Planta
Planta (1985)165:37-42
9 Springer-Verlag 1985
Phosphorylation of chloroplast membrane proteins partially protects against photoinhibition P. Horton* and P. Lee Research Institute for Photosynthesis and Department of Biochemistry, University of Sheffield, Sheffield S10 2TN, U K
Abstract. Thylakoids isolated from peas (Pisum sativum cv. Kelvedon Wonder) and phosphorylated by incubation with ATP have been compared with non-phosphorylated thylakoids in their sensitivity to photoinhibition by exposure to illumination in vitro. Assays of the kinetics of fluorescence induction at 20~ and the fluorescence emission spectra at -196~ indicate a proportionally larger decrease in fluorescence as a result of photoinhibitory treatment of non-phosphorylated compared with phosphorylated thylakoids. It is concluded that protein phosphorylation can afford partial protection to thylakoids exposed to photoinhibitory conditions. Key words: Chlorophyll fluorescence - Chloroplast membrane - Photoinhibition of photosynthesis Photosynthesis (photoinhibition) - Pisum (photosynthesis) - Protein phosphorylation.
Introduction
During growth and development of leaves, the chloroplast acquires a membrane composition which is adapted to the prevailing intensity and quality of light (for reviews see Anderson 1982; Melis 1984; Horton 1985). Thus, in shade conditions, which are deficient in light exciting photosystem II(PSII), the composition of lightharvesting chlorophyll protein (LHCP) and PSII is increased relative to PSI, the cytochrome b-f complex and ATP synthase. Conversely, in strong light * To whom correspondence should be addressed
Abbreviations and symbols: D C M U = 3-(3,4-dichlorophenyl)l,l-dimethylurea; F 0 = Level of chlorophyll fluorescence when photosystem 2 traps are open; F m = Level of chlorophyll fluorescence when photosystem 2 traps are closed; P = Maximum level of fluorescence reached in the absence of D C M U ; PSI (II) = photosystem I(II)
the LHCP and PSII content is lowered. When leaves are exposed to a light intensity in excess of that adapted to during growth, photoinhibition can result (Bjorkman and Holmgren 1963; Powles and Critchley 1980; Powles and Thorne 1981 ; Critchley 1981). Similarly, the balance between irradiance and consumption of photosynthetic "product" can be upset if the latter is reduced (by, for example, CO 2 deprivation) and photoinhibition can then occur even at moderate light intensity (Powles et al. 1979). Photoinhibition covers a broad range of deleterious effects although it is generally considered that the major site of primary damage resides in PSII (Osmond 1981; Powles 1984). Thus, photoinhibition results in a loss of the variable component of fluorescence emission from PSII (Critchley and Smillie 1981; Fork etal. 1981; Powles and Bjorkman 1982) and a loss of PSIIcatalysed electron-transfer capacity (Critchley 1981; Powles and Bjorkman 1982). The molecular mechanism for this loss of capacity in higher plants is not understood, but may involve a loss of the 32 000-M, polypeptide which catalyses electron transfer from QA t o the plastoquinone pool (Kyle et al. 1984). The susceptibility to high light seems to indicate that a limit is set on how well a plant can adapt to low light if photoinhibition is to be avoided during periods of excessive illumination. Therefore, there would seem to be a selective pressure for a mechanism which allows for short-term changes in the light-harvesting properties and electron-transfer capacity of PSII such that efficiency can be promoted in low light yet photoinhibition avoided in strong light (Horton 1985). It has been suggested that reversible phosphorylation of thylakoid proteins provides a mechanism for achieving short-term adaption to changes in light levels (for reviews, see Barber 1983;
38
P. Horton and P. Lee: Protein phosphorylation and photoinhibition in thylakoids
Bennett 1983; Horton 1983a). The activity ofthylakoid protein kinase responds to the level of reduced plastoquinone (Horton and Black 1980; Horton et al. 1981 ; Allen et al. 1981) and as such will tend to be high in strong light and low in weak light. Such switching of protein-kinase activity upon changing light intensity has been demonstrated by in vitro experiments (Horton and Foyer 1982). Since protein phosphorylation results in redirection of excitation away from PSII, it has been suggested that herein may lie a mechanism for protection against photoinhibition. In this paper data is presented which indeed indicates that phosphorylation of thylakoid proteins can partially offset photoinhibition of isolated thylakoids.
/ B.
,
f
//
Material and methods
Fluorescence
Chloroplast thylakoids were isolated from peas (P&um sativum L.) as previously described (Horton and Black 1980) and phosphorylated by illumination in the presence of 0.2 mM ATP for 20 rain with blue light defined by Coming ,~O6 filters (Coming Glass, Corning, N.Y., USA) and giving an energy fluence rate of 40 W m 2. A non-phosphorylated control was treated identically but without ATP. The incubation medium contained 0.33 M sorbitol, 10 mM NaC1, 5 mM MgC12 and 50 mM 4-(2hydroxyethyl)- 1-piperazineethanesulfonic acid (Hepes) (pH 7.6) and included 10 mM NaF to inhibit protein-phosphatase activity (Bennett 1980). Incubations done in the presence of 7-[32P] ATP followed by polyacrylamide-gel electrophoresis of thylakoid proteins showed that there was extensive labelling of several thylakoid proteins including LHCP, 32 000-M r and 9000-Mr. After phosphorylation, thylakoids were centrifuged and resuspended in the same medium. For photoinhibition, thylakoid suspensions (phosphorylated and non-phosphorytated) were illuminated with white light provided by tungsten lamps filtered through a 10-cm layer of circulating water. The thylakoids, diluted to a concentration of 50 gg chlorophyll m1-1, were contained to a depth of 9 cm in a 75-cmdiameter beaker which was immersed in a water bath to maintain the suspension at 21 ~: I~ The energy fluence rate was 200 Wm-2; because no electron acceptor was provided, photoinhibition could be observed at this relatively low fluence rate. After various illumination times samples were withdrawn for analysis. Fluorescence emission spectra at-196~ were recorded as described earlier (Fernyhough et al. 1984). Room-temperature fluorescence induction curves of dark-adapted samples were performed in the presence and absence of 3-(3',4'dichlorophenyl)-l,l-dimethylurea (DCMU) at a chlorophyll concentration of 10 p.g m1-1using apparatus described previously (Horton and Black 1980). Electron transport from water to methyl viologen was assayed in a modified Hansatech (Kings Lynn, Norfolk, UK) 02 electrode (Horton 1983b) in the presence of NH4Ct and using saturating red light (350 W m~2).
