Plant Physiol. (1991) 97, 197-2030032-0889/91/97/01 97/07/$01 .00/0

Received for publication February 26, 1991Accepted May 2, 1991

Effect of Diphenyl Ether Herbicides on Oxidation ofProtoporphyrinogen to Protoporphyrin in Organellar and

Plasma Membrane Enriched Fractions of Barley1

Judith M. Jacobs, Nicholas J. Jacobs*, Timothy D. Sherman, and Stephen 0. Duke

Department of Microbiology, Dartmouth Medical School, Hanover, New Hampshire 03756 (J.M.J., N.J.J.) andUnited States Department of Agriculture, Agricultural Research Service, Southern Weed Science Laboratory,

Stoneville, Mississippi 38776 (T.D.S., S.O.D.)

ABSTRACT

In barley (Hordeum vulgare L.) root cells, activity for oxidizingprotoporphyrinogen to protoporphyrin (protoporphyrinogen oxi-dase), a step in chlorophyll and heme synthesis, was found bothin the crude mitochondrial fraction and in a plasma membraneenriched fraction separated by a sucrose gradient techniqueutilized for preparing plasma membranes. The specific activity(expressed as nanomoles of protoporphyrin formed per hour permilligram protein) in the mitochondrial fraction was 8 and in theplasma membrane enriched fraction was 4 to 6. The plasmamembrane enriched fraction exhibited minimal cytochrome oxi-dase activity and no carotenoid content, indicating little contam-ination with mitochondrial or plastid membranes. Etioplasts frometiolated barley leaves exhibited a protoporphyrinogen oxidasespecific activity of 7 to 12. Protoporphyrinogen oxidase activityin the barley root mitochondrial fraction and etioplast extractswas more than 90% inhibited by assay in the presence of thediphenyl ether herbicide acifluorfen methyl, but the activity in theplasma membrane enriched fraction exhibited much less inhibi-tion by this herbicide (12 to 38% inhibition) under the same assayconditions. Acifluorfen-methyl inhibition of the organellar (mito-chondrial or plastid) enzyme was maximal upon preincubation ofthe enzyme with 4 mm dithiothreitol, although a lesser degree ofinhibition was noted if the organellar enzyme was preincubatedin the presence of other reductants such as glutathione or ascor-bate. Acifluorfen-methyl caused only 20% inhibition if the enzymewas preincubated in buffer without reductants. Incubation ofbarley etioplast extracts with the earlier tetrapyrrole precursorcoproporphyrinogen and acifluorfen-methyl resulted in the accu-mulation of protoporphyrinogen, which could be converted toprotoporphyrin even in the presence of the herbicide by theaddition of the plasma membrane enriched fraction from barleyroots. These findings have implications for the toxicity of diphenylether herbicides, whose light induced tissue damage is appar-ently caused by accumulation of the photoreactive porphyrinintermediate, protoporphyrin, when the organellar protoporphyri-nogen oxidase enzyme is inhibited by herbicides. Our resultssuggest that the protoporphyrinogen that accumulates as a resultof herbicide inhibition of the organellar enzyme can be oxidizedto protoporphyrin by a protoporphyrinogen oxidizing activity thatis located at sites such as the plasma membrane, which is muchless sensitive to inhibition by diphenylether herbicides.

'The study was supported by U.S. Department of AgricultureCompetitive Grant 9000705 (N.J., S.D.) with additional support fromNational Science Foundation Grant DCP 8918298 (N.J.J.).

