2.10. Assay of reactive oxygen species
Hydrogen peroxide (H2O2) was determined according to Sergiev et al. (1997) with some modifications. H2O2 was extracted from 0.5 g flag-leaf tissue with 0.1% (w/v) TCA. After centrifuged at 12,000 g for 15 min, the supernatant was incubated with 0.1 mM potassium phosphate buffer (pH 7.0) and 1 M potassium iodide solution and absorbance was measured at 390 nm. H2O2 content was obtained from a standard curve.
Superoxide anion (•O2−) was estimated as described by Chaitanya and Naithani (1994) with minor modifications. Fag-leaf samples were homogenized in ice cold 0.2 M phosphate buffer (pH 7.2) containing 10− 3 M diethyldithiocarbamate to inhibit SOD activity. The homogenate was centrifuged at 10,000 g for 10 min. The •O2− was measured in the supernatant by its capacity to reduce NBT (2.5 x 10− 4 M).Change in absorbance due to formation of •O2− was measured at 540 nm for 1 min.
2.11. Measurement of lipid peroxidation
Lipid peroxidation was measured in terms of malondealdehyde (MDA) content. The MDA content was determined by thiobarbituric acid (TBA) test according to Heath and Packer (1968). After extracting MDA from flag-leaf tissue with 0.5% TBA in 20% trichloroacetic acid (TCA) absorbance was measured at 532 nm. Values of non-specific absorption recorded at 600nm were subtracted from the values recorded at 532 nm. MDA content was calculated according to its extinction coefficient ε = 155mM− 1 cm− 1.
2.12. Estimation of total ascorbate and glutathione content
Total ascorbate (AsA + DHA) was quantified according to Law et al. (1983). Ascorbate was extracted from flag-leaf tissue with 6 % TCA and estimated with 2% dinitrophenyl hydrazine (DNPH) and 10% Thiourea. Absorbance recorded at 530 nm and concentration of ascorbate was calculated from the standard curve prepared with pure ascorbate.
Total glutathione (GSH + GSSG) was estimated according to Griffith (1980). Glutathione was extracted from flag-leaf tissue with 5% sulphosalicylic acid and estimated with 5,5′-dithiobis-(2-nitrobenzoic acid) and 3 units of glutathione reductase. Change in absorbance was measured at 412 nm. Total glutathione content was calculated from a standard curve with GSH.
2.13. Determination of antioxidant enzyme activities
Lyophilized plant tissue was homogenized with ice-cold phosphate buffer (pH 7.0) (Basu et al. 2017). Total protein was quantified in the plant tissue according to Bradford (1976). The absorbance of Coomassie Brilliant Blue after binding with the protein was recorded at 595 nm and was calculated from the BSA standard curve. The extracted protein was used to detect different antioxidant enzyme activities.
Superoxide dismutase (SOD) (EC22.214.171.124) activity was determined at 560 nm by its ability to reduce the formation of blue coloured formazone by NBT and •O2− radical (Dhindhsa et al. 1981).
Catalase (CAT) (EC 126.96.36.199) activity estimation was performed according to Aebi (1983). Change in absorbance with addition of H2O2 was recorded at 240 nm for 1 min. Enzyme activity was expressed as unit min− 1 mg− 1 protein and a change in absorbance by 0.01 corresponded to 1 unit of enzyme activity.
Peroxidase (POX) (EC 188.8.131.52) activity was determined at 436 nm by its ability to convert guaiacol to tetraguaiacol (Polle et al. 1994). The increase in absorbance was recorded by the addition of H2O2 at 436 nm for 1 min.
Ascorbate peroxidase (APX) (EC 184.108.40.206) activity was estimated following the method of Nakano and Asada (1981). Change in absorbance with was recorded at 290 nm for 1 min after addition of ascorbate and H2O2.
2.14. Native-PAGE and activity staining for antioxidant enzymes
Plant extracts containing equal amounts of protein were subjected to discontinuous polyacrylamide gel electrophoresis (PAGE) under non-denaturing and non-reducing conditions (Laemmli 1970).
SOD activity was detected following the method of Weisiger and Fridovich (1973) with some minor modifications. The gels were incubated in the dark in a reaction mixture containing 50 mM potassium phosphate buffer (pH 7.8), 2.45 mM NBT, 28 mM TEMED and 3 µM riboflavin. The gels were illuminated until colourless SOD bands appeared against a purple background.
CAT activity was determined by incubating the gels in 3.5 mM H2O2 followed by washing in de-ionized water. The gels were stained with 1% potassium ferricyanide and 1% ferric chloride (Scandalios 1968).
POX activity was detected using the method of Graham et al. (1964). Gels were incubated in darkness, till the POX activity-containing band visualized carefully.
Enzyme activities were estimated by measuring the relative intensities of bands with the Adobe Photoshop version 7.
2.15. Western blotting
Plant extracts containing equal amounts of proteins were electrophoresed on the SDS-Polyacrylamide gel (Laemmli 1970). A pre-stained marker was run along with to verify the transfer. The proteins were electro-blotted to the nitrocellulose membrane using a semi-dry trans-blot cell (Bio-Rad, India). The blotted membrane was blocked with 5% skimmed milk and was subsequently incubated with the anti-catalase primary antibody (Agrisera, Sweden) of 1:1500 dilutions. This was followed by 1 h incubation with 1:1000 diluted goat anti-rabbit IgG-Horse radish peroxidase conjugated secondary antibody (Agrisera, Sweden). Bands were observed by using DAB substrate. Similarly, primary antibody of anti-Cu/Zn superoxide dismutase (Agrisera, Sweden) of 1:1500 dilution was used. Rabbit anti-chicken IgY-conjugated with Horse radish peroxidase (Agrisera, Sweden) of 1:1000 dilution was used as secondary antibody for visualizing anti-Cu/Zn SOD.
2.16. Statistical analyses
All treatments were given three replications (n = 3) with proper randomization to eliminate bias, whatsoever. Each value is presented in the form of mean ± standard error (± SE) and the lowest standard deviations of mean with a reading considering at least three samples per experiment for each genotype and each condition. The data were statistically analysed for analysis of variance (ANOVA) as a 2×2 factorial completely randomized block design using SAS 9.3 software by SAS Institute Inc., USA. Influence of main and interaction effects of the treatments were carefully evaluated. The standard error of mean and differences between the treatments was compared pair wise by critical difference (CD) at 5% level of significance.