Arrangement of Photosystem II and ATP Synthase in Chloroplast Membranes of Spinach and Pea

We used cryoelectron tomography to reveal the arrangements of photosystem II (PSII) and ATP synthase in vitreous sections of intact chloroplasts and plunge-frozen suspensions of isolated thylakoid membranes. We found that stroma and grana thylakoids are connected at the grana margins by staggered lamellar membrane protrusions. The stacking repeat of grana membranes in frozen-hydrated chloroplasts is 15.7 nm, with a 4.5-nm lumenal space and a 3.2-nm distance between the flat stromal surfaces. The chloroplast ATP synthase is confined to minimally curved regions at the grana end membranes and stroma lamellae, where it covers 20% of the surface area. In total, 85% of the ATP synthases are monomers and the remainder form random assemblies of two or more copies. Supercomplexes of PSII and light-harvesting complex II (LHCII) occasionally form ordered arrays in appressed grana thylakoids, whereas this order is lost in destacked membranes. In the ordered arrays, each membrane on either side of the stromal gap contains a two-dimensional crystal of supercomplexes, with the two lattices arranged such that PSII cores, LHCII trimers, and minor LHCs each face a complex of the same kind in the opposite membrane. Grana formation is likely to result from electrostatic interactions between these complexes across the stromal gap.     

Jumping mode atomic force microscopy on grana membranes from spinach

The thylakoid membrane system is a complex membrane system that organizes and reorganizes itself to provide plants optimal chemical energy from sunlight under different and varying environmental conditions. Grana membranes are part of this system and contain the light-driven water-splitting enzyme Photosystem II (PSII) and light-harvesting antenna complexes. Here, we present a direct visualization of PSII complexes within grana membranes from spinach. By means of jumping mode atomic force microscopy in liquid, minimal forces were applied between the scanning tip and membrane or protein, allowing complexes to be imaged with high detail. We observed four different packing arrangements of PSII complexes, which occur primarily as dimers: co-linear crystalline rows, nanometric domains of straight or skewed rows, and disordered domains. Upon storing surface-adhered membranes at low temperature prior to imaging, large-scale reorganizations of supercomplexes between PSII and light-harvesting complex II could be induced. The highest resolution images show the existence of membrane domains without obvious topography extending beyond supercomplexes. These observations illustrate the possibility for diffusion of proteins and smaller molecules within these densely packed membranes.     

Core protein phosphorylation facilitates the repair of photodamaged photosystem II at high light

Phosphorylation of photosystem II (PSII) reaction center protein D1 has been hypothesised to function as a signal for the migration of photodamaged PSII core complex from grana membranes to stroma lamellae for concerted degradation and replacement of the photodamaged D1 protein. Here, by using the mutants with impaired capacity (stn8) or complete lack (stn7 stn8) in phosphorylation of PSII core proteins, the role of phosphorylation in PSII photodamage and repair was investigated. We show that the lack of PSII core protein phosphorylation disturbs the disassembly of PSII supercomplexes at high light, which is a prerequisite for efficient migration of damaged PSII complexes from grana to stroma lamellae for repair. This results in accumulation of photodamaged PSII complexes, which in turn results, upon prolonged exposure to high light (HL), in general oxidative damage of photosynthetic proteins in the thylakoid membrane.     

Probing the organization of photosystem II in photosynthetic membranes by atomic force microscopy

Efficient photosynthetic energy transduction and its regulation depend on a precise supramolecular arrangement of the plant photosystem II (PSII) complex in grana membranes of chloroplasts. The topography of isolated photosystem II supercomplexes and the supramolecular organization of this complex in grana membrane preparations are visualized by high-resolution atomic force microscopy (AFM) in air in tapping mode with an active feedback control to minimize tip-sample interactions. Systematic comparison between topographic characteristics of the protrusions in atomic force microscopic images and well-established high-resolution and freeze-fracture electron microscopic data shows that the photosystem II organization can be properly imaged by AFM in air. Taking the protruding water-splitting apparatus as a topographic marker for PSII, its distribution and orientation in isolated grana membrane were analyzed. For the latter a new mathematical procedure was established, which revealed a preference for a parallel alignment of PSII that resembles the organization in highly ordered semicrystalline arrays. Furthermore, by analyzing the height of grana membrane stacks, we conclude that lumenal protrusions of adjacent photosystem II complexes in opposing membranes are displaced relative to each other. The functional consequences for lateral migration processes are discussed.     

