Loss of the trans-thylakoid proton gradient is an early event during photoinhibitory illumination of chloroplast preparations

Biochimica et BiophysicaActa, 1183(1993) 315-322

315

© 1993 Elsevier Science Publishers B.V. All rights reserved 0005-2728/93/$06.00

BBABIO 43923

Loss of the trans-thylakoid proton gradient is an early event during photoinhibitory illumination of chloroplast preparations Staffan E. Tjus and Bertil Andersson * Department of Biochemistry, Arrhenius Laboratoriesfor Natural Sciences, Stockholm University, S-10691 Stockholm (Sweden)

(Received 31 March 1993)

Key words: pH gradient; Light stress; Photoinhibition;Photophosphorylation;Proton uptake; Uncoupling; (Chloroplast) During photoinhibitory illumination of isolated thylakoids or intact chloroplasts from spinach, an initial increase of Photosystem I electron transport was observed while proton uptake associated with PMS-mediated cyclic electron transport was rapidly lost. The latter reaction was at least as light-sensitive as Photosystem II electron transport, normally considered to be the primary target for light stress. Thus under both moderate and extreme light stress, loss of the proton gradient associated with cyclic electron transport around Photosystem I was an early event. In accordance with this observation, photoinhibitory light very rapidly caused inactivation of cyclic photophosphorylation. There was no kinetic correlation between light-induced degradation of the D1 protein and collapse of the proton gradient. Notably, under anaerobic conditions when D1 protein degradation does not occur, loss of proton uptake still occurred. Low temperatures (3°C) provided partial protection against the photodamage, but a subsequent increase of the temperature to 25°C resulted in a total loss of the proton uptake in total darkness. The proton gradient could not be re-established by addition of DCCD. Moreover, there were no changes in the polypeptide composition of CF 1 or any impairment of the ATPase activity during photoinhibitory illumination. The mechanism of the light-induced loss of the proton gradient and its correlation to other effects of light stress at the molecular and physiological levels are discussed.

Introduction

Reports on photoinhibition of photosynthesis have focused mainly on damage associated with Photosystem II [1-3]. It is well documented that high light intensities lead to inactivation of Photosystem II electron transport as well as to damage and degradation of the reaction centre, in particular the D1 protein [2-4]. Photosystem I, in contrast, requires very extreme light conditions for inactivation, a process which involves impairment of Fe-S clusters [5] and eventually photobleaching [1]. Studies concerning the effect of light stress on photophosphorylation and closely associated events in various photosynthetic systems are, however, contradictory [6-22]. For example, cold-sensitive plants have been shown to lose their CF 1 at excess light and

* Corresponding author. Fax: + 46 8 153679. Abbreviations: CF1 and CF0, catalytic and proton translocating moieties of the chloroplast ATP syathase, respectively; DAPP, 1,5-diadenosine pentaphosphate; DCIP, 2,6-dichlorophenol indophenol; DCCD, 1,3-dicyclohexylcarbodiimide; DCMU, 3-(3,4-dichlorophenyl)-l,l-dimethylurea; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; PMS, phenazine methosulphate; PpBQ, phenylp-benzoquinone; Tricine, (N-tris[hydroxymethyl]methylglycine.

thereby also their ability to form a trans-thylakoid proton gradient [10, 11], while Chlamydomonas cells show a rise in lumenal proton uptake during photoinhibitory illumination [12]. It has also been postulated that a rise in A p H during high light is involved in inducing so-called qE-quenching which is believed to protect against photoinhibition in vivo [14-22] (for reviews see Refs. 23,24). In this study, we present data showing that during photoinhibitory illumination of isolated spinach chloroplasts and thylakoid membranes, there is an initial stimulation of Photosystem I electron transport and a concomitant rapid loss both of the trans-thylakoid proton gradient and of cyclic photophosphorylation. The collapse of the proton gradient was compared to several other photoinhibitory damages of the photosynthetic apparatus and found to coincide with inactivation of Photosystem II electron transport but to precede light-induced D1 protein degradation. Material and Methods Preparation procedures Spinach (Spinacia oleracia L.) was grown under arti-

ficial light in nutrient solution as described in Ref. 25. Intact chloroplasts were prepared by Percoll density

316 centrifugation of spinach leaf homogenate essentially as in Ref. 26. The level of intactness (> 80%) was determined by comparing uncoupled Photosystem II electron transport before and after osmotic shock, with ferricyanide as electron acceptor [27]. Well-stacked thylakoid membranes were prepared according to Ref. 25. The preparations were kept on ice until use and all activities measured remained stable in the dark.

