Mémoire(M. Ress. Renouv.)--Université du Québec à Chicoutimi, 1997. / Document électronique également accessible en format PDF. CaQCU

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009. / Committee Chair: Bridgette Barry; Committee Member: El-Sayed, Mostafa; Committee Member: Fahrni, Christoph; Committee Member: Kröger, Nils; Committee Member: McCarty, Nael. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (Ph. D.)--University of Akron, Dept. of Chemistry, 2005. / "August, 2005." Title from electronic dissertation title page (viewed 09/24/2005). Advisor, David A. Modarelli; Committee members, Matthew Espe, Michael Taschner, Chrys Wesdemiotis, Stephanie Lopina; Department Chair, David Perry; Dean of the College, Charles B. Monroe; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.

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Οι μορφολογικές, φυσιολογικές και βιοχημικές προσαρμογές των φυτών (ή φύλλων) με διαφορετική έκθεση στην ηλιακή ακτινοβολία (σε συνθήκες πλήρους φωτός ή φυσικής σκίασης στο εσωτερικό της φυτικής κόμης) έχουν μελετηθεί και καταγραφεί λεπτομερώς. Στην παρούσα εργασία επανεξετάσαμε κάποιες από τις φυσιολογικές αυτές προσαρμογές, εκμεταλλευόμενοι τα πλεονεκτήματα της χρήσης σύγχρονων μεθόδων μέτρησης του in vivo φθορισμού της χλωροφύλλης. Επίσης, διερευνήθηκε η ύπαρξη προσαρμογών της φωτοσυνθετικής συσκευής σε διαφορετική ένταση φωτός, οι οποίες δεν ήταν είχαν μελετηθεί διεξοδικά. Πιο συγκεκριμένα, χρησιμοποιήθηκαν διαφορετικές τεχνικές φθορισμομετρίας, με τις οποίες μελετήθηκαν συγκριτικά αφ’ ενός οι κβαντικές αποδόσεις και οι περιορισμοί στα επιμέρους στάδια της φωτοσυνθετικής ροής ηλεκτρονίων και αφ’ ετέρου το ποσοστό συμμετοχής της κυκλικής ροής ηλεκτρονίων γύρω από το PSI και της φωτοαναπνοής στα φύλλα φωτός και σκιάς. Με βάση τα αποτελέσματά μας, θεωρούμε ότι το JIP-test μπορεί να προσφέρει αξιόπιστα και αξιοποιήσιμα αποτελέσματα. Πιο συγκεκριμένα, στα φύλλα που είναι προσαρμοσμένα στη σκιά φαίνεται να υπάρχει ένα εμπόδιο στη γραμμική ροή ηλεκτρονίων που εντοπίζεται γύρω από το φωτοσύστημα Ι. Οι παράμετροι που υποδεικνύουν αυτή την κατάσταση δείχνουν ότι περιοριστικός παράγοντας της ροής ηλεκτρονίων φαίνεται να είναι το μικρότερο ποσό των ενεργών κέντρων του PSI (1-VI) σε συνδυασμό με τα μικρότερα αποθέματα των τελικών αποδεκτών ηλεκτρονίων του (1/VI). Αυτό επιβεβαιώνεται από τη μικρότερη πιθανότητα μεταφοράς των ηλεκτρονίων από τους ενδιάμεσους φορείς στους τελικούς αποδέκτες ηλεκτρονίων του PSI (δRo) στα φύλλα σκιάς. Επιπρόσθετα, στην πλειοψηφία των φυτικών ειδών που εξετάστηκαν, η διαφορετική ποιότητα φωτός που δέχονται τα φύλλα σκιάς (φως εμπλουτισμένο σε βαθύ ερυθρό, που διεγείρει επιλεκτικά το PSI) σε σχέση με τα φύλλα φωτός φαίνεται να αυξάνει την κυκλική ροή ηλεκτρονίων γύρω από το PSI. Σε αυτά τα είδη οι προαναφερθείσες παράμετροι του JIP-test έχουν καλή συσχέτιση με το μέγεθος της κυκλικής ροής ηλεκτρονίων. Τέλος, μελετώντας τη φωτοαναπνοή, προκύπτει ότι στα φύλλα φωτός το ποσοστό του κύκλου C2 αυξάνεται με την αύξηση της φωτεινής ακτινοβολίας. Τα φύλλα σκιάς από την άλλη, φαίνεται να έχουν ένα όριο ως προς την προσαρμογή τους σε υψηλές εντάσεις φωτός. / The morphological and biochemical adjustments of plant leaf to high and low light intensity have been widely studied and recorded over the past years. In the present work, some of those physiological adjustments have been re-evaluated with the use of modern methods. In parallel, novel