MINIREVIEW Photosynthetic acclimation: Structural reorganisation of light harvesting antenna – role of redox-dependent phosphorylation of major and minor chlorophyll a/b binding proteins Joanna Kargul and James Barber Wolfson Laboratories, Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College London, UK Introduction Oxygenic photosynthesis is one of the most fundamen- tal processes sustaining life on Earth. During this pro- cess the solar energy is harnessed and converted into the chemical bonds of the energy-rich molecule ATP, and the reducing equivalents used for the conversion of CO 2 into carbohydrates (the building blocks of biomass) are generated. The first step in this process, light-driven charge separation, is conducted by photo- system I (PSI) and photosystem II (PSII), two multi- meric chlorophyll-binding protein complexes embedded in the thylakoid membranes of cyanobacteria, algae and plants (Fig. 1) [1,2]. PSI and PSII contain reaction centres that accept excitation energy from the chloro- phyll molecules bound to the light-harvesting antenna Keywords CP29; kinases; LHCII; phosphorylation; photosynthesis; photosystem I; photosystem II; single particle analysis; state transitions; X-ray structure Correspondence J. Kargul, Wolfson Laboratories, Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College London, London SW7 2AZ, UK Fax: +44 (0)20 7594 5267 Tel: +44 (0)20 7594 1774 E-mail: j.kargul@imperial.ac.uk (Received 21 September 2007, revised 11 December 2007, accepted 17 December 2007) doi:10.1111/j.1742-4658.2008.06262.x In order to carry out photosynthesis, plants and algae rely on the co-opera- tive interaction of two photosystems: photosystem I and photosystem II. For maximum efficiency, each photosystem should absorb the same amount of light. To achieve this, plants and green algae have a mobile pool of chlorophyll a ⁄ b-binding proteins that can switch between being light- harvesting antenna for photosystem I or photosystem II, in order to main- tain an optimal excitation balance. This switch, termed state transitions, involves the reversible phosphorylation of the mobile chlorophyll a ⁄ b-bind- ing proteins, which is regulated by the redox state of the plastoquinone- mediating electron transfer between photosystem I and photosystem II. In this review, we will present the data supporting the function of redox- dependent phosphorylation of the major and minor chlorophyll a ⁄ b-bind- ing proteins by the specific thylakoid-bound kinases (Stt7, STN7, TAKs) providing a molecular switch for the structural remodelling of the light- harvesting complexes during state transitions. We will also overview the latest X-ray crystallographic and electron microscopy-derived models for structural re-arrangement of the light-harvesting antenna during State 1-to- State 2 transition, in which the minor chlorophyll a ⁄ b-binding protein, CP29, and the mobile light-harvesting complex II trimer detach from the light-harvesting complex II–photosystem II supercomplex and associate with the photosystem I core in the vicinity of the PsaH ⁄ L ⁄ O ⁄ P domain. Abbreviations Cab proteins, chlorophyll a ⁄ b-binding proteins; cyt b 6 f, cytochrome b 6 f; EM, electron microscopy; LHCI, light-harvesting complex I; LHCII, light-harvesting complex II; NPQ, nonphotochemical quenching; PQ, plastoquinone; PQH 2, reduced plastoquinone or plastoquinol; PsaH ⁄ L ⁄ O ⁄ P ⁄ A ⁄ K ⁄ I, core subunits of photosystem I; PSI, photosystem I; PSII, photosystem II; TAK, thylakoid-associated kinase. 1056 FEBS Journal 275 (2008) 1056–1068 ª 2008 The Authors Journal compilation ª 2008 FEBS subunits. In response to photo-activation, PSII drives photo-induced oxidization of substrate water molecules to molecular di-oxygen (sustaining the aerobic atmo- sphere on Earth) and reducing equivalents in the form of water-derived electrons and protons. The electrons ejected from the PSII reaction centre cofactor P680 are rapidly transferred to the final electron acceptors, plas- toquinones (PQs) Q A and Q B . Following protonation of the doubly reduced PQ Q B , the final product, plas- toquinol (PQH 2 ), diffuses out of PSII into the thyla- koid membrane and provides protons and electrons to the cytochrome b 6 f (cyt b 6 f) complex at the quinol- binding site [3]. The oxidized form of P680 is reduced by electrons derived from substrate water molecules with the aid of a redox-active tyrosine Y z and a cata- lytic centre composed of four Mn ions and a Ca ion. Linear electron transfer proceeds with the soluble elec- tron carrier plastocyanin, which undergoes reduction by the cyt b 6 f complex and donates electrons to the oxidized reaction centre of PSI, P700 + . In this way, photo-activated PSI uses reducing equivalents derived from PSII to reduce the final acceptor ferrodoxin and ultimately convert NADP + to NADPH (see Fig. 1). Thus, the primary charge separation in the reaction centres of PSII and PSI triggers vectorial electron flow from PSII to PSI via the cyt b 6 f complex with the Fig. 1. Diagrammatic overview of the state transitions process. Under conditions of balanced light illumination of photosystems, a linear photosynthetic electron flow (solid arrows) is favoured resulting in the generation of reducing equivalents and NADPH 2 . The proton gradient concomitantly formed across the thylakoid membrane (light green area) drives the activity of ATP synthase to produce ATP. Both NADPH 2 and ATP are used for fixation and reduction of CO 2 into carbohydrates. State 1 is induced by excess PSI light (light 1) and State 2 by excess PSII light (light 2). State 1-to-State 2 transition occurs in response to illumination with excess light 2 (see the two hm bolts above PSII), when the PQ pool becomes over-reduced. Binding of the PQH 2 to the quinol-binding site of the cyt b 6 f complex activates a specific thylakoid- bound kinase that directly interacts with the cyt b 6 f complex and phosphorylates the mobile LHCII antenna (dark green; P, phosphate groups). The activity of LHCII kinase is regulated by co-operative redox control, both via PQ and cyt b 6 f, and through the thioredoxin ⁄ ferro- doxin system in the stroma. Phospho-LHCII, together with the phosphorylated linker subunit CP29 (and possibly TSP9 in plants) (blue tri- angles), detaches from PSII and docks onto PSI to redirect absorbed excitation energy to PSI