NaCl-free Medium Research Articles

High efficiency production of 5-hydroxyectoine using metabolically engineered Escherichia coli

The 5-hydroxyectoine is a natural protective agent with long-lasting moisturising and radiation resistance properties. It can be naturally synthesized by some extremophiles using the “bacterial milking” process, but this can corrode bioreactors and downstream purification may cause environmental pollution. In this study, an engineered Escherichia coli (E. coli) strain was constructed for the 5-hydroxyectoine production. First, three ectoine hydroxylases were characterised and the enzyme from Halomonas elongata was the most effective. The L-2,4-diaminobutyrate transaminase mutant was introduced into the engineered strain, which could accumulate 2.8 g/L 5-hydroxyectoine in shake flasks. By activating the glyoxylate cycle and balancing the α-ketoglutarate distribution, the 5-hydroxyectoine titer was further increased to 3.4 g/L. Finally, the optimized strain synthesized 58 g/L 5-hydroxyectoine via a semi-continuous feeding process in a NaCl-free medium. Overall, this study reported the highest titer of 5-hydroxyectoine synthesized by E. coli and established a low-salt fermentation process through the aforementioned efforts.

Antioxidant Response in the Salt-Acclimated Red Beet (Beta vulgaris) Callus

Callus cultures initiated from red beet tubers were acclimated to 75 or 100 mM NaCl salinity by exposing them to gradually increasing NaCl concentrations. The acclimated callus lines displayed growth rates comparable to the control culture cultivated on the NaCl-free medium. Several antioxidant system components were analyzed to assess the role of the antioxidant defense in the acclimated callus’s ability to proliferate on salt-supplemented media. It was found that proline and ascorbate concentrations were increased in salt-acclimated callus lines with respect to the control line. On the other hand, glutathione concentration was unchanged in all tested callus lines. Total activities of the antioxidant enzymes, namely superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and class III peroxidase (POX, EC 1.11.1.7) were increased in salt-acclimated cultures. The enzymatic components of the antioxidant systems were upregulated in a coordinated manner during the initial phases of the culture cycle when the increase in callus fresh mass occurs.

Halotolerant Terephthalic Acid-Degrading Bacteria of the Genus Glutamicibacter

Terephthalic acid (TPA) is an isomer of ortho-phthalic acid, which is widely used in the chemical industry to produce artificial fibers and plastics, including polyethylene terephthalate; it is a widespread environmental pollutant. The ability of two strains of Glutamicibacter spp. PB8-1 (=ВКМ Ac-2934D) and BO25 (=ВКМ Ac2935D) isolated from the salt mining area (Perm krai, Russia) to grow using terephthalic acid as the only source of carbon and energy was studied. The strains PB8-1 and BO25 could utilize high concentrations of TPA (30 g/L), which was shown for TPA-degrading bacteria for the first time. Strains PB8-1 and BO25 were halotolerant bacteria: they grew in the NaCl-free medium or at NaCl concentrations of up to 90 g/L in a rich medium and up to 60 g/L in a mineral medium supplemented with TPA. No bacteria capable of degrading TPA under saline conditions were previously described. The growth of the strain BO25 using TPA was accompanied by the accumulation and subsequent degradation of protocatechuic acid (PCA), suggesting that the TPA metabolism occurred through PCA, which was previously described for bacteria of different taxa, including actinobacteria. Thus, TPA-degrading strains Glutamicibacter spp. PB8-1 and BO25 are promising for the development of bioremediation methods for saline soils and wastewater contaminated with TPA.

Changes in Photosynthetic Pigments Content in Non-Transformed and AtCKX Transgenic Centaury (Centaurium erythraea Rafn) Shoots Grown under Salt Stress In Vitro

