Biochimica et Biophysica Acta 1807 (2011) 897–905

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Biochimica et Biophysica Acta

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a b i o

Review

State transitions—The molecular remodeling of photosynthetic supercomplexes that

controls energy flow in the chloroplast☆
Jun Minagawa ⁎
National Institute for Basic Biology, Okazaki 444-8585, Japan

a r t i c l e i n f o a b s t r a c t

Article history: In oxygen-evolving photosynthesis, the two photosystems—photosystem I and photosystem II—function in
Received 3 October 2010 parallel, and their excitation levels must be balanced to maintain an optimal photosynthetic rate under
Received in revised form 8 November 2010 natural light conditions. State transitions in photosynthetic organisms balance the absorbed light energy
Accepted 10 November 2010
between the two photosystems in a short time by relocating light-harvesting complex II proteins. For over a
Available online 23 November 2010
decade, the understanding of the physiological consequences, the molecular mechanism, and its regulation
Keywords:
has increased considerably. After providing an overview of the general understanding of state transitions, this
Acclimation review focuses on the recent advances of the molecular aspects of state transitions with a particular emphasis
Electron transfer on the studies using the green alga Chlamydomonas reinhardtii. This article is part of a Special Issue entitled:
Light-harvesting complex Regulation of Electron Transport in Chloroplasts.
Non-photochemical quenching © 2010 Elsevier B.V. All rights reserved.
Phosphorylation

1. Introduction their excitation levels to ensure optimal efficiency of electron flow.

State transitions occur under such conditions to redistribute the
In eukaryotes, photosynthesis is a process of photochemical harnessed energy to minimize its unequal distribution.
energy transduction, which occurs via the conductance of electron The phenomenon that we know today as “state transition” was
flow in the thylakoid membranes of chloroplasts, resulting in the discovered at the same time and independently by Murata [3] and
reduction of NADP+ in the stroma and the concomitant generation of Bonaventura and Myers [4,5] in early 1969. Norio Murata, who had
a proton motive force across the membranes. The NADPH generated been studying fluorescence properties of a unicellular red alga,
by the electron flow and the ATP synthesized by ATP synthase Porphyridium cruentim, observed that chlorophyll a (Chl) fluorescence
utilizing the proton motive force are used to fix carbon dioxide in the in the far-red region, which was primarily from the PSI antenna, was
Calvin–Benson cycle; the carbon dioxide is ultimately converted to increased upon the selective excitation of phycoerythrin, which
sugars. The two photosystems—photosystem I (PSI) and photosystem constitutes the PSII peripheral antenna in red algae [3,6]. Celia
II (PSII)—in the thylakoid membranes function as charge-separation Bonaventura and Jack Meyers had been studying another unicellular
devices. Each has a distinct pigment system with distinct absorption green alga, Chlorella pyrenoidosa, and observed that the yield of Chl
characteristics (PSI has a broad absorption peak in the far-red region fluorescence in the red region, which was primarily from the PSII
as well as peaks in the blue and red regions, whereas PSII has antenna, was decreased, and that the oxygen evolution rate was
absorption peaks in the blue and red, but not in the far-red region) increased due to the increased oxidation of PSII after the energy
and a distinct action spectrum. Thus, an imbalance of energy redistribution from PSII to PSI [4,5,7]. The two groups reached the
distribution between the two photosystems tends to occur in natural same conclusion that the light-harvesting ability of the two photo-
environments, where light quality and quantity fluctuate with time systems was regulated to balance the distribution of the excitation
[1,2]. Since the two photosystems are connected in series under between the two photosystems.
normal conditions, green plants and algae need to constantly balance In green plants and algae, State 1 is characterized by a higher Chl
fluorescence yield at room temperature, a higher ratio of fluorescence
intensity in the red region as compared with the far-red region at 77 K, a
Abbreviations: Chl, chlorophyll a; CEF, cyclic electron flow; DBMIB, 2,5-dibromo-3-
methyl-6-isopropylbenzoquinone; Cyt bf, cytochrome b6f complex; DCMU, 3-(3,4- higher quantum yield of PS II reactions, and a lower quantum yield of PS I
dichlorophenyl)-1,1-dimethylurea; Fd, ferredoxin; FNR, ferredoxin–NADPH oxidore- reactions. Conversely, State 2 is characterized by a lower Chl fluorescence
ductase; LHCI and LHCII, light-harvesting complex I and light-harvesting complex II; LEF, yield at room temperature, a lower ratio of fluorescence intensity in the
linear electron flow; PSI and PSII, photosystem I and photosystem II; PQ, plastoquinone red region as compared with the far-red region at 77 K, a lower quantum
☆ This article is part of a Special Issue entitled: Regulation of Electron Transport in
Chloroplasts.
yield of PS II, and a higher quantum yield of PS I reactions.
⁎ Tel.: +81 564 55 7515; fax: +81 564 55 7516. Although the core idea of state transitions has been established
E-mail address: minagawa@nibb.ac.jp. since 1969, our understanding of the molecular components and

