Photosynthesis Research 23: 291-296, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands.

Regular paper

Studies on the Hght-induced loss of the D1 protein in photosystem-II membrane fragments Matthias Kuhn & Peter B6ger

Lehrstuhl j~r Physiologie und Biochemie der Pflanzen, Universitiit Konstanz, D-7750 Konstanz, FRG Received 23 November 1988; accepted 17 September 1989

Key words: atrazine resistance, DI protein, herbicides, photoinhibition, photosystem-II membrane fragments, atrazine-resistant Chenopodium album Abstract

PS II membrane fragments produced from higher plant thylakoids by Triton X-100 treatment exhibit strong photoinhibition and concomitant fast degradation of the D 1 protein. Involvement of (molecular) oxygen is necessary for degradation of the D1 protein. The herbicides atrazine and diuron, but not ioxynil, partly protect the D1 protein against degradation. Binding of atrazine to the D1 protein is necessary to protect the DI polypeptide, as shown with PSII membrane fragments from an atrazine-resistant biotype of Chenopodium album which are protected by diuron not by atrazine.

Abbreviations; atrazine - 2-chloro-4-ethylamino-6-isopropylamino-l,3,5-triazine, Chl - chlorophyll, diuron (DCMU) - 3-(3,4-dichlorophenyl)-l,l-dimethylurea, DMBQ - 2,5-dimethyl-p-benzoquinone, DCIP - 2,6dichlorophenol indophenol, DPC - diphenylcarbazide, ioxynil - 4-cyano-2,6.diiodophenol, kb - binding constant, Mes - 4-morpholinoethanesulfonic acid, P-680 - reaction-center chlorophyll a of photosystem-II, PAGE - polyacrylamide gel electrophoresis, PS II - photosystem-II, QA and QB - primary and secondary quinone electron acceptors, Z - electron donor to the photosystem-II reaction center, SDS - sodium dodecylsulfate, Tricine - N-2-hydroxy- 1,1-bis(hydroxymethyl)ethylglycine

Introduction

Treatment with high-light intensities leads to inhibition of electron transport in higher plants (Trebst 1962, Chritchley 1981, Powles 1984). This phenomenon, called photoinhibition, was also observed in cell-free systems using thylakoids (Ohad et al. 1985, Virgin et al. 1988) and PS II membrane fragments (Cleland and Chritchley 1985). Photoinhibition is accompanied by degradation of the D 1 protein, a functional component of the PS II-core complex. This loss of the D 1 polypeptide was suggested to be responsible for inhibition of PS II activity (Kyle et al. 1984). However, some laboratories have obtained conflicting results, showing that

other reactions than damage to the D 1 protein lead to inhibition of PS II activity (Arntz and Trebst 1986, Theg et al. 1986) and the loss of the D1 protein is only a consequence not a cause. This complexity is poorly understood at the moment. The molecular mechanism of damage to the D 1 protein is not established yet. The D1 polypeptide, coded in the chloroplast genome by the psbA gene (Bedbrook et al. 1978), exhibits a rapid turnover in the light (Mattoo et al. 1984, Ohad et al. 1984). Arntzen et al. (1984) assumed that the Qff-semiquinone anion, formed transiently during the PS II photoreaction, may cause damage to the D1 protein. Alleviation of degradation by PS II herbicides appears feasible since such a herbicide is thought to

292 act by displacing QB (plastoquinone) from its binding site. The question is still unsolved whether a semiquinone causes significant damage to the D1 protein directly or indirectly by generating oxygen or hydroxyl radicals, probably by reacting with specific amino-acids side chains (Kyle 1987). This study demonstrates the effect of high-light intensities on the D1 protein in PS II membrane fragments by following its loss by SDS-PAGE. We have used this in vitro system with PS II-enriched membranes to show the dependence of D 1 protein degradation on the presence of molecular oxygen. It is demonstrated that protection by a herbicide depends on its ability to bind to the D1 protein using PS II membrane fragments prepared from an atrazine-resistant biotype of Chenopodium album. As shown further, ioxynil, a phenolic inhibitor of the PS II, does not protect the D1 protein as compared to diuron or atrazine.

0.3 M sucrose, 10mM NaC1, 5 MgC12 and 20mM Mes/NaOH, pH 6.5. After treatment with light the PS II membrane fragments were collected by centrifugation at 10,000 x g for 10min. The pellet was resuspended in 0.3M sucrose, 10mM NaCI, 5mM MgC12 and 20mM Mes/NaOH, pH6.5, adjusted to I mg Chl/ ml and used either for measuring PS II activity or for SDS-PAGE. Electrophoresis of proteins was carried out according to Laemmli (1970) with modifications as described by Kuhn et al. (1986). Binding studies with [14C]-labeled herbicides to determine kb-values and number of binding sites were performed according to Thiel and B6ger (1984), but with the centrifugation times prolonged to 10min (10,000 × g) after herbicide incubation for better sedimentation.

Results Materials and methods

Chenopodium album and Spinacia oleracea (strain Atlanta)

were cultivated in the greenhouse.

