574 research outputs found
The photosynthetic cytochrome c550 from the diatom Phaeodactylum tricornutum
The photosynthetic cytochrome c550 from the marine diatom Phaeodactylum tricornutum has been purified and characterized. Cytochrome c550 is mostly obtained from the soluble cell extract in relatively large amounts. In addition, the protein appeared to be truncated in the last hydrophobic residues of the C-terminus, both in the soluble cytochrome c550 and in the protein extracted from the membrane fraction, as deduced by mass spectrometry analysis and the comparison with the gene sequence. Interestingly, it has been described that the C-terminus of cytochrome c550 forms a hydrophobic finger involved in the interaction with photosystem II in cyanobacteria. Cytochrome c550 was almost absent in solubilized photosystem II complex samples, in contrast with the PsbO and Psb31 extrinsic subunits, thus suggesting a lower affinity of cytochrome c550 for the photosystem II complex. Under iron-limiting conditions the amount of cytochrome c550 decreases up to about 45% as compared to iron-replete cells, pointing to an iron-regulated synthesis. Oxidized cytochrome c550 has been characterized using continuous wave EPR and pulse techniques, including HYSCORE, and the obtained results have been interpreted in terms of the electrostatic charge distribution in the surroundings of the heme centre.España, MINECO BIO2012-35271, BIO2015-64169-P, MAT2011-23861 and CTQ2015-64486-
Alternative photosynthetic redox proteins in the diatom Phaeodactylum tricornutum
La fotosíntesis es la principal vía de entrada de energía en la biosfera y la ruta más importante de fijación y secuestro de dióxido de carbono. Aproximadamente la mitad de la productividad fotosintética ocurre en los océanos. Sin embargo, en estos ecosistemas nos encontramos grandes áreas pobres en fitoplancton debido a una limitación delhierro disponible como nutriente. Las algas diatomeas,microalgas eucariotas unicelulares fotosintéticas,son constituyentes fundamentales del fitoplancton oceánico, contribuyendoaproximadamente al 40 % de la fijación de carbono global marina, por lo que pueden ser consideradas así las mayores productorasprimariasen los océanos. Además,las diatomeastienen usos biotecnológicos relevantes. La limitación por hierro en el crecimiento de las diatomeas fue demostrada tras experimentos de fertilización masiva con hierroen el océano, donde, tras dicha fertilización, se produjeron explosiones de biomasaen las que predominabanlasdiatomeas. Phaeodactylum tricornutum(P. tricornutum),en la que se centra este trabajo de tesis, es una de las diatomeas modelos más usadas en investigación, debido tanto a su pequeño genoma (27,4 megabases), a su corto tiempo de crecimiento, a la facilidad de su cultivoy a las diferentes técnicas genéticasque se pueden emplear para su manipulación. Las diatomeas presentan un requerimiento extra de hierro en su cadena fotosintética, primero porque poseen citocromo c550(Cc550) asociado al fotosistema II (PSII), proteína ausente en algas verdes y plantas. Además, la mayoría de las diatomeas carecen de plastocianina (Pc) como alternativa al citocromo c6(Cc6) como transportador de electrones entre el citocromo f (Cf), del complejo b6f(Cb6f),y el fotosistema I (PSI). Una excepción a esto último es la diatomea Thalassiosira oceanica(T. oceanica), en la que se han encontrado evidencias de la presencia de una Pc “roja”. Por todo ello, en esta tesis se han establecido varios objetivos, enfocados al estudio de estas proteínas redox alternativas en fotosíntesis: i) caracterización del Cc550de la diatomea P. tricornutumy los efectos de la falta de hierro en su función como subunidad extrínseca del PSII; ii) efectos de la expresión heteróloga de la Pc del alga verdeChlamydomonas reinhardtii(C. reinhardtii) en P.
