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phase of leaf discs (up to 9 h) the increases in F~/Fm preceded that of the number of functional PS II centres, ... tion, both in-vivo and in-vitro, and that recovery of.
Photosynthesis Research21: 17-26, 1989. © 1989. KluwerAcademicPublishers.Printedin the Netherlands. Regular paper

Photosystem II function and herbicide binding sites during photoinhibition of spinach chloroplasts in-vivo and in-vitro W.S. Chow, 1 C.B. Osmond 2'3 and Lin Ke Huang. 2

~CSIRO Division of Plant Industry, G.P.O. Box 1600, Canberra, A.C.T. 2601, Australia; 2plant Environmental Biology, Research School of Biological Sciences, Australian National University, Box 475, Canberra, A.C.T. 2601, Australia; ~Present address: Botany Department, Duke University, Durham, NC 27706, U.S.A. Received 10 May 1988.

Key words: chlorophyll fluorescence, herbicide binding, photoinhibition, photosynthesis, photosystem II, temperature Abstract

The time courses of some Photosystem II (PS II) parameters have been monitored during in-vivo and in-vitro photoinhibition of spinach chloroplasts, at room temperature and at 10 °C or 0 °C. Exposing leaf discs of low-light grown spinach at 25 °C to high light led to photoinhibition of chloroplasts in-vivo as manifested by a parallel decrease in the number of functional PS II centres, the variable chlorophyll fluorescence at 77K (F~/Fm), and the number of atrazine-binding sites. When the photoinhibitory treatment was given at 10 °C, the former two parameters declined in parallel but the loss of atrazine-binding sites occurred more slowly and to a lesser extent. During in-vitro photoinhibition of chloroplast thylakoids at 25 °C, the loss of functional PS II centres proceeded slightly more rapidly than the loss of atrazine-binding sites, and this difference in rate was further increased when the thylakoids were photoinhibited at 0 °C. During the recovery phase of leaf discs (up to 9 h) the increases in F~/Fmpreceded that of the number of functional PS II centres, while only a further decline in the number of atrazine-binding sites was observed. The recovery of variable chlorophyll fluorescence and the concentration of functional PS II centres occurred more rapidly at 25 °C than at 10 °C. These results suggest that the photoinhibition of PS II function is a relatively temperatureindependent early photochemical event, whereas the changes in the concentration of herbicide-binding sites appear to be a more complex biochemical process which can occur with a delayed time course. Abbreviations: BSA - bovine serum albumin, Chl--chlorophyll, D 1-32 kDa herbicide-binding polypeptide in photosystem II and product of the psbA gene, D2-34 kDa polypeptide in photosystem II which is the product of the psbD gene, DCMU-3'-(3,4-dichlorophenyl)- 1,1-dimethylurea, DCPIP-2,6-dichlorophenolindophenol, F0, Fv, Fro--chlorophyll fluorescence with reaction centres open, variable and maximum fluorescence, respectively, LDS - lithium dodecyl sulfate, MES-2-(N-morpholino) ethanesulfonic acid; PS IIphotosystem II, QA, QB-first and second quinone-type PS II acceptor, respectively

Introduction

Recent studies of photoinhibition, the light dependent damage to the photosynthetic apparatus, have emphasized the sensitivity of photosystem II (PS II)

reactions (Powles 1984, Kyle et al. 1987). Two major themes as to mechanisms underlying photoinhibitory damage have emerged, one based on the central role played by damage and repair of D 1, the 32 kDa herbicide-binding protein (Kyle et al.