at the expense of PSII. Under the conditions of over-excitation of PSI (preferential illumination with light 1 or darkness; see the two hm bolts above PSI), oxidation of the PQ pool occurs followed by de-activation of LHCII-specific kinases and dephosphorylation of mobile LHCII by redox-independent constitutively active phos- phatase (although its activity may be regulated by immunophilin-like lumenal TLP40 protein). Dephosphorylated LHCII detaches from PSI and functionally couples with PSII (State 2-to-State 1 transition), favouring energy redistribution towards PSII. In Chlamydomonas, a switch between linear (State 1) and cyclic (State 2) electron flow around PSI occurs. Observed migration of the cyt b 6 f to the stroma lamellae in State 2 adaptation may promote preferential binding of ferrodoxin-NADP reductase with this complex and increase the rate of PQ reduction via the cyclic electron flow around PSI (dashed arrows), which exclusively generates ATP by driving protons across the membrane. Any met- abolic depletion of the cellular ATP level would switch between both types of photosynthetic electron transport and would therefore induce State 1-to-State 2 transition. A 0 , chlorophyll a; A 1 , phylloquinone; F x ,F A and F B, Fe 4 S 4 clusters; Fd, ferrodoxin; FNR, ferrodoxin-NADP oxido- reductase; OEC, oxygen-evolving complex (a CaMn 4 cluster oxidizing substrate water molecules); PC, plastocyanin; Phe, pheophytin (a pri- mary electron acceptor); PQ ⁄ PQH 2 , plastoquinone ⁄ plastoquinol (oxidized and reduced plastoquinone, respectively); Q A ,Q B , fixed and mobile electron carriers. J. Kargul and J. Barber Structure of photosystems in state transitions FEBS Journal 275 (2008) 1056–1068 ª 2008 The Authors Journal compilation ª 2008 FEBS 1057 concomitant formation of the proton gradient (or elec- trochemical potential gradient) across the thylakoid membrane, and in this way powers the activity of ATP synthase to convert ADP to ATP. Both ATP and NADPH produced in the light-driven redox reactions of photosynthesis are subsequently utilized for CO 2 assimilation during the photosynthetic dark reactions of the Calvin–Benson cycle. Considerable progress has been made in revealing the molecular organization of all the membrane com- plexes involved in photosynthetic electron flow, includ- ing their light-harvesting complexes (LHC), and their crystal structures are available at intermediate (3.0– 3.5 A ˚ ) or high (2.5 A ˚ ) resolutions (PSII: [4,5]; cyt b 6 f: [6,7]; PSI and the light-harvesting complex I (LHCI)– PSI: [8,9]; and light-harvesting complex II (LHCII): [10,11]). Recently, a 3.4 A ˚ X-ray structure of the higher plant PSI supercomplex provided an insight into the organization of the PSI core and the assembly of its associated LHCI together with the bound pig- ments and cofactors [9]. In this structure, four Lhca subunits of the LHCI complex form a crescent that binds asymmetrically to the core domain composed of 15 subunits. The organization of the plant and green algal PSII core dimer and its associated antenna, the LHCII, has been revealed by cryo-electron microscopy and single particle analysis [12]. As yet, no crystal structure of the eukaryotic LHCII–PSII supercomplex has been determined. Nevertheless, the recent X-ray structures of the cyanobacterial dimeric PSII core com- plex, together with the crystal structures of some sub- units of plant PSII, have been used to interpret a lower-resolution structure of the plant LHCII–PSII supercomplex derived from cryo-electron microscopy at 17 A ˚ [13]. In this model, the dimeric higher plant LHCII–PSII supercomplex binds two LHCII trimers together with two copies of the minor chloro- phyll a ⁄ b-binding (Cab) proteins CP29 and CP26, with each pair symmetrically related by the twofold axis of the core dimer. This is the basic highly conserved structural unit of the LHCII–PSII supercomplex, although more complex structures exist, in which two or three additional LHCII trimers and two copies of the minor subunit, CP24, associate with the dimeric PSII supercomplex and form complex crystalline arrays in thylakoid membranes of higher plants, depending on the light conditions and the species anal- ysed [12]. Environmental conditions can fluctuate on a time- scale of seconds, days and months. Photosynthetic organisms have evolved a number of ingenious short- term and long-term responses to changing environmen- tal conditions in order to maintain an optimal level of photosynthesis. As the light-driven reactions of photo- synthesis involve a complex chain of redox reactions, many environmental changes affect, directly or indi- rectly, the redox state of the components of the photo- synthetic electron flow, and thus photosynthetic efficiency [14]. Amongst the environmental changes affecting the quantum yield of photosynthesis are low and high temperatures, CO 2 availability, drought and mineral status (e.g. Mg 2+ and Fe 2+ that act as cofac- tors of the components of the photosynthetic electron transport chain). However, the most rapidly changing environmental factor is the quantity and spectral qual- ity of incident light, often leading to imbalanced exci- tation of the two photosystems. In order to ensure an optimal quantum yield of oxygen evolution during photosynthesis, PSII and PSI must be excitonically balanced. Overexcitation of photosystems occurs in high light intensity, which often results in the photo- inhibition of PSII and a rapid turnover of the reaction centre subunit D1 [15]. The rapid response to high irradiance is to dissipate excess light through heat via a mechanism known as nonphotochemical quenching (NPQ) [16–18] (reviewed by P. Horton et al. in this miniseries). In low light intensity, the imbalance in the excitation of both photosystems is counteracted by the rapid process of state transitions [19] followed by slower changes in photosystems stoichiometry, a long- term response occurring on a timescale of hours to days [20,21] (reviewed by T. Pfannschmidt et al. in this miniseries). The precise mechanisms and molecular components of state transitions appear to differ between the aquatic unicellular green alga Chlamydo- monas reinhardtii and land plants. In particular, the greater extent of state transitions in Chlamydomonas compared with higher plants, such as Arabidopsis, has been proposed to drive a