Update on Photosynthetic Apparatus
Assembly of the Photosynthetic Apparatus1
Jean-David Rochaix*
Departments of Molecular Biology and Plant Biology, University of Geneva, 1211 Geneva 4, Switzerland
The primary reactions of photosynthesis are mediated by three protein complexes embedded in the
thylakoid membranes of chloroplasts. These complexes are PSII, the cytochrome b6f complex (Cytb6f),
and PSI, which are connected in series through the
photosynthetic electron transport chain. Light energy
captured by the light-harvesting systems of PSII
(LHCII) and PSI (LHCI) is transferred to the reaction
center chlorophylls to create a charge separation across
the membrane. This leads to the formation of a strong
oxidant on the donor side of PSII capable of splitting
water into molecular oxygen, protons, and electrons.
The electrons are transferred stepwise from PSII to
the plastoquinone pool, Cytb6f, plastocyanin, and PSI
where a second charge separation creates a strong
reductant capable of reducing ferredoxin that subsequently reduces NADP+ to NADPH. Besides this
linear electron pathway there is a cyclic pathway in
which electrons are cycled around PSI through the
Cytb6f complex. The electron transfer reactions are
coupled to proton pumping into the lumen space of
the thylakoids and the resulting pH gradient drives
the ATP synthase for ATP production. Ultimately
NADPH and ATP are used for CO2 assimilation by
the Calvin-Benson cycle. Thylakoid membranes of
land plants are organized in two domains, the grana
stacks, comprising 80% of the membrane, and the
stromal nonappressed membranes that connect different grana stacks. The photosynthetic complexes with
exception of Cytb6f, are not distributed evenly through
the thylakoid membrane system. Whereas PSII is
mostly confined to the grana regions, PSI and ATP
synthase are located in the stroma lamellae. Electron
transfer between these complexes is mediated by the
diffusible electron carriers plastoquinone and plastocyanin.
A characteristic feature of the photosynthetic complexes is that they all consist of numerous chloroplast
and nucleus-encoded subunits. In the case of PSII, PSI,
and Cytb6f these complexes contain in addition pigments such as chlorophylls and xanthophylls as well
as hemes, quinones, and iron-sulfur (Fe-S) centers that
act as redox cofactors. Hence the biogenesis of the
photosynthetic apparatus involves a concerted interplay between the chloroplast and nucleocytosolic ge1
This work was supported by a grant from the Swiss National
Foundation (grant no. 3100AO–117712).
* E-mail [email protected].
www.plantphysiol.org/cgi/doi/10.1104/pp.110.169839
netic systems as well as a tight coordination between
protein and pigment synthesis and insertion into the
thylakoid membranes. Analysis of numerous mutants
affected in photosynthetic activity in Chlamydomonas
reinhardtii, Arabidopsis (Arabidopsis thaliana), and Zea
mays has provided many new insights into the assembly of the photosynthetic complexes (Barkan and
Goldschmidt-Clermont, 2000; Eberhard et al., 2008).
A remarkable feature of photosynthetic organisms is
their ability to adapt to changing light conditions,
which is reflected in changes in the organization of the
photosynthetic complexes. This Update reviews recent
advances in our understanding of the assembly of
these complexes and for some of them, their remodeling and/or reversible association into supercomplexes that depends on environmental conditions.
ASSEMBLY OF PHOTOSYNTHETIC COMPLEXES
A major problem in the assembly of photosynthetic
complexes is how the synthesis and insertion into the
membrane of their different subunits and cofactors is
coordinated. While the plastid protein synthesizing
system has been well characterized and includes components such as RNA polymerases, RNA processing
factors, ribosomes, and translation factors that have
a general role in gene expression, genetic analysis in
C. reinhardtii, Arabidopsis, and Z. mays has revealed a
surprisingly large number of nucleus-encoded factors
that act on specific plastid genes or RNAs. These
factors are required for different posttranscriptional
steps including RNA processing, RNA stability, RNA
editing, splicing, translation, and assembly of the
complexes (Barkan and Goldschmidt-Clermont, 2000;
Eberhard et al., 2008). In C. reinhardtii most of these
factors act in a gene-specific way whereas in land
plants they usually have a more pleiotropic role by
acting on more than one gene or its product. In the case
of factors involved in RNA processing and translation
in C. reinhardtii, the target site is very often located in
the 5#-untranslated region of a specific RNA. Many of
these factors have been identified, some are well conserved in all photosynthetic organisms while others
appear to be organism specific. In C. reinhardtii one of
these factors is usually required for RNA processing/
stability and one or two others for translation of a
given RNA. However, there are exceptions. As an
example, maturation of the chloroplast psaA mRNA
encoding a reaction center subunit of PSI and that is
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Rochaix
assembled from three independently transcribed
RNAs corresponding to three exons requires at least
14 factors involved in either of the two trans-splicing
reactions or in both (Fig. 1; see Barkan and GoldschmidtClermont, 2000).
