Effects of preconditioning on electrolyte leakasge and lipid composition in black spruce (Picea mariand) stressed with polyethylene giycol

Black spruce ( Picea mariana Mill. B. S. P.) rooted cuttings were grown in solution culture and preconditioned by osmotically stressing plants with polyethylene glycol. After relief from preconditioning stress, water relations, membrane leakiness, and the composition of lipids and fatty acids were compared in preconditioned and control, unconditioned plants. Both groups of plants were subsequently subjected to a severe osmotic stress with polyethylene glycol and examined again. Osmotic stress decreased shoot water potentials and increased the leakage of electrolytes from shoots of stressed, compared with unstressed, plants. However, both unstressed and stressed preconditioned plants leaked less electrolytes compared with unconditioned plants. Changes in sterol, phospholipid and glycolipid composition were observed in preconditioned unstressed and stressed plants. Sterol and phospholipid levels de clined as a result of stress, and preconditioning resulted in a decline in sterol: phospholipid ratios in plants.     

Early effects of triadimefon on water relations, sterol composition and plasma membrane ATPase in white spruce (Picea glauca) seedlings

Seedlings of white spruce ( Picea glauca [Moench] Voss.) were treated with triadimefon solution applied to the soil, and their early responses studied from 12 h to 7 days after treatment. Transpiration rates declined and respiration rates increased immediately after the commencement of triadimefon treatment. Photosynthetic rates declined less than transpiration rates, resulting in an increase in water use efficiency, whereas root and shoot water potentials remained unchanged during the first 5 days of triadimefon treatment. Triadimefon decreased root hydraulic conductivity and inhibited the activity of the plasma membrane ATPase. In addition, triadimefon-treated roots drastically increased the ratios between free sterols and sterol esters and decreased the ratios between sterol esters and acylated sterol glycosides.     

Hydrogen fluoride effects on plasma membrane composition and ATPase activity in needles of white pine (Pinus strobus) seedlings pretreated with 12 h photoperiod

 Eastern white pine (Pinus strobus L.) seedlings were pretreated with 12 h photoperiod to induce dormancy. Dormant plants were fumigated with 0.5 ppb (0.4 μg m^–3) or 2.0 ppb (1.6 μg m^–3) hydrogen fluoride (HF) for 2 – 28 days. Plasma membranes were isolated from needles of treated and control seedlings to determine their chemical composition and ATPase activity. For all analyses, only those plants which did not show needle necrosis were selected. The amount of plasma membrane phospholipid expressed on a plasma membrane protein basis was higher after 2 days in the 0.5 ppb HF treatment as compared to controls. After 2 days of 2.0 ppb HF treatment as well as after 8 and 28 days of both HF treatments phospholipid to protein ratios in fluoride treated seedlings were lower as compared to control levels. A decrease in sterol levels could be observed after 2 days in both HF treatments. A large increase in the ratio of sterols to proteins was observed in plasma membranes of eastern white pine seedlings treated with 0.5 ppb HF for 28 days. Increased sterol to phospholipid ratios were observed after 8 and 28 days in 0.5 ppb and after 2 and 8 days of 2.0 ppb HF treatment. A decrease in ATPase activity was observed after 8 days with both fluoride treatments. Drastic increase of ATPase activity was observed after 28 days of HF treated plants. Observed changes of sterol and phospholipid levels after only 2 days of fumigation suggest early fluoride effects on plasma membrane composition during plant dormancy. Received: 25 October 1995 / Accepted: 24 May 1996     

Hydrogen fluoride effects on plasma membrane composition,ATPase activity and cell structure in needles of eastern white pine (Pinus strobus) seedlings

