Dissertationen zum Thema „Rubisco“

Atmospheric CO2 fixation is catalysed by the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Despite the critical role Rubisco plays in the biosphere, it is a slow catalyst that poorly discriminates between substrate CO2 and O2, and is often the rate-limiting step of photosynthesis. These deficiencies have made improving Rubisco function a major target in steps towards enhancing leaf photosynthesis rate and plant growth. In pursuing this goal, one strategy is to identify solutions for improving the kinetics of plant Rubisco and introduce these altered Rubisco isoforms into crops (Sharwood, 2017). Unfortunately efforts to improve the performance of plant Rubiscos have so far proven unsuccessful. Success appears to be hindered by Rubisco’s complex catalytic mechanism and the extensive array of accessory proteins needed to assemble the eight large (L) and eight small (S) subunits into a L8S8 complex and to maintain it in a functional form. The complex catalytic chemistry of Rubisco is prone to inhibition by various sugar-phosphate ligands. These include catalytic misfire products as well as its own substrate, ribulose-1,5-bisphosphate (RuBP), which forms an inactive Rubisco-RuBP (ER) complex when bound to non-carbamylated (i.e. non-activated) catalytic sites. Release of these inhibitors is mediated by the AAA+ (ATPases associated with a variety of cellular activities) protein Rubisco activase (Rca); a Rubisco-specific metabolic repair chaperone for which there are convergently evolved structural isoforms found in most photosynthetic organisms (Bhat et al., 2017a; Mueller-Cajar, 2017). Rca is now also a promising target for manipulating photosynthetic performance, especially under elevated temperature stress and fluctuating light (Carmo-Silva & Salvucci, 2013; Kumar et al., 2009; Kurek et al., 2007; Scafaro et al., 2016; Yamori et al., 2012). Critical to future Rubisco and Rca engineering efforts in plants is a greater mechanistic understanding of how Rca interacts with Rubisco, and the extent to which the kinetics of Rca differ between plant species. Utilising chloroplast transformation in tobacco, this thesis investigated the H9 helix and N-terminal region of Rca, and the βC-βD loop and C terminus of the Rubisco large (L-) subunit (RbcL) to determine their role in the Rubisco-Rca interaction in plants using both in vivo and in vitro methods. Capitalising on the regulatory incompatibility between Rubisco and Rca enzymes from Solanaceae and non-Solanaceae species, this thesis examined how mutations in the tobacco (Solanaceae) and Flaveria (F. bidentis and F. floridana, non-Solanaceae) enzymes influenced their kinetic properties and the Rca activation potential for inhibited ER complexes. The ability to continuously monitor the rate of ER activation provided by a NADH-coupled spectrophotometric method made it preferential to the alternative two-step 14CO2-fixation assay method. Optimising the assay conditions, particularly the accurate determination of enzyme concentrations, was crucial for obtaining reproducible kinetic values. Normalising the measurements of ER activation rate relative to the Rca ATPase activity (kcatATP ) proved critical in providing a means to determine the effect the Rca and Rubisco mutations had on interactivity. Using these assay conditions, mutagenic analyses of the Rca H9 helix found residue 317 plays a dominant role in defining the Rubisco selectivity of NtRca, and is critical for functional FbRca. N-terminal domain swapping modifications to NtRca and FbRca revealed amino acids at the junction of the N-terminal extension and the AAA+ module (residues 50 to 85) significantly influenced both kcatATP and the potential to interact with their cognate Rubiscos, but had little influence on altering interaction with the non-native Rubisco. In vivo analysis of Rubisco-Rca interaction was undertaken using tobacco genotypes producing differing tobacco Rubisco or recombinant Flaveria Rubisco mutants generated by chloroplast transformation of the rbcL gene into the plastome. In vitro analysis using purified enzymes uniformly showed residues 89 and 94 of the βC-βD loop of the F. bidentis, F. floridana and tobacco RbcLs influence interaction with Rca. In contrast, in vivo leaf gas exchange measurements of photosynthetic induction in the tobacco genotypes producing mutant RbcL showed mutation of residues 89 and 94 had