The relation between membrane lipid phase separation and frost tolerance of cereals and other cool climate plant species

Abstract. The temperatures at which liposomes prepared from membrane phospholipids begin to phase separate were compared to the temperatures at which intact plants were damaged. Woody perennials tolerated temperatures below which their membrane phospholipids began to phase separate. By contrast, rye and wheat seedlings were damaged about 25°C above their phase separation temperature. Differences in tolerance among cultivars pre-hardened to frost were reflected by changes of the phase separation temperature. The results support the notion that alterations in membrane lipid composition are associated with frost hardening. A correlation between the temperature of phase separation and frost tolerance suggests that lipid properties may influence freezing tolerance of cereals; however, the lethal event is apparently not phase separation of the membrane phospholipids.     

NaCl胁迫对番茄嫁接苗生物量及离子含量的影响

以影武者为砧木,宝大903为接穗,研究了NaCl胁迫下番茄嫁接苗与接穗自根苗在生物量及Na 、K 、Ca2 、Mg2 、Cl-等含量方面的差异.结果显示,NaCl胁迫下,嫁接苗生长受抑制程度较轻;嫁接苗和自根苗Na 、Cl-含量增长幅度最大的部位均是叶片,嫁接苗增长幅度小于自根苗;K 、Ca2 、Mg2 含量下降幅度最大的部位均是根系,嫁接苗下降幅度小于自根苗;嫁接苗的K /Na 值、Ca2 /Na 值显著高于自根苗,说明嫁接苗选择性吸收K 、Ca2 能力显著强于自根苗.结果表明,NaCl胁迫下嫁接苗体内离子分布的区域化优于自根苗,耐盐性强于自根苗.     

Properties of native and solubilized plasma membrane ATPase from the halophyte Plantago crassifolia, grown under saline and non-saline conditions

Plasma membrane ATPase from the mediterranean halophyte Plantago crassifolia Forskal was analysed in the native and solubilized state. The enzyme revealed a broad pH optimum at 6.3 when analysed in plasma membrane preparations from roots. A further purified preparation of leaf plasma membrane ATPase was obtained by a three-step solubilization method. Final solubilization of the enzyme was achieved with 1% lysophosphatidylcholine. This enzyme showed a narrow pH optimum at 6.3, inhibition by vanadate, fluoride and N,N'-dicyclohexylcarbodiimide and a high specificity for ATP. The preparations contained a major polypeptide of 107 kDa. None of the parameters analysed in this enzyme changed upon transfer to saline conditions, although the leaves accumulated Na⁺ and Cl⁻ and an enhanced formation of the compatible organic osmoticum, sorbitol, was detected. It is concluded that at least in this halophyte, the plasma membrane ATPase undergoes no changes during the physiological adaptation of the plant to a saline environment.     

Salinity adaptation of plasma membrane H+-ATPase in the salt marsh plant Spartina patens: ATP hydrolysis and enzyme kinetics

Spartina patens, an intertidal C4 grass, grows in the upper salt marsh and tolerates coastal seawater salinity. The regulation of ion movement across the plasma membrane (PM) for plant salt tolerance is thought to be achieved by an electrochemical gradient generated by plasma membrane H⁺-ATPase. In this study, the change of PM H⁺-ATPase in response to NaCl was characterized for S. patens callus. Callus was cultured for 10 weeks under salinity levels of 0 mM, 170 mM, 340 mM, and 510 mM NaCl. Plasma membrane was isolated from a Dextran/PEG aqueous polymer two-phase system and the purity was demonstrated with membrane enzyme markers. There was a significant increase (up to 2-3 fold) of PM H⁺-ATPase activity when callus was grown on media containing NaCl. The incremental activation of PM H⁺-ATPase activity would enable the cell to tolerate higher cytoplasmic NaCl concentrations. PM H⁺-ATPase appeared to have a higher Vmax and a lower substrate concentration (Km to reach Vmax. When growth medium salinity increased from 0 mM to 170 and 340 mM, the Vmax of H⁺-ATPase increased from 0.64 to 1.00 and 1.73, respectively, while the Km decreased from 3.58 to 2.07 and 2.44 mM, respectively. In vitro NaCl inhibition kinetic data revealed a pattern of non-competitive inhibition by NaCl on PM H⁺-ATPase. The response of PM H⁺-ATPase in S. patens callus suggests that this species has evolved mechanisms that can regulate this important enzyme when cells are exposed to NaCl.     

