Effect of Salts on Growth and Pectinase Production by Halotolerant Yeast, <Emphasis Type="Italic">Debaryomyces nepalensis</Emphasis> NCYC 3413

In this study, Debaryomyces nepalensis NCYC 3413 isolated from rotten apple was studied for its halotolerance and its growth was compared with that of Saccharomyces cerevisiae in high salt medium. The specific growth rate of D. nepalensis was not affected by KCl even up to a concentration of 1 M, whereas NaCl and LiCl affected the growth of D. nepalensis. Among all tested salts, LiCl showed maximum inhibition on growth. At all conditions, halotolerance of D. nepalensis was much higher than that of S. cerevisiae. D. nepalensis showed maximum viability (80–100%) when grown in KCl, which was higher than with NaCl and LiCl. Pectinase production by D. nepalensis was noted at all high salt concentrations, namely, 2 M NaCl, 2 M KCl, and 0.5 M LiCl, and the maximum specific activity was observed when the strain was grown in 2 M NaCl.     

Osmotic adaptation in halotolerant yeast, Debaryomyces nepalensis NCYC 3413: role of osmolytes and cation transport

Debaryomyces nepalensis NCYC 3413, a food spoiling yeast isolated from rotten apple, has been previously demonstrated as halotolerant yeast. In the present study, we assessed its growth, change in cell size, and measured the intracellular polyol and cations (Na⁺ or K⁺) accumulated during growth in the absence and presence of different concentrations of salts (NaCl and KCl). Cells could tolerate 2 M NaCl and KCl in defined medium. Scanning electron microscopic results showed linear decrease in mean cell diameter with increase in medium salinity. Cells accumulated high amounts of K⁺ during growth at high concentrations of KCl. However, it accumulated low amounts of Na⁺ and high amounts of K⁺ when grown in the presence of NaCl. Cells grown in the absence of salt showed rapid influx of Na⁺/K⁺ on incubation with high salt. On incubation with 2 M KCl, cells grown at 2 M NaCl showed an immediate efflux of Na⁺ and rapid uptake of K⁺ and vice versa. To withstand the salt stress, osmotic adjustment of intracellular cation was accompanied by intracellular accumulation of polyol (glycerol, arabitol, and sorbitol). Based on our result, we hypothesize that there exists a balanced efflux and synthesis of osmolytes when D. nepalensis was exposed to hypoosmotic and hyperosmotic stress conditions, respectively. Our findings suggest that D. nepalensis is an Na⁺ excluder yeast and it has an efficient transport system for sodium extrusion.     

Effects of high concentration of salts on the esterase activity and structure of a kiwifruit peptidase, actinidain

Effects of salts on the activity and stability of actinidain were examined. With increasing salt concentration up to 0.5 M, the activity (kcat/Km) for N-alpha-Cbz-L-lysine p-nitrophenyl ester decreased to 40% of that in the absence of salt. The inhibitor constant Ki of LiCl, NaCl, and KCl was 0.16-0.43 M. With 3 M KCl and NaCl, the specificity constant kcat/Km recovered to 110 and 75%, respectively. No re-activation was observed with LiCl. The inhibition and re-activation were dependent on the changes in both Km and kcat, whereas no CD change was observed. The tryptophan fluorescence of actinidain was not affected by 0-0.5 M salt, but a considerable decrease in its intensity was observed with increasing salt concentration from 0.5 to 3.0 M. These results suggest that the inhibition observed with the lower salt concentration (<0.5 M) is due to attenuation of the electrostatic interaction between the enzyme and substrate, and the higher concentration (0.5-3.0 M) induces structural change in the states of tryptophan residues, which is associated with the re-activation. Actinidain keeps considerably high activity and stability even in the presence of 3 M salts.     

