Characterization of a H+/NO3- symport associated with plasma membrane vesicles of maize roots using 36ClO3- as a...

The mechanism of NO3- transport was examined in isolated plasma membrane vesicles from maize (Zea mays L., hybrid B73 X LH 51) roots using 36ClO3- as a radiotracer analog for NO3-. When an acid-exterior delta pH 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 ot the movement of another solute. Evidence supporting delta pH 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 delta pH, the occurrence of delta pH-driven 36ClO3- uptake in the presence of KSCN/valinomycin, and the ability of an imposed delta pH to drive 36ClO3- uptake when radiolabel was equilibrated across the membrane. When delta pH-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 delta pH-driven 36ClO3- uptake was examined as a function of 36ClO3- concentration and delta pH, saturation kinetics were observed and the magnitude of the imposed delta pH affected the Km but not the Vmax 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.