The trans-acting factors involved in chloroplast
gene expression fall into several classes. The first includes RNA-binding proteins derived from enzymes
involved in RNA metabolism such as for example
pseudouridine synthase (Perron et al., 1999) and
peptidyl-tRNA hydrolase (Jenkins and Barkan, 2001).
These proteins appear to have been recruited for novel
roles in chloroplast gene expression during evolution.
A second class comprises factors containing degenerate repeats of 34 to 38 amino acids. Repeat-containing
proteins are widespread in nature and are involved in
a broad range of cellular activities. The atomic structure of the 34 amino acid repeats, called tetratricopeptide repeats (TPRs) has revealed that each repeat
consists of a pair of antiparallel a-helices with adjacent
repeats packed together and forming a superhelix (Das
et al., 1998). Although TPR motifs are generally involved in protein-protein interactions, in some cases
they may also act as RNA-binding sites. In the case
of the helical repeat Pumilio protein from Drosophila
that contains Puf repeats, it was shown that the internal side of the superhelix acts as RNA-binding site
(Edwards et al., 2001). Unfortunately the chloroplast
TPR proteins are highly insoluble and attempts to test
for RNA binding in vitro have failed. In contrast
specific RNA binding could be demonstrated with
pentatricopeptide repeat (PPR) proteins, which are
structurally similar to TPR proteins except that the
repeats contain 35 residues (Small and Peeters, 2000).
The PPR family contains 450 members in Arabidopsis
that are involved in many steps of RNA metabolism,
especially RNA editing and translation (SchmitzLinneweber and Small, 2008). This large set of proteins
may provide an explanation for the specificity of the
chloroplast RNA targets and suggests the existence of
a PPR motif code in which specific RNA sequences are
recognized by specific PPR repeats. In C. reinhardtii
a large group of repeat-containing proteins are the
PPPEW/OPR (octatricopeptide repeats) proteins.
They were first identified in Tbc2, a factor required
for the translation of the psbC mRNA (Auchincloss
et al., 2002). These repeats were subsequently found in
other nucleus-encoded factors involved in chloroplast
gene expression. In contrast to the TPR and PPR
proteins, the PPPEW/OPR family appears to be restricted to Chlamydomonas among photosynthetic organisms but proteins of this type are also present in
protozoans such as Plasmodium and Theileria.
The existence of a large set of nucleus-encoded
factors required for proper chloroplast gene expression raises the question whether these factors are
constitutively required or whether they also play a
regulatory role. This problem was addressed with two
nucleus-encoded factors MCA1and TCA1 required for
the stable accumulation and translation, respectively,
of the chloroplast petA mRNA encoding cytochrome f
(Raynaud et al., 2007). Accumulation of petA mRNA
and translation of cytochrome f was reduced in transgenic strains producing decreased amounts of MCA1
Figure 1. Biosynthesis and CES cascade for the assembly of PSI in Chlamydomonas. The core PSI subunits are encoded by the
chloroplast genes psaA, psaB, and psaC, indicated by colored boxes. The corresponding RNAs are shown as wavy lines. The
psaA gene is fragmented into three exons whose transcripts are assembled into the mature psaA mRNA through two trans-splicing
reactions mediated in part by the nucleus-encoded factors Raa1, Raa2, Raa3, Rat1, and Rat2. Only those factors whose genes
have been identified are shown. 70S represents chloroplast ribosomes. Mab1 is specifically required for the processing/stability
of the psaB mRNA and Tab2 and Tab3 are necessary for its translation. PsaB is the first PSI subunit to be inserted into the
membrane. In its absence the CES subunit cannot assemble and inhibits directly or indirectly its own synthesis. Once CPI
consisting of PsaA and PsaB has been formed, the next subunit PsaC can assemble to form RCI. In the absence of CPI, PsaC
inhibits its own synthesis. The other PSI subunits are integrated subsequently into the complex. Ycf3, Ycf4, and Ycf37 are specific
PSI assembly factors that act at early stages of assembly.
