Revisiting the Supramolecular Organization of Photosystem II in Chlamydomonas reinhardtii

Photosystem II (PSII) is a multiprotein complex that splits water and initiates electron transfer in photosynthesis. The central part of PSII, the PSII core, is surrounded by light-harvesting complex II proteins (LHCIIs). In higher plants, two or three LHCII trimers are seen on each side of the PSII core whereas only one is seen in the corresponding positions in Chlamydomonas reinhardtii, probably due to the absence of CP24, a minor monomeric LHCII. Here, we re-examined the supramolecular organization of the C. reinhardtii PSII-LHCII supercomplex by determining the effect of different solubilizing detergents. When we solubilized the thylakoid membranes with n-dodecyl-β-d-maltoside (β-DM) or n-dodecyl-α-d-maltoside (α-DM) and subjected them to gel filtration, we observed a clear difference in molecular mass. The α-DM-solubilized PSII-LHCII supercomplex bound twice more LHCII than the β-DM-solubilized supercomplex and retained higher oxygen-evolving activity. Single-particle image analysis from electron micrographs of the α-DM-solubilized and negatively stained supercomplex revealed that the PSII-LHCII supercomplex had a novel supramolecular organization, with three LHCII trimers attached to each side of the core.     

Molecular remodeling of photosystem II during state transitions in Chlamydomonas reinhardtii

State transitions, or the redistribution of light-harvesting complex II (LHCII) proteins between photosystem I (PSI) and photosystem II (PSII), balance the light-harvesting capacity of the two photosystems to optimize the efficiency of photosynthesis. Studies on the migration of LHCII proteins have focused primarily on their reassociation with PSI, but the molecular details on their dissociation from PSII have not been clear. Here, we compare the polypeptide composition, supramolecular organization, and phosphorylation of PSII complexes under PSI- and PSII-favoring conditions (State 1 and State 2, respectively). Three PSII fractions, a PSII core complex, a PSII supercomplex, and a multimer of PSII supercomplex or PSII megacomplex, were obtained from a transformant of the green alga Chlamydomonas reinhardtii carrying a His-tagged CP47. Gel filtration and single particles on electron micrographs showed that the megacomplex was predominant in State 1, whereas the core complex was predominant in State 2, indicating that LHCIIs are dissociated from PSII upon state transition. Moreover, in State 2, strongly phosphorylated LHCII type I was found in the supercomplex but not in the megacomplex. Phosphorylated minor LHCIIs (CP26 and CP29) were found only in the unbound form. The PSII subunits were most phosphorylated in the core complex. Based on these observations, we propose a model for PSII remodeling during state transitions, which involves division of the megacomplex into supercomplexes, triggered by phosphorylation of LHCII type I, followed by LHCII undocking from the supercomplex, triggered by phosphorylation of minor LHCIIs and PSII core subunits.     

Light-harvesting complex II (LHCII) and its supramolecular organization in Chlamydomonas reinhardtii

LHCII is the most abundant membrane protein on earth. It participates in the first steps of photosynthesis by harvesting sunlight and transferring excitation energy to the core complex. Here we have analyzed the LHCII complex of the green alga Chlamydomonas reinhardtii and its association with the core of Photosystem II (PSII) to form multiprotein complexes. Several PSII supercomplexes with different antenna sizes have been purified, the largest of which contains three LHCII trimers (named S, M and N) per monomeric core. A projection map at a 13 Å resolution was obtained allowing the reconstruction of the 3D structure of the supercomplex. The position and orientation of the S trimer are the same as in plants; trimer M is rotated by 45° and the additional trimer (named here as LHCII-N), which is taking the position occupied in plants by CP24, is directly associated with the core. The analysis of supercomplexes with different antenna sizes suggests that LhcbM1, LhcbM2/7 and LhcbM3 are the major components of the trimers in the PSII supercomplex, while LhcbM5 is part of the “extra” LHCII pool not directly associated with the supercomplex. It is also shown that Chlamydomonas LHCII has a slightly lower Chlorophyll a/b ratio than the complex from plants and a blue shifted absorption spectrum. Finally the data indicate that there are at least six LHCII trimers per dimeric core in the thylakoid membranes, meaning that the antenna size of PSII of C. reinhardtii is larger than that of plants.     

