Characterization and regulation of ammonium transport systems in Citrus plants

We have investigated both the kinetics and regulation of 15NH4+ influx in roots of 3-month-old hydroponically grown Citrus (Citrus sinensis L. Osbeck x Poncirus trifoliata Blanco) seedlings. The 15NH4+ influx is saturable below an external ammonium concentration of 1 mM, indicating the action of a high-affinity transport system (HATS). The HATS is under feedback repression by the N status of the plant, being down-regulated in plants adequately supplied with N during growth, and up-regulated by N-starvation. When assayed between 1 and 50 mM [15NH4+]0, the 15NH4+ influx showed a linear response typical of a low-affinity transport system (LATS). The activity of the LATS increased in plants supplied with NH4+ as compared with plants grown on an N-free medium. Transfer of the plants to N-free solution resulted in a marked decrease in the LATS-mediated 15NH4+ influx. Accordingly, resupply of NH4+ after N-starvation triggered a dramatic stimulation of the activity of the LATS. These data provide evidence that in Citrus plants, the LATS or at least one of its components is inducible by NH4+. Even when up-regulated, both the HATS and the LATS displayed a limited capacity, as compared with that usually found in herbaceous species. The use of various metabolic uncouplers or inhibitors indicated that 15NH4+ influx mediated by the HATS is strongly dependent on energy metabolism and H+ transmembrane electrochemical gradient. By contrast, the LATS is not affected by protonophores or inhibitors of the H(+)-ATPase, suggesting that its activity is mostly driven by the NH4+/NH3 transmembrane gradient. In agreement with these hypotheses, the HATS-mediated 15NH4+ influx was strongly inhibited when the solution pH was raised from 4 to 7, whereas influx mediated by the LATS was slightly stimulated.