Plant Thylakoid Membranes Research Articles

Sublocalization of Cytochrome b6f Complexes in Photosynthetic Membranes.

It is well established that the majority of energy-converting photosynthetic protein complexes in plant thylakoid membrane are nonhomogenously distributed between stacked and unstacked membrane regions. Yet, the sublocalization of the central cytochrome b6f complex remains controversial. We present a structural model that explains the variation in cytochrome b6f sublocalization data. Small changes in the distance between adjacent membranes in stacked grana regions either allow or restrict access of cytochrome b6f complexes to grana. If the width of the gap falls below a certain threshold, then the steric hindrance prevents cytochrome b6f access to grana. Evidence is presented that the width of stromal gap is variable, demonstrating that the postulated mechanism can regulate the lateral distribution of the cytochrome b6f complexes.

Brassinolide alleviates the drought-induced adverse effects in barley by modulation of enzymatic antioxidants and ultrastructure

In the present study, possible role of exogenously applied brassinolide (BL) in alleviating the detrimental effects of polyethylene glycol (PEG)-inducted drought stress in barley was evaluated. Barley seedlings were subjected to two PEG levels (0 and 8% w/v) for 2 weeks followed by a foliar spray of BL (0, 0.01, 0.1 and 1 µM) to assess the changes in growth, gas exchange, chlorophyll contents, antioxidant enzyme activities and ultrastructural changes. PEG-induced drought considerably inhibited the barley growth in terms of shoot and root length, shoot, root fresh and dry weights, and chlorophyll contents. Moreover, extensive decrease by PEG-induced drought in gas exchange attributes i.e. net photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO2 were also recorded. However, exogenous application of BL remarkably improved the gas exchange attributes, chlorophyll contents and plant growth under either drought or control condition. Furthermore, foliar spray of BL decreased the production of malondialdehyde (MDA) and reactive oxygen species (ROS) in the leaves and roots by increasing enzymatic antioxidant activities under drought stress. The microscopic examination indicated that exogenous application of BL improved the cell structure and enabled a clean mesophyll cell having a well-developed chloroplast with thylakoid membranes of the drought stressed plants. On the basis of these findings, it can be concluded that exogenous application of BL could alleviate drought stress to barley by ameliorating photosynthetic parameters, improving antioxidants enzyme activities, reducing ultrastructural changes in leaf mesophyll and finally maintaining the plant growth.

Structure of the plant photosystem I supercomplex at 2.6 Å resolution.

Four elaborate membrane complexes carry out the light reaction of oxygenic photosynthesis. Photosystem I (PSI) is one of two large reaction centres responsible for converting light photons into the chemical energy needed to sustain life. In the thylakoid membranes of plants, PSI is found together with its integral light-harvesting antenna, light-harvesting complex I (LHCI), in a membrane supercomplex containing hundreds of light-harvesting pigments. Here, we report the crystal structure of plant PSI-LHCI at 2.6 Å resolution. The structure reveals the configuration of PsaK, a core subunit important for state transitions in plants, a conserved network of water molecules surrounding the electron transfer centres and an elaborate structure of lipids bridging PSI and its LHCI antenna. We discuss the implications of the structure for energy transfer and the evolution of PSI.

Low-pH induced reversible reorganizations of chloroplast thylakoid membranes — As revealed by small-angle neutron scattering

Energization of thylakoid membranes brings about the acidification of the lumenal aqueous phase, which activates important regulatory mechanisms. Earlier Jajoo and coworkers (2014 FEBS Lett. 588:970) have shown that low pH in isolated plant thylakoid membranes induces changes in the excitation energy distribution between the two photosystems. In order to elucidate the structural background of these changes, we used small-angle neutron scattering on thylakoid membranes exposed to low p2H (pD) and show that gradually lowering the p2H from 8.0 to 5.0 causes small but well discernible reversible diminishment of the periodic order and the lamellar repeat distance and an increased mosaicity — similar to the effects elicited by light-induced acidification of the lumen. Our data strongly suggest that thylakoids dynamically respond to the membrane energization and actively participate in different regulatory mechanisms.

