Extracellular polysaccharides and proteins of tobacco cell cultures and changes in composition associated with growth-limiting adaptation to water and saline stress

The chemical composition of extracellular polymers released by cells of tobacco (Nicotiana tabacum L. cv W38) adapted to a medium containing 30% polyethylene glycol 8000 (−28 bar) or 428 millimolar NaCl (−23 bar) was compared to the composition of those released by unadapted cells. Unadapted cells released uronic acid-rich material of high molecular weight, arabinogalactan-proteins, low molecular weight fragments of hemicellulosic polysaccharides, and a small amount of protein. Cells adapted to grow in medium containing NaCl released arabinogalactan and large amounts of protein but not the uronic acid-rich material, and cells adapted to grow in polyethylene glycol released only small amounts of an arabinogalactan of much lower molecular weight and some protein. Secretion of all material was nearly blocked by polyethylene glycol, but when cells were transferred to a medium containing iso-osmolar mannitol, they again released extracellular polymers at rates similar to those of unadapted cells. Like cells adapted to NaCl, however, these cells released arabinogalactan and large amounts of protein but only small amounts of the uronic acid-rich material. Media of NaCl-adapted cells were enriched in 40, 29, and 11 kilodalton polypeptides. CaCl₂ extracted the 40 and 11 kilodalton polypeptides from walls of unadapted cells, but the 29 kilodalton polypeptide was found only in the medium of the NaCl-adapted cells. Accumulation of low molecular weight polysaccharide fragments in the medium was also substantially reduced in both NaCl- and polyethylene glycol-adapted cells, and specifically, the material was composed of lower proportions of xyloglucan fragments. Our results indicate that adaptation to saline or water stress results in inhibition of both the hydrolysis of hemicellulosic xyloglucan and release of uronic acid-rich material into the culture medium.     

Proteins Associated with Adaptation of Cultured Tobacco Cells to NaCl

Cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) adapted to grow in medium containing high levels of NaCl or polyethylene glycol (PEG) produce several new or enhanced polypeptide bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The intensities of some of the polypeptide bands (molecular weights of 58, 37, 35.5, 34, 26, 21, 19.5, and 18 kilodaltons) increase with increasing levels of NaCl adaptation, while the intensities of other polypeptide bands (54, 52, 17.5, and 16.5 kilodaltons) are reduced. Enhanced levels of 43- and 26-kilodalton polypeptides are present in both NaCl and PEG-induced water stress adapted cells but are not detectable in unadapted cells. In addition, PEG adapted cells have enhanced levels of 29-, 17.5-, 16.5-, and 11-kilodalton polypeptides and reduced levels of 58-, 54-, 52-, 37-, 35.5-, 34-, 21-, 19.5-, and 18-kilodalton polypeptide bands.

Synthesis of 26-kilodalton polypeptide(s) occurs at two different periods during culture growth of NaCl adapted cells. Unadapted cells also incorporate ³⁵S into a 26-kilodalton polypeptide during the later stage of culture growth beginning at midlog phase. The 26-kilodalton polypeptides from adapted and unadapted cells have similar partial proteolysis peptide maps and are immunologically cross-reactive. During adaptation to NaCl, unadapted cells synthesize and accumulate a major 26-kilodalton polypeptide, and the beginning of synthesis corresponds to the period of osmotic adjustment and culture growth. From our results, we suggest an involvement of the 26-kilodalton polypeptide in the adaptation of cultured tobacco cells to NaCl and water stress.

     

Alteration of the physical and chemical structure of the primary cell wall of growth-limited plant cells adapted to osmotic stress

