Photosynthesis Research 10." 2 4 3 - 2 5 3 (1986) © Martinus N i j h o f f Publishers, Dordrecht - Printed in the Netherlands
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REGULATION OF ENERGY TRANSFER BY CATIONS AND PROTEIN PHOSPHORYLATION IN RELATION TO THYLAKOID MEMBRANE ORGANISATION J.
BARBER
Imperial College of Science and Technology, Department of Pure and Applied Biology, Prince Consort Road, London, SW7 2BB, United Kingdom
ABSTRACT
A brief review is given of the state of knowledge which indicates that the State I-State II transition in higher plants and green algae is due to the reversible phosphorylation of the chlorophyll a/b light harvesting complex. The importance of membrane reorganisational changes in this process is discussed in terms of changes in electrostatic parameters as emphasised by the interplay of the effect of phosphorylation and the background levels of cations surrounding the membrane. It is argued that recognition of this interplay is vital when using the bipartite or tripartite models of Butler to obtain quantitative information of energy transfer between the various pigment complexes. I. I N T R O D U C T I O N Without doubt, the bipartite and tripartite models developed by Warren Butler (25) to explain changes in chlorophyll fluorescence yields under different conditions form a firm basis for investigating energy distribution between the different pigment systems of photosynthetic organisms. The models are of particular value in attempting to account for the remarkable constancy of the steady-state quantum yield of photosynthesis over a broad spectral region where photosystem two (PSII) and photosystem one (PSI) have quite different absorption properties (71)o This phenomenon has been termed 'the quantum yield anomaly' and its explanation did not become clear until the discovery of the State IState II transition (24). This transitions represent a short term adaptation mechanism for finely tuning the distribution of light to PSII and PSI in order to achieve optimal rates of photosynthesis under the particular lighting conditions which prevail. Warren Butler set himself the task of formulating theoretical expressions which could be used to give quantitative information about energy distribution between PSII and PSI over a range of experimental conditions. For example, not only did he, in collaboration with A.C. Ley, confirm the existence of State IState II transitions in the red alga Porphyridium cruentum, but was also able to obtain for this organism estimates of changes in energy transfer between PSII and PSI (known as spillover) which correlated quantitatively with changes in quantum yields of photochemistry and oxygen evolution (25). This outstanding work was complemented by related studies using isolated thylakoid membranes of higher plant chloroplasts. It had been shown by Murata (70), from chlorophyll fluorescence measurements, that the distribution of l i ~ t between PSII and PSI could be altered by changing the level of Mg In a suspension of isolated thylakoids. This effect was considered to be reminiscent of the in vivo State changes and therefore was an ideal experimental system
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for Butler to test the analytical expressions derived from his kinetic models (25). By monitoring chlorophyll fluorescence, and by using the bipartite model he found that for wavelengths ranging from 540nm to 675nm, the distribution of energy between PSII and PSI was approximately 3+ 73% and 27%, respectively, when 6mM Mg was present. With the same ionic conditions the degree of energy transfer from PSII to PSI was 7% w h ~ the PSII traps were open but increased to 23% when fully closed. If Mg was absent the distribution was 68% to PSII and 32% to PSI, with spillover changing from 12% to 28% on closing the PSII traps. Of importance in this analysis is that B ~ l e r had shown, contrary to Murata's conclusions, that the effect of Mg was not only to change the extent of spillover but also to alter the absorption cross-sections of PSII and PSI. Later, 2+ when the tripartite model was adopted, the effect of Mg could be further interpreted in terms of changes in energy transfer between the light harvesting chlorophyll a/b complexes (LHC-II) and the PSII and PSI complexes (see ref. 25). Clearly the models and equations developed by Butler and the experimental approaches he adopted to exploit them provide an important framework for studying the regulation of energy distribution in quantitative terms. Indeed, today Butler's models find considerable usefulness in a range of different studies, including stress physiology (72,74). However, Warren Butler made no serious attempt to link his kinetic models with the structural organisation of pigment-protein complexes and with the organisation of the membrane in which they are located. This i T+ surprising because, as indicated above, he made extensive use of Mg to perturb fluorescence yields and alter the rate constants and yield parameters which occur in his mathematical expressions. But the addition of Mg to isolated thylakoids causes extensive conformational changes involving the lateral movements Of intramembrane particles (77) and restacking into granal and stromal lamellae (53). A thorough study of the relationships between conformational changes and chlorophyll fluorescence gave rise to a model in ~hich changes in energy distribution could be understood in terms of alterations in the spatial relationships between LHC-II, PSII and PSI complexes (9). Moreover, it was clearly demonstrated that these changes could be induced by a range of different cations and were controlled by the interplay of electrostatic and electrodynamic forces (10). Coupled with the emergence of a structural model based on surface electrical properties came the finding that protein phosphorylation, rather than changes in cation levels, underlie the mechanism by which higher plants and green algae regulate energy distribution between PSII and PSI as manifested in the State transitions (20,48). In this review paper I present, as concisely as possible, the evidence that it is protein phosphorylation which gives rise to the 'quantum yield anomaly' in higher plants and algae. The subject has been reviewed previously (2,3,13,14,19,40,45,78), but in this case I want to further emphasise the concept of a dynamic membrane system in which the lateral movements of various chlorophyll protein complexes is carefully regulated by the manipulation of their surface charges so as to alter the balance between attractive and repulsive forces. Consideration of these lateral movements and resulting changes in energy transfer between pigment complexes is in the spirit of the fluid-mosaic model of membrane structure but can, in principle, also be used for extrapolation of the parameters which appear in Butler's models.
