An NaCl-resistant line has been developed from suspension-cultured tobacco cells (Nicotiana tabacum/gossii) by stepwise increases in the NaCl concentration in the medium. Resistance showed stability through at least 24 generations in the absence of added NaCl.Above an external NaC concentration of 35 millimolar, proline concentration in the selected cells rose steeply with external NaC, particularly so above 100 millimolar NaCI. Proline accumulation in the wild type was far slighter. Selected cells which had been grown for 24 generations in the absence of added NaCI accumulated proline strongly on re-exposure to NaCl medium, indicating stability of this character. Proline accumulation was fully reversible with a half-time of about 6 hours. When selected cells were transferred sequentially to lower and lower NaCl concentrations, their proline content fell to the level corresponding to the new NaCl concentration. The NaCI-selected cells responded to water stress (i.e. added mannitol) by accumulating markedly more proline than did the wild type.

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Proton fluxes have been followed into and out of membrane vesicles isolated from the roots of the halophyte Atriplex nummularia and the glycophyte Gossypium hirsutum, with the aid of In view of their obvious potential role in the adaptation of plants to high ambient salt concentrations, we have been studying membrane transport mechanisms in plants of varying resistance to salinity (6, 7, 10). We have previously reported on proton-translocating ATPase activity in membrane vesicles isolated from the roots of the halophyte Atriplex nummularia and the glycophyte cotton, a crop plant resistant to mild saline stress (6, 10). We detected and reported on Na+/H+ antiporter activity in Atriplex root membranes (7). This was also recently observed in membrane from Atriplex leaf membranes (13). This paper presents evidence suggesting that two antiporters may operate in Atriplex, one for Na+ and one for K+. Further, we report on our search for such antiporter mechanisms in the glycophyte cotton.Na+/H+ antiport activity has been reported in two other higher plants as well as in Atriplex, both of them salt tolerant (1, 9), as well as in two algae (8,12). K+/H+ antiport has been deduced in the case of two glycophytes (16,17). In neither ' Supported by a grant from the U.S.-Israel Binational Agricultural Research and Development fund (BARD).case was the effect specific for K+, considerable activity being noted for other monovalent cations as well. In all the higher plants, apart from Atriplex roots (7) and zucchini hypocotyl (16), the antiporter activity described was present in the tonoplast. We have obtained evidence for the operation of antiporters in the plasmalemma as well as the tonoplast, both in cotton and in Atriplex. Some of these results have been briefly presented in a previous communication (5). MATERIALS AND METHODS Plant Material and Isolation of VesiclesAtriplex nummularia and Gossypium hirsutum L. var Acala San Jose 2 were grown as described before (6, 10) in aerated half-strength Hoagland-Snyder solution (1 1), with or without the addition of 400 mM NaCl in the case of Atriplex. Root tips were sampled as before (6) and homogenized in a medium containing 0.25 M sucrose, 2 mm EGTA, 5 mm MgSO4, 1% BSA, 25 mM BTP2-Mes (pH 7.2) with the addition of 100 mM ascorbic acid and 3% (w/v) PVP in the case of cotton. After centrifugation at 13,000g for 15 min the supernatant was recentrifuged at l00,OOOg (Beckman ultracentrifuge L5-50B, 50.2 Ti) for 30 min and the pellet resuspended with a syringe (25 gauge needle) in 2 mL suspension medium containing 1 mM MgSO4, 1% BSA, 5 mM BTP-Mes (pH 7.2). This hypoosmotic shock causes leakage of internal vesicular solutes, thereby decreasing buffering capacity and improving ApH measurements without affecting AI measurements. The crude microsome preparation thus obtained was layered on a 20 to 45% (w/v) six-step discontinuous sucrose gradient (6) containing 1 mM MgSO4, 1% BSA, 1 mM BTP-Mes (pH 7.2), and centrifuged at 80,000g for 2 h (SW 41). The membrane fractions collected at t...

Sorghum bicolor L. Moench, RS 610, was grown in liquid media salinized with NaCl, KCl, Na2SO4, K2SO4 or with variable mixtures of either NaCl/KCl or Na2SO4/K2SO4 at osmotic potentials ranging from 0 to ‐0.8 MPa. The purpose was to study the effects of different types and degrees of salinity in growth media on growth and solute accumulation. In 14‐day‐old plants the severity of leaf growth inhibition at any one level of osmotic potential in the medium increased according to the following order: NaCl < Na2SO4 < KCl = K2SO4. Inhibition of growth by mixtures of Na+ and K+ salts was the same as by K+ salts alone. Roots responded differently. Root growth was not affected by Na+ salts in the range of 0 to ‐0.2 MPa while it was stimulated by K+ salts. The major cation of leaves was K+ because S. bicolor is a Na+‐excluder, while Na+ was the major cation in roots except at low Na+/K+ ratios in media. Anions increased in tissues linearly in relation to total monovalent cation, but not with a constant anion/cation ratio. This ratio increased as the cation concentrations in tissues increased. Sucrose in leaf tissue increased 75 fold in Chloride‐plants (plants growing in media in which the only anion of the salinizing salts was Cl−) and 50 fold in Sulphate‐plants (the only anion of the salinizing salts was SO42‐). Proline increased 60 and 18 fold in Chloride‐ and Sulphate‐plants, respectively, as growth media potentials decreased from 0 to ‐0.8 MPa. The concentrations of both sucrose and proline were directly proportional to the amount of total monovalent cation in the tissue. Sucrose concentrations began increasing when total monovalent cations exceeded 100 μmol (g fresh weight)−1 (the monovalent cation level in non‐stressed plants), but proline did not start accumulating until monovalent cation concentrations exceeded 200 μmol (g fresh weight)−1. Therefore, sucrose seemed to be the solute used for osmotic adjustment under mild conditions of saline stress while proline was involved in osmotic adjustment under more severe conditions of stress. Concentrations of inorganic phosphate, glucose, fructose, total amino acids and malic acid fluctuated in both roots and leaves in patterns that could be somewhat correlated with saline stress and, sometimes, with particular salts in growth media. However, the changes measured were too small (at most a 2–3 fold increase) to be of importance in osmotic adjustment.

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