Plant PhysioL (1979) 64, 995-9990032-0889/79/64/0999/05/$0O.50/O

Modification of Herbicide Binding to Photosystem II in TwoBiotypes of Senecio vulgaris L.

Received for publication May 9, 1979 and in revised form July 18, 1979

KLAus Pl?IsTR1, STEvEN R. RADOSEVICH2, AND CHARLES J. ARNTZEN11 United States Department ofAgriculture, Science and Education Administration, Department of Botany,University ofIllinois, Urbana, Illinois 61801 and 2 Department of Botany, University of Caitfornia, Davis,California 95616

ABSTRACr

The pret study compares the lbding and i activity of twopbotosystem nh 3-(3,4ichorophenyl)-1,1-inethyur (diuronIDCMUI) and 2-chlor (ethylamie)-isopropyl amlne)-S.triazene(atazine). Cbroplasts isolated from naturly occurring triazn-ceptible and tiazin-resistant biotypes of common grdsl (Sscwo uAWiL.) sbowed the folowing c ttics. (a) Diuron strongly inhipbotosynthetic electron tasrt from H20 to >phenol in both biotypes. Strong inbitio by atrazine was observed onlywith the c asts (b) Hill plots of electron t ort

data inicate a noncooperatve bidng of one hibitor moleculeat the site of action for both diuron and atraie. (c) Scptbl chdoroplasts show a strong diur and atrazine bdg (14Cadoabel ays)with bing costnts (K) of IA x 10-i molar and 4 x 10-8 molar,reSPectilY. In the resstant cwoplasts the diuro binng was sllghtlydecreaed (K-5x 10-l molar), whereas no specfic atrazine binding wasdetected (d) In s ibe chlepsts, compete bing btweenradioactively labeled dbi ad non-labeled atrazine was observed. Thiscompetitin was absent In the resistant chloroplasts.We conclude that triazine resistance of both intact plats and isolated

choroplsts of Sewio w_ukvis L is based upon a minor iat ofthe protein In the photosystem II compxwch is r for ebinng. Ths cbhan results in a specfic loss of atrazine (triazne)-bnigcapadity.

Differential plant tolerance to various chemicals is the basis forcommercial utilization of herbicides in crops. The tolerance ofdesired crop species and susceptibility of undesirable weed speciesto herbicides have been explained in virtually all cases by differ-ential uptake of the applied chemicals by roots, leaves, or stems,differences in translocation and distribution of the herbicidesinside the plant, or metabolic reactions within the plant whichmodify the herbicide to produce nontoxic derivatives (4, 5).

Induced biological resistance to pesticides (other than herbi-cides) has frequently been observed. The appearance of houseffiesresistant to DDT [2,2-bis(p-chlorophenyl)-1,1,1-trichloroethaneIshortly after the introduction of this insecticide demonstrated thepossibility for rapid genetic modification leading to pesticidetolerance (12). Until recently there has been little evidence forinduced herbicide resistance in naturally occurring plant species.The first exception occurred in 1970 when Ryan (29) reported thatarazine and simazine no longer controlled common groundsel(Senecio vulgaris L.). The resistant biotype seeds for Ryan's studywere collected from a nursery where triazine herbicides had beenused annually for about 10 years. Since that time there have been

reports of atrazine3 resistance in five other weed species (Amar-anthus retroflexus L., Chenopodium album L., Ambrosia artemisi-£folia L., and Brassica campestris L.) in areas which have beensubjected to extensive and repeated triazine application (6, 7, 19).

Initial attempts to understand the mechanism of induced her-bicide resistance in weed biotypes have focused on altered herbi-cide metabolism by the resistant plants. The findings have pro-vided no evidence that differential metabolism of triazines in thesusceptible versus resistant biotypes could account for the appear-ance of the newly discovered triazine-resistant weeds. Differentialuptake or translocation of the herbicides has also been ruled outas a selection mechanism in S. vulgaris L., A. retroflexus L., andC. album L. (15, 16, 21-23).The first evidence that the newly developed herbicide resistance