Several laboratories have found the photobleaching DPE2herbicides to increase greatly the intracellular levels of thephotodynamic tetrapyrrole, protoporphyrin, causing light de-pendent phytotoxic effects (2-4, 6, 14-16, 18, 22-24, 26, 29).DPE herbicides also inhibit Protox, which is the last enzymeof the common branch of the heme and Chl biosyntheticpathways in plants and oxidizes protoporphyrinogen to pro-toporphyrin (1, 5, 12, 20, 21, 27, 30). These observations haveled to the hypothesis that DPE herbicides exert their primaryeffect by inhibiting Protox, causing an accumulation of itssubstrate, protoporphyrinogen. The question of how the ac-cumulated protoporphyrinogen is oxidized to the photoreac-tive protoporphyrin in the presence of DPE herbicides hasnot been addressed. It has been assumed that this is a non-enzymatic process since a chemical oxidation of protopor-phyrinogen to protoporphyrin does occur slowly upon expo-sure of protoporphyrinogen to air. In addition, the questionof how this oxidation occurs in a manner that prevents theprotoporphyrin formed from re-entering the biosyntheticpathway to Chl has not been studied. (Protoporphyrin is anintermediate in both the heme and Chl pathways.) Becausethis accumulation of protoporphyrin is apparently critical forDPE toxicity, it seemed important to investigate how proto-porphyrinogen could be oxidized to protoporphyrin withinthe plant cell.A possible explanation for protoporphyrin accumulation

from protoporphyrinogen in DPE treated plants might befound in the compartmentalization of Protox activity withinthe plant cell. Because organelles such as the chloroplast aresites for tetrapyrrole synthesis (and thus protoporphyrinogenformation) and because the Protox enzyme associated withchloroplast and mitochondrial membranes had been shownto be inhibited by DPE herbicides, we wanted to determinewhether other membranes within the plant cell can carry outprotoporphyrinogen oxidation and whether this oxidizingactivity is inhibited by DPE herbicides. Our findings dosuggest an additional location for protoporphyrinogen oxi-dation by other membranes such as the plasma membranewithin the plant cell. This plasma membrane activity is rela-tively insensitive to DPE herbicide inhibition. This couldexplain the rapid accumulation of protoporphyrin after ex-

Abbreviations: DPE. diphenylether; Protox. protoporphyrinogenoxidase; PM, plasma membrane; AFM. acifluorfen-methyl.

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Plant Physiol. Vol. 97, 1991

posure of plants to DPE herbicides (2, 19). In addition, thesefindings are compatible with previous in vivo localizationstudies in intact plant cells showing that DPE treated plantcells exhibit a rapid accumulation of porphyrins at non-plastidic sites, such as the plasma membrane or in the cyto-plasm (16). Taken together, these findings suggest that theprotoporphyrinogen, which is synthesized by organelles suchas the chloroplast and which accumulates in the presence ofDPE, may diffuse away from the chloroplast and be oxidizedto protoporphyrin at other membranous sites within the cell,such as the PM.

MATERIALS AND METHODS

Preparation of Crude Mitochondrial Fraction and PMEnriched Fraction from Barley Roots and Etioplasts fromBarley Leaves

The PM enriched fraction was prepared essentially by thetechniques of Hodges and Leonard (7) and Hodges and Mills(8) for the isolation of PMs. Barley seeds (Hordeum vulgareL. var Birka) (9) were germinated in the dark on moistenedpaper towels for 5 d and the roots (20-40 g) were ground ina mortar and pestle in homogenizing buffer (0.25 M sucrose,3 mM EDTA, 25 mm Tris Mes, pH 7.2) as described byHodges and Mills (8) with the addition of 2.5 mM DTT. Thefiltered homogenate was sedimented at 3000g for 5 min toremove debris and large plastids. The pellet was resuspendedin a small volume of homogenizing buffer without DTT andwas termed the plastid fraction. The supernatant was spun at1 3,000g for 15 min to sediment the mitochondria, which werewashed once in a small volume of the homogenizing bufferbut without added DTT. This layer is termed the crudemitochondrial or organellar fraction and contains mitochon-dria, but may also be contaminated with other organellefragments sedimenting under these conditions in root homog-enates (see "Results").To prepare the PM enriched fraction, the mitochondrial

