mode of action studies on a chiral ... - plant physiology · plant physiol. vol. 100, 1992 the...

Plant Physiol. (1992) 100, 1211 -2160032-0889/92/100/121 1/06/$0 1.00/0

Received for publication March 5, 1992Accepted May 13, 1992

Mode of Action Studies on a Chiral Diphenyl EtherPeroxidizing Herbicide

Correlation between Differential Inhibition of Protoporphyrinogen IX Oxidase Activity andInduction of Tetrapyrrole Accumulation by the Enantiomers

Beverly J. Hallahan1'2, Patrick Camilleri3, Alison Smith, and John R. Bowyer*Department of Biochemistry, Royal Holloway and Bedford New College, University of London, Egham Hill,

Egham, Surrey, TW20 OEX, United Kingdom (B.J.H., I.R.B.); Shell Research Ltd., Sittingbourne Research Centre,Sittingbourne, Kent, ME9 8AG, United Kingdom (P.C.); and Department of Plant Sciences, University of

Cambridge, Downing Street, Cambridge CB2 3EA, United Kingdom (A.S.)

ABSTRACT

The nitrodiphenyl ether herbicide 5-[2-chloro-4-(trifluorome-thyl)phenoxy]-2-nitroacetophenone oxime-O-(acetic acid, methylester) (DPEI) induced an abnormal accumulation of protoporphyrinIX in darkness in the green alga Chiamydomonas reinhardtii, as

determined by high-performance liquid chromatography and spec-trofluorimetry. It also inhibited the increase in cell density of thealga in light-grown cultures with an 150 (concentration required todecrease cell density increase to 50% of the noninhibited controlvalue) of 0.16 Mm. The relative ability of four peroxidizing diphenylether herbicides to cause tetrapyrrole accumulation in C. reinhard-tii correlated qualitatively with their ability to inhibit the increasein cell density in light-grown cultures. The purified S(-) enantiomerof the optically active phthalide DPE 5-[2-chloro-4-(trifluorome-thyl)phenoxyJ-3-methylphthalide (DPEIII), which has greater her-bicidal activity than the R(+) isomer, induces a 4- to 5-fold greatertetrapyrrole accumulation than the R(+) isomer. The 150 for inhibi-tion of increase in cell density in light-grown cultures of C. rein-hardtii by the S(-) isomer (0.019 uM) is less than 25% of that forthe R(+) isomer. DPEIII inhibits protoporphyrinogen IX oxidaseactivity in pea (Pisum sativum) etioplast lysates, with the S(-)enantiomer showing considerably greater potency than the R(+)isomer and the racemic mixture showing a potency intermediatebetween the two. The results indicate that the site at which DPEsinhibit protoporphyrinogen IX oxidase shows chiral discriminationand provide further evidence for the link between inhibition of thisenzyme, protoporphyrin IX accumulation, and the phytotoxicity ofDPE herbicides.

NitroDPE4 and related herbicides cause rapid light- and02-dependent lipid peroxidation and bleaching of Chl and

l B.J.H. gratefully acknowledges the support of a studentship fromthe Science and Engineering Research Council, UK, and Shell Re-search Ltd.

2 Present address: Department of Biology, Darwin Building, Uni-versity College London, Gower Street, London WClE 6BT, UK.

3 Present address: SmithKline Beecham Research Ltd., The Frythe,Welwyn, Herts. AL6 9AR, UK.

4 Abbreviations: DPE, diphenyl ether; DPEI, 5-[2-chloro-4-(tri-

carotenoids in leaves of higher plants (14, 16, 24). Similareffects have been demonstrated in the green algae Scenedes-mus obliquus (2, 15) and Chlamydomonas eugametos (6, 7). Theprimary mode of action of these herbicides, however, re-

mained unclear for many years.