Protein assembly of photosystem II and accumulation of subcomplexes in the absence of low molecular mass subunits PsbL and PsbJ.

The protein assembly and stability of photosystem II (PSII) (sub)complexes were studied in mature leaves of four plastid mutants of tobacco (Nicotiana tabacum L), each having one of the psbEFLJ operon genes inactivated. In the absence of psbL, no PSII core dimers or PSII-light harvesting complex (LHCII) supercomplexes were formed, and the assembly of CP43 into PSII core monomers was extremely labile. The assembly of CP43 into PSII core monomers was found to be necessary for the assembly of PsbO on the lumenal side of PSII. The two other oxygen-evolving complex (OEC) proteins, PsbP and PsbQ, were completely lacking in Delta psbL. In the absence of psbJ, both intact PSII core monomers and PSII core dimers harboring the PsbO protein were formed, whereas the LHCII antenna remained detached from the PSII dimers, as demonstrated by 77 K fluorescence measurements and by the lack of PSII-LHCII supercomplexes. The Delta psbJ mutant was characterized by a deficiency of PsbQ and a complete lack of PsbP. Thus, both the PsbL and PsbJ subunits of PSII are essential for proper assembly of the OEC. The absence of psbE and psbF resulted in a complete absence of all central PSII core and OEC proteins. In contrast, very young, vigorously expanding leaves of all psbEFLJ operon mutants accumulated at least traces of D2, CP43 and the OEC proteins PsbO and PsbQ, implying developmental control of the expression of the PSII core and OEC proteins. Despite severe problems in PSII assembly, the thylakoid membrane complexes other than PSII were present and correctly assembled in all psbEFLJ operon mutants.     

Unique organization of photosystem II supercomplexes and megacomplexes in Norway spruce

Photosystem II (PSII) complexes are organized into large supercomplexes with variable amounts of light‐harvesting proteins (Lhcb). A typical PSII supercomplex in plants is formed by four trimers of Lhcb proteins (LHCII trimers), which are bound to the PSII core dimer via monomeric antenna proteins. However, the architecture of PSII supercomplexes in Norway spruce[Picea abies (L.) Karst.] is different, most likely due to a lack of two Lhcb proteins, Lhcb6 and Lhcb3. Interestingly, the spruce PSII supercomplex shares similar structural features with its counterpart in the green alga Chlamydomonas reinhardtii [Kou?il et al. (2016) New Phytol. 210 , 808–814]. Here we present a single‐particle electron microscopy study of isolated PSII supercomplexes from Norway spruce that revealed binding of a variable amount of LHCII trimers to the PSII core dimer at positions that have never been observed in any other plant species so far. The largest spruce PSII supercomplex, which was found to bind eight LHCII trimers, is even larger than the current largest known PSII supercomplex from C. reinhardtii. We have also shown that the spruce PSII supercomplexes can form various types of PSII megacomplexes, which were also identified in intact grana membranes. Some of these large PSII supercomplexes and megacomplexes were identified also in Pinus sylvestris, another representative of the Pinaceae family. The structural variability and complexity of LHCII organization in Pinaceae seems to be related to the absence of Lhcb6 and Lhcb3 in this family, and may be beneficial for the optimization of light‐harvesting under varying environmental conditions.     

Biochemical Characterization of Photosystem II Antenna Polypeptides in Grana and Stroma Membranes of Spinach

The photosystem (PS) II antenna system comprises several biochemically and spectroscopically distinct complexes, including light-harvesting complex II (LHCII), chlorophyll-protein complex (CP) 29, CP26, and CP24. LHCII, the most abundant of these, is both structurally and functionally diverse. The photosynthetic apparatus is laterally segregated within the thylakoid membrane into PSI-rich and PSII-rich domains, and the distribution of antenna complexes between these domains has implications for antenna function. We report a detailed analysis of the differences in the polypeptide composition of LHCII, CP29, and CP26 complexes associated with grana and stroma thylakoid fractions from spinach (Spinacia oleracea L.), making use of a very high-resolution denaturing gel system, coupled with immunoblots using monospecific antibodies to identify specific antenna components. We first show that the polypeptide composition of the PSII antenna system is more complex than previously thought. We resolved at least five type I LHCII apoproteins and two to three type II LHCII apoproteins. We also resolved at least two apoproteins each for CP29 and CP26. In state 1-adapted grana and stroma thylakoid membranes, the spectrum of LHCII apoproteins is surprisingly similar. However, in addition to overall quantitative differences, we saw subtle but reproducible qualitative differences in the spectrum of LHCII apoproteins in grana and stroma membrane domains, including two forms of the major type II apoprotein. The implications of these findings for models of PSII antenna function in spinach are discussed.     

