Influence of a subinhibitory dose of antifungal fatty acids from Sporothrix flocculosa on cellular lipid composition in fungi.

Antifungal fatty acids produced by the biocontrol fungus Sporothrix flocculosa were studied on the basis of their effect on growth and cellular lipid composition of three fungi, Cladosporium cucumerinum, Fusarium oxysporum, and S. flocculosa, whose growth was decreased by 51, 33, and 5%, respectively, when exposed to 0.4 mg fatty acid per ml. The sensitivity to fatty acid antibiotics from S. flocculosa was related to a high degree of unsaturation of phospholipid fatty acids and a low proportion of sterols. The major responses of sensitive fungi to sublethal doses of antifungal fatty acids from liquid culture of S. flocculosa were: (i) a decrease in total lipid; (ii) an increase in the degree of fatty acid unsaturation (18:1 > 18:2 > 18:3); (iii) an increase in free fatty acids and phosphatidic acid and a decrease in total phospholipids; and (iv) an increase in sterol/phospholipid ratio. These modifications in lipid composition led to an increase in membrane fluidity in sensitive fungi as demonstrated by assessment of fluoresence anisotropy using liposomes and 1,6-diphenyl-1,3,5-hexatriene probe. This alteration in the physical state of lipids appears to be responsible for the previously demonstrated alteration of membrane structure and function in fungi confronted to S. flocculosa.

Chilling Injury Induces Lipid Phase Changes in Membranes of Tomato Fruit.

Wide-angle x-ray diffraction has provided evidence for lipid phase separations in microsomal membranes from chill-injured tomato (Lycopersicon esculentum Mill. cv Caruso) fruit. Mature-green fruit stored for 20 d at 5[deg]C had not begun to ripen and were essentially free of chilling injury symptoms. Within 4 d of being returned to 25[deg]C, however, the fruit displayed characteristic symptoms of chilling injury, including translucent water-soaked patches, surface pitting, and irregular pigmentation. Membrane damage measured as electrolyte leakage from pericarp discs intensified after the fruit were returned to ambient temperature. Wide-angle x-ray diffraction patterns recorded at 25[deg]C for microsomal membranes isolated from untreated, mature-green fruit indicated that the membrane bilayers were exclusively liquid-crystalline. Diffraction patterns for microsomal membranes from fruit stored for 20 d at 5[deg]C showed only trace amounts of gel phase lipid, but within 4 d of subsequent exposure of the fruit to ambient temperature, there was evidence for a pronounced lateral phase separation of lipids within the membranes that would render them leaky. Inas-much as the phase separations were detectable at 25[deg]C and became pronounced only subsequent to the chilling episode, they appear to be an indirect rather than direct effect of exposure to low temperature. The diffraction data thus support the notion that the lipid phase changes observed here are not directly induced by low temperature but rather reflect subsequent biochemical changes in the bilayers that may contribute to the development of chilling symptoms.

Modification of Phospholipid Catabolism in Microsomal Membranes of [gamma]-Irradiated Cauliflower (Brassica oleracea L.).

Acceleration of membrane deterioration has been observed recently during storage of [gamma]-irradiated cauliflower (Brassica oleracea L., Botrytis group). In the present study, the activity of microsome-associated lipolytic enzymes was investigated in cauliflower florets exposed to 0 or 4 kilograys of [gamma] radiation and stored for 8 d at 13[deg]C. Radiolabeled breakdown products obtained from the metabolism of (16:0/18:2*)-phosphatidylcholine and (16:0/16:0)-phosphatidyl-[N-methyl-3H]choline by microsomal membranes indicated that phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase were associated with the membranes. The rate of phosphatidylcholine catabolism by the membranes increased slowly in control cauliflower during storage. [gamma] irradiation caused an immediate rise in phosphatidylcholine catabolism that remained higher than that of the controls during subsequent storage. Collectively, the data suggest that enhancement of membrane lipolytic activity results from free-radical-induced stress. Rapid increase of the membrane-associated phospholipase D activity may be a key event leading to accelerated membrane deterioration following [gamma] irradiation.

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 CaCl(2) solutions at 15 degrees 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.

Induction of Senescence-Like Deterioration of Microsomal Membranes from Cauliflower by Free Radicals Generated during Gamma Irradiation.

