The effect of compatible solutes on proteins

Summary The ability of the compatible solute, proline, to affect the behavior of proteins has been examined in many different systems by many researches. In the present study of protein solvation, proline has been shown to prevent or diminish, in a concentration-dependent manner, the glutamine synthetase-precipitating ability of polyethylene glycol (PEG). The effects of PEG concentration and molecular weight are reduced by proline, and the interaction is strongly affected by pH.PEG causes precipitation of many proteins, and the ability of proline to reduce the precipitation of two non-enzymatic conjugated proteins, alfalfa mosaic virus and an³H-testosterone/antiserum complex, was also examined. Proline was effective in reducing the PEG-induced precipitation of both proteins. Virus precipitation by PEG and its alleviation by proline are influenced by pH. The increased virus-precipitating effect of PEG in the presence of salt (NaCl) is also alleviated by proline. The precipitation of the radioimmune complex by PEG is diminished by proline and by a mixture of free amino acids.These results indicate the generality of the three-way interaction between proline, protein and PEG. They may be of importance for extraction of proteins from biological systems and in studies of enzyme inactivation or protein denaturation in a cytoplasmic milieu. The results suggest that the protective effects of some amino acids are at least additive and are consistent with the conclusion that the compatible solutes protect protein-containing systems against the unfavorable consequences of dehydration and other stresses, by increasing the tendency of the system to maintain thestatus quo.     

Influence of exogenously applied paclobutrazol on some physiological traits and growth of Stevia rebaudiana under in vitro drought stress

This investigation was carried on to find out the changes occurred in Stevia rebaudiana in response to paclobutrazol (PBZ; 0–4 mg L^?1) treatment and drought stress. Polyethylene glycol (PEG; 0–6 % w/v) was used to stimulate drought stress. Drought stress reduced fresh and dry weight, water content, chlorophylls, carotenoids, anthocyanins, water soluble carbohydrates, reducing sugar and proline amounts. Electrolyte leakage, MDA, α-tocopherol and glycine betaine contents increased in drought-stressed plants. The activity of P5CS and PDH enzymes and protein content showed no significant changes under drought stress. PBZ (with or without PEG) treatments decreased fresh and dry weight and water content. In PBZ-treated plants, less pigments was damaged by drought stress. PBZ treatment reduced the negative effect of drought stress on lipid peroxidation which resulted in lower electrolyte leakage and MDA content, compared to the same PEG level without PBZ. PBZ (with or without PEG) treatments increased glycine betaine, α-tocopherol, proline and protein contents. The amount of water soluble carbohydrates, reducing sugar and activity of P5CS and PDH were not affected by PBZ treatments. SDS-PAGE analysis revealed that drought stress increased a 25 kD protein with a critical function in plant development under stresses. According to the results, PEG provoked a severe drought stress in S. rebaudiana that could partly be restored by PBZ treatment.     

Dithiothreitol and PEG Induced Combined Stress May Affect the Expressions of ABA Aldehyde Oxidase,Sucrose Synthase and Proline Metabolic Genes in Maize Seedlings

The endoplasmic reticulum (ER) is an organelle in the cell where proteins are created and folded. Folding is a very elaborate process that is often interrupted by various biotic and abiotic stresses, leading to the formation of unfolded and misfolded proteins called ER stress. Dithiothreitol (DTT)-induced unfolded protein response (UPR) in endoplasmic reticulum (ER) has been recently reported in plants. Also, previous studies demonstrated that treatment with polyethylene glycol (PEG₆₀₀₀) could stimulate water deficit in crops. However, further researches should be conducted to elucidate the molecular mechanism of ER stress response and the relationship between water deficiency and ER. In this study, we examined the expressions of sucrose synthase (SuS) gene, proline metabolic genes and abscisic aldehyde oxidase (AAO3) gene in maize seedlings that were subjected to DTT and PEG induced combined stresses by using quantitative real-time RT-PCR. Three weeks old detached maize seedlings were treated with or without DTT and PEG₆₀₀₀ for 12 h. The treatment with DTT increased about 2-fold the expression of gene encoding proline synthesis enzyme, pyrroline-5-carboxylate synthetase (P5CS) but no statistically affected the proline catabolism enzyme, proline dehydrogenase (ProDH) in comparison with un-treated seedlings. PEG treatment was also up-regulated P5CS while it was down-regulated ProDH. The relative expression levels of SuS and AAO3 genes statistically enhanced about 2.5 fold under the DTT-induced ER stress. Likewise, the expression levels of SuS and AAO3 genes were up-regulated in the detached seedlings exposed to PEG-induced water deficit. Conversely, the induced gene expressions were down-regulated under the combined stress, the DTT-induced ER stress and PEG-induced water deficit in comparison with the singular stress responses (DTT or PEG). The results indicated that the expressions of genes, related to the synthesis of some signal osmolyte compounds such as proline and sucrose can be suppressed when ER stress occurred under water deficiency in maize seedlings. The changes in the expressions of genes involved in osmolyte and ABA metabolism can be related to ER stress response as well as variations in water status.     

