Pyrazole phenyl ether herbicides inhibit protoporphyrinogen oxidase

PESTICIDE

BIOCHEMtSTRY

AND

PHYSIOLOGY

4,

236-245 (lwt)

Pyrazole Phenyl Ether Herbicides Inhibit Protoporphyrinogen Oxidase TIMOTHY D. SHERMAN,* MARY V. DUKE,* ROBERT D. CLARK,? ERNEST F.SANDERS,~ HIROSHI MATSUMOTO.' AND STEPHENO.DUKE*-~ *United Laboratory,

States Department of Agriculture, Agricultural Research Service, Southern Weed Science P.O. Box 350, Sroneville, Mississippi 38776. and fMonsanto Company, 800 North Lindbergh Boulevard. St. Louis, Missouri 63167

Received January 16. 1991; accepted April 17. 1991 Two isomeric pairs of pyrazole phenyl ether herbicides IAH 2.429. 4-chloro-I-methyl-5(4-nitrophenoxy)-3-(trifluoromethyB-Uf-pyrazole: AH 2.430, 4-chloro-1-methyl-3-(4-nitrophenoxy)-S(trifluoromethyl)-lH-pyrazole: AH 2.431. S-((4-chloro-l-methyl-5-(trifluoromethyl)-lH-pyrazol3-yl)oxy)-2-nitrobenzoic acid; and AH 2.432. 5-((4-chloro-1-methy!-3-(trifluoromethyll-IHpyrazol-5-yl)oxy)-2-nitrobenzoic acid were evaluated for herbicidal activity in both intact plants and in tissue sections. Their capacity to induce accumulation of porphyrins in tissue sections and to inhibit protoporphyrinogen oxidase (Protox) in vitro were determined. In whole plant tests, the order of herbicidal activity was AH 2.430 9 AH 2.431 > AH 2.429 > AH 2.432. AH 2.430 consistently caused light-dependent membrane leakage in both green and far-red light grown cucumber cotyledon and barley primary leaf tissue sections after incubation for 20 hr in darkness in 0.1 mM solutions. The same treatment caused marked increases in protoporphyrin 1X (PPIX) content during the 20.hr dark incubation. AH 2.429 and 2.431 were less effective and not effective in all tissues in causing herbicidal damage and PPIX accumulation. AH 2.432 was ineffective in tissue section assays. Mg-PPIX levels were not significantly affected by any of the compounds. Protochlorophyllide levels were decreased by AH 2.430 and 2.431 in barley and increased by AH 2.429, 2.431, and 2.432 in cucumber. A positive relationship was found between herbicidal activity and the amount of PPIX that was caused to accumulate by each compound. All of the compounds inhibited Protox activity. Positive correlations were found between herbicidal activity in planta over a 300-fold range and in vitro Protox inhibition and the amount of PPIX caused to accumulate in virw. These data support the view that the pyrazole phenyl ethers exert their herbicidal activity El 1991 Academic Prew Inc. entirely through inhibition of Protox.

INTRODUCTION

both heme and chlorophylls. These chemical classes include p-nitrodiphenyl ethers, oxadiazoles, N-phenylimides. and other polycyclic compounds. By inhibiting Protox activity, these herbicides greatly increase the levels of tetrapyrroles in plant cells (6-16). PPIX3 is the enzymatic product of Protox, yet it is the principal porphyrin to accumulate. PPIX is a photodynamic compound with an absorption spectrum that is similar to the action spectrum of these herbicides in yellow tissues (17-191, indicating that it is responsible for much of the herbicidal damage. In healthy tissues, enzymatically produced PPIX is normally channeled to the proper metal chelatase (to produce heme or Mg-PPIX3), thus preventing accumulation

Several chemical classes of herbicides that cause rapid peroxidative bleaching and/or desiccation of plant tissues have recently been found to inhibit Protox3 (l-3, the last common enzyme to the synthesis of ’ Current address: Institute of Applied Biochemistry, University of Tsukuba. Tsukuba, Ibaraki 305, Japan. ’ To whom correspondence should be addressed. ’ Abbreviations used: Mg-PPIX. magnesium-protoporphyrin IX: Mg-PPIXME, magnesium-protoporphyrin IX monomethyl ester; PAR, photosynthetically active radiation; PChlide, protochlorophyllide; PPIX. protoporphyrin IX; Protox, protoporphyrinogen oxidase; MES, 4-morpholineethanesulfonic acid; DTT, dithiothreitol; PMSF. phenylmethylsulfonyl fluoride. 236 0048-3575191 $3.00 Copyright All rights

0 1991 by Academic Press. Inc. of reproduction in any form reserved.

