Diphenyl Ether Herbicides' Received for Publication August 31, 1984 and in Revised Form January 3, 1985

Plant Physiol. (1985) 78, 46-50 0032-0889/85/78/0046/05/$0 1.00/0 Photosynthesis Involvement in the Mechanism of Action of Diphenyl Ether Herbicides' Received for publication August 31, 1984 and in revised form January 3, 1985 MICHAEL P. ENSMINGER2 AND F. DAN HESS* Department ofBotany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907 ABSTRACI initiating lipid peroxidation of the polyunsaturated fatty acids in the membrane of Scenedesmus acutus. Photosynthesis is not required for the toxicity of diphenyl ether The purpose of this study was to determine if photosynthesis herbicides, nor are chloroplast thylakoids the primary site of diphenyl is involved in DPE herbicide membrane disruption and cell ether herbicide activity. Isolated spinach (Spinacia oleracea L.) chloro- death as it is for cell death by paraquat and diuron (5). This plast fragments produced malonyl dialdehyde, indicating lipid peroxida- research used isolated spinach chloroplast fragments, the unicel- tion, when paraquat (1,1'-dimethyl4,4'-bipyridinium ion) or diuron 13- lular green alga Chlamydomonas, and excised cucumber cotyle- (3,4-dichlorophenyl)-I,I-dimethylureaI were added to the medium, but dons to evaluate the requirement of photosynthesis for DPE no malonyl dialdehyde was produced when chloroplast fragments were caused cell death. treated with the methyl ester of acifluorfen (methyl 5-12-chloro-4-(trifluoromethyl)phenoxy-2-nitrobenzoic acid), oxyfluorfen 12-chloro-l-(3- AND ethoxy4-nitrophenoxy)4-(trifluoromethyl)benzenel, or MC15608 MATERIALS METHODS (methyl 5-12-chloro44trifluoromethyl)phenoxyj2-chlorobenzoate). In Isolated Chloroplast Fragments. Spinach chloroplast frag- most cases the toxicity of acifluorfen-methyl, oxyfluorfen, or MC15608 ments were prepared by the method ofTakahama (24) with some to the unicellular green alga Chiamydomonas eugametos (Moewus) did modification. Large veins were removed from 25 to 30 g of not decrease after simultaneous treatment with diuron. However, diuron commercially grown spinach leaves, and then the leaves were cut significantly reduced cell death after paraquat treatment at all but the into 1 cm2 pieces. Leaf pieces were blended 30 s in a Waring highest paraquat concentration tested (0.1 millimolar). These data indi- Blendor with 125 ml of 17 mM Tris-HCI buffer (pH 7.4). The cate electron transport of photosynthesis is not serving the same function debris was filtered through eight layers of cheesecloth and cen- for diphenyl ether herbicides as for paraquat. Additional evidence for trifuged at 2000g for 1 min in a Sorvall HB-4 rotor. The super- differential action of paraquat was obtained from the superoxide scaven- natant was centrifuged 10,000g for 10 min, and the pellet was ger copper penicillamine (copper complex of 2-amino-3-mercapto-3- resuspended in 17 mm Tris - HCI buffer (pH 7.4) which was again methylbutanoic acid). Copper penicillamine eliminated paraquat toxicity centrifuged at 10,000g for 10 min. The chloroplast fragments in in cucumber (Cucumis sativus L.) cotyledons but did not reduce diphenyl the pellet were resuspended in 17 mM Tris-HCI buffer, pH 7.4. ether herbicide toxicity. All isolations were conducted at 4C, and Chl concentrations were determined using the absorption coefficients for Chl a and b as given by MacKinney (14). Photosynthetic electron transport activity in the chloroplasts was determined indirectly by measuring the reduction of DCPIP. The basic reaction mixture for each experiment contained chloroplast fragments (200 Mg of Chl) in 2 ml of 17 mm Tris. DPE3 herbicides initiate lipid peroxidation and eventually HCI buffer (pH 7.4) plus the appropriate herbicide. Nontreated disrupt cell membranes causing cell death (7, 13, 18). This cell samples contained the same amount of solvent as the treated death by DPE herbicides requires light (8, 15, 17); however, the samples (0.1 % [v/v] ethanol or 0.1 % [v/v] DMSO). To illumi- mechanism of initiation of lipid peroxidation of the polyunsat- nate the chloroplast fragments, a light intensity of 150 ME m-2 urated fatty acids in the membrane is unknown. Orr and Hess s-' (all light measurements were PAR) was provided by a bank (18) previously proposed that DPE herbicides directly become oftwelve General Electric F48T12/CW/VHO fluorescent lamps free radicals by interacting with carotenoids, and these activated and four 60-w incandescent bulbs. DPE herbicides initiate lipid peroxidation in the membrane. After treatment MDA formation, indicating lipid peroxida- However, this hypothesis can not be valid for all DPE herbicides. tion, was determined according to the method ofTakahama and For example, Cl and H atoms that replace the NO2 of AFM can Nishimura (25). To detect MDA, 0.5 ml of 40% TCA, 0.25 ml not undergo free radical formation yet have the same mode of of 5 N HCI, and 0.5 ml of 2% thiobarbituric acid were added to action as AFM (7). A different theory for DPE membrane 2 ml of chloroplast fragments. The thiobarbituric acid was first damage has been proposed by Sandmann and Boger (22), where dissolved in a small amount of 1 N NaOH (20) and brought up the DPE herbicide oxyfluorfen requires photosynthesis prior to to volume with distilled H20. Final NaOH concentration was 0.35 N. After mixing, the chloroplast fragments were placed in a 'Journal Paper no. 10,093 of the Purdue University Agricultural boiling water bath for 10 min, cooled on ice, and centrifuged at Experiment Station. 