Cells of tobacco (Nicotiana tabacum L.) adapted to grow in severe osmotic stress of 428 millimolar NaCl (−23 bar) or 30% polyethylene glycol 8000 (−28 bar) exhibit a drastically altered growth physiology that results in slower cell expansion and fully expanded cells with volumes only one-fifth to one-eighth those of unadapted cells. This reduced cell volume occurs despite maintenance of turgor pressures sometimes severalfold higher than those of unadapted cells. This report and others (NM Iraki et al [1989] Plant Physiol 90: 000-000 and 000-000) document physical and biochemical alterations of the cell walls which might explain how adapted cells decrease the ability of the wall to expand despite diversion of carbon used for osmotic adjustment away from synthesis of cell wall polysaccharides. Tensile strength measured by a gas decompression technique showed empirically that walls of NaCl-adapted cells are much weaker than those of unadapted cells. Correlated with this weakening was a substantial decrease in the proportion of crystalline cellulose in the primary cell wall. Even though the amount of insoluble protein associated with the wall was increased relative to other wall components, the amount of hydroxyproline in the insoluble protein of the wall was only about 10% that of unadapted cells. These results indicate that a cellulosic-extensin framework is a primary determinant of absolute wall tensile strength, but complete formation of this framework apparently is sacrificed to divert carbon to substances needed for osmotic adjustment. We propose that the absolute mass of this framework is not a principal determinant of the ability of the cell wall to extend.     

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.     

Enrichment of vitronectin- and fibronectin-like proteins in NaCl-adapted plant cells and evidence for their involvement in plasma membrane–cell wall adhesion

Cells of tobacco adapted to grow in high concentrations of NaCl develop tight zones of adhesion between the plasma membrane and cell wall, revealed by concave plasmolysis in osmotic solutions. Unadapted cells exhibit mostly convex plasmolysis and exhibit little or no adhesive character. Wall-less protoplasts isolated from the adapted cells retain the complementary adhesive character and adhere tightly to each other, whereas protoplasts from unadapted cells do not. The hexapeptide gly- arg-gly-asp -ser-pro, in which the arg-gly-asp represents the integrin-binding domain of several animal extracellular matrix proteins, specifically blocks adhesion of the protoplasts. A control hexapeptide, gly-arg-gly-glu-ser-pro, is ineffective in blocking adhesion. Tobacco proteins immunologically related to human vitronectin were found in cell walls and membranes of unadapted and NaCl-adapted cells, but the total extractable vitronectin-like protein was enriched in the adapted cells. Tobacco proteins immunologically related to human fibronectin were found in membranes and cell walls of NaCl-adapted cells but not in those from unadapted cells. Our observations indicate that plant cells possess cell-matrix adhesion complexes similar to animal cells, and these adhesion complexes accumulate in growth-limited cells adapted to saline stress.     

Effects of salt and osmotic shocks on unadapted and adapted callus lines of tobacco

Growth, viability and proline content of adapted and unadapted calluses of Nicotiana tabacum L. var. Jayasri, affected due to osmotic stresses and particularly to stress-shocks treated with different osmotica like NaCl (ionic-penetrating), mannitol (non-ionic-penetrating) and polyethylene glycol, (PEG) (non-ionic-non penetrating) were studied to evaluate the physiological differences of stress effects. The tissues adapted to a low concentration of NaCl (85 mM) showed low growth with high proline content compared to the tissues adapted to a low concentration of mannitol (165 mM). Proline content was similar in tissues adapted to high concentrations of NaCl (171 mM) and mannitol (329 mM) but growth in the latter case was relatively low. Growth and viability were subsequently correlated with the pattern of retention in or diffusion of proline out of the tissues after shock-treatments. The loss of tissue viability of the adapted calluses was comparatively less than the unadapted callus even after shock-treatments with 1282 mM NaCl and 823 mM mannitol. The former calluses retained the capability of regrowth though at a slow rate. Such adapted tissues also retained more proline. The mannitol-adapted tissues, when shocked with PEG (200 g l-1), showed low viability with more diffusion and a very little retention of proline while, in the unadapted tissue, all the proline was leached out. The results indicated that the effects of different osmotica on plant tissue varied depending upon the physico-chemical nature of the compounds used as stress-inducing-agents, and retention and diffusion of proline was altered when the tissues were shocked with high concentrations of all these compounds. This revised version was published online in June 2006 with corrections to the Cover Date.     

