Fujita, K., 2004. Effect of salinity on osmotic adjustment, glycinebetaine accumulation and the betaine aldehyde dehydrogenase gene expression in two halophyte plants, Salicornia europaea and Suaeda maritima. Plant Sci. 166, 1345–1349. Munns, R., 2002. Comparative physiology of salt and water stress. Plant Cell Environ. 25, 239–250. Munns, R., 2005. Genes and salt tolerance: bringing them together. New Phytol. 167, 645–663. Munns, R., Tester, M., 2008. Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59, 651–681. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880. Nyomora, A.M.S., Sah, R.N., Brown, P.H., Miller, R.O., 1997. Boron determination in biological materials by inductively coupled plasma atomic emission and mass spectrometry: effects of sample dissolution methods. J. Anal. Chem. 357, 1185–1191. Parida, A.K., Das, A.B., Mohanty, P., 2004. Defense potentials to NaCl in a mangrove Brugieara parviflora: differential changes of isoforms of some antioxidant enzymes. J. Plant Physiol. 161, 531–542. Perez-Lopez, U., Robredo, A., Lacuesta, M., Sgherri, C., Munoz-Ruedo, A., Navari-Izzo, F., Mena-Petite, A., 2009. The oxidative stress caused by salinity in two barley cultivars is mitigated by elevated by CO2 . Physiol. Plant. 135, 29–42. Polidoros, A.N., Scandalios, J.G., 1999. Role of hydrogen peroxide and different classes of antioxidants in the regulation of catalase and glutathione S-transferase gene expression in maize (Zea mays L.). Physiol. Plant. 106, 112–120. Redondo-Gomez, S., Mateos-Naranjo, E., Davy, A.J., Fernandez-Munoz, F., Castellanos, M.E., Luque, Figueroa, M.E., 2007. Growth and photosynthetic responses to salinity of the salt-marsh shrub Atriplex portulacoides. Ann. Bot. 100, 555–563.

76


Sairam, R.K., Rao, K.V., Srivastava, G.C., 2002. Differential response of wheat genotypes to long term salinity stress in relation to oxidative stress, antioxidant activity and osmolyte concentration. Plant Sci. 163, 1037–1046. Sairam, R.K., Tyagi, A., 2004. Physiology and molecular biology of salinity stress tolerance in plants. Curr. Sci. 86, 407–421. Santa-Cruz, A., Martinez-Rodriguez, M.M., Perez-Alfocea, F., Romero-Aranda, R., Bolarin, M.C., 2002. The rootstock effect on the tomato salinity response depends on the shoot genotype. Plant Sci. 162, 825–831. Satoh, K., Kondoh, H., Sasaya, T., Shimizu, T., Choi, I.R., Omura, T., Kikuchi, S., 2009. Selective modification of rice (Oryza sativa) gene expression by rice stripe virus infection. J. Gen. Virol. 91, 294–305. Scandalios, J.G., 1990. Response of plant antioxidant defense genes to environmental stress. Adv. Genet. 28, 1–41. Seckin, B., Sekmen, A.H., Turkan, I., 2009. An enhancing effect of exogenous mannitol on the antioxidant enzyme activities in roots of wheat under salt stress. J. Plant Growth Regul. 28, 12–20. Seckin, B., Turkan, I., Sekmen, A.H., Ozfidan, C., 2010. The role of antioxidant defense systems at differential salt tolerance of Hordeum marinum Huds. (sea barleygrass) and Hordeum vulgare L. (cultivated barley). Environ. Exp. Bot. 69, 76–85. Seevers, F.M., Daly, J.M., Catedral, F.F., 1971. The role of peroxidase isozymes in resistance to wheat stem rust. Plant Physiol. 48, 353–360. Sekmen, A.H., Ozdemir, F., Turkan, I., 2004. Effects of salinity, light, and temperature on seed germination in a Turkish endangered halophyte, Kalidiopsis wagenitzii (Chenopodiaceae). Isr. J. Plant Sci. 52, 21–30. Sekmen, A.H., Turkan, I., Takio, S., 2007. Differential responses of antioxidative enzymes and lipid peroxidation to salt stress in salt-tolerant Plantago maritima and salt-sensitive Plantago media. Physiol. Plant. 131, 399–411. Shalata, A., Tal, M., 1998. The effect of salt stress on lipid peroxidation and antioxidants in the leaf of the cultivated tomato and its wild salt-tolerant relative Lycopersicon pennellii. Physiol. Plant. 104, 169–174. Sharma, P., Dubey, R.S., 2005. Drought induces oxidative stress and enhances the activities of antioxidant enzymes in growing rice seedlings. Plant Growth Regul. 46, 209–221. Shigeoka, S., Ishikawa, T., Tamoi, M., Miyagawa, Y., Takeda, T., Yabuta, Y., Yoshimura, K., 2002. Regulation and function of ascorbate peroxidase isoenzymes. J. Exp. Bot. 53, 1305–1319. Slesak, I., Miszalski, Z., 2003. Superoxide dismutase-like protein from roots of the intermediate C3 -CAM plant Mesembryanthemum crystallinum L. in in vitro culture. Plant Sci. 164, 497–505.