Conditions for photoinhibitory illumination Intact chloroplasts were suspended to a concentration of 0.1 mg chlorophyll/ml in a buffer containing 50 mM Hepes-KOH (pH 8.0), 5 mM MgCI2, 1 mM MnC12, 2 mM Na2EDTA , 1 mM ascorbate and 350 mM sorbitol in a volume of 20 ml. Thylakoid membranes were suspended to a concentration of 0.1-0.2 mg chlorophyll/ ml in a buffer containing 5 mM Tricine (pH 7.4), 6 mM MgC12 and 300 mM sucrose in a volume of 10-20 ml. Photoinhibitory illumination was performed with UV-depleted white light at intensities of 190-7000 / ~ E m - 2 s - 1 at 25°C with continuous stirring as detailed in the figure legends. Control samples were kept at same conditions but in total darkness. Photoinhibitory illumination at low temperature was performed at 30C and proton translocation activity measured at both 3°C and 25°C. The samples were then transferred to 25°C at darkness for 40 min and proton translocation was measured again at 3°C. Photoinhibitory illumination under anaerobic conditions was performed, with 20 ml of thylakoids as above, in 50 ml rubber-stoppered vials from which air had been evacuated with a vacuum pump and replaced with argon [28]. Samples were withdrawn with a syringe through the membrane cap.

Measurement of photochemical activities Photosystem I electron transport was measured from ascorbate/DCIP to methylviologen as oxygen consumption using a Clark-type oxygen electrode. The assay medium was composed of; 40 mM sodium phosphate (pH 7.4), 1.0 mM NaC1, 10 mM sucrose, 10/zM DCMU, 5 mM sodium azide, 1.0 mM sodium ascorbate, 0.1 mM DCIP, 0.12 mM methylviologen and thylakoids corresponding to 10/zg of chlorophyll/ml. Photosystem II electron transport was measured from H 2 0 to PpBQ as oxygen evolution. The assay medium contained; 50 mM sodium phosphate (pH 6.5), 5 mM NaC1, 5 mM MgCI 2, 100 mM sucrose and 1 mM PpBQ (Figs. 2-5, 8) or; 50 mM Hepes (pH 7.4), 20 mM NaCI, 5 mM MgCI 2 200 mM sucroseand 0.6 mM PpBQ (Fig 1) and in both cases thylakoids corresponding to 10/~g of chlorophyll/ml. As an assay of the intactness of the thylakoid membrane, PMS-mediated total proton uptake kept at

steady state during Photosystem I cyclic electron transport [6] was measured with a pH electrode connected to a chart recorder. The assay medium was; 75 mM KCI, 5 mM MgCI2, 0.7 mM PMS, 70/~M DCMU, 150 mM sucrose and thylakoids corresponding to 25-50/zg chlorophyll in a total volume of 1.5 ml. Calibration was performed by adding known amounts of NaOH. Alternatively, proton uptake was detected in the above medium as absorbance change of the pH-indicating dye Bromocresol purple measured at 587 nm [29]. Lumenal acidification was measured likewise using Neutral red at 540 nm while buffering the external medium with BSA (1.3 m g / m l ) [30]. Rapid flash-induced electrochromic absorption changes at 522 nm and subsequent relaxation of the signal after a single-turnover flash were measured as in Ref. 31. Cyclic photophosphorylation was measured as ATP synthesis by the luciferin-luciferase bioluminescence method [32] in a medium (2.0 ml) composed of; 200 mM glycylglycine (pH 8.3), 10 mM sodium phosphate, 100 /~M ADP, 5 ~ M DAPP, 50 ~M PMS, 0.5 mM sodium ascorbate, 50/xM DCMU, 200/~l ATP-monitoring reagent (LKB-WALLAC), 50 mM sucrose and thylakoids corresponding to 10/zg of chlorophyll. The Mg2+-dependent ATPase activity was determined spectrophotometrically by coupling the reactions of the pyruvate kinase and lactate dehydrogenase and following the oxidation of NADH at 340 nm, as described in Ref. 33. Activation of the ATPase activity was performed by treatment with dithiothreitol in darkness for 2 h in the presence of ATP essentially as in Ref. 34.