Results
Photoinhibition both in vivo (Critchley and Smillie 1981) and in vitro (Malkin and Jones 1968) results in characteristic changes in the kinetics of fluores-
i
_ _
lores
Intensity
los
Fig. 1A, B. Fluorescence induction curves of non-phosphorylated (A) and phosphorylated (B) pea thylakoids recorded at 20~ before (--) and after 10 (- - -) min exposure to photoinhibitory conditions. Arrow indicates light on. Data were recorded on a dual time base as indicated
cence induction observed at 20 ~C. In Fig. 1 are shown fluorescence induction curves of phosphorylated and non-phosphorylated thylakoids. As documented by numerous studies the rise in fluorescence is complex (Lavorel and Etienne 1977); a level, F 0 (the level of chlorophyll fluorescence when PSII traps are open), is seen upon shutter opening and this is followed by a biphasic rise to a maximum level referred to as "P" (the maximum level of fluorescence reached in the absence of DCMU). As shown previously, the F o level is reduced by phosphorylation (Horton and Black 1981). The level at P is also reduced, not only because of the decrease in excitation transfer to PSII but due to a decreased level of Q reduction in the phosphorylated sample (Horton and Lee 1983). Photoinhibitory treatment had two effects on the induction curve of the nonphosphorylated sample; firstly there was an increase in Fo; secondly there was a large decrease in the level at P. In phosphorylated chloroplasts, the F 0 increase was observed but there was only a small diminution of the yield of fluorescence at "P". A time course for the changes in P is shown in Fig. 2. The level of fluorescence of the phosphorylated chloroplasts decayed slowly for the duration of the 60-rain treatment. The fluorescence level of the non-phosphorylated sample was consistently
P. Horton and P. Lee: Protein phosphorylation and photoinhibition in thylakoids
39
2ooT
200
15( NP > Co
8 100
%
P
-
100
o
$
Fo
O J Lt-
50
o
5O
'
2'o
4b
'
st;
20
T~me ( rnin )
Fig. 2. "P" level of chlorophyll fluorescence for phosphorylated ( ~ , P) and non-phosphorylated ( I , NP) pea thylakoids after exposure to between 0 and 60 min of photoinhibitory light. Fluorescence was measured as in Fig. 1, the "P" level corresponding to the maximum level seen after approx, t 0 s. illumination
found to decay more rapidly so that by 60 min the ~ levels of the phosphorylated and nonphosphorylated thylakoids were virtually identical despite a 35% difference in yield before photoinhibition. In Fig. 3, the levels of F 0 and F m (the level of chlorophyll fluorescence when PSII traps are closed) are plotted from induction curves recorded in the presence of DCMU. Photoinhibition of both chloroplast samples resulted in a parallel increase in F 0. However, the F m level decayed more extensively in the non-phosphorylated thylakoids. Again, after 60 min incubation F m levels of the two samples were almost identical. Photoinhibition in vivo has characteristic effects on fluorescence emission spectra recorded at -196~ An overall decrease in yield of fluorescence at both short-wavelength PSII (700 nm) has been reported but with PSII being preferentially quenched (Fork et al. 198l; Powles and Bjorkman 1982). Similar observations were made during photoinhibition of non-phosphorylated thylakoids (Fig. 4). After 30 min of photoinhibition a large decrease in yield of both PSI and PSII emission was observed. The ratio 685/734 dropped from 0.79 to 0-63. In contrast, phosphorylated thylakoids were remarkably resistant to pho-
/-,0 Time (mln)
60
Fig. 3. Fm and F0 levels of chlorophyll fluorescence recorded at 20~ in the presence of 10 pM DCMU tbr phosphorylated (A, O, -P) and non-phosphorylated (A, O, -NP) thylakoids after exposure to between 0 and 60 rain photoinhibitory light
550
70D
750
Phos
650
700 750 Wovelength (nm)
Fig. 4. Fluorescence emission spectra recorded at 196~ of phosphorylated (lowerspectrum) and non-phosphorylated (upper spectrum) thylakoids exposed to 0 (--) and 30 rain (- - -) photoinhibitory light
toinhibition. Overall yields only dropped by approx. 10% during the 30-min period of illumination, the 685/734 ratio changing from 0.51 to 0.47. Note that the 685/734 ratios of the phosphorylated thylakoids are always much lower than for nonphosphorylated thylakoids due to the redistribution of excitation between the two photosystems (Horton and Black 1980, 1981 ; Bennett et al. 1980).