PYRAZOLE

PHENYL

ETHERS INHIBIT

PROTOPORPHYRINOGEN

AB 2.429

AH 2.430

AH 2.432

AI3 2.431 FIG.

1. Structures

231

OXIDASE

of pyrazole

phenyl

of photodestructive PPIX (20). When Protox does not function in yeast or humans because of a genetic defect, PPIX accumulates due to loss of accumulated protoporphyrinogen from the porphyrin pathway, followed by uncontrolled autooxidation of protoporphyrinogen (21, 22). Furthermore, blockage of Protox also deregulates the porphyrin pathway by reducing feedback inhibition by heme (23-25). Thus, in cells affected by these herbicides, an unregulated flow of carbon into the porphyrin pathway results in massive accumulation of PPIX and, in some cases, PPIX products. Herbicide-induced PPIX accumulation is very rapid (13) and is apparently concentrated primarily in membranes outside the chloroplast where protection from toxic oxygen species is much lower than in the chloroplast (16). In this paper we demonstrate that a new class of herbicides, the pyrazole phenyl ethers (26) (Fig. I), have the same mechanism of action as other Protox inhibitors. That is, they cause rapid, light-dependent cellular leakage, accumulation of PPIX, and inhibition of Protox. Furthermore, we demonstrate that there are good correlations between efficacy at the molecular site, accumulation of PPIX, and herbicidal activity at the leaf disc and whole plant levels.

ether

herbicides

used in this study.

MATERIALS

AND METHODS

Plant material. For in vitro and tissue section experiments, barley (Hordeum vufgare L. [cv Morex]) and cucumber (Cucumis sativus L. [cv Straight Eight]) were grown in flats in a commercial greenhouse substrate (Jiffy-Mix; JPA, West Chicago, IL)4 and watered with distilled water. Green plants were grown at 25°C under white light of 500 l.J.mollm*/s PAR3 and ~90% relative humidity before tissues were harvested for use at 6 to 8 days of growth. Some plants were germinated in darkness at 25°C and grown in far-red light (27), and then exposed to 15-min red light followed by 12 hr of darkness prior to use at 6 to 8 days of growth. For studies on herbicidal effects on intact plants, barley was grown in growth chambers in IO-cm, square pots under a 12-hr, 20°C day/lS”C night cycle. Herbicide treatment. In tissue section experiments, tissues were treated with the herbicides depicted in Fig. 1 as before (28) by cutting 50 4-mm diameter cucumber cotyledon discs with a cork borer, or 4-mm 4 Mention of a trademark or product stitute endorsement of the product by partment of Agriculture and does not proval to the exclusion of other products be suitable.

does not conthe U.S. Deimply its apthat may also

238

SHERMAN

barley leaf squares with a razor blade, and then placing them in a 6 cm diameter polystyrene petri dish in 5 ml of 1% sucrose, 1 mM MES (pH 6.5) medium with or without technical-grade herbicide dissolved in absolute ethanol. The final concentration of ethanol in all dishes was 1%. The discs were then incubated at 25°C in darkness for 20 hr before exposure to 500 pmol/cm*/s PAR for up to 24 hr. In intact plant experiments, herbicides were applied at 0.28, 1.12, and 5.61 kg/ha to lo- to 13-cm barley seedlings (two-leaf stage) using a mechanical sprayer; AH 2.429 was also applied at 11.2 kg/ha. Spray formulations included up to 4% (v/v) acetone and 0.1% Triton AG-9g4 as a dispersant in 30 ml of water. Herbicidal activity. Cellular damage was measured by detection of electrolyte leakage into the bathing medium with a conductivity meter with the capability of assaying 1 ml of the bathing medium and returning it to the dish as before (28). Because of differences in background conductivity of different treatment solutions, results are expressed as change in conductivity after exposure to light. Previous studies have shown that photobleaching herbicides have no significant effect on cellular leakage in darkness. Photobleaching was determined by measuring chlorophyll content after 24 hr of exposure to 500 p,mol/m*/s PAR at 25°C. Chlorophyll was extracted and assayed by the methods of Hiscox and Israelstam (29). Discs from each dish were soaked in 5 ml of dimethyl sulfoxide in darkness at room temperature for 24 hr and total chlorophyll in extracts was determined spectrophotometrically. All treatments for electrolyte leakage and photobleaching measurements were triplicated. Herbicidal injury of intact plants was visually rated in quadruplicated treatments at 3, 7, and 14 days after treatment. Because variation between replicates did not differ significantly from the variation across time, scores were averaged globally for regres-

