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.     

Light acclimation involves dynamic re‐organization of the pigment–protein megacomplexes in non‐appressed thylakoid domains

Thylakoid energy metabolism is crucial for plant growth, development and acclimation. Non‐appressed thylakoids harbor several high molecular mass pigment–protein megacomplexes that have flexible compositions depending upon the environmental cues. This composition is important for dynamic energy balancing in photosystems (PS) I and II. We analysed the megacomplexes of Arabidopsis wild type (WT) plants and of several thylakoid regulatory mutants. The stn7 mutant, which is defective in phosphorylation of the light‐harvesting complex (LHC) II, possessed a megacomplex composition that was strikingly different from that of the WT. Of the nine megacomplexes in total for the non‐appressed thylakoids, the largest megacomplex in particular was less abundant in the stn7 mutant under standard growth conditions. This megacomplex contains both PSI and PSII and was recently shown to allow energy spillover between PSII and PSI (Nat. Commun., 6, 2015, 6675). The dynamics of the megacomplex composition was addressed by exposing plants to different light conditions prior to thylakoid isolation. The megacomplex pattern in the WT was highly dynamic. Under darkness or far red light it showed low levels of LHCII phosphorylation and resembled the stn7 pattern; under low light, which triggers LHCII phosphorylation, it resembled that of the tap38/pph1 phosphatase mutant. In contrast, solubilization of the entire thylakoid network with dodecyl maltoside, which efficiently solubilizes pigment–protein complexes from all thylakoid compartments, revealed that the pigment–protein composition remained stable despite the changing light conditions or mutations that affected LHCII (de)phosphorylation. We conclude that the composition of pigment–protein megacomplexes specifically in non‐appressed thylakoids undergoes redox‐dependent changes, thus facilitating maintenance of the excitation balance between the two photosystems upon changes in light conditions.     

Structural variability of plant photosystem II megacomplexes in thylakoid membranes

Plant photosystem II (PSII) is organized into large supercomplexes with variable levels of membrane‐bound light‐harvesting proteins (LHCIIs). The largest stable form of the PSII supercomplex involves four LHCII trimers, which are specifically connected to the PSII core dimer via monomeric antenna proteins. The PSII supercomplexes can further interact in the thylakoid membrane, forming PSII megacomplexes. So far, only megacomplexes consisting of two PSII supercomplexes associated in parallel have been observed. Here we show that the forms of PSII megacomplexes can be much more variable. We performed single particle electron microscopy (EM) analysis of PSII megacomplexes isolated from Arabidopsis thaliana using clear‐native polyacrylamide gel electrophoresis. Extensive image analysis of a large data set revealed that besides the known PSII megacomplexes, there are distinct groups of megacomplexes with non‐parallel association of supercomplexes. In some of them, we have found additional LHCII trimers, which appear to stabilize the non‐parallel assemblies. We also performed EM analysis of the PSII supercomplexes on the level of whole grana membranes and successfully identified several types of megacomplexes, including those with non‐parallel supercomplexes, which strongly supports their natural origin. Our data demonstrate a remarkable ability of plant PSII to form various larger assemblies, which may control photochemical usage of absorbed light energy in plants in a changing environment.     

Consequences of state transitions on the structural and functional organization of Photosystem I in the green alga Chlamydomonas reinhardtii

State transitions represent a photoacclimation process that regulates the light‐driven photosynthetic reactions in response to changes in light quality/quantity. It balances the excitation between photosystem I (PSI) and II (PSII) by shuttling LHCII, the main light‐harvesting complex of green algae and plants, between them. This process is particularly important in Chlamydomonas reinhardtii in which it is suggested to induce a large reorganization in the thylakoid membrane. Phosphorylation has been shown to be necessary for state transitions and the LHCII kinase has been identified. However, the consequences of state transitions on the structural organization and the functionality of the photosystems have not yet been elucidated. This situation is mainly because the purification of the supercomplexes has proved to be particularly difficult, thus preventing structural and functional studies. Here, we have purified and analysed PSI and PSII supercomplexes of C. reinhardtii in states 1 and 2, and have studied them using biochemical, spectroscopic and structural methods. It is shown that PSI in state 2 is able to bind two LHCII trimers that contain all four LHCII types, and one monomer, most likely CP29, in addition to its nine Lhcas. This structure is the largest PSI complex ever observed, having an antenna size of 340 Chls/P700. Moreover, all PSI‐bound Lhcs are efficient in transferring energy to PSI. A projection map at 20 Å resolution reveals the structural organization of the complex. Surprisingly, only LHCII type I, II and IV are phosphorylated when associated with PSI, while LHCII type III and CP29 are not, but CP29 is phosphorylated when associated with PSII in state2.     