40
P. Horton and P. Lee: Protein phosphorylation and photoinhibition in thylakoids
120
"7
lOC
T 0
~' ac
f-q C)
E 60 21_
z,0
20
0 Time
2'o (mini
'
3b
Fig. 5. Light saturated rates of uncoupled electron transfer (H20 ~ methyl viologen) of phosphorylated (O) and nonphosphorylated ( 0 ) pea thylakoids exposed to between 0 and 30 rain photoinhibitory light
Direct measurement of the rate of electron transfer from water to methyl viologen also revealed a decreased sensitivity to photoinhibition as a result of protein phosphorylation. As reported recently (Horton and Lee 1984), the light-saturated rate of electron transfer is reversibly inhibited by phosphorylation so that the zero time value in Fig. 5 for the phosphorylated sample is less than that of the non-phosphorylated thylakoids. However, photoinhibition causes a much larger loss of activity of the phosphorylated sample such that after 30 rain the rates of both phosphorylated and non-phosphorylated are approximately equal. Discussion
Photoinhibition in leaves has been shown to result whenever the rate of excitation exceeds the dissipative capacity of electron transport (Osmond 1981). The actual light intensity at which this occurs is therefore not absolute but depends upon the lightharvesting capacity and the presence of any factor which lowers either the rate of electron transfer or the consumption of ATP and NADPH. If plant leaves adapt to "average" light and metabolic conditions, then periods of high light or metabolic change may be potentially photoinhibitory (Osmond 1981 ; Horton 1984). It seems likely that PSII is the major site of photoinhibitory damage. There-
fore, any mechanism which can reversibly change the rate of photon delivery to PSII could provide a protective mechanism. It has been shown that phosphorylation of thylakoid proteins decreases the rate of excitation of PSII. In this paper, it has been demonstrated that protein phosphorylation can decrease the sensitivity of chloroplast thylakoids to photoinhibition. Photoinhibition was characterised by the decrease in fluorescence yield at room temperature and at-196 ~C. At room temperature, photoinhibition was associated with a decrease in yield at P as well as in the maximum yield (Fro). This loss of fluorescence is accompanied by a decrease in yield at -196 ~C, the emission from PSII decreasing more than from photosystem I and is characteristic of in vivo photoinhibition (Fork et al. 1981; Powles and Bjorkman 1982; Ogren and Oquist 1984). The decrease in fluorescence is probably related to accumulation of reduced phaeophytin in the PSII centres (Renger et al. 1983). It is of interest that the increase in F 0, which is also a characteristic of in vivo photoinhibition (Ogren and Oquist 1984) occurs at the same rate in both phosphorylated and non-phosphorylated thylakoids. The rates of decrease in fluorescence and electron-transfer capacity during photoinhibition are clearly diminished by prior phosphorylation of the thylakoid membranes. Interpretation of results is complicated by the differences in fluorescence that exist prior to photoinhibitory treatment due to the effects of phosphorylation on excitation distribution per se. Nevertheless, it is clear that the proportional decreases in fluorescence yield are much larger for non-phosphorylated thylakoids than for phosphorylated ones. Since the ratio Fv/ F m(Fv = Fro-F0) is independent ofphosphorylation state (Horton and Black 1981) it means that all the rate constants for dissipation of excitation in PSII are the same in phosphorylated and non-phosphorylated thylakoids. Therefore, the different extent of changes in the maximum fluorescence yield that accompany photoinhibition in phosphorylated and non-phosphorylated thylakoids cannot be a mathematical artefact but must represent real differences in the extent of photoinhibition. The simplest mechanism by which phosphorylation could prevent photoinhibition is by reducing the excess illumination reaching PSII. However, there may be additional effects of phosphorylation which may also be of benefit. Thus, the decrease in excitation rate of PSII may prevent over-reduction of intersystem carriers and allow better redox poise for PSI cyclic electron flow. There is some evidence that cyclic flow around PSI
P. Horton and P. Lee: Protein phosphorylation and photoinhibition in thylakoids
is necessary for prevention of photo-damage to chloroplasts (Ridley 1977; Ridley and Horton 1984). Also, there is considerable documentation of effects of protein phosphorylation on PSII other than on light harvesting. These include stimulation of NH2OH-sensitive Q oxidation (Horton and Lee 1983), increased stability of the secondary electron acceptor of PSII QB (Jursinic and Kyle 1983), increased sensitivity to DCMU (Shocat et al. 1982) and a decrease in the maximum rate of electron transfer (Horton and Lee 1984). All of these effects point to a reversible alteration of QA (the primary electron acceptor of PSII) to QB electron transfer which may serve to decrease the electron flow to QB (Horton and Lee 1984). It is noteworthy, therefore, that a deficiency in QB is thought to be the primary cause of photoinhibition (Kyle et al. 1984); the plastoquinol-dependent protein kinase, by suppressing both light harvesting and electron transfer provides a mechanism that decreases the delivery of electrons to QB. It should be added, however, that the relationship between photoinhibition and protein phosphorylation may not be as simple as described above; thus, loss of PSII function as a result of photoinhibition will decrease the flow of electrons into the plastoquinone pool and tend to decrease kinase activity. In fact, it has been observed that photoinhibition in Chlamydomonas is associated with complete dephosphorylation of thylakoid proteins (Ohad, I., Department of Biological Chemistry, The Hebrew University of Jerusalem, Israel personal communication). Moreover, the observation that a high ApH has an inhibitory effect on protein phosphorylation (Fernyhough et al. 1984) may mean that in some circumstances high light may reduce kinase activity with consequent diminution in the protective effect. The possible interplay between the "metabolic" and "protective" roles of protein phosphorylation needs further investigation. This work was funded by a grant from the United Kingdom Science and Engineering Research Council.