18 1984, Kyle 1987) and the other based on damage and repair of the photochemical reaction centre itself (Cleland and Critchley 1985, Cleland et al. 1986). Concurrently, homologies between the structures of the L and M subunits of the reaction centre of Rhodopseudomonas viridis (Deisenhofer et al. 1984) and the D1 and D2 polypeptides of higher plant chloroplasts (Trebst and Depka 1985, Barber 1987) have been used to argue that the reaction centre components of PS II may be bound by these polypeptides. As pointed out by Arntz and Trebst (1986), the possibility of intimate association between the reaction centre and the D 1 protein makes it difficult to exclude either of the above explanations as to the mechanism of photoinhibition. So far as we are aware, there have been no studies of photoinhibition in higher plants in which comparisons have been made of direct measurements of reaction centre primary function, and of the concentration of herbicide bindings sites in-vivo and in-vitro. We reasoned that such comparisons would be useful because repair mechanisms which might function in chloroplasts in-vivo might not function in isolated thylakoids. In this respect it is noteworthy that most studies favoring reaction centre damage as the primary site of photoinhibition were done with isolated thylakoids, and those favoring D 1 turnover were done with intact chloroplasts or with whole cells of algae. If two major contributory factors in photoinhibition are photochemical processes (responsible for reaction centre damage) and biochemical processes (responsible for removal and resynthesis of the D1 protein), they should be separable on the basis of their temperature coefficients. That is, we would expect reaction centre damage to have a Q~0 near one, as indeed was indicated for overall photoinhibition by Kok et al. (1965), and the turnover of the D1 protein to have a much larger Q~0. Temperature during photoinhibition, in-vivo and invitro should thus be an important determinant of relative changes in these processes. In this respect it is interesting that many studies favoring the view that reaction centre function is the primary site of photoinhibition were done at low temperature (05°C), whereas those favoring the turnover of D1 were done at 20-30°C. We have used a low temperature tolerant species to compare the effects of temperature on photoinhibition of intact leaves (shade-grown spinach),

and of isolated thylakoids (sun-grown spinach). In each case we have monitored the concentration of functional PSII reaction centres by an O2-flash yield technique, as well as the concentration of herbicide binding sites and chlorphyll fluorescence parameters at room temperature or 77 K. In some experiments changes in the quantum yield of 02 evolution in- vivo were followed, and changes in the concentration of thylakoid proteins were determined by gel electrophoresis. Our experiments show that changes in PS II reaction centre function can precede loss of D1 protein during photoinhibition, both in-vivo and in-vitro, and that recovery of PS II function may precede changes in D 1 protein concentration. Chlorophyll fluorescence changes at 77K, both in F0 and Fv/Fm, correlate well with changes in functional PS II concentrations. A preliminary report of related experiments has been published elsewhere (Osmond and Chow 1988). Materials and methods

Spinach plants were grown in aerated nutrient cultures containing 6mM KNO3, 5 mM Ca (NO3)2, 2mM MgSO4, 2mM KH2PO3, 0.1 mM FeEDTA and trace elements (2/~M MnC12, 0.3 #M ZnSO4, 0.2/~M CuSO4, 10#M H3BO3, 1/IM H2MoO4, and 0.1pMCo (NO3)2) in a temperature controlled glasshouse or in a growth chamber (25°C, 12h, day/17°C, 12h, night) in full sunlight or at 100 to 120 #mol photons m -2 s-l of PAR. In-vivo photoinhibitory treatments were applied in various ways as specified in the figure legends. Individual plants were sometimes transferred from the shaded part of the glasshouse into full sunlight (1500-1800 #mol photons m -2 s -l) in the same glasshouse, or placed under water-cooled xenon-arc lamps in the laboratory adjusted to deliver 1000 to 1500/~mol photons m -2 s -~ through glass heat filters. Leaf discs were sometimes cut from the shade grown plants and floated on tap water in containers in a thermostatically controlled waterbath under the water-cooled xenon-arc lamps. Light response curves of photosynthetic 02 evolution at 25°C and at C02 saturation (5% CO2) were determined in a leaf disc 02 electrode system, as described previously (Walker and Osmond 1986). Apparent quantum yield was calculated from the initial slope of the light response curve. Measurements of quantum yield in photoinhibited

