Lipid biosynthesis and composition in oil bodies and microsomes of olive fruit

Both lipid synthesis and composition in oil bodies and microsomes of olive fruit at the first stage of development have been studied. The rate of fatty-acid synthesis in isolated oil bodies was saturated by 4.0 microM [2-14C]-malonyl-CoA. The fatty-acids synthesized of phospholipids and neutral lipids were saturated and monounsaturated. Neutral lipids, galactolipids and, above all, phospholipids were the major acyl-lipid components of microsomal fraction, oleic and palmitic being their principal fatty-acids. When the lipids of microsomes were labelled in vivo with [1-14C]-acetate, phospholipids and neutral lipids exhibited a higher biosynthesis rate relative to the galactolipids. The increase in saturated and monounsaturated fatty-acid synthesis in microsomes, was also accompanied by an important [1-14C]-acetate incorporation into polyunsaturated acids. The data presented here, in conjunction with our previous morphological results, suggest the possibility that olive fruit oil bodies could contain the necessary enzymes for the reserve lipid biosynthesis.     

Influence of Etherel and Gibberellic Acid on Carbon Metabolism, Growth, and Essential Oil Accumulation in Spearmint (Mentha Spicata)

Changes in growth parameters and ¹⁴CO₂ and [U-¹⁴C]-sucrose incorporation into the primary metabolic pools and essential oil were investigated in leaves and stems of M. spicata treated with etherel and gibberellic acid (GA). Compared to the control, GA and etherel treatments induced significant phenotypic changes and a decrease in chlorophyll content, CO₂ exchange rate, and stomatal conductance. Treatment with etherel led to increased total incorporation of ¹⁴CO₂ into the leaves wheras total incorporation from ¹⁴C sucrose was decreased. When ¹⁴CO₂ was fed, the incorporation into the ethanol soluble fraction, sugars, organic acids, and essential oil was significantly higher in etherel treated leaves than in the control. However, [U-¹⁴C]-sucrose feeding led to decreased label incorporation in the ethanol-soluble fraction, sugars, organic acids, and essential oils compared to the control. When ¹⁴CO₂ was fed to GA treated leaves, label incorporation in ethanol-insoluble fraction, sugars, and oils was significantly higher than in the control. In contrast, when [U-¹⁴C]-sucrose was fed the incorporation in the ethanol soluble fraction, sugars, organic acids, and oil was significantly lower than in the control. Hence the hormone treatment induces a differential utilization of precursors for oil biosynthesis and accumulation and differences in partitioning of label between leaf and stem. Etherel and GA influence the partitioning of primary photosynthetic metabolites and thus modify plant growth and essential oil accumulation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of water stress on lipid metabolism in cotton leaves

After 2, 10 and 24 hr labelling with [1-¹⁴C] acetate, radioactivity incorporated into the lipids of cotton leaves is mainly found in phosphatidylcholine, phosphatidylglycerol and neutral lipids. Galactolipids are slowly synthesized and after 24 hr, account for only 10% of the total radioactivity. Under water stress, a marked decrease of precursor incorporation into leaf lipids occurs, particularly in phosphatidylcholine and galactolipids. Relative incorporation into neutral lipids, on the contrary, increases. Water deficits provoke an inhibition of the fatty acid desaturation, resulting in a sharp decrease of linoleic and linolenic acid biosynthesis. The decrease in unsaturated fatty acid biosynthesis occurs in all lipid classes, but is most pronounced in the galactolipid fractions. In the drought-resistant cotton variety (Mocosinho), the variations in lipid and fatty acid metabolism under water stress are less pronounced than in the drought-sensitive variety (Reba), and this attests a greater stability of the membrane system.     

