Energy flux and osmoregulation of Saccharomyces cerevisiae grown in chemostats under NaCl stress.

The energetics and accumulation of solutes in Saccharomyces cerevisiae were investigated for cells grown aerobically in a chemostat under NaCl stress and glucose limitation. Changed energy requirements in relation to external salinity were examined by energy balance determinations performed by substrate and product analyses, with the latter including heat measurements by microcalorimetry. In both 0 and 0.9 M NaCl cultures, the catabolism was entirely respiratory at the lowest dilution rates tested but shifted to a mixed respiratory-fermentative metabolism at higher dilution rates. This shift occurred at a considerably lower dilution rate for salt-grown cells. The intracellular solute concentrations, as calculated on the basis of intracellular soluble space determinations, showed that the internal Na+ concentration increased from about 0.02 molal in basal medium to about 0.18 molal in 0.9 M NaCl medium, while intracellular K+ was maintained around 0.29 molal despite the variation in external salinity. The intracellular glycerol concentration increased from below 0.05 molal at low salinity to about 1.2 molal at 0.9 M NaCl. The concentrations of the internal solutes, however, changed insignificantly with growth rate and energy metabolism. The additional maintenance energy expenditure for growth at 0.9 M NaCl was, depending on the growth rate, 14 to 31% of the total energy requirement for growth at 0 M NaCl. Including the energy conserved in glycerol, the total additional energy demand for growth at 0.9 M NaCl corresponded to 28 to 51% of the energy required for growth at 0 M NaCl.     

On the mechanism of salt tolerance. Production of glycerol and heat during growth of Debaryomyces hansenii.

As glycerol was suggested as an osmotic agent in the salt tolerant Debaryomyces hansenii the concentrations of total, intracellular, and extracellular glycerol produced by this yeast was followed during growth in 4 mM, 0.68 M, and 2.7 M NaCl media. The total amount of glycerol was not directly proportional to biomass production but to the cultural salinity with maximum concentrations just prior to or at the beginning of the stationary phase. In all cultures the cells lost some glycerol to the media, at 2.7 M NaCl the extracellular glycerol even amounted maximally to 80% of the total. A distinct maximum of intracellular glycerol, related to dry weight or cell number, appeared during the log phase at all NaCl concentrations. As the intracellular calculated glycerol concentrations amounted to 0.2 M, 0.8 M, and 2.6 M in late log phase cells at 4mM, 0.68 M, and 2.7 M NaCl, respectively, whereas the corresponding analysed values for the glycerol concentrations of the media were 0.7 mM, 2.5 mM, and 3.0 mM, glycerol contributes to the osmotic balance of the cells. During the course of growth all cultures showed a decreasing heat production related to cell substance produced, most pronounced at 2.7 M NaCl. At 2.7 M NaCl the total heat production amounted to--1690 kJ per mole glucose consumed in contrast to--1200 and--1130 kJ at 4 mM and 0.68 M NaCl, respectively. The Ym-values were of an inverse order, being 129, 120, and 93 at 4 mM, 0.68 M, and 2.7 M NaCl respectively.     

Regulation of internal solute concentrations of marine Vibrio alginolyticus in response to external NaCl concentration.

Slightly halophilic marine Vibrio alginolyticus grown in the range of NaCl from 0.2 to 1.5 M maintained the total internal solute concentration always higher than the external medium by about 0.25 osM. The concentrations of macromolecules such as DNA, RNA, and protein were little affected by the increase in medium NaCl. The internal K+ concentration was kept to about 400 mM in the range of medium NaCl from 0.4 to 0.8 M; it rose to 510 mM when the bacterium was grown in 1.5 M NaCl, indicating that K+ increased only slightly in response to the large increase in medium NaCl. Thus, in contrast to the case of nonhalophilic and extremely halophilic bacteria, K+ was unlikely to act as a major component to regulate the internal solute concentration of marine V. alginolyticus. The internal Na+ and Cl- concentrations were maintained always lower than those in the growth medium, but they increased in response to the increase in medium NaCl. The concentration of internal Na+ was close to that of K+ at the concentration of medium NaCl that supports the optimal growth of this organism. The total amino acid content of V. alginolyticus increased from 76 to 413 mM by the increase in medium NaCl from 0.2 to 1.5 M. The concentrations of glutamic acid and prolined were 254 and 72 mM, respectively, when grown in 1.5 M NaCl. These results indicated that Na+, Cl- and amino acids, especially glutamic acid and proline, contributed to the regulation of internal solute concentration of V. alginolyticus in response to the increased external NaCl.     

