2.1 Experimental Set-up
The experiment was conducted in pots under natural conditions in the Botanical garden of the Department of Botany, Banaras Hindu University, Varanasi. The dimensions of the pot were 27.5 cm X 21.5 cm X 12 cm. The soil was sandy loam in texture. The soil was mixed with the recommended dose of farm yard manure for Amaranthus. The seeds of Amaranthus var. Kalmi were purchased from IIVR, Varanasi. The seeds were sterilized in sodium hypochlorite for two minutes and then rinsed properly three times with ddH2O. Seeds were soaked in ddH2O for four hours before sowing. Twenty seeds of Amaranthus were sown in each pot. After the growth of the seedlings thinning was performed, ten seedlings per pot were grown. When the seedlings attained the four-leaf stage, foliar application of salicylic acid (purchased from Sigma-Aldrich, purity 99%) was performed three times after four days intervals. Spraying was done on both sides of the leaf until wet completely. The treatments applied were control (no SA), 5µM SA, 10µM SA, 20µM SA, 50µM SA, and 100µM SA. Each treatment had three replicates. Plant harvesting was done after forty days of seed germination.
2.2 Estimation of oxidative parameters
Hydrogen peroxide was measured according to Velikova et al. (2000). 40 mg of leaf sample was extracted in 3 mL of 0.1% TCA. The suspension was centrifuged at 15,000g for 15 minutes. 0.5 ml of the supernatant was added with 0.5 mL of potassium phosphate buffer (10 mM). Next, 1.0 mL of KI (1M) was added and the absorbance was measured at 390 nm by UV-VIS spectrophotometer (systronics 119).
Lipid peroxidation was measured in terms of malondialdehyde formation by the method of Heath and Packer (1968) using thiobarbituric acid (TBA). 0.1g leaf tissue was extracted in 2.0 mL of TCA (5%) and then centrifuged at 10,000 g for 15 minutes. Next, 0.5 mL of the extract was added with 2.0 mL of 20% TCA having 0.5% TBA. After incubation in boiling water bath for 20 minutes the mixture was cooled immediately in an ice-bath. Absorbance was recorded at 532 and 600 nm on UV-VIS spectrophotometer (systronics 119).
Superoxide radical was estimated according to Elstner and Heupel (1976). 200 mg leaf tissue was extracted in 2 mL of 65 mM phosphate buffer (pH 7.8). The homogenate was centrifuged at 10,000 rpm for 10 min. 1 mL of supernatant was taken and 0.9 mL phosphate buffer was added. 100 µL hydroxylamine hydrochloride (10 mM) was added to it and incubated in dark for 30 min at 25ºC. Then, 1 mL each of sulfanilamide and NEDD was added and left for incubation for 20 min. at 25ºC. The absorbance of the sample was recorded at 530 nm on UV-VIS spectrophotometer (systronics 119). The rate of SOR production was calculated from the standard curve.
2.3. Estimation of enzymatic and non-enzymatic antioxidants
Catalase activity was measured according to Aebi et al. (1984). 0.1g leaf tissue was extracted in phosphate buffer containing 1 mM EDTA. The homogenate was centrifuged at 6000g for 15 minutes. 0.2 mL of the enzyme extract was added to 0.5 mL H2O2 (40 mM), and 1.3 mL Phosphate buffer in quartz cuvette. The absorbance was recorded at 240 nM on UV-VIS spectrophotometer (systronics 119). One enzyme unit was described as the amount of catalase required to decompose 1µM of H2O2/min.
For APX measurement, 0.1g leaf tissue was extracted in 2.0 mL chilled phosphate buffer (50 mM, pH 7.0). The homogenate was centrifuged at 6000 rpm for 10 minutes. 0.2 mL of the enzyme extract was added to a reaction mixture constituting 0.8 mL phosphate buffer, 0.5 mL H2O2 (0.4 mM), and 0.5 mL ascorbate (2.0 mM). The absorbance was recorded for one minute at 290 nM on UV-VIS spectrophotometer (systronics 119). One enzyme unit was described as the amount of enzyme needed to oxidize 1nM of ascorbic acid/min.
