Involvement of phospholipids in triglyceride biosynthesis by developing soybean cotyledons

The incorporation of phospholipids specifically labeled with glycerol-2³H and acyl-¹⁴C by whole cell tissues of developing soybean cotyledons (Glycine max L.) reveals that phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, N-acylphosphatidylethanolamine, and phosphatidic acid can be metabolized to diglyceride. The diglyceride formed may be recylced into phospholipid or acylated to triglyceride. Diglyceride from phosphatidic acid and phosphatidylethanolamine is used readily in triglyceride biosynthesis compared to the other phospholipids. Incorporation of N-acylphosphatidylethanolamine having [9-10-³H(N)]oleic acid esterified at sn-3 in cotyledons shows rapid acyltransfer of ³H into triglyceride and therefore N-acylphosphatidylethanolamine appears to participate in triglyceride biosynthesis as an acyl donor. These studies emphasize phospholipid metabolism in developing soybean cotyledons is a dynamic process which plays a key role in triglyceride formation.     

Effect of retinoic acid on the acylation of phospholipids in granulocytes in vitro

The influence of retinoic acid on the incorporation of [1-14C]palmitic acid and [1-14C]arachidonic acid into phospholipids was examined in guinea pig peritoneal granulocytes. All-trans-retinoic acid inhibited the incorporation of both fatty acids into phosphatidic acid and phosphatidylinositol. However, it stimulated the incorporation of both fatty acids into phosphatidylcholine but not other phospholipids. All-trans-retinoic acid was more effective than 13-cis-retinoic acid. The influence of all-trans-retinoic acid on the acylation of phospholipids was concentration-dependent with significant effect occurring at 2.1 microM. The loss of labeled fatty acids from prelabeled phospholipids and the transport of labeled fatty acids into granulocytes were not responsive to the presence of retinoic acid in the incubation media. These results suggest that retinoic acid may affect the activities of acyltransferases involved in the synthesis of phosphatidic acid, phosphatidylinositol and phosphatidylcholine.     

Distribution of phospholipids around gramicidin and D-beta-hydroxybutyrate dehydrogenase as measured by resonance energy transfer

A resonance energy transfer method was developed to study the distribution of phospholipids around integral membrane proteins. The method involved measuring the extent of energy transfer from tryptophan residues of the proteins to different phospholipids labeled with a dansyl moiety in the fatty acid chain. No specific interactions were observed between gramicidin and dansyl-labeled phosphatidylcholine, phosphatidylethanolamine, or phosphatidic acid. The results were consistent with a random distribution of each phospholipid in the bilayer in the presence of gramicidin. However, a redistribution of both gramicidin and dansyl-labeled phospholipids was easily observed when a phase separation was induced by adding Ca2+ to vesicles made up of phosphatidylcholine and phosphatidic acid. Polarization measurements showed that in the presence of Ca2+ a rigid phosphatidic acid rich region and a more fluid phosphatidylcholine-rich region were formed. Energy-transfer measurements from gramicidin to either dansylphosphatidylcholine or dansylphosphatidic acid showed gramicidin preferentially partitioned into the phosphatidylcholine-rich regions. Energy-transfer measurements were also carried out with D-beta-hydroxybutyrate dehydrogenase reconstituted in a vesicle composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid. Although the enzyme has a specific requirement for phosphatidylcholine for activity, the extent of energy transfer decreased in the order dansylphosphatidic acid, dansylphosphatidylcholine, dansylphosphatidylethanolamine. Thus, the enzyme reorganized the phospholipids in the vesicle into a nonrandom distribution.     

The effect of corticotropin on phospholipid metabolism in isolated adrenocortical cells.