little to no influence on their capacity to be activated by the endogenous NtRca. Combinatorial in vitro analyses using the range of RbcL and Rca mutants generated in this study were undertaken to better understand their interaction mechanism. It was found the avidity of Rubisco-Rca interactions were primarily defined by residue 94 in RbcL and 317 in Rca, and less so by RbcL residue 89 and Rca residue 320. Mutations to the RbcL C terminus had little influence on plant Rubisco-Rca interactions except when coupled with mutations that disrupted the connection between the RbcL βC-βD loop and the Rca H9 helix. The cumulative effect of the RbcL βC-βD loop and C-terminal modifications on Rubisco-Rca interactivity of these mutants implies involvement of the RbcL C terminus in the activation mechanism of Rca. This thesis showcases a novel approach to study the mechanism of plant Rubisco-Rca interactions by generating mutant Rubisco variants through tobacco plastome transformation. The work highlights large variations in the kinetics of NtRca (Solanaceae) and FbRca (non-Solanaceae) and provides evidence for variations in their Rubisco activation mechanism. For instance, while a D317K substitution in NtRca significantly enhanced its ability to activate Flaveria Rubisco, the reciprocal K317D mutation in FbRca had little impact on improving its capacity to activate tobacco Rubisco. Improving our understanding of the mechanistic differences in plant Rubisco-Rca interactions is critical for future Rubisco engineering endeavours. It is especially relevant in the context of ongoing efforts to transplant more efficient Rubisco variants into leaf chloroplasts where co-transformation with an appropriate Rca may be needed. Potential transgenic strategies for co-engineering compatible Rubisco and Rca isoforms in planta are also discussed.

Rubisco activase operates as the chaperone responsible for maintaining the catalytic competency of Ribulose 1,5-bisphophate carboxylase oxygenase (Rubisco) in plants. Rubisco is notoriously inefficient, rapidly self-inactivating under physiological conditions. Rubisco activase uses the power released from the hydrolysis of ATP to power a conformational change in Rubisco, reactivating it. Rubisco activase has been previously shown to form a large range of species in solution; however, little has been done to relate the size of oligomeric species and physiological activity. In this thesis data is presented from a range of biophysical techniques including analytical ultracentrifugation, static light scattering, and small angle X-ray scattering combined with activity assays to show a strong relationship between oligomeric state and activity. The results suggest that small oligomers comprising 2-4 subunits are sufficient to attain full specific activity, a highly unusual property for enzymes from the AAA+ family. Studies utilising a number of Rubisco activase variants enabled the determination of how Rubisco and Rubisco activase may interact within a plant cell. A detailed characterisation of the α-, β-, and a mixture of isoforms further broadened our knowledge on the oligomerisation of Rubisco activase. Of particular importance was the discovery of a thermally stable hexameric Rubisco activase variant. It is hoped that these findings may contribute to development of more heat tolerant Rubisco activase and lead research into more drought resilient crop plants.

In this diploma work influence of needle age at Rubisco activity and content in Norway spruce (Picea abies) was studied. The plants were cultivated in conditions with ambient (A) CO2 concentration (350 µmol CO2/mol) and elevated (E) CO2 concentration (700 µmol CO2/mol). Sampling was done two times during the growing season (in the middle of June and in the end of September) were taken. Initial and total Rubisco activities were measured spectrophotometrically. Rubisco content was determined by SDS–PAGE method. Rubisco activity in 18-months-old needles was in E higher than in A. Rubisco contents in current-year needles and one-year-old needles were in A higher than in E in September. These differences were statistically significant that demonstrates the down-regulation of Rubisco content in conditions of elevated CO2 concentration. It seems the course of activities and content depending on age of the needles are antiparallel, that means that decrease of content is followed by increase of activity.