ET

AL

sion analysis. Relative herbicidal potencies (with AH 2.429 as unity) were determined by least-squares (R* = 0.951 with 12 degrees of freedom) analysis of logarithmically transformed data to a common slopes model. The relative herbicidal activities so obtained were fully consistent with direct concentration for concentration comparisons. Porphyrin determinations. All extractions for HPLC were made under a dim, green light source. Samples (50 discs) were homogenized in 3 ml of HPLC-grade methano1:O.l N NH,OH (9: 1, v/v) with a Brinkmann Polytron at 60% full power for 15 sec. The homogenate was centrifuged at 30,0001: for 10 min at 0°C and the supernatant was saved. The pellet was resuspended in 3 ml of methanol, sonicated for 5 min, and centrifuged at 30,OOOg for 10 min at 0°C. Supernatants were combined and evaporated to dryness at 40°C with a rotary evaporator. The residue was dissolved in 2 ml of HPLC-grade basic methanol and filtered through a 0.2~p,rn syringe filter. Samples were stored in light-tight (glass wrapped in aluminum foil) vials at -20°C until analysis by HPLC. HPLC determinations were made as before (30) with a system composed of Waters Associates4 components which included: two Model 510 pumps; a Model 712 autosampler; a Maxima 820 controller; and a Model 990 photodiode spectrophotometric detector. A Model 470 fluorescence detector preceded the Model 990 detector. The column was a 250 x 4.6 mm (i.d.) Spherisorb 5-pm ODS-1 reversed-phase column preceded by a Bio-Rad4 ODS-5S guard column. The solvent gradient was composed of 0.1 M ammonium phosphate (pH 5.8) (solvent A) and HPLC-grade methanol (solvent B) at a flow rate of 1.4 ml/min. The solvent delivery program was as follows: 20% A in B from 0 to 10 min, a linear transition from 20 to 0% A in B from 10 to 18 min, and B only from 18 to 35 min. The injection volume was 50 ~1. Commercial standards of PPIX (Sigma Chemical CO.~),

PYRAZOLE

PHENYL

ETHERS

INHIBIT

Mg-PPIX, and Mg-PPIXME3 (Porphyrin Products, Inc.4) were used. PChlide3 was obtained by extraction from etiolated tissues, quantified spectrophotometrically as before (14). and injected into the HPLC for calibration of the spectrofluorometric detector. Porphyrin detection was performed with fluorescence detector excitation and emission wavelength settings of 400 and 630 nm, respectively, for PPIX; 41.5 and 595 nm, respectively, for Mg-PPIX and MgPPIXME; and 440 and 630 nm, respectively, for PChlide. The photodiode array detector scanned from 300 to 700 nm to confirm all peaks. All porphyrin compound levels are expressed on a molar basis per gram of fresh weight. All treatments for porphyrin samples were triplicated. Protox assays. Cotyledons of farred-grown cucumber or barley or etiolated barley were homogenized with a Sorvall Omnimixer4 for 30 set at full speed using a fresh weight to volume ratio of 1:5. Homogenization buffer consisted of 10 mM HEPES (pH 7.7 at 2”C), 330 miV sorbitol, 1 mM EDTA, 1 mM MgCl,, and 5 mM cysteine. Homogenate was filtered through two layers of Miracloth, and crude cell debris was removed by centrifugation at 15Og for I min at 4°C. Plastids were pelleted from this supernatant by centrifugation at 6000g for 15 min at 4°C. On ice, plastids were resuspended in assay buffer and disrupted by sonication in two IO-set bursts. with intermittant cooling. The assay buffer was the same as the homogenization buffer except that 1 mM dithiothreitol (DTT) was substituted for 5 mM cysteine, and 1 mM PMSF, 10 p..M leupeptin, 10 ~.LM pepstatin, and 5 mM l,lO-phenanthroline were added as proteinase inhibitors. Protein concentration was determined using the method of Bradford (31), using bovine serum albumin as a standard. Extracts were resuspended to a concentration of 4 mg protein/ml in assay buffer and stored at -80°C until use. Protoporphyrinogen was prepared according to Jacobs and Jacobs (32). The preparation was adjusted to approximately

