Activity of Photosystem II Herbicides Is Related with Their Residence Times at the D 1 Protein 



This work has been digitalized and published in 2013 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution 4.0 International License.

Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Creative Commons Namensnennung 4.0 Lizenz.

Activity of Photosystem II Herbicides Is Related with Their Residence Times

at the D 1 Protein

J. Dirk Naber* and Jack J. S. van Rensen

Laboratory o f Plant Physiological Research, Agricultural University W ageningen, Gen. Foulkesw eg 72, 6703 BW Wageningen, The Netherlands

Z. N aturforsch. 4 6 c, 5 7 5 -5 7 8 (1991); received March 18, 1991

Chloroplasts, Photosystem II, D 1 Protein, Herbicides, Triazine Resistance

The reversible binding kinetics o f atrazine, diuron and ioxynil were measured via their bind­ ing and release parameters during steady state inhibition o f electron transport. The parameters were determined in isolated chloroplasts o f peas and o f triazine-resistant and -susceptible b io­ types o f Chenopodium album using a kinetic model. This model is based on the flash-induced oxygen evolution patterns o f isolated broken chloroplasts.

It was found that the binding parameters were always significantly higher in the case o f an oxidized acceptor quinone com plex as compared with a semi-reduced com plex. Triazine resist­ ance seems to originate from a significant increase o f the release kinetics. The release parame­ ters could be used to calculate the residence times o f the herbicides at the D 1 protein. The values o f these residence times were always much higher for the herbicides than for Q B; this explains the inhibition o f electron transport. The only exception was the residence time o f atra­ zine in the resistant biotype, where the value was close to that o f Q B.

It is concluded that the “on ” kinetics o f a com pound to its binding environm ent at the D 1 protein are determined principally by the accessibility o f the niche to the com pound. The dif­ ferences in activity between herbicides are mainly due to variations in the release kinetics.


Photosystem II herbicides are potent inhibitors of electron transport near photosystem II. They include a large num ber of (chemically) different com pounds. Two groups can be distinguished: the diuron-type herbicides, including the ureas and triazines, and the phenol-type herbicides [1-3]. The mode of action o f these herbicides is studied in detail. They interrupt electron transport between Qa and Q b. Interaction o f diuron-type herbicides with plastoquinone was first proposed by van Rensen [4], It is now widely accepted that the mechanism of action is a displacement of Q B from Abbreviations: Atrazine, 2-chloro-4-(ethylam ino)-6-(iso- propylam ino)-s-triazine; D C M U (diuron), 3-(3,4-di- chlorophenyl)-l,l-dim ethylurea; ioxynil, 3,5-diiodo- 4-hydroxybenzonitrile; / 50, inhibitor concentration caus­ ing 50% inhibition o f electron transport; p l 50, negative logarithm o f / 50; PS II, photosystem II; QA, primary qui­ none electron acceptor o f PS II; Q B, secondary quinone electron acceptor o f PS II; R , triazine-resistant biotype; S, triazine-susceptible biotype.

* Present address: Lehrstuhl Biochemie der Pflanzen, R uhr-Universität, Postfach 1021 48, D -4630 Bochum 1, Bundesrepublik D eutschland.

Reprint requests to Dr. J. J. S. van Rensen.

Verlag der Zeitschrift für N aturforschung, D-7400 Tübingen 0939-5075/91/0700-0575 $01.30/0

its binding site at the D 1 protein. This was in­ dependently and simultaneously proposed by Velthuys [5] for the PS II complex, and by W raight [6] for the reaction center of purple photosynthetic bacteria. It is thought that the inhibitors reside at the D 1 protein for a relatively long time instead of Q b; they cannot be reduced, and thus inhibit elec­ tron flow.