Eastern white pine (Pinus strobus L.) seedlings were grown in controlled environment growth cabinets and fumigated with 0.4 and 1.6 g m^–3 hydrogen fluoride for 2–28 days. Plasma membranes were isolated from needles of treated and control seedlings and their chemical composition and ATPase activity examined to determine early effects of hydrogen fluoride action. In plants treated for 2 days with both fluoride levels, ratios of plasma membrane free sterols:phospholipids and sterols:proteins were drastically higher than ratios in control plants. Seedlings treated with hydrogen fluoride for 8 days contained plasma membranes with elevated phospholipid:protein and sterol:protein ratios and their plasma membrane ATPase activity was higher than that of control plants. Prolonged, 28-day hydrogen fluoride treatment with 1.6 g m^–3 level was the only treatment which produced a drastic inhibition of plasma membrane ATPase activity. During the initial stages of hydrogen fluoride treatment, treated cells did not show alterations of ultrastructure which were previously shown in cells of plants treated with soil applied sodium fluoride. The results of the present study indicate that the plasma membranes may be among the initial sites of hydrogen fluoride injury to plants as well as initial sites of defense reaction.     

Effects of paclobutrazol and chilling temperatures on lipids, antioxidants and ATPase activity of plasma membrane isolated from green bell pepper fruits

The effects of paclobutrazol treatment on plasma membrane lipid composition and ATPase activity of bell pepper fruit ( Capsicum annuum ) subjected to chilling temperatures were assessed. Application of the growth regulator paclobutrazol affected plant growth and fruit morphology. The plants were more compact and the fruits were less elongated than control fruits. There was about 60% more plasma membrane on a fresh weight basis from treated fruits. At harvest there was no difference in sterol to phospholipid ratio, or in phospholipid fatty acid composition of control compared with paclobutrazol treated fruit. However, plasma membrane ATPase acitivity of treated fruit was two times higher than that of control fruit. After storage at chilling temperature (2°C), the control fruit developed more chilling iniury, and had greater weight loss and a higher rate of K⁺ leakage than paclobulrazol treated fruit. Plasma membrane phospholipid content decreased and saturation of phospholipid fatty acids was higher than in control fruit. These two changes were largely absent in plasma membrane from treated fruit. At harvest antioxidant levels in the plasma membrane of paclobutrazol treated peppers were higher than in those of controls and changed little during storage, whereas levels in control fruit plasma membrane decreased 66%. ATPase activity increased and then decreased in control fruit held at low temperature, whereas in treated fruit activity was constant. The protective effect of paclobutrazol against chilling injury of pepper fruit may result from a combination of its effect on fruit morphology, and protection of the lipids against oxidative stress.     

Lipid composition of plasma membranes isolated from sunflower seedlings grown under water-stress

Plasma membranes were isolated by aqueous two-phase-partitioning from sunflower ( Helianthus annuus cv. Isabel) seedlings grown both under field irrigation and dryland conditions. Water-stressed plants showed a decrease in the leaf water potential and in the osmotic potential at full turgor, with the turgor pressure remaining at positive values. Dryland conditions also induced a reduction in the bulk modulus of elasticity. Plasma membranes of irrigated plants were characterized by high contents of phospholipids (68% of total lipids), free sterols (15. 7%) and glycolipids (9. 1%), mainly glycosphingolipids and steryl glycosides. Diacylglycerols, triacylglycerols and free fatty acids were also present. The major phospholipids were phosphatidylcholine and phosphatidylethanolamine with smaller amounts of phosphatidylinositol and phosphatidylglycerol. Following water stress, the plasma membranes showed a reduction of about 24 and 31% in total lipids and phospholipids, respectively. Also the amounts of glycolipids and diacylglycerols decreased significantly upon water stress. There was no change in free fatty acids, however, and triacylglycerols and free sterols increased. As a consequence, the free sterol to phospholipid molar ratio increased from 0. 4 to 0. 7 under water deficit conditions. The ratio of phosphatidylcholine to phosphatidylethanolamine increased from 1. 1 (control plants) to 1. 6 (water-stressed plants), while phosphatidic acid rose to 4% of total phospholipids. Dehydration did not result in any substantial change in the unsaturation level of the individual lipid classes, however. The results show that dryland conditions resulted in a marked alteration in the lipid composition of the sunflower leaf plasma membrane     

Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel‐based and LC MS/MS‐based proteomics approaches

To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel‐based and a LC MS/MS‐based proteomics method. Two‐day‐old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two‐phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel‐based proteomics, four and eight protein spots were identified as up‐ and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up‐ and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low‐abundance proteins could be identified by the LC MS/MS‐based method. Three homologues of plasma membrane H⁺‐ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H⁺‐ATPase protein.     

