4.5. Detection of NH4+ and NO3- Concentrations

The NH₄⁺ and NO₃^- concentrations in roots were measured as described previously [27]. For NH₄⁺ determination, a 0.1-g sample was powdered in liquid nitrogen and extracted with 1 mL of 0.1 M HCl and 500 μL chloroform at 4 °C for 15 min. The solution was centrifuged at 12,000 rpm at 4 °C for 10 min, and the supernatant was transferred to a tube with 50 mg activated charcoal, mixed thoroughly, and centrifuged at 12,000 rpm at 4 °C for 5 min. About 100 μL supernatant was firstly well mixed with 500 μL solution containing 1% (v/v) phenol and 0.005% (w/v) sodium nitroprusside and then mixed with 500 μL solution containing 1% (w/v) sodium hypochlorite and 0.5% (w/v) NaOH. Thereafter, the solution was incubated in a 37 °C water bath for 30 min and the NH₄⁺ concentration was analyzed spectrophotometrically at 620 nm. For NO₃^- analysis, a 0.1-g fresh sample was incubated in 1 mL deionized water at 45 °C for 1 h, centrifuged at 6000 rpm at 20 °C for 15 min. The supernatant (0.2 mL) was initially thoroughly mixed with 0.8 mL of 5% (w/v) salicylic acid in concentrated H₂SO₄, and then added 19 mL of 2 M NaOH to raise the solution pH above 12. Thereafter, the solution was incubated at room temperature for 20 min, and allowed to cool to room temperature. The NO₃^- concentration was detected spectrophotometrically at 410 nm.