The effect of cadmium on lipid and fatty acid biosynthesis in tomato leaves

This research aims to examine the effect of cadmium uptake on lipid composition and fatty acid biosynthesis, in young leaves of tomato treated seedlings (Lycopersicon esculentum cv. Ibiza F1). Results in membrane lipids investigations revealed that high cadmium concentrations affect the main lipid classes, leading to strong changes in their composition and fatty acid content. Thus, the exposure of tomato plants to cadmium caused a concentration-related decrease in the unsaturated fatty acid content, resulting in a lower degree of fatty acid unsaturation. The level of lipid peroxides was significantly enhanced at high Cd concentrations. Studies of the lipid metabolism using radioactive labelling with [1-¹⁴C]acetate as a major precursor of lipid biosynthesis, showed that levels of radioactivity incorporation in total lipids as well as in all lipid classes were lowered by Cd doses. In total lipid fatty acids, [1-¹⁴C]acetate incorporation was reduced in tri-unsaturated fatty acids (C16:3 and C18:3); While it was enhanced in the palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0) and linoleic (C18:2) acids. [1-¹⁴C]acetate incorporation into C16:3 and C18:3 of galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] and some phospholipids [phosphatidylcholine (PC) and phosphatidylglycerol (PG)] was inhibited by Cd stress. Our results showed that in tomato plants, cadmium stress provoked an inhibition of polar lipid biosynthesis and reduced fatty acid desaturation process.     

Metabolic control analysis of developing oilseed rape (Brassica napus cv Westar) embryos shows that lipid assembly exerts significant control over oil accumulation

? Metabolic control analysis allows the study of metabolic regulation. We applied both single- and double-manipulation top-down control analysis to examine the control of lipid accumulation in developing oilseed rape (Brassica napus) embryos. ? The biosynthetic pathway was conceptually divided into two blocks of reactions (fatty acid biosynthesis (Block A), lipid assembly (Block B)) connected by a single system intermediate, the acyl-coenzyme A (acyl-CoA) pool. Single manipulation used exogenous oleate. Triclosan was used to inhibit specifically Block A, whereas diazepam selectively manipulated flux through Block B. ? Exogenous oleate inhibited the radiolabelling of fatty acids from [1-(14) C]acetate, but stimulated that from [U-(14) C]glycerol into acyl lipids. The calculation of group flux control coefficients showed that c. 70% of the metabolic control was in the lipid assembly block of reactions. Monte Carlo simulations gave an estimation of the error of the resulting group flux control coefficients as 0.27?±?0.06 for Block A and 0.73?±?0.06 for Block B. ? The two methods of control analysis gave very similar results and showed that Block B reactions were more important under our conditions. This contrasts notably with data from oil palm or olive fruit cultures and is important for efforts to increase oilseed rape lipid yields.     

Hydrocarbon accumulation and lipid biosynthesis during larval development in the cabbage looper,Trichoplusia ni

Larvae of the cabbage looper, Trichoplusia ni, were analyzed for the accumulation and biosynthesis of cuticular and internal hydrocarbon at closely spaced and accurately timed intervals during the fourth and fifth stadia. Large differences in the incorporation of [1-¹⁴C]acetate into hydrocarbon were observed at different times during larval development. Much higher incorporation was observed during feeding stages as compared to wandering stages, while lowest rates of biosynthesis occurred just prior to ecdysis. Fourth stadia wanderers accumulated increased amounts of internal hydrocarbon, which is apparently used to cover the newly forming cuticle. During the fourth to fifth stadium moult insects lost all cuticular hydrocarbon that was present on the old cuticle (about 8 μg/insect) and had about 8 μg/insect on the surface of the newly exposed cuticle. During the fourth stadium incorporation of [1-¹⁴C]acetate into total lipid declined between feeding and wandering stages from 24% of injected radiolabel to 7%. Similar decreases in lipid biosynthesis were observed between feeders and wanderers in fifth stadium larvae with the greatest decrease found in the triacylglycerol fraction. These results document dramatic changes in the accumulation and biosynthesis of hydrocarbon and other lipids during larval development.     

