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Mitochondrial involvement in the mode of action of acifluorfen
PESTICIDE
BIOCHEMISTRY
Mitochondrial STEPHEN Southern
Weed
Science
AND
PHYSIOLOGY
21, 368-376 (1984)
Involvement 0. DUKE, Laboratory, and Haas
in the Mode of Action of Acifluorfen
KEVIN
C. VAUGHN,
USDA, Company,
ARS, P.O. Box 22.5, Stoneville, Spring House. Pennsylvania
L. MEEUSEN*
AND RONALD
Mississippi 19477
38776,
and *Rohm
Received September 9, 1983; accepted November 10, 1983 The herbicidal action of acifluorfen {5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid} was studied with greened and expanded discs from cotyledons of cucumber (Cucumis sativus L.). Discs were floated on various treatment solutions for 20 hr in darkness before exposure to 400 pE m-’ set -’ of white light. Herbicide damage, as measured by electrolyte leakage, began in the light after a I- to 2-hr lag period. Cytochemical methods at the ultrastructural level indicated that acifluorfen caused marked increases in production of superoxide radical and hydrogen peroxide in the mitochondrion, but not in the plastid. The mitochondrial inhibitors antimycin A, rotenone, CCCP, and DNP antagonized the action of acifluorfen, lengthening the lag period and reducing the rate of electrolyte leakage. Ethanol, a-tocopherol, N-[2-(2-oxo-l-imidazolidinyl)ethyl]-N’-phenylurea, and copper-penicillin also lengthened the lag phase and slowed the rate of damage, indicating that acifluorfen damage involves toxic oxygen species. PS II-inhibiting levels of DCMU, atrazine, or bentazon did not affect acifluorfen-induced ion leakage. Yellow tissue produced by treatment with tentoxin was supersensitive to acifluorfen, but white tissue produced by treatment with nortlurazon was relatively insensitive. These data indicate that, after an initial carotenoid-acifluorfen interaction, the mitochondrion is involved in production of toxic oxygen species and that this process is closely tied to the mechanism of action of this herbicide.
information into a model describing a mechanism of action of acifluorfen which accounts for all major experimental results, but which does not designate the subcellular location of the proposed steps of the mechanism. Using a cytochemical procedure for the localization of hydrogen peroxide with which we previously studied paraquat mechanism of action (12), acifluorfen was found to cause high levels of hydrogen peroxide in mitochondria of green tissues. This finding, and subsequent studies which support the view that the mitochondrion and respiration are involved in the mechanism of action of acifluorfen, are reported in this paper. No previous studies have suggested involvement of the mitochondrion in the mechanism of action of acifluorfen.
INTRODUCTION
The mechanism of action of the diphenyl ether (DPE) herbicide, acifluorfen {5-[2chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid}, is unknown (1). The effects of acifluorfen and related ortholpara-substituted DPEs are unique in that light is required for herbicidal activity; however, photosynthesis is not involved (l-6). Apparently, carotenoids are the photoreceptors for this light-dependent activity because plants lacking carotenoids for genetic or chemical reasons are relatively insensitive to these DPE herbicides (2, 4, 7-9). Oxygen enhances the activity of DPE herbicides, and much of the etiology of damage, such as production of thiobarbituric acid-reacting materials (8) and ethane (IO), indicates that unsaturated lipid peroxidation by toxic oxygen species may be involved (1, 11). Protection against injury with a-tocopherol further supports this view (8). Orr and Hess (1) incorporated this 0048-3575/84 $3.00 Copyright All *:“Ltr
0 1984 by Academic Press. Inc. “FrG.^..,.A,.^r:l-” :” ““,, F..-- -m”o-.ioA
MATERIALS
AND METHODS
Plant material. Cucumber umis sativus L. (cv. Straight
seeds [CztcEight)] were planted in flats in a commercial greenhouse
368
MITOCHONDRIAL
INVOLVEMENT
IN ACIFLUORFEN ACTI3N
369
substrate (Jiffy-mix; JPA, West Chicago, Ill.)’ and watered with distilled water. Plants were treated with norflurazon [4chloro-Qmethylamino)-2-(cr,cY,a,-trifluorom-tolyl)-3-(2H)-pyridazinone] or tentoxin by soaking seeds in solutions (saturated and 100 mM, respectively) of the compounds before planting as reported earlier ( 13, 14). The seedlings were grown at 25 + 1°C under continuous white light 10 to 50 PE In-? set PI in a growth chamber at 90 t 5% relative humidity. Leaf discs (7 mm diameter) were punched from expanded cotyledons of 8- to I?-day-old seedlings with a sharpened cork borer. All discs for a particular experiment were floated, adaxial side up, on a solution of 2% (w/v) sucrose in I mM 2-(4-morpholino)ethane sulfonic acid (Mes) (pH 6.5) and swirled several times to randomize before selection of discs for each treatment. Trerrtment. Ten cotyledon discs were placed adaxial side up on 5 ml of 1 mM Mes (pH 6.5) with 2% (w/v) sucrose in a 6-cm diameter styrene Petri dish. All chemical treatments were prepared in this buffered sucrose solution with technical-grade reagents of better than 90% purity. The test level of a-tocopherol was dissolved in methanol and placed in the Petri dish, and the methanol was evaporated before buffer with or without acifluorfen was added. This treatment method was used because of the high degree of insolubility of a-tocopherol in aqueous solution. The discs were incubated on treatment solutions for 20 to 22 hr in complete darkness at 25°C and then transferred to continuous white light (400 p.E rn.-’ set ‘) at 25°C. Periodically conductivity of the treatment solution was assayed with a conductivity meter (Model 4503A. Amber Science)] having the capacity to assay and return 1 ml of solution to the Petri dish.
Conductivity for each treatment wa‘, plotted as the change in conductivity from that recorded at the beginning of the light exposure. All experiments were repeated two to six times and, in each repetition ol‘ an experiment, each treatment was repli.cated two or three times. All presented data are averages from a representative expcri.ment. The quantitative effects of a treat-ment often varied considerably betwcerl repetitions. However, the qualitative et’-fects and relative effects of various treatments were uniform between treatment\. Copper-penicillamine (CP) complex ti’at, prepared as described by Birker and Freeman ( IS). Its superoxide dismutatin:: activity was confirmed spectrophotometritally with the crocin-bleaching assay of Lengfelder and Elstner ( 16). Crocin was cxtracted from saffron according to Friend and Mayer (17). The more common assay4 for superoxide dismutating activity involving reduction of cytochrome c or autoxidation of epinephrine were avoided because free copper ions were found to imerfere (R. L. Meeusen. unpublished results). On a weight basis, this CP complex had virtually the same activity as commerciall! available bovine blood superoxide dismutase (SOD) (Sigma, St. Louis. Mo.)’ in protecting crocin from oxidation by enzyrnatically generated superoxide radical (data not shown). Fluorescent trvrnsients. In r*ilw fluorescence transients of cotyledon discs were measured with a Model SF-20 fluoromeref (Richard Brancker Research, Ltd.. Ottawa. Canada).’ A 30-sec. dark preincubation wasY used before the transient was determined. The technique was the same as that of Hetherington and Smillie (1X). Cytoc~lzevlistry. Tissue within 1 mm ot‘ the edge of the cotyledon disc was used for all cytochemical assays. Hydrogen pcroxide was visualized at the ultrastructuml ’ Mention of a trademark, proprietary product or level as reported earlier ( 12) by formation vendor does not constitute a guarantee or warranty of of cerium perhydroxide (19). CeCl, ( 1 m.M) the product by the U.S. Dep. Agric. and does not was added to treatment solutions present imply its approval to the exclusion of other products or vendors that may also be suitable.