Smart, R.E., Bingham, G.E., 1974. Rapid estimates of relative water content. Plant Physiol. 53, 258–260. Song, J., Feng, G., Tian, C.Y., Zhang, F.S., 2005. Strategies for adaptation of Suaeda physophora, Haloxylon ammodendron and Haloxylon persicum to saline environment during seed germination stage. Ann. Bot. 96, 399–405. Sreenivasulu, N., Grimm, B., Wobus, U., Weschke, W., 2000. Differential response of antioxidant compounds to salinity stress in salt-tolerant and saltsensitive seedlings of foxtail millet (Setaria italica). Physiol. Plant. 109, 435– 442. Tounekti, T., Vadel, A.M., Onate, M., Khemira, H., Munne-Bosch, S., 2011. Salt-induced oxidative stress in rosemary plants: Damage or protection? Environ. Exp. Bot. 71, 298–305. Turkan, I., Demiral, T., 2009. Recent developments in understanding salinity tolerance. Environ. Exp. Bot. 67, 2–9. Ungar, I.A., 1991. Ecophysiology of Vascular Halophytes. CRC Press, Boca Raton, FL. Ungar, I.A., 1995. Seed germination and seed-bank ecology of halophytes. In: Kigel, J., Galili, G. (Eds.), Seed Development and Germination. Marcel Dekker, New York, pp. 599–627. Ungar, I.A., 1996. Effect of salinity on seed germination, growth, and ion accumulation of Atriplex patula (Chenopodiaceae). Am. J. Bot. 83, 604–607. Uzilday, B., Turkan, I., Sekmen, A.H., Ozgur, R., Karakaya, H.C., 2011. Comparison of ROS formation and antioxidant enzymes in Cleome gynandra (C4 ) and Cleome spinosa (C3 ) under drought stress. Plant Sci., doi:10.1016/j.plantsci.2011.03.015. Verslues, P.E., Agarwal, M., Katiyar-Agarwal, S., Zhu, J., Zhu, J.K., 2006. Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status. Plant J. 45, 523–539. Vitória, A.P., Lea, P.J., Azevedo, R.A., 2001. Antioxidant enzymes responses to cadmium in radish tissues. Phytochemistry 57, 701–710. Voetberg, G.S., Sharp, R.E., 1991. Growth of the maize primary root at low water potentials. Plant Physiol. 96, 1125–1130. Woodbury, W., Spencer, A.K., Stahman, M.A., 1971. An improved procedure for using ferricyanide for detecting catalase isozymes. Anal. Biochem. 44, 301–305. Yıldıztugay, E., Sekmen, A.H., Turkan, I., Kucukoduk, M., 2011. Elucidation of physiological and biochemical mechanisms of an endemic halophyte Centaurea tuzgoluensis under salt stress. Plant Physiol. Biochem., doi:10.1016/j.plaphy.2011.01.021. Zheng, C., Jiang, D., Liu, F., Dai, T., Jing, Q., Cao, W., 2009. Effects of salt and water logging stresses and their combination on leaf photosynthesis, chloroplast ATP synthesis, and antioxidant capacity in wheat. Plant Sci. 176, 575–582.