Photosynthetic water splitting by the Mn4Ca2+OX catalyst of photosystem II: its structure, robustness and mechanism.

The biological energy cycle of our planet is driven by photosynthesis whereby sunlight is absorbed by chlorophyll and other accessory pigments. The excitation energy is then efficiently transferred to a reaction centre where charge separation occurs in a few picoseconds. In the case of photosystem II (PSII), the energy of the charge transfer state is used to split water into oxygen and reducing equivalents. This is accomplished by the relatively low energy content of four photons of visible light. PSII is a large multi-subunit membrane protein complex embedded in the lipid environment of the thylakoid membranes of plants, algae and cyanobacteria. Four high energy electrons, together with four protons (4H+), are used to reduce plastoquinone (PQ), the terminal electron acceptor of PSII, to plastoquinol (PQH2). PQH2 passes its reducing equivalents to an electron transfer chain which feeds into photosystem I (PSI) where they gain additional reducing potential from a second light reaction which is necessary to drive CO2 reduction. The catalytic centre of PSII consists of a cluster of four Mn ions and a Ca2+ linked by oxo bonds. In addition, there are seven amino acid ligands. In this Article, I discuss the structure of this metal cluster, its stability and the probability that an acid-base (nucleophilic-electrophilic) mechanism catalyses the water splitting reaction on the surface of the metal-cluster. Evidence for this mechanism is presented from studies on water splitting catalysts consisting of organo-complexes of ruthenium and manganese and also by comparison with the enzymology of carbon monoxide dehydrogenase (CODH). Finally the relevance of our understanding of PSII is discussed in terms of artificial photosynthesis with emphasis on inorganic water splitting catalysts as oxygen generating photoelectrodes.

Direct isolation of a functional violaxanthin cycle domain from thylakoid membranes of higher plants.

A special domain of the thylakoid membrane of higher plants has been isolated which carries out the de-epoxidation of the xanthophyll cycle pigment violaxanthin to zeaxanthin. Recent models indicate that in the chloroplast of higher plants, the violaxanthin (V) cycle takes place within specialized domains in the thylakoid membrane. Here, we describe a new procedure to directly isolate such a domain in functional state. The procedure consists of a thylakoid membrane isolation at a pH value of 5.2 which realizes the binding of the enzyme V de-epoxidase (VDE) to the membrane throughout the preparation process. Isolated thylakoid membranes are then solubilized with the very mild detergent n-dodecyl α-D-maltoside and the pigment-protein complexes are separated by sucrose gradient ultracentrifugation. The upper main fraction of the sucrose gradient represents a V cycle domain which consists of the major light-harvesting complex of photosystem II (LHCII), a special lipid composition with an enrichment of the galactolipid monogalactosyldiacylglycerol (MGDG) and the VDE. The domain is isolated in functional state as evidenced by the ability to convert the LHCII-associated V to zeaxanthin. The direct isolation of a V cycle domain proves the most important hypotheses concerning the de-epoxidation reaction in intact thylakoid membranes. It shows that the VDE binds to the thylakoid membrane at low pH values of the thylakoid lumen, that it binds to membrane regions enriched in LHCII, and that the domain contains high amounts of MGDG. The last point is in line with the importance of the galactolipid for V solubilisation and, by providing inverted hexagonal lipid structures, for VDE activity.

Fingerprinting the macro-organisation of pigment–protein complexes in plant thylakoid membranes in vivo by circular-dichroism spectroscopy