Chemical changes and O2˙− production in thylakoid membranes under water stress

Sunflower seedlings ( Helianthus annuus cv. Isabel) subjected to a moderate level of water stress showed a reduced growth and a 0. 1 MPa osmotic adjustment came into play. Thylakoid membranes isolated from stressed leaves showed decreased chlorophyll (Chl) and protein contents but the Chl al /Chl b and protein/Chl ratios were unchanged. Water stress caused a preferential hydrolysis in thylakoid proteins: the hydrophilic to hydrophobic protein ratio increased from 0. 8 in the control to 4. 5 in the stressed plants. However, the degree of unsaturation was unchanged and the electron spin resonance (ESR) measurements did not show an increased level of O₂^.-radical production by photosynthetic membranes.     

PLASMA AND PHAGOSOME MEMBRANES OF ACANTHAMOEBA CASTELLANII

Plasma membranes were isolated from the ameba Acanthamoeba castellanii by low-speed velocity centrifugation followed by equilibrium centrifugation in a sucrose gradient. The isolated membranes had a high ratio of sterol to phospholipid (0.98 moles/mole) and of phospholipid to protein (0.43 mg/mg). The plasma membranes had very low concentrations of DNA, RNA, lipid inositol, and glycerides. Glycolipids and glycoproteins were enriched in the plasma membranes relative to their concentrations in the whole cell. The plasma membranes were also judged to be of high purity by the absence, or very low level, of enzymatic activities considered to be indicative of other cell membranes, and by electron microscope examination. Alkaline phosphatase and 5'-nucleotidase activities were enriched in the plasma membranes 13-fold relative to the whole homogenate and had higher specific activities in the plasma membranes than in any other cell fractions. A Mg⁺⁺ adenosine triphosphatase (ATPase) was enriched sixfold in the plasma membranes relative to the whole homogenate. The phospholipids of the plasma membranes contained more phosphatidylethanolamine and phosphatidylserine and less phosphatidylcholine than did the phospholipids of the whole cells. There were differences in the fatty acid compositions of corresponding phospholipids in the plasma membranes and whole cells but no difference in the ratios of total saturated to unsaturated fatty acids. The membranes of phagosomes isolated from amebae that had ingested polystyrene latex had essentially the same phospholipid, sterol, and enzymatic composition as plasma membranes.     

Effect of Osmotic Stress on Ion Transport Processes and Phospholipid Composition of Wheat (Triticum aestivum L.) Mitochondria

The effect of osmotic stress on wheat (Triticum aestivum L.) mitochondrial activity and phospholipid composition was investigated. Preliminary growth measurements showed that osmotic stress (−0.25 or −0.5 megapascal external water potential) inhibited the rate of shoot dry matter accumulation while root dry matter accumulation was less sensitive. We have determined that differences in sensitivity to osmotic stress existed between tissues at the mitochondrial level. Mitochondria isolated from roots or shoots of stressed seedlings showed respiratory control and ADP/O ratios similar to control seedlings which indicates that stressed mitochondria were well coupled. However, under passive swelling conditions in a KCl reaction mixture, the rate and extent of valinomycin-induced swelling of shoot mitochondria were increased by osmotic stress while root mitochondria were largely unaffected. Active ion transport studies showed efflux transport by stressed-shoot mitochondria to be partially inhibited since mitochondrial contraction required the addition of N-ethylmaleimide or nigericin. Efflux ion transport by root mitochondria was not inhibited by osmotic stress which indicates that stress-induced changes in ion transport were largely limited to shoot mitochondria. Characterization of mitochondrial fatty acid and phospholipid composition showed an increase in the percentage of phosphatidylcholine in stressed shoot mitochondria compared to the control. Mitochondrial fatty acid composition was not markedly altered by stress. No significant changes in either the phospholipid or fatty acid composition of stressed root mitochondria were observed. Hence, these results suggest that a tissue-specific response to osmotic stress exists at the mitochondrial level.     