D1 protein determination SDS-polyacrylamide gel electrophoresis was run according to Laemmli [35] with modifications as in Ljungberg et al. [36]. Immunoblotting was performed essentially as described by Towbin et al. [37] in order to determine the relative amount of the D1 protein. Results

When isolated thylakoids were illuminated with strong light at 3500/~E m - 2 s - 1 the expected inactivation of Photosystem II electron transport was observed, followed by subsequent degradation of the D1 reaction centre protein (Fig. 1). At the same time, Photosystem I electron transport showed a pronounced initial increase with a maximum of about 230% after 30 min (Fig. 1). Only after prolonged illumination was impairment of the Photosystem I activity detectable, in accordance with [5]. To investigate whether the initial rise in Photosystem I electron transport was associated with an uncoupling event, the trans-thylakoid ApH was measured.

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Fig. 1. Thylakoids were subjected to light of 3500 p.E m - 2 s -1 and analysed for; steady-state total proton-uptake during PMS-mediated cyclic electron transport with Photosystem II blocked with DCMU ( n ) , Photosystem I electron transport from ascorbate/DCIP to methylviologen measured as oxygen consumption (0), Photosystem II electron transport from H 2 0 to PpBQ measured as oxygen evolution (e) and relative amount of D1 protein analysed by western blotting ( • ) . 100% activity values of dark controls were: proton uptake = 44 nmol H + (mg chlorophyll)-1; Photosystem I = 327/.¢mol 0 2 (mg chlorophyll) -1 h - t ; Photosystem I I = 1 8 0 ~mol 0 2 (mg chlorophyll)- 1 h - 1.

To avoid effects on proton translocation from Photosystem II being inactivated, we measured light-induced proton uptake of the illuminated samples in the presence of both PMS as a cofactor for cyclic electron transport around Photosystem I and DCMU as an inhibitor of Photosystem II. As can be seen in Fig. 1, the steady-state level of proton uptake decreased very rapidly during the photoinhibitory illumination. The inactivation half-time was approximately 10 min and total collapse was seen after 45 min. Strikingly, collapse of the proton gradient occurred even faster than the loss of Photosystem II electron transport (Fig. 1) which is normally considered to be the initial and major target for light stress [1]. Rapid loss of the trans-thylakoid proton gradient was also demonstrated spectrophotometrically using Bromocresol purple as an indicator of stromal alkalisation and Neutral red for measuring lumenal acidification (Fig. 2). In addition, we measured flash-induced electrochromic apsorption changes at 522 nm [31] during photoinhibition of thylakoids. The preliminary experiment revealed a loss of maximal electrochromic shift amplitude of 66%, while the half-time of the electric field relaxation was lowered from 11 ms to 1 ms at a stage when Photosystem II oxygen evolution was inactivated by 40% (unpublished data) This is giving evidence of lost membrane intactness in photoinhibited thylakoid membranes in agreement with the ApH measurements above. To see if loss of the ApH occurred only at extreme light intensities, we also illuminated thylakoid mem-

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illumination time (rain) Fig. 2. Thylakoids were subjected to light of 6500 or 7000/~E m - 2 s- 1 and analysed for; steady-state total proton-uptake during PMS-mediated cyclic electron transport. Absorbance change of Bromocresol purple (587 nm) was used as assay for stromal alkalisation ( n ) and absorbance change of Neutral red (540 nm, samples illuminated at 7000/zE m - 2 s - t ) showed acidification of the thylakoid lumen ( • ) as the external medium was buffered with BSA. Photosystem I] electron transport was also assayed (e). 100% activity value of dark control was: proton uptake with Bromocresol purple = 39 nmol H + (rag chlorophyll)- 1.