switch between linear and cyclic electron flow around PSI (see ‘Specificity of state transitions of C. reinhardtii’, below, and Fig. 1). In land plants, state transitions provide a fine-tuning reg- ulatory mechanism, allowing plants to optimize the quantum yield of linear electron flow under rapidly changing light conditions. In this review, we will present the current models for structural re-arrangement of the light-harvesting antenna during state transitions. In so doing, we will incorporate the recently published X-ray crystallo- graphic and electron microscopy (EM)-based visualiza- tion of green algal and plant photosynthetic complexes. Moreover, we will overview the data sup- porting the role of redox-dependent phosphorylation of major and minor LHCII subunits catalysed by LHCII-specific kinases, providing the trigger for the structural re-organization of LHCs in state transitions. Structure of photosystems in state transitions J. Kargul and J. Barber 1058 FEBS Journal 275 (2008) 1056–1068 ª 2008 The Authors Journal compilation ª 2008 FEBS Mechanism of state transitions General mechanism The process of state transitions represents a short-term adaptation of the photosynthetic apparatus to the con- ditions of imbalanced illumination of PSII or PSI. It occurs on a timescale of seconds to minutes (5–20 min) and it enables oxygenic phototrophs (higher plants, red and green algae, and cyanobacteria) to modulate the excitation energy of both photosystems, thus main- taining the optimal photosynthetic efficiency [19]. In higher plants and green algae, the basis of this phe- nomenon is the redistribution of LHCII complexes between PSII and PSI within the thylakoid membrane [19,22–24] (see Fig. 1). In cyanobacteria, which lack LHCII, the movement of phycobillisomes (the primary light-harvesting proteins in these organisms) may play a similar role [25]. In an ecological context, state tran- sitions may serve as a rapid response preceding a photoprotective adaptation by NPQ during exposure to excess illumination [26]. However, the most signifi- cant ecological relevance of this process occurs under shaded or light-limiting conditions, and during changes in spectral filtering properties of leaf canopies or water columns. In 1969, two laboratories reported independently that absorbed light energy could be redistributed between PSII and PSI to optimize the quantum yield of photosynthetic electron flow [27,28]. PSII and PSI have distinct light-harvesting properties with maxi- mum absorption at 680 nm (blue–green light) and 700 nm (red and far-red light), respectively. State 1 is induced by excess PSI light (light 1) and State 2 by excess PSII light (light 2). State 1-to-State 2 transition therefore occurs in response to over-reduction of the PQ pool, resulting in the activation of specific thyla- koid-bound kinase(s). This activation involves the binding of PQH 2 to the quinol-binding site of the cyt b 6 f complex and initiates the phosphorylation of the mobile LHCII antenna (see Fig. 1) [29–31, reviewed in ref. 32]. The phosphorylated LHCII has been pro- posed to transfer physically from PSII to PSI in order to redirect absorbed excitation energy to PSI at the expense of PSII. Thus, in State 2 the PSII antenna (or the PSII absorption cross-section) is reduced and the PSI antenna is increased compared with State 1 (Fig. 1). Under the conditions of over- excitation of PSI (or preferential illumination with light 1), oxidation of the PQ pool occurs followed by de-activation of LHCII-specific kinase(s) and dephos- phorylation of mobile LHCII by redox-independent constitutively active phosphatase(s) [33] (see Fig. 1). As a result, dephosphorylated LHCII detaches from PSI and functionally couples with PSII (State 2-to- State 1 transition), favouring energy redistribution towards PSII. Two main models have been proposed to explain the movement of the LHCII fraction during state transitions, although they both acknowledge a central role of reversible phosphorylation of LHCII for inducing transition to State 2. According to the sur- face charge model, redistribution of the surface charge at the periphery of the grana partition gaps upon phosphorylation may result in structural changes within the thylakoid membrane sufficient for the movement of phospho-LHCII away from the grana stacks towards nonappressed membrane regions (stromal lamellae) enriched with PSI [34]. A modifica- tion of this view suggests that phosphorylation does not induce lateral migration of LHCII, but rather causes partial unstacking of the thylakoid appressed regions and therefore some spillover of excitation energy from PSII to PSI [35]. The model of molecular recognition proposes that phospho-LHCII exhibits different binding specificity for both photosystems in that the phosphorylation of the mobile LHCII decreases its affinity for PSII and increases its affinity for PSI at the specific docking site [22,33]. Indeed, it has been shown that phosphorylation induces a con- formational change of the N-terminal domain of LHCII, leading to dissociation of the LHCII trimers into monomers, and therefore it may provide the mechanism for controlling functional interactions of LHCII in vivo [36]. Molecular components of state transitions Although the core mechanism of state transitions has been known since the late 1960s, significant progress in our understanding of the molecular components and structural basis for this phenomenon has been made only recently through genetic and structural studies in two model organisms: the green alga C. reinhardtii; and a higher plant, Arabidopsis thali- ana. The activity of the LHCII kinase was identified by John Bennett in 1977 [37]; however, biochemical attempts to isolate the specific enzymes have been unsuccessful to date. Nevertheless, by adopting an alternative approach, a small family of three thyla- koid-associated kinases (TAKs) have been identified in A. thaliana as candidates for LHCII kinases through screening for proteins that interact with the N-terminal domain of LHCII [38]. The antisense Ara- bidopsis plants with suppressed levels of the threonine kinase TAK1 showed increased sensitivity to high J. Kargul and J. Barber Structure of photosystems in state transitions FEBS Journal 275 (2008) 1056–1068 ª 2008 The Authors Journal compilation ª 2008 FEBS 1059 light intensity, a lower level