References Allen, J.F., Bennett, J., Steinback, K.E., Arntzen, C.J. (1981) Chloroplast protein phosphorylation couples plastoquinone redox state to distribution of excitation transfer between photosystems. Nature 291, 1-5 Anderson, J.M. (1982) The significance of grana stacking in chlorophyll b-containing chloroplasts. FEBS Lett. 124, 1-10 Barber, J. (1983) Membrane conformational changes due to phosphorylation and the control of energy transfer in photosynthesis. Photobiochem. Photobiophys. 5, 181-190
41
Bennett, J. (1980) Chloroplast phosphoproteins. Evidence for a thylakoid-bound phosphoprotein phosphatase. Eur. J. Biochem. 104, 85-89 Bennett, J. (1983) Regulation of photosynthesis by reversible phosphorylation of the light-harvesting a/b protein. Biochem. J. 212, 1-13 Bennett, J., Steinback, K.E., Arntzen, C.J. (1980) Chloroplast phosphoproteins: regulation of excitation energy transfer by phosphorylation of thylakoid membranes. Proc. Natl. Acad. Sci. USA 77, 5253-5257 Bjorkman, O., Holmgrcn, P. (1963) Adaptability of the photosynthetic apparatus to light intensity in ecotypes from exposed and shaded habitats. Physiol. Plant. 16, 889-894 Critchley, C. (1981) Studies on the mechanism of photoinhibition in higher plants. 1. Effects of high light intensity on chloroplast activities in cucumber adapted to low light. Plant Physiol. 67, 1161-1165 Critchlcy, C., Smillie, R.M. (1981) Leaf chlorophyll fluorescence as an indicator of photoinhibition in Cucumis sativus L. Aust. J. Plant Physiol. 8, 133 141 Fernyhough, P., Foyer, C.H., Horton, P. (1984) Increase in level of thylakoid protein phosphorylation in maize mesophyll chloroplasts by decrease in the transthylakoid proton gradient. FEBS Lett. 176, 351-353 Fork, D.C., Oquist, G., Powles, S.B. (1981) Photoinhibition in bean: A fluorescence analysis. Carnegie Inst. Washington Yearb. 80, 52-57 Horton, P. (1983a) Control of chloroplast electron transfer by phosphorylation of thylakoid proteins. FEBS Lett. 152, 47-52 Horton, P. (1983b) Relationships between electron transfer and carbon assimilation; simultaneous measurement of chlorophyll fluorescence transthylakoid pH gradient and 02 evolution in isolated chloroplasts. Proc. R. Soc. London Ser. B. 217, 405-416 Horton, P. (1984) Interactions between electron transfer and carbon assimilation. In: Photosynthetic mechanisms and the environment, Barber, J., Baker, N.R., eds. Elsevier Biomedical Press, Amsterdam, in press Horton, P., Allen, J.F., Black, M.T., Bennett, J. (1981) Regulation of phosphorylation of chloroplast membrane polypeptides by the redox state of plastoquinone. FEBS Lett. 125, 193-196 Horton, P., Black, M.T. (1980) Activation of adenosine 5'triphosphate induced fluorescence quenching by reduced plastoquinone. The basis of state 1 to state 2 transitions in chloroplasts. FEBS Lett. 119, 141-144 Horton, P., Black, M.T. (1981) Light dependent quenching of chlorophyll fluorescence in pea chloroplasts induced by adenosine 5'-triphosphate. Biochim. Biophys. Acta 635, 53-62 Horton, P., Foyer, C.H. (1982) Relationships between protein phosphorylation and electron transport in the reconstituted chloroplast system. Biochem. J. 210, 51%521 Horton, P., Lee, P. (1983) Stimulation of a cyclic electron transfer pathway around photosystem 2 by phosphorylation of chloroplast thylakoid proteins. FEBS Lett. 162, 81-84 Horton, P., Lee, P. (1984) Phosphorylation of chloroplast thylakoids decreases the maximum capacity of photosystem 2 electron transfer. Biochim. Biophys. Acta, 767, 563-567 Jursinic, P.A., Kyle, D.J. (1983) Changes in the redox state of the secondary acceptor of photosystem II associated with light-induced thylakoid protein phosphorylation. Biochim. Biophys. Acta 723, 37-44 Kyle, D.J., Ohad, I., Guy, R., Arntzen, C.J. (1984) Selective thylakoid protein damage and repair during photoinhibition. In: Advances in photosynthesis research, vol. 3, pp. 67 70, C. Sybesma, ed. Nijhoff/Junk Publishers, The Hague
42
P. Horton and P. Lee: Protein phosphorylation and photoinhibition in thylakoids
Lavorel, J., Etienne, A.-L. (1977) In vivo chlorophyll fluorescence. In: Primary process of photosynthesis, pp. 203-268, J. Barber, ed. Elsevier/North Holland Biomedical Press, Amsterdam Malkin, S., Jones, L.W. (1968) Photoinhibition and excitation quenching in photosystem 2 of photosynthesis from fluorescence induction measurements. Biochim. Biophys. Acta 162, 297-299 Melis, A. (1984) Light regulation of photosynthetic membrane structure, organisation and function. J. Cell. Biochem. 24, 271-285 Ogren, E., 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 Osmond, B. (1981) Photorespiration and photoinhibition. Some implications for the energetics of photosynthesis. Biochim. Biophys. Acta 639, 77-98 Powles, S.B. (1984) Photoinhibition of photosynthesis induced by visible light. Annu. Rev. Plant Physiol. 35, 15-44 Powles, S.B., Bjorkman, O. (1982) Photoinhibition of photosynthesis: effect on chlorophyll fluorescence at 77 K in intact leaves and in chloroplast membranes of Nerium oleander. Planta 156, 97-107 Powles, S.B., Critchley, C. (1980) Effect of light intensity during growth on photoinhibition of intact attached bean leaflets. Plant Physiol. 65, 1181-1187
Powles, S.B., Osmond, C.B,, Thorne, S.W. (1979) Photoinhibition of intact attached leaves of C3 plants illuminated in the absence of both carbon dioxide and of photorespiration. Plant Physiol. 64, 982-988 Powles, S.B., Thorne, S.W. (1981) Effect of high-light treatment in inducing photoinhibition of photosynthesis in intact leaves of low-light grown Phaseolus vulgaris and Lastreopsis microsora. Planta 152, 471M77 Renger, G., Koike, H., Yuasa, M., Inoue, Y. (1983) Studies on the mechanism of the fluorescence decline induced by strong actinic light in PSII particles under different redox conditions. FEBS Lett. 163, 89-93 Ridley, S.M. (1977) Interaction of chloroplasts with inhibitiors. Induction of chlorosis by diuron during prolonged illumination in vitro. Plant Physiol. 59, 724-732 Ridley, S.M., Horton, P. (1984) DCMU-induced photodestruction of pigments associated with an inhibition of photosystem 1 cyclic electron flow: a working hypothesis. Z. Naturforsch. Tell C 39, 351-353 Shocat, S., Owens, G.C., Hubert, P., Ohad, I. (1982) The dichlorophenyldimethylurea-binding site in thylakoids of Chlamydomonas reinhardi. Role of photosystem II reaction center and phosphorylation of the 32-35 kilodalton polypeptide in the formation of the high affinity binding site. Biochim. Biophys. Acta 681, 21-31 Received 19 July; accepted 20 December 1984
Planta (1985)165:37-42
9 Springer-Verlag 1985
Phosphorylation of chloroplast membrane proteins partially protects against photoinhibition P. Horton* and P. Lee Research Institute for Photosynthesis and Department of Biochemistry, University of Sheffield, Sheffield S10 2TN, U K
Abstract. Thylakoids isolated from peas (Pisum sativum cv. Kelvedon Wonder) and phosphorylated by incubation with ATP have been compared with non-phosphorylated thylakoids in their sensitivity to photoinhibition by exposure to illumination in vitro. Assays of the kinetics of fluorescence induction at 20~ and the fluorescence emission spectra at -196~ indicate a proportionally larger decrease in fluorescence as a result of photoinhibitory treatment of non-phosphorylated compared with phosphorylated thylakoids. It is concluded that protein phosphorylation can afford partial protection to thylakoids exposed to photoinhibitory conditions. Key words: Chlorophyll fluorescence - Chloroplast membrane - Photoinhibition of photosynthesis Photosynthesis (photoinhibition) - Pisum (photosynthesis) - Protein phosphorylation.
