Effects of in vitro treatment with ozone on the physical and chemical properties of membranes

125

Fluorescent products of lipid peroxidation accumulate with age in microsomal membranes from senescing cotyledons of Phaseolus wulgaris.The temporal pattern of accumulation is closely correlated with a rise in the lipid phase transition temperature reflecting the formation of gel phase lipid. Increased levels of fluorescent peroxidation products are also detectable in total lipid extracts of senescent cotyledons. Lipoxygenase activity increases with advancing age by about 3-fold on a fresh weight basis and 4-fold on a dry weight basis indicating that the tissue acquires elevated levels of lipid hydroperoxides. As well, levels of glutathione and superoxide dismutase activity decline on a dry weight basis as the cotyledons age, rendering the tissue more susceptible to oxidative damage. Catalase activity rises initially and then declines during senescence, but peroxidase activity rises steeply. Thus, apart from this increase in peroxidase, which would scavenge H202 only if appropriate cosubstrates were available, the defense mechanisms for coping with activated oxygen species (02', H202, OH') are less effective in the older tissue. The observations support the contention that formation of gel phase lipid in senescing membranes is attributable to lipid peroxidation and suggest that the reactions of lipid peroxidation are utilized by the cotyledons to mediate deteriorative changes accompanying the mobilization and transport of metabolites from the storage tissue to the developing embryo.Disruption of cell membrane integrity is an inherent feature of senescence in plants. This is evident from ultrastructural studies showing progressive deteriorative changes in the organelles and membranes of plant tissues and also from permeability studies indicating increased leakage ofsolutes (25). Large changes in the physical properties of membrane lipids accompanying senescence appear to contribute to this loss of selective permeability. In particular, increasing proportions of the membrane lipid assume the gel phase, resulting in a mixture of discrete liquid-crystalline and gel phase lipid domains in the membrane matrix (18)(19)(20). For microsomal membranes from senescing bean cotyledons, the lipid phase transition temperature increases by more than 50°C between days 2 and 9 after germination under etiolating conditions (18). By day 4 after germination, these membranes contain a mixture of gel and liquid crystalline lipid phases at the growth temperature, and this coexistence of lipid phases gives rise to discontinuities in the bilayer that result in greatly increased permeability (1 (27), and increased levels of oxygenated fatty acids have been observed in aged seeds of Cichorium and Crepis (26). Fluorescent products of lipid peroxidation have been reported in pear and banana pulp as well as in a mitochondrial fraction isolated from banana, and the levels of these pigments appear to increase with natural or ethylene-induced ripening (17). Senescing leaves also accumulate fluorescent pigments (29), and Dhindsa et al. (5...

mentioning

confidence: 99%

Evidence for the Accumulation of Peroxidized Lipids in Membranes of Senescing Cotyledons

Pauls¹,

Thompson²

1984

125

“…PAN reacts with double bonds leading to the production of oxidation products (7). Indeed, Pauls and Thompson (19,20) have reported that chemical and physical changes in membrane lipids such as the breakdown of phospholipids, the loss of sterols from the membrane, and the formation of gel phase lipid membrane were induced by treating the isolated microsomal membrane from bean leaves with ozone in solution. However, the results obtained from isolated systems have not been confirmed in plant tissues.…”

mentioning

confidence: 99%

Effects of Ozone and Peroxyacetyl Nitrate on Polar Lipids and Fatty Acids in Leaves of Morning Glory and Kidney Bean

Nouchi

Toyama²

1988

To compare the effects of ozone and peroxyacetyl nitrate (PAN) on leaf lipids, fatty acids and malondialdehyde (MDA), morning glory (Pharbitis nil Choisy cv Scarlet O'Hara) and kidney bean (Phaseolus vulgaris L. cv Gintebo) plants were exposed to either ozone (0.15 microliter per liter for 8 hours) or PAN (0.10 microliter per liter for up to 8 hours). Ozone increased phospholipids in morning glory and decreased in kidney bean at the initial stage (2-4 hours) of exposure, while it scarcely changed glycolipids, the unsaturated fatty acids, and MDA in both plants. A large reduction of glycolipids occurred 1 day after ozone exposure in both plants. PAN reacts with double bonds leading to the production of oxidation products (7). Indeed, Pauls and Thompson (19,20) have reported that chemical and physical changes in membrane lipids such as the breakdown of phospholipids, the loss of sterols from the membrane, and the formation of gel phase lipid membrane were induced by treating the isolated microsomal membrane from bean leaves with ozone in solution. However, the results obtained from isolated systems have not been confirmed in plant tissues.PAN reacts with the sulfhydryl group to form either disulfide or S-acetyl group and also reacts with olefine to form epoxides (14). Such reactions could affect the proteins and the lipids of membranes. Ozone not only destroys the double bond in unsaturated fatty acids, but also affects lipid biosynthesis (12, 13). Lipid synthesis from acetate and acetyl-CoA (12) and the incorporation of galactose from UDP-galactose into galactolipids (13) were inhibited by bubbling ozone through a chloroplast suspension. PAN in vitro also inhibited the synthesis of fatty acids from acetate (11). Thus, exposure to ozone and PAN seems to alter the membrane lipids in leaf tissues.The present study was initiated to compare the effects of ozone and PAN on the leaf lipids, fatty acids, and MDA of two different plants. Morning glory and 6 to 7 d old kidney bean plants were exposed to either ozone (0.15 ,ul/L for 8 h) or PAN (0.10 ,ul/L for 8 h) and then examined for changes in their fatty acids, polar lipids, and MDA that occurred immediately after or 24 h after the exposure periods.Morning glory and kidney bean are plants susceptible to ozone, and morning glory in particular has been used as an indicator plant of photochemical oxidants (ozone) in Japan (16 (65:25:4, v/v) and in the second direction with chloroform-methanol-isopropylamine-ammonium hydroxide (65:35:0.5:5, v/v) (1). The chromatogram was viewed under UV after spraying with 0.01% (w/ v) primuline in 80% acetone. Individual polar lipids were identified by co-chromatography with authentic standards and by their reaction with specific spray reagents of anthrone sulfuric acid, Dittmer, Dragendorff, ninhydrin, and iron (III) chloride specific to glycolipid, phospholipid, the choline group, the amino group, and sterol, respectively. Each fluorescent lipid area was scraped from the plate and subjected to methanolysis with 10% (w/v) sul...