supernatant was sedimented at 80,000g (1 h, 5°C) and theresidue was suspended in 1 to 2 mL of 25 mM Tris 7.5, 0.25M sucrose (without DTT). This was layered on a discontinuousgradient of 20, 30, 34, and 45% sucrose in 1 mM Tris Mesbuffer without DTT (7, 8, 25) and sedimented at 95, lOOg for2 h (SW 27 rotor, Beckman ultracentrifuge). By this proce-dure, the PM is reported to layer at the 34/45% interface (25),which was removed, diluted in an equal volume of homoge-nizing buffer, sedimented at 80,000g for 1 h, and resuspendedin a minimal volume of homogenizing buffer (without DTT)and stored at -70°C. This procedure is reported to yield PMpreparations of 70% purity (7). This fraction was termed thePM enriched fraction, although the presence of all othercontaminating membrane fragments was not critically evalu-ated. For the purposes of this study, it was important to assesscontamination of this fraction by either mitochondrial orplastid membranes. This was accomplished as indicated in"Results." The Triton X-100 extract of barley leaf etioplastswas prepared as described previously (9).

Assay for Protoporphyrinogen Oxidation

The direct spectrofluorometric assay described previously(12) was used. In brief, the reaction is followed directly in

spectrofluorometric cuvettes at room temperature by meas-uring the increase in fluorescence emission at the protopor-phyrin peak (630 nm) in an assay mixture containing Trisbuffer pH 7.5, EDTA, and Tween 20 detergent (12). DTT orother reductant was added to the assay mixture (where indi-cated) and the enzyme was preincubated in this mixture forat least 20 min at room temperature before addition ofprotoporphyrinogen. Protoporphyrinogen was prepared byreduction with sodium amalgam (10, 12) and neutralized bydilution with an equal volume of 0.5 M Tris and 50 mm DTTto keep the porphyrinogen reduced (12). A 10-gL aliquot ofthe protoporphyrinogen solution was added to the 250-,ALassay mixture. This addition brought the concentration ofprotoporphyrinogen in the assay to approximately 7 ,uM andadded 1 mm DTT to the assay. The formation of protopor-phyrin was followed immediately after addition of protopor-phyrinogen and the rate was calculated from the initial linearrate of increase in protoporphyrin fluorescence within the first15 min after substrate addition (12). Except where indicated,all rates are corrected for the low rate of protoporphyrinogenoxidation exhibited by a similar level of heated enzyme (12).This heated control was less than 10% of the rate of theunheated enzyme.

Assay for Conversion of Coproporphyrinogen toProtoporphyrin

The assay mixture was almost identical to that used forfollowing protoporphyrinogen oxidation and contained (in atotal assay volume of 500 ,uL): 100 mM Tris buffer pH 7.5; 1mM EDTA; 5 mM DTT; 384 mg of etioplast extract preparedas described above; and 10 Mm AFM (where indicated). Thereaction was started by the addition of 40 MuL of a solutioncontaining 6 nmol of coproporphyrinogen in 0.25 M Trisbuffer containing 25 mM DTT. Coproporphyrinogen wasprepared from a 300 Mm solution of coproporphyrin III (Por-phyrin Products, Logan, UT) by reduction with sodium amal-gam (approximately 1.7 g/mL) as described previously (12).The reduced porphyrin solution was neutralized by dilutioninto an equal volume of the Tris buffer plus DTT solution asdescribed previously for the direct assay for Protox (12). Theamount of protoporphyrin formed was followed spectrofluo-rometrically by scanning the reaction mixture over the emis-sion range from 580 to 700 nm using an excitation wavelengthof 420 nm after removal of a 50-,uL aliquot into 1.45 mL ofa measuring mixture containing buffer, EDTA, reductant,and detergent described previously (10). A recording spectro-fluorometer (9) with excitation and emission slits set at 2 nmwas used. Standard solutions ofprotoporphyrin IX (PorphyrinProducts) were used to calibrate the amount of protopor-phyrin formed, measured from the height of its emission peakat 633 nm. Any coproporphyrin formed could be detectedfrom its emission peak at 615, but we detected very littleincrease in coproporphyrin formed during the 4-h period ofthe assay, indicating little auto-oxidation of coproporphyri-nogen to coproporphyrin. In the experiment described in"Results," an addition of 140 MuL of PM enriched fraction(560 ,ug of protein, prepared from barley roots as describedabove) was added to the assay mixture 2 h after the additionof coproporphyrinogen, where indicated.