Membrane deterioration differs in aging and senescent tissues. Involvement of free radicals in the process is generally recognized. Little is known about the physiological effects of gamma irradiation on plant tissues. Degradation of microsomal membranes by the action of free radicals, generated in vivo by gamma rays, was investigated. Cauliflower florets (Brassica oleracea L., Botrytis group) were exposed to 2 or 4 kiloGray of gamma radiation. Membrane deterioration was assessed during 8-day storage at 13 degrees C. Some senescence was indicated in nonirradiated controls by a parallel depletion of lipid phosphate and protein. Irradiation caused an immediate increase in tissue electrolyte leakage and a small increase in the free fatty acid content of membranes. In irradiated samples, leakage of electrolytes and the ratios of sterol to phospholipid and of free fatty acid to phospholipid increased with storage. During this period, membrane protein was progressively lost and the lipid phosphate-to-protein ratio increased markedly. Polyunsaturated fatty acids were selectively depleted from the free fatty acid fraction for all treatments, suggesting lipoxygenase activity. No change in lipid saturation was observed in the polar lipid fraction. The results suggest an enzyme-catalyzed senescence-like membrane deterioration, probably induced by chemical deesterification of phospholipids by free radicals generated during irradiation.

Lipid content and cryotolerance of bakers' yeast in frozen doughs.

The relationship between lipid content and tolerance to freezing at -50 degrees C was studied in Saccharomyces cerevisiae grown under batch or fed-batch mode and various aeration and temperature conditions. A higher free-sterol-to-phospholipid ratio as well as higher free sterol and phospholipid contents correlated with the superior cryoresistance in dough or in water of the fed-batch-grown compared with the batch-grown cells. For both growth modes, the presence of excess dissolved oxygen in the culture medium greatly improved yeast cryoresistance and trehalose content (P. Gélinas, G. Fiset, A. LeDuy, and J. Goulet, Appl. Environ. Microbiol. 26:2453-2459, 1989) without significantly changing the lipid profile. Under the batch or fed-batch modes, no correlation was found between the cryotolerance of bakers' yeast and the total cellular lipid content, the total sterol content, the phospholipid unsaturation index, the phosphate or crude protein content, or the yeast cell morphology (volume and roundness).

Oleate desaturation in young winter wheat root tissue.

[1,2-(14)C]Acetate was incorporated into the lipids of young wheat (Triticum aestivum L. cv Kharkov 22 MC) root tissue, but predominantly into sterols. [1-(14)C]Ammonium oleate was initially incorporated mainly into phosphatidylcholine (PC), and later into triglycerides (TGs). Diglycerides (DGs) contained 16% of the lipid (14)C after 5 minutes and 8% after 40 minutes. The proportion of the label of each lipid group incorporated into linoleate during an 80-minute incubation increased at similar rates for each group, and was always highest in PC. Radioactivity was detected in PC-linoleate earlier than in linoleate of the other groups. During a prolonged incubation after a 15-minute pulse labeling, the percentage of the lipid (14)C incorporated into PC and DGs was high at the end of the pulse but decreased later, while that in TGs increased to 64% after 4 hours. The proportion of the label of each group recovered in linoleic acid peaked in all groups after 4 hours, except for the TGs where it increased slowly throughout the experiment. Only traces of radioactivity were detected in linolenate. The data are compatible with a pathway in which oleate is incorporated into PC, is desaturated to linoleate on PC, and where the linoleate-enriched DGs are transferred from PC to TGs.

Effect of BASF 13-338, a Substituted Pyridazinone, on Lipid Metabolism in Leaf Tissue of Spinach, Pea, Linseed, and Wheat.

A substituted pyridazinone (BASF 13-338) inhibited photosynthesis in spinach (Spinacia oleracea, Hybrid 102 Arthur Yates Ltd.) leaf discs and reduced the incorporation of [1-(14)C]acetate into trienoic acids of diacylgalactosylglycerol while causing radioactivity to accumulate in diacylgalac-tosylglycerol dienoic acids. Although BASF 13-338 inhibited photosynthesis in isolated spinach chloroplasts, it did not prevent dienoate desaturation. In discs, the labeling of fatty acids was affected by the inhibitor only in diacylgalactosylglycerol. Very little radioactivity was incorporated into trienes of phosphatidylcholine and the proportion of the label recovered in the fatty acids of phosphatidylcholine was not changed by BASF 13-338. The herbicides caused an increase in the proportion of the lipid (14)C incorporated into diacylgalactosylglycerol and a decrease in labeling of phosphatidylcholine, whereas the proportion of (14)C recovered in other lipids remained unchanged. Similar results were obtained with pea (Pisum sativum cv. Victory Freeze), linseed (Linum usitatissimum cv. Punjab), and wheat (Triticum aestivum cv. Karamu). With these species, a greater proportion of the label was incorporated into phosphatidylcholine and less into diacylgalactosylglycerol than with spinach. The data indicate that trienoate synthesis uses diacylgalactosylglycerol as substrate. BASF 13-338 appears to act at that step, and seems to cause in spinach a shift in polyenoate synthesis from the pathway involving microsomal phosphatidylcholine to the pathway operating inside the chloroplast.