0005-2728/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbabio.2010.11.005
898 J. Minagawa / Biochimica et Biophysica Acta 1807 (2011) 897–905

structural basis for this phenomenon has been advanced only same core architecture of the PSI–LHCI supercomplex, but the green
recently, through molecular genetic and biochemical studies in two algal PSI seems to have more LHCI subunits bound (Fig. 1B).
model organisms: the green alga Chlamydomonas reinhardtii and the
higher plant Arabidopsis thaliana. In particular, many of the recent 3. Regulation of state transitions
findings regarding state transitions were reported with C. reinhardtii;
examples of these recent findings include the involvement of the 3.1. Phosphorylation of LHCII
cytochrome b6f complex (Cyt bf) in the control of light-harvesting
complex II (LHCII) phosphorylation, the identification of LHCII kinase, Chloroplasts contain the highest concentration of plant phospho-
mapping phosphorylated residues in LHCII, the molecular reorgani- proteins [28]. Among these, LHCII phosphorylation was first observed
zation of the photosystem supercomplexes, and the interplay in illuminated pea chloroplasts supplied with [32P]orthophosphate;
between the other physiological responses, such as cyclic electron the Thr residues were reversibly phosphorylated upon illumination
flow (CEF) and excess energy dissipation. This is due, in part, to as [29]. Inhibition of LHCII phosphorylation by the PSII inhibitor 3-(3,4-
much as 80% of the LHCIIs being mobile during state transitions in this dichlorophenyl)-1,1-dimethylurea (DCMU) indicated that this light-
green alga [8], while only 20–25% of LHCIIs migrate in higher plants dependent phosphorylation was activated through electron flow
[1]. rather than a process mediated by a kinase directly activated by light
In this review, I present an overview of the current understanding [30]. The phosphorylation of LHCII coincided with a decrease in the
of state transitions with a particular emphasis on those recent yield of Chl fluorescence from PSII [31–33]. Low temperature
discoveries using C. reinhardtii. Readers should refer to other reviews fluorescence spectra showed that LHCII phosphorylation resulted in
providing complimentary information on LHCII phosphorylation a decreased yield of the PSII fluorescence band in the red region
[1,9,10], LHCII kinase [11], the mechanism of LHCII migration relative to that of the PSI band in the far-red region [31,32], indicating
[12,13], the role of Cyt bf [14], and switching between CEF and linear that LHCII phosphorylation increases the proportion of absorbed
electron flow (LEF) [15]. excitation energy transferred to PSI; this was further confirmed by the
increase of the Chl/P700 ratio and quantum efficiency of PSI (ΦI)
2. Photosystem supercomplexes [8,34,35].