was related to alterations at the site of action of the triazinesoccurred when Radosevich and colleagues (22, 23) found thatlight-induced electron trnsport by isolated chloroplasts fromthree different resistant weed biotypes was not inhibited by atra-zine. More extensive assays with stroma-free thylakoid membranesfrom susceptible and resistant weed biotypes have verified thatherbicide resistance lies at the level of the chloroplast membranes(3, 18, 24). In comparative studies ofdifferent classes ofherbicides,the degree of resistance was related to chemical structure. In somecases, 1,000-fold higher concentrations of various S-triazines wereneeded to inhibit electron transport to a similar extent in theresistant as compared to susceptible chloroplasts. Diuron (DCMU)was only slightly less effective in the triazine-resistant than in thesusceptible chloroplasts. This lack ofparallel behavior ofthylakoidmembranes in response to the triazines and diuron poses anapparent contradiction. It is generally accepted that both triazineand substituted urea inhibitors act on the same component and atthe same site in the photosynthetic electron transport chain (10,13). The present study was initiated to obtain further informationabout the herbicide-binding sites and, if possible, to learn howthese sites might be modified in resistant plants.

MATERIALS AND METHODSSeedlings of common groundsel were grown in soil for 6 to 8

weeks in a greenhouse. Stroma-free chloroplast thylakoids wereisolated from excised leaves as previously described (24). The Chlconcentration of the plastid suspension was calculated using theequations of Arnon (1). Kinetics of electron transport was moni-tored by detecting photoinduced A changes ofDCPIP spectropho-tometrically with a Hitachi model 100-60 spectrophotometer

3Abbreviations: atrazine: 2-chloro-4-(ethylamine)-6-(isopropylamine)-S-triazine; DCPIP: 2,6-dichlorophenolindophenol; Iso inhibitor concentra-tion giving 50% inhibition of the stated reaction; Q: the primary electronacceptor for photosystem II (quencher).

995

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PFISTER, RADOSEVICH, AND ARNTZEN

equipped for cross-illumination (24). Reaction mixtures contained5 ,ug Chl/ml, 0.1 M sorbitol, 10 mM MgC12, 10 mM NaCi, 10 mMTricine-NaOH (pH 7.8), 0.05 mm DCPIP, 1 mm NH4Cl, and 1tLM Gramicidin. Assays were conducted at 22 C.

Herbicide-binding analysis used the same suspension mediumused for electron transport measurements, except that DCPIP,ammonium chloride, and Gramicidin were omitted. All bindingstudies were conducted under room light at 22 C. Binding reac-tions were initiated by mixing 1 ml suspension medium, 25 p1chloroplasts (containing 50 ,ug Chl) and small amounts (5-20 pi)of either uniformly ring-labeled atrazine (5.37 Ci/mol) or diuron(0.99 Ci/mol). After 2-min incubation the samples were centri-fuged for 3 min at 12,000g in an Eppendorf 5415 centrifuge. One-half-ml aliquots of the clear supernatant were removed and addedto 9 ml of the PCS-scintillator fluid (Amersham-Buchler). Radio-activity of the samples was measured by liquid scintillation spec-trometry. The amount of bound inhibitor was calculated from thedifference between the total radioactivity added to the chloroplastsuspension and the amount of free inhibitor found in the super-natant after centrifugation. Further details of this procedure aredescribed by Tischer and Strotmann (30). Herbicide solutionswere prepared in methanol. For every experiment the final meth-anol concentration in the chloroplast suspension was less than 2%.

RESULTS

ANALYSIS OF ELECTRON TRANSPORT INHIBITION

Using isolated, stroma-free chloroplasts from susceptible andresistant biotypes of common groundsel seedlings, the effect ofdiuron and atrazine in PSII-mediated electron transport wasanalyzed. Addition of either atrazine or diuron reduced the rateof electron flow (as indicated by the decreased rate of dye reduc-tion) in the susceptible biotype chloroplasts (Fig. IA). In theresistant chloroplasts (Fig. 1B), diuron reduced electron transportwith slightly less efficiency than in the susceptible chloroplasts,whereas atrazine was much less effective in limiting electrontransport processes. These observations are consistent with resultspreviously reported by Radosevich et aL (24), who also showedthat differences in triazine inhibition are not due to reduced ratesof penetration of the inhibitor into the membrane. Using thisassay system, a concentration series of each herbicide was meas-ured for inhibitory activity. From these data, Lo values (a concen-tration of herbicide giving half-maximal inhibition of electrontransport) were calculated. The 150 values were 0.07 and 0.12 pMfor diuron in susceptible and resistant biotype chloroplasts, re-spectively, and 0.5 pm for atrazine in susceptible chloroplasts. Inresistant biotype chloroplasts, 50%o inhibition of electron transportcould not be achieved within the solubility range of atrazine(approximately 2 x 10-4 M).The measured values of electron transport inhibition at varying

