Proc. NatL Acad. Sci. USAVol. 78, No. 2, pp. 981-985, February 1981Botany

Photoaffinity labeling of an herbicide receptor protein inchloroplast membranes

(triazines/photosystem II/electron transport inhibitors/diuron)

KLAus PFISTER*, KATHERINE E. STEINBACKt, GARY GARDNERf, AND CHARLES J. ARNTZENtUnited States Department of Agriculture/Science and Education Administration/Agricultural Research, Department of Botany, University of Illinois,Urbana, Illinois 61801

Communicated by Anton Lang, October 28, 1980

ABSTRACT 2-Azido- 4 -ethylamino- 6 -isopropylamino-s- tria-zine (azido-atrazine) inhibits photosynthetic electron transport ata site identical to that affected by atrazine (2-chloro4-ethylamino-6-isopropylamino-s-triazine). The latter is a well-characterized in-hibitor of photosystem II reactions. Azido-atrazine was used as aphotoaffinity label to identify the herbicide receptor protein; UVirradiation of chloroplast thylakoids in the presence ofazido['4C]atrazine resulted in the covalent attachment of radio-active inhibitor to thylakoid membranes isolated from pea seed-lings and from a triazine-susceptible biotype of the weed Ama-ranthuw hybridus. No covalent binding of azido-atrazine wasobserved for thylakoid membranes isolated from a naturally oc-curring triazine-resistant biotype of A. hybridus. Analysis of thy-lakoid polypeptides from both the susceptible and resistant A. hy-bridus biotypes by sodium dodecyl sulfate/polyacrylamide gelelectrophoresis, followed by fluorography to locate "'C label,demonstrated specific association of the azido["'C]atrazine withpolypeptides of the 34- to 32-kilodalton size class in susceptible butnot in resistant membranes.

Many commercial herbicides inhibit photosynthetic electrontransport by interrupting electron flow at the reducing side ofphotosystem II (PS II) (1, 2). There are several lines of evidencewhich indicate that this inhibition occurs at the level of a pro-tein-bound plastoquinone called "B" (3). This electron carrieracts as the second stable electron acceptor of PS II (4, 5). It hasbeen proposed that the mode of action of compounds such asatrazine or diuron is via high-affinity binding to the PS II com-plex (6). Herbicide binding induces a change in the redox po-tential of the quinone cofactor of B, thus making the transferof electrons from the primary acceptor (Q) thermodynamicallyunfavorable (3, 5). In addition, binding may decrease kineticinteractions of Q and B.

Evidence that PS II inhibitors interact with a polypeptide ofthe PS II complex, possibly the apoprotein of B, comes fromstudies involving the enzyme trypsin (7). Proteolytic digestionof surface-exposed membrane polypeptides results in a destruc-tion of inhibitor binding sites with a concomitant inactivationof the secondary acceptor, B (8). Attempts have.been made tocorrelate trypsin-mediated changes in polypeptides of the chlo-roplast membrane or detergent-solubilized PS II particles withchanges in inhibitor binding properties (9). To date, however,the polypeptide(s) that determines the inhibitor binding site hasnot been identified.A variety of radiolabeled inhibitors of PS II have been used

for the characterization of the properties of the herbicide-bind-ing site; these studies have resulted in the demonstration of asingle binding site per electron transport chain (3, 10). In ad-dition, the affinity of various inhibitors for this site has beendetermined (6, 8). The association of PS II inhibitors with their

binding site is noncovalent. For this reason, attempts at physicalisolation of proteins labeled by a radioactive inhibitor havefailed because detergent fractionation or electrophoretic sepa-ration rapidly leads to a new equilibrium and dissociation of theacceptor-inhibitor complex. The approach we have used toovercome this difficulty in identification of the herbicide re-ceptor is to attach a radiolabeled photoaffinity azido derivativeof atrazine to its high-affinity receptor polypeptide in a covalentmanner. It is well established that activation of the azido func-tion of photoaffinity-labeled compounds by UV irradiation pro-duces a nitrene that is highly reactive (11). In preliminary in-vestigations this was found to covalently link the azido-atrazineto chloroplast membranes (12, 13).