19 leaves were commenced within 30 min of the end of the treatment, following a routine exposure to saturating light and subsequent measurement of dark respiration in the oxygen electrode chamber. Changes in the 77K fluorescence properties of leaves were measured using a device similar to that described by Powles and Bjrrkman (1982) as modified by Adams et al. (1987). Tissues were dark adjusted for at least 10rain before being frozen in liquid N2. The concentration of active PS II centres was measured in-vivo using a leaf disc O~ electrode as described by Chow et al. (1989). Single turnover flashes (approximately 3/~s duration at half peak height) were supplied by a xenon flash lamp (Stroboslave type 1539-A) via a condensing lens at a frequency of 4 Hz. Background far-red light (approximately 17 gmol quanta m-2 s- i, 700--730 nm) was present during flash illumination (and in the dark) in order to keep PSI turning over and the plastoquinone pool in a oxidized state. The rate of oxygen evolution (minus dark drift) was compared with the flash frequency to give the oxygen yield per flash. The number of O~molecules evolved per flash was multiplied by 4 to give the number of functional PS II centres. Changes in the composition of thylakoid membranes during photoinhibition in-vivo were examined by LDS polyacrylamide gel electrophoresis. Leaves were chopped into a grinding buffer (50g leaf per 200ml buffer) containing 0.33M sorbitol, 20mMMESpH6.5, 5 m M N a isoascorbate and 0.2mM MgCI2 and chloroplasts were extracted by 3 successive short bursts with a Polytron blender. The homogenate was filtered through cheesecloth and miracloth and centrifuged for I min at 2000 x g. The pellet was resuspended in 0.33M sorbitol, 20mMMES (pH6,5) and 5 mM MgC12 and sedimented again by centrifugation for 2 min at 2000 x g. The intact chlorplasts were burst by resuspension and 15 min incubation in 2 m M M E S (pH6.3), 5mMMgCI~ and 15mM NaCI. The thylakoids were recovered by 5 min centrifugation at 8000 x g and stored in a small volume of buffer containing 0.4 M sucrose, 20 mM MES (pH 6.5), 5 mM MgCI:, 15 mM NaC1 and 20% glycerol. Samples of thylakoids containing about 200/~g protein were resuspended in 0.3M Tris HC1 (pH8.8) with 13% glycerol, dithiothreitol (20/tg)

and LDS (20/~g). This solution was heated in boiling water for 90s to dissociate the membrane protein complexes. The proteins were separated on LDS polyacrylamide-gradient gels (10-22.5% acrylamide) in a BioRad "Protean" slab cell with standards (cytochrome c I I.7kDa; lysozyme 14.3kDa; myoglobin 17.2kDa; trypsin inhibitor 20. l kDa; carbonic anhydrase 29.0 kDa; ovalbumin 43 kDa; bovine serum albumin 67 kDa; phosphorylase a 94 kDa and fl galactosidase, 130 kDa). Gels were stained with 0.1% Coomassie blue, destained in ethanol/acetic acid/water and photographed with a Panacopy automatic slide maker to obtain maximum contrast. Western blots of unstained LDS gels were prepared using Whatman nitrocellulose sheets, and bands were located with amido black. The blot was then incubated for 90min with 3% (w/v) BSA in 150mMNaCl with 10mM Tds HCI (pH7.4) before overnight incubation in the same solution with a mixture of antisera to the 34 kDa, 23 kDa and 16kDa polypeptides of the water splitting complex of PS II (obtained from Professor B. Andersson). The blot was rinsed 5 x with buffer and then incubated for 2h with II2S-labeled protein A (l#Ci in 20ml). After exhaustive washing the blot was dried and put to X-ray film to locate labeled bands. The dried blot was then scanned with a gas-flow detector and the areas under the peaks traced by the detector were cut and determined. Strips of the blot were then cut out and counted in scintillant, using equal sized portions of the nitrocellulose sheet as background standards. Changes in the level of the atrazine binding protein following photoinhibition were qualitatively indicated by the photoaffinity technique of Pfister et aL (1981). Thylakoids from the same samples used for the LDS gels and Western blots were diluted to 80/~gChl in 1 ml buffer and incubated in 5#M azido (ethyl-l-~4C) atrazine at room temperature for 10min under long wavelength U¥ light from a CAMAG UV scanning lamp. The thylakoids were pelleted by centrifugation at 9000 x g and washed 3 x by resuspension in buffer. Aliquots of the resuspended thylakoids were counted in liquid scintillant. In one experiment this technique was compared with the estimate of [~4C]-atrazine binding by the kinetic method of Tischer and Strotmann (1977). The ratio of label incorporated by photoinhihited thylakoids

20 to control thylakoids was 0.68 for photoaffinity binding and that determined kinetically was 0.79. In most other experiments the concentration of herbicide binding protein was measured by extracting thylakoids from leaf discs at different times after commencement of photoinhibition. Thylakoids were prepared by grinding 750rag tissue in 5 ml buffer containing 400 mM sorbitol, 5 m M M E S (pH6.5), 5mMMgCl2, 10mMNaCl and 0.2% BSA in a glass homogeniser. After cenrifugation. At 5000 x g for 10min, the thylakoids were resuspended in a small volume of the extraction medium and frozen at 77 K until use. The number of herbicide-binding sites was determined according to the method of Tischer and Strotmann (1977) using [14C]-atrazine or [14C]-DCMU (Amersham, U.K.) at concentrations from 60 to 400nM, other details being given by Chow and Hope (1987).