Formation and role of glycine betaine in the moderate halophile Vibrio costicola

The moderate halophile Vibrio costicola, growing on a chemically-defined medium, transformed choline into glycine betaine (betaine) by the membrane-bound enzyme choline dehydrogenase and the cytoplasmic enzyme betainal (betaine aldehyde) dehydrogenase. Choline dehydrogenase was strongly induced and betainal dehydrogenase less strongly induced by choline. The formation of these enzymes was also regulated by the NaCl concentration of the growth medium, increasing with increasing NaCl concentrations. Intracellular betaine concentrations also increased with increasing choline and NaCl concentrations in the medium. This increase was almost completely blocked by chloramphenicol, which does not block the increase in salt-tolerant active transport on transfer from a low to a high salt concentration.Choline dehydrogenase was inhibited by chloride salts of Na⁺, K⁺, and NH _inf4 ^su+ , the inhibition being due to the Cl^- ions. Betainal dehydrogenase was stimulated by 0.5 M salts and could function in up to 2.0 M salts.Cells grew as well in the presence as in the absence of choline in 0.5 M and 1.0 M NaCl, but formed no intracellular betaine. Choline stimulated growth in 2.0 M NaCl and was essential for growth in 3.0 M NaCl. Thus, while betaine is important for some of the adaptations to high salt concentration by V. costicola, it by no means accounts for all of them.Abbreviations CDMM chemically-defined minimal medium - PPT proteose-peptone tryptone medium - SDS sodium dodecyl sulfate Deceased, 1987     

Use of a D-optimal design with constrains to quantify the effects of the mixture of sodium,potassium, calcium and magnesium chloride salts on the growth parameters of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The combined effect of NaCl, KCl, CaCl(2), and MgCl(2) on the water activity (a (w)) and the growth parameters of Saccharomyces cerevisiae was studied by means of a D-optimal mixture design with constrains (total salt concentrations < or = 9.0%, w/v). The a (w) was linearly related to the concentrations of the diverse salts; its decrease, by similar concentrations of salts, followed the order NaCl > CaCl(2) > KCl > MgCl(2), regardless of the reference concentrations used (total absence of salts or 5% NaCl). The equations that expressed the maximum specific growth (mu (max)), lag phase duration (lambda), and maximum population reached (N (max)) showed that the values of these parameters depended on linear effects and two-way interactions of the studied chloride salts. The mu (max) decreased as NaCl and CaCl(2) increased (regardless of the presence or not of previous NaCl); however, in the presence of a 5% NaCl, a further addition of KCl and MgCl(2) markedly increased mu (max). The lambda was mainly affected by MgCl(2) and the interactions NaCl x CaCl(2) and CaCl(2) x MgCl(2). The further addition of NaCl and CaCl(2) to a 5% NaCl medium increased the lag phase while KCl and MgCl(2) had negligible or slightly negative effect, respectively. N (max) was mainly affected by MgCl(2) and its interactions with NaCl, KCl, and CaCl(2); MgCl(2) stimulated N (max) in the presence of 5% NaCl while KCl, NaCl, and CaCl(2) had a progressive decreasing effect. These results can be of interest for the fermentation and preservation of vegetable products, and foods in general, in which this yeast could be present.     

Comparison of the effects of NaCl and KCl at the roots on seedling growth,cell death and the size,frequency and secretion rate of salt glands in leaves of <Emphasis Type="Italic">Limonium sinense</Emphasis>

Twenty days’ exposure to 50 or 100 mM NaCl in the rooting medium substantially increased fresh and dry weights of seedling shoots of the recretohalophyte Limonium sinense while 200 or 300 mM were increasingly inhibitory. KCl treatment was only slightly stimulating (50 mM) or strongly inhibitory (100–300 mM). Lesser effects on leaf area were also seen. Diameter of foliar salt glands was significantly larger than that of controls in 100 and 200 mM NaCl with the effect being reversed at higher concentrations. Gland enlargement was also observed in the presence of 100 mM KCl, while larger concentrations reduced gland size. Generally, gland diameter was larger in the presence of NaCl than in KCl. NaCl and KCl also increased gland number per leaf and secretion rate per gland. At 100 and 200 mM NaCl or KCl, Na⁺ secretion per leaf from NaCl-treated plants exceeded K⁺ secretion rate from KCl-treated plants while at 200 mM, Na⁺ secretion per gland was significantly higher for Na⁺ than for K⁺. Evidence of cell death in leaves of salt-treated plants using Evans blue staining indicates that release of cell contents through loss of membrane integrity contributed to the secretion values. We conclude that the greater tolerance of L. sinenseto to NaCl compared to KCl is linked to the more effective secretion of Na⁺ than of K⁺ and, in turn, to a greater stimulation of salt gland formation and activity and larger gland diameter.     