MATERIALS AND METHODS

Plant Material. Seedlings of dwarf pea (Pisum sativum Lin-naeus 'Progress No. 9,' Ferry Morse Seed, Mountain View, CA)were grown as described (14). Leaves were harvested from 12-to 20-day-old seedlings. Biotypes of Amaranthus hybridus sus-ceptible or resistant to PS II inhibitors were grown in sterilesoil. The seed stocks of A. hybridus were derived from plantsoriginally collected in Whatcon County, Washington, and re-ferred to previously as A. retroflexus (15); the correct taxonomicidentification for these plants has been provided by L. Wax(Agronomy Dept., Univ. of Illinois, Urbana, IL). Leaves wereharvested from 4- to 6-week-old seedlings.

Isolation of Stroma-Free Chloroplast Thylakoids. Leaf tis-sue (20 g) was homogenized in a blender for 5 s at high speed,with 100 ml of cold 100 mM N-[tris(hydroxy-methyl)methyl]glycine (Tricine)/NaOH pH 7.8 buffer con-taining 0.4 M sorbitol and 10 mM NaCl. Homogenizationbuffer for A. hybridus leaf tissue contained, in addition, 50 mMWodium ascorbate and 0.05% bovine serum albumin.The homogenate was filtered through two and then eight lay-

ers of cheesecloth, and the resulting filtrate was centrifuged at

Abbreviations: atrazine, 2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine; azido-atrazine, 2-azido-4-ethylamino-6-isopropylamino-s-tria-zine; B, a protein-bound quinone serving as secondary electron acceptorfor photosystem II; Chl, chlorophyll; CI2indophenol, 2,6-dichloroin-dophenol; diuron, 3-(3,4-dichlorophenyl)-1, 1-dimethylurea; kDal, kilo-dalton(s); PS II, photosystem II; Q, plastoquinone serving as primarystable electron acceptor from photosystem II; Tricine, N-[tris(hydroxymethyl)methyl]glycine.* Present address: Institut fur Botanik, Universitat Wurzburg, M. Dal-lenbergweg 64, -D-87 Wurzburg, Federal Republic of Germany.

t To whom reprint requests should be addressed at the present address:MSU-DOE Plant Research Laboratory, Michigan State University,East Lansing, MI 48824.

t Present address: Shell Development Co., P.O. Box 4248, Modesto,CA 95352.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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1000 X g for 10 min. The resulting pellet was resuspended in40 ml of 10 mM Tricine/NaOH, pH 7.8, containing 10 mMNaCl and recentrifuged at 3000 X g for 5 min. The pellet, con-

taining stroma-free chloroplast thylakoids, was resuspended to1-2 mg of chlorophyll (Chl) per ml in 50 mM Tricine/NaOHpH 7.8 buffer containing 100 mM sorbitol, 5 mM MgCl2, and10mM NaCl. Chl concentration was calculated by the equationsof Mackinney (16).

Assays for chloroplast photochemical activity with 2,6-di-chloroindophenol (Cl2indophenol) as the electron acceptorwere as described (10).

Binding Studies. Chloroplast membranes were diluted to a

final concentration of 50 kug of Chl per ml in a 1.0-ml volumeof the final resuspension buffer (20'C). Various concentrationsof 14C-labeled azido-atrazine (specific activity 37.1 MkCi/mg; 1

Ci = 3.7 x 1010 becquerels) were then added (0.07-7 ,uM finalconcentration). After an incubation time of 3 min at room tem-perature (under dim room light) the chloroplast suspension wascentrifuged in an Eppendorf 5415 centrifuge at 12,000 x g for3 min. From the supernatant, 0.8-ml samples were removedand the radioactivity of these samples was determined by liquidscintillation counting. From these measurements, the amountsof free and bound inhibitor were calculated.

