Salt tolerance mechanisms in three Irano-Turanian Brassicaceae halophytes relatives of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Salt tolerance mechanisms were studied in three Irano-Turanian halophytic species from the Brassicaceae ??(Lepidium latifolium, L. perfoliatum and Schrenkiella parvula) and compared with the glycophyte Arabidopsis thaliana. According to seed germination under salt stress, L. perfoliatum was the most tolerant species, while L. latifolium and S. parvula were rather susceptible. Contrastingly, based on biomass production L. perfoliatum was more salt sensitive than the other two species. In S. parvula biomass was increased up to 2.8-fold by 100 mM NaCl; no significant growth reduction was observed even when exposed to 400 mM NaCl. Stable activities of antioxidative defense enzymes, nil or negligible accumulation of superoxide anion and hydrogen peroxide, as well as stable membrane integrity in the three halophytes revealed that no oxidative stress occurred in these tolerant species under salt stress. Proline levels increased in response to salt treatment. However, it contributed only by 0.3?2.0% to the total osmolyte concentration in the three halophytes (at 400 mM NaCl) and even less (0.04%) in the glycophyte, A. thaliana (at 100 mM NaCl). Soluble sugars in all three halophytes and free amino acids pool in S. parvula decreased under salt treatment in contrast to the glycophyte, A. thaliana. The contribution of organic osmolytes to the total osmolyte pool increased by salt treatment in the roots, while decreased in halophyte and glycophyte, A. thaliana leaves. Interestingly, this reduction was compensated by a higher relative contribution of K in the leaves of the halophytes, but of Na in A. thaliana. Taken together, biomass data and biochemical indicators show that S. parvula is more salt tolerant than the two Lepidium species. Our data indicate that L. latifolium, as a perennial halophyte with a large biomass, is highly suitable for both restoration of saline habitats and saline agriculture.     

Chromosome number and DNA content of tobacco cells adapted to NaCl

Cultured tobacco (Nicotiana tabacum L. cv. Wisconsin 38) cells were found to have altered DNA contents and chromosome numbers after adaptation to NaCl. Cells adapted to 428 mM NaCl were predominately hexaploid compared to the normal tetraploid 2N(2C)=4X=48 chromosome number of unadapted cells. Enrichment of the cell population for hexaploid cells occurred only after exposure to higher NaCl (428 mM), not lower levels of NaCl (171 mM). The majority of adapted cells remain hexaploid for at least 25 cell generations after removal from NaCl exposure. Adapted cell populations were found to have fewer cells with highly polyploid (2N96) nuclei. Salt tolerance of hexaploid cells was not found to be significantly greater than that of tetraploid cells. Cells with higher ploidy levels were less salt tolerant. It is suggested that high levels of NaCl induce polyploidization and that exposure to NaCl selects against cells with very high ploidy levels.     

Intracellular compartmentation of ions in salt adapted tobacco cells

Na⁺ and Cl⁻ are the principal solutes utilized for osmotic adjustment in cells of Nicotiana tabacum L. var Wisconsin 38 (tobacco) adapted to NaCl, accumulating to levels of 472 and 386 millimolar, respectively, in cells adapted to 428 millimolar NaCl. X-ray microanalysis of unetched frozen-hydrated cells adapted to salt indicated that Na⁺ and Cl⁻ were compartmentalized in the vacuole, at concentrations of 780 and 624 millimolar, respectively, while cytoplasmic concentrations of the ions were maintained at 96 millimolar. The morphometric differences which existed between unadapted and salt adapted cells, (cytoplasmic volume of 22 and 45% of the cell, respectively), facilitated containment of the excited volume of the x-ray signal in the cytoplasm of the adapted cells. Confirmation of ion compartmentation in salt adapted cells was obtained based on kinetic analyses of ²²Na⁺ and ³⁶Cl⁻ efflux from cells in steady state. These data provide evidence that ion compartmentation is a component of salt adaptation of glycophyte cells.     