Chenopodium album, wild type, was collected from the fields close to Konstanz, the mutant from fields in Northern France. Both strains have been cultivated for several years in the Botanical Garden of the University of Konstanz. PS II membrane fragments were prepared according to Berthold et al. (1981) with some modification (Kuhn et al. 1988). Photoinhibition treatments were performed in 0.3M sucrose, 10mM NaC1, 5mM MgC12 and 20 mM Tricine/NaOH, pH 8.0. Samples equivalent to 50/~g of Chl/ml were illuminated with white light of a quartz-halogen lamp of an intensity of 4000/~E/m 2 x s (measured with a quantum sensor LI-190 SB, made by LI-COR, Lincoln, Nebraska, USA) for different periods at a constant temperature of 25°C. For anaerobic conditions PS II membrane fragments were flushed with nitrogen for 5 min. At start of illumination glucose (50/~g/ ml) and glucose oxidase (50/~g/ml, 29 U/mg, Serva, Heidelberg) were added. Photosynthetic activity was measured either as oxygen evolution with a Clark-type electrode (see Kuhn et al. 1986) or as DCIP reduction at 600 nm. With the latter system the assay mixture contained 15/~g Chl/ml, 0.06mM DCIP and 1 mM DPC in

PS II membrane fragments exhibit strong photoinhibition with concomitant fast degradation of the D1 protein. As described in earlier studies, identification of the D1 protein in Coomassie blue stained gels was performed either by autoradiography of the SDS-PAGE loaded with [t4C]-azidoatrazine labeld PS II membrane framents (Kuhn et al. 1986) or by Western blotting with a monospecific antiserum against the D I protein (Herrmann et al. 1985). Neither the intact D1 protein nor any of its degradation product(s) could be found in the supernatant of light-treated and pelleted PSII membrane fragments by SDS-PAGE and immunoblotting techniques using the antibodies directed against the whole D I protein (Herrmann et al. 1985). In the PS II membranes themselves no degradation product could be clearly identified, but after photoinhibition a diffuse antibody reaction was observed in the molecular-weight range of 23 to 33kDa and at a higher molecular-weight of approximately 54 kDa. This polypeptide at 54 kDa and another one at 50 kDa have been reported and discussed in earlier studies (Kuhn et al. 1988) and are shown in Fig. 1 (indicated as "X"). Degradation of the D1 protein was observed under aerobic conditions only. Under anaerobic conditions there was no significant loss of the D 1 protein after 15min (Fig. 1), while inhibition of PS II activity was also observed under anaerobic

293

Fig. 1. SDS-PAGEof spinach PS II membrane fragmentsstained by Coomassie-blue.The gel shows the influenceof molecularoxygen ondegradationoftheD1 protein. PSIImembranefragmentswerekeptinthedark(D)orilluminatedwith4000#E/m 2 x s for different periods as indicated in the presenceor the absence of molecularoxygen(see Materials and Methods). Under anaerobicconditions no degradation of the D1 protein was observedeven after a 15min high-light treatment. Proteins of 50 and 54 kDa which are generated during photoinhibition are marked by an "X" (see text).

conditions, but slightly less than in the aerobic control (not documented). Herbicides like diuron and atrazine, both binding to the D1 protein (Pfister et al. 1981, Boschetti et al. 1985), protect this protein against degradation induced by strong light (Kyle et al. 1984). The half-life time for disappearence of the D 1 protein in the SDS-PAGE protein pattern of PS II membrane fragments was found to be about 7 m i n / + 2min with photoinhibition conditions as described in Materials and Methods. With 50#M atrazine present the half-life time was prolonged to 15 min/ + 3 m i n (values are means of 4 experiments). Protection of the D1 protein varied with different herbicide classes. Atrazine and diuron exhibited a good protection at concentrations of 50pM and 5 pM, respectively. Apparently, the different con-

centrations for the same degree of protection are due to the different k b of the herbicides. (The k b for atrazine and diuron in PS II membrane fragments is about 0.1 #M and 0.02 pM, respectively; Kuhn et al. 1986.) Ioxynil, exhibiting a kb of about 0.008/~M (Kuhn et al. 1986), did not protect the D1 protein against degradation as effective as atrazine and diuron, even at a concentration up to 50/zM. The SDS-PAGE in Fig. 2 demonstrates the different protective effect of atrazine/diuron and ioxynil. Binding assays performed with [14C]-labeled herbicides and PS II membrane fragments almost depleted of the D1 protein by light treatment showed almost identical losses of binding capacity for all types of herbicides assayed. After 15 min of highlight treatment the number of chlorophyll per binding-site increased from 240 to 2560 for diuron and

294

Fig. 2. SDS-PAGE of spinach PS II membrane fragments stained by Coomassie-blue. The gel shows the light-dependent decrease of the DI protein and the different protective effect of different herbicides. PSII membrane fragments were kept in the dark (D) or illuminated (L) with 4000 #E/m 2 x s for 15 rain in the absence ( - ) or in the presence of the herbicides atrazine (ATR, 50 #M), diuron (DCMU, 5/~M) and ioxynil (IOX, 50#M).

