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The effects of a low temperature (13 degrees C) treatment known to provide protection against sulphur dioxide (SO2) injury were assessed on leaf lipid composition in two cultivars of Coleus blumei Benth, found previously to differ in sensitivity to SO2 and other environmental stresses. After 5 days growth at 13 degrees C, there were significant differences in membrane lipid fatty acid composition as well as in free fatty acid (FFA) levels between SO2-sensitive 'Buckley Supreme' ('BS') and SO2-insensitive 'Marty' ('M'). Molecular species of chloroplast galactolipids in 'M' contained increased levels of linolenic acid (18:3). In the leaf FFA pools, the saturated components, palmitic (16:0) and stearic (18:0) acids, were predominant at 20 degrees C. After temperature hardening at 13 degrees C, the total amount of FFAs decreased in 'M' but increased in 'BS.' These modifications in lipid composition suggest an additional mechanism for cultivar differences in tolerance to SO2 and other stressors in coleus.

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Techniques are described for the semi-preparative isolation of sulfoquinovosyldiacylglycerol from plant leaf tissue lipid extracts and the resolution and analysis of component molecular species. Sulfoquinovosyldiacylglycerol was resolved from phospholipids in a polar lipid fraction by isocratic normal phase HPLC with detection at 208 nm. The mobile phase was composed of heptane-isopropanol-0.001 M KCl 40:52:8 (v/v/v). Yields from spinach leaf lipid extracts were 1.8 mg.10 g-1 fresh wt leaf tissue. Molecular species components of purified sulfoquinovosyldiacylglycerol were separated by reversed-phase C18 HPLC, and fatty acid positional distribution was defined.

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A method is described for the determination of glutathione reductase activity (GR; EC 1.6.4.2) in plant extracts utilizing HPLC quantitation of NADP+ following the reduction of glutathione disulfides. After protein incubation, fluorescence of NADP+ was induced under strongly basic conditions, and the product was directly resolved from the reaction medium by isocratic reversed-phase elution on a silica-coated alumina support which took 2 min. The mobile phase was acetonitrile-water (50:50) delivered at a flow rate of 1.5 ml/min. The adduct (stable for at least 7 days) was detected fluorometrically and quantitated by direct integration of peak area.

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Although glycyrrhizic acid, a major constituent of licorice root, has important pharmacological effects in humans, the biological activity of glycyrrhizic acid and its aglycone glycyrrhetinic acid in plants is unknown. Here we report that these licorice-derived compounds and the analog carbenoxolone inhibit desaturation of linoleic acid (C18:2 omega 6) in soybean chloroplasts using monogalactosyldiacylglycerol and phosphatidylcholine substrates in an in vitro assay for desaturase activity. At 10 nM glycyrrhetinic acid, there is significant inhibition of desaturation of linoleic acid suggesting that licorice-derived compounds could prove useful in investigating biochemical pathways of linoleic acid desaturation in plant chloroplasts and plant desaturase regulation, which has application in modification of plant response to environmental stress, as well as optimization of oil seed composition.

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omega-3 fatty acids, alpha-tocopherol, ascorbic acid, beta-carotene and glutathione determined in leaves of purslane (Portulaca oleracea), grown in both a controlled growth chamber and in the wild, were compared in composition to spinach. Leaves from both samples of purslane contained higher amounts of alpha-linolenic acid (18:3w3) than did leaves of spinach. Chamber-grown purslane contained the highest amount of 18:3w3. Samples from the two kinds of purslane contained higher leaves of alpha-tocopherol, ascorbic acid and glutathione than did spinach. Chamber-grown purslane was richer in all three and the amount of alpha-tocopherol was seven times higher than that found in spinach, whereas spinach was slightly higher in beta-carotene. One hundred grams of fresh purslane leaves (one serving) contain about 300-400 mg of 18:3w3; 12.2 mg of alpha-tocopherol; 26.6 mg of ascorbic acid; 1.9 mg of beta-carotene; and 14.8 mg of glutathione. We confirm that purslane is a nutritious food rich in omega-3 fatty acids and antioxidants.