The osmoprotectant proline betaine is a major substrate for the binding-protein-dependent transport system ProU of Escherichia coli K-12

The ProP and ProU transport systems of Escherichia coli mediate the uptake of several osmoprotectants including glycine betaine. Here we report that both ProP and ProU are involved in the transport of the potent osmoprotectant proline betaine. A set of isogenic E. coli strains carrying deletions in either the proP or proU loci was constructed. The growth properties of these mutants in high osmolarity minimal media containing 1 mM proline betaine demonstrated that the osmoprotective effect of this compound was dependent on either an intact ProP or ProU uptake system. Proline betaine competes with glycine betaine for binding to the proU-encoded periplasmic substrate binding protein (ProX) and we estimate a K_D of 5.2 μM for proline betaine binding. This value is similar to the binding constant of the ProX protein determined previously for the binding of glycine betaine (K_D of 1.4 μM). Our results thus demonstrate that the binding-protein-dependent ProU transport system of E. coli mediates the efficient uptake of the osmoprotectants glycine betaine and proline betaine.     

Glycine betaine aldehyde dehydrogenase from Bacillus subtilis: characterization of an enzyme required for the synthesis of the osmoprotectant glycine betaine

Production of the compatible solute glycine betaine from its precursors choline or glycine betaine aldehyde confers a considerable level of tolerance against high osmolarity stress to the soil bacterium Bacillus subtilis. The glycine betaine aldehyde dehydrogenase GbsA is an integral part of the osmoregulatory glycine betaine synthesis pathway. We strongly overproduced this enzyme in an Escherichia coli strain that expressed a plasmid-encoded gbsA gene under T7φ10 control. The recombinant GbsA protein was purified 23-fold to apparent homogeneity by fractionated ammonium sulfate precipitation, ion-exchange chromatography on Q-Sepharose, and subsequent hydrophobic interaction chromatography on phenyl-Sepharose. Molecular sieving through Superose 12 and sedimentation centrifugation through a glycerol gradient suggested that the native enzyme is a homodimer with 53.7-kDa subunits. The enzyme was specific for glycine betaine aldehyde and could use both NAD⁺ and NADP⁺ as cofactors, but NAD⁺ was strongly preferred. A kinetic analysis of the GbsA-mediated oxidation of glycine betaine aldehyde to glycine betaine revealed K _m values of 125 μM and 143 μM for its substrates glycine betaine aldehyde and NAD⁺, respectively. Low concentrations of salts stimulated the GbsA activity, and the enzyme was highly tolerant of high ionic conditions. Even in the presence of 2.4 M KCl, 88% of the initial enzymatic activity was maintained. B. subtilis synthesizes high levels of proline when grown at high osmolarity, and the presence of this amino acid strongly stimulated the GbsA activity in vitro. The enzyme was stimulated by moderate concentrations of glycine betaine, and its activity was highly tolerant against molar concentrations of this osmolyte. The high salt tolerance and its resistance to its own reaction product are essential features of the GbsA enzyme and ensure that B. subtilis can produce high levels of the compatible solute glycine betaine under conditions of high osmolarity stress. Received: 2 May 1997 / Accepted: 2 July 1997     

Characteristics and osmoregulatory roles of uptake systems for proline and glycine betaine in Lactococcus lactis.