PROTOPORPHYRINOGEN

OXIDASE

239

pH 8 with 42% (v/v) phosphoric acid. To slow the rate of autooxidation, dithiothreito1 was added to a final concentration of 2 mM. Prior to assay, plastid extracts were thawed and held on ice. In the herbicide treatments, herbicides were added in a volume of 2 ~1 of ethanol to 200 ~1 of extract. Ethanol was added to control treatments. Herbicidal inhibition was maximal within 3 set after addition and was stable for at least 2 hr. The assay mixture consisted of 25 n-&Z HEPES (pH 7.5, 3O”C), 5 n&f EDTA, 2 mM DTT, and about 2 FM protoporphyrinogen IX. The reaction was initiated by addition of 0.2 ml of extract (kherbicide) to 0.8 ml of assay mixture and monitored for 2 min at 30°C. Fluorescence was monitored directly from the assay mixture using a Shimadzu RF-5000 U ,4 temperature-controlled, recording spectrofluorometer with excitation at 395 nm and emission monitored at 622 nm. The reaction rate was essentially constant over this 2 min period. Autooxidation in the presence of heatinactivated extract was negligible. RESULTS

Herbicidal activity. In all test systems used in our studies, AH 2.430 was significantly more herbicidal than the other three compounds tested. Intact, green barley plants were about lOO-fold more sensitive to AH 2.430 than to the isomeric AH 2.429 (Fig. 2). The 3-pyrazolyloxybenzoic analogue of acifluorfen (AH 2.431) was twice as active as AH 2.429, whereas the herbitidal activity of the isomeric benzoic acid AH 2.432 was about 3- to 4-fold lower (ca. 30% as potent as AH 2.429). In tissue sections of green cucumber or barley, the three compounds most active in planta (AH 2.429, AH 2.430, and AH 2.431) were significantly phytotoxic at 0.1 m&f in both green or far-red light-grown tissues (Fig. 3). However, neither AH 2.429 nor AH 2.43 1 were consistently herbicidal with all tissues. AH 2.432 was not herbicidal with this assay. Far-red light grown tissues

240

SHERMAN

ET

AL.

10 0 i herbicide

(kg/ha)

‘”

2. Herbicidal injury caused by different spray concentrations of four pyrazole phenyl ether herbicides one week after application. Numbers in rectangles associated with each curve are the relative efficaries (relative to AH 2.429) of the herbicides with the probable (95% level) range of efficacy in parentheses. The relative efficacies are based upon pooled injury data from 3, 7, and 14 days after treatment (see Muterials and Methods for method of computation). Error bars are 21 SE. FIG.

of both cucumber and barley were more sensitive to the herbicides than were green tissues. No effects of the herbicides could be detected during the dark incubation period. Upon exposure to light, after dark incubation, increases in leakage above control levels could usually be detected with AH 2.430 more rapidly than with the other two herbicidal compounds (data not shown). Cucumber was more sensitive than barley, so the electrolyte leakage data of 8 hr of exposure to light was used rather than that of 24 hr of exposure as for barley. Photobleaching of chlorophyll of green cucumber tissue was not significant in any of the treatments after 24 hr of light except for AH 2.430 (45% loss of chlorophyll). Effects on porphyrin contents. The most herbicidally active compounds (AH 2.430 and AH 2.431) greatly increased PPIX content of treated tissue sections of green cucumber and etiolated or far-red light-grown barley (Fig. 4). In barley, AH 2.429 caused a relatively small increase in PPIX content. There was no apparent effect on Mg-PPIX (data not shown) or PChlide, except a slight decrease in PChlide when the PPIX levels

GRLLN BdRLEY

CUCUMBER

3. Effects of four pyrazole phenyl ether herbicides on cellular leakage of four different plant tissues. in all experiments, the tissues were incubated in 0.1 mM herbicide for 20 hr in darkness and then e.xposed to light for 24 (barley) or 8 hr (cucumber). Cellular leakuge was determined by measuring the change in conductivity of the bathing solution that occurred during the period of illumination. Error bars are 21 SE. FIG.

were most strongly increased, probably due to blockage of the pathway. In green cucumber tissues, PChlide levels were increased by all compounds except for AH 2.430. There was a positive correlation between herbicidal activity in tissue sections and accumulation of PPIX in all tissues (Fig. 5). These graphs illustrate the greater susceptibility of far-red-light-grown than green tissues to PPIX. Previous studies have shown that yellow- or far-red-light-grown or tentoxin-treated tissues are hypersensitive to photobleaching herbicides (16, 27). We have speculated previously that these tissues are more sensitive because they do not have the photosynthetic ability to generate reductants that can protect against peroxidative damage. Effects on Protox. All of the compounds

