Plant PhysioL (1979) 64, 995-999 0032-0889/79/64/0999/05/$0O.50/O

Modification of Binding to Photosystem II in Two Biotypes 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. ARNTZEN1 1 United States Department ofAgriculture, Science and Education Administration, Department ofBotany, University ofIllinois, Urbana, Illinois 61801 and 2 Department ofBotany, University of Caitfornia, Davis, California 95616

ABSTRACr reports of atrazine3 resistance in five other weed species (Amar- anthus retroflexus L., Chenopodium album L., Ambrosia artemisi- The pret study compares the lbding and i activity of two £folia L., and Brassica campestris L.) in areas which have been pbotosystem nh 3-(3,4ichorophenyl)-1,1-inethyur (diuron subjected to extensive and repeated triazine application (6, 7, 19). IDCMUI) and 2-chlor (ethylamie)-isopropyl amlne)-S.triazene Initial attempts to understand the mechanism of induced her- (atazine). Cbroplasts isolated from naturly occurring triazn-cep bicide resistance in weed biotypes have focused on altered herbi- tible and tiazin-resistant biotypes of common grdsl (Sscwo uAWi cide metabolism by the resistant plants. The findings have pro- L.) sbowed the folowing c ttics. (a) Diuron strongly inhi vided no evidence that differential metabolism of triazines in the pbotosynthetic electron tasrt from H20 to > susceptible versus resistant biotypes could account for the appear- phenol in both biotypes. Strong inbitio by was observed only ance of the newly discovered triazine-resistant weeds. Differential with the c asts (b) Hill plots of electron t ort uptake or translocation of the has also been ruled out data inicate a noncooperatve bidng of one hibitor molecule as a selection mechanism in S. vulgaris L., A. retroflexus L., and at the site of action for both diuron and atraie. (c) Scptbl chdoro C. album L. (15, 16, 21-23). plasts show a strong diur and atrazine bdg (14Cadoabel ays) The first evidence that the newly developed herbicide resistance with bing costnts (K) of IA x 10-i molar and 4 x 10-8 molar, was related to alterations at the site of action of the triazines reSPectilY. In the resstant cwoplasts the diuro binng was sllghtly occurred when Radosevich and colleagues (22, 23) found that decreaed (K-5x 10-l molar), whereas no specfic atrazine binding was light-induced electron trnsport by isolated chloroplasts from detected (d) In s ibe chlepsts, compete bing btween three different resistant weed biotypes was not inhibited by atra- radioactively labeled dbi ad non-labeled atrazine was observed. This zine. More extensive assays with stroma-free thylakoid membranes competitin was absent In the resistant chloroplasts. from susceptible and resistant weed biotypes have verified that We conclude that triazine resistance of both intact plats and isolated herbicide resistance lies at the level of the chloroplast membranes choroplsts of Sewio w_ukvis L is based upon a minor iat of (3, 18, 24). In comparative studies ofdifferent classes ofherbicides, the protein In the photosystem II compx wch is r for e the degree of resistance was related to chemical structure. In some binng. Ths cbhan results in a specfic loss of atrazine (triazne)-bnig cases, 1,000-fold higher concentrations of various S-triazines were capadity. needed to inhibit electron transport to a similar extent in the resistant as compared to susceptible chloroplasts. Diuron (DCMU) was only slightly less effective in the triazine-resistant than in the susceptible chloroplasts. This lack ofparallel behavior ofthylakoid membranes in response to the triazines and diuron poses an apparent contradiction. It is generally accepted that both triazine Differential plant tolerance to various chemicals is the basis for and substituted inhibitors act on the same component and at commercial utilization of herbicides in crops. The tolerance of the same site in the photosynthetic electron transport chain (10, desired crop species and susceptibility ofundesirable weed species 13). The present study was initiated to obtain further information to herbicides have been explained in virtually all cases by differ- about the herbicide-binding sites and, if possible, to learn how