The effects of graded sodium chloride (NaCl) concentrations (0-, 50-, 100-, 150-, and 200-mM) on photosynthetic pigment contents in in vitro grown shoots of important medicinal plant species (Centaurium erythraea Rafn) were investigated. Non-transformed, one AtCKX1 and two AtCKX2 transgenic centaury lines, with altered cytokinin profiles, were used in this study. The chlorophyll (Chl) and carotenoid contents differed in the non-transformed and transgenic lines. In general, salinity significantly reduced the Chl a and Chl b contents in comparison to the NaCl-free medium. The lowest Chl content was observed in AtCKX2 transgenic shoots grown on all the culture media. The total carotenoid content was increased in shoots of non-transformed and both AtCKX2 transgenic lines grown in 50-mM NaCl. On the other hand, in concentrations >50-mM NaCl, the total carotenoid content was decreased in all analysed centaury shoots. The Chl a/Chl b ratio in all the shoots increased progressively in the graded NaCl concentrations. Contrarily, the addition of NaCl in the culture medium reduced the Chl/carotenoid ratio in centaury shoots. Taken together, the results of this study partly explained the mode of centaury plant adaptations to salt stress in vitro. Thus, the results on centaury shoots confirmed that the determination of the photosynthetic pigment contents can be a very useful non-destructive screening method in order to discriminate susceptible and resistant plant species/lines to salt stress conditions.

In vitro Propagation of Two Grapevine (Vitis vinifera L.) Cultivars under Conditions of Salt Stress

This study was aimed to investigate salt tolerance of two grapevine cultivars, ‘Chenine Blanc’ and ‘Canonannon’ through in vitro propagation on medium containing different concentrations of NaCl. Single-node shoots were cultured on MS with 1.0 mg/l BAP in combination with 0.1 mg/l IBA and containing 0.25, 0.50, 0.75, 1.00 or 1.50% NaCl. NaCl free medium was used as control. Shoots of both cultivars were cultured on the same MS containing 0.25, 0.50, 0.75 or 1.00% CaCl2 to reduce hyperhydricity. The shoots were transferred to rooting medium followed by acclimatization in greenhouse. Number and length of shoots and roots, number of leaves and nodes, length of nodes, fresh and dry weight of roots and shoots decreased significantly in consistent trend as the concentration of NaCl increased. ‘Canonannon’ cultivar was found to be significantly more tolerant to NaCl than ‘Chenine Blanc’ in all parameters. The lowest percentage of hyperhydric shoots were obtained on medium containing 0.25% CaCl2. Therefore, ‘Canonannon’ cultivar can be planted in relatively saline soils as it is more tolerant to salt than ‘Chenine Blanc’.
 Plant Tissue Cult. & Biotech. 30(1): 47-56, 2020 (June)

Tolerance of testate amoeba species to rising sea levels under laboratory conditions and in the South Pacific

Testate amoebae are ubiquitous unicellular eukaryotic micro-organisms. They occur in high abundances in terrestrial habitats and contribute to important ecological and biogeochemical processes like silicon cycling. Little is known about the growth of testate amoeba populations under hyperosmotic stress, as caused by salinity pulses due to coastal flooding. Given the increasing sea-levels in response to climate change we address this knowledge gap by jointly considering results from a laboratory experiment and a field study in Tuvalu (South Pacific). In the laboratory experiment, Trinema galeata cultures tolerated up to 1 g NaCl L−1 but showed a pronounced decrease in growth rate probably due to interactions with other micro-organisms. Tracheleuglypha dentata cultures had the highest growth rate at 0.5 g NaCl L−1. At higher salt concentrations (5–10 g NaCl L−1), a strongly reduced growth and rapid closing of the shell aperture with a plug occurred, which can be considered as rapid pre-encystment. Euglypha rotunda cultures had the highest growth rates at 1 g NaCl L−1. At higher salt concentrations (5–10 g NaCl L−1), a strongly reduced growth and spherical shrinking of cell plasma occurred, which is again considered as rapid pre-encystment. After seven weeks of exposure, a replacement of the 5 and 10 g NaCl L−1 culture medium by NaCl-free medium resulted in a quick recovery of T. dentata and E. rotunda cultures, but not of T. galeata cultures. Generally, cultures of T. dentata showed the highest growth rates and shortest generation times (up to 0.37/1.91 day−1, respectively). In the field study, different flooding regimes resulted in fundamental changes of the composition of soil amoeba communities. In conclusion, terrestrial testate amoebae can tolerate at least short periods of exposure to moderate salinity caused by flooding stress due to their ability to form cysts and fast growth rates.

