Regulation of membrane fatty acid composition by temperature in mutants of Arabidopsis with alterations in membrane lipid composition

Background  

A wide range of cellular responses occur when plants are exposed to elevated temperature, including adjustments in the unsaturation level of membrane fatty acids. Although membrane bound desaturase enzymes mediate these adjustments, it is unknown how they are regulated to achieve these specific membrane compositions. Furthermore, the precise roles that different membrane fatty acid compositions play in photosynthesis are only beginning to be understood. To explore the regulation of the membrane composition and photosynthetic function in response to temperature, we examined the effect of temperature in a collection of mutants with altered membrane lipid fatty acid composition.     

Alteration of the fatty acid composition of membrane phospholipids in mouse lymphoid cells

A simple method is described for introducing exogenous fatty acids into the membrane phospholipids of the murine leukemia cell EL-4, and into the membrane phospholipids of resting mouse lymphocytes. The method involves culturing of the cells with free or methylated fatty acids at concentrations up to 50 microgram/ml. The presence of serum in the culture medium does not interfere with fatty acid uptake, but does increase the growth rate and viability of the cells. Membrane lipid composition returns to normal after the cells are grown in medium without exogenous fatty acid. Fractionation of the cell membranes confirmed that exogenous fatty acids were incorporated into the phospholipids of the plasma membrane.     

Fatty acid desaturation and microsomal lipid fatty acid composition in experimental hypothyroidism.

We have studied the influence of experimental hypothyroidism in the rat on the synthesis of unsaturated fatty acids and on liver microsomal lipid fatty acid composition. Hypothyroid rats demonstrated an 80% decrease in delta 9 (stearate) desaturation and a 43% decrease in delta 6 (linoleate) desaturation. Liver microsomal fatty acid composition was altered in the hypothyroid animals with a significantly decreased proportion of arachidonate and increased proportions of linoleate, eicosa-8,11,14-trienoate, eicosapentaenoate and docosahexaenoate. The bulk of these changes occurred in both of the two major phospholipid components, phosphatidylcholine and phosphatidylethanolamine. All of the changes were corrected by treatment of the hypothyroid rat with 25 micrograms of tri-iodothyronine/100 g body wt. twice daily. The diminished delta 9 desaturation did not lead to any changes in fatty acid composition. The increased linoleate and decreased arachidonate levels may be due to the diminished delta 6 desaturase activity, the rate-controlling step in the conversion of linoleate into arachidonate. The increases in the proportions of the other polyunsaturated fatty acid components cannot be explained by changes in the synthesis of unsaturated fatty acids, but are probably due to diminished utilization of these fatty acids.     

Perturbation of lipid metabolism in L-M cultured cells by elaidic acid supplementation: formation of fatty alcohols

Supplementation of culture medium with elaidic acid (400 μg/flask) in L-M cells results in the formation of an otherwise undetected lipid component. We have identified this lipid component to be a mixture of free fatty alcohols containing primarily elaidyl alcohol with cetyl, stearoyl, and oleoyl alcohols as minor constituents. Formation of fatty alcohols by fatty acid supplementation seems to be specific with $\text{trans}$ fatty acids (i.e., elaidate, $\text{trans}$ vaccenate, and linolelaidate); addition of stearate and oleate to the L-M cells does not produce fatty alcohols. The fatty alcohols accumulated by the $\text{trans}$ fatty acid supplementation are associated with both the particulate and supernatant fractions of the cells.     

Alteration of the fatty acid composition of Ehrlich ascites tumor cell lipids.

The fatty acid composition of Ehrlich ascites tumor lipids was altered markedly $\text{in vivo}$ by changing the type of fat fed to the tumor-bearing mice. As compared with regular chow, large differences were produced in polar and neutral lipid fatty acyl groups when the tumor cells were grown in mice fed coconut oil, sunflower oil or fat deficient diets. Subcellular membrane fractions obtained from these cells exhibited similar variations in fatty acyl composition. This experimental system provides large quantities of malignant cells for study of the relationships between membrane lipid structure and function.     