First evidence for the salt-dependent folding and activity of an esterase from the halophilic archaea Haloarcula marismortui

A gene encoding an esterase from Haloarcula marismortui, a halophilic archaea from the Dead Sea, was cloned, expressed in Escherichia coli, and the recombinant protein (Hm EST) was biochemically characterized. The enzymatic activity of Hm EST was shown to exhibit salt dependence through salt-dependent folding. Hm EST exhibits a preference for short chain fatty acids and monoesters. It is inhibited by phenylmethylsulfonyl fluoride, diethyl-p-nitrophenyl phosphate, and 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one, confirming the conclusion from sequence alignments that Hm EST is a serine carboxylesterase belonging to the hormone-sensitive lipase family. The activity of Hm EST is optimum in the presence of 3 M KCl and no activity was detected in the absence of salts. Far–UV circular dichroism showed that Hm EST is totally unfolded in salt-free medium and secondary structure appears in the presence of 0.25–0.5 M KCl. After salt depletion, the protein was able to recover 60% of its initial activity when 2 M KCl was added. A 3D model of Hm EST was built and its surface properties were analyzed, pointing to an enrichment in acidic residues paralleled by a depletion in basic residues. This peculiar charge repartition at the protein surface supports a better stability of the protein in a high salt environment.     

Occurrence of Salt, pH, and Temperature-tolerant, Phosphate-solubilizing Bacteria in Alkaline Soils

An ecological survey was conducted to characterize 4800 bacterial strains isolated from the root-free soil, rhizosphere, and rhizoplane of Prosopis juliflora growing in alkaline soils. Of the 4800 bacteria, 857 strains were able to solubilize phosphate on plates. The incidence of phosphate-solubilizing bacteria (PSB) in the rhizoplane was highest, followed by rhizosphere and root-free soil. Eighteen bacterial strains out of 857 PSB were able to produce halo at 30°C in a plate assay in the presence of 5% salt (NaCl) and solubilize tricalcium phosphate in National Botanical Research Institute's phosphate growth medium (NBRIP) broth, in the presence of various salts, pHs, and temperatures. Among the various bacteria tested, NBRI4 and NBRI7 did not produced halo in a plate assay at 30°C in the absence of salt. Contrary to indirect measurement of phosphate solubilization by plate assay, the direct measurement of phosphate solubilization in NBRIP broth assay always resulted in reliable results. The phosphate solubilization ability of NBRI4 was higher than in the control in the presence of salts (NaCl, CaCl₂, and KCl) at 30°C. Phosphate solubilization further increased in the presence of salts at 37°C as compared with 30°C. At 37°C, CaCl₂ reduced phosphate solubilization ability of NBRI4 compared with the control. The results indicated the role of calcium salt in the phosphate solubilization ability of NBRI4. Received: 9 March 1999 / Accepted: 16 April 1999     

Conformational change of poly-N epsilon-glutaryl-L-lysine and poly-N epsilon-succinyl-L-lysine in aqueous salt solutions

The conformational changes of poly-N^ε-glutaryl-L -lysine (PGL) and poly-N^ε-succinyl-L -lysine (PSL) in various salt solutions were studied by use of ORD and potentiometric titration measurements. The addition of alkali metal salts to the fully ionized PGL or PSL solution caused helix formation. The helical content of the polymers increases in the following sequences: at salt concentration 0–2 M, CsCl < KCl < LiCl < NaCl; and at 2–3 M, LiCl < CsCl < KCl ～ NaCl. The preferential binding of the solvent components with various alkali metal salts of PGL or PSL was measured in LiCl, NaCl, and KCl solutions by means of equilibrium dialysis and differential refractometry. It was found that with increasing salt concentration, the polymers were preferentially hydrated in NaCl and KCl soultions; however the salt was preferentially bound to the polymers in LiCl solution. Such preferential binding was suggested to be closely related to conformational change. The addition of CaCl₂ to polymer solutions led to the stabilization of the helical structure of PGL or PSL.     

Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress

The comparative analysis of growth, intracellular content of Na⁺ and K⁺, and the production of trehalose in the halophilic Debaryomyces hansenii and Saccharomyces cerevisiae were determined under saline stress. The yeast species were studied based on their ability to grow in the absence or presence of 0.6 or 1.0 M NaCl and KCl. D. hansenii strains grew better and accumulated more Na⁺ than S. cerevisiae under saline stress (0.6 and 1.0 M of NaCl), compared to S. cerevisiae strains under similar conditions. By two methods, we found that D. hansenii showed a higher production of trehalose, compared to S. cerevisiae; S. cerevisiae active dry yeast contained more trehalose than a regular commercial strain (S. cerevisiae La Azteca) under all conditions, except when the cells were grown in the presence of 1.0 M NaCl. In our experiments, it was found that D. hansenii accumulates more glycerol than trehalose under saline stress (2.0 and 3.0 M salts). However, under moderate NaCl stress, the cells accumulated more trehalose than glycerol. We suggest that the elevated production of trehalose in D. hansenii plays a role as reserve carbohydrate, as reported for other microorganisms.     

