The Role of Secondary Quinone Acceptor QB of Photosystem II in Photoinhibition of Isolated Wheat Chloroplasts

J. Plant Physiol. Vol. 138. pp. 602 - 607 (1991)

The Role of Secondary Quinone Acceptor QB of Photosystem II in Photoinhibition of Isolated Wheat Chloroplasts RANJIT

K.

MISHRA, NEELAM

P.

CHAUHAN,

and GAURI

S. SINGHAL

*

School of Life Sciences, Jawaharlal Nehru University, New Delhi-ll0 067, India Received December 29,1990 . Accepted May 14,1991

Summary The role of semiquinone anion radicals in photoinhibition of isolated wheat ( Triticum aestivum) chloroplasts was investigated by subjecting the chloroplasts to high light stress in the presence or absence of DCMU and DBMIB. The decrease in the efficiency of PS II photochemistry measured as FJFm ratio and oxygen evolution after photoinhibition of isolated wheat chloroplasts was less in the presence of DCMU. We suggest that the protective effect of DCMU is due to its binding to the 32 kDa QB-binding protein and reducing the probability of formation of semiquinone anion and other free radical species that have been suggested to be involved in photoinhibition damage. The hypothesis was also tested by using DBMIB during photoinhibition. DBMIB is known to reduce the plastoquinone pool, resulting in an increase in the semiquinone ion population. A greater extent of reduction of FJFm and oxygen evolution was observed when chloroplasts were photoinhibited in the presence of DBMIB. The results suggest an involvement of reduced semiquinones in the photoinhibition of wheat chloroplasts. A partial recovery of variable chlorophyll fluorescence in the presence of 20 mM hydroxylamine was also observed in chloroplasts subjected to light stress. Key words: Wheat, chloroplast, photo inhibition, DeMu, DBMIB, chlorophyll fluorescence.

Abbreviations: DCMU = 3-(3,4-dichlorophenyl)-1,1-dimethyl urea; DBMIB = 2,5-dibromo-6-isopropyl-3-methyl-l,4-benzoquinone; Hepes = N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]; Fm = maximal chlorophyll fluorescence; Fo = intrinsic chlorophyll fluorescence; Fv = variable chlorophyll fluorescence; KDT = rate constant for non-radiative energy dissipation and energy transfer to PSI; Kp = rate constants for PS II photochemistry; PS II = photosystem II.

Introduction Exposure of green leaves or isolated chloroplasts to high light intensities damages the photosynthetic apparatus specifically by impairing the photosystem II activity (Barenyi and Krause, 1985; Cleland and Critchley, 1985; Critchley, 1981; Critchley and Smillie, 1981; Krause et aI., 1985; Nedbal et al., 1986). Studies of the inhibition of QB-dependent and QB-independent activities of higher plant thylakoids (Cleland and Critchley, 1985; Ohad et al., 1985) have been used to identify the primary site of damage. The results indicate that the QB-dependent activity is lost prior to the

* Author for correspondence. © 1991 by Gustav Fischer Verlag, Stuttgart

QB-independent reaction center activity, which suggests that the initial site of photoinhibition damage is probably at the QB position. The damage at the QB position is followed rapidly by damage to the photosystem II reaction center (Kyle, 1987). However, there is no agreement over the primary site of photo inhibition damage, and several other sites between Z and QA have been proposed (Allakhverdiev et aI., 1987; Callahan et aI., 1986; Demeter et aI., 1987; Styring et aI., 1990). The involvement of semiquinone anion radicals along with other free radical species in the photoinhibition damage of chloroplasts has been suggested (Kyle, 1987; Ohad et aI., 1985). However, there is no conclusive evidence to prove the involvement of semiquinone anion radicals in photoinhibi-

The role of QB in photoinhibiton of isolated wheat chloroplasts

tion. We used DCMU during photo inhibition of chloroplasts to investigate the extent of damage when the QB-binding site on the D1 protein is occupied by DCMU. DCMU binding to the D 1 protein is likely to keep the site blocked that might reduce the formation of semiquinone anions, which have been suggested to be potentially damaging to the PS II complex of chloroplasts. On the other hand, DBMIB has been shown to inhibit the oxidation of plastohydroquinone (Trebst, 1980). Such an inhibition is likely to increase the population of reduced plastoquinones and semiquinone anions. We have also used DBMIB during photo inhibition of chloroplasts to study the effect of accumulation of reduced semiquinones on the photoinhibition of chloroplasts when the QB-binding sites on the D 1 proteins are not occupied by DCMU.

