Photosynthesis Research 15:221 232 (1988) © Kluwer Academic Publishers, Dordrecht Printed in the Netherlands

Regular p a p e r

Changes in the flash-induced oxygen yield pattern by thylakoid membrane phosphorylation N . K . P A C K H A M , l M. H O D G E S , A . L . E T I E N N E & J.M. BRIANTAIS

Laboratorie de photosynthese, CNRS, 91190 Gif-sur-Yvette, France;1 author for correspondence: Department of Biochemistry', The University, Newcastle- Upon- Tyne, NE1 7RU, UK Received 7 September 1987; accepted in revised form 16 October 1987

Key words: Photosystem 2, oxygen evolution, chloroplast, thylakoid membrane phosphorylation Abstract. Phosphorylation of thylakoid membrane proteins results in a partial inhibition (approximately 15-20%) of the light-saturated rate of oxygen evolution. The site of inhibition is thought to be located on the acceptor side of photosystem 2 (PS2) between the primary, QA, and secondary, QB, plastoquinone acceptors (Hodges et al. 1985, 1987). In this paper we report that thylakoid membrane phosphorylation increases the damping of the quaternary oscillation in the flash oxygen yield and increases the extent of the fast component in the deactivation of the $2 oxidation state. These results support the proposal that thylakoid membrane protein phosphorylation decreases the equilibrium constant for the exchange of an electron between QA and QB- An analysis of the oxygen release patterns using the recurrence matrix model of Lavorel (1976) indicates that thylakoid membrane phosphorylation increases the probability that PS2 miss a S-state transition by 20%. This is equivalent, however, to an insignificant inhibition (approximately 2.4%) of the light-saturated oxygen evolution rate. If a double miss in the S-state transitions is included when the PS2 centres are in $2 the fit between the experimental and theoretical oxygen yield sequences is better, and sufficient to account for the 15-20% inhibition in the steady-state oxygen yield. A double miss in the S-state transition is a consequence of an increased population of PS2 centres retaining Qf, : not only will these PS2 centres fail to catalyse photochemical charge transfer until QT, is reoxidized, but the re-oxidation reaction will also result in the deactivation of $2 to S1. Abbreviations: Chl Chlorophyll, PS2 - Photosystem 2, Si The oxidation states of PS2 (where i can be from 0 to 4), Q~ and Qff : the anionic semiquinone forms of the primary and secondary plastoquione acceptors of PS2

Introduction I n c u b a t i o n of osmotically shocked chloroplasts with A T P u n d e r conditions w h i c h r e d u c e the p l a s t o q u i n o n e p o o l results i n the p h o s p h o r y l a t i o n o f

222 several thylakoid membrane polypeptides (Bennett 1977). The three major phosphopolypeptides have been identified as the 26kDa and 24kDa polypeptides of the chlorophyll a/b light-harvesting pigment protein (LHC2) (Bennett 1983) and a 9kDa protein associated with the photosystem 2 (PS2) in the appressed thylakoid lamellae (Owens and Ohad 1982). Two distinct thylakoid membrane protein kinases are thought to be involved in the labelling of the LHC2 and 9kDa polypeptides (Farchaus et al. 1985). The reversible phosphorylation of LHC2, controlled by the redox state of the plastoquinone pool, is thought to be the mechanism by which chloroplasts can redistribute the incident excitation energy between PS2 and PS1 by altering the partitioning of LHC2 between PS2 and PS 1 (Allen et al. 198 l, Horton and Black 1982). The degree of inhibition of PS2 photochemistry by thylakoid membrane phosphorylation is, however, not reversed at saturating light intensities (Horton and Lee 1984, Hodges et al. 1985). Thus, thylakoid membrane protein phosphorylation, in addition to changing the size of PS2 antenna, has a direct inhibitory effect on photosynthetic electron transport. Recent studies indicate that this inhibition is possibly due to the phosphorylation of the 9kDa polypeptide of the appressed thylakoid membranes (Packham 1987). Electron transport on the acceptor side of PS2 proceeds through a twoelectron gating mechanism (Bouges-Bocquet 1973, Velthuys and Amesz 1974). This mechanism involves a primary plastoquinone acceptor, QA, which can only be reduced to the anionic semiquinone form QA, in series with a secondary plastoquinone acceptor, Qa. The semiquinone generated by the first turnover is sequestered on QB until the arrival of the second electron which results in the formation of the fully-reduced plastohydroquinone. The plastohydroquinone is displaced at the Qs site by a fully oxidized form. The equilibrium constant for the exchange of the electron between QA and QB has been measured to be about 20 (Robinson and Crofts 1983): thus, 95 percent of the PS2 centres will generate a longlived Q~ species after single flash excitation. Our studies (Hodges et al. 1985, 1987) have shown that thylakoid membrane protein phosphorylation decreases the stability of the secondary plastoquinone acceptor, Q~, of PS2, presumably by altering the equilibrium constant for the exchange of the electron between QAand QB- The aim in this report was to determine the effect of an increased level of QA, caused by thylakoid membrane protein phosphorylation, on the quaternary oscillation in the flash oxygen release pattern. The quaternary oscillation in oxygen release upon flash excitation was first observed by Joliot and colleagues (1969) and interpreted by Kok and colleagues (1970) in terms of the S-state cycle. In this model the PS2 centres must accumulate the oxidising equivalents from four turnovers before oxy-