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A general method for identification of fatty acids covalently bound to acylated proteins following their electrophoretic transfer onto nitrocellulose paper is described. As demonstrated for [3H]palmitoylated RAS1 protein of Saccharomyces cerevisiae and the acylated acyl carrier protein of Spirodela oligorrhiza, this procedure alleviates the need for elution of proteins from polyacrylamide gel slices. Fatty acid ligands of such proteins are hydrolyzed directly from their immobilized state on the nitrocellulose paper, then derivatized with p-nitrophenacyl bromide, and finally resolved by reversed-phase high-performance liquid chromatography. The amount of acylated protein required for identification of acyl groups is minimized compared to that required for more conventional approaches by coupling a radioactive flow detector with the HPLC system.

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We have examined the effects of the substituted pyridazinone herbicide, 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)pyridazinone (BASF 13-338, Sandoz 9785), on the desaturation of linoleic acid (18:2) on different molecular species of monogalactosyldiacylglycerol (MGDG) and phosphatidylcholine (PC) in leaf tissue of Arabidopsis thaliana (L.) Heynh. Specific changes in lipid composition allowed identification of different substrates for desaturation of 18:2 to linolenic acid (18:3). 18:2/16:2 MGDG was desaturated in the chloroplast to form 18:3/16:3 MGDG. Levels of 18:3/16:3 MGDG were reduced by treatment with BASF 13-338, suggesting that both the formation of 18:3 at the sn-1 position, and the formation of 16:3 at the sn-2 position of 18:2/16:2 MGDG were inhibited by this compound. Kinetic studies using exogenously incorporated [(14)C] 18:1 indicated that 18:2/18:3 MGDG originated from an 18:2/18:3 diglyceride precursor derived from PC. The formation of 18:3 at the sn-1 position of 18:2/18:3 MGDG was also inhibited by BASF 13-338. In contrast the desaturation of 18:2 proposed to occur at the sn-2 position of PC outside the chloroplast, was not affected.

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A method is described for the direct quantitation of phosphatidylcholine molecular species by reverse phase high-performance liquid chromatography employing flame ionization detection. The method is shown to be applicable to plan phosphatidylcholine. The molecular species are separated with a C18 column eluted in an isocratic mode. Detection by a commercially available flame ionization detector overcomes the problems of detecting underivatized naturally occurring lipids using ultraviolet detectors, and allows direct and rapid mass determination of the resolved molecular species. Detection limits for quantitation are defined.

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Synthesis of unsaturated monogalactosyldiacylglycerol (MGDG) was examined in a mutant of Arabidopsis thaliana (L.) Heynh. containing reduced levels of hexadecatrienoic (16:3) and linolenic (18:3) acids in leaf lipids. Molecular species composition and labeling kinetics following the incorporation of exogenous [(14)C]fatty acids suggest that at least two pathways and multiple substrates are involved in desaturation of linoleic acid (18:2) to 18:3 for production of unsaturated galactolipids. A reduction in 18:3/16:3 MGDG and an increase in 18:2/16:2 MGDG, together with labeling kinetics of these molecular species following the incorporation of exogenous [(14)C]12:0 fatty acids, suggests that a chloroplastic pathway for production of 18:3 at the sn-1 position of MGDG utilizes 18:2/16:2 MGDG as a substrate. This chloroplastic (prokaryotic) pathway is deficient in the mutant. When exogenous [(14)C]18:1 was supplied, a eukaryotic (cytoplasmic) pathway involving the desaturation of 18:2 to 18:3 on phosphatidylcholine serves as the source of 18:3 for the sn-2 position of MGDG. This eucaryotic pathway predominates in the mutant.

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Conditions are described for the quantitative analysis of phosphatidylglycerol and plant glycolipid molecular species by reversed-phase high-performance liquid chromatography employing a commercially available flame ionization detector. Direct detection on a mass basis overcomes the problem of poor detectability found with most natural lipids. Effective mobile phases composed primarily of volatile solvents are described. Splitting of the column eluate stream allows a portion of each individual molecular species to be recovered for other types of analysis.