PYRAZOLE

PHENYL

ETHERS

INHIBIT

PROTOPORPHYRINOGEN

2.0

3.5

,

I

3.0

-

CUCUMBER

R

241

OXIDASE r

I

\

1.8 1.6 1.4 1.2 I.0 0.8 0.5 0.4 0.2 0.0 4.0 3.6 3.2 I

2.8 2.4

4.5

-

4.0

-

2.0 1.6

II

I

BARLEY

green t

1.2

3.5

0.8

3.0

0.4

2.5

0.0

t -

2.0

4. Effects of four pyrazole phenyl ether herbicides on porphyrin accumutkation in four different plan1 tissues. In all experiments, the tissues nvere incubated in 0. I mM herbicide for 20 hr in darkness and then extracted. Error bars are 51 SE. FIG.

1.5

-

1.0

-

-500

inhibited Protox activity in an in vitro assay (Fig. 6). However, there was a broad range in Z,, values (ca. 400-fold), with the most potent herbicides having the lowest values. None of the compounds were as active in inhibiting Protox activity as was acifluorfen-methyl (I,, s 0.1 td4’). The Z,, values for acifluorfen-methyl in our preparations are about 4- and 25fold higher than those reported earlier for cucumber (3) and corn (1) plastid preparations, respectively. Such differences could be the result of many factors, including differences in specific activities, assay conditions, and tissue differences. Our specific activities were higher than those of these earlier papers (see Fig. 6 legend), so one might expect the I,, values to be somewhat higher. Assay conditions can greatly influence both specific activity and susceptibility of the enzyme to the herbicide (5). There was a rough, negative relationship between I,, values for Protox and the effect of the herbicide on PPIX accumulation I’n

far-red

0 cellular

500 leakage

1000 (gmho/cm/g

J 2000

1500 fr

wt)

5. Relalionship between qffcls of four pyrazole phenyl ethers on PPIX content and herbicidal activity (data are taken from Figs. 3 and 4). Error bars are tl SE. FIG.

vivo (Fig. 7), although

there was a consistent discontinuity in the relationship between AH 2.431 and AH 2.429. Better correlations were found in green barley between the relative in plnnta herbicidal activities and the in vitro I,, values for Protox and the in viva accumulation of PPIX in excised tissues (Fig. 8). DISCUSSION

The effects of these compounds on plant injury, cellular leakage, PPIX accumulation, and Protox activity are very similar to those previously described for acifluorfen and acifluorfen-methyl (1, 3-9. 11, 13, 14. 28). The pyrazole phenyl ethers that we used had a broader range of herbicidal activities than other Protox inhibitors that we

242

SHERMAN

ET

AL. 10

r-

CUCUMBER -A

1

T

0.1

I 01 \

BARLEY

z 0 E .5 x a a

AH 2.4JL ?\T AH 2.429 0

i\

.

green

v

far-red

. A

BARLEY

H 2.43 a

1

0.1

0.01

0.1

101 0.01

",-

0.1

",,-

1 herbicide

I,,,,,/

10

,,(,,,,I 100

--!

1000

(PM)

1

10

100

Protox

Iso (@A)

1000

10000

6. Dose-response curves for effects of four pyrazole phenyl ethers on Protox activity from plastids of far-red hght-grown cucumber and barley leaves. The control (zero herbicide) Protox activity rates were 20.5 and 10.8 nmollhrlmg protein for barley and cucumber, respectively. Error bars are tl SE.

FIG. 7. Relationships between the Protox I,, values for four pyrazole phenyl ether herbicides and their capacity to induce accumulation of PPIX in far-red lightgrown and green cucumber cotyledon and bariey leaf tissues. The herbicide is listed between the two data points that were generated with that herbicide (data are taken from Figs. 4 and 6).

have previously used in studies in which we have tried to correlate PPIX accumulation with herbicidal activity (9, 10, 13). The small range of analogues used in this study does not permit an extensive analysis of structure/activity relationships; however, some observations can be made. The 3pyrazole ethers, AH 2.430 and AH 2.431. were much more herbicidally active than the corresponding Spyrazole ether isomers, AH 2.429 and AH 2.432, confirming previous phytotoxicity data (26). Like most other Protox inhibitors, the pyrazole phenyl ethers are bicyclic compounds (33). Like commercial diphenyl ethers, these compounds are p-nitro in phenyl substitution. Other Protox-inhibiting herbicides have been reported to be competitive inhib-

itors (34,35). If this is correct, the two rings of these inhibitors may simulate the binding niches of two (either adjacent or opposite) of the pyrrole rings of protoporphyrinogen or PPIX. TNPP-ethyl (2-[ 1-(2,3,4-trichlorophenyl)-4-nitropyrazolyl-Soxylpropionate), a phenyl pyrazole, is the most closely related herbicide to the pyrazole phenyl ethers with the capacity to cause PPIX accumulation in plant tissues (36). In this compound, the nitro group is attached to the pyrazole ring and the halogens are on the phenyl ring. Furthermore, the two rings are not attached with an ether linkage. The oxypropionate is adjacent to the nitro group, as with some p-nitro diphenyl ethers. The phytotoxicity of these herbicides

FIG.

