NO3− transport across the plasma membrane of Arabidopsis thaliana root hairs: Kinetic control by pH and membrane voltage

High-affinity nitrate transport was examined in intact root hair cells of Arabidopsis thaliana using electrophysiological recordings to characterise the response of the plasma membrane to NO3−challenge and to quantify transport activity. The NO3−-associated membrane current was determined using a three-electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in the roots of seedlings grown in the absence of a nitrogen source, but only 4–6 days postgermination. In 6-day-old seedlings, additions of 5–100 μm NO3−to the bathing medium resulted in membrane depolarizations of 8–43 mV, and membrane voltage (Vm) recovered on washing NO3−from the bath. Voltage clamp measurements carried out immediately before and following NO3−additions showed that the NO3−-evoked depolarizations were the consequence of an inward-directed current that appeared across the entire range of accessible voltages (−300 to +50 mV). Both membrane depolarizations and NO3−-evoked currents recorded at the free-running voltage displayed quasi-Michaelian kinetics, with apparent values for Km of 23 ± 6 and 44 ± 11 μm, respectively and, for the current, a maximum of 5.1 ± 0.9 μA cm−2. The NO3−current showed a pronounced voltage sensitivity within the normal physiological range between −250 and −100 mV, as could be demonstrated under voltage clamp, and increasing the bathing pH from 6.1 to 7.4–8.0 reduced the current and the associated membrane depolarizations 3- to 8-fold. Analyses showed a well-defined interaction between the kinetic variables of membrane voltage, pHo and [NO3−]o. At a constant pHo of 6.1, depolarization from −250 to −150 mV resulted in an approximate 3-fold reduction in the maximum current but a 10% rise in the apparent affinity for NO3−. By contrast, the same depolarization effected an approximate 20% fall in the Km for transport as a function in [H+]o. These, and additional characteristics of the transport current implicate a carrier cycle in which NO3−binding is kinetically isolated from the rate-limiting step of membrane charge transit, and they indicate a charge-coupling stoichiometry of 2(H+) per NO3−anion transported across the membrane. The results concur with previous studies showing a high-affinity NO3−transport system in Arabidopsis that is inducible following a period of nitrogen-limiting growth, but they underline the importance of voltage as a kinetic factor controlling NO3−transport at the plant plasma membrane.