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Biogenesis of Photosystem II in the Model Cyanobacterium Synechocystis sp. PCC 6803 - The Role of Selected Auxiliary Protein Factors and Subcellular LocalisationKNOPPOVÁ, Jana January 2016 (has links)
This thesis explores localisations and roles of three auxiliary protein factors involved in the biogenesis of Photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803 and contributes to subcellular localisation of the initial steps of PSII biogenesis and repair-related D1 synthesis. The main results consist in i) identification of a functional interaction of the protein factor Psb27 with a lumenal domain of the Photosystem II subunit CP43, ii) discovery of a novel pigment binding complex formed by the Ycf39 protein and high-light-inducible proteins implicated in photoprotection and delivery of recycled chlorophyll to newly synthesized D1 protein during the PSII reaction centre formation, iii) providing evidence that the early steps of PSII assembly and the repair-related D1 synthesis occur in the thylakoid membrane of Synechocystis, and iv) revealing that the cyanobacterial PsbP orthologue, CyanoP, assists in the early phase of PSII biogenesis as an assembly factor facilitating the association of D2 and D1 assembly modules.
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The regulatory role of cyanobacterial High light inducible proteinsSHUKLA, Mahendra Kumar January 2018 (has links)
The aim of the thesis was to elucidate the role of High light inducible proteins (Hlips) in the protection/regulation of the biogenesis of photosynthesis machinery. During the project two Hlip proteins (HliC and HliD) were isolated from the cyanobacterium Synechocystis PCC 6803; either as a pure oligomer (HliC protein) or as a small complex with a putative Photosystem II assembly factor Ycf39 (HliD protein). Pigments bound to purified Hlips were analyzed by state-of-art spectroscopic techniques to elucidate the mechanism of thermal energy dissipation. In addition, this work explained the mechanism of how the HliC protein regulates the interaction between chlorophyll synthase enzyme and the Ycf39 protein. This conceptually new mechanism is based on the replacement of HliD dimers in chlorophyll synthase complexes by stress-induced HliD-HliC heterodimers, which changes the affinity of Ycf39 towards chlorophyll synthase.
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Nucleotide-Dependent Processes in the Thylakoid Lumen of Plant ChloroplastsLundin, Björn January 2008 (has links)
Plants, algae and photosynthetic bacteria are able to harvest the sunlight and use its energy to transform water and carbon dioxide to carbohydrate molecules and oxygen, both important to sustain life on Earth. This process is called photosynthesis and is the route by which almost all energy enters the biosphere. As most simple things in life, the process of photosynthesis is easily explained but unfortunately not that easy to reproduce. If we could, we would be living in a much different world with almost unlimited energy. Light energy is harvested by chlorophyll molecules, bound to proteins in the chloroplast thylakoid membrane and drives the oxygen-evolving complex, to extract electrons from water. Electrons are then transferred to NADPH through photosystem II (PSII) to cytochrome b6f and photosystem I, the major photosynthetic protein complexes. The cytochrome b6f complex also transfers protons into the lumenal space of the thylakoid. These protons together with those from water oxidation create an electrochemical gradient across the thylakoid membrane, which fuels the ATP synthase to produce ATP. ATP, NADPH and carbon dioxide are used during the dark reactions to produce sugars in the chloroplast stroma. The thylakoid lumenal space where the water oxidation occurs has until recently been viewed as a proton sink with very few proteins. With the publication of the genome of Arabidopsis thaliana it seems to be a much more complex compartment housing a wide variety of biochemical processes. ATP is a nucleotide and the major energy currency, but there are also other nucleotides such as AMP, ADP, GMP, GDP and GTP. Chloroplast metabolism has mostly been associated with ATP, but GTP has been shown to have a role in integration of light harversting complexes into the thylakoid. In this work, we have demonstrated the occurrence of nucleotide-dependent processes in the lumenal space of spinach by bringing evidence first for nucleotide (ATP) transport across the thylakoid membrane, second for nucleotide inter-conversion (ATP to GTP) by a nucleoside diphosphate kinase, and third the discovery that the PsbO extrinsic subunit of PSII complex can bind and hydrolyse GTP to GDP. The active PSII complex functions as a dimer but following light-induced damage, it is monomerised allowing for repair of its reaction center D1 protein. PsbO is ubiquitous in all oxygenic photosynthetic organisms and together with other extrinsic proteins stabilises the oxygen-evolving complex. We have modelled the GTP-binding site in the PsbO structure and showed that the GTPase activity of spinach PsbO induces changes in the protein structure, dissociation from the complex and stimulates the degradation of the D1 protein, possibly by inducing momerisation of damaged PSII complexes. As compared to spinach, Arabidopsis has two isoforms of PsbO, PsbO1 and PsbO2, expressed in a 4:1 ratio. A T-DNA insertion knockout mutant of PsbO1 showed a retarded growth rate, pale green leaves and a decrease in the oxygen evolution while a PsbO2 knockout mutant did not show any visual phenotype as compared to wild type. Unexpectedly, during growth under high light conditions the turnover rate of the D1 protein was impaired in the PsbO2 knockout, whereas it occurred faster in the PsbO1 knockout as compared to wild type. We concluded that the PsbO1 protein mainly functions in stabilizing the oxygen evolving complex, whereas the PsbO2 protein regulates the turnover of the D1 protein. The two PsbO proteins also differ in their GTPase-activity (PsbO2 >> PsbO1). Although their amino acid sequences are 90% identical, they differ in the GTP-binding region which could explain the difference in their GTPase activity. Based on these data, we propose that the GTPase activity of PsbO(2) leads to structural changes in interacting loops and plays a role in the initial steps of D1 turnover such as the PSII monomerisation step. The nucleotide-dependent processes we discovered in the thylakoid lumen raise questions of transporters to facilitate these processes. As stated earlier, we provided biochemical evidence of an ATP thylakoid transporter, and most recently have identified a transporter that may be important for the export of lumenal phosphate back to the stroma. More transporters for GDP, metal ions and others solutes have still to be identified.