“…Membranes damaged by peroxidation of the polyunsaturated fatty acids by aging, ozone, or chemical oxidizing agents show marked increases in the temperature at which this change in molecular ordering occurs (4,20,21). Concomitant with this change in molecular ordering are marked decreases in the fluidity of the membrane (7,9), increases in the ratio of saturated to unsaturated fatty acids (9,21), and increases in the permeability of the membrane (11,19).…”

Section: Discussionmentioning

confidence: 99%

“…In plants, data obtained by x-ray diffraction, fluorescence polarization, and studies on the temperature dependence of fatty acid spin labels indicate that cooperative changes in molecular ordering occur in liposomes, microsomes, and chloroplast and mitochondrial membranes at 10 to 20°C (20)(21)(22). Membranes damaged by peroxidation of the polyunsaturated fatty acids by aging, ozone, or chemical oxidizing agents show marked increases in the temperature at which this change in molecular ordering occurs (4,20,21). Concomitant with this change in molecular ordering are marked decreases in the fluidity of the membrane (7,9), increases in the ratio of saturated to unsaturated fatty acids (9,21), and increases in the permeability of the membrane (11,19).…”

Section: Discussionmentioning

confidence: 99%

Changes in Tobacco Cell Membrane Composition and Structure Caused by Cercosporin

Daub

Briggs²

1983

Cercosporin, a toxin produced by Cercospora species, rapidly kills plant cells in the Light. Previous work has shown that cercosporin treatment causes products of lipid peroxidation to be released. We have found that the unsaturated acyl chains of lipids in tobacco (Niotiana tabacum) cell membranes are destroyed when cells are treated with cercosporin. Concomitant with this change in composition is a change in structure of the membranes as detected by two different fatty acid spin labels, 2-(3-carboxypropyl)4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl (denoted 1112,31) and 2-(1'-carboxytetradecyl)-2-ethyl-4,4-dmethyl-3-oxazolidinyloxyl (denoted 111,141). Cercosporin causes the membranes to become more rigid at all temperatures tested and increases the membrane phase transformation temperature from 12.70C to 20.80C.Cercosporin, a toxic compound from Cercospora species, affects both hosts and non-hosts of the producing fungi. Cercosporin was first isolated in 1957 by Kuyama and Tamura (14) from C. kikuchii, a soybean pathogen, and has since been isolated from a large number of Cercospora species (1,8,16) and from Cercosporainfected plants (8,14). The structure of cercosporin was determined independently by Lousberg et al. (15) and Yamazaki and Ogawa (25).Cercosporin is a photosensitizing agent capable of generating both singlet oxygen and superoxide during irradiation (M. Daub and R. Hangarter, unpublished). Plant cells, bacteria, and mice are killed rapidly by cercosporin in the presence of light (5,26). Cercosporin causes electrolytes to leak from plant tissues (6, 17), destroys plant protoplasts (6), and causes peroxidation of plant membrane lipids both in vitro (3) and in vivo (6). These data suggest that cercosporin acts directly on membranes in situ.We report here that cercosporin causes a large increase in the ratio of saturated to unsaturated fatty acids extracted from membranes of toxin-treated cells. Concomitant with this change is a decreased membrane fluidity and the possible appearance of a phase separation in the membranes at the growth temperature of the cells. Electrolyte leakage and cell death may be accounted for by these perturbations of membrane composition and structure. MATERIALS AND METHODS Cercosporin. Cercosporin was isolated and purified from cultures of Cercospora nicotianae as previously described (5). Stock solutions were prepared in acetone and stored at -20°C in the dark. Control cultures were either treated with the same concentration of acetone or treated with cercosporin but incubated in the dark. Final acetone concentrations in both treated and control cultures did not exceed 1% (v/v).Host Material. All experiments were conducted with Nicotiana tabacum cv. 'Wisconsin 38' cell culture line NT575. Liquid suspension cultures were maintained as previously described (5).Protoplast Isolation. Protoplasts were isolated from NT575 suspension cultures 2 d after subculture (early log phase). The cells were preplasmolyzed in 0.6 M mannitol for 2 h followed by centrifugation. Packed ...