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.     

Arrangement of photosystem II supercomplexes in crystalline macrodomains within the thylakoid membrane of green plant chloroplasts

The chloroplast thylakoid membrane of green plants is organized in stacked grana membranes and unstacked stroma membranes. We investigated the structural organization of Photosystem II (PSII) in paired grana membrane fragments by transmission electron microscopy. The membrane fragments were obtained by a short treatment of thylakoid membranes with the mild detergent n-dodecyl-alpha, d-maltoside and are thought to reflect the grana membranes in a native state. The membranes frequently show crystalline macrodomains in which PSII is organized in rows spaced by either 26.3 nm (large-spaced crystals) or 23 nm (small-spaced crystals). The small-spaced crystals are less common but better ordered. Image analysis of the crystals by an aperiodic approach revealed the precise positions of the core parts of PSII in the lattices, as well as features of the peripheral light-harvesting antenna. Together, they indicate that the so-called C(2)S(2) and C(2)S(2)M supercomplexes form the basic motifs of the small-spaced and large-spaced crystals, respectively. An analysis of a pair of membranes with a well-ordered large-spaced crystal reveals that many PSII complexes in one layer face only light-harvesting complexes (LHCII) in the other layer. The implications of this type of organization for the efficient transfer of excitation energy from LHCII to PSII and for the stacking of grana membranes are discussed.     

Dimeric and monomeric organization of photosystem II. Distribution of five distinct complexes in the different domains of the thylakoid membrane

The supramolecular organization of photosystem II (PSII) was characterized in distinct domains of the thylakoid membrane, the grana core, the grana margins, the stroma lamellae, and the so-called Y100 fraction. PSII supercomplexes, PSII core dimers, PSII core monomers, PSII core monomers lacking the CP43 subunit, and PSII reaction centers were resolved and quantified by blue native PAGE, SDS-PAGE for the second dimension, and immunoanalysis of the D1 protein. Dimeric PSII (PSII supercomplexes and PSII core dimers) dominate in the core part of the thylakoid granum, whereas the monomeric PSII prevails in the stroma lamellae. Considerable amounts of PSII monomers lacking the CP43 protein and PSII reaction centers (D1-D2-cytochrome b559 complex) were found in the stroma lamellae. Our quantitative picture of the supramolecular composition of PSII, which is totally different between different domains of the thylakoid membrane, is discussed with respect to the function of PSII in each fraction. Steady state electron transfer, flash-induced fluorescence decay, and EPR analysis revealed that nearly all of the dimeric forms represent oxygen-evolving PSII centers. PSII core monomers were heterogeneous, and a large fraction did not evolve oxygen. PSII monomers without the CP43 protein and PSII reaction centers showed no oxygen-evolving activity.     

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.     

Quality control of photosystem II: FtsH hexamers are localized near photosystem II at grana for the swift repair of damage

The reaction center-binding D1 protein of Photosystem II is oxidatively damaged by excessive visible light or moderate heat stress. The metalloprotease FtsH has been suggested as responsible for the degradation of the D1 protein. We have analyzed the distribution and subunit structures of FtsH in spinach thylakoids and various membrane fractions derived from the thylakoids using clear native polyacrylamide gel electrophoresis and Western blot analysis. FtsH was found not only in the stroma thylakoids but also in the Photosystem II-enriched grana membranes. Monomeric, dimeric, and hexameric FtsH proteases were present as major subunit structures in thylakoids, whereas only hexameric FtsH proteases were detected in Triton X-100-solubilized Photosystem II membranes. Importantly, among the membrane fractions examined, hexameric FtsH proteases were most abundant in the Photosystem II membranes. In accordance with this finding, D1 degradation took place in the Photosystem II membranes under light stress. Sucrose density gradient centrifugation analysis of thylakoids and the Photosystem II membranes solubilized with n-dodecyl-β-d-maltoside and a chemical cross-linking study of thylakoids showed localization of FtsH near the Photosystem II light-harvesting chlorophyll-protein supercomplexes in the grana. These results suggest that part of the FtsH hexamers are juxtapositioned to PSII complexes in the grana in darkness, carrying out immediate degradation of the photodamaged D1 protein under light stress.     