branes at a moderately high light of 190 /~E m - 2 s - 1 (Fig. 3). Photosystem I electron transport followed the same pattern of an initial increase. Photosystem II electron transport followed a steady route of inactivation with a half-time of 60 min. At this moderately high light intensity, the trans-thylakoid proton uptake showed a short initial stable phase or even an increase, but then rapidly declined (Fig. 3). The decline closely followed Photosystem II inactivation, as seen under the very high light conditions (Fig. 1). We also studied the effect of different light intensities, above saturation level, on steady-state proton up-

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illumination time (rain) Fig. 3. Thylakoids were subjected to moderately high light of 190 /.¢E m -2 s -1 and analysed for; steady-state total proton-uptake during PMS-mediated cyclic electron transport ([]), Photosystem I electron transport ( 0 ) and Photosystem II electron transport (e). 100% activity values of dark controls were: proton uptake = 140 nmol H + (mg chlorophyll)- 1; Photosystem I = 257/~mol 0 2 (mg chlorophyll)- 1 h-1; Photosystem II = 66/~mol 0 2 (rag chlorophyll)-1 h-1.

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light intensity ( g E / m 2 s ) Fig. 4. Thylakoids were subjected to photoinhibitory illumination at an interval of light intensities for 35 min and analysed for; steady-state total proton-uptake during PMS-mediated cyclic electron transport ([]) and Photosystem II electron transport (e). 100% activity values of dark controls were: proton uptake = 106 nmol H + (mg chlorophyll)- I; Photosystem II = 56/~mol 0 2 (mg chlorophyll)- 1 h - 1.

take as compared to Photosystem II electron transport (Fig. 4). Thylakoid membranes illuminated for 35 min at different light intensities from the moderately high light of 275/~E m -2 s -1 to the very strong light of 2300 /zE m -2 s -1 showed very parallel inactivation of Photosystem II electron transport and Photosystem-Imediated proton uptake. The above effects were seen when isolated thylakoid membranes were illuminated during photoinhibitory conditions. To verify these observations under more native conditions, intact chloroplasts were also subjected to photoinhibitory light. After illumination at 7000 /zE m - 2 s -1, the intact chloroplasts were ruptured osmotically and the thylakoids assayed for photochemical activities. The same pattern as observed with thylakoids was obtained, i.e., a rapid loss of ApH which paralleled or even preceded inactivation of Photosystem II electron transport (Fig. 5). These findings clearly show that the trans-membrane proton gradient associated with cyclic electron transport around Photosystem I is rapidly lost during photoinhibitory illumination not only of isolated thylakoids but also of intact chloroplasts. Various experiments were designed to investigate the mechanism behind the collapse of the proton gradient induced by the high-light treatment. Initially, the temperature dependency of the photoinhibitory damage was investigated. After treatment of thylakoids with light of 4500/zE m -2 s -1 at 3°C, the PMS mediated proton uptake was measured directly at 3°C. As can be seen in Fig. 6, under these experimental conditions the proton gradient was lost with a half-time of approx. 40 min and after 1 h of strong light about 20% of the steady-state proton uptake still remained. These inactivation values for the steady-state proton uptake

20 40 60 illumination time (rain)

Fig. 5. Intact chloroplasts were subjected to strong illumination of 7000 / z E m - 2 s -1. Thylakoids were isolated and analysed for; steady-state total proton uptake during PMS-mediated cyclic electron transport ( D ) and Photosystem II electron transport (e). 100% activity values of dark controls were: proton uptake = 62 nmol H + (nag chlorophyll)-1; Photosystem II = 97 ~mol 0 2 (mg chlorophyll)- I h - 1.

level should be compared to those obtained after illumination at 25°C and 3500 /zE m - 2 s -1 where the inactivation half-time was approx. 10 min and total collapse of the proton gradient was observed after 45 min (Fig. 1). Strikingly, however, when transferring samples illuminated at low temperature to room temperature (25°C) for 40 min in total darkness, the ApH of the illuminated thylakoids decreased further to a level similar to that obtained after illumination at room temperature (Fig. 6). These observations clearly demonstrate temperature-dependency for the lightstress induced impairment of the proton translocation. Further, the damage appears to be triggered by the high light, while its completion can readily occur in darkness at room temperature but not in cold conditions. Such a temperature dependency is not seen for

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i l l u m i n a t i o n t i m e (rain) Fig. 6. Thylakoids were subjected to high light of 4500/~E m - 2 s - 1 at low temperature (3°C) and analysed for; steady-state total protonuptake during PMS-mediated cyclic electron transport, before (O) and after ( o ) dark incubation at room temperature (25°(2) for 40 min of illuminated samples. 100% activity value of dark control was: proton uptake = 206 nmol H + (rag chlorophyll)-1.