Salt-tolerance in plants. I. Ions, compatible organic solutes and the stability of plant ribosomes

Abstract Polysomes and ribosomes recovered from a number of plant species were tested for stability when incubated at 25°C in salt solutions in the absence of ATP and initiation factors. Stability was assessed by sucrose density gradient analysis. The stability was inversely proportional to salt concentrations above 125 mol m⁻³ KCl. Polysomes were less stable in the presence of Na⁺ than K⁺ salts, and were much less stable in Cl⁻ than in acetate salts. Polysomes from Triticum aestivum. Hordeum vulgare, Capsicum annuum, Helianthus annuus. Pisum sativum, Atriplex nummularia, Beta vulgaris, Cladophora sp., Enteromorpha sp. and Corallina cuvieri were similarly sensitive to KCl. Polysomes from Ulva lactuca were more sensitive than the other species. Cytoplasmic and plastid polysomes from T. aestivum were similarly unstable in 500 mol m⁻³ KCl. Unprogrammed ribosomal subunit couples from T. aestivum, B. vulgaris and U. lactuca showed Mg²⁺-dependent conformational instability and dissociation in KCl. Slight differences in ribosomal stability were observed between species, but these were unrelated to the salt tolerances of the plants. The ‘compatible’ organic solutes, glycinebetaine and proline, failed to reduce ion-induced instability. Ribosome yield and polysome profiles were similar in leaves of B. vulgaris containing significantly different levels of both Na⁺ and Cl⁻ after growth in media containing 50 or 200 mol m⁻³ NaCl. The results are consistent with the hypothesis that plants maintain a cytoplasmic solute environment that is compatible with ribosomal stability.     

Activity,stability and enantioselectivity of lipase-coated microcrystals of inorganic salts in organic solvents

Lipase-coated microcrystals of inorganic salts were prepared by dissolving enzymes in buffers and then mixing with 3 volumes of saturated salt solutions followed by drop-wise addition into polar precipitating organic solvents. The Mucor javanicus lipase-coated microcrystals did not show any activity for esterification of lauric acid with 1-propanol in isooctane when NaCl and Na₂SO₄ were used as the salts but showed much higher activity than the enzyme powder when KCl (10.0 times) and K₂SO₄ (5.8 times) were used as the salts and precipitated in 1-propanol. Acetonitrile was found to be the best precipitating solvent for preparing M. javanicus lipase-coated microcrystals, with enzyme activities 26.2 and 22.4 times higher than that of the enzyme powder when KCl and K₂SO₄ were used as precipitating salts, respectively. The presence of water in the precipitating solvents markedly decreased the enzyme activity. The M. javanicus lipase-coated microcrystals prepared using K₂SO₄ as the salt and acetonitrile as the precipitating solvent was as active at 80°C as at 40°C. No significant improvement in enantioselectivity of Candida rugosa lipase-coated microcrystals was observed for transesterification of 1-phenylethanol with vinyl acetate in hexane when the microcrystals were prepared by dissolving the enzymes in salt solutions containing 25% (v/v) of acetone or 2-propanol before precipitating in polar solvents.     

Salt-dependent monomer-dimer equilibrium of bovine beta-lactoglobulin at pH 3

Although bovine beta-lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer-dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO(4) was more effective than other salts by about 20-fold, suggesting that anion binding is responsible for the salt-induced dimer formation, as observed for acid-unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO(4), the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (DeltaH(D)) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric beta-lactoglobulin at pH 3.0 with NaClO(4). The net heat effects after subtraction of the heat of salt dilution, corresponding to DeltaH(D), were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the DeltaH(D) value at 20 degrees C and the heat capacity change (DeltaC(p)) of dimer formation. In both NaCl and NaClO(4), the obtained DeltaC(p) value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed DeltaC(p) values were consistent with the calculated value from the X-ray dimeric structure using a method of accessible surface area. These results indicated that monomer-dimer equilibrium of beta-lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.     