The PSII–LHCII supercomplex is a large chlorophyll–protein complex 3.2. Phosphorylated residues in LHCII
comprising more than 30 subunits, which collects light energy, converts
it into electrochemical energy, and drives electron flow from water to Information about the phosphorylated residues was fragmented
plastoquinone (PQ). Light energy is captured by the peripheral antenna until the genome sequences became available; then the proteomic
and is transferred to the dimeric core complex, where it is trapped. In approach using mass spectrometry could be applied. Vener and his
green plants, the peripheral antenna is formed by two layers of protein colleagues mapped the phosphorylated amino acid residues in the
complexes, i.e., (1) major “more abundant” trimeric LHCII proteins, and thylakoids in C. reinhardtii during state transitions [36,37]. Two
(2) minor “less abundant” monomeric LHCII proteins [16]. In the higher phospho-LHCII proteins were found in State 2 cells, including LhcbM1
plant A. thaliana, there are three major trimeric LHCII proteins (types I– (major LHCII type IV) and CP29. CP29 was heavily phosphorylated;
III) encoded by 5, 4, and 1 duplicated genes (Lhcb1.1–1.5; Lhcb2.1–2.4; Thr-7 and -33 were phosphorylated irrespective of the state, and Thr-
and Lhcb3.1), respectively [17]. In contrast, in the green alga C. 17 and Ser-103 were phosphorylated only in State 2 [37,38]. Thr-27 in
reinhardtii, there are four major LHCII proteins (types I–IV) encoded the major LHCII and Thr-7 in CP29 were commonly phosphorylated in
by 5, 1, 2, and 1 duplicated genes (LhcbM3, -4, -6, -8, and -9; LhcbM5; C. reinhardtii and higher plants [39,40]. These redox-dependent and
LhcbM2 and -7; LhcbM1), respectively [18]. The three minor monomeric the other light-dependent phosphorylated residues in D1, D2, CP43,
LHCII polypeptides CP29, CP26, and CP24 are encoded by the genes CP26, and LhcbM4, -6, -9, and -11 (major LHCII type I) clustering at
Lhcb4, -5, and -6, respectively, in A. thaliana [17], whereas C. reinhardtii the interface between the PSII core and the peripheral LHCII proteins
only contains the first two [19]. may suggest their role in a mechanism to undock peripheral LHCII
Single-particle image analysis of electron micrographs revealed proteins during state transitions as well as energy dissipation under
that these peripheral antenna proteins are bound to both sides of the high light conditions [37].
PSII core, each of which consists of one LHCII trimer residing at the
outer edge of the PSII–LHCII supercomplex, and two LHCII monomers, 3.3. Regulation of LHCII phosphorylation
CP26 and CP29, which border the core complex, as well as the LHCII
trimer in spinach [20] and C. reinhardtii [21] (Fig. 1A). In this protein 3.3.1. Redox control
complex organization, called the C2S2 supercomplex, “C” and “S” refer Imbalances in the excitation of the two photosystems are sensed
to the PSII core complex and strongly bound LHCII trimer, respectively by the redox state of the intersystem electron transfer component in
[16]. In higher plants, there are also C2S2M1 and C2S2M2 complexes, the thylakoid membranes, which gives rise to LHCII phosphorylation
where two moderately (hence the “M”) bound LHC II trimers along (Fig. 2). Several initial studies suggested PQ as a redox regulator of the
with CP24 are associated with the C2S2 supercomplex [22] (Fig. 1C). phosphorylation of LHCII. Reduced ferredoxin (Fd) [41], dithionite
To date, the crystal structure of no PSII–LHCII supercomplex has been [42], and duroquinol [43], which are known to reduce PQ in isolated
determined. thylakoids, activated LHCII phosphorylation in the dark, whereas
All eukaryotic PSI cores characterized so far are monomers [23], DCMU, which is known to block PSII, inhibited light-dependent LHCII
but trimeric PSI cores have been observed in cyanobacteria [24,25]. phosphorylation [32], indicating that LHCIIs are phosphorylated when
Attached to the core are peripheral light-harvesting antennas the PQ pool is reduced. Further analysis revealed that it is not the
composed of several light-harvesting complex I (LHCI) proteins. The reduced PQ molecule (PQH2) per se that is critical for the activation of
results of single-particle analysis suggested that the LHCI proteins are the kinase but the binding of PQH2 to Cyt bf. Wollman and Lemaire
asymmetrically bound to the PSI core in C. reinhardtii [26] (Fig. 1B) [44] reported that C. reinhardtii mutants lacking Cyt bf were unable to
and spinach [27] (Fig. 1D). The crescent-shaped “LHCI belt” was phosphorylate LHCII while the other phosphoproteins in the thyla-
demonstrated to be attached to the side of PsaJ/F/G subunits in a 4.4 Å koids were unaffected. Bennett et al. [45] reported that while an
crystal structure of the PSI–LHCI supercomplex from pea [23]. The inhibitor of the stromal side of Cyt bf (Qi-site), 2-heptyl-4-hydro-
other side with PsaH/L/O subunits is open, at least under normal xyquinoline-N-oxide (HQNO) did not affect activation of LHCII
conditions. It is likely that green algae and higher plants share the phosphorylation by PQH2, a lumenal side inhibitor of Cyt bf, 2,5-
 J. Minagawa / Biochimica et Biophysica Acta 1807 (2011) 897–905 899

Fig. 1. Subunit composition of PSII–LHCII and PSI–LHCI supercomplexes in higher plants and green algae. A, Top view of a C2S2-type PSII–LHCII supercomplex, based on the single-
particle image of PSII–LHCII supercomplexes from C. reinhardtii [116]. B, Top view of a PSI–LHCI supercomplex, based on the single-particle image of PSI–LHCI supercomplexes from
C. reinhardtii [26]. C, Top view of a C2S2M2-type PSII–LHCII supercomplex, based on the single-particle image of PSII–LHCII supercomplexes from spinach [16]. D, Top view of a PSI–
LHCI supercomplex, based on the crystallographic image of PSI–LHCI supercomplex from pea [117]. All top view images are from the luminal sides. The PSII and PSI cores were taken
from the coordinates for the crystallographic structures 3BZ1/2.pdb and 2WSC.pdb, respectively.

dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), inhibited ylated [54], they may be part of a signal cascade; unfortunately, no
LHCII phosphorylation, suggesting that the binding of PQH2 to the further investigations have been conducted.
Qo-site of Cyt bf activates LHCII kinase(s) (Fig. 2). Consistent results Probably the most likely candidate for the LHCII kinase was
were provided by later studies using spectroscopy [46] and site- identified in a genetic screen using C. reinhardtii. Using the
directed mutants around the Qo-site [47]. There are also reports characteristic fluorescence quenching due to a State 1-to-2 transition,
claiming that LHCII kinase(s) is not only activated by the reduced PQ Rochaix and his coworkers isolated several mutants that lack state
pool, but also deactivated by the reduced thioredoxin pool that is transitions [56]. One such mutant, stt7, was demonstrated to be
downstream of PSI in the stroma of the chloroplasts [48,49]. deficient in a Ser/Thr kinase in the chloroplast thylakoid membranes
[57], where LHCIIs were barely phosphorylated. Subsequently, a
3.3.2. LHCII kinases mutant lacking an ortholog of Stt7 in A. thaliana, STN7, also showed an
Although the activity of the LHCII kinase was identified in 1977 impairment in state transitions in a land plant [2]. The Stt7/STN7
[29], biochemical attempts to isolate the specific LHCII kinases have kinase contains a single membrane-spanning domain and is localized
been unsuccessful [50–53]. The identities of the kinase and phospha- in the thylakoid membranes. Immunoprecipitation assays indicated
tase of LHCII had thus remained elusive until recently. Snyders and that Stt7 could interact with LHCII and Cyt bf, and PSI subunits. The
Kohorn [54] screened for proteins able to interact with the N-terminal two Cys-residues (which are conserved in all Stt7/STN7 orthologs in
region of LHCII in A. thaliana and identified TAK kinases, which form a various species) near the lumen-exposed N-terminus of Stt7 are
protein family with the human TGFβ1 receptor. The TAK kinases, critical for its activity and might be potential targets for the
which are not conserved among green algae and could be specific to thioredoxin-mediated inhibition [58].
land plants, were shown to be associated with PSII and Cyt bf [55]. The
antisense lines led to a decrease in LHCII phosphorylation, increased 3.3.3. LHCII phosphatases
sensitivity to high light, and partial deficiency in the ability to perform The reversible phosphorylation of LHCII observed with thylakoids
state transitions [55]. Because TAK kinases are themselves phosphor- during the state transitions implies that the phosphatases participate
900 J. Minagawa / Biochimica et Biophysica Acta 1807 (2011) 897–905

phosphatase rather improves the photosynthetic performance and

plant growth under constant low-light (State 2-favoring) condi-
tions [64], implying that the fitness advantage is expected specifically
under State 1-favoring conditions in higher plants. The LHCII
phosphatase(s) in C. reinhardtii, which has no TAP38/PPH1 counter-
part [64], need to be clarified.

4. Molecular remodeling of PSI

4.1. LHCII migration

How can LHCII phosphorylation decrease the energy distribution

to PSII and increase the energy distribution to PSI? Lateral heteroge-
neity in the distribution of the photosystems between appressed and
non-appressed membranes has been observed: the non-appressed
region (stromal lamella) is highly enriched in PSI, and the appressed
region (grana lamella) is highly enriched in PSII and LHCII [65]. LHCII
in the non-appressed region is more phosphorylated than in the
appressed region [66,67], suggesting that the phosphorylation of
LHCII in the appressed region causes the migration of LHCII from the
PSII-enriched appressed region to the PSI-enriched non-appressed
region [68–70]. Initially, the association of LHCII proteins with PSI was
shown indirectly as changes in the quantum yield of PSI and PSII
during state transitions [35].

