Phycobilisome Dotaz Zobrazit nápovědu
Primary photosynthetic reactions take place inside thylakoid membrane where light-to-chemical energy conversion is catalyzed by two pigment-protein complexes, photosystem I (PSI) and photosystem II (PSII). Light absorption in cyanobacteria is increased by pigment-protein supercomplexes--phycobilisomes (PBSs) situated on thylakoid membrane surfaces that transfer excitation energy into both photosystems. We have explored the localization of PSI, PSII and PBSs in thylakoid membrane of native cyanobacteria cell Anabaena sp. 7120 by means of cryogenic confocal microscopy. We have adapted a conventional temperature controlling stage to an Olympus FV1000 confocal microscope. The presence of red shifted emission of chlorophylls from PSI has been confirmed by spectral measurements. Confocal fluorescence images of PSI (in a spectral range 710-750 nm), PSII (in a spectral range 690-705 nm) and PBSs (in a spectral range 650-680 nm) were recorded at low temperature. Co-localization of images showed spatial heterogeneity of PSI, PSII and PBSs over the thylakoid membrane, and three dominant areas were identified: PSI-PSII-PBS supercomplex area, PSII-PBS supercomplex area and PSI area. The observed results were discussed with regard to light-harvesting regulation in cyanobacteria.
- MeSH
- Anabaena cytologie enzymologie metabolismus MeSH
- fotosystém I - proteinový komplex chemie metabolismus MeSH
- fotosystém II - proteinový komplex chemie metabolismus MeSH
- fykobilizomy chemie metabolismus MeSH
- konfokální mikroskopie * MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- transport proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- biliverdin analogy a deriváty MeSH
- Eukaryota genetika MeSH
- fotosyntéza MeSH
- fykoerythrin biosyntéza MeSH
- fykokyanin biosyntéza MeSH
- proteiny MeSH
- Publikační typ
- přehledy MeSH
The orange carotenoid protein (OCP) is a structurally and functionally modular photoactive protein involved in cyanobacterial photoprotection. Recently, based on bioinformatic analysis and phylogenetic relationships, new families of OCP have been described, OCP2 and OCPx. The first characterization of the OCP2 showed both faster photoconversion and back-conversion, and lower fluorescence quenching of phycobilisomes relative to the well-characterized OCP1. Moreover, OCP2 is not regulated by the fluorescence recovery protein (FRP). In this work, we present a comprehensive study combining ultrafast spectroscopy and structural analysis to compare the photoactivation mechanisms of OCP1 and OCP2 from Tolypothrix PCC 7601. We show that despite significant differences in their functional characteristics, the spectroscopic properties of OCP1 and OCP2 are comparable. This indicates that the OCP functionality is not directly related to the spectroscopic properties of the bound carotenoid. In addition, the structural analysis by X-ray footprinting reveals that, overall, OCP1 and OCP2 have grossly the same photoactivation mechanism. However, the OCP2 is less reactive to radiolytic labeling, suggesting that the protein is less flexible than OCP1. This observation could explain fast photoconversion of OCP2.
The mobility of photosynthetic proteins represents an important factor that affects light-energy conversion in photosynthesis. The specific feature of photosynthetic proteins mobility can be currently measured in vivo using advanced microscopic methods, such as fluorescence recovery after photobleaching which allows the direct observation of photosynthetic proteins mobility on a single cell level. The heterogeneous organization of thylakoid membrane proteins results in heterogeneity in protein mobility. The thylakoid membrane contains both, protein-crowded compartments with immobile proteins and fluid areas (less crowded by proteins), allowing restricted diffusion of proteins. This heterogeneity represents an optimal balance as protein crowding is necessary for efficient light-energy conversion, and protein mobility plays an important role in the regulation of photosynthesis. The mobility is required for an optimal light-harvesting process (e.g., during state transitions), and also for transport of proteins during their synthesis or repair. Protein crowding is then a key limiting factor of thylakoid membrane protein mobility; the less thylakoid membranes are crowded by proteins, the higher protein mobility is observed. Mobility of photosynthetic proteins outside the thylakoid membrane (lumen and stroma/cytosol) is less understood. Cyanobacterial phycobilisomes attached to the stromal side of the thylakoid can move relatively fast. Therefore, it seems that stroma with their active enzymes of the Calvin-Benson cycle, are a more fluid compartment in comparison to the rather rigid thylakoid lumen. In conclusion, photosynthetic protein diffusion is generally slower in comparison to similarly sized proteins from other eukaryotic membranes or organelles. Mobility of photosynthetic proteins resembles restricted protein diffusion in bacteria, and has been rationalized by high protein crowding similar to that of thylakoids.
