NaCl Adaptation Research Articles

Study on the intracellular adaptative mechanism of Acidithiobacillus caldus MTH-04 to NaCl stress

To understand the adaptive mechanism of bioleaching microorganism Acidithiobacillus caldus MTH-04, its physiology and metabolic changes at the transcriptional level were systemically studied. The results of growth curves, SO42− content, pH and flow cytometry analyses indicated that the higher the NaCl concentration, the more the strain was inhibited. The transcriptome response of A. caldus to elevated NaCl concentrations included changes in carbon flux, elevated glutathione synthesis, alterations in cell wall and membrane composition, the down-regulation in genes involved in flagellar synthesis and rotation, the reduced energy generation through sulfur oxidation, and the up-regulation in genes involved in DNA and protein repair. Based on the transcriptome results, the effects of proline and glutathione on NaCl adaptation in A. caldus were analyzed separately. We found that either the exogenous addition of proline and glutathione or the intracellular overexpression of the enzymes responsible for the synthesis of these two substances contributed to the enhancement of the adaptive capacity of A. caldus under NaCl stress. The findings offer insight into the design of chloride-based techniques for the bioprocessing of minerals.

Transcriptomic analysis reveals the molecular adaptation to NaCl stress in Zostera marina L.

The seagrass Zostera marina L. shows optimal growth in marine water and reduced growth under low salinity conditions. However, little is known about the molecular mechanisms underlying its adaptation to high salinity in Z. marina. In this study, transcriptomic analyses were performed using RNA-seq of the following two groups with different NaCl content: the CK group (seagrasses grown in the absence of NaCl) and the NaCl group (seagrasses grown in the presence of 400 mM NaCl for 6 h). Approximately 316 million high-quality reads were generated, and 87.9% of the data were mapped to the reference genome. Moreover, differentially expressed genes between the CK and NaCl groups were identified. According to a functional analysis, the up-regulated genes after the NaCl treatment were significantly enriched in nitrogen metabolism, calcium signalling and DNA replication while the down-regulated genes were significantly enriched in photosynthesis. A comparative transcriptomic analysis detected many differentially expressed genes and pathways required for adaptation to NaCl stress, providing a foundation for future studies investigating the molecular mechanisms of salt adaptation in Z. marina. We discuss how molecular changes in these processes may have contributed to the NaCl adaptation.

The Correlation between NaCl Adaptation and Heat Sensitivity of Listeria monocytogenes, a Foodborne Pathogen through Fresh and Processed Meat.

This study examined the relationship between NaCl sensitivity and stress response of Listeria monocytogenes. Nine strains of L. monocytogenes (NCCP10805, NCCP10806, NCCP10807, NCCP10808, NCCP10809, NCCP10810, NCCP10811, NCCP10920 and NCCP 10943) were exposed to 0%, 1%, 2% and 4% NaCl, and then incubated at 60℃ for 60 min to select strains that were heat-sensitized (HS) and non-sensitized (NS) by NaCl exposure. After heat challenge, L. monocytogenes strains were categorized as HS (NCCP 10805, NCCP10806, NCCP10807, NCCP10810, NCCP10811 and NCCP10920) or NS (NCCP10808, NCCP10809 and NCCP10943). Total mRNA was extracted from a HS strain (NCCP10811) and two NS strains (NCCP10808 and NCCP10809), and then cDNA was prepared to analyze the expression of genes (inlA, inlB, opuC, betL, gbuB, osmC and ctc) that may be altered in response to NaCl stress, by qRT-PCR. The expression levels of two invasion-related genes (inlA and inlB) and two stress response genes (opuC and ctc) were increased (p<0.05) in NS strains after NaCl exposure in an NaCl concentration-dependent manner. However, only betL expression was increased (p<0.05) in the HS strains. These results indicate that the effect of NaCl on heat sensitization of L. monocytogenes is strain dependent and that opuC and ctc may prevent NS L. monocytogenes strains from being heat sensitized by NaCl. Moreover, NaCl also increases the expression of invasion-related genes (inlA and inlB).

Characterization of NaCl tolerance in Desulfovibrio vulgaris Hildenborough through experimental evolution