The effects of salt stress on physio-biochemical traits, total phenolic and mucilage content of Plantago ovata Forsk under in vitro conditions

Plantago ovata Forsk (psyllium) is an important source of mucilage which is an ingredient in certain drugs and foodstuffs. The calli of 14 genotypes of psyllium were cultured on MS (Murashige and Skoog, 1962) medium containing 0, 100, and 200 mM NaCl, for four weeks, and the effects of salt stress on the following callus traits were evaluated: growth rate (CGR), relative growth rate (RGR), relative water content (RWC), Na+ concentration, K+ concentration, K+/Na+ ratio, proline content, total phenolic compounds (TPC), and mucilage content. A reducing trend was observed in GR, RGR, RWC and K+/ Na+ of the callus cultured in the medium with 100 mM NaCl, comparing to NaCl-free medium, while an increasing trend was observed in Na+ content, proline content, and TPC under the same conditions. Mucilage content of callus was found to increase in the medium containing 100 mM NaCl (0.13 g g-1 DW) but decreased afterwards at 200 mM NaCl (0.117 g g-1 DW), albeit with significant variations among genotypes. The results showed that among evaluated genotypes, Isfahan-1 was the most salt tolerant genotype at cellular level. In addition, the highest mucilage content was obtained in Khor-Biabanak genotype when the calli grown at 100 mM NaCl. It was postulated that mucilage content likely to be associated with salt tolerance and could be exploited to counteract the negative osmotic potential in callus affected by salt stress in P. ovata.

Mutation of a Salmonella serogroup-C1-specific gene abrogates O7-antigen biosynthesis and triggers NaCl-dependent motility deficiency.

Several molecular detection marker genes specific for a number of individual Salmonella serogroups have been recently identified in our lab by comparative genomics for the genotyping of diverse serogroups. To further understand the correlation between serotype and genotype, the function of a Salmonella serogroup-C1-specific gene (SC_2092) was analyzed in this study. It was indicated from the topological prediction using the deduced amino acid sequence of SC_2092 that this putative protein was highly similar to the confirmed Wzx flippases. Furthermore, SDS-PAGE revealed that lipopolysaccharide (LPS) biosynthesis, specifically O-antigen synthesis, was incomplete in an SC_2092 in-frame deletion mutant, and no agglutination reaction with the O7 antibody was exhibited in this mutant. Therefore, it was revealed that this Salmonella serogroup-C1-specific gene SC_2092 encoded a putative flippase, which was required for O7-polysaccharide biosynthesis, and was designated here as wzxC1. Subsequently, the effects of the deletion of wzxC1 on bacterial motility and sodium chloride (NaCl) tolerance were evaluated. The wzxC1 mutant lacked swarming motility on solid surfaces and was impaired in swimming motility in soft agar. Moreover, microscopic examination and RT-qPCR exhibited that an increased auto-aggregation and a strong defect in flagella expression, respectively, were responsible for the reduced motility in this mutant. In addition, the wzxC1 mutant was more sensitive than the wild-type strain to NaCl, and auto-aggregation of mutant cells was observed immediately up on the addition of 1% NaCl to the medium. Interestingly, the motility deficiency of the mutant strain, as well as the cell agglomeration and the decrease in flagellar expression, were relieved in a NaCl-free medium. This is the first study to experimentally demonstrate a connection between a Salmonella serogroup specific gene identified by comparative genomics with the synthesis of a specific O-antigen biosynthesis. Also, our results show that the mutation of wzxC1 triggers a NaCl-dependent motility deficiency.

Quantification of metabolic activity of cultured plant cells by vital staining with fluorescein diacetate

The metabolic activity of suspension cultures of Sonneratia alba cells was quantified by measurement of the hydrolysis of fluorescein diacetate (FDA). FDA is incorporated into live cells and is converted into fluorescein by cellular hydrolysis. Aliquots (0.1–0.75g) of S. alba cells were incubated with FDA at a final concentration of 222μg/ml suspension for 60min. Hydrolysis was stopped, and fluorescein was extracted by the addition of acetone and quantified by measurement of absorbance at 490nm. Fluorescein was produced linearly with time and cell weight. Cells of S. alba are halophilic and proliferated well in medium containing 50 and 100mM NaCl. Cells grown in medium containing 100mM NaCl showed 2- to 3-fold higher FDA hydrolysis activity than those grown in NaCl-free medium. When S. alba cells grown in medium supplemented with 50mM NaCl were transferred to fresh medium containing 100mM mannitol, cellular FDA hydrolysis activity was down-regulated after 4days of culture, indicating that the moderately halophilic S. alba cells were sensitive to osmotic stress. Quantification of cellular metabolic activity via the in vivo FDA hydrolysis assay provides a simple and rapid method for the determination of cellular activity under differing culture conditions.