aspects of the light/shade acclimation syndrome were sought. Our results indicate that the JIP-test is quite useful for assessing parameters related to the function of both photosystems. In shade leaves, a lower content of PSI reaction centers (1-VI) combined with a smaller pool size of final electron acceptors of PSI seem to create an obstacle in linear electron flow around photosystem I. This is confirmed by the lower efficiency of electron transfer between intermediate carriers to the reduction of end electron acceptors of PSI (δRo). In addition, in the majority of plant species examined, shade leaves have higher rates of cyclic electron flow around PSI, which is probably caused by the quality of incident light (enriched in far red, FR). In these species there is good correlation between cyclic electron flow and the JIP-test parameters mentioned above. Photorespiration is known to be higher when the plant is under stress. Light acclimated leaves seem confirm that theory as they have higher oxygenase activity of Rubisco with increasing light intensity. On the other hand, shade leaves seem unable to fully adjust to very high light intensity.

Little is known about the evolution and ecology of crassulacean acid metabolism (CAM) in the genus Puya Molina. CAM is a photosynthetic pathway typified by nocturnal CO2 fixation and is regarded as a water-saving mechanism. Puya is one of the largest genera in the pineapple family (Bromeliaceae), with 226 species distributed across the Andes to Costa Rica and the Guiana Shield, and from sea level to 5000 m. About 21% of Puya species are CAM and at least 10 of these CAM species occur above 3000 m. The main aim of this thesis was to uncover new evidence to understand the ecophysiology and evolution of CAM in the montane genus Puya. The prevalence of CAM and C₃ species in Puya was estimated from carbon isotope values of 161 species. The climatic niche of constitutive CAM species and C₃ species of Puya was modelled using georeferenced herbarium records and climatic variables to evaluate the differences between their niches. The evolution of CAM in Puya was investigated by reconstructing the ancestral photosynthetic pathway on an AFLP phylogeny and by studying positive selection in the genes encoding the key enzyme phosphoenolpyruvate carboxylase (PEPC). The coldresistance and the thermal lability of PEPC was investigated for high- and low- elevation CAM species of Puya to explore the potential molecular adaptations of CAM plants in high-elevation environments. The present study concludes that the common ancestor of Puya was a cold-resistant plant. This is suggested to explain the prevalence of Puya at highelevations. The evolution of CAM was correlated with changes in the climatic niche, and occurred multiple times in Puya. These multiple origins were not independent because the common ancestor of Puya was likely to be a weak CAM plant (based on a diagnostic Arg679 residue in the PEPC sequence). It is likely that populations of P. chilensis gained CAM by introgression with P. alpestris ssp. zoellneri. Weak CAM photosynthesis and coldxv resistance allowed Puya to colonise the Andes from the south to the north; and, in the process, constitutive CAM and C₃ evolved. The later-evolving species in the genus are suggested to have lost their capacity for CAM as they radiated into more mesic habitats during their colonisation of the northern Andes.