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DIPHENYL ETHER HERBICIDES AND PROTOPORPHYRINOGEN OXIDASE

Table I. Protox Activity of Barley Root Mitochondrial and PM-Enriched Fractions Assayed after Preincubation of Enzyme in 4 mMDTT

The fractions were prepared from two different batches of barleyroots, and the data represent the mean and range of three determi-nations on batch 1 and two determinations on batch 2. The enzymepreparations were preincubated for at least 20 min in 4 mm DTTbefore substrate addition. AFM was present at 10 Mm where indicated.The reaction was started by adding a protoporphyrinogen solutioncontaining sufficient DTT to bring the final DTT concentration to 5mm in the assay mixture (see "Materials and Methods"). The activityof the heated controls (see "Materials and Methods") for both mito-chondria and PM-enriched fractions was equivalent (0.17 ± 0.05nmol/h/mg) and less than 5% of the rate of unheated enzyme.

Protox Activity

Uninhibited Plus AFM % Inhibition

nmol protoporphyrinformed h- mg-' protein

Mitochondrial fractionBatch 1 8.1 ± 0.6 0.8 ± 0.4 91Batch2 7.8 ± 1.5 0.4 ± 0.1 94

PM-enriched fractionBatch 1 4.1 ± 0.4 3.6 ± 0.2 12Batch 2 6.5 ± 0.1 4.0 ± 0.3 38

Other Methods

Cyt c oxidase activity was measured by the method ofHodges and Leonard (7). AFM, obtained as described previ-ously (12), was dissolved in acetone and added to the assay in2.5-,uL volumes, with equivalent amounts of acetone addedto the assays without inhibitor. The carotenoid content ofmembrane fractions was utilized as a marker for plastidcontamination of the PM enriched fraction (28). A smallvolume of the plastid or PM enriched fraction (50-100 ,L)was extracted with 400 ,L of acetone by vigorous agitationfor 5 min. The precipitated protein was removed by centrif-ugation and the pellet was re-extracted with 300 ,L ofacetone.The combined acetone layers were mixed with 1 mL ofpetroleum ether. The ether layer was transferred to a -mLcurvette and scanned for typical carotenoid spectrum between550 and 400 nm using the 0.1 absorbancy scale on a Cary 17spectrophotometer. Absorbancy values as low as 0.002 couldbe reliably detected by this technique.

RESULTS

Protoporphyrinogen Oxidation by the Mitochondrial(Organellar) Fraction and the PM Enriched Fraction ofBarley Roots

Both the mitochondrial and the PM enriched fraction ex-hibited high levels of Protox activity (Table I), with the PMenriched fraction containing more than half the levels shownby mitochondria. Protox activity in the mitochondrial frac-tion was more than 90% inhibited by AFM, whereas the PMenriched fraction exhibited a much lower but significantinhibition by AFM under identical assay conditions (Table I).As indicated, this low level of inhibition varied between 12

and 38% inhibition in two different PM enriched preparationsfrom two different root plantings (Table I). We also examineda third PM enriched preparation and found it to be 33 ± 0%inhibited by AFM (average of duplicate assays). Further stud-ies are underway in our laboratory to explain the variabilityin the low levels of AFM inhibition exhibited by the PMenriched fractions (see "Discussion").

For the purposes of this study, it was important to examinethe possibility that the Protox activity in the PM enrichedfraction was due to significant contamination by mitochon-drial membranes that exhibit high levels of AFM sensitiveProtox activity. Contamination by plastid membrane frag-ments was also evaluated, since our previous studies showedetioplasts from barley leaf to contain high levels of AFMsensitive Protox activity (12). Although the sucrose gradientprocedure we utilized to prepare the PM enriched fraction isreported to yield preparations containing 70% PM, the PMcontent of our preparation was not established and other rootmembranes might have been present. Mitochondrial contam-ination of the PM enriched fraction was evaluated with themitochondrial marker enzyme, cytochrome oxidase, whichwas present at high levels in the mitochondrial preparations(Table II). The PM enriched fraction exhibited less than 10%