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Assembly of Photosynthetic Complexes
or TCA1, indicating that these factors are limiting.
Moreover because MCA1 is a short-lived protein, its
abundance varies rapidly under physiological conditions where the demand for Cytb6f changes. Such a
case occurs under nitrogen starvation or in aging
cultures where a decrease of both factors can be correlated with a loss of petA mRNA and the disappearance of Cytb6f (Raynaud et al., 2007).
The assembly of the photosynthetic complexes
within the thylakoid membrane requires both general
and complex-specific proteins. In vascular plants and
Chlamydomonas, the ALB3 protein is required for the
integration of the LHC proteins into the thylakoid
membrane (Sundberg et al., 1997; Bellafiore et al.,
2002). Mutants deficient in ALB3 are albino in Arabidopsis. This protein is related to the mitochondrial
Oxa1p and YidC proteins from Escherichia coli that are
essential components for inserting proteins into membranes. There are two ALB3-related genes in Chlamydomonas Alb3.1 and Alb3.2. Mutants deficient in
Alb3.1 accumulate reduced levels of LHCII. Moreover
the insertion of D1 into functional PSII complexes is
retarded and Alb3.1 is associated with D1 upon its
insertion into membranes. As Alb3.1, Alb3.2 is also
located in the thylakoids. Coimmunoprecipitation experiments reveal that Alb3.2 interacts with Alb3.1 and
with the reaction center polypeptides of PSI and PSII,
and also with VIPP1, a protein involved in thylakoid
formation (Göhre et al., 2006). Although Alb3.1 and
Alb3.2 are related, with 37% sequence identity and
57% sequence similarity, the phenotypes of the respective mutants differ drastically. A decrease of Alb3.2 to
25% to 40% of wild-type levels leads to a reduction in
PSII and PSI and several other photosynthetic proteins, indicating that Alb3.2 is limiting for the assembly and/or maintenance of these complexes in
thylakoids. However other proteins such as VIPP1
and the chloroplast chaperone pair Hsp70/Cdj2 are
overproduced under the same conditions. Moreover
the size of the vacuoles increases considerably and,
after a prolonged period, cell death occurs, indicating
that in contrast to Alb3.1, Alb 3.2 has an essential
function probably related to thylakoid biogenesis
and/or function (Göhre et al., 2006). This raises new
questions on the role of thylakoids in cell growth and
survival.
Besides these general assembly factors, a large
number of complex-specific assembly factors have
been identified. They include Ycf3, Ycf4, and Ycf37
in the case of PSI (Fig. 1). In mutants deficient in these
proteins, the PSI proteins are still synthesized, but they
do not accumulate presumably because of a defect in
the assembly of the complex. These proteins are also
present in cyanobacteria. However in several cases
their role appears to have changed during evolution as
the loss of these proteins generally has more dramatic
effects in plants and algae than in cyanobacteria. It
is not clear how these factors act. Ycf3 contains TPR
repeats and was shown to interact with PsaA and
PsaD (Naver et al., 2001). Ycf4 of C. reinhardtii is part of
a high molecular complex of more than 1,500 kD that
associates with newly synthesized PSI subunits partially assembled as a chlorophyll-containing subcomplex, suggesting that the Ycf4 complex may act as a
scaffold for PSI assembly (Ozawa et al., 2009). Ycf37 is
essential for PSI assembly in land plants whereas in
cyanobacteria PSI still accumulates, although in lower
amounts, in mutants lacking Ycf37 (Dühring et al.,
2007).