of LHCII phosphoryla- tion and partial deficiency in the ability to perform state transitions [39]. As TAKs are themselves phos- phorylated [38], they may be part of a signalling cas- cade involving other kinase(s) directly regulated by the reduced cyt b 6 f complex. Recent studies of the mutants that were blocked in State 1 revealed that the thylakoid-associated serine– threonine protein kinase, Stt7, of the green alga C. reinhardtii, and its higher plant orthologue, STN7, are required for the phosphorylation of several major LHCII polypeptides [40–42], thus providing further evidence that protein phosphorylation is essential for state transitions. Interestingly, phosphorylation of other thylakoid proteins, such as PSII core subunits CP43, D1, D2 and PsbH, still occurs in the stn7 mutant background, demonstrating the specificity of the STN7 kinase for state transitions [41–43]. Nota- bly, Arabidopsis mutants deficient in STN7 showed inhibited phosphorylation of not only major LHCII, but also of the minor light-harvesting protein, CP29, at the Thr6 residue [43]. However, the direct sub- strates of these two protein kinases remain to be determined. The common structural features of all the LHCII kinases characterized to date are a putative single transmembrane domain and a large hydrophilic loop oriented to the stromal side of the thylakoid mem- brane where the catalytic kinase domain is located [32]. Considerable progress has been made in determin- ing the mechanisms of controlling the activity of the LHCII kinases [24,32]. It is clear that LHCII phos- phorylation and the redox state of PQ are not tightly coupled, as there are numerous reports of down-regu- lation of LHCII phosphorylation at high irradiance, when the PQ pool is reduced [24]. Conversely, maxi- mum phosphorylation of LHCII polypeptides in vivo occurs at low light intensities [24,44]. It now seems that the phosphorylation of LHCII proteins is regu- lated by a complex network involving co-operative redox control both via PQ and the cyt 6 f complex, and through the thioredoxin ⁄ ferrodoxin system in the stroma of the chloroplasts [44]. Rochaix has recently reported that mutations of either of the two conserved cysteine residues at the N-termini of Stt7 and STN7 kinases abolish state transitions and LHCII phosphor- ylation [32]. These two cysteine residues may be poten- tial targets for thioredoxin-mediated inhibition of LHCII kinase activity. The identification of LHCII-specific phosphatases has been unsuccessful to date. Although it has been suggested that the LHCII phosphatase is constitutively active [33], there is evidence that its activity may be regulated by the immunophilin-like lumenal TLP40 protein [45,46]. Docking site for mobile LHCII Another important issue has been to identify the struc- tural basis for state transitions, in particular the postu- lated docking site for the association of the mobile LHCII with PSI under State 2 conditions. The evi- dence for the lateral migration of a fraction of LHCII and the cyt b 6 f complex from the grana stacks (enriched in PSII) to the stromal lamella (enriched in PSI) has been known for some time through a number of spectroscopic, biochemical and in situ immuno- localization studies [12,47–50]. The elegant chemical cross-linking and double-stranded RNA interference approaches of Scheller and co-workers provided bio- chemical evidence for the docking domain for LHCII binding to be the PsaI ⁄ H ⁄ O region at the tip of the PSI core [51,52]. Arabidopsis plants devoid of the PsaO core subunit showed 50% reduction in state transitions [52], indicating the role of this protein in putative binding of mobile LHCII. An even more drastic effect on state transitions was demonstrated by Lunde et al. who suppressed the expression of the PsaH and PsaL core subunits in Arabidopsis [53]. Plants lacking PsaH were essentially unable to perform state transitions and were locked in State 1, indicating direct involvement of PsaH as a docking site for the mobile phospho-LHCII under State 2 conditions. Importantly, in the absence of PsaH, nonphotochemical fluorescence quenching was identical upon illumination with light 1 and light 2, and LHCII still underwent phosphorylation in State 2. These results suggest that the majority of LHCII in the PsaH null plants remains attached to PSII in spite of the unaffected LHCII phosphorylation. Similarly, Delosme et al. observed that phospho-LHCII remains part of the PSII antenna in PSI-deficient mutants of Chlamydomonas [48]. These observations support the concept of molecular recognition where the relative binding affinity of the phospho-LHCII pool for PSII and PSI changes during state transitions. The postulate of a critical role of the PsaH subunit, which, together with PsaL and PsaO, may form a docking site for mobile LHCII during state transitions, was reinforced by the recent X-ray crystallographic studies of the higher plant PSI (see Section 4). In the latest X-ray structures of the LHCI–PSI supercomplex, the PsaH protein was shown to be located at an exposed hydro- phobic surface of the PSI core and to bind a single chlorophyll molecule [9,54], which may aid energy transfer from the bound LHCII to the PSI reaction centre. Structure of photosystems in state transitions J. Kargul and J. Barber 1060 FEBS Journal 275 (2008) 1056–1068 ª 2008 The Authors Journal compilation ª 2008 FEBS Specificity of state transitions in C. reinhardtii Chlamydomonas reinhardtii provides a unique system for analysis of the mechanism of state transitions, in particular, dissecting molecular components involved in this process. In this green alga, the degree of state transitions is often much larger than in higher plants, with up to 85% of LHCII antenna reported to become displaced from PSII in State 2 [48] in comparison to a relatively small fraction (20–33%) of LHCII in green plants [33,53]. It has been proposed that the extensive nature of the state transitions in Chlamydomonas pro- vides a unique adaptive mechanism that allows a switch between linear (State 1) and cyclic (State 2) electron flow around PSI [49,55–57] (see Fig. 1). The observed accumulation of the cyt b 6 f in the stroma lamellae following State 2 adaptation has been sug- gested to promote preferential binding of ferrodoxin– NADP oxidoreductase with this complex and thus increase the rate