Characterization of a H⁺ NO₃^- symport associated with plasma membrane vesicles of maize roots using ³⁶CIO₃^- as a radiotracer analog

The mechanism of NO₃^- transport was examined in isolated plasma membrane vesicles from maize (Zea mays L., hybrid B73 × LH 51) roots using ³⁶ClO₃^- as a radiotracer analog for NO₃^-. When an acid-exterior ΔpH was imposed across the vesicle membrane, uptake of ³⁶ClO₃^- was stimulated and the time course of radiolabel uptake displayed an overshoot phenomenon characteristic of the coupling of one solute gradient to the movement of another solute. Evidence supporting ΔpH as the driving force for ³⁶ClO₃^- uptake included a dependence of the overshoot peak and initial rate of ³⁶ClO₃^- uptake on the magnitude of the imposed ΔpH, the occurrence of ΔpH-driven ³⁶ClO₃^- uptake in the presence of KSCN/valinomycin, and the ability of an imposed ΔpH to drive ³⁶ClO₃^- uptake when radiolabel was equilibrated across the membrane. When ΔpH-driven ³⁶ClO₃^- transport was examined in the presence of NO₃^- radiolabel uptake was inhibited in a competitive manner. This was consistent with the carrier having the capacity to use either ClO₃^- or NO₃^- and supports the use of this radiotracer as an analog for NO₃^- in transport studies. When ΔpH-driven ³⁶ClO₃^- uptake was examined as a function of ³⁶ClO₃^- concentration and ΔpH, saturation kinetics were observed and the magnitude of the imposed ΔpH affected the K_m but not the V_max for ³⁶ClO₃^- uptake. This suggested an ordered binding mechanism where ³⁶ClO₃^- would bind to the protonated form of the carrier prior to translocation. Radiolabeled ³⁶ClO₃^- uptake was inhibited by treatment of the vesicles with phenylglyoxal, suggesting the involvement of arginine moieties in the process of transport. Taken together, these results support the presence of a H⁺ NO₃^- symport carrier at the plasma membrane which could be involved in mediating energy-dependent NO₃^- uptake into plant cells.