Cadmium stress induces changes in the lipid composition and biosynthesis in tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.) leaves

The effects of cadmium (Cd) stress on lipid composition and biosynthesis were investigated in young leaves of ten-day-old tomato seedlings (Lycopersicon esculentum Mill. cv. Ibiza F1). Cd was found to be mainly accumulated in roots, but a severe inhibition of biomass production occurred in leaves, even at its low concentration (1.0 μM). Seven days after Cd treatment, the membrane lipids were extracted and separated on silica-gel thin layer chromatography (TLC). Fatty acid methyl esters were analyzed by FID-GC on a capillary column. Our results showed that Cd stress decreased the quantities of all lipids classes (phospholipids, galactolipids and neutral lipids). Likewise, there was also a decline in the levels of tri-unsaturated fatty acids, such as linolenic (C18:3) and hexadecatrienoic (C16:3) acids. The linolenic acid (C18:3) decreased in monogalactosyldiacylglycerol (MGDG) and all phospholipids, while hexadecatrienoinic acid (C16:3) declined mainly in MGDG. Moreover, Cd at high concentrations (25.0 and 50.0 μM) significantly enhanced the levels of lipid peroxides. Radiolabelling experiments were carried out by laying down microdroplets of [1-¹⁴C]acetate–a major precursor of lipid biosynthesis–on attached leaves of the control and Cd-treated plants. After incubation for 1, 2, 12 and 24 h, the leaves were harvested and lipids extracted and analysed. Cd stress was found to decrease the incorporation of [1-¹⁴C]acetate in total lipids. The biosynthesis of total lipids was altered with 25.0 and 50.0 μM Cd. The decline in the incorporation of [1-¹⁴C]acetate due to Cd stress was observed in all lipid classes. There was also a substantial decline in the incorporation of [1-¹⁴C]acetate in tri-unsaturated fatty acids. The results indicate that Cd treatment induces an oxidative stress by inhibiting the chloroplastic and extrachloroplastic lipid-biosynthesis pathways as well as lipid peroxidation.     

Effects of a squalene epoxidase inhibitor, terbinafine, on ether lipid biosyntheses in a thermoacidophilic archaeon, Thermoplasma acidophilum

The archaeal plasma membrane consists mainly of diether lipids and tetraether lipids instead of the usual ester lipids found in other organisms. Although a molecule of tetraether lipid is thought to be synthesized from two molecules of diether lipids, there is no direct information about the biosynthetic pathway(s) or intermediates of tetraether lipid biosynthesis. In this study, we examined the effects of the fungal squalene epoxidase inhibitor terbinafine on the growth and ether lipid biosyntheses in the thermoacidophilic archaeon Thermoplasma acidophilum. Terbinafine was found to inhibit the growth of T. acidophilum in a concentration-dependent manner. When growing T. acidophilum cells were pulse-labeled with [2-(14)C]mevalonic acid in the presence of terbinafine, incorporation of radioactivity into the tetraether lipid fraction was strongly suppressed, while accumulation of radioactivity was noted at the position corresponding to diether lipids, depending on the concentration of terbinafine. After the cells were washed with fresh medium and incubated further without the radiolabeled substrate and the inhibitor, the accumulated radioactivity in the diether lipid fraction decreased quickly while that in the tetraether lipids increased simultaneously, without significant changes in the total radioactivity of ether lipids. These results strongly suggest that terbinafine inhibits the biosynthesis of tetraether lipids from a diether-type precursor lipid(s). The terbinafine treatment will be a tool for dissecting tetraether lipid biosynthesis in T. acidophilum.     

Lipid biosynthesis in synchronized cultures of the photosynthetic bacterium Rhodopseudomonas sphaeroides.

Lipid biosynthesis has been studied in photosynthetic cultures of Rhodopseudomonas sphaeroides that had been synchronized by stationary-phase cycling or by a centrifugation selection procedure. Synchrony index values in the range 0.70-0.80 were obtained for the first cell cycle with both synchronization methods. The major membrane lipids phosphatidylethanolamine and phosphatidylglycerol were accumulated discontinuously during the cell cycle, their mass doubling immediately before cell division. This accumulation of lipid corresponded to peaks in incorporation of radioactivity from either [1-14C]acetate or [2-3H]glycerol into individual acyl lipids as measured in individual portions of bacteria. For phosphatidylglycerol an additional peak of incorporation of radioactivity from [2-3H]glycerol was found midway through the cell cycle. In spite of their rather similar endogenous fatty acid compositions, the individual phosphoacylglycerols showed distinctive patterns of incorporation of radioactivity from [1-14C]acetate into their acyl moieties. The discontinuous synthesis of acyl lipids observed in cultures of Rhodopseudomonas sphaeroides synchronized by either stationary-phase cycling or centrifugation selection procedures contrasted with the accumulation of chlorophyll-protein complexes whose amounts were found to increase throughout the cell cycle. The implications of these findings for the control of lipid synthesis in bacterial photosynthetic membranes are discussed.     