Trypsin-dispersed cat adrenocortical cells were incubated at 37 degrees C in modified Eagle's medium containing [14C]arachidonic acid of sodium [14C]-acetate and then in non-radioactive medium. Radioactive incorporation was obtained in all phospholipids, with the greatest amount of radioactivity in phosphatidylcholine, followed by phosphatidylethanolamine, phosphatidyl-serine, and phosphatidylinositol. Concentrations of individual phospholipids generally paralleled the relative amounts of corresponding radiolabeled phospholipids, although the percentage of phosphatidylinositol was considerably lower than its radioactive counterpart, resulting in a high specific activity of this particular phospholipid. Although a potently steroidogenic concentration of corticotropin failed to enhance release of label from any particular phospholipid, analysis of specific activity showed that corticotropin stimulation was accompanied by an increased turnover of phosphatidylinositol and phosphatidic acid. These studies demonstrate that isolated cortical cells have the capacity to synthesize phospholipids from radioactive precursors. The finding that the acute effects of corticotropin are associated with changes in specific phospholipids, including phosphatidylinositol and phosphatidic acid, conforms to the general pattern observed in other secretory systems.     

Biosynthesis of triacylglycerols containing very long chain monounsaturated acyl moieties in developing seeds

Particulate (15,000g) fractions from developing seeds of honesty (Lunaria annua L.) and mustard (Sinapis alba L.) synthesize radioactive very long chain monounsaturated fatty acids (gadoleic, erucic, and nervonic) from [1-¹⁴C]oleoyl-CoA and malonyl-CoA or from oleoyl-CoA and [2-¹⁴C]malonyl-CoA. The very long chain monounsaturated fatty acids are rapidly channeled to triacylglycerois and other acyl lipids without intermediate accumulation of their CoA thioesters. When [1-¹⁴C]oleoyl-CoA is used as the radioactive substrate, phosphatidylcholines and other phospholipids are most extensively radiolabeled by oleoyl moieties rather than by very long chain monounsaturated acyl moieties. When [2-¹⁴C]malonyl-CoA is used as the radioactive substrate, no radioactive oleic acid is formed and the newly synthesized very long chain monounsaturated fatty acids are extensively incorporated into phosphatidylcholines and other phospholipids as well as triacylglycerols. The pattern of labeling of the key intermediates of the Kennedy pathway, e.g. lysophosphatidic acids, phosphatidic acids, and diacylglycerols by the newly synthesized very long chain monounsaturated fatty acids is consistent with the operation of this pathway in the biosynthesis of triacylglycerols.     

Effect of freezing and cold storage on phospholipids in developing soybean cotyledons

Freezing of plant tissue adversely affects lipid composition. Immature soybean cotyledons (Glycine max L. Merr.) var. “Harosoy 63” were frozen with liquid N₂, dry ice, or stored in a freezer (−20 C) before lipid extraction. The effects of freezing temperature, thawing rate, and cold storage on the lipid composition of frozen tissue revealed significantly higher levels of phosphatidic acid, and diminished levels of phosphatidylcholine, phosphatidylethanolamine, and N-acylphosphatidylethanolamine from the control. Regardless of freezing temperature, phosphatidic acid levels increased from 4.7 mole% to nearly 50 mole% of the total phospholipid when frozen tissues were stored 10 days at −20 C. During the same period, N-acylphosphatidylethanolamine decreased from 54.1 mole% to 6.6 mole% phospholipid. At least 8 mole% of the phosphatidic acid increase occurred during slow thawing of the frozen tissues. In autoclaved samples, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, and N-acylphosphatidylethanolamine levels were not different from the control. Labeling of the lipid-glycerol with ³H, and fatty acids with ¹⁴C, demonstrated the degradation product was primarily phosphatidic acid. Apparently enzymic destruction of the phospholipids occurred during freezing, cold storage, and thawing.     