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Two-minute exposures to exogenous [14C]palmitic, [14C]oleic, or [14C]lauric acid differentially labeled the lipids of Dunaliella salina microsomes and chloroplasts. Changes in fatty acid desaturation and intracellular movement during a subsequent 16-h incubation in nonradioactive medium indicated a slow transfer of lipids into the chloroplast from other organelles. Since Dunaliella lacks the massive traffic of microsomally produced glycerolipids into chloroplast galactolipids that dominates chloroplast-microsome lipid relations in most plant cells, it affords a sensitive system for studying more subtle intracellular lipid fluxes. Lowering the culture temperature from 30 to 12 degrees C was more inhibitory toward glycerolipid biosynthesis in chloroplasts than in microsomes. The ability of Dunaliella chloroplasts to utilize microsomal lipids may be essential for their systematic acclimation to low temperature.

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A detailed analysis was made of individual phosphatidylglycerol (PG) molecular species isolated from microsomes and chloroplasts at various times after labeling Dunaliella salina cells with [14C]palmitic, [14C]oleic, or [14C]lauric acid. The patterns of [14C]fatty acid incorporation were in agreement with PG being formed by the "eucaryotic" type pathway in microsomes and the "procaryotic" type pathway in chloroplasts. In Dunaliella, which lacks a quantitatively significant flux of eucaryotic-type lipids from microsomes into chloroplast glycolipids, indications were found for a more subtle movement of microsomally synthesized PG into the chloroplasts. This transfer was more evident in cells stressed by exposure to 12 degrees C than it was at 30 degrees C, and may afford a mechanism for recruiting key microsomal PG molecular species toward low-temperature acclimation in chloroplasts.

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A method for isolating viable protoplasts in high yield from the aleurone layers of developing wheat grains is described, and the techniques for their subsequent culture outlined. Protoplasts from untreated tissue do not produce α-amylase in response to gibberellic acid (GA3) if the incubation temperature is left at 25°C. However, pre-treatment of the protoplast preparation at temperatures above 27°C for at least 8 h followed by a short incubation at 25°C induces sensitivity to the growth regulator such that α-amylase is produced. The requirements of the sensitisation process are similar to those for intact aleurone tissue although additional adjustment to the calcium ion is beneficial. Pre-treatment of aleurone layers with the sensitising temperature regimes prior to protoplast isolation have the advantage of increasing protoplast viability. Once sensitised, the protoplasts respond to a GA3 concentration as low as 10(-11) mol dm(-3) with a maximal response at 10(-9) mol dm(-3). The successful isolation of wheat aleurone protoplasts whose sensitivity to GA3 can be manipulated represents a useful step towards investigating the role of cell membranes in growth-regulator action.

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Aleurone layers from immature developing wheat grains (Triticum aestivum L. cvs. Sappo. and Champlein), though normally insensitive, can be made to produce α-amylase in response to gibberellic acid by subjecting the grains to a period of enforced dehydration prior to introduction to the hormone. The change in sensitivity appears to depend upon the water content of the tissue, water levels of below approximately 25% being critical for the effect. Grain detachment or duration of drying apparently do not qualitatively influence the development of sensitivity to gibberellic acid. Enhanced sensitivity resulting from drying is not caused by changes in gibberellic acid uptake. A possible mechanism for the change in sensitivity of aleurone cells might be through structural alterations in cell membranes.

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Aleurone tissue from undried immature developing wheat grains (Triticum aestivum L. cv. Sappo), normally insensitive to gibberellic acid, can be made to respond to the hormone by a series of temperature treatments. Incubation of the de-embryoed grains at temperatures above 27° C for at least 8 h causes the tissue to become sensitive. Prolonged incubation at temperatures below 27° C does not effect a change in sensitivity. In addition to the requirement for exposure to an elevated temperature for a period of several hours the tissue must also subsequently be subjected to a period at a lower temperature for just a few seconds for the response to be observed. Once sensitized, the tissue remains responsive to gibberellic acid for substantial periods of time. Exposure of the tissue to temperatures which induce sensitivity to gibberellic acid also results in an increased leakage of amino acids. It is suggested that the increase in sensitivity to gibberellin requires two separate processes to take place. One could be a homeoviscous adaptation of the cell membranes in response to elevated temperature, the other a subsequent, permanent change in conformation of membrane components.