Manipulation of fatty acid composition of membrane phospholipid and its effects on cell growth in mouse LM cells

Fatty acid composition of the phospholipids of mouse LM cells grown in suspension culture in serum-free chemically defined medium was modified by supplementing the medium with various fatty acids bound to bovine serum albumin.Following supplementation with saturated fatty acids of longer than 15 carbons (100 μM) profound inhibition of cell growth occurred; this inhibitory effect was completely abolished when unsaturated fatty acids were added at the same concentration. Supplementing with unsaturated fatty acids such as linoleic acid, linolenic acid or arachidonic acid had no effect on the cell growth.Fatty acid composition of membrane phospholipids could be manipulated by addition of different fatty acids. The normal percentage of unsaturated fatty acids in LM cell membrane phospholipids (63%) was reduced to 35–41% following incorporation of saturated fatty acids longer than 15 carbon atoms and increased to 72–82% after addition of unsaturated fatty acids.A good correlation was found between the unsaturated fatty acid content of membrane phospholipids and cell growth. When incorporated saturated fatty acids reduced the percentage of unsaturated fatty acids in membrane phospholipids to less than 50%, severe inhibition of the cell growth was found. Simultaneous addition of an unsaturated fatty acid completely abolished this effect of saturated fatty acids.     

Effect of glutamic acid on the fatty acid and lipid composition of Choanephora cucurbitarum.

The fatty acid composition of the total, neutral, sterol, free fatty acid, and polar-lipid fractions in the mycelium of Choanephora curcurbitarum was determined. The major fatty acids in all lipid fractions were palmitic, oleic, linoleic, and gamma-linolenic acid. Different lipid fractions did not show any particular preference for any individual fatty acid; however, the degree of unsaturation was different in different lipid fractions. Free fatty acid and polar lipid fractions contained a higher proportion of gamma-linolenic acid than did triglyceride and sterol fractions. Addition of glutamic acid to the malt-yeast extract and medium resulted in the biosynthesis of a number of long-chain fatty acids beyond the gamma-linolenic acid. These fatty acids, e.g., C22:1, C24:0, and C26:0, were never observed to be present in the fungus when grown on a malt-yeast extract medium without glutamic acid. Furthermore, thin-layer chromatographic analysis showed a larger and denser spot of diphosphatidyl glycerol from the mycelium grown on glutamic acid medium than from the control mycelium. The possible significance of this finding is discussed.     

Effect of temperature on fatty acid composition in each lipid fraction of Spirometra erinaceieuropaei plerocercoids

The effects of temperature and host fatty acids on the fatty acid contents of Spirometra erinaceieuropaei plerocercoids were investigated to clarify their role in sparganosis. After 24 hr incubation at 18 C in host snake serum, omega6 series fatty acids, especially arachidonic acid in the phospholipid fraction of the plerocercoids, increased compared with those of plerocercoids incubated at 37 C. The changes in the ratio of polyunsaturated to saturated fatty acids in the phospholipid fraction of plerocercoids incubated in physiological saline for 6 hr at 10 C were almost the same as the changes at 37 C. The ratio of polyunsaturated to saturated fatty acids of the triglyceride fraction showed almost opposite change versus the phospholipid fraction. The percentage of arachidonic acid in the phospholipid fraction of plerocercoids increased during the first 3 hr of incubation and then decreased, regardless of temperature. At 37 C, the percentage of arachidonic acid in the free fatty acid fraction fell for the first 3 hr of incubation and was significantly elevated at the end of the 6-hr incubation. At 10 C, however, arachidonic acid in the free fatty acid fraction decreased for the first hour of incubation, increased at 3 hr of incubation, then decreased again. These results suggest that fatty acids of the plerocercoids are frequently exchanged between fractions. Plerocercoids can mobilize arachidonic acid to the free fatty acid fraction more quickly at lower temperature than at higher temperature. They may utilize mobilized arachidonic acid early in the infection stage to produce prostaglandins. Alternatively, they can incorporate arachidonic acid into the phospholipid fraction again when arachidonic acid is readily available in the environment.     