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Localization and function of small protein subunits of the cyanobacterial Photosystem II complexDOBÁKOVÁ, Marika January 2008 (has links)
The Photosystem II complex (PSII) contains besides D1, D2, CP43, CP47 and several lumenal extrinsic proteins also a large number of small intrinsic subunits. These subunits are found in all kinds of oxygenic phototrophs and their sequences are usually highly conserved. It is assumed that the small subunits are involved in stabilization, assembly or dimerization of the PSII complex but their exact function remains largely unknown. Thus, we chose four small PSII proteins: cytochrome b-559, PsbH, PsbI and Psb28, and we studied their function and location in PSII of the cyanobacterium Synechocystis sp. PCC 6803. We attempted: (i) to better define the role of cyt b-559 in the assembly of PSII using various mutant strains lacking one or more PSII core protein subunits; (ii) to clarify the location of the PsbH subunit using NTA gold labelling methodology; (iii) to improve the available knowledge on function of the PsbI protein in the assembly and repair of the cyanobacterial PSII; and (iv) to confirm the presence of the Psb28 protein in PSII, to localize it and to establish its importance for the cyanobacterial PSII.
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Investigation of the regulation of photosynthesis at the molecular level for improvement of plant growth and productivity under limiting light conditions / Investigation of the regulation of photosynthesis at the molecular level for improvement of plant growth and productivity under limiting light conditionsKhuong, Thi thu huong 17 January 2013 (has links)
La lumière est indispensable à la survie des plantes via le processus photosynthétique, pourtant les plantes doivent s'adapter à différentes conditions environnementales où la quantité et la qualité de la lumière peuvent être non optimales pour la photosynthèse. Cela peut provoquer des dégâts photo-induits par formation d'espèces réactives de l'oxygène (ROS), qui sont dangereux pour la plante. Pour limiter la formation des ROS, les plantes mettent en place une régulation importante qui est la dissipation thermique de l'énergie absorbée en excès, appelé Non photochemical quenching (NPQ). Il est connu que la protéine PsbS joue le rôle clé de senseur du pH bas du lumen thylacoïdal, qui est le signal initial pour activer le NPQ. Dans le contexte de cette thèse, on propose d'étudier l'hypothèse que l'absence de la protéine PsbS (diminué NPQ) pourrait augmenter la croissance et la productivité des plantes en conditions contrôlées de faible lumière par l'éminilation de la protéine PsbS chez Arabidopsis thaliana et chez la tomate. Les résultats obtenus indiquent qu'en lumière faible les plantes mutantes montrent une augmentation du rendement de photosystème II conduisant une croissance et un nombre de fleurs significativement augmentés par rapport aux plantes sauvages.De plus, une autre régulation de la photosynthèse, nommée « transitions d'état », est importante pour optimiser la photosynthèse en réponse aux variations de la quantité et de la qualité de la lumière, grâce à la migration réversible des antennes collectrices d'énergie LHCII phosphorylées du PSII au PSI, c'est aussi étudié dans ma thèse. / Light is indispensable for plant survival, but plants have to cope with different environmental situations where light quantity and quality can be not optimal for photosynthesis. This can cause photodamage due to the formation of harmful reactive oxygen species (ROS). To limit ROS formation, plants developed a mechanism important as the dissipation of excess absorbed energy as heat and is called Non Photochemical Quenching (NPQ). The PsbS protein plays the key role of sensor of the low lumenal pH, the signal to activate NPQ. In this thesis, we proposed and investigated the hypothesis that PsbS absence (NPQ decrease) would improve growth under controlled low light upon elimination of the PsbS in Arabidopsis and tomato plants. Results showed that the increase of photosystem II yield in mutant plants leaded to a significant improvement of growth and flower number in mutants as compared with wild type plants under low light, suggesting that this mutation could be useful to improve plant performances in controlled conditions where light is strongly limiting. In addition, another photosynthetic regulation, called “state transitions”, which is important to optimize photosynthesis under variable light for intensity and quality thank to reversible migration of phosphorylated light harvesting complexes LHCII from PSII to PSI also investigated in my thesis.
