Plant Physiol. (1988) 86, 672-6760032-0889/88/86/0676/05/$Ol .00/0

The Generation of Singlet Oxygen (102) by the NitrodiphenylEther Herbicide Oxyfluorfen Is Independent of Photosynthesis

Received for publication July 17, 1987 and in revised form October 5, 1987

PHIL HAWORTH* AND F. DAN HESSZoecon Research Institute, Sandoz Crop Protection Corporation, 975 California Avenue, Palo Alto,California 94304

ABSTRACT

The mechanism of action of the p-nitrodiphenyl ether herbicides hasremained ambiguous because of conflicting reports in the literature. Thediphenyl ether herbicide oxyfluorfen causes a light induced consumptionof oxygen which resembles the electron acceptor reaction of paraquat.However, this reaction is not linked to the transport of electrons throughphotosystem I. This conclusion is based on the observation that the rateof oxygen consumption, in the presence of oxyfluorfen, does not dem-onstrate a first order rate dependence on light intensity. Using the bleach-ing of N,N-dimethyl p-nitrosoaniline as a specific detector of singlet oxy-gen, we demonstrate that oxyfluorfen is a potent generator of this toxicradical. The production of singlet oxygen occurs in the presence of in-hibitors of photosynthetic electron transport (oxyfluorfen at 10-4 molarand paraquat) and also under temperature conditions (3'C) which preventelectron transport. This light induced reaction results in oxygen con-sumption and is the primary cause of lethality for oxyfluorfen. The pro-duction of singlet oxygen occurs rapidly and at low herbicide concentra-tions (10-' molar). The reaction occurs without photosynthetic electrontransport but does require an intact thylakoid membrane.

The widely used DPE' herbicides cause rapid chlorosis andnecrosis of sensitive plant species, which is triggered by a lightdependent oxidative destruction of membrane components (16-18). The mechanism of the light activation and the role of thephotosynthetic membrane remain unclear since evidence existsfor and against the involvement of photosynthetic electron trans-port in the initiation of the oxidative process (5, 6).Because the symptoms of DPE injury resemble those of the

dipyridinium herbicides (e.g., paraquat), the initial search for amechanism of action focused on photosynthetic electron trans-port (see Refs. 1 and 6 for review). Although some involvementwith the photosynthetic process has been suggested (6, 11, 29),the mechanism of action of DPE herbicides and paraquat areclearly different. Lambert et al. (17) demonstrated, by electronspin resonance spectroscopy, that the peroxidative activity ofparaquat and diphenyl ethers are caused by different mecha-nisms. Dipyridinium herbicides act through the generation ofsuperoxide (02-) (4), however, scavengers of 02-, SOD (16)and copper penicillamine (8), do not protect against the toxiceffects of DPE. Other mechanisms proposed for DPEs includea direct chemical role of the herbicide in lipid peroxidation throughabstraction of hydrogen from unsaturated lipids, but such a hy-

' Abbreviations: DPE, p-nitrodiphenyl ether; RNO, N,N-dimethyl p-nitrosoaniline; SOD, superoxide dismutase; DCPIP, 2,4-dichlorophenol-indophenol.

pothesis is inconsistent with available physical-chemical data (9).Despite the confusion, there is broad agreement that the lethaleffect of DPE herbicides is the result of lipid peroxidation whichleads to extensive membrane destruction (4-6). The peroxida-tion of membrane lipid ultimately depends on the generation ofa toxic oxygen species (4, 12, 23, 26); 02-, singlet oxygen ('02),peroxide (022-) or hydroxyl radical (,OH). Since the involve-ment of 02- in the activity of DPEs has already been eliminatedan alternative species must be identified.Two significant questions on the mechanism of action of these

herbicides remain unanswered; what is the nature of the toxicoxygen species generated and through what route is the lightrequirement mediated? In this report we demonstrate that theDPE herbicide oxyfluorfen is a potent generator of '0,. Fur-thermore the generation of 102 does not require photosyntheticelectron transport.