Thylakoid protein phosphorylation in dynamic regulation of photosystem II in higher plants

In higher plants, the photosystem (PS) II core and its several light harvesting antenna (LHCII) proteins undergo reversible phosphorylation cycles according to the light intensity. High light intensity induces strong phosphorylation of the PSII core proteins and suppresses the phosphorylation level of the LHCII proteins. Decrease in light intensity, in turn, suppresses the phosphorylation of PSII core, but strongly induces the phosphorylation of LHCII. Reversible and differential phosphorylation of the PSII-LHCII proteins is dependent on the interplay between the STN7 and STN8 kinases, and the respective phosphatases. The STN7 kinase phosphorylates the LHCII proteins and to a lesser extent also the PSII core proteins D1, D2 and CP43. The STN8 kinase, on the contrary, is rather specific for the PSII core proteins. Mechanistically, the PSII-LHCII protein phosphorylation is required for optimal mobility of the PSII-LHCII protein complexes along the thylakoid membrane. Physiologically, the phosphorylation of LHCII is a prerequisite for sufficient excitation of PSI, enabling the excitation and redox balance between PSII and PSI under low irradiance, when excitation energy transfer from the LHCII antenna to the two photosystems is efficient and thermal dissipation of excitation energy (NPQ) is minimised. The importance of PSII core protein phosphorylation is manifested under highlight when the photodamage of PSII is rapid and phosphorylation is required to facilitate the migration of damaged PSII from grana stacks to stroma lamellae for repair. The importance of thylakoid protein phosphorylation is highlighted under fluctuating intensity of light where the STN7 kinase dependent balancing of electron transfer is a prerequisite for optimal growth and development of the plant. This article is part of a Special Issue entitled: Photosystem II.     

PsbI affects the stability, function, and phosphorylation patterns of photosystem II assemblies in tobacco

Photosystem II (PSII) core complexes consist of CP47, CP43, D1, D2 proteins and of several low molecular weight integral membrane polypeptides, such as the chloroplast-encoded PsbE, PsbF, and PsbI proteins. To elucidate the function of PsbI in the photosynthetic process as well as in the biogenesis of PSII in higher plants, we generated homoplastomic knock-out plants by replacing most of the tobacco psbI gene with a spectinomycin resistance cartridge. Mutant plants are photoautotrophically viable under green house conditions but sensitive to high light irradiation. Antenna proteins of PSII accumulate to normal amounts, but levels of the PSII core complex are reduced by 50%. Bioenergetic and fluorescence studies uncovered that PsbI is required for the stability but not for the assembly of dimeric PSII and supercomplexes consisting of PSII and the outer antenna (PSII-LHCII). Thermoluminescence emission bands indicate that the presence of PsbI is required for assembly of a fully functional Q(A) binding site. We show that phosphorylation of the reaction center proteins D1 and D2 is light and redox-regulated in the wild type, but phosphorylation is abolished in the mutant, presumably due to structural alterations of PSII when PsbI is deficient. Unlike wild type, phosphorylation of LHCII is strongly increased in the dark due to accumulation of reduced plastoquinone, whereas even upon state II light phosphorylation is decreased in delta psbI. These data attest that phosphorylation of D1/D2, CP43, and LHCII is regulated differently.     

The effect of zeaxanthin as the only xanthophyll on the structure and function of the photosynthetic apparatus in Arabidopsis thaliana