330 to 2930 for ioxynil (data from a typical experiment), corroborating our previous findings that also the ioxynil binding-site is located on the D1 protein (Thiel and B6ger 1986). Noteworthy, the k:values of the herbicides were altered slightly after photoinhibition. The binding constant increased from about 0.02 gM to 0,04 #M for diuron and from about 0.008#M to 0.02#M for ioxynil, respectively. Our experiments with a triazine-resistant (exhibiting a resistance factor for atrazine of about 190; Thiel and Bfger 1984) and with a susceptible biotype of Chenopodium album corroborate the hypothesis of Kyle et al. (1984) that protection by a herbicide depends on its ability to bind to the QB-site. In the resistant biotype the D1 protein could not be protected against damage by atrazine as seen with the wild.type (Fig. 3). Both biotypes, however, were protected from degradation of the D 1 polypeptide by diuron to the same degree. Physiological data as growth, electron-transport activity and sensitivity to photoinhibition of isolated

PS II membrane fragments were found nearly identical in both forms of Chenopodium album (data not shown),

Discussion

Our data indicate that PS II membrane fragments are advantageous to study degradation of the D I protein induced by strong light. Using a high resolution SDS-PAGE system there is no need to specifically label the DI protein before light treatment to monitor the loss of this polypeptide. In our gels the D1 protein migrates as a distinct band above the 33 kDa protein of the oxygen-evolving complex and can be detected directly after Coomassie-blue staining. The role of molecular oxygen in D1 protein degradation was also estimated in our system. Comparison between aerobic and anaerobic conditions showed molecular oxygen being necessary for degradation of the D 1 protein. Inhibition of electron-

295

Fig. 3. SDS-PAGEof PS II membranefragmentsfroma triazine-resistant biotype (MU) and a susceptible one (WT) of Chenopodium album. The Commassie-bluestainded gel shows the light dependent decrease of the D1 protein and the lost protectiveeffectof atrazinein the resistantbiotype(MU). PSII membrane fragmentswere kept in the dark (D) or illuminated (L) for 15min with4000#E/m~ x s in the absence(-) or in the presence (+) of atrazine (ATR, 50#M).

transport activity in light was lowered in the absence of molecular oxygen. Protection against light-induced D1 protein degradation by herbicides was first observed by Kyle et al. (1984) and explained by displacement of the quinone QB from its binding niche (Vermaas et al. 1983). Thereby production of semiquinones is inhibited, the latter being candidates for primary damage to the D1 protein (Kyle 1987). Our data show protection by atrazine and diuron but not by ioxynil, indicative of a different binding site for ioxynil at the D 1 protein compared to atrazine and diuron. This is in agreement with previous data from our laboratory showing a different binding

behavior of ioxynil. However, this effective inhibitor of PS II is assumed to act at the DI protein like atrazine and diuron (Thiel and Brger 1986). Protection against D1 protein degradation by atrazine is lost in a triazine-resistant biotype of Chenopodium album while protection by diuron and sensitivity to diuron is still observed. This finding corroborates the report of Kyle et al. (1984), who used an atrazine-resistant mutant of the alga Chlamydomonas reinhardii exhibiting a higher degree of photoinhibition in the presence of atrazine than the wildtype. Their conclusions are based on the different decrease in variable fluorescence after photoinhibition. We could show that binding of atrazine is necessary for protection against photoinhibition and D1 protein degradation. There is general accordance that (reactive) plastoquinone is replaced by a herbicide molecule at the D1 protein (Vermaas et al. 1983). Accordingly, our findings strengthen the hypothesis of Kyle et al. (1984) that a species of a bound quinone together with oxygen is responsible for damage to the protein which leads to degradation of the D1 protein. The degradation process is highly active in isolated PS II membrane fragments. If degradation depends on a protease, this should be a membrane associated protein of PSII, since all soluble proteins of the thylakoid are lost during the preparation steps. Experiments failed to detect degradation products of the D I polypeptides by immunoblotting. After photoinhibition treatment the antibodies reacted in a wide range from 23 to 33 kDa and additionally at about 55 kDa. Proteins of higher molecular weights, generated during photoinhibition as observed by us (Kuhn et al. 1988) have been reported earlier by Marder et al. (1987) using non-illuminated PS II reaction center preparations. These authors discussed these 55kDa species as an aggregate of the D1 and D2 protein. We could not follow the fate of the D2 protein during photoinhibition since up to now we are not able to reliably identify the D2 protein in the SDSPAGE, since a reactive antibody is not yet in our hands. Further studies will deal with the involvement of the D2 protein in photoinhibition. Whether the D1 polypeptide breakdown is responsible for all photoinhibition phenomena cannot be concluded from the data presented. It should be noted that the 9-kDa phosphoprotein of PSII shows a similar behavior after high-light treatment a~ the DI protein (Kuhn et al. 1988).

296

Acknowledgement T h i s s t u d y was s u p p o r t e d b y the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t a n d the F o n d s d e r C h e m i s chen Industrie.

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