NaCl-supplemented Medium Research Articles

Lethality caused by ADP-glucose accumulation is suppressed by salt-induced carbon flux redirection in cyanobacteria.

Cyanobacteria are widely distributed photosynthetic organisms. During the day they store carbon, mainly as glycogen, to provide the energy and carbon source they require for maintenance during the night. Here, we generate a mutant strain of the freshwater cyanobacterium Synechocystis sp. PCC 6803 lacking both glycogen synthases. This mutant has a lethal phenotype due to massive accumulation of ADP-glucose, the substrate of glycogen synthases. This accumulation leads to alterations in its photosynthetic capacity and a dramatic decrease in the adenylate energy charge of the cell to values as low as 0.1. Lack of ADP-glucose pyrophosphorylase, the enzyme responsible for ADP-glucose synthesis, or reintroduction of any of the glycogen synthases abolishes the lethal phenotype. Viability of the glycogen synthase mutant is also fully recovered in NaCl-supplemented medium, which redirects the surplus of ADP-glucose to synthesize the osmolite glucosylglycerol. This alternative metabolic sink also suppresses phenotypes associated with the defective response to nitrogen deprivation characteristic of glycogen-less mutants, restoring the capacity to degrade phycobiliproteins. Thus, our system is an excellent example of how inadequate management of the adenine nucleotide pools results in a lethal phenotype, and the influence of metabolic carbon flux in cell viability and fitness.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry identification of Vibrio (Listonella) anguillarum isolated from sea bass and sea bream.

Vibrio (Listonella) anguillarum is a pathogenic bacterium causing septicaemia in a wide range of marine organisms and inducing severe mortalities, thus it is crucial to conduct its accurate and rapid identification. The aim of this study was to assess MALDI-TOF MS as a method of choice for identification of clinical V. anguillarum isolates from affected marine fish. Since the method accuracy might be influenced by the type of the medium used, as well as by the incubation conditions, we tested V. anguillarum isolates grown on standard media with and without the addition of NaCl, cultured at three incubation temperatures, and at three incubation periods. The best scores were retrieved for V. anguillarum strains grown on NaCl-supplemented tryptone soy agar (TSA) at 22°C and incubated for 48h (100% identification to species level; overall score 2.232), followed by incubation at 37°C and 48h (100% to species level; score 2.192). The strains grown on non-supplemented TSA gave the best readings when incubated at 22°C for 72h (100% identification to species level; overall score 2.182), followed by incubation at 15°C for 72h (100% to species level; score 2.160). Unreliable identifications and no-identifications were growing with the incubation duration at 37°C, on both media, amounting to 88.89% for 7d incubation on supplemented TSA, and 92.60% for 7d incubation on non-supplemented TSA. The age of the cultured strains and use of media significantly impacted the mass spectra, demonstrating that for reliable identification, MALDI-TOF MS protein fingerprinting with the on-target extraction should be performed on strains grown on a NaCl-supplemented medium at temperatures between 15 and 22°C, incubated for 48–72 hours.

Halogenated cladosporols produced by the sodium halide-supplemented fermentation of the plant-associated fungus Cladosporium sp. TMPU1621

A new cladosporol derivative, 2-chloro-cladosporol D (6), was isolated together with known cladosporol (1), cladosporols B–D (2–4), and cladosporol F (5) from the culture broth of plant-associated Cladosporium sp. TMPU1621. The structure of 6 was elucidated from spectroscopic analyses including NMR and MS data. The productivity of 6 increased in 3% NaCl-supplemented medium; therefore, fermentation of the producing fungus TMPU1621 with 3% NaBr was investigated, leading to the induced production of a new halogenated cladosporol, 2-bromo-cladosporol D (7). Compound 2 showed the lowest MIC values of 3.13 and 12.5 μg/mL against two MRSA strains, ATCC43300 and ATCC700698, respectively.

Wallemia peruviensis sp. nov., a new xerophilic fungus from an agricultural setting in South America.