In green plants, the xanthophyll carotenoid zeaxanthin is synthesized transiently under conditions of excess light energy and participates in photoprotection. In the Arabidopsis lut2 npq2 double mutant, all xanthophylls were replaced constitutively by zeaxanthin, the only xanthophyll whose synthesis was not impaired. The relative proportions of the different chlorophyll antenna proteins were strongly affected with respect to the wild-type strain. The major antenna, LHCII, did not form trimers, and its abundance was strongly reduced as was CP26, albeit to a lesser extent. In contrast, CP29, CP24, LHCI proteins, and the PSI and PSII core complexes did not undergo major changes. PSII-LHCII supercomplexes were not detectable while the PSI-LHCI supercomplex remained unaffected. The effect of zeaxanthin accumulation on the stability of the different Lhc proteins was uneven: the LHCII proteins from lut2 npq2 had a lower melting temperature as compared with the wild-type complex while LHCI showed increased resistance to heat denaturation. Consistent with the loss of LHCII, light-state 1 to state 2 transitions were suppressed, the photochemical efficiency in limiting light was reduced and photosynthesis was saturated at higher light intensities in lut2 npq2 leaves, resulting in a photosynthetic phenotype resembling that of high light-acclimated leaves. Zeaxanthin functioned in vivo as a light-harvesting accessory pigment in lut2 npq2 chlorophyll antennae. As a whole, the in vivo data are consistent with the results obtained by using recombinant Lhc proteins reconstituted in vitro with purified zeaxanthin. While PSII photoinhibition was similar in wild type and lut2 npq2 exposed to high light at low temperature, the double mutant was much more resistant to photooxidative stress and lipid peroxidation than the wild type. The latter observation is consistent with an antioxidant and lipid protective role of zeaxanthin in vivo.     

Quenching in Arabidopsis thaliana mutants lacking monomeric antenna proteins of photosystem II

The minor light-harvesting complexes CP24, CP26, and CP29 have been proposed to play a key role in the zeaxanthin (Zx)-dependent high light-induced regulation (NPQ) of excitation energy in higher plants. To characterize the detailed roles of these minor complexes in NPQ and to determine their specific quenching effects we have studied the ultrafast fluorescence kinetics in knockout (ko) mutants koCP26, koCP29, and the double mutant koCP24/CP26. The data provide detailed insight into the quenching processes and the reorganization of the Photosystem (PS) II supercomplex under quenching conditions. All genotypes showed two NPQ quenching sites. Quenching site Q1 is formed by a light-induced functional detachment of parts of the PSII supercomplex and a pronounced quenching of the detached antenna parts. The antenna remaining bound to the PSII core was also quenched substantially in all genotypes under NPQ conditions (quenching site Q2) as compared with the dark-adapted state. The latter quenching was about equally strong in koCP26 and the koCP24/CP26 mutants as in the WT. Q2 quenching was substantially reduced, however, in koCP29 mutants suggesting a key role for CP29 in the total NPQ. The observed quenching effects in the knockout mutants are complicated by the fact that other minor antenna complexes do compensate in part for the lack of the CP24 and/or CP29 complexes. Their lack also causes some LHCII dissociation already in the dark.     

The role of light-harvesting complex I in excitation energy transfer from LHCII to photosystem I in Arabidopsis

Photosynthesis powers nearly all life on Earth. Light absorbed by photosystems drives the conversion of water and carbon dioxide into sugars. In plants, photosystem I (PSI) and photosystem II (PSII) work in series to drive the electron transport from water to NADP⁺. As both photosystems largely work in series, a balanced excitation pressure is required for optimal photosynthetic performance. Both photosystems are composed of a core and light-harvesting complexes (LHCI) for PSI and LHCII for PSII. When the light conditions favor the excitation of one photosystem over the other, a mobile pool of trimeric LHCII moves between both photosystems thus tuning their antenna cross-section in a process called state transitions. When PSII is overexcited multiple LHCIIs can associate with PSI. A trimeric LHCII binds to PSI at the PsaH/L/O site to form a well-characterized PSI–LHCI–LHCII supercomplex. The binding site(s) of the “additional” LHCII is still unclear, although a mediating role for LHCI has been proposed. In this work, we measured the PSI antenna size and trapping kinetics of photosynthetic membranes from Arabidopsis (Arabidopsis thaliana) plants. Membranes from wild-type (WT) plants were compared to those of the ΔLhca mutant that completely lacks the LHCI antenna. The results showed that “additional” LHCII complexes can transfer energy directly to the PSI core in the absence of LHCI. However, the transfer is about two times faster and therefore more efficient, when LHCI is present. This suggests LHCI mediates excitation energy transfer from loosely bound LHCII to PSI in WT plants.

The light-harvesting antennae of photosystem I facilitate energy transfer from trimeric light-harvesting complex II to photosystem I in the stroma lamellae membrane.     