The Effect of Various Alkaline Salts on the Glycolate Oxidase of Salicornia europaea and Pisum sativum in vitro

The response of glycolate oxidase from shoots of Salicornia europaea L. and from leaves of Pisum sativum L. to salt treatment during assay was studied by DCPIP reduction and O₂ uptake. In Pisum there was found up to five times more glycolate oxidase activity per gram fresh weight than in Salicornia. However, the calculation of the specific activity pointed out that this result was caused only by the high level of enzyme protein in Pisum, and that specific activity from both species was of equal size. By the DCPIP method it was shown that in test media containing up to 1.0 M NaCl or KCl glycolate oxidase of Salicornia was of equal size compared with the control (medium without additional salts). With 2.0 M NaCl or KCl the activity decreased by about 80 and 30% respectively. Glycolate oxidase of Pisum was somewhat more salt sensitive. 1.0 M NaCl or KCl reduced the activity by about 35%. In the presence of 2.0 M NaCl or KCl the enzyme activity from Pisum was inhibited to about 80 and 60% respectively. By substituting sulfates for chlorides the activity of glycolate oxidase from both Salicornia and Pisum was stimulated strongly. 1.5 M Na₂SO₄ and 0.5 M K₂SO₄ (both are saturated solutions) caused an increase of glycolate activity from Salicornia of about 225 and 185% respectively, and from Pisum of about 50 and 30% respectively. Studying the response of glycolate oxidase to salt treatment by O₂ uptake one must establish that with this method the degree of inhibition of enzyme activity at higher salt concentrations was always more severe than with dye reduction. Addition of 1.0 M NaCl or KCl to the assay medium caused an inhibition of glycolate oxidase activity from Salicornia of about 50% and from Pisum of about 60%. 2.0 M NaCl or KCl reduced the enzyme activity of both Salicornia and Pisum to nearly 10% of control activity. Furthermore, in contrast to DCPIP reduction no stimulating effect of sulfates on glycolate oxidase activity was detectable. Indeed, the inhibitory effect of sulfates was very slight. 1.0 M Na₂SO₄ caused a mean inhibition of glycolate oxidase activity of only 15% with both species, and in the presence of 1.5 M Na₂SO₄ 50% of control activity was measured. At maximal K₂SO₄ concentrations (0.5 M) glycolate oxidase from both Salicornia and Pisum was also unaffected. It is supposed that the described salt tolerance of glycolate oxidase in vitro, possibly is due to an adaptation of the enzyme to high salt levels within peroxisomes in vivo.     

A thermophilic amyloglucosidase fromHalobacterium sodomense,a halophilic bacterium from the Dead Sea

Halobacterium sodomense, a halophilic bacterium from the Dead Sea, degraded starch to glucose by means of an extracellular amyloglucosidase with a temperature optimum of around 65°C in the presence of 1.4 M NaCl, and around 75°C in the presence of 3.9 M NaCl. The enzyme required salt concentrations higher than 1 M for optimal activity, NaCl, KCl, and MgCl₂ being equally suitable as activators. The optimum pH was 7.5.H. sodomense culture supernatants showed only a very low maltose degrading activity. H. sodomense excreted amyloglucosidase constitutively, and relatively high activities were found in cultures grown in the absence of starch; when glucose was added to the growth medium, the amount of enzyme excreted into the medium decreased.     

ELECTRICAL CHARGES OF COLLOIDAL PARTICLES AND ANOMALOUS OSMOSIS : II. INFLUENCE OF THE RADIUS OF THE ION.

1. When solutions of KCl, NaCl, or LiCl are separated from water without salt by a collodion-gelatin membrane and when the pH of both salt solution and water are on the acid side of the isoelectric point of gelatin, water diffuses from the side of pure water into the salt solution at a rate increasing inversely with the radius of the cations. 2. The adsorption theory would lead us to assume that this influence of the cations is due to an increase of the P.D. between the liquid and the membrane inside the pores of the gelatin film of the membrane, but direct measurements of this P.D. contradict such an assumption, since they show that the influence of the three salts on this P.D. is identical at pH 3.0. 3. It is found, however, that the P.D. across the membrane is affected in a similar way by the three cations as is the transport of water through the membrane. 4. This P.D. across the membrane varies inversely as the relative mobility of the three cations which suggests that the influence of the three cations on the diffusion of liquid through the membrane is partly if not essentially due to a diffusion potential.     