Polyhydric alcohol production and intracellular amino acid pool in relation to halotolerance of the yeast Debaryomyces hansenii

Changes in polyol production and the intracellular amino acid pool were followed during the growth cycle of Debaryomyces hansenii in 4 mM and 2.7 M NaCl media. The intracellular levels of polyols were markedly enhanced by high salinity, the dominant solutes being glycerol in log phase cells and arabinitol in stationary phase cells. At low salinity arabinitol was the most prominent intracellular solute throughout the growth cycle. There were no major changes in the composition of the total amino acid pool with changes in cultural salinity. The amount of total free amino acids related to cell dry weight was 15–50% lower in cells cultured in 2.7 M NaCl as compared to 4 mM NaCl media.After subtraction of contributions from intracellular polyols the calculated cellular C/N ratio was found to be unaffected by cultural age and salinity during the late log and early stationary phase. On prolonged incubation of stationary phase cells, this ratio decreased, particularly at high salinity. The sensitivity of cells towards exposure to high salinity was measured in terms of the length of the lag phase after transference to 2.7 M NaCl media. This lag phase decreased with increasing intracellular polyol concentrations. At a given polyol content, stationary phase cells were considerably less sensitive than were log phase cells.When cultured at high salinity the mutant strain, 26-2b, grew more slowly and retained less of the total polyol produced during the early growth stages than did the wildtype. Exogenously supplied mannitol, arabinitol, and glycerol stimulated the growth of the mutant in saline media. Erythritol was without effect.Abbreviations GLC gas-liquid chromatography - TCA trichloroacetic acid     

The ATP pool in relation to the production of glycerol and heat during growth of the halotolerant yeast Debaryomyces hansenii

In a study of the halotolerant yeast Debarymyces hansenii cultured in 4 mM and 2.7 M NaCl the intracellular ATP pool, the heat production, the oxygen uptake, and, in the high culture salinity also, the intracellular glycerol concentration were found to be correlated. The intracellular ATP in the 2.7 M NaCl culture had a constant concentration of 3.5 mM ATP during the second half of the lag phase, while in 4 mM NaCl it rose to a maximum of 3.1 mM during the late log phase. The intracellular glycerol concentration in 2.7 M NaCl was about 1.3M during the entire exponential growth phase. Sine the glycerol concentration of the medium was not more than 0.23 mM, glycerol must contribute to the osmotic balance of the cells in high salinity. The corresponding maximum values for the 4 mM NaCl culture were 0.16 M and 0.08 mM. The experimental enthalpy changes were approximately the same for the two salinities, viz. about-1200 kJ per mole consumed glucose. The Y _m-values for the 4 mM and 2.7 M NaCl cultures were 91 and 59, respectively, the difference being a consequence of the decreased efficiency of growth in high salinity.Abbreviations CFU colony-forming units - PCA perchloric acid - TCA trichloroacetic acid     