Coexistence of Fluid and Crystalline Phases of Proteins in Photosynthetic Membranes

Photosystem II (PSII) and its associated light-harvesting complex II (LHCII) are highly concentrated in the stacked grana regions of photosynthetic thylakoid membranes. PSII-LHCII supercomplexes can be arranged in disordered packings, ordered arrays, or mixtures thereof. The physical driving forces underlying array formation are unknown, complicating attempts to determine a possible functional role for arrays in regulating light harvesting or energy conversion efficiency. Here, we introduce a coarse-grained model of protein interactions in coupled photosynthetic membranes, focusing on just two particle types that feature simple shapes and potential energies motivated by structural studies. Reporting on computer simulations of the model’s equilibrium fluctuations, we demonstrate its success in reproducing diverse structural features observed in experiments, including extended PSII-LHCII arrays. Free energy calculations reveal that the appearance of arrays marks a phase transition from the disordered fluid state to a system-spanning crystal. The predicted region of fluid-crystal coexistence is broad, encompassing much of the physiologically relevant parameter regime; we propose experiments that could test this prediction. Our results suggest that grana membranes lie at or near phase coexistence, conferring significant structural and functional flexibility to this densely packed membrane protein system.     

Coexistence of Fluid and Crystalline Phases of Proteins in Photosynthetic Membranes

Photosystem II (PSII) and its associated light-harvesting complex II (LHCII) are highly concentrated in the stacked grana regions of photosynthetic thylakoid membranes. PSII-LHCII supercomplexes can be arranged in disordered packings, ordered arrays, or mixtures thereof. The physical driving forces underlying array formation are unknown, complicating attempts to determine a possible functional role for arrays in regulating light harvesting or energy conversion efficiency. Here, we introduce a coarse-grained model of protein interactions in coupled photosynthetic membranes, focusing on just two particle types that feature simple shapes and potential energies motivated by structural studies. Reporting on computer simulations of the model’s equilibrium fluctuations, we demonstrate its success in reproducing diverse structural features observed in experiments, including extended PSII-LHCII arrays. Free energy calculations reveal that the appearance of arrays marks a phase transition from the disordered fluid state to a system-spanning crystal. The predicted region of fluid-crystal coexistence is broad, encompassing much of the physiologically relevant parameter regime; we propose experiments that could test this prediction. Our results suggest that grana membranes lie at or near phase coexistence, conferring significant structural and functional flexibility to this densely packed membrane protein system.     

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.     

Knockdown of the PsbP protein does not prevent assembly of the dimeric PSII core complex but impairs accumulation of photosystem II supercomplexes in tobacco

The PsbP protein is an extrinsic subunit of photosystem II (PSII) specifically found in land plants and green algae. Using PsbP-RNAi tobacco, we have investigated effects of PsbP knockdown on protein supercomplex organization within the thylakoid membranes and photosynthetic properties of PSII. In PsbP-RNAi leaves, PSII dimers binding the extrinsic PsbO protein could be formed, while the light-harvesting complex II (LHCII)-PSII supercomplexes were severely decreased. Furthermore, LHCII and major PSII subunits were significantly dephosphorylated. Electron microscopic analysis showed that thylakoid grana stacking in PsbP-RNAi chloroplast was largely disordered and appeared similar to the stromally-exposed or marginal regions of wild-type thylakoids. Knockdown of PsbP modified both the donor and acceptor sides of PSII; In addition to the lower water-splitting activity, the primary quinone Q_A in PSII was significantly reduced even when the photosystem I reaction center (P700) was noticeably oxidized, and thermoluminescence studies suggested the stabilization of the charged pair, S₂/Q_A⁻. These data indicate that assembly and/or maintenance of the functional MnCa cluster is perturbed in absence of PsbP, which impairs accumulation of final active forms of PSII supercomplexes.     