concentrations of diuron and atrazine were analyzed by Hill plotsto characterize the binding properties of these herbicides. It isknown that binding equilibria of small molecules interacting withmacromolecules may take place by either independent or coop-erative binding (35). Binding properties are defined by the Hillequation:

l =K.[L]n

where 0 is the fraction of binding sites occupied, K equals thebinding constant, [LI is the concentration of herbicide molecules,and n is the Hill coefficient. Assuming that binding is equivalentto inhibition, it is possible to plot log [% inhibition/(100-% inhi-bition)] versus log [herbicide] for diuron or atrazine. These Hillplots (Fig. 2) show linear relationships with slope values near 1(values in parentheses in Fig. 2). The slope defines the Hillcoefficient; the values of unity indicate independent binding of a

FIG. 1. Inhibition of PSII-dependent electron transport (H20 -DDCPIP) by atrazine or diuron. Data presented are direct tracings of light-induced absorption changes recorded on a strip-chart recorder. Decreasesin A (580 nm) indicate photoreduction of DCPIP. Ih values presentedwere calculated from similar experiments using a concentration series ofthe two inhibitors.

_N ~~~~~~~It ~~~~~~~~~~~(1.14)

Q +1.0 _ (1.09)(1.07)

a+0.5

_

Diuron

-z5-rtrazine-z0.5-

K-1.0

8 7 6 5

- log [Herbicide]

FIG. 2. Hill plots of data obtained from analysis of electron transport(H20-) DCPIP) in the presence of various concentrations of herbicides.

Values in parentheses are calculated Hill coefficients. (U, 0): Data pointsfrom experiments using triazine-susceptible chloroplasts; (0): experimentsusing triazine-resistant chloroplasts.

single herbicide molecule. Hill plots are highly sensitive to chlo-roplast concentrations used during the herbicide/electron trans-port assays. Low Chl levels must be used to measure herbicideinhibition constants accurately (32). The value of the Hill coeffi-

Susceptible Chloroplasts

-;;~~~~~~~tLM ~~0.51,tMDiurontrazine

I50 (Atrazine) = 0.5 ILM

I 50 (Diuron) = 0.07 HM

I50 (Diuron) = 0.12 kLM20 sec

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HERBICIDE BINDING TO PSII

ment determined at very low Chl levels (as we have used) must beaccepted as more accurate than the previously reported Hillcoefficient of 2 for diuron in assays where higher Chl concentra-tions were used (36).

INHIBITOR-BINDING STUDIES

In electron transport inhibition experiments, a known quantityof inhibitor is added to the reaction mixture. The observed degreeof inhibition of photoreactions is then measured and related tothe total inhibitor concentration. Because of partitioning or bind-ing of the inhibitors to the chloroplast membranes, the actualamount of inhibitor in solution (free inhibitor) can be much lowerthan the initially added concentration. This equilibrium dependsupon the amount of chloroplasts, the amount of inhibitor present,and the affinity of the inhibitor to its binding site (14). Bindingstudies with radioactively labeled herbicides can be used to char-acterize directly the amounts of bound inhibitor, affinity, andnumber of binding sites for the inhibitor at true equilibriumconditions (30-32).Diuron Bhiding. The amount of diuron found to bind to chlo-

roplast membranes increased as the concentration of free, un-bound diuron was increased (Fig. 3A). From these plots (boundversus free diuron) it is apparent that the resistant chloroplastsshow a slightly lower affinity for diuron than the susceptiblechloroplasts. A more quantitative analysis of the binding data ispossible in a double reciprocal plot (Fig. 3B), in which the dataare transformed to linear relationships. The intercept on theordinate is a measure of the maximum number of availablebinding sites (on a Chl basis) and thus allows the determinationof the amount of bound inhibitor per "photosynthetic unit." Thishas been previously discussed in more detail by Tischer andStrotmann (32). The double reciprocal plot for diuron in thesusceptible chloroplasts clearly shows two different absorptionprocesses: one occurring at low inhibitor concentrations withhighaffinity, and another at higher inhibitor concentrations with loweraffinity. Tischer and Strotmann have called the second process"unspecific absorption," because this inhibitor binding was notcorrelated with the inhibition of electron transport. Only the datapoints fitting the high specific absorption process were used forour calculation of binding constants and the number of bindingsites (Table I). Using the calculated constants, the hyperboliccurves shown in Figure 3A were fitted to the actually measureddata points according to the equation:

