Alterations of wheat root plasma membrane lipid composition induced by copper stress result in changed physicochemical properties of plasma membrane lipid vesicles

A response when wheat is grown in excess copper is an altered lipid composition of the root plasma membrane (PM). With detailed characterisation of the root PM lipid composition of the copper-treated plants as a basis, in the present study, model systems were used to gain a wider understanding about membrane behaviour, and the impact of a changed lipid composition.PMs from root cells of plants grown in excess copper (50 μM Cu²⁺) and control (0.3 μM Cu²⁺) were isolated using the two-phase partitioning method. Membrane vesicles were prepared of total lipids extracts from the isolated PMs, and also reference vesicles of phosphatidylcholine (PC). In a series of tests, the vesicle permeability for glucose and for protons was analysed. The vesicles show that copper stress reduced the permeability for glucose of the lipid bilayer barrier. When vesicles from stressed plants were modified by addition of lipids to resemble vesicles from control plants, the permeability for glucose was very similar to that of vesicles from control plants. The permeability for protons did not change upon stress.Electron paramagnetic resonance (EPR) of the lipid vesicles spin probed with n-doxylstearic acid (nDSA) was used to explore the lipid rotational freedom at different depth of the bilayer. The EPR measurements supported the permeability data, indicating that the copper stress resulted in more tightly packed bilayers of the PMs with reduced acyl chain motion.     

Changes in plasma-membrane lipid composition: a strategy for acclimation to copper stress

Wheat seedlings were grown hydroponically in the presence of 50 microM Cu2+. The copper stress resulted in plasma-membrane (PM) changes of the root cells as altered lipid composition, a decreased phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio from 0.7 to 0.3, a decreased fatty acyl unsaturation and a decrease in the lipid/protein ratio. Membrane vesicles made from total lipid extracts of isolated PMs of wheat grown under copper excess showed a remarkably low permeability to polar molecules like glucose, as compared with the control, and no difference in proton permeability. Permeability studies of vesicles of plasma-membrane lipids, which were selectively modified by addition of specific lipids such as PC and PE, were also performed. The results are discussed with emphasis on the role of the increased PE proportion.     

Permeability behaviour of lipid vesicles prepared from plant plasma membranes--impact of compositional changes

Exposure of oat seedlings to repeated moderate water deficit stress causes a drought acclimation of the seedlings. This acclimation is associated with changes in the lipid composition of the plasma membrane of root cells. Here, plasma membranes from root cells of acclimated and control plants were isolated using the two-phase partitioning method. Membrane vesicles were prepared of total lipids extracted from the plasma membranes. In a series of tests the vesicle permeability for glucose and for protons were analysed and compared with the permeability of model vesicles. Further, the importance of critical components for the permeability properties was analysed by modifying the lipid composition of the vesicles from acclimated and from control plants. The purpose was to add specific lipids to vesicles from acclimated plants to mimic the composition of the vesicles from control plants and vice versa. The plasma membrane lipid vesicles from acclimated plants had a significantly increased permeability for glucose and decreased permeability for protons as compared to control vesicles. The results point to the importance of the ratio phosphatidylcholine (PC)/phosphatidylethanolamine (PE), the levels of cerebrosides and free sterols and the possible interaction of these components for the plasma membrane as a permeability barrier.     

Lipids and NADPH-dependent superoxide production in plasma membrane vesicles from roots of wheat grown under copper deficiency or excess