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2. P R O T E I N P H O S P H O R Y L A T I O N
In 1977, Bennett (15) reported t ~ t when isolated intact chloroplasts were incubated in the light with --P-orthophosphate, several thylakoid proteins became phosphorylated. The most conspicuous were the polypeptides of LHC-II which have apparent molecular weights of approximately 27kDa and 25kDa although other phosphoproteins with molecular weights of about 9kDa, 33-35kDa and 45kDa were also detected. Since then considerable knowledge of the phosphorylation properties of these polypeptides, especially LHC-II, has accumulated and is summarised below. 2.1 (a) LHC-II (i) The kinase responsible for phosphorylating LHC-II is membrane bound (17), is removable by a detergent mixture of cholate and octylglucoside (1,62) and may have a molecular weight of 50kDa (31). (ii) When in the thylakoid membrane the kinase is activated by light below 700nm (82) or in darkness in the presence of reducing agents such as ferredoxin (17), dithionite (4) or duroquinol (5). (iii) The kinase can be fully activated in the presence of uncouplers but can be partially inhibited by the formation of a pH gradient across the thylakoid membrane (33). (iv) The kinase requires Mg 2+ for its activation with a C for maximum activity of 0.3mM for isolated pea thylakoids stacked in ~he presence of 10mM lysyl-lysine (81). (v) The kinase has a K for ATP of about 90uM (20) although its m activity is modified by the relative levels of other adenylates (8). (vi) The kinase probably has thiol groups at its active site (68). (vii) The phosphorylation site is at the threonine residues of the surface exposed N-terminal segment of the LHC-II polypeptides and mild trypsin treatment removes a 2kDa fragment containing the phosphorylatable threonines (16,69). (viii) The ability of the kinase to phosphorylate LHC-II seems to be enhanced when the thylakoids are stacked, as compared with the unstacked conformation (81). (ix) The kinase activity can be stimulated by low levels of Zn 2+ (64). (x) With spinach LHC-II it has been found that there is a preferential phosphorylation of the 25kDa relative to the 27kDa polypeptide (59). (xi) In contrast to the kinase, the phosphatase, which brings about dephosphorylation of LHC-II is insensitive to illumination conditions or to redox potentials, although it is inhibited by molybdate and fluoride (18). (xii) Like the LHC-II kinase, the phosphatase is membrane bound (18). (xiii) The kinase(s) responsible for the phosphorylation of other membrane polypeptides differ to that _acting on LHC-II in having a different conCentration requirement for Mg 2+ (81) and ATP (22), sensitivity to Zn 2+ (64) and being less sensitive to the nucleotide affinity inhibitor, 5'-p-fluorosulphonyl-benzyl-adenosine (31). Recently, two different kinases have been isolated. These kinase did not phosphorylate LHC-II but a crude isolate did (61,62). (xiv) As with the LHC-II kinase, the additional kinase activity is stimulated by light and reducing conditions in the dark (16,17). 2.2. Other polypeptides The identity of the
other
phosphoproteins
is
still
a
matter
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of investigation. It seems likely that the 9kDa polypeptide is neither the DCCD-binding subunit of the coupling factor (80) nor the apoprotein of cytochrome b-559 (88). However, like the other main phosphoproteins, this low molecular weight polypeptide is probably a component of PSII. The protein(s) in the region of 33kDa has been suggested to be the Q_ herbicide-binding protein often referred to as the psbA gene product or the D1 protein (76,87). However, very recently new evidence has emerged that the 33kDa phosphoprotein is the product of the psbD gene, known as the D2 protein (29,89). To my knowledge there have been no attempts to identify the origin of the higher molecular weight phosphoproteins in the region of 45kDa, although they do seem to form a part of the PSII core complex (67). 3o F U N C T I O N A L R O L E FOR THE PHOSPHORYLATION OF LHC-II There are a number of key observations which indicate that the phosphorylation of LHC-II is the underlying mechanism for controlling energy distribution between PSII and PSI during the State transitions.
(i) The light induced phosphorylation of LHC-II is inhibited by DCMU but not by low concentrations of DBMIB (4) (ii) The phosphorylation of LHC-II occurs in light which is preferentially absorbed by PSII and is inhibited by far-red PSI absorbing light (48,82). (iii) The redox potential for the dark phosphorylation of LHC-II has a midpoint potential of 0mV and titrates with a requirement of 2e/2H + (47,48). These observations led to the conclusion that the phosphorylation of LHC-II only occurs when the plastoquinone pool is in a reduced state (see Fig. I). Further observations listed below link the LHC-II phosphorylation process with changes in the quantum yields of PSI and PSII activities, indicative of an in vivo regulatory process. (i) LHC-II phosphorylation reduces the quantum yield of PSII mediated electron flow (32,52,79) and the chlorophyll fluorescence with maxima below 700nm at both room temperature (20,31,48,49) and 77K (20,54). (ii) LHC-II phosphorylation increases the quantum yield of PSI mediated electron flow (32,50), primary charge separation in PSI (83) and 77K chlorophyll fluorescence above 700nm (20,54). (iii) After LHC-II phosphorylation there is an increase in the ability of chlorophyll-b to excite PSI fluorescence at room temperature (56) and 77K (57). (iv) The effect of LHC-II phosphorylation is also to reduce overall connectivity between LHC-II-PSII complexes (55,73,86). All these findings support a scheme in which a population of LHC-II, which on undergoing phosphorylation due to the over-reduction of the plastoquinone pool, can redirect excess excitation energy from PSII to PSI (4,20,48). In this way the redox poise of the plastoquinone pool (or another intersystem component with a similar redox potential) can delicately regulate the distribution 6f quanta to PSII and PSI and therefore maintain a maximum quantum yield of electron transport for any particular lighting conditions. As suggested in refs. (4) and (48) the optimisation of energy distribution by the LHC-II phosphorylation/ dephosphorylation process could be the basis of the in vivo State I-State II phenomenon. Indeed, studies with intact leaves (26,28) and with the green unicellular alga Chlorella (75) support this relationship. However, to-date the links between the in vitro and in vivo mechanisms have been
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qualitative and, indeed, some have claimed that LHC-II phosphorylation does not bring about any increase in the quantum yield of PSI photochemistry (41) as would be required for a State I to State II transition to occur. 4.