PYRAZOLE

0

0.1

-

Proto,x

‘I”0.1

PHENYL

INHIBIT

inhibition

,‘,1 relative

ETHERS

herbicidol

10

,,,,,,,,I

100

effect

FIG. 8. Relationships between the relative efficacies of the four pyrazole phenyl ether herbicides on intact, green barley seedlings and their effects on Protox from barley tissues and on PPIX accumulation in green barley leaf sections (data are from Figs. 2, 4, and 6).

correlated positively with their capacity to stimulate accumulation of PPIX in vivo and the PPIX levels induced to accumulate by these herbicides correlated with their capacity to inhibit Protox in vitro (Figs. 5, 7, and 8). It is particularly remarkable that the 300-fold range of activity seen in intact barley plants correlates so well with PPIX accumulation in tissue sections and to in vitro Protox inhibition (Fig. 8). If the pK, values of the two carboxylate analogues had been sufficiently low or the pH of the spray solution sufficiently high enough for significant proportions of these herbicides to exist as ions, drastic differences in uptake (both cuticular and cellular) could have confounded this relationship. We and others have previously found similar correlations between herbicidal activity of acifluorfen and induction of PPIX accumulation (13, 37, 38). This is the first report to further correlate Protox inhibition with these effects. Others (12,39,40) have found no correlation between PPIX content and activity of peroxidizing herbicides. In tissue sections, ‘the poorer correlation between capacity to inhibit Protox in vitro

PROTOPORPHYRINOGEN

OXIDASE

243

and in vivo PPIX accumulation than the correlation between PPIX accumulation and herbicidal activity is not unexpected. The in vitro activity is not influenced by herbicide uptake or metabolic alteration, whereas the in vivo effect could be influenced by both factors. PPIX is the actual phytotoxic compound, and once accumulated, its herbicidal effects should be the same, regardless of the herbicide that caused its accumulation. The better correlation of Protox-inhibiting capacity and herbicidal activity of foliar-applied compounds (Fig. 8) would indicate that foliar uptake may be more uniform among compounds than their uptake from liquid media by tissue sections. The stimulation of PChlide accumulation by these compounds was unexpected. Others have reported induction of PChlide accumulation by Protox inhibitors (16, 40, 41). We (16) attribute this effect to reentry of PPIX into the porphyrin pathway after very high levels of PPIX accumulated. However, the results reported here indicate that PChlide levels can be increased in some tissues by Protox inhibitors that have little effect on PPIX accumulation. Whether this is due to an effect on Protox or to an effect at another site in the porphyrin pathway cannot be determined from our data. If the increased PChlide is not converted to chlorophyll and incorporated into the photosynthetic apparatus, it could contribute to the photodynamic damage caused by these herbicides. Our data do not support the view that PChlide plays a significant part in the herbicidal activity of these compounds because the most active of the four compounds had the least effect on PChlide accumulation. In summary, our data strongly indicate that a new class of herbicides, the pyrazole phenyl ethers, exert their herbicidal damage solely through inhibition of Protox. This is the first study to physiologically link Protox inhibition, PPIX accumulation, and herbicidal activity.

244

SHERMAN

ACKNOWLEDGMENTS

We thank Alfred D. Lane for his technical assistance. R. Scalla and M. Matringe generously provided preprints describing their recent research. We thank Judy and Nick Jacobs for their valuable technical advice. REFERENCES

1. M. Matringe. J.-M. Camadro, P. Labbe, and R. Scalla, Protoporphyrinogen oxidase as a molecular target for diphenyl ether herbicides, Biothem.