Introduction
During growth and development of leaves, the chloroplast acquires a membrane composition which is adapted to the prevailing intensity and quality of light (for reviews see Anderson 1982; Melis 1984; Horton 1985). Thus, in shade conditions, which are deficient in light exciting photosystem II(PSII), the composition of lightharvesting chlorophyll protein (LHCP) and PSII is increased relative to PSI, the cytochrome b-f complex and ATP synthase. Conversely, in strong light * To whom correspondence should be addressed
Abbreviations and symbols: D C M U = 3-(3,4-dichlorophenyl)l,l-dimethylurea; F 0 = Level of chlorophyll fluorescence when photosystem 2 traps are open; F m = Level of chlorophyll fluorescence when photosystem 2 traps are closed; P = Maximum level of fluorescence reached in the absence of D C M U ; PSI (II) = photosystem I(II)
the LHCP and PSII content is lowered. When leaves are exposed to a light intensity in excess of that adapted to during growth, photoinhibition can result (Bjorkman and Holmgren 1963; Powles and Critchley 1980; Powles and Thorne 1981 ; Critchley 1981). Similarly, the balance between irradiance and consumption of photosynthetic "product" can be upset if the latter is reduced (by, for example, CO 2 deprivation) and photoinhibition can then occur even at moderate light intensity (Powles et al. 1979). Photoinhibition covers a broad range of deleterious effects although it is generally considered that the major site of primary damage resides in PSII (Osmond 1981; Powles 1984). Thus, photoinhibition results in a loss of the variable component of fluorescence emission from PSII (Critchley and Smillie 1981; Fork etal. 1981; Powles and Bjorkman 1982) and a loss of PSIIcatalysed electron-transfer capacity (Critchley 1981; Powles and Bjorkman 1982). The molecular mechanism for this loss of capacity in higher plants is not understood, but may involve a loss of the 32 000-M, polypeptide which catalyses electron transfer from QA t o the plastoquinone pool (Kyle et al. 1984). The susceptibility to high light seems to indicate that a limit is set on how well a plant can adapt to low light if photoinhibition is to be avoided during periods of excessive illumination. Therefore, there would seem to be a selective pressure for a mechanism which allows for short-term changes in the light-harvesting properties and electron-transfer capacity of PSII such that efficiency can be promoted in low light yet photoinhibition avoided in strong light (Horton 1985). It has been suggested that reversible phosphorylation of thylakoid proteins provides a mechanism for achieving short-term adaption to changes in light levels (for reviews, see Barber 1983;
38
P. Horton and P. Lee: Protein phosphorylation and photoinhibition in thylakoids
Bennett 1983; Horton 1983a). The activity ofthylakoid protein kinase responds to the level of reduced plastoquinone (Horton and Black 1980; Horton et al. 1981 ; Allen et al. 1981) and as such will tend to be high in strong light and low in weak light. Such switching of protein-kinase activity upon changing light intensity has been demonstrated by in vitro experiments (Horton and Foyer 1982). Since protein phosphorylation results in redirection of excitation away from PSII, it has been suggested that herein may lie a mechanism for protection against photoinhibition. In this paper data is presented which indeed indicates that phosphorylation of thylakoid proteins can partially offset photoinhibition of isolated thylakoids.
/ B.
,
f
//
Material and methods
Fluorescence
Chloroplast thylakoids were isolated from peas (P&um sativum L.) as previously described (Horton and Black 1980) and phosphorylated by illumination in the presence of 0.2 mM ATP for 20 rain with blue light defined by Coming ,~O6 filters (Coming Glass, Corning, N.Y., USA) and giving an energy fluence rate of 40 W m 2. A non-phosphorylated control was treated identically but without ATP. The incubation medium contained 0.33 M sorbitol, 10 mM NaC1, 5 mM MgC12 and 50 mM 4-(2hydroxyethyl)- 1-piperazineethanesulfonic acid (Hepes) (pH 7.6) and included 10 mM NaF to inhibit protein-phosphatase activity (Bennett 1980). Incubations done in the presence of 7-[32P] ATP followed by polyacrylamide-gel electrophoresis of thylakoid proteins showed that there was extensive labelling of several thylakoid proteins including LHCP, 32 000-M r and 9000-Mr. After phosphorylation, thylakoids were centrifuged and resuspended in the same medium. For photoinhibition, thylakoid suspensions (phosphorylated and non-phosphorytated) were illuminated with white light provided by tungsten lamps filtered through a 10-cm layer of circulating water. The thylakoids, diluted to a concentration of 50 gg chlorophyll m1-1, were contained to a depth of 9 cm in a 75-cmdiameter beaker which was immersed in a water bath to maintain the suspension at 21 ~: I~ The energy fluence rate was 200 Wm-2; because no electron acceptor was provided, photoinhibition could be observed at this relatively low fluence rate. After various illumination times samples were withdrawn for analysis. Fluorescence emission spectra at-196~ were recorded as described earlier (Fernyhough et al. 1984). Room-temperature fluorescence induction curves of dark-adapted samples were performed in the presence and absence of 3-(3',4'dichlorophenyl)-l,l-dimethylurea (DCMU) at a chlorophyll concentration of 10 p.g m1-1using apparatus described previously (Horton and Black 1980). Electron transport from water to methyl viologen was assayed in a modified Hansatech (Kings Lynn, Norfolk, UK) 02 electrode (Horton 1983b) in the presence of NH4Ct and using saturating red light (350 W m~2).