Production,purification, and characterization of two extremely halotolerant,thermostable, and alkali-stable α-amylases from Chromohalobacter sp. TVSP 101

The halophilic bacterial strain Chromohalobacter sp. TVSP 101 was shown to produce extracellular, halotolerant, alkali-stable and moderately thermophilic α-amylase activity. The culture conditions for higher amylase production were optimized with respect to NaCl, pH, temperature and substrates. Maximum amylase production was achieved in a medium containing 20% NaCl or 15% KCl at pH 9.0 and 37 °C in the presence of 0.5% rice flour and tryptone. Addition of 50 mM CaCl₂ to the medium increased amylase production by 29%. Two kinds of amylase activity, designated amylase I and amylase II, were purified from culture filtrates to homogeneity with molecular masses of 72 and 62 kDa, respectively. Both enzymes had maximal activity at pH 9.0 and 65 °C in the presence of 0–20% (w/v) NaCl but amylase I was much more stable in the absence of NaCl than amylase II. The enzymes efficiently hydrolyzed carbohydrates to yield maltotetraose, maltotriose, maltose, and glucose as the end products.     

Freezing of isolated thylakoid membranes in complex media: I. The effect of potassium and sodium chloride,nitrate, and sulfate

Thylakoid membranes isolated from spinach leaves (Spinacia oleracea L. cv. Monatol) were subjected to a freeze-thaw cycle in the presence of a buffered medium containing sorbitol as a cryoprotectant and various combinations of potassium and sodium chloride, nitrate, and sulfate. Above a certain total salt concentration, an increase in the concentration of a single electrolyte, or of potassium plus sodium salts with identical anions, always led to a decrease in photophosphorylation activity. A similar effect was obtained with combinations of nitrate plus chloride with identical cations and of KNO₃ plus NaCl. By contrast, in the presence of suitable combinations of NaNO₃ plus KCl, NaNO₃ plus sulfates, and chlorides plus sulfates, inactivation of photophosphorylation was diminished, sometimes dramatically, at initial molarities of nitrate or chloride which alone caused partial or complete membrane damage. When NaNO₃, KCl, and potassium or sodium sulfate were simultaneously present during freezing, thylakoids were affected very little over a wide range of concentration. Diminution or prevention of inactivation of photophosphorylation by suitable combinations of two or more cryotoxic inorganic salts can be explained by postulating that the different solutes act on different sites and that each reduces the concentration of the others by colligative action, together with specific effects of the various electrolytes on individual membrane sites.     

Reduced stomatal responses to light, carbon dioxide and abscisic acid in the presence of sodium ions

Abstract Responses of stomata to light and CO₂ were smaller when detached epidermis of Commelina communis L. was incubated on a medium containing 50 mol m^?3 NaCl than when an equimolar KCl solution was used. Although opening in the light in the absence of CO₂ seemed to be the same whichever salt was present, apertures on KCl solutions were smaller in the dark or with CO₂-containing air. The response to 10^?7 mol dm^?3 ABA was similarly reduced in the presence of NaCl. If there is an optimal NaCl concentration for stomatal CO₂ and light responses it is at or below 25 mol m^?3. These findings point towards control of stomatal movements by light, CO₂ and ABA at the level of cation uptake or extrusion.     

Ion fluxes and abscisic Acid-induced proline accumulation in barley leaf segments

The increase in proline induced by ABA, a process stimulated by NaCl or KCl in barley leaves, did not occur when Na⁺ (or K⁺) was present in the external medium as the gluconate salt, namely with an anion unable to permeate the plasma membrane. However, proline increase was restored, to different extents, by the addition of various chloride salts but not by ammonium chloride. Moreover, it was shown that the stimulation of the process by NaCl (or KCl) was variously affected by the presence of different salts; all the ammonium salts (10 millimolar NH₄⁺ concentration) inhibited this stimulation almost completely. Inhibition by NH₄⁺ was accompanied by a decreased Na⁺ influx (−40%). Also, in the case of Na-gluconate, Na⁺ uptake was reduced and the addition of Cl⁻ as the calcium or magnesium salt (but not as ammonium salt) restored both the ion influxes and the increase in proline typical of NaCl treatments. Both 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS), an anion transport inhibitor, and tetraethylammonium chloride (TEA), a K⁺ channels-blocking agent, caused, as well as with a reduction of ion influxes, an inhibition of the proline accumulation. The inhibition was practically total with 1 millimolar DIDS and about 80% with 20 millimolar TEA. A possible role of ion influxes in the process leading to the increase in proline induced by ABA is proposed.