Structure,function and assembly of Photosystem II and its light-harvesting proteins

Photosystem II (PSII) is a multisubunit chlorophyll–protein complex that drives electron transfer from water to plastoquinone using energy derived from light. In green plants, the native form of PSII is surrounded by the light-harvesting complex (LHCII complex) and thus it is called the PSII–LHCII supercomplex. Over the past several years, understanding of the structure, function, and assembly of PSII and LHCII complexes has increased considerably. The unicellular green alga Chlamydomonas reinhardtii has been an excellent model organism to study PSII and LHCII complexes, because this organism grows heterotrophically and photoautotrophically and it is amenable to biochemical, genetic, molecular biological and recombinant DNA methodology. Here, the genes encoding and regulating components of the C. reinhardtii PSII–LHCII supercomplex have been thoroughly catalogued: they include 15 chloroplast and 20 nuclear structural genes as well as 13 nuclear genes coding for regulatory factors. This review discusses these molecular genetic data and presents an overview of the structure, function and assembly of PSII and LHCII complexes.     

Repressible chloroplast gene expression in Chlamydomonas: A new tool for the study of the photosynthetic apparatus

A repressible/inducible chloroplast gene expression system has been used to conditionally inhibit chloroplast protein synthesis in the unicellular alga Chlamydomonas reinhardtii. This system allows one to follow the fate of photosystem II and photosystem I and their antennae upon cessation of chloroplast translation. The main results are that the levels of the PSI core proteins decrease at a slower rate than those of PSII. Amongst the light-harvesting complexes, the decrease of CP26 proceeds at the same rate as for the PSII core proteins whereas it is significantly slower for CP29, and for the antenna complexes of PSI this rate is comprised between that of CP26 and CP29. In marked contrast, the components of trimeric LHCII, the major PSII antenna, persist for several days upon inhibition of chloroplast translation. This system offers new possibilities for investigating the biosynthesis and turnover of individual photosynthetic complexes in the thylakoid membranes. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Monomeric light harvesting complexes enhance excitation energy transfer from LHCII to PSII and control their lateral spacing in thylakoids

Proper assembly of plant photosystem II, in the appressed region of thylakoids, allows for both efficient light harvesting and the dissipation of excitation energy absorbed in excess. The core moiety of wild type supercomplex is associated with monomeric antennae that, in turn, bind peripheral trimeric LHCII complexes. Acclimation to light environment dynamics involves structural plasticity within PSII-LHCs supercomplexes, including depletion in LHCII and CP24. Here, we report on the acclimation of NoM, an Arabidopsis mutant lacking monomeric LHCs but retaining LHCII trimer. Lack of monomeric LHCs impaired the operation of both photosynthetic electron transport and state transitions, despite the fact that NoM underwent a compensatory over-accumulation of the LHCII complement compared to the wild type. Mutant plants displayed stunted growth compared to the wild type when probed over a range of light conditions. When exposed to short-term excess light, NoM showed higher photosensitivity and enhanced singlet oxygen release than the wild type, whereas long-term acclimation under stress conditions was unaffected. Analysis of pigment-binding supercomplexes showed that the absence of monomeric LHCs did affect the macro-organisation of photosystems: large PSI-LHCII megacomplexes were more abundant in NoM, whereas the assembly of PSII-LHCs supercomplexes was impaired. Observation by electron microscopy (EM) and image analysis of thylakoids highlighted impaired granal stacking and membrane organisation, with a heterogeneous distribution of PSII and LHCII compared to the wild type. It is concluded that monomeric LHCs are critical for the structural and functional optimisation of the photosynthetic apparatus.     

The mobile thylakoid phosphoprotein TSP9 interacts with the light-harvesting complex II and the peripheries of both photosystems