Competition binding experiments were performed in a man-ner similar to the binding studies described above. A suspensionof chloroplasts (13 ml) (50 ,g of Chl per ml) was first incubatedwith either 0.4 AM ['4C]atrazine (specific activity 27.2 ,uCi/mg) or 0.33 ,uM azido[14C]atrazine (specific activity 37.1 AOCi/mg). Samples were either UV irradiated or incubated in thedark for 10 min. After this incubation, various amounts of[12C]atrazine (10-7 to 2 x 10-4 M final concentrations) were

added in small volumes to each sample. The samples were in-cubated for 3 min and then centrifuged as described above.

All inhibitor stock solutions were made up in methanol; thefinal methanol concentration in any chloroplast suspension was

always below 1%. Unlabeled and [ 4C]atrazine were gifts of H.LeBaron, CIBA-GEIGY Agricultural Chemical Division. Un-labeled and azido[14C]atrazine were synthesized by Shell De-velopment, Modesto, CA.UV irradiation was carried out for 10 min with two Sylvania

G15T8 germicidal UV lamps located 10 cm above a 5.8-cm di-ameter glass petri dish containing 13 ml of chloroplast mem-brane suspension (50 ug of Chl per ml).

100

O 0

'4-

0 rO.= cQ

0.

N4 °

V C.)

AO

50

10-8 10-7 10-6Herbicide concentration, M

FIG. 1. Inhibition of Cl2indophenol photoreduction in isolated peachloroplasts by azido-atrazine (o) and atrazine (o). Rates are expressedas percent of control rate in the absence of added inhibitor. The controlrate for these experiments was 400 Aeq/mg of Chl per hr.

Polyacrylamide Gel Electrophoresis. Analysis of mem-

brane polypeptides by NaDodSOJpolyacrylamide gel electro-phoresis was carried out by using the discontinuous buffer sys-

tem of Laemmli (17). Electrophoresis was performed in a slabgel apparatus (18) on a 12-20% (wt/vol) linear polyacrylamidegradient gel and a 5% stacking gel. Membrane samples (50 ,mgof Chl) were solubilized in 25 ,ul of sample buffer containing195 mM Tris-HCl, pH 6.8, 30% (vol/vol) glycerol, 6% (vol/vol) 2-mercaptoethanol, 6% (vol/vol) NaDodSO4, for 30 min atroom temperature. Samples (7-12 ,ug of Chl) were applied tosample wells. Electrophoresis and staining of gels for proteinwere carried out as described (19).

Polyacrylamide gels were analyzed for covalent 14C-labeledtriazine by x-ray fluorography (20) at -80°C, using Kodak SB-5 x-ray film.

RESULTS AND DISCUSSIONCharacterization of the Azido-Atrazine as an Inhibitor of

PS II Photoreactions. It was previously demonstrated that a

variety of 2-azido-4-alkylamino-6-alkylamino-s-triazines blocklight-induced electron transport dependent upon PS II activity(21). Inhibition of PS II-mediated photoreduction ofCl2indophenol by atrazine and azido-atrazine is shown in Fig.1 for chloroplast membranes isolated from pea seedlings. Theconcentrations of inhibitor required for 50% inhibition (Iso con-

centrations) of this reaction were very similar for the two tria-zines (0. 12pM for azido-atrazine and 0. 19 ,uM for atrazine). Thereplacement of the chloro group of atrazine by the photolabileazido group does not result in markedly changed inhibitoryproperties.The modification of fluorescence induction kinetics by azido-

atrazine is shown in Fig. 2. The induction curve for chloroplastmembranes treated with azido-atrazine was identical to that ofatrazine-treated samples. Because the relative area above theinduction curve is a measure of the site of electron transportinhibition (see ref. 3), these data establish that the actions ofazido-atrazine and atrazine are at the same step in the electrontransport chain.