Matringe and Scalla (23) proposed that the phytotoxicityof DPE herbicides is due to their ability to induce abnormalaccumulation of photosensitizing tetrapyrroles, specificallyprotoporphyrin IX. This pigment is known to sensitize light-dependent singlet oxygen generation and is thus capable ofinitiating the peroxidizing activity associated with DPE tox-icity (26). DPE-induced tetrapyrrole accumulation has beendemonstrated in a number of higher plants (19, 23, 31) andin the green algae S. obliquus (2) and Bumilleriopsis filiformis(27).Lydon and Duke (19) and Witkowski and Halling (31)

suggested that the primary site of action of these herbicidesis magnesium chelatase, the enzyme responsible for the in-sertion of Mg2" into the tetrapyrrole ring during Chl synthe-sis. More recently, Matringe et al. (21, 22) and Witkowskiand Halling (32) showed that certain peroxidizing herbicides,including DPEs, inhibit protoporphyrinogen oxidase, but notmagnesium chelatase, in chloroplasts and etioplasts (10, 11).These compounds appear to bind to the same or closelyoverlapping sites on protoporphyrinogen oxidase, in com-

petition with the substrate, protoporphyrinogen IX (29).Protoporphyrin IX accumulation is thought to result fromstimulation of synthesis of the porphyrin precursor, 5-ami-nolevulinic acid (13, 20), and herbicide-insensitive oxidationof protoporphyrinogen IX in nonplastid cell compartments,which sequester the resulting protoporphyrin IX away fromthe magnesium and iron chelatases (18).There are many examples in the literature of drugs with

chiral centers in which one enantiomer is more active than

fluoromethyl)phenoxy]-2-nitroacetophenone oxime-O-(acetic acid,methyl ester); DPEIII, 5-[2-chloro-4-(trifluoromethyl)phenoxy]-3-methylphthalide; PIC, paired ion chromatography; TAP, Tris-ace-tate-phosphate; 150, concentration required to decrease cell densityincrease to 50% of the noninhibited control value.

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Plant Physiol. Vol. 100, 1992

the other (5), but there are few examples from herbicidebiochemistry (e.g. PSII inhibitors [1] and aryloxyphenoxypro-pionates inhibiting fatty acid biosynthesis [30]). Camilleri etal. (3) recently described a novel chiral phthalide DPE-per-oxidizing herbicide in which the S(-) enantiomer showedmarkedly higher herbicidal activity on Vicia faba and Hordeumvulgare seedlings than the R(+) form. In this paper, we haveshown that this differential effect can also be observed ineffects on increase in cell density and tetrapyrrole accumu-lation in Chlamydomonas reinhardtii and inhibition of proto-porphyrinogen IX oxidase activity in etioplast lysates fromPisum sativum.

MATERIALS AND METHODS

Materials

PIC reagent A was obtained from Waters ChromatographyDivision and made up in water according to the manufactur-er's instructions. Standard protoporphyrin IX was obtainedfrom Sigma. DPEI (purity >99%) was obtained from P.P.G.Industries Inc. (Philadelphia, PA), nitrofen (purity >95%)was from Shell Research Ltd. (Sittingboume, UK), and oxy-

fluorfen (purity 99%) was from Rohm and Haas Company(Spring House, PA). They were dissolved in DMSO such thatthe final concentration of DMSO in algal cultures was 0.1%(v/v). DPEIII (purity >99%) and its enantiomers were pro-

vided by Shell Research Ltd. The S(-) and R(+) isomers wereseparated using a chiral stationary phase HPLC column (3)giving an enantiomeric purity of 95 ± 2%, based on HPLCanalysis. They were dissolved in propanol such that theconcentration of propanol in algal cultures was 0.1% (v/v).

Algal Culture

Suspension cultures of Chlamydomonas reinhardtii were

grown in TAP medium (9) on an orbital shaker at 250C undera light intensity of 200 Wm-2 and subcultured every 5 d.

