A kaleidoscope of photosynthetic antenna proteins and their emerging roles.

Photosynthetic light-harvesting antennae are pigment-binding proteins that perform one of the most fundamental tasks on Earth, capturing light and transferring energy that enables life in our biosphere. Adaptation to different light environments led to the evolution of an astonishing diversity of light-harvesting systems. At the same time, several strategies have been developed to optimize the light energy input into photosynthetic membranes in response to fluctuating conditions. The basic feature of these prompt responses is the dynamic nature of antenna complexes, whose function readily adapts to the light available. High-resolution microscopy and spectroscopic studies on membrane dynamics demonstrate the crosstalk between antennae and other thylakoid membrane components. With the increased understanding of light-harvesting mechanisms and their regulation, efforts are focusing on the development of sustainable processes for effective conversion of sunlight into functional bio-products. The major challenge in this approach lies in the application of fundamental discoveries in light-harvesting systems for the improvement of plant or algal photosynthesis. Here, we underline some of the latest fundamental discoveries on the molecular mechanisms and regulation of light harvesting that can potentially be exploited for the optimization of photosynthesis.

Cryo-EM structure of a tetrameric photosystem I from Chroococcidiopsis TS-821, a thermophilic, unicellular, non-heterocyst-forming cyanobacterium.

Photosystem I (PSI) is one of two photosystems involved in oxygenic photosynthesis. PSI of cyanobacteria exists in monomeric, trimeric, and tetrameric forms, in contrast to the strictly monomeric form of PSI in plants and algae. The tetrameric organization raises questions about its structural, physiological, and evolutionary significance. Here we report the ∼3.72 Å resolution cryo-electron microscopy structure of tetrameric PSI from the thermophilic, unicellular cyanobacterium Chroococcidiopsis sp. TS-821. The structure resolves 44 subunits and 448 cofactor molecules. We conclude that the tetramer is arranged via two different interfaces resulting from a dimer-of-dimers organization. The localization of chlorophyll molecules permits an excitation energy pathway within and between adjacent monomers. Bioinformatics analysis reveals conserved regions in the PsaL subunit that correlate with the oligomeric state. Tetrameric PSI may function as a key evolutionary step between the trimeric and monomeric forms of PSI organization in photosynthetic organisms.

Revealing the architecture of the photosynthetic apparatus in the diatom Thalassiosira pseudonana.

Diatoms are a large group of marine algae that are responsible for about one-quarter of global carbon fixation. Light-harvesting complexes of diatoms are formed by the fucoxanthin chlorophyll a/c proteins and their overall organization around core complexes of photosystems (PSs) I and II is unique in the plant kingdom. Using cryo-electron tomography, we have elucidated the structural organization of PSII and PSI supercomplexes and their spatial segregation in the thylakoid membrane of the model diatom species Thalassiosira pseudonana. 3D sub-volume averaging revealed that the PSII supercomplex of T. pseudonana incorporates a trimeric form of light-harvesting antenna, which differs from the tetrameric antenna observed previously in another diatom, Chaetoceros gracilis. Surprisingly, the organization of the PSI supercomplex is conserved in both diatom species. These results strongly suggest that different diatom classes have various architectures of PSII as an adaptation strategy, whilst a convergent evolution occurred concerning PSI and the overall plastid structure.

PSI of the Colonial Alga Botryococcus braunii Has an Unusually Large Antenna Size.

PSI is an essential component of the photosynthetic apparatus of oxygenic photosynthesis. While most of its subunits are conserved, recent data have shown that the arrangement of the light-harvesting complexes I (LHCIs) differs substantially in different organisms. Here we studied the PSI-LHCI supercomplex of Botryococccus braunii, a colonial green alga with potential for lipid and sugar production, using functional analysis and single-particle electron microscopy of the isolated PSI-LHCI supercomplexes complemented by time-resolved fluorescence spectroscopy in vivo. We established that the largest purified PSI-LHCI supercomplex contains 10 LHCIs (∼240 chlorophylls). However, electron microscopy showed heterogeneity in the particles and a total of 13 unique binding sites for the LHCIs around the PSI core. Time-resolved fluorescence spectroscopy indicated that the PSI antenna size in vivo is even larger than that of the purified complex. Based on the comparison of the known PSI structures, we propose that PSI in B. braunii can bind LHCIs at all known positions surrounding the core. This organization maximizes the antenna size while maintaining fast excitation energy transfer, and thus high trapping efficiency, within the complex.