4.2. The role of PsaH

Scheller and his colleagues studied the association of the mobile

LHCII with PSI via crosslinking and the antisense approaches. A.
thaliana plants without PsaH and PsaL [71], as well as those without
PsaO [72], were highly deficient in state transitions. Since these small
PSI subunits are located on a vacant side of the PSI core, which is on
the opposite side of the LHCI belt [23] (Fig. 1), these PSI subunits were
hypothesized to constitute a specific binding site for the mobile LHCII.
Fig. 2. Schematic representation of the regulation of state transitions. State 1, when PSI Later, binding of a LHCII trimer, and possibly a few LHCII monomers,
is preferentially excited, the PQ pool is oxidized. In this state, LHCIIs are bound to PSII. was observed near this site in an electron microscopic study of
The photosynthetic electron flow proceeds in LEF mode generating a proton gradient partially solubilized thylakoids from A. thaliana by digitonin [73]. In
across the thylakoid membrane that is used for ATP production, as well as NADPH. In
the transient state at the onset of the preferential excitation of PSII, the PQ pool gets
the absence of the docking site protein PsaH, LHCII still underwent
reduced. Docking of PQH2 to the Qo-site of Cyt bf leads to the activation of the Stt7 phosphorylation in State 2, and a significant portion of the phospho-
kinase, which is required for the phosphorylation of LHCIIs, causing the undocking of LHCII remained attached to PSII. Similarly, phospho-LHCIIs were also
the mobile LHCIIs (orange) from PSII. State 2, the reassociation of mobile LHCII occurs observed to be part of the PSII antenna in PSI-deficient C. reinhardtii
on the PsaH side of PSI that is on the opposite side of the LHCI belt. Cyt bf and FNR are
mutants [8].
also bound to PSI to form a super-supercomplex (CEF supercomplex), which is required
to establish CEF between PSI and the PQ pool generating mostly ATP. bf, Cyt bf; PQ/
PQH2, plastoquinone/plastoquinol; Pc, plastocyanin; Fd, ferredoxin. 4.3. Mobile LHCII and the role of minor LHCII

Despite an accumulation of physiological observations, the

in the process and that at least a portion of those are membrane structural basis for state transitions has remained obscure because
associated. The activities of such thylakoid protein phosphatases have of the lack of information about the identity of the mobile LHCII
been reported to be redox independent and constitutively active [59]. polypeptide(s) [12]. The two obstacles toward this identification were
A biochemical approach demonstrated that protein phosphatases of as follows: 1) possible loose binding of the mobile LHCII protein(s) to
different families were involved in the reversible phosphorylation of PSI under State 2 conditions, which could lead to their detachment
thylakoid phosphoproteins [60–62]. The LHCII phosphatase activity during biochemical manipulation, and 2) most of the biochemical
was shown to depend on the presence of divalent cations, and it was studies were carried out in higher plants, where the amount of energy
not inhibited by microcystin and okadaic acid [60,62]. These findings redistribution is small, making it difficult to detect a correspondingly
strongly suggested the involvement of a PP2C-type phosphatase in small amount of mobile LHCII polypeptides. Only recently have
phospho-LHCII dephosphorylation. Recently, two groups reported a several lines of direct biochemical evidence shown the binding of
putative thylakoid-bound phosphatase in A. thaliana, called TAP38/ LHCII proteins to PSI [74–76]. Such an association was first observed in
PPH1, which is specifically required for the LHCII dephosphorylation, the A. thaliana psae1-1 mutant; a fraction of LHCII was associated with
but not for that of the PSII core proteins [63,64]. This phosphatase, PSI when the mutant plants were exposed to low-light (State 2-
which belongs to a family of monomeric PP2C type phosphatases, is favoring) conditions, giving rise to a high-molecular-mass protein-
mainly associated with the stromal lamellae of the thylakoid pigment complex [74]. This large complex, however, seemed to be an
membranes. Loss of the TAP38/PPH1 phosphatase leads to an increase aggregated product, because the mutant did not show state transi-
in the antenna size of PSI and impairs the State 2-to-1 transition [63], tions, probably owing to a low level of PsaH. The next attempt was via
and overexpression of the gene decreases LHCII phosphorylation and the crosslinking of the mobile LHCII proteins with the PSI–LHCI
inhibits State 1-to-2 transitions [63,64]. Interestingly, the LHCII supercomplex in A. thaliana [75]. In State 2, more major LHCII
hyperphosphorylation caused by the inactivation of the LHCII proteins, including Lhcb1 and -2, were crosslinked to the PsaH, PsaI,
 J. Minagawa / Biochimica et Biophysica Acta 1807 (2011) 897–905 901

