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Photosystem II particles were exposed to 800 W m -2 white light at 20 °C under anoxic conditions. The Fo level of fluorescence was considerably enhanced ...
PhotosynthesisResearch 46: 213-218, 1995. (~) 1995KluwerAcademicPublishers. Printedin the Netherlands. Regular paper

Comparative EPR and thermoluminescence study of anoxic photoinhibition in Photosystem II particles S~indor D e m e t e r 1, J o n a t h a n H. A. N u g e n t 2, L~iszl6 Kov~ics 1, G f i b o r Bernfit I & M i c h a e l C.W. E v a n s 2

l lnstitute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary; 2Department of Biology (Darwin Building), University College London, London, UK Received 6 March 1995;acceptedin revisedform3 July 1995

Key words: Photoinhibition, Photosystem II, quinone-iron complex, electron paramagnetic resonance (EPR), thermoluminescence (TL)

Abstract

Photosystem II particles were exposed to 800 W m -2 white light at 20 °C under anoxic conditions. The Fo level of fluorescence was considerably enhanced indicating formation of stable-reduced forms of the primary quinone electron acceptor, QA. The Fm level of fluorescence declined only a little. The g = 1.9 and g = 1.82 EPR forms characteristic of the bicarbonate-bound and bicarbonate-depleted semiquinone-iron complex, QA-Fe 2+, respectively, exhibited differential sensitivity against photoinhibition. The large g = 1.9 signal was rapidly diminished but the small g = 1.82 signal decreased more slowly. The S2-state multiline signal, the oxygen evolution and photooxidation of the high potential form of cytochrome b-559 were inhibited approximately with the same kinetics as the g = 1.9 signal. The low potential form of oxidized cytochrome b-559 and Signal Ilslow arising from TyrD+ decreased considerably slower than the g = 1.9 semiquinone-iron signal. The high potential form of oxidized cytochrome b-559 was diminished faster than the low potential form. Photoinhibition of the g = 1.9 and g = 1.82 forms Of QA was accompanied with the appearance and gradual saturation of the spin-polarized triplet signal of P 680, The amplitude of the radical signal from photoreducible pheophytin remained constant during the 3 hour illumination period. In the thermoluminescence glow curves of particles the Q band (S2QA- charge recombination) was almost completely abolished. To the contrary, the C band (TyrD+Q~, charge recombination) increased a little upon illumination. The EPR and thermoluminescence observations suggest that the Photosystem II reaction centers can be classified into two groups with different susceptibility against photoinhibition.

Abbreviations: C band - thermoluminescence band associated with Tyr-D+QA charge recombination; Chl chlorophyll; DCMU - 3-(3,4-dichlorophenyl)- 1,1-dimethylurea; EPR - electron paramagneric resonance; Fo initial fluorescence; Fm - maximum fluorescence; Q band - thermoluminescence band originating from S2QAcharge recombination; QA - the primary quinone electron acceptor of PS II; P 680 - the primary electron donor chlorophyll ofPS II; $2 - oxidation state of the water-splitting system; P h e - pheophytin; T L - thermoluminescence; TyrD - redox active tyrosine-160 of the D2 protein. Introduction

The majority of data suggests that during 'acceptor side' photoinhibition the primary irreversible damage occurs at the QA quinone electron acceptor (Cleland et al. 1986; Styring et al. 1990; Setlik et al. 1990). The

sequence of events can be easily followed under anaerobic conditions when the D 1 protein is not degraded. It has been observed that illumination results in the formarion of stable-reduced and protonated QA species and finally in a loss of QA from its binding site (Vass et al. 1992).