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 [Kouril 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.

A Technical Introduction to Transmission Electron Microscopy for Soft-Matter: Imaging, Possibilities, Choices, and Technical Developments.

With a significant role in material sciences, physics, (soft matter) chemistry, and biology, the transmission electron microscope is one of the most widely applied structural analysis tool to date. It has the power to visualize almost everything from the micrometer to the angstrom scale. Technical developments keep opening doors to new fields of research by improving aspects such as sample preservation, detector performance, computational power, and workflow automation. For more than half a century, and continuing into the future, electron microscopy has been, and is, a cornerstone methodology in science. Herein, the technical considerations of imaging with electrons in terms of optics, technology, samples and processing, and targeted soft materials are summarized. Furthermore, recent advances and their potential for application to soft matter chemistry are highlighted.

Physiological and evolutionary implications of tetrameric photosystem I in cyanobacteria.

Photosystem I (PSI) is present as trimeric complexes in most characterized cyanobacteria and as monomers in plants and algae. Recent reports of tetrameric PSI have raised questions regarding its structural basis, physiological role, phylogenetic distribution and evolutionary significance. Here, we examined PSI in 61 cyanobacteria, showing that tetrameric PSI, which correlates with the psaL gene and a distinct genomic structure, is widespread among heterocyst-forming cyanobacteria and their close relatives. Physiological studies revealed that expression of tetrameric PSI is favoured under high light, with an increased content of novel PSI-bound carotenoids (myxoxanthophyll, canthaxanthan and echinenone). In sum, this work suggests that tetrameric PSI is an adaptation to high light intensity, and that change in PsaL leads to monomerization of trimeric PSI, supporting the hypothesis of tetrameric PSI being the evolutionary intermediate in the transition from cyanobacterial trimeric PSI to monomeric PSI in plants and algae.

A LHCB9-dependent photosystem I megacomplex induced under low light in Physcomitrella patens.

Photosystem I of the moss Physcomitrella patens has special properties, including the capacity to undergo non-photochemical fluorescence quenching. We studied the organization of photosystem I under different light and carbon supply conditions in wild-type moss and in moss with the lhcb9 (light-harvesting complex) knockout genotype, which lacks an antenna protein endowed with red-shifted absorption forms. Wild-type moss, when grown on sugars and in low light, accumulated LHCB9 proteins and a large form of the photosystem I supercomplex, which, besides the canonical four LHCI subunits, included a LHCII trimer and four additional LHC monomers. The lhcb9 knockout produced an angiosperm-like photosystem I supercomplex with four LHCI subunits irrespective of the growth conditions. Growth in the presence of sublethal concentrations of electron transport inhibitors that caused oxidation or reduction of the plastoquinone pool prevented or promoted, respectively, the accumulation of LHCB9 and the formation of the photosystem I megacomplex. We suggest that LHCB9 is a key subunit regulating the antenna size of photosystem I and the ability to avoid the over-reduction of plastoquinone: this condition is potentially dangerous in the shaded and sunfleck-rich environment typical of mosses, whose plastoquinone pool is reduced by both photosystem II and the oxidation of sugar substrates.

Solvent Mixing To Induce Molecular Motor Aggregation into Bowl-Shaped Particles: Underlying Mechanism, Particle Nature, and Application To Control Motor Behavior.

Control over dynamic functions in larger assemblies is key to many molecular systems, ranging from responsive materials to molecular machines. Here we report a molecular motor that forms bowl-shaped particles in water and how confinement of the molecular motor affects rotary motion. Studying the aggregation process in a broader context, we provide evidence that, in the case of bowl-shaped particles, the structures are not the product of self-assembly, but a direct result of the mixing a good solvent and a (partial) non-solvent and highly independent of the molecular design. Under the influence of the non-solvent, droplets are formed, of which the exterior is hardened due to the increase in the glass transition temperature by the external medium, while the interior of the droplets remains plasticized by the solvent, resulting in the formation of stable bowl-shaped particles with a fluid interior, a glass-like exterior, and a very specific shape: dense spheres with a hole in their side. Applying this to a bulky first-generation molecular motor allowed us to change its isomerization behavior. Furthermore, the motor shows in situ photo-switchable aggregation-induced emission. Strong confinement prohibits the thermal helix inversion step while altering the energy barriers that determine the rotary motion, such that it introduces a reverse trans- cis isomerization upon heating. These studies show a remarkable control of forward and backward rotary motion by simply changing solvent ratios and extent of confinement.