and PsaL subunits than in State 1. A further report was provided from showed that the megacomplex was predominant in State 1, whereas
a study on C. reinhardtii, where the PSI–LHCI/II supercomplex isolated the core complex was predominant in State 2, indicating LHCIIs are
from State 2 cells contained two minor monomeric LHCII (CP26 and dissociated from PSII upon a State 1-to-2 transition. Moreover, in State
CP29) and one major trimeric LHCII (LhcbM5), suggesting a pivotal 2, strongly phosphorylated LHCII type I (LhcbM3, -4, -6, and -9) was
role for the minor monomeric LHCII in state transitions [76]. Mobile found in the supercomplex, but not in the megacomplex. Phosphor-
LHCII proteins were also observed in single-particle images of the PSI– ylated minor LHCIIs (CP26 and CP29) were only found in the unbound
LHCI/II supercomplexes. As described above, Boekema and his- form. The PSII subunits including the CP43 and D2 proteins were most
colleagues [73] reported a large density along the side of PsaH/L/A/K phosphorylated in the core complex. Based on these observations, I
in electron micrographs of A. thaliana PSI, which they assigned to the propose the following 3-step model for the PSII remodeling during a
LHCII trimer. Barber and his-colleagues located a smaller density near State 1-to-2 transition (Fig. 3): Initially, unphosphorylated LHCIIs
PsaH in C. reinhardtii, which they assigned to CP29 [77]. stabilize the megacomplex (State 1); 1) the phosphorylation of LHCII
The significance of the minor LHCII in state transitions in C. type I in the major LHCII trimer triggers the division of the
reinhardtii was further supported by an RNA interference (RNAi) megacomplex, resulting in individual C2S2 supercomplexes; 2) the
study, where one of the two minor LHCII proteins, CP29 or CP26, was phosphorylation of CP26 and CP29, as well as the PSII core subunits D2
knocked down [78]. Both the CP29 and CP26 RNAi mutants and CP43, induces the undocking of all the LHCIIs from PSII; and 3)
underwent reductions in the PSII antenna size during a State 1-to-2 the dissociated LHCIIs reassociate with the PSI–LHCI supercomplex
transition, as reflected by the non-photochemical quenching of yielding State 2.
fluorescence, low temperature fluorescence spectra, and functional
absorption cross section. However, the undocked LHCIIs from PSII did 5.2. Phosphorylation of the major LHCII
not reassociate with PSI in the CP29-RNAi mutant because the
antenna size of PSI was not complementarily increased. However, the It has been hypothesized that phosphorylated major LHCIIs do not
mobile LHCIIs in the CP26-RNAi mutant reassociated with PSI, whose necessarily dissociate themselves from PSII in A. thaliana [71]. In C.
PSI–LHCI/II supercomplex was visualized on a sucrose density reinhardtii, LhcbM3, -4, -6, and -9 (LHCII type I) remain associated
gradient, thus clarifying that CP29, not CP26, is crucial when mobile with PSII upon phosphorylation [79]. The phosphorylation of LHCII
LHCIIs reassociate with PSI under State 2 conditions in C. reinhardtii. type I is induced earlier in the course of state transition, and it is
mostly localized in the supercomplex, not in the megacomplex. This
5. Molecular remodeling of PSII suggests that the phosphorylation of LHCII type I probably causes the
division of the megacomplex into single supercomplexes. As has been
5.1. LHCII dissociation previously described by Dekker and Boekema [16], the two C2S2
supercomplexes constituting the megacomplex are bridged by an
Studies on the migration of LHCII proteins have focused primarily LHCII trimer in C. reinhardtii. Therefore, the phosphorylation of the
on their reassociation with PSI. However, the polypeptide composi- most abundant trimeric LHCII type I may induce the division of the
tion, supramolecular organization, and phosphorylation of PSII megacomplex due to its altered conformation [80,81].
complexes under State 1 and State 2 conditions were recently studied LHCII types III (LhcbM2 and -7) and IV (LhcbM1) were not
in C. reinhardtii [79]. Three PSII fractions—the PSII core complex, the associated with PSII when they were phosphorylated. These unbound
PSII–LHCII supercomplex, and the multimer of PSII–LHCII super- phosphorylated polypeptides were detected not only in the State 2
complex (PSII megacomplex)—were affinity-purified from a mutant samples but also in the State 1 samples, implying that the
carrying a His-tagged CP47. Gel filtration and electron microscopy phosphorylation-induced undocking of LHCII types III and IV occurs

Fig. 3. A current model of molecular remodeling of PSI and II during a State 1-to-2 transition. 1) Initially, unphosphorylated LHCIIs stabilize the PSII–LHCII megacomplex; 1) the
phosphorylation of LHCII type I in the major LHCII trimer triggers the division of the megacomplex, resulting in discrete PSII–LHCII supercomplexes; 2) the phosphorylation of CP26
and CP29, as well as the PSII core subunits D2 and CP43, induces the displacement of LHCIIs from the PSII core complex; several dissociated phospho-LHCII complexes then form an
energy-dissipative aggregation; and 3) several LHCIIs (including minor LHCII) reassociate with PSI, thereby completing the State 1-to-2 transition. A top-down view from the
lumenal side of the membrane is provided. The crystal structures of the PSII core complex and PSI–LHCI supercomplex are from 3BZ1/2.pdb and 2WSC.pdb, respectively.
Phosphorylations are indicated by red circles with a letter “P”.
902 J. Minagawa / Biochimica et Biophysica Acta 1807 (2011) 897–905