In pea stipules a functional photosynthetic electron flow occurs despite a reduced dynamicity of LHCII association with photosystems

The flexible association of the light harvesting complex II (LHCII) to photosystem (PS) I and PSII to balance their excitation is a major short-term acclimation process of the thylakoid membrane, together with the thermal dissipation of excess absorbed energy, reflected in non-photochemical quenching of chlorophyll fluorescence (NPQ). In Pisum sativum, the leaf includes two main photosynthetic parts, the basal stipules and the leaflets. Since the stipules are less efficient in carbon fixation than leaflets, the adjustments of the thylakoid system, which safeguard the photosynthetic membrane against photodamage, were analysed. As compared to leaflets, the stipules experienced a decay in PSII photochemical activity. The supramolecular organization of photosystems in stipules showed a more conspicuous accumulation of large PSII-LHCII supercomplexes in the grana, but also a tendency to retain the PSI-LHCI-LHCII state transition complex and the PSI-LHCI-PSII-LHCII megacomplexes probably located at the interface between appressed and stroma-exposed membranes. As a consequence, stipules had a lower capacity to perform state transitions and the overall thylakoid architecture was less structurally flexible and ordered than in leaflets. Yet, stipules proved to be quite efficient in regulating the redox state of the electron transport chain and more capable of inducing NPQ than leaflets. It is proposed that, in spite of a relatively static thylakoid arrangement, LHCII interaction with both photosystems in megacomplexes can contribute to a regulated electron flow.     

Supramolecular organization of thylakoid membrane proteins in green plants

The light reactions of photosynthesis in green plants are mediated by four large protein complexes, embedded in the thylakoid membrane of the chloroplast. Photosystem I (PSI) and Photosystem II (PSII) are both organized into large supercomplexes with variable amounts of membrane-bound peripheral antenna complexes. PSI consists of a monomeric core complex with single copies of four different LHCI proteins and has binding sites for additional LHCI and/or LHCII complexes. PSII supercomplexes are dimeric and contain usually two to four copies of trimeric LHCII complexes. These supercomplexes have a further tendency to associate into megacomplexes or into crystalline domains, of which several types have been characterized. Together with the specific lipid composition, the structural features of the main protein complexes of the thylakoid membranes form the main trigger for the segregation of PSII and LHCII from PSI and ATPase into stacked grana membranes. We suggest that the margins, the strongly folded regions of the membranes that connect the grana, are essentially protein-free, and that protein-protein interactions in the lumen also determine the shape of the grana. We also discuss which mechanisms determine the stacking of the thylakoid membranes and how the supramolecular organization of the pigment-protein complexes in the thylakoid membrane and their flexibility may play roles in various regulatory mechanisms of green plant photosynthesis.     

Proteomic approach to characterize the supramolecular organization of photosystems in higher plants

