Characteristics of ammonium uptake by rice cells in suspension culture

As a model system that does not involve long distance transport, cell cultures of rice (Oryza sativa L. cv Sasanishiki) were used to investigate the characteristics of NH₄⁺uptake. The cells, which had been subcultured for 8 d in normal R2 medium containing 5 mm NH₄⁺and 40 mm NO₃, were pre-cultured for another 2.5 d without nitrogen and then used as the materials. In some experiments, the cells were further treated with 10.μm of methionine sulfoximine (MSX) for 3 h in the medium lacking nitrogen to inhibit the endogenous glutamine synthetase activity. Uptake of NH₄⁺was determined by measuring the consumption of NH₄⁺from 25 mL of an uptake medium containing 25 mm 2-morpholinoethanesulfonic acid-NaOH (pH 5.6), 100 μm CaCl₂, 100 μm NH₄Cl, and 0.5 g fresh weight cells. When the uptake was followed as a function of time, the cells treated with MSX rapidly absorbed NH₄⁺during the first 20 min and then the absorption leveled off. The cells without MSX treatment absorbed the ion at the same rate for 40 min and then the absorption leveled off. The uptake rate during the first 20 min was calculated to be 90 nmol NH₄⁺per min per g fresh weight. Intracellular concentrations of NH₄⁺were at least several times higher than those of the medium regardless of MSX treatment throughout the period of incubation. A biphasic saturation curve for the uptake of NH₄⁺, with an apparent K_mof 240 μm and 1.7 mm, was obtained when the cells were incubated in the medium containing 0 to 500 μm of the ion. The rice cells showed a pH optimum of about 7.0 for the uptake of NH₄⁺. The uptake was strongly inhibited by a proton conductor and an inhibitor of H⁺-ATPase. The results of these kinetic studies suggest that NH₄⁺is transported via a carrier system across the cell membrane, but not via a simple diffusion mechanism, in rice cell cultures.