CD

q)

._

a)

a)

4-0

C-:

3

2

1

0

Time of illumination, s

FIG. 2. Modification of Chl fluorescence induction transients ofisolated pea chloroplasts by 10 gM azido-atrazine (----) or atrazine( ). The stimulation in rate of the fast fluorescence rise by azido-atrazine or atrazine in comparison to control ( ) indicates a blockin electron transfer at the reducing side of PS II. The data presentedare direct tracings of the plotted data obtained from a digital storageoscilloscope; the transient changes observed for atrazine and azido-atrazine were exactly superimposed in plotted form.

I II

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I t a

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Evidence for Covalent Binding of Azido-Atrazine to Mem-branes After UVIrradiation. Binding of atrazine to chloroplastmembranes is noncovalent and thus reversible. This can bedemonstrated either by washing procedures to remove the in-hibitor from its binding site or by competition experiments inwhich the bound inhibitor is replaced by increasing concentra-tions of an added second inhibitor (see refs. 6 and 10). Evidencefor covalent binding of a photoaffinity inhibitor to its receptorsite therefore is: (i) that transfer of membranes with bound in-hibitor to herbicide-free solution (membrane washing) does notresult in release of bound inhibitor and restoration of electrontransport activity, and (ii) that the inhibitor is not replaced bya competitive inhibitor.We have compared the reversibility of electron transport in-

hibition for atrazine and azido-atrazine in samples of pea thy-lakoids that were either incubated in the dark or treated withUV (Table 1). In the absence of inhibitor, electron transport wasaffected by UV treatment as reported (22). A 10-min UV irra-diation resulted in a 33% decrease of PS II activity. For exper-iments with atrazine and azido-atrazine as inhibitors, the her-bicide concentration was adjusted to give a similar degree ofinhibition (70%). In samples that were not irradiated or thatwere irradiated in the presence of atrazine, the inhibition couldbe largely reversed by washing (see Table 1). In contrast, in-hibition of samples incubated with azido-atrazine and treatedwith UV could not be reversed by washing. This irreversibleinhibition observed in the UV-treated azido-atrazine samplesindicates irreversible attachment of the inhibitor to its receptorsite.

Herbicide Binding and Competition Experiments. The re-sults of binding studies with '4C-labeled azido-atrazine areshown in Fig. 3. These data, plotted in double reciprocal form,allow a calculation of the number of binding sites (1 bound in-hibitor per 430 Chl molecules) and of a binding constant (Kb)of 32 nM. Both values are in good agreement with the data

Table 1. Measurement of reversible herbicide bindingto isolated thylakoids

Ratio ofCl2indophenol

Time of UV Control rate of reduction ratesInhibitor irradiation, Cl2indophenol (after wash/added min reduction before wash)

None 0 225 0.8710 150 0.98

Atrazine, 0 66 2.1820M 10 42 2.28

Azido-atrazine, 0 66 2.5412 AM 10 36 1.00

Chloroplast membranes (13-ml samples, 50 jig of Chl per ml) wereincubated for 10 min in the dark or under UV irradiation with no ad-ditions, 20 1AM atrazine, or 12 pM azido-atrazine. Aliquots were thenremoved, diluted to 5 ug of Chl per ml in the reaction mixture usedto monitor Cl2indophenol photoreduction, and illuminated in a spec-trophotometer as described (10). The remaining portion of the 13-mlvolume was centrifuged at 3000 x g. The resultant pellet was washedtwice by suspension and recentrifugation in the resuspension buffer.Aliquots of the washed membranes were then removed and assayedfor Cl2indophenol photoreduction. All assay mixtures contained, in 2-ml volumes, 10 1g of Chl, 50 mM sodium phosphate buffer (pH 6.8),10 mM MgCl2, 100 mM sorbitol, 60 ,uM Cl2indophenol, 100 pM di-phenylcarbazide, 2 mM NH4Cl, and 1 MM gramicidin D. The controlrates presented were determined for samples prior to washing; valuespresented are ,umol of Ci2indophenol reduced permg of Chl per hr. Theextent of reversal of herbicide inhibition caused by washing (i.e., re-lease of bound herbicide) is indicated by the ratio of Cl2indophenolreduction rate after washing to that before washing; values greaterthan 1.0 indicate herbicide release.

bO

N'U

Free azido-atrazine, ,uM

FIG. 3. Binding of azidol[4Clatrazine to pea chloroplast mem-branes. Analysis of these data by a double reciprocal plot gave a bind-ing constant (Kb) of 32 nM and a value of 1 inhibitor binding site per430 Chl molecules.