A proper spatial distribution of photosynthetic pigment-protein complexes - PPCs (photosystems, light-harvesting antennas) is crucial for photosynthesis. In plants, photosystems I and II (PSI and PSII) are heterogeneously distributed between granal and stromal thylakoids. Here we have described similar heterogeneity in the PSI, PSII and phycobilisomes (PBSs) distribution in cyanobacteria thylakoids into microdomains by applying a new image processing method suitable for the Synechocystis sp. PCC6803 strain with yellow fluorescent protein-tagged PSI. The new image processing method is able to analyze the fluorescence ratios of PPCs on a single-cell level, pixel per pixel. Each cell pixel is plotted in CIE1931 color space by forming a pixel-color distribution of the cell. The most common position in CIE1931 is then defined as protein arrangement (PA) factor with xy coordinates. The PA-factor represents the most abundant fluorescence ratio of PSI/PSII/PBS, the 'mode color' of studied cell. We proved that a shift of the PA-factor from the center of the cell-pixel distribution (the 'median' cell color) is an indicator of the presence of special subcellular microdomain(s) with a unique PSI/PSII/PBS fluorescence ratio in comparison to other parts of the cell. Furthermore, during a 6-h high-light (HL) treatment, 'median' and 'mode' color (PA-factor) of the cell changed similarly on the population level, indicating that such microdomains with unique PSI/PSII/PBS fluorescence were not formed during HL (i.e. fluorescence changed equally in the whole cell). However, the PA-factor was very sensitive in characterizing the fluorescence ratios of PSI/PSII/PBS in cyanobacterial cells during HL by depicting a 4-phase acclimation to HL, and their physiological interpretation has been discussed.
The oceanic unicellular diazotrophic cyanobacterium Crocosphaera watsonii WH8501 exhibits large diel changes in abundance of both Photosystem II (PSII) and Photosystem I (PSI). To understand the mechanisms underlying these dynamics, we assessed photosynthetic parameters, photosystem abundance and composition, and chlorophyll-protein biosynthesis over a diel cycle. Our data show that the decline in PSII activity and abundance observed during the dark period was related to a light-induced modification of PSII, which, in combination with the suppressed synthesis of membrane proteins, resulted in monomerization and gradual disassembly of a large portion of PSII core complexes. In the remaining population of assembled PSII monomeric complexes, we detected the non-functional version of the D1 protein, rD1, which was absent in PSII during the light phase. During the dark period, we also observed a significant decoupling of phycobilisomes from PSII and a decline in the chlorophyll a quota, which matched the complete loss of functional PSIIs and a substantial decrease in PSI abundance. However, the remaining PSI complexes maintained their photochemical activity. Thus, during the nocturnal period of nitrogen fixation C. watsonii operates a suite of regulatory mechanisms for efficient utilization/recycling of cellular resources and protection of the nitrogenase enzyme.
- MeSH
- chlorofyl a metabolismus MeSH
- chlorofyl metabolismus MeSH
- fixace dusíku MeSH
- fotosyntéza * MeSH
- fotosystém I - proteinový komplex metabolismus MeSH
- fotosystém II - proteinový komplex metabolismus MeSH
- fykobilizomy metabolismus MeSH
- oceány a moře MeSH
- sinice metabolismus MeSH
- tma MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- oceány a moře MeSH
The Orange Carotenoid Protein (OCP) is a photoactive water soluble protein that is crucial for photoprotection in cyanobacteria. When activated by blue-green light, it triggers quenching of phycobilisome fluorescence and regulates energy flow from the phycobilisome to the reaction center. The OCP contains a single pigment, the carotenoid 3'-hydroxyechinenone (hECN). Binding to the OCP causes a conformational change in hECN leading to an extension of its effective conjugation length. We have determined the S(1) energy of hECN in organic solvent and compared it with the S(1) energy of hECN bound to the OCP. In methanol and n-hexane, hECN has an S(1) energy of 14,300cm(-1), slightly higher than carotenoids with shorter conjugation lengths such as zeaxanthin or β-carotene; this is consistent with the proposal that the presence of the conjugated carbonyl group in hECN increases its S(1) energy. The S(1) energy of hECN in organic solvent is independent of solvent polarity. Upon binding to the OCP, the S(1) energy of hECN is further increased to 14,700cm(-1), underscoring the importance of protein binding which twists the conjugated carbonyl group into s-trans conformation and enhances the effect of the carbonyl group. Activated OCP, however, has an S(1) energy of 14,000cm(-1), indicating that significant changes in the vicinity of the conjugated carbonyl group occur upon activation.