from the beginning of the experiment. Ami,-
370
DUKE,
VAUGHN,
AND
MEEUSEN
0 c
.-
0 E
I50m c ," u ,.
c 0 0
FIG.
change
0
0.3
I concentration
3
IO
30
IO0
(PM)
FIG. 1. Dose-response of acifluorfen on ion leakage of cucumber cotyledon discs preincubated 20 hr in darkness and then exposed to 21 hr of white light.
notriazole was not used to block catalase activity as in a previous study (12). Tissue pieces (1 mm*) for superoxide localization from treated leaves were fixed for 30 min in 4% (w/v) paraformaldehyde at 4”C, washed with 0.1 M phosphate buffer, pH 7.2, with 5% sucrose, and incubated in the medium of Karnovsky and Robinson (20). Postfixation was carried out in 1% 0~0, for 2 hr at room temperature. Dehydration and embedding were carried out as for H,O, localization (12).
2. Structure in position
of RH 5349. The arrow from acifluorfen.
shon*s
the
on ion leakage. Results for each compound were similar to those shown for atrazine (Fig. 4). Increased variable fluorescence at the end of the 20-hr dark period indicated that electron flow from PS II to PS I was effectively blocked by each of the PS II inhibitors (inset to Fig. 4) Acifluorfen itself had little or no effect on the fluorescence transfer after the 20-hr dark period; however, on illumination, the variable portion of the fluorescence transient decreased with time (Fig. 5). Cytochemical evidence of acifuorfencaused mitochondrial superoxide and hydrogen peroxide production. Cerium perhydroxide precipitates, indicators of hydrogen peroxide presence, were observed in the mitochondria of acifluorfen-treated tissue in the light, whereas no precipitates were seen in RH 5349-treated tissues (Figs.
RESULTS
Evidence of nonphotosynthetic light requirement. In preliminary experiments we found that 30 PM acifluorfen caused little or no ion leakage in the dark, but caused rapid leakage in the light (Figs. 1 & 3) after a l- to 3-hr lag period. All further experiments were conducted with this concentration of the herbicide. RH 5349, an herbicidally inactive isomer of acifluorfen, (Fig. 2) had no effect on ion leakage (Fig. I). Tentoxin, a fungal toxin which prevents chlorophyll, but not carotenoid, accumulation (14), made tissues more sensitive to acifluorfen (Fig. 3). Norflurazon, which inhibits carotenoid synthesis and thereby causes photobleaching of chlorophyll in white light (2 l-23)) prevents acifluorfen-induced ion leakage. The PS II inhibitors DCMU, bentazon, and atrazine, each at 10 FM, had no effect
I
I
0
2
4 TIME
6 (h)
FIG. 3. Effects of acifluorfen (darkened symbols) on ion leakage from control, tentoxin-treated, or norfurazon-treated cucumber cotyledon discs in white light after a 20-hr dark incubation. The open symbols are treatments without acijluorfen.
MITOCHONDRIAL
time
INVOLVEMENT
(h)
-
FIG. 4. Ion leakage during white light exposure of control (open symbols) und 30 @I acijluorfen-treated (darkened symbols) cotyledon discs xsith (squares) or M’ithout (circles) IO )LM atrazine. The inset contains thejluorescence transients ofacijluorfen-treated disc;, treated (upper curise) or not treated (lotiler curve) with atrazine after the 20-hr dark incubation. but before e.uposwe to white light.
6A-C). After 3 hr of light exposure extensive cerium perhydroxide depositions were noted (Fig. 6C). Although acifluorfen caused thylakoid dilation, no precipitate was observed in chloroplasts or any other part of the cell (Fig. 6A). Localization of superoxide by cytochemical techniques in acifluorfen-treated tissues resulted in staining in the mitochondrial matrix (Fig. 6D). Tissue preincubated in the inactive analog was unstained during the brief incubation in the superoxide detection medium (Fig. 6E). No cytochemical stain for hydrogen peroxide or superoxide was observed after the 20-hr dark incubation in the presence of acifluorfen (not shown).
FIG. 5. Fluorescence transients of control and acifluorfen-treated (30 )LM) cucumber cotyledon discs after 20 hr of darkness (0 hr) and then after exposure to 400 p.E mm3 .tec’ n,hite light .for 3 or 5 hr.
IN
ACIFLUORFEN
ACTION
3-1
Evidence oftoxic oxygen species involvement. Copper-penicillamine complex has ;I high level of superoxide dismutating activity and has been shown to protect planr tissue from paraquat-generated toxic oxygen radical (IO, 24). We found 1 r*.iLI copper-penicillamine afforded virtually complete protection from paraquat damage to cucumber cotyledons as measured by ion leakage (Fig. 7A). Partial protection by Cl’ complex was seen with acifluorfen-treated discs (Figs. 7B). N-[2-(2-oxo-l-imidazolidinyl)ethyl]-N’-phenylurea (EDU). which is known to induce high levels of super’ oxide dismutase and catalase in plant tissues (25). also reduced the effect of acifluoren on leakage (Fig. 8). A number of toxic oxygen radical scavengers and reducing agents were tested for antidotal propertie> (Table 1). Only ethanol and a-tocopherol were effective at the dosages used. possihl!V because of difficulties in uptake of these: compounds into intact plant tissue. Inhibitors of mitochondrial ATP prodirction. Antimycin A and rotenone increased the lag time and reduced the rate of leakage from acifluorfen-treated disks (data noi shown). The efficacy of antimycin A increased with concentration up to about 100 )*M (Fig. 9). The oxidative phosphorylatlon uncouplers dinitrophenol (DNP) and cyanide-r?z-chlorophenylhydrazone (CCC’P I had similar but less pronounced effeifb, (Fig. IO). DISCUSSION
Plant mitochondria are sites of superoxide and, by dismutation, H,O, production (26-3 1). Under normal physiological conditions this process apparently causes no cellular damage. Mitochondrial superoxide and H,Oz are detoxified by mitochondrial superoxide dismutase, glutathione peroxidase, and cytochrome c peroxidase (26). Most cell membranes, including those of the mitochondria, arc very permeable to H,O, (27), so that much H,O, detoxification may occur outside the mitochondrion. Our cytochemical result,, suggest that acifluorfen increases the mi’
372
DUKE,
VAUGHN,
AND
MEEUSEN
FIG. 6. (A) Cytochemicul localization of H,O, (arrows) in a mitochondrion of acif7uorfentreated (30 PM) cucumber cotyledon discs after I-‘lz hr in the light. Note thylakoid dilations, but no precipitation in chloroplast. (B) Same experimental procedure as in (A), but RH 5349 was used in place of acifluorfen. (C) Localization of H,O, (asterisks) in mitochondria of acifluorfen-treated tissue after 3 hr of light. Arrows indicate mitochondrial membrane. (D) Cytochemical localization of superoxide radical (arrows) in the mitochondria of acifluorfen-treated (30 PM) cucumber cotyledon discs after I-t11 hr of exposure to white light after 20 hr dark incubation. (E) Same experimental procedures as in (D), but RH 5349 was used in place of acifluorfen. m, mitochondrion; pj plastid; bar. I pm in A, B, and E, and 0.5 u.rn in C and D.