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The influence of phytoplankton pigments composition and dominant cell size on fluorescence-derived photophysiological parameters and implications for primary production rates / Influência da composição de pigmentos e tamanho celular dominante do fitoplâncton em parâmetros fotofisiológicos derivados da fluorescência e implicações nas taxas de produção primáriaGiannini, Maria Fernanda Colo 19 August 2016 (has links)
Phytoplankton chlorophyll-a fluorescence, measured in situ, can be applied as a tool to estimate primary production in the ocean over a large range of temporal and spatial scales. This non-invasive technique allows for fast assessments of photo-physiological parameters in contrast to the traditional methodologies (14C uptake and O2 evolution). The main photo-physiological parameters derived by the available instruments are yields, and as such, require careful interpretation. The comprehension of the main sources of variability of the photochemical and the light absorption efficiencies in marine phytoplankton has increased in the past years, largely by studies using monospecific cultures. In natural communities, however, the development of primary production models based on chlorophyll-a fluorescence remain limited as they are simultaneously subjected to a wide range of environmental and biological factors. This study will test the hypothesis that photo-physiological models for primary production estimates can be improved when key phytoplankton features, such as the pigments composition and dominant cell size, are taking into account. The approach was to contrast the photo-physiological parameters derived from measurements in distinct oceanographic regions, as well as those derived in a specific environment with presented different nutrient concentration according to the time of sampling. In addition, we showed for monospecific cultures, how the photo-physiological parameters are quantitatively related to the production of carbon under the interactive effects of taxonomic composition and cell size. The proportions of photosynthetic and photoprotective pigments present in the community were related to the bulk photochemical efficiency and the cross-section of light absorption, but varied among oceanographic regions and the depth of the water column. A parameterization of fluorescence-derived primary production rates, using four dominant size classes, was derived for natural phytoplankton communities under different nutrients conditions in a coastal environment, showing that the parameters differed among size classes above a threshold of nutrient concentration. The direct conversion rates between fluorescence-derived primary production and carbon assimilation rates, computed for two distinct phytoplankton cell sizes grown in controlled laboratorial conditions, showed that cell size strongly influences the efficiency of light absorption and photochemistry, however species-specific responses in photosynthetic energy allocation dominated the differences observed in how absorbed light is utilized to carbon assimilation, i.e., in the electron requirements for carbon assimilation. The results highlighted the importance of the tight coupling of nutrients availability and phytoplankton communities, as well as for measurements of chlorophyll-a fluorescence in the ocean and primary production models. This work presents a novel contribution to the increasing efforts to apply fluorescence-based techniques to understand and parameterize primary production estimates in marine systems, especially at highly dynamic environments. / A fluorescência da clorofila-a do fitoplâncton, medida in situ, pode ser uma ferramenta para estimar produção primária no oceano em grande escala temporal e espacial. Esta técnica não-invasiva permite análises rápidas de parâmetros foto-fisiológicos ao contrário de metodologias tradicionais (assimilação de 14C e produção de O2). Os principais parâmetros foto-fisiológicos de instrumentos disponíveis hoje tratam-se de eficiências, e como tal, requerem cuidados em serem interpretados. A compreensão das principais fontes de variabilidade da eficiência fotoquímica e de absorção de luz no fitoplâncton marinho tem aumentado nos últimos anos, em sua maioria em cultivos monoespecíficos. Em comunidades naturais, entretanto, o desenvolvimento de modelos de produção primária baseados na fluorescência da clorofila-a ainda é limitado uma vez que estão sujeitos à uma ampla gama de fatores ambientais e biológicos. Esse estudo testa a hipótese de que modelos foto-fisiológicos para estimar produção primária podem ser aprimorados considerando-se características fundamentais do fitoplâncton, como a composição de pigmentos e tamanho celular dominante. A estratégia foi contrastar parâmetros foto-fisiológicos derivados de medidas em regiões oceanográficas distintas, assim como medidas em um ambiente específico com diferentes concentrações de nutrientes ao longo do período amostrado. Adicionalmente, apresentamos através de cultivos monoespecíficos, como parâmetros foto-fisiológicos são quantitativamente relacionados à produção de carbono e os efeitos interativos da composição taxonômica e tamanho celular nessa relação. A proporção de pigmentos fotossintéticos e fotoprotetores da comunidade foram relacionados à eficiência fotoquímica e seção transversal de absorção de luz, porém variaram de acordo com a região oceanográfica e profundidade na coluna d\'água. Uma parameterização de taxas de produção primária derivadas da fluorescência, usando quatro classes de tamanho dominantes, foi proposta para comunidades naturais de fitoplâncton sob condições de nutrientes diferentes em um ambiente costeiro, mostrando que os parâmetros diferiram entre as classes de tamanho acima de um limiar de concentração de nutrientes. As taxas de conversão diretas entre produção primária derivada da fluorescência e taxas de assimilação de carbono, computadas para dois tamanhos de fitoplâncton crescidos em condições controladas em laboratório, mostraram que tamanho celular influencia as eficiências de absorção de luz e fotoquímica, porém respostas espécie-específicas na alocação de energia fotossintética dominaram as diferenças observadas em como a luz absorvida é utilizada para assimilação de carbono, ou seja, na razão de elétrons exigidos para assimilação de carbono. Os resultados destacam a importância do acoplamento da disponibilidade de nutrientes com a comunidade fitoplanctônica, assim como das medidas de fluorescência da clorofila-a no oceano e nos modelos de produção primária. Este trabalho apresenta uma contribuição inédita nos esforços crescentes em aplicar técnicas baseadas na fluorescência para entender e parameterizar estimativas de produção primária nos sistemas marinhos, especialmente em ambientes altamente dinâmicos.