Isolation and characterization of a vacuolar Na+/H+ antiporter gene from Cucumis melo L.

We isolated a vacuolar Na+/H+ antiporter gene (CmNHX1) from melon using the rapid amplification of cDNA ends approach. Sequence analysis indicated that the full-length cDNA of CmNHX1 was 2534 bp, including an open reading frame (ORF) of 1659 bp, which encoded a deduced polypeptide of 553 amino acids. The deduced protein contained conserved structural domains and shared a high degree of homology with putative vacuolar Na+/H+ antiporters from other higher plants. Subcellular localization revealed that this protein located to the vacuolar membrane. These results indicated that, CmNHX1 is a vacuolar Na+/H+ antiporter. As shown by RT-PCR, expression of CmNHX1 was detected in roots, stems and leaves. In addition, the expression increased in the root but decreased in the leaves with increasing NaCl concentration. Similarly, expression increased with increasing time in plants treated with 100 mM NaCl. Expression of CmNHX1 in ATX3 yeast Na+/H+antiporter mutants showed functional complementation. No differences in yeast cell growth were detected in the presence or absence of CmNHX1 on a NaCl-free medium. However, control yeast growth was noticeably suppressed on medium containing NaCl, whereas, yeasts overexpressing CmNHX1 showed increased population growth rates. These results indicate that the CmNHX1 protein enhanced AXT3 salt tolerance. Keywords: Melon (Cucumis melo L.), Na+/H+ antiporter, salt tolerance, vacuolar membrane, yeast.

Comparative study on the effects of NaCl on selected moss and fern representatives

Salt demonstrates various osmotic and ionic effects on vascular plant growth, development and function, but very few data can be found on how salt affects non-tracheophytes. To explore this, gametophytes of two moss – Bryum argenteum Hedw. and Atrichum undulatum (Hedw.) P. Beauv., and three fern species – Asplenium viride Britton, Ceterach officinarum DC, and Phyllitis scolopendrium (L.) Newman, were treated for 3 days with different NaCl concentrations in growth medium under in vitro controlled conditions. Subsequently, these plants recovered for 18 days on NaCl-free medium, after which the following parameters were measured for mosses: presence of secondary protonema and shoots, protonemal radius and index of multiplication. Survival, chlorophyll a, b, total and a/b ratio were determined as well as total phenolic content, both for ferns and mosses. All species tolerated 50 and 100 mM of NaCl-enriched media, quite well. On higher salt concentrations in the substrata, measured morphological parameters and chlorophyll content were reduced. In general, mosses exhibited higher NaCl tolerance than ferns. Change of phenolic content in ferns suggests these plants use antioxidative properties of phenolics as a mechanism of salt tolerance, in contrast with mosses whose phenolic content was stable.

IN VITRO SELECTION OF ANTHURIUM ANDREANUM FOR SALT STRESS RESISTANCE

The youngest leaves, petioles, and roots of micropropagated anthurium explants of cv. Bolero were regenerated in vitro on media consisting of ½ Murashige-Skoog salts with the ammonium nitrate content lowered to 1/4, and with different combinations of growth regulators. In all the experiments, NaCl was included in the medium at a concentration range of 0, 10, 20, 40, 80, 160 and 320 mM, or 0, 25 and 50 mM. The most effective medium for shoot regeneration contained 0.5 mg/L thidiazuron and 0.5 mg/L NAA. The earliest regeneration was from leaves, then from petioles and roots. The highest NaCl concentration that the explants could survive and regenerate in was below 40 mM when BAP and 2,4-D were used, and 50 mM when TDZ and NAA were used. The shoot regeneration was generally preceded by a globular callus that was light green on the medium without NaCl and yellow with scattered green points on the medium with NaCl. The green shoots on the NaCl medium regenerated 2-3 months later than those on the NaCl-free medium. Most of these shoots yellowed and proved to be escapers, but five survived prolonged selection.