J. 260, 231 (1989).

2. M. Matringe. J.-M. Camadro, P. Labbe, and R. Scalla, Protoporphyrinogen oxidase inhibition by 3 peroxidizing herbicides: Oxadiazon. LS 82-556 and M and B 39279, FEES Left. 245, 35 (1989). 3. D. A. Witkowski and B. P. Halling, Inhibition of plant protoporphyrinogen oxidase by the herbicide acifluorfen-methyl, Plant Physiol. 90, 1239 (1989). 4. R Scalla and M. Matringe. Recent advances in the mode of action of diphenyl ethers and related herbicides, Z. Naturforsch.. C: Biasci. 45, 502 (1990). 5. J M. Jacobs, N. J. Jacobs, S. E. Borotz, and M. L. Guerinot, Effects of the photobleaching herbicide, acifluorfen-methyl, on protoporphyrinogen oxidation in barley organelles, soybean root mitochondria, soybean root nodules, and bacteria, Arch. Biochem. Biophys. 280, 369 (1990). 6. M. Matringe and R. Scalla. Induction of tetrapyroles by diphenylether-type herbicides, Proc. Br. Crop Prot. Conf. 9B, 981 (1987). 7. M. Matringe and R. Scalla. Studies on the mode of action of acifluorfen-methyl in non-chlorophyllous soybean cells: Accumulation of tetrapyroles. Plant Physiol. 86, 619 (1988). 8. M. Matringe and R. Scalla. Effects of acifluorfenmethyl on cucumber cotyledons: Protoporphyrin accumulation, Pesric. Biochem. Physiol. 32, 164 (1988). 9. J. Lydon and S. 0. Duke, Porphyrin synthesis is required for photobleaching activity of the pnitrosubstituted diphenyl ether herbicides. Pestic. Biochem. Physiol. 31, 74 (1988). IO. S. 0. Duke, J. Lydon, and R. N. Paul, Oxadiazon activity is similar to that of p-nitro-diphenyl ether herbicides, Weed Sci. 37, 152 (1989). Il. D. A. Witkowski and B. P. Halling, Accumulation of photodynamic tetrapyrroles induced by acifluorfen-methyl, P/ant Physiol. 87, 632 (1988). 12. G. Sandmann and P. Boger, Accumulation of protoporphyrin 1X in the presence of peroxidizing herbicides. Z. Naturf~orsclr., Cc Biosci. 43, 699 (1988).

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AL.

13. J. M. Becerril and S. 0. Duke, Protoporphyrin IX content correlates with activity of photobleaching herbicides, Plant Physiol. 90, 1175 (1989). 14. J. M. Becerril and S. 0. Duke, Acifluorfen effects on intermediates of chlorophyll synthesis in green cucumber cotyledon tissues. Pestic. Biothem. Physio/. 35, I I9 (1989). 15. B. Nicolaus, G. Sandmann. H. Watanabe. K. Wakabayashi, and P. Boger, Herbicide-induced peroxidation: Influence of light and diuron on protoporphyrin IX formation. Peck. Biochem. Physiol. 35, 192 (1989). 16. L. P. Lehnen, T. D. Sherman, J. M. Becerril, and S. 0. Duke, Tissue and cell localization of acifluorfen-induced porphyrins in cucumber cotyledons. Pestic. Biochem. Physiol. 37, 239 (1990). 17. R. Sato. E. Nagano, H. Oshio, and K. Kamoshita, Activities of the N-phenylimide S-23142 in carotenoid-deficient seedlings of rice and cucumber. Pestic. Biochem. Physiol. 31, 213 (1988). 18. R. Sato. E. and H. Furuya, Wavelength effect on the action of a N-phenylimide S-23142 and a diphenylether acifluorfen-ethyl in cotyledons of cucumber (Cucumis sarivus L.) seedlings, Plant Physiol. 85, 1146 (1987). 19. S. 0. Duke. J. M. Becerril, T. D. Sherman, J. Lydon, and H. Matsumoto, The role of protoporphyrin IX in the mechanism of action of diphenyl ether herbicides, Pesric. Sci.. 30, 367 (1990). 20. G. C. Ferreira, T. L. Andrew, S. W. Karr, and H. A. Dailey, Organization of the terminal two enzymes of the heme biosynthetic pathway, f. Eiol. Chem. 263, 3835 (1988). 21. J. M. Camadro. D. Urban-Grimal, and P. Labbe. A new assay of protoporphyrinogen oxidase: Evidence for a total deficiency in that activity in a heme-less mutant of Saccharomcyces cerevisiae.

22.

23.

24.

25. 26.

Biachem.

Biophys.

Res.

Commun.

