Plant growing conditions and treatments
Maize seeds (Zea mays L.) were obtained from Black Sea Agricultural Research Institute, Samsun, Turkey. The seeds were put in small pots (5.5 cm x 6.0 cm x 6.0 cm) filled with vermiculite and then the pots were placed into larger pots (17.0 cm x 24.5 cm x 8.0 cm) containing 1.5 L of 0.1 X Hoagland Nutrient solution (pH 5.8). When the seedlings were appeared after germination, 1X Hoagland Nutrient solution (pH 5.8) were used for growing of the seedlings. The seedlings were grown in a growth chamber (temperature 25°C±2; relative humidity 60% ±2; light intensity 400 µmol m-2 s-1) for 21 days. Hoagland Nutrient solution in the larger pots were refreshed every second day. After that, the pots were divided into nine groups: 1) a non-stressed group (NS), the seedlings were only grown in Hoagland Nutrient solution during the experiment, 2) Mock group (PEG alone), gradually increased osmotic stress treatment, at 3% polyethylene glycol6000 (PEG6000) for 1 days, at 6% PEG6000 for 3 day and at 10% PEG6000 for 3 days, (3) LA group, the treatment with 12 µM LA for 24 h and then the PEG treatment as mentioned above, 4) DMTU group, the treatment with 5mM DMTU for 12 h and Hoagland solution for 24 h and then the PEG treatment, 5) BSO group, the treatment with 1 mM BSO for 12 h and Hoagland solution for 24 h and then the PEG treatment, 6) AF group, the treatment with 1 mM AF for 12 h and Hoagland solution for 24 h and then the PEG treatment, 7) DMTU+LA group, the treatment with 5mM DMTU for 12 h and 12 µM LA for 24 h and the PEG treatment, 8) BSO+LA group, the treatment with 5mM BSO for 12 h and 12 µM LA for 24 h and then the PEG treatment, 9) AF+LA group, the treatment with 5mM AF for 12 h and 12 µM LA for 24 h and then the PEG treatment. LA, DMTU, BSO and AF were prepared using Hoagland Nutrient Solution. Samples taken from the second leaves of the nine treatment groups were used for the following analysis.
Determination of ROS content
H2O2
H2O2 content was determined according to modified method of Velikova et al. (2000). The extract obtained from leaf samples crushed with activated 0.1 g charcoal in 5% TCA was centrifuged 12 000 g for 15 min. To 0.5 ml aliquot of the supernatant, 0.5 ml of 10 mM potassium phosphate buffer (pH 7.0) and 0.75 ml of 1 M KI were added. The absorbance was measured at 390 nm.
Superoxide
Superoxide content was analyzed spectrophotometrically according to Frahry and Schopfer (2001). Superoxide radical was determined by monitoring the reduction of the tetrazolium salt XTT by NADPH or NADH at 470 nm for 25 min.
Ascorbate and glutathione contents
Ascorbate content was estimated as described by Liso et al. (1984). After homogenization of the leaf samples (0.1 g) with 5 ml of 5% (w/v) metaphosphoric acid, the homogenate was centrifuged at 10 000 g for 10 min. The supernatant (70 µl) was added to 3 ml of reaction medium consisting of 0.1 M citrate-0.2 M phosphate buffer (pH 6.2). After initial absorbance read at 265 nm, ASC content was determined by reading the reduction occurring 5 min after the addition of 2 U ascorbate oxidase to the reaction medium. After completion of ASC oxidation, ascorbate oxidase was inhibited by addition of 10 mM sodium azide. After that 2.5 mM dithiothreitol was added to the mixture. After the reduction with dithiothreitol for 3 min, the absorbance was read again at 265 nm.
Glutathione content was determined by extraction of leaf samples (0.5 g) in 5 ml of 5% metaphosphoric acid with 1 mM ethylene diamine tetra acetic acid (EDTA). After centrifugation GSH content was determined with a “Total Glutathione Assay Kit (Northwest Life Sci Spec)” according to the manufacturer’s instructions. GSH content was measured using a reaction mixture containing 250 mM K2HPO4/KH2PO4 (pH 7.5), 200 µM NADPH, 600 µM 5,5’-dithiobis- (2-nitrobenzoic acid), 25 µl extract and 0.3 U GR.
Antioxidant enzyme activities
SOD (EC 1.15.1.1) activity was measured (25°C) based on the method of Beauchamp and Fridovich (1973). APX (EC 1.11.1.11) activity was determined according to the method of Nakano and Asada (1987), which is based on ascorbate oxidation at 290 nm (25°C, ε=2.8 mM-1 cm-1). CAT (EC 1.11.1.6) activity was measured according to the method of Aebi (1983) (25°C, ε=39.4 mM-1cm-1) at 240 nm. GR (EC 1.6.4.2) activity was determined according to Foyer and Halliwell (1976) by following the decrease in absorbance at 340 nm (25°C, ε=6.22 mM-1 cm-1) associated with NADPH oxidation. MDHAR (EC 1.6.5.4) activity was determined by following NADH oxidation at 340 nm (25°C, ε=6.22 mM-1 cm-1) (Hossain et al. 1984). DHAR activity was measured as described by Hossain and Asada (1984) by following the increase in absorbance at 265 nm (25°C, ε=7 mM-1cm-1). The protein content was measured (25°C) according to the method of Bradford (1976). Bovine serum albumin was used as a standard. Enzyme activities were expressed as units per milligram of protein.
Gene expression analysis
Total RNA isolation was performed using a Plant Total RNA Purification Kit (FavorPrep) to determine relative expression levels of SOD, APX1 and CAT1 genes. For the cDNA synthesis, High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was used. The obtained cDNAs were used in Real Time PCR assays to determine transcript level. For analysis, SsoFast EvaGreen Supermix with Low ROX and CFX Connect Real Time PCR System (BioRad) were used. RT-PCR process steps were modified by Solis BioDyne instructions: 95 °C for 12 min, 45 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s, and a melt curve was held in 0.5 °C increments from 60 °C to 95 °C. Actin gene (ACT) was used as the reference gene for Zea mays L. Data were normalized according to reference gene expression level and expressed as relative gene expression. Designs of primers were presented in Table.
Lipoic acid content
Leaves (0.1 g) were extracted with 1800 μl of extraction buffer (70% ethanol + 0.5% activated carbon) and centrifuged 15 000 g for 10 min. The supernatant (125 μl) was taken and 1.5 ml of MBTH solution and 2 ml of FeCl3 solution were added. After waiting for 10 min, the mixture was made up to 10 ml with distilled water. The absorbance was recorded at 450 nm. LA standards were prepared by modified method by Deephti et al. (2012).
Statistical analysis
All analysis was conducted three times with three replicates. The obtained data was presented as means ± standard deviation. Statistical analyses were carried out with one-way ANOVA variance analysis tests (Duncan Multiple Comparison Test) (the significant level, P < 0.05) using Statistical Package for Social Sciences (SPSS). Also, for qRT-PCR analysis, the relative gene expression level was analyzed using Bio-Rad CFX Manager 3.1. The expression levels were assayed by SPSS software. SPSS software assay was performed to determine the expression level.