of PQ reduction via the cyclic electron flow around PSI [3]. From the metabolic point of view, state transitions in Chlamydomonas can be understood as a shift from linear electron transport, generating reducing equivalents and ATP (State 1), to a cyclic electron flow that exclusively generates ATP (Fig. 1). In this way, any conditions leading to depletion of the cellular level of ATP would switch between both types of photosynthetic electron transport [3,57] and would therefore induce State 1-to-State 2 transition. Because of the large amplitude of state transitions, as monitored by changes in relative absorption cross- section in both photosystems, C. reinhardtii provided an excellent model system for developing simple fluo- rescence video imaging screening assays for identifica- tion of mutants affected in the signalling cascade of this process. These types of screening have led to iso- lation of the series of stm [58] and stt [59] mutants deficient in state transitions. The stt7 mutant [59] has been shown to be of particular importance, as the cor- responding gene whose mutation was responsible for the mutant phenotype (blocking in State 1 and defi- ciency in phosphorylation of LHCII), as discussed above, has been shown to encode a thylakoid-bound protein kinase specific for phosphorylation of LHCII [40]. Structural remodelling of light-harvest- ing antenna during state transitions Although it is widely recognized that during state tran- sitions a pool of LHCII shuttles between PSII and PSI, direct structural evidence for the physical associa- tion of this mobile antenna with PSI has been revealed only recently. During the last 2 years, three important papers have highlighted various possible models for this association. We demonstrated, for the first time, a novel structural role of the Cab ⁄ minor light- harvest- ing subunit, CP29, in State 1-to-State 2 transition in Chlamydomonas [60]. A well-established primary func- tion of the monomeric CP29 protein is to stabilize the binding of the outer antenna LHCII trimers with the PSII reaction centre core complex [61–63]. Through the combination of MS, phosphopeptide mapping, EM and single particle analysis of the LHCI–PSI super- complexes isolated from the State 2-induced Chlamydo- monas cells, we showed that CP29 dissociates from PSII and binds with the core domain of PSI [60] (see Fig. 2). This redistribution of CP29 was correlated with a quadruple phosporylation of unique Thr and Ser residues at its N-terminal domain [60]. The binding site was in the vicinity of the PsaH subunit (see Fig. 4a), a region previously suggested to bind mobile LHCII during state transitions [53] (see above, under ‘Docking site for mobile LHCII’). The protein density assigned to phospho-CP29 in the projection map derived from EM and single particle analysis of State 2 LHCI–PSI was absent in the corresponding particles isolated from State 1-induced cells (compare Fig. 2a,d with Fig. 2b,e) [60,64]. We have obtained further struc- tural evidence for the direct binding of CP29 to PSI by EM analysis of the LHCI–PSI particles isolated from the CP29 null mutant of Chlamydomonas induced to State 2 (J Kargul, J Nield, S Benson, A Kanno, J Min- agawa & J Barber, unpublished results). When the expression of CP29 is silenced by the interference with double-stranded RNA, the additional protein density in the proximity of PsaH detected in the State 2 wild- type PSI particles is completely missing from the whole population of the LHCI–PSI supercomplex particles analysed (see Fig. 2c,f). Moreover, the mutant depleted of CP29 lacked any detectable 35 kDa phosphor-CP29 in the State 2 LHCI–PSI supercomplex, even though the phosphorylation of major LHCII remained unaf- fected. Thus, for the first time, direct structural evi- dence for association of the LHCII-like component with PSI was obtained in support of the previous indi- rect biochemical, spectroscopic and immunolocaliza- tion data. We postulate that under some conditions CP29 acts as a sole monomeric Cab protein that increases the absorption cross-section of PSI. Alterna- tively, under more extreme State 2 conditions, it pro- vides a linker domain for binding the additional LHCII subunits associated with PSI [60]. This latter possibility is not excluded by our work [60] because it is likely that such PSI–CP29–LHCII supercomplexes J. Kargul and J. Barber Structure of photosystems in state transitions FEBS Journal 275 (2008) 1056–1068 ª 2008 The Authors Journal compilation ª 2008 FEBS 1061 are too labile to be successfully purified by standard fractionation procedures in the presence of the dode- cyl-maltoside detergent. Whether hyperphosphoryla- tion of CP29 occurs in higher plants, and whether this phosphoprotein binds to the plant PSI in State 2, remains to be determined, although Tikkanen et al. have recently demonstrated the STN7 kinase-depen- dent phosphorylation of the Arabidopsis CP29 isoform Lhcb4.2 [43]. Importantly, one phosphorylation site (Thr16) identified in our studies [60,65] under State 2 conditions is fully conserved between higher plant (Arabidopsis and maize) CP29 and its Chlamydomonas orthologue. In plants, the absence of CP29 prevents the assembly of LHCII–PSII supercomplexes [63], whereas inactivation of other minor light-harvesting components (CP26 and CP24) does not inhibit the for- mation of a basic structural unit of the LHCII–PSII supercomplex [63,66]. Therefore, it is feasible that in green algae, the large amplitude of state transitions may be, to some extent, caused by a substantial desta- bilization of the LHCII–PSII supercomplex upon dissociation of hyperphosphorylated CP29, triggering a large increase of the PSI antenna absorption cross- section as a result of the availability of an increased pool of mobile LHCII (see Fig. 3). Subsequent to our discovery of the novel function of CP29 in algal state transitions, Takahashi et al. dem- onstrated, through biochemical analysis, that under State 2 conditions, another minor light-harvesting component, CP26, together with the major LHCII sub- unit (LhcbM5) and CP29 may also become displaced from PSII and associate with PSI [67]. However, it is important to emphasize that the direct structural evi- dence for such putative binding of LhcbM5 and CP26 remains to be established, as does the precise mapping of the phosphorylation sites