Distribution and dynamics of electron transport complexes in cyanobacterial thylakoid membranes.
ABSTRACT: The cyanobacterial thylakoid membrane represents a system that can carry out both oxygenic photosynthesis and respiration simultaneously. The organization, interactions and mobility of components of these two electron transport pathways are indispensable to the biosynthesis of thylakoid membrane modules and the optimization of bioenergetic electron flow in response to environmental changes. These are of fundamental importance to the metabolic robustness and plasticity of cyanobacteria. This review summarizes our current knowledge about the distribution and dynamics of electron transport components in cyanobacterial thylakoid membranes. Global understanding of the principles that govern the dynamic regulation of electron transport pathways in nature will provide a framework for the design and synthetic engineering of new bioenergetic machinery to improve photosynthesis and biofuel production. This article is part of a Special Issue entitled: Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux.
Project description:Thylakoid membranes are the specialized internal membrane system produced in plants, algae, and cyanobacteria to convert sunlight to chemical energy via oxygenic photosynthesis. Cyanobacterial thylakoid membranes harbor the protein complexes and electron transport molecules that are necessary for photosynthetic light reactions and respiratory electron flow. The thylakoid membranes sit between the plasma membrane and the central cytoplasm, leading to intricate cellular compartmentalization. How thylakoid membranes are generated to form the functional network and how protein complexes are recruited into thylakoids remain elusive. Here, we developed a method to modulate thylakoid biogenesis in the model cyanobacterium Synechococcus elongatus PCC7942 and probed the spatial-temporal stepwise biogenesis process of cyanobacterial thylakoid membranes, using electron microscopy, in situ cryo-electron tomography, confocal microscopy, mass spectroscopy, and biochemical approaches. Our results revealed that the plasma membrane and regularly-arranged concentric thylakoid layers have no physical connections. The newly synthesized thylakoid membrane fragments commerce between the plasma membrane and pre-existing thylakoids, where the initial biogenesis of Photosystem II occurs. Photosystem I monomers appear in thylakoid membranes earlier than other photosystem assemblies. Redistribution of photosynthetic protein complexes during thylakoid biogenesis ensures establishment of the spatial organization of the functional thylakoid membrane network. This study provides insights into the molecular processes of the photosynthetic machinery biosynthesis and organization.
Project description:The machinery that conducts the light-driven reactions of oxygenic photosynthesis is hosted within specialized paired membranes called thylakoids. In higher plants, the thylakoids are segregated into two morphological and functional domains called grana and stroma lamellae. A large fraction of the luminal volume of the granal thylakoids is occupied by the oxygen-evolving complex of photosystem II. Electron microscopy data we obtained on dark- and light-adapted Arabidopsis thylakoids indicate that the granal thylakoid lumen significantly expands in the light. Models generated for the organization of the oxygen-evolving complex within the granal lumen predict that the light-induced expansion greatly alleviates restrictions imposed on protein diffusion in this compartment in the dark. Experiments monitoring the redox kinetics of the luminal electron carrier plastocyanin support this prediction. The impact of the increase in protein mobility within the granal luminal compartment in the light on photosynthetic electron transport rates and processes associated with the repair of photodamaged photosystem II complexes is discussed.
Project description:Cyanobacteria are photosynthetic prokaryotes that make major contributions to the production of the oxygen in the Earth atmosphere. The photosynthetic machinery in cyanobacterial cells is housed in flattened membrane structures called thylakoids. The structural organization of cyanobacterial cells and the arrangement of the thylakoid membranes in response to environmental conditions have been widely investigated. However, there is limited knowledge about the internal dynamics of these membranes in terms of their flexibility and motion during the photosynthetic process. We present a direct observation of thylakoid membrane undulatory motion in vivo and show a connection between membrane mobility and photosynthetic activity. High-resolution inelastic neutron scattering experiments on the cyanobacterium Synechocystis sp. PCC 6803 assessed the flexibility of cyanobacterial thylakoid membrane sheets and the dependence of the membranes on illumination conditions. We observed softer thylakoid membranes in the dark that have three-to four fold excess mobility compared to membranes under high light conditions. Our analysis indicates that electron transfer between photosynthetic reaction centers and the associated electrochemical proton gradient across the thylakoid membrane result in a significant driving force for excess membrane dynamics. These observations provide a deeper understanding of the relationship between photosynthesis and cellular architecture.