Lactococcus lactis subsp. lactis ML3 contains high pools of proline or betaine when grown under conditions of high osmotic strength. These pools are created by specific transport systems. A high-affinity uptake system for glycine betaine (betaine) with a Km of 1.5 microM is expressed constitutively. The activity of this system is not stimulated by high osmolarities of the growth or assay medium but varies strongly with the medium pH. A low-affinity proline uptake system (Km, > 5 mM) is expressed at high levels only in chemically defined medium (CDM) with high osmolarity. This transport system is also stimulated by high osmolarity. The expression of this proline uptake system is repressed in rich broth with low or high osmolarity and in CDM with low osmolarity. The accumulated proline can be exchanged for betaine. Proline uptake is also effectively inhibited by betaine (Ki of between 50 and 100 microM). The proline transport system therefore probably also transports betaine. The inhibition of proline transport by betaine results in low proline pools in cells grown in high-osmotic-strength, betaine-containing CDM. The energy and pH dependency and the influence of ionophores on the activity of both transport systems suggest that these systems are not proton motive force driven. At low osmolarities, proline uptake is low but significant. This low proline uptake is also inhibited by betaine, although to a lesser extent than in cells grown in high-osmotic-strength CDM. These data indicate that proline uptake in L. lactis is enzyme mediated and is not dependent on passive diffusion, as was previously believed.     

The effect of polyethylene glycol-induced drought stress on photosynthesis,carbohydrates and cell membrane in <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> grown in greenhouse

Drought stress is one of the major environmental stresses that limit crop production in arid regions. A greenhouse culture experiment was conducted to evaluate the response of an agronomically and economically important sweet medical herb (Stevia rebaudiana) to polyethylene glycol (PEG 6000)-induced drought stress (5, 10, and 15% (w/v) PEG, equivalent to leaf water potentials of ??0.49, ??1.40 and ??2.93 MPa, respectively) for 1 month. Plant mass, a major determinant of Stevia yield, showed a reduction after PEG treatments. PEG-reduced photosynthesis traits included the maximal quantum yield of photosystem II (F_v/F_m), efficiency of photosystems I and II (PI_abs), intercellular CO₂, net photosynthesis, chlorophylls, carotenoids and water use efficiency, followed by the reduction of carbohydrates. Under PEG treatment, the reactive oxygen species (ROS) accumulation occurred and plants exhibited an increase in H₂O₂ generation. Consequently, an increase in malondialdehyde and electrolyte leakage was evident in PEG treatment, indicating membrane lipid peroxidation. In PEG-treated plants, the ROS accumulation was accompanied by an increase in activity of some enzymatic and non-enzymatic antioxidants. Leaf extracts of PEG-treated plants showed lower superoxide anion, hydroxyl and nitric oxide radical scavenging activity than control plants. Drought stress also caused the accumulation of the compatible solutes proline and glycine betaine. Collectively, the results demonstrated that PEG-induced oxidative stress, due to insufficient antioxidant mechanisms, provoked damages to cell membrane and photosynthetic apparatus, with consequently reduced carbohydrates and plant growth. These results are of basic importance as vegetative growth is the major determining criterion for Stevia crops and adequate irrigation is crucial for obtaining higher yield.     

Direct Regeneration of Shoots from Hairy Root Cultures of Centaurium erythraea Inoculated with Agrobacterium rhizogenes

Effect of free radical scavengers and metal chelators on polyethylene glycol (PEG, osmotic potential −1.5 MPa) induced oxidative damage in detached rice leaves was investigated. PEG treatment resulted in a decrease in relative water content and an increase in proline content, and lipid peroxidation. PEG treatment also decreased chlorophyll and protein contents. Free radical scavengers (ascorbate, sodium benzoate, reduced glutathione, and thiourea) retarded and metal chelators [2,2′-bipyridine (BP), 8-hydroxyquinoline, and 1,10-phenanthroline] prevented PEG-induced oxidative damage. Furthermore, the protective effect of BP was reversed by adding Fe²⁺ and Cu²⁺, but not by Mn²⁺ or Zn²⁺. The protective effect of BP is most likely mediated through chelation of iron. It seems that oxidative damage induced by PEG may require the participation of iron. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression and substrate specificity of betaine/proline transporters suggest a novel choline transport mechanism in sugar beet