We obtained four isolates of the xerophilic genus Wallemia from the rooftop of a house made of red brick and cement in an agronomic field planted with common beans and maize in Pachacamac, Lima, Peru. Bayesian phylogenetic analysis with rDNA gene sequences showed these Wallemia isolates form a distinct and strongly supported clade closely related to W. hederae. We examined the macro and micromorphology, growth rate and production of exudates of isolates on media containing different amounts of glucose and NaCl (water activity from 0.9993 to 0.8480). Their chaotropic and kosmotropic tolerance were tested on media with multiple molar concentrations of MgCl2 and MgSO4 (water activity from 0.9880 to 0.7877). Isolates are xerophilic and halotolerant, growing on 17% NaCl-supplemented media (water activity=0.8480). Maximum concentrations of MgCl2 and MgSO4 at which growth was observed were 1.7 and 3.5M, respectively. Isolates were shown to represent a novel species, described as Wallemia peruviensis sp. nov. In contrast to W. hederae, W. peruviensis does not produce exudates on malt extract agar+17% NaCl media. An updated dichotomous key to Wallemia species is provided. This is the first new species of Wallemia described from South America and the first association of a Wallemia species with an agricultural environment on this continent.

Role of theAFRD1-encoded fumarate reductase in hypoxia and osmotolerance inArxula adeninivorans

Fumarate reductase is an enzyme involved in maintaining redox balance through regeneration of reduced cofactors during oxygen deficiency conditions. This work reports the identification and characterization of the gene and its promoter and terminator elements that encodes cytosolic fumarate reductase enzyme in the nonconventional yeast, Arxula adeninivorans. The gene harbours an ORF of 1446 bp, encoding a 482-amino acid protein. The deduced amino acid sequence is similar to those of fumarate reductases from other yeast and fungi, such as the two fumarate reductases of Saccharomyces cerevisiae, Frd1p (44%) and Osm1p (41%). This enzyme is located in the cytosol and has a pH optimum of ca. 7.5 and a Michaelis constant (K(M)) of 2.9 mM with fumarate as the substrate. Expression of AFRD1 is regulated by the cultivation conditions. A shift from NaCl-free to NaCl-supplemented media and aerobic to hypoxic growth conditions leads to reduced AFRD1 transcription levels, but not to alteration in the concentration of Afrd1p. The functional analyses of Afrd1p were performed in A. adeninivorans and S. cerevisiae disruption mutants. The A. adeninivorans fumarate reductase is capable of functional complementation of the missing S. cerevisiae genes during anoxia; however, it is not involved in yeast growth under osmotic stress.

Expression and stress tolerance of PR10 genes from Panax ginseng C. A. Meyer

Pathogenesis-related 10 protein families (PgPR10 proteins) from ginseng are reported to have ribonuclease activity, conferring defense-related resistance against various stresses. Homology-based PCR using PgPR10-2 specific primers allowed for the isolation of two additional PgPR10 genes. PgPR10-1 is identical to the previously reported ribonuclease 1, while PgPR10-3 is a newly-discovered protein, suggesting that the PgPR10s are a multi-gene family. Differential organ-specific transcripts of PgPR10-1 and PgPR10-2 in the flower bud and root, respectively, indicate that there are tissue-specific functional roles for this gene family. Overexpression of PgPR10-2 in Arabidopsis conferred longer root length and a tolerant growth phenotype on NaCl-supplemented media. Further changes in transcriptional levels against sets of abiotic stressors suggest similar functional roles of PgPR10-1 in the root and predominantly in the flower organ based on its higher expression levels. Overall, this suggests that the manipulation of PgPR10 genes in plants can be used as valuable tool to enhance its physiological status.

Regeneration of plantlets of guava (Psidium guajava L.) from somatic embryos developed under salt-stress condition

The present study demonstrates the regeneration of plantlets of guava (Psidium guajava L.) from somatic embryos developed under salt-stress conditions. With increasing concentrations of NaCl in induction medium (MS + 4.52 μM 2,4-d + 5% sucrose) from 0 to 200 mM, the number of somatic embryos per responsive explant decreased. Somatic embryos induced on 0–100 mM NaCl containing medium developed into torpedo stages, whereas, the development of somatic embryos that differentiated on 150 and 200 mM NaCl-supplemented medium was arrested prior to torpedo stage and did not undergo maturation phase. Somatic embryos that developed on NaCl-containing medium, showed better germination in the presence of NaCl as compared with those developed on medium without NaCl. The effect of increasing salt-stress was also investigated on plant growth, chlorophyll and carotenoids, Na+ and K+, and proline and glycine betaine accumulation in in vitro grown plantlets. The level of Na+ in leaves increased with increasing concentrations of NaCl in the medium. Accumulation of free proline and glycine betaine in leaves significantly increased with increasing salinity. The results suggest that accumulation of proline and glycine betaine may be important for osmotic adjustment in guava under salinity stress.