A project to investigate the supramolecular structure of photosystems was initiated, which is based on protein solubilizations by digitonin, protein separations by Blue native (BN)-polyacrylamide gel electrophoresis (PAGE) and protein identifications by mass spectrometry (MS). Under the conditions applied, nine photosystem supercomplexes could be described for chloroplasts of Arabidopsis, which have apparent molecular masses between 600 and 3200 kDa on BN gels. Identities of the supercomplexes were determined on the basis of their subunit compositions as documented by 2D BN/SDS-PAGE and BN/BN-PAGE. Two supercomplexes of 1060 and approximately 1600 kDa represent dimeric and trimeric forms of photosystem I (PSI), which include tightly bound LHCI proteins. Compared to monomeric PSI, these protein complexes are of low abundance. In contrast, photosystem II mainly forms part of dominant supercomplexes of 850, 1000, 1050 and 1300 kDa. According to our interpretation, these supercomplexes contain dimeric PSII, 1-4 LHCII trimers and additionally monomeric LHCII proteins. The 1300-kDa PSII supercomplex (containing four LHCII trimers) is partially converted into the 1000-kDa PSII supercomplex (containing two LHCII trimers) in the presence of dodecylmaltoside on 2D BN/BN gels. Analyses of peptides of the trypsinated 1300-kDa PSII supercomplex by mass spectrometry allowed to identify known subunits of the PSII core complex and additionally LHCII proteins encoded by eight different genes in Arabidopsis. Further application of this experimental approach will allow new insights into the supermolecular organization of photosystems in plants.     

Light‐dependent reversible phosphorylation of the minor photosystem II antenna Lhcb6 (CP24) occurs in lycophytes

Evolution of vascular plants required compromise between photosynthesis and photodamage. We analyzed representative species from two divergent lineages of vascular plants, lycophytes and euphyllophytes, with respect to the response of their photosynthesis and light‐harvesting properties to increasing light intensity. In the two analyzed lycophytes, Selaginella martensii and Lycopodium squarrosum, the medium phase of non‐photochemical quenching relaxation increased under high light compared to euphyllophytes. This was thought to be associated with the occurrence of a further thylakoid phosphoprotein in both lycophytes, in addition to D2, CP43 and Lhcb1‐2. This protein, which showed light intensity‐dependent reversible phosphorylation, was identified in S. martensii as Lhcb6, a minor LHCII antenna subunit of PSII. Lhcb6 is known to have evolved in the context of land colonization. In S. martensii, Lhcb6 was detected as a component of the free LHCII assemblies, but also associated with PSI. Most of the light‐induced changes affected the amount and phosphorylation of the LHCII assemblies, which possibly mediate PSI–PSII connectivity. We propose that Lhcb6 is involved in light energy management in lycophytes, participating in energy balance between PSI and PSII through a unique reversible phosphorylation, not yet observed in other land plants.     

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.     

Biogenesis of water splitting by photosystem II during de‐etiolation of barley (Hordeum vulgare L.)

Etioplasts lack thylakoid membranes and photosystem complexes. Light triggers differentiation of etioplasts into mature chloroplasts, and photosystem complexes assemble in parallel with thylakoid membrane development. Plastids isolated at various time points of de‐etiolation are ideal to study the kinetic biogenesis of photosystem complexes during chloroplast development. Here, we investigated the chronology of photosystem II (PSII) biogenesis by monitoring assembly status of chlorophyll‐binding protein complexes and development of water splitting via O₂ production in plastids (etiochloroplasts) isolated during de‐etiolation of barley (Hordeum vulgare L.). Assembly of PSII monomers, dimers and complexes binding outer light‐harvesting antenna [PSII‐light‐harvesting complex II (LHCII) supercomplexes] was identified after 1, 2 and 4 h of de‐etiolation, respectively. Water splitting was detected in parallel with assembly of PSII monomers, and its development correlated with an increase of bound Mn in the samples. After 4 h of de‐etiolation, etiochloroplasts revealed the same water‐splitting efficiency as mature chloroplasts. We conclude that the capability of PSII to split water during de‐etiolation precedes assembly of the PSII‐LHCII supercomplexes. Taken together, data show a rapid establishment of water‐splitting activity during etioplast‐to‐chloroplast transition and emphasize that assembly of the functional water‐splitting site of PSII is not the rate‐limiting step in the formation of photoactive thylakoid membranes.     