106,

724 (1982). D. A. Brenner and J. R. Bloomer. Enzymatic defect in variegate porphyria: Studies with human cultured skin fibroblasts. N. Engl. J. Med. 302, 765 (1980). S. I. Beale and J. D. Weinstein, Tetrapyrrole metabolism in photosynthetic organisms. in “Biosynthesis of Heme and Chlorophylls” (H. A. Dailey, Ed.), pp. 287-391, McGraw-Hill, New York, 1990. T. Masuda. H. Kouji. and S. Matsunaka, Diphenyl ether herbicide-decreased heme contents stimulate 5-aminolevulinic acid synthesis, Pesric. Biochem. Physiol. 36, I06 (1990). G. Padmanaban. V. Ventkateswar. and P. N. Rangarajan. Haem as a multifunctional regulator, Trend3 Biochem. Sci. 14, 492 (1990). K. Moedritter. S. G. Allgood. P. Charumilind. R. D. Clark, B. J. Gaede. M. L. Kurtzweil,

PYRAZOLE

27.

28.

29.

30.

31.

32. 33.

34.

PHENYL

ETHERS

INHIBIT

D. A. Mischke, J. J. Parlow. M. C. Rogers, R. K. Singh, and G. L. Stikes. Pyrazole phenyl ethers: A new class of highly active herbicides, Int. Congr. Pesiic. Chem.. 7th OIA-57 (1990). S. 0. Duke and W. H. Kenyon. Photosynthesis is not involved in the mechanism of action of acifluorfen in cucumber (Cucumis satiraus L.). Plant Physiol. 81, 882 (1986). W. H. Kenyon, S. 0. Duke, and K. C. Vaughn, Sequence of herbicidal effects of acifluorfen on ultrastructure and physiology of cucumber cotyledons, Pestic. Biochem. Physiol. 24, 240 (1985). J. D. Hiscox and G. F. Israelstam, A method for the extraction of chlorophyll from leaf tissues without maceration. Can. J. Bof. 57, 1332 (1979). H. Matsumoto. and S. 0. Duke, Acifluorfenmethyl effects on porphyrin synthesis in Lemna paucicostafa Hegelm. 6746, J. Agric. Food Chem. 38, 2066 (1990). M. M. Bradford, A rapid and sensitive method for the quantification of microgram quantities of protein-dye binding, Anal. Biochem. 72, 248 t 1976). N. J. Jacobs and J. M. Jacobs, Assay for enzymatic protoporphyrinogen oxidase, a late step in heme synthesis, Enzyme 28, 206 (1982). S. 0. Duke. J. M. Becerril, J. Lydon. H. Matsumoto. and T. D. Sherman, Proporphorinogen oxidase-inhibiting herbicides, Weed Sci. in press (1991). J.-M. Camadro. M. Matringe, R. Scalla. and P. Labbe, Kinetic studies on protoporphyrinogen

PROTOPORPHYRINOGEN

35.

36.

37.

38.

39.

OXIDASE

245

oxidase by diphenyl ether herbicides, Biochem. J. in press (1991). R. Varsano, M. Matringe, N. Magnin. R. Momet, and R. Scalla. Competitive interaction of three peroxidizing herbicides with the binding of H3 acifluorfen to corn etioplast membranes. FEBS Leff. 272, 106 (1990). D. Yanase and A. Andoh, Porphyrin synthesis involvement in diphenyl ether-like mode of action of TNPP-ethyl, a novel phenylpyrazole herbicide, Pestic. Biochem. Physiol. 35, 70 (1989). S. 0. Duke. J. M. Becerril, H. Matsumoto, and T. D. Sherman, Photosensitizing porphyrins as herbicides, Am. Chem. Sot., Symp. Ser. No. 449, 371 (1990). M. Matringe, D. Clair, and R. Scalla, Effects of peroxidizing herbicides on protoporphyrin IX levels in non-chlorophyllous soybean cell culture, Pestic. Biochem. Physiol. 36, 300 (1990). G. Sandmann, B. Nicolaus, and P. Boger, Typical peroxidative parameters verified with mungbean seedlings. soybean cells and duckweed, 2. Naturforsch..

C: Biosci.

45, 512 (1990).

40. J. M. Mayasich. U. B. Nandihalli, R. A. Liebl. and C. A. Rebeiz. The primary mode of action of acifluorfen-Na in intact seedlings is not via tetrapyrrole accumulation during the first dark period following treatment, Pestic. Biochem. Physiol. 36, 259 (1990). 41. H. Kouji, T. Masuda. and S. Matsunaka. Action mechanism of diphenyl ether herbicides; stimulation of 5-aminolevulinic acid synthesizing system activity, Pesric. Biochem. Physiol. 33, 230 (1989).