Organization of Plant Photosystem II and Photosystem I Supercomplexes.

In nature, plants are continuously exposed to varying environmental conditions. They have developed a wide range of adaptive mechanisms, which ensure their survival and maintenance of stable photosynthetic performance. Photosynthesis is delicately regulated at the level of the thylakoid membrane of chloroplasts and the regulatory mechanisms include a reversible formation of a large variety of specific protein-protein complexes, supercomplexes or even larger assemblies known as megacomplexes. Revealing their structures is crucial for better understanding of their function and relevance in photosynthesis. Here we focus our attention on the isolation and a structural characterization of various large protein supercomplexes and megacomplexes, which involve Photosystem II and Photosystem I, the key constituents of photosynthetic apparatus. The photosystems are often attached to other protein complexes in thylakoid membranes such as light harvesting complexes, cytochrome b 6 f complex, and NAD(P)H dehydrogenase. Structural models of individual supercomplexes and megacomplexes provide essential details of their architecture, which allow us to discuss their function as well as physiological significance.

A general mechanism of ribosome dimerization revealed by single-particle cryo-electron microscopy.

Bacteria downregulate their ribosomal activity through dimerization of 70S ribosomes, yielding inactive 100S complexes. In Escherichia coli, dimerization is mediated by the hibernation promotion factor (HPF) and ribosome modulation factor. Here we report the cryo-electron microscopy study on 100S ribosomes from Lactococcus lactis and a dimerization mechanism involving a single protein: HPFlong. The N-terminal domain of HPFlong binds at the same site as HPF in Escherichia coli 100S ribosomes. Contrary to ribosome modulation factor, the C-terminal domain of HPFlong binds exactly at the dimer interface. Furthermore, ribosomes from Lactococcus lactis do not undergo conformational changes in the 30S head domains upon binding of HPFlong, and the Shine-Dalgarno sequence and mRNA entrance tunnel remain accessible. Ribosome activity is blocked by HPFlong due to the inhibition of mRNA recognition by the platform binding center. Phylogenetic analysis of HPF proteins suggests that HPFlong-mediated dimerization is a widespread mechanism of ribosome hibernation in bacteria.When bacteria enter the stationary growth phase, protein translation is suppressed via the dimerization of 70S ribosomes into inactive complexes. Here the authors provide a structural basis for how the dual domain hibernation promotion factor promotes ribosome dimerization and hibernation in bacteria.

Subunit and chlorophyll organization of the plant photosystem II supercomplex.

Photosystem II (PSII) is a light-driven protein, involved in the primary reactions of photosynthesis. In plant photosynthetic membranes PSII forms large multisubunit supercomplexes, containing a dimeric core and up to four light-harvesting complexes (LHCs), which act as antenna proteins. Here we solved a three-dimensional (3D) structure of the C2S2M2 supercomplex from Arabidopsis thaliana using cryo-transmission electron microscopy (cryo-EM) and single-particle analysis at an overall resolution of 5.3 Å. Using a combination of homology modelling and restrained refinement against the cryo-EM map, it was possible to model atomic structures for all antenna complexes and almost all core subunits. We located all 35 chlorophylls of the core region based on the cyanobacterial PSII structure, whose positioning is highly conserved, as well as all the chlorophylls of the LHCII S and M trimers. A total of 13 and 9 chlorophylls were identified in CP26 and CP24, respectively. Energy flow from LHC complexes to the PSII reaction centre is proposed to follow preferential pathways: CP26 and CP29 directly transfer to the core using several routes for efficient transfer; the S trimer is directly connected to CP43 and the M trimer can efficiently transfer energy to the core through CP29 and the S trimer.

Transmission Electron Microscopy as a Tool for the Characterization of Soft Materials: Application and Interpretation.