The effects of in vivo copper on the lipid composition of root plasma membrane and the activities of membrane-bound enzymes, such as NADPH-dependent oxidases and lipoxygenase, were studied. Plants were grown in hydroponic culture for 11 d without Cu supply or in the presence of 50 microM Cu. Control plants were supplied with 0.3 microM Cu. Growth of roots was severely affected in the 50 microM Cu-grown plants, whereas roots grown in Cu-deficient solution did not show any difference in comparison with the control. The 50 microM Cu concentration caused an increase in the leakage of K(+) ions as well. Excess metal supply resulted in a decrease in the total lipid content of plasma membrane, a higher phospholipid amount and a reduction of steryl lipids (free sterols, steryl glycosides and acylated steryl glycosides). Cu depletion in the growth solution had only a slight effect on the plasma membrane lipid composition. In comparison with the control, only the excess of Cu caused a decrease in the lipid to protein ratio as well as a change in the phospholipid composition, with a lower phosphatidylcholine to phosphatidylethanolamine ratio. The degree of unsaturation of root plasma membranes decreased following the 0 Cu treatment and even more after the 50 microM Cu supply. Plasma membranes of wheat grown under metal deficiency and excess showed increased NADPH-dependent superoxide-producing oxidase activities, whereas membrane-bound lipoxygenase was not increased or activated due to Cu treatments. The consequences of changes in plasma membrane lipid composition and activated oxygen production as a result of Cu treatments are discussed.     

Growth in excess copper induces changes in the lipid composition and fluidity of PSII-enriched membranes in wheat

Changes in the lipid composition and fluidity of PSII-enriched thylakoids were studied in seedlings of wheat ( Triticum durum Desf. cv. Adamello) grown in nutrient solution supplemented with CuSO₄ to achieve a final concentration of 10 and 50 μ M Cu. Metal content increased in the chloroplasts of the 50 μ M Cu-grown plants. PSII isolated from wheat supplied with 10 μ M Cu did not show any alteration in the lipid composition or in the lipid and protein levels of the membranes, nor was any change in the ultrastructure of the membranes detected. The 50 μ M Cu-grown plants showed thylakoid swelling, particularly in the stroma and terminal grana thylakoids. Furthermore, an alteration in the lipid composition of PSII preparations was observed together with a decrease in the lipid content, which resulted in a reduction in the lipid to protein ratio. The monogalactosyldiacylglycerol (MGDG) to digalactosyldiacylglycerol (DGDG) molar ratio decreased, whereas the degradation of the polar lipids caused an accumulation of free fatty acids (FFA). The total amount of unsaturated lipids associated with the PSII-enriched membranes of wheat was not affected by excess copper supplies, even though changes in the individual fatty acids occurred. The effect of copper on the fluidity of PSII membranes was evaluated by electron paramagnetic resonance (EPR) measurements, using spin-probed fatty acids as probes. The PSII membranes, spin probed by means of 5- and 16-doxylstearic acids, showed that only the fluidity of the surface region of the bilayer close to the polar head group was reduced following the 50 μ M Cu supply. In contrast, the fluidity of the inner membrane region of the bilayer did not show any change. The implications of changes in the lipid composition and lipid-protein interactions on the fluidity of specific transversal membrane regions are discussed.     

Modification of the root plasma membrane lipid composition of cadmium-treated Pisum sativum

The effects of in vivo Cd treatments on pea root plasma membrane(PM) lipid composition were studied. In the long-term experiment,plants were supplied with Cd: moderate stress (10 µM)or strong stress (50 µM) for 10 d. Growth of root andshoot was severely affected in 50 µM Cd-treated plants,as evidenced by the approximately 7-fold reduction in theirRelative Growth Increment (RGI). Treatment with Cd (10 µM)resulted in changes to the lipid composition of the pea rootPM, including increases in the degree of unsaturation of phospholipid-associatedfatty acids and in the relative amount of stigmasterol (30–42%).This change was accompanied by a reduction in sitosterol content(26.8 to 17.4 µg mg^–1 protein). However, the sterolcomposition was not altered in plants treated with 50 µMCd for 10 d. The content of phosphatidylethanolamine and phosphatidylcholine(major phospholipids present in pea root PM) decreased as Cdlevel increased, but the ratio between them remained unaffected.In the short-term experiment, plants exposed to Cd (50 µM)accumulated less sitosterol (from 27.7 to 14.0 µg g mg^–1protein) over 72 h, but no significant effect on other measuredlipids was observed. The physiological repercussions of changesin plasma membrane lipid composition, as a result of Cd exposureare discussed. Key words: Cadmium, lipids, pea, Pisum sativum, plasma membranes     