ential uptake of the applied chemicals by roots, leaves, or stems, these sites might be modified in resistant plants. differences in translocation and distribution of the herbicides inside the plant, or metabolic reactions within the plant which MATERIALS AND METHODS modify the herbicide to produce nontoxic derivatives (4, 5). Induced biological resistance to (other than herbi- Seedlings of common groundsel were grown in soil for 6 to 8 cides) has frequently been observed. The appearance ofhouseffies weeks in a greenhouse. Stroma-free chloroplast thylakoids were resistant to DDT [2,2-bis(p-chlorophenyl)-1,1,1-trichloroethaneI isolated from excised leaves as previously described (24). The Chl shortly after the introduction of this insecticide demonstrated the concentration of the plastid suspension was calculated using the possibility for rapid genetic modification leading to equations of Arnon (1). Kinetics of electron transport was moni- tolerance (12). Until recently there has been little evidence for tored by detecting photoinduced A changes ofDCPIP spectropho- induced herbicide resistance in naturally occurring plant species. tometrically with a Hitachi model 100-60 spectrophotometer The first exception occurred in 1970 when Ryan (29) reported that arazine and no longer controlled common groundsel 3Abbreviations: atrazine: 2-chloro-4-(ethylamine)-6-(isopropylamine)- (Senecio vulgaris L.). The resistant biotype seeds for Ryan's study S-triazine; DCPIP: 2,6-dichlorophenolindophenol; Iso inhibitor concentra- were collected from a nursery where triazine herbicides had been tion giving 50% inhibition of the stated reaction; Q: the primary electron used annually for about 10 years. Since that time there have been acceptor for photosystem II (quencher). 995 996 PFISTER, RADOSEVICH, AND ARNTZEN Plant Physiol. Vol. 64, 1979 equipped for cross-illumination (24). Reaction mixtures contained 5 ,ug Chl/ml, 0.1 M sorbitol, 10 mM MgC12, 10 mM NaCi, 10 mM Susceptible Chloroplasts Tricine-NaOH (pH 7.8), 0.05 mm DCPIP, 1 mm NH4Cl, and 1 tLM Gramicidin. Assays were conducted at 22 C. - Herbicide-binding analysis used the same suspension medium used for electron transport measurements, except that DCPIP, LM ;;~~~~~~~t~~0.51,tM ammonium chloride, and Gramicidin were omitted. All binding Diuron studies were conducted under room light at 22 C. Binding reac- trazine tions were initiated by mixing 1 ml suspension medium, 25 p1 chloroplasts (containing 50 ,ug Chl) and small amounts (5-20 pi) of either uniformly ring-labeled atrazine (5.37 Ci/mol) or diuron I50 (Atrazine) = 0.5 ILM (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- I 50 (Diuron) = 0.07 HM half-ml aliquots of the clear supernatant were removed and added to 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 the difference between the total radioactivity added to the chloroplast suspension and the amount of free inhibitor found in the super- natant after centrifugation. Further details of this procedure are described by Tischer and Strotmann (30). Herbicide solutions were 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 and resistant biotypes of common groundsel seedlings, the effect of diuron and atrazine in PSII-mediated electron transport was 20 sec I50 (Diuron) = 0.12 kLM analyzed. Addition of either atrazine or diuron reduced the rate of electron flow (as indicated by the decreased rate of dye reduc- tion) in the susceptible biotype chloroplasts (Fig. IA). In the FIG. 1. Inhibition of PSII-dependent electron transport (H20 -D resistant chloroplasts (Fig. 1B), diuron reduced electron transport DCPIP) by atrazine or diuron. Data presented are direct tracings of light- with slightly less efficiency than in the susceptible chloroplasts, induced absorption changes recorded on a strip-chart recorder. Decreases whereas atrazine was much less effective in limiting electron in A (580 nm) indicate photoreduction of DCPIP. Ih values presented transport processes. These observations are consistent with results were calculated from similar experiments using a concentration series of previously reported by Radosevich et aL (24), who also showed the two inhibitors. that differences