Subcellular NH4+ flux analysis in leaf segments of wheat (Triticum aestivum)

We report the first use of tracer (13) NH4 (+) (N-13-ammonium) efflux and retention data to analyse subcellular fluxes and compartmentation of NH4 (+) in the leaves of a higher plant (wheat, Triticum aestivum). Leaf segments, 1-2 mm, were obtained from 8-d-old seedlings. The viability of the segments, and stability of NH4 (+) acquisition over time, were confirmed using oxygen-exchange and NH4 (+)-depletion measurements. Fluxes of NH4 (+) and compartment sizes were estimated using tracer efflux kinetics and retention data. Influx and efflux across the plasma membrane, half-lives of exchange and cytosolic pool sizes were broadly similar to those in root systems. As the external concentration of NH4 (+) ([NH4 (+)](o)) increased from 10 mum to 10 mm, both influx and efflux greatly increased, with a sixfold increase in the ratio of efflux to influx. Half-lives were similar among treatments, except at [NH4 (+)](o) = 10 mm, where they declined. Concentrations of NH4 (+) in the cytosol ([NH4 (+)](c)) increased from 2.6 to 400 mm. Although [NH4+](c) became large as [NH4+](o) increased, the ratio of [NH4+](c) to [NH4+](o) decreased more than sixfold. The apparently futile cycling of NH4+ at high [NH4+](o) suggested by the large fluxes of NH4+ in both directions across the membrane indicate that leaf cells respond to potentially toxic NH4+ concentrations in a manner similar to root cells.