Changes in pectin structure and localization during the growth of unadapted and NaCl-adapted tobacco cells

Tobacco cells adapted to grow in high concentrations of NaCl exhibit a drastically altered growth physiology that results in cells whose fully expanded volume is only one-fifth to one-eighth those of unadapted cells. Comparison between NaCl-adapted and unadapted tobacco cells provides an opportunity to evaluate current concepts of the structural and mechanical determinants of cell wall expansion. Both biochemical studies of pectic polymers and the ultrastructural localization of pectic epitopes at three specific phases of cell culture, maximal cell division, maximal elongation, and stationary phase are reported here. One-half of the galactosyluronic acid units in wall polymers of NaCl-adapted cells are esterified throughout the culture period, while wall polymers of unadapted cells show a rise in esterified polygalacturonic acid from 50 to 80% during elongation and then a decrease to 70% at stationary phase. Methyl esters account for only a proportion of the total esterified polygalacturonic acid at any stage in both unadapted and NaCl-adapted cell walls. Using monoclonal antibodies, we show differences in the localization of relatively methyl-esterified and unesterified pectic epitopes at different stages of growth and corroborate the chemical determinations. Fourier transform infrared (FTIR) microspectroscopy of representative walls of both NaCl-adapted and unadapted cells confirms, at the single cell wall level, that results obtained from chemical analysis of bulk samples are applicable to the entire cell population. FTIR microspectroscopy also reveals an increase in wall protein in the walls of adapted cells. Images obtained by the fast-freeze, deep-etch, rotary-shadowed replica technique show clearly different cell wall architectures in NaCl-adapted compared with unadapted cells; walls of elongating unadapted cells contain long, thin fibres that show a net orientation with respect to the long axis of the cell, whereas walls of adapted cells have thicker, flatter bundles of fibres with no clear net orientation. Polarized FTIR microspectroscopy indicates that, in unadapted tobacco cells during elongation, pectin molecules may be oriented within the wall in a similar manner to cellulose. Possible ways in which pectin structure and conformation may affect the behaviour of the cellulose-xyloglucan network are discussed.     

Effect of hypertonicity on survival of unprotected human cultured cells following freezing and thawing

An investigation was carried out on the post-thaw survival of unprotected human heteroploid EUE cells, either maintained in isotonic medium (0.137 M NaCl) or adapted to hypertonicity (0.356 M NaCl) and frozen in medium with an increased concentration of NaCl. A fivefold increase in the survival fraction of the adapted cells in comparison with the unadapted ones was observed when cells were frozen in isotonic medium. When cells were frozen in hypertonic medium (0.356 M NaCl), the two cell types exhibit comparable survival values. The results are discussed, with special attention to cell defense mechanisms against freezing injury.     

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.     

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.

     

Salt stress in cultured rice cells: effects of proline and abscisic acid

Abstract. The presence of 1 and 10 mol m⁻³ proline in media containing 100 and 200 mol m⁻³ of NaCl, had little effect on the growth of salt-adapted callus of rice. However, in such callus proline accumulation was stimulated by 10 mol m⁻³ proline in the presence of 100 mol m⁻³ NaCl. On the other hand, with 100 mol m⁻³ NaCl, both 1 and 10 mol m⁻³ proline significantly increased both the growth and proline content of salt-unadapted callus. On replacing NaCl with KCl (100 and 200 mol m⁻³), growth of saltadapted as well as unadapted callus was inhibited, but the presence of 10 mol m⁻³ proline had an ameliorating effect. Abscisic acid (ABA) supressed the growth of both salt-adapted and unadapted callus of rice in the absence of salt stress. ABA inhibited the growth of callus adapted to and grown in 100 and 200 mol m⁻³ of NaCl or when it was replaced by equimolar concentrations of KCl. Growth of 100 mol m⁻³ NaCl adapted cells was inhibited when they were transferred to a medium containing 200 mol m⁻³ of NaCl, but in the presence of ABA it was stimulated. ABA increased the growth of unadapted cells when subjected to different salts. Also, ABA accelerated the adaptation of cells exposed to salt but not to water deficits imposed by nonionic solutes.     