in triazine inhibition are not due to reduced rates _N ~~~~~~~I of penetration of the inhibitor into the membrane. Using this t ~~~~~~~~~~~(1.14) assay system, a concentration series of each herbicide was meas- ured for inhibitory activity. From these data, Lo values (a concen- Q +1.0 _ (1.09) tration of herbicide giving half-maximal inhibition of electron (1.07) were calculated. The values were 0.07 and 0.12 transport) 150 pM +0.5 re- a _ for diuron in susceptible and resistant biotype chloroplasts, Diuron spectively, and 0.5 pm for atrazine in susceptible chloroplasts. In resistant biotype chloroplasts, 50%o inhibition of electron transport could not be achieved within the solubility range of atrazine -z5-rtrazine (approximately 2 x 10-4 M). The measured values of electron transport inhibition at varying -z0.5- concentrations of diuron and atrazine were analyzed by Hill plots to characterize the binding properties of these herbicides. It is K-1.0 known that binding equilibria of small molecules interacting with macromolecules may take place by either independent or coop- erative binding (35). Binding properties are defined by the Hill equation: 8 7 6 5 - log [Herbicide] l =K.[L]n FIG. 2. Hill plots of data obtained from analysis of electron transport (H20-) DCPIP) in the presence of various concentrations of herbicides. where 0 is the fraction of binding sites occupied, K equals the Values in parentheses are calculated Hill coefficients. (U, 0): Data points is the concentration of herbicide from experiments using triazine-susceptible chloroplasts; (0): experiments binding constant, [LI molecules, triazine-resistant and n is the Hill coefficient. Assuming that binding is equivalent using chloroplasts. to inhibition, it is possible to plot log [% inhibition/(100-% inhi- bition)] versus log [herbicide] for diuron or atrazine. These Hill single herbicide molecule. Hill plots are highly sensitive to chlo- plots (Fig. 2) show linear relationships with slope values near 1 roplast concentrations used during the herbicide/electron trans- (values in parentheses in Fig. 2). The slope defines the Hill port assays. Low Chl levels must be used to measure herbicide coefficient; the values of unity indicate independent binding of a inhibition constants accurately (32). The value of the Hill coeffi- Plant Physiol. Vol. 64, 1979 HERBICIDE BINDING TO PSII 997 ment determined at very low Chl levels (as we have used) must be accepted as more accurate than the previously reported Hill coefficient of 2 for diuron in assays where higher Chl concentra- tions were used (36). INHIBITOR-BINDING STUDIES In electron transport inhibition experiments, a known quantity of inhibitor is added to the reaction mixture. The observed degree of inhibition of photoreactions is then measured and related to the total inhibitor concentration. Because of partitioning or bind- ing of the inhibitors to the chloroplast membranes, the actual amount of inhibitor in solution (free inhibitor) can be much lower than the initially added concentration. This equilibrium depends upon the amount of chloroplasts, the amount of inhibitor present, and the affinity of the inhibitor to its binding site (14). Binding studies with radioactively can to labeled herbicides be used char- 1.2 acterize directly the amounts of bound inhibitor, affinity, and c° DIURON (DCMU) number of binding sites for the inhibitor at true equilibrium conditions (30-32). 1.0 / Diuron Bhiding. The amount of diuron found to bind to chlo- / roplast membranes increased as the concentration of free, un- .8 9' Susceptible bound diuron was increased (Fig. 3A). From these plots (bound versus free diuron) it is apparent that the resistant chloroplasts .6 68 ZResistant show a slightly lower affinity for diuron than the susceptible A more quantitative analysis chloroplasts. of the binding data is .4 possible in a double reciprocal plot (Fig. 3B), in which the data are transformed to linear relationships. The intercept on the /2 ordinate is a measure of the maximum number of available I/ binding sites (on a Chl basis) and thus allows the determination of the amount of bound inhibitor per "photosynthetic unit." This 0 20 40 60 