X1.fb=KK + f

where b denotes the concentration of bound inhibitor/mg CM fis the concentration of free inhibitor in the solution; XI is theconcentration of inhibitor binding sites; and K is the bindingconstant. This method of curve fitting,ie. using only the constantsforhigh specific absorption, explains the apparent difference ofthe fitted curve and actually measured values for susceptiblechloroplasts athigh diuron concentrations (Fig. 3A).Atrzine Binding. Atrazine binding to susceptible chloroplasts

(Fig. 4, A and B) was analyzed and data are presented in the samefashion as was described for diuron. The calculated values for thebinding constant (K = 5.0 x 10- m) and the number of bindingsites (450 Chl/inhibitor) are presented in TableI. Over the sameatrazine concentration range, a completely different binding pat-tern was observed between the two chloroplast samples. Suscep-tible chloroplasts bound atrazine, but no binding was detected inthe resistant chloroplast sample. Because of the extremely lowatrazine affinity to thylakoids of resistant chloroplasts, an analysisin a reciprocal plot was not possible.

In other experiments (data not presented) higher concentrationsof atrazine were used in binding studies to analyze "unspecificabsorption." This process was observed only in the susceptiblechloroplasts.

0 20 40 60/Free Diuron (/iM)

80

FIG. 3. A: binding of ['4Cqdiuron to susceptible and resistant chloro-plast membranes. Fitting of curve to data points was accomplished usingthe constants (K and Xi) from Table I. B: double recprocal plot of thedata from Figure 3A used for detmination of XI (ordinate-intercept) andK (abscissa-intercept).

Table I. Calculated Binding Constants (K) and Number of Binding Sites(X; Chlorophylls per One Bound Inhibitor Molecule) for Diuron and

Atrazine

The values were determined by regression analysis of reciprocal plotssimilar to those shown in Figures 3B and 4B (correlation coefficients werebetween 0.997 and 0.970). The data are averages of six experiments.

Inhibitor Suseptbl ResisantK Xl K

Diuron 1.4 x 10-8M 420 ChM/in- 5 x 10-8 M 500 ChM/in-hibitor hibitor

Atrazine 4X 10-8 M 450 Chl/in- No binding detectedhibitor

It is not possible to determine from these data whether thebinding site for atrazine is totally lost in the resistant chloroplastsor if the affinity between the inhibitor and the membrane isstrongly diminished. The definition of a structure which is able tobind a molecule implies a certain detectable affinity between thesetwo. In the case of atrazine, however, the loss in affinity was sopronounced that at low concentration ranges specific binding tomembrane components was not detectable. The presence of weakbinding for atrazine in the resistant chloroplasts was suggested bythe observed slight inhibition of photosynthetic electron transportat very high atrazine concentrations (Fig. lB and ref. 24). Thisinhibition occurred only at much higher inhibitor concentrationsthan those used for binding assays in Figure 4. A qualitativeindication of weakatrazine binding to the membranes of resistantchloroplasts was obtained by analysis of binding competition

1.2 c° DIURON (DCMU)

1.0 //

.8 9' Susceptible

6 ZResistant.6 8

.4

/2I/

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PFISTER, RADOSEVICH, AND ARNTZEN

0 20 40 60 80/Free Atrozine (/zM)

FIG. 4. A: binding of ['4Clatrazine to susceptible and resistant chloro-plast membranes. Note that no atrazine binding to resistant chloroplastswas observed at these inhibitor concentrations. Curve fitting was asdescribed for Figure 3 and in text. B: double reciprocal plot of atrazinebinding to susceptible chloroplasts.

between ['4Cjdiuron and atrazine. The release of previouslybound, radioactively labeled diuron from chloroplast membranesin the presence of increasing concentrations of unlabeled atrazineis shown in Figure 5. In the susceptible chloroplasts, unlabeledatrazine effectively displaced diuron from the membranes. Theremaining amount of bound [14C]diuron, which was not releasedby high atrazine concentrations in susceptible chloroplasts, rep-resents unspecifically bound diuron. With resistant chloroplasts,high concentrations of unlabeled atrazine caused slight competi-tion against [14CJdiuron. It should be pointed out that the concen-tration scales in Figures 4 and 5 cannot be compared directly,since the extent of competition between inhibitors depends on theconcentration of I'4Cldiuron used in membrane pretreatments, itsaffmity, and on binding properties of atrazine. In other experi-ments using low diuron concentrations, a small but significantcompetition by atrazine became more apparent (data not pre-sented). This observation indicates a low triazine affinity, inagreement with the above-mentioned slight electron transportinhibition at concentrations near 0.1 mm atrazine.