ASSEMBLY OF LIPIDS INTO MEMBRANES IN ACANTHAMOEBA PALESTINENSIS : I. Observations on the Specificity and Stability of Choline-14C and Glycerol-3H as Labels for Membrane Phospholipids

In order to determine the feasibility of using radioactive precursors as markers for membrane phospholipids in Acanthamoeba palestinensis, the characteristics of phospholipids labeled with choline-¹⁴C and glycerol-³H were examined. Choline-¹⁴C was found to be a specific label for phosphatidyl choline. There was a turnover of the radioactive moiety of phosphatidyl choline at a rate that varied with the concentration of nonradioactive choline added to the growth medium. Radioactivity was lost from labeled phosphatidyl choline into the acid-soluble intracellular pool and from the pool into the extracellular medium. This loss of radioactivity from cells leveled off and an equilibrium was reached between the label in the cells and in the medium. Radioactive choline was incorporated into phosphatidyl choline by cell-free microsomal suspensions. This incorporation leveled off with the attainment of an equilibrium between the choline-¹⁴C in the reaction mixture and the choline-¹⁴C moiety of phosphatidyl choline in the microsomal membranes. Therefore, a choline exchange reaction may occur in cell-free membranes, as well as living A. palestinensis. In contrast to choline-¹⁴C, the apparent turnover of glycerol-³H-labeled phospholipids was not affected by large concentrations of nonradioactive choline or glycerol in the medium. The radioactivity in lipids labeled with glycerol-³H consisted of 33% neutral lipids and 67% phospholipids. Phospholipids labeled with glycerol-³H turned over slowly, with a concomitant increase in the percentage of label in neutral lipids, indicating a conversion of phospholipids to neutral lipids. Because most (~96%) of the glycerol-³H recovered from microsomal membranes was in phospholipids, whereas only a minor component (~2%) of the glycerol-³H was in the phospholipids isolated from nonmembrane lipids, glycerol-³H was judged to be a specific marker for membrane phospholipids.     

Extensive segregation of acidic phospholipids in membranes induced by protein kinase C and related proteins

Protein kinase C and two other proteins with molecular masses of 64 and 32 kDa, purified from bovine brain, constitute a type of protein that binds a large number of calcium ions in a phospholipid-dependent manner. This study suggested that these proteins also induced extensive clustering of acidic phospholipids in the membranes. Clustering of acidic phospholipids was detected by the self-quenching of a fluorescence probe that was attached to acidic phospholipids (phosphatidic acid or phosphatidylglycerol). Addition of these proteins to phospholipid vesicles containing 15% fluorescently labeled phosphatidic acid dispersed in neutral phosphatidylcholine resulted in extensive, rapid, and calcium-dependent quenching of the fluorescence signal. Fluorescence-quenching requirements coincided with protein-membrane binding characteristics. As expected, the addition of these proteins to phospholipid vesicles containing fluorescent phospholipids dispersed with large excess of acidic phospholipids produced only small fluorescence changes. In addition, association of these proteins with vesicles composed of 100% fluorescent phospholipids resulted in no fluorescence quenching. Protein binding to vesicles containing 5-50% fluorescent phospholipid showed different levels of fluorescence quenching that closely resemble the behavior expected for extensive segregation of the acidic phospholipids in the outer layer of the vesicles. Thus, the fluorescence quenching appeared to result from self-quenching of the fluorophores that become clustered upon protein-membrane binding. These results were consistent with protein-membrane binding that was maintained by calcium bridges between the proteins and acidic phospholipids in the membrane. Since each protein bound eight or more calcium ions in the presence of phospholipid, they may each induce clustering of a related number of acidic phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bradykinin Activates a Phospholipase D that Hydrolyzes Phosphatidylcholine in PC12 Cells