Materials and Methods

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Fig. 1: Effect of the presence of DCMU and DBMIB on the Fv/Fm ratio of wheat chloroplasts during photoinhibition. Chloroplasts (100!!g Chi · mL - I) were pre-illuminated at 1716 W' m -2 in the absence (open circles) and presence of l!!M DCMU (closed circles) or l!!M DBMIB (open triangles). The chloroplast samples were dark adapted for 10 min before recording the fluorescence induction. See materials and methods for details.

Plant material Chloroplasts were prepared from 10-day-old wheat (Triticum aes· tivum var HD-2329, Indian Agricultural Research Institute, New Delhi, India) seedlings grown at 25°C under 14/10 h light-dark cycles. Chloroplasts were isolated in the dark at 0 - 4 °C in 0.4 M sucrose, 20mM Hepes, pH 7.6, 15mM NaCI and 5mM MgCh, and were resuspended in the same medium. Chlorophyll concentration was determined according to Arnon (1949).

Photoinhibition of chloroplasts Photoinhibitory treatment to the isolated chloroplasts (100!!g Chi· mL - I) was given in a jacketed glass container. The temperature of the samples was maintained at 25°C by circulating water from a temperature controlled water bath. Photo inhibitory light at 1716 W' m - 2 at the center of the jacketed glass chamber was provided by a slide projector (Parkeo automat S-250, Germany, 24 VI 250 W). Light treatment to chloroplasts was given for different periods of time up to 40 min in the presence or absence of 1 !!M DCMU or l!!M DBMIB.

Measurement of electron transport activity Measurement of rates of photosynthetic electron transport was performed with an oxygen electrode (CB1D, Hansatech, England) at 25°C in the presence of silicomolybdate as acceptor. The reaction mixture contained 100 mM sucrose, 40 mM Hepes-KOH (pH 7.0), 15 mM NaCl, 5 mM MgCh, 5 mM CaCh and chloroplasts equivalent to 50!!g Chi· mL - I. DCMU, silicomolybdate and potassium ferricyanide were also added to the reaction mixture just before light treatment to give final concentrations of 5 !!M, 0.1 mM and 0.5 mM, respectively.

Recording of Fluorescence Transients Fluorescence induction curves of chloroplasts in the presence of 20 itM DCMU were recorded at room temperature for 6 sec. on a transient recorder (TR1, Hansatech, England). Samples were dark adapted for 10 min before recording fluorescence induction. The samples were excited with blue actinic light using a Corning CS 4-96 glass filter. Emission kinetics was recorded through a Wratten 89B filter. The rate constants for PS II photochemistry (Kp), nonradiative energy dissipation and energy transfer to PS I (K DT), and energy dissipation as fluorescence (KF ) were calculated according to

Bjorkman (1987 a). The rate constants for non-radiative energy dissipation in the pigment bed (K D) and for energy transfer to PS I (KT ) were merged into a single rate constant (KDT = KD + KT)' The assumptions made for the calculations were that in healthy leaves not subjected to any stress, the ratio of FvlFm is equal to 0.864; KF was assumed to be constant and its value was set to 1 (Bjorkman, 1987 a). The values of Fm in untreated control chloroplasts were set at 100 and all other readings were normalized accordingly. All fluorescence induction measurements were (performed) in the presence of 20!!M DCMU. Hydroxylamine (20mM) was added to the sample as an electron donor wherever indicated.

