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: email@example.com
In order to carry out photosynthesis, plants and algae rely on the co-operative 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 maintain an optimal excitation balance. This switch, termed state transitions, involves the reversible phosphorylation of the mobile chlorophyll a/b-binding 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-binding 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.
Oxygenic photosynthesis is one of the most fundamental processes sustaining life on Earth. During this process 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 CO2 into carbohydrates (the building blocks of biomass) are generated. The first step in this process, light-driven charge separation, is conducted by photosystem I (PSI) and photosystem II (PSII), two multimeric 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 chlorophyll molecules bound to the light-harvesting antenna subunits. In response to photo-activation, PSII drives photo-induced oxidization of substrate water molecules to molecular di-oxygen (sustaining the aerobic atmosphere 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, plastoquinones (PQs) QA and QB. Following protonation of the doubly reduced PQ QB, the final product, plastoquinol (PQH2), diffuses out of PSII into the thylakoid membrane and provides protons and electrons to the cytochrome b6f (cyt b6f) complex at the quinol-binding site . The oxidized form of P680 is reduced by electrons derived from substrate water molecules with the aid of a redox-active tyrosine Yz and a catalytic centre composed of four Mn ions and a Ca ion. Linear electron transfer proceeds with the soluble electron carrier plastocyanin, which undergoes reduction by the cyt b6f 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 b6f complex with the concomitant formation of the proton gradient (or electrochemical 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 CO2 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 complexes involved in photosynthetic electron flow, including their light-harvesting complexes (LHC), and their crystal structures are available at intermediate (3.0–3.5 Å) or high (2.5 Å) resolutions (PSII: [4,5]; cyt b6f: [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 Å 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 pigments and cofactors . 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 . 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 complex, together with the crystal structures of some subunits 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 Å . In this model, the dimeric higher plant LHCII–PSII supercomplex binds two LHCII trimers together with two copies of the minor chlorophyll 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 analysed .
Environmental conditions can fluctuate on a timescale of seconds, days and months. Photosynthetic organisms have evolved a number of ingenious short-term and long-term responses to changing environmental conditions in order to maintain an optimal level of photosynthesis. As the light-driven reactions of photosynthesis involve a complex chain of redox reactions, many environmental changes affect, directly or indirectly, the redox state of the components of the photosynthetic electron flow, and thus photosynthetic efficiency . Amongst the environmental changes affecting the quantum yield of photosynthesis are low and high temperatures, CO2 availability, drought and mineral status (e.g. Mg2+ and Fe2+ that act as cofactors of the components of the photosynthetic electron transport chain). However, the most rapidly changing environmental factor is the quantity and spectral quality of incident light, often leading to imbalanced excitation 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 . 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  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 Chlamydomonas 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 regulatory 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 crystallographic and electron microscopy (EM)-based visualization of green algal and plant photosynthetic complexes. Moreover, we will overview the data supporting 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.
Mechanism of state transitions
The process of state transitions represents a short-term adaptation of the photosynthetic apparatus to the conditions 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 maintaining the optimal photosynthetic efficiency . In higher plants and green algae, the basis of this phenomenon 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 . In an ecological context, state transitions may serve as a rapid response preceding a photoprotective adaptation by NPQ during exposure to excess illumination . However, the most significant 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 maximum 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 thylakoid-bound kinase(s). This activation involves the binding of PQH2 to the quinol-binding site of the cyt b6f complex and initiates the phosphorylation of the mobile LHCII antenna (see Fig. 1) [29–31, reviewed in ref. 32]. The phosphorylated LHCII has been proposed 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 overexcitation of PSI (or preferential illumination with light 1), oxidation of the PQ pool occurs followed by de-activation of LHCII-specific kinase(s) and dephosphorylation of mobile LHCII by redox-independent constitutively active phosphatase(s)  (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 surface 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 . A modification 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 . 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 conformational 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 .
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 thaliana. The activity of the LHCII kinase was identified by John Bennett in 1977 ; however, biochemical attempts to isolate the specific enzymes have been unsuccessful to date. Nevertheless, by adopting an alternative approach, a small family of three thylakoid-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 . The antisense Arabidopsis plants with suppressed levels of the threonine kinase TAK1 showed increased sensitivity to high light intensity, a lower level of LHCII phosphorylation and partial deficiency in the ability to perform state transitions . As TAKs are themselves phosphorylated , they may be part of a signalling cascade involving other kinase(s) directly regulated by the reduced cyt b6f 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]. Notably, 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 . However, the direct substrates 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 membrane where the catalytic kinase domain is located . Considerable progress has been made in determining the mechanisms of controlling the activity of the LHCII kinases [24,32]. It is clear that LHCII phosphorylation and the redox state of PQ are not tightly coupled, as there are numerous reports of down-regulation of LHCII phosphorylation at high irradiance, when the PQ pool is reduced . Conversely, maximum phosphorylation of LHCII polypeptides in vivo occurs at low light intensities [24,44]. It now seems that the phosphorylation of LHCII proteins is regulated by a complex network involving co-operative redox control both via PQ and the cyt 6f complex, and through the thioredoxin/ferrodoxin system in the stroma of the chloroplasts . 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 phosphorylation . These two cysteine residues may be potential 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 , 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 structural basis for state transitions, in particular the postulated docking site for the association of the mobile LHCII with PSI under State 2 conditions. The evidence for the lateral migration of a fraction of LHCII and the cyt b6f 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 immunolocalization studies [12,47–50]. The elegant chemical cross-linking and double-stranded RNA interference approaches of Scheller and co-workers provided biochemical 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 , 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 . 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 . 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 hydrophobic 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.
