Báo cáo khoa học: Photosynthetic acclimation: Structural reorganisation of light harvesting antenna – role of redox-dependent phosphorylation of major and minor chlorophyll a/b binding proteins pot

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:
(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.
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