Isolation and preliminary function analysis of a Na + /H + antiporter gene from Malus zumi

A full-length cDNA Na+/H+ antiporter gene (MzNHX1) was isolated from Malus zumi according to the homologous Na+/H+ antiporter gene region in plants. Sequence analysis indicated that the cDNA was 2062 bp in length, including an open reading frame (ORF) of 1629 bp, which encoded a predicted polypeptide of 542 amino acids. The MzNHX1 protein shared high identity with other reported plant vacuolar Na+/H+ antiporters. Southern blot analysis detected multiple copies of MzNHX1 in the M. zumi genome. Northern blot analysis showed negligible expression of the gene in roots, but expression was detected in stems and leaves. To test the function of MzNHX1, we expressed the gene in the saltsensitive AXT3 yeast mutant. No differences in yeast cell growth were detected given the presence or absence of MzNHX1 on a NaCl free medium. However, on a 70 mM NaCl medium, growth in the control transformant was noticeably suppressed, and yeast overexpression of MzNHX1 showed increased population growth rates. These results indicated that the MzNHX1 protein increased AXT3 salt tolerance. Alignments of the deduced Na+/H+ antiporter amino acid sequence of different plants from NCBI revealed that MzNHX1 shared high identity (>86%) with vacuolar Na+/H+ antiporters, including RhNHX1 from rose, cNHX1 from citrus, and AtNHX1 from Arabidopsis thaliana. However, MzNHX1 shared very low identity ( thaliana. These results indicated that MzNHX1 was localized to the vacuolar membrane.

Proteomic and Transcriptomic Analysis of Grapevine PR10 Expression During Salt Stress and Functional Characterization in Yeast

Proteomic and transcriptomic analyses of pathogenesis-related proteins, PR10, were carried out by subjecting adult plants of the salt-tolerant grapevine cv. Razegui to 100 mM NaCl for 6 and 24 h under controlled greenhouse conditions. Within 6 h of salt exposure, an increase in levels of RzPR10 was observed in the roots. PR10 mRNA levels in leaves were significantly different than those in roots; moreover, significant differences for mRNA levels were found between treated and control plants. Within 24 h of salt treatment, roots exhibited the highest levels of RzPR10 protein, but no differences were observed for mRNA levels in both leaves and roots. Overexpression of Vvpr10 cDNA, under the control of the Gal promoter, in the yeast Saccharomyces cerevisiae revealed that growth of transformed cells on a minimal medium containing 500 mM NaCl is similar to that of cells cultivated on NaCl-free medium. This indicated that Vvpr10 gene was functional in yeast and conferred tolerance to high salt concentrations.

Activator Protein-1 Contributes to High NaCl-induced Increase in Tonicity-responsive Enhancer/Osmotic Response Element-binding Protein Transactivating Activity

Tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP) is a Rel protein that activates transcription of osmoprotective genes at high extracellular NaCl. Other Rel proteins NFAT1-4 and NF-kappaB complex with activator protein-1 (AP-1) to transactivate target genes through interaction at composite NFAT/NF-kappaB.AP-1 sites. TonEBP/OREBP target genes commonly have one or more conserved AP-1 binding sites near TonEBP/OREBP cognate elements (OREs). Also, TonEBP/OREBP and the AP-1 proteins c-Fos and c-Jun are all activated by high NaCl. We now find, using an ORE.AP-1 reporter from the target aldose reductase gene or the same reporter with a mutated AP-1 site, that upon stimulation by high extracellular NaCl, 1) the presence of a wild type, but not a mutated, AP-1 site contributes to TonEBP/OREBP-dependent transcription and 2) AP-1 dominant negative constructs inhibit TonEBP/OREBP-dependent transcription provided the AP-1 site is not mutated. Using supershifts and an ORE.AP-1 probe, we find c-Fos and c-Jun present in combination with TonEBP/OREBP. Also, c-Fos and c-Jun coimmunoprecipitate with TonEBP/OREBP, indicating physical association. Small interfering RNA knockdown of either c-Fos or c-Jun inhibits high NaCl-induced increase of mRNA abundance of the TonEBP/OREBP target genes AR and BGT1. Furthermore, a dominant negative AP-1 also reduces high NaCl-induced increase of TonEBP/OREBP transactivating activity. Inhibition of p38, which is known to stimulate TonEBP/OREBP transcriptional activity, reduces high NaCl-dependent transcription of an ORE.AP-1 reporter only if the AP-1 site is intact. Thus, AP-1 is part of the TonEBP/OREBP enhanceosome, and its role in high NaCl-induced activation of TonEBP/OREBP may require p38 activity.