S T R U C T U R A L R E O R G A N I S A T I O N IN R E S P O N S E T O L H C - I I PHOSPHORYLATION Accepting that the phosphorylation/dephosphorylation of LHC-II is the basis of the State transitions in higher plants and green algae, how can this be understood in terms of our modern concepts of thylakoid membrane structure? The answer to this question comes from considering three important features of the chloroplast thylakoid: (i) Under non-phosphorylating conditions (dark or far-red light) the majority of LHC-II, together with the PSII core complexes, are located in the appressed lamellae of the grana while PSI is predominantly found in the non-appressed membranes which constitute the stromal and endgranal lamellae (6,10,). The LHC-II-PSII inter-connecting domains within the appressed regions probably give rise to PSII -units while the low levels of separate LHC-II-PSII complexes in the non-appressed regions can be equated with PSIIunits (65,66). (ii) The lateral separation of complexes, as described above, is due mainly to differences in the electrical charge properties of their outward facing surfaces (9,10). Arguments have already been presented based on the principles of electrostatic and electrodynamic interactions (11,12) that those components in the appressed regions carry a low net surface charge density while those in the non-appressed regions are more electrically charged. (iii) The thylakoid is a dynamic system having a lipid matrix of high fluidity (35) such that perturbation of the electrical characteristics of the exposed surfaces of the intrinsic protein complexes will induce changes in their spatial relationships in response to alterations in coulombic forces (12). Indeed, the fatty acids of the polar lipids are extremely unsaturated (36) and variation in coulombic forces due to changes in ionic levels do bring about lateral movements of protein complexes (9,77). If these three features are taken at face value then it is reasonable to speculate that the introduction of additional negative charges onto the surface of LHC-II complexes by phosphorylation will destabilize their aggregation within the appressed regions and force them to migrate laterally into the more fluid non-appressed membranes where they will statistically interact with PSI via energy transfer mechanisms (10,13,14). There may be no requirement for them to become physically attached to PSI and there is no evidence for this. They could, however, transfer energy to PSI via PSII -units. The removal of a fraction of the LHC-II population from the appressed regions would be expected to decrease energy transfer between LHC-II-PSII as is observed (55,73). Dephosphorylation of LHC-II would lead to a return to its original surface charge properties and thus to its re-establishment as a part of the LHC-II-PSII domain in the appressed regions. There are a number of observations which give strong support to this lateral mobility model and its dependence on surface charge properties and membrane fluidity. (i) Titration of the yield of chlorophyll fluorescence with mono-, diand tri-valent cations in the presence of DCMU indicates an overall increase in the surface charge density after LHC-II phosphorylation (81, 85).
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(ii) After LHC-II phosphorylation the chlorophyll a/b ratio decreases in the non-appressed lamellae (28) due to an increase in the LHC-II level in this membrane r e ~ o n (57). (iii) Radiolabelling with --P indicates higher specific activities of LHC-II in the non-appressed membranes (7) which are time dependent in a pulse-chase experiment (57,59) and which occur mainly on the 25kDa apopolypeptide (59) (iv) Freeze-fracture analyses show that LHC-II phosphorylation is accompanied by changes in the distribution of particles in fracture faces indicative of a proportion of LHC-II complexes migrating from appressed to non-appressed membranes (58). (v) The phosphorylation induced lateral movement of LHC-II and not the phosphorylation process itself, is dependent on the fluidity of the thylakoid membrane (37). (vi) The extent of reorganisation of the membrane, and therefore degree of interactions between different chlorophyll complexes, is dependent on the background cation levels as would be expected for a process under electrostatic control. At sub-optimal background levels of • 2+ cations (e.g. 1-2mM Mg ), the effect of LHC-II phosphorylation is to cause considerable membrane unstacking (84) and intermixing of PSI, PSII and LHC-II complexes as indicated by changes in both absorption crosssections and spillover, as monitored by chlorophyll fluorescence (50, 51,84). At higher levels of cations (e.g. above 5mM) the degree of membrane unstacking is much less, about 10% (28,39,58,85) and it seems that the absorption cross-section changes predominate, indicative of only LHC-II movement (43,50,84) Figure I presents a diagrammatic representation of the structural changes which seem to occur when LHC-II is phosphorylated in the presence of a high level of cations which effectively screen surface electrical charges (e.g. above 5mM). Under these conditions a pool of LHC-II acts as a mobile antenna system able to regulate the absorption cross-section of PSII and PSI. Although absorption cross-section changes dominate there is also evidence for some changes in the extent of spillover (27,39,55,86). The pool of LHC-II involved in this process is probably only weakly associated with PSII in the appressed membranes and contains a high level of the 25kDa apoprotein compared with the 27kDa (59). In contrast, the more tightly held LHC-II is enriched in the 27kDa apoprotein (59). At lower ca ion levels, when electrostatic screening is poorer (e.g. below 5mM Mg +), the effect of LHC-II phosphorYlation is more dramatic leading to a considerable degree of unstacking and intermixing of complexes (85). Under these conditions both absorption crosssection and spillover changes occur (38). All evidence to-date, whether obtained with higher plants (27,63) or with green algae (42) indicate that the cation level of chloroplasts is sufficiently high that the in vivo State transitions involve mainly changes in absorption crosssections due to LHC-II movement. The only exception to this is with chloroplasts isolated from developing leaves where the LHC-II phosphorylation seems to induce _~pillover, as well as absorption cross-section changes, even at high Mg 2 levels (23). Although the model given in Figure I indicates the control of LHC-II phosphorylation/dephosphorylation b y the redox state of plastoquinone, it does not take into account the possible role of chloroplast adenylate levels, transmembrane pH gradients and NADPH/ATP requirements, all of which effect kinase activity and may also be important in controlling excitation distribution to PSII and PSI (3,46). The model also does not
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Unstacking and randomisation of protein complexes due toincreased coulomblc repulsion resulting from poor electrostatic screening of surface electrical charges. Energy transfer between PSII and PSI is increased and absorption crosssection changes also occur.
