Na⁺/H⁺ Antiport in Isolated Tonoplast Vesicles from Storage Tissue of Beta vulgaris

Abstract

The pH-dependent fluorescence quenching of acridine orange was used to study the Na'-and KI-dependent H' fluxes in tonoplast vesicles isolated from storage tissue of red beet and sugar beet (Beta vulgaris L.). The Na'-dependent H' flux across the tonoplast membrane could be resolved into two components: (a) a membrane potential-mediated flux through conductive pathways; and (b) an electroneutral flux which showed Michaelis-Menten kinetics relationship to Na' concentration and was competitively inhibited by amiloride (K1 = 0.1 millimolar). The potentialdependent component of H' flux showed an approximately linear dependence on Na' concentration. In contrast, the K-dependent H' flux apparently consisted of a single component which showed an approximately linear dependence on K concentration, and was insensitive to amiloride. Based on the Na'-and Ktdependent H' fluxes, the passive permeability of the vesicle preparation to Na was about half of that to K+.The apparent K,for Na of the electroneutral Nat/H+ exchange varied by more than 3-fold (7.5-26.5 millimolar) when the internal and external pH values were changed in parallel. The results suggest a simple kinetic model for the operation of the Na+/H+ antiport which can account for the estimated in vivo accumulation ratio for Na into the vacuole.Plant cells typically maintain a high K+/Na+ ratio in the cytoplasm, and there is evidence that Na+ is actively transported out ofthe cytoplasm at both the plasmalemma and the tonoplast membranes (10,14,15,17,24,25). While some moderately sodium-tolerant species depend mainly on exclusion of sodium at the plasma membrane, halophytes such as Beta vulgaris may accumulate large amounts of sodium which are sequestered into the vacuole to serve as osmoticum. Thus, transport of sodium at the tonoplast may be particularly important in these species (9).The mechanism of sodium transport at the tonoplast has not yet been elucidated. There is evidence supporting an H+/Na+ antiport mechanism at the plasma membrane of barley roots (19). The existence of a proton transporting ATPase at the tonoplast of various species (5, 7) including B. vulgaris (2,18,27) suggests the possibility of a similar mechanism for sodium transport into the vacuole. In the present work, we present evidence for an Na+/H+ antiport in tonoplast vesicles isolated from B. vulgaris. Membrane Preparations. Tonoplast vesicles were isolated as described elsewhere (20). For the identification of the membrane preparation as tonoplast, see "Discussion." The vesicles were preloaded with a buffer of desired ionic composition by suspension and sedimentation at 100,OOOg for 30 min. Final membrane pellets were resuspended in the same media to 6 mg protein/ml and incubated at 4°C for 3 h.Fluorescence Assays. The fluorescence quenching of acridine orange was used to monitor the formation and dissipation of inside-acid pH gradients (3, 18) across the membranes of the tonoplast vesicles. In all experiments, tonoplast vesicles (60 yg protein) were added to 2 ml of buffer con...

Salt Tolerance in Suspension Cultures of Sugar Beet

“…The effect of Na+ on the dissipation of a transmembrane pH gradient was tested in tonoplast vesicles basically as described before (3,5). The addition of tonoplast vesicles equilibrated at pH 5.8 to a pH 8.0 buffer (pH jump) caused quenching of acridine orange fluorescence.…”

“…In a previous study (3) we characterized a Na+/H+ antiport in tonoplast vesicles isolated from storage tissue of red beet and sugar beet. Since the vacuolar accumulation of sodium is characteristic of salt-tolerant species (7,8) this tonoplast Na+/H+ antiport may be one of the principal physiological factors conferring salt tolerance on the plant.…”

Sodium as nutrient and toxicant

“…However, a specific demonstration in guard cell opening and closing has not been made in any study of which we are aware. At the tonoplast, vacuolar sequestration of Na + has been attributed to the function of Na + /H + exchangers (NHX; Blumwald and Poole 1985;Apse et al 1999Apse et al , 2003 and both slow-vacuolar (SV) (Hedrich and Neher 1987;Schönknecht et al 2002;Ivashikina and Hedrich 2005) and fast-vacuolar (FV) channels (Isayenkov et al 2010). It has further been suggested that members of the cation exchanger (CAX) family could transport Na + (Luo et al 2005; see also Zhao et al 2008), but, again, none of these candidates is a part of current models of guard cell function.…”

The genomic organization and transcriptional pattern of genes encoding nitrate transporters 1 (NRT1) in cucumber

“…It has been established that nitrate uptake by plants is an active, H + -coupled process facilitated through the highand low-affinity transport systems (HATS and LATS) which could be constitutive or inducible (Glass and Siddiqi 1995;Glass et al 2001Glass et al , 2002Zhao et al 1999;Tischner 2000;Touraine and Gojon 2001;Forde 2002). Also the nitrate influx into vacuole (Blumwald and Poole 1985;Schumaker and Sze 1987;Kabała et al 2003;de Angeli et al 2006;von der Fecht-Bartenbach et al 2010) as well as its efflux out of the plant cell (Segonzac et al 2007;Lin et al 2008) have been shown to be driven by a proton motive force. Active nitrate transport across plant cell membranes has been attributed to three families including the proton-coupled symporters and antiporters belonging to NRT1, NRT2 (Tsay et al 2007) and CLC multigenic families (de Angeli et al 2006Angeli et al , 2009von der Fecht-Bartenbach et al 2010).…”