postulated for both pro- teins based on immunodetection with anti-phospho- threonine serum [67]. Fig. 2. Minor Cab protein CP29 associates with LHCI–PSI in State 2. EM top-view projections of State 1 and State 2 LHCI–PSI supercom- plexes of wild-type (A, B, D, E) and the CP29-less mutant (C, F) of Chlamydomonas reinhardtii viewed from the stromal side. (A) Projection of wild-type State 1 LHCI–PSI. (B), Projection of wild-type State 2 LHCI–PSI. (C), Projection of LHCI–PSI from the State 2-induced CP29-less mutant. (D–F), Modelling of the projection maps for the LHCI–PSI supercomplex isolated from wild-type (D, E) and CP29-less (F) C. rein- hardtii cells placed in State 1 (D) and State 2 (E, F). Modelling is based on higher plant coordinates 1QZV.pdb for the higher plant LHCI–PSI [54] and 1RWT.pdb for the LHCII monomer [11]. PSI core (green), LHCI antenna (red), PsaJ (yellow), PsaK (magenta), PsaG (purple), PsaI (orange), PsaL (cyan) and PsaH (white, arrowed in d and f). Chlorophylls are shown in yellow and for clarity were excluded from LHCI and CP29 subunits. The additional density observed in the State 2 LHCI–PSI supercomplex, which is able to accommodate an additional Cab sub- unit, is indicated with a white arrow in (B) and coloured in red in (E) and corresponds to phospho-CP29 (see the text). The detergent shell surrounding the particles is assigned as a wide outer contour (yellow) of  15 A ˚ . Scale bar represents 50 A ˚ . Data in (A) and (B) were taken from Kargul et al. [60]. Structure of photosystems in state transitions J. Kargul and J. Barber 1062 FEBS Journal 275 (2008) 1056–1068 ª 2008 The Authors Journal compilation ª 2008 FEBS The physical association of the major LHCII trimer with PSI in State 2 has been recently demonstrated in A. thaliana. Kouril et al. used digitonin to solubilize thylakoid membranes in the State 2 conformation fol- lowed by EM and single particle analysis of the resul- tant mildly solubilized protein complexes [68]. This type of analysis allowed, for the first time, visualiza- tion of the higher plant LHCI–PSI supercomplex and the associated LHCII trimer. By constructing the 2D projection map of the State 2 PSI supercomplex at 15 A ˚ , the authors propose that a single LHCII trimer binds asymmetrically to the PSI core domain contain- ing PsaH ⁄ L ⁄ O ⁄ P subunits on the PsaA ⁄ H ⁄ L ⁄ K side of the complex [68,69] (Fig. 4b). This interpretation is in line with the crystallographic modelling of Amunts et al. who also postulate binding of a single LHCII tri- mer on the PsaK side near the PsaH subunit [9] (see Fig. 4c,d). The LHCII–PSI interaction site is suggested to be composed of PsaH, PsaL, PsaA and PsaK core subunits [9] (Fig. 4d). Importantly, Amunts et al. emphasize that only one LHCII monomer within the LHCII trimer is likely to be excitonically coupled with the PSI reaction centre under State 2 conditions (Fig. 4d), suggesting that binding of a single LHCII monomer is also feasible in higher plants [9]. It is tempting to speculate whether CP29 is, in fact, the monomeric subunit and therefore functions to anchor the mobile LHCII trimer not only in green alga but also in higher plants. Lunde et al. determined a 33% relative increase of the antenna size of PSI in intact leaves of Arabidopsis upon State 1-to-State 2 transition [53]. Bearing in mind that each LHCII monomer binds 14 chlorophyll molecules [11], and that the absolute number of chlorophyll molecules functionally associ- ated with LHCI–PSI in State 1 has been measured as  160–200 [24] and assigned as 168 in the latest 3.4 A ˚ crystal structure of LHCI–PSI [9], the increase in the antenna size of PSI in State 2 corresponds to one to four LHCII monomers (or one LHCII-like monomer, such as CP29, and a single LHCII trimer). Notably, in the recently modified overlay projection map of the Arabidopsis LHCII–LHCI–PSI supercomplex, Jensen et al. have modelled in two additional protein densi- ties, the largest one being at the interface between the LHCII trimer and the PsaH ⁄ L ⁄ K side of the PSI core [69] (see Fig. 4). The identity of these protein densities remains to be established. Future outlook The recent progress in unravelling the structural basis of state transitions has not only advanced our knowl- edge of the direct components involved in this process, but has also raised some questions that remain to be addressed. There is an urgent need to obtain high- resolution structures of the LHCII–LHCI–PSI and, possibly, LHCII–CP29–LHCI–PSI supercomplexes to determine the precise molecular and excitonic interac- tion between the mobile LHCII and the PSI core dur- ing state transitions. Another important question arises from the cross-linking results of Zhang & Scheller [51], who postulate an alternative binding site for the LHCII trimer on the PsaI ⁄ B ⁄ G side of the PSI core tip, although this has been recently questioned by Amunts et al., who, based on their modelling of the LHCII trimer crystal structure [10] into the 3.4 A ˚ X-ray structure of higher plant LHCI–PSI, postulate the most likely position of LHCII to be on the PsaH ⁄ L ⁄ K side [9]. Further work is required to vali- date the possibility of this putative, albeit of weaker affinity, binding site for the mobile LHCII. Further research is needed to identify the origin of the mobile LHCII trimers migrating towards PSI in State 2. Recent EM and functional analyses of the Fig. 3. Role of CP29 in state transitions and in thermal energy dissipation. (A) Schematic representation of the subunit organiza- tion in the Chlamydomonas LHCII–PSII supercomplex taken from Turkina et al. [65]. (B) Proposed mechanism for the regulation of light harvesting in Chlamydomonas by sequential phosphorylation of the CP29 linker protein [65]. The open and closed circles mark the phosphorylation sites identified in cells induced to State 2 or exposed to high irradiance, respectively. Quadruple phosphoryla- tion of CP29 upon State 1-to-State 2 transition causes detachment of phospho-CP29–LHCII from PSII and its docking onto PSI in the vicinity of the PsaH core subunit, as proposed previously [60,65]. High light illumination induces phosphorylation of CP29 at seven residues, leading to the dissociation of phospho-CP29–LHCII from PSII. This detachment may promote thermal energy dissipation within the LHCII trimers. In green plants, phosphorylated