Peroxidation of Tobacco Membrane Lipids by the Photosensitizing Toxin, Cercosporin

Abstract

Cercosporin, a nonspecific toxin from Cercospora species, is a photosensitizing compound which rapidly kills plant cells in the light. Cell death appears to be due to a cercosporin-mediated peroxidation of membrane lipids. Tobacco leaf discs treated with cercosporin showed a large increase in electrolyte leakage I to 2 minutes after irradiation with light. All tobacco protoplasts exposed to cercosporin in the light were damaged within 45 minutes. Chloroform:methanol extracts of toxin-treated suspension cultures gave positive reactions for lipid hydroperoxides in the thiobarbituric acid test. Cercosporin-treated leaf discs emitted high concentrations of ethane 12 to 24 hours after incubation in the light. Cercosporin also oxidized solutions of methyl linolenate as determined by the thiobarbituric acid assay and the emission of ethane. a-Tocopherol had an inhibitory effect on the cercosporin-mediated lipid peroxidation.Cercosporin [1,12-bis(2-hydroxypropyl)-2,1 1-dimethoxy-6,7-methylenedioxy-4,9-dihydroxyperylene-3, 10-quinoneI is a nonspecific phytotoxin produced by members ofthe genus Cercospora. It was first isolated in 1957 (9) from Cercospora kikuchii, a soybean pathogen, and has since been isolated from a large number of Cercospora species (1,2,7,11,14,23) and from Cercosporainfected plants (7,9,23). Its structure was determined independently by Lousberg et al. (10) and Yamazaki and Ogawa (25).Cercosporin is a photosensitizing compound (5, 26), thus, it is not toxic to cells in the dark. In the presence of light, however, photosensitizers absorb the light energy to form electronically excited states and then transfer this energy to oxygen (8,18). This results in the production of toxic oxygen species such as singlet oxygen or superoxide ions, which can damage living cells. Yamazaki et al. (26) showed that mice and bacteria were killed by cercosporin only when they were exposed to light, and that oxygen was involved in the process. Previous work in this laboratory (5) has determined that the action spectrum for the killing of tobacco cells by cercosporin is in close agreement with the absorption spectrum of cercosporin, and that the toxic effect of cercosporin can be inhibited by several quenchers of singlet oxygen.The toxic effects of photosensitizing compounds are well known (8, 18). One of the most common of these effects is damage to cellular membranes. Macri and Vianello (12) MATERIALS AND METHODSCercosporin. Cercosporin was isolated and purified from cultures of Cercospora nicotianae as previously described (5). Stock solutions were prepared in either acetone (for assays that involved cell cultures) or methanol (for assays that utilized leaf tissues). Stock solutions were stored at -20°C in the dark. The final concentration of acetone or methanol in control and cercosporintreated cultures did not exceed 1%.Host Material. Tissue discs for electrolyte leakage and ethane studies were cut from fully expanded leaves of Nicotiana tabacum cv 'Wisconsin 38' grown in the greenhouse. The N. tabacum cv 'W...

Changes in Tobacco Cell Membrane Composition and Structure Caused by Cercosporin

“…Inasmuch as cercosporin requires light for its action (5,17,26), it is likely that these changes were caused by the toxin. These data confirm earlier reports of the production of products of lipid peroxidation both in vitro and in vivo by cercosporin (3,6).…”

“…The presence of gel phase lipid domains in a fluid membrane matrix is correlated with a breakdown of membrane semipermeability (11,19). This may account for the cell leakiness and death observed following cercosporin treatment (6,17). While changes in membrane permeability were observed soon after treatment, changes in fatty acid composition and membrane fluidity required more time to be detected.…”

“…Cercosporin treatment caused a marked increase in the ratio of saturated to unsaturated fatty acids, a decrease in the fluidity of the membrane, and raised the membrane phase transformation temperature, as determined by fatty acid spin label mobility. Cercosporin also caused large increases in membrane permeability (6,17). Untreated cells and protoplasts or those which were treated and held in the dark did not show these changes.…”

“…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.…”

Electrolyte Leakage, Lipoxygenase, and Lipid Peroxidation Induced in Tomato Leaf Tissue by Specific and Nonspecific Elicitors from Cladosporium fulvum

“…Lipoxygenase catalyzes the addition of oxygen to several molecules including fatty acids, esters, and alcohols that contain a cis-1,4-pentadiene system (3). Substrates include linoleic and linolenic acids but not monounsaturated fatty acids such as oleic acid (16,32 elicitors (29), fungal toxins (6), and in incompatible bacterialplant interactions (20,21). The development of a hypersensitive response, lipid peroxidation or an increase in electrolyte leakage was reduced in a bacteria-plant interaction with the addition of the free radical scavenger Tiron or the enzyme superoxide dismutase, suggesting that oxygen radical species were involved in the initiation of this peroxidation (20,22).…”