Proline transporters (ProTs) originally described as highly selective transporters for proline, have been shown to also transport glycinebetaine (betaine). Here we examined and compared the transport properties of Bet/ProTs from betaine accumulating (sugar beet, Amaranthus, and Atriplex,) and non-accumulating (Arabidopsis) plants. Using a yeast mutant deficient for uptake of proline and betaine, it was shown that all these transporters exhibited higher affinity for betaine than proline. The uptake of betaine and proline was pH-dependent and inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). We also investigated choline transport by using a choline transport-deficient yeast mutant. Results revealed that these transporters exhibited a higher affinity for choline uptake rather than betaine. Uptake of choline by sugar beet BvBet/ProT1 was independent of the proton gradient and the inhibition by CCCP was reduced compared with that for uptake of betaine, suggesting different proton binding properties between the transport of choline and betaine. Additionally, in situ hybridization experiments revealed the localization of sugar beet BvBet/ProT1 in phloem and xylem parenchyma cells.     

Salt Tolerance in the Halophyte, Suaeda maritima (L.) Dum.: Properties of Malic Enzyme and PEP carboxylase

Malic enzyme and phosphenol pyruvate carboxylase activitieshave been isolated and characterized from the shoots of Suaedamaritima plants grown in culture solution (with and withoutNaCl) or in tap water. The enzymes isolated from the lattershowed increases in both specific activity and K_m values incomparison with plants grown in culture solution: however, theaddition of NaCl to the culture solution had no significanteffect on either enzyme. Malate levels were high in plants grownin tap water, reduced by an ordeT of magnitude by the additionof culture solution and then halved by the addition of NaCl. Both enzymes were inhibited in vitro by NaCl, although the additionof high concentrations of betaine and proline to the assay mediumdid not further inhibit enzyme activity. The significance ofthese results is discussed in relation to the proposed roleof betaine and proline as cytoplasmic osmoregulators. Suaeda maritima, halophyte, salt tolerance, malic enzyme, PEP carboxylase     

Functional Expression of Sinorhizobium meliloti BetS, a High-Affinity Betaine Transporter, in Bradyrhizobium japonicum USDA110

Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Østerås, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of B. japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na⁺/H⁺ antiporters in B. japonicum could explain its very high Na⁺ sensitivity.     

Betaine Accumulation and [C]Formate Metabolism in Water-stressed Barley Leaves

Barley (Hordeum vulgare L.) plants at the three-leaf stage were water-stressed by flooding the rooting medium with polyethylene glycol 6000 with an osmotic potential of −19 bars, or by withholding water. While leaf water potential fell and leaf kill progressed, the betaine (trimethylglycine) content of the second leaf blade rose from about 0.4 micromole to about 1.5 micromoles in 4 days. The time course of betaine accumulation resembled that of proline accumulation. Choline levels in unstressed second leaf blades were low (<0.1 micromole per blade) and remained low during water stress. Upon relief of stress, betaine-like proline—remained at a high concentration in drought-killed leaf zones, but betaine did not disappear as rapidly as proline from viable leaf tissue during recovery.

When [methyl-¹⁴C]choline was applied to second leaf blades of intact plants in the growth chamber, water-stressed plants metabolized 5 to 10 times more ¹⁴C label to betaine than control plants during 22 hours. When infiltrated with tracer quantities of [¹⁴C]formate and incubated for various times in darkness or light, segments cut from water-stressed leaf blades incorporated about 2- to 10-fold more ¹⁴C into betaine than did segments from unstressed leaves. In segments from stressed leaves incubated with [¹⁴C]formate for about 18 hours in darkness, betaine was always the principal ¹⁴C-labeled soluble metabolite. This ¹⁴C label was located exclusively in the N-methyl groups of betaine, demonstrating that reducing equivalents were available in stressed leaves for the reductive steps of methyl group biosynthesis from formate. Incorporation of ¹⁴C from formate into choline was also increased in stressed leaf tissue, but choline was not a major product formed from [¹⁴C]formate.

These results are consistent with a net de novo synthesis of betaine from 1- and 2-carbon precursors during water stress, and indicate that the betaine so accumulated may be a metabolically inert end product.