Dietary fatty acid composition influences tissue lipid profiles and regulation of body temperature in Japanese quail

Many avian species reduce their body temperature (T _b) to conserve energy during periods of inactivity, and we recently characterized how ambient temperature (T _a) and nutritional stress interact with one another to influence physiologically controlled hypothermic responses in Japanese quail (Coturnix japonica). In the present study, we examined how the fatty acid (FA) composition of the diet influences the FA composition of phospholipids in major organs and how these affect controlled hypothermic responses and metabolic rates in fasted birds. For 5 weeks prior to fasting, quail were fed a standard diet and gavaged each morning with 0.7 ml of water (control), or a vegetable oil comprising saturated fatty acids (SFA; coconut oil), or unsaturated fatty acids (UFA; canola oil). Birds were then fasted for 4 days at a T _a of 15°C. We found that, while fasting, both photophase and scotophase T _b decreased significantly more in the SFA treatment group than in the control group; apparently the former down-regulated their T _b set point. This deeper hypothermic response was correlated with changes in the phospholipid composition of the skeletal muscle and liver, which contained significantly more oleic acid (18:1) and less arachidonic acid (20:4), respectively. Our data imply that these two FAs may be associated with thermoregulation.     

Self-regulation of membrane fluidity. The effect of saturated normal and methoxy fatty acid supplementation on Tetrahymena membrane physical properties and lipid composition.

Tetrahymena cells elongated and desaturated massive supplements of palmitic or lauric acid at nearly twice the rates employed by unfed cells, thereby maintaining constant the physical properties of their membrane lipids. However, when a mixture of the 9- and 10-monomethoxy derivatives of stearic acid was administered, these compounds were incorporated without further metabolism. The marked fluidizing effect of the phospholipid-bound methoxy-fatty acids elicited an immediate reduction in fatty acid desaturase activity, the pattern of change being very similar to that induced by supplements of polyunsaturated fatty acids. The modulation of fatty acid desaturase activity by methoxy-acids clearly seems to be governed by membrane fluidity rather than by some form of end product inhibition of the type which might have been postulated to explain the similar effect caused by polyunsaturated fatty acids.     

Changes in lipid fluidity and fatty acid composition with altered culture temperature in Tetrahymena pyriformis-NT1

Cultures of T. pyriformis-NT1 were grown at 20 degrees C (Tg 20 degrees C) and 38 degrees C (Tg 38 degrees C). G.L.C. analysis and D.P.H. fluorescence polarization measurements in extracted phospholipids indicated that there was increased saturation of fatty acids and relatively reduced fluidity as growth temperature was increased. Breakpoints occurred in the Arrhenius plots of fluorescence polarization at 16 degrees C for Tg 38 degrees C total extracted phospholipids and 9 degrees C for Tg 20 degrees C lipids.     

Alteration of the fatty acid composition of Escherichia coli by growth in the presence of normal alcohols.

The addition of normal alcohols in the series n-butanol to n-octanol to cultures of Escherichia coli ML308 grown on defined or lipid-free medium (at 17, 27, and 37 degrees C) caused an alteration in the fatty acid composition of this organism: the ratio of saturated to unsaturated fatty acids increased. Changes in the relative quantities of individual fatty acid species elicited by increasing concentrations of these alcohols were as follows: (i) myristic acid remained constant: (ii) palmitic acid increased; and (iii) the combined amount of palmitoleic plus cis-methylene hexadecanoic acids changed in a way which was reflected inversely by changes in the amount of cis-vaccenic acid. Comparable changes were not observed when cells were grown in the presence of n-nonanol and n-decanol in the concentration range tested. The changes observed upon addition of normal alcohols (n-butanol to n-octanol) paralleled, in part, the alterations in fatty acid composition observed when growth temperature was increased.     