A novel in vitro tissue culture approach to study salt stress responses in citrus

In citrus, a major crop throughout the world, growth and yield are seriously affected by salinity. Different approaches, including agronomical, physiological and molecular methods, have been used to address this problem. In this work, an in vitro experimental system has been developed to study the toxic effect of NaCl on three citrus genotypes, avoiding the ion filter that represents the root system. To carry out the experiments, shoots were obtained from nodal segments of Cleopatra mandarin, Carrizo citrange and citrumelo CPB4475 plants growing in a greenhouse. Shoots were cultured in control or NaCl-supplemented media. After testing several salt concentrations, 60 mM NaCl was selected as saline treatment. Shoots accumulated similar levels of chloride when cultured without roots and exhibited similar leaf damage. No increases in malondialdehyde levels were observed in any genotype (as a measure of oxidative stress). Similar patterns of hormonal signalling (in terms of abscisic acid and salicylic acid contents) were exhibited in the three genotypes, despite their different tolerance under field conditions. All data together indicate that, without root system, all genotypes had the same behaviour under salt stress. The in vitro culture system has been proved as a useful tool to study biochemical processes involved in the response of citrus to salt stress.

Tolerance to NaCl induces changes in plasma membrane lipid composition, fluidity and H+-ATPase activity of tomato calli.

Two tomato (Lycopersicon esculentum Mill. cv. Pera) callus lines tolerant to NaCl were obtained by successive subcultures of NaCl-sensitive calli in 50 and 100 mM NaCl-supplemented medium. Growth and ion content, as well as plasma membrane lipid composition, fluidity and H+-ATPase (EC 3.6.1.35) activity, were studied in both NaCl-sensitive and NaCl-tolerant calli. Although calli tolerant to 100 mM NaCl exhibited a reduced growth relative to calli sensitive to NaCl or tolerant to 50 mM NaCl, growth of calli tolerant to 100 mM NaCl was higher than that of NaCl-sensitive calli grown for one subculture in 100 mM NaCl. Growth in the presence of 100 mM NaCl provoked an increase of Na+ and Cl- content, but no significant changes in K+ and Ca2+. As compared with NaCl-sensitive and 50 mM NaCl-tolerant calli, plasma membrane vesicles isolated from calli tolerant to 100 mM NaCl exhibited a higher phospholipid and sterol content as well as a lower phospholipid/free sterol ratio and a lower double bond index (DBI) of phospholipid fatty acids. The changes in plasma membrane lipid composition were correlated with a decrease of plasma membrane fluidity in calli tolerant to 100 mM NaCl, as indicated by fluorimetric studies using diphenylhexatriene (DPH) as probe. Plasma membrane-enriched vesicles isolated from calli tolerant to 100 mM NaCl showed lower ATP hydrolysis and ATP-dependent H+-pumping activities, as well as a lower passive permeability to H+ than plasma membrane from NaCl-sensitive and 50 mM NaCl-tolerant calli. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H+-ATPase activity in salt tolerance of tomato calli is discussed.

Viability of dried vegetative trichomes, formation of akinetes and heterocysts and akinete germination in some blue-green algae under water stress.