Obtained for atrazine binding in Senecio and Amaranthus chlo-roplasts (one inhibitor per 440-450 Chl and a Kb of 40-70 nM)(3, 10).

Competition experiments demonstrated that noncovalentlybound radiolabeled atrazine could be displaced by nonlabeled

10-7 10-6 10-5 10-4

Unlabeled atrazine, M

FIG. 4. Competition of [Cl4Catrazine with unlabeled atrazine incontrol pea chloroplast thylakoid membranes Mo and membranes thathave been treated with UV for 10 min in the presence of [14C~atrazine(0).

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-

ut2bo2E0.

0

-o

._

coQ

0 10-7 10-6 10-5 10-4Unlabeled atrazine, M

FIG. 5. Competition of azido['4C]atrazine with unlabeled atrazinein control pea chloroplast thylakoid membranes (o) and membranesthat had been treated with UV for 10 min in the presence ofazido[14C]atrazine (e).

atrazine in dark-treated as well as UV-treated membranes (Fig.4). The extent to which UV damage affected atrazine bindingsites is seen from the decrease in binding of ['4C]atrazine in theabsence of unlabeled atrazine. The loss of binding sites cor-responded closely with the loss of PS II activity in UV-treatedmembranes. In either control or UV-irradiated samples, how-ever, unlabeled atrazine caused similar displacement of the[I4C]atrazine. UV irradiation also caused loss of binding sitesfor azido['4C]atrazine (Fig. 5). Bound radioactive azido-atrazinecould be displaced by nonlabeled atrazine in the sample incu-bated in the dark. This result further establishes that both in-hibitors act at the same chloroplast membrane receptor site. Incontrast to the sample incubated in the dark, the azido-atrazinewas not displaced from its binding site by unlabeled atrazineafter 10-min UV irradiation. The specificity of the photoaffinitylabeling has been further supported by the observations thatatrazine protects the site from the UV-induced nitrene, and thatphotolysis of nonradioactive azido-atrazine at the binding sitereduces subsequent [14C]atrazine binding (12).

To test the specificity of azido-atrazine binding to the PS IIinhibitor site, we compared inhibitor binding to triazine-re-sistant and susceptible chloroplasts of A. hybridus biotypes. Itis now established that, in the chloroplasts of the resistant bio-type, the binding site for triazines is selectively altered so thataffinity for these inhibitors is greatly diminished (3, 8, 10). The

Table 2. Binding of azido-atrazine to chloroplasts from triazine-resistant and triazine-susceptible Amaranthus weed biotypes

nmol bound inhibitorChloroplasts Treatment per mg ChlResistant Dark <0.1

UV <0.1

Susceptible Dark 2.3UV 1.25

Chloroplasts were incubated with 0.5 ,uM azido-atrazine (50 ,g ofChl per ml).

data presented in Table 2 show that the UV-treated resistantchloroplasts bind only a negligible amount of azido-atrazine, ascompared to chloroplasts isolated from the susceptible biotypes.This demonstrates that the azido-atrazine binding occurs onlyin the susceptible membranes and specifically at the high-af-finityMinhibitor-binding site of the PS II complex.