Results and Discussion Increase in the duration of photo inhibition of chloroplasts resulted in a gradual reduction in FJFm ratio (Fig. 1). Fv/Fm ratio is a quantitative measure of the photochemical efficiency of photosystem II complex (Kitajima and Butler, 1975) and photon yield of oxygen evolution at high light intensities (Bjorkman and Demmig, 1987; Demmig and Bjorkman, 1987; Adams III et al., 1990). The decrease in Fv/Fm was more when the thylakoids were exposed to high light intensity in the presence of DBMIB and it was less when DCMU was present during photo inhibition. This decrease coincided well with the reduction in variable fluorescence (Fig. 2). The reduction in variable fluorescence was brought about by the decrease in maximal chlorophyll fluorescence as there was no significant change in F o • Reduction in F y upon exposure of chloroplasts to high light intensity reflects a decrease in photoreduction of the primary quinone acceptor QA (Bjorkman, 1987 a). Hydroxylamine has been shown to donate electrons to the reaction center bypassing the oxygen evolving complex. A partial recovery of variable chlorophyll fluorescence in photo inhibited chloroplasts was observed in the presence of hydroxylamine (Fig. 2). The hydroxylamine induced recovery, however, accounted for a small part of the total photoinhibition, which might rule out any major damage prior to the site of electron donation by hydroxylamine. The extent

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Fig. 3: Effect of photoinhibition of isolated wheat chloroplasts on the rate constant for primary photochemistry of PS II (Kp). Open circles: no addition; filled circles: 111M DCMU; open triangles: 111M DBMIB.

Fig.4: Effect of photoinhibition of isolated wheat chloroplasts on KDT . Open circles: no addition; filled circles: 111M DCMU; open triangles: 111M DBMIB.

of recovery was minimal in the presence of DCMU when the photo inhibition damage was least and maximal in the presence of DBMIB when the damage was greatest. These results might suggest that damage to the electron transport chain prior to the electron donation site by hydroxylamine was small but comparatively more in the presence of DBMIB compared with DCMU and without any addition. Reduction in the FJFm ratio upon exposure of chloroplasts to excessive light may be caused by two concomitant events: (i) a decrease in the rate of photochemistry of PS II, and (ii) an increase in the non-radiative (thermal) dissipation or preferential transfer of excitation energy to PS I centers. The rate constants for PS II photochemistry (Kp), nonradiative energy dissipation and energy transfer to PS I (KDT) were affected upon photoinhibition of chloroplasts in our experiments, irrespective of the presence or absence of DCMU or DBMIB. Fig. 3 shows a reduction in K p, which in dicates a decrease in the efficiency of primary photochemistry of PS II complex (Ogren and Oquist, 1984; Demmig and Bjorkman, 1987). The increase in KOT (Fig. 4) indicates either an increased rate of de-excitation by means of non-radiative decay mechanisms or transfer of excitation energy, preferen-

tially to the PS I complex at high light intensities (Bjorkman, 1987 a, 1987 b). However, according to the (Schatz and Holzwarth, 1987; Holzwarth et al., 1987; Holzwarth, 1990), the exciton equilibration between the antenna and reaction center could be very fast, so that changes in KOT may not allow for discrimination between effects on the antenna or reaction center. Hence, an increase in KOT may also indicate a disorder in the PS II reaction center. It is apparent from Figs. 3 and 4 that the reduction in Kp and increase in KOT were less when chloroplasts were photoinhibited in the presence of DCMU. On the other hand, the presence of DBMIB during photoinhibition results in enhancement of the damage. The results thus suggest that DCMU extends a protection of chloroplasts from high light stress. Our results are also in agreement with those of Kyle et al. (1984), who have demonstrated that when Chlamydomonas reinhardtii cells are photoinhibited in the presence of atrazine the resulting photoinhibition damage is significantly reduced. The relationship between rate constants and Fv/Fm of control and photoinhibited chloroplasts is shown in Fig. 5. The photo inhibition damage is clearly reflected by a decrease in Kp, which results from inactivation of PS II com-

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Fig. 6: Electron transport activity of isolated wheat chloroplasts photoinhibited in the absence (open circles) and presence of l/lM DCMU (closed circles) or l/lM DBMIB (open triangles). Chloroplasts (100/lgChl·mL -1) were pre-illuminated at 1716W·m- 2 at 25°C for 0 to 40 min before measuring oxygen evolution. The assay mixture contained 100mM sucrose, 40mM Hepes-KOH (pH 7.0) 15mM NaCl, 5mM MgCh, 5mM CaCh, 5/lM DCMU, O.lmM potassium ferricyanide, 0.5 mM silicomolybdate and chloroplasts equivalent to 50 /lg Chi· mL - 1.