223 gen is released. The damping in the quaternary oscillation arises from the probability that the PS2 centres either fail to catalyse an S-state transition or undergo two turnovers during flash excitation; resulting in a randomised distribution in the S-states with increasing flash number. Jursinic and Kyle (1983) were unable to detect a difference between the oxygen release patterns of "phosphorylated" and "non-phosphorylated" thylakoid membranes. Their study can be criticized, however, on the grounds that they compared light-activated phosphorylated thylakoid membranes with dark-adapted non-phosphorylated control samples. In this present study the nonphosphorylated "control" thylakoid membranes were subjected to the same pre-treatment conditions as the phosphorylated samples to ensure that no other effect (such as photo-inhibition) would interfere with our comparative study.

Materials and methods

Osmotically shocked chloroplasts were isolated from market lettuce, or greenhouse-grown pea plants as previously described (Packham and Ford 1986). The membranes were suspended at 0.2 mg Chl/ml in 330mM sorbitol, 20mM Hepes (pH 7.8), 100mM KC1, 5mM NaF and 5mM MgC12. Phosphorylated thylakoid membranes were generated by the addition of 0.4raM ATP to the incubation medium. The thylakoid membrane protein kinases were either dark-activated by the addition of 0.5mM NADPH and 5 pM Spirulina maxima ferredoxin, or were light-activated (Hodges et al. 1984, 1985). After 30 minutes treatment, the thylakoid membranes were recovered by centrifugation and resuspended at 0.4mg Chl/ml concentration in incubation buffer depleted of ATP and ferredoxin/NADPH. The nonphosphorylated "control" thylakoid membranes were given identical activation treatments. The flash-induced oxygen yield patterns of phosphorylated and non-phosphorylated thylakoid membranes were obtained using a rate oxygen electrode as described elsewhere (Lemasson and Etienne 1975), and analysed by the recurrence matrix model of Lavorel (1976). The thylakoid membranes were dark-adapted on the platinum electrode for 10 minutes before excitation by a train of 20 flashes at a frequency of 2Hz.

Results and discussion

The phosphorylation of thylakoid membrane proteins was indirected monitored by the measurement of a 15 % inhibition in the light-saturated oxygen evolution rate (Hodges et al. 1985), a 20% decrease in the Fm level of room

224 temperature chlorophyll a fluorescence (Allen and Horton 1981), or by increase in the F735/F685 quotient of 77K chlorophyll fluorescence (Krause et al. 1983) with respect to non-phosphorylated thylakoid membranes treated identically except for the depletion of ATP. The changes in the chlorophyll a fluorescence yield are thought to monitor the phosphorylation of LHC2 (Telfer et al, 1983), whereas the partial inhibition of the oxygen evolution rate is believed to monitor the labelling of the 9kDa PS2-associated polypeptide (Packham 1987). The flash oxygen yield patterns of non-phosphorylated (open circles) and "phosphorylated" (closed circles) thylakoid membranes, normalised to the oxygen release on the third flash, are shown in Fig. 1. It is evident that thylakoid membrane phosphorylation increases the damping of the quaternary oscillation in oxygen yield; as characterised by a decrease in the Y3/Y4 quotient (i.e. the oxygen yield of the third flash divided by that of the fourth flash), and also the Y7/Y8 and Y1 l/Y12 quotients when the two oxygen release patterns are compared. A statistical analysis of the oxygen yield patterns from four chloroplast preparations confirms that the Y8 yields of non-phosphorylated and phosphorylated thylakoid membranes are significantly different (Paired Student's T-Test; P < 0.05). We have analyzed the oxygen yield patterns by applying the recurrence matrix model of Lavorel (1976) to the oxygen yields of the first twenty flashes. This algorithm computes S0/S 1 ratio of the dark-adapted thylakoid membranes and the probabilities that the PS2 centres undergo either a "miss" (~), "single-hit" (/~), or a "double-hit" (~) during flash excitation. The computed analysis of the two oxygen yield patterns of Fig. 1 is shown in Table 1. Although phosphorylated thylakoid membranes have a 20% increase in the "miss" probability, this would account for only an insignificant 2.4% attenuation in the light-saturated rate of oxygen evolution. This analysis therefore suggests that the 15-20% decrease in the oxygen evolution rate in phosphorylated thylakoid membranes is due to a complete inhibition of 15-20% of the PS2 population. The fit between the oxygen yield pattern predicted by the recurrence matrix model and the experimental data is, however, not satisfactory for either the non-phosphorylated (Fig. 2A) or phosphorylated (Fig. 3A) thylakoid membranes. A much closer fit (compare Figs. 2B and 3B with Figs. 2A and 3A, respectively) is achieved by a modification of the Kok S-state model to include a probability that PS2 centres can undergo a double-miss (i.e. fail to accumulate the oxidizing equivalents of two charge separation events) when in the $2 state. An increased single-miss when PS2 centres are in the $2 state has already been analyzed by Delrieu (1983). A double miss is a

