Biological and polymeric self-assembled hybrid systems: structure and properties of thylakoid/polyelectrolyte complexes

A novel hybrid system composed of biological components and synthetic polymer, thylakoid/polycation complex, has been formed and studied. Effects of complex formation on the structure, electrostatics and functioning of thylakoid membranes have been examined. Thylakoids from bean leaves were used to form complexes with polycation polyallylamine hydrochloride (PAAH) in two systems: (i) thylakoid/polycation complexes formed in an aqueous bulk phase, and (ii) immobilized thylakoid/polycation planar complexes. Immobilized on a solid substrate surface, thylakoid/polycation complexes were prepared using layer-by-layer stepwise alternate adsorption technique, i.e., via the sequential alternate adsorption of thylakoids and polycation molecules. The morphology of built up structures was investigated by scanning electron microscopy. Light-induced electron transport in chloroplasts was studied by the electron paramagnetic resonance (EPR) method. Spin probe technique was employed to study the structural and electrostatic characteristics of thylakoid membranes. We have found that efficiency of light-induced electron transport in thylakoid membranes and membrane structure were not changed noticeably by PAAH binding to thylakoids in a wide range of PAAH concentrations. The data obtained indicate the physiologically-soft character of polycation interactions with thylakoid membranes and demonstrate effectiveness of interfacial self-assembly approach to fabrication of complex planar functional nanostructures from biological components and synthetic polymers.     

Functional and topological aspects of pH-dependent regulation of electron and proton transport in chloroplasts in silico

In this work, we summarize results of computer simulation of electron and proton transport processes coupled to ATP synthesis in chloroplasts performed within the frames of a mathematical model developed as a system of differential equations for concentrations of electron carriers and hydrogen ion inside and outside the granal and stromal thylakoids. The model takes into account topological peculiarities and lateral heterogeneity of the chloroplast lamellar system. This allowed us to analyze the influence of restricted diffusion of protons inside small compartments of a chloroplast (e.g., in the narrow inter-thylakoid gap) on electron transport processes. The model adequately describes two modes of pH-dependent feedback control of electron transport associated with: (i) the acidification of the thylakoid lumen, which causes the slowing down of plastoquinol oxidation and stimulates an increase in dissipation of excess energy in PS2, and (ii) the alkalization of stroma, inducing the activation of the BBC (Bassham-Benson-Calvin) cycle and intensified consumption of ATP and NADPH. The influence of ATP on electron transport is mediated by modulation of the thylakoid membrane conductivity to protons through the ATP synthase complexes. We also analyze the contribution of alternative electron transport pathways to the maintenance of optimal balance between the energy donating and energy consuming stages of the light-induced photosynthetic processes.     

Oxygen reduction in chloroplast thylakoids results in production of hydrogen peroxide inside the membrane

Hydrogen peroxide production in isolated pea thylakoids was studied in the presence of cytochrome c to prevent disproportionation of superoxide radicals outside of the thylakoid membranes. The comparison of cytochrome c reduction with accompanying oxygen uptake revealed that hydrogen peroxide was produced within the thylakoid. The proportion of electrons from water oxidation participating in this hydrogen peroxide production increased with increasing light intensity, and at a light intensity of 630 μmol quanta m^− 2 s^− 1 it reached 60% of all electrons entering the electron transport chain. Neither the presence of a superoxide dismutase inhibitor, potassium cyanide or sodium azide, in the thylakoid suspension, nor unstacking of the thylakoids appreciably affected the partitioning of electrons to hydrogen peroxide production. Also, osmolarity-induced changes in the thylakoid lumen volume, as well as variation of the lumen pH induced by the presence of Gramicidin D, had negligible effects on such partitioning. The flow of electrons participating in lumen hydrogen peroxide production was found to be near 10% of the total electron flow from water. It is concluded that a considerable amount of hydrogen peroxide is generated inside thylakoid membranes, and a possible mechanism, as well as the significance, of this process are discussed.     

