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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 , 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  for the PS II complex, and by W raight  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 . Broken chloroplast thylakoid m embranes were isolated
from the leaves according to a previously de scribed procedure . 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 , 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  and for the meas urement of flash-induced oxygen production a laboratory-designed Joliot-type apparatus  was used.
The exchange param eters were m easured using a m ethod, initiated by Vermaas et al.  and adapted by N aber , 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:
E,S n' Qa' Qb + I ^ Sn • Qa • I + Q b
2Sn • (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. ).
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. , 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 , 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
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