It is im portant to realize that there is a reversible exchange of Q B and an added PS II herbicide at their com m on binding site at the D 1 protein. We have measured the kinetics of reversible binding of herbicides via their binding and release param e­ ters. We com pared the diuron-type herbicides D C M U and atrazine with the phenol-type herbi­ cide ioxynil. In addition, we measured the kinetics of their binding in thylakoids of a triazine-resist­ ant plant. We conclude that a strong inhibition is related to a long residence time at the D 1 protein. Materials and Methods

Peas (Pisum sativum L. cv. Finale) and lambs- quarters (Chenopodium album L.) were grown in growth chambers. The growth of the plants and the origin of the triazine-resistant and -susceptible lam bsquarters were described earlier [7]. Broken chloroplast thylakoid m embranes were isolated



from the leaves according to a previously de­ scribed procedure [8]. The thylakoids were sus­ pended in a medium, which contained 0.3 m sorbi­

tol, 50 m M tricine-K O H (pH 7.5), 100 m M KC1,

10 m M NaCl and 2 m M M gCl2. Total chlorophyll

concentration was determined spectrophotom etri- cally according to [9], Aliquots of 0.5 ml were stored at - 8 0 °C, and slowly thawed on ice prior to use.

Oxygen evolution in continuous light was meas­ ured with a Gilson oxygraph [10] and for the meas­ urement of flash-induced oxygen production a laboratory-designed Joliot-type apparatus [11] was used.

The exchange param eters were m easured using a m ethod, initiated by Vermaas et al. [12] and adapted by N aber [11], It is based on the flash- induced oxygen evolution patterns o f isolated broken chloroplasts, which are m easured in the absence and in the presence of herbicides. The ex­ change param eters are obtained by fitting experi­ mental data to those calculated with a kinetic model. This model is derived from the following equations:


S n' Qa' Qb + I ^ Sn • Qa • I + Q b





Sn • (Qa • Q b)- + I ^ Sn ■ (Qa • I)- + Q B



In these equations, Sn (where n = 0, 1,2, 3) rep­ resents the redox state o f the oxygen-evolving complex. In the presence of slowly exchanging her­ bicides, having residence times on the D 1 protein of the same order of m agnitude as the duration of the flash train or longer, the oscillation is hardly altered com pared to the control. In this case only the am plitude o f the signal is diminished. H ow ­ ever, when the herbicide exchange is occurring with the same or higher frequency than the firing of the flashes, the dam ping o f the oscillation is considerably stronger. This is caused by the fact that then reaction centers are blocked for a certain time span, and start m aking turnovers at the m o­ ment the herbicide is displaced by a plastoquinone molecule. Thus, centers can get out o f phase with each other, and produce oxygen at different flash­ es. By com paring flash patterns with different flash frequencies and herbicide concentrations, the

1 Protein exchange param eters E, to E4 can be calculated. In Fig. 1 the results o f a typical m easurem ent are illustrated.

Fig. 1. Oxygen evolution patterns at different flash fre­ quencies with and w ithout atrazine. O, 4 Hz flash fre­ quency, no inhibitor; A , 0.5 Hz flash frequency, no inhibitor; • , 4 Hz flash frequency, 0.5 (im atrazine;

▲, 0.5 Hz flash frequency, 0.5 (im atrazine.

Results and Discussion

The inhibitory activity of the herbicides a tra ­ zine, D C M U and ioxynil on oxygen evolution in continuous light as measured in chloroplasts from peas and lam bsquarters is illustrated in Table I. The /?/50-values are between 6.5 and 7.5 for all three herbicides; in the triazine-resistant m aterial there is very little activity of atrazine, a little de­ crease in activity of D C M U and a little increase in activity of ioxynil. This is in agreement with what is generally observed (e.g. [13]).

The results of the following tables are different from the action kinetics of PS II herbicides on

thy-Table I. Values o f p l 50 for the herbicides atrazine, D C M U and ioxynil measured in chloroplasts from peas and triazine-resistant ( R ) and -susceptible (S) Chenopo- dium album plants.

Pea C. album S C. album R

Atrazine 7.0 6.5 < 4

D C M U 7.5 7.5 6.7

Ioxynil 6.5 6.6 7.0


J. D. N a b e r an d J. J. S. van R ensen • Residence Tim es o f PS II H erbicides a t the D 1 P ro tein 577

lakoids th at were reported by Ducruet et al. [14], These authors added a herbicide to a thylakoid suspension and m easured the kinetics of the pro­ gress of inhibition of electron transport by chloro­ phyll fluorescence; the time needed to reach 50% inhibition was defined as apparent half-time (t 1/2). In our experiments binding and release ki­ netics were determ ined while a steady state inhibi­ tion was obtained of about 50% inhibition o f elec­ tron transport.