Almost every carbon atom that our bodies are made of and clothed in, at one point or another saw the active site of the enzyme, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). This enzyme, one of the largest in nature at ~550 kDa, is also said to be the most abundant in nature, constituting up to 50% of soluble protein in land plants. It is however, notoriously inefficient at fixing carbon dioxide, due to its slow catalytic turnover, low affinity for atmospheric CO2, and its use of both CO2 and O2 as substrates for competing reactions. For this reason, Rubisco has also been one of the most intensively studied enzymes. The potential harvest yield of crops has been shown to be limited by the rate at which atmospheric carbon is fixed by Rubisco. As a result much focus is on studying and engineering Rubisco in order to increase the overall efficiency of biomass production, to cope with the fuel, fibre and food needs of a growing global population. Since Rubisco is one component of a system, the demand for developing tools to study and engineer Rubisco in light of its wider interactome and regulatory networks, is ever increasing. This thesis describes the development of tools to study the function of Rubisco. It includes the development of a novel method for purifying the enzyme using hydrophobic interaction chromatography. Different populations of Rubisco were isolated with distinct maximal rates of carboxylation, possibly reflecting differences in hydrophobic characteristics. It also contains the development of a spectroscopic activity assay for Rubisco activity, and an enzyme-linked immunosorbent assay (ELISA) for Rubisco and its catalytic chaperone, Rubisco Activase. These tools were then applied to investigate the influence of cellulose biosynthesis inhibition (CBI) on Rubisco in Arabidopsis seedlings, to investigate any potential sink-regulation of the activity or levels of Rubisco. It was found that specific Rubisco activity was reduced as a result of CBI, and rescued by providing osmotic support, thus implicating turgor pressure as a potential mechanism. Finally, preliminary results were obtained which demonstrate the potential of mass spectrometry for measuring relative levels of Rubisco ions generated by MALDI (matrix-assisted laser desorption ionisation), showing that this can be a useful method for use in interactome studies in conjunction with other tools.

La nécessité de coordonner l’expression des gènes provenant de génomes différents chez les plantes a conduit à l’émergence de mécanismes imposant un contrôle nucléaire sur l’expression génétique de l’organelle. Des signaux antérogrades, exercés par des protéines reconnaissant des séquences spécifiques, existent en parallèle avec un contrôle des synthèses chloroplastiques dépendant de l’assemblage (CES). Ensemble, ils coordonnent la formation stoichiométrique des complexes photosynthétiques.La Ribulose bisphosphate carboxylase/oxygénase (Rubisco) est une enzyme localisée dans le chloroplaste qui contient deux sous-unités. La grande sous-unité (LSU) et la petite sous-unité (SSU) sont codées par les génomes chloroplastique et nucléaire respectivement. Elles s’assemblent dans le stroma du chloroplaste pour former une holoenzyme hexadécamérique (LSU8SSU8). Pendant mon travail au laboratoire, j’ai tenté de décrire les étapes régulatrices majeures de la synthèse de la Rubisco chez Chlamydomonas reinhardtii en me focalisant sur la régulation post-transcriptionelle de la LSU.J’ai montré que la protéine PPR – MRL1 est un facteur limitant pour l’accumulation de l’ARN messager de rbcL. Bien qu’il ait été décrit précédemment comme un facteur stabilisateur du transcrit susnommé, MRL1 s’est révélé avoir un rôle dans la traduction.J’ai par ailleurs démontré que chez Chlamydomonas, l’expression de la Rubisco est contrôlée par la présence de la SSU. En son absence, la traduction de rbcL est inhibée par son propre produit – la grande sous-unité non assemblée. J’ai pu montrer qu’un intermédiaire d’assemblage, constitué de LSU en complexe avec sa chaperonne RAF1, est nécessaire pour cette régulation, ce qui prouve que ce processus dépend de l’état d’oligomérisation de la LSU. Parallèlement, j’ai caractérisé le devenir de la LSU non assemblée quand la régulation CES est perturbée, et grâce à cela ait contribué à améliorer la connaissance de son processus de repliement et d’assemblage