80 has been previously discussed in more detail by Tischer and /Free Diuron Strotmann (32). The double reciprocal plot for diuron in the (/iM) FIG. 3. A: binding of to susceptible and resistant susceptible chloroplasts clearly shows two different absorption ['4Cqdiuron chloro- plast membranes. Fitting of curve to data points was accomplished using processes: one occurring at low inhibitor concentrations withhigh the constants (K and Xi) from Table I. B: double recprocal plot of the affinity, and another at higher inhibitor concentrations with lower data from Figure 3A used for detmination of XI (ordinate-intercept) and affinity. Tischer and Strotmann have called the second process K (abscissa-intercept). "unspecific absorption," because this inhibitor binding was not correlated with the inhibition of electron Only the data transport. Table I. Calculated Binding Constants (K) and Number of Binding Sites points fitting the high specific absorption process were used for (X; Chlorophylls per One Bound Inhibitor Molecule)for Diuron and our calculation of binding constants and the number of binding Atrazine sites (Table I). Using the calculated constants, the hyperbolic curves shown in Figure 3A were fitted to the actually measured The values were determined by regression analysis of reciprocal plots data points according to the equation: similar to those shown in Figures 3B and 4B (correlation coefficients were between 0.997 and 0.970). The data are averages of six experiments. X1.f b=KK + f Inhibitor Suseptbl Resisant where b denotes the concentration of bound inhibitor/mg CM f K Xl K is the concentration of free inhibitor in the solution; XI is the Diuron 1.4 x 10-8M 420 ChM/in- 5 x 10-8 M 500 ChM/in- concentration of inhibitor binding sites; and K is the binding hibitor hibitor constant. This method of curve fitting,ie. using only the constants Atrazine 4X 10-8 M 450 Chl/in- No binding detected forhigh specific absorption, explains the apparent difference of hibitor the fitted curve and actually measured values for susceptible chloroplasts athigh diuron concentrations (Fig. 3A). Atrzine Binding. Atrazine binding to susceptible chloroplasts It is not possible to determine from these data whether the (Fig. 4, A and B) was analyzed and data are presented in the same binding site for atrazine is totally lost in the resistant chloroplasts fashion as was described for diuron. The calculated values for the or if the affinity between the inhibitor and the membrane is binding constant (K = 5.0 x 10- m) and the number of binding strongly diminished. The definition of a structure which is able to sites (450 Chl/inhibitor) are presented in TableI. Over the same bind a molecule implies a certain detectable affinity atrazine between these concentration range, a completely different binding pat- two. In the case of atrazine, however, the loss in affinity was so tern was observed between the two chloroplast samples. Suscep- pronounced that at low concentration ranges specific binding to tible chloroplasts bound atrazine, but no binding was detected in membrane components was not detectable. The presence of weak the resistant chloroplast sample. Because of the extremely low binding for atrazine in the resistant chloroplasts was suggested by atrazine affinity to thylakoids of resistant chloroplasts, an analysis the observed slight inhibition of photosynthetic electron transport in a reciprocal plot was not possible. at very high atrazine concentrations (Fig. lB and ref. 24). This In other experiments (data not presented) higher concentrations inhibition occurred only at much higher inhibitor concentrations of atrazine were used in binding studies to analyze "unspecific than those used for binding assays in Figure 4. A qualitative absorption." This process was observed only in the susceptible indication of weak atrazine binding to the membranes of resistant chloroplasts. chloroplasts was obtained by analysis of binding competition 998 PFISTER, RADOSEVICH, AND ARNTZEN Plant Physiol. Vol. 64, 1979 on the oxidizing site of PSII) (8, 26, 29) are now thought to play a minor role at the diuron concentrations where high specificity binding is observed. The