Modification of fatty acid composition of lipid accumulating yeasts with cyclopropene fatty acid desaturase inhibitors

Summary The effect of cyclopropene fatty acids, sterculic and malvalic, on the lipids of yeasts grown under nitrogen limiting, lipid accumulating, conditions was studied. The ratio of stearic to oleic acid showed a dose response effect, with an increase in stearic acid content as the dose of cyclopropene fatty acid increased, and a corresponding reduction in oleic acid. Linoleic and linolenic acids were not affected to the same extent. These effects are shown for the yeasts Candida sp. 107, Trichosporon cutaneum, and Rhodosporidium toruloides.     

Effects of ambient temperature on lipid and fatty acid composition in the oviparous lizards, Phrynocephalus przewalskii

This study was designed to assess the effect of ambient temperature on lipid content, lipid classes and fatty acid compositions of heart, liver, muscle and brain in oviparous lizards, Phrynocephalus przewalskii, caught in the desert area of China. Significant differences could be observed in the contents of the total lipid and fatty acid compositions among different temperatures (4, 25 and 38 degrees C). The study showed that liver and muscle were principal sites of lipid storage. Triacylglycerol (TAG) mainly deposited in the liver, while phospholipids (PL) was identified as the predominant lipid class in the muscle and brain. Palmitic and stearic acid generally occupied the higher proportion in saturated fatty acids (SFA), while monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) consisted mainly of 16:1n-7, 18:1n-9, 18:2n-6, 18:3n-3, 20:4n-6 and 22:6n-3 regardless of tissue and temperature. These predominant fatty acids proportion fluctuations caused by temperature affected directly the ratio of unsaturated to saturated fatty acids. There was a tendency to increase the degree of unsaturation in the fatty acids of TAG and PL as environmental temperature dropped from 38 to 4 degrees C, although the different extent in different tissues. These results suggested that lipid characteristics of P. przewalskii tissues examined were influenced by ambient temperature.     

Determination of fatty acid content and composition in ultramicro lipid samples by gas-liquid chromatography

Simplified quantitative manipulations of very small amounts (30 micrograms) of lipids for determination of fatty acid content and composition by gas-liquid chromatography after (a) methanolysis (b) reduction and acetylation are described.     

Temperature-dependent lipid content and fatty acid composition of three thermophilic bacteria

The lipid content and fatty acid composition of a strain ofBacillus caldolyticus and of two facultative thermophiles (B. flavothermus and strain NZ-2) were analysed after growth at different temperatures. In all three strains the amount of membrane, as a fraction of total cellular dry mass, was found to increase with temperature, however, in varying degrees. Changes of lipid content and protein/lipid ratio inB. caldolyticus between 60°C and 100°C and in strain NZ-2 between 45°C and 70°C were minor; inB. f avothermus the alterations in the 50°C–70°C range were more pronounced. The same was found for changes observed in the phospholipid/total lipid and phospholipid/membrane ratios, and also in the amounts of individual phospholipids. The alterations of the fatty acid composition were most significant inB. caldolyticus, especially between 80°C and 95°C. In contrast, the main changes inB. flavothermus and NZ-2 were found to occur between 30°C and 50°C, and between 45°C and 60°C, respectively. Based on these data, strain NZ-2 could be characterized as the least andB. flavothermus as the most versatile of the three organisms.     