MATERIALS AND METHODSIntact thylakoids were isolated from 10 to 14 d old pea seed-

lings (Pisum sativum L.) according to previously published pro-cedures (13), before final resuspension in buffer consisting of 25mM tricine-NaOH (pH 7.8), 10 mm NaCl, 5 mM MgCl,, and 0.1M sorbitol. Gramicidin (1 uM) was included as an uncoupler.Photosynthetic electron transport in these membranes was meas-ured by an oxygen electrode (YSI Model 53, Yellow Springs,Ohio). Triton-solubilization of the thylakoid membrane into threemajor pigment protein complexes (light-harvesting complex-I1,PSII, and PSI), was achieved according to documented proce-dures (20-22). Chl determination was performed according tothe method of Mackinney (19).

Singlet oxygen was detected chemically by monitoring thebleaching (decrease in A at 440 nm) of RNO. The procedurewas adapted from the published protocol of Joshi and Pathak(14). Intact thylakoids were diluted in resuspension buffer con-taining 0.01 M L-histidine and approximately 300 ltM RNO, thenilluminated with white light (1.3 x 103 ,uEm-2 s- 1 PAR). Ali-quots (1 ml) were removed at various times from these samplesand centrifuged at 3000g to pellet the membranes. The super-natant was decanted and the absorption at 440 nm measured.Once removed from direct illumination the A0440 nm remainedconstant for several hours. Cercosporin a known generator of102 (3), was included as a positive control. All the herbicidesused in this study were obtained from Chem Service (West Ches-ter, PA).

RESULTS

Oxygen Consumption in the Presence of Oxyfluorfen. PSI elec-tron transport in isolated thylakoids can be estimated by oxygenconsumption via the Mehler reaction, with ascorbate/DCPIP aselectron donor and methyl viologen (paraquat) as acceptor (Fig.

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SINGLET OXYGEN GENERATION BY OXYFLUORFEN

1A, trace 1). Assuming that reducing equivalents are in excess,then the rate of PSI electron transport is a first order reactionwith respect to light intensity (10). When paraquat is replacedby (10-7 M) oxyfluorfen, a proposed alternate acceptor (16), aqualitatively similar consumption of oxygen occurs (Fig. 1A,trace 2). This observation suggests oxyfluorfen acts as a PSIelectron acceptor in a manner similar to paraquat. The data inFigure 1B illustrates that the PSI electron transport rate, as de-fined by ascorbate/DCPIP to paraquat, saturates at low levels ofascorbate. The rate of oxygen consumption is the same at 0.3mM ascorbate (trace 1) and 10 mm ascorbate (trace 3). A similarexperiment using oxyfluorfen (Fig. 1C) shows that oxygen con-sumption increases with ascorbate concentration (cf. trace 1 andtrace 3). Since PSI electron transport saturates at low levels ofascorbate, this observation suggests oxyfluorfen dependent oxy-gen consumption is not linked to the flow of electrons throughPSI. This conclusion does not agree with previously suggestedhypotheses which link oxyfluorfen to PSI electron transport aseither a primary or secondary electron acceptor (1, 11). Furthersupport for the hypothesis that PSI electron transport and oxy-fluorfen activity are separate is given in Figure 2. Since the rateof PSI electron transport is 1st order with respect to actinic light(10), a light saturation curve of the type seen in Figure 2 (closedcircles) is expected when paraquat is the electron acceptor. Theoxyfluorfen light saturation curve (closed diamonds) does notshow the same first-order dependency on light but continues toincrease beyond the light saturation point of PSI electron trans-port. Because the oxyfluorfen experiment was carried out at 10mM ascorbate, a comparison curve of PSI electron transport withparaquat (open symbols) was included with ascorbate at 10 mM.This curve saturates at a lower light intensity than seen with 1mM ascorbate because of an increased dark rate at the higherconcentration, but the data still demonstrate a first order de-pendency. In all of these experiments the thylakoid membraneswere isolated under identical condition so that the relative ab-

1 2

1.01moles12

100 s*conds11

D light on

* light oft

1

2 3

V~~~~

FIG. 1. PSI electron transport, measured with pea thylakoids. Chlconcentration 30,g/ml, volume 3 ml, temperature 30°C, actinic lightintensity 1.3 x 103, E-m- 2-S-' PAR. Resuspension buffer, 25 mm tri-cine-NaOH (pH 7.8), 10 mM NaCl, 5 mM MgCl2, 0.1 M sorbitol, 1 AMgramicidin S, and 10-5 M DCMU. Electron donor: 1 mM ascorbate +0.3 mm DCPIP. A, Electron acceptor: 1, 0.25 mM paraquat; 2, 10-7 Moxyfluorfen (rates = 1.12 and 0.58 mmol 02/mg Chl-h, respectively). B,Electron acceptor: 0.25 mm paraquat; 1, 0.3 mM ascorbate, 2, 1 mMascorbate; 3, 10 mm ascorbate. C, Electron acceptor: 10- M oxyfluorfen1, 2 and 3 as in B.