TSP9 protein may perform a similar role as a linker subunit, shuttling between PSII and PSI during state transitions, as proposed previ- ously [72]. J. Kargul and J. Barber Structure of photosystems in state transitions FEBS Journal 275 (2008) 1056–1068 ª 2008 The Authors Journal compilation ª 2008 FEBS 1063 plant LHCII–PSII particles assembled in the absence of the minor light-harvesting subunits suggest that at least some of these trimers may originate from the so- called M-LHCII pool representing the LHCII trimers that are bound to the PSII core dimer close to the CP29 and CP24 minor subunits [66,68]. However, it is possible that the mobile LHCII may also originate from the tightly bound S-type LHCII trimer that Fig. 4. Models for association of the mobile LHCII antenna with type LHCI–PSI supercomplex during state transitions, as derived from EM and X-ray crystallography. (A) EM top-view projection of LHCI–PSI supercomplexes of State 2-induced Chlamydomonas reinhardtii viewed from the stromal side (data taken from Kargul et al. [60]). Modelling is based on coordinates 1QZV.pdb for the higher plant LHCI–PSI [54] and 1RWT.pdb [11] for CP29. The scale bar represents 50 A ˚ . Association of the CP29 minor LHCII protein (blue) with the LHCI–PSI super- complex close to the PsaH core subunit (white) in State 2-induced thylakoids of Chlamydomonas may provide an anchor for transient binding of mobile LHCII trimer or may be the sole monomeric light-harvesting subunit increasing absorption cross-section of PSI. PSI core (green), LHCI antenna (red), PsaJ (yellow), PsaK (magenta), PsaG (purple), PsaI (orange), PsaL (cyan). Chlorophylls are shown in yellow and for clarity were excluded from LHCI and CP29 subunits. (B) Overlay of the EM top-view projection of the Arabidopsis LHCII–LHCI–PSI supercomplex with the X-ray structures of plant LHCI–PSI [54] and trimeric LHCII [11] (data taken from Kouril et al. [68]). Positions of the LHCI subunits Lhca1–4 and the small core subunits are indicated. Additional protein density detected in the vicinity of the PsaH ⁄ L ⁄ O domain is postulated to accommodate a single LHCII trimer [68,69]. Note the additional protein densities (pink) between the PSI core and the postulated LHCII tri- mer, which may accommodate additional linker proteins or small core subunits. (C, D) X-ray crystallography-derived modelling of the possible association of the LHCII trimer (coordinates 2BHW.pdb [10]) with LHCI–PSI (coordinates 2O01.pdb [9]) under State 2 conditions (images taken from Amunts et al. [9]). (C), Top view of the putative LHCII–LHCI–PSI supercomplex from the stromal side of the membrane. The pro- posed LHCII–PSI interaction site viewed from the stromal side and depicted in (D) is formed between LHCII (red) and the PSI core domain composed of PsaH (magenta), PsaL (cyan), PsaA (orange) and PsaK (brown). Note the postulated excitonic coupling between two chlorophyll molecules of the LHCII monomer (blue) and two chlorophyll molecules co-ordinated by the PsaA reaction centre subunit (green). Structure of photosystems in state transitions J. Kargul and J. Barber 1064 FEBS Journal 275 (2008) 1056–1068 ª 2008 The Authors Journal compilation ª 2008 FEBS dissociates together with the hyperphosphorylated CP29 from the LHCII–PSII supercomplex [60,65], possibly following sequential disassembly of the LHCII–PSII supercomplex into PSII monomers, as recently reported by Iwai & Minagawa [70]. It cannot be excluded that the mobile phospho-LHCII may origi- nate from the free LHCII complexes located in a part of the thylakoid membrane, as argued by Dekker & Boekema [12]. Although it is now well established that phosphory- lation of the mobile LHCII triggers conformational changes in the components of this complex, leading to their dissociation from PSII, it is still debatable whether phosphorylation is required for the docking of LHCII to PSI in State 2 [51,60]. In particular, redox- induced quadruple phosphorylation of a minor light- harvesting subunit, CP29, in green alga [60], and triple phosphorylation of the TSP9 protein in higher plants [71,72], could regulate dynamic redistribution of LHCII from PSII to PSI during state transitions by providing a linker domain for binding LHCII trimers. The Arabidopsis TSP9 knockout mutant exhibits altered state transitions and NPQ responses in compar- ison to the wild-type plant, supporting the role of this protein in stabilizing the interaction between the LHCII antenna and the PSII core, as well as between mobile LHCII and PSI in State 2 (A. Vener, Univer- sity of Linko ¨ ping, Sweden, personal communication). The recent precise mapping of the phosphorylation sites within the thylakoid proteome of green algae and higher plants during state transitions and high-light acclimation pinpointed a number of discrete Ser and Thr residues whose phosphorylation is up-regulated in both types of adaptation [65,73]. Moreover, most of the light-induced and redox-induced phosphorylation events cluster at the interface between the PSII core and its associated LHCII antenna [65] (see Fig. 3). This indicates that multiple and sequential phosphory- lation events within the discrete components of the PSII core and LHCII induce conformational changes sufficient for dissociation of the LHCII–PSII super- complex and diffusion of the mobile LHCII pool. Understanding the precise regulation of this process, in particular identification of the specific kinases and their substrates involved in these sequential phosphory- lation events, provide a great challenge for future research. Acknowledgements JK and JB are supported by grants from the UK Biotechnology and Biological Sciences Research Council. We wish to thank our collaborators Jon Nield (QMUL) and Alexander Vener (Linko ¨ ping) for fruitful discussions and for sharing some unpublished data. References 1 Allen JF & Martin W (2007) Evolutionary biology: out of thin air. Nature 445, 610–612. 2 Barber J (2007) Biological solar energy. Philos Transact A Math Phys Eng Sci 365, 1007–1023. 3 Finazzi G (2005) The central role of the green alga Chlamydomonas reinhardtii in revealing the mechanism of state transitions. J Exp Bot 56, 383–388. 4 Ferreira KN, Iverson TM, Maghlaoui K, Barber J & Iwata S (2004) Architecture of the photosynthetic oxy- gen-evolving center. Science 303, 1831–1838. 