During biogenesis of the photosynthetic complexes,
the first step in the assembly is the insertion of an
anchor protein that acts as a scaffold for the following
assembly steps. These anchor proteins (also called
dominant subunits) are D2 for PSII, PetB for Cytb6f,
and PsaB for PSI (Fig. 1). In the absence of these
proteins translation of the next protein to be inserted in
the complex, called a control by epistasy of synthesis
(CES) subunit, is inhibited (see Choquet and Vallon,
2000). In this CES control, the unassembled CES subunit inhibits its own translation through a negativefeedback mechanism. Thus translation of the CES
subunits only occurs if the previous step in the assembly pathway has been accomplished. In the case of PSI
a CES cascade has been identified that proceeds first
by the insertion of the PsaB subunit into the membrane
(Wostrikoff et al., 2004). The PsaA subunit that forms
the PSI core together with PsaA is translated as long
as the partner of its assembly, PsaB is present. Similarly, the following subunit to be inserted, PsaC is only
translated in the presence of PsaA and PsaB, but not in
their absence (Fig. 1). Similar CES cascades have been
found for PSII, the Cytb6f complex, and ATP synthase.
PSII ASSEMBLY, DEGRADATION, AND REPAIR
PSII exists mainly in dimeric form with the monomer containing at least 27 to 28 subunits (Dekker and
Boekema, 2005). The core includes the D1 and D2
reaction center proteins that bind the redox cofactors,
the a- and b-subunits of cytochrome b559, PsbI, CP43,
and CP47 that coordinate chlorophyll a molecules and
constitute the inner antenna, and numerous small Mr
subunits. The products of the PsbO, PsbP, and PsbQ
genes form the oxygen-evolving complex on the lumen side. Assembly of PSII starts with the formation of
the reaction center complex followed by the association of the intrinsic inner antenna proteins CP43/
CP47, the integration of the small subunits, and the
assembly of the water-splitting complex and finally
the dimerization of PSII monomers. Several PSII assembly factors have been identified such as HCF136,
LPA1, LPA2, and LPA3. In the absence of HCF136, PSII
proteins are normally synthesized but they do not
assemble into stable PSII complexes (Meurer et al.,
1998). In the lpa1 mutant of Arabidopsis, assembly of
the newly synthesized PSII proteins is less efficient
than in wild-type plants (Peng et al., 2006). LPA1
appears to be a membrane chaperone that interacts
specifically with D1. LPA2 and LPA3 have overlapping
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Rochaix
functions and interact with ALB3. Together these
proteins appear to be specifically involved in the
folding and assembly of CP43 within PSII (Cai et al.,
2010). In the absence of LPA2 newly synthesized CP43
is rapidly degraded and there is a defect in PSII
supercomplex formation (Ma et al., 2007). LPA2 is
only present in plants and absent in Chlamydomonas
and cyanobacteria, suggesting that it evolved after the
divergence of the land plant lineage or that it was lost
in the other lineages. FKBP-2, a lumen-localized immunophilin, appears to play a role in the formation of
PSII supercomplexes as in its absence the levels of PSII
monomers and dimers are increased whereas accumulation of PSII supercomplexes is diminished (Lima
et al., 2006). Thus a large number of factors participate
in the assembly of a functional PSII complex. How
they act mechanistically is still largely unknown.
Among photosynthetic complexes PSII is particularly
prone to photooxidative damage because the watersplitting reaction catalyzed by this complex inevitably
leads to the formation of reactive oxygen species that
damage the complex. Thus PSII is constantly damaged
and needs to be repaired. Indeed a very efficient repair
system has evolved in which the D1 subunit of photodamaged PSII is predominantly degraded and the
complex moves from the grana to the stromal region
for repair (see Nixon et al., 2010). Possibly, chlorophyll
released during this process is transiently stored in
small CAB-like proteins of cyanobacteria containing a
single transmembrane helix with high similarity to
some transmembrane domains of the light-harvesting
proteins (Kufryk et al., 2008). Newly synthesized D1
precursor with a C-terminal extension is inserted into
PSII in the stromal thylakoid region followed by cleavage of the extension by the C-terminal peptidase CtpA
and assembly of the catalytic manganese cluster (see
Nixon et al., 2010). The reconstituted PSII complex
moves then back to the grana. Several factors have been
identified that play a role in this repair cycle among
which the ATP-dependent zinc metalloprotease FtsH
present in all species from cyanobacteria to plants (see
Nixon et al., 2010). FtsH forms a multisubunit complex
exposed to the outer surface of thylakoid membranes
and is enriched in stroma thylakoids and grana margins. Deg proteases also appear to be involved in D1
degradation (Sun et al., 2010). Interestingly the lumenally exposed thylakoid Deg1 protease is involved both
in D1 degradation and in PSII assembly presumably
through its direct interaction with D2. Besides proteases, other factors have been discovered such as LPA19
that facilitates D1 protein precursor processing and
interacts with the C-terminal region of the mature and
precursor form of D1 (Wei et al., 2010). LPA19 is related
to Psb27 present in both cyanobacteria and plants that
appears to function in the assembly of the manganese
cluster (Roose and Pakrasi, 2004). A second Psb27
homolog of Arabidopsis was identified as a PSII component and it is required for efficient repair of photodamaged PSII but is nonessential for PSII accumulation
(Chen et al., 2006).