abstract: Rhodoferax antarcticus strain ANT.BR, a purple nonsulfur bacterium isolated from a microbial mat in Ross Island, Antarctica, is the first described anoxygenic phototrophic bacterium that is adapted to cold habitats and is the first beta-proteobacterium to undergo complete genome sequencing. R. antarcticus has unique absorption spectra and there are no obvious intracytoplasmic membranes in cells grown phototrophically, even under low light intensity. Analysis of the finished genome sequence reveals a single chromosome (3,809,266 bp) and a large plasmid (198,615 bp) that together harbor 4,262 putative genes. The genome contains two types of Rubiscos, Form IAq and Form II, which are known to exhibit quite different kinetic properties in other bacteria. The presence of multiple Rubisco forms could give R. antarcticus high metabolic flexibility in diverse environments. Annotation of the complete genome sequence along with previous experimental results predict the presence of structural genes for three types of light-harvesting (LH) complexes, LH I (B875), LH II (B800/850), and LH III (B800/820). There is evidence that expression of genes for the LH II complex might be inhibited when R. antarcticus is under low temperature and/or low light intensity. These interesting condition-dependent light-harvesting apparatuses and the control of their expression are very valuable for the further understanding of photosynthesis in cold environments. Finally, R. antarcticus exhibits a highly motile lifestyle. The genome content and organization of all putative polar flagella genes are characterized and discussed. / Dissertation/Thesis / M.S. Molecular and Cellular Biology 2011

Chez les eucaryotes, la photosynthèse a lieu dans le chloroplaste, un organite spécifique de la cellule végétale et caractérisé par différents compartiments : (i) l'enveloppe, la double membrane qui délimite le chloroplaste ; (ii) le stroma, phase aqueuse principalement composée de protéines solubles et (iii) un système membranaire interne, les thylacoïdes, qui contiennent les complexes photosynthétiques. Les thylakoïdes forment un réseau tridimensionnel continu et sont différenciés en deux domaines physiques distincts : des empilements de vésicules de membrane (appelés granas ou BBY) et des extensions de membrane simple (lamelles stromales). Les complexes majeurs de la photosynthèse ne sont pas distribués de manière égale dans cette membrane à cause de contraintes électrostatiques et stériques. Ainsi, le photosystème I et l'ATP-synthétase sont enrichis dans les granas, le photosystème II dans les lamelles stromales alors que d'autres complexes, comme le cytochrome b6f, auraient une répartition équivalente entre granas et lamelles stromales. Pour faire face aux variations environnementales de lumière (en qualité et quantité), les plantes ont développé des processus pour moduler leur capacité d'absorption et d'utilisation de la lumière par les photosystèmes, processus regroupés sous le terme de « quenching non photosynthétique ou NPQ ». Dans le cadre de ma thèse, je me suis intéressé à deux composants du NPQ, les états de transition et la dissipation sous forme de chaleur (partie qE).Le premier objectif de ma thèse a été d'identifier de nouveaux acteurs impliqués dans les transitions d'état et ceci en étudiant la relocalisation de protéines au sein des sous-compartiments des thylacoïdes par une approche protéomique. En effet, il a été montré que certaines antennes collectrices de lumière sont réorganisées dans les membranes photosynthétiques lors des transitions d'état. Jusqu'à présent, aucune description exhaustive de la composition et distribution des protéines dans les sous-compartiments de thylacoïdes n'avait été réalisée. J'ai donc dans un premier temps développé des protocoles de purification des sous-compartiments des thylakoïdes (granas et lamelles stromales) à partir de chloroplastes de plantes sauvage d'Arabidopsis thaliana. Ensuite, grâce à une approche d'analyse protéomique semi-quantitative, nous avons pu déterminer la localisation d'environ 300 protéines des thylacoïdes. Les résultats suggèrent que la localisation de complexes photosynthétiques est beaucoup plus dynamique que celle jusqu'à lors proposée. En effet, même s'ils sont préférentiellement identifiés dans un sous-compartiment, certains complexes photosynthétiques présentent une double localisation qui était inattendue. De plus, la composition en sous-unités de ces complexes diffère selon leur localisation, dans les granas et dans les lamelles stromales, suggérant l'existence de processus de régulation de la photosynthèse jusqu'à lors insoupçonnés. Cette approche a ensuite été appliquée sur des plantes mutantes d'Arabidopsis affectées dans les transitions d'état afin d'identifier des protéines pouvant être impliquées dans ce processus d'adaptation. En parallèle, je me suis intéressé au qE . L'activation de ce mécanisme n'est pas constitutive et nécessite la formation