Results and discussion

Early experiments indicated that leaves of shadegrown spinach experience substantial photoinhibition if exposed to bright light at room temperature, as judged by the decreased quantum yield of 02 evolution and loss of the variable components of room temperature chlorophyll fluorescence (Walker and Osmond 1986). Thylakoids prepared from the photoinhibited spinach leaves appeared to lack a polypeptide with a molecular weight of 32 kDa (Fig. 1). All other membrane polypeptides appeared to be present in approximately the same proportions in thylakoids of both control and photoinhibited leaves. Quantitative assessment of the 16, 23 and 34 kDa polypeptides of the water splitting components of PS II, for example, showed no differences between controls and photoinhibited leaves. When thylakoids from 3 sets of experiments

Fig. 1. The polypeptide pattern of thylakoid membranes isolated from control or photoinhibited spinach leaves resolved by LDS polyacrylamide gel electrophoresis and stained with Coomassie blue. Spinach plants grown under shade in a glasshouse were exposed to full sunlight at ambint temperature (approx. 25°C) for 4h. The loss of a polypeptide of apparent molecular weight 32 kDa in photoinhibited samples is indicated by an arrow in each experiment.

21 were separated on the same gel, blotted and challenged with labeled antibodies the mean distribution of radioactivity a m o n g these three polypeptides was 26% (16kDa), 36% (23kDa) and 38% (34kDa) in control and 26%, 34% and 40% respectively in the photoinhibited preparations. These experiments illustrate, incidentally, that the 33 k D a band lost during photoinhibition (Fig. 1) was not that of the water-splitting complex. The missing band was almost certainly the D 1 herbicide binding protein, because in 3 experiments there was a 21 to 69% decrease in the a m o u n t of labeled azido atrazine which could be photo-affinity bound to these thylakoids. The D1 protein does not stain well on gels with Coomassie blue, and although the binding of labeled azido-atrazine to controls was similar in all 3 experiments (91-107 x 103 d p m m g -l Chl), the variable results obtained by this method with thylakoids from photoinhibited leaves suggested more direct approaches were necessary. Preliminary experiments, again with leaves of shade-grown spinach exposed to bright light at room temperature, confirmed that the atrazine binding protein was lost during photoinhibition. Table 1 shows that, under these conditions, the loss of the atrazine binding sites was approximately proportional to the decrease in functional PS II centres as measured by 02-yield per single turnover flash in-vivo or in-vitro, and to the decrease in quantum yield o f 02 evolution. There was no appreciable decrease in the affinity for atrazine binding after photoinhibition; in four separate experiments performed at r o o m temperature, the mean K,~ + s.e.m, was 35 ___ 1 nM for controls and 40 + 3 nM for leaves exposed to highlight

(about 1500 #mol m -2 s-~ ) for 4 h. It is impossible to determine cause and effect on the basis of such data, as emphasized in our preliminary report (Osmond and Chow 1988). These experiments were repeated using leaf discs of shade-grown spinach which were floated on tap water maintained at 10°C under a xenon-arc lamp. As shown in Fig. 2, 4 h treatment under these photoinhibitory conditions led to a 75% decrease in quantum yield and 41% decrease in the m a x i m u m photosynthetic rate. The treatment led to an increase in F0 and a decrease in Fv/Fm for 77k fluorescence (Fig. 2). A series of time course experiments was done in- vivo at 10°C (with 1300 #mol photons m - 2 s- l ) in which 77 K flurescence parameters, atrazine binding sites and functional P S I I centres were measured (Fig. 3). The results show that 7 7 K fluorescence parameters change in much the same way as described in other comparable systems (t~gren and t~quist 1984, Greer et al. 1986). An initial increase in F0 during the first 1 h is sustained throughout the period of the experiment, whereas Fv/Fm declines with an approximately first order rate. The concentration of atrazine binding sites increases slightly in the first 1-2 h and then declines to reach a steady level of approximately 70-75% of the starting value. The concentration of functional P S I I centres, like the ratio Fv/F,~ of chlorophyll fluorescence at 77 K declines throughout the experiment with apparently first order kinetics. Recovery from these effects of photoinhibition in-vivo was studied by returning leaf discs to water baths at either 10°C or 25°C in weak white light (5#mol photons m-2s-~). As has been reported previously (Greer et al. 1986, t~quist et al. 1987)