Cytochrome b 6 f complex: Dynamic molecular organization,function and acclimation

The cytochrome b ₆ f complex occupies a central position in photosynthetic electron transport and proton translocation by linking PS II to PS I in linear electron flow from water to NADP⁺, and around PS I for cyclic electron flow. Cytochrome b ₆ f complexes are uniquely located in three membrane domains: the appressed granal membranes, the non-appressed stroma thylakoids and end grana membranes, and also the non-appressed grana margins, in contrast to the marked lateral heterogeneity of the localization of all other thylakoid multiprotein complexes. In addition to its vital role in vectorial electron transfer and proton translocation across the membrane, cytochrome b ₆ f complex is also involved in the regulation of balanced light excitation energy distribution between the photosystems, since its redox state governs the activation of LHC II kinase (the kinase that phosphorylates the mobile peripheral fraction of the chlorophyll a/b-proteins of LHC II of PS II). Hence, cytochrome b ₆ f complex is the molecular link in the interactive co-regulation of light-harvesting and electron transfer.The importance of a highly dynamic, yet flexible organization of the thylakoid membranes of plants and green algae has been highlighted by the exciting discovery that a lateral reorganization of some cytochrome b ₆ f complexes occurs in the state transition mechanism both in vivo and in vitro (Vallon et al. 1991). The lateral redistribution of phosphorylated LHC II from stacked granal membrane regions is accompanied by a concomitant movement of some cytochrome b ₆ f complexes from the granal membranes out to the PS I-containing stroma thylakoids. Thus, the dynamic movement of cytochrome b ₆ f complex as a multiprotein complex is a molecular mechanism for short-term adaptation to changing light conditions. With the concept of different membrane domains for linear and cyclic electron flow gaining credence, it is thought that linear electron flow occurs in the granal compartments and cyclic electron flow is localised in the stroma thylakoids at non-limiting irradiances. It is postulated that dynamic lateral reversible redistribution of some cytochrome b ₆ f complexes are part of the molecular mechanism involved in the regulation of linear electron transfer (ATP and NADPH) and cyclic electron flow (ATP only). Finally, the molecular significance of the marked regulation of cytochrome b ₆ f complexes for long-term regulation and optimization of photosynthetic function under varying environmental conditions, particularly light acclimation, is discussed.Abbreviations Chl chlorophyll - cyt cytochrome - PS Photosystem     

The grana margins of plant thylakoid membranes

Plant thylakoid membranes contain three structurally distinct domains: the planar appressed membranes of the grana; the planar non-appressed stroma thylakoids; and the highly curved, non-appressed margins of the grana. Evidence is presented to suggest that the grana margins form a significant structural domain, which has hitherto been neglected. If indeed the grana margins contain some of the cytochrome b/f complex, photosystem (PS) I complex and ATP synthase, they form a third functional domain of the laterally heterogeneous continuous thylakoid membrane network. The consequences of grana margins containing complexes are explored with respect to linear electron transport under light-saturating and light-limiting conditions, non-cyclic vs cyclic photophorylation, and the regulation of light energy distribution to both PS I and PS II.     

Production of reactive oxygen species by photosystem II

Photosysthetic cleavage of water molecules to molecular oxygen is a crucial process for all aerobic life on the Earth. Light-driven oxidation of water occurs in photosystem II (PSII) — a pigment-protein complex embedded in the thylakoid membrane of plants, algae and cyanobacteria. Electron transport across the thylakoid membrane terminated by NADPH and ATP formation is inadvertently coupled with the formation of reactive oxygen species (ROS). Reactive oxygen species are mainly produced by photosystem I; however, under certain circumstances, PSII contributes to the overall formation of ROS in the thylakoid membrane. Under limitation of electron transport reaction between both photosystems, photoreduction of molecular oxygen by the reducing side of PSII generates a superoxide anion radical, its dismutation to hydrogen peroxide and the subsequent formation of a hydroxyl radical terminates the overall process of ROS formation on the PSII electron acceptor side. On the PSII electron donor side, partial or complete inhibition of enzymatic activity of the water-splitting manganese complex is coupled with incomplete oxidation of water to hydrogen peroxide. The review points out the mechanistic aspects in the production of ROS on both the electron acceptor and electron donor side of PSII.     