Results
Photoinhibition both in vivo (Critchley and Smillie 1981) and in vitro (Malkin and Jones 1968) results in characteristic changes in the kinetics of fluores-
i
_ _
lores
Intensity
los
Fig. 1A, B. Fluorescence induction curves of non-phosphorylated (A) and phosphorylated (B) pea thylakoids recorded at 20~ before (--) and after 10 (- - -) min exposure to photoinhibitory conditions. Arrow indicates light on. Data were recorded on a dual time base as indicated
cence induction observed at 20 ~C. In Fig. 1 are shown fluorescence induction curves of phosphorylated and non-phosphorylated thylakoids. As documented by numerous studies the rise in fluorescence is complex (Lavorel and Etienne 1977); a level, F 0 (the level of chlorophyll fluorescence when PSII traps are open), is seen upon shutter opening and this is followed by a biphasic rise to a maximum level referred to as "P" (the maximum level of fluorescence reached in the absence of DCMU). As shown previously, the F o level is reduced by phosphorylation (Horton and Black 1981). The level at P is also reduced, not only because of the decrease in excitation transfer to PSII but due to a decreased level of Q reduction in the phosphorylated sample (Horton and Lee 1983). Photoinhibitory treatment had two effects on the induction curve of the nonphosphorylated sample; firstly there was an increase in Fo; secondly there was a large decrease in the level at P. In phosphorylated chloroplasts, the F 0 increase was observed but there was only a small diminution of the yield of fluorescence at "P". A time course for the changes in P is shown in Fig. 2. The level of fluorescence of the phosphorylated chloroplasts decayed slowly for the duration of the 60-rain treatment. The fluorescence level of the non-phosphorylated sample was consistently
P. Horton and P. Lee: Protein phosphorylation and photoinhibition in thylakoids
39
2ooT
200
15( NP > Co
8 100
%
P
-
100
o
$
Fo
O J Lt-
50
o
5O
'
2'o
4b
'
st;
20
T~me ( rnin )
Fig. 2. "P" level of chlorophyll fluorescence for phosphorylated ( ~ , P) and non-phosphorylated ( I , NP) pea thylakoids after exposure to between 0 and 60 min of photoinhibitory light. Fluorescence was measured as in Fig. 1, the "P" level corresponding to the maximum level seen after approx, t 0 s. illumination
found to decay more rapidly so that by 60 min the ~ levels of the phosphorylated and nonphosphorylated thylakoids were virtually identical despite a 35% difference in yield before photoinhibition. In Fig. 3, the levels of F 0 and F m (the level of chlorophyll fluorescence when PSII traps are closed) are plotted from induction curves recorded in the presence of DCMU. Photoinhibition of both chloroplast samples resulted in a parallel increase in F 0. However, the F m level decayed more extensively in the non-phosphorylated thylakoids. Again, after 60 min incubation F m levels of the two samples were almost identical. Photoinhibition in vivo has characteristic effects on fluorescence emission spectra recorded at -196~ An overall decrease in yield of fluorescence at both short-wavelength PSII (700 nm) has been reported but with PSII being preferentially quenched (Fork et al. 198l; Powles and Bjorkman 1982). Similar observations were made during photoinhibition of non-phosphorylated thylakoids (Fig. 4). After 30 min of photoinhibition a large decrease in yield of both PSI and PSII emission was observed. The ratio 685/734 dropped from 0.79 to 0-63. In contrast, phosphorylated thylakoids were remarkably resistant to pho-
/-,0 Time (mln)
60
Fig. 3. Fm and F0 levels of chlorophyll fluorescence recorded at 20~ in the presence of 10 pM DCMU tbr phosphorylated (A, O, -P) and non-phosphorylated (A, O, -NP) thylakoids after exposure to between 0 and 60 rain photoinhibitory light
550
70D
750
Phos
650
700 750 Wovelength (nm)
Fig. 4. Fluorescence emission spectra recorded at 196~ of phosphorylated (lowerspectrum) and non-phosphorylated (upper spectrum) thylakoids exposed to 0 (--) and 30 rain (- - -) photoinhibitory light
toinhibition. Overall yields only dropped by approx. 10% during the 30-min period of illumination, the 685/734 ratio changing from 0.51 to 0.47. Note that the 685/734 ratios of the phosphorylated thylakoids are always much lower than for nonphosphorylated thylakoids due to the redistribution of excitation between the two photosystems (Horton and Black 1980, 1981 ; Bennett et al. 1980).