Salt stress alters membrane lipid content and lipid biosynthesis pathways in the plasma membrane and tonoplast

Plant cell membranes are the sites of sensing and initiation of rapid responses to changing environmental factors including salinity stress. Understanding the mechanisms involved in membrane remodeling is important for studying salt tolerance in plants. This task remains challenging in complex tissue due to suboptimal subcellular membrane isolation techniques. Here, we capitalized on the use of a surface charge-based separation method, free flow electrophoresis, to isolate the tonoplast (TP) and plasma membrane (PM) from leaf tissue of the halophyte ice plant (Mesembryanthemum crystallinum L.). Results demonstrated a membrane-specific lipidomic remodeling in this plant under salt conditions, including an increased proportion of bilayer forming lipid phosphatidylcholine in the TP and an increase in nonbilayer forming and negatively charged lipids (phosphatidylethanolamine and phosphatidylserine) in the PM. Quantitative proteomics showed salt-induced changes in proteins involved in fatty acid synthesis and desaturation, glycerolipid, and sterol synthesis, as well as proteins involved in lipid signaling, binding, and trafficking. These results reveal an essential plant mechanism for membrane homeostasis wherein lipidome remodeling in response to salt stress contributes to maintaining the physiological function of individual subcellular compartments.

Charge-based membrane fractionation techniques and tandem mass spectrometry combined with proteomic and lipidomic approaches reveal membrane-specific lipid remodeling in plants during salt stress.     

Changes in plasma membrane fluidity of corn (Zea mays L.) roots after Brij 58 treatment

Summary The detergent Brij 58 has been introduced to reverse plasma membrane (PM) vesicles from the right-side-out to the inside-out form. The aim of the present work was to investigate the effect of Brij 58 on the formation of an ATP-dependent proton gradient and on the fluidity of the lipid phase of PM vesicles. PMs of corn (Zea mays L.) roots were isolated by phase-partitioning. The fluidity of PMs was estimated by measurement of fluorescence polarization with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The PMs of corn roots were relatively rigid. The hydrophobic part of the lipid bilayer was more fluid than the hydrophilic part. After intercalation of Brij 58 into the lipid bilayer the membrane fluidity changed in a concentration-dependent manner. Treatment with the detergent Brij 58 increased the degree of fluorescence polarization for TMA-DPH, while it decreased it for DPH. This effect was saturated at a detergent-to-protein ratio of 1 4 for both fluorescence probes. Although the biophysical characteristics of the membrane were changed after Brij 58 treatment, the formation of ATP-dependent proton gradients could still be measured with those vesicles. The generation of an ATP-dependent proton gradient with Brij 58-treated PM vesicles suggests that the detergent treatment indeed turned the originally right-side-out vesicles to sealed inside-out vesicles. The limits of the effect caused by Brij 58 in the context of PM enzyme activities are discussed.Abbreviations Brij 58 polyoxyethylene 20 cetyl ether - DPH 1,6-diphenyl-1,3,5-hexatriene - HCF III hexacyanoferrate (III) - ISO inside-out - PM plasma membrane - RSO right-side-out - TMA-DPH 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene     

Improving our picture of the plasma membrane: Rafts induce ordered domains in a simplified model cytoplasmic leaflet

By study of asymmetric membranes, models of the cell plasma membrane (PM) have improved, with more realistic properties of the asymmetric lipid composition of the membrane being explored. We used hemifusion of symmetric giant unilamellar vesicles (GUVs) with a supported lipid bilayer (SLB) to engineer bilayer leaflets of different composition. During hemifusion, only the outer leaflets of GUV and SLB are connected, exchanging lipids by simple diffusion. aGUVs were detached from the SLB for study. In general these aGUVs are formed with one leaflet that phase-separates into Ld (liquid disordered) + Lo (liquid ordered) phases, and another leaflet with lipid composition that would form a single fluid phase in a symmetric bilayer. We observed that ordered phases of either Lo or Lβ (gel phase) induce an ordered domain in the apposed fluid leaflet that lacks high melting lipids. Results suggest both an inter-leaflet and an intra-leaflet redistribution of cholesterol. We used C-Laurdan spectral images to investigate the lipid packing/order of aGUVs, finding that cholesterol partitions into the induced ordered domains. We suggest this behavior to be commonplace, that when Ld + Lo phase separation occurs in a cell PM exoplasmic leaflet, an induced order domain forms in the cytoplasmic leaflet.     

