Effects of frost hardening on the composition of galactolipids and phospholipids occurring during isolation of chloroplast thylakoids from needles of scots pine

Frost hardening of seedlings of Scots pine (Pinus sylvestris) at a non-freezing temperature of 4°C resulted in a 2-fold increase of the acyl lipids of the needles. This was because of increases in phospholipids and triglycerides. The galactolipid content of the needles was almost the same in unhardened and frost-hardened seedlings. In unhardened seedlings the mol ratio of monogalactosyl diacylglycerol (MGDG) to digalactosyl diacylglycerol (DGDG) was 1.7 ± 0.3 and 0.9 ± 0.2 in needles and isolated thylakoids, respectively. Corresponding ratios for frost-hardened seedlings were 1.5 ± 0.2 and 0.3 ± 0.03. The lower ratios found in isolated thylakoids, particularly in thylakoids from frost-hardened seedlings, are suggested to depend on the enzyme galactolipid: galactolipid galactosyltransferase being active during the isolation procedure. This is deduced from the result that the content of MGDG decreased and that of DGDG and 1.2 diglycerides increased. Needles of Scots pine also contain phospholipidase D. This enzyme was active during thylakoid preparation, particularly after frost hardening, as judged from the large amount of phosphatidic acid found the in thylakoid fraction isolated from frost-hardening needles. The fatty acid composition of the acyl lipids showed no major changes due to hardening at non-freezing temperature.     

Greening-related lipid changes in leaves,protoplasts and a plasmamembrane-enriched fraction of pea

Light-induced changes in the membrane lipid compositions were studied in pea leaves and in protoplasts and a plasmamembrane-enriched fraction (PMEF)* of pea leaves. PC, PE, PI, PG, PA, MGDG, DGDG and SL were identified as the glycerolipids. The relative levels of various membrane lipids changed due to light-induced greening. There was an increase in the galactolipids of leaves and leaf protoplasts. The galactolipid constituent of the PMEF was very low and showed no change. Among the plasmamembrane phospholipids, PI increased with a concomitant decrease in PC.     

Cyanobacterial monogalactosyldiacylglycerol-synthesis pathway is involved in normal unsaturation of galactolipids and low-temperature adaptation of Synechocystis sp. PCC 6803

We characterized certain physiological functions of cyanobacterial monoglucosyldiacylglycerol using a Synechocystis sp. PCC 6803 mutant in which the gene for monoglucosyldiacylglycerol synthase had been disrupted and its function complemented by inclusion of an Arabidopsis monogalactosyldiacylglycerol synthase gene. By using this method, we prepared the first viable monoglucosyldiacylglycerol-deficient mutant of cyanobacterium and found that monoglucosyldiacylglycerol is not essential for its growth and photosynthesis under a set of “normal growth conditions” when monogalactosyldiacylglycerol is adequately supplied by the Arabidopsis monogalactosyldiacylglycerol synthase. The mutant had healthy thylakoid membranes and normal pigment content. The membrane lipid composition of the mutant was similar with that of WT except lack of monoglucosyldiacylglycerol and a slight increase in the level of phosphatidylglycerol at both normal and low temperatures. However, the ratio of unsaturated fatty acids in monogalactosyldiacylglycerol and digalactosyldiacylglycerol was reduced in the mutant compared with WT. Although the growth of the mutant was indistinguishable with that of WT at normal growth temperature, it was markedly retarded at low temperature compared with that of WT. Our data indicated the possibility that cyanobacterial monogalactosyldiacylglycerol-synthesis pathway might be required for the adequate unsaturation level of fatty acids in galactolipids and affect the low-temperature sensitivity.     

Aluminum mediates compositional alterations of polar lipid classes in maize seedlings