Macro-organisation of the protein complexes in plant thylakoid membranes plays important roles in the regulation and fine-tuning of photosynthetic activity. These delicate structures might, however, undergo substantial changes during isolating the thylakoid membranes or during sample preparations, e.g., for electron microscopy. Circular-dichroism (CD) spectroscopy is a non-invasive technique which can thus be used on intact samples. Via excitonic and psi-type CD bands, respectively, it carries information on short-range excitonic pigment–pigment interactions and the macro-organisation (chiral macrodomains) of pigment–protein complexes (psi, polymer or salt-induced). In order to obtain more specific information on the origin of the major psi-type CD bands, at around (+)506, (−)674 and (+)690nm, we fingerprinted detached leaves and isolated thylakoid membranes of wild-type and mutant plants and also tested the effects of different environmental conditions in vivo. We show that (i) the chiral macrodomains disassemble upon mild detergent treatments, but not after crosslinking the protein complexes; (ii) in different wild-type leaves of dicotyledonous and monocotyledonous angiosperms the CD features are quite robust, displaying very similar excitonic and psi-type bands, suggesting similar protein composition and (macro-) organisation of photosystem II (PSII) supercomplexes in the grana; (iii) the main positive psi-type bands depend on light-harvesting protein II contents of the membranes; (iv) the (+)506nm band appears only in the presence of PSII–LHCII supercomplexes and does not depend on the xanthophyll composition of the membranes. Hence, CD spectroscopy can be used to detect different macro-domains in the thylakoid membranes with different outer antenna compositions in vivo.

Excitation energy transfer between Light-harvesting complex II and Photosystem I in reconstituted membranes

Light-harvesting complex II (LHCII), the major peripheral antenna of Photosystem II in plants, participates in several concerted mechanisms for regulation of the excitation energy and electron fluxes in thylakoid membranes. In part, these include interaction of LHCII with Photosystem I (PSI) enhancing the latter's absorption cross-section – for example in the well-known state 1 – state 2 transitions or as a long-term acclimation to high light. In this work we examined the capability of LHCII to deliver excitations to PSI in reconstituted membranes in vitro. Proteoliposomes with native plant thylakoid membrane lipids and different stoichiometric ratios of LHCII:PSI were reconstituted and studied by steady-state and time-resolved fluorescence spectroscopy. Fluorescence emission from LHCII was strongly decreased in PSI–LHCII membranes due to trapping of excitations by PSI. Kinetic modelling of the time-resolved fluorescence data revealed the existence of separate pools of LHCII distinguished by the time scale of energy transfer. A strongly coupled pool, equivalent to one LHCII trimer per PSI, transferred excitations to PSI with near-unity efficiency on a time scale of less than 10ps but extra LHCIIs also contributed significantly to the effective antenna size of PSI, which could be increased by up to 47% in membranes containing 3 LHCII trimers per PSI. The results demonstrate a remarkable competence of LHCII to increase the absorption cross-section of PSI, given the opportunity that the two types of complexes interact in the membrane.

'Photosystem II: the water splitting enzyme of photosynthesis and the origin of oxygen in our atmosphere'.

About 3 billion years ago an enzyme emerged which would dramatically change the chemical composition of our planet and set in motion an unprecedented explosion in biological activity. This enzyme used solar energy to power the thermodynamically and chemically demanding reaction of water splitting. In so doing it provided biology with an unlimited supply of reducing equivalents needed to convert carbon dioxide into the organic molecules of life while at the same time produced oxygen to transform our planetary atmosphere from an anaerobic to an aerobic state. The enzyme which facilitates this reaction and therefore underpins virtually all life on our planet is known as Photosystem II (PSII). It is a pigment-binding, multisubunit protein complex embedded in the lipid environment of the thylakoid membranes of plants, algae and cyanobacteria. Today we have detailed understanding of the structure and functioning of this key and unique enzyme. The journey to this level of knowledge can be traced back to the discovery of oxygen itself in the 18th-century. Since then there has been a sequence of mile stone discoveries which makes a fascinating story, stretching over 200 years. But it is the last few years that have provided the level of detail necessary to reveal the chemistry of water oxidation and O-O bond formation. In particular, the crystal structure of the isolated PSII enzyme has been reported with ever increasing improvement in resolution. Thus the organisational and structural details of its many subunits and cofactors are now well understood. The water splitting site was revealed as a cluster of four Mn ions and a Ca ion surrounded by amino-acid side chains, of which seven provide direct ligands to the metals. The metal cluster is organised as a cubane structure composed of three Mn ions and a Ca2+ linked by oxo-bonds with the fourth Mn ion attached to the cubane. This structure has now been synthesised in a non-protein environment suggesting that it is a totally inorganic precursor for the evolution of the photosynthetic oxygen-evolving complex. In summary, the overall structure of the catalytic site has given a framework on which to build a mechanistic scheme for photosynthetic dioxygen generation and at the same time provide a blue-print and incentive to develop catalysts for artificial photo-electrochemical systems to split water and generate renewable solar fuels.