In PC12 pheochromocytoma cells whose phospholipids had been prelabelled with [3H]palmitic acid, bradykinin increased the production of [3H]phosphatidic acid. The increase in [3H]phosphatidic acid occurred within 1-2 min. before the majority of the increase in [3H]diacylglycerol. When the phospholipids were prelabeled with [3H]choline, bradykinin increased the intracellular release of [3H]choline. The production of phosphatidic acid and choline suggests that bradykinin was increasing the activity of phospholipase D. Transphosphatidylation is a unique property of phospholipase D. In cells labeled with [3H]palmitic acid, bradykinin stimulated the transfer of phosphatidyl groups to both ethanol and propanol to form [3H]phosphatidylethanol and [3H]phosphatidylpropanol, respectively. The effect of bradykinin on [3H]phosphatidic acid and [3H]phosphatidylethanol formation was partially dependent on extracellular Ca2+. In cells treated with nerve growth factor, carbachol also increased [3H]phosphatidylethanol formation. To investigate the substrate specificity of phospholipase D, cells were labeled with [14C]stearic acid and [3H]palmitic acid, and then incubated with ethanol in the absence or presence of bradykinin. The 14C/3H ratio of the phosphatidylethanol that accumulated in response to bradykinin was almost identical to the 14C/3H ratio of phosphatidylcholine. The 14C/3H ratio in phosphatidic acid and diacylglycerol was higher than the ratio in phosphatidylcholine. These data provide additional support for the idea that bradykinin activates a phospholipase D that is active against phosphatidylcholine. The hydrolysis of phosphatidylcholine by phospholipase D accounts for only a portion of the phosphatidic acid and diacylglycerol that accumulates in bradykinin-stimulated cells: bradykinin evidently stimulates several pathways of phospholipid metabolism in PC12 cells.     

Influence of proteins on the reorganization of phospholipid bilayers into large domains.

Using large (5-10 microns) vesicles formed in the presence of phospholipids fluorescently labeled on the acyl chain and visualized using a fluorescence microscope, charge-coupled-device camera, and digital image processor, we examined the effects of membrane proteins on phospholipid domain formation. In vesicles composed of phosphatidic acid and phosphatidylcholine, incubation with cytochrome c induced the reorganization of phospholipids into large phosphatidic acid-enriched domains with the exclusion of phosphatidylcholine. Cytochrome c binding was demonstrated to be highest in the phosphatidic acid-enriched domain of the vesicle using the absorbance of the heme moiety for visualization. Both binding of cytochrome c and phospholipid reorganization were blocked by pretreatment of the vesicles with 0.1 M NaCl. The pore forming peptide gramicidin was examined for the effects of an integral protein on domain formation. Initially, gramicidin distributed randomly within the vesicle and showed no phospholipid specificity. Phosphatidic acid domain formation in the presence of 2.0 mM CaCl2 or 100 microM cytochrome c was not affected by the presence of 5 mol % gramicidin within the vesicles. In both cases, gramicidin was preferentially excluded from the phosphatidic acid-enriched domain and became associated with phosphatidylcholine-enriched areas of the vesicle. Thus, cytochrome c caused a major reorganization of both the phospholipids and the proteins in the bilayer.     

Phosphatidylglycerol synthesis in spinach chloroplasts: characterization of the newly synthesized molecule

Intact chloroplasts from spinach (Spinacia oleracea L., hybrid 424) readily incorporate [¹⁴C]glycerol-3-phosphate and [¹⁴C]acetate into diacylglycerol, monoacylglycerol, diacylglycrol, free fatty acids (only when acetate is the precursor), phosphatidic acid, phosphatidylcholine, and most notably phosphatidylglycerol. The fraction of phosphatidylglycerol synthesized is greatly increased by the presence of manganese chloride in the reaction mixture. Glycerol-3-phosphate-labeled phosphatidylglycerol is equally labeled in the two glycerol moieties of the molecule. Acetate-labeled phosphatidylglycerol is equally labeled in both acyl groups. Position one contains primarily oleate, linoleate and small amounts of palmitate. Position two contains primarily palmitate. No radioactive trans-Δ³-hexadecenoate was detected. The labeling patterns indicate that the radioactive phosphatidylglycerol is the product of de novo chloroplast lipid biosynthesis and furthermore, phosphatidylglycerol may be a substrate for fatty acid desaturation.     