Transmission electron microscopy (TEM) provides direct structural information on nano-structured materials and is popular as a characterization tool in soft matter and supramolecular chemistry. However, technical aspects of sample preparation are overlooked and erroneous image interpretations are regularly encountered in the literature. There are three most commonly used TEM methods as we derived from literature: drying, staining and cryo-TEM, which are explained here with respect to their application, limitations and interpretation. Since soft matter chemistry relies on a lot of indirect evidence, the role of TEM for the correct evaluation of the nature of an assembly is very large. Mistakes in application and interpretation can therefore have enormous impact on the quality of present and future studies. We provide helpful background information of these three techniques, the information that can and cannot be derived from them and provide assistance in selecting the right technique for soft matter imaging. This essay warns against the use of drying and explains why. In general cryo-TEM is by far the best suited method and many mistakes and over-interpretations can be avoided by the use of this technique.

Interaction between the photoprotective protein LHCSR3 and C2S2 Photosystem II supercomplex in Chlamydomonas reinhardtii.

Photosynthetic organisms can thermally dissipate excess of absorbed energy in high-light conditions in a process known as non-photochemical quenching (NPQ). In the green alga Chlamydomonas reinhardtii this process depends on the presence of the light-harvesting protein LHCSR3, which is only expressed in high light. LHCSR3 has been shown to act as a quencher when associated with the Photosystem II supercomplex and to respond to pH changes, but the mechanism of quenching has not been elucidated yet. In this work we have studied the interaction between LHCSR3 and Photosystem II C2S2 supercomplexes by single particle electron microscopy. It was found that LHCSR3 predominantly binds at three different positions and that the CP26 subunit and the LHCII trimer of C2S2 supercomplexes are involved in binding, while we could not find evidences for a direct association of LHCSR3 with the PSII core. At all three locations LHCSR3 is present almost exclusively as a dimer.

Atypical composition and structure of the mitochondrial dimeric ATP synthase from Euglena gracilis.

Mitochondrial respiratory-chain complexes from Euglenozoa comprise classical subunits described in other eukaryotes (i.e. mammals and fungi) and subunits that are restricted to Euglenozoa (e.g. Euglena gracilis and Trypanosoma brucei). Here we studied the mitochondrial F1FO-ATP synthase (or Complex V) from the photosynthetic eukaryote E. gracilis in detail. The enzyme was purified by a two-step chromatographic procedure and its subunit composition was resolved by a three-dimensional gel electrophoresis (BN/SDS/SDS). Twenty-two different subunits were identified by mass-spectrometry analyses among which the canonical α, ß, γ, δ, ε, and OSCP subunits, and at least seven subunits previously found in Trypanosoma. The ADP/ATP carrier was also associated to the ATP synthase into a dimeric ATP synthasome. Single-particle analysis by transmission electron microscopy of the dimeric ATP synthase indicated that the structures of both the catalytic and central rotor parts are conserved while other structural features are original. These new features include a large membrane-spanning region joining the monomers, an external peripheral stalk and a structure that goes through the membrane and reaches the inter membrane space below the c-ring, the latter having not been reported for any mitochondrial F-ATPase.

Conservation of core complex subunits shaped the structure and function of photosystem I in the secondary endosymbiont alga Nannochloropsis gaditana.

Photosystem I (PSI) is a pigment protein complex catalyzing the light-driven electron transport from plastocyanin to ferredoxin in oxygenic photosynthetic organisms. Several PSI subunits are highly conserved in cyanobacteria, algae and plants, whereas others are distributed differentially in the various organisms. Here we characterized the structural and functional properties of PSI purified from the heterokont alga Nannochloropsis gaditana, showing that it is organized as a supercomplex including a core complex and an outer antenna, as in plants and other eukaryotic algae. Differently from all known organisms, the N. gaditana PSI supercomplex contains five peripheral antenna proteins, identified by proteome analysis as type-R light-harvesting complexes (LHCr4-8). Two antenna subunits are bound in a conserved position, as in PSI in plants, whereas three additional antennae are associated with the core on the other side. This peculiar antenna association correlates with the presence of PsaF/J and the absence of PsaH, G and K in the N. gaditana genome and proteome. Excitation energy transfer in the supercomplex is highly efficient, leading to a very high trapping efficiency as observed in all other PSI eukaryotes, showing that although the supramolecular organization of PSI changed during evolution, fundamental functional properties such as trapping efficiency were maintained.