40
P. Horton and P. Lee: Protein phosphorylation and photoinhibition in thylakoids
120
"7
lOC
T 0
~' ac
f-q C)
E 60 21_
z,0
20
0 Time
2'o (mini
'
3b
Fig. 5. Light saturated rates of uncoupled electron transfer (H20 ~ methyl viologen) of phosphorylated (O) and nonphosphorylated ( 0 ) pea thylakoids exposed to between 0 and 30 rain photoinhibitory light
Direct measurement of the rate of electron transfer from water to methyl viologen also revealed a decreased sensitivity to photoinhibition as a result of protein phosphorylation. As reported recently (Horton and Lee 1984), the light-saturated rate of electron transfer is reversibly inhibited by phosphorylation so that the zero time value in Fig. 5 for the phosphorylated sample is less than that of the non-phosphorylated thylakoids. However, photoinhibition causes a much larger loss of activity of the phosphorylated sample such that after 30 rain the rates of both phosphorylated and non-phosphorylated are approximately equal. Discussion
Photoinhibition in leaves has been shown to result whenever the rate of excitation exceeds the dissipative capacity of electron transport (Osmond 1981). The actual light intensity at which this occurs is therefore not absolute but depends upon the lightharvesting capacity and the presence of any factor which lowers either the rate of electron transfer or the consumption of ATP and NADPH. If plant leaves adapt to "average" light and metabolic conditions, then periods of high light or metabolic change may be potentially photoinhibitory (Osmond 1981 ; Horton 1984). It seems likely that PSII is the major site of photoinhibitory damage. There-
fore, any mechanism which can reversibly change the rate of photon delivery to PSII could provide a protective mechanism. It has been shown that phosphorylation of thylakoid proteins decreases the rate of excitation of PSII. In this paper, it has been demonstrated that protein phosphorylation can decrease the sensitivity of chloroplast thylakoids to photoinhibition. Photoinhibition was characterised by the decrease in fluorescence yield at room temperature and at-196 ~C. At room temperature, photoinhibition was associated with a decrease in yield at P as well as in the maximum yield (Fro). This loss of fluorescence is accompanied by a decrease in yield at -196 ~C, the emission from PSII decreasing more than from photosystem I and is characteristic of in vivo photoinhibition (Fork et al. 1981; Powles and Bjorkman 1982; Ogren and Oquist 1984). The decrease in fluorescence is probably related to accumulation of reduced phaeophytin in the PSII centres (Renger et al. 1983). It is of interest that the increase in F 0, which is also a characteristic of in vivo photoinhibition (Ogren and Oquist 1984) occurs at the same rate in both phosphorylated and non-phosphorylated thylakoids. The rates of decrease in fluorescence and electron-transfer capacity during photoinhibition are clearly diminished by prior phosphorylation of the thylakoid membranes. Interpretation of results is complicated by the differences in fluorescence that exist prior to photoinhibitory treatment due to the effects of phosphorylation on excitation distribution per se. Nevertheless, it is clear that the proportional decreases in fluorescence yield are much larger for non-phosphorylated thylakoids than for phosphorylated ones. Since the ratio Fv/ F m(Fv = Fro-F0) is independent ofphosphorylation state (Horton and Black 1981) it means that all the rate constants for dissipation of excitation in PSII are the same in phosphorylated and non-phosphorylated thylakoids. Therefore, the different extent of changes in the maximum fluorescence yield that accompany photoinhibition in phosphorylated and non-phosphorylated thylakoids cannot be a mathematical artefact but must represent real differences in the extent of photoinhibition. The simplest mechanism by which phosphorylation could prevent photoinhibition is by reducing the excess illumination reaching PSII. However, there may be additional effects of phosphorylation which may also be of benefit. Thus, the decrease in excitation rate of PSII may prevent over-reduction of intersystem carriers and allow better redox poise for PSI cyclic electron flow. There is some evidence that cyclic flow around PSI
P. Horton and P. Lee: Protein phosphorylation and photoinhibition in thylakoids
is necessary for prevention of photo-damage to chloroplasts (Ridley 1977; Ridley and Horton 1984). Also, there is considerable documentation of effects of protein phosphorylation on PSII other than on light harvesting. These include stimulation of NH2OH-sensitive Q oxidation (Horton and Lee 1983), increased stability of the secondary electron acceptor of PSII QB (Jursinic and Kyle 1983), increased sensitivity to DCMU (Shocat et al. 1982) and a decrease in the maximum rate of electron transfer (Horton and Lee 1984). All of these effects point to a reversible alteration of QA (the primary electron acceptor of PSII) to QB electron transfer which may serve to decrease the electron flow to QB (Horton and Lee 1984). It is noteworthy, therefore, that a deficiency in QB is thought to be the primary cause of photoinhibition (Kyle et al. 1984); the plastoquinol-dependent protein kinase, by suppressing both light harvesting and electron transfer provides a mechanism that decreases the delivery of electrons to QB. It should be added, however, that the relationship between photoinhibition and protein phosphorylation may not be as simple as described above; thus, loss of PSII function as a result of photoinhibition will decrease the flow of electrons into the plastoquinone pool and tend to decrease kinase activity. In fact, it has been observed that photoinhibition in Chlamydomonas is associated with complete dephosphorylation of thylakoid proteins (Ohad, I., Department of Biological Chemistry, The Hebrew University of Jerusalem, Israel personal communication). Moreover, the observation that a high ApH has an inhibitory effect on protein phosphorylation (Fernyhough et al. 1984) may mean that in some circumstances high light may reduce kinase activity with consequent diminution in the protective effect. The possible interplay between the "metabolic" and "protective" roles of protein phosphorylation needs further investigation. This work was funded by a grant from the United Kingdom Science and Engineering Research Council.