214 The QA acceptor has two EPR detectable forms (Vermaas and Rutherford 1984). When the non-heme iron of the quinone-iron complex has a bound bicarbonate the QA acceptor exhibits the g = 1.9 EPR signal. Depletion of bicarbonate by formate converts this signal into the g = 1.82 form with about a 10-fold increase in its amplitude. In order to increase the signal to noise ratio of spectra, photoinhibition of samples used in EPR measurements is often carded out in the presence of formate (Vass and Styring 1993). This procedure assumes that photoinhibition is the same in bicarbonate-containing and -depleted membranes. However, it has been recently observed that bicarbonate depletion provides partial protection against photoinhibitory damage (Demeter et al. 1995). Addition of formate after photoinhibition preceding the EPR measurement also has the shortcoming that only the changes of the g = 1.82 form of QA can be measured (Styring et al. 1990). Therefore, the aim of the present EPR study was to reinvestigate the photoinhibition of PS II enriched thylakoid fragments under anoxic conditions in the absence of formate (bicarbonate-containing samples in the presence of carbondioxide). In this way not only the behaviour of the g = 1.82 EPR form but that of the g = 1.9 form of QA could also be followed during photoinhibition. The EPR measurements were accompanied by thermoluminescence (TL) measurements because any change in the amount and redox state of QA is reflected in the amplitude and peak position of the Q (SEQA charge recombination) and C (Tyr+Qg charge recombination) TL bands (Demeter et al. 1993; Johnson et al. 1994). In the present work it was observed that under anoxic conditions in bicarbonate-containing PS II particles the g = 1.82 form of the semiquinone-iron complex was photoinhibited slower than the g -- 1.9 form. While the Q TL band was gradually abolished the C band slightly increased during illumination. It was also observed that contrary to aerobic photoinhibition (Styring et al. 1990) the high potential form of cytochrome b-559 was not converted to the low potential form and was photoinhibited faster than the low potential form.

Materials and methods Oxygen-evolving PS II particles were isolated from pea chloroplasts (V61ker et al. 1985). Photoinhibition of samples and all of the measurements were carried out in the suspension medium containing 0.4 M

sucrose, 15 mM NaC1, 5 mM MgC12, 5 mM EDTA, 20 mM MES at pH 6.0. Photoinhibitory treatment of PS II particles was performed at 20 °C in EPR tubes (5 mg Chl/ml) at a light intensity of 800 W m -2. It was observed that the oxygen content of the samples stored in EPR tubes at room temperature gradually decreased during incubation. After 2 hour dark storage no oxygen could be polarographically detected in a Clark electrode (not shown). The rate of oxygen consumption increased with the chlorophyll concentration of the sample. This means that dense samples in EPR tubes became anoxic during short dark incubation. Based on this experience anoxic conditions were established by keeping the dense sample for two hours at room temperature in the dark in EPR tube before photoinhibitory illumination in the tube. The validity of this procedure was proved in a control series of photoinhibitory measurements using the glucose (10 mM)/glucose oxidase (50 units m l - 1)/catalase (1000 units m l - t) trap method (Setlik et al. 1990) for the establishment of anoxic conditions in the samples. Both procedures resulted in similar fluorescence, EPR and TL characteristics. Samples taken at different times of photoinhibition were stored at room temperature in dark at least for two hours to allow relaxation of the photoinhibitioninduced reversible changes. In order to determine possible dark inactivation of samples in each experiment a control sample was kept in dark under conditions identical to those of the illuminated sample. Fluorescence induction transients were measured in a home-built setup (Demeter et al. 1995). The TL glow curves were measured at a heating rate of 20 °C/min using an apparatus described in (Demeter et al. 1984). PS 1I electron transport at 20 °C in saturating light was measured as Oe-evolution in a Clark electrode in the presence of 200/~M phenyl-para-benzoquinone. Fluorescence, oxygen evolution and TL measurements were carried out on aerobic samples diluted from anoxic samples after photoinhibition in EPR tubes. X-band low-temperature EPR spectra were recorded with a JEOL RE1X spectrometer equipped with an Oxford Instruments ESR 9 liquid helium cryostat. Chemical reduction of QA was performed by incubation of samples in dark at room temperature for 10 min in the presence of 50 mM dithionite. For measurements of the amount of photoreducible pheophytin (Phe) samples were incubated for 10 min in the presence of 50 mM dithionite in dark at room temperature and cooled to 77 K during continuous illumination.

