Passive nitrate transport by root plasma membrane vesicles exhibits an acidic optimal pH like the H(+)-ATPase

Abstract

The net initial passive flux (J(Ni)) in reconstituted plasma membrane (PM) vesicles from maize (Zea mays) root cells was measured as recently described (P. Pouliquin, J.-P. Grouzis, R. Gibrat ¿1999 Biophys J 76: 360-373). J(Ni) in control liposomes responded to membrane potential or to NO(3)(-) as expected from the Goldman-Hodgkin-Katz diffusion theory. J(Ni) in reconstituted PM vesicles exhibited an additional component (J(Nif)), which was saturable (K(m) for NO(3)(-) approximately 3 mM, with J(Nifmax) corresponding to 60 x 10(-9) mol m(-2) s(-1) at the native PM level) and selective (NO(3)(-) = ClO(3)(-) > Br(-) > Cl(-) = NO(2)(-); relative fluxes at 5 mM: 1:0.34:0.19). J(Nif) was totally inhibited by La(3+) and the arginine reagent phenylglyoxal. J(Nif) was voltage dependent, with an optimum voltage at 105 mV at pH 6.5. The activation energy of J(Nif) was high (129 kJ mol(-1)), close to that of the H(+)-ATPase (155 kJ mol(-1)), and J(Nif) displayed the same acidic optimal pH (pH 6.5) as that of the H(+) pump. This is the first example, to our knowledge, of a secondary transport at the plant PM with such a feature. Several properties of the NO(3)(-) uniport seem poorly compatible with that reported for plant anion channels and to be attributable instead to a classical carrier. The physiological relevance of these findings is suggested.

Figure 1

NO₃⁻-dependent dissipation of K⁺-valinomycin diffusion potentials across reconstituted PM vesicles and control liposomes. Reconstituted PM vesicles (A) and control liposomes (B) were prepared as described in “Materials and Methods.” The fluorescent dye oxonol VI was used to determine the K⁺-valinomycin diffusion potential (E_m), as detailed elsewhere (Pouliquin et al., 1999), after the addition of 100 mm K₂SO₄ (lines a) or K₂SO₄ plus KNO₃ to make final concentration of K⁺ and NO₃⁻ equal to 200 and 15 mm, respectively (lines b). Both the assay medium and the vesicle lumen contained 100 mm Li⁺ (see “Materials and Methods”), final addition of the Li⁺-ionophore eth 149 clamped E_m to 0 (short-circuiting effect).

Figure 2

Net initial passive flux of NO₃⁻ in reconstituted PM vesicles and control liposomes as a function of NO₃⁻ concentration. The net initial passive flux of NO₃⁻ (J_Ni) was determined from the NO₃⁻-dependent depolarization rate measured as indicated in the precedent figure and detailed previously (Pouliquin et al., 1999). J_Ni in liposomes (○) was linear with the external NO₃⁻ concentration ([Ni_o]), as expected from the Goldman-Hodgkin-Katz relation (see text). The slope (k = 7.0 × 10⁻¹¹ m s⁻¹) of the linear regression of J_Ni versus [Ni_o] gave the mean permeability coefficient of liposomes to NO₃⁻ (P_N = −k[RT/(−FE_m)][1 − exp {−(F/RT)E_m}] = 1.8 × 10⁻¹¹ m s⁻¹). J_Ni across reconstituted PM vesicles (●) exhibited two components: J_Ni was linear for [Ni_o] higher than 15 mm, with the same slope as for control liposomes, making this component attributable to NO₃⁻ diffusion across the lipidic bilayer; correction of J_Ni for the latter component gave a saturable one (J_Nif, ▵) with K_m for NO₃⁻ and J_Nifmax of 3 mm and 3.8 × 10⁻⁹ mol m⁻² s⁻¹, respectively (inset, Scatchard plot). Dashed lines were calculated for diffusion (liposomes) or both diffusion and catalyzed (saturable) transport (reconstituted PM vesicles) with parameters indicated above.

Figure 3

. pH dependence of the net initial passive flux of NO₃⁻ in reconstituted PM vesicles and control liposomes. Reconstituted PM vesicles (●, ○) or control liposomes (▴, ▵) were equilibrated for 20 min in a medium containing 50 mm Li₂SO₄, 0.5 mm K₂SO₄, 50 nm oxonol VI, and 5 mm MES-Li (●, ▴) or 5 mm HEPES-Li (○, ▵) at the indicated pH before imposition of the indicated initial K⁺-valinomycin diffusion E_m (□). J_Ni was determined from the NO₃⁻-dependent (15 mm) depolarization rate.

Figure 4

Voltage dependence of the net initial passive flux of NO₃⁻ in reconstituted PM vesicles and control liposomes. E_m was adjusted by adding variable K⁺ concentrations to reconstituted PM vesicles (●, ○) or liposomes (▵). J_Ni was determined from the NO₃⁻-dependent (15 mm) depolarization rate at pH 6.5 (closed symbol) or 7.5 (open symbols). J_Ni in control liposomes was fitted (dashed line) using the Goldman-Hodgkin-Katz relation for ion diffusion (Eq. 2) with P_N = 1.8 × 10⁻¹¹ m s⁻¹.

Figure 5

Temperature dependence of NO₃⁻ and H⁺ transport in reconstituted PM vesicles. The net initial passive fluxes of NO₃⁻ (facilitated component J_Nif at 15 mm NO₃⁻, ●) or H⁺ (J_H, ▵) in reconstituted PM vesicles were measured at pH 6.5, with the initial E_m close to 100 mV and at the indicated temperatures measured in the assay cuvette. The initial rate of H⁺ pumping (V_H) of the H⁺-ATPase was also determined (○) using the permeant fluorescent pH probe ACMA (1 μm) in an assay medium containing 60 mm BTP-SO₄ (pH 6.5), 1 mm ATP-Mg, 50 mm K₂SO₄; vesicles were loaded with 50 mm K₂SO₄ in place of Li₂SO₄, and valinomycin (0.1 μm) was used to short-circuit the H⁺-pump, ensuring the maximum V_H value.