High yield of mannosylglycerate production by upshock fermentation and bacterial milking of trehalose-deficient mutant Thermus thermophilus RQ-1

A production process, using upshock fermentation and osmotic downshock, for the effective production/excretion of mannosylglycerate (MG) by the trehalose-deficient mutant of the strain Thermus thermophilus RQ-1 has been developed. In the first phase of fed-batch fermentation, the knockout mutant was grown at 70 degrees C on a NaCl-free medium. After the culture reached the end of the exponential growth phase, upshift in temperature and NaCl concentration was applied. The temperature was increased to 77 degrees C, and NaCl was added up to 3.0% and kept constant during the second phase of fermentation. Although this shift in cultivation parameters caused a dramatic drop of cell density, a significant improvement in accumulation of MG up to 0.64 micromol/mg protein compared to batch fermentations (0.31 micromol/mg protein) was achieved. A total yield of 4.6 g MG/l of fermentation broth was obtained in the dialysis bioreactor with a productivity of 0.29 g MG l(-1) h(-1). The solute was released from the harvested biomass by osmotic downshock using demineralized water at 70 degrees C. More than 90% of the intracellularly accumulated solute was recovered from the water fraction. The process was very efficient, as hyperosmotic shock, release of the solute, and reiterative fed-batch fermentation could be repeated at least four times.

Development of NaCl-tolerant line in Chrysanthemum morifolium Ramat. through shoot organogenesis of selected callus line

Plants were regenerated successfully through shoot organogenesis of a NaCl-selected callus line of Chrysanthemum morifolium Ramat. cv. Maghi Yellow (a salt sensitive cultivar), developed through stepwise increase in NaCl concentration (0-100mM) in the MS medium. The stepwise increase in NaCl concentration from a relatively low level to cytotoxic level was found to be a better way to isolate NaCl-tolerant callus line, since direct transfer of callus to high saline medium was detrimental to callus survival and growth. The selected callus line exhibited significant increase in superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) activities compared to control callus (grown in medium devoid of NaCl). Stability of salt tolerance character of the selected callus line was checked by growing the calli in NaCl-free medium for 3 consecutive months followed by re-exposure to higher salinity stress (120mM NaCl). Among different growth regulator treatments, a combination of 5mgl(-1) TDZ (Thidiazuron) along with 0.25mgl(-1) NAA and 0.5mgl(-1) GA(3) was found to be the most effective for shoot organogenesis in selected callus line. The regeneration potential of the NaCl-tolerant callus ranged from 20.8% to 0% against 62.4% to 0% in control callus line. Under elevated stress condition (medium supplemented with 250mM NaCl), selected calli derived regenerants (S1 plants) exhibited significantly higher SOD and APX activities over both PC (positive control: control callus derived plants grown on MS medium devoid of NaCl) and NC (negative control: control callus derived plants subjected to 250mM NaCl stress) plants. In addition, the NC plants showed stunted growth, delayed root initiation, and had lesser number of roots as compared to S1 plants. Based on growth performance and antioxidant capacity, the S1 plants could be considered as NaCl-tolerant line showing all positive adaptive features towards the salinity stress. Further study on agronomic performance of these S1 plants under saline soil condition need to be undertaken to check the genetic stability of the induced salt-tolerance.

Regulation of the P2X7 Receptor Permeability to Large Molecules by Extracellular Cl– and Na+