Rapid kinetic labeling of Arabidopsis cell suspension cultures: implications for models of lipid export from plastids

Cell cultures allow rapid kinetic labeling experiments that can provide information on precursor-product relationships and intermediate pools. T-87 suspension cells are increasingly used in Arabidopsis (Arabidopsis thaliana) research, but there are no reports describing their lipid composition or biosynthesis. To facilitate application of T-87 cells for analysis of glycerolipid metabolism, including tests of gene functions, we determined composition and accumulation of lipids of light- and dark-grown cultures. Fatty acid synthesis in T-87 cells was 7- to 8-fold higher than in leaves. Similar to other plant tissues, phosphatidylcholine (PC) and phosphatidylethanolamine were major phospholipids, but galactolipid levels were 3- to 4-fold lower than Arabidopsis leaves. Triacylglycerol represented 10% of total acyl chains, a greater percentage than in most nonseed tissues. The initial steps in T-87 cell lipid assembly were evaluated by pulse labeling cultures with [(14)C]acetate and [(14)C]glycerol. [(14)C]acetate was very rapidly incorporated into PC, preferentially at sn-2 and without an apparent precursor-product relationship to diacylglycerol (DAG). By contrast, [(14)C]glycerol most rapidly labeled DAG. These results indicate that acyl editing of PC is the major pathway for initial incorporation of fatty acids into glycerolipids of cells derived from a 16:3 plant. A very short lag time (5.4 s) for [(14)C]acetate labeling of PC implied channeled incorporation of acyl chains without mixing with the bulk acyl-CoA pool. Subcellular fractionation of pea (Pisum sativum) leaf protoplasts indicated that 30% of lysophosphatidylcholine acyltransferase activity colocalized with chloroplasts. Together, these data support a model in which PC participates in trafficking of newly synthesized acyl chains from plastids to the endoplasmic reticulum.     

Localization of sites of lipid biosynthesis in mammalian epidermis

The end-product of epidermal differentiation is a stratified layer of corneocytes whose extracellular lipid bilayers provide a permeability barrier. It is generally accepted that the epidermis synthesizes most if not all of the lipids found in this tissue and that extra-epidermal tissues contribute very little to this lipid content. Moreover, the individual epidermal strata in which epidermal lipid biosynthesis occurs are not known. To address this question, we examined [3H]H2O incorporation into nonsaponifiable and saponifiable lipids in individual epidermal cell layers 3 hr after intraperitoneal injection into neonatal mice, and compared this to protein and DNA synthesis using intraperitoneal [3H]leucine and [3H]thymidine incorporation, respectively. Lipid biosynthesis was also assessed by [14C]acetate incorporation into lipid fractions in organ cultured skin and in epidermal subpopulations. The in vivo studies demonstrated that the biosynthetic activity of both saponifiable and nonsaponifiable lipids was comparable to, if not greater, in the stratum granulosum (SG) than in basal/spinous (SB + SS) layer, despite significantly lower levels of both protein and DNA synthesis in the SG. On a mass basis, the SG accounts for about four times the biosynthetic activity of the combined SB + SS layers. The lipid biosynthetic activity in vitro also was two- to fivefold higher in the SG, regardless of whether the epidermis was separated into individual cell layers before or after incubations with radiolabel. Moreover, this difference could not be ascribed to increased acetate pools or to elevated metabolism in the SG versus the SB + SS since the rates of CO2 production were much lower in the SG fraction. The increase in lipid biosynthesis in SG over SB + SS was greatest for phospholipids, followed by glycosphingolipids, and free sterols but was observed in almost all lipid classes. These studies show not only that mammalian epidermis is an active site of de novo lipid biosynthesis, but also that this activity remains high in the stratum granulosum, while other forms of metabolic activity are diminishing. These observations are consistent with the knowledge that lipids extruded from the stratum granulosum layer provide the hydrophobic permeability barrier, and further suggest that elevated synthetic activity in the stratum granulosum would allow rapid replenishment in the event that the barrier is damaged.     