5 Loll B, Kern J, Saenger W, Zouni A & Biesiadka J (2005) Towards complete cofactor arrangement in the 3.0 A ˚ resolution structure of photosystem II. Nature 438, 1040–1044. 6 Kurisu G, Zhang H, Smith JL & Cramer WA (2003) Structure of the cytochrome b 6 f complex of oxygenic photosynthesis: tuning the cavity. Science 302, 1009– 1014. 7 Stroebel D, Choquet Y, Popot JL & Picot D (2003) An atypical haem in the cytochrome b 6 f complex. Nature 426, 413–418. 8 Jordan P, Fromme P, Klukas O, Witt HT, Saenger W & Krauß N (2001) Three-dimensional structure of cyanobacterial photosystem I at 2.5 A ˚ resolution. Nature 411, 909–917. 9 Amunts A, Drory O & Nelson N (2007) The structure of a plant photosystem I supercomplex at 3.4 A ˚ resolu- tion. Nature 447, 58–63. 10 Standfuss J, Terwisscha van Scheltinga AC, Lamborgh- ini M & Kuhlbrandt W (2005) Mechanisms of photo- protection and nonphotochemical quenching in pea light-harvesting complex at 2.5 A ˚ resolution. EMBO J 24, 919–928. 11 Liu Z, Yan H, Wang K, Kuang T, Zhang J, Gui L, An X & Chang W (2004) Crystal structure of spinach major light-harvesting complex at 2.72 A ˚ resolution. Nature 428, 287–292. 12 Dekker JP & Boekema EJ (2005) Supramolecular orga- nization of thylakoid membrane proteins in green plants. Biochim Biophys Acta 1706, 12–39. 13 Nield J & Barber J (2006) Refinement of the structural model for the photosystem II supercomplex of higher plants. Biochim Biophys Acta 1757, 353–361. 14 Pfannschmidt T (2003) Chloroplast redox signals: how photosynthesis controls its own genes. Trends Plant Sci 8, 33–41. 15 Murata N, Takahashi S, Nishiyama Y & Allakhverdiev SI (2007) Photoinhibition of photosystem II under envi- ronmental stress. Biochim Biophys Acta 1767, 414–421. J. Kargul and J. Barber Structure of photosystems in state transitions FEBS Journal 275 (2008) 1056–1068 ª 2008 The Authors Journal compilation ª 2008 FEBS 1065 [...]... (2005) Molecular basis of photoprotection and control of photosynthetic light- harvesting Nature 436, 13 4–1 37 18 Horton P & Ruban A (2005) Molecular design of the photosystem II light- harvesting antenna: photosynthesis and photoprotection J Exp Bot 56, 36 5–3 73 19 Allen JF (2003) State transitions – a question of balance Science 299, 153 0–1 532 20 Fan DY, Hope AB, Smith PJ, Jia H, Pace RJ, Anderson JM & Chow... Structural characterization of a complex of photosystem I and light- harvesting complex II of Arabidopsis thaliana Biochemistry 44, 1093 5–1 0940 Jensen PE, Bassi R, Boekema EJ, Dekker JP, Jansson S, Leister D, Robinson C & Scheller HV (2007) Structure, function and regulation of plant photosystem I Biochim Biophys Acta 1767, 33 5–3 52 Iwai M & Minagawa J (2007) Dissociation of Light- harvesting Complex II from... 100, 75 7–7 62 1068 72 Hansson M, Dupuis T, Stromquist R, Andersson B, Vener AV & Carlberg I (2007) The mobile thylakoid phosphoprotein TSP9 interacts with the light- harvesting complex II and the peripheries of both photosystems J Biol Chem 282, 1621 4–1 6222 73 Vener AV (2007) Environmentally modulated phosphorylation and dynamics of proteins in photosynthetic membranes Biochim Biophys Acta 1767, 44 9–4 57... transitions reveal the dynamics and flexibility of the photosynthetic apparatus EMBO J 20, 362 3–3 630 24 Haldrup A, Jensen PE, Lunde C & Scheller HV (2001) Balance of power: a view of the mechanism of photosynthetic state transitions Trends Plant Sci 6, 30 1–3 05 25 Mullineaux CW & Emlyn-Jones D (2005) State transitions: an example of acclimation to low -light stress J Exp Bot 56, 38 9–3 93 26 Miroslavina Y, Nilkens... transitions revisited – a buffering system for dynamic low light acclimation of Arabidopsis Plant Mol Biol 62, 77 9–7 93 Rintamaki E, Martinsuo P, Pursiheimo S & Aro EM (2000) Cooperative regulation of light- harvesting complex II phosphorylation via the plastoquinol and ferredoxin–thioredoxin system in chloroplasts Proc Natl Acad Sci USA 97, 1164 4–1 1649 FEBS Journal 275 (2008) 105 6–1 068 ª 2008 The Authors... energy transduction J Biol Chem 274, 913 7–9 140 Snyders S & Kohorn BD (2001) Disruption of thylakoid-associated kinase 1 leads to alteration of light harvesting in Arabidopsis J Biol Chem 276, 3216 9–3 2176 Depege N, Bellafiore S & Rochaix J-D (2003) Role of chloroplast protein kinase STT7 in LHCII phosphorylation and state transition in Chlamydomonas Science 299, 157 2–1 575 Bellafiore S, Barneche F, Peltier... Dekker JP, Andersson J, Jansson S, Ruban AV & Horton P (2003) The structure of photosystem II in Arabidopsis: localization of the CP26 and CP29 antenna complexes Biochemistry 42, 60 8–6 13 Kargul J, Nield J & Barber J (2003) Three-dimensional reconstruction of a light- harvesting complex Iphotosystem I (LHCI-PSI) supercomplex from the green alga Chlamydomonas reinhardtii Insights into light harvesting. .. distribution of excitation energy between photosystems Nature 291, 2 5–2 9 Zito F, Finazzi G, Delosme R, Nitschke W, Picot D & Wollman FA (1999) The Qo site of cytochrome b6f complexes controls the activation of the LHCII kinase EMBO J 18, 296 1–2 969 Rochaix J-D (2007) Role of thylakoid protein kinases in photosynthetic acclimation FEBS Lett 581, 276 8– 2775 Allen JF (1992) How does protein phosphorylation. .. chloroplasts is not homogeneous Biochim Biophys Acta 1188, 38 0–3 90 Nilsson A, Stys D, Drakenberg T, Spangfort MD, Forsen S & Allen JF (1997) Phosphorylation controls the three-dimensional structure of plant light harvesting complex II J Biol Chem 272, 1835 0–1 8357 Bennett J (1977) Phosphorylation of chloroplast membrane polypeptides Nature 269, 34 4–3 46 Snyders S & Kohorn BD (1999) TAKs, thylakoid membrane... subunit of photosystem I is essential for state transitions in plant photosynthesis Nature 408, 61 3–6 15 54 Ben-Shem A, Frolow F & Nelson N (2003) Crystal structure of plant photosystem I Nature 426, 63 0–6 35 55 Finazzi G, Furia A, Barbagallo RP & Forti G (1999) State transitions, cyclic and linear electron transport and photophosphorylation in Chlamydomonas reinhardtii Biochim Biophys Acta 1413, 11 7–1 29 . MINIREVIEW Photosynthetic acclimation: Structural reorganisation of light harvesting antenna – role of redox-dependent phosphorylation of major and minor chlorophyll. same amount of light. To achieve this, plants and green algae have a mobile pool of chlorophyll a ⁄ b -binding proteins that can switch between being light- harvesting