The localization of the plant-specific thylakoid-soluble phosphoprotein of 9 kDa, TSP9, within the chloroplast thylakoid membrane of spinach has been established by the combined use of fractionation, immunoblotting, cross-linking, and mass spectrometry. TSP9 was found to be exclusively confined to the thylakoid membranes, where it is enriched in the stacked grana membrane domains. After mild solubilization of the membranes, TSP9 migrated together with the major light-harvesting antenna (LHCII) of photosystem II (PSII) and with PSII-LHCII supercomplexes upon separation of the protein complexes by either native gel electrophoresis or sucrose gradient centrifugation. Studies with a cleavable cross-linking agent revealed the interaction of TSP9 with both major and minor LHCII proteins as identified by mass spectrometric sequencing. Cross-linked complexes that in addition to TSP9 contain the peripheral PSII subunits CP29, CP26, and PsbS, which form the interface between LHCII and the PSII core, were found. Our observations also clearly suggest an interaction of TSP9 with photosystem I (PSI) as shown by both immunodetection and mass spectrometry. Sequencing identified the peripheral PSI subunits PsaL, PsaF, and PsaE, originating from cross-linked protein complexes of around 30 kDa that also contained TSP9. The distribution of TSP9 among the cross-linked forms was found to be sensitive to conditions such as light exposure. An association of TSP9 with LHCII as well as the peripheries of the photosystems suggests its involvement in regulation of photosynthetic light harvesting.     

Isolation and characterization of a large photosystem I–light‐harvesting complex II supercomplex with an additional Lhca1–a4 dimer in Arabidopsis

The biological conversion of light energy into chemical energy is performed by a flexible photosynthetic machinery located in the thylakoid membranes. Photosystems I and II (PSI and PSII) are the two complexes able to harvest light. PSI is the last complex of the electron transport chain and is composed of multiple subunits: the proteins building the catalytic core complex that are well conserved between oxygenic photosynthetic organisms, and, in green organisms, the membrane light‐harvesting complexes (Lhc) necessary to increase light absorption. In plants, four Lhca proteins (Lhca1–4) make up the antenna system of PSI, which can be further extended to optimize photosynthesis by reversible binding of LHCII, the main antenna complex of photosystem II. Here, we used biochemistry and electron microscopy in Arabidopsis to reveal a previously unknown supercomplex of PSI with LHCII that contains an additional Lhca1–a4 dimer bound on the PsaB–PsaI–PsaH side of the complex. This finding contradicts recent structural studies suggesting that the presence of an Lhca dimer at this position is an exclusive feature of algal PSI. We discuss the features of the additional Lhca dimer in the large plant PSI–LHCII supercomplex and the differences with the algal PSI. Our work provides further insights into the intricate structural plasticity of photosystems.     

Different Localization of Two Ca2+ in Spinach Oxygen-Evolving Photosystem II Membranes. Evidence for Involvement of Only One Ca2+ in Oxygen Evolution

Localization of the two Ca²⁺ bound to oxygen-evolving photosystemII (PSII) membranes from spinach was investigated by fractionatingthe membranes into the PSII reaction center core complexes andperipheral antenna Chl a/b-proteins after solubilization withn-heptylthioglucoside. The core complex fraction contained oneCa²⁺ per PSII, while another Ca²⁺ was found in the solubilizedmajor light-harvesting Chl a/b-proteins (LHCII). LHCII isolatedwith Triton X-100 or dodecylmaltoside also contained Ca²⁺ inan amount corresponding to one per PSII. The Ca²⁺ bound to LHCIIcould not be removed by treatment with Chelex 100, which effectivelysequestered extraneous Ca²⁺ bound to LHCII, or by preparationof LHCII in the presence of 40 mM citrate. Localization of thetwo Ca²⁺ in different functional domain of PSII membranes conclusivelyindicates that the number of the bound Ca²⁺ that can functionin oxygen evolution is one per PSII. The results also suggestthat one Ca²⁺ has a structural role in the peripheral antennaassembly. (Received July 21, 1992; Accepted March 9, 1993)     

Involvement of protein phosphorylation in the sensitivity of photosystem II to strong illumination

Four types of differently phosphorylated hylakoids isolated from field grown spinach ( Spinacia oleracea L.) were tested for the sensitivity of photosystem II (PSII) to photoinactivation. Phosphorylation of light-harvesting II complexes (LHCII) protected PSII electron transfer from photoinhibitory damage, while the phosphorylation of the PSII core polypeptides slightly accelerated the decline of electron transfer during high irradiance treatment. Dephosphorylation of the CP43 apoprotein and PsbH protein by an alkaline phosphatase resulted in an extreme sensitivity of the thylakoids to strong illumination. The PSII photoinactivation of thylakoids with the impaired oxygen-evolving complex was found to be independent of phosphorylation.
The thylakoids of the thermophilic cyanobacterium Synechococcus elongates were used in order to compare the plants with an organism where LHCII complexes are missing and the PSII core proteins are not phosphorylated.     