Analysis of membrane polypeptides from resistant and sus-ceptible Amaranthus chloroplasts by NaDodSOjpolyacryla-mide gel electrophoresis is shown in Fig. 6 (Coomassie blue-stained polypeptides in lanes A). Analysis of the gel by a fluo-rographic technique showed no detectable bound radiolabel insamples from either resistant or susceptible chloroplast mem-branes incubated in the dark with azido['4C]atrazine beforeelectrophoretic analysis (data not shown). In samples that wereUV irradiated in the presence of azido[14C]atrazine prior to sol-ubilization in NaDodSO4, only the susceptible membranesshowed specific covalent attachment of the azido['4C]atrazineto a polypeptide migrating close to stained polypeptides of34-32 kDal (lanes B). Lack of covalent binding to this poly-

SusceptibleA B

ResistantA B

ams

32 -*- 4S

25 --

16 _-P *w j

5_I

FIG. 6. Polyacrylamide slab gel electrophoresis of thylakoid mem-brane polypeptides from susceptible and resistant biotypes of A.-hy-bridus, stained for protein (lanes A) and by fluorography (lanes B).Susceptible and resistant membranes were incubated with 0.5 ,uMazido[(4C]atrazine under UV light for 10 min prior to solubilizationwith NaDodSO4. Arrows indicate polypeptides of 32, 25, and 16 kilo-daltons (kDal). The predominant location of the radiolabel as shownby fluorography is over the 34- to 32-kDal polypeptide size classes.

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peptide in the resistant membranes indicates the loss of thetriazine receptor region of this polypeptide.Two polypeptides of 25 and 16 kDal were labeled to a minor

extent in the susceptible membranes. The polypeptide at 25kDal corresponds to the major polypeptide of the light-har-vesting pigment-protein complex, which is structurally andfunctionally associated with PS II. It is improbable that the 25-kDal polypeptide contains the atrazine-binding site because itis absent from chloroplast membranes of a Chl b-less barleymutant (23) and partially developed chloroplasts recovered frompea seedlings grown under intermittent light (24), though bothof these chloroplasts retain sensitivity to atrazine and other in-hibitors of PS II reactions. We have determined that prepa-rations of purified light-harvesting pigment-protein complexdo not bind atrazine (data not shown). The fact that the 25-kDalpolypeptide does become slightly labeled in susceptible mem-branes may indicate its close structural proximity to the high-affinity binding site.The significance of the slight labeling of a 16-kDal polypep-

tide is not clear. It is possible that this polypeptide is also a com-ponent of the PS II complex that is in proximity to the triazine-binding site. Alternatively, it is possible that the 16-kDal poly-peptide is structurally related to the 32-kDal component via amembrane-localized processing step; this concept will be dis-cussed in a subsequent manuscript.

CONCLUSIONSWe have demonstrated that azido-atrazine inhibits PS II pho-toreactions by binding at the receptor site that confers atrazinesusceptibility. UV irradiation resulted in the irreversible bind-ing of azido-atrazine to polypeptides of 34-32 kDal. The factthat binding did not occur in triazine-resistant chloroplasts isan important control showing specificity of labeling of the pro-tein containing the high-affinity binding site.The identification of 34- to 32-kDal polypeptides as the tria-

zine herbicide receptor is consistet with several other obser-vations. Chloroplasts from a maize mutant lacking the 32-kDalpolypeptide are blocked in PS II function (25) and lack bindingsites for radioactive atrazine (unpublished observations). A 32-kDal protein is present in isolated PS II particles and is removedby trypsin digestion in parallel with loss of diuron (9) bindingsites.

It has been demonstrated that triazine resistance in intactplants is maternally inherited in Brassica campestris Linnaeus(26); this was correlated to the maternal inheritance of PS IIcomponents that confer atrazine sensitivity (27). Chloroplast-directed synthesis of a precursor polypeptide to a 32-kDal pro-tein has been reported (28). In the latter case, the functionalrole of the chloroplast gene product has not been determined;

however, preliminary evidence indicates that the chloroplast-directed polypeptide and the membrane polypeptide labeledby azido['4C]atrazine are structurally identical.

We thank Jan Watson for patient technical assistance. This researchwas supported, in part, by U.S. Department of Agriculture BinationalAgricultural Research and Development Grant US 80-79 and a travelgrant to K. P. by the Deutsche Forshungsgemeinschaft.

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