C3+>cZ+>c+ STATE I Maximum stacking and maximum separation of complexes due to differences In surface charge densities and therefore dlfferentlal electrostatic screening. Mobile LHC-II is nonphosphorylated and acts as antenna to PSII within appressed regions.
kinast PQ
,phosphatase +K
EXCESS
]
PQH2
STATE I I Partial unstacklng and lateral movement of LHC-II in response to protein phosphorylatlondue to increased coulomblc repulsion. The effect is to increase absorption cross section of PSI at ~he expense of PSII. At Intermedlatecation levels the phosphorylatlonof LHC-II can bring about a more extensive unstacklng and intermixing of complexes.
(D
LHC-II+ PSII core complex
Q
PSI complex
LHC-II mobile (unphosphorylated)
?
CFo-CF I complex
LHC-II mobile (phosphorylated)
O
Cyt b6-f complex
P F I G U R E I. Diagrammatic representation organisation in response to protein cation levels.
of changes in thylakoid membrane p h o s p h o r y l a t i o n a n d m o d i f i c a t i o n of
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include the possible regulatory function of the other phosphoproteins or the membrane locations of the kinase(s) and phosphatase(s) involved. Work to understand these latter considerations has started (34,44,46,52,87) but no clear picture has emerged which can match the clarity of the relatively simple concepts presented in Figure I. 5,
CONCLUSIONS The segregation of LHC-II PSII and PSI into the appressed and nonappressed regions of the thylakoids of higher plants and green algal chloroplasts allows these photosynthetic organisms to regulate excitation distribution in such a way as to obtain optimal rates of electron flow under limiting light conditions. The process of changing surface charge densities by protein phosphorylation/dephosphorylation and therefore inducing reshuffling of LHC-II between PSII and PSI is elegant, not only in its conceptional simplicity, but also because it can be described in physico-chemical terms. However, what is sadly lacking at present is a rigorous quantitative analysis of energy transfer changes which occur between the various pigment beds. It is in this context that the bipartite and tripartite models, devised by Warren Butler, have such a lot to offer. Analyses using Butler's models of the LHC-II phosphorylation phenomenon has been restricted to a few studies (23,38). Further work is needed, but one word of caution is that before undertaking such a study it is important to recognise that the interactions between the pigment complexes is an interplay of the increase in surface charge density by LHC phosphorylation and the background cation levels screening these surface electrica~ charges. Therefore both parameters must be given consideration. Also because the thylakoid membrane structure is dynamic and under the control of electrostatic forces great care should be taken in isolating and manipulating chloroplasts for experiments. Leaves should be pretreated in order to standardise the phosphorylation and organisation of their thylakoids (e.g. preillumination with far-red ligh~l. If the thylakoids are then isolated into media containing 5mM Mg-- they are likely to maintain their in vivo organisation. However, if they are allowed to unstack in low salt containing media and then restacked by adding 5mM ~+ Mg they may not precisely regain the configuration of the in vivo state. Unfortunately as yet the 'phosphorylation-mobile antenna' properties of LHC-II cannot be extrapolated to those organisms which show State transitions but do not contain LHC-II. Studies by Biggins and colleagues (21) concluded that no phosphorylation processes can be correlated with State I-State II transitions in Porphyridium cruentum and previous to this Ley and Butler (60) had shown that in this organism the State transitions involved large changes in extent of spillover rather than alterations in absorption cross-sections. Unfortunately then, the 'quantum yield anomaly' is not understood in structural terms for the phycobilin containing organisms having no obvious differentiation into appressed and nonappressed membranes. Nevertheless, Warren Butler's thorough studies with Porphyridium established without doubt that this organism can regulate energy transfer between PSII and PSI and that this regulation can be described by his models. We can look forward to using this knowledge as a basis for obtaining explanations about the conformational changes which occur and Comparing the similarities and differences with higher plant and green algal systems.
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6.
ACKNOWLEDGEMENTS I wish to thank both the Science and Engineering Research Council (SERC) and the Agricultural and Food Research Council (AFRC) for financial support. I would also like to thank my colleagues for their support during the course of the writing of this review, particularly Alison Teller and Julian whitelegge. REFERENCES
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78. 79° 80° 81. 82. 83° 84
o
85. 86° 87° 88° ~9°
Staehelin LA, Arntzen CJ (1983) J. Cell Biol. 9 7 : 1 3 2 7 - 1 3 3 7 Steinback KE, Bose S, Kyle DJ (1982) Arch. Biochem. Biophys. 216: 356-361 Suss, KH (1980) FEBS Lett. 112:255-259 Telfer A (1986) Biochem Soc. Trans. 14:52-53 Teller A, Allen JF, Barber J, Bennett J (1983) Biochim. Biophys. Acta 722:176-181 Telfer A, Bottin H, Barber J, Mathis P (1984) Biochim. Biophys. Acta 764: 324-330 Telfer A, Hodges M, Barber J (1983) Biochim. Biophys. Acta 724: 167-175 Telfer A, Hodges M, Millner PA, Barber J (1984) Biochim Biophys Acta 766: 554-562 Torti F, Gerola PD, Jennings RC (1984) Biochim. Biophys. Acta 767: 321-325 Vermaas WFJ, Steinback KE, Arntzen CJ (1984) Arch. Biochem. Biophys. 231: 226-237 Widger WR, Farchaus JW, Cramer WA, Dilley RA (1984) Arch. Biochem° Biophys. 233:72-79 wildner GF, Dmoch R, Fiebig C, Dedner N (1985) Quinones in Photosynthetic Membranes. Abstract of meeting held in Saint Lambert des Bois •
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REGULATION OF ENERGY TRANSFER BY CATIONS AND PROTEIN PHOSPHORYLATION IN RELATION TO THYLAKOID MEMBRANE ORGANISATION J.
