Ascorbate and glutathione contents
Ascorbate concentration was determined according to Liso et al. (1984). S. hieraciifolia shoots (0.25 gr) were homogenized with 5 ml, 5 % (w/v) m-phosphoric acid and the extract was centrifuged at 10,000g for 4 min. Sample (70 µl) was added to 3 ml of reaction medium containing 0.1 M citrate-0.2 M phosphate buffer (pH 6.2). The initial absorbance was recorded at 265 nm and then the ascorbate concentration was determined by reading the reduction of 5 min after the addition of two units of ascorbate oxidase to the reaction medium. After ascorbate oxidation was completed, ascorbate oxidase was inhibited with 10 mM sodium azide. Dithiothreitol (DTT) (2.5 mM) was added to the medium and following reduction (3 min) with DTT, the absorbance was recorded again at 265 nm.
Glutathione content was determined according to the total glutathione assay kit (Northwest Life Science Specialties, LLC.) according to the instructions of the producer firm. Glutathione was measured using a reaction mixture containing 250 mM K2HPO4/KH2PO4 (pH 7.5), 200 μM NADPH, 600 μM DTNB, 25 μl extract and 0.3 U GR. The change in absorbance was observed at 412 nm for 3 min. GSH concentration was calculated on the standard graph obtained by using GSH at 0-5 μM concentrations.
 
Antioxidant enzyme assays and protein determination
For superoxide dismutase, catalase and guaiacol peroxidase extractions, the shoot tissues (0.1 g) were homogenized with a 50 mM sodium phosphate buffer (pH 7.8) with 1 mM ethylenediaminetetraacetic acid (EDTA) and polyvinylpolypyrrolidone (1%). For determination of APX activity, 2 mM of ascorbate was added into the sodium phosphate buffer. The samples were centrifuged at 15,000g for 15 min.
Superoxide dismutase activity was determined according to the method of Beauchamp and Fridovich (1971). Reaction was initiated by the addition of 2 μM riboflavin to 1 ml reaction medium containing 50 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM L-methionine, 75 μM nitro blue tetrazolium (NBT), and 50 μl extract. The absorbance values at 560 nm were determined after the mixture was exposed to white light at 375 μmol m−2 s−1 for 10 min. One unit (U) of SOD activity was defined as the amount of enzyme needed to bring about 50% inhibition of the NBT photoreduction rate.  
Catalase activity was determined by measuring the decrease in reaction time of 1 ml at 240 nm for 5 min, containing 50 mM potassium phosphate buffer (pH 7.0), 30 mM H2O2 and 20 μl enzyme extract. Catalase activity was calculated using the 39.4 mM−1cm−1 epsilon coefficient for H2O2 (Bergmeyer and Graßl 1983).
Ascorbate peroxidase activity was determined with decrease at 290 nm (Nakano and Asada 1987). APX activity was determined by measuring a 1 ml reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 250 μM ascorbate, 5 mM H2O2 and 20 μl enzyme extract. APX activity was calculated using the 2.8 mM−1cm−1 epsilon coefficient for ASC.
Guaiacol peroxidase activity was measured by increase in absorbance at 470 nm (25°C, e = 26.6 mM-1cm-1) in a 100 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA, 5 mM guaiacol, 15 mM H2O2 and 50 μl of enzyme extract (Urbanek et al. 1991).
Glutathione reductase activity was determined spectrophotometrically according to Foyer and Halliwell (1976). GR was assayed by the fall in absorbance at 340 nm as NADPH was oxidized. The assay contained 50 mM Tris-HCl (pH 7.8), 150 μM NADPH, 500 μM oxidized glutathione (GSSG) and 50 μl extract. The activity of GR was calculated using an extinction coefficient of 6.22 mM−1cm−1 for NADPH at 340 nm.
Antioxidant enzyme activities were presented on a protein basis. Protein content was determined according to Bradford (1976), using BSA as a standard.
 
Western blot analysis of antioxidant enzymes
Protein extractions of SOD, CAT, glutathione peroxidase (GPX; E.C. 1.11.1.9) and GR were performed using 4X PEB (protein extraction buffer, AS08 300 Agrisera Inc.). Samples were dissolved in an equal volume of sample buffer (2X Laemmli sample buffer 1610737 Bio-Rad) and heated at 95 °C for 5 min. Separation of total proteins (30 μg) by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, (TGX Stain-Free Precast Protein Gels, 4568095 Bio-Rad) was performed by electrophoresis (Mini-PROTEAN Tetra Cell system, 165800 Bio- Rad) at room temperature. The marker (Precision Plus Protein Western C Blotting Standards, 1610376 Bio-Rad) was used to determine the molecular weight of the proteins. Proteins separated in gel electrophoresis were transferred to the PVDF membrane (Trans-Blot Turbo Mini PVDF Transfer Packs, 1704156 Bio-Rad) using the Trans-Blot Turbo Transfer System (1704155 Bio-Rad). After transfer, the membrane was blocked at 4 °C with 2.5% milk powder in TBS. Fe-SOD Chloroplastic Fe-Dependent Superoxide Dismutase (A S06 125) for SOD, Glutathione Peroxidase Chloroplastic (A S04 055) for GPX, and, Catalase (A S09 501) for CAT were primary used antibodies and they incubated overnight at 4 °C. After incubation, the membranes were incubated with goat anti-rabbit IgG-HRP secondary antibodies (AS09 602 Agrisera) for 4 h at room temperature. The density of the scanned protein bands was calculated with Image Lab Software (1709690, Bio-Rad).
Statistical analysis
All experiments were carried out five times with five biological replicates. All results were presented as means ± standard deviation. All physiological data were processed with one-way analysis of variance (α = 0.05) using the SPSS Ver. 15.0 software for Microsoft Windows (SPSS Inc., Chicago, USA). Mean differences were determined with the Duncan multiple comparison test at α = 0.05.