The necessity to coordinate the expression of genes originating from different genomes within the plant cell resulted in the appearance of mechanisms imposing nuclear control over organelle gene expression. Anterograde signaling through sequence-specific trans-acting proteins (OTAFs) coexists in the chloroplast with an assembly dependent control of chloroplast synthesis (CES process) that coordinates the stoichiometric formation of photosynthetic complexes.Ribulose bisphosphate carboxylase/oxygenase (Rubisco) is a chloroplast-located carbon fixing enzyme constituted of two subunits. Large subunit (LSU) and small subunit (SSU) are encoded in the chloroplast and nuclear genomes respectively. In the stroma they assemble to form a hexadecameric holoenzyme (LSU8SSU8). In this study I tried to highlight major regulatory points of its synthesis in Chlamydomonas reinhardtii focusing on the posttranscriptional regulation of LSU.I showed that the MRL1 PPR protein is a limiting factor for rbcL mRNA accumulation. Whereas it has been previously designated as a stabilization factor for the abovementioned transcript, MRL1 appeared also to have a function in rbcL translation.Most notably, I have demonstrated that in Chlamydomonas reinhardtii Rubisco expression is controlled by the small subunit (SSU) presence. In its absence rbcL undergoes an inhibition of translation through its own product – the unassembled Rubisco large subunit. This process depends on LSU-oligomerization state as I was able to show that the presence of a high order LSU assembly intermediate bound to the RAF1 assembly chaperone is essential for the regulation to occur. In parallel I shed light on the fate of unassembled LSU in a deregulated CES context, thereby improving our understanding of the process of its folding and assembly

La nécessité de coordonner l’expression des gènes provenant de génomes différents chez les plantes a conduit à l’émergence de mécanismes imposant un contrôle nucléaire sur l’expression génétique de l’organelle. Des signaux antérogrades, exercés par des protéines reconnaissant des séquences spécifiques, existent en parallèle avec un contrôle des synthèses chloroplastiques dépendant de l’assemblage (CES). Ensemble, ils coordonnent la formation stoichiométrique des complexes photosynthétiques.La Ribulose bisphosphate carboxylase/oxygénase (Rubisco) est une enzyme localisée dans le chloroplaste qui contient deux sous-unités. La grande sous-unité (LSU) et la petite sous-unité (SSU) sont codées par les génomes chloroplastique et nucléaire respectivement. Elles s’assemblent dans le stroma du chloroplaste pour former une holoenzyme hexadécamérique (LSU8SSU8). Pendant mon travail au laboratoire, j’ai tenté de décrire les étapes régulatrices majeures de la synthèse de la Rubisco chez Chlamydomonas reinhardtii en me focalisant sur la régulation post-transcriptionelle de la LSU.J’ai montré que la protéine PPR – MRL1 est un facteur limitant pour l’accumulation de l’ARN messager de rbcL. Bien qu’il ait été décrit précédemment comme un facteur stabilisateur du transcrit susnommé, MRL1 s’est révélé avoir un rôle dans la traduction.J’ai par ailleurs démontré que chez Chlamydomonas, l’expression de la Rubisco est contrôlée par la présence de la SSU. En son absence, la traduction de rbcL est inhibée par son propre produit – la grande sous-unité non assemblée. J’ai pu montrer qu’un intermédiaire d’assemblage, constitué de LSU en complexe avec sa chaperonne RAF1, est nécessaire pour cette régulation, ce qui prouve que ce processus dépend de l’état d’oligomérisation de la LSU. Parallèlement, j’ai caractérisé le devenir de la LSU non assemblée quand la régulation CES est perturbée, et grâce à cela ait contribué à améliorer la connaissance de son processus de repliement et d’assemblage
The necessity to coordinate the expression of genes originating from different genomes within the plant cell resulted in the appearance of mechanisms imposing nuclear control over organelle gene expression. Anterograde signaling through sequence-specific trans-acting proteins (OTAFs) coexists in the chloroplast with an assembly dependent control of chloroplast synthesis (CES process) that coordinates the stoichiometric formation of photosynthetic complexes.Ribulose bisphosphate carboxylase/oxygenase (Rubisco) is a chloroplast-located carbon fixing enzyme constituted of two subunits. Large subunit (LSU) and small subunit (SSU) are encoded in the chloroplast and nuclear genomes respectively. In the stroma they assemble to form a hexadecameric holoenzyme (LSU8SSU8). In this study I tried to highlight major regulatory points of its synthesis in Chlamydomonas reinhardtii focusing on the posttranscriptional regulation of LSU.I showed that the MRL1 PPR protein is a limiting factor for rbcL mRNA accumulation. Whereas it has been previously designated as a stabilization factor for the abovementioned transcript, MRL1 appeared also to have a function in rbcL translation.Most notably, I have demonstrated that in Chlamydomonas reinhardtii Rubisco expression is controlled by the small subunit (SSU) presence. In its absence rbcL undergoes an inhibition of translation through its own product – the unassembled Rubisco large subunit. This process depends on LSU-oligomerization state as I was able to show that the presence of a high order LSU assembly intermediate bound to the RAF1 assembly chaperone is essential for the regulation to occur. In parallel I shed light on the fate of unassembled LSU in a deregulated CES context, thereby improving our understanding of the process of its folding and assembly