Fatty acid composition and lipid synthesis in developing safflower seeds

Linoleic acid predominated in every lipid class during the whole period of seed development of safflower, while linolenic acid decreased with increasing maturation and it was not detected in mature seeds. Just before the initiation of triacylglycerol accumulation, the fatty acid composition of triacylglycerols changed more rapidly than those of phospholipids and glycolipids. Saturated fatty acids tended to accumulate at the 1- and 3-positions of the glycerol molecule and the more highly unsaturated acids at the 2-position. The fatty acid compositions at the 1- and 3-positions were similar in all cases investigated, but in none of the triacylglycerols was the distribution completely symmetrical. The positional distribution of linolenic acid in triacylglycerols prepared from the immature seeds 2 days after flowering and from the leaves was unusual; in spite of its highest degree of unsaturation, it was preferentially esterified at the 1- and 3-positions. When triacylglycerol was most rapidly accumulated (14–18 days after flowering), the incorporation of acetate-[U- ¹⁴C] into total lipids was also maximum and dienoic fatty acids were the principal acids labelled. Diacylglycerols and compound lipids reached the highest rate of synthesis 15 days after flowering, and then a maximum incorporation into triacylglycerol occurred 18 days after flowering. Incubation temperature affected the synthesis of individual lipid classes. Triacylglycerol was more rapidly synthesized at 32° than at 10°, while diacylglycerols and compound lipids were accumulated under the low-temperature condition. A rise of incubation temperature caused a depression in dienoic acid synthesis.     

Effect of Zn deficiency on net photosynthetic rate, 14C partitioning,and oil accumulation in leaves of peppermint

Changes in growth, CO₂ exchange rate, and distribution of photosynthetically fixed ¹⁴CO2 into the primary photosynthetic metabolic pool (sugars, amino acids and organic acids) and essential oil accumulation were determined in leaves (leaf positions 1-6 from apex) of developing peppermint grown in a solution culture at Zn concentrations of 0 and 0.05 g m-3. There was a significant decrease in ¹⁴C incorporation in total, ethanol-soluble and ethanol-insoluble fractions in Zn deficient plants at all leaf positions. 14C incorporated in essential oil and in sugars were significantly higher in leaf pairs 1 to 3 than in leaf pairs 4 to 6. ¹⁴C incorporation into amino acids and organic acids was higher in all leaf pairs in Zn deficient plants. Statistical analysis showed a positive significant association between Zn content of leaf and ¹⁴C incorporation into ethanol-soluble fraction and sugars and a negative correlation with ¹⁴C incorporation into amino acids and organic acids. Hence the content of sugars in leaves significantly influences essential oil accumulation under Zn stress. This revised version was published online in September 2006 with corrections to the Cover Date.     

The effect of 3-methylindole on the rates of phospholipid and neutral lipid synthesis in cultured fibroblasts