225 -

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Fig. 1. Flash-induced oxygen yield pattern of phosphorylated (closed symbols) and nonphosphorylated (open symbols) thylakoid membranes. The thylakoid membranes (25ug Chl) were dark-adapted on the rate oxygen electrode for 10 minutes before flash excitation. The oxygen yields, obtained at a flash frequency of 2 Hz and normalised to the oxygen yield at the third flash (Y3), are the average of sequences from 13 separate non-phosphorylated and 11 separate phosphorylated samples from four chloroplast preparations. For clarity, the averaged yield pattern for the phosphorylated thylakoid sample has been displaced vertically.

226 Table 1. Analysis of the damped quaternary oscillation of the oxygen yield patterns of phosphorylated and non-phosphorylated thylakoid membranes: applying the equal miss on all S-state transitions model

S0/(S0 + Sl) single miss (~) single hit (#) double hit (7) Predicted oxygen yield (a.u)

Non-phosphorylated

Phosphorylated

0.29 0.10 0.83 0.07 4.66 (100%)

0.31 0.12 0.81 0.07 4.55 (97.6%)

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NON- PHOSPHORYLATED

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227 consequence of a population of PS2 centres which retain Q~, following photochemical charge separation. The PS2 centres which remain in the [$2 QT,] state will not only miss the next S-state transition, owing to the inability to catalyze a charge separation due to the presence of Q~,, but will also decay to the [S1 QA] state. Thus, these PS2 centres will have failed to stabilize at least two charge transfer turnovers. Table 2 shows the computed initial SO population, the single miss, double turnover and double hit probabilities which give the best fits for the non-phosphorylated and phosphorylated thylakoid membranes. Thylakoid membrane phosphorylation is inferred to increase the population of PS2 centres that undergo a double-miss from 5% to 16%. Moreover, the increased double miss is sufficient to account for the observed 15-20% decrease in the light-saturated oxygen evolution rate. Figure 4 shows the kinetics of the S2-to-S1 deactivation in the nonphosphorylated and phosphorylated thylakoid membranes, as measured from the decrease in Y3 as the dark-time between the first and second flash is lengthened (following the proecedure described in Vermaas et al. 1984). The semilogarithm plot of fig. 4 indicates that $2 deactivation is biphasic. Under the conditions used in this report, we assign the fast phase, with a 2s halftime, to the re-reduction of $2 by Q~, (Diner 1977, Robinson and Crofts 1983), and the slow phase, with a 30s halftime, to the re-reduction of $2 by Qff (Robinson and Crofts 1983 Rutherford et al. 1984). Contrary to the report of Jursinic and Kyle (1983), thylakoid membrane protein phosphorylation appears to modify $2 deactivation by increasing the proportion of the fast component from 5% to 16% without affecting the 2s halftime. This is consistent with an increased level Of Q~, in phosphorylated thylakoid membranes. From the increased extent of the fast phase in $2 deactivation, we calculate that the equilibrium constant for the exchange of the electron from QA to QB would have to be decreased from the value of 20.0 in the non-phosphorylated thylakoid membranes to 6.25 in the phosphorylated membranes. As a consequence of the increased level of Q~,, thylakoid membrane phosphorylation results in an increased cyclic electron transport around PS2: in line with the proposal by Horton and Lee (1983). Figure 5 shows the deactivation kinetics of the $3 oxidation state in phosphorylated and non-phosphorylated thylakoid membranes. The $3

Fig. 2. The fit between the experimental oxygen yield patterns from non-phosphorylated chloroplasts with the theoretical sequences obtained from equal misses on all S-state transitions (A) or from a double miss on $2 (B). The experimental data are presented as open circles, and the computer-simulated data shown as closed circles. The Chi-squares for the fit between the experimental data and equal miss sequence (traces A) is 0.11, and for the fit between the experimental data and the double miss on $2 sequence (traces B) is 0.029.