     

Relationships between polyamines, ethylene, osmoprotectants and antioxidant enzymes activities in wheat seedlings after short-term PEG- and NaCl-induced stresses

Contents of ethylene, osmoprotectants, levels and forms of polyamines (PAs) and activities of antioxidant enzymes in the leaves and roots were investigated for five wheat cultivar seedlings (differing in drought tolerance) exposed to osmotic stress (?1.5 MPa). Stress was induced by 2-day-long treatment of plants with polyethylene glycol 6000 (PEG) or NaCl added to hydroponic cultures. Nawra, Parabola and Manu cv. (drought tolerant) showed a marked increase in osmoprotectors (proline and soluble carbohydrates, mainly glucose, saccharose and maltose), free PAs (putrescine Put, spermidine Spd and spermine Spm) and Spd-conjugated levels, in both leaves and roots, after PEG-treatments. Radunia and Raweta (drought sensitive) exhibited smaller changes in the content of these substances. The analysis of enzymes involved in proline metabolism revealed the glutamate as a precursor of proline synthesis in PEG-induced stress conditions. The increase in the activity of antioxidative enzymes, especially catalase and peroxidases, was characteristic for tolerant wheat plants, but for sensitive ones, a decrease in superoxide dismutase and an increase in mainly glutathione reductase activities were observed. After NaCl-treatment smaller changes of all biochemical parameters were registered in comparison with PEG-induced stress. Exceptions were the higher values of ethylene content and a significant increase in saccharose, raffinose and maltose levels (only in stress sensitive plants). The proline synthesis pathway was stimulated from both glutamate and ornithine precursors. These results suggest that the accumulation of inorganic ions in NaCl-stressed plants may be involved in protective mechanisms as an additional osmoregultor. Thus, a weaker stressogenic effect as determined as water deficit by leaf relative water content and relative dry weight increase rate and differences in metabolite synthesis in comparison with PEG stress was observed. Proline seems to be the most important osmo-protector in osmotic stress initiated by both PEG and NaCl. The synthesis of sugars and PAs may be stimulated in a stronger stress conditions (PEG).     

Corynebacterium glutamicum Is Equipped with Four Secondary Carriers for Compatible Solutes: Identification, Sequencing, and Characterization of the Proline/Ectoine Uptake System, ProP, and the Ectoine/Proline/Glycine Betaine Carrier, EctP

Gram-positive soil bacterium Corynebacterium glutamicum uses the compatible solutes glycine betaine, proline, and ectoine for protection against hyperosmotic shock. Osmoregulated glycine betaine carrier BetP and proline permease PutP have been previously characterized; we have identified and characterized two additional osmoregulated secondary transporters for compatible solutes in C. glutamicum, namely, the proline/ectoine carrier, ProP, and the ectoine/glycine betaine/proline carrier, EctP. A ΔbetP ΔputP ΔproP ΔectP mutant was unable to respond to hyperosmotic stress, indicating that no additional uptake system for these compatible solutes is present. Osmoregulated ProP consists of 504 residues and preferred proline (K_m, 48 μM) to ectoine (K_m, 132 μM). The proP gene could not be expressed from its own promoter in C. glutamicum; however, expression was observed in Escherichia coli. ProP belongs to the major facilitator superfamily, whereas EctP, together with the betaine carrier, BetP, is a member of a newly established subfamily of the sodium/solute symporter superfamily. The constitutively expressed ectP codes for a 615-residue transporter. EctP preferred ectoine (K_m, 63 μM) to betaine (K_m, 333 μM) and proline (K_m, 1,200 μM). Its activity was regulated by the external osmolality. The related betaine transporter, BetP, could be activated directly by altering the membrane state with local anesthetics, but this was not the case for EctP. Furthermore, the onset of osmotic activation was virtually instantaneous for BetP, whereas it took about 10 s for EctP.     