Integrating on-grid immunogold labeling and cryo-electron tomography to reveal photosystem II structure and spatial distribution in thylakoid membranes

A long-standing challenge in cell biology is elucidating the structure and spatial distribution of individual membrane-bound proteins, protein complexes and their interactions in their native environment. Here, we describe a workflow that combines on-grid immunogold labeling, followed by cryo-electron tomography (cryoET) imaging and structural analyses to identify and characterize the structure of photosystem II (PSII) complexes. Using an antibody specific to a core subunit of PSII, the D1 protein (uniquely found in the water splitting complex in all oxygenic photoautotrophs), we identified PSII complexes in biophysically active thylakoid membranes isolated from a model marine diatom Phaeodactylum tricornutum. Subsequent cryoET analyses of these protein complexes resolved two PSII structures: supercomplexes and dimeric cores. Our integrative approach establishes the structural signature of multimeric membrane protein complexes in their native environment and provides a pathway to elucidate their high-resolution structures.     

Photosystem II solubilizes as a monomer by mild detergent treatment of unstacked thylakoid membranes*

We studied the aggregation state of Photosystem II in stacked and unstacked thylakoid membranes from spinach after a quick and mild solubilization with the non-ionic detergent n-dodecyl-α,D-maltoside, followed by analysis by diode-array-assisted gel filtration chromatography and electron microscopy. The results suggest that Photosystem II (PS II) isolates either as a paired, appressed membrane fragment or as a dimeric PS II-LHC II supercomplex upon mild solubilization of stacked thylakoid membranes or PS II grana membranes, but predominantly as a core monomer upon mild solubilization of unstacked thylakoid membranes. Analysis of paired grana membrane fragments reveals that the number of PS II dimers is strongly reduced in single membranes at the margins of the grana membrane fragments. We suggest that unstacking of thylakoid membranes results in a spontaneous disintegration of the PS II-LHC II supercomplexes into separated PS II core monomers and peripheral light-harvesting complexes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Dynamics of photosystem II: a proteomic approach to thylakoid protein complexes

Oxygenic photosynthesis produces various radicals and activeoxygen species with harmful effects on photosystem II (PSII).Such photodamage occurs at all light intensities. Damaged PSIIcentres, however, do not usually accumulate in the thylakoidmembrane due to a rapid and efficient repair mechanism. Theexcellent design of PSII gives protection to most of the proteincomponents and the damage is most often targeted only to thereaction centre D1 protein. Repair of PSII via turnover of thedamaged protein subunits is a complex process involving (i)highly regulated reversible phosphorylation of several PSIIcore subunits, (ii) monomerization and migration of the PSIIcore from the grana to the stroma lamellae, (iii) partial disassemblyof the PSII core monomer, (iv) highly specific proteolysis ofthe damaged proteins, and finally (v) a multi-step replacementof the damaged proteins with de novo synthesized copies followedby (vi) the reassembly, dimerization, and photoactivation ofthe PSII complexes. These processes will shortly be reviewedpaying particular attention to the damage, turnover, and assemblyof the PSII complex in grana and stroma thylakoids during thephotoinhibition–repair cycle of PSII. Moreover, a two-dimensionalBlue-native gel map of thylakoid membrane protein complexes,and their modification in the grana and stroma lamellae duringa high-light treatment, is presented. Key words: Arabidopsis thylakoid membrane proteome, assembly of photosystem II, D1 protein, light stress, photosystem II photoinhibition, repair of photosystem II     

The structure of photosystem II in Arabidopsis: localization of the CP26 and CP29 antenna complexes

A genetic approach has been adopted to investigate the organization of the light-harvesting proteins in the photosystem II (PSII) complex in plants. PSII membrane fragments were prepared from wild-type Arabidopis thaliana and plants expressing antisense constructs to Lhcb4 and Lhcb5 genes, lacking CP29 and CP26, respectively (Andersson et al. (2001) Plant Cell 13, 1193-1204). Ordered PS II arrays and PS II supercomplexes were isolated from the membranes of plants lacking CP26 but could not be prepared from those lacking CP29. Membranes and supercomplexes lacking CP26 were less stable than those prepared from the wild type. Transmission electron microscopy aided by single-particle image analysis was applied to the ordered arrays and the isolated PSII complexes. The difference between the images obtained from wild type and antisense plants showed the location of CP26 to be near CP43 and one of the light-harvesting complex trimers. Therefore, the location of the CP26 within PSII was directly established for the first time, and the location of the CP29 complex was determined by elimination. Alterations in the packing of the PSII complexes in the thylakoid membrane also resulted from the absence of CP26. The minor light-harvesting complexes each have a unique location and important roles in the stabilization of the oligomeric PSII structure.     