Effects of NaCl and Na2SO4 on Water Relations ofPlantago maritima (L.) andPlantago lanceolata (L.)

Transpiration and water absorption rates, stomatal and cuticular resistances to water vapour diffusion, were measured onPlantago maritima (halophyte) andPlantago lanceolata (glycophyte) grown in the presence of NaCl or Na₂SO₄. Water absorption was reduced in the presence of Na₂SO₄ and transpiration rate was increased when NaCl was added to the nutrient solution. The glycophyte daily water balance was more disturbed than that of the halophyte in the presence of Na₂SO₄.     

Growth Yields and Maintenance Coefficients of Unadapted and NaCl-Adapted Tobacco Cells Grown in Semicontinuous Culture

Comparison of carbon utilization between unadapted and NaCl (428 millimolar) adapted tobacco (Nicotiana tabacum L.) cells under substrate limited growth conditions was facilitated using semicontinuous culture. Growth yields (Y_g) and maintenance coefficients (m) of unadapted and NaCl adapted cells were similar, indicating that the efficiency of carbon utilization for growth was not altered as a result of salt adaptation and that no additional metabolic costs were associated with growth of adapted cells in the presence of a high concentration (428 millimolar) of NaCl. The Y_g (0.588 grams organic dry weight gain per gram sugar uptake) and m values (0.117 grams sugar uptake per gram organic dry weight per day) were comparable in spite of substantial physiological and biochemical differences that exist between unadapted and NaCl adapted cells. Apparently, a metabolic homeostasis governs biomass production of cells before and after adaptation to salinity.     

Peroxidase activity and isoenzymes in the culture medium of NaCl adapted tomato suspension cells

The medium of tomato (Lycopersicon esculentum) cells adapted to grow in the presence of 15 g l^–1 NaCl had a higher peroxidase activity than the medium of an unadapted tomato cell line. When the adapted cells were cultured in a medium without NaCl, the value found for peroxidase activity was intermediate. The increase in peroxidase activity was parallel to an increase of lignin-like compounds in the cell walls, as well as to an increased content or appearance of neutral and basic peroxidase isoenzymes. Apparently, the high values of peroxidase activity in the medium of the salt-adapted cells reflect the changed mechanical properties of the cell wall which, in turn, could be related to the salt adaptation process.Abbreviations LO Control tomato cell line unable to grow in the presence of 15 g 1^–1 of NaCl - L15 tomato cell line adapted to 15 g 1^–1 of NaCl and growing in this salt concentration - L15-0 tomato cell line adapted to 15 g 1^–1 of NaCl and growing in the absence of this salt - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - PBS phosphate buffer saline     

Enhanced Net K Uptake Capacity of NaCl-Adapted Cells

Maintenance of intracellular K⁺ concentrations that are not growth-limiting, in an environment of high Na⁺, is characteristic of NaCl-adapted cells of the glycophyte, tobacco (Nicotiana tabacum/gossii). These cells exhibited a substantially greater uptake of ⁸⁶Rb⁺ (i.e. an indicator of K⁺) relative to unadapted cells. Potassium uptake into NaCl-adapted cells was 1.5-fold greater than unadapted cells at 0 NaCl and 3.5-fold greater when cells were exposed to 160 millimolar NaCl. The difference in net K⁺ uptake between unadapted and NaCl-adapted cells was due primarily to higher rates of entry rather than to reduced K⁺ leakage. Presumably, enhanced K⁺ uptake into adapted cells is a result of electrophoretic flux, and a component of uptake may be linked to vanadate-sensitive H⁺ extrusion.     