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Fig. 7. (a) Thylakoids were subjected to high light of 3500 / ~ E m - e s -1 under anaerobic conditions and analysed for; steady-state total proton-uptake during PMS-mediated cyclic electron transport ( [] ), Photosystem I electron transport ((3) and relative amount of D1 protein ( • ). 100% activity values of dark controls were: proton uptake = 55 nmol H + (mg chlorophyll)- 1; Photosystem I = 287/z mol 0 2 (rag chlorophyll)- 1 h - 1 (b) Results from (a), but with an enlargement of the scale of the Y-axis to emphasize the difference between D1 protein degradation and loss of proton uptake.

the inactivation of the Photosystem II electron transport [38]. Low temperature is known to have a general stabilising effect on biomembranes such as thylakoids, but it is also known to slow down the degradation of the D1 protein [38,39]. In order to investigate whether the light-induced collapse of the proton gradient was associated with damages to the membrane integrity caused by events associated with D1 protein degradation, changes in the proton permeability of the membrane was measured relative to loss of the D1 protein. As shown in Fig. 1, loss of proton uptake is a very early event, in contrast to D1 protein degradation which occurs quite slowly. Thus, under conditions when 75% of the steady-state level of proton uptake has been lost, as much as 90% of the D1 protein remains. Photoinhibitory experiments were also performed at anaerobic conditions, where no degradation of the D1 protein occurs [3,28,40,41] despite pronounced photoinactivation of Photosystem II electron transport. As shown in Fig. 7, the proton gradient collapsed very quickly during photoinhibitory illumination of thylakoids under anaerobic conditions at 3500 /~E m -2 s -1. Thus, the changes in proton permeability occur in the absence of oxygen and without irreversible oxidative damage to the Photosystem II reaction centre. Notably, even under anaerobic conditions, there is a marked stimulation of the Photosystem I electron transport induced by high light (Fig. 7a). The increased rate reached a plateau after 60 min of illumination and no decrease of the activity was observed even after prolonged illumination. This is consistent with the previously shown oxygen-dependency for photoinactivation of Photosystem I [42]. Moreover, there was good correlation between collapse of the proton gradient and stimulation of Photosystem I activity. Once the total collapse of the proton gradient had occurred,

Photosystem I had reached and remained at a steady and high rate of electron transport. It has earlier been shown that photoinhibitory inactivation of Photosystem II electron transport under anaerobic conditions can recover in the dark without protein turnover [28]. The recovery of the proton uptake after the anaerobic inactivation was therefore investigated. However, in contrast to the Photosystem II electron transport, there was no recovery of the lost proton uptake (not shown). Since no correlation was found between the lost proton uptake capacity and inhibitory events associated with Photosystem II, some properties of the ATP-synthase were investigated after subjecting the thylakoids to strong illumination. As expected, the light-induced loss of ApH was accompanied by rapid deactivation of

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i l l u m i n a t i o n t i m e (min) Fig. 8. Thylakoids were subjected to strong light of 5500/.~E m -2 s-1 and analysed for; PMS-mediated cyclic photophosphorylation (o), MgZ+-dependent ATPase activity (13) and Photosystem II electron transport (e). 100% rates of dark controls were: ATP synthesis = 158 /zmol ATP (mg chlorophyll) -1 h - l ; ATP hydrolysis = 52 ~mol Pi released (rag chlorophyll)-1 h - i ; Photosystem II = 47 izmol 0 2 (mg chlorophyll)- i h - 1.

