4.3.3. Measurement of Glutamine Synthetase (GS) Enzyme Activity

GS activity was assayed according to a published protocol [55], with some modifications. Briefly, ~500 mg frozen leaf samples were homogenized in a 6 mL extraction buffer (pH 8.0) containing 50 mM Tris–HCl, 2 mM MgCl₂, 2 mM DTT, and 0.4 M sucrose at 4 °C. The homogenate was centrifuged at 15,000 g for 20 min at 4 °C and the supernatant was used as enzyme extract. To initiate the reaction, 0.7 mL of the enzyme extract was introduced into the reaction mixture. This mixture was composed of 0.7 mL of 40 mM ATP, combined with 1.6 mL of 0.1 M Tris-HCl buffer (pH 7.4). The buffer contained 20 mM Na-glutamate, 80 mM MgSO4, 20 mM cysteine, 2 mM EGTA, and 80 mM NH₂OH. The reaction was carried out at 37 °C for 30 min, before terminating by adding 0.5 mL of stop solution (370 mM FeCl₃, 200 mM trichloroacetic acid, and 700 mM HCl). Following centrifugation at 5000 g for 15 min at 4 °C, the absorbance of the supernatant was measured at 540 nm. The unit of GS enzyme activity (U) expressed as μmol γ-glutamyl hydroxamate formed per gram fresh weight per minute (U = μmol·g⁻¹·min⁻¹ FW⁻¹) based on a standard curve of γ-glutamyl hydroxamate. Each independent experiment consisted of three biological replicates and three independent experiments were performed to obtain the results.