Desulfovibrio vulgaris Hildenborough strains with significantly increased tolerance to NaCl were obtained via experimental evolution. A NaCl-evolved strain, ES9-11, isolated from a population cultured for 1200 generations in medium amended with 100 mM NaCl, showed better tolerance to NaCl than a control strain, EC3-10, cultured for 1200 generations in parallel but without NaCl amendment in medium. To understand the NaCl adaptation mechanism in ES9-11, we analyzed the transcriptional, metabolite and phospholipid fatty acid (PLFA) profiles of strain ES9-11 with 0, 100- or 250 mM-added NaCl in medium compared with the ancestral strain and EC3-10 as controls. In all the culture conditions, increased expressions of genes involved in amino-acid synthesis and transport, energy production, cation efflux and decreased expression of flagellar assembly genes were detected in ES9-11. Consistently, increased abundances of organic solutes and decreased cell motility were observed in ES9-11. Glutamate appears to be the most important osmoprotectant in D. vulgaris under NaCl stress, whereas, other organic solutes such as glutamine, glycine and glycine betaine might contribute to NaCl tolerance under low NaCl concentration only. Unsaturation indices of PLFA significantly increased in ES9-11. Branched unsaturated PLFAs i17:1 ω9c, a17:1 ω9c and branched saturated i15:0 might have important roles in maintaining proper membrane fluidity under NaCl stress. Taken together, these data suggest that the accumulation of osmolytes, increased membrane fluidity, decreased cell motility and possibly an increased exclusion of Na(+) contribute to increased NaCl tolerance in NaCl-evolved D. vulgaris.

Growth, photosynthesis and H+-ATPase activity in two Jerusalem artichoke varieties under NaCl-induced stress

In order to evaluate differential growth, photosynthesis and H+-ATPase activity responses to salt-induced stress, two Jerusalem artichoke (Helianthus tuberosus L.) genotypes (Nanyu No. 1 and Qingyu No. 2) were used in sand-culture experiment with different concentrations of NaCl (0, 30, 60, 90, 120 and 150mM). After 20 days of growth, the NaCl stress resulted in a decrease of biomass accumulation, relative leaf expansion rate and photosynthetic rate, but an increase of proline content in both genotypes. Compared with Qingyu No. 2, Nanyu No. 1 had lower biomass, photosynthetic rate, gas exchange and transpiration rate, but higher proline content, activities of plasma membrane H+-ATPase (PM H+-ATPase) and vacuolar membrane H+-ATPase (VM H+-ATPase). Hence, the NaCl adaptation strategy in Nanyu No. 1 was by lowering photosynthetic rate, stomatal conductance and transpiration rate while maintaining high H+-ATPase activities, whereas the adjustment of Qingyu No. 2 was by keeping much higher rate of proline accumulation and concentration of chlorophyll. The differences in salt tolerance showed that different adaptation mechanisms existed between cultivars of Jerusalem artichoke. The findings offered the possibility of selecting salt-tolerant genotypes of Jerusalem artichoke.

Structural adaptation of endonuclease I from the cold-adapted and halophilic bacteriumVibrio salmonicida

The crystal structure of the periplasmic/extracellular endonuclease I from Vibrio salmonicida has been solved to 1.5 A resolution and, in comparison to the corresponding endonucleases from V. cholerae and V. vulnificus, serves as a model system for the investigation of the structural determinants involved in the temperature and NaCl adaptation of this enzyme class. The overall fold of the three enzymes is essentially similar, but the V. salmonicida endonuclease displays a significantly more positive surface potential than the other two enzymes owing to the presence of ten more Lys residues. However, if the optimum salt concentrations for the V. salmonicida and V. cholerae enzymes are taken into consideration in the electrostatic surface-potential calculation, the potentials of the two enzymes become surprisingly similar. The higher number of basic residues in the V. salmonicida protein is therefore likely to be a result, at least in part, of adaptation to the more saline habitat of V. salmonicida (seawater) than V. cholerae (brackish water). The hydrophobic core of all three enzymes is almost identical, but the V. salmonicida endonuclease has a slightly lower number of internal hydrogen bonds. This, together with repulsive forces between the basic residues on the protein surface of V. salmonicida endonuclease I and differences in the distribution of salt bridges, probably results in higher flexibility of regions of the V. salmonicida protein. This is likely to influence both the catalytic activity and the stability of the protein.

Expression and Ca 2+ Dependency of Plasma Membrane K + Channels of Tobacco Suspension Cells Adapted to Salt Stress

The expression of plasma membrane K+ channels of NaCl-adapted tobacco suspension cells and effects of extracellular Ca2+ on plasma membrane K+ channels were investigated. Reverse transcription-PCR (RT-PCR) analysis showed that expression of TORK1, which encodes a K+ channel, was much lower in NaCl-adapted cells than in NaCl-unadapted cells. The magnitude of the outward K+ currents of NaCl-adapted as well as NaCl-unadapted cells decreased with increasing extracellular Ca2+ but there is no significant difference in Ca2+ dependency of the K+ current. These analyses suggest that reduction of the number of K+ channels might cause NaCl adaptation of cells through the decrease of outward K+ currents.