COFACTOR ASSEMBLY IN
PHOTOSYNTHETIC COMPLEXES
An important step in the assembly of photosynthetic
complexes is the insertion of chlorophyll, carotenoids,
cytochromes, and Fe-S centers that need to be assembled in a coordinate way with the proteins of these
complexes. It is not possible within this short Update
to cover all these aspects. One area that has progressed
rapidly in recent years is the identification of several
novel factors required for the maturation of c-type
cytochromes in which heme is covalently attached to
the protein most often by two thioether bonds between
the heme vinyl groups and the thiols of two Cys
residues in a conserved CXXCH motif in which His
acts as one of the axial ligands to heme Fe. Covalent
heme binding plays a major role during assembly of
the Cytb6f complex that consists of four large subunits
(cytochrome b6, subunit IV, cytochrome f, and the
Rieske protein) and four small subunits (PetG, PetL,
PetM, and PetN). Additionally it contains a chlorophyll a molecule, b-carotene, one Fe2S2 cluster, two
b-hemes, and two c-hemes. Two maturation pathways
for heme c attachment are operating in the chloroplast
(see de Vitry and Kuras, 2009). The first, system II
(c-type cytochrome synthesis), comprises one chloroplast- and six nucleus-encoded proteins required for
heme attachment to the apoforms of cytochrome f and
cytochrome c6 on the lumen side of the thylakoid
membrane. The latter replaces plastocyanin under copper deprivation for transferring electrons from the
Cytb6f complex to PSI. In Arabidopsis the thioredoxinlike protein HCF164 and a protein related to the bacterial thiol disulfide transporter CCDA are involved in
cytochrome f maturation (Lennartz et al., 2001; Page
et al., 2004). They have been proposed to be part of a
redox relay system that shuttles thiol-reducing equivalents across the thylakoid membrane. The second,
system IV (cofactor assembly on complex C subunit B
[CCB]), comprises at least four factors, CCB1 to CCB4,
identified through genetic screens in Chlamydomonas
that are required for the attachment of heme ci’ to
apocyochrome b6 of the Cytb6f complex on the stromal
face of the thylakoid membrane (see de Vitry and
Kuras, 2009). Heme ci’ is only covalently bound to a
single Cys residue close to the Qi site where quinone is
reduced during the Q cycle. The CCB factors are
conserved in all photosynthetic organisms performing
oxygenic photosynthesis.
Fe-S clusters play a key role in photosynthetic
electron transport, mostly at the level of the Cytb6f
complex and of PSI that contain one 2Fe-2S and three
4Fe-4S centers, respectively. Although it is known that
isolated chloroplasts are able to form Fe-S clusters,
it is only recently that components of the plastid
Fe-S assembly machinery have been identified (see
Balk and Lobréaux, 2005). The finding of six SUF-like
proteins in chloroplasts suggest the existence of a
plastid system related to the bacterial SUF system that
is required in E. coli under Fe-limiting and oxidative
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Assembly of Photosynthetic Complexes
stress conditions. The SUF machinery is known to be
more robust to oxidative stress than the common ISC
system of bacteria and appears therefore to be the
appropriate system in the plastid environment (Balk
and Lobréaux, 2005). Only few of the plastid SUF
components have been characterized and the regulation of this machinery in response to oxidative stress,
Fe status, and light is still poorly understood. Another
key player in plastid Fe homeostasis is ferritin, which
is able to store large amounts of Fe. Interestingly in
response to reduced Fe availability in C. reinhardtii, PSI
is degraded, its LHC is remodeled, and ferritin levels
increase in contrast of what is observed in animal or
plant systems in which ferritin is repressed (Busch
et al., 2008). In this way ferritin is used to buffer the Fe
released by PSI during its degradation and the Fe can
be reutilized efficiently upon Fe repletion. The adaptation to Fe deficiency involves also a remodelling of
the LHCI antenna with N-terminal truncation of the
Lhca3 subunit and the specific depletion of some LHCI
subunits coupled to the up-regulation of others, resulting in the disconnection of LHCI from PSI and thus
diminished energy transfer to PSI (Moseley et al., 2002;
Naumann et al., 2005).