Free and conjugated desmethylsterol composition of Zea mays hybrids exposed to mild osmotic stress

Five hybrids of Zea mays (A619 × H60, B73 × MO17, B73 × PA91, B73 × VA17. A632 × H96) were grown hydroponically at osmotic potentials of -0.1 (control), -0.4 and -0.6 MPa. Dry weights of hybrids B73 × VA17 and A632 × H96 decreased significantly at -0.4 and -0.6 MPa. Few changes were observed in the free sterol, steryl glycoside and steryl ester fractions. However, steryl esters of the roots and stems of osmotically treated plants declined in most of the hybrids. The stigmasterol to sitosterol ratio increased in all three steryl fractions in the roots of B73×VA17 and A632×H96 as a result of the osmotic treatments. B73×VA17 and A632×H96 also exhibited the greatest initial electrolyte leakage when leaf dises were subjected to osmotic stress. A632×H96 and possibly B73×VA17 were less tolerant to osmotic treatments than the other hybrids. Modification of sterols may reflect initial events that lead to the stabilization or destabilization of cellular membrances which in turn may affect the tolerance of plants to stressful environments.     

Sterol carrier protein-2 expression alters plasma membrane lipid distribution and cholesterol dynamics

Although sterol carrier protein-2 (SCP-2) binds, transfers, and/or enhances the metabolism of many membrane lipid species (fatty acids, cholesterol, phospholipids), it is not known if SCP-2 expression actually alters the membrane distribution of lipids in living cells or tissues. As shown herein for the first time, expression of SCP-2 in transfected L-cell fibroblasts reduced the plasma membrane levels of lipid species known to traffic through the HDL-receptor-mediated efflux pathway: cholesterol, cholesteryl esters, and phospholipids. While the ratio of cholesterol/phospholipid in plasma membranes of intact cells was not changed by SCP-2 expression, phosphatidylinositol, a molecule important to intracellular signaling and vesicular trafficking, and anionic phospholipids were selectively retained. Only modest alterations in plasma membrane phospholipid percent fatty acid composition but no overall change in the proportion of saturated, unsaturated, monounsaturated, or polyunsaturated fatty acids were observed. The reduced plasma membrane content of cholesterol was not due to SCP-2 inhibition of sterol transfer from the lysosomes to the plasma membranes. SCP-2 dramatically enhanced sterol transfer from isolated lysosomal membranes to plasma membranes by eliciting detectable sterol transfer within 30 s, decreasing the t(1/2) for sterol transfer 364-fold from >4 days to 7-15 min, and inducing formation of rapidly transferable sterol domains. In summary, data obtained with intact transfected cells and in vitro sterol transfer assays showed that SCP-2 expression (i) selectively modulated plasma membrane lipid composition and (ii) decreased the plasma membrane content cholesterol, an effect potentially due to more rapid SCP-2-mediated cholesterol transfer from versus to the plasma membrane.     

Lipids of plasma membranes prepared from oat root cells : effects of induced water-deficit tolerance

Plasma membranes were isolated from oat (Avena sativa) roots by the phase-partitioning method. The membranes were exposed to repeated periods of moderate water-deficit stress, and a water-deficit tolerance was induced (acclimated plants). The plasma membranes of the controls (nonacclimated plants) were characterized by a high phospholipid content, 79% of total lipids, cerebrosides (9%) containing hydroxy fatty acids (>90% 24:1-OH) and free sterols, acylated sterylglucosides, sterylglucosides, and steryl esters, together amounting to 12%. Major phospholipids were phosphatidylcholine and phosphatidylethanolamine with lesser amounts of phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid. After the membranes were acclimated to dehydration, the lipid to protein ratio decreased from 1.3 to 0.7 micromoles per milligram. Furthermore, the cerebrosides decreased to 5% and free sterols increased from 9% (nonacclimated plants) to 14%. Because the total phospholipids did not change significantly, the free sterol to phospholipid ratio increased from 0.12 to 0.19. There was no change in the relative distribution of sterols after acclimation. The ratio of phosphatidylcholine to phosphatidylethanolamine changed from 1.1 in the nonacclimated plants to 0.69 in the acclimated plants. The results show that acclimation to dehydration implies substantial alterations in the lipid composition of the plasma membrane.     