On the mechanism of salt tolerance

As glycerol was suggested as an osmotic agent in the salt tolerantDebaryomyces hansenii the concentrations of total, intracellular, and extracellular glycerol produced by this yeast was followed during growth in 4 mM, 0.68 M, and 2.7 M NaCl media. The total amount of glycerol was not directly proportional to biomass production but to the cultural salinity with maximum concentrations just prior to or at the beginning of the stationary phase. In all cultures the cells lost some glycerol to the media, at 2.7 M NaCl the extracellular glycerol even amounted maximally to 80% of the total. A distinct maximum of intracellular glycerol, related to dry weight or cell number, appeared during the log phase at all NaCl concentrations. As the intracellular calculated glycerol concentrations amounted to 0.2 M, 0.8 M, and 2.6 M in late log phase cells at 4 mM, 0.68 M, and 2.7 M NaCl, respectively, whereas the corresponding analysed values for the glycerol concentrations of the media were 0.7 mM, 2.5 mM, and 3.0 mM, glycerol contributes to the osmotic balance of the cells.During the course of growth all cultures showed a decreasing heat production related to cell substance produced, most pronounced at 2.7 M NaCl. At 2.7 M NaCl the total heat production amounted to-1690 kJ per mole glucose consumed in contrast to-1200 and-1130 kJ at 4 mM and 0.68 M NaCl, respectively. TheY _m -values were of an inverse order, being 129, 120, and 93 at 4 mM, 0.68 M, and 2.7 M NaCl, respectively.     

Choline Derivatives Involved in Osmotolerance of Penicillium fellutanum

Penicillium fellutanum is osmotolerant and xerotolerant when cultured in a low-phosphate medium containing 3 M NaCl. Glycerol and erythritol accumulated in cultures with NaCl concentrations up to 2 M; glycerol was the only detectable polyol in cultures containing 3 M NaCl. In cultures with 3 M NaCl, the intracellular levels of glycine betaine and choline-O-sulfate were 22- and 2.6-fold greater (70 and 46 mM), respectively, than those of cultures without added NaCl. The levels of glycine betaine and glycerol decreased in mycelia transferred from a medium containing 3 M NaCl into a fresh medium without added NaCl. NaCl at 3 M inhibited mycelial mass accumulation; this inhibition was partially corrected by supplementation of cultures with glycine betaine (2 mM) or choline-O-sulfate (10 mM). The presence of exogenous choline chloride (2 mM) in plate cultures protected the cells from stress from 3 M NaCl. The data suggest that glycine betaine and choline-O-sulfate are secondary osmoprotectants which are effective at the point that the cell is incapable of synthesizing more glycerol.     

The intracellular Na+ and K+ composition of the moderately halophilic bacterium, Paracoccus halodenitrificans

The intracellular concentrations of Na+ and K+ in exponentially growing Paracoccus halodenitrificans were independent of the NaCl concentration of the growth medium. The observed values were approximately 100 and 300 mM for Na+ and K+, respectively. In stationary phase cells, the ultimate values for Na+ depended on the NaCl concentration of the growth medium. With cells grown in the presence of 1 M NaCl, the value was about 500 mM; for cells grown in the presence of 3 M NaCl, the value was about 1.1 M. The K+ concentration in stationary phase cells was unaffected by the NaCl concentration in the growth medium. The final value was about 100 mM. Associated with these changes were changes in the ATP pool and decreases in the activities of the NADH oxidase system and the membrane-bound ATPase. It is proposed that the decrease in the activities of these enzymes may account for the ion flows observed in stationary phase cells.     

Regulation of intracellular osmotic pressure during the initial stages of salt stress in a salt-tolerant yeast, Zygosaccharomyces rouxii.

The accumulation of glycerol and inorganic ions as it related to osmotic pressure, and the regulation of intracellular osmotic pressure in a salt-tolerant yeast, Zygosaccharomyces rouxii, were examined for several hours after salt stress. Intracellular contents of glycerol increased for up to 6 h in media supplemented with 1 M and 2 M NaCl and did not increase in medium containing 3 M NaCl. Intracellular contents of Na+ and Cl- reached a maximum value within 1 and 3 h, respectively, in all NaCl-containing media and increases were proportional to the concentration of NaCl in the medium. As glycerol was accumulated in cells, the intracellular contents of Na+ and Cl- gradually decreased in media containing 1 M and 2 M NaCl. After salt stress, cell volume decreased within 1 h and the original volume was re-established for 3 to 6 h in media with 1 M and 2 M NaCl but not in medium with 3 M NaCl. Intracellular concentrations of solutes, which were calculated from the total contents of glycerol and inorganic ions and the cell volume, became almost equivalent to the external osmotic pressure within 1 h after salt stress. Experiments using various inhibitors showed that a large amount of ATP was required not only for the synthesis and accumulation of glycerol but also for the exclusion of Na+ and Cl- from cells under salt-stressed conditions.     