Salinity tolerance of Kosteletzkya virginica. II. Root growth, lipid content, ion and water relations

Abstract. Kosteletzkya virginica (L.) Presl., a dicot halophyte native to brackish tidal marshes, was grown on nutrient solution containing 0. 85, 170 or 255 mol m ³ NaCl, and the effects of external salinity on root growth, ion and water levels, and lipid content were examined in successive harvests. Root growth paralleled shoot growth trends, with some enhancement observed at 85 mol m ³ NaCl and a reduction noted at the higher salinities. Root Na⁺ content increased with increasing external NaCl, but remained constant with time for each treatment. K⁺ content, although lower in salt-grown plants after 14 d salinization, subsequently increased to levels comparable to unsalinized plants. A strong K⁺ affinity was reflected in the increased K⁺/Na⁺ selectivity of salt-grown plants and by their low Na⁺/K⁺ ratios. Cl levels rose in salinized plants and values were double or more those for Na⁺, indicating the possibility of a sodium-excluding mechanism in roots. Root phospholipids and sterols, principal membrane constituents, were maintained or elevated and the free sterol/phospholipids ratio increased in salinized K. virginica plants, suggesting retention of overall membrane structure and decreased permeability. This response, considered in light of root calcium maintenance and high potassium levels, suggests that salinity-induced changes in membrane lipid composition may be important in preventing K⁺ leakage from cells.     

Effect of auxin concentration and growth phase on the plasma membrane H-ATPase of tobacco calli

The activity of plasma membrane H⁺-ATPase increases up to 3 fold during the growth of tobacco calli for 6 weeks. In medium with optimal auxin this activity is always greater than in medium with suboptimal hormone. The amount of H⁺-ATPase measured with specific antibody only experiences small changes under the same conditions. Therefore, auxin level and growth phase mostly influence the molecular activity of the enzyme and not its amount. Despite the increase in H⁺-ATPase activity, there is a decrease in K⁺ content and ths decrease is greater in medium with suboptimal auxin. Apparently, there is a progressive deenergization of the plasma membrane which is only partially compensated by an auxin-dependent increase in activity of plasma membrane H⁺-ATPase.     

The induction of freezing tolerance in jack pine seedlings: The role of root plasma membrane H+- ATPase and redox activities

The acquired freezing tolerance of jack pine seedlings (Pinus banksiana Lamb.) conditioned at low nonfreezing temperatures and short photoperiods was determined by comparison of seedling survival to that of nonconditioned (control) seedlings following exposure to ?5 and ?10°C. Compared to that of controls, survival of conditioned seedlings was markedly increased following exposure to freezing temperatures. A 1-week conditioning treatment significantly increased the survival of the seedlings after exposure to ?5°C, but was less effective on seedlings exposed to ?10°C. Conditioning periods of 2 and 4 weeks resulted in higher survival of seedlings exposed to both ?5 and ?10°C. The changes of two root-plasma-membrane-associated enzyme activities, H⁺-ATPase and NADH-dependent ferricyanide reductase, were studied in enriched plasma membrane fractions during conditioning and after freezing. Post-freezing activities of both enzymes were enhanced by conditioning at low temperatures and short photoperiods. These changes may be related to the increased frost hardiness also induced by conditioning.     

