4.4. Analysis of the Biochemical Parameters of the Plants

Accurately weighted 0.5 g plant sample was taken and homogenized in tissue homogenizer (Ultra-Turrax Tube Drive, IKA, Staufen, Germany) with 10 mL of 80% acetone. The homogenized sample mixture was centrifuged at 10,000 rpm for 15 min at 4 °C. The supernatant was separated. Then, 0.5 mL of it was mixed with 4.5 mL of the solvent. Optical absorbance of the above mixture was determined at 470 nm, 646.8 nm, and 663.2 nm. Pigments content was calculated according to [59]. Finally, contents of chlorophyll a, chlorophyll b, and carotenoids in plant were converted to mg per gram dry weight. The total chlorophyll content was calculated by adding contents of chlorophyll a and chlorophyll b.

Homogenization of the plant material (0.5 g) was performed using a cooled mortar and pestle in a mixture of 20% polyvinylpyrrolidone and 0.1% trichloroacetic acid. The homogenate was centrifuged at 10,000× g for 10 min under 4 °C. The malondialdehyde concentration in the supernatant was determined by reaction with thiobarbituric acid as described in [60]. Briefly, the supernatant was added to 5% thiobarbituric acid solution in 20% trichloroacetic acid solution and incubated in a water bath at 95 °C for 30 min. Then, the reaction was stopped by placing the tubes on ice for 10 min. The mixture was centrifuged at 10,000× g for 10 min. Optical absorbance of the supernatant was determined at 532 and 600 nm. Malondialdehyde content in the plant material was calculated using an extinction coefficient of 155 mM⁻¹ cm⁻¹ and expressed in nmol per gram dry weight.

Hydrogen peroxide content in rye shoots was determined according to [61]. The plant material was homogenized in trichloroacetic acid in an ice bath. The homogenate was centrifuged at 16,000× g for 15 min. The supernatant was added to the reaction mixture containing 10 mM of phosphate buffer (pH 7.0) and 1 M of potassium iodine (KI). The mixture was incubated in the dark for 1 h. Optical absorbance was measured at 390 nm. Hydrogen peroxide content in the plant material was determined using a calibration graph and expressed in nmol H₂O₂ per gram dry weight.

Proline content in rye shoots was determined spectrophotometrically using the acid-ninhydrin method as described in [62] with some modifications. The plant material (0.5 g) was homogenized in 10 mL of 3% sulfosalicylic acid. The homogenate was filtered, and the filtrate was used for further analysis. The filtrate was mixed with 2 mL of ninhydrin reagent (1.25 g of ninhydrin, 30 mL of glacial acetic acid, 20 mL of 6 M H₃PO₄ solution) and 2 mL of glacial acetic acid. The reaction mixture was incubated for 1 h in a water bath at 100 °C, after which it was rapidly cooled on ice. The mixture was extracted with toluene, and optical absorbance was determined at 520 nm. Standard solutions of L-proline were used to make the calibration graph. Proline content in the plant material was expressed in µmol per gram dry weight.

Non-protein thiols content was determined using 5,5′-dithiobis- (2-nitrobenzoic acid) as described in [63]. The plant material (1 g) was homogenized in 10 mL of 5% sulfosalicylic acid. The homogenate was centrifuged at 20,000× g for 20 min. The reaction mixture contained supernatant, 0.1 M sodium phosphate buffer (pH 7.0), 0.5 mM EDTA, and 0.25 mM 5,5′-dithiobis- (2-nitrobenzoic acid). The mixture was incubated for 10 min at room temperature. Optical absorbance was measured at 412 nm. Non-protein thiols content was determined using a calibration graph. Glutathione was used as a standard. Non-protein thiols content in plants was expressed in μmoles per g dry weight.

Contents of ascorbic acid, dehydroascorbic acid, and 2,3-diketogulonic acid were measured spectrophotometrically by using the reaction of DHA and DKGA with 2,4-dinitrophenylhydrazine as described in [44]. To find the total content of all acids, AsA was oxidized with 2,6-dichlorophenolindophenol reagent (2% solution in 4.5 M sulphuric acid, containing 0.25% of thiourea). In order to determine DHA and DKGA separately, DHA in the plant extract was reduced to AsA with 2·10⁻³ M unithiol solution prepared in phosphate buffer (pH 7.0). This step made it possible to determine DKGA content. DHA content was calculated as difference between results obtained without the “reduction” step and with it. Finally, ascorbic acid content was calculated as difference between the total contents of the three acids, and the sum of contents of DHA and DKGA. Ascorbic acid solutions of fixed concentration were used to make a calibration curve. Contents of AsA, DHA and DKGA were expressed in μg of per gram dry weight.