Are glucocerebrosides the predominant sphingolipids in plant plasma membranes?

Long-chain sphingobases have been analyzed in various fractions prepared from different organs (leaf, root, storage tissue) from five dicotyledoneous plants (Arabidopsis thaliana, Brassica oleracea, Nicotiana tabacum, Pisum sativum, Spinacia oleracea). The resulting sphingobase profiles from cerebrosides and plasma membranes (PMs) show large qualitative and quantitative differences. Assuming that cerebrosides from all cellular membranes have similar sphingobase profiles, these data suggest that cerebrosides, considered to be characteristic glycolipids of plant PMs and specified by large proportions of sphingobases with an 8Z-double bond motif, do not represent the major sphingolipids of PMs. The fraction of unidentified complex sphingolipids, containing mainly 8E-phytosphingenine, exceeds the cerebroside proportion in PMs by several factors and may be as abundant as diacylglycerol-based phospholipids. These results are discussed with respect to the distribution of various lipids between the bilayer halves of plant PM.     

Effects of different cultivation temperatures on plasma membrane ATPase activity and lipid composition of sugar beet roots.

Sugar beet seedlings (Beta vulgaris L. cv. Monohill) were cultivated for 3 weeks at different root and shoot temperatures and the plasma membranes (PM) from roots were purified by aqueous two-phase partitioning and analyzed for lipid composition and ATPase activities. Lipid analyses, undertaken immediately after PM purification from the roots, showed that a low root zone temperature (10 degrees C) decreased the ratio between the major lipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). A low temperature in the root environment increased the mol% of PE and decreased the mol% of phosphatidic acid (PA), independent on the shoot growth temperature. A low temperature also decreased the mol% of linoleic acid (18:2) and increased mol% of linolenic acid (18:3) in the analyzed lipid classes, especially in PC and PE. The ratio between acyl chain lipids and protein generally increased in PM from roots grown at 10 degrees C, compared with higher temperature. The changes in lipid composition correlated with changes in ATPase activities, detected as hydrolyses of MgATP. The kinetic parameters, K(m) and V of the PM H(+)ATPase in roots increased at a low cultivation temperature, independent on shoot temperature. Moreover, Arrhenius analyses showed that the transition temperature was independent of both root or shoot growth temperature at 10-24 degrees C, whereas the activation energy of the ATPase was dependent on the growth temperature of the root, and independent on shoot temperature. Thus, acclimation processes can take place in roots, irrespective of the shoot temperature.     

Characterization of lipid rafts from Medicago truncatula root plasma membranes: a proteomic study reveals the presence of a raft-associated redox system

Several studies have provided new insights into the role of sphingolipid/sterol-rich domains so-called lipid rafts of the plasma membrane (PM) from mammalian cells, and more recently from leaves, cell cultures, and seedlings of higher plants. Here we show that lipid raft domains, defined as Triton X-100-insoluble membranes, can also be prepared from Medicago truncatula root PMs. These domains have been extensively characterized by ultrastructural studies as well as by analysis of their content in lipids and proteins. M. truncatula lipid domains are shown to be enriched in sphingolipids and Delta(7)-sterols, with spinasterol as the major compound, but also in steryl glycosides and acyl-steryl glycosides. A large number of proteins (i.e. 270) have been identified. Among them, receptor kinases and proteins related to signaling, cellular trafficking, and cell wall functioning were well represented whereas those involved in transport and metabolism were poorly represented. Evidence is also given for the presence of a complete PM redox system in the lipid rafts.     