FIG. 2. Intensity dependence of oxygen consumption in the presenceof, (@ 0) 0.25 mm paraquat + 1 mm ascorbate (rate = 0.80m/mol O2mg Chl-h), 10 -7M oxyfluorfen (* *) + 10 mM ascorbate(rate = 0.40) and (A A) 0.25 mm paraquat + 10 mm ascorbate(rate = 0.82). Maximum illumination 1.3 x 103,uE m -2- 1 PAR, otherexperimental conditions are as described in Figure 1.

sorption cross sectons (i.e., Chl/P700) are identical. Althoughthese observations argue against direct involvement of PSI elec-tron transport, they do demonstrate the light dependent natureof oxyfluorfen activity.

Singlet Oxygen Production by Oxyfluorfen. Scavengers of 02-do not protect against the toxic effects of DPE herbicides (8,16). If 02- is not the toxic oxygen species involved, the mostlikely alternative is 102. Accordingly, we have investigated theability of oxyfluorfen to generate '02. To carry out this inves-tigation a positive control (i.e. a known generator of 102) isrequired. For this study we have used the fungal toxin cerco-sporin (2, 3). In the presence of lights cercosporin will generate'02 and rapidly bleach RNO, this reaction occurs directly viathe cercosporin chromaphore (2). Host plants when infected withCercospora fungal cells show membrane disruption induced bytoxic oxygen species (3), similar to that caused by paraquat andthe DPEs. Cercosporin therefore, provides an alternative chem-ical model on which to base mechanism of action studies foroxyfluorfen.

Figure 3 compares the ability of cercosporin, oxyfluorfen andparaquat to generate '02 in the presence of isolated thylakoidmembranes. As expected cercosporin generates 'O indepen-dently of the amount of photosynthetic pigment, compare (closedcircles) Figure 3A (300 ,ug Chl/ml) and Figure 3B (30 ,ug Chl/ml), since the light activation is direct. Oxyfluorfen (10 M) isalso a potent generator of '02 (closed triangles), but does showsome dependence on the amount of photosynthetic membranepresent. Singlet oxygen production by oxyfluorfen is not ob-served when thylakoid membranes are omitted from the reactionmedium (data not shown). Paraquat shows little or no generationof 102 under either Chl concentration (closed squares), whichstrongly supports the earlier conclusion of Lambert et al. (17).A significant amount of RNO bleaching i.e. 102 production,occurs in the thylakoid control experiments (open circles), par-ticularly at the higher Chl concentration. Because the assay sys-tem used in this experiment does not contain electron acceptors,the production of 102 by the pigment bed under strong illumi-nation is predictable (4, 24). Under conditions where an electronacceptor is present (i.e. paraquat), little or no 102 generationfrom the pigment bed is observed. Since the inclusion of anelectron acceptor most closely resembles the intact system, ex-periments where paraquat is present represent the most appro-priate control.

In many mechanism of action studies the concentration ofherbicide used does not accurately reflect the low levels knownto cause in vivo injury. Ensminger et al. (9) have determined I.,,

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Plant Physiol. Vol. 86, 1988

illumination time (minutes)

illumination time (minutes)

FIG. 3. Bleaching of RNO by singlet oxygen (102) A = 300,g Chl/ml, B = 30 ,ug Chi/ml (U U) + 0.25 mm paraquat, (0 O) =thylakoid membranes alone, (A A) = 10-7 M oxyfluorfen. (0*) = 10-5 M cercosporin. Samples resuspended in 25mM tricine-NaOH(ph 7.6) 10 mm NaCl 5 mM MgCl2 and 0.1 M sorbitol at room temper-ature. The assay mixture contained 0.1 M L-histidine and approximately300 ,UM RNO.