d'un gradient de pH entre le stroma et le lumen des thylacoïdes (ΔpH). L'objectif de l'étude a été d'identifier des acteurs pouvant contrôler la formation de ce gradient de pH. Pour cela, nous nous somme focalisés sur le rôle d'un transporteur de potassium récemment caractérisé, TPK3. Grâce à des approches biophysiques et biochimiques, nous avons démontré que TPK3 est impliqué, in vivo, dans la modulation des deux composantes de la force proton motrice (pmf), le gradient de pH (ΔpH) et la différence de potentiel (Δψ). En contrôlant la répartition de la force proton motrice,TPK3, permet une utilisation correcte de la lumière en dissipant l'excès d'énergie. / Within higher plants and algae, photosynthesis is carried out in the chloroplast. Structurally, chloroplasts are organized in (i) the envelope, a double membrane system surrounding the chloroplast (ii) the stroma, the aqueous space which mainly contains soluble proteins and the (iii) thylakoids, a three-dimensional membrane network where photosynthetic electron transport reactions occur. Thylakoids are non-homogeneously folded, and comprise two major domains: (i) the grana-BBY, which are stacks of thylakoids particularly enriched in photosystem II, LHCII (the antenna-protein complex responsible for light harvesting) and (ii) the stroma lamellae, which are unstacked thylakoids connecting grana stacks enriched in photosystem I and ATP synthase. Plants can respond to changes in the environmental light conditions by several means as those which are collectively called non-photochemical quenching or NPQ. During my thesis, I mainly focused on two components of the NPQ: state transition (qT) and high-energy state quenching (qE).State transitions is the process by which PSII-antenna proteins are re-organized between stroma-lamellae and grana-BBY following changes in ambient light both of intensity and spectral composition. State transitions play a key role in the plant adaptation but many aspects of this process remain unclear. The main objective of my thesis was to study the thylakoid protein re-localization between stroma-lamellae and grana-BBY during state transitions using a proteomic-based approach. At this aim I firstly focused on the sub-thylakoid protein localization in Arabidopsis WT and I developed different protocols for the purification of the two sub-compartments (stroma-lamellae and grana-BBY) starting from intact chloroplasts. Later, thanks to a semi-quantitative proteomic approach, I determined the precise localization of around 300 thylakoid proteins in Arabidopsis WT. Results suggested that the localization of the different photosynthetic complexes is much more dynamics than previously hypothesized. In fact, even if characterized by a preferential localization, some photosynthetic complexes displayed an unexpected double localization. Moreover the subunit composition of these complexes was found to vary according to their localization (BBY or stroma-lamellae) suggesting the existence of mechanisms of regulation which have never been evidenced before. Later, we used the same mass-spectrometry-based approach on two different Arabidopsis mutants unable to perform state transitions. The objective was to highlight the involvement of other proteins (other than LHCII) which could possibly be re-localized within the photosynthetic membrane during state transitions. In the second part of my thesis, I focused on the high-energy state quenching component of the NPQ. qE allows the plant to dissipate excessive light energy as heat. This process it's not constitutive but need to be activated by the formation of a difference in the pH between the stroma and the thylakoid lumen (ΔpH). The objective of the study was to identify new possible actors in the regulation of the ΔpH formation. At this purpose I focused on a recently characterized potassium channel, TPK3. Thanks to a biophysical and biochemical approach, we demonstrated that TPK3 is involved, in vivo, in the modulation of the two components of the proton motive force (pmf), the ΔpH and the difference in the electric field Δψ. By controlling the repartition of the pmf, TPK3, controls also the formation of the NPQ and directly affects light utilization and dissipation in vivo. This avoids serious damages to the photosynthetic chain when plants are exposed to high-light conditions