Regulation of transbilayer distribution of a fluorescent sterol in tumor cell plasma membranes

The lipid composition and transbilayer distribution of plasma membrane isolated from primary tumor (L-929, LM, A-9 and C3H) and nine metastatic cell lines cultured under identical conditions was examined. Cultured primary tumor and metastatic cells differed two-fold in sterol/phospholipid molar ratios. There was a direct correlation between plasma membrane anionic phospholipid (phosphatidylinositol and phosphatidylserine) content and plasma membrane sterol/phospholipid ratio. This finding may bear on the possible link between oncogenes and inositol lipids. The fluorescent sterol, dehydroergosterol, was incorporated into primary tumor and metastatic cell lines. Selective quenching of outer monolayer fluorescence by covalently linked trinitrophenyl groups demonstrated an asymmetric transbilayer distribution of sterol in the plasma membranes. The inner monolayer of the plasma membranes from both cultured primary and metastatic tumor cells was enriched in sterol as compared with the outer monolayer. Consistent with this, the inner monolayer was distinctly more rigid as determined by the limiting anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Dehydroergosterol fluorescence was temperature dependent and sensitive to lateral phase separations in phosphatidylcholine vesicles and in LM cell plasma membranes. Dehydroergosterol detected phase separations near 24 degrees C in the outer monolayer and at 21 degrees C and 37 degrees C in the inner monolayer of LM plasma membranes. Yet, no change in transbilayer sterol distribution was detected in ascending or descending temperature scans between 4 and 45 degrees C. Alterations in plasma membrane phospholipid polar head group composition by choline analogues (N,N-dimethylethanolamine, N-methylethanolamine, and ethanolamine) also did not perturb transbilayer sterol asymmetry. Treatment with phenobarbital or prilocaine, drugs that selectively fluidize the outer and inner monolayer of LM plasma membranes, respectively, did not change dehydroergosterol transbilayer distribution.     

Phospholipid and sterol analysis of plasma membranes of azole-resistant Candida albicans strains

The phospholipid and sterol composition of the plasma membranes of five fluconazole-resistant clinical Candida albicans isolates was compared to that of three fluconazole-sensitive ones. The three azole-sensitive strains tested and four of the five resistant strains did not exhibit any major difference in their phospholipid and sterol composition. The remaining strain (R5) showed a decreased amount of ergosterol and a lower phosphatidylcholine:phosphatidylethanolamine ratio in the plasma membrane. These changes in the plasma membrane lipid and sterol composition may be responsible for an altered uptake of drugs and thus for a reduced intracellular accumulation of fluconazole thereby providing a mechanism for azole resistance.     

Fluidity and lipid composition of oat and rye shoot plasma membrane: effect of sterol perturbation by xenobiotics

Oat and rye plants were treated with either tetcyclacis (an experimental plant growth regulator), nuarimol (a fungicide) or gamma-ketotriazole (an experimental herbicide). These treatments reduced shoot growth and changed the lipid composition of the shoot plasma membranes. In oat, both tetcyclacis and nuarimol treatments increased plasma membrane cholesterol and increased the phosphatidylethanolamine/phosphatidylcholine (PE/PC) ratio, whereas gamma-ketotriazole treatment reduced cholesterol and the PE/PC ratio. In rye, all treatments reduced the PE/PC ratio. Generally, the sterol/phospholipid ratio was less in oat than in rye but the cholesterol/phospholipid ratio was greater. With all treatments in oat and rye, increases were observed in unsaturation of the phospholipid acyl chains. The fluidity of membranes was measured by steady-state fluorescence polarisation of the probe diphenylhexatriene; oat membranes were more fluid than rye. Membrane fluidity was greater in plasma membranes from plants treated with the xenobiotics than the controls. The results are discussed in the context of the effect of plasma membrane lipid composition on membrane fluidity, and it is concluded that there appears to be no overall simple relationship between membrane lipid composition and fluidity that holds for all treatments in both species.     