The neural code for taste in the nucleus of the solitary tract of the rat: effects of adaptation

Adaptation of the tongue to NaCl, HCl, quinine or sucrose was used as a tool to study the stability and organization of response profiles in the nucleus of the solitary tract (NTS). Taste responses in the NTS were recorded in anesthetized rats before and after adaptation of the tongue to NaCl, HCl, sucrose or quinine. Results showed that the magnitude of response to test stimuli following adaptation was a function of the context, i.e., adaptation condition, in which the stimuli were presented. Over half of all taste responses were either attenuated or enhanced following the adaptation procedure: NaCl adaptation produced the most widespread, non-stimulus-selective cross-adaptation and sucrose adaptation produced the least frequent cross-adaptation and the most frequent enhancement of taste responses. Adaptation to quinine cross-adapted to sucrose and adaptation to HCl cross-adapted to quinine in over half of the units tested. The adaptation procedure sometimes unmasked taste responses where none were present beforehand and sometimes altered taste responses to test stimuli even though the adapting stimulus did not itself produce a response. These effects demonstrated a form of context-dependency of taste responsiveness in the NTS and further suggest a broad potentiality in the sensitivity of NTS units across taste stimuli. Across unit patterns of response remained distinct from each other under all adaptation conditions. Discriminability of these patterns may provide a neurophysiological basis for residual psychophysical abilities following adaptation.

Alterations in cell membrane structure and expression of a membrane‐associated protein after adaptation to osmotic stress

Cultured tobacco cells (Nicotiana tabacum L. cv. Wisconsin 38) adapted to NaCl exhibited significant morphological and physiological changes. Adapted cells remained smaller and more isodiametric at maturity than unadapted cells. The vacuole increased in size relative to the cytoplasm and an extensive network of transvacuolar membrane strands developed. These changes altered the surface contact area between the cytoplasm and the vacuole substantially. In addition, the network of Hechtian strands that anchor the cortical structure to the cell wall became more extensively branched possibly facilitating surface contact of the cytoplasm to the extracellular matrix. Many changes in membrane proteins could also be identified after NaCl adaptation. In particular, a 50‐kDa protein that is associated with the plasma membrane and tonoplast was induced during adaptation. Immunocytochemical localization indicated that this 50‐kDa protein is associated with Golgi vesicles. By immunoscreening using anti‐50‐kDa antibody, a 1.71‐kb cDNA clone (p50C) was isolated from a λ‐ZAP cDNA expression library. The sequence of p50C did not show any significant identity with other genes. Because of the very low abundance of the p50C message, quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) was used to analyze p50C gene expression. Immunoblot and quantitative RT‐PCR analyses indicated that the expression of this gene is regulated developmentally since the mRNA and protein increased with age in salt‐adapted cells but decreased with age in unadapted cells. Also in tobacco plants, p50C mRNA was more abundant in younger leaves than in older leaves. The gene was responsive to NaCl in tobacco cells and to ABA in tobacco seedlings.

NaCl-Induced Alterations in Both Cell Structure and Tissue-Specific Plasma Membrane H+ -ATPase Gene Expression.

NaCl-induced plasma membrane H+-ATPase gene expression, which occurs in roots and fully expanded leaves of the halophyte Atriplex nummularia L. (X. Niu, M.L. Narasimhan, R.A. Salzman, R.A. Bressan, P.M. Hasegawa [1993] Plant Physiol 103: 713-718), has been differentially localized to specific tissues using in situ RNA hybridization techniques. Twenty-four-hour exposure of plants to 400 mM NaCl resulted in substantial accumulation of H+ pump message in the epidermis of the root tip and the endodermis of the root elongation/differentiation zone. In expanded leaves, NaCl induction of plasma membrane H+-ATPase message accumulation was localized to bundle-sheath cells. Ultrastructural analyses indicated that significant cytological adaptations in root cells included plasmolysis that is accompanied by plasma membrane invaginations, formation of Hechtian strands and vesiculation, and vacuolation. These results identify specific tissues that are involved in the regulation of Na+ and Cl- uptake into different organs of the halophyte A. nummularia and provide evidence of the intercellular and interorgan coordination that occurs in the mediation of NaCl adaptation.

Quinine-HCI-Induced Modification of Receptor Potentials for Taste Stimuli in Frog Taste Cells

After frog taste cells were adapted to 1 mM quinine-HCl (Q-HCl) for 10 sec, modification of receptor potentials in the taste cells induced by salt, acid, sugar and bitter stimuli was studied with microelectrodes. The phasic component of receptor potentials induced by 0.1 M NaCl, KCl, NH4Cl and MgCl2 was enhanced following adaptation to Q-HCl. The rate of rise of receptor potentials in response to the salts was increased after Q-HCl adaptation. The amplitude and the rate of rise of receptor potentials induced by 1 mM acetic acid were larger after Q-HCl adaptation than after water adaptation. The amplitude of phasic component and rate of rise of receptor potentials for 0.5 M sucrose after Q-HCl were the same as those after water. The amplitudes of tonic receptor potentials for 1 mM Q-H2SO4, brucine and picric acid after Q-HCl adaptation were the same as those after 1 mM NaCl adaptation. Correlation coefficient between taste cell responses induced by 1 mM Q-HCl and 1 mM Q-H2SO4 was very high, but those between 1 mM Q-HCl and 1 mM brucine responses and between 1 mM Q-HCl and 1 mM picric acid responses were low. This indicates that Q-HCl and Q-H2SO4 bind to the same receptor site, but brucine and picric acid bind to different receptor sites to which Q-HCl does not bind.