Changes in lipid composition were investigated on maize roots and shoots under aluminum stress. After 4d exposure to 100 microM Al, root growth was inhibited while shoot growth was not affected. In roots, the decrease of the DBI (double bond index) of total fatty acids may signal a decrease in membrane fluidity. The total lipids (TL) decreased by 49%, but phospholipids (PL), phosphatidylcholine (PC) and phosphatidylinositol (PI) increased to approximately 3-fold. The MGDG increased to 2-fold but no significant change was found in the DGDG. The steryl lipids (SL) increased by 69%. The SL/PL ratio decreased from 2.64 to 1.52 and the MGDG/DGDG ratio increased from 0.45 to 1.06 in roots of Al-stressed plants. Al leads to oxidative stress in roots of treated plants as indicated by the increase of malondialdehyde (MDA) concentrations. In shoots, changes in fatty acid composition were associated with an increase of the DBI in all lipid classes except that of the DGDG decreased. The PG was the lipid class which shows the large variation of fatty acid composition. No significant changes were found either for TL, PL, SL or MDA concentrations in shoots of Al-treated plants. While PE levels did not show significant change, PI and PG increased and PC decreased. However, the Al caused 87% decrease in the GL levels. The MGDG and DGDG decreased to 19- and 8-fold, respectively. The deleterious effects of Al on polar lipids could be caused by a direct intervention of Al on plasma membrane and/or alteration of cell metabolism.     

Functional and topological analysis of phosphatidylcholine synthase from Sinorhizobium meliloti

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of eubacteria. It can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation pathway or the phosphatidylcholine synthase (Pcs) pathway. Pcs belongs to the CDP-alcohol phosphotransferase superfamily and synthesizes PC and CMP in one step from CDP-diacylglycerol and choline. In this study, we aligned sequences of characterized Pcs enzymes to identify conserved amino acid residues. Alanine scanning mutagenesis was performed on 55 of these conserved residues. The mutation of nine residues caused a drastic to complete loss (< 20% of wild type activity) of Pcs activity. Six of these essential residues were subjected to further mutagenesis studies replacing them by amino acids with similar properties or size. A topological analysis of sinorhizobial Pcs showed the presence of eight transmembrane helices, with the C- and N-terminus located in the cytoplasm. The majority of the conserved residues is predicted to be either located within the cytoplasmic loops or on the cytoplasmic side of the membrane which can be expected for an enzyme using one membrane-associated and one soluble substrate.     

Activation of the CF0-CF1, ATP synthase from spinach chloroplasts by chloroplast lipids

The interactions of CF₀-CF₁ with different lipids were studied by following the stimulation of Mg-ATPase and of P_i-ATP exchange activities of reconstituted CF₀-CF₁ proteoliposomes. The following results were obtained: (1) Both P_i-ATP exchange and Mg-ATPase activities are stimulated by lipids. Furthermore, the inhibition of Mg-ATPase by N,N′-dicyclohexylcarbodiimide is dependent on the interactions of CF₀-CF₁ with lipids. (2) A polar lipid extract of thylakoid membranes stimulates Mg-ATPase activity of CF₀-CF₁ more efficiently than phospholipids. The relative effectiveness of Mg-ATPase stimulation is: chloroplast lipids > soybean phospholipids > phosphatidylcholine/phosphatidylserine (4: 1) > phosphatidylcholine. The rate of P_i-ATP exchange in chloroplast lipids CF₀-CF₁ proteoliposomes is, however, lower than in soybean lipids CF₀-CF₁ proteoliposomes, due to their higher permeability to protons. Addition of 10% phosphatidylserine to chloroplast lipids reduces their permeability to protons and stimulates P_i-ATP exchange. (3) The kinetic mechanism of ATPase stimulation by chloroplast lipids is by decreasing the K_m (ATP) and by increasing V_max in comparison to soybean lipid proteoliposomes. This may explain the low affinity for ATP and the slow turnover rate of the purified enzyme in artificial lipids in comparison to the native enzyme in chloroplast thylakoids. (4) Chloroplast lipids lacking monogalactosyldiacylglycerols only poorly activate CF₀-CF₁. A large stimulation of P_i-ATP exchange is obtained by a mixture of 60% monogalactosyldiacylglycerol and 40% of the rest of the chloroplast lipids, but not by mixtures of monogalactosyldiacylglycerol with phospholipids. Hydrogenation of the unsaturated fatty acids of monogalactosyldiacylglycerol inhibits the activation of CF₀-CF₁. (5) The results suggest that: (a) interactions of specific chloroplast lipids with CF₀-CF₁ activates the enzyme by increasing its turnover and its affinity for ATP; (b) specific requirements for CF₀-CF₁ activation are the presence of monogalactosyldiacylglycerols together with another chloroplast lipid component and of highly unsaturated fatty acids.     