Specific phospholipid fatty acid composition of brain regions in mice. Effects of n-3 polyunsaturated fatty acid deficiency and phospholipid supplementation

This study examined the effects of dietary alpha-linolenic acid deficiency followed or not by supplementation with phospholipids rich in n;-3 polyunsaturated fatty acid (PUFA) on the fatty acid composition of total phospholipids in 11 brain regions. Three weeks before mating, mice were fed a semisynthetic diet containing both linoleic and alpha-linolenic acid or deficient in alpha-linolenic acid. Pups were fed the same diet as their dams. At the age of 7 weeks, a part of the deficient group were supplemented with n;-3 polyunsaturated fatty acids (PUFA) from either egg yolk or pig brain phospholipids for 2 months. Saturated and monounsaturated fatty acid levels varied among brain regions and were not significantly affected by the diet. In control mice, the level of 22:6 n-3 was significantly higher in the frontal cortex compared to all regions. alpha-Linolenic acid deficiency decreased the level of 22:6 n-3 and was compensated by an increase in 22:5 n-6 in all regions. However, the brain regions were affected differently. After the pituitary gland, the frontal cortex, and the striatum were the most markedly affected with 40% reduction of 22:6 n-3. Supplementation with egg yolk or cerebral phospholipids in deficient mice restored a normal fatty acid composition in brain regions except for the frontal cortex. There was a regional distribution of the fatty acids in the brain and the impact of deficiency in alpha-linolenic acid was region-specific. Dietary egg yolk or cerebral phospholipids are an effective source of n-3 PUFA for the recovery of altered fatty acid composition induced by a diet deficient in n-3 PUFA.     

Alterations of characteristic temperatures for lectin interactions in LM cells with altered lipid composition

Alteration of the fatty acid composition of mouse LM cell lipids dramatically affected the concanavalin A binding and concanavalin A-mediated hemadsorption properties of these cells. A critical temperature for these two concanavalin A related phenomena observed at 15–19° in cells with unaltered fatty acid composition was shifted to 22–28° for cells containing a higher proportion of saturated fatty acids and lowered to 7–11° for cells containing polyunsaturated fatty acids substituted for monoenoic unsaturated fatty acids. In contrast, a second critical temperature (at 5–7°) observed for concanavalin A binding and concanavalin A-mediated hemadsorption to LM cells was essentially unchanged by alterations in cellular lipid fatty acid composition. We conclude that a change in membrane lipid freezing point is responsible for the higher critical temperature (15–19°), and factors other than lipid melting properties, perhaps cytoskeleton structure, contribute to the lower critical temperature (5–7°) for lectin interactions with the exposed surface of LM cells.     

Alteration of seed fatty acid composition by an ethyl methanesulfonate-induced mutation in Arabidopsis thaliana affecting diacylglycerol acyltransferase activity.

In characterizing the enzymes involved in the formation of very long-chain fatty acids (VLCFAs) in the Brassicaceae, we have generated a series of mutants of Arabidopsis thaliana that have reduced VLCFA content. Here we report the characterization of a seed lipid mutant, AS11, which, in comparison to wild type (WT), has reduced levels of 20:1 and 18:1 and accumulates 18:3 as the major fatty acid in triacylglycerols. Proportions of 18:2 remain similar to WT. Genetic analyses indicate that the fatty acid phenotype is caused by a semidominant mutation in a single nuclear gene, designated TAG1, located on chromosome 2. Biochemical analyses have shown that the AS11 phenotype is not due to a deficiency in the capacity to elongate 18:1 or to an increase in the relative delta 15 or delta 12 desaturase activities. Indeed, the ratio of desaturase/elongase activities measured in vitro is virtually identical in developing WT and AS11 seed homogenates. Rather, the fatty acid phenotype of AS11 is the result of reduced diacylglycerol acyltransferase activity throughout development, such that triacylglycerol biosynthesis is reduced. This leads to a reduction in 20:1 biosynthesis during seed development, leaving more 18:1 available for desaturation. Thus, we have demonstrated that changes to triacylglycerol biosynthesis can result in dramatic changes in fatty acid composition and, in particular, in the accumulation of VLCFAs in seed storage lipids.