Almost all dried vegetative trichomes ofAnabaena iyengarii, Westiellopsis prolifica andNostochopsis lobatus died within 1 h, while those ofOscillatoria acuminata retained viability to some extent for 1 d under similar storage conditions. The viability of dried vegetative trichomes ofO. acuminata decreased about equally on storage at 20 degrees C in the light or in the dark, but dropped rapidly at 12 and 0 degrees C in the dark. Vegetative trichomes ofA. iyengarii, N. lobatus andW. prolifica were more sensitive to frost than those ofO. acuminata, and this correlated with their low resistance to desiccation because both types of exposure involved osmotic stress. Both dried and wet akinetes ofA. iyengarii, W. prolifica andN. lobatus were about equally viable when stored at 20 degrees C in the light or the dark or at 12 and 0 degrees C in the dark, but their germination ability decreased on storage at 0 degrees C. The water stress imposed on growing vegetative trichomes either in high-agar media or in NaCl-supplemented liquid media reduced the survival ofO. acuminata trichomes, decreased or totally suppressed akinete and heterocyst formation and akinete germination inA. iyengarii, W. prolifica andN. lobatus. The sensitivity decreased in the sequenceA. iyengarii <W. prolifica ~N. lobatus. The akinete germination in all three blue-green algae was more sensitive to physiological water stress than their formation. In all of them, akinetes formed under water stress were equally viable as those formed under normal conditions. Trichomes ofO. acuminata became broader when grown in 0.5-0.8 mol/L NaCl-supplemented media, probably due to polyol accumulation, and they also developed a thin sheath-like structure.

Expression of the yeast HAL2 gene in tomato increases the in vitro salt tolerance of transgenic progenies

Agrobacterium-mediated transformation has been used to introduce the yeast halotolerant HAL2 as well as the nptII and uidA marker genes into tomato ( Lycopersicon esculentum) cv. UC82B. Five to six percent of the explants produced transgenic plants. HAL2 expressing transformants were allowed to self-pollinate and salt tolerance assays were performed in vitro on progenies from two independent transgenic plants with different levels of expression of the transgene. In vitro salt tolerance was evaluated according to the level of growth of hypocotyl-derived calli as well as the rooting capability of isolated shootlets on MS-modified medium supplemented with NaCl. Under salt stress, callus formation from hypocotyl explants was higher on both transgenic-derived progenies than in the control. In addition, progenies from the plant with the highest expression of the transgene (2H20b), also showed a higher level of root production on NaCl-supplemented medium. These results suggested a positive effect of the yeast HAL2 gene on the level of salt tolerance in progenies derived from transgenic plants.

Saline stress alters the temporal patterns of xylem differentiation and alternative oxidase expression in developing soybean roots

We conducted a coordinated biochemical and morphometric analysis of the effect of saline conditions on the differentiation zone of developing soybean (Glycine max L.) roots. Between d 3 and d 14 for seedlings grown in control or NaCl-supplemented medium, we studied (a) the temporal evolution of the respiratory alternative oxidase (AOX) capacity in correlation with the expression and localization of AOX protein analyzed by tissue-print immunoblotting; (b) the temporal evolution and tissue localization of a peroxidase activity involved in lignification; and (c) the structural changes, visualized by light microscopy and quantified by image digitization. The results revealed that saline stress retards primary xylem differentiation. There is a corresponding delay in the temporal pattern of AOX expression, which is consistent with the xylem-specific localization of AOX protein and the idea that this enzyme is linked to xylem development. An NaCl-induced acceleration of the development of secondary xylem was also observed. However, the temporal pattern of a peroxidase activity localized in the primary and secondary xylem was unaltered by NaCl treatment. Thus, the NaCl-stressed root was specifically affected in the temporal patterns of AOX expression and xylem development.

P38 Kinase Activity Is Essential for Osmotic Induction of mRNAs for HSP70 and Transporter for Organic Solute Betaine in Madin-Darby Canine Kidney Cells