Influence of growth conditions on the composition of the plasma membrane from yeast and on kinetic properties of two membrane functions

The influence of different growth conditions on the phospholipid composition and on two membrane functions, the Mg-ATPase and the purine transport system, was investigated. Addition of cholinechloride to the growth medium led to a certain rise in the amount of phosphatidylcholine, whereas supplementation with ethanolamine resulted in a considerably higher portion of phosphatidylethanolamine. When yeast cells were cultured at lower temperatures we found more short-chain fatty acids with a higher content of monounsaturated chains as compared to higher growth temperatures. Addition of paraquat, a herbicide which enhances lipid peroxidation by free radicals, reduced the amount of unsaturated fatty acids without influencing their chain length. The altered membrane composition had no influence on the basic mechanism of interaction between ATPase, MgATP, and free Mg2+ ions. However, several kinetic constants such as Km, Vmax, Ka, and especially Ki were influenced to some extent. Whereas the affinity of the purine transport system to its substrate was not significantly changed by the growth conditions, an effect on Vmax could be seen. Lower growth temperatures clearly led to higher maximal uptake velocities. The presence of paraquat during growth resulted in a considerable decrease of Vmax.     

Relationship between effect of ethanol on proton flux across plasma membrane and ethanol tolerance, in Pichia stipitis

Pichia stipitis efficiently converts glucose or xylose into ethanol but is inhibited by ethanol concentrations exceeding 30 g/L. In Saccharomyces cerevisiae, ethanol has been shown to alter the movement of protons into and out of the cell. In P. stipitis the passive entry of protons into either glucose- or xylose-grown cells is unaffected at physiological ethanol concentrations. In contrast, active proton extrusion is affected differentially by ethanol, depending on the carbon source catabolized. In fact, in glucose-grown cells, the H(+)-extrusion rate is reduced by low ethanol concentrations, whereas, in xylose-grown cells, the H(+)-extrusion rate is reduced only at non-physiological ethanol concentrations. Thus, the ethanol inhibitory effect on growth and ethanol production, in glucose-grown cells, is probably caused by a reduction in H(+)-extrusion. Comparison of the rates of H(+)-flux with the related in vitro H(+)-ATPase activity suggests a new mechanism for the regulation of the proton pumping plasma membrane ATPase (EC 3.6.1.3) of P. stipitis, by both glucose and ethanol. Glucose activates both the ATP hydrolysis and the proton-pumping activities of the H(+)-ATPase, whereas ethanol causes an uncoupling between the ATP hydrolysis and the proton-pumping activities. This uncoupling may well be the cause of ethanol induced growth inhibition of glucose grown P. stipitis cells.     

Differential sensitivity of astrocyte primary cultures and derived spontaneous transformed cell lines to 70-hydroxycholesterol: effect on plasma membrane lipid composition and fluidity,and on cell surface protein expression

Summary The cytotoxicity of 7-hydroxycholesterol (7-OHC) was investigated on rat astrocyte primary cultures and spontaneously transformed cell lines derived from them. Confluent astrocyte primary cultures (normal cells) were unaffected by 20 µM 7(3-OHC over a period of 72 h whereas 30 µM markedly affected the viability of the transformed cells within the first 72 h. Both cell types incorporated 18% of the total amount of 7-OHC added to the cultures at concentrations of 20 µM or 30 µM. Cellular fractionation after incubation with 20 µM or 30 µM 7-OHC indicated that the plasma membrane incorporated 2 or 6 fold more 7-OHC than the intracellular one's respectively. Plasma membrane cholesterol (CH) and phospholipid (PL) analysis showed that 20 µM 7-OHC did not affect CH/PL in normal cells; in contrast, plasma membranes of transformed cells displayed a significant CH/PL decrease, which was more pronounced with 30 µM 7-OHC treatment. Fluorescence anisotropy measurements indicated that 20 µM 7-OHC slightly fluidified the plasma membrane of normal cells whereas it has not effect on that of the transformed cells one; however, an increase in plasma membrane fluidity was observed when the transformed cells were treated with 30 µM 7-OHC. Lactoperoxidase catalyzed radioiodination of cell surface proteins and subsequent autoradioelectrophoretic analysis demonstrated that the labelled protein pattern was unchanged when both cell types were incubated with 30 µM 7-OHC.     