Table 1. Changes in photosynthetic parameters of shade-grown (120gmol photons m-2s-') spinach leaves following photoinhibition in bright light (1500-1800#mol photons m-2s -~) at 25°C for 4h. Atrazine binding sites were measured in one batch of isolated thylakoids, whereas the other measurements were made with 4-5 leaf discs. Numbers in parentheses indicate % of control.

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2.41 _+ 0.27

1.32 _ 0.24 (55)

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recovery of fluorescence parameters was temperature dependent. Thus F,/Fm measured at both 7 7 K and r o o m temperature recovered more rapidly at 25°C than at 10°C (Fig. 4). Recovery of fluorescence was more rapid when measured at r o o m temperature than that at 77 K, presumably reflecting the much more complex interactions which underlie fluorescence quenching at r o o m temperature (see later). During recovery of fluorescence properties, there was no evidence of restoration of the herbicide binding protein concentration at either 25°C or 10°C. Measurements based on ]4C-DCMU binding gave slightly higher concentrations [control, 2.82 _ 0.09 (n = 6) m m o l (molChl) -~] than those based on ]4C-atrazine [control, 2.29 _ 0.08(n = 6) m m o l (molChl) -~] but the relative trend in the experiment was the same (Fig. 4). The concentration of functional PS II centres, which closely followed F,/Fm during photoinhibition, did not follow fluorescence parameters during recovery. Instead, the concentration seemed to continue to decline for 2-3 h after removal from bright light and possibly showed a belated partial recovery, which was even further delayed at 10°C (Fig. 4). The above observations contrast with those of Cleland et al. (1988) who reported a rapid loss of photosynthetic 02 evolution in the quantum yield region (tl/2 approx. 30 min) during photoinhibifion of mangrove leaves, without significant change in

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remain constant during recovery primarily because of the changes in functional PS II centres. However, the ratio returns to about one in the latter stages of recovery, even though the concentration remains at only about 80% of the control. This could mean that our measurements of total D1 protein hide some complex relationship between functional and non-functional D1 protein of the sort implied by Wettern (1986). These in-vivo experiments show many complex interactions between onset and recovery of photosynthetic processes during photoinhibition which are discussed below. The basic issue, the

relationship between PS II reaction centre function and the concentration of herbicide binding sites may be more directly tested in-vitro using isolated thylakoids in which recovery due to ongoing protein synthesis is unlikely. The time courses for changes in these parameters during photoinhibitio~_ of thylakoids from sun-grown spinach leaves are shown in Fig. 5. At low temperature (0°C) the loss of functional PS II centres commences immediately on exposure to bright light (5000/~mol photons m-2s - ' ) and proceeds in an approximately first order manner. The variable chlorophyll fluorescence (Fv/F,,) declined with the same time-course

24 as for functional PS II centres (data not shown). However, the loss of herbicide binding sites is more or less linear, and proceeds rather more slowly. Similar results were obtained when treatments and measurements were both done at 0°C (data shown Fig. 5a) and when measurements were done at 25°C (data not shown). The changes are thus due to the conditions of treatment, not contingent upon conditions of assay. At 25°C the rates of both processes were slower, but the dark controls also declined slowly throughout the experiment. When corrected for the changes in controls, loss of functional PS II centres proceeded more rapidly than loss o f herbicide binding (Fig. 5b), at least initially. These data on photoinhibition in-vitro at 0°C