values (the molar concentration causing 50% inhibition of Chla-mydomonas cell growth) as low as 10-9 M for some DPE her-bicides. If the generation of '02 is the primary cause of oxy-fluorfen lethality, we would expect to see activity at or below10-9 M in this in vitro system. The data in Figure 4A shows thatoxyfluorfen can produce '02 over a concentration range from10-4 M to 10-9 M. No bleaching of RNO was observed at 10- 10M oxyfluorfen (data not shown). The same figure demonstratesthe effect of paraquat addition on the ability of oxyfluorfen togenerate 102 (closed symbols, Fig. 4A). The addition of paraquatreduces, but does not abolish generation of 102 by oxyfluorfen.The effect of paraquat (0.25 mM) does not depend on the con-centration of oxyfluorfen present. This clearly demonstrates thatoxyfluorfen and paraquat do not act through a competing mech-anism, i.e. paraquat and oxyfluorfen cannot both be acting aselectron acceptors at either the same or different sites (11).The difference in the rate of RNO bleaching between oxy-

fluorfen (open symbols) and oxyfluorfen plus paraquat (closedsymbols) in Figure 4A can in part be explained by the reductionof background '02 production within the pigment bed. The levelof this background production can be estimated. Figure 4B dem-

illumination time (minues)

FIG. 4. Effect of paraquat on singlet oxygen production by (A) oxy-fluorfen and (B) 10-5 M cercosporin. Open symbols oxyfluorfen (orcercosposin) alone, closed symbols oxyfluorfen (or cercosposin) plus 0.25mM paraquat. A (0 O) 10-4 M oxyfluorfen, (O C]) 10-7 Moxyfluorfen, (A A\) 10-9 M oxyfluorfen. Experimental conditionsdescribed in Figure 3.

onstrates the effect of paraquat addition on the generation of102 by cercosporin when thylakoid membranes are present. Thesmall difference between the open and closed symbols in thisexperiment reflects the production of '02 within the pigmentbed, a reaction which occurs independently of the cercosporinproduced 102. The production of '02 in the pigment bed, esti-mated from Figure 4B, does not explain the entire differenceseen between the open symbols and the closed symbols observedin the oxyfluorfen experiment Figure 4A. One possible expla-nation for the additional effect upon the oxyfluorfen dependentproduction of 102 could be linked to the overall redox state ofthe photosynthetic membrane in the presence or absence of anelectron acceptor. In the absence of an electron acceptor thethylakoid membrane will develop a higher redox (more negative)potential than when the acceptor is present, due to a build upof electrons in the plastoquinone pool (10). Thus experimentsusing oxyfluorfen alone utilize a membrane system with morereducing power than a system in which paraquat is included. Asseen in Figure 1C, the activity of oxyfluorfen does appear to besensitive to the redox state of this system. Therefore, the inclu-sion of paraquat could slow the rate of '02 production indirectly,by changing the redox potential of the activating complex as-

674 HAWORTH AND HESS

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SINGLET OXYGEN GENERATION BY OXYFLUORFEN

sociated with this membrane. Because the RNO assay system issensitive to chemical reducing agents (e.g. ascorbate) we cannottest this hypothesis directly.

Elstner and Zeller (7) were the first investigators to report theuse of RNO to investigate generation of excited oxygen speciesby chloroplast membranes. In their study a number of inhibitorsof photosynthetic electron transport prevented the bleaching ofRNO. As a result, these workers concluded that excited oxygenspecies were not involved in the light dependent bleaching ofRNO observed in the presence of chloroplast membranes (7).As an alternative hypothesis they proposed that the bleachingof RNO occurred directly at the reducing side of PSI. In ourstudy we have conducted a number of experiments to investigatethis possibility. In agreement with these authors (7), inhibitorsof PSII electron transport (diuron and atrazine) also completelyinhibited RNO bleaching by oxyfluorfen. However, atrazine anddiuron also inhibited bleaching induced by cercosporin, even inthe absence of thylakoid membranes (data not shown). Sincecercosporin bleaching is independent of photosynthetic electrontransport, the effect of atrazine and diuron does not relate totheir herbicidal properties. We attribute this surprising obser-vation to either a direct effect of these herbicides on the assaysystem or the ability of atrazine and diuron to quench '02. Noinhibition of RNO bleaching was observed when paraquat (0.25mM) was included with cercosporin in the absence of thylakoidmembranes. More importantly the bleaching of RNO observedin the presence of either cercosporin or oxyfluorfen is inhibitedby known singlet oxygen quenchers, quercetin and azide (25, 27)see Figure 5. The degree to which quercetin and azide inhibitthis bleaching is consistent with their previously reported 'O.quenching ability (25, 27).Kenyon et al. (15) have recently reported that the activity of