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The influence of phytoplankton pigments composition and dominant cell size on fluorescence-derived photophysiological parameters and implications for primary production rates / Influência da composição de pigmentos e tamanho celular dominante do fitoplâncton em parâmetros fotofisiológicos derivados da fluorescência e implicações nas taxas de produção primáriaMaria Fernanda Colo Giannini 19 August 2016 (has links)
Phytoplankton chlorophyll-a fluorescence, measured in situ, can be applied as a tool to estimate primary production in the ocean over a large range of temporal and spatial scales. This non-invasive technique allows for fast assessments of photo-physiological parameters in contrast to the traditional methodologies (14C uptake and O2 evolution). The main photo-physiological parameters derived by the available instruments are yields, and as such, require careful interpretation. The comprehension of the main sources of variability of the photochemical and the light absorption efficiencies in marine phytoplankton has increased in the past years, largely by studies using monospecific cultures. In natural communities, however, the development of primary production models based on chlorophyll-a fluorescence remain limited as they are simultaneously subjected to a wide range of environmental and biological factors. This study will test the hypothesis that photo-physiological models for primary production estimates can be improved when key phytoplankton features, such as the pigments composition and dominant cell size, are taking into account. The approach was to contrast the photo-physiological parameters derived from measurements in distinct oceanographic regions, as well as those derived in a specific environment with presented different nutrient concentration according to the time of sampling. In addition, we showed for monospecific cultures, how the photo-physiological parameters are quantitatively related to the production of carbon under the interactive effects of taxonomic composition and cell size. The proportions of photosynthetic and photoprotective pigments present in the community were related to the bulk photochemical efficiency and the cross-section of light absorption, but varied among oceanographic regions and the depth of the water column. A parameterization of fluorescence-derived primary production rates, using four dominant size classes, was derived for natural phytoplankton communities under different nutrients conditions in a coastal environment, showing that the parameters differed among size classes above a threshold of nutrient concentration. The direct conversion rates between fluorescence-derived primary production and carbon assimilation rates, computed for two distinct phytoplankton cell sizes grown in controlled laboratorial conditions, showed that cell size strongly influences the efficiency of light absorption and photochemistry, however species-specific responses in photosynthetic energy allocation dominated the differences observed in how absorbed light is utilized to carbon assimilation, i.e., in the electron requirements for carbon assimilation. The results highlighted the importance of the tight coupling of nutrients availability and phytoplankton communities, as well as for measurements of chlorophyll-a fluorescence in the ocean and primary production models. This work presents a novel contribution to the increasing efforts to apply fluorescence-based techniques to understand and parameterize primary production estimates in marine systems, especially at highly dynamic environments. / A fluorescência da clorofila-a do fitoplâncton, medida in situ, pode ser uma ferramenta para estimar produção primária no oceano em grande escala temporal e espacial. Esta técnica não-invasiva permite análises rápidas de parâmetros foto-fisiológicos ao contrário de metodologias tradicionais (assimilação de 14C e produção de O2). Os principais parâmetros foto-fisiológicos de instrumentos disponíveis hoje tratam-se de eficiências, e como tal, requerem cuidados em serem interpretados. A compreensão das principais fontes de variabilidade da eficiência fotoquímica e de absorção de luz no fitoplâncton marinho tem aumentado nos últimos anos, em sua maioria em cultivos monoespecíficos. Em comunidades naturais, entretanto, o desenvolvimento de modelos de produção primária baseados na fluorescência da clorofila-a ainda é limitado uma vez que estão sujeitos à uma ampla gama de fatores ambientais e biológicos. Esse estudo testa a hipótese de que modelos foto-fisiológicos para estimar produção primária podem ser aprimorados considerando-se características fundamentais do fitoplâncton, como a composição de pigmentos e tamanho celular dominante. A estratégia foi contrastar parâmetros foto-fisiológicos derivados de medidas em regiões oceanográficas distintas, assim como medidas em um ambiente específico com diferentes concentrações de nutrientes ao longo do período amostrado. Adicionalmente, apresentamos através de cultivos monoespecíficos, como parâmetros foto-fisiológicos são quantitativamente relacionados à produção de carbono e os efeitos interativos da composição taxonômica e tamanho celular nessa relação. A proporção de pigmentos fotossintéticos e fotoprotetores da comunidade foram relacionados à eficiência fotoquímica e seção transversal de absorção de luz, porém variaram de acordo com a região oceanográfica e profundidade na coluna d\'água. Uma parameterização de taxas de produção primária derivadas da fluorescência, usando quatro classes de tamanho dominantes, foi proposta para comunidades naturais de fitoplâncton sob condições de nutrientes diferentes em um ambiente costeiro, mostrando que os parâmetros diferiram entre as classes de tamanho acima de um limiar de concentração de nutrientes. As taxas de conversão diretas entre produção primária derivada da fluorescência e taxas de assimilação de carbono, computadas para dois tamanhos de fitoplâncton crescidos em condições controladas em laboratório, mostraram que tamanho celular influencia as eficiências de absorção de luz e fotoquímica, porém respostas espécie-específicas na alocação de energia fotossintética dominaram as diferenças observadas em como a luz absorvida é utilizada para assimilação de carbono, ou seja, na razão de elétrons exigidos para assimilação de carbono. Os resultados destacam a importância do acoplamento da disponibilidade de nutrientes com a comunidade fitoplanctônica, assim como das medidas de fluorescência da clorofila-a no oceano e nos modelos de produção primária. Este trabalho apresenta uma contribuição inédita nos esforços crescentes em aplicar técnicas baseadas na fluorescência para entender e parameterizar estimativas de produção primária nos sistemas marinhos, especialmente em ambientes altamente dinâmicos.