site of action of the triazine inhibitors is thought to be similar to that of diuron. This conclusion is based on similar patterns 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 have been less carefully examined than diuron with regard to specific mechanisms of action. The fact that diuron and atrazine appear to inhibit the same step in electron transport does not necessarily indicate that the two inhibitors act at the same binding site. This information can only be obtained by direct measurements of binding and analysis of competition between inhibitors. Tischer and Strotmann (32) have analyzed competition between these herbicides in spinach chloroplasts and indicate that both inhibitors act at the same site. This conclusion has now been verified for chloroplasts isolated from the triazine-susceptible biotype of common groundsel (Fig. 5). In contrast, very weak competition-between diuron and atrazine was observed in the resistant chloroplasts. This latter observation is directly related to the fact that a high affinity binding site for diuron but not atrazine was detected in the resistant chloroplasts. A contradiction is, therefore, apparent: how can lack of binding of atrazine occur (resistant chloroplasts) while diuron still binds if both act at an identical site? A number of structure-activity studies with different classes of inhibitors have shown the important role of a single structural element common to all PSII inhibitors (11, 34). The proposed structures for this element are the configurations: -CO-NH , -C-N<, or a nitrogen with a lone electron pair adjacent to an 0 20 40 60 80 electron deficient carbon (sp2) atom. Besides this essential element, /Free Atrozine (/zM) the efficiency of PSII inhibitors is influenced by different lipo- FIG. 4. A: binding of ['4Clatrazine to susceptible and resistant chloro- philic substituents. Apparently two (or more) substructures of the plast membranes. Note that no atrazine binding to resistant chloroplasts inhibitor molecule form noncovalent bonds to a defined compo- was observed at these inhibitor concentrations. Curve fitting was as described for Figure 3 and in text. B: double reciprocal plot of atrazine nent(s) within the chloroplast membrane. binding to susceptible chloroplasts. A correlation between structural specificity of PSII inhibitors and selective resistance to these inhibitors in the triazine-resistant weed chloroplasts is summarized in Figure 6. In this highly between ['4Cjdiuron and atrazine. The release of previously schematic diagram, the asymmetrical organization of electron bound, radioactively labeled diuron from chloroplast membranes transport carriers in the chloroplast thylakoid is indicated in in the presence of increasing concentrations of unlabeled atrazine general terms. More specific details of the organization of thyla- is shown in Figure 5. In the susceptible chloroplasts, unlabeled koid membrane electron transport constituents have been previ- atrazine effectively displaced diuron from the membranes. The ously reviewed (2, 33). It is known that mild trypsin digestion of remaining amount of bound [14C]diuron, which was not released chloroplast membranes results in the appearance of diuron insen- by high atrazine concentrations in susceptible chloroplasts, rep- sitive PSII activity (25, 27, 28). These data suggest that PSII resents unspecifically bound diuron. With resistant chloroplasts, herbicides interact with a target protein that is localized near the high concentrations of unlabeled atrazine caused slight competi- membrane surface. It is also thought that the electron transport tion against [14CJdiuron. It should be pointed out that the concen- tration scales in Figures 4 and 5 cannot be compared directly, *E since the extent of competition between inhibitors depends on the concentration of I'4Cldiuron used in membrane pretreatments, its I ~~~~~~0It> affmity, and on binding properties of atrazine. In other experi- ments using low diuron concentrations, a small but significant 2 Resistant competition by atrazine became more apparent (data not pre- sented). This observation indicates a low triazine affinity, in .a: agreement with the above-mentioned