tochondrial production of superoxide radical rather than inhibiting the protective mechanisms against superoxide radical and its products. If mitochondrial SOD were inhibited by acifluorfen, little or no H,O, should have been observed cytochemically. If acifluorfen reduced the detoxification of H,O,, unusually high levels of superoxide
would not necessarily accompany high H,O, levels. Thus, our cytochemical results suggest that acifluorfen increases mitochondrial production of superoxide anion. Two sites of superoxide production in mitochondria have been identified (26,28, 30); b site, and (i) the ubiquinone-cytochrome (ii) the NADH dehydrogenase site. Our re-
MITOCHONDRIAL TABLE Activity
INVOLVEMENT
IN ACIFLUORFEN
ACTION
373
I
of Various Possible Antidotes Acij7uorfen Activity
against
Reduction (%)
Treatment a-Tocopherol (60 kg/ml) Ethanol (l-2.5%. v/v) DABCO (IO- 100 /.LM) NADH (100 pn/n NAD ( 100 PM) NADPH ( 100 FM) Glutathione (1 Q4) Azide (10-100 )IM) Ascorbate (0. l- I )~kf)
75-80
55-75 0 0 0 0 0 0 0
Note. Results are given as the percentage reduction of acifluorfen damage after 7 to 8 hr of light.
I 2
0
I 4
/ 6
I
TIME (h)
suits are not compatible with either of these as a site of increased superoxide production due to acifluorfen. Antimycin A has been shown to increase superoxide production at the ubiquinone-cytochrome b site (27, 31). Antimycin A apparently lessened the effect of acifluorfen on superoxide production in our system. Rotenone had a similar effect. Our data suggest that electron flow through the mitochondrial electron transport chain is necessary for full activity of acifluorfen.
FIG. 8. Reduction ofthe ejfect of30 FM uc(flrrrwfen on ion leukage by inclusion of 50 )*M EDU fi)r 20 hr in darkness.
These results rule out involvement of the cyanide-resistant alternative respiratory pathway, which is resistant to antimycin A and is not known to produce hydrogen peroxide (29). We reasoned that if superoxide production in this system is dependent on mitochondrial electron flow, uncouplers
‘“O’
0
20
40
time
FIG. 7. Reduction
0
20
40
(h)
of ion leakage from 0.1 )LM parayuat (A) and 30 PM acifluorfen (B) treatment by 1 )IM copper-penicillamine in the light following 19 hi durk incubation. 0. controi: 0, paraquat; n , acijluorfen; A. CP complex: LI. herbicide plus CP comph.
antfmyclo
FIG. 9. Effect of vaqing
A (PM)
concentration.\ c$antinr?c,in A (circles) or of rotenone (squares) on ion leahtrge of 30 PM cr~l~uorfen-trecrted (darkened symbols) or (WItrol (open symbols) c,ucumbrr cotyledon di.~c~.c. Thd discs ,l’ere a.\-posed to 24 hr light after a 2O-hr (iarX incubation.
374
DUKE,
VAUGHN,
AND
MEEUSEN
bicidal injury. Pritchard et al. (5) found oxyfluorfen to have no effect on photosynthetic electron transport, and its action to be independent of photosynthetic electron trans,’ . IOuMCCCP ,/.” port. Furthermore, Yoshimoto and Matsunaka (32) found DPEs to have no effect on 1 superoxide or H,O, production in isolated spinach chloroplasts, although paraquat greatly accelerated production. Taken together, these data indicate that photosynthesis is not involved in acifluorfen-induced damage as with other herbicides, such as bipyridiliums, which cause their effect through photoreduction of the herbicide by PS I, followed by reduction of molecular oxygen by the herbicide radical (11, 12). TIME (h) Leong and Briggs (33) have shown that FIG. 10. Effects ofDNPand CCCPon ion leakage of acifluorfen enhances the blue light-induced 30 PM acijluorfen-treated (darkened symbols) and unabsorbance change of a flavin-cytochrome treated (open symbols) cucumber cotyledon discs. complex in crude membrane preparations might increase acifluorfen-stimulated ion of oat coleoptiles as well as increasing pholeakage. However, both DNP and CCCP totropic responses. The meaning of these slightly reduced leakage, indicating that results in relation to the mechanism of acATP may be involved in the superoxidetion of acifluorfen is clouded by the fact generating process. that norflurazon eliminates the herbicidal Although light is required for acifluorfen activity of acifluorfen, but has no effect on damage, photosynthesis is apparently not phototropism of coleoptiles (34). involved in the light requirement. The only Partial reversal of the effects of aciinvolvement of the plastid previously estab- fluorfen with CP complex, EDU, cr-tocophlished is that of its carotenoids (I). Our erol, and ethanol strongly implicate toxic finding that plants with carotenoids, but oxygen species in the herbicidal action of with very low levels of chlorophyll and no acifluorfen. Except for a-tocopherol, radphotosynthetic function (tentoxin treated), ical scavengers such as DABCO, SOD, are supersensitive to acifluorfen supports DPPD, BHA, or BHT have not effectively this view. No effect of acifluorfen was de- protected against acifluorfen damage (1). In tected in tissues without carotenoids (nor- our system and in theirs, inadequate peneflurazon treated). These results substantration to the site of action may have pretiate previous findings from experiments vented some of these compounds from utilizing achlorophyllous mutants and ca- acting as protectants. rotenoid synthesis-inhibiting herbicides (2, The implication of the mitochondrion in 4, 5, 7-9). the mode of action of acifluorfen compliFurthermore, inhibitors of PS II (DCMU, cates the model for its mechanism. We proatrazine, and bentazon) had little or no ef- pose the following mechanism to explain fect on acifluorfen activity. Orr and Hess our results. Apparently, in higher plants, (8) obtained similar results with DCMU. light causes the production of a toxic speThey also found DBMIB (2,5 - bromo - 3 - cies by the herbicide using a carotenoid as methyl - 6 - isopropyl - p - benzoquinone), a the photoreceptor. The toxic species apcyclic photophosphorylation inhibitor, to parently then acts in the mitochondrion to have no effect on acifluorfen-caused her- cause excessive superoxide production. If
MITOCHONDRIAL
INVOLVEMENT
so, the site of activation is likely to be the outer plastid envelope, a site of high carotenoid levels (35) that is often closely appressed to adjacent mitochondria. This interpretation fits the etiology of ultrastructural damage, because the plastid envelope, mitochondrion, plasmalemma, and tonoplast show structual damage before the thylakoids are noticeably affected ((1, 8); and Vaughn, unpublished). Acifluorfen-induced production of high levels of toxic oxygen species in the mitochondrion apparently leads to autocatalytic levels of lipid peroxides. as occurs with other bleaching herbicides (11). In this case, however, the source of these extremely destructive species is the mitochondrion rather than the chloroplast. Because two organelles appear to be involved in this mechanism, cell-free systems to determine the molecular site(s) of action of acifluorfen may be complicated. ACKNOWLEDGMENTS We thank Al Lane. Louise Giachelli, and Susan Lokey for their expert technical assistance. Technicalgrade norflurazon was generously provided by the Sandoz Corporation.