320 photophosphorylation (Fig. 8) in accordance with recent work [9]. The Mg2+-dependent ATPase activity was however not damaged to any significant extent (Fig. 8). It is well established that protons can leak through the CF0-channel of the ATP-synthase if the CF 1 extrinsic part is lost from the complex or disturbed otherwise [43]. However, no loss of CF 1 subunits could be detected by immunoblotting of photoinhibited thylakoid membranes (not shown). Nor could we see reversal of the photoinhibitory uncoupling by incubating photodamaged thylakoids with DCCD, known to block leakage of protons through the CF0-channel [44]. Our photoinhibitory illumination experiments were carried out under non-phosphorylating conditions. It could therefore be argued that a continuous build-up of ApH could not be released by ATP synthesis, which in itself could be damaging to the thylakoid membrane [7]. However, adding ADP and inorganic phosphate to the medium during the photoinhibitory illumination gave no protection against the loss of the proton uptake (not shown). Altogether, these observations suggest that the observed loss of ApH is not associated with the ATP-synthase complex. Discussion

The present data demonstrate a very rapid loss of the PMS mediated trans-thylakoid proton gradient and of cyclic photophosphorylation concomitant with a stimulation of Photosystem I electron transport during photoinhibitory illumination of intact and broken chloroplasts. In fact, the loss of Photosystem-I-mediated proton uptake was often faster than the inactivation of Photosystem II electron transport normally considered to be the primary target for photoinhibition. The mechanism behind this rapid impairment of the trans-thylakoid proton gradient is not yet understood. Even though there is relatively close kinetic correlation between the inactivation of Photosystem II activity and loss of the proton gradient, it is not easy to find any obvious mechanistic cause and effect relationship between the two impairment events. In particular, after strong illumination under anaerobic conditions the lost Photosystem II electron transport can be restored in the dark [28,45] which is not the case for the impaired proton translocation reaction. Moreover, the two inactivation events respond differently to low temperatures. The experiments also suggest that reduction and loss of the proton gradient is not induced by any phase of the process that leads to degradation of photodamaged D1 protein. In contrast to D1 protein degradation, the inactivation of proton uptake is a very early consequence of the illumination and is not dependent

on the presence of oxygen, since it readily proceeds under anaerobic conditions (Fig. 7). The latter observation also discriminates between the inhibitory effect on the proton gradient and photoinactivation of Photosystem I which does not occur under anaerobic conditions [42] (Fig. 7). However, most importantly, the ability to photo-induce a complete collapse of the proton gradient under anaerobic conditions suggests that the damage is not mediated by molecular oxygen. Studies on the subunit composition of CF 1 and the effects of DCCD on the CF0 proton channel did not show that damage to the ATP-synthase was responsible for the increased proton permeability of the thylakoid membranes. Still, interpretations must be made with caution, since only a few 'holes' due to some very minor release of CF 1 from the intrinsic part of the ATP-synthase could facilitate significant leakage of translocated protons back over the membrane. For comparison, Lill et al. [46] have shown that leakage through one single open CF0-channel is enough to dissipate the proton motive force of one thylakoid vesicle containing about 100 CF 0 - CF 1 complexes. The same argumentation could also be applied if a few damaged or degrading copies of the D1 protein could lead to extensive proton leakage. The temperature dependency of the high-light-induced collapse of the proton gradient is quite complicated. It involves a significant loss of activity at low temperature, but its completion occurs readily during subsequent incubation in the dark at room temperature (Fig. 6). It can therefore be concluded that the damage is induced by high-light, but that an actual increase of proton permeability of the thylakoid membrane can subsequently occur in the dark. Earlier studies have noted a heat-induced stimulation of Photosystem I electron transport [47,48] that resembles the high-light-induced stimulation shown here (Figs. 1, 3, 7a). However, this effect was shown not to be the result of any thermal uncoupling of Photosystem I [47,48]. This notion is also consistent with the work of Emmett and Walker [49] who did not observe any decrease in d p H during increased temperature (50°C), although photophosphorylation was lost. These observations were interpreted as loss of d~b due to changed permeability of the compensating ions, thereby making the created proton gradient insufficient to drive photophosphorylation. We therefore conclude that heat-induced effects do not contribute to the loss of the proton gradient seen under the present photoinhibitory conditions. Taken together, all our available data point to a collapse of the proton gradient due to general destabilisation of the membrane induced by a light-damage targeted to a site which does not necessarily have to involve Photosystem II and which remains to be identified. Possibly, the high-light may induce a modification