slight electron transport inhibition at concentrations near 0.1 mm atrazine. DISCUSSION Competition * Susceptible It is commonly believed that diuron acts as a specific PSII * inhibitor by interrupting electron transfer between the primary 1C-Atrazine vs Bound 14C-Diuron and the for a review electron acceptor Q pool (9; LL// see 13). The most recent speculation about the mode of action of 0 10-7 lo-6 10-5 1o-4 diuron is that it interacts directly with the second carrier on the of 12C-Atrazine (M) reducing side of PSII, thereby altering its redox properties (37). Concentration Other effects of diuron (direct interaction with the reaction center FIG. 5. Competition between [14Cldiuron and atrazine in susceptible of PSII, inhibition of cyclic electron flow around PSI, or effects and ,resistant chloroplasts. Concentration of ['4C]diuron: 0.5 uM. Plant Physiol. Vol. 64, 1979 HERBICIDE BINDING TO PSII 999 3. ARNrmZ CJ, C DSrro, P BREwEl 1979 Choroplast membne alttions in triazine-reastant Amaranthus biotypes Proc Nat Acad Sci USA 76: 278-282 4. AsHToN FM, AS CaoFrs 1963 Mode of Action of Herbicids John Wiley & Sons, New York 5. AUDUS Ul 1976 Herbiides, Vold Academic Press, New York 6. BAsDrEm ID, RD McLARIN 1976 Reatne of Chuspods. alm L. to triazine herbidea. CanJ Plant Sci 56:411412 7. BANDENm JD, IV PARocsxrT, GI RYAN, B MALTmS, DV PEABODY 1979 Discovery and distribution of triazine reaistant weeds in North America. Abstr 229, 1979 Meeting Weod Sdence Society of America 8. BENNOUN P, YS Li 1973 New resuks on the mode of action of 3-(3,4-dichlorophenyl)-1,1- dimethylurea in spinach chlropl Biochim Biophys Acta 292: 162-168 9. DYsES LNM, HE SwEs 1963 M i of two photochemical reacuions in algae as studied by means of fluorescence. In J Ashida, ed, Microalge and Photosynthetic Bacteria. © SUSCEPTIBLE Univ of Tokyo Press, Tokyo, pp 353-372 10. EERT E, SW DuwORD 1976 Effects oftriaine herbcides on the physiology of plants. In FA atrazine - type inhibitors diuron- type inhibitors Gunther, JD Gunther, ads, Residue Revicws, Vol 65. Springer, New York 1 1. HANSCH C 1969 Theoretical consideations of the uacture-activity relationship in photosyn- thesis inhibitors. In H Metzner, ed, Progres on Photos Res, Vol III. pp 1685-1692 12. HosmNs WM, AS PERRY 1950 Thedetoxifica of DDT by reaistnt houseflies and inhibition of this process by piperonyl cycloene. Science 111: 600 13. IZAWA S 1977 Inhibitors of electon ransport In A Trebat, M Avron, eds, Encyclopodia of Plant Physiology, New Series Vol 5, Photosyntdhsis . Springer, New York, pp 266-282 14. IzAwAS, N GOOD 1965 The number of sites sensitive to 3-(3,4ichlorophenyl)-1,1-dimethyl- urea, 3-(4-chlorophenyl)-1,1-dimethylura and 2-chkoo-4-(2-propylamino)6-hylamine-s- triazine in isolated chbropls Bioim Biophys Acta 102: 20-38 rr') 15. imasN KIN, JD BANDEEN, V SouzA-MAcsAo 1977 Studies on the differential tolerance of W-~!j RESISTANT two mbequate ion of S-riazine herbicides Can I Plant Sci 57: 1169-1177 16. IJEsEN KIN, ID BAmDEN, V SouzA-MAcHso 1979 Role of trizine herbicide uptake, nlion, acumulton and metabolisms in plantselecivity. Abstr 224, 1979 Meetin Weed Scienc Society of America 17. JuNoE W 1975 Physical aspet of the eleo urnsport and in green p l Ber Deutche Bot Gas 88: 283-301 no 18. MAcHAsDO VS, Cl AasTzs N,ID BAssEsi, GR STEPHENSON 1978 Compartive triazine effecs /-t inhi upon systemI[photcemity inchiroplasts of two common lambsquarters (Chenopodium a )biotpes. Weed Sci 26: 318-322 19. MACHADO SV, JD BANDEEN, WD TAYLOR, P LAviGNE 1977 Atrazine resistant biotypes of no inhibition inhibition commonrgweed and bird!srape. Can Weed Comm East Sect Report FIG. 6. Model of inhibitor binding to chloroplast membranes. Model 20. MoaAID DE, WA GENThE, VL HILTON, KL HuiL 1959 Studies on the mechanism of presents the following concepts: A: asymmetrical orientation of photosyn- habicidal action of 2-chloro-4,6-bis(thylamino)-S-trzine. Plant Physiol 34: 432435 theticclectron transport chains; external localization of herbicide-binding 21. RADOsVICH SR, AP APPLEY 1973 Studies on the mchanismn of resistance to simazine in component in thylakoid membrane. B: existence of two different regions common