Control of lipid membrane stability by cholesterol content

Cholesterol has a concentration-dependent effect on membrane organization. It is able to control the membrane permeability by inducing conformational ordering of the lipid chains. A systematic investigation of lipid bilayer permeability is described in the present work. It takes advantage of the transmembrane potential difference modulation induced in vesicles when an external electric field is applied. The magnitude of this modulation is under the control of the membrane electrical permeability. When brought to a critical value by the external field, the membrane potential difference induces a new membrane organization. The membrane is then permeable and prone to solubilized membrane protein back-insertion. This is obtained for an external field strength, which depends on membrane native permeability. This approach was used to study the cholesterol effect on phosphatidylcholine bilayers. Studies have been performed with lipids in gel and in fluid states. When cholesterol is present, it does not affect electropermeabilization and electroinsertion in lipids in the fluid state. When lipids are in the gel state, cholesterol has a dose-dependent effect. When present at 6% (mol/mol), cholesterol prevents electropermeabilization and electroinsertion. When cholesterol is present at more than 12%, electropermeabilization and electroinsertion are obtained under milder field conditions. This is tentatively explained by a cholesterol-induced alteration of the hydrophobic barrier of the bilayer core. Our results indicate that lipid membrane permeability is affected by the cholesterol content.     

Changes in plasma membrane lipids, aquaporins and proton pump of broccoli roots, as an adaptation mechanism to salinity

Salinity stress is known to modify the plasma membrane lipid and protein composition of plant cells. In this work, we determined the effects of salt stress on the lipid composition of broccoli root plasma membrane vesicles and investigated how these changes could affect water transport via aquaporins. Brassica oleracea L. var. Italica plants treated with different levels of NaCl (0, 40 or 80 mM) showed significant differences in sterol and fatty acid levels. Salinity increased linoleic (18:2) and linolenic (18:3) acids and stigmasterol, but decreased palmitoleic (16:1) and oleic (18:1) acids and sitosterol. Also, the unsaturation index increased with salinity. Salinity increased the expression of aquaporins of the PIP1 and PIP2 subfamilies and the activity of the plasma membrane H⁺-ATPase. However, there was no effect of NaCl on water permeability (P_f) values of root plasma membrane vesicles, as determined by stopped-flow light scattering. The counteracting changes in lipid composition and aquaporin expression observed in NaCl-treated plants could allow to maintain the membrane permeability to water and a higher H⁺-ATPase activity, thereby helping to reduce partially the Na⁺ concentration in the cytoplasm of the cell while maintaining water uptake via cell-to-cell pathways. We propose that the modification of lipid composition could affect membrane stability and the abundance or activity of plasma membrane proteins such as aquaporins or H⁺-ATPase. This would provide a mechanism for controlling water permeability and for acclimation to salinity stress.     

Plasma membrane permeability as an indicator of salt tolerance in plants

There is evidence that the plasma membrane (PM) permeability alterations might be involved in plant salt tolerance. This review presents several lines of evidence demonstrating that PM permeability is correlated with salt tolerance in plants. PM injury and hence changes in permeability in salt sensitive plants is brought about by ionic effects as well as oxidative stress induced by salt imposition. It is documented that salinity enhances lipid peroxidation as well as protein oxidative damage, which in turn induces permeability impairment. PM protection, and thus retained permeability, in tolerant plants under salt imposition could be achieved through increasing antioxidative systems and thereby reducing lipid peroxidation and protein oxidative damage of PM. It appears that specific membrane proteins and/or lipids are constitutive or induced under salinity, which may contribute to maintenance of membrane structure and function in salt tolerant plant species. Furthermore, protecting agents (e.g., glycinebetaine, proline, polyamines, trehalose, sorbitol, mannitol) accumulated in salt tolerant species/cultivars may also contribute to PM stabilization and protection under salinity. Based on the presented evidence that PM permeability correlates with plant salt tolerance, we suggest that PM permeability is an easy and useful parameter for selection of genotypes of agriculture crops adapted to salt stress.     