IN
8.
9.
10.
I I.
12.
13.
14.
IS.
REFERENCES I. G. L. Orr and F. D. Hess, Proposed site(s) of action of new diphenyl ether herbicides. in “Biochemical Responses Induced by Herbicides” (D. E. Moreland. J. B. St. John, and F. D. Hess, Eds.). p. 131. Amer. Chem. Sot.. Washington, D.C.. 1982. 2. 0. Fadayomi and G. F. Warren, The light requirement for herbicidal activity of diphenyl ethers. Weed Sci. 24, 598 (1976). 3. S. Matsunaka, Acceptor of light energy in photoactivation of diphenyl ether herbicides, J. Agric. Food Chum. 17, 171 (1969). 4. G. L. Orr and E D. Hess. Characterization of herbicidal injury by acifluorfen-methyl in excised cucumber (Cucrrmis sativus L.) cotyledons. Pestic. Biochrm. Physiol. 16, 171 (1981). 5. M. K. Pritchard, G. F. Warren, and R. A. Dilley. Site of action of oxyfluorfen, Weed Sci. 28, 640 ( 1980). 6. D. E. Vanstone and E. H. Stobbe, Light requirement of the diphenylether herbicide oxyfluorfen, Weed Sci. 27, 88 (1979). 7. R. M. Devlin, S. J. Karczmarczyk, and I. I.
16.
17.
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20.
ACIFI.UORFEN
ACTION
\7i
Zbiec, Influence of norflurazon on the actlccttion of substituted diphenylether herbicide\ hi light. Weed Sci. 31, 109 (1983). G. L. Orr and F. D. Hess, Mechanism of actior! of the diphenyl ether herbicide acitluorfen methyl in excised cucumber (Cucumi.\ strtii~u~ L.) cotyledons, Planr Pllysio[. 69, 502 (19X?). H. Mohr and H. Drumm-Herrel. Coaction be tween phytochrome and blue/UV light in anthocyanin synthesis in seedling?. Phsiol. f’lr,ni 58. 408 (1983). K. J. Kunert and P. BBger. The bleaching effec! 01’ the diphenyl ether oxyfluorfen. l+‘c~& SC,;. 29 169 (1981). B. Halliwell. The toxic effects of oxygen on plan1 tissues. in “Superoxide Dismutase“ !I,. 1 Oh erley. Ed.). Vol. I. p. 89. CRC Pre\%. Boc;. Raton. Florida. 1982. K. C. Vaughn and S. 0. Duke. In ,\itu locali~atior of the \ites of paraquat action. Plrrnl (‘rs// 5 \,ircv~. 6, I3 I 1983). S. 0. Duke. K. C. Vaughn. and S. H. Duke. Ef fects of norflurazon (San 97891 on light-In creased extractable nitrate reducta<;e attic I[? iI> soybean [(;/ycine tnc1.r IL.) Merr. / \ecdling\ Plnrrt (‘e/I Eri~~inm. 5, 155 (1982). S. 0. Duke. J. L. Wickliff. K. C. Vaughn. ;,nd R. N. Paul. Tentoxin does not cause chloroylk in greening mung bean leaves by inhibiting pho tophosphorylation. Phvsiol. Plr~ot. 56, 48(1983). P. J. Birker and H. C. Freeman. Structure. pr,,p erties and function of a copper (I)-copper ill’ complex of D-penicillamine. J. .-tr~<,r. (-I;( f?i .So(~. 99, 6890 (1977). E. Lengfelder and E. Elstner, Determination tr: the superoxide dismutating activity cjf D-pcm cillamine copper. Hoppr-Sr\,/er’5 /. Ph~,;:rjl Cl7rm. 359, 751 (1978). J. Friend and A. Mayer. The enzymatic de\truc tion of carotenoids by isolated chloropla~t~ Bioc~him. Biophys. A<,ttr 41, 421 ( 1960). S. E. Hetherington and R. M. Smillie. HumidIt\sensitive degreening and regreening of ff~t!:vci nitidtr Labill. as followed by change\ In chlo rophyll fluorescence. .4!r.~t. ./. Pltrfrr Pl:\,\iii/ 0 5x7 11982). R. 1‘. Briggs. D. B. Drath. M. L. Karnobsky. ,~nd M. T. Karnovsky. Localization of Ni\DH oxi dase on the surface of human polymorphonuclear leucocyte5 by a new cytocheml
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21. R. M. Devlin, M. J. Kisiel, and S. J. Karczmarezyk, Chlorophyll production and chloroplast development in norflurazon-treated plants, Weed Res. 16, 125 (1976). 22. H. W. Ktimmel and L. H. Grimme, Inhibition of carotenoid biosynthesis in green algae by SAN 6706: Accumulation of phytoene and phytofluene in Chlorella jicsca. Z. Naturforsch. 3Oc, 333 (1975). 23. P. C. Bartels and C. W. Watson, Inhibition of carotenoid synthesis by fluridone and norflurazon, Weed Sci. 26, 198 (1978). 24. R. J. Youngman and A. D. Dodge, Mechanism of paraquat action: Inhibition of the herbicidal effect by a copper chelate with superoxide dismutating activity, Z. Naturforsch. 34c, 1032 (1979). 25. E. H. Lee and J. H. Bennett. Superoxide dismutase: A possible protective enzyme against ozone injury in snap beans (Phaseolus vulgaris L.), Plant Physiol. 69, 1444 (1982). 26. A. Boveris and E. Cadenas, Production of superoxide radicals and hydrogen peroxide in mitochondria, in “Superoxide Dismutase” (L. L. Oberley, Ed.), Vol. 2. p. 15, CRC Press, Boca Raton, Florida 1982. 27. H. J. Forman and A. Boveris, Superoxide radical and hydrogen peroxide in mitochondria. in “Free Radicals in Biology” (W. A. Pryor, Ed.), Vol. V, p. 65, Academic Press, New York. 1982.
AND MEEUSEN 28. T. Ramasarma, Generation of Hz02 in biomembranes, Biochim. Biophys. Acta 694, 69 (1982). 29. P. R. Rich, Quinol oxidation in Arum maculatum mitochondria and its application to the assay, solubilization and partial purification of the alternative oxidase, FEBS Lett. 96, 252 (1978). 30. P. R. Rich and W. D. Bonner, Jr., The sites of superoxide anion generation in higher plant mitochondria, Arch. Biochem. Biophys. 188, 206 (1978). 31. P. R. Rich, A. Boveris. W. D. Bonner, Jr., and A. L. Moore, Hydrogen peroxide generation by the alternate oxidase of higher plants, Biochem. Biophys. Res. Commun. 71, 695 (1976). 32. T. Yoshimoto and S. Matsunaka. The mode of action of diphenylether herbicides: The scavenging of active oxygens is not inhibited by diphenylethers. in “Abstracts of the 5th International Congress of Pesticide Chemistry,” Vol. 5, p. 62, 1982. 33. T.-L. Leong and W. R. Briggs. Evidence from studies with acifluorfen for participation of a flavin-cytochrome complex in blue light photoreception for phototropism of oat coleoptiles. Plant Physiol. 70, 875 (1982). 34. R. D. Vierstra and K. L. Poff. Role of carotenoids in the phototropic response of corn seedlings, Plant Physiol. 68, 798 (1981). 35. R. J. Deuce and J. Joyard, Structure and function of the plastid envelope, Advan. Rot. Res. 7, 1 (1979).