irrespective of the state and has a unique role in photosynthesis other free LHCII shows the same fluorescence lifetime, this 250 ps
than state transitions. It should be noted that a mutant of the LhcbM1 component was ascribed to dissociated and free LHCII. Single-cell
gene in C. reinhardtii (npq5) has been shown to undergo normal state FLIM further indicated that the dissociated LHCII spreads through the
transitions [82]. Interestingly, the npq5 mutant was originally isolated cell during State 2 transitions and forms several large spotted areas.
as exhibiting little qE quenching [83]. This implies that the Further biochemical analyses indicated that dissociated phospho-
phosphorylated and dissociated LHCII type IV (LhcbM1) may, in LHCII formed a large aggregated structure, whereas unphosphory-
fact, be involved in qE quenching in addition to their role in state lated LHCII did not. Thus, the free phospho-LHCII aggregates
transitions. appearing during State 2 transitions are in energy-dissipative form,
which has previously been suggested [78,95]. There is a substantial
5.3. Phosphorylation of minor LHCII difference between the fluorescence lifetime of phospho-LHCII in vivo
(~250 ps) and that in vitro (~1300 ps) in the study by Iwai et al. [94],
In C. reinhardtii, the minor monomeric LHCIIs are shuttled to PSI, which suggests that additional factor(s) must be involved in the
thus acting as a linker between PSI and major trimeric LHCII during a stronger energy dissipation observed in vivo. An unidentified
transition to State 2 [76–78]. Although both CP26 and CP29 are not component must be associated with the phospho-LHCII aggregates
phosphorylated while associated with the PSII–LHCII megacomplex or during State 2 in C. reinhardtii cells to make the fluorescence lifetime
PSII–LHCII supercomplex, they were phosphorylated when dissociat- of LHCII aggregates even shorter.
ed from PSII, suggesting that their dissociation was caused by their The molecular mechanism for qE quenching has been a heated
phosphorylation. The minor monomeric LHCIIs border the major issue during the last two decades, and it remains controversial (for a
LHCII trimers and the PSII core [84,85] as shown in Fig. 1C, and the review, see [96]). Because the unexpectedly short fluorescence
hyperphosphorylated residues in CP29 were indeed mapped at the lifetime from the phospho-LHCII aggregates during a state transition
interface of the PSII core and the peripheral antenna proteins [37]. described above were not caused by high light illumination, they are
Therefore, the phosphorylation of CP26 and CP29 likely triggers the not exhibiting qE quenching, but rather exhibiting qT (state transition)
undocking of the entire peripheral antenna during the State 2 quenching [97]. However, in the light of a recent report suggesting
transition. that qE quenching could also involve LHCII dissociation [98,99], it is
tempting to speculate that LHCII aggregates are a common site of
5.4. Phosphorylation of PSII core subunits energy dissipation, i.e., that both qE and qT quenching are causally
related by the energy-dissipative LHCII aggregates [94,100]. If such
Since phosphorylation of the PSII core subunits and LHCIIs are both energy-dissipative aggregation is facilitated or reinforced by PsbS
controlled by the redox state of the PQ pool [9,86], the two (higher plants and moss [98,101,102]) or LHCSR (moss and eukaryotic
phosphorylation events could be coincident. In fact, phosphorylation algae [102–104]), the quenching could be classified as qE; if it is
of two of the PSII core subunits CP43 and D2 protein was observed in induced by redox-controlled LHCII phosphorylation, the quenching
State 2 C. reinhardtii along with LHCII phosphorylation [79]. Unlike could be classified as qT. Whether there is a pool of unbound LHCII at
LHCII phosphorylation, however, PSII core phosphorylation is inde- all times and how much undocked LHCIIs actually reassociate with PSI
pendent of Cyt bf [9,87]. are points that require further investigation.
Although controversial [88–90], phosphorylation of the D1 protein
has been suggested to have a key function (the damaged D1 is marked 6.2. State transitions and cyclic electron flow
by phosphorylation, which then functions as a signal for migration of
the damaged PSII from grana to stroma lamellas where D1 is State transitions have long been considered as a mechanism by
degraded) during the PSII repair cycle [91–93]. However, the which the distribution of light excitation between the two photo-
significance of D2 and CP43 phosphorylation is not clear. The systems is regulated. However, the performance of PSI tends to
simultaneous phosphorylation of both minor LHCIIs and PSII core overwhelm PSII under State 2 conditions in C. reinhardtii because of its
subunits may facilitate the dissociation of peripheral antenna via extensive ability to relocate LHCII proteins; this implies that state
coulombic repulsion [37,79]. transitions might represent a mechanism by which the electron
transfer chain in the thylakoid membranes is switched to the
6. Interplay between state transitions, cyclic electron flow, and mechanism exclusively employed by PSI [105] (Apparently, this is
energy dissipation not the case in A. thaliana [106]). This type of electron flow—CEF
around PSI (Fig. 2)—has been known for years [68] in addition to LEF
6.1. State transitions and energy dissipation from water to NADP+ via PSII and PSI in series [107]. The dual
organization of the electron transfer chain in the thylakoid mem-
Although state transitions have been widely accepted as a short- branes has been well documented in plants and algae [108].
term response in plants to acclimate to the fluctuating light The hypothesis that state transitions switch the electron flow was
conditions, most of the previous investigations were conducted in tested spectroscopically by assessing the relative contribution of LEF
vitro, implying that the real impact on photosynthesis remains to be and CEF to electron flow to PSI [109]. When the light-induced
characterized. Recently, phospho-LHCII dissociation was visualized in reduction of Cyt bf was probed in State 1 and State 2 adapted cells, a
vivo during state transitions using fluorescence lifetime imaging differential sensitivity to the addition of the PSII inhibitor DCMU was
microscopy (FLIM) [94]. Real-time monitoring of protein interactions observed. DCMU blocked reduction of Cyt bf in State 1, but not in State
in plants using fluorescence probes has been often hampered by Chl 2, suggesting that PSII activity was not required for Cyt bf reduction in
autofluorescence from chloroplasts. However, Iwai et al. made use of State 2 [109]. In contrast, an identical sensitivity to the addition of an
such Chl fluorescence by differentiating the particular component inhibitor of the Qo-site of Cyt bf, DBMIB, was observed in both State 1
from the mobile LHCII by its fluorescence lifetime. In their study, the and State 2 conditions [109]. The switching of electron flow by state
fluorescence lifetime in live C. reinhardtii cells was monitored under a transitions was further confirmed with the stt7 mutant of C.
fluorescence microscope during a transition from State 1 to 2. Initially, reinhardtii, which is locked in State 1 because of the lack of LHCII
the average lifetime of fluorescence emitted between 680 and 700 nm phosphorylation [57]. In this mutant, electron flow remained sensitive
was 170 ps, which was largely due to the PSII-bound LHCII, but it to DCMU under conditions promoting both State 1 and State 2 [110].
shifted to 250 ps when the cells were in transition to State 2 after 5 Thus, the DCMU-sensitive CEF can be switched on only when State 1-
min. Because a mutant lacking both photosystems but having retained to-2 transitions occur. Upon preferential excitation of PSII (State 2),
 J. Minagawa / Biochimica et Biophysica Acta 1807 (2011) 897–905 903