228

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Fig. 3. The fit between the experimental oxygen yield patterns from phosphorylated chloroplasts with the theoretical sequences obtained from equal misses on all S-state transitions (A) or from a double miss on $2 (B). As for Fig. 2, the experimental data are presented as open circles and the computer-stimulated data shown as closed circles. The Chi-squares for the fit between the experimental data and the equal miss sequence (traces A) is 0.059 and for the fit between experimental data and the double miss on $2 (traces B) is 0.03.

deactivation is m o n o p h a s i c , with a halftime o f 70s, and thylakoid m e m b r a n e protein p h o s p h o r y l a t i o n has no effect. This result is in a g r e e m e n t with Jursinic and Kyle (1983). U n d e r the conditions e m p l o y e d in this study we were unable to dete~t a c o m p o n e n t in the decay o f either $2 or $3 due to the oxidation o f the auxiliary d o n o r , D (see V e r m a a s et al. 1984).

229 Table 2. Analysis of the damped quaternary oscillation of the oxygen yield patterns of phosphorylated and non-phosphorylated thylakoid membranes assuming a probability of "double-misses" when PS2 centres are in the $2 oxidation state.

S0/(S0 + S1) single miss (ct) double hit (7) double miss (3) Predicted oxygen yield (a.u.)

Non-phosphorylated

Phosphorylated

0.30 0.12 0.06 0.05 4.26 (100%)

0.24 0.14 0.06 0.16 3.64 (85.4%)

100

[$2] 80 60

~0

20

20

40

60

80 TIME (s)

Fig. 4. The deactivation kinetics of the $2 oxidation state in phosphorylated (closed symboles)

and non-phosphorylated (open symbols) thylakoid membranes. Experimental conditions as for Fig. 1, except that the dark time between the first and second flashes was varied between 0.2s and 90s. The [$2] concentration was determined from the oxygen yield of the third flash, subtracted from the yield of the second flash in samples treated at 5 Hz. This value was divided by the total oxygen released from flashes 2 to 5, and treated as a percentage of the [$2] at 0.5s. The extent and time constant for the slow phase in the $2 deactivation was determined by linear regression analysis. O u r a n a l y s i s o f t h e flash o x y g e n y i e l d p a t t e r n s s u g g e s t s t h a t t h e p r e d i c t e d d o u b l e m i s s in t h e S - s t a t e t r a n s i t i o n s u p o n the p h o s p h o r y l a t i o n o f t h y l a k o i d m e m b r a n e p r o t e i n s is a s s o c i a t e d w i t h P S 2 c e n t r e s in t h e $2 s t a t e a n d n o t t h e

230 100 [S3]

80

60

40

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I

I

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20

40

60

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120

Fig. 5. The deactivation kinetics of the $3 oxidation state of PS2 in phosphorylated (closed symbols) and non-phosphorylated (open symbols) thylakoid membranes. Experimental conditions as for Fig. I, except that the dark time between the second and third flashes was varied between 0.2s and 60s. The [$3] concentration was determined from the oxygen yield on the third flash divided by the total oxygen release from flashes 3 to 6, and treated as a percentage of the [$3] at 0.2s.

SO state. A difference between SO and $2 in the stabilisation of QA might reflect the increased net positive charge of $2 (Saygin and Witt 1984) causing an increased coulombic attraction between the water-splitting complex with QA in phosphorylated thylakoid membranes. The observed increase damping in the flash oxygen yield pattern and an augmented extent of the fast kinetic component in $2 deactivation by thylakoid membrane phosphorylation, is reminiscent of the reported differences in the oxygen yield pattern detected in atrazine-resistant chloroplasts (Holt et al. 1981, Vermass et al. 1984). PS2 centres which are resistant to atrazine, due to a single amino acid substitution in the "D 1" polypeptide (Hirschberg and Mclntosh 1981), also display a decreased equilibrium constant for the exchange of the electron between QA and QB (Bowes et al 1980). Recent studies have, however, correlated the inhibition of the steady-state oxygen yield to the phosphorylation of the 9kDa thylakoid membrane protein and not with the "DI" and "D2" polypeptides (Packham 1987). Although the 9kDa polypeptide is

231

associated with the PS2 complexes of the appressed thylakoid lamellae (Owens and Ohad 1982), it is not considered to be constituent of the PS2 reaction centre (Nanba and Satoh 1987). The functional role of the 9kDa protein in QA to QB electron transport, and the reason why it is phosphorylated, remains unclear.