DYNAMICS OF PSII-LHCII COMPLEXES MEDIATED
BY PROTEIN PHOSPHORYLATION
PSII and LHCII form large supercomplexes in the
thylakoid membranes. The peripheral antenna system
of PSII consists of six different complexes that coordinate Chl a, Chl b, and several xanthophylls (Jansson,
1999). Whereas the major LHCII complex of land plants
is organized in heterotrimers containing the products of
the Lhcb1-3 genes, the other three minor LHCIIs, CP29,
CP26, and CP24, are present as monomers (Dekker and
Boekema, 2005). The structure of these complexes has
been intensively studied by electron microscopy and
single-particle analysis after mild solubilization of membranes (Dekker and Boekema, 2005). The larger supercomplex of Arabidopsis contains a dimeric PSII core
(C2), two LHCII trimers (trimer S) strongly bound to
the complex on the side of CP43 and CP26, and two
additional trimers moderately bound (trimer M) to
CP29 and CP24 (C2S2M2 complex). A series of PSII
supercomplexes with different antenna sizes, ranging
from the core complex to C2S2M2 was isolated and
functionally characterized (Caffarri et al., 2009). The
study of Lhcb-deficient mutants provided insights into
the assembly steps and into the role of the individual
subunits in the supramolecular organization. Thus CP24
binds the M trimer through interaction with Lhcb3.
Interestingly CP24 and Lhcb3 are only present in land
plants where they could have been responsible for the
increase in the PSII antenna size. In marked contrast
only C2S2 complexes were found in green algae
(Minagawa, 2010). CP26 is important for the stable
binding of trimer S while CP29 is mostly involved in
stabilizing the PSII dimer (Caffarri et al., 2009).
Phosphorylation of PSII and LHCII profoundly affects both the formation/dissociation of PSII-LHCII
supercomplexes in the grana and the association of
LHCII to PSI in the stromal lamellae. The two protein
kinases STN7/Stt7 and STN8/Stl1 that are tightly
associated with thylakoid membranes and conserved
in oxygenic photosynthetic organisms play a major
role in these phosphorylations (see Lemeille and
Rochaix, 2010). The former is involved in LHCII phosphorylation whereas the latter phosphorylates the PSII
core. As STN7, STN8 is associated with thylakoid
membranes and activated by moderate light. The
availability of mutants of Arabidopsis and Chlamydomonas lacking either STN7, STN8, or both kinases and
therefore deficient in LHCII and PSII core protein
phosphorylation has made it possible to test to what
extent surface charges are responsible for thylakoid
membrane folding. Earlier studies indicated that unappressed regions carry a higher negative surface
charge than the appressed regions and that destacking of thylakoid membranes can be induced in vitro
through depletion of cations presumably because of
the unmasking of repulsive negative charges (Barber,
1982). Analysis of the membranes from the stn8 mutant deficient in PSII core protein phosphorylation
revealed a more compact thylakoid organization with
increased thylakoid membrane folding as compared to
wild-type plants (Fristedt et al., 2009). The size of the
stacked thylakoid membranes was significantly larger.
This phenotype was only detected in the absence of the
STN8 kinase but not in plants lacking STN7. Hence
PSII core protein phosphorylation plays a major role in
the folding of thylakoid membranes whereas the
phosphorylation status of LHCII appears to be less
important in this respect.