In vivo system for analyzing the function of the PsbP protein using <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The PsbP protein is an extrinsic subunit of photosystem II (PSII) specifically developed in green-plant species including land plants and green algae. The protein–protein interactions involving PsbP and its effect on oxygen evolution have been investigated in vitro using isolated PSII membranes. However, the importance of those interactions needs to be examined at the cellular level. To this end, we developed a system expressing exogenous PsbP in the background of the Chlamydomonas BF25 mutant lacking native PsbP. Expression of His-tagged PsbP successfully restored the oxygen-evolving activity and photoautotrophic growth of the mutant, while PsbP-?15 lacking the N-terminal 15 residues, which are crucial for the oxygen-evolving activity of spinach PSII in vitro, only partially did. This demonstrated the importance of N-terminal sequence of PsbP for the photosynthetic activity in vivo. Furthermore, the PSII–LHCII supercomplex can be specifically purified from the Chlamydomonas cells having His-tagged PsbP using a metal affinity chromatography. This study provides a platform not only for the functional analysis of PsbP in vivo but also for structural analysis of the PSII–LHCII supercomplex from green algae.     

PHOTOSYSTEM II SUBUNIT R Is Required for Efficient Binding of LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 to Photosystem II-Light-Harvesting Supercomplexes in Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii, the LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3) protein is crucial for efficient energy-dependent thermal dissipation of excess absorbed light energy and functionally associates with photosystem II-light-harvesting complex II (PSII-LHCII) supercomplexes. Currently, it is unknown how LHCSR3 binds to the PSII-LHCII supercomplex. In this study, we investigated the role of PHOTOSYSTEM II SUBUNIT R (PSBR) an intrinsic membrane-spanning PSII subunit, in the binding of LHCSR3 to PSII-LHCII supercomplexes. Down-regulation of PSBR expression diminished the efficiency of oxygen evolution and the extent of nonphotochemical quenching and had an impact on the stability of the oxygen-evolving complex as well as on PSII-LHCII-LHCSR3 supercomplex formation. Its down-regulation destabilized the PSII-LHCII supercomplex and strongly reduced the binding of LHCSR3 to PSII-LHCII supercomplexes, as revealed by quantitative proteomics. PHOTOSYSTEM II SUBUNIT P deletion, on the contrary, destabilized PHOTOSYSTEM II SUBUNIT Q binding but did not affect PSBR and LHCSR3 association with PSII-LHCII. In summary, these data provide clear evidence that PSBR is required for the stable binding of LHCSR3 to PSII-LHCII supercomplexes and is essential for efficient energy-dependent quenching and the integrity of the PSII-LHCII-LHCSR3 supercomplex under continuous high light.Plant photosynthetic electron transfer is conducted by a series of reactions at the chloroplast thylakoid membrane, resulting in light-dependent water oxidation, NADP reduction, and ATP formation (Whatley et al., 1963). Two separate photosystems (PSI and PSII) and an ATP synthase catalyze these reactions. PSI and PSII are multiprotein complexes that are mainly embedded in unstacked and stacked regions of the thylakoid membrane, respectively. PSI consists of more than 10 subunits and a number of cofactors such as chlorophyll a, β-carotene, phylloquinone, and three iron-sulfur (4Fe-4S) clusters (Busch and Hippler, 2011). PSI catalyzes light-driven electron transfer from luminal plastocyanin to stromal ferredoxin. The latter reduces the ferredoxin-NADP reductase that, in turn, leads to the formation of NADPH. PSII catalyzes light-induced electron transfer from water to the plastoquinone pool by using chlorophyll a, carotenoids, as well as redox-active cofactors, causing the release of oxygen and protons (Pagliano et al., 2013). The core complex is organized as a dimer. Monomers are composed of the reaction center subunits PSBA (D1) and PSBD (D2), the inner antenna proteins PSBB (CP47) and PSBC (CP43), the α- and β-subunits (PSBE and PSBF) of cytochrome b₅₅₉, as well as a number of intrinsic low-molecular-mass subunits. The core monomer is further associated with an inorganic Mn₄O₅Ca cluster and a few