Correlation of tumor metastasis with sterol carrier protein and plasma membrane sterol levels

Squalene and sterol carrier protein (SCP) levels and sterol/phospholipid molar ratios of whole cells and plasma membranes were measured in cultured primary tumor and metastatic cell lines. SCP is abundant in all cell lines. However, metastatic lines have significantly lower SCP levels and plasma membrane sterol/phospholipid ratios than do primary lines. The results indicate that extremely malignant, metastatic cells are unable to produce or maintain adequate levels of both SCP and plasma membrane sterols when grown in lipoprotein deficient media. This defect, in vivo, probably causes excess uptake of SCP and lipid.     

Modulation of yeast plasma membrane composition of a yeast sterol auxotroph as a function of exogenous sterol

Plasma membranes isolated from a yeast sterol auxotroph (RD5-R) grown on 1, 5, and 15 micrograms ml-1 exogenous concentrations of sterol showed no discontinuity in plots of steady-state fluorescence anisotropy. Liposomes constructed from phospholipid and sterol extracted from RD5-R grown on different sterols indicated that exogenously supplied sterol modulated cellular phospholipids such that lipid-phase transitions were avoided. Liposomes derived from sterol and phospholipid extracted from the same culture exhibited no lipid-phase transitions. However, when phospholipid extracted from a culture grown on a specific sterol was mixed with sterol extracted from a heterologous culture grown on a different sterol to form liposomes, discontinuities were detected in the anisotropy measurements of the liposomes produced. Quantitative analyses revealed that the exogenously supplied sterol coordinately regulated specific phospholipid species, fatty acid composition, and sterol to phospholipid ratios in yeast auxotrophs.     

Endogenous hormonal and enzymatic responses of Catharanthus roseus with triadimefon application under water deficits

The effect of triadimefon was investigated in a medicinal plant, Catharanthus roseus subjected to water deficit stress. The abscisic acid (ABA) level, DNA and RNA contents and activities of ATPase and protease were found varying in different parts of the plants under treatment. Drought treatment increased the ABA level more than twofold in all parts of the plants. TDM treatment to the drought stressed plants showed highest contents. In roots, stem and leaves, drought stress caused a decrease in the DNA and RNA contents when compared with control and other treatments. TDM treatment with drought increased the nucleic acid contents to the level of the control roots. The activity of ATPase and protease were increased under drought treatment and lowered due to TDM applications. This information could be useful in the field of soil water deficits reclamation efforts by using plant growth regulators.     

Comparison of the Lipid Composition of Oat Root and Coleoptile Plasma Membranes : Lack of Short-Term Change in Response to Auxin

The total lipid composition of plasma membranes (PM), isolated by the phase partitioning method from two different oat (Avena sativa L.) tissues, the root and coleoptile, was compared. In general, the PM lipid composition was not conserved between these two organs of the oat seedling. Oat roots contained 50 mole percent phospholipid, 25 mole percent glycolipid, and 25 mole percent free sterol, whereas comparable amounts in the coleoptile were 42, 39, and 19 mole percent, respectively. Individual lipid components within each lipid class also showed large variations between the two tissues. Maximum specific ATPase activity in the root PM was more than double the activity in the coleoptile. Treatment of coleoptile with auxin for 1 hour resulted in no detectable changes in PM lipids or extractable ATPase activity. Differences in the PM lipid composition between the two tissues that may define the limits of ATPase activity are discussed.