BARBER
Imperial College of Science and Technology, Department of Pure and Applied Biology, Prince Consort Road, London, SW7 2BB, United Kingdom
ABSTRACT
A brief review is given of the state of knowledge which indicates that the State I-State II transition in higher plants and green algae is due to the reversible phosphorylation of the chlorophyll a/b light harvesting complex. The importance of membrane reorganisational changes in this process is discussed in terms of changes in electrostatic parameters as emphasised by the interplay of the effect of phosphorylation and the background levels of cations surrounding the membrane. It is argued that recognition of this interplay is vital when using the bipartite or tripartite models of Butler to obtain quantitative information of energy transfer between the various pigment complexes. I. I N T R O D U C T I O N Without doubt, the bipartite and tripartite models developed by Warren Butler (25) to explain changes in chlorophyll fluorescence yields under different conditions form a firm basis for investigating energy distribution between the different pigment systems of photosynthetic organisms. The models are of particular value in attempting to account for the remarkable constancy of the steady-state quantum yield of photosynthesis over a broad spectral region where photosystem two (PSII) and photosystem one (PSI) have quite different absorption properties (71)o This phenomenon has been termed 'the quantum yield anomaly' and its explanation did not become clear until the discovery of the State IState II transition (24). This transitions represent a short term adaptation mechanism for finely tuning the distribution of light to PSII and PSI in order to achieve optimal rates of photosynthesis under the particular lighting conditions which prevail. Warren Butler set himself the task of formulating theoretical expressions which could be used to give quantitative information about energy distribution between PSII and PSI over a range of experimental conditions. For example, not only did he, in collaboration with A.C. Ley, confirm the existence of State IState II transitions in the red alga Porphyridium cruentum, but was also able to obtain for this organism estimates of changes in energy transfer between PSII and PSI (known as spillover) which correlated quantitatively with changes in quantum yields of photochemistry and oxygen evolution (25). This outstanding work was complemented by related studies using isolated thylakoid membranes of higher plant chloroplasts. It had been shown by Murata (70), from chlorophyll fluorescence measurements, that the distribution of l i ~ t between PSII and PSI could be altered by changing the level of Mg In a suspension of isolated thylakoids. This effect was considered to be reminiscent of the in vivo State changes and therefore was an ideal experimental system
[97]
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[98]
for Butler to test the analytical expressions derived from his kinetic models (25). By monitoring chlorophyll fluorescence, and by using the bipartite model he found that for wavelengths ranging from 540nm to 675nm, the distribution of energy between PSII and PSI was approximately 3+ 73% and 27%, respectively, when 6mM Mg was present. With the same ionic conditions the degree of energy transfer from PSII to PSI was 7% w h ~ the PSII traps were open but increased to 23% when fully closed. If Mg was absent the distribution was 68% to PSII and 32% to PSI, with spillover changing from 12% to 28% on closing the PSII traps. Of importance in this analysis is that B ~ l e r had shown, contrary to Murata's conclusions, that the effect of Mg was not only to change the extent of spillover but also to alter the absorption cross-sections of PSII and PSI. Later, 2+ when the tripartite model was adopted, the effect of Mg could be further interpreted in terms of changes in energy transfer between the light harvesting chlorophyll a/b complexes (LHC-II) and the PSII and PSI complexes (see ref. 25). Clearly the models and equations developed by Butler and the experimental approaches he adopted to exploit them provide an important framework for studying the regulation of energy distribution in quantitative terms. Indeed, today Butler's models find considerable usefulness in a range of different studies, including stress physiology (72,74). However, Warren Butler made no serious attempt to link his kinetic models with the structural organisation of pigment-protein complexes and with the organisation of the membrane in which they are located. This i T+ surprising because, as indicated above, he made extensive use of Mg to perturb fluorescence yields and alter the rate constants and yield parameters which occur in his mathematical expressions. But the addition of Mg to isolated thylakoids causes extensive conformational changes involving the lateral movements Of intramembrane particles (77) and restacking into granal and stromal lamellae (53). A thorough study of the relationships between conformational changes and chlorophyll fluorescence gave rise to a model in ~hich changes in energy distribution could be understood in terms of alterations in the spatial relationships between LHC-II, PSII and PSI complexes (9). Moreover, it was clearly demonstrated that these changes could be induced by a range of different cations and were controlled by the interplay of electrostatic and electrodynamic forces (10). Coupled with the emergence of a structural model based on surface electrical properties came the finding that protein phosphorylation, rather than changes in cation levels, underlie the mechanism by which higher plants and green algae regulate energy distribution between PSII and PSI as manifested in the State transitions (20,48). In this review paper I present, as concisely as possible, the evidence that it is protein phosphorylation which gives rise to the 'quantum yield anomaly' in higher plants and algae. The subject has been reviewed previously (2,3,13,14,19,40,45,78), but in this case I want to further emphasise the concept of a dynamic membrane system in which the lateral movements of various chlorophyll protein complexes is carefully regulated by the manipulation of their surface charges so as to alter the balance between attractive and repulsive forces. Consideration of these lateral movements and resulting changes in energy transfer between pigment complexes is in the spirit of the fluid-mosaic model of membrane structure but can, in principle, also be used for extrapolation of the parameters which appear in Butler's models.