Simultaneous Inhibition of Linolenic Acid Synthesis in Winter Wheat Roots and Frost Hardening by BASF 13-338, a Derivative of Pyridazinone.

Treatment of 12-day-old winter wheat (Triticum aestivum) plants with BASF 13-338 {4-chloro-5 (dimethylamino)-2-phenyl-3(2H)-pyridazinone} 36 hours before frost hardening simultaneously and completely inhibits accumulation of linolenic acid in the roots during the hardening period and the acquisition of frost resistance. Increased unsaturation of fatty acids is therefore probably an important part of the mechanism of cold adaptation in winter wheat.BASF 13-338 also prevents the increase in per cent dry weight in roots and shoots during hardening and causes a decrease in root lipid phosphorus and total fatty acids.The concurrent increase in linoleic acid and decrease in linolenic acid in the treated plants, while the level of the other fatty acids is but little affected, suggest that BASF 13-338 specifically inhibits linoleic acid desaturase.

Sodium-1,2-C Acetate Incorporation in Roots of Frost-hardy and Less Hardy Alfalfa Varieties under Hardening Conditions.

When the temperature of incorporation of sodium acetate-1, 2-(14)C into lipids of alfalfa (Medicago media Pers. var. Rambler and Medicago sativa L. var. Caliverde) roots was lowered from 22 C to 1 C, elongation and desaturation of fatty acids and the labeling of phosphatidylcholine were strongly stimulated.Controlled hardening of alfalfa stimulated strongly the incorporation of sodium acetate-1, 2-(14)C into root lipids of the hardy variety Rambler, but only slightly in the case of the frost-sensitive variety Caliverde. When incorporation was done at 1 C at various times of hardening, labeling decreased significantly in linoleic acid with a corresponding increase in oleic acid. Hardening, therefore, repressed specifically the initial low temperature stimulation of oleic acid desaturation, without affecting the stimulation of elongation of palmitic acid and the desaturation of stearic acid at low temperature. The radioactivity in linoleic acid was slightly greater in hardy Rambler than in Caliverde throughout hardening.When feedings were done at 1 C at various times of hardening, labeling of phosphatidylcholine increased in Rambler while it decreased in Caliverde. Throughout the hardening period, when incorporation was done at 1 C, linoleic acid represented a higher percentage of the label in phosphatidylcholine than in phosphatidylethanolamine or triglycerides and its specific radioactivity was much greater in phosphatidylcholine than in phosphatidylethanolamine and triglycerides and in Rambler than in Caliverde. Phosphatidylcholine seems, therefore, to play a special part in linoleic acid synthesis and in its control during the acquisition of frost hardiness.

Stimulation of Phospholipid Biosynthesis during Frost Hardening of Winter Wheat.

Lipids were labeled with (33)P during frost hardening of two varieties of winter wheat (Triticum aestivum), hardy Kharkov and much less hardy Champlein. The main labeled compounds were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylglycerol. With time of incorporation the proportion of the radioactivity incorporated into the lipids increased in phosphatidylcholine, especially in Kharkov and at 1 C. During hardening, phospholipid synthesis was greatly stimulated in Kharkov, but much less in Champlein. The proportion of the phospholipids synthesized changed only little with hardening, with a trend towards an increase in phosphatidylcholine. Increased phospholipid synthesis does not seem to be a prerequisite to hardening in winter wheat. However, a high rate of phospholipid synthesis may be required to maintain frost resistance.

Fat metabolism in higher plants. XXXIV. Development of fatty acid synthetase as a function of protein synthesis in aging potato tuber slices.

Experiments with inhibitors of protein synthesis (actinomycin D, puromycin, actidione) showed that the increase and the change in fatty acid synthetase activity, observed during the aging of potato disks, were accompanied by and related to a temporary rise in the rate of protein and RNA synthesis. These results concur with the earlier suggestion by Click and Hackett that the aging process involves a type of derepression. A possible course of events during aging, and possible derepression mechanisms are suggested and discussed.