Phosphorylation of the Light-Harvesting Complex II Isoform Lhcb2 Is Central to State Transitions

Light-harvesting complex II (LHCII) is a crucial component of the photosynthetic machinery, with central roles in light capture and acclimation to changing light. The association of an LHCII trimer with PSI in the PSI-LHCII supercomplex is strictly dependent on LHCII phosphorylation mediated by the kinase STATE TRANSITION7, and is directly related to the light acclimation process called state transitions. In Arabidopsis (Arabidopsis thaliana), the LHCII trimers contain isoforms that belong to three classes: Lhcb1, Lhcb2, and Lhcb3. Only Lhcb1 and Lhcb2 can be phosphorylated in the N-terminal region. Here, we present an improved Phos-tag-based method to determine the absolute extent of phosphorylation of Lhcb1 and Lhcb2. Both classes show very similar phosphorylation kinetics during state transition. Nevertheless, only Lhcb2 is extensively phosphorylated (>98%) in PSI-LHCII, whereas phosphorylated Lhcb1 is largely excluded from this supercomplex. Both isoforms are phosphorylated to different extents in other photosystem supercomplexes and in different domains of the thylakoid membranes. The data imply that, despite their high sequence similarity, differential phosphorylation of Lhcb1 and Lhcb2 plays contrasting roles in light acclimation of photosynthesis.Light capture and its conversion to chemical energy occur in a set of transmembrane protein complexes of the thylakoid membrane. PSII, the cytochrome b₆f complex, and PSI drive photosynthetic electron flow and the creation of a proton gradient across the thylakoid membrane. ATP synthase couples the dissipation of this gradient to the synthesis of ATP. The light-harvesting antennae play an important role in collecting light and transferring energy to the photosystems. Light-Harvesting Complex I (LHCI) exclusively transfers light energy to PSI, with which it is tightly associated (Croce and van Amerongen, 2014). In contrast, LHCII, which is the most abundant complex of the thylakoid membrane, can transfer energy to PSI or PSII (Grieco et al., 2015). Light is highly variable in natural environments, and plants experience continuous changes in both the spectrum and intensity of light on timescales as short as seconds. Changes in light quality may unbalance the activity of the two photosystems since their absorption spectra differ, whereas high light intensity can lead to overexcitation and induce photodamage. At low or moderate light intensities, the LHCII complex differentially associates with PSII or PSI, in a phosphorylation-dependent process known as state transitions, to rapidly respond to changes in the spectrum of light. In brief, under light quality that activates PSII more than PSI (e.g. blue light), LHCII is phosphorylated, and as a consequence, its binding to PSI is favored (state 2). Conversely, under light that preferentially excites PSI (enriched in far-red), this association can be reverted by dephosphorylation of the LHCII antenna, which favors its binding to PSII (state 1; Goldschmidt-Clermont and Bassi, 2015; Kim et al., 2015). A protein kinase, STATE TRANSITION7 (STN7), and a protein phosphatase, PROTEIN PHOSPHATASE1 (PPH1)/THYLAKOID-ASSOCIATED PHOSPHATASE38 (TAP38), are essential for the rapid phosphorylation and dephosphorylation of the LHCII antenna that regulates its differential association to PSI or PSII (Bellafiore et al., 2005; Pribil et al., 2010; Shapiguzov et al., 2010). Only a relatively small fraction of the LHCII antenna (<20%) is estimated to participate in state transitions in Arabidopsis (Arabidopsis thaliana; Allen, 1992). However, the process is conserved across the green eukaryotes and is relevant to plant