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2. P R O T E I N P H O S P H O R Y L A T I O N
In 1977, Bennett (15) reported t ~ t when isolated intact chloroplasts were incubated in the light with --P-orthophosphate, several thylakoid proteins became phosphorylated. The most conspicuous were the polypeptides of LHC-II which have apparent molecular weights of approximately 27kDa and 25kDa although other phosphoproteins with molecular weights of about 9kDa, 33-35kDa and 45kDa were also detected. Since then considerable knowledge of the phosphorylation properties of these polypeptides, especially LHC-II, has accumulated and is summarised below. 2.1 (a) LHC-II (i) The kinase responsible for phosphorylating LHC-II is membrane bound (17), is removable by a detergent mixture of cholate and octylglucoside (1,62) and may have a molecular weight of 50kDa (31). (ii) When in the thylakoid membrane the kinase is activated by light below 700nm (82) or in darkness in the presence of reducing agents such as ferredoxin (17), dithionite (4) or duroquinol (5). (iii) The kinase can be fully activated in the presence of uncouplers but can be partially inhibited by the formation of a pH gradient across the thylakoid membrane (33). (iv) The kinase requires Mg 2+ for its activation with a C for maximum activity of 0.3mM for isolated pea thylakoids stacked in ~he presence of 10mM lysyl-lysine (81). (v) The kinase has a K for ATP of about 90uM (20) although its m activity is modified by the relative levels of other adenylates (8). (vi) The kinase probably has thiol groups at its active site (68). (vii) The phosphorylation site is at the threonine residues of the surface exposed N-terminal segment of the LHC-II polypeptides and mild trypsin treatment removes a 2kDa fragment containing the phosphorylatable threonines (16,69). (viii) The ability of the kinase to phosphorylate LHC-II seems to be enhanced when the thylakoids are stacked, as compared with the unstacked conformation (81). (ix) The kinase activity can be stimulated by low levels of Zn 2+ (64). (x) With spinach LHC-II it has been found that there is a preferential phosphorylation of the 25kDa relative to the 27kDa polypeptide (59). (xi) In contrast to the kinase, the phosphatase, which brings about dephosphorylation of LHC-II is insensitive to illumination conditions or to redox potentials, although it is inhibited by molybdate and fluoride (18). (xii) Like the LHC-II kinase, the phosphatase is membrane bound (18). (xiii) The kinase(s) responsible for the phosphorylation of other membrane polypeptides differ to that _acting on LHC-II in having a different conCentration requirement for Mg 2+ (81) and ATP (22), sensitivity to Zn 2+ (64) and being less sensitive to the nucleotide affinity inhibitor, 5'-p-fluorosulphonyl-benzyl-adenosine (31). Recently, two different kinases have been isolated. These kinase did not phosphorylate LHC-II but a crude isolate did (61,62). (xiv) As with the LHC-II kinase, the additional kinase activity is stimulated by light and reducing conditions in the dark (16,17). 2.2. Other polypeptides The identity of the
other
phosphoproteins
is
still
a
matter
246
[100]
of investigation. It seems likely that the 9kDa polypeptide is neither the DCCD-binding subunit of the coupling factor (80) nor the apoprotein of cytochrome b-559 (88). However, like the other main phosphoproteins, this low molecular weight polypeptide is probably a component of PSII. The protein(s) in the region of 33kDa has been suggested to be the Q_ herbicide-binding protein often referred to as the psbA gene product or the D1 protein (76,87). However, very recently new evidence has emerged that the 33kDa phosphoprotein is the product of the psbD gene, known as the D2 protein (29,89). To my knowledge there have been no attempts to identify the origin of the higher molecular weight phosphoproteins in the region of 45kDa, although they do seem to form a part of the PSII core complex (67). 3o F U N C T I O N A L R O L E FOR THE PHOSPHORYLATION OF LHC-II There are a number of key observations which indicate that the phosphorylation of LHC-II is the underlying mechanism for controlling energy distribution between PSII and PSI during the State transitions.
(i) The light induced phosphorylation of LHC-II is inhibited by DCMU but not by low concentrations of DBMIB (4) (ii) The phosphorylation of LHC-II occurs in light which is preferentially absorbed by PSII and is inhibited by far-red PSI absorbing light (48,82). (iii) The redox potential for the dark phosphorylation of LHC-II has a midpoint potential of 0mV and titrates with a requirement of 2e/2H + (47,48). These observations led to the conclusion that the phosphorylation of LHC-II only occurs when the plastoquinone pool is in a reduced state (see Fig. I). Further observations listed below link the LHC-II phosphorylation process with changes in the quantum yields of PSI and PSII activities, indicative of an in vivo regulatory process. (i) LHC-II phosphorylation reduces the quantum yield of PSII mediated electron flow (32,52,79) and the chlorophyll fluorescence with maxima below 700nm at both room temperature (20,31,48,49) and 77K (20,54). (ii) LHC-II phosphorylation increases the quantum yield of PSI mediated electron flow (32,50), primary charge separation in PSI (83) and 77K chlorophyll fluorescence above 700nm (20,54). (iii) After LHC-II phosphorylation there is an increase in the ability of chlorophyll-b to excite PSI fluorescence at room temperature (56) and 77K (57). (iv) The effect of LHC-II phosphorylation is also to reduce overall connectivity between LHC-II-PSII complexes (55,73,86). All these findings support a scheme in which a population of LHC-II, which on undergoing phosphorylation due to the over-reduction of the plastoquinone pool, can redirect excess excitation energy from PSII to PSI (4,20,48). In this way the redox poise of the plastoquinone pool (or another intersystem component with a similar redox potential) can delicately regulate the distribution 6f quanta to PSII and PSI and therefore maintain a maximum quantum yield of electron transport for any particular lighting conditions. As suggested in refs. (4) and (48) the optimisation of energy distribution by the LHC-II phosphorylation/ dephosphorylation process could be the basis of the in vivo State I-State II phenomenon. Indeed, studies with intact leaves (26,28) and with the green unicellular alga Chlorella (75) support this relationship. However, to-date the links between the in vitro and in vivo mechanisms have been
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qualitative and, indeed, some have claimed that LHC-II phosphorylation does not bring about any increase in the quantum yield of PSI photochemistry (41) as would be required for a State I to State II transition to occur. 4.