In renal cells, hypertonicity induces genes for heat shock proteins (HSP70, alpha B-crystallin), as well as enzymes and transporters directly involved in the metabolism and transport of protective organic osmolytes. While heat shock proteins are induced by many stresses including osmotic stress, the induction of the osmolytes genes appears to be specific to osmotic stress. These two adaptive mechanisms allow kidney cells to survive and function in the hypertonic environment that exists on routine basis in kidney medulla. In mammalian cells, hypertonicity induces three mitogen-activated protein kinase pathways: ERK (extracellular regulated kinase), JNK (Jun N-terminal kinase), and p38. ERK activation by osmotic stress is a consistent finding in many cells, but it is not essential for transcriptional regulation of mRNA for transporter of organic osmolyte betaine. While the growth of yeast cells on NaCl-supplemented medium is dependent on HOG1 pathway, it is still unclear which pathway mediates the adaptation to osmotic stress in mammalian cells. Here, we show that inhibition of p38 kinase activity, using the specific inhibitor SB203580 (4-(fluorophenyl)-2-(4-methylsulfonyl-phenyl)-5-(4-pyridyl) imidazole), abolishes the hypertonicity-mediated induction of mRNAs for HSP70 and betaine transporter in Madin-Darby canine kidney cells. The inhibition is dose-dependent and correlates with the in situ activity of native p38 kinase, determined as MAPKAPK-2 activity in cell extracts. As reported previously, the activities of ERK-1 and -2 were not affected by SB203580, but surprisingly, inhibition of native p38 kinase activity correlates with up-regulation of native JNK-1 activity in osmotically stressed cells. p38 mRNA is induced by hypertonic stress and is attenuated with p38 kinase inhibition. We also find that thermal induction of HSP70 mRNA is not affected by p38 kinase inhibition. Such findings suggest that p38 kinase activity is essential for the induction of genes involved in the adaptation of mammalian cells to osmotic stress and that the increased activity of JNK-1 during p38 kinase inhibition is consistent with regulation of JNK-1 by p38 kinase in osmotically stressed cells. In addition, the transduction pathways mediating HSP70 mRNA induction by different stresses appear to be divergent; osmotic induction of HSP70 is p38 kinase-dependent, while thermal induction is not.

Response of a rice field cyanobacterium Anabaena sp. to physiological stressors

The impact of physiological factors such as heat, salinity and L-methionine-DL-sulfoximine (MSO) on growth and total protein profile was studied in a rice field cyanobacterium, Anabaena sp. There was a gradual decrease in growth rate of the organism with increase in incubation time at 47°C; by 168 h of incubation there was essentially no growth. NaCl concentration > 5 mM inhibited growth of the organism, and there was apparently no growth at > 200 mM NaCl-supplemented media. Similarly, growth was inhibited with MSO concentration > 5 μM and there was no growth in cultures treated with > 100 μM MSO. SDS-PAGE protein profile after heat stress revealed a decline in the synthesis of several proteins but at the same time, synthesis of a new set of proteins of approximately 60–65 kDa was induced after 12 and 24 h of incubation and the same was completely eliminated after 96 h of incubation. Cultures treated with 10 mM of NaCl did not show any change in the protein pattern. In contrast, 100 mM NaCl treated cultures elicited a number of new proteins at around 29, 32, 40 and 70 kDa. Most of the protein bands disappeared in the cultures treated with 500 mM NaCl. Cultures treated with 10 μM MSO did not show any change in the appearance of the protein bands, whereas the cultures treated with 100 μM MSO showed a drastic decline in the intensity of all protein bands. The results indicate that different stressors exert specific effects on cyanobacterial protein synthesis.

Rice anther culture: callus initiation and androclonal variation in progenies of regenerated plants

Anthers from rice (Oryza sativa L.) subspecies japonica initiated more callus than their indica or indica x japonica counterparts. A mild stress, either by slow desiccation or heat shock, prior to the plating of anthers enhanced the ability to initiate callus. Slow dessication of anthers enhanced the ability of the japonica anthers to initiate callus even in medium that was supplemented with NaCl. The ability to initiate callus by the anthers plated on NaCl-supplemented medium decreased as the NaCl level in the medium increased. Among the regenerated plants 2.5% were albino and another 2% were haploid. Androclonal variation for tiller numbers, shoot height, plant dry matter and flowering were noticed in the progenies of the regenerated plants.

Exceptions to mutual exclusion among cell surface i-antigens of Tetrahymena thermophila during salt stress and stationary phase.

In ciliates, only one of the alternative forms of the immunodominant membrane glycoprotein usually coats the external surface of the cell. Such mutual exclusion is regulated at the pretranslational level by mechanisms that result in the expression of a single protein gene. In the holotrich Tetrahymena thermophila five alternative cell surface immobilization proteins (i-antigens) are expressed under different conditions of temperature (L, H, T) and culture media (I, S). Using polyclonal and monoclonal antibodies to these proteins and a cDNA probe derived from the SerH3 gene, we have reinvestigated expression of i-antigens in media supplemented with 0.2 M NaCl. We find that in addition to S, the H and L antigens are also present on the cell surface. While all three i-antigens may be simultaneously present on the cell surface, the combinations S/L and S/H are more frequent. Compared to cells expressing H and L singly, the level of H3 mRNA is diminished, and a subset of the L family of polypeptides is variably expressed. The expression of S begins within 30 min after transfer to NaCl-supplemented medium, while the expression of L begins three days to several weeks after transfer. When cells are transferred out of NaCl-supplemented medium, S is turned off within 24 h, and L is expressed for at least 1 wk prior to the return of full H expression. Although these differences in kinetics suggest differences in control mechanism(s), the absence of I and T on the surface of NaCl-grown cells suggests that there is also a common regulatory link among H, S and L.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of osmolytes in MDCK cells by solutes, inhibitors, and vasopressin