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Συγκριτική μελέτη φωτοσυνθετικών και φωτοπροστατευτικών χαρακτηριστικών σε φύλλα με υψηλό και χαμηλό περιεχόμενο ανθοκυανινώνΖέλιου, Κωνσταντίνα 07 October 2011 (has links)
Στα φύλλα κάποιων φυτικών ειδών παρουσιάζεται παροδική ερυθρότητα, λόγω συσσώρευσης ανθοκυανινών, η οποία μπορεί να είναι τόσο έντονη που να καλύπτεται το χρώμα της χλωροφύλλης. Δεδομένου ότι απορροφούν και στο ορατό φάσμα (κυρίως στην πράσινη και ελαφρώς στη μπλε και κίτρινη περιοχή) οι ανθοκυανίνες πρακτικώς λειτουργούν ως φίλτρα, αφού απορροφούν μέρος της προσπίπτουσας φωτοσυνθετικά ενεργής ακτινοβολίας και ανταγωνίζονται τις χλωροφύλλες στην απορρόφηση του φωτός. Εξ αιτίας αυτής της ιδιότητάς τους, ένας από τους ρόλους που τους έχει αποδοθεί είναι η φωτοπροστατευτική δράση, ενώ παράλληλα (και ανεξαρτήτως ρόλου που επιτελούν) έχει προταθεί ότι διαμορφώνουν ένα ιδιαίτερο μικροπεριβάλλον σκιάς στο εσωτερικό του φύλλου. Προκειμένου να διερευνηθεί η πιθανή συνεισφορά των ανθοκυανινών στη φωτοπροστασία αλλά και στην επαγωγή σκιόφιλων χαρακτήρων, στην παρούσα διατριβή παρακολουθήσαμε την πορεία των φωτοσυνθετικών και φωτοπροστατευτικών χαρακτηριστικών, παράλληλα με τη μεταβολή της ερυθρότητας, σε φύλλα του ίδιου είδους που διαφέρουν ως προς το ανθοκυανικό περιεχόμενο.
Εξετάστηκαν πέντε είδη, τρία ως κύρια πειραματόφυτα (Cistus creticus L., Photinia x fraseri Dress και Quercus coccifera L.) και δύο ως συμπληρωματικά στον προσδιορισμό της στοιχειομετρίας των δύο φωτοσυστημάτων (Ricinus communis L., Rosa sp. L.). Στο C. creticus, η παροδική ερυθρότητα εμφανίζεται στα ώριμα φύλλα και σχετίζεται με αβιοτικούς περιβαλλοντικούς παράγοντες, συγκεκριμένα το συνδυασμό χαμηλών θερμοκρασιών και υψηλών εντάσεων φωτός. Στα υπόλοιπα, η παροδική συσσώρευση ανθοκυανινών επάγεται από οντογενετικούς παράγοντες (νεαρά κόκκινα φύλλα που πρασινίζουν με την ωρίμανση). Στα P. x fraseri και R. communis, η χρωματική ποικιλομορφία αφορά φύλλα διαφορετικής ηλικίας, που ανήκουν όμως στο ίδιο άτομο (νεαρά κόκκινα-ώριμα πράσινα). Tα υπόλοιπα τρία είδη εμφανίζουν ενδο-ειδική ποικιλομορφία ως προς τον ανθοκυανικό χαρακτήρα, και επομένως συγκρίνονται φύλλα ίδιας κάθε φορά φυσιολογικής ηλικίας, που ανήκουν σε άτομα διαφορετικών φαινοτύπων.
Στην περίπτωση που οι ανθοκυανίνες λειτουργούν φωτοπροστατευτικά, αναμένεται τα φύλλα που τις περιέχουν να αντιμετωπίζουν μικρότερο κίνδυνο φωτοαναστολής και να έχουν μικρότερη ανάγκη για μη-φωτοχημική απόσβεση σε σύγκριση με τα αντίστοιχα πράσινα. Ωστόσο, από τις μετρήσεις του in vivo φθορισμού της χλωροφύλλης σε προ-σκοτεινιασμένα δείγματα (OJIP-ανάλυση) δεν διαπιστώθηκε τα ανθοκυανικά φύλλα να διαθέτουν κάποιο συγκριτικό πλεονέκτημα όσον αφορά στη φωτοσυνθετική τους λειτουργία. Αντιθέτως, τα κόκκινα φύλλα των ειδών P. x fraseri και C. creticus εκτός του ότι παρουσιάζουν ελαφρώς, αλλά στατιστικώς σημαντικά, χαμηλότερες τιμές μέγιστης φωτοχημικής απόδοσης του PSII (Fv/Fm) συναντούν και σαφώς μεγαλύτερους περιορισμούς στα μετέπειτα στάδια ηλεκτρονιακής ροής και μετατροπής της ενέργειας σε σχέση με τα αντίστοιχα πράσινα. Και στις δυο περιπτώσεις είναι πιθανό να συμβαίνει απενεργοποίηση κάποιων λειτουργικών ενεργών κέντρων του PSII. Στο Q. coccifera, τα όμοιας ηλικίας φύλλα των δύο φαινοτύπων δεν παρουσιάζουν διαφορές στις ανωτέρω παραμέτρους.