S T R U C T U R A L R E O R G A N I S A T I O N IN R E S P O N S E T O L H C - I I PHOSPHORYLATION Accepting that the phosphorylation/dephosphorylation of LHC-II is the basis of the State transitions in higher plants and green algae, how can this be understood in terms of our modern concepts of thylakoid membrane structure? The answer to this question comes from considering three important features of the chloroplast thylakoid: (i) Under non-phosphorylating conditions (dark or far-red light) the majority of LHC-II, together with the PSII core complexes, are located in the appressed lamellae of the grana while PSI is predominantly found in the non-appressed membranes which constitute the stromal and endgranal lamellae (6,10,). The LHC-II-PSII inter-connecting domains within the appressed regions probably give rise to PSII -units while the low levels of separate LHC-II-PSII complexes in the non-appressed regions can be equated with PSIIunits (65,66). (ii) The lateral separation of complexes, as described above, is due mainly to differences in the electrical charge properties of their outward facing surfaces (9,10). Arguments have already been presented based on the principles of electrostatic and electrodynamic interactions (11,12) that those components in the appressed regions carry a low net surface charge density while those in the non-appressed regions are more electrically charged. (iii) The thylakoid is a dynamic system having a lipid matrix of high fluidity (35) such that perturbation of the electrical characteristics of the exposed surfaces of the intrinsic protein complexes will induce changes in their spatial relationships in response to alterations in coulombic forces (12). Indeed, the fatty acids of the polar lipids are extremely unsaturated (36) and variation in coulombic forces due to changes in ionic levels do bring about lateral movements of protein complexes (9,77). If these three features are taken at face value then it is reasonable to speculate that the introduction of additional negative charges onto the surface of LHC-II complexes by phosphorylation will destabilize their aggregation within the appressed regions and force them to migrate laterally into the more fluid non-appressed membranes where they will statistically interact with PSI via energy transfer mechanisms (10,13,14). There may be no requirement for them to become physically attached to PSI and there is no evidence for this. They could, however, transfer energy to PSI via PSII -units. The removal of a fraction of the LHC-II population from the appressed regions would be expected to decrease energy transfer between LHC-II-PSII as is observed (55,73). Dephosphorylation of LHC-II would lead to a return to its original surface charge properties and thus to its re-establishment as a part of the LHC-II-PSII domain in the appressed regions. There are a number of observations which give strong support to this lateral mobility model and its dependence on surface charge properties and membrane fluidity. (i) Titration of the yield of chlorophyll fluorescence with mono-, diand tri-valent cations in the presence of DCMU indicates an overall increase in the surface charge density after LHC-II phosphorylation (81, 85).
248
[102]
(ii) After LHC-II phosphorylation the chlorophyll a/b ratio decreases in the non-appressed lamellae (28) due to an increase in the LHC-II level in this membrane r e ~ o n (57). (iii) Radiolabelling with --P indicates higher specific activities of LHC-II in the non-appressed membranes (7) which are time dependent in a pulse-chase experiment (57,59) and which occur mainly on the 25kDa apopolypeptide (59) (iv) Freeze-fracture analyses show that LHC-II phosphorylation is accompanied by changes in the distribution of particles in fracture faces indicative of a proportion of LHC-II complexes migrating from appressed to non-appressed membranes (58). (v) The phosphorylation induced lateral movement of LHC-II and not the phosphorylation process itself, is dependent on the fluidity of the thylakoid membrane (37). (vi) The extent of reorganisation of the membrane, and therefore degree of interactions between different chlorophyll complexes, is dependent on the background cation levels as would be expected for a process under electrostatic control. At sub-optimal background levels of • 2+ cations (e.g. 1-2mM Mg ), the effect of LHC-II phosphorylation is to cause considerable membrane unstacking (84) and intermixing of PSI, PSII and LHC-II complexes as indicated by changes in both absorption crosssections and spillover, as monitored by chlorophyll fluorescence (50, 51,84). At higher levels of cations (e.g. above 5mM) the degree of membrane unstacking is much less, about 10% (28,39,58,85) and it seems that the absorption cross-section changes predominate, indicative of only LHC-II movement (43,50,84) Figure I presents a diagrammatic representation of the structural changes which seem to occur when LHC-II is phosphorylated in the presence of a high level of cations which effectively screen surface electrical charges (e.g. above 5mM). Under these conditions a pool of LHC-II acts as a mobile antenna system able to regulate the absorption cross-section of PSII and PSI. Although absorption cross-section changes dominate there is also evidence for some changes in the extent of spillover (27,39,55,86). The pool of LHC-II involved in this process is probably only weakly associated with PSII in the appressed membranes and contains a high level of the 25kDa apoprotein compared with the 27kDa (59). In contrast, the more tightly held LHC-II is enriched in the 27kDa apoprotein (59). At lower ca ion levels, when electrostatic screening is poorer (e.g. below 5mM Mg +), the effect of LHC-II phosphorYlation is more dramatic leading to a considerable degree of unstacking and intermixing of complexes (85). Under these conditions both absorption crosssection and spillover changes occur (38). All evidence to-date, whether obtained with higher plants (27,63) or with green algae (42) indicate that the cation level of chloroplasts is sufficiently high that the in vivo State transitions involve mainly changes in absorption crosssections due to LHC-II movement. The only exception to this is with chloroplasts isolated from developing leaves where the LHC-II phosphorylation seems to induce _~pillover, as well as absorption cross-section changes, even at high Mg 2 levels (23). Although the model given in Figure I indicates the control of LHC-II phosphorylation/dephosphorylation b y the redox state of plastoquinone, it does not take into account the possible role of chloroplast adenylate levels, transmembrane pH gradients and NADPH/ATP requirements, all of which effect kinase activity and may also be important in controlling excitation distribution to PSII and PSI (3,46). The model also does not
[1031
249
Unstacking and randomisation of protein complexes due toincreased coulomblc repulsion resulting from poor electrostatic screening of surface electrical charges. Energy transfer between PSII and PSI is increased and absorption crosssection changes also occur.