BIOCHEMISTRY
Mitochondrial STEPHEN Southern
Weed
Science
AND
PHYSIOLOGY
21, 368-376 (1984)
Involvement 0. DUKE, Laboratory, and Haas
in the Mode of Action of Acifluorfen
KEVIN
C. VAUGHN,
USDA, Company,
ARS, P.O. Box 22.5, Stoneville, Spring House. Pennsylvania
L. MEEUSEN*
AND RONALD
Mississippi 19477
38776,
and *Rohm
Received September 9, 1983; accepted November 10, 1983 The herbicidal action of acifluorfen {5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid} was studied with greened and expanded discs from cotyledons of cucumber (Cucumis sativus L.). Discs were floated on various treatment solutions for 20 hr in darkness before exposure to 400 pE m-’ set -’ of white light. Herbicide damage, as measured by electrolyte leakage, began in the light after a I- to 2-hr lag period. Cytochemical methods at the ultrastructural level indicated that acifluorfen caused marked increases in production of superoxide radical and hydrogen peroxide in the mitochondrion, but not in the plastid. The mitochondrial inhibitors antimycin A, rotenone, CCCP, and DNP antagonized the action of acifluorfen, lengthening the lag period and reducing the rate of electrolyte leakage. Ethanol, a-tocopherol, N-[2-(2-oxo-l-imidazolidinyl)ethyl]-N’-phenylurea, and copper-penicillin also lengthened the lag phase and slowed the rate of damage, indicating that acifluorfen damage involves toxic oxygen species. PS II-inhibiting levels of DCMU, atrazine, or bentazon did not affect acifluorfen-induced ion leakage. Yellow tissue produced by treatment with tentoxin was supersensitive to acifluorfen, but white tissue produced by treatment with nortlurazon was relatively insensitive. These data indicate that, after an initial carotenoid-acifluorfen interaction, the mitochondrion is involved in production of toxic oxygen species and that this process is closely tied to the mechanism of action of this herbicide.
information into a model describing a mechanism of action of acifluorfen which accounts for all major experimental results, but which does not designate the subcellular location of the proposed steps of the mechanism. Using a cytochemical procedure for the localization of hydrogen peroxide with which we previously studied paraquat mechanism of action (12), acifluorfen was found to cause high levels of hydrogen peroxide in mitochondria of green tissues. This finding, and subsequent studies which support the view that the mitochondrion and respiration are involved in the mechanism of action of acifluorfen, are reported in this paper. No previous studies have suggested involvement of the mitochondrion in the mechanism of action of acifluorfen.
INTRODUCTION
The mechanism of action of the diphenyl ether (DPE) herbicide, acifluorfen {5-[2chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid}, is unknown (1). The effects of acifluorfen and related ortholpara-substituted DPEs are unique in that light is required for herbicidal activity; however, photosynthesis is not involved (l-6). Apparently, carotenoids are the photoreceptors for this light-dependent activity because plants lacking carotenoids for genetic or chemical reasons are relatively insensitive to these DPE herbicides (2, 4, 7-9). Oxygen enhances the activity of DPE herbicides, and much of the etiology of damage, such as production of thiobarbituric acid-reacting materials (8) and ethane (IO), indicates that unsaturated lipid peroxidation by toxic oxygen species may be involved (1, 11). Protection against injury with a-tocopherol further supports this view (8). Orr and Hess (1) incorporated this 0048-3575/84 $3.00 Copyright All *:“Ltr
0 1984 by Academic Press. Inc. “FrG.^..,.A,.^r:l-” :” ““,, F..-- -m”o-.ioA
MATERIALS
AND METHODS
Plant material. Cucumber umis sativus L. (cv. Straight
seeds [CztcEight)] were planted in flats in a commercial greenhouse
368
MITOCHONDRIAL
INVOLVEMENT
IN ACIFLUORFEN ACTI3N
369
substrate (Jiffy-mix; JPA, West Chicago, Ill.)’ and watered with distilled water. Plants were treated with norflurazon [4chloro-Qmethylamino)-2-(cr,cY,a,-trifluorom-tolyl)-3-(2H)-pyridazinone] or tentoxin by soaking seeds in solutions (saturated and 100 mM, respectively) of the compounds before planting as reported earlier ( 13, 14). The seedlings were grown at 25 + 1°C under continuous white light 10 to 50 PE In-? set PI in a growth chamber at 90 t 5% relative humidity. Leaf discs (7 mm diameter) were punched from expanded cotyledons of 8- to I?-day-old seedlings with a sharpened cork borer. All discs for a particular experiment were floated, adaxial side up, on a solution of 2% (w/v) sucrose in I mM 2-(4-morpholino)ethane sulfonic acid (Mes) (pH 6.5) and swirled several times to randomize before selection of discs for each treatment. Trerrtment. Ten cotyledon discs were placed adaxial side up on 5 ml of 1 mM Mes (pH 6.5) with 2% (w/v) sucrose in a 6-cm diameter styrene Petri dish. All chemical treatments were prepared in this buffered sucrose solution with technical-grade reagents of better than 90% purity. The test level of a-tocopherol was dissolved in methanol and placed in the Petri dish, and the methanol was evaporated before buffer with or without acifluorfen was added. This treatment method was used because of the high degree of insolubility of a-tocopherol in aqueous solution. The discs were incubated on treatment solutions for 20 to 22 hr in complete darkness at 25°C and then transferred to continuous white light (400 p.E rn.-’ set ‘) at 25°C. Periodically conductivity of the treatment solution was assayed with a conductivity meter (Model 4503A. Amber Science)] having the capacity to assay and return 1 ml of solution to the Petri dish.
Conductivity for each treatment wa‘, plotted as the change in conductivity from that recorded at the beginning of the light exposure. All experiments were repeated two to six times and, in each repetition ol‘ an experiment, each treatment was repli.cated two or three times. All presented data are averages from a representative expcri.ment. The quantitative effects of a treat-ment often varied considerably betwcerl repetitions. However, the qualitative et’-fects and relative effects of various treatments were uniform between treatment\. Copper-penicillamine (CP) complex ti’at, prepared as described by Birker and Freeman ( IS). Its superoxide dismutatin:: activity was confirmed spectrophotometritally with the crocin-bleaching assay of Lengfelder and Elstner ( 16). Crocin was cxtracted from saffron according to Friend and Mayer (17). The more common assay4 for superoxide dismutating activity involving reduction of cytochrome c or autoxidation of epinephrine were avoided because free copper ions were found to imerfere (R. L. Meeusen. unpublished results). On a weight basis, this CP complex had virtually the same activity as commerciall! available bovine blood superoxide dismutase (SOD) (Sigma, St. Louis. Mo.)’ in protecting crocin from oxidation by enzyrnatically generated superoxide radical (data not shown). Fluorescent trvrnsients. In r*ilw fluorescence transients of cotyledon discs were measured with a Model SF-20 fluoromeref (Richard Brancker Research, Ltd.. Ottawa. Canada).’ A 30-sec. dark preincubation wasY used before the transient was determined. The technique was the same as that of Hetherington and Smillie (1X). Cytoc~lzevlistry. Tissue within 1 mm ot‘ the edge of the cotyledon disc was used for all cytochemical assays. Hydrogen pcroxide was visualized at the ultrastructuml ’ Mention of a trademark, proprietary product or level as reported earlier ( 12) by formation vendor does not constitute a guarantee or warranty of of cerium perhydroxide (19). CeCl, ( 1 m.M) the product by the U.S. Dep. Agric. and does not was added to treatment solutions present imply its approval to the exclusion of other products or vendors that may also be suitable.