2000g for 5 min. Absorbance of the supernatant, containing 2 Present address: Stauffer Chemical Company, Mountain View, CA MDA, was recorded by a spectrophotometer at 532 nm. 94042. Algal Bioassay. All experiments with algae were conducted 3Abbreviations: DPE, diphenyl ether, AFM, acifluorfen-methyl; using the unicellular green alga Chlamydomonas eugametos DCPIP, 2,6-dichlorophenol-indophenol; MDA, malonyl dialdehyde; (Moewus). Chlamydomonas cells were maintained on agar slants, ESR, electron spin resonance. and liquid cultures were initiated and grown as described previ- 46 PHOTOSYNTHESIS INVOLVEMENT IN DIPHENYL ETHER TOXICITY 47 ously (7). The population of Chlamvdomonas cells at the start of Table I. Chl Degradation by Paraquat, Diuron, and DPE Herbicides in each experiment was adjusted to I x 106 cells ml-'. To initiate Spinach LeafSegments a bioassay, 10 ml of the cells were added to 25-ml flasks. Cotton Spinach leaf segments were floated on distilled H20 in the light (600 plugs were placed in the neck of each flask. These flasks were ALE m-2 s-') for 48 h with paraquat, AFM, MC15608, or oxyfluorfen or aerated and placed on a shaker (60 rpm) in a growth chamber. for 64 h with diuron. Nonherbicide-treated samples contained the same Light intensity was 175 ME m 2 s-'. The Chiamydomonas cells amount of solvent as the herbicide-treated samples; different solvents were pretreated for 5 min with 1 x 10-6 M diuron prior to adding (1.0% [v/vJ DMSO or 1.0% [v/v] ethanol) were used depending on the the other herbicides for 90 min. solvent the herbicide was dissolved in. Herbicide toxicity induced by the Cell death was recorded using the fluorescein diacetate proce- herbicides was determined by the degradation ofChl. dure, which has previously been shown to be a good indicator of cell death (7, 27). Relative fluorescence intensity was measured Treatment Chl Content by an Aminco fluoro-colorimeter (model number JA-7439; ,gg Chl ml-' g-'fresh wt American Instrument Company, Silver Springs, MD), using Control (water) 1370 excitation filter 47B (360-500 nm) and cutoff filter 8 (transmit- Paraquat (I x 10-5 M) 27 tance greater than 470 nm). Control (ethanol) 1111 Spinach and Cucumber Bioassays. To evaluate herbicide-in- Diuron (5 x IO-' M) 637 duced Chl destruction in spinach, commercial spinach leaves Control (ethanol) 1423 were cut into I cm2 pieces, avoiding major veins. Eight leaf AFM(5x I05 M) 119 segments were floated on 40 ml of distilled H20 in a Petri dish AFM (I xlO-5 M) 450 (9 cm diameter) with the appropriate herbicide. Nontreated Control (DMSO) 1325 samples contained the same amount of solvent as the treated MC15608 (I x 10-4M) 456 samples. Spinach leaf segments were illuminated with 600 ME Oxyfluorfen (5 x lo-5 M) 642 m-2 s-'. Solutions were changed after 24 h and Chl was extracted at 48 or 64 h by grinding the spinach leaf segments in 80% acetone with a hand homogenizer. The homogenate was centri- (5 x 10-' M) was needed to observe Chl degradation (Table I). fuged at 2000g for 5 min, and Chl was determined by measuring In this study, Chl degradation caused by treating leaf segments A at 645 and 663 nm. Chl concentrations in the supernatant with I x 10-' M paraquat was visually evident by 24 h, and by were determined using the absorption coefficients of MacKinney 48 h more than 98% of the Chl had been degraded (Table I). In (14). spinach leaf segments, Chl degradation by 1 x 10-4 M paraquat, For the cucumber bioassays, cucumber seeds were planted in the paraquat concentration used in isolated chloroplast frag- vermiculite and grown in the growth chamber with a 12 h diurnal ments, was similar to the breakdown of Chl caused by I x 10-5 day (220 ME m 2 s-') and night. Vermiculite was kept moist with M paraquat, except the bleaching occurred earlier (data not deionized H20. After 7 d, eight greened cucumber cotyledons shown). Bleaching symptoms induced by diuron occurred later were placed in a Petri dish (9 cm diameter) containing 40 ml of than paraquat induced bleaching. After 48 h, little Chl degrada- distilled H20. These cotyledons were pretreated 24 h (220 MuE tion was evident (data not shown), but by 64 h the Chl content m-2 s-') with the superoxide scavenger copper penicillamine.

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