Effects of human apolipoprotein E isoforms on the amyloid beta-protein concentration and lipid composition in brain low-density membrane domains

Apolipoprotein E4 (apoE4) encoded by epsilon 4 allele is a strong genetic risk factor for Alzheimer's disease (AD). ApoE4 carriers have accelerated amyloid beta-protein (A beta) deposition in their brains, which may account for their unusual susceptibility to AD. We hypothesized that the accelerated A beta deposition in the brain of apoE4 carriers is mediated through cholesterol-enriched low-density membrane (LDM) domains. Thus, the concentrations of A beta and various lipids in LDM domains were quantified in the brains of homozygous apoE3 and apoE4 knock-in (KI) mice, and in the brains of those mice bred with beta-amyloid precursor protein (APP) transgenic mice (Tg2576). The A beta 40 and A beta 42 concentrations and the A beta 42 proportions in LDM domains did not differ between apoE3 and apoE4 KI mice up to 18 months of age. The A beta 40 concentration in the LDM domains was slightly, but significantly higher in apoE3/APP mice than in apoE4/APP mice. The lipid composition of LDM domains was modulated in an apoE isoform-specific manner, but its significance for A beta deposition remains unknown. These data show that the apoE isoform-specific effects on the A beta concentration in LDM domains do not occur in KI mouse models.     

Surface architecture of the plant cell: Biogenesis of the cell wall,with special emphasis on the role of the plasma membrane in cellulose biosynthesis

Cell wall structure and biogenesis in the unicellular green alga, Oocystis apiculata, is described. The wall consists of an outer amourphous primary layer and an inner secondary layer of highly organized cellulosic microfibrils. The primary wall is deposited immediately after cytokinesis. Golgi-derived products contribute to this layer. Cortical microtubules underlie the plasma membrane immediately before and during primary wall formation. They function in maintaining the elliptical cell shape. Following primary wall synthesis, Golgi-derived materials accumulate on the cell surface to form the periplasmic layer. This layer functions in the deposition of coating and cross-linking substances which associate with cellulosic microfibrils of the incipient secondary wall. Secondary wall microfibrils are assembled in association with the plasma membrane. Freeze-etch preparations of untreated, living cells reveal linear terminal complexes in association with growing cellulosic microfibrils. These complexes are embedded in the EF fracture face of the plasma membrane. The newly synthesized microfibril lies in a groove of the outer leaflet of the plasma membrane. The groove is decorated on the EF fracture face by perpendicular structures termed “ridges.” The ridges interlink with definitive rows of particles associated with the PF fracture face of the inner leaflet of the plasma membrane. These particles are termed “granule bands,” and they function in the orientation of the newly synthesized microfibrils. Microfibril development in relation to a coordinated multienzyme complex is discussed. The process of cell wall biogenesis in Oocystis is compared to that in higher plants.     

Effects of exogenously applied plant growth regulators in combination with PGPR on the physiology and root growth of chickpea (Cicer arietinum ) and their role in drought tolerance

Both the plant growth promoting rhizobacteria (PGPR) and plant growth regulators (PGR) exert beneficial effects on plant growth even under stress, but combined effect of both of them has not been evaluated yet. Present investigation was aimed to determine the responses of 2 chickpea varieties (differing in drought tolerance) to 3 PGPR viz. Bacillus subtilis, Bacillus thuringiensis and Bacillus megaterium and PGR (SA and Putrescine) on physiology of chickpea grown in sandy soil. The PGR, Salicylic acid (SA) and Putrescine (Put) were sprayed on the seedling 20 days after germination. Results revealed, synergistic effects of PGPR and PGR on chlorophyll, protein and sugar contents. Addition of PGR to PGPR inoculated plants assisted the plant in osmoregulation and amelioration of oxidative stresses and in induction of new proteins. Combined application of PGR and PGPR decreased lipid peroxidation more effectively but increased the leaf area. It is inferred that PGPR and PGR work synergistically to promote growth of plants under moisture and nutrient deficit condition of sandy soil. Since, SA induces Systemic Acquired Resistance (SAR) in plants hence the addition of SA along with PGPR may render the plant more productive and better tolerant to diseases/pathogen attack.     