Glycine betaine, carnitine, and choline enhance salinity tolerance and prevent the accumulation of sodium to a level inhibiting growth of Tetragenococcus halophila

Natural-abundance (13)C-nuclear magnetic resonance was used to probe the intracellular organic solute content of the moderately halophilic bacterium Tetragenococcus halophila. When grown in complex growth media supplemented or not with NaCl, T. halophila accumulates glycine betaine and carnitine. Unlike other moderate halophiles, T. halophila was not able to produce potent osmoprotectants (such as ectoines and glycine betaine) through de novo synthesis when cultured in defined medium under hyperosmotic constraint. Addition of 2 mM carnitine, glycine betaine, or choline to defined medium improved growth parameters, not only at high salinity (up to 2.5 M NaCl) but also in media lacking NaCl. These compounds were taken up when available in the surrounding medium. The transport activity occurred at low and high salinities and seems to be constitutive. Glycine betaine and carnitine were accumulated by T. halophila in an unmodified form, while exogenously provided choline led to an intracellular accumulation of glycine betaine. This is the first evidence of the existence of a choline-glycine betaine pathway in a lactic acid bacterium. An assay showed that the compatible solutes strikingly repressed the accumulation of glutamate and slightly increased the intracellular potassium level only at high salinity. Interestingly, osmoprotectant-treated cells were able to maintain the intracellular sodium concentration at a relatively constant level (200 to 300 nmol/mg [dry weight]), independent of the NaCl concentration of the medium. In contrast, in the absence of osmoprotectant, the intracellular sodium content increased sharply from 200 to 2,060 nmol/mg (dry weight) when the salinity of the medium was raised from 1 to 2 M. Indeed, the imported compatible solutes play an actual role in regulating the intracellular Na(+) content and confer a much higher salt tolerance to T. halophila.     

Short- and Long-Term Effects of Salinity on Leaf Growth in Rice (Oryza sativa L.)

Addition of 50 mM NaCl to Oryza sativa L. had little effectupon the time of leaf initiation, but leaf mortality prior tothe normal phase of senescence was increased and the onset ofsenescence was advanced. There was no significant effect uponthe day-to-day pattern of growth, nor upon the ultimate length,of leaves that were developing at the time of, or shortly after,salinization with 50 mM NaCI. Leaves that developed after prolongedexposure of the plants to salinity were shorter. Addition ofNaCl, KC₁ or mannitol to the root medium brought about a cessationof leaf elongation within one minute. Growth at a reduced raterestarted abruptly after a lag period that depended upon theexternal concentration. Elongation rate recovered its originalvalue within 24 h after exposure to 50 mM NaCl, though not athigher concentrations. Addition of NaCl at concentrations upto 100 mM elicited no short-term effects upon photosyntheticgas exchange. Na uptake contributed to osmotic adjustment ofthe growing zone. When plants were rapidly exposed to 50 mMNaCl, no change in turgor pressure was detectable in the growingzone with the resolution of the miniature pressure probe used(about 70 kPa). It is concluded that the initial growth reductionin rice caused by salinization is due to a limitation of watersupply. A clear distinction is made between the initial effectsof low salinity which are recoverable and the long-term effectswhich result from the accumulation of salt within expanded leaves. Key words: Leaf elongation, gas exchange, photosynthesis, water relations     

Osmotic regulation in the marine alga, Codium decorticatum. I. Regulation of turgor pressure by control of ionic composition.