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  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 provides 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 b6f in the stroma lamellae following State 2 adaptation has been suggested 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 . 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 fluorescence video imaging screening assays for identification of mutants affected in the signalling cascade of this process. These types of screening have led to isolation of the series of stm  and stt  mutants deficient in state transitions. The stt7 mutant  has been shown to be of particular importance, as the corresponding gene whose mutation was responsible for the mutant phenotype (blocking in State 1 and deficiency in phosphorylation of LHCII), as discussed above, has been shown to encode a thylakoid-bound protein kinase specific for phosphorylation of LHCII .
Structural remodelling of light-harvesting antenna during state transitions
Although it is widely recognized that during state transitions a pool of LHCII shuttles between PSII and PSI, direct structural evidence for the physical association 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- harvesting subunit, CP29, in State 1-to-State 2 transition in Chlamydomonas . A well-established primary function 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 supercomplexes isolated from the State 2-induced Chlamydomonas cells, we showed that CP29 dissociates from PSII and binds with the core domain of PSI  (see Fig. 2). This redistribution of CP29 was correlated with a quadruple phosporylation of unique Thr and Ser residues at its N-terminal domain . 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  (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 structural 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 Minagawa & 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 unaffected. Thus, for the first time, direct structural evidence for association of the LHCII-like component with PSI was obtained in support of the previous indirect biochemical, spectroscopic and immunolocalization data. We postulate that under some conditions CP29 acts as a sole monomeric Cab protein that increases the absorption cross-section of PSI. Alternatively, under more extreme State 2 conditions, it provides a linker domain for binding the additional LHCII subunits associated with PSI . This latter possibility is not excluded by our work  because it is likely that such PSI–CP29–LHCII supercomplexes are too labile to be successfully purified by standard fractionation procedures in the presence of the dodecyl-maltoside detergent. Whether hyperphosphorylation 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-dependent phosphorylation of the Arabidopsis CP29 isoform Lhcb4.2 . 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 , whereas inactivation of other minor light-harvesting components (CP26 and CP24) does not inhibit the formation 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 destabilization 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. demonstrated, through biochemical analysis, that under State 2 conditions, another minor light-harvesting component, CP26, together with the major LHCII subunit (LhcbM5) and CP29 may also become displaced from PSII and associate with PSI . However, it is important to emphasize that the direct structural evidence for such putative binding of LhcbM5 and CP26 remains to be established, as does the precise mapping of the phosphorylation sites postulated for both proteins based on immunodetection with anti-phosphothreonine serum .
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 followed by EM and single particle analysis of the resultant mildly solubilized protein complexes . This type of analysis allowed, for the first time, visualization 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 Å, the authors propose that a single LHCII trimer binds asymmetrically to the PSI core domain containing 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 trimer on the PsaK side near the PsaH subunit  (see Fig. 4c,d). The LHCII–PSI interaction site is suggested to be composed of PsaH, PsaL, PsaA and PsaK core subunits  (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 . 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 . Bearing in mind that each LHCII monomer binds 14 chlorophyll molecules , and that the absolute number of chlorophyll molecules functionally associated with LHCI–PSI in State 1 has been measured as ∼ 160–200  and assigned as 168 in the latest 3.4 Å crystal structure of LHCI–PSI , 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 densities, the largest one being at the interface between the LHCII trimer and the PsaH/L/K side of the PSI core  (see Fig. 4). The identity of these protein densities remains to be established.
The recent progress in unravelling the structural basis of state transitions has not only advanced our knowledge 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 interaction between the mobile LHCII and the PSI core during state transitions. Another important question arises from the cross-linking results of Zhang & Scheller , 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  into the 3.4 Å X-ray structure of higher plant LHCI–PSI, postulate the most likely position of LHCII to be on the PsaH/L/K side . Further work is required to validate 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 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 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 . It cannot be excluded that the mobile phospho-LHCII may originate from the free LHCII complexes located in a part of the thylakoid membrane, as argued by Dekker & Boekema .
Although it is now well established that phosphorylation 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 , 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 comparison 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, University of Linköping, 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  (see Fig. 3). This indicates that multiple and sequential phosphorylation events within the discrete components of the PSII core and LHCII induce conformational changes sufficient for dissociation of the LHCII–PSII supercomplex 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 phosphorylation events, provide a great challenge for future research.
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 (Linköping) for fruitful discussions and for sharing some unpublished data.