On the presence of inside-out plasma membrane vesicles and vanadate-inhibited K+,Mg2+-ATPase in microsomal fractions from wheat and maize roots

We have estimated the amount of inside-out plasma membrane (PM) vesicles in microsomal fractions from wheat (Triticum aestivum L. cv. Drabant) and maize (Zea mays L.) roots; non-latent activities of the PM markers vanadate-inhibited K⁺, Mg²⁺-ATPase (ΔVO₄-ATPase) and glucan synthase II (GS II, EC 2.4.1.34) were used as markers for inside-out PM vesicles, latent activities as markers for right-side-out PM vesicles, and specific staining with silicotungstic acid (STA) as a general marker for the PM. Separation of presumptive inside-out PM vesicles from right-side-out ones was achieved by counter-current-distribution (CCD) in an aqueous polymer two-phase system. Most of the GS II activity was latent and was found in material partitioning into the upper phase; a distribution which correlated well with that of STA-stained vesicles. Thus, most of the PM vesicles had a right-side-out orientation. ΔVO₄-ATPase, on the other hand, had a dual distribution (particularly pronounced in wheat) and was recovered both in material partitioning into the lower phase and into the upper phase. This indicates that ΔVO₄-ATPase activity was present also in membranes other than the PM. Additional evidence for this interpretation came from sucrose gradient centrifugation of wheat root material. This produced two peaks of ΔVO₄-ATPase activity with the membranes partitioning into the lower phase, none of which coincided with the peak obtained with right-side-out PM vesicles. Taken together, these results indicate that only very few inside-out PM vesicles are present in the microsomal fraction, and that ΔVO₄-ATPase as a marker for the PM, in contrast to GS II, may give quite misleading results with some plant materials. This stresses the need to use well-defined preparations of scaled, inside-out PM vesicles in solute uptake studies. The distribution of Ca²⁺-inhibited ATPase, on the other hand, agreed well with those of GS II and STA-stained vesicles both after CCD and sucrose gradient centrifugation, which suggests that Ca²⁺ inhibition may be a more specific property of the PM H⁺-ATPase than vanadate inhibition.     

RNA--induced silencing of the plasma membrane Ca2+-ATPase 2 in neuronal cells: effects on Ca2+ homeostasis and cell viability

Intraneuronal calcium ([Ca(2+)](i)) regulation is altered in aging brain, possibly because of the changes in critical Ca(2+) transporters. We previously reported that the levels of the plasma membrane Ca(2+)-ATPase (PMCA) and the V(max) for enzyme activity are significantly reduced in synaptic membranes in aging rat brain. The goal of these studies was to use RNA(i) techniques to suppress expression of a major neuronal isoform, PMCA2, in neurons in culture to determine the potential functional consequences of a decrease in PMCA activity. Embryonic rat brain neurons and SH-SY5Y neuroblastoma cells were transfected with in vitro--transcribed short interfering RNA or a short hairpin RNA expressing vector, respectively, leading to 80% suppression of PMCA2 expression within 48 h. Fluorescence ratio imaging of free [Ca(2+)](i) revealed that primary neurons with reduced PMCA2 expression had higher basal [Ca(2+)](i), slower recovery from KCl-induced Ca(2+) transients, and incomplete return to pre-stimulation Ca(2+) levels. Primary neurons and SH-SY5Y cells with PMCA2 suppression both exhibited significantly greater vulnerability to the toxicity of various stresses. Our results indicate that a loss of PMCA such as occurs in aging brain likely leads to subtle disruptions in normal Ca(2+) signaling and enhanced susceptibility to stresses that can alter the regulation of Ca(2+) homeostasis.     