C3+>cZ+>c+ STATE I Maximum stacking and maximum separation of complexes due to differences In surface charge densities and therefore dlfferentlal electrostatic screening. Mobile LHC-II is nonphosphorylated and acts as antenna to PSII within appressed regions.
kinast PQ
,phosphatase +K
EXCESS
]
PQH2
STATE I I Partial unstacklng and lateral movement of LHC-II in response to protein phosphorylatlondue to increased coulomblc repulsion. The effect is to increase absorption cross section of PSI at ~he expense of PSII. At Intermedlatecation levels the phosphorylatlonof LHC-II can bring about a more extensive unstacklng and intermixing of complexes.
(D
LHC-II+ PSII core complex
Q
PSI complex
LHC-II mobile (unphosphorylated)
?
CFo-CF I complex
LHC-II mobile (phosphorylated)
O
Cyt b6-f complex
P F I G U R E I. Diagrammatic representation organisation in response to protein cation levels.
of changes in thylakoid membrane p h o s p h o r y l a t i o n a n d m o d i f i c a t i o n of
250
[104]
include the possible regulatory function of the other phosphoproteins or the membrane locations of the kinase(s) and phosphatase(s) involved. Work to understand these latter considerations has started (34,44,46,52,87) but no clear picture has emerged which can match the clarity of the relatively simple concepts presented in Figure I. 5,
CONCLUSIONS The segregation of LHC-II PSII and PSI into the appressed and nonappressed regions of the thylakoids of higher plants and green algal chloroplasts allows these photosynthetic organisms to regulate excitation distribution in such a way as to obtain optimal rates of electron flow under limiting light conditions. The process of changing surface charge densities by protein phosphorylation/dephosphorylation and therefore inducing reshuffling of LHC-II between PSII and PSI is elegant, not only in its conceptional simplicity, but also because it can be described in physico-chemical terms. However, what is sadly lacking at present is a rigorous quantitative analysis of energy transfer changes which occur between the various pigment beds. It is in this context that the bipartite and tripartite models, devised by Warren Butler, have such a lot to offer. Analyses using Butler's models of the LHC-II phosphorylation phenomenon has been restricted to a few studies (23,38). Further work is needed, but one word of caution is that before undertaking such a study it is important to recognise that the interactions between the pigment complexes is an interplay of the increase in surface charge density by LHC phosphorylation and the background cation levels screening these surface electrica~ charges. Therefore both parameters must be given consideration. Also because the thylakoid membrane structure is dynamic and under the control of electrostatic forces great care should be taken in isolating and manipulating chloroplasts for experiments. Leaves should be pretreated in order to standardise the phosphorylation and organisation of their thylakoids (e.g. preillumination with far-red ligh~l. If the thylakoids are then isolated into media containing 5mM Mg-- they are likely to maintain their in vivo organisation. However, if they are allowed to unstack in low salt containing media and then restacked by adding 5mM ~+ Mg they may not precisely regain the configuration of the in vivo state. Unfortunately as yet the 'phosphorylation-mobile antenna' properties of LHC-II cannot be extrapolated to those organisms which show State transitions but do not contain LHC-II. Studies by Biggins and colleagues (21) concluded that no phosphorylation processes can be correlated with State I-State II transitions in Porphyridium cruentum and previous to this Ley and Butler (60) had shown that in this organism the State transitions involved large changes in extent of spillover rather than alterations in absorption cross-sections. Unfortunately then, the 'quantum yield anomaly' is not understood in structural terms for the phycobilin containing organisms having no obvious differentiation into appressed and nonappressed membranes. Nevertheless, Warren Butler's thorough studies with Porphyridium established without doubt that this organism can regulate energy transfer between PSII and PSI and that this regulation can be described by his models. We can look forward to using this knowledge as a basis for obtaining explanations about the conformational changes which occur and Comparing the similarities and differences with higher plant and green algal systems.
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6.
ACKNOWLEDGEMENTS I wish to thank both the Science and Engineering Research Council (SERC) and the Agricultural and Food Research Council (AFRC) for financial support. I would also like to thank my colleagues for their support during the course of the writing of this review, particularly Alison Teller and Julian whitelegge. REFERENCES
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78. 79° 80° 81. 82. 83° 84
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85. 86° 87° 88° ~9°
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