References Allen, J.F., Bennett, J., Steinback, K.E., Arntzen, C.J. (1981) Chloroplast protein phosphorylation couples plastoquinone redox state to distribution of excitation transfer between photosystems. Nature 291, 1-5 Anderson, J.M. (1982) The significance of grana stacking in chlorophyll b-containing chloroplasts. FEBS Lett. 124, 1-10 Barber, J. (1983) Membrane conformational changes due to phosphorylation and the control of energy transfer in photosynthesis. Photobiochem. Photobiophys. 5, 181-190
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Bennett, J. (1980) Chloroplast phosphoproteins. Evidence for a thylakoid-bound phosphoprotein phosphatase. Eur. J. Biochem. 104, 85-89 Bennett, J. (1983) Regulation of photosynthesis by reversible phosphorylation of the light-harvesting a/b protein. Biochem. J. 212, 1-13 Bennett, J., Steinback, K.E., Arntzen, C.J. (1980) Chloroplast phosphoproteins: regulation of excitation energy transfer by phosphorylation of thylakoid membranes. Proc. Natl. Acad. Sci. USA 77, 5253-5257 Bjorkman, O., Holmgrcn, P. (1963) Adaptability of the photosynthetic apparatus to light intensity in ecotypes from exposed and shaded habitats. Physiol. Plant. 16, 889-894 Critchley, C. (1981) Studies on the mechanism of photoinhibition in higher plants. 1. Effects of high light intensity on chloroplast activities in cucumber adapted to low light. Plant Physiol. 67, 1161-1165 Critchlcy, C., Smillie, R.M. (1981) Leaf chlorophyll fluorescence as an indicator of photoinhibition in Cucumis sativus L. Aust. J. Plant Physiol. 8, 133 141 Fernyhough, P., Foyer, C.H., Horton, P. (1984) Increase in level of thylakoid protein phosphorylation in maize mesophyll chloroplasts by decrease in the transthylakoid proton gradient. FEBS Lett. 176, 351-353 Fork, D.C., Oquist, G., Powles, S.B. (1981) Photoinhibition in bean: A fluorescence analysis. Carnegie Inst. Washington Yearb. 80, 52-57 Horton, P. (1983a) Control of chloroplast electron transfer by phosphorylation of thylakoid proteins. FEBS Lett. 152, 47-52 Horton, P. (1983b) Relationships between electron transfer and carbon assimilation; simultaneous measurement of chlorophyll fluorescence transthylakoid pH gradient and 02 evolution in isolated chloroplasts. Proc. R. Soc. London Ser. B. 217, 405-416 Horton, P. (1984) Interactions between electron transfer and carbon assimilation. In: Photosynthetic mechanisms and the environment, Barber, J., Baker, N.R., eds. Elsevier Biomedical Press, Amsterdam, in press Horton, P., Allen, J.F., Black, M.T., Bennett, J. (1981) Regulation of phosphorylation of chloroplast membrane polypeptides by the redox state of plastoquinone. FEBS Lett. 125, 193-196 Horton, P., Black, M.T. (1980) Activation of adenosine 5'triphosphate induced fluorescence quenching by reduced plastoquinone. The basis of state 1 to state 2 transitions in chloroplasts. FEBS Lett. 119, 141-144 Horton, P., Black, M.T. (1981) Light dependent quenching of chlorophyll fluorescence in pea chloroplasts induced by adenosine 5'-triphosphate. Biochim. Biophys. Acta 635, 53-62 Horton, P., Foyer, C.H. (1982) Relationships between protein phosphorylation and electron transport in the reconstituted chloroplast system. Biochem. J. 210, 51%521 Horton, P., Lee, P. (1983) Stimulation of a cyclic electron transfer pathway around photosystem 2 by phosphorylation of chloroplast thylakoid proteins. FEBS Lett. 162, 81-84 Horton, P., Lee, P. (1984) Phosphorylation of chloroplast thylakoids decreases the maximum capacity of photosystem 2 electron transfer. Biochim. Biophys. Acta, 767, 563-567 Jursinic, P.A., Kyle, D.J. (1983) Changes in the redox state of the secondary acceptor of photosystem II associated with light-induced thylakoid protein phosphorylation. Biochim. Biophys. Acta 723, 37-44 Kyle, D.J., Ohad, I., Guy, R., Arntzen, C.J. (1984) Selective thylakoid protein damage and repair during photoinhibition. In: Advances in photosynthesis research, vol. 3, pp. 67 70, C. Sybesma, ed. Nijhoff/Junk Publishers, The Hague
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P. Horton and P. Lee: Protein phosphorylation and photoinhibition in thylakoids
Lavorel, J., Etienne, A.-L. (1977) In vivo chlorophyll fluorescence. In: Primary process of photosynthesis, pp. 203-268, J. Barber, ed. Elsevier/North Holland Biomedical Press, Amsterdam Malkin, S., Jones, L.W. (1968) Photoinhibition and excitation quenching in photosystem 2 of photosynthesis from fluorescence induction measurements. Biochim. Biophys. Acta 162, 297-299 Melis, A. (1984) Light regulation of photosynthetic membrane structure, organisation and function. J. Cell. Biochem. 24, 271-285 Ogren, E., 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 Osmond, B. (1981) Photorespiration and photoinhibition. Some implications for the energetics of photosynthesis. Biochim. Biophys. Acta 639, 77-98 Powles, S.B. (1984) Photoinhibition of photosynthesis induced by visible light. Annu. Rev. Plant Physiol. 35, 15-44 Powles, S.B., Bjorkman, O. (1982) Photoinhibition of photosynthesis: effect on chlorophyll fluorescence at 77 K in intact leaves and in chloroplast membranes of Nerium oleander. Planta 156, 97-107 Powles, S.B., Critchley, C. (1980) Effect of light intensity during growth on photoinhibition of intact attached bean leaflets. Plant Physiol. 65, 1181-1187
Powles, S.B., Osmond, C.B,, Thorne, S.W. (1979) Photoinhibition of intact attached leaves of C3 plants illuminated in the absence of both carbon dioxide and of photorespiration. Plant Physiol. 64, 982-988 Powles, S.B., Thorne, S.W. (1981) Effect of high-light treatment in inducing photoinhibition of photosynthesis in intact leaves of low-light grown Phaseolus vulgaris and Lastreopsis microsora. Planta 152, 471M77 Renger, G., Koike, H., Yuasa, M., Inoue, Y. (1983) Studies on the mechanism of the fluorescence decline induced by strong actinic light in PSII particles under different redox conditions. FEBS Lett. 163, 89-93 Ridley, S.M. (1977) Interaction of chloroplasts with inhibitiors. Induction of chlorosis by diuron during prolonged illumination in vitro. Plant Physiol. 59, 724-732 Ridley, S.M., Horton, P. (1984) DCMU-induced photodestruction of pigments associated with an inhibition of photosystem 1 cyclic electron flow: a working hypothesis. Z. Naturforsch. Tell C 39, 351-353 Shocat, S., Owens, G.C., Hubert, P., Ohad, I. (1982) The dichlorophenyldimethylurea-binding site in thylakoids of Chlamydomonas reinhardi. Role of photosystem II reaction center and phosphorylation of the 32-35 kilodalton polypeptide in the formation of the high affinity binding site. Biochim. Biophys. Acta 681, 21-31 Received 19 July; accepted 20 December 1984