Structural determinants of water permeability through the lipid membrane

Despite intense study over many years, the mechanisms by which water and small nonelectrolytes cross lipid bilayers remain unclear. While prior studies of permeability through membranes have focused on solute characteristics, such as size, polarity, and partition coefficient in hydrophobic solvent, we focus here on water permeability in seven single component bilayers composed of different lipids, five with phosphatidylcholine headgroups and different chain lengths and unsaturation, one with a phosphatidylserine headgroup, and one with a phosphatidylethanolamine headgroup. We find that water permeability correlates most strongly with the area/lipid and is poorly correlated with bilayer thickness and other previously determined structural and mechanical properties of these single component bilayers. These results suggest a new model for permeability that is developed in the accompanying theoretical paper in which the area occupied by the lipid is the major determinant and the hydrocarbon thickness is a secondary determinant. Cholesterol was also incorporated into DOPC bilayers and X-ray diffuse scattering was used to determine quantitative structure with the result that the area occupied by DOPC in the membrane decreases while bilayer thickness increases in a correlated way because lipid volume does not change. The water permeability decreases with added cholesterol and it correlates in a different way from pure lipids with area per lipid, bilayer thickness, and also with area compressibility.     

Lipid composition of integral purple membrane by 1H and 31P NMR

In the purple membrane (PM) of halobacteria, lipids stabilize the trimeric arrangement of bacteriorhodopsin (BR) molecules and mediate the packing of the trimers in a regular crystalline arrangement. To date, the identification and quantification of these lipids has been based either on lipid extraction procedures or structural models. By directly solubilizing PMs from Halobacterium salinarum in aqueous detergent solutions (SDS or Triton X-100), we avoided any separation or modification steps that might modify the lipid composition or even the lipid molecules themselves. Our analysis of integral PM preparations should resolve partially conflicting literature data on the lipid composition of the PM. Using 31P and 1H NMR of detergent-solubilized but otherwise untreated samples, we found two glycolipids and 6.4 +/- 0.1 phospholipids per BR molecule, 4.4 +/- 0.1 of the latter being the phosphatidylglycerophosphate methyl ester. The only glycolipid detected was S-TGD-1. For an additional glycolipid, glycocardiolipin, that was recently identified in lipid extracts, we show that it was produced mainly during the lipid extraction procedure but also was partially dependent on the preparation of the PM suspensions.     

Characterization of plasma membrane domains enriched in lipid metabolites.

A subpopulation of plasma membrane vesicles enriched in membrane lipid metabolites has been isolated from petals of carnation flowers and leaves of canola seedlings. This was achieved by immunopurification from a microsomal membrane preparation using region-specific antibodies raised against a recombinant polypeptide of the plasma membrane H(+)-ATPase. The properties of this subpopulation of vesicles were compared with those of purified plasma membrane isolated by partitioning in an aqueous dextran-polyethylene glycol two-phase system. The lipid composition of the immunopurified vesicles proved to be clearly distinguishable from that of phase-purified plasma membrane, indicating that they represent a unique subpopulation of plasma membrane vesicles. Specifically, the immunopurified vesicles are highly enriched in lipid metabolites, including free fatty acids, diacylglycerol, triacylglycerol and steryl and wax esters, by comparison with the phase-purified plasma membrane. These findings can be interpreted as indicating that lipid metabolites generated within the plasma membrane effectively phase-separate by moving laterally through the plane of the membrane to form discrete domains within the bilayer. It is also apparent that these domains, once formed, are released as vesicles into the cytosol, presumably by microvesiculation from the surface of the plasmalemma. Such removal may be part of normal membrane turnover.     

The ins and outs in membrane dynamics: tubulation and vesiculation

Living cells constantly adjust the composition and size of their membrane systems to accommodate the demands for the housekeeping activities, to expand and reduce cell size, and to commit the cell for division. Although it is well known that vesicles are the vehicles to deliver and retrieve lipids and proteins to and from the membranes, the mechanisms allowing vesicles to pinch off from membranes or fuse into a flat lipid bilayer have been poorly understood, particularly in plants. Recent studies on dynamins and dynamin-related proteins in animals and plants now allow new concepts in membrane dynamics to be considered.