Οι μετρήσεις φθορισμού της χλωροφύλλης κάτω από συνθήκες ακτινικού φωτός έδειξαν ότι τα κόκκινα φύλλα έχουν όμοιες (P. x fraseri και C. creticus) ή χαμηλότερες (Q. coccifera) τιμές τρέχουσας φωτοχημικής απόδοσης (YII) και αντίστοιχα όμοιες ή υψηλότερες τιμές μη-φωτοχημικής απόσβεσης (YNPQ). Ωστόσο, λόγω της απορρόφησης των ανθοκυανινών, οι χλωροπλάστες των κόκκινων φύλλων δέχονται πραγματική ένταση φωτός χαμηλότερη της προσπίπτουσας, και επομένως τα ανωτέρω αποτελέσματα είναι αντίθετα των αναμενόμενων. Διαφωτιστικές προς αυτή την κατεύθυνση είναι οι μετρήσεις φθορισμού της κάτω (πράσινης) φυλλικής επιφάνειας σε φωτιζόμενα φύλλα των δύο φαινοτύπων του C. creticus. Σ’ αυτήν την περίπτωση, έχοντας παρακάμψει το πρόβλημα της απορρόφησης των ανθοκυανινών, τα φύλλα του κόκκινου φαινοτύπου εμφανίζουν υψηλότερη ανάγκη μη-φωτοχημικής απόσβεσης (YNPQ) και υψηλότερη ευαισθησία έναντι της φωτοαναστολής (YNΟ), παρ’ όλη τη θεωρούμενη προστασία των υπερκείμενων ανθοκυανινών της άνω επιφανείας. Η μεγαλύτερη ανάγκη μη φωτοχημικής απόσβεσης των κόκκινων φύλλων διαφαίνεται και από την υψηλότερη επένδυση σε συστατικά του κύκλου των ξανθοφυλλών (P. x fraseri) ή την καλύτερη λειτουργικότητα του κύκλου (C. creticus), σε συνθήκες έντονου φωτισμού. Μάλιστα, κατά την καταπονητική περίοδο του χειμώνα ο κόκκινος φαινότυπος του C. creticus εμφανίζει μείωση των περιεχομένων χλωροφυλλών, πιθανόν σε μια προσπάθεια περαιτέρω ελάττωσης της απορροφούμενης ενέργειας διεγέρσεως, παρ’ όλη την ύπαρξη του ανθοκυανικού φίλτρου.
Με βάση τα ανωτέρω ευρήματα, η υπόθεση του φωτοπροστατευτικού ρόλου των ανθοκυανινών δεν ενισχύεται, εκτός εάν υποτεθεί είτε ότι ο στόχος για την προστασία δεν συνδέεται με τη φωτοσύνθεση είτε ότι τα κόκκινα φύλλα εάν δεν τις διέθεταν θα ήταν σε πολύ πιο δυσμενή θέση, σε σύγκριση πάντα με τα πράσινα.
Όσον αφορά στη συνεισφορά των ανθοκυανινών στην επαγωγή σκιόφιλων χαρακτήρων βρέθηκαν κάποιες ισχυρές ενδείξεις προς αυτήν την κατεύθυνση, ανεξάρτητα από τον παράγοντα που επάγει τη συσσώρευσή τους. Έτσι, και στα πέντε είδη που εξετάστηκαν, παρατηρήθηκε αύξηση της σχετικής αναλογίας PSII/PSI στα κόκκινα φύλλα, η οποία είναι στατιστικά σημαντική (εκτός από την περίπτωση του Q. coccifera) και βαίνει μειούμενη με τη μείωση των περιεχομένων ανθοκυανινών. Αύξηση της αναλογίας PSII/PSI έχει συσχετισθεί με την ποιοτική αλλοίωση του φωτός σε συνθήκες σκιάς και στοχεύει στην εξισορροπημένη διέγερση των δύο φωτοσυστημάτων. Από τον τρόπο προσδιορισμού της (ως F686/F735), η ανωτέρω αναλογία δεν κάνει διάκριση μεταξύ φωτοσυλλεκτικής κεραίας και ενεργών κέντρων των δύο φωτοσυστημάτων. Μία άλλη παράμετρος, που θεωρείται τυπικός δείκτης σκιοφιλίας και μετρήθηκε στο σύνολο των πειραματοφύτων, είναι η αναλογία Chl a/b. Μειωμένη αναλογία Chl a/b σε συνθήκες σκιάς υποδεικνύει αύξηση των φωτοσυλλεκτικών κεραιών σε σχέση με τα ενεργά κέντρα. Στατιστικά σημαντική μείωση της ανωτέρω αναλογίας βρέθηκε στα κόκκινα φύλλα των ειδών C. creticus, Q. coccifera και Rosa sp.. Στα P. x fraseri και R. communis δεν παρατηρήθηκαν στατιστικά σημαντικές διαφορές μεταξύ κόκκινων και πράσινων φύλλων. Τέλος, στα είδη C. creticus, P. x fraseri και Q. coccifera προσδιορίσθηκε και η παράμετρος ABS/RC (όπως προκύπτει από την OJIP-ανάλυση), όπου παρατηρήθηκε στατιστικώς σημαντική αύξηση στα κόκκινα φύλλα των δύο πρώτων ειδών. Στο Q. coccifera η ίδια τάση δεν είναι στατιστικά σημαντική. Αύξηση της αναλογίας ABS/RC, η οποία αποτελεί θα λέγαμε έναν λειτουργικό περισσότερο δείκτη, μπορεί να αποδοθεί σε αυξημένο μέγεθος της φωτοσυλλεκτικής κεραίας ή/και σε μείωση του αριθμού των λειτουργικών ενεργών κέντρων του PSII. Από το συνδυασμό των παραπάνω αποτελεσμάτων διαφαίνεται ότι στα είδη C. creticus, Rosa sp. και Q. coccifera η επίδραση των ανθοκυανινών εντοπίζεται κυρίως σε αύξηση του μεγέθους της φωτοσυλλεκτικής κεραίας. Στα P. x fraseri και R. communis, η αύξηση του λόγου PSII/PSI αντανακλά πιθανώς μια αύξηση των ενεργών κέντρων του PSII. / Leaves of some plant species appear transiently red because of the accumulation of anthocyanins, at levels sufficient to mask the green chlorophyll color. Given that leaf anthocyanins absorb also in the visible spectrum (strongly in the green and less in the blue-violet and yellow region), they act as sunscreens attenuating part of the photosynthetically active radiation and hence they compete with chlorophylls for photon capture. Due to this attribute, one of the ascribed roles of leaf anthocyanins is the photoprotection of the photosynthetic mesophyll. Additionally (and regardless of the proposed role) it has been suggested that they shape a particular shade microenvironment in the leaf interior. In order to investigate these two hypotheses, in the present study we monitored the course of photosynthetic and photoprotective characteristics in leaves of the same species with different anthocyanic content.