The phosphorylation of LHCII by the STN7/Stt7
kinase affects its association with PSII and PSI within
the thylakoid membrane. The activity of this kinase is
sensitive to changes in light quality and quantity that
can lead to unequal excitation of the two photosystems
because of differences in the light absorption properties of their corresponding antennae. This kinase is at
the heart of state transitions, a process that rebalances
the absorbed light energy between two photosystems
by adjusting the size of their LHCs (Wollman, 2001;
Lemeille and Rochaix, 2010). Upon excess excitation of
PSII and reduction of the plastoquinone pool, this
kinase induces, directly or indirectly, the phosphorylation of LHCII that leads to the detachment of the
mobile antenna from PSII and its movement and
binding to PSI (state 2). The process is reversible as
preferential excitation of PSI with far-red light inactivates the kinase and the phosphatase PPH1/TAP38
dephosphorylates LHCII that then moves back to PSII
(state 1; Fig. 2; see Lemeille and Rochaix, 2010). At high
light, when the acceptor side of PSI is reduced, the
LHCII kinase is inactivated through the ferredoxin/
thioredoxin system (Rintamäki et al., 1997). Two conserved Cys residues in the N-terminal domain of the
kinase are obvious candidate targets for the high-light-
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Rochaix
mediated inactivation of the kinase. In this respect the
trans-thylakoid thiol-reducing pathway mediated by
the thiol disulfide transporter CcdA and thioredoxinlike Hcf164 could be involved in this process (see
Lemeille and Rochaix, 2010). Site-directed mutagenesis in C. reinhardtii showed that these two Cys are
essential for kinase activity (Lemeille and Rochaix,
2010).
A key feature of state transitions is the movement
of the mobile part of the LHC from PSII to PSI. This
movement was first documented in C. reinhardtii
through spectroscopic measurements that indicated
that 80% of the LHCII antenna is mobile (Wollman,
2001). Biochemical evidence for this antenna mobility
was provided through the isolation of a PSI-LHCILHCII supercomplex in state 2 from Arabidopsis
(Zhang and Scheller, 2004). This supercomplex consists of one PSI-LHCI complex and one LHCII trimer
(Kouril et al., 2005). Cross-linking studies further
revealed that the docking site of LHCII on PSI is
formed by the PsaH, PsaI, and PsaO subunits and is
consistent with the finding that Arabidopsis mutants
lacking any of these subunits are deficient in state
transitions (Lunde et al., 2000). In C. reinhardtii a PSILHCI-LHCII supercomplex could also be isolated that
includes the three LHCII proteins CP29, CP26, and
Lhcbm5 (Minagawa, 2010). Lhcbm5 appears to have a
similar role as CP24 in land plants as it is present in
similar amounts as CP29 and CP26 but at a lower level
than the major LHCII proteins. These three monomeric
LHCII proteins migrate to the PsaH side of PSI in state
2 where they form a binding site for the LHCII trimers
on PSI. Interestingly, the crystal structure of the PSILHCI complex indicates that four Lhca subunits form
a belt on the opposite side of PsaH (Ben-Shem et al.,
Figure 2. LHCII cycle during state transitions. Under state 1 conditions
PSII and LHCII form megacomplexes within the thylakoid grana. Upon
phosphorylation of the major LHCII by the Stt7 kinase, the megacomplex dissociates into PSII-LHCII supercomplexes. After further phosphorylation of the minor monomeric LHCII (CP26 and CP29) and of
PSII core proteins possibly mediated by the Stl1 kinase, phosphorylated
LHCII is released from PSII and migrates to the stroma region of the
thylakoids where it associates with PSI (state 2; see Minagawa, 2010).
Upon deactivation of the STN7 kinase, LHCII is dephosphorylated by
the PPH1 phosphatase and released from PSI, it then migrates back to
PSII in the grana region.