Tetrapyrrole Formation

For induction of tetrapyrrole accumulation, 5-d-old cul-tures of C. reinhardtii were harvested and the cells resus-

pended in fresh TAP medium to 0.03 g wet weight cellsmL-1. Aliquots (5 mL) were transferred to 10-mL conicalflasks, and appropriate herbicide additions made. The flaskswere then incubated in the dark for 24 h at 250C on an

orbital shaker. Tetrapyrroles were extracted using a modifi-cation of the method of Rebeiz et al. (25). After a 24-hincubation, the cells were collected by centrifugation at 3000gand resuspended in 10 mL of acetone:0.1 M NH40H (9:1, v/v). After 30 min on ice in darkness, the suspensions were

centrifuged at 39,000g for 10 min, the supematant fractionswere washed twice with hexane in a 1:1 ratio by volume,and the washed supernatant fluids were made up to 5 mLwith acetone:0.1 M NH40H.

For routine assay of tetrapyrrole content, fluorescenceemission spectra were recorded using a Perkin-Elmer fluores-cence spectrometer with an excitation wavelength of 398 nm.

When our instrument was used, protoporphyrin IX had an

emission maximum at 628 nm. The magnitude of the 628 nmemission band in the C reinhardtii extracts was related to astandard curve prepared using known concentrations of pro-toporphyrin IX. To prepare samples for HPLC, the ace-tone:NH40H extracts (5 mL) were mixed with 1:17 volumesaturated NaCl and 1:16 volume 0.25 M maleic acid, and thepH was adjusted to 6.8 with concentrated Na2HPO4 solution.The tetrapyrroles were then extracted into peroxide-freeether. The ether extract was washed three times with waterand concentrated to 1 mL under a stream of N2.

HPLC Analysis of Tetrapyrroles

The ether extracts were analyzed by ion-pair HPLC (8) ona Spherisorb S5 ODS2-EXL C18 reverse phase column (250 x4.6 mm, i.d.) coupled to a Waters 600E HPLC system. Theether extract (5 ,L) was injected onto the column. Tetrapyr-roles were eluted using 70% methanol:30% 5 mm PIC Areagent, which was changed to 70% methanol:30% H20 after3 min. PIC A reagent (tetrabutylammonium hydrogen sul-fate) enables acids normally separated by ion exchange chro-matography to be resolved using reverse phase chromatog-raphy. The flow rate was 0.75 mL/min at room temperature(approximately 200C). Tetrapyrroles were detected with aphotodiode array detector (Waters Associates model 990). Forfurther identification, peak fractions were collected and flu-orescence emission spectra measured using a Perkin-Elmerfluorescence spectrometer with an excitation wavelength of398 nm.

Growth Inhibition in C. reinhardtii

Five-day-old cultures were harvested and the cells resus-pended in fresh TAP medium to a density of 106 cells mL-1.Aliquots (5 mL) were transferred to 10-mL conical flasks andappropriate additions of herbicide (0.01-10 $M final concen-tration) made. The cells were incubated for 24 h in the darkon an orbital shaker at 250C followed by 24-h incubation ata light intensity of 200 Wm-2. The cell density in each cultureafter this treatment was determined using a hemocytometer,with appropriate dilution of the suspension before counting.150 values were determined using cultures treated with0.1% (v/v) DMSO as the control. The results presented arebased on the means of five incubations at each herbicideconcentration.

Growth of Peas

Pea (Pisum sativum, var Feltham First) seeds were rinsedfor 15 min in a solution of 10% Milton's sterilizing fluid,sown in autoclaved soil, and left to germinate in completedarkness at 220C.

Preparation of Pea Etioplasts

Seven-day-old etiolated pea seedlings (10 g fresh weight)were chopped with a razor blade in 20 mL of 0.3 M sucrose,25 mm Hepes/NaOH (pH 7.6), 1 mm EDTA, and 0.1 mm DTTfor 10 to 15 min at 40C and filtered through eight layers ofmuslin. After centrifugation at 10,000g for 15 min, the crudeorganelle pellet was carefully resuspended in 5 mL of a

1212 HALLAHAN ET AL.



STEREOSPECIFIC EFFECTS OF A CHIRAL DIPHENYL ETHER HERBICIDE

23.7 mlim

10.01 A1

21.4 mhL_ ~~~~~~~~~~AProtoporphpyrn IX

-A A- -.. C

0 10 20 30 0

Retention tim (ml)

Figure 1. HPLC analysis of tetrapyrroles. Pigment extracts from C.reinhardtii were separated by ion-pair HPLC. Elution of chromo-phores was monitored at 405 to 460 nm. A, Cells incubated 24 hin the dark in the presence of 10 Mm DPEI. B, Untreated cells. C,Protoporphyrin IX standard (approximately 80 pmol).

solution consisting of 0.3 M sucrose, 25 mm Hepes/NaOH(pH 7.6), 1 mm EDTA, 0.1 mm DTT with a small paintbrush.