Cyanamide mediated syntheses under plausible primitive earth conditions

Summary A mixture of ammonium palmitate,¹⁴C-sn-glycero-1(3)-phosphate, cyanimide and imidazole when heated for several hours formed significant quantities of phospholipids. These reaction products were shown by chromatographic, chemical and enzymatic procedures to be monopalmitoylglycerophosphate (MPGP), dipalmitoylglycerophosphate (DPGP) and monopalmitoyl cyclic glycerophosphate (cMPGP). A portion of the MPGP and DPGP possessed the same steric configuration as naturally occurring lysophosphatidic acid and phosphatidic acid. The yield of total phospholipid was maximal at temperatures between 60° and 90° after 8 h. When ratios of reactants were varied, up to 45% of radioactive glycerophosphate was converted into phospholipids. The average proportions of individual phosphatidic acids were: 60% MPGP, 27% DPGP and 13% cMPGP. Evidence was obtained for a synergistic relationship between cyanamide and imidazole in promoting the formation of phosphatidic acids. These results suggest that phosphatidic acids, which are essential precursors for the biochemical synthesis of more complex membrane phospholipids, could have been produced on the primitive Earth.The following abbreviations are employed: for phospholipid standards with designated steric configuration LPA lysophosphatidic acid (1-acyl-sn-glycero-3-phosphate) - PA phosphatidic acid (1, 2-di-acyl-sn-glycero-3-phosphate) - MPGP monopalmitoylglycerophosphate - cMPGP monopalmitoyl cyclic glycerophosphate (1(3)-acyl-sn-glycero-2,3 (1,2)-cyclic phosphate) - DPGP dipalmitoylglycerophosphate - GP glycerophosphate - cGP cyclic glycerophosphate (sn-glycero-2,3 (1,2)-cyclic phosphate) - TLC thin layer chromatography To whom reprint requests should be addressed.     

Epidermal growth factor stimulates the incorporation of phosphate into phosphatidic acid and phosphoinositides but does not affect phosphoinositide breakdown by phospholipase C in renal cortical slices

The effects of epidermal growth factor (EGF) on the metabolism of phosphatidic acid and phosphoinositides were examined using renal cortical slices labelled with either sodium [³²P]orthophosphate or myo-[³H]inositol. EGF was found to increase the incorporation of phosphate into phosphatidic acid and phosphoinositides. This effect is not dependent on external calcium and is inhibited by 12-O-tetradecanoylphorbol 13-acetate (TPA). When phospholipids were prelabelled, EGF did not decrease the level of ³²P in phosphatidic acid and phosphoinositides, and EGF did not affect the formation of inositol phosphates or the concentration of cAMP and cGMP in renal tissue. The results show that EGF stimulates the incorporation of phosphate into phosphatidic acid and phosphoinositides, but does not affect breakdown of phosphoinositides by phospholipase C in renal cortical slices.     

Lipid metabolism in Aspergillus niger under conditions of heat shock

The processes of lipid synthesis and decomposition in Aspergillus niger under conditions of heat shock (HS) were studied in a pulse-chase experiment with ¹⁴C-labeled sodium acetate. HS (60 min) resulted in the synthesis of phospholipids and sphingolipids intensified compared to the control, as was evident from incorporation of the labeled substrate. The same pattern was observed for neutral lipids, especially for triacylglycerides, while incorporation of the label into sterols remained almost the same. Further cultivation for 3 h in the medium without the labeled substrate resulted in a significant decrease of the label content in the membrane lipids of both the control and the experiment, although under HS conditions this decrease was much more pronounced, especially for phosphatidylcholines and phosphatidylethanolamines. A threefold increase of the label content in phosphatidic acids was observed only under HS conditions. These results indicate more intense metabolism of the membrane lipids under heat shock and suggest the degradation of the major cell phospholipids as the factor responsible for the increased level of phosphatidic acids in A. niger mycelium.     

