N metabolism in plants is closely associated with the activities of several key enzymes, such as NR and GS [62 (link)]. For NR activity determination, the roots and leaves harvested were frozen in liquid N immediately, and then stored at − 80 °C until further analysis. Samples were ground to a fine powder (~ 100 mg), extracted, and analyzed spectrophotometrically [57 (link), 63 , 64 (link)]. The activity of GS was assayed as reported by Wang et al. [65 (link)]. The activity of NADH-GOGAT was quantified using a NADH-GOGAT measurement kit (Solarbio Bioengineering Institute). Glutamate and glutamine were quantified using a glutamic acid measurement kit and a glutamine measurement kit, respectively (both from Nanjing Jiancheng Bioengineering Institute). Enzyme activities were expressed as moles of metabolite generated/consumed per milligram of fresh weight or protein per unit of time. The protein concentration was determined by the Coomassie brilliant blue method with Modified BCA Protein Assay Kit, C503051, Sangon Biotech.
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