fitness (Frenkel et al., 2007). Under high light, energy-dependent quenching of LHCII predominates, and furthermore, this antenna can uncouple from PSII (Wientjes et al., 2013b).The differential association of photosystems, LHCII, and other components of the thylakoid membrane gives rise to a set of supercomplexes that are central in ensuring photosynthetic efficiency and a rapid response to environmental cues (Caffarri et al., 2009; Duffy et al., 2013; Pietrzykowska et al., 2014; Fristedt et al., 2015). Fine tuning the dynamic assembly of these supercomplexes involves the association of antennae containing specific sets of Lhcb proteins. The major LHCII antenna comprises homo- and heterotrimers of Lhcb1 to Lhcb3 (Jackowski et al., 2001), whereas the minor LHCII isoforms (Lhcb4–Lhcb6) are monomeric (de Bianchi et al., 2008). Lhcb1 and Lhcb2 share a very similar primary structure and associated pigments (Formaggio et al., 2001; Zhang et al., 2008), whereas Lhcb3 appears to have slightly different features (Standfuss and Kühlbrandt, 2004). In Arabidopsis, five genes encode Lhcb1 isoforms, three genes encode Lhcb2 isoforms, and a single gene encodes Lhcb3. The principal discriminant between these classes is a short stretch of residues at the N-terminal end, which is of particular importance since it contains the Thr that is reversibly phosphorylated during light-acclimation processes (Goldschmidt-Clermont and Bassi, 2015). During evolution, land plants have maintained a major LHCII composed of different classes of Lhcb subunits. The phosphorylated N terminus of Lhcb2 was particularly well conserved (Alboresi et al., 2008; Zhang et al., 2008).PSII-LHCII supercomplexes have been isolated from Arabidopsis with up to four LHCII trimers bound to a PSII dimer, as well as the three minor monomeric antennae (Lhcb4–Lhcb6; Caffarri et al., 2009; Kouřil et al., 2012). In the LHCII trimers of these supercomplexes, different classes of Lhcb subunits are distributed differently, suggesting a specific role in light acclimation for each of them (Damkjaer et al., 2009; Pietrzykowska et al., 2014). In the stably bound S trimer, Lhcb1 and Lhcb2 are more abundant, whereas the moderately bound M trimer contains mostly Lhcb1 and Lhcb3 (Galka et al., 2012). PSII supercomplexes isolated from spinach (Spinacia oleracea) showed the presence of an extra LHCII trimer (L trimer); therefore, it is possible that, in Arabidopsis, other trimers are associated with the PSII dimer in a more labile supercomplex that cannot be isolated (Boekema et al., 1999). A single LHCII trimer, containing Lhcb1 and Lhcb2, stably associates with PSI to constitute the PSI-LHCII supercomplex, whose formation is dependent on LHCII phosphorylation by STN7 in state 2 (Kouřil et al., 2005; Galka et al., 2012).Previous reports have shown that the relative phosphorylation of Lhcb1 and Lhcb2 isoforms differs among thylakoid supercomplexes (Galka et al., 2012; Leoni et al., 2013). Here, we address the specific roles of Lhcb1 and Lhcb2 phosphorylation in photosynthetic acclimation. The improved protocol for SDS-PAGE in the presence of Phos-tag (Wako Chemicals) that we present allows quantification of the extent of phosphorylation for each class of antenna isoforms. We report that, in the PSI-LHCII supercomplex that is assembled in state 2, only the phosphorylated form of Lhcb2 is present, whereas the phosphorylated form of Lhcb1 is excluded. In contrast, both Lhcb1 and Lhcb2 are phosphorylated to different levels in other supercomplexes. This quantitative information on the level of phosphorylation of Lhcb1 and Lhcb2 offers new insights into the specific roles of the two classes of LHCII isoforms in light acclimation and supercomplex formation.     

