Rapid Induction of Na⁺/H⁺ Exchange Activity in Barley Root Tonoplast

Abstract

Na+/H exchange activity in barley (Hordeum vulgare cv CM-72) root tonoplast was induced by Na+ even in the presence of inhibitors of protein synthesis. Induction occurred with a half-time of only 15 minutes. When salt-treated roots were transferred to a nutrient solution containing no Nat, the activity disappeared with a similar time course. The data suggest that Na+/H+ exchange was due to activation of an existing protein rather than to de novo protein synthesis.Buildup of salts in the soil is a major problem on irrigated farmland and in semiarid regions where winter rainfall is not sufficient to flush accumulated salts from the top soil (18). To grow in saline soils, plants must maintain a much lower ratio of Na+/K+ in their cytoplasm than is present in their surroundings (10,13,14). Multiple mechanisms, including morphological and biochemical adaptations, are probably involved in maintaining this low cytoplasmic Na+/K+ ratio. In barley (Hordeum vulgare), two such mechanisms are extrusion of Na+ into the vacuole across the tonoplast membrane and extrusion of Na+ into the external medium across the plasma membrane (13, 16). The energy for Na+ extrusion could be provided by the proton gradients generated by the plasma membrane or tonoplast H+-ATPases. Recently, using the tonoplast ATPase to generate the H+ gradient, we discovered a Na+/H+ exchange in vesicles from a tonoplast-enriched fraction isolated from barley roots (9). Na+/H+ antiports have also been reported in tonoplast fractions from red beet (Beta vulgaris) (2) and sugar beet (3). The Na+/H+ exchange activity in cell suspension cultures of sugar beet, a halophyte, was partly constitutive (3). In contrast, that of barley, a relatively salt-tolerant glycophyte, was undetectable unless the plants were grown in the presence of Na+. In this paper, induction of the tonoplast Na+/H+ exchange activity in barley is characterized in more detail. MATERIALS AND METHODS Plant MaterialA salt-tolerant cultivar of barley, Hordeum vulgare L. cv CM-72 (1), was grown as described previously (7) Membrane PreparationsA tonoplast-enriched membrane fraction was isolated as described previously (6, 9). Roots (10 gm) were excised into ice water, rinsed, and then ground in homogenization buffer (67 mL) containing 250 mm sucrose, 50 mm Tris, 8 mM EDTA, and 4 mm DTT (pH 8.0). The fraction collected from the 22/30% interface of a sucrose step gradient was washed and resuspended in buffer containing 250 mM sorbitol, 1 mM DTT, and 5 mm Mes adjusted to pH 7.0 with Tris. Aliquots containing 0.5 to 1.5 mg protein/mL were stored at -70C. Enzyme AssaysThe generation and dissipation of pH gradients across the membrane vesicles were followed by measuring quenching and recovery of acridine orange fluorescence as described previously (6,9

Characterization of a rice gene showing organ-specific expression in response to salt stress and drought.

“…Within 15 min of exposure to saline conditions, an Na+/H+ exchange process is activated so that K+ can be pumped across the cell membrane (Watad et ai., 1986) and Na+ can be pumped into tonoplasts (Binzel et al, 1988;Garbarino and DuPont, 1989). Over the course of several days, the ratio of glycolipids to phospholipids can increase (Hirayama and Mihara, 1987), permitting more solutes to enter the cell and thus facilitate water entry, while the overall concentration of osmoprotectants including sugars, proline, and organic acids rises (Binzel et al, 1987;LaRosa et al, 1987).…”

An in Vivo Nuclear Magnetic Resonance Investigation of Ion Transport in Maize (Zea mays) and Spartina anglica Roots during Exposure to High Salt Concentrations

“…In some studies, for example, in Atriplex nummularia (Braun et al, 1988) and Dunaliella salina (Katz et al, 1991), the Na+/H+-antiport appears to be associated with the plasmalemma, but in the majority of studies the antiport activity has been found in the tonoplast, where it appears to be responsible for the uptake and sequestration of sodium into the vacuole, thus preventing the accumulation of toxic ions in the cytosol. Evidence for the importance of the tonoplast Na+/H+-antiport in salt tolerance is provided by the discovery that the antiport can be activated by salt treatment in barley (Gabarino and Dupont, 1989) and in P. maritima (Staal et al, 1991), although it is not yet clear how activation is achieved.…”

Estimation of Ammonium Ion Distribution between Cytoplasm and Vacuole Using Nuclear Magnetic Resonance Spectroscopy

“…However, Streeter (27) estimated the NH4+ concentration in soybean nodule cytosol to be 'essentially nil. ' The cytoplasmic NH4' concentration may also be important with respect to pH gradients between intracellular compartments; millimolar concentrations of NH4' can collapse transmembrane pH gradients in vitro (3). Solutions of NH3 have been shown to rapidly and dramatically increase intracellular pH (15,21).…”