Glycerolipid synthesis in Chlorella kessleri 11h: I. Existence of a eukaryotic pathway

The fatty acid distributions at the sn-1 and sn-2 positions in major chloroplast lipids of Chlorella kessleri 11h, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), were determined to show the coexistence of both C₁₆ and C₁₈ acids at the sn-2 position, i.e. of prokaryotic and eukaryotic types in these galactolipids. For investigation of the biosynthetic pathway for glycerolipids in C. kessleri 11h, cells were fed with [¹⁴C]acetate for 30 min, and then the distribution of the radioactivity among glycerolipids and their constituent fatty acids during the subsequent chase period was determined. MGDG and DGDG were labeled predominantly as the sn-1-C₁₈-sn-2-C₁₆ (C₁₈/C₁₆) species as early as by the start of the chase, which suggested the synthesis of these lipids within chloroplasts via a prokaryotic pathway. On the other hand, the sn-1-C₁₈-sn-2-C₁₈ (C₁₈/C₁₈) species of these galactolipids gradually gained radioactivity at later times, concomitant with a decrease in the radioactivity of the C₁₈/C₁₈ species of phosphatidylcholine (PC). The change at later times can be explained by the conversion of the C₁₈/C₁₈ species of PC into galactolipids through a eukaryotic pathway. The results showed that C. kessleri 11h, distinct from most of other green algal species that were postulated mainly to use a prokaryotic pathway for the synthesis of chloroplast lipids, is similar to a group of higher plants designated as 16:3 plants in terms of the cooperation of prokaryotic and eukaryotic pathways to synthesize chloroplast lipids. We propose that the physiological function of the eukaryotic pathway in C. kessleri 11h is to supply chloroplast membranes with 18:3/18:3-MGDG for their functioning, and that the acquisition of a eukaryotic pathway by green algae was favorable for evolution into land plants.     

Thermal stability of trimeric light-harvesting chlorophyll a/b complex (LHCIIb) in liposomes of thylakoid lipids

The major light-harvesting chlorophyll a/b complex (LHCIIb) of photosystem (PS) II functions by harvesting light energy and by limiting and balancing the energy flow directed towards the PSI and PSII reaction centers. The complex is predominantly trimeric; however, the monomeric form may play a role in one or several of the regulatory functions of LHCIIb. In this work the dissociation temperature was measured of trimeric LHCIIb isolated from Pisum thylakoids and inserted into liposomes made of various combinations of thylakoid lipids at various protein densities. Dissociation was measured by monitoring a trimer-specific circular dichroism signal in the visible range. The LHCIIb density in the membrane significantly affected the trimer dissociation temperature ranging from 70 °C at an LHCIIb concentration comparable to or higher than the one in thylakoid grana, to 65 °C at the density estimated in stromal lamellae. Omitting one thylakoid lipid from the liposomes had virtually no effect on the thermal trimer stability in most cases except when digalactosyl diacylglycerol (DGDG) was omitted which caused a drop in the apparent dissociation temperature by 2 °C. In liposomes containing only one lipid species, DGDG and, even more so, monogalactosyl diacylglycerol (MGDG) increased the thermal stability of LHCIIb trimers whereas phosphatidyl diacylglycerol (PG) significantly decreased it. The lateral pressure exerted by the non-bilayer lipid MGDG did not significantly influence LHCII trimer stability.     

Lipid variation of the green alga Fritschiella tuberosa during growth in axenic batch culture

The lipid and pigment pattern of the chaetophoralean green alga Fritschiella tuberosa was studied during different growth periods. Four phases in axenic batch culture are observed. The first phase commences at the end of logarithmic growth and results in a decrease of phosphatidyl inositol and hexadecatetraenoic acid.The second phase coincides with the end of linear growth and is characterized by a decrease in glycolipids, phospholipids and α-linolenic acid, and an increase of linoleic and oleic acid. The third phase starts with the beginning of the stationary phase. During this time glycolipids, phospholipids, and α-linolenic acid decrease and oleic acid, linoleic acid and triacylglycerol increase. The fourth phase begins about one week after the onset of the stationary growth phase. A marked accumulation of triacylglycerol took place, secondary carotenoids were detectable and the long filamentous cells of Fritschiella changed into slightly rotund short cells.     