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ctional PS II reaction centres (o, e) during photoinhibition of spinach thylakoids in-vitro at (a) 0°C and (b) 25°C. Thylakoids were isolated from spinach grown in full glasshouse light and suspended in a medium containing 330mM sorbitol, 10mM Hepes/NaOH (pHT.8), 10mMNaC1 and 5mMMgCI2 at a chlorophyll concentration of 0.1 mM. The suspension was illuminated with white projector light (5000#mol photons m-2s-t). At a given time aliquots were withdrawn for the determination of atrazine-binding sites or oxygen-yieldper single turnover flash (ferricyanideas electron aeceptor). The samples were assayed at the same temperature as for the treatment. Closed symbols, samples kept in the dark.

resemble those of Cleland and Critchley (1985) in which spinach thylakoids illuminated in bright light for 10min showed more than 85% loss of silicomolybdate-dependent 02 evolution apparently without loss of 35S-methionine labeled D1 protein. They do not agree with the experiments of Ohad et al. (1985) in which photoinhibition of pea thylakoids in-vitro produced 90% decrease in PS II dependent electron transport (H20 ~ DCPIP) which proceeded only slightly more rapidly than the loss of 35S-methionine labeled D1 protein. N o r do they agree with observations of Bradbury and Baker (1986) with intact pea chloroplasts, in which the concentration of atrazine binding sites declined more rapidly than PS II dependent electron transport. The more rapid rate of photoinhibition of PS II centres at 0°C than at 25°C is at first sight surprising, but serves to illustrate the complexity of the process, even in isolated thylakoids. It may imply that some recovery processes functional at 25°C may persist in thylakoids. On the other hand, it is possible that the imbalance between primary photochemical processes, and subsequent biochemical processes is greater at 0°C, leading to more rapid loss of PS II reaction centre function. Interestingly, the loss of herbicide binding sites was faster at 25°C, after the initial lag, which is possibly consistent with the involvement of membrane bound enzymic processes in the removal of the polypeptide. Interpretation of these experiments is difficult because the precise relationships between the concentrations of the D1 protein and of functional P S I I centres are not known. However, in our material the stoichiometry of these components prior to photoinhibition is similar, at about 2 per 103 Chl. This may imply a one to one relationship between functional reaction centres and D I proteins in the thylakoid. The fact that the rates of decline in the concentration of these two components can be different in the early stages of photoinhibition implies a measure of independence. Because the rate of decline in functional PS II centres is initially greater than that of herbicide binding during photoinhibition in-vivo at 10°C and in-vitro at 0°C, our data suggest that P S I I reaction centre function is the primary site of photoinhibition under these conditions. Whether this is so because damage to

25 reaction centre function is accelerated by slower removal of non-functional herbicide binding protein at low temperature cannot be determined from our data. However, we note that during the early stages of photoinhibition in-vivo when D1 protein concentrations remain high, there is no significant change in the affinity of this protein for atrazine or DCMU. In five separate experiments, the mean Km for atrazine was 44 + 7nM for controls, and 4 7 _ 6nM for samples photoinhibited in-vivo for 4h at 10°C. That is, the herbicide-binding protein appears to be unchanged in its properties. In contrast, Cleland et al. (1988) found that the affinity for DCMU declined (7 to 30 nM) without change in concentration of binding sites during photoinhibition of isolated thylakoids at 25°C, while electron transport declined 60% at the same time. It is interesting to consider the changes in fluorescence properties which accompany this rapid loss in functional PS II centres. An increase in F0 and a decline in Fv/Fm have been observed by Ogren and Oquist (1984) during analogous low temperature photoinhibition experiments with intact Lemna gibba. Bjrrkman (1987) used a model to calculate that approximately 80% of the decline in Fv/Fm in t3gren and Oquist's data was due to a decrease in the rate constant for deexcitation through functional PS II. According to this model, a rise in F0 is directly proportional to the decrease in quantum efficiency of primary photochemistry of PS II. Thus in our experiments the initial rise in F0 and the subsequent decline in F~/Fm are both consistent with impaired PS II function as measured by a decrease in functional PS II centres. Demmig and Bjrrkman (1987) found that at room temperature, photoinhibition of a shade-grown leaf of Monstera deliciosa was accompanied by a slow increase in F0 and a rapid initial decline in Fv and F~/Fm. In this case, and in other shade plants, Bjrrkman's model suggests that reaction centre damage is a minor component of the fluorescence change (Bjrrkman 1987). Thus it seems that photoinhibition in our experiments may well stem from slower repair to damaged PS II centres at low temperature, but this evidently is independent of changes in the total pool of D1 protein. This is especially so during recovery. An inspection of Fv/Fm measured at 77 K or at room temperature during the onset of, and

