Model ub 1800

Total chlorophyll was estimated in the fresh leaves according to the method of Arnon [40 (link)]. Leaves were chopped in small pieces (0.1 g) and 10 ml of dimethyl sulfoxide (DMSO) was added to each test tube. The incubation was completed at 65°C in oven for 120 minutes to release the whole chlorophyll in DMSO. The tubes were taken out from the oven and absorbance of the solution was recorded at 663 and 645 nm on a UV-vis spectrophotometer (Model UB-1800, Shimadzu, Japan). The content of chlorophyll was calculated as mg/g fresh weight.

The CAT activity was determined by estimating H₂O₂ degradation according to the method of Aebi [42 (link)]. The reaction was performed in 3.0 ml of reaction mixture containing 10 mMH₂O₂, 100 mM potassium phosphate buffer solution (pH 7.0), and 100 µl of enzyme extract. The decrease in absorbance of H₂O₂ was recorded at 240 nm using the UV-vis spectrophotometer (Model UB-1800, Shimadzu, Japan). The enzyme activity was calculated using the extinction coefficient (0.036 mM⁻¹ cm⁻¹). One unit of CAT determines the amount necessary to decompose 1 µmol of H₂O₂ per min at 25°C.

The TBARS content was estimated in fresh leaves using the method described by Cakmak and Horst [43 (link)]. 0.5 g of fresh leaves was ground in 5.0 ml of 0.1% (w/v) trichloroacetic acid (TCA) at 4°C. The reaction mixture was taken in falcon tube and centrifuged at 12,000 ×g for 5 min. The 4.0 ml of 0.5% (w/v) TBA in 20% (w/v) TCA was added in 1.0 ml of supernatant taken from the above step. The mixture was kept for 30 min at 90°C in water bath. After incubation of mixture, the reaction was terminated by keeping it on ice bath. The reaction mixture was centrifuged at 10,000 ×g for 5 min, and absorbance of the supernatant was taken at 532 and 600 nm on a spectrophotometer (Model UB-1800, Shimadzu, Japan).
The amount of TBARS was calculated using an extinction coefficient of 155 mM cm⁻¹ as follows: $\begin{array}{c}\text{TBARS}\left(\text{nmol\hspace{0.17em}}{\text{g}}^{-\mathrm{1}}\text{\hspace{0.17em}fw}\right)=\frac{\left({\mathit{\text{A}}}_{\mathrm{532}}-{\mathit{\text{A}}}_{\mathrm{600}}\right)\times \mathit{\text{V}}\times \mathrm{1000}}{\mathrm{155}\left(\text{extinction\hspace{0.17em}\hspace{0.17em}coff.}\right)\times W},\end{array}$ where

 
A₅₃₂ is absorbance at 532 nm

 
A₆₀₀ is absorbance at 600 nm

 
V is extraction volume

 
W is fresh weight of tissues and methods.

Total chlorophyll was estimated according to the Arnon method [59 (link)]. The leaves were separated and washed with DDW; 0.1 g of chopped leaves was placed in the test tubes for each treatment, and 10 ml of DMSO was added to each test tube. The tubes were kept in an oven at 65°C. After 120 minutes, the tubes were taken out and the absorbance of the solution was recorded immediately at 663 nm and 645 nm on a UV-vis spectrophotometer (Model UB-1800, Shimadzu, Japan). The pigment concentration was calculated in μg/ml for treated and untreated samples.

The activity of superoxide dismutase (EC 1.15.1.1) was measured according to the method developed by Dhindsa et al. [60 (link)]. A fresh sample (0.05 g) was homogenized in 2 ml of extraction mixture containing phosphate buffer (0.5 M, pH 7.3), 0.3 mM-EDTA, 1% Triton × 100 (w/v), and 1% PVP (w/v). The mixture was centrifuged for 10 min at 4°C at 10,000 ×g. The supernatant was taken after centrifugation for the assay of SOD activity. The assay mixture, consisting of 1.5 ml reaction buffer, 0.2 ml of methionine, 0.1 ml of each (1 M-NaCO₃, 2.25 mM-NBT solution, 3 mM-EDTA, riboflavin, and enzyme extract), and 1 ml of DDW, was incubated under the light. The blank mixture containing all substances was kept in the dark. Absorbance of samples along with the blank mixture was read at 560 nm using the UV-vis spectrophotometer (Model UB-1800, Shimadzu, Japan). A 50% reduction in color was considered as one enzyme unit (EU). The activity of SOD was calculated in EU (mg⁻¹ protein min⁻¹).

The proline was estimated using the method developed by Hanson et al. [58 (link)]. Fresh leaves (0.3 g) were ground in 10 ml of aqueous sulphosalicylic acid (3%). The mixture was centrifuged for 15 min at 9000 ×g. The supernatant (2 ml) from the above step was taken and mixed with an equal volume of acid ninhydrin (1.25 g ninhydrin in 30 ml acetic acid and 20 ml of 6 N H₃PO₄) and acetic acid. The mixture was placed for 1 h in boiling water for incubation. After incubation, the mixture was taken out from the boiling water and immediately placed in an ice bath. 4 ml of toluene was added in the mixture (4 ml) after taking it from the ice water bath. The mixture was vortexed, and chromatophore-containing toluene was separated from the aqueous phase. The absorbance was taken at 520 nm (Model UB-1800, Shimadzu, Japan) to determine proline content.

The superoxide dismutase (SOD) activity was measured using the method reported by Dhindsa et al. [24 (link)]. The fresh leaf samples (0.25 g) were ground in 2 mL of phosphate buffer containing 1% Triton ×100 (w/v), 0.3 mM EDTA, and 1% PVP (w/v). The sample was centrifuged at 10,000 × g for 10 min, after which the supernatant was collected for assaying SOD activity. The enzyme assay was performed in 1.5 mL of reaction buffer containing 0.2 mL of methionine and 0.1 mL of each of 1 M NaCO₃, 2.25 mM NBT solution, riboflavin, 3 mM EDTA, enzyme extract, and 1 mL of DDW incubated in the light. The blank was kept in the dark while containing all components as in the treated samples. The sample absorbance along with the blank was recorded at 560 nm using a UV-Vis spectrophotometer (Model UB-1800; Shimadzu, Japan). A 50% reduction in color was considered one enzyme unit (EU), and activity of SOD was calculated in mg⁻¹ protein min⁻¹.