Analysis of photosystem II biogenesis in cyanobacteria

Photosystem II (PSII), a large multisubunit membrane protein complex found in the thylakoid membranes of cyanobacteria, algae and plants, catalyzes light-driven oxygen evolution from water and reduction of plastoquinone. Biogenesis of PSII requires coordinated assembly of at least 20 protein subunits, as well as incorporation of various organic and inorganic cofactors. The stepwise assembly process is facilitated by numerous protein factors that have been identified in recent years. Further analysis of this process requires the development or refinement of specific methods for the identification of novel assembly factors and, in particular, elucidation of the unique role of each. Here we summarize current knowledge of PSII biogenesis in cyanobacteria, focusing primarily on the impact of methodological advances and innovations. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux.

The TatC component of the twin-arginine protein translocase functions as an obligate oligomer.

SummaryThe Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in E scherichia coli is composed of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate‐bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild‐type tatC. Single substitutions that confer dominant negative TatC activity were localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue‐native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild‐type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate‐induced disulphide crosslink in transmembrane helix 5 of TatC. Our findings show that TatC functions as an obligate oligomer.

Preparation of Thylakoid/Polyaniline/Reduced Graphene Oxide/Glassy Carbon Integrated System and Photocurrent Enhancement

Solar energy is enormous, inexhaustible and environmentally friendly. Photosynthesis is a highly developed system that converts solar energy into chemical energy, taking place in thylakoid membrane of plants, algae and cyanobacteria. In the light reaction, solar energy is absorbed by light-harvesting complexes (LHC) containing antenna pigments. Absorbed solar energy is transferred to the reaction center, where the water photolysis into oxygen, protons and electrons occurs by oxygen evolving complex (OEC) attached to photosystem II (PSII). The generated electrons are transferred along the electron transfer pathway called Z-scheme and eventually stored in the form of NADPH. Since quantum yields of photosystem I (PSI) and photosystem II are very high, numerous attempts have been made to integrate these photosynthetic units (PSUs) into photoelectrochemical cells to directly convert light into electricity. Rather than using PSI or PSII, here we present our preliminary results on light energy conversion with thylakoid membrane that is installed on the electrode surface. Thylakoid membrane was isolated from spinach whose oxygen evolving activity was measured by a Clark-type oxygen sensor under the illumination. For the effective light energy conversion, we have developed an integrated system containing thylakoid multilayers on the glassy carbon (GC) electrode. First, 4-aminophenyl (4-AP)-modified GC surfaces were prepared. Isolated thylakoid was mixed with polyaniline (PANI) and graphene oxide (GO), and this composite material was applied onto the 4-AP-modified GC. Since GO has poor electric conductivity, we electrochemically reduced GO to highly conductive reduced graphene oxide (RGO) form. Thus prepared integrated system is denoted as Thyl/PANI/RGO/GC. This integrated system was proven effective in solar energy conversion. To achieve high photocurrent, we prepared the same system with different GO/chlorophyll ratios. After cycling potential to reduce GO to RGO, photocurrent was measured. The ratio of 3:1 (GO:chlorophyll) turned out to give highest photocurrent. The electron transfer pathways have been identified from the blocking experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of QB site in PSII. Thylakoid suspension was treated with DCMU before immobilization and then applied to the electrode. Virtually no photocurrent enhancement was observed proving that the electron transfer was blocked. A complete solar cell composed of Thyl/PANI/RGO/GC anode and Pt/C air cathode was constructed. From the polarization curve, the maximum power density of 10.5 μW/cm2 with current density of 24.7 μA/cm2 under one sun illumination was obtained. The turnover frequency was calculated to be 5.8 water molecule oxidation per PSII unit per second, indicating that the immobilized thylakoid in the form of the multilayer films maintained its activity. This result shows that larger number of thylakoid units are needed for enhanced photocurrent and our method of integrating thylakoid using polyaniline and graphene oxide could be a useful way to this end.