plex; this does not seem to be rapidly reversible and recovery from photoinhibition requires synthesis of chloroplast encoded D1 protein (Ohad et al., 1984; Greer et al., 1986). Photosystem II mediated electron transport activity of chloroplasts, measured as O 2 evolution with silicomolybdate as the electron acceptor, was observed to decrease with increase in duration of photo inhibition. The damage to electron transport activity was reduced when chloroplasts were photo inhibited in the presence of DCMU whereas the activity was reduced to a greater extent when DBMIB was present during illumination (Fig. 6). It has been suggested that the QB site is excluded in measurements using silicomolybdate, but benzoquinones and phenylenediamines could not be used for the measurements because DCMU present in the reaction mixture would block the transfer of electrons between QA and QB. Our results indicate that a protection from high light stress is provided to the chloroplasts by DCMU. On the contrary, DBMIB shows the abil-

605

ity to enhance the effect of high light stress on the chloroplasts. The results also suggest a damage in the region from water to QA, which might indicate that photoinhibition damage is not only localized to the acceptor side of the PS II complex but also spreads to the donor side. It may be mentioned here that all these measurements were done in a steady-state condition and distinction of damage due to primary and secondary quinones is only possible after a kinetic study. Photo-oxidation of chlorophylls upon exposure of leaves or isolated chloroplasts to high photon flux densities for a long period has been reported by many workers (Kislyuk, 1979; Ludlow, 1987). Although the bleaching of pigments is not observed in photoinhibition, it was observed in our experiments and accounted for 10 % loss of pigments during the first 20 min of photoinhibition. Photobleaching of chlorophylls, however, increased to about 30 % in the latter half of photo inhibition (Table 1), which could be due to illumination of chloroplasts with very high light intensity. Although photo-oxidation of chlorophylls might drastically affect the fluorescence characteristics of chloroplasts, the observation that the extent of bleaching was not significantly different either in the presence or absence of DCMU or DBMIB suggests that the observed differences in FjFm and electron transport activities may not be caused by pigment loss. It may also be mentioned that the differences due to DCMU and DBMIB were apparent in the first 20 min of photoinhibition when not more than 10 % of the chlorophylls were degraded. Kyle (1987) has put forward a hypothesis suggesting a domination of the plastoquinol population over the oxidized quinones under high light conditions, which could result in the damage of the D 1 protein. Herbicides have been shown to compete with the quinones for binding sites on the QBprotein (Pfister and Arntzen, 1979). We used DCMU during photoinhibitory treatment to provide a competitive inhibitor to the dioxygen for the QB-binding site. The decrease in the extent of photoinhibition observed in the presence of DCMU suggests that high light induced damage to chloroplasts occurs at the QB binding site. One of the mechanisms Table 1: Changes in the pigment content of isolated wheat chloroplasts on photoinhibition at 1716 W m - 2 at 25°C for 40 min. Light treatments were given in the absence or presence of l/lM DCMU or l/lM DBMIB. Control

DCMU

DBMIB

Chlorophyll a Omin 20 min 40min

100± 1 91±2 71±5

100±1 90±0 70±4

100± 1 91±1 78±5

Chlorophyll b Omin 20 min 40 min

28±1 28±0 27±1

30±1 28±0 27±1

28±1 26±1 26±1

Chla + Chlb Omin 20 min 40 min

128±2 119±2 81±6

130±2 118±0 80±5

128±2 117±2 90±5

606

RANJIT K. MiSHRA, NEELAM P. CHAUHAN, and GAURI S. SINGHAL

by which DCMU may reduce the photodamage could be that it does not leave the QB-binding site unoccupied even at the time of overstimulation, thus reducing the probability of any damage at that position. The results are in agreement with the reports on the decreased turnover rate of D 1 protein in the presence of herbicides (Mattoo et al., 1984). Our results also show an enhancement of damage in the presence of DBMIB. DBMIB, at low concentrations, has been shown to inhibit the reoxidation of plastohydroquinones (Trebst, 1980), which could, under high light conditions, result in the accumulation of reduced plastoquinones and semiquinone anions. The semiquinone anions are considered to be of major interest in photo inhibition because of their high affinity to the QB binding site, whereas the doubly reduced and protonated plastoquinol has a very low binding affinity to the QB binding site (Velthuys, 1981; Kyle, 1987). The increase in photoinhibition damage in the presence of DBMIB may be explained by the accumulation· of reduced semiquinones, as proposed by Kyle (1987). The observation that only a little recovery was observed by addition of hydroxylamine suggests that the major photoinhibitory damage was localized on the acceptor side of PS II. The mechanism of damage is still not clear and identification of the actual free radical species involved in photoinhibition damage to the D 1 protein is in progress using a number of free radical scavengers specific to different free radicals. A quantitative estimation of the local population of free radical species in the vicinity of the QB-binding pocket would also be useful for a better understanding of the mechanism of high light induced damage to the QB-protein. Acknowledgements R. K. Mishra gratefully acknowledges the award of junior/senior research fellowship from the Council of Scientific and Industrial Research, India. This work was in part supported by the ICAR-USDA project grant no FG-In-678 (IN-ARS-401).