Cryoprotectin protects thylakoids during a freeze-thaw cycle by a mechanism involving stable membrane binding

Chloroplast thylakoid membranes of higher plants are damaged by freezing both in vivo and in vitro. The resulting inactivation of photosynthetic electron transport has been related to transient membrane rupture, leading to the loss of soluble electron transport proteins and osmotically active solutes from the thylakoid lumen. We have recently purified and sequenced a protein from cold acclimated cabbage, that protects thylakoids from this freeze-thaw damage. The protein belongs to the WAX9 family of nonspecific lipid transfer proteins, but has no detectable lipid transfer activity. Conversely, other transport-active lipid transfer proteins show no cryoprotective activity. We show here that cryoprotectin binds to thylakoid membranes. Both cryoprotective activity and membrane binding were inhibited in the presence of specific sugars, most effectively by Glc-6-S. The binding of cryoprotectin to thylakoids reduced the fluidity of the membrane lipids close to the membrane/solution interface, but not in the hydrophobic core region. Using immobilized liposomes we could show that cryoprotectin was able to bind to pure lipid membranes.     

Plastocyanin redox kinetics in spinach chloroplasts: evidence for disequilibrium in the high potential chain

Reduction kinetics of cytochrome f, plastocyanin (PC) and P₇₀₀ (‘high-potential chain’) in thylakoids from spinach were followed after pre-oxidation by a saturating light pulse. We describe a novel approach to follow PC redox kinetics from deconvolution of 810-860 nm absorption changes. The equilibration between the redox-components was analyzed by plotting the redox state of cytochrome f and PC against that of P₇₀₀. In thylakoids with (1) diminished electron transport rate (adjusted with a cytochrome bf inhibitor) or (2) de-stacked grana, cytochrome f and PC relaxed close to their thermodynamic equilibriums with P₇₀₀. In stacked thylakoids with non-inhibited electron transport, the equilibration plots were complex and non-hyperbolic, suggesting that during fast electron flux, the ‘high-potential chain’ does not homogeneously equilibrate throughout the membrane. Apparent equilibrium constants <5 were calculated, which are below the thermodynamic equilibrium known for the ‘high potential chain’. The disequilibrium found in stacked thylakoids with high electron fluxes is explained by restricted long-range PC diffusion. We develop a model assuming that about 30% of Photosystem I mainly located in grana end-membranes and margins rapidly equilibrate with cytochrome f via short-distance transluminal PC diffusion, while long-range lateral PC migration between grana cores and distant stroma lamellae is restricted. Implications for the electron flux control are discussed.     

Structural and functional self-organization of Photosystem II in grana thylakoids

The biogenesis of the well-ordered macromolecular protein arrangement of photosystem (PS)II and light harvesting complex (LHC)II in grana thylakoid membranes is poorly understood and elusive. In this study we examine the capability of self organization of this arrangement by comparing the PSII distribution and antenna organization in isolated untreated stacked thylakoids with restacked membranes after unstacking. The PS II distribution was deduced from freeze-fracture electron microscopy. Furthermore, changes in the antenna organization and in the oligomerization state of photosystem II were monitored by chlorophyll a fluorescence parameters and size analysis of exoplasmatic fracture face particles. Low-salt induced unstacking leads to a randomization and intermixing of the protein complexes. In contrast, macromolecular PSII arrangement as well as antenna organization in thylakoids after restacking by restoring the original solvent composition is virtually identical to stacked control membranes. This indicates that the supramolecular protein arrangement in grana thylakoids is a self-organized process.     