recovery from, photoinhibition revealed a dependence on temperature during measurement. Whilst F~/F,~ (approx. 0.81) was similar for controls measured at both temperatures, the value measured at room temperature declined to a greater extent during the course of photoinhibition. During recovery, on the other hand, it increased more rapidly (Fig. 4). Since F~/Fm is equivalent to the efficiency of photochemistry when PS II centres are open (Bjrrkman 1987), we propose the following explanation. Upon photoinhibition, it is possible that the co-operativity among PS II centres is diminished; excitation energy arriving at a photoinhibited PS II may not be redirected to a neighbouring functional PS II, thus resulting in a loss of efficiency of photochemistry (F~/Fm). On the other hand, freezing at 77 K may help to enhance the co-operativity among adjacent PS II centres, thus conferring some improvement of Fv/F,~in photoinhibited samples. During recovery, the relatively rapid increase of Fv/Fmmeasured at room temperature may partly reflect the recovery in cooperativity. In summary, the independent changes in the concentration of functional PS II reaction centres and of atrazine-binding sites may be explained by noting that a D 1 protein with an atrazine-binding moiety may not necessarily carry a founctional reaction centre. On the other hand, since the measurement of the funcitonal PSII reaction centre involves electron flow through QA, QB and the plastoquinone pool, an atrazine/QB binding site is a necessary feature of PS II function. Indeed, during the onset and recovery phases, the concentration of functional PS II reaction centres (expressed as % of control) never exceeded that of atrazine-binding sites. We have no evidence of which or how PS II reaction centre components are impaired during low temperature photoinhibition or restored during recovery. At present, controversy exists in the literature (Demeter et al. 1987, Allakhverdiev et al. 1987) as to whether the photoreduction of pheophytin is impaired following photoinhibition in-vitro. Clearly, further experiments are needed to ascertain the exact site of primary damage.

Acknowledgements We are grateful to Sue Young for her skilled assistance in electrophoresis, and to Prudence Kell and

26

Stephanie Hossack-Smith for help with quantum yield, 02 flash yield and atrazine-binding measurements. Ichiro Terashima and Robyn Cleland gave advice during the experiments and during the preparation of the manuscript. References Adams WW III, Smith SD and Osmond CB (1987) Photoinhibition of the CAM succulent Opuntia basilaris growing in Death Valley: evidence from 77 K fluorescence and quantum yield. Oecologia 71:221-228 Allakhverdiev SI, Setlikovfi E, Klimov VV and Setlik I (1987) In photoinhibited photosystem II particles pheophytin photoreduction remains unimpaired. FEBS Lett 226:186-190 Arntz B and Trebst A (1986) On the role of the QB protein of PS II in photoinhibition. FEBS Lett 194:43-49 Barber J (1987) Photosynthetic reaction centres: a common link. TIBS 12:321-326 Bj6rkman O (1987) Low temperature fluorescence in leaves and its relationship to photon yield of photosynthesis in photoinhibition. In: Kyle, Osmond and Arntzen (eds) Photoinhibition, pp. 123-144. Amsterdam: Elsevier Bradbury M and Baker NR (1986) The kinetics of photoinhibition of the photosynthetic apparatus in pea chloroplasts. Plant Cell Environment 9:289-297 Chow WS and Hope AB (1987) The stoichiometries of supramolecular complexes in thylakoid membranes from spinach chloroplasts. Aust J Plant Physiol 14:21-28 Chow WS, Hope AB and Anderson JM (1989) Oxygen per flash from leaf disks quantifies Photosystem II. Biochim. Biophys. Acta, 973: 105-108. Cleland RE and Critchley C (1985) Studies on the mechanism of photoinhibition in higher plants. II. Inactivation by highlight of photosystem II reaction centre function in isolated spinach thylakoids and 02 evolving particles, photobiochem Photobiophys 10:83-89 Cleland RE, Kalling M and Critchley C (1988) The cause and consequences of photoinhibition. Biochem Biophys Acta, submitted Cleland RE, Melis A and Neale PJ (1986) Mechanism of photoinhibition: photochemical reaction centre inactivation of chloroplasts. Photosyn Res 9:79-88 Deisenhofer J, Epp O, Miki K, Huber R and Michel H (1984) X-ray structure analysis of a membrane protein complex: electron density map at 3 A resolution and a model of the chromophores of the photosynthetic reaction centre from Rhodopseudomonas viridis. J Mol Biol 180:385-398 Demeter S, Neale PJ and Melis A (1987) Photoinhibition: impairment of the primary charge separation between P-680 and pheophytin in photosystem II of chloroplasts. FEBS Lett 214: 370-374 Demmig B and Bj6rkman O (1987) Comparison of the effect of excessive light on chlorophyll fluorescence (77 K) and photon yield of O2 evolution in leaves of higher plants. Planta 171: 171-184