from the beginning of the experiment. Ami,-
370
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MEEUSEN
0 c
.-
0 E
I50m c ," u ,.
c 0 0
FIG.
change
0
0.3
I concentration
3
IO
30
IO0
(PM)
FIG. 1. Dose-response of acifluorfen on ion leakage of cucumber cotyledon discs preincubated 20 hr in darkness and then exposed to 21 hr of white light.
notriazole was not used to block catalase activity as in a previous study (12). Tissue pieces (1 mm*) for superoxide localization from treated leaves were fixed for 30 min in 4% (w/v) paraformaldehyde at 4”C, washed with 0.1 M phosphate buffer, pH 7.2, with 5% sucrose, and incubated in the medium of Karnovsky and Robinson (20). Postfixation was carried out in 1% 0~0, for 2 hr at room temperature. Dehydration and embedding were carried out as for H,O, localization (12).
2. Structure in position
of RH 5349. The arrow from acifluorfen.
shon*s
the
on ion leakage. Results for each compound were similar to those shown for atrazine (Fig. 4). Increased variable fluorescence at the end of the 20-hr dark period indicated that electron flow from PS II to PS I was effectively blocked by each of the PS II inhibitors (inset to Fig. 4) Acifluorfen itself had little or no effect on the fluorescence transfer after the 20-hr dark period; however, on illumination, the variable portion of the fluorescence transient decreased with time (Fig. 5). Cytochemical evidence of acifuorfencaused mitochondrial superoxide and hydrogen peroxide production. Cerium perhydroxide precipitates, indicators of hydrogen peroxide presence, were observed in the mitochondria of acifluorfen-treated tissue in the light, whereas no precipitates were seen in RH 5349-treated tissues (Figs.
RESULTS
Evidence of nonphotosynthetic light requirement. In preliminary experiments we found that 30 PM acifluorfen caused little or no ion leakage in the dark, but caused rapid leakage in the light (Figs. 1 & 3) after a l- to 3-hr lag period. All further experiments were conducted with this concentration of the herbicide. RH 5349, an herbicidally inactive isomer of acifluorfen, (Fig. 2) had no effect on ion leakage (Fig. I). Tentoxin, a fungal toxin which prevents chlorophyll, but not carotenoid, accumulation (14), made tissues more sensitive to acifluorfen (Fig. 3). Norflurazon, which inhibits carotenoid synthesis and thereby causes photobleaching of chlorophyll in white light (2 l-23)) prevents acifluorfen-induced ion leakage. The PS II inhibitors DCMU, bentazon, and atrazine, each at 10 FM, had no effect
I
I
0
2
4 TIME
6 (h)
FIG. 3. Effects of acifluorfen (darkened symbols) on ion leakage from control, tentoxin-treated, or norfurazon-treated cucumber cotyledon discs in white light after a 20-hr dark incubation. The open symbols are treatments without acijluorfen.
MITOCHONDRIAL
time
INVOLVEMENT
(h)
-
FIG. 4. Ion leakage during white light exposure of control (open symbols) und 30 @I acijluorfen-treated (darkened symbols) cotyledon discs xsith (squares) or M’ithout (circles) IO )LM atrazine. The inset contains thejluorescence transients ofacijluorfen-treated disc;, treated (upper curise) or not treated (lotiler curve) with atrazine after the 20-hr dark incubation. but before e.uposwe to white light.
6A-C). After 3 hr of light exposure extensive cerium perhydroxide depositions were noted (Fig. 6C). Although acifluorfen caused thylakoid dilation, no precipitate was observed in chloroplasts or any other part of the cell (Fig. 6A). Localization of superoxide by cytochemical techniques in acifluorfen-treated tissues resulted in staining in the mitochondrial matrix (Fig. 6D). Tissue preincubated in the inactive analog was unstained during the brief incubation in the superoxide detection medium (Fig. 6E). No cytochemical stain for hydrogen peroxide or superoxide was observed after the 20-hr dark incubation in the presence of acifluorfen (not shown).
FIG. 5. Fluorescence transients of control and acifluorfen-treated (30 )LM) cucumber cotyledon discs after 20 hr of darkness (0 hr) and then after exposure to 400 p.E mm3 .tec’ n,hite light .for 3 or 5 hr.
IN
ACIFLUORFEN
ACTION
3-1
Evidence oftoxic oxygen species involvement. Copper-penicillamine complex has ;I high level of superoxide dismutating activity and has been shown to protect planr tissue from paraquat-generated toxic oxygen radical (IO, 24). We found 1 r*.iLI copper-penicillamine afforded virtually complete protection from paraquat damage to cucumber cotyledons as measured by ion leakage (Fig. 7A). Partial protection by Cl’ complex was seen with acifluorfen-treated discs (Figs. 7B). N-[2-(2-oxo-l-imidazolidinyl)ethyl]-N’-phenylurea (EDU). which is known to induce high levels of super’ oxide dismutase and catalase in plant tissues (25). also reduced the effect of acifluoren on leakage (Fig. 8). A number of toxic oxygen radical scavengers and reducing agents were tested for antidotal propertie> (Table 1). Only ethanol and a-tocopherol were effective at the dosages used. possihl!V because of difficulties in uptake of these: compounds into intact plant tissue. Inhibitors of mitochondrial ATP prodirction. Antimycin A and rotenone increased the lag time and reduced the rate of leakage from acifluorfen-treated disks (data noi shown). The efficacy of antimycin A increased with concentration up to about 100 )*M (Fig. 9). The oxidative phosphorylatlon uncouplers dinitrophenol (DNP) and cyanide-r?z-chlorophenylhydrazone (CCC’P I had similar but less pronounced effeifb, (Fig. IO). DISCUSSION
Plant mitochondria are sites of superoxide and, by dismutation, H,O, production (26-3 1). Under normal physiological conditions this process apparently causes no cellular damage. Mitochondrial superoxide and H,Oz are detoxified by mitochondrial superoxide dismutase, glutathione peroxidase, and cytochrome c peroxidase (26). Most cell membranes, including those of the mitochondria, arc very permeable to H,O, (27), so that much H,O, detoxification may occur outside the mitochondrion. Our cytochemical result,, suggest that acifluorfen increases the mi’
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FIG. 6. (A) Cytochemicul localization of H,O, (arrows) in a mitochondrion of acif7uorfentreated (30 PM) cucumber cotyledon discs after I-‘lz hr in the light. Note thylakoid dilations, but no precipitation in chloroplast. (B) Same experimental procedure as in (A), but RH 5349 was used in place of acifluorfen. (C) Localization of H,O, (asterisks) in mitochondria of acifluorfen-treated tissue after 3 hr of light. Arrows indicate mitochondrial membrane. (D) Cytochemical localization of superoxide radical (arrows) in the mitochondria of acifluorfen-treated (30 PM) cucumber cotyledon discs after I-t11 hr of exposure to white light after 20 hr dark incubation. (E) Same experimental procedures as in (D), but RH 5349 was used in place of acifluorfen. m, mitochondrion; pj plastid; bar. I pm in A, B, and E, and 0.5 u.rn in C and D.