Adaptation of Tobacco Cells to NaCl

Cell lines of tobacco (Nicotiana tabacum L. var Wisconsin 38) were obtained which are adapted to grow in media with varying concentrations of NaCl, up to 35 grams per liter (599 millimolar). Salt-adapted cells exhibited enhanced abilities to gain both fresh and dry weight in the presence of NaCl compared to cells which were growing in medium without NaCl (unadapted cells). Tolerance of unadapted cells and cells adapted to 10 grams per liter NaCl was influenced by the stage of growth, with the highest degree of tolerance exhibited by cells in the exponential phase. Cell osmotic potential and turgor varied through the growth cycle of unadapted cells and cells at all levels of adaptation, with maximum turgor occurring at approximately the onset of exponential fresh weight accumulation.

Adaptation to NaCl led to reduced cell expansion and fresh weight gain, while dry weight gain remained unaffected. This reduction in cell expansion was not due to failure of the cells to maintain turgor since cells adapted to NaCl underwent osmotic adjustment in excess of the change in water potential caused by the addition of NaCl to the medium. Tolerance of the adapted cells, as indicated by fresh or dry weight gain, did not increase proportionately with the increase in turgor. Adaptation of these glycophytic cells to NaCl appears to involve mechanisms which result in an altered relationship between turgor and cell expansion.

     

Carbon Use Efficiency and Cell Expansion of NaCl-Adapted Tobacco Cells

Carbon use efficiencies (gram cell organic dry weight accumulated per gram sugar assimilated from the medium) of unadapted and NaCl-adapted (428 millimolar) cells of tobacco (Nicotiana tabacum L. var Wisconsin 38) were determined to evaluate metabolic costs associated with growth and survival in a saline environment. No net increase in carbon costs was associated with salt adaptation. At low substrate levels, carbon use efficiencies of unadapted and NaCl-adapted cells were not appreciably different (0.495 and 0.422, respectively) and at higher substrate levels carbon use efficiency of NaCl-adapted cells was clearly higher than that of unadapted cells. These results indicate that a homeostasis of metabolic efficiency is established after cells have adapted to NaCl. Altered carbon availability does not cause the reduced cell volume that results from adaptation to NaCl. This does not preclude, however, the possibility that altered intracellular partitioning of carbon affects cell expansion.     

Loss of arabinogalactan-proteins from the plasma membrane of NaCl-adapted tobacco cells

Cultured cells of tobacco (Nicotiana tabacum L.) adapted to 428 mM NaCl exhibited a reduced rate of cell enlargement, which is probably due to decreased cell-wall extensibility. Arabinogalactan-protein (AGP) has been implicated as a cell-wall-loosening factor (Schopfer 1990). Levels of plasma membrane and extracellular AGPs that react with Yariv reagent were measured and compared between NaCl-adapted and unadapted tobacco cells. Unadapted cells contained a very high level of AGPs on the plasma membrane, which amounted to 0.16 g·g^–1 membrane protein. In contrast, AGPs were virtually undetectable on the plasma membrane of NaCl-adapted cells. Accumulation of AGPs was also decreased in culture media of NaCl-adapted cells. These data support the hypothesis that AGPs participate in cell expansion. Possible mechanisms of the proposed cell-expansion role of AGPs are discussed.Abbreviations AGP arabinogalactan-protein - S₀, S₂₅ cells un-adapted, NaCl-adapted tobacco cells This work was supported in part by a Mcknight Foundation fellowship to J.K.Z. This is journal paper No. 13,569 of Purdue University Agricultural Experimental Station. The authors thank Dr. Eugene A. Nothnagel for the Yariv reagent gift and for helpful discussion. The authors also thank Glenda McClatchey for excellent technical assistance.     

