Lipid Peroxidation in Higher Plants

Abstract

To study the role of glutathione reductase in lipid peroxidation, bean leaves (Phaseolus vulgaris) cv Fori were treated with the herbicide acifluorfen-sodium (sodium 5-12-chloro4-(trifluoromethyl)phenoxy-2-nitrobenzoic acid). Acifluorfen is a potent inducer of lipid peroxidation. In beans, decrease of acid-soluble SH-compounds and lipid peroxidation, measured as ethane evolution, were the toxic events after treatment of leaves with acifluorfen. As a primary response to peroxidation, increased production of antioxidants, such as vitamin C and glutathione, was found. This was followed by elevation of glutathione reductase activity. Enhanced activity of the enzyme prevented both further decline of acid-soluble SH-compounds and lipid peroxidation. Increased production of antioxidants and elevated activity of antioxidative enzymes, like glutathi-one reductase, seem to be a general strategy to limit toxic peroxidation in plants. The deleterious effect of many xenobiotics, including certain p-nitrodiphenyl ether herbicides, is believed to be strong oxidation ofcell components, such as peroxidation ofpolyunsaturated fatty acids in biomembranes via free radical reactions (10, 13). Research to date has shown that peroxidation of lipids is responsible for damage of proteins, DNA, and pigments (11, 12, 24, 26). Protection against phytotoxic peroxidation is achieved by several antioxidants, such as the vitamins E and C or glutathione (3, 26). The lipophilic vitamin E, however, seems to be the most effective radical chain-breaking substance (2). It has to be reduc-tively regenerated by water-soluble GSH3 either directly or via a system consisting of GSH and the water-soluble vitamin C (18). To maintain a high level of active GSH, GSSG has to be rapidly reduced. This reaction is catalyzed by the enzyme GR (EC 1.6.4.2) in the presence of NADPH. GR has been isolated from a number of different organisms including higher plants and bacteria (9, 17). Little is known, however, about the physiological role of GR in plants under peroxidative conditions. Halliwell and Foyer (9) proposed that the enzyme is involved in the ascorbate-glutathi-one cycle to reduce GSSG to GSH. Ascorbate is necessary to remove phytotoxic H202 in chloroplasts via the ascorbate/ascor-bate peroxidase system (5, 14). A similar physiological function of GR has also been proposed by other researchers working with I Supported by Deutsche Forschungsgemeinschaft. 3Abbreviations: GSH, reduced glutathione; GLO, galactonolactone oxidase; GR, glutathione reductase. blue-green algae (25). Recently, Gillham and Dodge (7) found that GR is located in both the cytoplasm and the chloroplast. This paper presents results showing some new aspects of the role of GR under peroxidative conditions in higher plants. For comparison, the enzyme GLO, which converts L-galactonolac-tone to vitamin C (16), was also investigated to differentiate between specific responses of enzymes either responsible for antioxidant recycling or antioxidant biosynthesis. Peroxidation was initiated in beans by the p-nitrodiphenyl ether acifluorfen. This compound has been shown to induce peroxidation of lipids (10). The peroxidation process was measured by ethane evolution , a decomposition product of w-3-unsaturated fatty acid hydroperoxides (26). MATERIALS AND METHODS Materials. Acifluorfen-sodium was supplied by Rohm and Haas; L-galactono-1,4-lactone was purchased from Sigma. Plant Cultivation. Seeds of beans (Phaseolus vulgaris) cv Fori were planted in moistened vermiculite. They remained in a darkened growth chamber with a constant temperature of 20TC and 80% RH. After 6 d, the etiolated seedlings were exposed for 7 d to a 12-h photoperiod (170 W/m2 supplemental light) with d and night temperatures of 22 and 18°C, respectively. Chemical Treatment of Plants. In the short-term experiment, primary bean leaves were treated 7 d after light exposure with 50 pld/leaf of an aqueous solution containing 0.5 mM acifluorfen. In the long-term experiment, primary leaves were treated 7 d after light exposure with 50 ,l/1eaf of an aqueous solution containing different herbicide concentrations. This procedure was repeated once after 2 d before leaves were excised for analysis after 4 d. The herbicide was applied on the surface of the leaves with a small brush. Enzyme Assay. GR activity was assayed spectrophotomet-rically at 340 nm by oxidation of NADPH as described by Halliwell and Foyer (9). GLO activity was determined by floating disks of primary bean leaves on 10 ml incubation medium in the dark for 18 h. The incubation medium consists of 10 mm sodium phosphate buffer (pH 7.2) containing 20 mm galactono-lactone. The activity of the enzyme was then determined by measuring the vitamin C content of the galactonolactone-incu-bated disks. Analysis. Total glutathione content was determined according to the method of Law et al. (14). The vitamin E and vitamin C content of primary leaves were determined by HPLC technique and dye titration with 2,6-dichlorophenolindophenol, respectively , as described by Finckh and Kunert (3). To determine herbicide-induced ethane evolution, disks of acifluorfen-pretreated primary leaves (0.2 g) were placed into 10 ml vials and sealed with rubber septa. Incubation followed for 18 h at 20°C under slight shaking and continuous light of 40 W/ mI. The vials contained 2.5 ml medium consisting of 10 mm 700 https://plantphysiol.org Downloaded on January 1, 2021.-Published by