Diurnal changes of initial and total activity and content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) under conditions of ambient (350 µmol mol-1) and elevated (E = 700 µmol mol 1) concentration CO2 were measured in needles of Norway spruce. Needles of Norway spruce were taken on 22th July in two-hours from 3:30 a. m. till 10:30 p. m. The Rubisco activity was determined by spectrophotometry, the Rubisco content was detected by SDS-PAGE. The Rubisco activity was in most samples statistically significantly lower in needles cutlivated under ambient carbon dioxide then in needles cultivated under elevated carbon dioxide. Diagrams of the Rubisco activity shows absence of night inhibitor CA1P. The Rubisco activity was directly proportional to the intensity of photosynthetically active radiation in time from 7:00 a. m. till 7:30 p. m. The Rubisco content was statistically significantly higher in needles of Norway spruce cultivated under ambient carbon dioxide than in needles cultivated under elevated CO2, which shows acclimation of the Rubisco content during long-term effect of elevated CO2 concentration.

Rubisco, the enzyme that catalyzes the assimilation of atmospheric CO2 and sustains the vast majority of food chains in the Biosphere, presents functional inefficiencies limiting the photosynthetic process. Rubisco is a slow enzyme and can not fully discriminate between CO2 and O2. When oxygen catalysis takes place, CO2 is released and energy is dissipated in the process of photorespiration. The existence of interspecific species variability in the catalytic properties of Rubisco suggests that this enzyme has been adapting to the prevailing environmental conditions, in particular to changes in CO2 concentration at the catalytic site and different thermal conditions. These findings offer new alternatives on genetic manipulation of Rubisco in order to overcome their inefficiencies and improve crop yields. However, the great structural and biochemical complexity of Rubisco is actually limiting a successful improvement. This is precisely the aim of this thesis: to expand the molecular and biochemical knowledge on the evolution of Rubisco to further understand and manipulate the enzyme. In this sense, the objectives of this thesis were: i) to investigate the genetic variability of Rubisco large subunit (L-subunit) and its evolution in different groups of plants, and ii) to explore the temperature dependence of Rubisco kinetic constants. Four experiments were done to answer these objectives. Experiments 1 and 2 shared a common pattern that consisted in selecting phylogenetically related species (174 species of Fagales in experiment 1, and 60 species of bromeliads and orchids in experiment 2) to describe Rubisco L-subunit sequence variability and to investigate the existence of positively selected sites related to particular environments and leaf traits. Experiments 3 and 4 were focused on the study of Rubisco kinetic response to temperature. Thus, experiment 3 consisted in a bibliographic compilation to study the thermal sensitivity of Rubisco turnover rate for carboxylation (kcat c). In experiment 4, the kinetic parameters of Rubisco and their response to temperature in 20 economically important crops were measured. In both experiments we modeled the effect of the observed kinetic variability on the ability of Rubisco to assimilate CO2 under different scenarios of CO2 concentration. Experiments 1 and 2 showed, first, that there was a great variability in Rubisco L-subunit in closely related species. Specifically, the 174 Fagales species were distributed into 29 haplotypes (groups of species with the same sequence), the 158 Quercus species in 21 haplotypes, and 19 haplotypes in orchids and 23 haplotypes in bromeliads. In all studied groups, the analysis showed that large part of this variability was determined by natural selection, corroborating the hypothesis of processes of adaptive change in Rubisco. Furthermore, in experiment 1, evidences of positive selection were found in terms of leaf habit and climate, both traits influencing the CO2 concentration at the site of carboxylation. This finding is a clear evidence that species adjust their Rubisco depending on the prevailing environmental conditions. By contrast, in experiment 2, positive selection processes could not be related to CAM photosynthetic mechanism. In any case, in all studied groups appeared co-evolving pairs of amino acid sites, located in important Rubisco regions for the functionality and structure of the enzyme. The implementation of a mathematical model of decision trees (DT) for sequence analysis permitted to relate sites variability to environmental and leaf traits. DT results corroborated, in part, observations of positive selection tests, thus validating the use