DISCUSSION

It is commonly believed that diuron acts as a specific PSIIinhibitor by interrupting electron transfer between the primaryelectron acceptor Q and the plastoquinone pool (9; for a reviewsee 13). The most recent speculation about the mode of action ofdiuron is that it interacts directly with the second carrier on thereducing side of PSII, thereby altering its redox properties (37).Other effects of diuron (direct interaction with the reaction centerof PSII, inhibition of cyclic electron flow around PSI, or effects

on the oxidizing site of PSII) (8, 26, 29) are now thought to playa minor role at the diuron concentrations where high specificitybinding is observed.The site of action of the triazine inhibitors is thought to be

similar to that of diuron. This conclusion is based on similarpatterns of inhibition of PSII-dependent electron transport reac-tions and on the same pattern of modification of the Chl fluores-cence induction curve (3, 20, 37). However, the triazines havebeen less carefully examined than diuron with regard to specificmechanisms of action.The fact that diuron and atrazine appear to inhibit the same

step in electron transport does not necessarily indicate that thetwo inhibitors act at the same binding site. This information canonly be obtained by direct measurements of binding and analysisof competition between inhibitors. Tischer and Strotmann (32)have analyzed competition between these herbicides in spinachchloroplasts and indicate that both inhibitors act at the same site.This conclusion has now been verified for chloroplasts isolatedfrom the triazine-susceptible biotype of common groundsel (Fig.5). In contrast, very weak competition-between diuron and atrazinewas observed in the resistant chloroplasts. This latter observationis directly related to the fact that a high affinity binding site fordiuron but not atrazine was detected in the resistant chloroplasts.A contradiction is, therefore, apparent: how can lack of bindingof atrazine occur (resistant chloroplasts) while diuron still binds ifboth act at an identical site?A number of structure-activity studies with different classes of

inhibitors have shown the important role of a single structuralelement common to all PSII inhibitors (11, 34). The proposedstructures for this element are the configurations: -CO-NH ,-C-N*E ResistantCompetition * Susceptible

1C-Atrazine vs Bound 14C-Diuron *

LL//0 10-7 lo-6 10-5 1o-4

Concentration of 12C-Atrazine (M)FIG. 5. Competition between [14Cldiuron and atrazine in susceptible

and resistant chloroplasts. Concentration of ['4C]diuron: 0.5 uM.

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HERBICIDE BINDING TO PSII

SUSCEPTIBLE

diuron- type inhibitors

W-~!j RESISTANT

no /-t inhi

no inhibition inhibition

FIG. 6. Model of inhibitor binding to chloroplast membranes. Modelpresents the following concepts: A: asymmetrical orientation of photosyn-theticclectron transport chains; external localization of herbicide-bindingcomponent in thylakoid membrane. B: existence of two different regionswithin the herbicide binding component: an "essential region" of thebinding constituent which interacts with a common structural element ofthe different herbicides to produce a conformational change of the con-stituent, thus interrupting electrontransport on the reducing side of PSII;and, differentdomains which are necessary for specific acnt of theinhibitors to the binding constituent. C: modification of one substructureof the binding constituent in the ristant chloroplast leads to a loss ofatrazine-binding capability, whereas DCMU binding to the same constit-uent is not substantially affected.

carriers acting near the reducing side of PSII are also surface-localized in chloroplast membranes (33). In Figure 6 we haveindicated how specific changes in the herbicide-binding proteinmay selectively control herbicide activity. It is apparent thatstudies comparing normal susceptible chloroplast preparationsand plastids containing a modified PSI1 complex (resistant chlo-roplasts) will open a new area of research allowing further clari-fication ofthe molecularmechanisms which regulate the selectivityand mode of action of PSII-directed herbicides.AnaWet k Dr. Homer LeBaon, CIBA-GEIGY Corp, Raleigh, N.C., for

the radioactive atrane used ths study and for support and of this earch. We

aho Dr. James Risglman E. L du Pon de Nenoura and Co., Wilmington, Delawarefor the radioactive diuron. Te excellent technical asistance of Ms. Cathy Ditto and Ms Jan

Watson grateflly ecated. We thank Dr. K. Stenback for her cution regrding this

research.

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©atrazine - type inhibitors

999

rr')

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