of the Cyt oxidase activity of the mitochondria (Table II),ruling out significant mitochondrial contamination. Contam-ination of the PM enriched fraction with fragments of plastidmembrane was evaluated with the use of carotenoids as a

specific marker for plastid membranes (29). The carotenoidcontent of the plastid fraction from the barley root homoge-nate (prepared by low speed centrifugation as indicated in"Materials and Methods") was compared with the carotenoidcontent of the PM enriched fraction. When 185 mg of plastidmembrane protein (from batch 2, Table I) was extracted forcarotenoids as indicated in "Materials and Methods," a typicalcarotenoid spectrum was readily visible with peaks at 468,435, and 417 nm. The absorbancy at the 468 nm peak was0.007. When 370 mg of PM enriched protein (from batch 2,Table I) was extracted, no carotenoid peaks were seen, indi-cating no significant contamination of the PM enriched frac-tion with plastid membrane fragments.The demonstration of a Protox activity in the PM enriched

fraction, which is much less sensitive to AFM inhibition than

Table II. Cyt Oxidase Activity of Mitochondrial and PM-EnrichedFractions from Barley Root

The values shown are nmol of reduced Cyt c oxidized/min/mgprotein of the membrane preparations used in Table I, and representthe mean and SD of triplicate determinations for batch 1 and twodeterminations for batch 2.

Cyt Oxidase Activity

nmol min-' mg 1 proteinMitochondrial fraction

Batch 1 1481 ± 74Batch 2 1958 ± 120

PM-enriched fractionBatch 1 108 ± 36Batch 2 225 ± 61

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the mitochondrial Protox activity, suggests that protopor-phyrinogen can be oxidized by either an AFM sensitive or arelatively AFM resistant mechanism at different locationswithin the plant cell. This observation has implications bothfor the mechanism by which DPE herbicides cause accumu-lation of the phytotoxic protoporphyrin within the plant celland for the possible mechanisms involved in protoporphyri-nogen oxidation by the plant cell (see "Discussion").

Effect of Preincubation with DTT on AFM Inhibition ofProtox

As shown in Table III, there is a much decreased level ofAFM inhibition in the mitochondrial fraction if the enzymeis preincubated in the assay mixture in the absence of 4 mMDTT before substrate addition (compare Table III with TableI). We have previously reported this phenomenon (12), al-though its significance will require further studies into themechanism of Protox and its inhibition by DPE herbicides.A comparison of Table I and Table III also shows that theuninhibited rate of oxidation of protoporphyrinogen is de-creased more than threefold by preincubation with 4 mmDTT. The reason for this marked decrease in Protox activityafter preincubation with DTT is not understood, but couldhave important implications for the mechanism of Protoxaction.

Effect of Other Reductants on AFM Inhibition

Because the effects of preincubation with DTT on AFMinhibition could have important physiological implications,and since DTT is not a physiological reductant, we deter-mined the effect of naturally occurring reductants on AFMinhibition. For these studies, we utilized detergent extracts ofetioplasts isolated from etiolated barley leaves as the source

of Protox activity. We have previously shown these extractsto have high Protox activity (9). Table IV illustrates the resultsof experiments comparing the ability of several physiologicalreductants to substitute for DTT in potentiating AFM inhi-bition. As illustrated, reductants such as glutathione, ascor-

bate, and NADPH can potentiate AFM inhibition, especiallywhen used at high concentrations. However, none of thesewas as effective as DTT. These results suggest that the reduc-tant concentration within the plant organelle may have a

significant role in AFM-mediated inhibition of Protox (see"Discussion"). It seems possible that only under conditions

Table Ill. AFM Inhibition of Protox Activity in Mitochondrial and PM-Enriched Fractions from Barley Root Assayed without Preincubationin the Presence of DTT

All conditions are as shown in Table I, except that the enzyme wasnot preincubated in 4 mm DTT. Data represent mean and SD of threedeterminations.