of alternative models in the search for variable sites. Furthermore, in bromeliads and orchids, DT revealed a relation between sites variability and variables indicative of the degree of expression of CAM (δ13C and leaf thickness). In bromeliads and orchids interspecific variability in the catalytic constants of Rubisco was observed, part of which explained by the existence of carbon concentration mechanism in CAM plants. However, this catalytic variability could not be directly related to sites under positive selection or resolved by DT. In experiment 3, distinct kcatc photosynthetic response to temperature appeared among the studied groups. Thus, the highest and lowest activation energy (ΔHa) values for kcatc were observed in Rhodophyta and Chlorophyta, respectively. In terrestrial plants, C3 species from warm habitats and C4 species showed a higher ΔHa for kcatc than C3 plants from cold habitats. These results suggest that Rubisco evolved by adjusting the sensitivity of their kinetic properties to temperature indicating thermal adaptation to local conditions. In experiment 4, Rubisco kinetic parameters (Michaelis-Menten constant for CO2, specificity factor and kcatc) from the 20 studied crops showed interspecies variability at three temperatures of measurement (15, 25 and 35 °C). Variability in ΔHa of kinetic parameters was significant among all crop species and photosynthetic mechanisms. This variability could not be related to their domestication region climate, possibly due to subsequent processes of artificial selection. The use of measured values in mathematical models of photosynthesis quantified the importance of improving Rubisco in different crops at changing temperature and CO2 availability conditions. This thesis presents, under an evolutionary background, relevant information to improve Rubisco by bioengineering processes. The combined analysis of positive selection and coevolution are useful to elucidate interactions between amino acids that should be taken into account when designing better Rubiscos. In addition, the application of DT helps in the construction of relationships between the amino acid variability and the environment in which species evolve. Moreover, this thesis represents the most convincing evidence that Rubiscos of different species differ not only in catalytic constants at 25 °C, but also exhibit different response to temperature. The data published here offer the possibility to increase the accuracy of leaf photosynthesis models and addressed the need for improvement of Rubisco in species of high agricultural interest.
La Rubisco, enzima que cataliza la asimilación de CO2 atmosférico y sustenta la inmensa mayoría de cadenas tróficas de la Biosfera, presenta ineficiencias que limitan el proceso fotosintético. La Rubisco es lenta y puede “confundirse” de sustrato catalizando la fijación de O2 en el proceso de fotorrespiración, que termina liberando CO2 y disipando energía. La existencia de variabilidad interespecífica en las características catalíticas de la Rubisco sugiere que esta enzima se ha sido adaptando a las condiciones ambientales reinantes, en particular, a cambios en la concentración de CO2 en el sitio de catálisis y a las diferentes condiciones térmicas. Estos hallazgos han renovado la esperanza de manipulación genética de la Rubisco con el objetivo de superar sus ineficiencias y mejorar el rendimiento de los cultivos. Sin embargo, el desconocimiento aún presente en aspectos derivados de la gran complejidad estructural y bioquímica de la Rubisco limita, hoy en día, el éxito de mejora. Éste es, precisamente, el enfoque de la presente tesis: ampliar el conocimiento de la evolución molecular y bioquímica de la Rubisco para avanzar en la comprensión y manipulación de la enzima con mayores garantías de éxito. En este sentido, los objetivos de esta tesis fueron: i) investigar la variabilidad genética de la subunidad grande de la Rubisco y su evolución en diferentes grupos de plantas, y ii) explorar la dependencia térmica de las constantes cinéticas de la Rubisco. Para dar respuesta a estos objetivos, se llevaron a cabo 4 experimentos. Los experimentos 1 y 2 presentaron un patrón común consistente en seleccionar especies próximas filogenéticamente (174 especies de Fagales en el experimento 1, y 60 especies de bromelias y orquídeas en el experimento 2) para descifrar la variabilidad en la secuencia de la subunidad grande de la Rubisco e investigar la existencia de sitios aminoacídicos seleccionados positivamente en función del ambiente y caracteres morfológicos y fisiológicos foliares. Los experimentos 3 y 4 se centraron en el estudio de la respuesta de la cinética de la Rubisco a la temperatura. Así, en el experimento 3 se llevó a cabo una compilación bibliográfica para el estudio de la sensibilidad térmica del número de recambio catalítico para la reacción de carboxilación de la Rubisco, kcat c (el parámetro cinético más