Photosynthetic Membranes of Synechocystis or Plants Convert Sunlight to Photocurrent through Different Pathways due to Different Architectures.

Thylakoid membranes contain the redox active complexes catalyzing the light-dependent reactions of photosynthesis in cyanobacteria, algae and plants. Crude thylakoid membranes or purified photosystems from different organisms have previously been utilized for generation of electrical power and/or fuels. Here we investigate the electron transferability from thylakoid preparations from plants or the cyanobacterium Synechocystis. We show that upon illumination, crude Synechocystis thylakoids can reduce cytochrome c. In addition, this crude preparation can transfer electrons to a graphite electrode, producing an unmediated photocurrent of 15 μA/cm2. Photocurrent could be obtained in the presence of the PSII inhibitor DCMU, indicating that the source of electrons is QA, the primary Photosystem II acceptor. In contrast, thylakoids purified from plants could not reduce cyt c, nor produced a photocurrent in the photocell in the presence of DCMU. The production of significant photocurrent (100 μA/cm2) from plant thylakoids required the addition of the soluble electron mediator DCBQ. Furthermore, we demonstrate that use of crude thylakoids from the D1-K238E mutant in Synechocystis resulted in improved electron transferability, increasing the direct photocurrent to 35 μA/cm2. Applying the analogous mutation to tobacco plants did not achieve an equivalent effect. While electron abstraction from crude thylakoids of cyanobacteria or plants is feasible, we conclude that the site of the abstraction of the electrons from the thylakoids, the architecture of the thylakoid preparations influence the site of the electron abstraction, as well as the transfer pathway to the electrode. This dictates the use of different strategies for production of sustainable electrical current from photosynthetic thylakoid membranes of cyanobacteria or higher plants.

Pigment Interactions in Light-harvesting Complex II in Different Molecular Environments

Extraction of plant light-harvesting complex II (LHCII) from the native thylakoid membrane or from aggregates by the use of surfactants brings about significant changes in the excitonic circular dichroism (CD) spectrum and fluorescence quantum yield. To elucidate the cause of these changes, e.g. trimer-trimer contacts or surfactant-induced structural perturbations, we compared the CD spectra and fluorescence kinetics of LHCII aggregates, artificial and native LHCII-lipid membranes, and LHCII solubilized in different detergents or trapped in polymer gel. By this means we were able to identify CD spectral changes specific to LHCII-LHCII interactions, at (-)-437 and (+)-484 nm, and changes specific to the interaction with the detergent n-dodecyl-β-maltoside (β-DM) or membrane lipids, at (+)-447 and (-)-494 nm. The latter change is attributed to the conformational change of the LHCII-bound carotenoid neoxanthin, by analyzing the CD spectra of neoxanthin-deficient plant thylakoid membranes. The neoxanthin-specific band at (-)-494 nm was not pronounced in LHCII in detergent-free gels or solubilized in the α isomer of DM but was present when LHCII was reconstituted in membranes composed of phosphatidylcholine or plant thylakoid lipids, indicating that the conformation of neoxanthin is sensitive to the molecular environment. Neither the aggregation-specific CD bands, nor the surfactant-specific bands were positively associated with the onset of fluorescence quenching, which could be triggered without invoking such spectral changes. Significant quenching was not active in reconstituted LHCII proteoliposomes, whereas a high degree of energetic connectivity, depending on the lipid:protein ratio, in these membranes allows for efficient light harvesting.

