Preliminary experiments were carried out to determine the threshold concentration of salinity (which caused the lowest detrimental effect) and the most effective concentration of phenolic compounds on L. sativum growth parameters. In the preliminary experiments, the effects of different concentrations of NaCl (0, 25, 50, 100, 150, 200, 250, 300 and 350 mM), β-carotene (0, 0.5, 2.5 and 5 mM) and gallic acid (0, 0.5, 1, 2.5, 5, 10, 12.5, 15 and 20 mM) were evaluated on seed germination and seedling growth for 5 days. According to the findings of the preliminary experiments, 25 mM NaCl salinity, 0.5 mM β-carotene, and 5 mM gallic acid were chosen for the main experiment (Data not shown, Figure Suppl number 1–13).
The main experiment was carried out in a completely randomized design with three replications. Ten surface-sterilized seeds were placed in the 90 mm petri dish containing 30 ml of distilled water and incubated in a growth chamber (25 °C, 50% RH, 16/8 h day/night photoperiod, 120 µmol m−2 s−1 light intensity) for 5 days. Five-day-old seedlings were transferred to culture trays containing perlite and fertigated with 1/2 Hoagland solution (Peralta-Videa et al. 2002). The seedlings were transferred to a greenhouse (16/8 h day/night photoperiod, 27 °C). β-carotene (0.5 mM) and gallic acid (5 mM) solutions were sprayed on the seedlings for three consecutive days (three times a day). After antioxidant treatment, 8-day-old seedlings received 1/2 Hoagland solution containing 25 mM NaCl.
Treatments included: (1) Control (2) plants sprayed with β-carotene (3) plants sprayed with gallic acid (4) plants received 25 mM NaCl (5) plants sprayed with β-carotene and received 25 mM NaCl (6) plants sprayed with gallic acid and received 25 mM NaCl.
Fifteen-day-old seedlings were harvested, and their fresh (FW) and dry weights (DW) were measured and then stored at − 80 °C for further analysis.
Determination of total chlorophyll content
The total chlorophyll content was determined spectrophotometrically using 0.1 g FW of leaf tissue ground with mortar and pestle in 10 ml of acetone 80% (v/v). After centrifugation and reading the absorbance values at 663 and 645 nm, the values in the following equations were used (Arnon 1949). The contents were expressed as mg total chlorophyll g−1 FW.
$$Total \, chlorophyll \left( {mg/gFW} \right) = \left[ {\left( {8.02 \times A663} \right) + \left( {20.2 \times A645} \right)} \right] \times \frac{{volume \, of \, acetone\; \left( {ml} \right)}}{{weight \, of \, sample \;\left( {mg} \right) \times 1000}}$$
(1)
Determination of total phenolic content and DPPH radical scavenging activity
Using the Singleton and Rossi (1965) methods, the total soluble phenolic compounds were estimated. The frozen leaf tissue (0.1 g) was ground with 3 ml of 80% methanol in a cooled mortar. The obtained extract was centrifuged at 15,000 rpm for 15 min. The supernatant was used to measure the amount of phenolic compounds. The absorbance of the reaction mixture consisting of 30 μl of extract, 120 μl of sodium carbonate (Na2CO3), and 150 μl of Folin–Ciocalteu reagent was read at 765 nm after exposure to darkness for 30 min.
Utilizing the Kulisic et al. (2004) method, radical scavenging activity was estimated by a spectrophotometric method based on the reduction of a methanol solution of 2,2‐diphenyl‐1‐picryl‐hydrazyl‐hydrate (DPPH). An aliquot of 1 ml of the plant extract was added to 1 ml DPPH solution in a concentration of 100 µM in methanol. The control sample was prepared without any extract. The absorbance was measured at 517 nm, after 30 min incubation in darkness at ambient temperature. Radical scavenging activity was calculated using the following equation.
$$Inhibition \left( \% \right) = \frac{A\,control - A \, sample}{{A \, control}} \times 100$$
(2)
Measurement of oxidative stress markers
Determination of H2O2 and malondialdehyde (MDA) content
H2O2 was measured spectrophotometrically (λ = 390 nm) by a reaction with 1 M KI according to Alexieva et al. (2001). 0.1 g of frozen leaf tissue was ground in a cooled mortar with 1.5 ml of TCA (trichloroacetic acid) 0.1%. The obtained extract was centrifuged at 15,000g for 4 min at 4 °C. Five hundred μl of supernatant was added to 500 μl of 10 mM phosphate buffer and 1 ml of 1 M KI solution. The absorption rate was read by a spectrophotometer at 390 nm. The malondialdehyde (MDA) content was detected by commercially available kits (KIA ZIST, Hamadan, Iran). The MDA content was expressed as nmol MDA/gFW.
Determination of glutathione (GSH) level, catalase (CAT), superoxide dismutase (SOD) guaiacol peroxidase (GPX), glutathione reductase (GR) and ascorbate peroxidase (APX) enzymes activities
Glutathione (GSH) level and glutathione reductase (GR) were detected by commercially available kits (KIA ZIST, Hamadan, Iran). The GSH levels and GR activity were measured according to the manufacturer’s instructions. The absorption of both was measured using a spectrophotometer at 405 nm.
The CAT, SOD and GPX, APX enzyme activities were determined by the following method. 0.1 g of each sample was homogenized in the extraction phosphate buffered saline (PBS) pH 7.8. The catalase activity was measured utilizing the method described by Aebi (1984). The activity was estimated via monitoring the decrease in absorbance due to H2O2 decomposition [extinction coefficient (ε = 39 µmol−1 cm−1)] at 240 nm. The reaction mixture contained 50 µl plant extract, 50 mM phosphate buffer (pH 7.0), and 10 mM H2O2. The SOD activity was measured spectrophotometrically, as described by Beyer and Fridovich (1987), and assayed by monitoring the inhibition of photochemical reduction of nitro blue tetrazolium (NBT). For the amount of enzyme which causes 50% inhibition of the NBT photo-reduction rate in 1 min at 560 nm, a unit of SOD activity was defined. The specific activity of SOD was expressed as unit/mg FW. For GPX activity estimation (Lin and Kao 1999), the reaction was initiated by adding the plant extract (50 µl) to a reaction mix comprising 50 mM phosphate buffer, 19 mM H2O2, and 9 mM guaiacol. The absorbance was recorded at 470 nm for the amount of enzyme which forms 1 μM tetraguaiacol per minute at 470 nm. According to the Nakano and Asada (1987) method for the measurement of APX activity, 50 µl plant extract, 50 mM phosphate buffer containing 50 mM phosphate buffer comprising 0.5 mM ascorbic acid and 0.25 M H2O2 were utilized. Using a decrease in adsorption at 290 nm, hydrogen peroxide-dependent oxidation of ascorbate was followed.
Determination of Na+ and K+ concentration
The ash sample of shoots and roots were acid digested and filtered to determine Na+ and K+ concentration by a flame photometer (Corning, UK). Na+ and K+ content were recorded in mg/g DW.
Statistical analysis
Data was analyzed using SPSS ver. 17.0 software and means were compared using Duncan test at 5% probability level.