Specord bio 200

The soluble protein content of tissue extracts was measured by Bradford’s method^{40 (link)} to express enzyme activities in terms of the unit amount of protein. Bradford reagent (Genetix) was added to the plant extract prepared earlier, followed by vortexing. After 2 min of incubation, absorbance was measured at 595 nm using UV–a visible spectrophotometer (Specord Bio-200, Analytik Jena, Germany).

The tissue nitrate content of the leaf and root samples was measured following the hydrazine sulphate reduction method^{41 (link)}. Oven-dried samples were finely ground for estimation. An equivalent amount of sample and nitrate-free charcoal were taken in a conical flask containing 15 ml of double-distilled water and mixed thoroughly. It was then boiled for 3–4 min and filtered through Whatman filter paper-42 followed by re-extracted and refiltration and volume made up to 50 ml. After 20–30 min of incubation, absorbance was recorded at 540 nm using UV–a visible spectrophotometer (Specord Bio-200, Analytik Jena, Germany).

NR activity of leaf and root samples were determined by the in-vitro assay^{36 (link)}. For enzyme extraction, freshly harvested samples were homogenized in refrigerated 0.1 M phosphate buffer (pH 7.5) containing 5 mM EDTA and 5 mM cysteine. Extracts were centrifuged (sigma 3K30) at 10,000 rpm for 15 min at 4 °C^{37 (link)}. The supernatant was collected and kept on ice. A reaction mixture containing 0.1 M phosphate buffer (pH 7.5), 0.1 M KNO3 and 10 mM NADH was prepared for assaying enzyme activity. Enzyme extract was added in test samples only to start the reaction, whereas in the blank sample, the extract was substituted with deionized water. To stop the reaction, 1 M zinc acetate was added with 75% ethanol, followed by centrifugation at 2000 rpm for 5 min at room temperature. The supernatant was collected, and 1% (w/v) sulfanilamide solution and 0.02% (w/v) N-(1-naphthyl) ethylene diamine solution were added to it^{38 (link)}. After 20 min of incubation, absorbance was measured at 540 nm using UV–a visible spectrophotometer (Specord Bio-200, Analytik Jena, Germany). The enzyme activity was expressed as μmol nitrite formed mg⁻¹ protein h⁻¹.