Protein translocation without specific quality control in a computational model of the Tat system

The twin-arginine translocation (Tat) system transports folded proteins of various sizes across both bacterial and plant thylakoid membranes. The membrane-associated TatA protein is an essential component of the Tat translocon, and a broad distribution of different sized TatA-clusters is observed in bacterial membranes. We assume that the size dynamics of TatA clusters are affected by substrate binding, unbinding, and translocation to associated TatBC clusters, where clusters with bound translocation substrates favour growth and those without associated substrates favour shrinkage. With a stochastic model of substrate binding and cluster dynamics, we numerically determine the TatA cluster size distribution. We include a proportion of targeted but non-translocatable (NT) substrates, with the simplifying hypothesis that the substrate translocatability does not directly affect cluster dynamical rate constants or substrate binding or unbinding rates. This amounts to a translocation model without specific quality control. Nevertheless, NT substrates will remain associated with TatA clusters until unbound and so will affect cluster sizes and translocation rates. We find that the number of larger TatA clusters depends on the NT fraction f. The translocation rate can be optimized by tuning the rate of spontaneous substrate unbinding, . We present an analytically solvable three-state model of substrate translocation without cluster size dynamics that follows our computed translocation rates, and that is consistent with in vitro Tat-translocation data in the presence of NT substrates.

A tandem mass spectrometric method for singlet oxygen measurement.

Singlet oxygen, a harmful reactive oxygen species, can be quantified with the substance 2,2,6,6-tetramethylpiperidine (TEMP) that reacts with singlet oxygen, forming a stable nitroxyl radical (TEMPO). TEMPO has earlier been quantified with electron paramagnetic resonance (EPR) spectroscopy. In this study, we designed an ultra-high-performance liquid chromatographic-tandem mass spectrometric (UHPLC-ESI-MS/MS) quantification method for TEMPO and showed that the method based on multiple reaction monitoring (MRM) can be used for the measurements of singlet oxygen from both nonbiological and biological samples. Results obtained with both UHPLC-ESI-MS/MS and EPR methods suggest that plant thylakoid membranes produce 3.7 × 10(-7) molecules of singlet oxygen per chlorophyll molecule in a second when illuminated with the photosynthetic photon flux density of 2000 μmol m(-2 ) s(-1).

Mass spectroscopy locates the extrinsic proteins of photosystem II

The photosynthetic oxidation of water is catalyzed by photosystem II (PSII), a multisubunit pigment-protein embedded in the thylakoid membranes of cyanobacteria, algae, and plants. This remarkable photoenzyme is capable of generating the highly oxidizing chemical species required to extract four tightly bound electrons of two substrate water-molecules, yielding biosynthetically useful reductant and by-product O2. PSII is a large homodimeric complex in vivo, with a combined mass of ∼700 kDa. Each PSII monomer comprises more than 20 different proteins collectively coordinating ∼60 cofactors, including 35 chlorophylls, 2 pheophytins, 2 plastoquinone molecules, and the Mn4Ca cluster, responsible for H2O-oxidation (1, 2). Despite much progress, including the 1.9 A crystal structure of cyanobacterial PSII (1, 3), crucial structural information is missing. This lack includes extrinsic proteins affecting the Mn4Ca cluster, not observed in the PSII crystal structure because they are lost during the purification and crystallization process. In PNAS, Liu et al. use complementary technical approaches to construct a model for the binding of one of the lost extrinsic proteins, PsbQ, to the PSII, complex, and in doing so provide an example for the solution of the more general structural problem of defining the 3D structure of separately crystallized proteins that assemble into larger macromolecular complexes (4).

Photosystem II: its function, structure, and implications for artificial photosynthesis.