tricornutum; iii) modelado del complejo de transferencia electrónica [Cf:aceptor] en las diatomeas P. tricornutumy T. oceanica. Nuestros resultados, respecto al Cc550de P. tricornutum, muestran que dicha proteína es purificada en una forma truncada, careciendo de las dos últimas tirosinas, cuando se purifica siguiendo protocolos estándares de purificación. Sin embargo, se ha demostrado, a través de un nuevo protocolo diseñado para purificar el Cc550completo, que el truncamiento es un proceso no fisiológico. Además, la limitación por hierro en los cultivos de esta diatomea induce una drástica bajada en el contenido tanto del Cc550como dela actividad delPSII. Por otro lado, se ha comprobado quela correcta expresión heteróloga de la Pc de C. reinhardtiien P. tricornutumincrementa el crecimiento de esta diatomea en condiciones de deficiencia de hierro. Estos mutantes presentan una mejora en las actividades tanto del PSI como del PSII. Por último, hemos podido ver que el mejor modelo de docking del complejo [Cf:Cc6] de P. tricornutumtiene una orientación diferente al mismocomplejo en C. reinhardtii, siendo más similaral descrito previamente en cianobacterias. Los modelos de docking del complejo [Cf:Pc] de la diatomea T. oceanicamuestran que el complejo no está optimizado para una sola configuración, lo quepodríaindicarque aún no se ha alcanzado una configuración optima para la transferencia de electrones. Además, las configuraciones de energía más favorablede este complejo corresponden a orientaciones aparentemente no productivas, pero que podrían constituiruna posible vía alternativa de transferencia de electrones del Cf a la P
The photosynthetic cytochrome c 550 from the diatom Phaeodactylum tricornutum
The photosynthetic cytochrome c550 from the marine diatom Phaeodactylum tricornutum has been purified and characterized. Cytochrome c550 is mostly obtained from the soluble cell extract in relatively large amounts. In addition, the protein appeared to be truncated in the last hydrophobic residues of the C-terminus, both in the soluble cytochrome c550 and in the protein extracted from the membrane fraction, as deduced by mass spectrometry analysis and the comparison with the gene sequence. Interestingly, it has been described that the C-terminus of cytochrome c550 forms a hydrophobic finger involved in the interaction with photosystem II in cyanobacteria. Cytochrome c550 was almost absent in solubilized photosystem II complex samples, in contrast with the PsbO and Psb31 extrinsic subunits, thus suggesting a lower affinity of cytochrome c550 for the photosystem II complex. Under iron-limiting conditions the amount of cytochrome c550 decreases up to about 45% as compared to iron-replete cells, pointing to an iron-regulated synthesis. Oxidized cytochrome c550 has been characterized using continuous wave EPR and pulse techniques, including HYSCORE, and the obtained results have been interpreted in terms of the electrostatic charge distribution in the surroundings of the heme centre.This work was supported by the Spanish Ministry of Economy and Competitiveness (BIO2012-35271, BIO2015-64169-P, MAT2011-23861 and CTQ2015-64486-R) the Andalusian Government (PAIDI BIO-022) and the Aragón Government (Grupo consolidado B-18). All these grants were partially financed by the EU FEDER ProgramPeer reviewe
The photosynthetic cytochrome c550 from the diatom Phaeodactylum tricornutum
The photosynthetic cytochrome c550 from the marine diatom Phaeodactylum tricornutum has been purified and characterized. Cytochrome c550 is mostly obtained from the soluble cell extract in relatively large amounts. In addition, the protein appeared to be truncated in the last hydrophobic residues of the C-terminus, both in the soluble cytochrome c550 and in the protein extracted from the membrane fraction, as deduced by mass spectrometry analysis and the comparison with the gene sequence. Interestingly, it has been described that the C-terminus of cytochrome c550 forms a hydrophobic finger involved in the interaction with photosystem II in cyanobacteria. Cytochrome c550 was almost absent in solubilized photosystem II complex samples, in contrast with the PsbO and Psb31 extrinsic subunits, thus suggesting a lower affinity of cytochrome c550 for the photosystem II complex. Under iron-limiting conditions the amount of cytochrome c550 decreases up to about 45% as compared to iron-replete cells, pointing to an iron-regulated synthesis. Oxidized cytochrome c550 has been characterized using continuous wave EPR and pulse techniques, including HYSCORE, and the obtained results have been interpreted in terms of the electrostatic charge distribution in the surroundings of the heme centre