ampliamente estudiado en todos los grupos fotosintéticos). En el experimento 4 se midieron los parámetros cinéticos de la Rubisco y su respuesta a la temperatura en 20 cultivos económicamente importantes. En ambos experimentos se modelizó el impacto de la variabilidad observada sobre la capacidad asimiladora de la Rubisco bajo diferentes escenarios de disponibilidad de CO2. Los resultados de los experimentos 1 y 2 mostraron, por primera vez, la existencia de una gran variabilidad en la secuencia de la subunidad grande de la Rubisco en especies próximas taxonómicamente. En concreto, las 174 especies estudiadas de Fagales se distribuyeron en 29 haplotipos (grupos de especies con la misma secuencia), 21 haplotipos en las 158 especies de Quercus, 19 haplotipos en orquídeas y 23 haplotipos en bromelias. En todos los grupos estudiados, los análisis demostraron que una parte importante de esta variabilidad había sido fijada por la selección natural, corroborando la hipótesis de la existencia de procesos de cambio adaptativo en la Rubisco. Además, en el experimento 1, se encontraron evidencias de selección positiva en función del hábito foliar y del clima, ambos caracteres determinantes de la concentración de CO2 en el sitio de carboxilación. Este hallazgo supone la primera prueba inequívoca de que las especies ajustan su Rubisco en función de las condiciones ambientales prevalentes. Por el contrario, en el experimento 2, los procesos de selección positiva no se relacionaron aparentemente con el mecanismo fotosintético CAM. En cualquier caso, en todos los grupos estudiados aparecieron pares de sitios aminoacídicos con tendencia a coevolucionar, la mayoría ubicados en regiones importantes para la función y estructura de la Rubisco. Se implementó, de forma pionera, el modelo matemático de árboles de decisión (DT) para el análisis de secuencias y su relación con variables externas. Los resultados del DT corroboraron, en parte, las observaciones de los análisis de selección positiva, validando de esta forma la aplicación de modelos alternativos en la búsqueda de sitios variables. Además, en bromelias y orquídeas, los DT revelaron la existencia de cambios aminoacídicos específicos en función de variables indicadoras del grado de expresión de CAM (δ13C y espesor de hoja). En bromelias y orquídeas se observó variabilidad interespecífica en las constantes catalíticas de la Rubisco, parte de la cual se explicó por la existencia de mecanismos de concentración de carbono en plantas CAM. No obstante, esta variabilidad catalítica no se relacionó directamente con los cambios aminoacídicos detectados por los DT o bajo selección positiva. En el experimento 3, la respuesta de kcat c a la temperatura mostró diferencias entre los grupos fotosintéticos estudiados. Así, los valores más altos y más bajos para la energía de activación (ΔHa) de kcat c se observaron en Rhodophyta y Chlorophyta, respectivamente. En las plantas terrestres, las especies C3 de hábitats cálidos y las especies C4 presentaron una ΔHa de kcat c mayor que las plantas C3 de hábitats fríos. Estos resultados sugieren que la Rubisco ha evolucionado ajustando la sensibilidad de sus propiedades cinéticas a la temperatura e indican adaptación a las condiciones térmicas locales. En el experimento 4, los parámetros cinéticos de la Rubisco (constante de Michaelis-Menten para el CO2, factor específico y kcat c ) de los 20 cultivos estudiados mostraron variabilidad interespecífica a las tres temperaturas de ensayo (15, 25 y 35 °C). La variabilidad en la ΔHa de los parámetros cinéticos fue significativa entre todas las especies de cultivo y tipos fotosintéticos, si bien dicha variabilidad no se relacionó con el clima en la región de domesticación, posiblemente debido a procesos posteriores de selección artificial. La aplicación de datos específicos en los modelos matemáticos de fotosíntesis cuantificó la importancia de mejora de la Rubisco en los diferentes cultivos según condiciones cambiantes de temperatura y disponibilidad de CO2. Bajo un trasfondo evolutivo, esta tesis presenta información relevante para procesos de bioingeniería direccionados a la mejora de la Rubisco. Los análisis combinados de selección positiva y coevolución resultan útiles para resolver interacciones entre aminoácidos que deberían tenerse en cuenta a la hora de diseñar mejores Rubiscos. Además, la aplicación de los DT ayuda a generar relaciones entre la variabilidad aminoacídica y el ambiente en el que evolucionan las especies. Por otra parte, esta tesis supone la evidencia más convincente de que Rubiscos de diferentes especies no sólo presentan diferencias en las constantes catalíticas a 25 ºC, sino que también presentan diferente respuesta al rango fisiológico de temperaturas. Los datos aquí publicados ofrecen la posibilidad de aumentar la precisión de los modelos de fotosíntesis en hoja, y direccionan la mejora de Rubisco en especies de alto interés agrícola.