In Table II the E, to E4 exchange param eters are presented for the herbicides atrazine, D CM U and ioxynil, measured in chloroplasts of pea, triazine- resistant and -susceptible C. album. The param e­ ters and E 3 represent the exchange rates of her­ bicides to an oxidized binding environment. They are supposed to be much higher than the corre­ sponding param eters for a reduced complex, E2 and E4. This is caused by the fact that the second­ ary acceptor Q B binds very strongly to its binding niche when it is in the semiquinone form, whereas both the fully reduced and the oxidized forms are easily exchanged [5, 6]. In the semireduced state it is then difficult to replace the quinone by a herbi­ cide molecule.

In all cases the binding param eters E, were sig­ nificantly higher than E2, which is in agreement with the expectation as described above. However, the release param eters E4 were not always lower than E3. In fact, in m any experiments E4 is found

Table II. Values for the exchange parameters measured in isolated chloroplasts. Pea E, e2 e3 e4 Atrazine 0.24 0.02 0.100 0.141 D C M U 0.01 0.001 0.135 0.141 Ioxynil 0.1 0.01 0.03 1.0 C. album S E, e2 e3 e4 Atrazine 0.1 0.03 0.11 0.04 D C M U 0.056 0.0015 0.008 0.0035 Ioxynil 0.4 0.02 2.0 2.0 C. album R E, e2 E3 e4 Atrazine 0.05 0.002 15 2.25 D C M U 0.064 0.002 0.001 0.001 Ioxynil 0.22 0.04 0.2 0.05

R = triazine-resistant; 5 = triazine-susceptible; E, and E2: (im_ 1 • s -1; E3 and E4: s “1.

to be higher than E3. This may be explained by the reasoning that a bound herbicide destabilizes a negative charge at the acceptor complex. M utual­ ly, the presence o f an electron on Q A accelerates the release of the herbicide from its binding site, leading to higher E4 values.

Atrazine proved to bind faster than D CM U (E, param eter). In the resistant biotype o f Chenopo­ dium, the atrazine-binding constant E, is only slightly decreased as com pared to the wild type. However, because the values of the release param e­ ters E3 and E4 are much higher in the resistant chloroplasts com pared to the wild type ones, tria- zine resistance seems to originate from a signifi­ cant increase in the release kinetics. This can be ex­ plained on the molecular level. The “o n ” kinetics of a com pound to the binding environm ent are de­ termined principally by the accessibility of the niche to the com pound. This is determ ined by the chemical structure of the herbicide, especially its molecular dimensions, charges and hydrophobici- ty. These properties are, o f course, the same when atrazine is added to resistant or to susceptible chloroplasts. A very slight change o f hydrophobic- ity of the binding pocket can be expected as a re­ sult of the serine to glycine substitution at position 264 in the triazine-resistant biotype. However, the atrazine molecule cannot be stabilized in its bind­ ing environm ent in the m utant protein, probably because the ser-OH group provides an im portant H -bonding possibility in the wild type. The result is a decrease in herbicidal activity of 2 to 3 orders of m agnitude (Table I). In the case o f ioxynil the situation is reversed, though the difference in ac­ tivity in both biotypes is far less as com pared with atrazine. F or ioxynil only a slight difference in binding to the D 1 protein is observed, but now the release from the resistant biotype is about 10-fold slower than from the wild type protein. The hy­ droxyl group o f ser-264 apparently has a destabi­ lizing effect on the binding of ioxynil.

The dissociation rates E3 and E4 can be used to calculate the time a herbicide stays at its binding site at the D 1 protein. This residence time equals the inverse of the param eters E3 + E4. In Table III residence times are presented for atrazine, D C M U and ioxynil in chloroplasts from peas and from triazine-resistant and -susceptible C. album bio­ types. Com pared with the residence time of Q B, which is about 20 ms [5], those of the herbicides


578 J. D. N a b e r an d J. J. S. van R ensen • R esidence Tim es o f PS II H erbicides a t th e D 1 P ro tein

Table III. Residence times o f herbicides (in seconds) at the D 1 protein.

