Conny Liljenberg

Phosphate‐deficient oat replaces a major portion of the plasma membrane phospholipids with the galactolipid digalactosyldiacylglycerol

The plasma membranes of oat normally resemble those of other eukaryotes in containing mainly phospholipids and sterols. We here report the novel finding that the galactolipid digalactosyldiacylglycerol (DGDG) can constitute a substantial proportion of oat plasma membrane lipids, in both shoots and roots. When oat was cultivated under severe phosphate limitation, up to 70% of the plasma membrane phosphoglycerolipids were replaced by DGDG. Our finding not only reflects a far more developed potential for plasticity in plasma membrane lipid composition than often assumed, but also merits interest in the context of the limited phosphate availability in many soils.

Phosphate-limited Oat THE PLASMA MEMBRANE AND THE TONOPLAST AS MAJOR TARGETS FOR PHOSPHOLIPID-TO-GLYCOLIPID REPLACEMENT AND STIMULATION OF PHOSPHOLIPASES IN THE PLASMA MEMBRANE

We recently reported that cultivation of oat (Avena sativa L.) without phosphate resulted in plasma membrane phosphoglycerolipids being replaced to a large extent by digalactosyldiacylglycerol (DGDG) (Andersson, M. X., Stridh, M. H., Larsson, K. E., Liljenberg, C., and Sandelius, A. S. (2003) FEBS Lett. 537, 128–132). We report here that DGDG is not the only non-phosphorous-containing lipid that replaces phospholipids but that also the content of glucosylceramides and sterolglycosides increased in plasma membranes as a response to phosphate starvation. In addition, phosphate deficiency induced similar changes in lipid composition in the tonoplast. The phospholipid-to-glycolipid replacement apparently did not occur to any greater extent in endoplasmic reticulum, Golgi apparatus, or mitochondrial inner membranes. In contrast to the marked effects on lipid composition, the polypeptide patterns were largely similar between root plasma membranes from well-fertilized and phosphate-limited oat, although the latter condition induced at least four polypeptides, including a chaperone of the HSP80 or HSP90 family, a phosphate transporter, and a bacterial-type phosphoesterase. The latter polypeptide reacted with an antibody raised against a phosphate deficiency-induced phospholipase C from Arabidopsis thaliana (Nakamura, Y., Awai, K., Masuda, T., Yoshioka, Y., Takamiya, K., and Ohta, H. (2005) J. Biol. Chem. 280, 7469–7476). In plasma membranes from oat, however, a phospholipase D-type activity and a phosphatidic acid phosphatase were the dominant lipase activities induced by phosphate deficiency. Our results reflect a highly developed plasticity in the lipid composition of the plasma membrane and the tonoplast. In addition, phosphate deficiency-induced alterations in plasma membrane lipid composition may involve different sets of lipid-metabolizing enzymes in different plant tissues or species, at different stages of plant development and/or at different stages of stress adjustments.

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 microM Cu(2+)) and control (0.3 microM Cu(2+)) 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.

Glucosylceramides of oat root plasma membranes--physicochemical behaviour in natural and in model systems.

Glucosylceramides from oat root plasma membranes have been characterized using HPLC-particle beam-mass spectrometry, differential scanning calorimetry, low angle X-ray diffraction and surface balance technique. 24:1-OH was dominating fatty acid (90%) together with 24:0-OH and 22:1-OH. The sphingosine base was sphingadienine isomers and the monosaccharide alpha and beta glucose. Differential scanning calorimetry of an aqueous dispersion of glucosylceramide revealed during heating an endothermic gel-liquid crystalline transition with double peaks at 47 degrees and 51 degrees C, the lowest known for naturally occurring glucosylceramides. A cooling scan after the endothermic gel-liquid transition showed one exotherm at 15 degrees C and if this was followed by another heating scan a large exotherm appeared with a peak at 18 degrees C. During the second heating the matrix was hydrated and the exotherm at 18 degrees C reflects then the transition between the supercooled metastable gel phase and the corresponding hydrated form. The calorimetric data indicate a lamellar phase which during the cooling scan appeared as an supercooled liquid crystalline phase. Low angle X-ray diffraction confirmed these calorimetric data. The surface pressure-area-curves of pure oat glucosylceramides were more expanded than those of bovine origin. Mixtures of oat glucosylceramides and phosphatidylcholine species similar to those present in oat root plasma membranes showed molecular miscibility but no interaction.