POD activity was measured after crushing 40 mg leaf in 2.5mL phosphate buffer. The reaction mixture was prepared with 0.8mL phosphate buffer, 1mL H2O2 (1.2%), and 1 mL guiacol (3%). Finally, 0.2 mL enzyme extract was added and the increase in O.D was recorded for 3 minutes at 470nm on UV-VIS spectrophotometer (systronics 119). One unit of POD was defined as the amount of enzyme needed to change the absorbance at 470nm equal to 0.01/min.
SOD activity was measured according to Gianopolitis and Ries (1977). 100 mg fresh leaves were crushed in 2 mL phosphate buffer (100 mM, pH- 7.8) containing 1 mM EDTA. The reaction mixture comprised 0.5 mL sodium carbonate (0.05 M), 0.5 mL methionine (13 mM), 0.9 mL phosphate buffer, and 0.1 mL enzyme extract. Lastly, 0.5 mL NBT (63µM), and 0.5 mL riboflavin (13µM) were added. The mixture was exposed to light for 30 minutes. The intensity of the blue color formation was measured at 560 nm on UV-VIS spectrophotometer (systronics 119). One unit of the enzyme is defined as the amount of enzyme that inhibits the rate of formation of formazan by 50%.
The total antioxidant activity was estimated by Ferric Reducing Antioxidant Power (FRAP) assay according to Vijayalaxmi and Ruckmani (2016). The O.D. was taken at 700 nm on UV-VIS spectrophotometer (systronics 119). The activity was compared with standard ascorbic acid.
The estimation of proline was measured according to Bates et al. (1973). 500 mg leaf tissue was homogenized in 10 mL sulfosalicylic acid (3%). After centrifugation, 2 mL of supernatant was added to 2 mL glacial acetic acid, 2 mL ninhydrin solution. After incubating in water bath for 1 hr. The reaction was then stopped by placing it in low temperature (10 ºC) for 15 minutes. 4 mL toluene was added, and the O.D. of the upper red layer was measured at 520 nm on UV-VIS spectrophotometer (systronics 119). Toluene was used as a reference for the O.D. measurement.
The total phenol was estimated according to Bray and Thorpe following the Folin - Ciocalteau method, 1954. The leaf sample was extracted in acetone (70%). The formation of blue color due to the reduction of folin reagent was recorded at 650 nm on UV-VIS spectrophotometer (systronics 119) and the total phenolics was quantified using a standard curve prepared using gallic acid.
2.4 Oxalic acid, Minerals, and protein determination
The determination of oxalic acid was done by the modified method of Fitriani et al. (2016). Two grams of Amaranthus leaves were crushed and heated in 100 mL DDW. After 20 minutes of heating, the extract was filtered. 50 mL of the solution was taken and 1 mL H2SO4 was added. The solution was titrated with KMnO4.
The leaves were dried in an oven at 70 ºC for 24 hours. The dried leaves were crushed finely and heat digested in a mixture of nitric acid, sulfuric acid, and perchloric acid (5:1:1). Calcium, magnesium, and zinc were estimated through atomic absorption spectrophotometer.
To estimate the protein content 500mg leaf was used and the Lowry et al. method (1951) was adopted and the content was calculated from the standard curve of protein.
2.5 Estimation of Chlorophyll Content and Fluorescence Matrix
The chlorophyll fluorescence was performed by PAM-2500 (Heinz Walz GmbH, Germany). Prior to the study, the plants were dark-adapted for 30 minutes at room temperature. The photosynthetic response to rapid light increase (every 30 s) was recorded to light intensities between 0-2000 µmol photons m− 2s− 1. Different parameters such as the maximum photochemical quantum yield of photosystem II (Fv/Fm), effective photochemical quantum yield (Y(II)), the rate of electron transport (ETR), and non-photochemical fluorescence quenching (NPQ).
2.6 Estimation of free salicylic acid
The free form of salicylic acid was quantified by HPLC following the method of Huang et al. (2015) with some modifications. 2.0 g of leaf was crushed in liquid nitrogen and was extracted in methanol by soaking overnight. After adding ethyl acetate on the following day it was vortexed and then centrifuged at 4ºC for 10 min. at 10000 RPM. The collected supernatant was cleaned using PSA and GCB before analysis through HPLC.
2.7 Statistical analysis
The statistical analysis for mean, ANOVA, and standard error was performed using IBM SPSS software (ver. 21.0). The different superscript letters in the graph and table show significant differences according to the Tukeys’-b test at P < 0.05 significant level.