tochondrial production of superoxide radical rather than inhibiting the protective mechanisms against superoxide radical and its products. If mitochondrial SOD were inhibited by acifluorfen, little or no H,O, should have been observed cytochemically. If acifluorfen reduced the detoxification of H,O,, unusually high levels of superoxide
would not necessarily accompany high H,O, levels. Thus, our cytochemical results suggest that acifluorfen increases mitochondrial production of superoxide anion. Two sites of superoxide production in mitochondria have been identified (26,28, 30); b site, and (i) the ubiquinone-cytochrome (ii) the NADH dehydrogenase site. Our re-
MITOCHONDRIAL TABLE Activity
INVOLVEMENT
IN ACIFLUORFEN
ACTION
373
I
of Various Possible Antidotes Acij7uorfen Activity
against
Reduction (%)
Treatment a-Tocopherol (60 kg/ml) Ethanol (l-2.5%. v/v) DABCO (IO- 100 /.LM) NADH (100 pn/n NAD ( 100 PM) NADPH ( 100 FM) Glutathione (1 Q4) Azide (10-100 )IM) Ascorbate (0. l- I )~kf)
75-80
55-75 0 0 0 0 0 0 0
Note. Results are given as the percentage reduction of acifluorfen damage after 7 to 8 hr of light.
I 2
0
I 4
/ 6
I
TIME (h)
suits are not compatible with either of these as a site of increased superoxide production due to acifluorfen. Antimycin A has been shown to increase superoxide production at the ubiquinone-cytochrome b site (27, 31). Antimycin A apparently lessened the effect of acifluorfen on superoxide production in our system. Rotenone had a similar effect. Our data suggest that electron flow through the mitochondrial electron transport chain is necessary for full activity of acifluorfen.
FIG. 8. Reduction ofthe ejfect of30 FM uc(flrrrwfen on ion leukage by inclusion of 50 )*M EDU fi)r 20 hr in darkness.
These results rule out involvement of the cyanide-resistant alternative respiratory pathway, which is resistant to antimycin A and is not known to produce hydrogen peroxide (29). We reasoned that if superoxide production in this system is dependent on mitochondrial electron flow, uncouplers
‘“O’
0
20
40
time
FIG. 7. Reduction
0
20
40
(h)
of ion leakage from 0.1 )LM parayuat (A) and 30 PM acifluorfen (B) treatment by 1 )IM copper-penicillamine in the light following 19 hi durk incubation. 0. controi: 0, paraquat; n , acijluorfen; A. CP complex: LI. herbicide plus CP comph.
antfmyclo
FIG. 9. Effect of vaqing
A (PM)
concentration.\ c$antinr?c,in A (circles) or of rotenone (squares) on ion leahtrge of 30 PM cr~l~uorfen-trecrted (darkened symbols) or (WItrol (open symbols) c,ucumbrr cotyledon di.~c~.c. Thd discs ,l’ere a.\-posed to 24 hr light after a 2O-hr (iarX incubation.
374
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bicidal injury. Pritchard et al. (5) found oxyfluorfen to have no effect on photosynthetic electron transport, and its action to be independent of photosynthetic electron trans,’ . IOuMCCCP ,/.” port. Furthermore, Yoshimoto and Matsunaka (32) found DPEs to have no effect on 1 superoxide or H,O, production in isolated spinach chloroplasts, although paraquat greatly accelerated production. Taken together, these data indicate that photosynthesis is not involved in acifluorfen-induced damage as with other herbicides, such as bipyridiliums, which cause their effect through photoreduction of the herbicide by PS I, followed by reduction of molecular oxygen by the herbicide radical (11, 12). TIME (h) Leong and Briggs (33) have shown that FIG. 10. Effects ofDNPand CCCPon ion leakage of acifluorfen enhances the blue light-induced 30 PM acijluorfen-treated (darkened symbols) and unabsorbance change of a flavin-cytochrome treated (open symbols) cucumber cotyledon discs. complex in crude membrane preparations might increase acifluorfen-stimulated ion of oat coleoptiles as well as increasing pholeakage. However, both DNP and CCCP totropic responses. The meaning of these slightly reduced leakage, indicating that results in relation to the mechanism of acATP may be involved in the superoxidetion of acifluorfen is clouded by the fact generating process. that norflurazon eliminates the herbicidal Although light is required for acifluorfen activity of acifluorfen, but has no effect on damage, photosynthesis is apparently not phototropism of coleoptiles (34). involved in the light requirement. The only Partial reversal of the effects of aciinvolvement of the plastid previously estab- fluorfen with CP complex, EDU, cr-tocophlished is that of its carotenoids (I). Our erol, and ethanol strongly implicate toxic finding that plants with carotenoids, but oxygen species in the herbicidal action of with very low levels of chlorophyll and no acifluorfen. Except for a-tocopherol, radphotosynthetic function (tentoxin treated), ical scavengers such as DABCO, SOD, are supersensitive to acifluorfen supports DPPD, BHA, or BHT have not effectively this view. No effect of acifluorfen was de- protected against acifluorfen damage (1). In tected in tissues without carotenoids (nor- our system and in theirs, inadequate peneflurazon treated). These results substantration to the site of action may have pretiate previous findings from experiments vented some of these compounds from utilizing achlorophyllous mutants and ca- acting as protectants. rotenoid synthesis-inhibiting herbicides (2, The implication of the mitochondrion in 4, 5, 7-9). the mode of action of acifluorfen compliFurthermore, inhibitors of PS II (DCMU, cates the model for its mechanism. We proatrazine, and bentazon) had little or no ef- pose the following mechanism to explain fect on acifluorfen activity. Orr and Hess our results. Apparently, in higher plants, (8) obtained similar results with DCMU. light causes the production of a toxic speThey also found DBMIB (2,5 - bromo - 3 - cies by the herbicide using a carotenoid as methyl - 6 - isopropyl - p - benzoquinone), a the photoreceptor. The toxic species apcyclic photophosphorylation inhibitor, to parently then acts in the mitochondrion to have no effect on acifluorfen-caused her- cause excessive superoxide production. If
MITOCHONDRIAL
INVOLVEMENT
so, the site of activation is likely to be the outer plastid envelope, a site of high carotenoid levels (35) that is often closely appressed to adjacent mitochondria. This interpretation fits the etiology of ultrastructural damage, because the plastid envelope, mitochondrion, plasmalemma, and tonoplast show structual damage before the thylakoids are noticeably affected ((1, 8); and Vaughn, unpublished). Acifluorfen-induced production of high levels of toxic oxygen species in the mitochondrion apparently leads to autocatalytic levels of lipid peroxides. as occurs with other bleaching herbicides (11). In this case, however, the source of these extremely destructive species is the mitochondrion rather than the chloroplast. Because two organelles appear to be involved in this mechanism, cell-free systems to determine the molecular site(s) of action of acifluorfen may be complicated. ACKNOWLEDGMENTS We thank Al Lane. Louise Giachelli, and Susan Lokey for their expert technical assistance. Technicalgrade norflurazon was generously provided by the Sandoz Corporation.
IN
8.
9.
10.
I I.
12.
13.
14.
IS.