Codium decorticatum regulates its internal ionic composition and osmotic pressure in response to changes in external salinity. Over a salinity range of 23 to 37% (675 to 1120 mosmol/kg) Codium maintains a constant turgor pressure of 95 mosmol/kg (2.3 atm), observed as a constant difference between internal and external osmotic pressures. The changes in internal osmotic pressure are due to changes in intracellular inorganic ions. At 30 0/00 salinity the major intracellular ions are present in the following concentrations (mmol/kg cell H20): K+, 295; Na+, 255; Cl-, 450. At different salinities intracellular ion concentrations remain in constant proportion to the external ion concentrations, and thus the equilibrium potentials are approximately constant. The potential difference between the vacuole and seawater (-76 mV), whici is predominantly a K+ diffusion potential, is also constant with changing salinity. Comparison of the equilibrium potentials with the vacuole potential suggests that Cl- is actively absorbed and Na+ actively extruded, whereas K+ may be passively distributed between the vacuole and seawater. Turgor pressure does not change with environmental hydrostatic pressure, and increasing the external osmotic pressure with raffinose elicits a response similar to that obtained by increasing the salinity. These two results suggest that the stimulus for turgor regulation is a change in turgor pressure rather than a change in internal hydrostatic pressure or ion concentrations.     

Osmoadaptation in representatives of haloalkaliphilic bacteria from soda lakes

The adaptation of microorganisms to life in brines allows two strategies: the accumulation of organic osmoregulators in the cell (as in many moderate halophiles, halomonads in particular) or the accumulation of inorganic ions at extremely high intracellular concentrations (as, for example, in haloanaerobes). To reveal the regularities of osmoregulation in haloalkaliphiles developing in soda lakes, Halomonas campisalis Z-7398-2 and Halomonas sp. AIR-2 were chosen as representatives of halomonads, and Natroniella acetigena, as a representative of haloanaerobes. It was established that, in alkaliphilic halomonads, the intracellular concentrations of inorganic ions are insufficient for counterbalancing the environmental osmotic pressure and balance is attained due to the accumulation of organic osmoregulators, such as ectoine and betaine. On the contrary, the alkaliphilic haloanaerobe N. acetigena employs K+, Na+, and Cl- ions for osmoregulation. High intracellular salt concentrations increasing with the content of Na+ in the medium were revealed in this organism. At a concentration of 1.91 M Na+ in the medium, N. acetigena accumulated 0.83 M K+, 0.91 M Na+, and 0.29 M Cl- in cells, and, with an increase in the Na+ content in the medium to 2.59 M, it accumulated 0.94 M K+, 1.98 M Na+, and 0.89 M Cl-, which counterbalanced the external osmotic pressure and provided for cell turgor. Thus, it was shown that alkaliphilic microorganisms use osmoregulation strategies similar to those of halophiles and these mechanisms are independent of the mechanism of pH homeostasis.     

Effect of salinity and temperature on germination, growth and ion relations of Panicum turgidum Forssk

Seed germination of Panicum turgidum was significantly affected by salinity levels, temperature and their interaction. Maximum germination was noted in the lowest saline media (25-50 mM) and distilled water at the temperature of 15-25 degrees C and 20-30 degrees C. Seeds germination was substantially delayed and reduced with an increase in NaCl to levels above 50mM. This trend was much pronounced under high levels of NaCl and incubation temperature. Low levels of NaCl (25-50 mM) stimulated shoot and root dry weights of P. turgidum seedlings. However, the highest NaCl levels (>100 mM) resulted in a significant decrease in shoot, root and total dry weights of seedlings. Intermediate degrees of temperature, 15-25 and 20-30 degrees C, resulted in a significant increase in biomass accumulation. The Na+ concentration in shoots and roots significantly increased as NaCl concentration increased. The K+ concentration in roots and K/Na ratio in shoots and roots was significantly reduced as salinity concentration increased. The K/Na ratio was greatly affected by higher NaCl concentration and incubation temperatures.     