CEF becomes predominant, but upon preferential excitation of PSI Furthermore, it is not known whether there is a relationship between
(State 1), LEF becomes predominant. state transitions and qE quenching. The driving force for the formation
Since CEF generates only ATP, not NADPH, it is crucial to achieve a of the CEF supercomplex remains to be identified, and it is not known
proper balance of NADPH and ATP in the thylakoid stroma of how the major redox players PSI, Cyt bf, and FNR form a super-
photosynthetic organisms; this balance cannot be realized by LEF supercomplex. These issues need to be further investigated if we are
alone [14]. In fact, State 2 can be induced by intracellular ATP to fully understand how plants adapt to changing light environments.
depletion [111]. State transitions could thus be a regulation
mechanism aimed at controlling the ATP/NADPH ratio via the balance Acknowledgements
between LEF and CEF.
Recently, a biochemical study clarified the molecular basis of how I thank Dr. Govindjee for his insightful discussion. Research in my
state transitions switch electron flow [112] (Fig. 1). Iwai et al. used laboratory was supported by Grants-in-Aid for Scientific Research
solubilized thylakoid membranes from C. reinhardtii cells under State from the Ministry of Education, Culture, Sports, Science, and
2 conditions; they were loaded onto a sucrose density gradient. A Technology (18GS0318, 2120006309, and 2157003109), the Strategic
super-supercomplex composed of the PSI–LHCI supercomplex with International Cooperative Program by the Japan Science and Tech-
LHCIIs, Cyt bf, Fd–NADPH oxidoreductase (FNR), and the integral nology Agency, and a Research Grant in the Natural Sciences by the
membrane protein PGRL1 [113] was detected in a fraction heavier Mitsubishi Foundation.
than the PSI–LHCI supercomplex. Spectroscopic analyses indicated
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