the DPE herbicide acifluorfen is independent of temperatureover a range of3°C to 30°C. They conclude from this observationthat metabolism is not required for the activity of acifluorfen.The temperature dependence of '02 production by oxyfluorfenis depicted in Figure 6, which compares the degree of RNObleaching by cercosporin, oxyfluorfen and paraquat at 30°C (closedsymbols) and 3°C (open symbols). At 30°C both cercosporin(closed circles) and oxyfluorfen (closed triangles) display a rapidrate of bleaching, which is slowed, but not abolished at 3°C. Wehave measured whole chain photosynthetic electron transport(water to methylviologen) at 30°C, electron transport was notdetectable at the lower temperature.The dependence of oxyfluorfen activity on the absorption of

light by photosynthetic pigments is a hypothesis which can betested simply. An absolute dependence on the intact membranesystem can be demonstrated by separating the thylakoid into itsmajor pigment-protein complexes (20-22); PSI, PSII and light-harvesting complex. When thylakoid membranes are replacedwith any of the isolated complexes, no light dependent '02 pro-duction by oxyfluorfen is observed (data not shown). This finalobservation suggests that the activation of oxyfluorfen is de-pendent on the integrity of a membrane associated complex ratherthan a simple pigment, which is not preserved under the deter-gent isolation conditions.

DISCUSSION

Although we report in this study that oxyfluorfen does causea light dependent oxygen consumption, the activity of DPE her-bicides is distinct from a similar phenomenon caused by paraquat.The lethal effects of paraquat are associated with the productionof 02-, resulting from direct chemical reduction at PSI. Theproduction of O.- results in massive lipid peroxidation and celldeath (4). This injury can be protected by SOD (16) and copperpenicillamine (9). The lethal effects of oxyfluorfen, however, arenot inhibited by these agents (6, 9). Since 0- is not involved

JAIl0 ecl. i D

60

%A440mn40

-f l~~~~~~~~~~~~~-\ lW~~~~~~~~k

201

nI i 1U A a2 R--bU

%A440mn

u 10 15 20 25 30 60Eakato thng (muv~tes)

bnmo t (m*itm)

FIG. 5. Inhibition of RNO bleaching by singlet oxygen quenchers.A, 10-5 M cercosporin (0 0). + 100 AM quercetin (O- O),+ 5 mM sodium azide (0 0). B. 10-7 M oxyfluorfen (U U),+ 100 AM quercetin (El Ol) + 5 mm sodium azide (E] E). Ex-perimental condition described in Figure 3.

in the toxicity of oxyfluorfen an alternative toxic oxygen speciesmust be considered. Using the fungal toxin cercosporin as amodel, we have demonstrated that oxyfluorfen will generate 'O.in the presence of thylakoid membranes and light. The oxyfluor-fen induced generation of '02 occurs at 10-9 M oxyfluorfen (Fig.4), a concentration which is consistent with the IS, values deter-mined by Ensminger et al. (9) for the inhibition of Chlamnvdo-inonas growth. Production of '02 by oxyfluorfen occurs withoutphotosynthesis as demonstrated by the data of Figures 4 and 5,but the reaction does- require an intact membrane system. Theobserved consumption of oxygen (Fig. 1) can be explained bythe production of '0. as demonstrated by Reszka et al. (25).We are able to detect oxygen consumption under the RNO assayconditions (data not shown) which supports our hypothesis. Theoxygen consumed under these conditions, however, can arisedirectly from '0. or indirectly through oxidative events initiatedby 'O. (4, 12, 24). Although '02 quenchers will prevent thebleaching of RNO at 440 nm (Fig. 5) these molecules do notprevent oxygen consumption since the quenching mechanismitself consumes 02 (25). The RNO assay system used in thisstudy eliminates the possibility of OH as the toxic species. Al-though the RNO is sensitive to OH we only observe bleachingin the presence of an imidazole derivative (histidine). Bleaching