The production of hydroxyl radicals by adriamycin in red blood cells

Spin trapping of the free radicals formed from the interaction between adriamycin and red blood cells resulted in the formation of a hydroxyl spin adduct. The formation of hydroxyl radicals was found to be inhibited by mannitol. Hemoglobin was found not to be obligatory for the formation of hydroxyl radicals which probably result from the reduction of hydrogen peroxide by adriamycin semiquinone.     

Analysis of molecular species of plant polar lipids by high-performance and gas liquid chromatography

Total lipid extracts from potato tubers and tobacco leaves are separated into lipid classes by two step HPLC using a silicic column. Elution is first performed for 20 min with a programmed linear gradient of two mixed solvents running from 100% of solution A (isopropanol-hexane, 4:3) to 100% of solution B (isopropanol-hexane-water, 8:6:1.5); the column is then eluted with pure solution B in an isocratic mode for 20 min more. The main polar lipids (MGDG, DGDG, PC, PE, PG) from both plant tissues can be collected and further separated into component molecular species on a simplified HPLC system with a C₁₈ column eluted in an isocratic mode with a polar solvent. Molecular species separations are achieved within 35 min; quantifications are made through GLC analysis of attached fatty acids. Three to five main molecular species are thus clearly identified in each lipid class. In potato tuber, phospholipids (PC, PE) 18:2/18:2 species are predominant. In tobacco leaf, six double bond species (18:3/18:3 and 16:3/18:3) are predominant in galactolipids, whereas PC contains a greater number of molecular species varying by their degree of unsaturation (from 18:3/18:3 to 16:0/18:2). Only certain molecular species of PG contain Δ³-trans-hexadecenoic acid.     

Thermal phase transitions in the polar lipids of plant membranes. Their induction by disaturated phospholipids and their possible relation to chilling injury

The phase behaviour of leaf polar lipids from three plants, varying in their sensitivity to chilling, was investigated by differential scanning calorimetry. For the lipids from mung bean (Vigna radiata L. var. Berken), a chilling-sensitive plant, a transition exotherm was detected beginning at $\text{10 ± 2°}\text{C}$. No exotherm was evident above 0°C with polar lipids from wheat (Triticum aestivum cv. Falcon) or pea (Pisum sativum cv. Massey Gem), plants which are insensitive to chilling. The enthalpy for the transition in the mung bean polar lipids indicated that only about 7% w/w of the lipid was in the gel phase at ?8°C. The thermal transition of the mung bean lipids was mimicked by wheat and pea polar lipids after the addition of 1 to 2% w/w of a relatively high melting-point lipid such as dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol or dimyristoylphosphatidylcholine. Analysis of the polar lipids from the three plants showed that a dipalmitoylphosphatidylglycerol was present in mung bean (1.7% w/w) and pea (0.3% w/w) but undetected in wheat, indicating that the transition exotherm temperature of 10°C in mung bean, 0°C in pea and about ?3°C in wheat correlates with the proportion of the high melting-point disaturated component in the polar lipids. The results indicate that the transition exotherm, observed at temperatures above 0°C in the membranes of chilling-sensitive plants, could be induced by small amounts of high melting-point lipids and involves only a small proportion of the membrane polar lipids.     

Interaction of a polymeric biguanide biocide with phospholipid membranes

Differential scanning calorimetry (DSC) and fluorescence polarization methods have been used to study the interactions between phospholipid membranes and a polymeric biocide, poly(hexamethylene biguanide hydrochloride) (PHMB). It was found that PHMB had very little effect on neutral lipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE), whereas it greatly reduced the phase transition temperature of phosphatidylglycerol (PG), an acidic lipid found in bacteria. Although the corresponding monomeric biocide had a similar effect on the PG bilayer, the behaviour towards mixed lipid bilayers of PC and PG has been shown to be completely different for the polymeric and monomeric biocides: viz. the former can induce isothermal phase separation into a PHMB-PG complex domain and a PC-enriched domain, whilst the latter cannot. This may account for the great difference in bactericidal activity between them. It is suggested that PHMB interacts primarily with negatively charged species in the membranes, inducing aggregation of acidic lipids in the vicinity of the adsorption site, where higher fluidity and higher permeability are expected. The results have shown that two factors might be crucial in the cidal activity of such types of cationic disinfectants as biguanides: phase separation and interaction with the hydrocarbon interior of the membranes. Polymeric biocides could be particularly effective by virtue of their ability to combine hydrophobic character and multiple charges within a single molecule.