## 2 Materials and Methods
### Data Recorded
#### 2.5.4 Enzymatic Antioxidants and H\({}_{2}\)O\({}_{2}\) Content  
Leaf samples were harvested at 60 DAS, kept in a disposable zipper bag and stored at \(-80\)\({}^{\circ}\)C for the determination of antioxidant enzyme activity. The activity of SOD was measured by following the procedure of [29]. For this, 200 mg leaf sample was homogenized in 2 mL of extraction buffer (0.5 mM EDTA + 0.1 M phosphate pH 7.5) with precooled mortar and pestle. The homogenate was centrifuged at 10,000 rmp at 4 \({}^{\circ}\)C and the supernatant was stored at 4 \({}^{\circ}\)C. SOD activity in the supernatant was assayed by its ability to inhibit the photochemical reduction of nitrobluetetrazolium. A 3 mL assay mixture, containing 0.2 mL of 200 mM methionine + 1.5 M sodium carbonate + 0.1 mL 3 mM EDTA + 0.1 mL 2.25 mM NBT + 0.1 mL riboflavin (60 uM) + 1.5 mL 100 mM potassium phosphate buffer + 1 mL distilled water and 0.1 mL of enzyme, was incubated under two 15 W inflorescent lamps for 15 min; illuminated and nonilluminated reactions without supernatant served as calibration. The absorbance of the samples along with the blank were recorded at 560 nm wavelength in a spectrophotometer (UV-4000, ORI, Hille, Germany). One unit of enzyme activity was defined as the quantity of enzyme that reduced the absorbance reading of samples to 50% in comparison with tubes lacking enzymes (supernatant) and was calculated as follows. \[Enzymes\ units\ (EU)=\frac{\text{Enzyme}*(\text{light})-[\text{Enzyme}\#( \text{light})-\text{ighte}\#*(\text{dark})]}{\text{Enzyme}*\frac{(\text{light} )}{2}}\]  
where \(*\) sample without enzyme, # sample with enzyme. Fresh leaf samples (0.5 g) were ground in a 5 mL of 50 mM phosphate buffer (pH 7.8) with the help of a pestle and mortar. The homogenates were centrifuged at 15,000 rpm for 20 min at 4 \({}^{\circ}\)C. The supernatant was used to assess the peroxidase and catalase activity. POD activity was determined by following procedure of [30] with slight modifications. The reaction mixture contained 10 mM guaiacol + 5 mM H\({}_{2}\)O\({}_{2}\) and 50 mM phosphate buffer (pH 7.0). The reaction mixture was preheated at 20 \({}^{\circ}\)C in a water bath. Then, 2.8 mL reaction solution + 0.2 Ml enzyme was added in 10 mL centrifuged tube and mix thoroughly. Absorbance was recorded with blank and with reaction mixture. Four absorbance readings were recorded at 470 nm wavelength with 1 min time interval using spectrophotometer (UV-4000, ORI, Hille, Germany). \[POD\ Activity=\frac{\triangle 470\times\text{Vt}}{0.1\times V\times T\times W} \tag{1}\]  
where \(V\), Vt, \(T\) and \(W\) are the volume of the sample solution (mL), the measured liquid volume (mL), the measurement time and the fresh weight of the sample, respectively. Catalase activity in fresh leaves was measured following the method of [31] with slight modification. A total of 100 Mm H\({}_{2}\)O\({}_{2}\) + 50 mM phosphate buffer (pH 7.8) solution was preheated in water bath at 25 \({}^{\circ}\)C. In a 10 mL tube, 0.2 mL phosphate buffer + 0.2 mL enzyme solution was added and preheated in water bath for 3 min. Then, 0.3 mL (100 mM H\({}_{2}\)O\({}_{2}\)) solution was added in 10 mL tube. The control tube was heated in a boiling water bath for 5 min to kill the enzyme solution. After mixing, the absorbance at the 240 nmwavelength was calculated at interval of 1 min. With continuous determination for 4 min, 1 unit of enzyme activity (U) was considered as a decrease of 0.1 of A240 within 1 min. \[CAT\ activity=\frac{\triangle\Delta 240\times\ Vt}{0.1\times V\times T\times W} \tag{2}\]  
where \(V\), Vt, \(T\) and \(W\) are the volume of the sample solution (mL), the measured liquid volume (mL), the measurement time and the fresh weight of the sample, respectively. H\({}_{2}\)O\({}_{2}\) contents were determined with the help of the procedure described by [32]. A total of 500 mg of fresh leaves were homogenized with 5 mL of 0.1% (\(w/v\)) trichloroacetic acid in a pre-chilled pestle and mortar. The homogenate was centrifuged at 12,000 rpm for 15 min.