Characterization of glucosylceramide from plasma membranes of plant root cells

Plasma membranes of oat root cells were isolated from intracellular membranes by subfractionation of the microsomal fraction using an aqueous polymer two-phase system. The plasma membranes originated from oat plants which were acclimated to dehydration by exposure to a repeated water-deficit stress program. Glucosylceramides was a major component of the plasma membrane lipids and amounted to 9% of the lipid of control plants and 5% of the lipid of acclimated plants. Structural analysis using FAB-MS showed only one type of glucosylceramides. The constituent monosaccharide was exclusively glucose and the sphingosine base was 4,8-sphingadienine. The fatty acid composition was determined to 24:1-OH, with only trace levels of non-hydroxy acids. The decrease in the level of glucosylceramides during acclimation to dehydration was accompanied by a corresponding decrease in phospholipids and increase in free sterols.

Phase behaviour and molecular species composition of oat root plasma membrane lipids. Influence of induced dehydration tolerance

Tolerance to dehydration induced by repeated water-deficit stress is well correlated to changes in the lipid composition of oat root cell plasma membranes. The molecular species of the two dominant phospholipids phosphatidylcholine and phosphatidylethanolamine were determined. The four major species were 16:0/18:2, 16:0/18:3, 18:2/18:2 and 18:2/18:3. In contrast to the large changes in plasma membrane lipid composition in other respects, induced tolerance resulted in very weak alterations concerning the phospholipid molecular species pattern. Only minor alterations, appearing as a decrease in the 18:3-containing lipids, occurred. Total lipids of microsomes and isolated plasma membranes of root cells were analysed by X-ray crystallography at different degrees of hydration. The lipid phase behaviour at different degrees of hydration was further confirmed by polarization microscopy. In the presence of excess water all membrane lipids adopted a reversed micellar configuration. The plasma membrane lipids from root cells with induced dehydration tolerance formed upon dehydration two coexisting lamellar structures. The importance of the phase behaviour at different degrees of hydration for the membrane properties and the relation to membrane composition is discussed.

Effects of pH and Mineral Nutrition Supply on Lipid Composition and Protein Pattern of Plasma Membranes from Sugar Beet Roots

Summary Plasma membranes (PM) were isolated by two-phase-partitioning from roots of 21-day-old sugar beet ( Beta vulgaris L. cv. Monohill) seedlings cultivated under different conditions. The seedlings were either grown in a complete nutrient solution with a high concentration of nutrients (H-s roots) and high (6.5) or low (5.3) pH, or in a low concentration medium (L-s roots) with a 15% daily relative addition rate of nutrients at pH 5.3. The protein pattern, the phospholipids, the glycolipids and the free sterols were analysed. The protein pattern of plasma membranes did not change, but the composition of lipids changed under the different conditions of cultivation. After cultivation at a low concentration of nutrients and low pH, the free sterols dominated while after cultivation at a high concentration of nutrients, the phospholipids were the dominant lipids. The Δ 7 -sterols were the most abundant free sterols representing more than 60% of the free sterols in PM of roots growing in a low concentration of nutrients and more than 70 to 80% in plasma membranes of roots growing in a high concentration. Other free sterols present were stigmasterol, sitosterol, campesterol and brassicasterol. The ratio of more planar/to less planar sterols increased with high salt treatment. Phosphatidylethanolamine and phosphatidylcholine, which were the major phospholipids, increased with high salt concentration in the growth medium. The glycolipid levels remained substantially unchanged. However, the ratio of phospholipids to glycolipids increased with high salt and high pH. The relative distribution of fatty acids in the lipid classes also changed after cultivation of the seedlings under different conditions. The results show that the different growth conditions used in these experiments caused important changes to the plasma membrane lipid composition, which were well correlated with differences in ATPase activities and K + ( 86 Rb + ) influx observed under the same experimental conditions.

Drought Stress Effects on Root Cell Membranes

Drought stress or consecutive periods of drought stress implies drastic changes in the root cell membranes of higher plants. We have found, in previous investigations on lipid analysis of whole roots, changes in the total amount of acyl lipids, the unsaturation degree and the dry weight ratio root/shoot, the molar ratio free sterols/phospholipids and the level of abscisic acid. These investigations were performed on oats and rape (Liljenberg and Kates, 1982; Liljenberg and Kates, 1985; Svenningsson and Liljenberg, 1986).