Different response of photosystem II to short and long‐term drought stress in Arabidopsis thaliana

Short‐ and long‐term drought stress on photosystem II (PSII) and oxidative stress were studied in Arabidopsis thaliana. Under drought stress, chlorophyll (Chl) content, Chl fluorescence, relative water content and oxygen evolution capacity gradually decreased, and the thylakoid structure was gradually damaged. Short‐term drought stress caused a rapid disassembly of the light‐harvesting complex II (LHCII). However, PSII dimers kept stable under the short‐term drought stress and significantly decreased only after 15 days of drought stress. Immunoblotting analysis of the thylakoid membrane proteins showed that most of the photosystem proteins decreased after the stress, especially for Lhcb5, Lhcb6 and PsbQ proteins. However, surprisingly, PsbS significantly increased after the long‐term drought stress, which is consistent with the substantially increased non‐photochemical quenching (NPQ) after the stress. Our results suggest that the PSII–LHCII supercomplexes and LHCII assemblies play an important role in preventing photo‐damages to PSII under drought stress.     

Quantification of energy-converting protein complexes in plant thylakoid membranes

Knowledge about the exact abundance and ratio of photosynthetic protein complexes in thylakoid membranes is central to understanding structure-function relationships in energy conversion. Recent modeling approaches for studying light harvesting and electron transport reactions rely on quantitative information on the constituent complexes in thylakoid membranes. Over the last decades several quantitative methods have been established and refined, enabling precise stoichiometric information on the five main energy-converting building blocks in the thylakoid membrane: Light-harvesting complex II (LHCII), Photosystem II (PSII), Photosystem I (PSI), cytochrome b₆f complex (cyt b₆f complex), and ATPase. This paper summarizes a few quantitative spectroscopic and biochemical methods that are currently available for quantification of plant thylakoid protein complexes. Two new methods are presented for quantification of LHCII and the cyt b₆f complex, which agree well with established methods. In addition, recent improvements in mass spectrometry (MS) allow deeper compositional information on thylakoid membranes. The comparison between mass spectrometric and more classical protein quantification methods shows similar quantities of complexes, confirming the potential of thylakoid protein complex quantification by MS. The quantitative information on PSII, PSI, and LHCII reveal that about one third of LHCII must be associated with PSI for a balanced light energy absorption by the two photosystems.     

Supramolecular organization of photosystem II in green plants

Green plant photosystem II (PSII) is involved in the light reactions of photosynthesis, which take place in the thylakoid membrane of the chloroplast. PSII is organized into large supercomplexes with variable amounts of membrane-bound peripheral antenna complexes. These supercomplexes are dimeric and contain usually 2-4 copies of trimeric LHCII complexes and have a further tendency to associate into megacomplexes or into crystalline domains, of which several types have been characterized. This review focuses on the overall composition and structure of the PSII supercomplex of green plants and its organization and interactions within the photosynthetic membrane. Further, we present the current knowledge how the thylakoid membrane is three-dimensionally organized within the chloroplast. We also discuss how the supramolecular organization in the thylakoid membrane and the PSII flexibility may play roles in various short-term regulatory mechanisms of green plant photosynthesis. This article is part of a Special Issue entitled: Photosystem II.     

Photosystem I-LHCII megacomplexes respond to high light and aging in plants

Photosystem II is known to be a highly dynamic multi-protein complex that participates in a variety of regulatory and repair processes. In contrast, photosystem I (PSI) has, until quite recently, been thought of as relatively static. We report the discovery of plant PSI-LHCII megacomplexes containing multiple LHCII trimers per PSI reaction center. These PSI-LHCII megacomplexes respond rapidly to changes in light intensity, as visualized by native gel electrophoresis. PSI-LHCII megacomplex formation was found to require thylakoid stacking, and to depend upon growth light intensity and leaf age. These factors were, in turn, correlated with changes in PSI/PSII ratios and, intriguingly, PSI-LHCII megacomplex dynamics appeared to depend upon PSII core phosphorylation. These findings suggest new functions for PSI and a new level of regulation involving specialized subpopulations of photosystem I which have profound implications for current models of thylakoid dynamics.     

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.     

Redox signalling and the structural basis of regulation of photosynthesis by protein phosphorylation