Measurement of Protoporphyrinogen IX Oxidase Activity

Protoporphyrinogen IX oxidase activity was determinedspectrofluorimetrically at 300C, essentially as described byLabbe et al. (17), by measuring the rate of formation ofprotoporphyrin IX from chemically reduced protoporphyri-nogen IX (12). Protoporphyrinogen IX was stored underparaffin at -700C and used within 2 to 4 weeks. It waschecked for the absence of fluorescence emission before use.The assays were done using a Perkin-Elmer MPF 44B spec-trofluorimeter, and the excitation and emissions wavelengthswere 410 and 632 nm, respectively. The reaction was carriedout in a total volume of 3 mL and contained 100 mm potas-sium phosphate (pH 7.6) saturated with air, 1 mm EDTA, 6mM DTT, 2 mg Tween 80, and 3.3 Mm protoporphyrinogen.The reaction was started by the addition of an aliquot ofcrude organelle suspension (200-500 MAg of protein).DPEs at the appropriate concentrations were added in

propanol (final propanol concentration, 0.1%, v/v) to thereaction mixture after an initial rate of fluorescence increase(control rate) had been obtained. The rates, which weremeasured between 2 and 6 min after addition of the organellesuspension, are expressed as percentages of the individualcontrol rates.

Protein Estimations

These were carried out by the Bradford method using theBio-Rad kit and BSA (Sigma) as the protein standard.

RESULTS AND DISCUSSION

Incubation of C reinhardtii cells in the dark with the nitroDPE herbicide DPEI resulted in the accumulation of tetra-pyrrole compounds during a period of at least 30 h (data notshown). Figure 1 shows the HPLC profiles of extracts oftreated (A) and untreated (B) cells after a 24-h incubation. Itcan be seen that addition of the herbicide induced abnormalaccumulation of at least four components. The major pig-ment, with a retention time of 23.7 min under these condi-tions, was identified as protoporphyrin IX by comparison ofits retention time, absorption, and fluorescence spectra withstandard protoporphyrin IX (Fig. 1, A and C; other data notshown). When the DPEI-treated extract and standard proto-porphyrin IX samples were cochromatographed, the majorpeak was enhanced, although it ran at a slightly differentretention time. Slight variations in retention time were gen-erally observed with this solvent system. Our results are inagreement with the findings of Witkowski and Halling (31),who demonstrated that protoporphyrin IX was the majortetrapyrrole accumulated when cucumber cotyledons incu-bated with 5-aminolevulinic acid were treated in the darkwith acifluorfen-methyl.A dose-response curve for DPEI-induced tetrapyrrole ac-

cumulation during 24 h in the dark is shown in Figure 2. Theconcentration required to give a half-maximal tetrapyrroleaccumulation is 0.45 Mm, and tetrapyrrole accumulation as afunction of DPEI concentration is approximately linear at low(<0.25 Mm) herbicide concentrations. We decided to use thelatter parameter as a method for comparing the relativeefficacy of compounds because this avoids ambiguities arisingfrom herbicide solubility limiting the maximal level of tetra-pyrrole accumulation. For each compound, the dependence

pM DPEI

Figure 2. Dose-response curve for tetrapyrrole accumulation in C.reinhardtii incubated with DPEI in darkness for 24 h. Tetrapyrroleconcentrations in cell extracts were measured as described in"Materials and Methods." The initial linear component in the dose-response curve is indicated by the dashed line.