Effect of salt stress on growth and osmotic regulation in Thellungiella and Arabidopsis callus

The response of Thellungiella (Thellungiella holophila) and Arabidopsis (Arabidopsis thaliana) callus to salt stress was investigated. The relative growth rate of Arabidopsis calli decreased with increased levels of NaCl. However, the relative growth rate of Thellungiella calli increased with higher levels of NaCl, reaching maximal growth at 100 mM NaCl, but then subsequently declined. A similar pattern of accumulation of proline, glycine betaine, and total flavonoid was observed; whereas, accumulation of treholase continued to increase with increasing NaCl levels in both Thellungiella and Arabidopsis calli. Overall, with increasing NaCl levels, accumulation of glycine betaine, total flavonoid, and treholase was higher in Thellungiella than in Arabidopsis calli; while, proline and sucrose contents were higher in Arabidopsis than in Thellungiella calli. These results indicated that compatible solutes were involved in the response of plant calli to salt stress, and that the halophyte Thellungiella and glycophyte Arabidopsis selected different compatible solutes to adapt to salt stress environments. X. Zhao and H. J. Tan have contributed equally to the paper.     

Stable NaCl Tolerance of Tobacco Cells Is Associated with Enhanced Accumulation of Osmotin

Osmotin is a major protein which accumulates in tobacco cells (Nicotiana tabacum L. var Wisconsin 38) adapted to low water potentials. Quantitation of osmotin levels by immunoblots indicated that cells adapted to 428 millimolar NaCl contained 4 to 30 times the level of osmotin found in unadapted cells, depending on the stage of growth. Unadapted cells accumulated low levels of osmotin with apparent isoelectric points, (pl) of 7.8 and >8.2. Upon transfer of NaCl-adapted cells to medium without NaCl and subsequent growth for many cell generations, the amount of osmotin declined gradually to a level intermediate between that found in adapted and unadapted cells. NaCl-adapted cells grown in the absence of NaCl accumulated both pl forms; however, the form accumulated by cells adapted to NaCl (pl > 8.2) was most abundant. Adapted cells grown in the absence of NaCl exhibited absolute growth rates and NaCl tolerance levels which were intermediate to those of NaCl-adapted and unadapted cells. The association between osmotin accumulation and stable NaCl tolerance indicates that cells with a stable genetic change affecting the accumulation of osmotin are selected during prolonged exposure to high levels of NaCl. This stable alteration in gene expression probably affects salt tolerance.     

Chromosome number and nuclear DNA content of plants regenerated from salt adapted plant cells

Plants regenerated from tobacco (Nicotiana tabacum L. cv. Wisconsin 38) cells that were adapted to 428 mM NaCl were found to have hexaploid or near-hexaploid chromosome numbers compared to the normal tetraploid, 2N(2C)=4X=48 chromosome numbers of plants regenerated from unadapted cells. Even though cells with chromosome numbers other than hexaploid were found in the cell population only hexaploid plants were regenerated. The hexaploid condition may impart some karyotypic stability that allows more efficient morphogenic activity. The hexaploid condition could not be correlated with several phenotypic alterations associated with plants regenerated from adapted cells, including male sterility and increased salt tolerance.     

A method for screening rice plants for salt tolerance

A number of varieties of rice, a halophyte, Sesuvium portulacastrum and a glycophyte, Phaseolus vulgaris were grown in culture solution containing a range of concentrations of NaCl. Growth of the plants and internal sodium concentrations of the roots were measured after 14 days. The electrical potential difference (PD) between the external solution and the vacuole of the outer cells of the root was also measured. This enabled the driving force on sodium at the cell membranes to be calculated using the Nernst equation. It was found that Sesuvium and those varieties of rice that had previously shown salt tolerance generated relatively negative PDs and large driving forces tending to exclude sodium from the root. This suggested that a simple measurement of PD for plants grown in a given concentration of NaCl over a given period of time would provide a fairly rapid screening method for salt tolerance in rice and possibly other species also. T J Flowers Section editor     

Seasonal changes in the germination responses of Hordeum maritimum and H. murinum seeds in relation to salinity,temperature and after‐ripening time