The singular properties of photosynthetic cytochrome c 550 from the diatom Phaeodactylum tricornutum suggest new alternative functions
Cytochrome c 550 is an extrinsic component in the luminal side of photosystem II (PSII) in cyanobacteria, as well as in eukaryotic algae from the red photosynthetic lineage including, among others, diatoms. We have established that cytochrome c 550 from the diatom Phaeodactylum tricornutum can be obtained as a complete protein from the membrane fraction of the alga, although a C-terminal truncated form is purified from the soluble fractions of this diatom as well as from other eukaryotic algae. Eukaryotic cytochromes c 550 show distinctive electrostatic features as compared with cyanobacterial cytochrome c 550 . In addition, co-immunoseparation and mass spectrometry experiments, as well as immunoelectron microscopy analyses, indicate that although cytochrome c 550 from P. tricornutum is mainly located in the thylakoid domain of the chloroplast – where it interacts with PSII –, it can also be found in the chloroplast pyrenoid, related with proteins linked to the CO 2 concentrating mechanism and assimilation. These results thus suggest new alternative functions of this heme protein in eukaryotes.Ministerio de Economía, Industria y Competitividad BIO2015-64169-PJunta de Andalucía PAIDI BIO-02
Plastid thylakoid architecture optimizes photosynthesis in diatoms
Photosynthesis is a unique process that allows independent colonization of the land by plants and of the oceans by phytoplankton. Although the photosynthesis process is well understood in plants, we are still unlocking the mechanisms evolved by phytoplankton to achieve extremely efficient photosynthesis. Here, we combine biochemical, structural and in vivo physiological studies to unravel the structure of the plastid in diatoms, prominent marine eukaryotes. Biochemical and immunolocalization analyses reveal segregation of photosynthetic complexes in the loosely stacked thylakoid membranes typical of diatoms. Separation of photosystems within subdomains minimizes their physical contacts, as required for improved light utilization. Chloroplast 3D reconstruction and in vivo spectroscopy show that these subdomains are interconnected, ensuring fast equilibration of electron carriers for efficient optimum photosynthesis. Thus, diatoms and plants have converged towards a similar functional distribution of the photosystems although via different thylakoid architectures, which likely evolved independently in the land and the ocean.ISSN:2041-172
Biochemical characterization of Fucoxanthin Chlorophyll a/c binding proteins in the diatom Phaeodactylum tricornutum
Diatoms contribute largely to the total primary production of the ecosphere and are key players in global biogeochemical cycles. Their chloroplasts are surrounded by four membranes owing to their secondary endosymbiotic origin. Their thylakoids are arranged into three parallel bands and differentiation of thylakoid membranes into grana or stroma is not observed. The fucoxanthin chlorophyll a/c binding proteins act as the light harvesting proteins and play a role in photoprotection during excess light as well. The diatom genome encodes three different families of antenna proteins. Family I are the classical light harvesting proteins called "Lhcf". Family II are the red algae related Lhca-R1/2 proteins called "Lhcr" and family III are the photoprotective LI818 related proteins called "Lhcx".
All known Fcps have a molecular weight in the range of 17-23 kDa. They are membrane proteins and have shorter loops and termini compared to LHCs of higher plants and are therefore extremely hydrophobic. This makes the isolation of single specific Fcps using routine protein purification techniques difficult.