o

1213




of tetrapyrrole accumulation on herbicide concentration isexpressed relative to the value for DPEI, as a reference,measured on the same batch of cells. The results are shownin Table I. Camilleri et al. (4) introduced the phthalide DPEsand showed that these compounds have phytotoxic effectssimilar to those of the nitro DPEs. The results in Table Idemonstrate that phthalide DPEIII was more effective ateliciting tetrapyrrole accumulation in Chlamydomonas culturesthan the nitro DPEs, DPEI, and nitrofen. Oxyfluorfen was

the most potent compound.As a measure of toxicity of the DPEs on Chlamydomonas,

we tested their ability to inhibit an increase in cell density.Other symptoms normally used to monitor DPE activity(light-induced ethane formation, pigment bleaching, and celldeath monitored using loss of fluorescein diacetate hydrolysis[2, 6, 7, 15]) were not observed in illuminated C. reinhardtiicells (data not shown). 150 values for a range of DPEs are

shown in Table I. The ability to inhibit growth correlatesqualitatively with the ability to induce tetrapyrrole accumu-

lation, suggesting that the two effects are linked.To examine this possibility further, we tested the relative

abilities of the purified S(-) and R(+) enantiomers of thephthalide DPEIII to inhibit growth of C reinhardtii and theirability to induce tetrapyrrole formation in darkness. Bothparameters indicate that the S(-) isomer is 4.4-fold more

potent than the R(+) isomer (Table I, Fig. 3), consistent withthe differential herbicidal effects of the two enantiomers on

intact plants (3). HPLC analysis of extracts of cells treatedwith the isomers confirmed that the major species showingenhanced accumulation upon treatment with the S(-) isomerwas protoporphyrin IX (data not shown).

3030i

0 20

/ R(+)E 10

E

A

0 0.025 0.05 0.075PM phthalide DPEIII

Figure 3. Dose-response curves for tetrapyrrole accumulation in C.reinhardtii incubated with the R(+) and S(-) enantiomers of DPEIIIin darkness for 24 h. Tetrapyrrole was assayed as described in"Materials and Methods."

These results suggest that growth inhibition and tetrapyr-role accumulation result from DPEIII binding to the same

binding site showing chiral discrimination. They cannot,however, distinguish between chiral discrimination at theactive site of an enzyme, which differentially metabolizesDPEIII to an inactive form, and discrimination at the sitewhere DPEIII exerts its primary herbicidal action. Protopor-

Table I. Comparison of the Effects of DPE Herbicides on C. reinhardtii Cells and ProtoporphyrinogenIX Oxidase Activity in Crude Etioplast Lysates from P. sativum

150 for Inhibition ofRelative Induction of ko for Growth Protoporphyrinogen

Herbicide Tetrapyrrole 50.forGw IX Oxidase Activity inAccumulation in C reinhardtiio Crude EtioplastC. reinhardtii' Suspension from

P. sativum'

AM AM

Nitrofen 0.223 0.34DPEI 1.00" 0.16Phthalide DPEIII 3.76 0.023 0.025Phthalide DPEIII 1.06 0.083 >0.3R(+) isomerPhthalide DPEIII 4.67 0.019 0.01S(-) isomerOxyfluorfen 12.55 0.0026

The values shown represent tetrapyrrole accumulation activity relative to DPEI (= 1.00). Tetra-pyrrole accumulation was determined at low herbicide concentrations where accumulation increaseslinearly with concentration (see Fig. 2). Dividing the slope of such plots for each herbicide by thatobtained with DPEI yielded the figures shown. b 150 is for growth of the culture in TAP medium,monitored by the effect on increase in cell density after a 24-h dark preincubation and 24-h incubationunder illumination of 200 Wm-2, following subculturing. Cultures treated with 0.1% (v/v) DMSOwere used as controls. ' 150 is for a decrease in the in vitro rate of protoporphyrinogen IX oxidation.The values are estimated from the data presented in Figure 4. d The concentration of DPEIrequired to give half-maximal accumulation of tetrapyrrole was 0.45 uM (see Fig. 2).