chloride ions (Rivalta et al., 2011; Umena et al., 2011) required for photosynthetic water oxidation. To optimize this process, the oxygen-evolving complex is formed at the luminal side by the extrinsic polypeptides PSBO, PSBP, PSBQ, and PSBR (for review, see Pagliano et al., 2013).To enhance the light-harvesting capacity of PSII, various light-harvesting proteins bind to dimeric PSII core complexes (Dekker and Boekema, 2005). A common structure found for vascular plants and green algae is the C₂S₂ supercomplex, where two copies of monomeric Lhcb4 and Lhcb5 and two LHCII trimers (S-trimer; Boekema et al., 1995) bind to the dimeric PSII core. In vascular plants, larger but less stable PSII supercomplexes, known as C₂S₂M₂, are composed of two extra copies of the monomeric Lhcb6 with two additional LHCII trimers (M-trimer) bound through Lhcb4 and Lhcb5 (Dekker and Boekema, 2005; Caffarri et al., 2009). Even larger complexes containing two additional LHCII trimers (L-trimer), bound via Lhcb6, are found and are known as C₂S₂M₂L_1–2 (Boekema et al., 1999). A recent study in Chlamydomonas reinhardtii identified PSII-LHCII supercomplexes with three LHCII trimers attached to each side of the core (C₂S₂M₂L₂; Tokutsu et al., 2012). Interestingly, such PSII-LHCII supercomplexes associate with LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3; Tokutsu and Minagawa, 2013), an ancient light-harvesting protein required for efficient energy-dependent (qE) quenching in the alga (Peers et al., 2009). The qE component of nonphotochemical quenching (NPQ) is an energy-dependent constituent of NPQ and regulates the thermal dissipation of excess absorbed light energy (Li et al., 2000; Peers et al., 2009). The qE capacity in C. reinhardtii increases proportionally to the light-dependent accumulation of the LHCSR3 protein (Peers et al., 2009). In contrast, in vascular plants, qE is constitutively active and dependent on PSBS, a PSII polypeptide (Li et al., 2000). Mass spectrometric analyses of isolated C₂S₂M₂ PSII supercomplexes revealed the presence of extrinsic subunits PSBP, PSBQ, and PSBR, while PSBS was not identified, suggesting that PSBS does not influence the association of the PSII core with the outer light-harvesting complex system (Pagliano et al., 2014). In line with the proteomic findings, recent data suggest that subunits PSBP, PSBQ, and PSBR contribute to the stability of PSII-LHCII supercomplexes in vascular plants (Caffarri et al., 2009; Ifuku et al., 2011; Allahverdiyeva et al., 2013). A recent quantitative proteomic study performed with C. reinhardtii identified PSBR as the only PSII subunit to be induced upon the shift from photoheterotrophic to photoautotrophic growth conditions similar to LHCSR3 (Höhner et al., 2013).In vascular plants and green algae, PSBR is nucleus encoded and has a mass of about 10 kD. The mature protein has a predicted 70-amino acid luminal N-terminal part and a C-terminal transmembrane span (Ljungberg et al., 1986; Lautner et al., 1988; Webber et al., 1989). An association of PSBR with the oxygen-evolving complex has been suggested, as its presence is required for the stable assembly of PSBP with the PSII core and its absence also impacts the binding of PSBQ to the core (Suorsa et al., 2006; Liu et al., 2009). For stable association with the PSII core complex, PSBR needs the presence of PSBJ (Suorsa et al., 2006). Functionally, the depletion of PSBR protein expression decreased rates of oxygen evolution (Allahverdiyeva et al., 2007, 2013) and quinone reoxidation (Allahverdiyeva et al., 2007). PSBR phosphorylation is known for Arabidopsis (Arabidopsis thaliana; Reiland et al., 2009, 2011; Nakagami et al., 2010) and in the green alga C. reinhardtii (Turkina et al., 2006), although phosphorylation sites are not conserved between the alga and the vascular plant.In this work, we addressed the question of whether down-regulation of PSBR expression would affect LHCSR3 binding to the PSII-LHCII supercomplex in C. reinhardtii. To this end, we took advantage of artificial microRNA (amiRNA) technology to down-regulate PSBR expression and investigated the impact of PSBR down-regulation on photosynthetic performance as well as on PSII-LHCII-LHCSR3 supercomplex formation. Our data provide evidence that PSBR is required for the stable binding of LHCSR3 to PSII-LHCII supercomplexes.