Unique thylakoid membrane architecture of a unicellular N2-fixing cyanobacterium revealed by electron tomography

Cyanobacteria, descendants of the endosymbiont that gave rise to modern-day chloroplasts, are vital contributors to global biological energy conversion processes. A thorough understanding of the physiology of cyanobacteria requires detailed knowledge of these organisms at the level of cellular architecture and organization. In these prokaryotes, the large membrane protein complexes of the photosynthetic and respiratory electron transport chains function in the intracellular thylakoid membranes. Like plants, the architecture of the thylakoid membranes in cyanobacteria has direct impact on cellular bioenergetics, protein transport, and molecular trafficking. However, whole-cell thylakoid organization in cyanobacteria is not well understood. Here we present, by using electron tomography, an in-depth analysis of the architecture of the thylakoid membranes in a unicellular cyanobacterium, Cyanothece sp. ATCC 51142. Based on the results of three-dimensional tomographic reconstructions of near-entire cells, we determined that the thylakoids in Cyanothece 51142 form a dense and complex network that extends throughout the entire cell. This thylakoid membrane network is formed from the branching and splitting of membranes and encloses a single lumenal space. The entire thylakoid network spirals as a peripheral ring of membranes around the cell, an organization that has not previously been described in a cyanobacterium. Within the thylakoid membrane network are areas of quasi-helical arrangement with similarities to the thylakoid membrane system in chloroplasts. This cyanobacterial thylakoid arrangement is an efficient means of packing a large volume of membranes in the cell while optimizing intracellular transport and trafficking.     

Interaction of methylamine with extrinsic and intrinsic subunits of photosystem II

The interaction of methylamine with chloroplasts' photosystem II (PSII) was studied in isolated thylakoid membranes. Low concentration of methylamine (mM range) was shown to affect water oxidation and the advancement of the S-states. Modified kinetics of chlorophyll fluorescence rise and thermoluminescence in the presence of methylamine indicated that the electron transfer was affected at both sides of PSII, and in particular the electron transfer between Y_Z and P680⁺. As the concentration of methylamine was raised above 10 mM, the extrinsic polypeptides associated with the oxygen-evolving complex were lost and energy transfer between PSII antenna complexes and reaction centers was impaired. It was concluded that methylamine is able to affect both extrinsic and intrinsic subunits of PSII even at the lowest concentrations used where the extrinsic polypeptides of the OEC are still associated with the luminal side of the photosystem. As methylamine concentration increases, the extrinsic polypeptides are lost and the interaction with intrinsic domains is amplified resulting in an increased F₀.     

Electron and proton transport in chloroplasts taking into account lateral heterogeneity of thylakoids. Mathematical model]

A mathematical model of a chloroplast was constructed, which takes into account the inhomogeneous distribution of complexes of photosystems I and II between granal and intergranal thylakoids. The structural and functional complexes of photosystems I and II, which are localized in intergranal and granal thylakoids, respectively, and the b/f complex, which is uniformly distributed in thylakoid membranes, are assumed to be immobile. The interactions between spatially distant electron transport complexes are provided by plastoquinone and plastocyanine, which diffuse in the thylakoid membrane and intrathylakoid space, respectively. The main stages of proton transport associated with the functioning of photosystem II and oxidation-reduction transformations of plastoquinone are considered. The model takes into account the interactions of protons with membrane-bound buffer groups, the lateral diffusion of hydrogen ions in the intrathylakoid space and in the lumen between adjacent granal thylakoids, and the transmembrane proton transport associated with the function of ATP synthase and passive leakage of protons from thylakoids outside. The numerical integration of two systems of differential equations describing the behavior of some variables in two different regions: granal and intergranal thylakoids was performed. The model describes adequately the kinetics of processes being studied and predicts the occurrence of inhomogeneous lateral profiles of proton potentials and redox state of electron carriers. Modeling the electron and proton transport with allowance for the topological features of chloroplasts (lateral heterogeneity of thylakoids) is important for correct interpretation of "power-flux" interactions and the experimentally measured kinetic parameters averaged over the entire spatially inhomogeneous thylakoid system.     