Membrane transformations in aging potato tuber slices

When potato tuber slices (Solanum tuberosum L.) are incubated with radioactive choline, labeled membrane-bound phospholipids are formed. If potato slices are aged for 0 to 24 hours before exposure to radioactive choline, the distribution of the labeled phospholipids undergoes both quantitative and qualitative changes. Quantitatively, there is a marked increase in the total lipoidal radioactivity with aging time. Qualitatively, there is a shift in the kinds of subcellular fractions that are being labeled. Fresh slices incorporate most of the lipoidal radioactivity in the microsomes. Slices aged for 9 hours incorporate most of the label in a fraction consisting of single membrane-bound cisternae, which are presumed to be dictyosomal fragments. Slices aged for 24 hours before incubation with radioactive choline incorporate the greater portion of the label in this same fraction, but a significant portion of the label is found in a heavier, mitochondria-containing fraction.     

Turnover of Phospholipids in Normal and Phosphorus-deficient Spirodela

When ³²P₁ was supplied as a 15-minute pulse to normal Spirodela oligorrhiza plants, the first phospholipid to become fully labeled was phosphatidic acid. Phosphatidyl glycerol reached maximum labeling before the other major phospholipids. In phosphorus-deficient plants, however, phosphatidyl glycerol became labeled much more slowly than either phosphatidyl choline or phosphatidyl ethanolamine, and also the proportion of phosphatidyl glycerol present was smaller. Thus, phosphatidyl glycerol synthesis is sensitive to phosphorus deficiency. Since most of the phosphatidyl glycerol present in Spirodela was localized in the chloroplast, this effect appeared to be specifically one on chloroplast composition. The phosphorus-deficient chloroplast had a 60% lower phospholipid content and a normal phospholipid pattern, but the phospholipid which was present was apparently cycling much less rapidly. Zeatin, which ameliorates the visual symptoms of phosphorus deficiency, also reduces the effect of phosphorus deficiency on phospholipid synthesis.     

Changes in conformation of spin-labeled calmodulin by phospholipids

Spin-labeled calmodulin was synthesized and the effects of phospholipids on its conformation were examined by ESR spectroscopy. Phosphatidylserine (0.1-1.0 mM) increased the signal intensity of the ESR spectrum of spin-labeled calmodulin and decreased the apparent rotational correlation time in the presence of 0.1 mM CaCl2. This change was reversed by addition of excess calcium, and in the absence of calcium phosphatidylserine did not change the spectrum, suggesting that the change in spin-labeled calmodulin brought about by phosphatidylserine was not induced by a hydrophobic interaction of the two, but by inhibition of the binding of calcium to calmodulin. L-Serine and O-phospho-L-serine had no effect on the ESR signals of spin-labeled calmodulin. The effects of various other phospholipids were also examined. Their inhibitory activities were in the order phosphatidic acid greater than phosphatidylserine greater than phosphatidylglycerol = phosphatidylinositol; phosphatidylethanolamine and phosphatidylcholine had no effect on the spectra. The effects of these phospholipids were dependent on their binding activities toward calcium. Furthermore, phosphatidic acid and phosphatidylserine at 1 mM reduced the activity of calmodulin-dependent phosphodiesterase by 16.4 and 8.7%, respectively. These findings indicate that spin-labeled calmodulin did not interact with the phospholipids by a hydrophobic interaction, but that calcium binding to spin-labeled calmodulin interfered with phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol, and some of these phospholipids inactivated calmodulin. Thus the activity of calmodulin may be regulated in part by some phospholipids.     