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676 HAWORTH AND HESS

ilklmination time (minutes)FIG. 6. Temperature dependency of RNO bleaching by singlet oxy-

gen. Closed symbols, 30°C; open symbols, 3°C; circles, 10 5 Mcercos-porin; triangles, 10 7 M oxyfluorfen; squares, 0.25 mM paraquat.

of RNO by OH occurs in the absence of histidine (7). Theconclusion is in contrast to the results of Upham and Matzios(28) who observed production of -OH in the presence of oxy-fluorfen. The reported production of OH only occurred underconditions where photosynthesis was active. It is possible thatthe production of OH occurred as a secondary effect of 10,

production and lipid peroxidation (12, 24).As a consequence of these studies, we feel confident in pro-

posing a mechanism to explain the lethal effects of oxyfluorfenwhich may be applicable to other DPE herbicides. The gener-ation of 102 by oxyfluorfen occurs through the interaction of theherbicide with an in situ pigment complex in the thylakoid mem-brane. This complex is not preserved during triton solubilizationof thylakoid membranes. At this stage of our investigation it is

not possible to describe the nature of this in situ pigment complexand it may exist in systems other than the thylakoid. All the datapresented in this study are consistent with the light dependentnature of this reaction. In addition, the results presented in Fig-ures 1 and 4 suggest the ability of the in situ oxyfluorfen-pigmentcomplex to generate 10 might depend on the redox potentialof the membrane, at least in the immediate environment of thecomplex. Addition of ascorbate (Fig. IC) and omission of pa-raquat (Fig. 4A) both serve to increase the redox potential ofthe membrane and also stimulate 10, production. This associa-tion with redox state does not imply a requirement for activemetabolism (Fig. 6) but does suggest active metabolism (pho-tosynthesis) might enhance production of 10, by the in situ oxy-fluorfen-pigment complex (1). Active photosynthesis will reducethe plastoquinone pool and elevate the redox potential of themembrane. It is this relationship between the production of 10,

and the redox potential of the cell which has confused mechanismof action studies with DPE herbicides in general and oxyfluorfenin particular (6).

Singlet oxygen is known to poise a toxic threat to immediatebiological systems because of its role in lipid peroxidation (12,23, 26). It is proposed to be a direct initiator of lipid peroxidationby a concerted addition-abstraction reaction with the diene bondsof unsaturated lipids, giving rise to lipid hydroperoxides (12, 23,26). The inevitable consequences of this peroxidation are mem-

Plant Physiol. Vol. 86. 1988

brane destruction and cell death, a pattern of injury which isentirely consistent with the effects of the DPE herbicides (6).

Ackniowi,ledgmnemits-Cercosporin used in this study was a gift of Northrup Kill.Stanton MN. The authors ssish to acknowledge the technical assistance of W. Miller.

LITERATURE CITED

1. BOGER P 1984 Multiple modes of action of diphenvl ethers. Z Naturforsch 39c:468-475

2. DUAB ME 1982 Cercosporin. a photosensitizing toxin fromii (Cercospor species.Phytopathology 72: 370-374

3. DAUB ME. RP HANGARTER 1983 Light indueed production of singlct oxygenand superoxide by the fungal toxin cercosporin. Plant Physiol 73: 85-5-857

4. DODGiE AD 1983 Toxic oxygcn species and herbicide action. In S Matsunaka;.D Hutson. SE Murphy. eds. Pesticidc Chemistry. Human Wclfarc and theEnvironment. Vol 3. Mode of Action. Metabolism aind Toxicology. Pega-mon Press. New York, pp 59-66

5. DUKE SO, WH KENYON 1986 Photosynthesis is not involvcd in the mechanisnof action of acifluorfen in cucumber (C04cunits sautivs L.). Plant PhNsiol 81:882- 888

6. DUKE SO. WH KENYON 1987 A non-metabolic model of acifluorteni actis its.Z Naturforsch 42c: 813-818

7. ELSITNER EF. H ZELLER 1978 Bleaching of p-nitrosodimethylaatlane bs pho-tosvstcm I of chloroplast lamellac. Plant Sci Lctt 13: 15-2(0

8. ENSMIGER MP. FD HESS 1985 Photosvnthesis involvement in the mechanismof action of diphenylethcr hcrbicides. Plant Physiol 78: 46-50

9. ENSMINGER MP. FD HESS. LT BAHR 1985 Nitrofrec radical formation otdiphenyl ether herbicides is not necessary for their toxic action. Pest BiochcmPhysiol 23: 163-170

