GS activity in E. indica tissue extracts and in yeast recombinant EiGS1-1 proteins was measured by the γ-transferase assay (GS-dependent formation of γ-glutamyl hydroxamate) (Pateman 1969 ) using a commercial detection kit (Solabio, Beijing, China). Assays were conducted in the presence of glufosinate at final concentrations of 0, 0.001, 0.01, 0.025, 0.05, 0.1, 0.5, and 1 mM. The reaction product was measured spectrophotometrically for absorbance at 540 nm. Protein concentration was determined for sample calibration. Assays using E. indica tissue extracts contained three biological replicates and those using yeast recombinant EiGS1-1 proteins contained three technical replicates.
Kinetic characterization (Km and Vmax) of recombinant EiGS1-1 proteins was performed for the two substrates, glutamate and ATP, by the biosynthetic assay (Jalaludin et al., 2017) . The reaction mixture consisted of 100 mM Tris-HCl (pH 7.5), 10 mM ATP, 20 mM MgCl2, 30 mM hydroxylamine and 20 mM glutamate.