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Results and discussion

Fig. 2. Effectof anoxic photoinhibition on the g = 1.9 and g = 1.82 EPR forms of the quinone-iron complex, QAFe2+. The photoinhibited samples were dark adapted for two hours. In order to avoid the overlapping of the g = 1.9 and g = 1.82 EPR signals with the S2-state multiline signal the samples were excited at 77 K where only the photoreductionof QA was induced. EPR conditions: temperature, 4.2 K; microwavepower 20 mW; modulationamplitude 1.6 roT; microwave frequency 9.055 GHz. Curves represent light minus dark difference spectra.

Illumination of oxygen evolving PS II particles under anoxic conditions resulted in a rapid and irreversible rise of the Fo level and in a small decline of the Fm level of fluorescence (Fig. 1). Similar irreversible Fo increase could be induced by illumination of anaerobic samples (Setlik et al. 1990) and was attributed to double-reduction and protonation of the QA molecules which in turn probably dissociated from the binding sites (Vass et al. 1992). Formation of stable-reduced QA forms promotes the generation of long-lived triplet state of P 680 and results in a lowered fluorescence yield (van Mieghem et al. 1989). In the photoinhibited samples (Fig. 1) the Fm level (about 82%) was close to the fluorescence yield (86-94%) calculated by Vass and Styring (1992) for triplet forming reaction centers. This suggests that the observed small Fm decrease can mainly be attributed to triplet formation. Consequently, fluorescence quenchers, which were thought to cause the typical fast and large decline of Fm fluorescence during photoinhibition (Kirilovsky et al. 1994) and were identified as carotenoid and chlorophyll cations in donor side photoinhibited samples (Bluhaugh et al. 1991), were formed in a very small amount in our anoxic samples.

Illumination of the PS II particles at 77 K induced a large g = 1.9 EPR signal, characteristic of the semiquinone-iron complex, QA Fe2+ with bound bicarbonate (Fig. 2). Since the majority of centers were in the bicarbonate-containing native state the g = 1.82 signal associated with bicarbonate-free quinone-iron complexes appeared in the spectrum with a smaller amplitude (Fig. 2). It has to be mentioned that without 77 K illumination the QAFe 2+ signals were not present in the spectrum (not shown) because after photoinhibitory illumination the short-lived reduced forms of QA completely decayed during the 2 hour dark adaptation and the double-reduced form of QA is EPR silent (Vass et al. 1992). During photoinhibitory illumination the g = 1.9 signal was progressively and irreversibly diminished (Fig. 2) suggesting the formation of EPR silent doublereduced and protonated QA molecules and their probable loss from the binding sites (Vass and Styring 1993). It was apparent that the small g = 1.82 EPR form of QA- was less susceptible to photoinhibition and after 120 min illumination its amplitude was about the same as that of the decreasing g = 1.9 form (Fig. 2). Although, it still requires further experimental support

Fig, 1. Effect of anoxic photoinhibition on the Fm and Fo level of

fluorescence in PS II particles. Before measurements the photoinhibited samples were diluted to a chlorophyll concentration of 10 #g/ml and dark adapted for 2 h.