References ADAMS Ill, W. W., B. DEMMIG-ADAMS, K. WINTER, and U. SCHREIBER: The ratio of variable to maximum chlorophyll fluorescence from photosystem II, measured in leaves at ambient temperature and at 77K, as an indicator of the photon yield of photosynthesis. Planta 180, 166-174 (1990). ALLAKHVERDIEV, S. I., E. SEnlKOVA, V. V. KLIMOV, and I. SETLIK: In photoinhibited photosystem II particles pheophytin photoreduction remains unimpaired. FEBS Lett. 226, 186-190 (1987). ARNON, D. I.: Copper enzymes in isolated chloroplasts. Polyphenol oxidase in &ta vulgaris. Plant Physiol. 24, 1-15 (1949). BARENYI, B. and G. H. KRAUSE: Inhibition of photosynthetic reactions by light. A study with isolated spinach chloroplasts. Planta 163,218-226 (1985). BJORKMAN, 0.: Low-temperature chlorophyll fluorescence in leaves and its relationship to photon yield of photosynthesis in photoinhibition. In: KYLE, D. J., C. B. OSMOND, and C. J. ARNTZEN (eds.): Photoinhibition, Elsevier Science Publishers, Amsterdam, pp. 123 -144 (1987 a). - High irradiance stress in higher plants and interaction with other stress factors. In: SYBESMA, C. (ed.): Prog. Photosynth. Res.

Vol. 4, Martinus Nijhoff Junk Publishers, The Netherlands, pp. 11-18 (1987 b). BJORKMAN, O. and B. DEMMIG: Photon yield of O 2 evolution and chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origins. Planta 170, 489-504 (1987). CALLAHAN, F. E., D. W. BECKER, and G. M. CHENIAE: Studies on the photoactivation of the water-oxidizing enzyme. II. Characterization of weak light photoinhibition of photosystem II and its light-induced recovery. Plant Physiol. 82, 261-269 (1986). CLELAND, R. E. and C. CRITCHLEY: Studies on the mechanism of photoinhibition in higher plants. II. Inactivation by high light of photosystem II in thylakoids and O 2 evolving particles. Photobiochem. Photobiophys. 10, 83-92 (1985).