Project description:Nature's solar energy converters, the Photosystem I (PSI) and Photosystem II (PSII) reaction center proteins, flawlessly manage photon capture and conversion processes in plants, algae, and cyanobacteria to drive oxygenic water-splitting and carbon fixation. Herein, we utilize the native photosynthetic Z-scheme electron transport chain to drive hydrogen production from thylakoid membranes by directional electron transport to abiotic catalysts bound at the stromal end of PSI. Pt-nanoparticles readily self-assemble with PSI in spinach and cyanobacterial membranes as evidenced by light-driven H2 production in the presence of a mediating electron shuttle protein and the sacrificial electron donor sodium ascorbate. EPR characterization confirms placement of the Pt-nanoparticles on the acceptor end of PSI. In the absence of sacrificial reductant, H2 production at PSI occurs via coupling to light-induced PSII O2 evolution as confirmed by correlation of catalytic activity to the presence or absence of the PSII inhibitor DCMU. To create a more sustainable system, first-row transition metal molecular cobaloxime and nickel diphosphine catalysts were found to perform photocatalysis when bound in situ to cyanobacterial thylakoid membranes. Thus, the self-assembly of abiotic catalysts with photosynthetic membranes demonstrates a tenable method for accomplishing solar overall water splitting to generate H2, a renewable and clean fuel. This work benchmarks a significant advance toward improving photosynthetic efficiency for solar fuel production.
Project description:While photosynthetic processes have become increasingly understood in cyanobacterial model strains, differences in the spatial distribution of thylakoid membranes among various lineages have been largely unexplored. Cyanobacterial cells exhibit an intriguing diversity in thylakoid arrangements, ranging from simple parietal to radial, coiled, parallel, and special types. Although metabolic background of their variability remains unknown, it has been suggested that thylakoid patterns are stable in certain phylogenetic clades. For decades, thylakoid arrangements have been used in cyanobacterial classification as one of the crucial characters for definition of taxa. The last comprehensive study addressing their evolutionary history in cyanobacteria was published 15 years ago. Since then both DNA sequence and electron microscopy data have grown rapidly. In the current study, we map ultrastructural data of >200 strains onto the SSU rRNA gene tree, and the resulting phylogeny is compared to a phylogenomic tree. Changes in thylakoid architecture in general follow the phylogeny of housekeeping loci. Parietal arrangement is resolved as the original thylakoid organization, evolving into complex arrangement in the most derived group of heterocytous cyanobacteria. Cyanobacteria occupying intermediate phylogenetic positions (greater filamentous, coccoid, and baeocytous types) exhibit fascicular, radial, and parallel arrangements, partly tracing the reconstructed course of phylogenetic branching. Contrary to previous studies, taxonomic value of thylakoid morphology seems very limited. Only special cases such as thylakoid absence or the parallel arrangement could be used as taxonomically informative apomorphies. The phylogenetic trees provide evidence of both paraphyly and reversion from more derived architectures in the simple parietal thylakoid pattern. Repeated convergent evolution is suggested for the radial and fascicular architectures. Moreover, thylakoid arrangement is constrained by cell size, excluding the occurrence of complex architectures in cyanobacteria smaller than 2 ?m in width. It may further be dependent on unknown (eco)physiological factors as suggested by recurrence of the radial type in unrelated but morphologically similar cyanobacteria, and occurrence of special features throughout the phylogeny. No straightforward phylogenetic congruences have been found between proteins involved in photosynthesis and thylakoid formation, and the thylakoid patterns. Remarkably, several postulated thylakoid biogenesis factors are partly or completely missing in cyanobacteria, challenging their proposed essential roles.
Project description:The chloroplast is the chlorophyll-containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live-cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three-dimensional structured illumination microscopy (3D-SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D-SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild-type and mutant strains. Using 3D-SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D-SIM. This study demonstrates that 3D-SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.
Project description:All cyanobacterial membranes contain diesel-range C15-C19 hydrocarbons at concentrations similar to chlorophyll. Recently, two universal but mutually exclusive hydrocarbon production pathways in cyanobacteria were discovered. We engineered a mutant of Synechocystis sp. PCC 6803 that produces no alkanes, which grew poorly at low temperatures. We analyzed this defect by assessing the redox kinetics of PSI. The mutant exhibited enhanced cyclic electron flow (CEF), especially at low temperature. CEF raises the ATP:NADPH ratio from photosynthesis and balances reductant requirements of biosynthesis with maintaining the redox poise of the electron transport chain. We conducted in silico flux balance analysis and showed that growth rate reaches a distinct maximum for an intermediate value of CEF equivalent to recycling 1 electron in 4 from PSI to the plastoquinone pool. Based on this analysis, we conclude that the lack of membrane alkanes causes higher CEF, perhaps for maintenance of redox poise. In turn, increased CEF reduces growth by forcing the cell to use less energy-efficient pathways, lowering the quantum efficiency of photosynthesis. This study highlights the unique and universal role of medium-chain hydrocarbons in cyanobacterial thylakoid membranes: they regulate redox balance and reductant partitioning in these oxygenic photosynthetic cells under stress.