Somewhere in the region of 3 billion years ago an enzyme emerged which would dramatically change the chemical composition of our planet and set in motion an unprecedented explosion in biological activity. This enzyme used solar energy to power the thermodynamically and chemically demanding reaction of water splitting. In so doing it provided biology with an unlimited supply of hydrogen equivalents needed to convert carbon dioxide into the organic molecules of life. The enzyme, which facilitates this reaction and therefore underpins virtually all life on our planet, is known as Photosystem II (PSII). It is a multisubunit enzyme embedded in the lipid environment of the thylakoid membranes of plants, algae, and cyanobacteria. Over the past 10 years, crystal structures of a 700kDa cyanobacterial dimeric PSII complex have been reported with ever increasing improvement in resolution with the latest being at 1.9 Å. Thus the organizational and structural details of its many subunits and cofactors are now well understood. The water splitting site was revealed as a cluster of four Mn ions and a Ca ion surrounded by amino acid side chains, of which seven provide ligands to the metals. The metal cluster is organized as a cubane-like structure composed of three Mn ions and the Ca2+ linked by oxo-bonds with the fourth Mn attached to the cubane via one of its bridging oxygens together with another oxo bridge to a Mn ion of the cubane. The overall structure of the catalytic site is providing a framework on which to develop a mechanistic scheme for the water splitting process and gives a blue print and confidence for the development of catalysts for mimicking the reaction in an artificial photo-electrochemical system to generate solar fuels.

Monitoring thylakoid ultrastructural changes in vivo using small-angle neutron scattering

The light reactions of oxygenic photosynthesis take place in the thylakoid membranes, flattened vesicles, which contain the two photosystems and also embed the cytochrome b6f complex and the ATP synthase. In general, the thylakoid membranes are assembled into multilamellar membrane systems, which warrant an optimal light capturing efficiency. In nature, they show astounding variations, primarily due to large variations in their protein composition, which is controlled by multilevel regulatory mechanisms during long-term acclimation and short-term adaptation processes and also influenced by biotic or abiotic stresses – indicating a substantial degree of flexibility in the membrane ultrastructure. The better understanding of the dynamic features of this membrane system requires the use of non-invasive techniques, such as small angle neutron scattering (SANS), which is capable of providing accurate, statistically and spatially averaged information on the repeat distances of periodically organized thylakoid membranes under physiologically relevant conditions with time resolutions of seconds and minutes. In this review, after a short section on the basic properties of neutrons, we outline the fundamental principles of SANS measurements, its strengths and weaknesses in comparison to complementary structure investigation techniques. Then we overview recent results on isolated plant thylakoid membranes, and on living cyanobacterial and algal cells as well as on whole leaves. Special attention is paid to light-induced reversible ultrastructural changes in vivo, which, in cyanobacterial and diatom cells, were uncovered with the aid of SANS measurements; we also discuss the role of membrane reorganizations in light adaptation and photoprotection mechanisms.

The ultrastructure and flexibility of thylakoid membranes in leaves and isolated chloroplasts as revealed by small-angle neutron scattering

We studied the periodicity of the multilamellar membrane system of granal chloroplasts in different isolated plant thylakoid membranes, using different suspension media, as well as on different detached leaves and isolated protoplasts—using small-angle neutron scattering. Freshly isolated thylakoid membranes suspended in isotonic or hypertonic media, containing sorbitol supplemented with cations, displayed Bragg peaks typically between 0.019 and 0.023Å−1, corresponding to spatially and statistically averaged repeat distance values of about 275–330Å. Similar data obtained earlier led us in previous work to propose an origin from the periodicity of stroma thylakoid membranes. However, detached leaves, of eleven different species, infiltrated with or soaked in D2O in dim laboratory light or transpired with D2O prior to measurements, exhibited considerably smaller repeat distances, typically between 210 and 230Å, ruling out a stromal membrane origin. Similar values were obtained on isolated tobacco and spinach protoplasts. When NaCl was used as osmoticum, the Bragg peaks of isolated thylakoid membranes almost coincided with those in the same batch of leaves and the repeat distances were very close to the electron microscopically determined values in the grana. Although neutron scattering and electron microscopy yield somewhat different values, which is not fully understood, we can conclude that small-angle neutron scattering is a suitable technique to study the periodic organization of granal thylakoid membranes in intact leaves under physiological conditions and with a time resolution of minutes or shorter. We also show here, for the first time on leaves, that the periodicity of thylakoid membranes in situ responds dynamically to moderately strong illumination. This article is part of a Special Issue entitled: Photosynthesis research for sustainability: Keys to produce clean energy.