Abstract

The effects of temperature and salinity (NaCl) on germination of Hordeum maritimum With, (halophyte) and H. murinum L. (glycophyte) seeds were investigated. Dehulled caryopses were used for monthly germination trials, starting from November (120 days of after‐ripening in darkness at 20±1°C). Trials were continued for one year. Differences in germination response between the two species were observed, confirming that H. Maritimum is better adapted to high salinity levels and to variations in external temperature than H. murinum. H. maritimum showed a germination control mechanism related to after‐ripening time and based on seed dormancy break/resumption. At higher temperature (30°C), thermodormancy was also recorded. No germination strategies were observed in H. murinum that is relatively insensitive to the combined effects of temperature and salinity. Thus, in virtually all treatments, H. murinum exhibited a higher germination rate compared with H. maritimum, as early as 72 h after imbibition, suggesting that dormancy, both in the presence or absence of salt, is totally abolished by early fall or at the latest in winter.     

Enhanced H Transport Capacity and ATP Hydrolysis Activity of the Tonoplast H-ATPase after NaCl Adaptation

Tonoplast enriched membrane vesicle fractions were isolated from unadapted and NaCl (428 millimolar) adapted tobacco cells (Nicotiana tabacum L. var Wisconsin 38). Polypeptides from the tonoplast enriched vesicle fractions were separated by SDS-PAGE and analyzed by Western blots using polyclonal antibodies to the 70 kilodalton subunit of the red beet tonoplast H⁺-ATPase. These antibodies cross-reacted exclusively to a tobacco polypeptide of an apparent molecular weight of 69 kilodaltons. The antibodies inhibited ATP-dependent, NO₃⁻ sensitive H⁺ transport into vesicles in tonoplast enriched membrane fractions from both unadapted and NaCl adapted cells. The relative H⁺ transport capacity per unit of 69 kilodalton subunit of the tonoplast ATPase of vesicles from NaCl adapted cells was fourfold greater than that observed for vesicles from unadapted cells. The increase in specific H⁺ transport capacity after adaptation was also observed for ATP hydrolysis.     

Effects of salt stress on the reproductive biology of the halophyte <Emphasis Type="Italic">Plantago crassifolia</Emphasis>

Floral phenology, pollen quality and seed set of Plantago crassifolia plants, grown in the presence of increasing NaCl concentrations, were studied to test how this Mediterranean halophyte responded to salt stress during the reproductive phase of its life cycle. Reproductive success was maximal in plants grown in non-saline conditions, or in the presence of 100 mM NaCl, but it was negatively affected by higher salinities, due to a progressive reduction of pollen fertility, seed set, and seed viability.     

Abscisic Acid accelerates adaptation of cultured tobacco cells to salt

Adaptation of tobacco (Nicotiana tabacum L. var Wisconsin 38) cells to NaCl was accelerated by (±) abscisic acid (ABA). In medium with 10 grams per liter NaCl, ABA stimulated the growth of cells not grown in medium with NaCl (unadapted, S-0) with an increasing response from 10⁻⁸ to 10⁻⁴ molar. ABA (10⁻⁵ molar) enhanced the growth of unadapted cells in medium with 6 to 22 grams per liter NaCl but did not increase the growth of cells previously adapted to either 10 (S-10) or 25 (S-25) grams per liter NaCl unless the cells were inoculated into medium with a level of NaCl higher than the level to which the cells were adapted. The growth of unadapted cells in medium with Na₂SO₄ (85.5 millimolar), KCl (85.5 or 171 millimolar), K₂SO₄ (85.5 millimolar) was also stimulated by ABA. ABA (10⁻⁸-10⁻⁴ molar) did not accelerate the growth of unadapted cells exposed to water deficits induced by polyethylene glycol (molecular weight 8000) (5-20 grams per 100 milliliters), sorbitol (342 millimolar), mannitol (342 millimolar) or sucrose (342 millimolar). These results suggest that ABA is involved in adaptation of cells to salts, and is not effective in promoting adaptation to water deficits elicited by nonionic osmotic solutes.