Adaptation of yeasts to salt stress (review)]

Mechanisms underlying adaptation of various yeasts to salt stress were summarized. Stress response involves modulation of enzymatic activities and changes in gene expression. Elevated salinity of the environment can be regarded as a two-factor stress process that includes an osmotic component leading to loss of cellular turgor and a toxic component inhibiting a set of functions due to an increase in the intracellular concentration of Na+. Adaptation of yeast cells to these stress conditions obligatorily involves the accumulation of osmotically active compounds (mainly glycerol) to counterbalance an increased external osmotic pressure and the modification of plasma membrane transport systems to extrude Na+ from the cell.     

Characterization of putrescine production in nongrowing Vibrio parahaemolyticus cells in response to external osmolality

Nongrowing Vibrio parahaemolyticus cells rapidly produced putrescine (Put) from added arginine when subjected to a low osmotic stress. This phenomenon was characterized in connection with a regulatory mechanism of the responsible enzymes, arginine decarboxylase (ADC) and agmatine ureohydrolase (AUH). NaCl, KCl, LiCl, sucrose, and glycerol were used as solutes to prepare the resuspending media with various osmolalities. Regardless of whether the solutes were electrolytes or non-electrolytes, exposure of cells to low osmolality brought about instantaneous increases in both intra- and extracellular Put contents without significant changes in the contents of other polyamines. This acceleration in Put production was accompanied by no increases in the specific activities of ADC and AUH. On the other hand, when cells were exposed to the osmolality equivalent to 2 or 5% NaCl, all solutes except for glycerol did not cause a remarkable variation in the intracellular Put content, while the amount of Put in the medium varied depending on the solute used; sucrose and glycerol still greatly prompted Put production, as judged by high Put contents in the media, even at the osmolality equivalent to 5% NaCl. The cation efflux from cells, measured as the K+ release, was observed whenever the increase in Put production occurred. Furthermore, in vitro experiments showed that NaCl and KCl inhibited ADC to a similar extent, about 70% inhibition being observed at 200 mM. However, AUH was not affected by these compounds. These results suggest that the reduction in the concentrations of Na+ and K+ predominantly present in cells may cause the increase in activity of the preexisting ADC, which leads to the enhancement of Put production.     

Alterations in Ca2+-binding on plasma membrane after adaptation to salt stress of tobacco cells in suspension

Electrophoresis was used to study effects of salinity on the characteristics of Ca2+ binding to the outer surface of plasma membrane (PM) of protoplasts isolated from two types of tobacco (Nicotiana tabacum L., cv. Bright Yellow) cultured cells that were adapted (tolerant) and unadapted (sensitive) to 50 mM NaCl stress. Electrophoretic analysis of salt-sensitive NaCl-unadapted cells shows that Na+ induced an appreciably higher degree of reduction in the amount of Ca2+ bound to PM compared with K+ with increasing concentration from 0.1 to 30 mM. In salt-tolerant NaCl-adapted cells, however, both Na+ and K+ ions induced almost the same degree of reduction in the amount of Ca2+ bound to PM in the physiological concentration range of Ca2+ in the medium between 2 and 4 mM. These results suggest that, under the physiological conditions, PM of salt-sensitive NaCl-unadapted cells has an appreciable amount of PM-bound Ca2+ that is desorbed much easier by Na+ than K+, whereas PM of salt-tolerant NaCl-adapted cells has the PM-bound Ca2+ that can be equally desorbed by Na+ and K+.     

