## 4 Materials and Methods
### Determination of Net Fluxes of NH4\({}^{+}\), NO3\({}^{-}\), and H\({}^{+}\)  
The net fluxes of NH4\({}^{+}\), NO3\({}^{-}\), and H\({}^{+}\) in the roots were measured by using a non-invasive micro-test technique (NMT, system BIO-IM, Amherst, MA, USA) as described in our previous study [23]. Briefly, silanized glass micropipettes were first filled with a backfilling solution (100 mM NH4 Cl for the NH4\({}^{+}\) electrode; 10 mM KNO3 for the NO3\({}^{-}\) electrode; and 15 mM NaCl and 40 mM KH2 PO4 for H\({}^{+}\) electrode). Then, 50 \(\upmu\)m columns of selective liquid ion-exchange cocktails (NH4\({}^{+}\) LIX, #09879, Sigma, Amherst, MA, USA; NO3\({}^{-}\) LIX, #72549, Sigma; H\({}^{+}\), #95293, Sigma) were front-filled. An Ag/AgCl wire electrode holder (XY-DJGD, Younger USA, LLC, Amherst, MA, USA) was inserted into the back of the electrode. The microelectrodes were then calibrated and used for measurement. A fine root was equilibrated for 20 min in measuring solution (0.1 mM KCl, 0.1 mM CaCl2, pH 5.5) with 0.01, 1, or 10 mM NH4 NO3 according to the N treatment of the selected root. Six white fine roots were selected from the root system for each treatment. Ion gradients near the root surface (approximately 5 \(\upmu\)m above the root surface) were measured by moving an ion-selective microelectrode between two positions that were 30 \(\upmu\)m apart and perpendicular to the root axis. The recordings were performed every 6 s for an average of 10 min at each measurement point. Determination of Contents of NH4\({}^{+}\), NO3\({}^{-}\), NO2\({}^{-}\), \(\delta^{15}\)N, and Total N  
NH4\({}^{+}\) content in the roots and leaves was spectrophotometrically analysed as previously described [14]. Approximately 100 mg of the fine powder was homogenized in extraction solution containing 1 mL of 100 mM HCl, and 500 \(\upmu\)L of chloroform. The mixture was rotated for 15 min at 4 \({}^{\circ}\)C and then centrifuged (10,000\(\times\)\(g\), 4 \({}^{\circ}\)C, 10 min). The supernatant was transferred to a 2 mL tube, and 50 mg of activated charcoal was added and mixed thoroughly. After centrifugation (12,000\(\times\)\(g\), 4 \({}^{\circ}\)C, 5 min), the NH4\({}^{+}\) content in the supernatant was measured at 620 nm. NO3\({}^{-}\) content was quantified based on the method of [PERSON] et al. [51]. First, 100 mg of the frozen powder was extracted in 1 mL of deionized water (45 \({}^{\circ}\)C, 1 h). The solution was then centrifuged (5000\(\times\)\(g\), 20 \({}^{\circ}\)C, 15 min) and 0.2 mL of the supernatant was transferred to a 2 mL tube containing 0.8 mL of 5% (\(w/v\)) salicylic acid in concentrated H2 SO4 and incubated for 20 min at 25 \({}^{\circ}\)C. Then, 2 M NaOH was added to raise the pH to above 12. The NO3\({}^{-}\) content in the solution was measured spectrophotometrically at 410 nm. NO2\({}^{-}\) content was assayed as described by [PERSON] et al. [52]. Approximately 100 mg of the fine powder was homogenized in extraction buffer containing 50 mM TRIS-HCl (pH 7.9), 5 mM cysteine, and 2 mM EDTA and centrifuged (10,000\(\times\)\(g\), 20 \({}^{\circ}\)C, 20 min). Then, 500 \(\upmu\)L of the supernatant, 250 \(\upmu\)L of 1% sulfanilamide and 250 \(\upmu\)L of 0.02% N-(1-naphthyl) ethylenediamine dihydrochloride in 3.0 M HCl were combined and mixed well. The NO2\({}^{-}\) content was spectrophotometrically determined at 540 nm. \(\delta^{15}\)N and total N concentration in roots and leaves were analyzed as proposed by [PERSON] et al. [21]. The samples were dried in an oven at 80 \({}^{\circ}\)C. The isotopic ratio and total N concentration were analysed by using an elemental analyser (NA 1110, CE Instruments, Rodano, Italy) coupled to a GVI IsoPrime isotope ratio mass spectrometer (IRMS). Stable N isotope composition was calculated as:  
\[\delta^{15}\text{N}=(\text{R}_{\text{san}}-\text{R}_{\text{sdn}})/\text{R}_{ \text{sdn}}\times 1000\]  
where R\({}_{\text{san}}\) and R\({}_{\text{sdn}}\) are the ratios of \({}^{15}\)N to \({}^{14}\)N in the sample and the standard, respectively.