321 that leads to an alteration of the optimal packing of the membrane components that can progress laterally through the lipid-bilayer once initiated. Such changes in the optimal packing could involve specific proteinprotein, protein-lipid as well as lipid-lipd interactions. Still, the alteration of the thylakoid membrane must be quite subtle, since electronmicrographs of photoinhibited thylakoids did not reveal any major structural change or rupture (not shown). Obviously, the understanding of the molecular mechanism behind the photoinhibitory uncoupling requires further experimental studies involving a determination of the action spectrum for the induced photo-damage. It should be emphasised that, in the present study, care was taken to minimise contribution from UV light, which recently has been shown to induce thylakoid membrane permeability [50] The high-light-induced collapse of the protein gradient and the inactivation of cyclic photophosphorylation was induced mostly in isolated thylakoid membranes using quite high light intensities and any physiological relevance of the present observations therefore remains to be determined. Still, light-induced loss of proton uptake can be obtained at intensities around 200 /zE m -2 s -1 (Figs. 3, 4) which are not far above the saturation level of photochemical activities and which normally occur in nature. Moreover, the proton gradient was not only lost in isolated thylakoids, but also under more native conditions as revealed by the photoinhibitory illumination of intact chloroplasts (Fig. 5). In preliminary experiments, we have observed a collapse of the trans-thyalkoid proton gradient after strong illumination of intact spinach leaves. Earlier studies of various photosynthetic organisms and experimental systems have shown contradictory results concerning the effects on ApH and photophosphorylation during photoinhibitory conditions [6-24). For example, Critchley reported [6], from studies of low-light adapted cucumber, that during photoinhibition of Photosystem II electron transport in vivo, there was also inactivation of Photosystem I mediated cyclic photophosphorylation but not of proton uptake. There are observations that low temperature during excess light makes thylakoids leaky to protons due to loss of CF 1 in cold-sensitive plants [10-11]. Our present findings of light-induced collapse of the proton gradient are, however, based upon studies on cold-tolerant spinach and are not easily related to damage to the ATP-synthase. Our results are in accordance with those of Wu et al. [8], who reported stimulation of wholechain electron transport from water to ferricyanide and gradual uncoupling when intact spinach chloroplasts were subjected to photoinhibitory conditions. In contrast to these reports, there are several proposals for a ApH rise during photoinhibition [12-22]. Such an increased trans-thylakoid proton gradient has

been reported to occur in vivo, in Chlamydomonas reinhardtii cells during photoinhibition, which is suppressed in mutants lacking cyclic Photosystem I electron transport ability [12]. In the work of Dujardyn and Foyer [13], it has also been proposed that photoinhibition in vivo initially leads to a phase of increased pH induced by stimulation of cyclic electron transport. In particular, the present results appear to be in apparent contradiction to the rise in trans-thylakoid proton gradient, associated with the so-called qE2 quenching, that is proposed to protect the photosynthetic apparatus by thermal dissipation of excess excitation energy [14-24]. Possibly, the collapse of the proton gradient seen in the present study may represent a later event during light-stress when the qE" quenching protection mechanism has been oversaturated and inactivation and damage to the photosynthetic apparatus occurs. This possibility is supported by fluorescence studies of intact spinach chloroplasts under our present photoinhibitory conditions revealing no significant changes in the qE-quenching (Van Wijk, K.J., personal communication). Moreover, Barenyi and Krause [16] reported that, during strong photoinhibitory illumination of intact chloroplasts from spinach, there was no increased ApH observed, but Photosystem II electron transport and photo-phosphorylation were suppressed to the same degree. Photoinhibition work on the same material in Ref. 19, demonstrated a clear decrease of the ApH, although uncoupling was not reported. In the present work, we observed an initial transient phase of ApH rise during the moderately high light treatment of thylakoids (Fig. 3) prior to the induction of the gradual uncoupling. This observation may be indicative of an early protective phase being overruled, resulting in rapid inactivation of photosynthesis involving not only Photosystem II electron transport but also Photosystem-I-mediated proton translocation.

Acknowledgements This project was supported by the Swedish Natural Science Research Council and the G6ran Gustafsson Foundation for Research in Natural Sciences and Medicine. We gratefully acknowledge Wolfgang Junge and Markus Weichselbaum for assistance in measurements of electrochromic shift. We also thank K.J. van Wijk for fluorescence measurements.

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