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.

Supercomplexes of plant photosystem I with cytochrome b6f, light-harvesting complex II and NDH.

Photosystem I (PSI) is a pigment-protein complex required for the light-dependent reactions of photosynthesis and participates in light-harvesting and redox-driven chloroplast metabolism. Assembly of PSI into supercomplexes with light harvesting complex (LHC) II, cytochrome b6f (Cytb6f) or NAD(P)H dehydrogenase complex (NDH) has been proposed as a means for regulating photosynthesis. However, structural details about the binding positions in plant PSI are lacking. We analyzed large data sets of electron microscopy single particle projections of supercomplexes obtained from the stroma membrane of Arabidopsis thaliana. By single particle analysis, we established the binding position of Cytb6f at the antenna side of PSI. The rectangular-shaped Cytb6f dimer binds at the side where Lhca1 is located. The complex binds with its short side rather than its long side to PSI, which may explain why these supercomplexes are difficult to purify and easily disrupted. Refined analysis of the interaction between PSI and the NDH complex indicates that in total up to 6 copies of PSI can arrange with one NDH complex. Most PSI-NDH supercomplexes appeared to have 1-3 PSI copies associated. Finally, the PSI-LHCII supercomplex was found to bind an additional LHCII trimer at two positions on the LHCI side in Arabidopsis. The organization of PSI, either in a complex with NDH or with Cytb6f, may improve regulation of electron transport by the control of binding partners and distances in small domains.

Cryo-EM structure of a tetrameric cyanobacterial photosystem I complex reveals novel subunit interactions.

Photosystem I (PSI) of the thermophilic cyanobacterium Chroococcidiopsis sp. TS-821 (TS-821) forms tetramers Li et al. (2014). Two-dimensional maps obtained by single particle electron microscopy (EM) clearly show that the tetramer lacks four-fold symmetry and is actually composed of a dimer of dimers with C2 symmetry. The resolution of these negative stain 2D maps did not permit the placement of most of the small PSI subunits, except for PsaL. Therefore cryo-EM was used for 3D reconstruction of the PSI tetramer complex. A 3D model at ~11.5Å resolution was obtained and a 2D map within the membrane plane of ~6.1Å. This data was used to build a model that was compared with the high-resolution structure of the PSI of Thermosynechococcus elongatus (T. elongatus) at 2.5Å. This comparison reveals key differences in which subunits are involved in the two different interfaces, interface type 1 within a dimer and interface type 2 between dimers. The type 1 interface in TS-821 is similar to the monomer interface in the trimeric PSI from T. elongatus, with interactions between subunits PsaA, -B, -I, -L and M. In type 2 the interaction is only between PsaA, -B and -L. Unlike the trimeric PSI, the central cavity of the complex is not filled with the PsaL-derived helical bundle, but instead seems filled with lipids. The physiological or evolutionary advantage of the tetramer is unknown. However, the presence of both dimers and tetramers in the thylakoid membrane suggest a dynamic equilibrium that shifts towards the tetramers in high light.

Evolutionary loss of light-harvesting proteins Lhcb6 and Lhcb3 in major land plant groups--break-up of current dogma.

Photosynthesis in plants and algae relies on the coordinated function of photosystems (PS) I and II. Their efficiency is augmented by finely-tuned light-harvesting proteins (Lhcs) connected to them. The most recent Lhcs (in evolutionary terms), Lhcb6 and Lhcb3, evolved during the transition of plants from water to land and have so far been considered to be an essential characteristic of land plants. We used single particle electron microscopy and sequence analysis to study architecture and composition of PSII supercomplex from Norway spruce and related species. We have found that there are major land plant families that lack functional lhcb6 and lhcb3 genes, which notably changes the organization of PSII supercomplexes. The Lhcb6 and Lhcb3 proteins have been lost in the gymnosperm genera Picea and Pinus (family Pinaceae) and Gnetum (Gnetales). We also revealed that the absence of these proteins in Norway spruce modifies the PSII supercomplex in such a way that it resembles its counterpart in the alga Chlamydomonas reinhardtii, an evolutionarily older organism. Our results break a deep-rooted concept of Lhcb6 and Lhcb3 proteins being the essential characteristic of land plants, and beg the question of what the evolutionary benefit of their loss could be.