Expression of Osmotin-Like Genes in the Halophyte Atriplex nummularia L.

A peptide (molecular mass 50 kilodaltons) that is immunologically related to tobacco osmotin was detected in cells of the halophyte Atriplex nummularia. This protein was constitutively expressed in both unadapted and NaCl-adapted cells. A predominant osmotin-like peptide (molecular mass 24 kilodaltons) was also found in culture media after cell growth. Two unique A. nummularia cDNA clones, pA8 and pA9, encoding osmotin-like proteins have been isolated. The pA8 and pA9 inserts are 952 and 792 base pairs and encode peptides of 222 and 224 amino acids, respectively. The peptide deduced from pA8 has a molecular mass of 23,808 daltons and theoretical isoelectric point of 8.31, whereas the peptide derived from pA9 has a molecular mass of 23,827 daltons and an isoelectric point of 6.88. Unique transcripts were detected by the inserts of the cDNA clones, two (1.2 and 1.0 kilobases) by pA8 and one (0.9 kilobase) by pA9. The pA8 transcripts were constitutively accumulated in unadapted and NaCl-adapted cells, whereas the mRNA levels were up-regulated by abscisic acid treatment. The level of pA9 mRNA was induced by NaCl treatment and increased in cells as a function of NaCl adaptation. Southern analysis of the genomic DNA indicated the presence of osmotin-like multigene families in A. nummularia.

Active urea transport independent of H+ and Na+ transport in frog skin epithelium

We investigated the relationship between H+ secretion (JH), Na+ absorption (JNa), and urea transport (Ju) in skin of frogs (Rana esculenta) adapted to running tap water, NaCl (100 mM), and KCl (100 mM). In addition, cell morphological changes, particularly in the mitochondria-rich cells (MRC), were followed. NaCl adaptation stimulated an active Ju, reduced JNa and JH, and caused a decrease in the apical surface of MRC. After KCl adaptation, JNa and JH were increased and highly correlated, with a twofold increase in Ju, whereas the numerous MRC developed infoldings on their apical membranes. No correlation was found between JH and Ju. Clamping the skins in a range of +/- 50 mV or changing the external pH from 7.4 to 5.4 (at high cellular buffering power) had no effect on Ju. Depolarization of the basolateral membranes (serosal KCl-Ringer) had no effect on Ju. Ju was reversibly blocked by acidification of the cells by oxygen-free solution and sulfhydryl reagents (Hg2+, p-chloromercuribenzenesulfonic acid, and N-ethylmaleimide). Diethylstilbestrol, a proton transport blocker, had no effect on Ju. Apical addition of amiloride and derivatives (phenamil and ethylisopropyl amiloride) reversibly blocked Ju, whereas ouabain had no effect. We conclude that a cation (Na+ or H+)-dependent process is unlikely to exist in R. esculenta skin. A primary active transport in a two-step process is the simplest hypothesis to account for the energy-dependent Ju that develops in NaCl-adapted frogs.

Hormonal regulation of protein synthesis associated with salt tolerance in plant cells

Cultured tobacco cells (Nicotiana tabacum L. cv. Wisconsin 38) synthesize a predominant 26-kDa protein upon exposure to abscisic acid (ABA). ABA also accelerates the rate of adaptation of unadapted cells to NaCl stress. The ABA-induced 26-kDa protein is immunologically cross-reactive to, and produces a similar pattern of peptides after partial proteolysis as, the major 26-kDa protein associated with NaCl adaptation. Both have pI values of >8.2. The synthesis of the ABA-induced 26-kDa protein is transient unless the cells are simultaneously exposed to NaCl stress. There is an association between increased intracellular accumulation of ABA during cell growth and commencement of synthesis of the 26-kDa protein. ABA induces the synthesis of an immunologically cross-reactive 26-kDa protein in cultured cells of several plant species. In tobacco plants, synthesis of the 26-kDa protein could be detected in several tissues but the highest level of expression was seen in outer stem tissue. In root tissues, exogenous ABA greatly stimulated the synthesis of 26-kDa protein as compared to outer stem tissue and leaf. We suggest that ABA is involved in the normal induction of the synthesis of 26-kDa protein and that the presence of NaCl is necessary for the protein to accumulate.

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 (35)S 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.