Project description:Plant chloroplasts originated from an endosymbiotic event by which an ancestor of contemporary cyanobacteria was engulfed by an early eukaryotic cell and then transformed into an organelle. Oxygenic photosynthesis is the specific feature of cyanobacteria and chloroplasts, and the photosynthetic machinery resides in an internal membrane system, the thylakoids. The origin and genesis of thylakoid membranes, which are essential for oxygenic photosynthesis, are still an enigma. Vipp1 (vesicle-inducing protein in plastids 1) is a protein located in both the inner envelope and the thylakoids of Pisum sativum and Arabidopsis thaliana. In Arabidopsis disruption of the VIPP1 gene severely affects the plant's ability to form properly structured thylakoids and as a consequence to carry out photosynthesis. In contrast, Vipp1 in Synechocystis appears to be located exclusively in the plasma membrane. Yet, as in higher plants, disruption of the VIPP1 gene locus leads to the complete loss of thylakoid formation. So far VIPP1 genes are found only in organisms carrying out oxygenic photosynthesis. They share sequence homology with a subunit encoded by the bacterial phage shock operon (PspA) but differ from PspA by a C-terminal extension of about 30 amino acids. In two cyanobacteria, Synechocystis and Anabaena, both a VIPP1 and a pspA gene are present, and phylogenetic analysis indicates that VIPP1 originated from a gene duplication of the latter and thereafter acquired its new function. It also appears that the C-terminal extension that discriminates VIPP1 proteins from PspA is important for its function in thylakoid formation.
Project description:The structural dynamics and flexibility of cell membranes play fundamental roles in the functions of the cells, i.e., signaling, energy transduction, and physiological adaptation. The cyanobacterial thylakoid membrane represents a model membrane that can conduct both oxygenic photosynthesis and respiration simultaneously. In this study, we conducted direct visualization of the global organization and mobility of photosynthetic complexes in thylakoid membranes from a model cyanobacterium, Synechococcus elongatus PCC 7942, using high-resolution atomic force, confocal, and total internal reflection fluorescence microscopy. We visualized the native arrangement and dense packing of photosystem I (PSI), photosystem II (PSII), and cytochrome (Cyt) b6f within thylakoid membranes at the molecular level. Furthermore, we functionally tagged PSI, PSII, Cyt b6f, and ATP synthase individually with fluorescent proteins, and revealed the heterogeneous distribution of these four photosynthetic complexes and determined their dynamic features within the crowding membrane environment using live-cell fluorescence imaging. We characterized red light-induced clustering localization and adjustable diffusion of photosynthetic complexes in thylakoid membranes, representative of the reorganization of photosynthetic apparatus in response to environmental changes. Understanding the organization and dynamics of photosynthetic membranes is essential for rational design and construction of artificial photosynthetic systems to underpin bioenergy development. Knowledge of cyanobacterial thylakoid membranes could also be extended to other cell membranes, such as chloroplast and mitochondrial membranes.
Project description:A potassium channel (SynK) of the cyanobacterium Synechocystis sp. PCC 6803, a photoheterotrophic model organism for the study of photosynthesis, has been recently identified and demonstrated to function as a potassium selective channel when expressed in a heterologous system and to be located predominantly to the thylakoid membrane in cyanobacteria. To study its physiological role, a SynK-less knockout mutant was generated and characterized. Fluorimetric experiments indicated that SynK-less cyanobacteria cannot build up a proton gradient as efficiently as WT organisms, suggesting that SynK might be involved in the regulation of the electric component of the proton motive force. Accordingly, measurements of flash-induced cytochrome b(6)f turnover and respiration pointed to a reduced generation of ?pH and to an altered linear electron transport in mutant cells. The lack of the channel did not cause an altered membrane organization, but decreased growth and modified the photosystem II/photosystem I ratio at high light intensities because of enhanced photosensitivity. These data shed light on the function of a prokaryotic potassium channel and reports evidence, by means of a genetic approach, on the requirement of a thylakoid ion channel for optimal photosynthesis.