Protox Activity

Uninhibited Plus AFM % Inhibition

nmol h- mg 1 protein

Mitochondria fraction (batch 1) 28.0 ± 3.2 21.7 ± 3.4 20PM-enriched fraction (batch 1) 20.2 ± 1.8 18.0 ± 2.0 10

Table IV. Effect of Other Reductants on AFM Inhibition of ProtoxActivity in Barley Etioplast Extracts

Detergent extracts of etioplasts (0.15 mg protein/250 Ail assay vol)prepared as described in "Materials and Methods" were preincubated,either in buffer with no reductant or with the reductant indicated forat least 20 min before addition of protoporphyrinogen to start theenzymatic reaction. All data represent the mean and range of at leasttwo determinations. Different preparations of etioplast extract pre-pared from two different batches of etioplasts were used in experi-ments 1 and 2.

Preincubation Protox ActivityConditions Uninhibited Plus AFM % Inhibition

nmol protoporphyrin formedh mg1 protein

Experiment 1No reductant 32.7 ± 2.5a 30.1 ± 3.3a 8.14 mM DTT 7.0 ± 0.8a 1.1 ± 0.1a 84.74 mM glutathione 16.6 ± 0.0 11.3 ± 0.5 32.54 mM NADPH 13.7 ± 0.0 7.8 ± 0.5 43.0

Experiment 2No reductant 64.2 ± 3.8 53.1 ± 1.0 17.55 mM DTT 12.5 ± 1.0 0.5 ± 0.5 96.520 mM glutathione 17.7 ± 3.1 6.3 ± 2.1 65.520 mM ascorbate 16.9 ± 0.8 2.4 ± 1.4 86.0

a Average of three determinations.

in which reductant concentrations are high will there besignificant AFM inhibition of Protox within the plant organ-elle (see "Discussion"). Table IV also shows that preincuba-tion with these other reductants, in addition to potentiatingAFM inhibition, also caused a significant decrease in unin-hibited Protox activity.

Demonstration That the Protoporphyrinogen ThatAccumulates from Earlier Precursors during AFMTreatment of Etioplasts Can be Oxidized toProtoporphyrin by the PM Enriched Fraction in thePresence of AFM

Extracts of barley etioplasts are capable of forming proto-porphyrin from the earlier porphyrin precursor, copropor-phyrinogen (Fig. 1). It can be assumed that this requires aconcerted reaction of the two enzymes, coproporphyrinogenoxidase (which converts coproporphyrinogen to protopor-phyrinogen) and Protox. In this coupled system, AFM causeda marked inhibition of protoporphyrin formation from co-proporphyrinogen (Fig. 1). It seems likely that this inhibitionof protoporphyrin accumulation was caused by AFM inhibi-tion of Protox.

Figure 1 also shows that the protoporphyrinogen that ac-cumulated in etioplasts in the presence of AFM could berapidly converted to protoporphyrin upon the addition of aPM enriched fraction derived from barley root. The latterfraction does not exhibit coproporphyrinogen oxidase activityand therefore cannot form protoporphyrin from copropor-phyrinogen (data not shown), although it can oxidize proto-porphyrinogen to protoporphyrin by a largely AFM-resistantmechanism (see Table I). This finding has significant impli-cations for the mechanism by which protoporphyrin accu-

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DIPHENYL ETHER HERBICIDES AND PROTOPORPHYRINOGEN OXIDASE

1.2

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Figure 1. Effect ofto protoporphyrin bfrom barley roots.Tprotoporphyrin formterials and Methods.with coproporphyrinand the formation c

and Methods"). AftEenriched fraction frcvolume of H20 was,,

mulates when plhtreated with AFMthat accumulatesinhibited by AF1membranes elsewAFM (see "Discu:

The finding th.,protoporphyrin itthe PM enrichedfor the mechanisidamage within ththat DPE herbicidelles such as the c

leading to the ac(nogen. However,gen is converted tis important to un

protoporphyrinogcule responsible fDPE herbicides. (in which the prot

tetrapyrrole synthbition diffuses awrelatively DPE re

branes such as the PM. An attractive feature of this hypothesisis that it would explain the observation that the protopor-

no AFM phyrin formed in DPE treated plants does not readily re-enter

AFM the Chl or heme synthesis pathways, since the protoporphyrinAFM + PM / is formed at a membranous site within the cell that is separated

from the enzymes in the chloroplast, which are involved inconverting protoporphyrin to later intermediates in the mag-nesium branch of the pathway. In other words, our hypothesissuggests that in DPE treated plants the oxidation of protopor-phyrinogen to protoporphyrin has become spatially separatedfrom the other enzymes of Chl synthesis.Another attractive feature of this hypothesis is that it is

compatible with previous in vivo observations that the cyto-plasmic membranes or cytoplasm of the plant cell is an earlysite of protoporphyrin accumulation in DPE treated plants(16). These previous in vivo observations were made by ex-posing intact, achlorophyllous plant tissue to DPE and ob-

addition of PM or H2O serving the appearance of the typical porphyrin fluorescence2. , , . ,.. in the intact cell by fluorescence microscopy. In these studies,

0 1 2 3 4 the PM or cytoplasm was the first to exhibit fluorescencewithin a brief period following DPE exposure, and it was

Time (h) concluded that protoporphyrin accumulated outside the plas-

AFM on the conversion of coproporphyrinogen tid (16).iybarley etioplasts and the PM enriched fraction It is possible that the protoporphyrin that is formed from7he assay conditions and procedure for following protoporphyrinogen at extraplastidic membrane sites can ac-iation from coproporphyrinogen are given in "Ma- cumulate to a high concentration and subsequently diffuse."Etioplast extract (384 Ag protein) was incubated back into the chloroplast and be converted to later Chlogen either in the presence or absence of AFM, intermediates. In fact, this previous study (16) has shown that)f protoporphyrin was monitored (see "Materials when intact, achlorophyllous plant tissue is treated with DPEer 2 h (as indicated by the arrow) either the PM . ' .

zm barley roots (560zg protein) or an equivalent in the dark, there iS an initial accumulation of protoporphyrinadded to thetwo assay tubes inhibited with AFM. that increases to a high level after several hours. This increase

is followed by a subsequent DPE-dependent slow accumula-tion of the later Chl intermediate, protochlorophyllide (16).

ants that are actively synthesizing Chl are To summarize our conclusions about the effect of DPE her-It seems likely that the protoporphyrinogen bicides on the partitioning of porphyrin accumulation withinwhen the plastid or mitochondrial Protox is the plant cell, we propose the model shown in Figure 2. Thisd can be converted to protoporphyrin by model emphasizes maximum accumulation of the phototoxicthere within the cell even in the presence of protoporphyrin at extra plastidic sites such as the PM. Thession"). model offers an explanation for the observation that photoac-

tive DPE herbicides cause damage first to the PM and otherDISCUSSION extraplastidic membranes before damage to the plastid mem-

branes occurs (see ref. 16 and references therein).at protoporphyrinogen can be oxidized to We have also examined, for the first time, some of then a relatively AFM insensitive reaction by factors involved in the coupled conversion of coproporphyr-fraction of the plant cell has implications inogen to protoporphyrinogen followed by the oxidation ofm by which DPE herbicides cause cellular protoporphyrinogen to protoporphyrin in plants. Apparently,te plant cell. Previous studies had indicated AFM causes protoporphyrinogen to accumulate from copro-[es block tetrapyrrole synthesis within organ- porphyrinogen, as would be expected if AFM specificallyhloroplast by inhibiting the Protox enzyme, inhibited the Protox enzyme. The demonstration that thecumulation of its substrate, protoporphyri- accumulated protoporphyrinogen could be rapidly oxidizedthis did not explain how protoporphyrino- to protoporphyrin even in the presence of AFM by the addi-to protoporphyrin in DPE treated plants. It tion of the PM enriched fraction represents a convincingderstand this, since protoporphyrin and not argument in favor of our hypothesis that the PM plays anen is apparently the photodynamic mole- important role in the formation of protoporphyrin from theor the light-activated destruction caused by protoporphyrinogen that accumulates when the organellarDur findings are compatible with a process Protox enzyme is blocked by AFM in vivo.toporphyrinogen that accumulates within a Our findings also have implications for the mechanismkesizing organelle as the result of DPE inhi- involved in protoporphyrinogen oxidation within the planttay from the organelle and is oxidized by a cell. One question that arises is the difference between thesistant mechanism at other cellular mem- Protox activity in barley organelles, which is very sensitive to

