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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;

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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 CO2 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 photophoto-system 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 b6f; 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.

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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) QA and QB Following protonation

of the doubly reduced PQ QB, the final product,

plas-toquinol (PQH2), diffuses out of PSII into the

thyla-koid membrane and provides protons and electrons to

the cytochrome b6f (cyt b6f) 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 Yz 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 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

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 NADPH2 The proton gradient concomitantly formed across the thylakoid membrane (light green area) drives the activity of ATP synthase to produce ATP Both NADPH2 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 PQH2to the quinol-binding site of the cyt b6f 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 b6f, 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 b6f 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 A0, chlorophyll a; A1, phylloquinone; Fx, FAand FB,Fe4S4clusters; 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 , QB, fixed and mobile electron carriers.

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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 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

com-plexes involved in photosynthetic electron flow,

includ-ing their light-harvestinclud-ing 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 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 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-short-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, CO2 availability, drought and mineral status (e.g Mg2+ and Fe2+ 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

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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 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

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 phosphorylamodifica-tion 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

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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 b6fcomplex

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 controlldetermin-ing 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 phosphorydown-regu-lation 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 cyt6fcomplex, 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 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 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

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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 b6f 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 durlight-harvest-ing 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 condiimmunolocaliza-tions 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

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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].

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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 transiphosphoryla-tion 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].

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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).

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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

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