WT, OSA1-ox and osa1 mutant seedlings were grown in IRRI nutrient solution containing 2 mM NH4+ for 4 weeks in a growth chamber (NC-410HC, Nippon Medical & Chemical Instruments Co., Ltd.) under ~150 μmol m−2 s−1 fluorescent light at 30 °C/24 °C (12 h/12 h) and 60–80% relative humidity. To determine 15NH4+ absorption rates within 30 min, seedlings were rinsed in 0.1 mM CaSO4 for 1 min, transferred to modified IRRI nutrient solution containing 2 mM (15NH4)2SO4 (atom% 15N: 98%) incubated with mock 5 μM FC (Fig. 1a ) or 350 μM vanadate (Supplementary Fig. 5 ) for 30 min53 (link) and rinsed again with 0.1 mM CaSO4 for 1 min48 (link). To determine 15NH4+ absorption rates within 5 min in roots of WT, OSA1-ox and osa1 mutant plants under different NH4+ concentrations, seedlings were incubated with 0.5, 1, 2, 4 and 8 mM 15NH4+ for 5 min (Figs. 2f and 3f ).
Roots and shoots were separated for weighing, and then immediately frozen in liquid N2. After grinding, an aliquot of the powder was dried to a constant weight at 70 °C, and 10 mg of each sample was analysed using the MAT253-Flash EA1112-MS system (Thermo Fisher Scientific, Inc., USA). Each experiment was performed with three independent biological replicates, and statistical analyses were performed using two-tailed Student’s t tests.
Roots and shoots were separated for weighing, and then immediately frozen in liquid N2. After grinding, an aliquot of the powder was dried to a constant weight at 70 °C, and 10 mg of each sample was analysed using the MAT253-Flash EA1112-MS system (Thermo Fisher Scientific, Inc., USA). Each experiment was performed with three independent biological replicates, and statistical analyses were performed using two-tailed Student’s t tests.