Changes in Phospholipid Composition of a Winter Wheat Cultivar during Germination at 2 C and 24 C

Evaluation of various solvent systems for lipid extraction of wheat Triticum aestivum L. cv. Rideau seeds showed that boiling 2-propanol followed by the Bligh-Dyer procedure was the most efficient method, with respect to lipid yield and ability to inactivate lipolytic enzymes. Ten phospholipids were identified in dry seeds; the major components being phosphatidylcholine, lysophosphatidylcholine, N-acyl lysophosphatidyl-ethanolamine, N-acylphosphatidylethanolamine, and phosphatidylethanolamine. After growth for 1 week (2 C) or 31 hours (24 C), the proportions of phosphatidylethanolamine + lysophosphatidic acid and phosphatidic acid increased, lysophosphatidylcholine decreased, and the remaining phospholipids showed little change. At 5 weeks (2 C) or 72 hours (24 C), the seedlings showed 5-fold increases in the proportion of phosphatidic acid largely at the expense of phosphatidylcholine, small decreases in N-acyl lysophosphatidylethanolamine and N-acylphosphatidylethanolamine, and significant increases in lysophosphatidylcholine. The changes in phosphatidic acid and phosphatidylcholine are interpreted as being partially due to increasing phospholipase D activity during germination. In general, the phospholipid composition was similar in morphologically equivalent seedlings grown at 2 C or 24 C. The increased membrane content in seedlings grown at 2 C does not reflect any preferential synthesis of individual phospholipids.     

Dynamics of Phospholipid Content in the Vacuolar Membrane of Red Beet Taproots Exposed to Abiotic Stress

The composition of vacuolar membrane phospholipids in the taproot of red beet (Beta vulgaris L.), cv. Modana, was determined at normal conditions and under different types of stress (hypo- and hyperosmotic and oxidative stress). The experiments have shown that, among vacuolar membrane phospholipids in red beet taproot, phosphatidylcholines and phosphatidylethanolamines dominated and accounted for 70% of total phospholipids. It is interesting that the content of phosphatidic acid was high (20% of total phospholipids of the vacuolar membrane). Stress effects brought about changes in the composition of membrane phospholipids, which may be an element of phenotypic adaptation. Under hypoosmotic stress, reliable changes in the content of phosphatidic acid were observed, hyperosmotic stress was associated with changes in the level of phosphatidylcholines and phosphatidylinositols, and oxidative stress was notable for changes in the content of phosphatidylethanolamines and phosphatidylserines. The most significant changes were observed in the classes of phospholipids that may be involved in structural modification of membranes associated with transformation of their bilayer lamellar structure into hexagonal. These phospholipids comprise phosphatidic acid, phosphatidylcholines, and phosphatidylethanolamines. Revealed changes in the content of these phospholipids may alter the ratio between lamellar bilayer and nonbilayer hexagonal lipid structures in the vacuolar membrane and act as an important adaptation mechanism ensuring protection against stress.     

Delay of Membrane Lipid Degradation by Calcium Treatment during Cabbage Leaf Senescence

Cabbage leaf discs (Brassica oleracea L., Capitata group) were floated adaxial side up in 0, 0.05, or 0.25 m CaCl₂ solutions at 15°C for 14 d in the dark. To assess whether the delay of senescence by calcium treatment involved protection of membrane lipids, chlorophyll and protein content and the lipid composition of the membranes were determined during incubation. Chlorophyll and protein content decreased with time, in correlation with a reduction in the amount of phospholipids. The degree of unsaturation of phospholipids and free fatty acids decreased, whereas the ratio of sterol to phospholipid increased. The proportions of phospholipid classes did not change during senescence. The catabolism of phospholipids was delayed by 0.05 m calcium, but accelerated by 0.25 m, as compared to the untreated control. Based on the levels of the lipid intermediates, phospholipase D, phosphatidic acid phosphatase, lipolytic acyl hydrolase, and lipoxygenase appeared to be involved in the breakdown of phospholipids during senescence. Phospholipase D and phosphatidic acid phosphatase may be directly influenced by calcium. The calcium treatment apparently did not affect the activity of acyl hydrolase. Lipoxygenase, responsible for the peroxidation of the polyunsaturated fatty acids, was probably indirectly influenced by calcium. We conclude that the delay of senescence of cabbage leaf discs by calcium treatment involved protection of membrane lipids from degradation.