Molecular Cloning of Osmotin and Regulation of Its Expression by ABA and Adaptation to Low Water Potential

In response to adaptation to NaCl, cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) synthesize a major 26 kilodalton protein which has been named osmotin due to its induction by low water potentials. To help characterize the expression of osmotin in adapted cells, a cDNA clone for osmotin has been isolated. Abscisic acid induces messenger RNA encoding osmotin. Levels of this mRNA in adapted cells are approximately 15-fold higher than in unadapted cells. Message for osmotin is present at constant levels through the growth cycle of adapted cells, while in unadapted cells, the level decreases during exponential phase of growth and increases again when the cells approach stationary phase. While abscisic acid induces the message for osmotin, a low water potential environment appears to be required for accumulation of the protein. An osmotic shock to unadapted cells does not increase the amount of message or protein present most likely because this treatment does not induce immediately the accumulation of abscisic acid. The increased expression of osmotin in adapted cells is not correlated with an increase in osmotin gene copy number. Osmotin is homologous to a 24 kilodalton NaCl-induced protein in tomato, as well as thaumatin, maize α-amylase/trypsin inhibitor and a tobacco mosaic virus-induced pathogenesis-related protein.     

Effects of sodium chloride and polyethylene glycol on root-hair infection and nodulation of Vicia faba L. plants by Rhizobium leguminosarum

The effects of sodium chloride and polyethylene glycol (PEG) on the interaction between Rhizobium leguminosarum strain 29d and root hairs of field bean (Vicia faba L. cv. Maris Bead) plants were investigated. Two levels each of NaCl (50 and 100 mol·m^–3) and PEG (100 and 200 mol·m^–3) were given at the time of root-hair formation. Scanning electron microscopy showed rhizobial attachment and colonization on root-hair tips. Adhesion of rhizobia in both lateral and polar orientation, sometimes associated with microfibrils, occurred mainly in crooks at the root-hair tips; most of the infections also occurred here. Bacterial colonization and root-hair curling were both reduced by stress treatments. Polyethylene glycol but not NaCl significantly reduced root-hair diameter. The proportion of root hairs containing infection threads was reduced by 30% under NaCl and by 52% under PEG. The structure of some of the root hairs, epidermal and hypodermal cells, as seen by light microscopy in ultrasections, was distorted as a result of NaCl and PEG treatments; cells showed plasmolysis and folded membranes. After three weeks of treatment, both NaCl and PEG inhibited nodule number by about 50% and nodule weight by more than 60%. It is concluded that the root-hair infection process in Vicia faba is impaired by NaCl and PEG treatments and this in turn results in fewer nodules being produced.Abbreviation PEG polyethylene glycol     

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.     

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.     

Cell growth and water relations of the halophyte,Atriplex nummularia L., in response to NaCl

Summary Growth reduction or cessation is an initial response of Atriplex nummularia L. cells to NaCl. However, A. nummularia L. cells that are adapted to 342 and 428 mM NaCl are capable of sustained growth in the presence of salt. Cells that are adapted to NaCl exhibit a reduced rate of division compared to unadapted cells. Unlike salt adapted cells of the glycophyte Nicotiana tabacum L., A. nummularia L. cells do not exhibit reduced rate of cell expansion after adaptation. However, the cell expansion rate of unadapted A. nummularia L. cells is considerably slower than that of unadapted glycophyte cells and this normally low rate of cell expansion may contribute to the enhanced capacity of the halophyte to tolerate salt. Turgor of NaCl adapted cells was equivalent to unadapted cells indicating that the cells of the halophyte do not respond to salt by osmotic over adjustment as reported for the glycophyte tobacco (Binzel et al. 1985, Plant Physiol. 79:118–125).     

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.     

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     

Proteins Produced during Salt Stress in Tobacco Cell Culture

The protein pattern of cultured tobacco (Nicotiana tabacum L. var Wisconsin 38) cells that have become adapted to a medium containing 10 grams NaCl per liter was compared to that of unadapted cells on one-dimensional sodium dodecyl sulfate gels. Two protein bands (32,000 and 20,000 daltons) were much more abundant in the salt-adapted cells, and one protein (26,000 daltons) was unique to the salt cells. This protein pattern did not change during the growth cycle of the cells. When salt-adapted cells are transferred to control medium, their ability to grow in the salt-containing medium returns to that of control cells after one passage in the control medium (Hasegawa, Bressan, Handa 1980 Plant Cell Physiol 21: 1347). Within this time the levels of the 32,000 and 20,000 dalton proteins also return to that of the control cells, but the 26,000 dalton protein does not disappear until after at least two passages in control medium. Amino acid analyses of these three proteins revealed that they all contain some hydroxyproline.