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Plant Physiol. Vol. 97, 1991

Figure 2. Scheme for porphyrin metabolism in diphenyl ether herbi-cide (DPE)-affected plant cells. We propose that in cells exposed toDPE herbicides in darkness the following sequence of events occur:1, Protox is inhibited; 2, this causes protoporphyrinogen (Protogen)to accumulate and to leave the pathway to arrive at a PM-associatedProtogen-oxidizing factor, which may or may not be enzymic; 3, thisfactor generates high protoporphyrin (Proto) levels in extraplastidicsites such as the PM; and 4, the accumulated Proto can subsequentlyre-enter the plastid-localized porphyrin pathway, especially after ex-traplastidic sites are saturated. The exact location of many of theenzymatic steps of Chl synthesis has not been established (1).Coprogen = coproporphyrinogen, PChlide = protochlorophyllide.

AFM inhibition, and the activity in the PM enriched fraction,which is relatively resistant to AFM. One explanation couldbe that the Protox activity in the latter fraction is a reflectionof a relatively nonspecific but membrane dependent proto-porphyrinogen oxidizing capacity. This activity may have nobiosynthetic function, whereas the organellar Protox activityoccurs through the mediation of a specific biosynthetic en-zyme. It is possible that a nonspecific protoporphyrinogenoxidizing activity is associated with a variety of other plantmembranes such as the PM, the tonoplast membrane, andthe Golgi and endoplasmic reticulum, although we have onlyexamined the PM enriched fraction. Although it is prematureto designate the activity in the PM enriched fraction as

enzymic or nonenzymic, it should be emphasized that theactivity in this fraction is heat labile. Heated controls exhibitless than 10% of the activity observed in the presence of theunheated membrane (see Table I and ref. 12).Another possible explanation for the difference in AFM

sensitivity of the different membrane preparations is thatprotoporphyrinogen oxidation is carried out by the sameProtox enzyme in both the PM enriched fraction and theorganelle, but the activity in the former fraction is in a

different form that lacks AFM sensitivity but retains theoxidizing capacity. It is possible that the Protox activity in thePM fraction is partially AFM sensitive, but is much moreeasily converted to an AFM resistant form than is the mito-chondrial Protox activity. To examine this possibility, we

have attempted to convert the Protox activity in barley rootmitochondria to an AFM resistant form merely by dilutionand recentrifugation in dilute buffer without reductants or by

purification by gradient centrifugation in the absence of re-ductants. These preliminary experiments failed to demon-strate conversion of mitochondrial Protox to an AFM resist-ant form. Further work comparing the characteristics of Pro-tox activity from plant mitochondria and chloroplasts (11-13, 17) to that in other plant membranes is needed to resolvethese questions.Another observation that may have significance for herbi-

cide action concerns the effect of reductants in potentiatingAFM inhibition of Protox activity in barley etioplasts andmitochondria. Nearly complete inhibition is noted only whenthe enzyme is preincubated with 5 mm DTT. Lesser degreesof inhibition occur in the presence of physiological reductantssuch as glutathione and ascorbate, and little inhibition isobserved if the enzyme is preincubated in the assay bufferwithout reductant. Further studies on the mechanism of theProtox enzyme will be needed to clarify the role of reductants.However, this observation may have implications for explain-ing the variable effect of DPE herbicides in different plantspecies, if species differ in reductant concentrations at theorganellar site of Protox activity.

ACKNOWLEDGMENTS

AFM (99.5% pure) was generously provided by Rhone-PoulencAgricultual Company, Research Triangle Park, NC. We thank KevinC. Vaughn for helpful discussions and critical advice during prepa-ration of the manuscript.

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