Acknowledgements This study was supported by a short-term EMBO fellowship awarded to NKP. Other financial support has come from the CNRS, PIRSEM, the Royal Society (MH) and the SERC (NKP). We would also wish to thank Dr K.K. Rao for the gift of Spirulina maxima ferredoxin.

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232 Hodges M, Packham NK and Barber J (1985) Modification of photosystem 2 by protein phosphorylation. FEBS Letts. 181:83-87 Holt JS, Stemler AJ and Radowitch SR (1981) Differential light responses of photosynthesis by triazine-resistant and triazine-susceptiable Senecio vulgaris biotypes. Plant Physiol 67: 744-748 Horton P and Black MT (1982) On the nature of fluorescence decrease due to phosphorylation of chloroplast membrane proteins. Biochim Biophys Acta 680:22-27 Horton P and Lee P (1983) Stimulation of cyclic electron transfer pathway around photosystem 2 by phosphorylation of chloroplast thylakoid proteins. FEBS Letts 162:81-84 Horton P and Lee P (1984) Phosphorylation of chloroplast thylakoids decreases maximal capaicty of photosystem 2 electron transfer. Biochim Biophys Acta 767:563-567 Jursinic PA and Kyle DJ (1983) Changes in the redox state of the secondary electron acceptor of photosystem 2 associated with light-induced thylakoid membrane phosphorylation. Biochim Biophys Acta 723:37-44 Joliot P, Barbieri G and Chabaund R (1969) Un nouveau modele des centres photochimiques du systeme II. Photochem Photobiol 10:309-329 Kok B, Forbush B and McGloin M (1970) Cooperativity of charges in photosynthetic oxygen evolution. Photochem Photobiol 11:457-475 Krause GH, Briantais JM and Vernotte C (1983) Characterisation of chlorophyll fluorescence quenching in chloroplasts by fluorescence spectroscopy at 77K. Biochim Biophys Acta 723: 169-175 Lavorel J (1976) Matrix analysis of the oxygen evolving system of photosynthesis. J Theor Biol 57:171-185 Lemasson C and Etienne AM (1975) Photo-inactivation of system 2 centres by carbonyl cyanide m-chlorophenylhydrazone in Chlorella pyrenoidosa. Biochim Biophys Acta 408: 135-142 Nanba O and Satoh K (1987) Isolation of a Photosystem 2 reaction centre consisting of D1 and D2 polypeptides and cytochrome b-559. Proc Natl Acad Sci USA 84:109-112 Owens GC and Ohad I (1982) Phosphorylation of Chlamydornonas reinhardi chloroplast membrane proteins in vivo and in vitro. J Cell Biol 93:712-718 Packham NK (1987) Phosphorylation of the 9kDa photosystem 2-associated polypeptide and the inhibition of photosynthetic oxygen evolution. Biochim Biophys Acta 983:259-266 Packham NK and Ford RC (1986) Deactivation of the photosystem 2 oxidation (S) states by ANT2p and the putative role of carotenoid. Biochim Biophys Acta 852:183-190 Robinson H and Crofts AR (1983) Kinetics of the oxidation-reduction reactions of the photosystem 2 quinone acceptor complex, and the pathway for deactivation. FEBS Letts 153:221-226 Rutherford AW Govindjee and Inoue Y (1984) Charge accumulation and photochemistry in leaves studied by thermoluminescence and delayed light emission. Proc Natl Acad Sci USA 81:1107-1111 Saygin O and Witt HT (1984) On the change of charges in the four photo-induced oxidation steps of the water-splitting system S. FEBS Letts 176:83-87 Telfer A, Allen JF, Barber J and Bennett J (1983) Thylakoid protein phosphorylation during state 1-state 2 transitions in osmotically-shocked pea chloroplasts. Biochim Biophys Acta 722:176-181 Velthuys BR and Amesz J (1974) Charge accumulation at the reducing side of system 2 of photosynthesis. Biochim Biophys Acta 333:85-94 Vermaas WFJ, Renger G and Dohnt G (1984) Reduction of the oxygen-evolving system in chloroplasts by thylakoid components. Biochim Biophys Acta 764:194-202