The present study was designed to test the hypothesis that a pneumotoxin, 3-methylindole, alters the basic metabolic pathways involved in phospholipid and neutral lipid synthesis in cultured fibroblasts. Rat skin fibroblasts were obtained from day-old pups. Confluent monolayers were preincubated for up to 24 h with a range of concentrations (0-0.76 mM) of 3-methylindole. Following these treatments, the cell lipids were labelled by incubation for 6 h with [14C]glycerol. The lipids were extracted, separated by thin layer chromatography, and the radioactivity in each fraction was determined. 3-Methylindole had no effect on the total incorporation of [14C]glycerol into lipids, but significantly altered the distribution among lipid fractions. Incubation with 3-methylindole caused a decrease in the incorporation of [14C]glycerol into phosphatidylcholine, while radioactivity accumulated in the neutral lipid fraction. The other lipid fractions responded variably. Similarily, Flow 2000 human diploid lung fibroblasts were incubated for 24 h with 3-methylindole followed by treatment with [14C]glycerol, resulting in a 74% decrease in the incorporation of [14C]glycerol into phosphatidylcholine and a 50% increase in its accumulation in neutral lipid. The results indicate that 3-methylindole inhibits the synthesis of phosphatidylcholine from diacylglycerol precursors on the endoplasmic reticulum in cultured fibroblasts. This is an important observation as it shows that 3-methylindole affects the synthesis of phospholipids required for membrane turnover in cells that are not specialized for the production of phospholipids for surfactant.     

Changes in the Amount of Complex Lipids in the Seeds and in the Pericarp during the Development of Ivy Fruit (Hedera helix)

By a combination of thin-layer chromatography and gas liquid chromatography, a complete study of the development of the different lipid classes and of their fatty acids, during the development of the fruit of Hedera helix L., the English Ivy, has been achieved. In any part of the fruit observed, at any particular stage, the phospholipids and the neutral lipids are the most abundant lipid classes. They accumulate during the entire process of maturation, whereas significant changes occur in their relative proportions, phospholipids being largely dominant until fruit blackening. The accumulation of fatty acids during maturation is characterized by large amounts of C_18:1 in the neutral lipids, especially in the seed, where petroselinic acid (C_18:1Δ⁶) reaches 86% of the total fatty acids. To a smaller extent, the phospholipids also accumulate and thus have the character of reserve molecules. However, their composition remains more stable, which relates them to the “structural lipids” such as galactolipids that maintain their characteristic fatty acid composition, despite the radical changes occurring in the fatty acid metabolism during fruit ripening.     

Lipid metabolism of Pleurotus sajor caju

The biosynthesis of lipids in the mycelium and sporophore of Pleurotus sajor caju was studied. Whereas in the mycelium the biosynthesis of lipids was directly primarily towards storage (e.g. tri-acylglycerols), in the sporophore it was directed towards structural components (e.g. sterols). The incorporation of ¹⁴C precursors into non-polar and polar lipid fractions was generally similar for ¹⁴C acetate, ¹⁴C palmitate, ¹⁴C oleate and ¹⁴C linoleate in the case of mycelium and sporophore. It appears that linoleic acid was utilised as a source of acetate for lipid biosynthesis in the sporophore. A significantly higher incorporation of label was seen in sporophore sterol than in mycelial sterol. Malate dehydrogenase activity increased in the mycelium grown in the presence of lipids. Lipase of P. sajor caju was inducive. The growth of P. sajor caju was enhanced by increased lipid utilisation. The implications of these results on commercial cultivation of this fungus are discussed.     

Comparison of acetate and glucose incorporation into rat and horse skin lipids

The relative efficiency of acetate and glucose as substrates for the biosynthesis of lipids in the skin of the rat and horse was examined using in vivo pulse labelling of skin with [1-14C]acetate and [U-14C]glucose by intradermal injections. The resulting radiolabelled lipids were recovered in the rat by punch biopsy as well as by daily, long-term skin surface lipid collections and in the horse by punch biopsy of the injection sites. The lipids were examined by liquid scintillation and by a combination of thin-layer chromatography and autoradiography. In both species the recovery of radiolabel in the non-polar lipids was much higher after a pulse of [1-14C]acetate than after a pulse of [U-14C]glucose. In the rat, the skin surface lipids labelled through acetate contained sufficient radiolabel to allow observation of the time course of excretion of 14C in the major non-polar lipid classes. The results suggest that the biosynthesis of these lipid classes in the sebaceous glands of the rat are not entirely synchronous. In the skin lipid extracts of the horse, all of the major lipid classes, including phospholipids and glycolipids, were labelled through acetate. In contrast, none of the non-polar lipids and very little of the polar lipids were labelled through glucose.