## 4 Materials and Methods
### Determination of Activities of Enzymes Involved in N Metabolism in Roots and Leaves  
NR activity (EC 1.7.99.4) in the roots and leaves was determined as previously described [53]. Fine powder (1 g) was homogenized in 4 mL of ice cold extraction buffer (25 mM phosphate buffer (K\({}_{2}\)HPO\({}_{4}\) and KH\({}_{2}\)PO\({}_{4}\), pH 7.5), 5 mM cysteine and 5 mM EDTA-Na\({}_{2}\)). After centrifugation (4000\(\times\)\(g\), 4 \({}^{\circ}\)C, 15 min), 0.4 mL of supernatant and 1.6 mL of the assay solution (1.2 mL of 0.1 M KNO\({}_{3}\)-phosphate buffer and 0.4 mL of 2.0 mg mL\({}^{-1}\) NADH) were mixed well and incubated at 25 \({}^{\circ}\)C for 30 min). The reaction was terminated by adding 1 mL of 1% (\(w/v\)) sulfanilamide in 3 N HCl and 0.02% N-naphthylthediamine in water. After incubation for 15 min, the solution was then centrifuged for 10 min at 4000\(\times\)\(g\) followed by spectrophotometric measurement of the supernatant at 540 nm and determination of the NO\({}_{2}\)\({}^{-}\) concentration by using a standard curve of known NO\({}_{2}\)\({}^{-}\) concentrations.  
The activity of NiR (EC 1.7.2.1) was measured according to the methods described by [PERSON] et al. [54]. Briefly, 0.5 g of the fine powder was extracted in the extraction buffer consisting of 25 mM phosphate buffer (K\({}_{2}\)HPO\({}_{4}\) and KH\({}_{2}\)PO\({}_{4}\), pH 7.5), 5 mM cysteine, and 5 mM EDTA-Na\({}_{2}\). The crude extract was mixed with 0.1 M potassium phosphate buffer (pH 6.8), 0.4 mM NaNO\({}_{2}\), 2.3 mM methyl viologen, and 4.3 mM sodium dithoinite in 100 mM NaHCO\({}_{3}\). The mixture was incubated for 30 min at 27 \({}^{\circ}\)C, and the reaction was stopped by boiling for 5 min. The concentration of NO\({}_{2}\)\({}^{-}\) remaining in the reaction mixture was determined at 540 nm; one unit of NiR activity was defined as 1 mmol of NO\({}_{2}\)\({}^{-}\) reduced mg\({}^{-1}\) protein h\({}^{-1}\).  
The activity of GS (EC 6.3.1.2) was analysed spectrophotometrically, as proposed by [PERSON] et al. [55]. Briefly, frozen fine powder (approximately 1 g) was homogenized in ice cold 50 mM Tris-HCl extraction buffer (pH 8.0) containing 2 mM MgCl\({}_{2}\), 2 mM DTT, and 0.4 M sucrose. The solution was centrifuged (15,000\(\times\)\(g\), 4 \({}^{\circ}\)C, 20 min) and the supernatant was mixed with the assay solution (0.35 mL of 40 mM ATP and 0.8 mL of 0.1 M Tris-HCl buffer (pH 7.4) with 20 mM Na-glutamate, 80 mM MgSO\({}_{4}\), 20 mM cysteine, 2 mM EGTA, and 80 mM NH\({}_{2}\)OH). After incubation for 30 min at 37 \({}^{\circ}\)C, the stop reagent (0.37 M FeCl\({}_{3}\), 0.2 M trichloroacetic acid, 0.6 M HCl) was added. After centrifugation (5000\(\times\)\(g\), 4 \({}^{\circ}\)C, 15 min), the supernatant was spectrophotometrically measured at 540 nm; one unit of GS activity was defined as 1\(\upmu\)mol of \(\gamma\)-glutamyl hydroxamate formed per min.  
GOGAT (EC 1.4.7.1) and GDH (EC 1.4.1.2) activities were determined as suggested by [PERSON] and [PERSON] [56]. Briefly, 0.5 g of the fine powder was extracted in 0.2 M sodium phosphate buffer (pH 7.5) containing 2 mM EDTA, 50 mM KCl, 0.1% (\(v/v\)) mercaptoethanol and 0.5% (\(v/v\)) Triton X 100. After centrifugation (6000\(\times\)\(g\), 4 \({}^{\circ}\)C, 15 min), the supernatant was used for measurement of GOGAT and GDH activities. For GOGAT, 0.3 mL of enzyme extract was mixed with 0.2 mL of 20 mM L-glutamine, 50 \(\upmu\)L of 0.1 M 2-oxoglutate, 100 \(\upmu\)L of 10 mM KCl, and 0.1 mL of 3 mM NADH. The absorbance was measured at 340 nm for 5 min. For GDH, 0.15 mL of 0.1 M 2-oxoglutate, 0.15 mL of 1 M NH\({}_{4}\)Cl, 0.1 mL of 3 mM NADH, and 0.6 mL of 0.2 M Tris-HCl buffer (pH 8.0) were mixed and 0.5 mL of the supernatant was added. The decrease in absorbance at 340 nm was measured.