Greer DH, Berry JA and Bj6rkman O (1986) Photoinhibition of photosynthesis in intact bean leaves: role of light and temperature, and requirement for chloroplast protein synthesis during recovery. Planta 168:253-260 Kok B, Gassner EB and Rurainski HJ (1965) Photoinhibition of chloroplast reactions. Photochem Photoibiol 4:215-227 Kyle DJ, Ohad I and Arntzen CJ (1984) Membrane protein damage and repair: selective loss of a quinone-protein fraction in chloroplast membranes. Proc Natl Acad Sci USA 81: 4070-4074 Kyle DJ (1987) The biochemical basis for photoinhibition of photosystem II. In: Kyle D J, Osmond CB and Arntzen CJ (eds) Photoinhibition, pp 197-226. Amsterdam: Elsevier Kyle D J, Omond CB and Arntzen CJ (1987) (eds) Photoinhibition, Topics in Photosynthesis Vol. 9, pp315. Amsterdam: Elsevier Ogren E and Oquist G (1984) Photoinhibition of photosynthesis in Lemna gibba as induced by the interaction between light and temperature. III Chlorophyll fluorescence at 77 K. Physiol Plant 62:193-200 Ohad I, Kyle DJ and Arntzen CJ (1984) Membrane protein damage and repair: removal and replacement of inactivated 32-kilodalton polypeptides in chloroplast membranes. J Cell Biol 99:481-485 Ohad I, Kyle DJ and Hirschberg J (1985) Light-dependent degradation of the QB protein in isolated pea thylakoids. EMBO J. 4:1655-1659 Oquist G, Greer DH and Ogren E (1987) Light stress at low temperature. In: Kyle DJ, Osmond CB and Arntzen CJ (eds) Photoinhibition, pp. 67-87, Amsterdam: Elsevier Osmond CB and Chow WS (1988) Ecology of photosynthesis in the sun and shade: summary and prognostications Aust J Plant Physiol, 15:1-9 Pfister K, Steinback KE, Gardner G and Arntzen CJ (1981) Photoaifinity labeling of an herbicide receptor protein in chloroplast membranes. Proc Natl Acad Sci USA 78:981-985 Powles SB (1984) Photoinhibition of photosynthesis induced by visible light. Annu Rev Plant Physiol 35:15-44 Powles SB and Bj6rkman O (1982) Photoinhibition of photosynthesis: effect on chlorophyll fluorescence at 77 K in intact leaves and chloroplast membranes of Nerium oleander. Planta 156:97-107 Tischer W and Strotmann H (1977) Relationship between inhibitor binding by chloroplasts and inhibition of photosynthetic electron transport. Biochim Biophys Acta 460:113-125 Trebst A and Depka B (1985) The architecture of photosystem II in plant photosynthesis. Which peptide subunits carry the reaction centre of PS II? In: Michel-Beyerle ME (ed) Current Physics Series: Antennas and Reaction Centres of Photosynthetic Bacteria-Structure, interactions and Dynamics, pp215-223. Heidelberg: Springer Verlag Walker DA and Osmond CB (1986) Measurements of photosynthesis in vivo with a leaf disc electrode: correlations between light dependence of steady state photosyntheti~ 02 evolution and chlorophyll a fluorescence transients. Pr6c R Soc London B227:267-280 Wettern M (1986) Localization of 32000 dalton chloroplast protein pools in thylakoids: significance in atrazine binding. Plant Sci 43:173-177