Glycine Betaine, Carnitine, and Choline Enhance Salinity Tolerance and Prevent the Accumulation of Sodium to a Level Inhibiting Growth of Tetragenococcus halophila

Natural-abundance ¹³C-nuclear magnetic resonance was used to probe the intracellular organic solute content of the moderately halophilic bacterium Tetragenococcus halophila. When grown in complex growth media supplemented or not with NaCl, T. halophila accumulates glycine betaine and carnitine. Unlike other moderate halophiles, T. halophila was not able to produce potent osmoprotectants (such as ectoines and glycine betaine) through de novo synthesis when cultured in defined medium under hyperosmotic constraint. Addition of 2 mM carnitine, glycine betaine, or choline to defined medium improved growth parameters, not only at high salinity (up to 2.5 M NaCl) but also in media lacking NaCl. These compounds were taken up when available in the surrounding medium. The transport activity occurred at low and high salinities and seems to be constitutive. Glycine betaine and carnitine were accumulated by T. halophila in an unmodified form, while exogenously provided choline led to an intracellular accumulation of glycine betaine. This is the first evidence of the existence of a choline-glycine betaine pathway in a lactic acid bacterium. An assay showed that the compatible solutes strikingly repressed the accumulation of glutamate and slightly increased the intracellular potassium level only at high salinity. Interestingly, osmoprotectant-treated cells were able to maintain the intracellular sodium concentration at a relatively constant level (200 to 300 nmol/mg [dry weight]), independent of the NaCl concentration of the medium. In contrast, in the absence of osmoprotectant, the intracellular sodium content increased sharply from 200 to 2,060 nmol/mg (dry weight) when the salinity of the medium was raised from 1 to 2 M. Indeed, the imported compatible solutes play an actual role in regulating the intracellular Na⁺ content and confer a much higher salt tolerance to T. halophila.     

Distinction of two components of passive Ca2+ transport into human erythrocytes by Ca2+ entry blockers

The nature of downhill Ca2+ net-transport into human erythrocytes was investigated using the experimental models of Ca2+ pump inhibition by vanadate and of intracellular chelation of Ca2+ by quin2. Ca2+ uptake by erythrocytes loaded with 0.5 mM vanadate and suspended in 145 mM Na+ -5 mM K+ media was reduced by about 60% when medium K+ was raised to 80 mM. Organic and inorganic Ca2+ entry blockers such as nifedipine (10(-5) M), verapamil (10(-4) M), diltiazem (10(-4) M), Co2+ (1.5 mM) and Cu2+ (0.1 mM) as well as the K+ channel blocker quinidine (1mM) inhibited Ca2+ uptake in 145 mM Na+ -5 mM K+ media by 60-75%. Flunarizine was less effective. In vanadate-loaded cells suspended in 70 mM Na+ -80 mM K+ media, in contrast, flunarizine exerted a dose-dependent inhibition of Ca2+ uptake by up to 80% at 10(-5) M, the other blockers being ineffective (except for verapamil at 10(-4) M). A similar pattern of inhibition was seen in quin2-loaded erythrocytes. The different susceptibility towards inhibitors may indicate that passive Ca2+ uptake by vanadate-loaded erythrocytes suspended in 145 mM Na+ -5 mM K+ media, on the one hand, and by vanadate-loaded erythrocytes suspended in 70 mM Na+ -80 mM K+ media as well as by quin2-loaded erythrocytes, on the other hand, is mediated by two different transport components.     

Response of the pear rootstock to boron and salinity in vitro

The effects of boron and NaCl induced salinity on growth and mineral composition of the pear (Pyrus communis L.) rootstock OH × F 333 shoots cultured in vitro were investigated. Shoots were grown in vitro for seven weeks on a Murashige and Skoog medium containing two B concentrations (0.1 and 2 mM) combined with five NaCl concentrations (0, 10, 20, 40, and 80 mM). The longest shoots were produced at 0.1 mM B and 80 mM NaCl, but highest number of shoots were produced at 0.1 mM B and 0–20 mM NaCl. Inclusion of 20 and 40 mM NaCl in the culture medium significantly increased fresh mass of cultures compared to 0 mM NaCl for all B concentrations tested. The concentrations of P, K, Ca, Mg, Na, Fe, Mn and Zn of plants were affected by B and NaCl concentration of the medium.