Exogenous proline and glycinebetaine increase NaCl-induced ascorbate-glutathione cycle enzyme activities, and proline improves salt tolerance more than glycinebetaine in tobacco Bright Yellow-2 suspension-cultured cells

Up-regulation of the antioxidant system provides protection against NaCl-induced oxidative damage in plants. Antioxidants and activity of enzymes involved in the ascorbate-glutathione (ASC-GSH) cycle in tobacco Bright Yellow-2 (BY-2) were investigated to assess the antioxidant protection offered by exogenous proline and glycinebetaine (betaine from now on) against salt stress using cells grown in suspension culture. Reduced ascorbate (ASC) was detected in BY-2 cells but dehydroascorbate (DHA) was not. Large quantities of a reduced form of glutathione (GSH) and smaller quantities of an oxidized form of glutathione (GSSG) were detected in BY-2 cells. Salt stress significantly reduced the contents of ASC and GSH as well as activities of ASC-GSH cycle enzymes such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR). Exogenous proline or betaine increased the activities of all enzymes except MDHAR involved in NaCl-induced ASC-GSH cycle. Levels of ASC and GSH in BY-2 cells under salt stress were lower in the presence of proline or betaine than in the absence of proline or betaine whereas there was no difference in redox status. Proline proved more effective than betaine in maintaining the activity of enzymes involved in NaCl-induced ASC-GSH cycle. Neither proline nor betaine had any direct protective effect on NaCl-induced enzyme activity involved in the antioxidant system; however, both improved salt tolerance by increasing enzyme activity. The present study, together with our earlier findings [Hoque MA, Okuma E, Banu MNA, Nakamura Y, Shimoishi Y, Murata Y. Exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant enzyme activities. J Plant Physiol 2006;164:553-61.], suggests that proline offered greater protection against salt stress than betaine did because proline was more effective in increasing the activity of enzymes involved in the antioxidant system.     

Mechanism of Osmotic Activation of the Quaternary Ammonium Compound Transporter (QacT) of Lactobacillus plantarum

The accumulation of quaternary ammonium compounds in Lactobacillus plantarum is mediated via a single transport system with a high affinity for glycine betaine (apparent K_m of 18 μM) and carnitine and a low affinity for proline (apparent K_m of 950 μM) and other analogues. Mutants defective in the uptake of glycine betaine were generated by UV irradiation and selected on the basis of resistance to dehydroproline (DHP), a toxic proline analogue. Three independent DHP-resistant mutants showed reduced glycine betaine uptake rates and accumulation levels but behaved similarly to the wild type in terms of direct activation of uptake by high-osmolality conditions. Kinetic analysis of glycine betaine uptake and efflux in the wild-type and mutant cells is consistent with one uptake system for quaternary ammonium compounds in L. plantarum and a separate system(s) for their excretion. The mechanism of osmotic activation of the quaternary ammonium compound transport system (QacT) was studied. It was observed that the uptake rates were inhibited by the presence of internal substrate. Upon raising of the medium osmolality, the QacT system was rapidly activated (increase in maximal velocity) through a diminished inhibition by trans substrate as well as an effect that is independent of intracellular substrate. We also studied the effects of the cationic amphipath chlorpromazine, which inserts into the cytoplasmic membrane and thereby influences the uptake and efflux of glycine betaine. The results provide further evidence for the notion that the rapid efflux of glycine betaine upon osmotic downshock is mediated by a channel protein that is responding to membrane stretch or tension. The activation of QacT upon osmotic upshock seems to be brought about by a turgor-related parameter other than membrane stretch or tension.     

Effects of Salinity on Metabolic Profiles, Gene Expressions, and Antioxidant Enzymes in Halophyte Suaeda salsa

Halophyte Suaeda salsa is native to the saline soil in the Yellow River Delta. Soil salinity can reduce plant productivity and therefore is the most important factor for the degradation of wetlands in the Yellow River Delta. In this work we characterized the salinity-induced effects in S. salsa in terms of metabolic profiling, antioxidant enzyme activities, and gene expression quantification. Our results showed that salinity inhibited plant growth of S. salsa and upregulated gene expression levels of myo-inositol-1-phosphate synthase (INPS), choline monooxygenase (CMO), betaine aldehyde dehydrogenase (BADH), and catalase (CAT), and elevated the activities of superoxide dismutase (SOD), peroxidase (POD), CAT, and glutathione peroxidase (GPx). The significant metabolic responses included the depleted amino acids malate, fumarate, choline, phosphocholine, and elevated betaine and allantoin in the aboveground part of S. salsa seedlings as well as depleted glucose and fructose and elevated proline, citrate, and sucrose in root tissues. Based on these significant biological markers, salinity treatments induced clear osmotic stress (for example, INPS, CMO, BADH, betaine, proline) and oxidative stress (for example, SOD, POD, CAT, GPx activities), disturbed protein biosynthesis/degradation (amino acids and total protein) and energy metabolism (for example, glucose, sucrose, citrate) in S. salsa.     