Upon continuous stimulation, the pore of the monovalent cation-selective P2X7 receptor (P2X7R) expands to accommodate large molecules such as N-methyl-D-glucamine (NMDG+). How the change in P2X7R permeability is regulated is not known. Here we report that extracellular Cl- (Cl-(o)) regulates the outward current, whereas extracellular Na+ (Na+(o)) regulates the inward current of large molecules by P2X7Rs. The P2X7R-mediated current was measured in parotid acinar and duct cells of wild type and P2X7R-/- mice and in HEK293 cells expressing the human or mouse P2X7R isoforms. In symmetrical NaCl, triethylammonium chloride, and NMDG+ chloride solutions, the P2X7R current followed a linear current/voltage relationship. In symmetrical NaCl, removal of Cl-(o) reduced the inward Na+ current by approximately 35% and the outward Na+ current by only 10%. By contrast, in the absence of Na+(i) and the presence of Na+(o) or NMDG+(o), the removal of Cl-(o) reduced the inward Na+ or NMDG+ currents by 35% but the outward NMDG+ current by >95%. The effect of Cl-(o) was half-maximal at approximately 60 mm. Reducing Cl-(i) from 150 to 10 mm did not reproduce the effects of Cl-(o). All currents were eliminated in P2X7R-/- cells and reproduced by expressing the P2X7Rs in HEK cells. These findings suggest that Cl-(o) primarily regulates the outward P2X7R current of large molecules. When cells dialyzed with NMDG+ were stimulated in the presence of Na+(o), subsequent removal of Na+(o) resulted in a strongly outward rectifying NMDG+ current, indicating maintained high selectivity for Na+ over NMDG+. During continuous incubation in Na+-free medium, the permeability of the P2X7Rs to NMDG+ gradually increased. On the other hand, when the cells were incubated in symmetrical NMDG+ and only then stimulated with ATP, the NMDG+ current by P2X7Rs followed a linear current/voltage relationship and did not change with time. These findings suggest that the P2X7R has a "Na+(o) memory" and that Na+(o) regulates the inward permeability of P2X7Rs to large molecules. The novel regulation of P2X7R outward and inward permeability to large molecules by Cl-(o) and Na+(o), respectively, may have an important protective function, particularly in secretory epithelial cells.

Salt tolerance of Casuarina equisetifolia and Frankia Ceq1 strain isolated from the root nodules of C. equisetifolia

Effects of NaCl on the seed germination and growth of Casuarina equisetifolia seedlings and multiplication of the Frankia Ceq1 strain isolated from the root nodules of C. equisetifolia were examined. The germination rate of the seeds markedly decreased as the NaCl concentration increased and germination did not occur at 300 mM NaCl. The fresh weight of both shoots and roots of the seedlings treated with NaCl for 6 weeks apparently decreased as the NaCl concentration increased. However, root nodules were formed by inoculation with the Frankia Ceq1 strain in some seedlings treated with 300 mM NaCl and the viability of the seedlings at 500 mM NaCl was almost the same as that of the seedlings not subjected to the NaCl treatment. The Na+ concentration in the shoots sharply increased with the elevation of the NaCl concentration in the ambient solution, but the level was approximately 300 mM even in the seedlings treated with 500 mM NaCl for 6 weeks. On the other hand, the increase of the Na+ concentration in the roots by the NaCl treatment was much smaller than that in the shoots and the level was less than 150 mM. The growth of the free-living Frankia Ceq1 strain was approximately linearly suppressed as the NaCl concentration in the medium increased and the hyphae became somewhat thicker and shorter or disintegrated in the medium containing NaCl at a concentration above 150 mM. The Na+ concentration in the cells increased as the NaCl concentration in the medium increased, but the level was maintained at less than 30 mM even in the medium containing 500 mM NaCl. The cells whose growth was suppressed by the NaCI treatment grew actively again at almost the same rate as the control cells (not subjected to the NaCl treatment) when they were transferred to NaCl-free medium. These results strongly suggested that both C. equisetifolia seedlings and Frankia Ceq1 strain are highly tolerant to salt and this symbiotic system is useful for the recovery of the vegetation in areas with severe salt accumulation.

Selection, Regeneration and Protein Profile Characteristics of NaCl-Tolerant Callus of Robinia pseudoacacia L.

Changes in callus growth, differentiation potency and protein profile induced by salt stress were investigated in the calli induced from a hypocotyl ofRobinia pseudoacacia L. The NaCl treatment evidently obstructed the callus growth and the influences seemed to continue to be present in the selected calli even after they were restored in NaCl-free medium that could partially recover the callus growth, and some polypeptides were found to restore. The calli selected and restored intermittently every two weeks for one year completely lost their differentiation potency. The NaCl-tolerance test of the regenerated plantlets revealed that part of the plantlets that directly regenerated from the selected NaCl-tolerant calli were able to survive in 0.2 M NaCl, while most of the plantlets regenerated from the NaCl-nonselected calli or from restored calli were obviously damaged by this treatment. The SDS-PAGE assay indicated that a 54 kDa polypeptide increased distinctly to a very high level while a 41 kDa polypeptide decreased to a lower level in almost all the NaCl-tolerant plantlets. This observation led to the suggestion that the changes may play an adaptive role in allowing plantlets to survive under saline conditions.