The purification of a specific Fcp containing complex has not been achieved so far and until this is done several questions concerning light harvesting antenna systems of diatoms cannot be answered. For e.g. Which proteins interact specifically? Are various Fcps differently pigmented? Which pigments interact with each other and how? Which proteins contribute to photosystem specific antenna systems? Can pure Fcps be reconstituted into crystals like LHCII proteins? In order to answer these questions specific Fcp containing complexes have to be purified. ...Diatomeen tragen maßgeblich zu der Primärproduktion der Ökosphäre bei und sind Hauptakteure in den globalen biogeochemischen Stoffkreisläufen. Ihre Chloroplasten sind aufgrund ihrer Entstehung durch sekundäre Endosymbiose von vier Membranen umgeben. Die Thylakoide sind in drei parallelen Bändern angeordnet und eine Differenzierung der Thylakoidmembranen in Grana- und Stromathylakoide liegt nicht vor. Die Fucoxanthin – Chlorophyll a/c – Bindeproteine fungieren hauptsächlich als Lichtsammelproteine und besitzen darüber hinaus auch eine Schutzfunktion bei überschüssiger Lichteinstrahlung.
Die Genomsequenzen von Diatomeen kodieren drei verschiedene Familien von Antennenproteinen: Proteine der Familie I sind die klassischen Lichtsammelproteine und werden als "Lhcf" bezeichnet. Familie II Proteine sind durch ihre strukturelle Ähnlichkeit zu dem einzigen membranintrinsischen Lichtsammelprotein der Rotalgen, lhca-R1/2, charakterisiert. Sie werden demnach auch als "Lhcr"-Proteine bezeichnet. Die Proteine der Familie III besitzen Lichtschutzfunktion und werden als "Lhcx" bezeichnet. Sie zeichnen sich durch ihre nahe Verwandtschaft zu den LI818-Proteinen der Grünalgen aus.
Alle bekannten Fcps besitzen eine relative ähnliche molekulare Masse in der Größenordnung von 17 – 21 kDa. Als Membranproteine sind sie zudem stark hydrophob, was eine Trennung spezifischer Fcp-Komplexe voneinander über die üblichen Proteinisolationsmethoden zusätzlich erschwert.
Die Isolation spezifischer Fcp-Komplexe konnte bisher nicht erreicht werden und aus diesem Grund sind bestimmte Fragestellungen betreffend der Lichtsammelproteine der Diatomeen immer noch unbeantwortet. Zum Beispiel, welche Proteine interagieren spezifisch miteinander? Sind die verschiedenen Fcps unterschiedlich pigmentiert? Welche Pigmente interagieren miteinander und auf welche Art und Weise? Welche Proteine sind Bestandteil von photosystem-spezifischen Antennensystemen? Ist es möglich, isolierte Fcp-Komplexe zu kristallisieren, um ihre dreidimensionale Struktur aufzuklären, wie es bereits bei LHCII-Proteinen gelungen ist? Um all diese Fragen beantworten zu können, ist es unabdingbar, Fcp-Komplexe spezifisch zu isolieren. ..