Pea C. album S C. album R

Atrazine 4.1 6.7 0.058

D C M U 3.6 86.9 500

Ioxynil 0.97 0.25 4.0

are much higher; they vary from about 10-fold (ioxynil in C. album S) to about 25,000-fold (D C M U in C. album R ). It thus appears that herbi­ cides stay much longer at their binding site on the D 1 protein than Q B; since they cannot be reduced by Qa they interrupt electron transport at the site o f

Qb-A special case is the residence time o f atrazine in chloroplasts o f the triazine-resistant C. album bio­ type. This time is 58 ms, which is in the same order as that of Q B. The fact that the residence times of atrazine and Q B are very close to each other may be the explanation for the resistance.

In Table IV the ratios of the resistant (R ) over susceptible (S) values o f the activity of the herbi­ cides (in ^5o) are com pared with the ratios of their residence times. It appears that a high ratio of R /S in activity is correlated with a low R /S ratio in resi­ dence time (atrazine); a low R /S ratio in activity is

correlated with a high R /S ratio in residence time (ioxynil). DCM U has an interm ediate position for both R /S ratios. This means that the inhibition of a herbicide is stronger when the time it stays at the D 1 protein is longer.

In conclusion, it appears that the “on” kinetics of a com pound to a binding environm ent are de­ termined principally by the accessibility o f the niche to the compound. This is determined by the properties of the herbicide: its chemical structure, especially its molecular dimensions, charges and hydrophobicity. The differences in activity be­ tween herbicides are mainly due to variations in the release kinetics, which determine the residence times. A stationary binding, resulting in a signifi­ cant electron transport inhibition, requires a strict molecular shape.

Table IV. Comparison o f the activity o f herbicides with their residence times at the D 1 protein in triazine-resist- ant ( R ) and -susceptible (5) biotypes o f C. album .

R /S o f R /S o f / 50 values residence times

Atrazine > 3 2 2 0.0087

D C M U 6.5 5.8

Ioxynil 0.4 16.0

[1] A. Trebst, Z. N aturforsch. 4 2 c, 7 4 2 -7 5 0 (1987). [2] J. J. S. van Rensen, in: Herbicides and Plant M etab­

olism (A. D. D odge, ed.), pp. 2 1 - 3 6 , Cambridge University Press, Cambridge 1989.

[3] J. J. S. van Rensen, in: Photosynthesis and Plant Productivity (Y. P. Abrol, P. M ohanty, and G ovin- djee, eds.), Oxford U niversity Press, Oxford 1991 (in press).

[4] J. J. S. van Rensen, Meded. Landbouw hogeschool W ageningen 71-9, 1 -8 0 (1 9 7 1 ).

[5] B. R. Velthuys, FEBS Lett. 126, 2 7 7 -2 8 1 (1981). [6] C. A. Wraight, Isr. J. Chem. 21, 3 4 8 -3 5 4 (1981). [7] M. A. K. Jansen, J. H. H obe, J. C. W esselius, and

J. J .S .v a n Rensen. Physiol. Veg. 2 4 ,2 7 5 -2 8 4 (1986). [8] J. J. S. van Rensen, D. W ong, and Govindjee, Z.

Naturforsch. 3 3 c, 413 - 4 2 0 (1978).

[9] J. Bruinsma, Photochem . Photobiol. 2, 2 1 4 -2 4 9 (1963).

[10] J. J. S. van Rensen, W. van der Vet, and W. P. A. van Vliet, Photochem. Photobiol. 25, 5 7 9 -5 8 3 (1977).

[11] D. Naber, Ph.D. thesis, Agricultural U niversity, Wageningen 1989.

[12] W. F. J. Vermaas, G. D ohnt, and G. Renger, B io­ chim. Biophys. Acta 765, 7 4 - 8 3 (1984).

[13] W. Oettmeier, K. M asson, C. Fedtke, J. Konze, and R. R. Schmidt, Pest. Biochem. Physiol. 18, 3 5 7 -3 6 7 (1982).

[14] J.-M. Ducruet, S. Creuzet, and J. V ienot, Z. Natur- forsch. 45c, 3 4 8 -3 5 2 (1990).