CRITCHLEY, c.: Studies on the mechanism of photoinhibition in higher plants. Plant Physiol. 67, 1161-1165 (1981). CRITCHLEY, C. and R. M. SMILLIE: Leaf chlorophyll fluorescence as an indicator of high light stress (photoinhibition) in Cucumis sativus L. Aust. J. Plant Physiol. 8, 133-141 (1981). DEMETER, S., P. J. NEALE, and A. MELIS: Photoinhibition: Impairment of the primary charge separation between P-680 and pheophytin in photosystem II of chloroplasts. FEBS Lett. 214, 370-374 (1987). DEMMIG, B. and O. BJORKMAN: Comparison of the effect of excessive light on chlorophyll fluorescence (77K) and photon yield of O 2 evolution in leaves of higher plants. Planta 171, 171-184 (1987). GREER, D., J. A. BERRY, and O. BJORKMAN: Photoinhibition of photosynthesis in intact bean leaves: Role of light and temperature and requirement for chloroplast-protein synthesis during recovery. Planta 168, 253 -260 (1986). HOLZWARTH, A. R.: The functional organization of the antenna systems in higher plants and green algae as studied by timeresolved fluorescence techniques. In: BALTSCHEFFSKY, M. (ed.): Current Res. Photosynth. Vol. II, Kluwer Academic Publishers, The Netherlands, pp. 223-230 (1990). HOLZWARTH, A. R., H. BROCK, and G. H. SCHATZ: Picosecond transient absorbance spectra and fluorescence decay kinetics in photosystem II particles. In: BIGGINS, J. (ed.): Prog. Photosynth. Res. Vol. 1, Martinus Nijhoff Publishers, Dordrecht, pp. 61- 65 (1987). KISLYUK, I. M.: Protecting and injurious effects of light on photosynthetic apparatus during and after heat treatment of leaves. Photosynthetica 13, 386-391 (1979). KITAJIMA, M. and W. L. BUTLER: Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone. Biochim. Biophys. Acta 376, 105 -115 (1975). KRAUSE, G. H., S. KOSTER, and S. C. WONG: Photoinhibition of photosynthesis under anaerobic conditions studied with leaves and chloroplasts of Spinacia oleracea L. Planta 165, 430-438 (1985). KYLE, D. J.: The biochemical basis for photoinhibition of photosystem II. In: KYLE, D. J., C. B. OSMOND, and C. J. ARNTZEN (eds.): Photoinhibition, Elsevier Science Publishers, Amsterdam, pp. 197-226 (1987). KYLE, D. J., I. ORAD, and C. J. ARNTZEN: Membrane protein damage and repair: Selective loss of a quinone-protein fraction in chloroplast membrane. Proc. Nat!. Acad. Sci. USA 81, 4070-4074 (1984). LUDLOW, M. M.: Light stress at high temperature. In: KYLE, D. J., C. B. OSMOND, and C. J. ARNTZEN (eds.): Photoinhibition, Elsevier Science Publishers, Amsterdam, pp. 89-109 (1987).

The role of QB in photoinhibiton of isolated wheat chloroplasts MArroo, A. K., H. HOFFMAN-FALK, J. B. MARDER, and M. EDELMAN: Regulation of protein metabolism: Coupling of photosynthetic electron transport to in vivo degradation of the rapidly metabolized 32-kDa protein of the chloroplast membrane. Proc. Natl. Acad. Sci. USA 81, 1380-1384 (1984). NEDBAL, L., E. SETLlKOVA, J. MASO]IDEK, and 1. SETLlK: The nature of photoinhibition in isolated thylakoids. Biochim. Biophys. Acta 848, 108-119 (1986). OGREN, E. and G. OQUIST: Photoinhibition of photosynthesis in Lemna gibba as induced by the interaction between light and temperature. III. Chlorophyll fluorescence at 77K. Physiol. Plant. 62, 193-200 (1984). ORAD, 1., D. J. KYLE, and C. J. ARNTZEN: Membrane protein damage and repair: Removal and replacement of 32-kilodalton polypeptides in chloroplast membranes. J. Cell BioI. 99, 481-485 (1984). ORAD, I., D. J. KYLE, and J. HIRSCHBERG: Light-dependent degradation of the QB-protein in isolated pea thylakoids. EMBO Jour. 4, 1655-1659 (1985).

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PFISTER, K. and C. J. ARNTZEN: The mode of action of photosystem II-specific inhibitors in herbicide resistant weed biotypes. Z. Naturforsch. 34c, 996-1009 (1979). POWLES, S. B.: Photo inhibition of photosynthesis induced by visible light. Annu. Rev. Plant Physiol. 35, 15-44 (1984). SCHATZ, G. H. and A. R. HOLZWARTH: Picosecond time resolved chlorophyll fluorescence spectra from pea chloroplast thylakoids. In: BIGGINS, J. (ed.): Prog. Photosynth. Res. Vol. 1, Martinus Nijhoff Publishers, Dordrecht, pp. 67 - 69 (1987). STYRlNG, S., I. VIRGIN, A. EHRENBERG, and B. ANDERSSON: Strong light photoinhibition of electron transport in photosystem II. Impairment of the function of the first quinone acceptor QA. Biochim. Biophys. Acta 1015, 269-278 (1990). TREBST, A.: Inhibitors of electron flow: Tools for functional and structural localization of carriers and energy conservation sites. Methods Enzymol. 69, 675-715 (1980). VELTHUYS, B. R.: Electron-dependent competition between plastoquinone and inhibitors for binding to photosystem II. FEBS Lett. 126,277-281 (1981).