Characterization of a trimeric light-harvesting complex in the diatom Phaeodactylum tricornutum built of FcpA and FcpE proteins
Fucoxanthin chlorophyll proteins (Fcps), the light-harvesting antennas of heterokont algae, are encoded by a multigene family and are highly similar with respect to their molecular masses as well as to their pigmentation, making it difficult to purify single Fcps. In this study, a hexa-histidine tag was genetically added to the C-terminus of the FcpA protein of the pennate diatom Phaeodactylum tricornutum. A transgenic strain expressing the recombinant His-tagged FcpA protein in addition to the endogenous wild type Fcps was created. This strategy allowed, for the first time, the purification of a specific, stable trimeric Fcp complex. In addition, a pool of various trimeric Fcps was also purified from the wild-type cells using sucrose density gradient ultracentrifugation and gel filtration. In both the His-tagged and the wild-type Fcps, excitation energy coupling between fucoxanthin and chlorophyll a was intact and the existence of a chlorophyll a/fucoxanthin excitonic dimer was demonstrated using circular dichroism spectroscopy. Mass spectrometric analyses of the trimeric His-tagged complex indicated that it is composed of FcpA and FcpE polypeptides. It is confirmed here that a trimer is the basic organizational unit of Fcps in P. tricornutum. From circular dichroism spectra, it is proposed that the organization of the pigments on the polypeptide backbone of Fcps is a conserved feature in the case of chlorophyll a/c containing algae
Biochemistry of diatom photosynthetic membranes and pigment-protein complexes
Thesis (M.S.) University of Alaska Fairbanks, 1996Diatoms are an ecologically important group of algae in both marine and freshwater systems, but in spite of their significance little is known about the structure of their photosynthetic apparatus. This is due in part to the lack of a highly purified, oxygen evolving thylakoid membrane preparation. Since thylakoid membranes purified from diatoms using methods developed for green plants did not evolve oxygen, a new procedure was developed for use with diatoms. An oxygen-evolving thylakoid membrane preparation is crucial for the study of photosynthetic pigment-protein complexes from these algae because the stability of the Photosystem I (PS I) and Photosystem II reaction centers was shown to be greatly reduced in thylakoid preparations that did not retain electron transport activity. As a result of the instability of PS I in some thylakoid preparations, a novel chlorophyll-binding complex was isolated that contained only the PsaA polypeptide. The isolation of this complex should prove useful in elucidating the structure of the PS I reaction center in all plants. Immunological and N-terminal protein sequencing methods were used to identify several photosynthetic proteins in the purified thylakoid preparation. These results provided evidence for posttranslational modification of two light-harvesting polypeptides (LHCPs) as well as of the PsaB subunit of the PS I reaction center core. Posttranslational modification of LHCPs and/or of PsaB has not been observed in green plants. In contrast to green plants, PS I in diatoms has been shown to be located in the inner thylakoid membranes. It was hypothesized that proteolytic processing of the C-terminus of PsaB in diatoms may be necessary for the PS I holocomplex to be present in the inner membranes, and that this processing may be responsible for the instability of PS I in purified diatom thylakoids. The existence of a functional, highly purified, and extensively characterized thylakoid preparation from diatoms will promote our understanding of the photosynthetic apparatus in these algae
Glyphosate-Based Herbicide Toxicophenomics in Marine Diatoms: Impacts on Primary Production and Physiological Fitness
Glyphosate is the main active component of the commercial formulation Roundup®, the most widely used chemical herbicide worldwide. However, its potential high toxicity to the environment and throughout trophic webs has come under increasing scrutiny. The present study aims to investigate the application of bio-optical techniques and their correlation to physiological and biochemical processes, including primary productivity, oxidative stress, energy balance, and alterations in pigment and lipid composition in Phaeodactylum tricornutum, a representative species of marine diatoms, using the case study of its response to the herbicide glyphosate-based Roundup® formulation, at environmentally relevant concentrations. Cultures were exposed to the herbicide formulation representing effective glyphosate concentrations of 0, 10, 50, 100, 250, and 500 μg L−1. Results showed that high concentrations decreased cell density; furthermore, the inhibition of photosynthetic activity was not only caused by the impairment of electron transport in the thylakoids, but also by a decrease of antioxidant capacity and increased lipid peroxidation. Nevertheless, concentrations of one of the plastidial marker fatty acids had a positive correlation with the highest concentration as well as an increase in total protein. Cell energy allocation also increased with concentration, relative to control and the lowest concentration, although culture growth was inhibited. Pigment composition and fatty acid profiles proved to be efficient biomarkers for the highest glyphosate-based herbicide concentrations, while bio-optical data separated controls from intermediate concentrations and high concentrations.info:eu-repo/semantics/publishedVersio
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