In photosynthesis in chloroplasts and cyanobacteria, redox control of thylakoid protein phosphorylation regulates distribution of absorbed excitation energy between the two photosystems. When electron transfer through chloroplast photosystem II (PSII) proceeds at a rate higher than that through photosystem I (PSI), chemical reduction of a redox sensor activates a thylakoid protein kinase that catalyses phosphorylation of light-harvesting complex II (LHCII). Phosphorylation of LHCII increases its affinity for PSI and thus redistributes light-harvesting chlorophyll to PSI at the expense of PSII. This short-term redox signalling pathway acts by means of reversible, post-translational modification of pre-existing proteins. A long-term equalisation of the rates of light utilisation by PSI and PSII also occurs: by means of adjustment of the stoichiometry of PSI and PSII. It is likely that the same redox sensor controls both state transitions and photosystem stoichiometry. A specific mechanism for integration of these short- and long-term adaptations is proposed. Recent evidence shows that phosphorylation of LHCII causes a change in its 3-D structure, which implies that the mechanism of state transitions in chloroplasts involves control of recognition of PSI and PSII by LHCII. The distribution of LHCII between PSII and PSI is therefore determined by the higher relative affinity of phospho-LHCII for PSI, with lateral movement of the two forms of the LHCII being simply a result of their diffusion within the membrane plane. Phosphorylation-induced dissociation of LHCII trimers may induce lateral movement of monomeric phospho-LHCII, which binds preferentially to PSI. After dephosphorylation, monomeric, unphosphorylated LHCII may trimerize at the periphery of PSII.     

Functional Analyses of the Plant Photosystem I-Light-Harvesting Complex II Supercomplex Reveal That Light-Harvesting Complex II Loosely Bound to Photosystem II Is a Very Efficient Antenna for Photosystem I in State II

State transitions are an important photosynthetic short-term response that allows energy distribution balancing between photosystems I (PSI) and II (PSII). In plants when PSII is preferentially excited compared with PSI (State II), part of the major light-harvesting complex LHCII migrates to PSI to form a PSI-LHCII supercomplex. So far, little is known about this complex, mainly due to purification problems. Here, a stable PSI-LHCII supercomplex is purified from Arabidopsis thaliana and maize (Zea mays) plants. It is demonstrated that LHCIIs loosely bound to PSII in State I are the trimers mainly involved in state transitions and become strongly bound to PSI in State II. Specific Lhcb1-3 isoforms are differently represented in the mobile LHCII compared with S and M trimers. Fluorescence analyses indicate that excitation energy migration from mobile LHCII to PSI is rapid and efficient, and the quantum yield of photochemical conversion of PSI-LHCII is substantially unaffected with respect to PSI, despite a sizable increase of the antenna size. An updated PSI-LHCII structural model suggests that the low-energy chlorophylls 611 and 612 in LHCII interact with the chlorophyll 11145 at the interface of PSI. In contrast with the common opinion, we suggest that the mobile pool of LHCII may be considered an intimate part of the PSI antenna system that is displaced to PSII in State I.     

Structural and functional differentiation of the light‐harvesting protein Lhcb4 during land plant diversification

About 475 million years ago, plants originated from an ancestral green alga and evolved first as non‐vascular and later as vascular plants, becoming the primary producers of biomass on lands. During that time, the light‐harvesting complex II (LHCII), responsible for sunlight absorption and excitation energy transfer to the photosystem II (PSII) core, underwent extensive differentiation. Lhcb4 is an ancestral LHCII that, in flowering plants, differentiated into up to three isoforms, Lhcb4.1, Lhcb4.2 and Lhcb4.3. The pivotal position of Lhcb4 in the PSII‐LHCII supercomplex (PSII‐LHCIIsc) allows functioning as linker for either S‐ or M‐trimers of LHCII to the PSII core. The increased accumulation of Lhcb4.3 observed in PSII‐LHCIIsc of plants acclimated to moderate and high light intensities induced us to investigate, whether this isoform has a preferential localization in a specific PSII‐LHCIIsc conformation that might explain its light‐dependent accumulation. In this work, by combining an improved method for separation of different forms of PSII‐LHCIIsc from thylakoids of Pisum sativum L. grown at increasing irradiances with quantitative proteomics, we assessed that Lhcb4.3 is abundant in PSII‐LHCIIsc of type C₂S₂, and, interestingly, similar results were found for the PsbR subunit. Phylogenetic comparative analysis on different taxa of the Viridiplantae lineage and structural modeling further pointed out to an effect of the evolution of different Lhcb4 isoforms on the light‐dependent modulation of the PSII‐LHCIIsc organization. This information provides new insight on the properties of the Lhcb4 and its isoforms and their role on the structure, function and regulation of PSII.