2003). The fact that phosphorylated CP29 and Lhcbm5
were found associated with PSI in state 2 suggests that
the affinities of CP29 and Lhcbm5 for PSII and PSI are
modulated by phosphorylation (Minagawa, 2010). Remarkably, four sites of CP29 are phosphorylated in
state 2. Taken together these results show that reversible phosphorylation at the interface between the PSII
core and LHCII play an important role in state transitions. More recently the large PSI supercomplex was
characterized further and shown to comprise in addition to LHCI, the major trimeric LHCII and the minor
monomeric LHCII CP29 and CP26, Cytb6f, including
PetO, a subunit that is specific to C. reinhardtii, and the
proteins PGRL1 and FNR (Minagawa, 2010). Spectroscopic measurements showed that upon illumination
reducing equivalents generated by PSI are transferred
to Cytb6f and that oxidized PSI can be reduced by
reducing equivalents from Cytb6f, indicating that this
complex is capable of driving cyclic electron flow. The
location of this PSI supercomplex is still unknown
within the thylakoid membrane system. However it is
expected to localize with a fraction of the mobile
electron carriers plastoquinone, plastocyanin, and ferredoxin in its vicinity in such a way that cyclic and
linear electron flow can operate independently from
each other to avoid disturbance of the redox poise of
cyclic by reduced linear electron flow components.
The dissociation of LHCII from PSII that occurs
during a transition from state 1 to state 2 was examined by purifying PSII-containing complexes through
a His tag inserted in CP47 by nickel-affinity chromatography (Minagawa, 2010). In this way a PSII core
complex, a PSII-LHCII supercomplex, and a multimer
of the PSII supercomplex called PSII megacomplex
were identified. Moreover the megacomplex was predominant in state 1 whereas the core complex was
detected in state 2, indicating a dissociation of LHCII
from PSII during a state 1 to state 2 transition. Phosphorylated LHCII was mostly associated with the
supercomplex and less with the megacomplex while
phosphoryated CP26 and CP29 were only detected in
unbound form. A model was derived from these
observations in which PSII remodeling during a transition from state 1 to state 2 proceeds in two steps.
First, as a result of LHCII phosphorylation, the megacomplex dissociates, giving rise to supercomplexes,
and second, LHCII is released from the supercomplex
by phosphorylation of the monomeric complexes CP29
and CP26 and the PSII core proteins. According to this
model CP29 and CP26 play an essential role because
their phosphorylation leads to the release of the entire
peripheral antenna during a transition from state 1 to
state 2. The released LHCII will then reassociate with
the PSI-LHCI complex. The role of CP29 and CP26 was
further tested by RNA interference experiments in C.
reinhardtii (Minagawa, 2010). These studies showed
that in the absence of CP29 the mobile LHCII antenna
is still phosphorylated and detached from PSII, but it is
unable to bind to the PSI-LHCI complex. Therefore
state transitions no longer occur in the absence of
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Assembly of Photosynthetic Complexes
CP29. However they are not affected by the loss of
CP26. Thus CP29 plays a key role in the docking of
LHCII to PSI.
PERSPECTIVES
The picture that emerges from these studies is a
dynamic thylakoid membrane network in which the
assembly of photosynthetic complexes is mediated by
numerous nucleus- and a few chloroplast-encoded
factors. The dynamics of this system is influenced to a
large extent by environmental factors such as light and
nutrients. Some of these factors such as ALB3 appear
to have a general role in thylakoid membrane biogenesis whereas others are specifically required for the
efficient assembly of the different photosynthetic complexes. Other factors function mainly during the repair
of photodamaged complexes. We still know very little
how these factors act mechanistically and how their
activity is influenced by environmental conditions.
This will undoubtedly form an important part of
future research. A more ambitious goal will be to
reconstitute the assembly process in vitro, a daunting
task given the fact that most of the components and the
redox cofactors are highly hydrophobic. Once they
have been assembled, several of the photosynthetic
complexes can form supercomplexes or even megacomplexes. Some of these complexes occur only transiently or are unstable and are therefore difficult to
study. Posttranslational protein modifications such as
phosphorylations mediated by the thylakoid membrane protein kinases STN7/Stt7 and STN8/Stl1 appear to play a major role in these associations/
dissociations. Furthermore some of these phosphorylations also affect the folding of the thylakoid membrane and the movement of the complexes. Additional
factors are likely to be involved in these processes and
their identification represents a further challenge.
ACKNOWLEDGMENTS
I thank N. Roggli for drawings and M. Goldschmidt-Clermont for helpful
comments.
Received November 22, 2010; accepted January 8, 2011; published January 14,
2011.
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