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Magnetic field (roT) Fig. 3. Effect of anoxic photoinlfibition on the amount of oxidized cytochrome b-559. After various times of photoinhibition the sampies were dark adapted for 2 h at room temperature and measured before (lower curves) and after 5 rain illumination at 200 K (upper curves). The high potential form appears at g = 3.06 and the low potential form at about g = 2.94 value. EPR conditions: temperature 12 K; microwave power 5 roW; modulation amplitude 1.6 mT; microwave frequency 9.055 GHz.

from the present experiments we assume that the small g = 1.9 form remaining after 120 min photoinhibition is also associated with less susceptible centers. It is to note that the amplitudes of the g = 1.9 and g = 1.82 forms which were reduced chemically in the dark decreased with similar kinetics as the photoinduced g = 1.9 and g = 1.82 signals (not shown). Under anoxic conditions photoinhibition of the high and low potential forms of the oxidized cytochrome b-559 (Fig. 3) was completely different as in aerobic samples (Styring et al. 1990). In the dark adapted sample only the low potential form of cytochrome b-559 was present in oxidized form. After 200 K illumination the high potential form also became oxidized and formed about 60-70% of the total signal. Photoinhibitory illumination without 200 K additional illumination did not result in an increased oxidation of the cytochrome b-559 (Fig. 3, dark spectra). During photoinhibitory illumination the high potential form was diminished faster than the low potential form (Figs. 3 and 4A). Contrary to the aerobic pho-

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Illumination time (rain) Fig. 4. Effect of anoxic photoinhibition on the various EPR signals (A) and TL bands (B) arising from PS II. (A) free-radical signal from Phe- (B--~), spin-polarized triplet signal from P 680 (~7--x7), g = 1.82 signal OfQA-Fe 2+ (~)--0), Signal IIs]ow from TyrD + (o--o), low potential form of cytochrome b-559 ( T - - T ) , high potential form of cytochrome b-559 ( n - - O ) , g = 1.9 signal of Q A - F e 2+ 0 - - ~ , S2-state multiline signal (/x-/x). For the triplet signal of P 680 the saturation level of the signal reached after 180 min illumination was taken as 100%. EPR conditions for the free radical signal from P h e - : temperature 15 K; microwave power 1 ~W; modulation amplitude 0.25 mT. EPR conditions for the 3p 680 signal: temperature 4 K; microwave power 50/zW; modulation amplitude 2.0 mT. The signal was measured during continuous illumination at 4 K in the EPR cavity. EPR conditions for the S2-state multiline signal: temperature 10 K; microwave power 20 mW; modulation amplitude 1.6 roT. The signal was excited at 200 K. The microwave frequency was 9.055 GHz in all cases. (B) C TL band (e--o), Q TL band (+-----+),oxygen evolution ( & - - A ) .

toinhibition (Styring et al. 1990) interconversion of the high potential form to low potential form could not be observed under anoxic conditions. The decay course of the high potential form of oxidized cytochrome b-559 was about the same as that of the S2-state multiline signal, g = 1.9 signal (Fig. 4A) and oxygen evolution (Fig. 4B). The parallel decrease of these signals without concomitant appearance of other donor signals suggests that the S2-state multiline and the high potential cytochrome b-559 signals probably disappear due to the impairment of the function of QA. The low potential form of cytochrome b-559 was diminished slower in parallel with the decrease of the Signal Ilslow

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Fig. 5. Effect of anoxic photoinhibition on the Q and C thermoluminescence bands in PS II particles. The photoinhibited samples were diluted to a chlorophyll concentration of 125/zg/ml and dark adapted for 2 h. After addition of 10/~M DCMU thermoluminescence was excited at 200 K and measured at a heating rate of 20 ° C/min.

of the Tyro+ radical (Fig. 4A). Similar slow decrease of Signal IIs]ow was also observed in anaerobic sampies by Setlik et al. (1990). The decrease of the low potential form of oxidized cytochrome b-559 and that of Signal Ilslow can be attributed to the development of a largely reduced donor side of PS II during anaerobic photoinhibition (Vass and Styring 1993). Concomitantly with the decrease of the QA signals the triplet signal of P 680 gradually increased and reached saturation at about 60 rain illumination (Fig. 4A). Similarly as in aerobic samples (Styring et al. 1990) the pheophytin radical signal remained almost constant during the 3 h illumination period (Fig. 4A). The behaviour of the triplet P 680 and pheophytin radical signal is in agreement with the conclusion of Setlik et al. (1990) according to which in anaerobic samples the primary charge separation is not affected in the first so called 'fast phase' of photoinhibition. The effect of photoinhibition on the QA acceptor was also investigated by TL measurements because in the presence of DCMU charge recombination of the semiquinone-iron complex, QAFe 2+ with the donor side of PS II gives rise to the characteristic Q and