REFERENCES I. G. L. Orr and F. D. Hess, Proposed site(s) of action of new diphenyl ether herbicides. in “Biochemical Responses Induced by Herbicides” (D. E. Moreland. J. B. St. John, and F. D. Hess, Eds.). p. 131. Amer. Chem. Sot.. Washington, D.C.. 1982. 2. 0. Fadayomi and G. F. Warren, The light requirement for herbicidal activity of diphenyl ethers. Weed Sci. 24, 598 (1976). 3. S. Matsunaka, Acceptor of light energy in photoactivation of diphenyl ether herbicides, J. Agric. Food Chum. 17, 171 (1969). 4. G. L. Orr and E D. Hess. Characterization of herbicidal injury by acifluorfen-methyl in excised cucumber (Cucrrmis sativus L.) cotyledons. Pestic. Biochrm. Physiol. 16, 171 (1981). 5. M. K. Pritchard, G. F. Warren, and R. A. Dilley. Site of action of oxyfluorfen, Weed Sci. 28, 640 ( 1980). 6. D. E. Vanstone and E. H. Stobbe, Light requirement of the diphenylether herbicide oxyfluorfen, Weed Sci. 27, 88 (1979). 7. R. M. Devlin, S. J. Karczmarczyk, and I. I.
16.
17.
18.
19.
20.
ACIFI.UORFEN
ACTION
\7i
Zbiec, Influence of norflurazon on the actlccttion of substituted diphenylether herbicide\ hi light. Weed Sci. 31, 109 (1983). G. L. Orr and F. D. Hess, Mechanism of actior! of the diphenyl ether herbicide acitluorfen methyl in excised cucumber (Cucumi.\ strtii~u~ L.) cotyledons, Planr Pllysio[. 69, 502 (19X?). H. Mohr and H. Drumm-Herrel. Coaction be tween phytochrome and blue/UV light in anthocyanin synthesis in seedling?. Phsiol. f’lr,ni 58. 408 (1983). K. J. Kunert and P. BBger. The bleaching effec! 01’ the diphenyl ether oxyfluorfen. l+‘c~& SC,;. 29 169 (1981). B. Halliwell. The toxic effects of oxygen on plan1 tissues. in “Superoxide Dismutase“ !I,. 1 Oh erley. Ed.). Vol. I. p. 89. CRC Pre\%. Boc;. Raton. Florida. 1982. K. C. Vaughn and S. 0. Duke. In ,\itu locali~atior of the \ites of paraquat action. Plrrnl (‘rs// 5 \,ircv~. 6, I3 I 1983). S. 0. Duke. K. C. Vaughn. and S. H. Duke. Ef fects of norflurazon (San 97891 on light-In creased extractable nitrate reducta<;e attic I[? iI> soybean [(;/ycine tnc1.r IL.) Merr. / \ecdling\ Plnrrt (‘e/I Eri~~inm. 5, 155 (1982). S. 0. Duke. J. L. Wickliff. K. C. Vaughn. ;,nd R. N. Paul. Tentoxin does not cause chloroylk in greening mung bean leaves by inhibiting pho tophosphorylation. Phvsiol. Plr~ot. 56, 48(1983). P. J. Birker and H. C. Freeman. Structure. pr,,p erties and function of a copper (I)-copper ill’ complex of D-penicillamine. J. .-tr~<,r. (-I;( f?i .So(~. 99, 6890 (1977). E. Lengfelder and E. Elstner, Determination tr: the superoxide dismutating activity cjf D-pcm cillamine copper. Hoppr-Sr\,/er’5 /. Ph~,;:rjl Cl7rm. 359, 751 (1978). J. Friend and A. Mayer. The enzymatic de\truc tion of carotenoids by isolated chloropla~t~ Bioc~him. Biophys. A<,ttr 41, 421 ( 1960). S. E. Hetherington and R. M. Smillie. HumidIt\sensitive degreening and regreening of ff~t!:vci nitidtr Labill. as followed by change\ In chlo rophyll fluorescence. .4!r.~t. ./. Pltrfrr Pl:\,\iii/ 0 5x7 11982). R. 1‘. Briggs. D. B. Drath. M. L. Karnobsky. ,~nd M. T. Karnovsky. Localization of Ni\DH oxi dase on the surface of human polymorphonuclear leucocyte5 by a new cytocheml
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21. R. M. Devlin, M. J. Kisiel, and S. J. Karczmarezyk, Chlorophyll production and chloroplast development in norflurazon-treated plants, Weed Res. 16, 125 (1976). 22. H. W. Ktimmel and L. H. Grimme, Inhibition of carotenoid biosynthesis in green algae by SAN 6706: Accumulation of phytoene and phytofluene in Chlorella jicsca. Z. Naturforsch. 3Oc, 333 (1975). 23. P. C. Bartels and C. W. Watson, Inhibition of carotenoid synthesis by fluridone and norflurazon, Weed Sci. 26, 198 (1978). 24. R. J. Youngman and A. D. Dodge, Mechanism of paraquat action: Inhibition of the herbicidal effect by a copper chelate with superoxide dismutating activity, Z. Naturforsch. 34c, 1032 (1979). 25. E. H. Lee and J. H. Bennett. Superoxide dismutase: A possible protective enzyme against ozone injury in snap beans (Phaseolus vulgaris L.), Plant Physiol. 69, 1444 (1982). 26. A. Boveris and E. Cadenas, Production of superoxide radicals and hydrogen peroxide in mitochondria, in “Superoxide Dismutase” (L. L. Oberley, Ed.), Vol. 2. p. 15, CRC Press, Boca Raton, Florida 1982. 27. H. J. Forman and A. Boveris, Superoxide radical and hydrogen peroxide in mitochondria. in “Free Radicals in Biology” (W. A. Pryor, Ed.), Vol. V, p. 65, Academic Press, New York. 1982.
AND MEEUSEN 28. T. Ramasarma, Generation of Hz02 in biomembranes, Biochim. Biophys. Acta 694, 69 (1982). 29. P. R. Rich, Quinol oxidation in Arum maculatum mitochondria and its application to the assay, solubilization and partial purification of the alternative oxidase, FEBS Lett. 96, 252 (1978). 30. P. R. Rich and W. D. Bonner, Jr., The sites of superoxide anion generation in higher plant mitochondria, Arch. Biochem. Biophys. 188, 206 (1978). 31. P. R. Rich, A. Boveris. W. D. Bonner, Jr., and A. L. Moore, Hydrogen peroxide generation by the alternate oxidase of higher plants, Biochem. Biophys. Res. Commun. 71, 695 (1976). 32. T. Yoshimoto and S. Matsunaka. The mode of action of diphenylether herbicides: The scavenging of active oxygens is not inhibited by diphenylethers. in “Abstracts of the 5th International Congress of Pesticide Chemistry,” Vol. 5, p. 62, 1982. 33. T.-L. Leong and W. R. Briggs. Evidence from studies with acifluorfen for participation of a flavin-cytochrome complex in blue light photoreception for phototropism of oat coleoptiles. Plant Physiol. 70, 875 (1982). 34. R. D. Vierstra and K. L. Poff. Role of carotenoids in the phototropic response of corn seedlings, Plant Physiol. 68, 798 (1981). 35. R. J. Deuce and J. Joyard, Structure and function of the plastid envelope, Advan. Rot. Res. 7, 1 (1979).