Five species have been used, three of them as main experimental material (Cistus creticus L., Photinia x fraseri Dress και Quercus coccifera L.) and two additional (Ricinus communis L., Rosa sp. L.) for the assessment of the stoichiometry of the two photosystems. In Cistus creticus (L.) transient redness is induced in mature leaves by abiotic environmental factors, i.e. the combination of low temperatures and high light intensity during winter. In the rest species redness is developmentally determined (young red leaves which become red upon maturation). In P. x fraseri and R. communis the color variation concerns leaves of different age from the same individual (comparison of young red and mature green). The rest three species show intra-species variation of the anthocyanic trait. Thus, leaves of the same physiological age from different individuals and phenotypes (occupying the same habitat) were compared.
The photoprotective hypothesis of leaf anthocyanins entails that red leaves would be less sensitive against photoinhibition and have a reduced need of non-photochemical quenching, compared to their green counterparts. However, according to our chlorophyll fluorescence measurements with dark adapted samples (OJIP-analysis), anthocyanic leaves did not display a comparative advantage in their photosynthetic function. On the contrary, red leaves of P. x fraseri and C. creticus, apart from their slightly, yet statistically significant, lower maximum PSII yield (Fv/Fm), they also confront higher limitations in the further steps of excitation energy processing and its transformation compared to greens. In both species, a transformation of active PSII centers to non-QA -reducing ones, could be inferred. In Q. coccifera, no differences were observed between leaves of the same physiological age from the two phenotypes in the above parameters.
Moreover, chlorophyll fluorescence measurements in light acclimated samples have shown that red leaves have similar (P. x fraseri και C. creticus) or lower (Q. coccifera) PSII effective yield (YII) and similar or higher corresponding non-photochemical energy quenching (YNPQ). It has to be noted, however, that the actual PAR levels penetrating to the red leaf mesophyll are in fact lower compared to their green counterparts, due to anthocyanin absorption of the white and blue actinic light used during these measurements. Accordingly, for the same incident PAR, the opposite trend was expected in red leaves. In C. creticus, measurements performed in the lower leaf side by-pass this problem, since anthocyanins accumulate only in the palisade mesophyll (of the upper side) and the abaxial leaf surface is always green. In that case, red leaves showed a higher need for non-photochemical quenching (YNPQ) and a higher vulnerability to photoinhibition (YNΟ), in spite of the supposed photoprotective function of the overlying anthocyanins. The trend for an enhanced need of non-photochemical dissipation in red leaves is indicated also by the higher investment in “xanthophyll cycle” components (P. x fraseri) and/or the lower EPS values (P. x fraseri and C. creticus) during midday. In addition, during the superimposed winter stress, red leaves of C. creticus proceed to a Chl loss, possibly as an attempt to decrease further the absorbed excitation energy, in spite of the anthocyanic screen.
The above results weaken the photoprotective hypothesis for leaf anthocyanins, unless it is assumed either that photosynthesis is not the target for protection or that red leaves would be in a worse position, compared to greens, if anthocyanins were absent.
Some of our results support the shade acclimation hypothesis in red leaves, irrespectively of the factor that induces the accumulation of anthocyanins. In all tested species, red leaves display a higher, statistically significant, relative PSII/PSI ratio compared to greens (except of Q. coccifera, where the same was observed as a trend). PSII/PSI ratio declines in parallel with the decrease of anthocyanin accumulation. A higher PSII/PSI ratio is considered as an acclimation to the altered light quality under shade conditions in order to adjust the excitation pressure of two photosystems.
Chl a/b was statistically lower in red leaves of C. creticus, Q. coccifera και Rosa sp., while in P. x fraseri and R. communis no differences were observed. A decrease in Chl a/b ratio is a typical shade acclimation feature, indicating an increased ratio of light harvesting antennae per reaction center. Finally, another parameter indicative of shade acclimation is the mean antenna size (calculated as ABS/RC ratio), which was considerably higher in red leaves of C. creticus and P. x fraseri (again in Q. coccifera only a similar trend was observed). Yet, according to the JIP-analysis the antenna size is expressed per active RC, hence the increased ABS/RC ratio could be the combined effect of an increase in the antenna size and a decrease in active PSII centers.
The combination of the above results indicates that anthocyanin accumulation in C. creticus, Rosa sp. and Q. coccifera contributes mainly to a higher relative size of LHC. In the case of P. x fraseri and R. communis, the enhanced PSII/PSI ratio of red leaves reflects rather an increase of PSII centers.