C termoluminescence bands. In the TL glow curves of bicarbonate-containing PS II particles the Q and C band appeared at about +10 and +50 °C, respectively (Fig. 5). During photoinhibition the Q band was rapidly and irreversibly diminished but the C band increased a little with prolonged illumination (Figs. 4B and 5). The decrease of the Q band can be attributed to formation of stable reduced QA molecules which cannot undergo fast charge recombination reaction with the donor side required to the generation of the Q band (tl/2 of the S2QA charge pair is about 1-3 s (Demeter and Vass 1984)). It has been observed that in the anaerobically photoinhibited PS II center, simultaneously with the formation of stable-reduced QA forms, the wateroxidizing complex is trapped in the So and $1 states and formation of the S2-state is inhibited even in the presence of stable singly-reduced QA (Vass and Styring 1993). This phenomenon can also cause the disappearance of the Q band (S2Q~, charge recombination) but at the same time can facilitate the enlargement of the C band (TyrD+QA charge recombination) which originates from centers possessing inactive water-splitting system or from centers which are in the So and $1 states before charge recombination (Demeter et al. 1984). The simultaneous decrease of the Q band andthe g = 1.9 EPR signal (Fig. 4A and B) suggests that in bicarbonate-containing particles the major part of the g = 1.9 form of QA (susceptible to photoinhibition) is associated with the Q TL band (S2QA charge recombination). This suggestion is supported by our earlier observation (Demeter et al. 1993) according to which the dark relaxation half-time of the Q band and that of the g = 1.9 form is approximately the same. However, it cannot be excluded that a small part of the g = 1.9 form remained after 120 min photoinhibition participates in the generation of the C band (TyrD÷QA charge recombination) resistant to photoinhibition. About 30% of the g = 1.82 signal was abolished during 120 min photoinhibition. It can be assumed that this fraction of the g = 1.82 form is associated with the rapidly decreasing part of the Q band. The remaining 70% of the g = 1.82 form which survived 180 min of photoinhibitory illumination can contribute to the generation of the C band. The similar decay half-time (tl/2 is about 10 min) of the g = 1.82 EPR form and that of the C band substantiates this assumption (Demeter et al. 1993; Johnson et al. 1994). We cannot explain at present why is a certain fraction of the PS II reaction centers (in bicarbonate containing particles represented mainly by the g = 1.82

218 signal) less susceptible to anoxic photoinhibition. It can be assumed that in these centers the water-splitting system is in the So state and the electron transfer is inhibited between QA and QB. Consequently, charge recombination would result in the appearance of the C TL band. At the same time interruption of electron transport between QA and QB could provide partial protection (as it has been shown for QB non-reducing, bicarbonate-depleted, DCMU or atrazin inhibited centers) against both donor (Callahan et al. 1986) and acceptor side (Demeter et al. 1995) photoinhibition. To clarify the real underlying mechanism futher investigations are required.

Acknowledgements This work was supported by the US-Hungarian Science and Technology Joint Fund in cooperation with the USDA and the Hungarian Academy of Sciences under Project HU-AES-25 (J.F. No. 087/91). Additional support was provided by the Hungarian National Committee for Technological Development in the PHARE ACCORD programmes, No. H 9112--0147 and No. H 9112-0150 as well as by the Hungarian National Science Foundation OTKA I/3 2667/1991, OTKA 1/3 2668/1991 and OTKA T 016449/1995.

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