Increasing NaCl and CaCl<Subscript>2</Subscript> concentrations in the growth medium of quince leaves: I. Effects on somatic embryo and root regeneration

Summary The effects of increasing concentrations of NaCl and CaCl₂ on quince (Cydonia oblonga Mill. BA 29 clone) somatic embryogenesis and adventitious root regeneration were investigated. Leaves collected from in vitro-grown shoots were used as explants and induced for 2d in liquid Murashige and Skoog medium containing 11.3 μM 2,4-dichlorophenoxyacetic acid. Explants were then cultured on semisolid Murashige and Skoog medium enriched with 4.7 μM kinetin and 0.5 μM naphthaleneacetic acid under red light for 25 d and under white light for another 25 d. Two experiments were performed: in the first, NaCl was used at 0,25, 50, 100, and 200 mM in factorial combination with CaCl₂ at 3, 9, and 27 mM; in the second, NaCl was applied at 0, 5, 10, 20, 40, and 80 mM in combination with CaCl₂ at 0.3, 1.0, and 3.0 mM. Quince leaves revealed the capacity to regenerate somatic embryos and/or adventitious roots. Quantitative and qualitative regeneration from leaves was affected by NaCl treatments: increasing NaCl concentrations, in combination with CaCl₂ at 1 mM, led to an increase in the proportion of leaves producing somatic embryos only, and to a decrease of both leaves regenerating roots only and leaves simultaneously producing somatic embryos and adventitious roots. This suggests a beneficial effect of salt stress on the embryogenic process. The regeneration response decreased with increasing salt concentrations and was almost totally inhibited above 50 mM NaCl and 9 mM CaCl₂. The presence of CaCl₂ in the culture medium apparently mitigated the effects of salt stress, but only when NaCl was applied at 40 mM. NaCl at 5 mM, in the presence of 0.3 or 1 mM CaCl₂, was favorable both to somatic embryo and root production. No value of the ratio Na⁺/Ca²⁺ was found to be optimal for the regeneration processes.     

Regulation of the cancer cell membrane lipid composition by NaCHOleate : Effects on cell signaling and therapeutical relevance in glioma

This review summarizes the cellular bases of the effects of NaCHOleate (2-hydroxyoleic acid; 2OHOA; Minerval) against glioma and other types of tumors. NaCHOleate, activates sphingomyelin synthase (SGMS) increasing the levels of cell membrane sphingomyelin (SM) and diacylglycerol (DAG) together with reductions of phosphatidylethanolamine (PE) and phosphatidylcholine (PC). The increases in the membrane levels of NaCHOleate itself and of DAG induce a translocation and overexpression of protein kinase C (PKC) and subsequent reductions of Cyclin D, cyclin-dependent kinases 4 and 6 (CDKs 4 and 6), hypophosphorylation of the retinoblastoma protein, inhibition of E2F1 and knockdown of dihydrofolate reductase (DHFR) impairing DNA synthesis. In addition in some cancer cells, the increases in SM are associated with Fas receptor (FasR) capping and ligand-free induction of apoptosis. In glioma cell lines, the increases in SM are associated with the inhibition of the Ras/MAPK and PI3K/Akt pathways, in association with p27Kip1 overexpression. Finally, an analysis of the Repository of Molecular Brain Neoplasia Data (REMBRANDT) database for glioma patient survival shows that the weight of SM-related metabolism gene expression in glioma patients' survival is similar to glioma-related genes. Due to its low toxicity and anti-tumoral effect in cell and animal models its status as an orphan drug for glioma treatment by the European Medicines Agency (EMA) was recently acknowledged and a phase 1/2A open label, non-randomized study was started in patients with advanced solid tumors including malignant glioma. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.