groundseL Weed Sci 21: 497-500 22. RADOsVICH SR, OT DEvu.us 1976 Studies on the mechanism ofS-triazine resistance in within the herbicide binding component: an "essential region" of the binding common groundseL Weed Sci 24: 229-232 constituent which interacts with a common structural element of 23. RADOSICH SR 1977 Mechanism of atrazine restane inlambequartera and pigweed. Weed the different herbicides to produce a conformational change of the con- Sci 25: 316-318 stituent, thus interrupting electrontransport on the reducing side of PSII; 24. RADOVICH SR, KE STnaAcx, Cl AssrzEN 1979 Effect of photystemII inhibitors on and, differentdomains which are necessary for specific acnt of the thylakoid membranes of two common grounde (Senao ulgarls) biotypes. Weed Sci 27: inhibitors to the binding constituent. C: modification of one substructure 216-217 of the binding constituent in the ristant chloroplast leads to a loss of 25. Rwmrrz G, I OHAD 1974 Changes in the proteinorgnizatini developing thylakoids of as atrazine-binding capability, whereas DCMU binding to the same constit- Cklanydomua renadi Y-l shown by senitivity to typsin. In M Avron, ed, Proc III Congr Elsevier, pp 1616-1625 uent is not substantially affected. Int Photosynth Rehovot. Amsterdam, 26. RiNGERG 1973The action of 3-(3,4dihkorophenyl)-1,1-dimethylurea on the water splitting enzyme system Y of Biochim Biophys Acts 314:113-116 carriers acting near the reducing side of PSII are also surface- 27. RENvEo G 1976 Studies on the structural and functional organization of sytem II of photosynthesis Theuse of as a sewleive inibitor at the outer surface of in membranes (33). In Figure 6 we have typin suctury localized chloroplast the thylakoid membrmne. Biochim Biophys Acta 440: 287-300 indicated how specific changes in the herbicide-binding protein 28. RINGERG, K EIxoN, 0 D6aRNo, C Wo.u 1976 Studies on the nature of the inhibitory may selectively control herbicide activity. It is apparent that effect of tryphn on the photosynt eectntransport of system Iin spinachchroplasts. studies comparing normal susceptible chloroplast preparations Bichim Biophys Acta 440: 278-286 29. RYAN GE 1970 Resistance of common groundsel to smazine and atrzine. Weed Sci 18: 614- and plastids containing a modified PSI1 complex (resistant chlo- 616 roplasts) will open a new area of research allowing further clari- 30. STomoMANN H, W Txscta, K EDnmNs 1974 Spezifische Bindung vonInhibitoren durch fication ofthe molecularmechanisms which regulate the selectivity Elekronsnabertrige der etih E i tro . Ber Deutsche Bot and mode of action of PSII-directed herbicides. Ges 87: 457463 31. TzcHm W 1977B "nhugw zwischenHe tbndmung durch Chioroplasten und Hem- t AnaWe k Dr. Homer LeBaon, CIBA-GEIGY Corp, Raleigh, N.C., for mug des photosntheiche E rn s Thesis Univ Hannover, Germany 32. W, H 1977R ip the radioactive atrane used ths study and for support and of this earch. We TIcHs SamoTmAw betwe inhibitorbnding bychioropl and aho Dr. James Risglman E. L du Pon de Nenoura and Co., Wilmington, Delaware inhibitio of celtro tansport Biochim Biophys Acta 460: 113-125 for the radioactive diuron. Te excellent technical asistance of Ms. Cathy Ditto and Ms Jan 33. TSar A 1974 Energy conservation in photsthetic eleo transport ofchrop Annu Watson grateflly ecated. We thank Dr. K. Stenback for her cution regrding this Rev Plat Ptysli 25: 423458 research. 34. Tas A, E HARTIH 1974 Herbcidal N-alkyed- and ringdoed N-acyluada as inhibito ofp II. Z Naturoruch 29c 232-235 LrIJTURE CITED 35. VAN HOME KE 1971 Physical Biochemisry. Prentice-Hal Lodon, 246 p 36. VAN RINSEN S, D WoNo, Goviwa 1978 ar of the inhibition of photosyn- 1. AssoN DI 1949 Copper enzymes in isoatd polyphenooxidae in Beta vdgaris thedc electo tansport in peach by the herbicide 4,6-dinitro-o-cresol by compar- L Plant Physl 24: 1-15 ative studies with 3-(3,4-dchlorophenyl)-1,1-dimethylurea. Naturfoch 33c:413420 2. Aw4TzEN CI,IM BrANrAis 1975 Chlropat sucture and fUncto In Govindjee, ed, 37. VzLTHuys BR 1976 Charge acumulation andrcombintion system 2 of photosynthesis Bioenergetics of Photosynthsis. Academic Press, San Frncisco, pp 51-113 Thesis UnivLaide, The Netherlands