Photosynthesis research employs several biophysical methods, including the detection of fluorescence. Even though fluorescence is a key method to detect photosynthetic efficiency, it has not been applied/adapted to single-cell confocal microscopy measurements to examine photosynthetic microorganisms. Experiments with photosynthetic cells may require automation to perform a large number of measurements with different parameters, especially concerning light conditions. However, commercial microscopes support custom protocols (through Time Controller offered by Olympus or Experiment Designer offered by Zeiss) that are often unable to provide special set-ups and connection to external devices (e.g., for irradiation). Our new system combining an Arduino microcontroller with the Cell⊕Finder software was developed for controlling Olympus FV1000 and FV1200 confocal microscopes and the attached hardware modules. Our software/hardware solution offers (1) a text file-based macro language to control the imaging functions of the microscope; (2) programmable control of several external hardware devices (light sources, thermal controllers, actuators) during imaging via the Arduino microcontroller; (3) the Cell⊕Finder software with ergonomic user environment, a fast selection method for the biologically important cells and precise positioning feature that reduces unwanted bleaching of the cells by the scanning laser. Cell⊕Finder can be downloaded from http://www.alga.cz/cellfinder. The system was applied to study changes in fluorescence intensity in Synechocystis sp. PCC6803 cells under long-term illumination. Thus, we were able to describe the kinetics of phycobilisome decoupling. Microscopy data showed that phycobilisome decoupling appears slowly after long-term (>1 h) exposure to high light.
Thylakoids are the place of the light-photosynthetic reactions. To gain maximal efficiency, these reactions are conditional to proper pigment-pigment and protein-protein interactions. In higher plants thylakoids, the interactions lead to a lateral asymmetry in localization of protein complexes (i.e. granal/stromal thylakoids) that have been defined as a domain-like structures characteristic by different biochemical composition and function (Albertsson P-Å. 2001,Trends Plant Science 6: 349-354). We explored this complex organization of thylakoid pigment-proteins at single cell level in the cyanobacterium Synechocystis sp. PCC 6803. Our 3D confocal images captured heterogeneous distribution of all main photosynthetic pigment-protein complexes (PPCs), Photosystem I (fluorescently tagged by YFP), Photosystem II and Phycobilisomes. The acquired images depicted cyanobacterial thylakoid membrane as a stable, mosaic-like structure formed by microdomains (MDs). These microcompartments are of sub-micrometer in sizes (~0.5-1.5 μm), typical by particular PPCs ratios and importantly without full segregation of observed complexes. The most prevailing MD is represented by MD with high Photosystem I content which allows also partial separation of Photosystems like in higher plants thylakoids. We assume that MDs stability (in minutes) provides optimal conditions for efficient excitation/electron transfer. The cyanobacterial MDs thus define thylakoid membrane organization as a system controlled by co-localization of three main PPCs leading to formation of thylakoid membrane mosaic. This organization might represent evolutional and functional precursor for the granal/stromal spatial heterogeneity in photosystems that is typical for higher plant thylakoids.
- MeSH
- bakteriální proteiny metabolismus MeSH
- fotosyntéza fyziologie MeSH
- fotosystém I - proteinový komplex metabolismus MeSH
- fotosystém II - proteinový komplex metabolismus MeSH
- fykobilizomy metabolismus MeSH
- konfokální mikroskopie MeSH
- membránové mikrodomény metabolismus MeSH
- Synechocystis MeSH
- tylakoidy metabolismus MeSH
- zobrazování trojrozměrné MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In dark-adapted plants and algae, chlorophyll a fluorescence induction peaks within 1s after irradiation due to well documented photochemical and non-photochemical processes. Here we show that the much slower fluorescence rise in cyanobacteria (the so-called "S to M rise" in tens of seconds) is due to state 2 to state 1 transition. This has been demonstrated in particular for Synechocystis PCC6803, using its RpaC(-) mutant (locked in state 1) and its wild-type cells kept in hyperosmotic suspension (locked in state 2). In both cases, the inhibition of state changes correlates with the disappearance of the S to M fluorescence rise, confirming its assignment to the state 2 to state 1 transition. The general physiological relevance of the SM rise is supported by its occurrence in several cyanobacterial strains: Synechococcus (PCC 7942, WH 5701) and diazotrophic single cell cyanobacterium (Cyanothece sp. ATCC 51142). We also show here that the SM fluorescence rise, and also the state transition changes are less prominent in filamentous diazotrophic cyanobacterium Nostoc sp. (PCC 7120) and absent in phycobilisome-less cyanobacterium Prochlorococcus marinus PCC 9511. Surprisingly, it is also absent in the phycobiliprotein rod containing Acaryochloris marina (MBIC 11017). All these results show that the S to M fluorescence rise reflects state 2 to state 1 transition in cyanobacteria with phycobilisomes formed by rods and core parts. We show that the pronounced SM fluorescence rise may reflect a protective mechanism for excess energy dissipation in those cyanobacteria (e.g. in Synechococcus PCC 7942) that are less efficient in other protective mechanisms, such as blue light induced non-photochemical quenching. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.
- MeSH
- fluorescence MeSH
- sinice chemie MeSH
- Synechocystis chemie MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