10. GHIRARDI M. A MELIS 1984 Photosystem electron transport capacity aind lightharvesting antenna size in maize chloroplasts. Plant Phvsiol 74: 993-998

11. GILLMAN DJ. MW EVANS. AD DODGE 1985 Oxyfluorfen actisation bv pho-tosynthetic eljectron transport. Proc Br Crop Protect Conf Wecds 7: 1195-12()1

12. HALLIWEII B, JMC GUTiTERIDGE 1984 Oxygen toxicity. oxxgcn radicals.transition metals and diseasc. Biochem J 219: 1-14

13. HAWOR1yI P 1983 Protein phosphorvlation induced state I-statc 11 triansitionsare dependent on thylakoid membrane microviscositv. Arch Biochemii Bio-phy 226: 145 154

14. JosHi PC. MA PATHAK 1984 The role of active oxygen (10, and O) inducedbv crude coal tar and its ingrcdients used in photochcmothcrapy of skindiseases. J Invest Dermatol 82: 67-73

15. KENYON WH. SO DUKE, RN PAUL 1987 Effects of temperature on the actisitvof the p-nitrosubstituted diphenvl ether herbicide acifluorfen in cucmnbetl(Cucutnis sativus L.) Pest Biochem Physiol (in press)

16. KUNERT KJ. P BOGER 1987 The bleaching effect of the diphcnyl ethcr herbicidcoxyfluorfen. Weed Sci 29: 169- 173

17. LAMBERT R. PMH KRONECK P BOGER 1984 Radical formation and peroxi-dative activity of phytotoxic diphenyl ethers. Z Naturforsch 39c: 486-491

18. LAMBERT R. G SANDMANN, P BOGER 1983 Mode of action of nitrodiphenyl-ethers affecting pigments and membrane Integrity. In S Matsunaka. D Hut-son. SD Murphy. eds. Pesticide Chemistry. Human Welfare and the Ensi-ronment. Vol 3. Mode of Action. Metabolism and Toxicology. PergamonPress, Ncw York. pp 97- 10(2

19. MACKINNEY G 1941 Absorption of light by chlorophyll solutions. J Biol Chcm14(1: 544-522

21). MULLET JE, JJ BURKE. CJ ARNTZEN 1980 Chlorophvll proteins of photosys-tem 1. Plant Physiol 65: 814-822

21. MULLET JE. CJ ARNTZEN 1981) Stimulation of grana staicking in a mnodclmcmbrane system. Mediation by a purified light-harvesting pigment protcincomplex from chloroplasts. Biochim Biophys Acta 589: I()-1 17

22. MULLET JE. CJ ARNTZEN 1981 Identification of a 32-kilodalton polvpeptidcsas herbicide receptor protein in photosystem 11. Biochim Biophvs Acta 635:236-238

23. PERCIVAL MP, AD DODGE 1983 Photodynamic damage to chloroplast milemil-branes. photosensitizcd oxidation of chloroplast acyl lipid. Plant Sci. Lctt29: 255-264

24. RABINOWITCH HD, I FRIDOVICH 1983 Superoxide radicals. supcroxide dis-mutases and oxygen toxicits in plants. Photochem Photobiol 37: 679-69(0

25. RESZKA K. PG TsoUNCAS. JW LoWN 1986 Photosensitization b1s iantituImoragents. 1. Production of singlet oxygen during irradation of anthrapyrazolcswith visiblc light. Photochem Photobiol 43: 499-504

26. SVINGEN BA. FO O'NEAL. SD AUST 1978 The role of superoxidc and singlctoxygen lipid peroxidation. Photochem Photobiol 28: 81)3-809

27. TAKAHAMA U 1983 Supression of lipid photoperoxidation by quercctin and its

glycosides in spinach chloroplasts. Photochem Photobiol 38: 363-36728. UPHAM BL. HC HATZIOS 1987 Potential involvement of alkoxvl aInd hvdroxs

radicals in the peroxidativc reaction of oxyfluorfcn. Pcst Biocheml Phvsiol28: 248-256

29. WETTI-AUFER SHI. R ALSCHER. C STRICK 1985 Chloroplast-diphcslll cthli-interactions 11. Plant Physiol 78: 215-"221

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