Low pH Modulates the Macroorganization and Thermal Stability of PSII Supercomplexes in Grana Membranes

Protonation of the lumen-exposed residues of some photosynthetic complexes in the grana membranes occurs under conditions of high light intensity and triggers a major photoprotection mechanism known as energy dependent nonphotochemical quenching. We have studied the role of protonation in the structural reorganization and thermal stability of isolated grana membranes. The macroorganization of granal membrane fragments in protonated and partly deprotonated state has been mapped by means of atomic force microscopy. The protonation of the photosynthetic complexes has been found to induce large-scale structural remodeling of grana membranes—formation of extensive domains of the major light-harvesting complex of photosystem II and clustering of trimmed photosystem II supercomplexes, thinning of the membrane, and reduction of its size. These events are accompanied by pronounced thermal destabilization of the photosynthetic complexes, as evidenced by circular dichroism spectroscopy and differential scanning calorimetry. Our data reveal a detailed nanoscopic picture of the initial steps of nonphotochemical quenching.     

Loss‐of‐function of OsSTN8 suppresses the photosystem II core protein phosphorylation and interferes with the photosystem II repair mechanism in rice (Oryza sativa)

STN8 kinase is involved in photosystem II (PSII) core protein phosphorylation (PCPP). To examine the role of PCPP in PSII repair during high light (HL) illumination, we characterized a T–DNA insertional knockout mutant of the rice (Oryza sativa) STN8 gene. In this osstn8 mutant, PCPP was significantly suppressed, and the grana were thin and elongated. Upon HL illumination, PSII was strongly inactivated in the mutants, but the D1 protein was degraded more slowly than in wild‐type, and mobilization of the PSII supercomplexes from the grana to the stromal lamellae for repair was also suppressed. In addition, higher accumulation of reactive oxygen species and preferential oxidation of PSII reaction center core proteins in thylakoid membranes were observed in the mutants during HL illumination. Taken together, our current data show that the absence of STN8 is sufficient to abolish PCPP in osstn8 mutants and to produce all of the phenotypes observed in the double mutant of Arabidopsis, indicating the essential role of STN8‐mediated PCPP in PSII repair.     

LPA2 is required for efficient assembly of photosystem II in Arabidopsis thaliana

To elucidate the molecular mechanism of photosystem II (PSII) assembly, we characterized the low psii accumulation2 (lpa2) mutant of Arabidopsis thaliana, which is defective in the accumulation of PSII supercomplexes. The levels and processing patterns of the RNAs encoding the PSII subunits are unaltered in the mutant. In vivo protein-labeling experiments showed that the synthesis of CP43 (for chlorophyll a binding protein) was greatly reduced, but CP47, D1, and D2 were synthesized at normal rates in the lpa2-1 mutant. The newly synthesized CP43 was rapidly degraded in lpa2-1, and the turnover rates of D1 and D2 were higher in lpa2-1 than in wild-type plants. The newly synthesized PSII proteins were assembled into PSII complexes, but the assembly of PSII was less efficient in the mutant than in wild-type plants. LPA2 encodes an intrinsic thylakoid membrane protein, which is not an integral subunit of PSII. Yeast two-hybrid assays indicated that LPA2 interacts with the PSII core protein CP43 but not with the PSII reaction center proteins D1 and D2. Moreover, direct interactions of LPA2 with Albino3 (Alb3), which is involved in thylakoid membrane biogenesis and cell division, were also detected. Thus, the results suggest that LPA2, which appears to form a complex with Alb3, is involved in assisting CP43 assembly within PSII.