Watching the components of photosynthetic bacterial membranes and their in situ organisation by atomic force microscopy

The atomic force microscope has developed into a powerful tool in structural biology allowing information to be acquired at submolecular resolution on the protruding structures of membrane proteins. It is now a complementary technique to X-ray crystallography and electron microscopy for structure determination of individual membrane proteins after extraction, purification and reconstitution into lipid bilayers. Moving on from the structures of individual components of biological membranes, atomic force microscopy has recently been demonstrated to be a unique tool to identify in situ the individual components of multi-protein assemblies and to study the supramolecular architecture of these components allowing the efficient performance of a complex biological function.Here, recent atomic force microscopy studies of native membranes of different photosynthetic bacteria with different polypeptide contents are reviewed. Technology, advantages, feasibilities, restrictions and limits of atomic force microscopy for the acquisition of highly resolved images of up to 10 Å lateral resolution under native conditions are discussed. From a biological point of view, the new insights contributed by the images are analysed and discussed in the context of the strongly debated organisation of the interconnected network of membrane-associated chlorophyll-protein complexes composing the photosynthetic apparatus in different species of purple bacteria.     

Electron tomography of plant thylakoid membranes

For more than half a century, electron microscopy has been a main tool for investigating the complex ultrastructure and organization of chloroplast thylakoid membranes, but, even today, the three-dimensional relationship between stroma and grana thylakoids, and the arrangement of the membrane protein complexes within them are not fully understood. Electron cryo-tomography (cryo-ET) is a powerful new technique for visualizing cellular structures, especially membranes, in three dimensions. By this technique, large membrane protein complexes, such as the photosystem II supercomplex or the chloroplast ATP synthase, can be visualized directly in the thylakoid membrane at molecular (4-5 nm) resolution. This short review compares recent advances by cryo-ET of plant thylakoid membranes with earlier results obtained by conventional electron microscopy.     

Evidence for a close spatial location of the binding sites for CO2 and for photosystem II inhibitors

1. CO2-depletion of thylakoid membranes results in a decrease of binding affinity of the Photosystem II (PS II) inhibitor atrazine. The inhibitory efficiency of atrazine, expressed as I50-concentration (50% inhibition) of 2,6-dichlorophenolindophenol reduction, is the same in CO2-depleted as well as in control thylakoids. This shows that CO2-depletion results in a complete inactivation of a part of the total number of electron transport chains. 2. A major site of action of CO2, which had previously been located between the two electron acceptor quinone molecule B (or R) and Photosystem II inhibitor atrazine as suggested by the following observations: (a) CO2-depletion results in a shift of the binding constant (kappa b) of [14C]atrazine to thylakoid membranes indicating a decreased affinity of atrazine to membrane; (b) trypsin treatment, which is known to modify the Photosystem II complex at the level of B, strongly diminishes CO2 stimulation of electron transport reactions in CO2-depleted membranes; and (c) thylakoids from atrazine-resistant plants, which contain a Photosystem II complex modified at the inhibitor binding site, show an altered CO2-stimulation of electron flow. 3. CO2-depletion does not produce structural changes in enzyme complexes involved in Photosystem II function of thylakoid membranes, as shown by freeze-fracture studies using electron microscopy.     

Correlation between thylakoid stacking and cation-induced decrease in photosystem I electron transport at saturating light intensity

A Mg²⁺-induced decrease of the rate of photosystem I (PS I) electron transport (DCIPH₂ → methyl viologen) in thylakoids under saturated light intensities has been reported earlier (S. Bose, J. E. Mullet, G. E. Hoch, and C. J. Arntzen, 1981, Photobiochem. Photobiophys.2, 45–52). A similar effect is observed with Na⁺, although the concentration required for half-maximal inhibition was higher by about two orders of magnitude. The cation effect was gradually abolished as the thylakoids were aged by incubation at 30 °C for 6 h. The loss of cation effect on PS I electron transport rate during aging was parallel to the corresponding loss of cation effect on thylakoid stacking. The cation concentration required for thylakoid stacking and the degree of inhibition as a function of cation concentration correlated strongly with the degree of thylakoid stacking. These observations indicated that the inhibition of the rate of PS I electron transport by cations is a consequence of cation-induced stacking of thylakoid membranes. The observed inhibition of the rate of PS I electron transport is discussed in terms of two hypotheses: (i) a fraction (20–30%) of the PS I complexes is trapped in the appressed region of grana and becomes unavailable to the electron donor (DCIPH₂) and (ii) the membrane structure is altered by the cations in such a manner that the rate constant of electron donation by the donor to the electron transport chain in the thylakoid is decreased.     