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Investigations of the structural dynamics of the water and proton channels in Photosystem IIAli, Rana Emadeldin Hussein 12 April 2022 (has links)
Bei der lichtinduzierten Oxidation von Wasser im Photosystem II (PSII) werden zwei wassermoleküle im katalytischen Zyklus des Metallclusters (Mn4CaO5) benötigt, und vier Protonen aus dem Cluster in den Lumen abgegeben. Daher ist es für das Verständnis des Mechanismus´ der Wasseroxidation von entscheidender Bedeutung, die Veränderung der Protonierungszustände am cluster während der Katalyse zu untersuchen. Hierbei sollten sowohl die Wasserkanäle für die Zuführung der Substratwassermoleküle als auch die Transportwege für die Freisetzung der Protonen untersucht werden. Deshalb wurde in meiner ersten Veröffentlichung ein neues Protokoll entwickelt, um einzelne große Kristalle von dPSII mit einer Länge von ~3 mm in der Längsachse zu züchten. Diese Kristalle mit einer Auflösung von ca. 8 Å gemessen. Um eine höhere Auflösung zu erzielen, ist die Verbesserung der Kristallqualität essenziell. Daher wurde in meiner zweiten Veröffentlichung die Struktur des Detergens-Protein-Komplexes von dPSII mit βDM, durch Anwendung von SANS in Kombination mit SAXS untersucht. Die Ergebnisse zeigten, dass βDM eine monomolekulare Schicht um dPSII bildet. Darüber hinaus konnten freie Mizellen von βDM in der Lösung nachgewiesen werden. Damit ist eine weitere Optimierung der βDM-Konzentration in der Proteinlösung erforderlich, um die Bildung von freien Mizellen zu minimieren. In meiner dritten Veröffentlichung wurde die strukturelle Dynamik in den Wasserkanälen, während des S2-S3 Übergangs mit Hilfe der XFEL untersucht. Ein Datensatz mit einer hohen Auflösung von 1,89 Å wurde durch die Zusammenführung von Daten gewonnen, die während des S2-S3 Übergangs gesammelt wurden. In Anbetracht der Analyse der zusammengeführten Daten und der einzelnen Zeitpunkte, die während des S2-S3 Übergangs gesammelt wurden, ist es wahrscheinlich, dass ein Substratwasser durch den O1-Kanal geliefert wird. Im Gegensatz dazu wird ein Proton aus dem Cluster durch den Cl1 Transportweg in Richtung Lumen freigesetzt. / The light-induced oxidation of water in Photosystem II (PSII) requires incorporating two water molecules in the catalytic cycle of the active metal cluster (Mn4CaO5). Furthermore, four protons are released towards the bulk from the cluster. Therefore, tracking the change of protonation states at the active catalytic site and the surrounding protein side chains during catalysis and elucidating the pathways of water substrate insertion and proton release are crucial to understanding the water oxidation mechanism. Therefore, in the first study of my work, a new protocol was developed to grow single large dPSIIcc crystals with a length of ~3 mm in the long axis. These crystals, soaked in D2O containing buffer, diffracted to about 8 Å resolution. Improving the crystal quality is crucial for achieving a better resolution. Consequently, in the second study of my work, the structure of the detergent-protein complex of βDM-dPSIIcc has been investigated by applying SANS in combination with SAXS. The results showed that βDM is forming a monomolecular layer around the dimeric PSII core complex (dPSIIcc). Moreover, the SAXS data detected a peak assigned to the free micelles of βDM. These results raise the necessity to optimize the βDM concentration in the protein solution to avoid the possible excess of free micelles. In the third study of my work, the structural dynamics in the water channels connecting the cluster to the lumen during the S2 S3 transition were investigated using serial femtosecond XFEL. A high-resolution data set was obtained at a resolution of 1.89 Å by combining data collected at RT. Considering the analysis of the combined data and the individual time points collected during the S2 S3 transition, it is likely that the substrate water insertion into the open coordination site of the Mn1 ion is delivered through the O1 channel. In contrast, a proton from the cluster is released towards the bulk through the Cl1 A channel.
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Broken symmetry density functional theory studies of multinuclear manganese metalloproteinsBeal, Nathan James January 2017 (has links)
The photosynthetic water splitting reaction performed in green plants, algae and cyanobacteria is perhaps one of the most important reactions on the planet. The reaction is catalysed by a tetranuclear manganese cluster that is driven by sunlight and as such has received a high degree of interest in the context of solar fuels research. Due to the intricacy of the bioinorganic systems, the molecular mechanisms of the reactions are unknown and structural elucidation of the active sites is complicated. Computational techniques can provide considerable support in the analysis and interpretation of the complex EPR spectra of such biological systems. In this work, the molecular and electronic structures of several multinuclear manganese containing bioinorganic systems are investigated using BS-DFT. A particular focus of this work is the provision of high quality calculated EPR parameters yielding structural and mechanistic insight. In the first results chapter, the MnIIIMnIV superoxidised state of manganese catalase as well as azide inhibited manganese catalase is studied. Several variants are proposed and analysed on the basis of their calculated EPR parameters. The results presented in this chapter offer a new interpretation of previous experimental assignments. Chapter 6 features investigations on the S2 state of the Oxygen Evolving Complex of Photosystem II. In this chapter both the native OEC as well as the strontium substituted OEC are studied, in order to investigate how replacement of the calcium ion affects the structure of the OEC. The final results chapter presents calculations on the split signal S2Yz dot radical formed on the transition from the S2 to S3 state, as well as studying the S3 state. The calculation of various EPR hyperfine couplings and their comparison with available experimental data has provided key insights into the electronic structure of the OEC.
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