abstract: Most of the sunlight powering natural photosynthesis is absorbed by antenna arrays that transfer, and regulate the delivery of excitation energy to reaction centers in the chloroplast where photosynthesis takes place. Under intense sunlight the plants and certain organisms cannot fully utilize all of the sunlight received by antennas and excess redox species are formed which could potentially harm them. To prevent this, excess energy is dissipated by antennas before it reaches to the reaction centers to initiate electron transfer needed in the next steps of photosynthesis. This phenomenon is called non-photochemical quenching (NPQ). The mechanism of NPQ is not fully understood, but the process is believed to be initiated by a drop in the pH in thylakoid lumen in cells. This causes changes in otherwise nonresponsive energy acceptors which accept the excess energy, preventing oversensitization of the reaction center. To mimic this phenomenon and get insight into the mechanism of NPQ, a novel pH sensitive dye 3'6'-indolinorhodamine was designed and synthesized which in a neutral solution stays in a closed (colorless) form and does not absorb light while at low pH it opens (colored) and absorbs light. The absorption of the dye overlaps porphyrin emission, thus making energy transfer from the porphyrin to the dye thermodynamically possible. Several self-regulating molecular model systems were designed and synthesized consisting of this dye and zinc porphyrins organized on a hexaphenylbenzene framework to functionally mimic the role of the antenna in NPQ. When a dye-zinc porphyrin dyad is dissolved in an organic solvent, the zinc porphyrin antenna absorbs and emits light by normal photophysical processes. Time resolved fluorescence experiments using the single-photon-timing method with excitation at 425 nm and emission at 600 nm yielded a lifetime of 2.09 ns for the porphyrin first excited singlet state. When acetic acid is added to the solution of the dyad, the pH sensitive dye opens and quenches the zinc porphyrin emission decreasing the lifetime of the porphyrin first excited singlet state to 23 ps, and converting the excitation energy to heat. Under similar experimental conditions in a neutral solution, a model hexad containing the dye and five zinc porphyrins organized on a hexaphenylbenzene core decays exponentially with a time constant of 2.1 ns, which is essentially the same lifetime as observed for related monomeric zinc porphyrins. When a solution of the hexad is acidified, the dye opens and quenches all porphyrin first excited singlet states to <40 ps. This converts the excitation energy to heat and renders the porphyrins kinetically incompetent to readily donate electrons by photoinduced electron transfer, thereby mimicking the role of the antenna in photosynthetic photoprotection. / Dissertation/Thesis / Ph.D. Chemistry 2012

abstract: The photosynthetic reaction center is a type of pigment-protein complex found widely in photosynthetic bacteria, algae and higher plants. Its function is to convert the energy of sunlight into a chemical form that can be used to support other life processes. The high efficiency and structural simplicity make the bacterial reaction center a paradigm for studying electron transfer in biomolecules. This thesis starts with a comparison of the primary electron transfer process in the reaction centers from the Rhodobacter shperoides bacterium and those from its thermophilic homolog, Chloroflexus aurantiacus. Different temperature dependences in the primary electron transfer were found in these two type of reaction centers. Analyses of the structural differences between these two proteins suggested that the excess surface charged amino acids as well as a larger solvent exposure area in the Chloroflexus aurantiacus reaction center could explain the different temperature depenence. The conclusion from this work is that the electrostatic interaction potentially has a major effect on the electron transfer. Inspired by these results, a single point mutant was designed for Rhodobacter shperoides reaction centers by placing an ionizable amino acid in the protein interior to perturb the dielectrics. The ionizable group in the mutation site largely deprotonated in the ground state judging from the cofactor absorption spectra as a function of pH. By contrast, a fast charge recombination assoicated with protein dielectric relaxation was observed in this mutant, suggesting the possibility that dynamic protonation/deprotonation may be taking place during the electron transfer. The fast protein dielectric relaxation occuring in this mutant complicates the electron transfer pathway and reduces the yield of electron transfer to QA. Considering the importance of the protein dielectric environment, efforts have been made in quantifying variations of the internal field during charge separation. An analysis protocol based on the Stark effect of reaction center cofactor spectra during charge separation has been developed to characterize the charge-separated radical field acting on probe chromophores. The field change, monitored by the dynamic Stark shift, correlates with, but is not identical to, the electron transfer kinetics. The dynamic Stark shift results have lead to a dynamic model for the time-dependent dielectric that is complementary to the static dielectric asymmetry observed in past steady state experiments. Taken together, the work in this thesis emphasizes the importance of protein electrostatics and its dielectric response to electron transfer. / Dissertation/Thesis / Ph.D. Physics 2012