Action of polyethylene glycol on the fusion of human erythrocyte membranes

Summary Factors affecting the polyethylene glycol (PEG)-induced membrane fusion were examined. Human erythrocyte membrane ghosts, cytoskeleton-free vesicles budded from erythrocytes, mechanically disrupted erythrocyte vesicles, and recombinant vesicles from glycophorin and egg phosphatidylcholine were used as models. Fusion was monitored by darkfield light microscopy and by freeze-fracture electron microscopy. Osmotic swelling was found necessary for fusion between membrane ghosts following PEG treatment. The sample with the highest fusion percentage was sealed ghosts incubated in hypotonic media after at least 5 min of treatment in <25% PEG. At similar osmolarity, glycerol, dextran and PEG produced progressively more pronounced intramembranous particle (IMP) patching, correlating with their increasing fusion percentages. The patching of IMP preceded cell-cell contact, and occurred without direct PEG-protein interaction. The presence of cytoskeletal elements in small vesicles had no significant effect on fusion, nor on the aggregation of intramembranous particle (IMP) upon PEG treatment. Disrupting the membrane by lysolecithin, dimethylsulfoxide, retinol or mild sonication resulted in the fragmentation of ghosts without an increase in fusion percentage. The purity of the commercial PEG used had no apparent effect on fusion. We concluded that the key steps in PEG-induced fusion of cell membrane are the creation of IMP-free zones, and the osmotic swelling of cells after the formation of bilayer contacts during the PEG treatment. Cell cytoskeleton affects PEG-induced fusion only to the extent of affecting IMP patching.     

Partitioning of Differently Sized Poly(ethylene glycol)s into OmpF Porin

To understand the physics of polymer equilibrium and dynamics in the confines of ion channel pores, we study partitioning of poly(ethylene glycol)s (PEGs) of different molecular weights into the bacterial porin, OmpF. Thermodynamic and kinetic parameters of partitioning are deduced from the effects of polymer addition on ion currents through single OmpF channels reconstituted into planar lipid bilayer membranes. The equilibrium partition coefficient is inferred from the average reduction of channel conductance in the presence of PEG; rates of polymer exchange between the pore and the bulk are estimated from PEG-induced conductance noise. Partition coefficient as a function of polymer weight is best fitted by a “compressed exponential” with the compression factor of 1.65. This finding demonstrates that PEG partitioning into the OmpF channel pore has sharper dependence on polymer molecular weight than predictions of hard-sphere, random-flight, or scaling models. A 1360-Da polymer separates regimes of partitioning and exclusion. Comparison of its characteristic size with the size of a 2200-Da polymer previously found to separate these regimes for the α-toxin shows good agreement with the x-ray structural data for these channels. The PEG-induced conductance noise is compatible with the polymer mobility reduced inside the OmpF pore by an order of magnitude relatively to its value in bulk solution.     

Exogenous glycine betaine and proline play a protective role in heat-stressed barley leaves (Hordeum vulgare L.): A chlorophyll a fluorescence study

Abstract

Effects of drought and exogenous glycine betaine and proline on Photosystem II (PSII) photochemistry were studied in barley leaves under heat stress induced by exposing them to 45°C for 10 min. Polyphasic fluorescence transient (OJIP) was used to evaluate PSII photochemistry in leaves treated with either glycine betaine or proline, combined or not with heat treatment. A distinct K step in the fluorescence transient OJIP appeared in control leaves, indicating an inactivation of the oxygen evolving complex (OEC). Drought stress and exogenous glycine betaine and proline modified the shape of the OJIP curve of leaves heated at 45°C and the K step was not as pronounced. Increased thermostability of PSII may be associated with the resistance of OEC and increased energy connectivity between PSII units. The thermostability of PSII was also reflected by a lower decrease in maximum quantum yield of primary photochemistry (?_Po = F _V/F _M) and performance index (PI). Exogenous application of glycine betaine or proline can play an important role in enhancing plant stress tolerance and may help reduce effects of environmental stresses.