HALLAHAN ET AL.1214



STEREOSPECIFIC EFFECTS OF A CHIRAL DIPHENYL ETHER HERBICIDE

phyrinogen IX oxidase has been implicated as the primaryherbicide target for peroxidizing DPE herbicides (21, 22, 29,32), possibly arising through the DPE mimicking two of thepyrroles of the protoporphyrinogen macrocycle at the activesite. We tested the effect of the enantiomers of DPEIII onprotoporphyrinogen IX oxidase activity in a crude etioplastpreparation from P. sativum. Such an assay system could beexpected to preclude significant herbicide metabolism with areasonable degree of certainty. The results of this experimentare shown graphically in Figure 4. The S(-) isomer is clearlya more potent inhibitor than the R(+) isomer, with theracemic mixture showing an intermediate potency.The concentration of the S(-) isomer of DPEIII required to

inhibit protoporphyrinogen oxidation in the P. sativum etio-plast lysate assay system by 50% (approximately 0.01 uM;Table I) is approximately half of that required to inhibit thecell density increase in C reinhardtii by 50%. In the etioplastassay, chiral discrimination between the S(-) and R(+) iso-mers is considerably greater than for growth inhibition andtetrapyrrole accumulation in C. reinhardtii cells (Table I). Thedata in Figure 4 suggest that approximately 60% of theprotoporphyrinogen IX oxidase activity in the crude P. sati-vum etioplast preparation is highly sensitive to inhibition bythe S(-) isomer of DPEIII, and the remaining activity is muchless sensitive. This suggests that in the crude P. sativumetioplast preparation there are two different catalytic siteswith at least one site showing a high level of chiral discrimi-nation in binding the enantiomers of DPEIII.Our demonstration that the differential effects of the enan-

100

800

c60

, 40._

20

0 25 50 75 100 125 600 1000

Phthalide DPE conc (nM)

Figure 4. Inhibition of pea protoporphyrinogen IX oxidase activityby DPEIII. A, R(+) enantiomer; U, S(-) enantiomer; *, racemicmixture of the two enantiomers. The in vitro assays were carriedout as described in "Materials and Methods." Activities are ex-

pressed as the percentage of the control rate measured beforeaddition of the inhibitor. The results presented are a composite ofthree separate experiments with at least two replicates per point.The control rate of protoporphyrinogen IX oxidase activity (= 100%)was 5.48 ± 0.90 (SE) nmol/h/mg of protein (10.95 ± 1.80 [SE] nmol/h/g fresh weight). Note the interruption of the x axis and change ofscale between 125 and 600 nm, so that the lines joining points on

either side of the interruption are illustrative only.

tiomers of DPEIII on protoporphyrinogen oxidase activity ina cell-free system can be correlated with their differentialeffects on tetrapyrrole accumulation in intact cells of C.reinhardtii provides strong supporting evidence for the linkbetween inhibition of protoporphyrinogen IX oxidase activityand protoporphyrin IX accumulation.Our results also show that the relative abilities of the DPEs

and enantiomers of DPEIII to inhibit growth in illuminatedC reinhardtii cultures can be correlated with their abilities toinduce tetrapyrrole accumulation in these cells in darkness.Such a correlation would result either from DPE inhibitionof Chl (and/or heme) biosynthesis having a direct effect ongrowth and cell division or from a more indirect effect ontetrapyrrole accumulation leading to differential photosensi-tization of singlet oxygen formation and oxidative damage.In studies of various higher plant species, Sherman et al. (28)observed a correlation between the extent of DPE-inducedtetrapyrrole accumulation in darkness and subsequent elec-trolyte leakage and Chl photobleaching after exposure tolight, consistent with the latter explanation.

ACKNOWLEDGMENTS

We would like to thank Dr. D.L. Hallahan for assistance in thepreparation of the manuscript, Chris Gerrish for skillful technicalassistance, PPG Industries Inc., Shell Research Ltd., and Rohm andHaas Company for kind gifts of the agrochemicals used in this work,and Drs. P. Labbe and J.-M. Camadro for their generous gift ofprotoporphyrinogen (chemically reduced) and protoporphyrin IXused to calibrate the spectrofluorimeter.

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