Total phenolics content was determined using Folin–Ciocalteu method as described in [44]. Briefly, the plant material (0.1 g) was homogenized in 10 mL of 70% ethanol. The homogenate was centrifuged at 4500× g for 30 min. The reaction mixture contained 100 µL of supernatant, 300 µL of Folin–Ciocalteu reagent, and 6 mL of 6.75% solution of sodium carbonate. The mixture was incubated for 30 min in the dark at room temperature. Optical absorbance was measured at 720 nm. Content of total phenolic compounds was determined using a calibration curve with gallic acid as standard and expressed in mg gallic acid equivalents per g dry weight.

Samples of frozen shoots (approximately 0.4 g of fresh weight) were ground in liquid nitrogen and homogenized in 2.0 mL of ice-cold 100 mM phosphate buffer containing 0.1 mM EDTA and 1.0% polyvinylpyrrolidone. The homogenate was centrifuged at 12,000× g for 20 min at 4 °C. The supernatant was used to analyze the activities of antioxidative enzymes and protein content.

Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined by its ability to inhibit the photochemical reduction of nitro blue tetrazolium (NBT) as described in [64] with some modifications. The reaction mixture contained 63 µM NBT, 13 mM L-methionine, 0.1 mM EDTA, 0.05 M sodium carbonate and 0.5 mL enzyme extract (or distilled water in control). The reaction was started by adding 20 μL of 0.025% riboflavin. The tubes were then quickly placed under fluorescent lamps (18 W). The reaction time was 15 min. After the time elapsed, the reaction was stopped by placing the tubes in the dark. Optical absorbance of solutions was determined at 560 nm. The amount of enzyme necessary to inhibit the photoreduction of 50% nitro blue tetrazolium at 25 °C was taken as a unit of SOD activity and was expressed per mg of protein.

Catalase (CAT, EC 1.11.1.6) activity was determined by the decrease in optical absorbance at 240 nm caused by H₂O₂ decomposition [65]. The reaction mixture contained 2.95 mL of 50 mM K, Na-phosphate buffer (pH 7.0), and 30 μL of the plant extract. The reaction was started by introducing 20 μL of 0.6 M hydrogen peroxide into the reaction mixture. The control cuvette contained the same reagents, but no hydrogen peroxide was added. Catalase activity was determined by the change in optical absorbance at 240 nm every second for 100 s. Extinction coefficient (ε = 39.4 mM^–1 cm^–1) was used for calculations.

Ascorbate peroxidase (APX, EC 1.11.1.11) activity was determined by monitoring the oxidation rate of H₂O₂-dependent ascorbate as described in [66]. The reaction mixture included 80 mM phosphate buffer (pH 7.0), 0.6 mM H₂O₂, 0.1 mM Na₂EDTA, 0.5 mM ascorbic acid and 50 μL of the plant extract. H₂O₂ was added to start the reaction at 25 °C. The decrease in optical absorbance of the mixture was measured at 290 nm every second for 120 s. Enzyme activity was calculated based on extinction coefficient equal to 2.8 mM⁻¹ cm⁻¹.

Glutathione peroxidase (GPX, EC 1.11.1.9) activity was determined according to [67] using hydrogen peroxide as a substrate. To perform the enzymatic reaction, 200 μL of the plant extract was placed into the reaction tube containing 400 μL of 0.1 mM solution of reduced glutathione and 200 μL of 0.067 M solution of KNaHPO₄. The mixture containing all the reagents except the plant extract was used as a control. After incubating the mixture in a water bath at 25 °C for 5 min, 0.2 mL of 1.3 mM hydrogen peroxide solution was added to the mixture. The reaction time was 10 min. The reaction was stopped by adding 1 mL of 1% trichloroacetic acid solution and placing the reaction mixture in an ice bath. Then, the mixture was centrifuged and used to determine glutathione content in it. For determining glutathione content, 0.48 mL of the supernatant was mixed with 2.2 mL of 0.32 M Na₂HPO₄ and 0.32 mL of 1.0 mM 5,5′-dithiobis-(2-nitrobenzoic) acid. The mixture was incubated for 10 min at room temperature. Optical absorbance was measured at 412 nm. Enzyme activity was calculated based on the decrease in content of reduced glutathione.

Peroxidase (POD, EC 1.11.1.7) activity was determined spectrophotometrically using guaiacol as a phenolic substrate and hydrogen peroxide [68]. The reaction mixture contained 0.15 mL of 4% guaiacol, 0.15 mL of 1% (v/v) H₂O₂, 2.66 mL of 0.1 M phosphate buffer (pH 7.0), and 40 μL of the plant extract. The control sample contained all the reagents but the plant extract. Optical absorbance of the mixture was measured at 470 nm. Enzyme activity was calculated based on extinction coefficient of tetraguaiacol equal to 26.6 mM^–1 cm^–1.

Activities of all studied antioxidative enzymes were converted to mg of protein. Total soluble protein was estimated according to the Bradford method [69] with bovine serum albumin (BSA) as a standard. Shimadzu UV-3600 spectrophotometer (Shimadzu, Kyoto, Japan) was used for spectrophotometric analyses.