MDCK cells accumulate organic osmolytes in response to hyperosmotic NaCl-supplemented medium. We examined time course and inhibitor sensitivity of myo-inositol, sorbitol, and glycerophosphorylcholine (GPC) accumulation in MDCK cells exposed to hyperosmotic NaCl-, D-glucose-, or mannitol-supplemented media. In NaCl medium, cells preferentially accumulated inositol and GPC. In comparison, in glucose medium cells preferentially accumulated sorbitol and GPC. Inositol demonstrated a late (72-96 h) accumulation in glucose medium, although less than in NaCl medium. Mannitol medium did not significantly stimulate accumulation of any of these three osmolytes at 24 h, suggesting that hyperosmolality alone is not sufficient stimulus for their accumulation in this time frame. GPC accumulation was very rapid in glucose medium, and fell to the level induced by NaCl medium at 96 h (approximately 50 nmol/mg protein). Inositol and sorbitol accumulated more gradually, each reaching greater than 400 nmol/mg protein after 96 h. Sorbitol was still accumulating at 96 h, whereas inositol plateaued at 72-96 h. Phlorizin or sorbinil blocked accumulation of inositol or sorbitol, respectively. Sorbitol and GPC accumulation in glucose medium were partially inhibited in absence of serum or in presence of 1 microM vasopressin. Thus NaCl and glucose appear to stimulate specific cellular mechanisms responsible for accumulation of inositol, sorbitol, and GPC in MDCK cells. This accumulation is also modulated by constituents of serum.

Isolation of sodium chloride-tolerant plantlets of Kickxia ramosissima under in vitro conditions

Summary Salt-tolerant plantlets of Kickxia ramosissima have been obtained as spontaneous variants under in vitro conditions. These variants comprised shoot buds which differentiated from cultured internodal segments and leaves. They could grow on a medium supplemented with sodium chloride (up to 120 mM). The variants had thick, fleshy, pale-green leaves in compact clusters, and highly reduced internodes. With subsequent passages, better internodal elongation was obtained. Flowering occurred after four passages of 8–10 wk each, on NaCl-supplemented media. The tolerance persisted even after three passages (8–10 wk each) in the absence of selective agent, namely, sodium chloride. Some of the selected variants were able to grow when transferred to a medium with 240 mM sodium chloride. Proline and phenylalanine, when supplemented singly at 100 μM to a medium with 120 mM sodium chloride, counteracted the inhibitory growth effect of sodium chloride.

Cell growth and division in hypertonic medium

A comprehensive investigation has been made into the effects of raising medium strength on the growth and division of HeLa and other cell lines in tissue culture. Tonicity was raised by addition of concentrated solutions of NaCl, KCl, CaCl 2, choline chloride etc., and of substances such as urea, raffinose and glucose. It was established that osmotic effects were particularly important in arresting cells in metaphase, but some ions, e.g. K + and Mg 2+, more actively arrested metaphase than could be accounted for by osmotic effects alone. Some substances, particularly NaCl, allowed cells to progress into mitosis at concentrations which were adequate to prevent their exit from metaphase whereas other substances, such as MgCl 2, inhibited both entry into and exit from mitosis at about the same tonicity. Arrest of metaphases in hypertonic medium was not due to interference with the production of spindle but might have been attributable to excessive stickiness of the over-condensed chromosomes. Cells exposed to NaCl-supplemented medium exhibit a concentration dependent inhibition of macromolecular synthesis, protein synthesis being most affected, DNA synthesis less so and RNA synthesis least, except at slightly elevated tonicities which often produced a slight stimulation of the rate of synthetic activity.