Entropy-assisted stacking of thylakoid membranes

Chloroplasts in plants and some green algae contain a continuous thylakoid membrane system that is structurally differentiated into stacked granal membranes interconnected by unstacked thylakoids, the stromal lamellae. Experiments were conducted to test the hypothesis that the thermodynamic tendency to increase entropy in chloroplasts contributes to thylakoid stacking to form grana. We show that the addition of bovine serum albumin or dextran, two very different water-soluble macromolecules, to a suspension of envelope-free chloroplasts with initially unstacked thylakoids induced thylakoid stacking. This novel restacking of thylakoids occurred spontaneously, accompanied by lateral segregation of PSII from PSI, thereby mimicking the natural situation. We suggest that such granal formation, induced by the macromolecules, is partly explained as a means of generating more volume for the diffusion of macromolecules in a crowded stromal environment, i.e., greater entropy overall. This mechanism may be relevant in vivo where the stroma has a very high concentration of enzymes of carbon metabolism, and where high metabolic fluxes are required.     

The role of chlorophyll-protein complexes in the function and structure of chloroplast thylakoids

Summary The photosynthetic pigments of chloroplast thylakoid membranes are complexed with specific intrinsic polypeptides which are included in three supramolecular complexes, photosystem I complex, photosystem II complex and the light-harvesting complex. There is a marked lateral heterogeneity in the distribution of these complexes along the membrane with photosystem II complex and its associated light-harvesting complex being located mainly in the stacked membranes of the grana partitions, while photosystem I complex is found mainly in unstacked thylakoids together with ATP synthetase. In contrast, the intermediate electron transport complex, the cylochrome b-f complex, is rather uniformly distributed in these two membrane regions. The consequences of this lateral heterogeneity in the location of the thylakoid complexes are considered in relation to the function and structure of chloroplasts of higher plants.     

Modulation of the light-harvesting chlorophyll antenna size in Chlamydomonas reinhardtii by TLA1 gene over-expression and RNA interference

Truncated light-harvesting antenna 1 (TLA1) is a nuclear gene proposed to regulate the chlorophyll (Chl) antenna size in Chlamydomonas reinhardtii. The Chl antenna size of the photosystems and the chloroplast ultrastructure were manipulated upon TLA1 gene over-expression and RNAi downregulation. The TLA1 over-expressing lines possessed a larger chlorophyll antenna size for both photosystems and contained greater levels of Chl b per cell relative to the wild type. Conversely, TLA1 RNAi transformants had a smaller Chl antenna size for both photosystems and lower levels of Chl b per cell. Western blot analyses of the TLA1 over-expressing and RNAi transformants showed that modulation of TLA1 gene expression was paralleled by modulation in the expression of light-harvesting protein, reaction centre D1 and D2, and VIPP1 genes. Transmission electron microscopy showed that modulation of TLA1 gene expression impacts the organization of thylakoid membranes in the chloroplast. Over-expressing lines showed well-defined grana, whereas RNAi transformants possessed loosely held together and more stroma-exposed thylakoids. Cell fractionation suggested localization of the TLA1 protein in the inner chloroplast envelope and potentially in association with nascent thylakoid membranes, indicating a role in Chl antenna assembly and thylakoid membrane biogenesis. The results provide a mechanistic understanding of the Chl antenna size regulation by the TLA1 gene.