La Rubisco, l’enzim que catalitza l’assimilació del CO2 atmosfèric i sustenta la immensa majoria de cadenes tròfiques de la Biosfera, presenta ineficiències que limiten el procés fotosintètic. La Rubisco és lenta i es pot “confondre” de substrat, catalitzant la fixació d’O2 en el procés de fotorespiració, que acaba alliberant CO2 i dissipant energia. L’existència de variabilitat interespecífica en les característiques catalítiques de la Rubisco suggereix que aquest enzim ha anat adaptant-se a les condicions ambientals predominants, en particular, a canvis a la concentració de CO2 al lloc de catàlisi i a les diferents condicions tèrmiques. Aquests descobriments han renovat l’esperança de manipulació genètica de la Rubisco amb l’objectiu de superar les seves ineficiències i millorar així el rendiment dels cultius. No obstant, el desconeixement encara present a alguns aspectes derivats de la gran complexitat estructural de la Rubisco limita, avui en dia, l’èxit de millora. Aquest és precisament l’enfocament de la present tesi: ampliar el coneixement de l’evolució molecular i bioquímica de la Rubisco per avançar en la comprensió i manipulació de l’enzim, i així obtenir majors garanties d’èxit. En aquest sentit, els objectius d’aquesta tesi foren: 1) investigar la variabilitat genètica de la subunitat gran de la Rubisco i la seva evolució dins diferents grups de plantes, 2) explorar la dependència tèrmica de les constants cinètiques de la Rubisco. Per a poder donar resposta a aquests objectius, es varen dur a terme quatre experiments. Els experiments 1 i 2 presentaren un patró basat en la selecció d’espècies pròximes filogenèticament (174 espècies de Fagals en el experiment 1 i 60 espècies d’orquídies i bromèlies en el experiment 2) per tal de desxifrar la variabilitat en la seqüència de la subunitat gran de la Rubisco i investigar l’existència de llocs aminoacídics seleccionats positivament en funció de l’ambient i dels caràcters morfològics i fisiològics foliars. Els experiments 3 i 4 es varen centrar en l’estudi de la resposta de la cinètica de la Rubisco a la temperatura. Així, en el experiment 3 es va dur a terme una compilació bibliogràfica per a l’estudi de la sensibilitat tèrmica del nombre de recanvi catalític per a la reacció de carboxilació de la Rubisco, kcat c (el paràmetre cinètic més àmpliament estudiat en tots els grups fotosintètics). En el experiment 4 es varen mesurar els paràmetres cinètics de la Rubisco i la seva resposta a la temperatura dins 20 cultius econòmicament importants. En ambdós experiments es va modelitzar l’impacte de la variabilitat observada sobre la capacitat assimiladora de la Rubisco sota diferents escenaris de disponibilitat de CO2. Els resultats dels experiments 1 i 2 varen mostrar, per primer cop, l’existència d’una gran variabilitat dins la seqüència de la subunitat gran de la Rubisco a espècies pròximes taxonòmicament. En concret, les 174 espècies estudiades de Fagals es varen distribuir en 29 haplotips (grups d’espècies amb la mateixa seqüència), 21 haplotips dins les 158 espècies de Quercus, 19 haplotips dins orquídies i 23 haplotips dins les bromèlies. Dins tots els grups estudiats, les anàlisis demostraren que una part important d’aquesta variabilitat havia estat fixada per la selecció natural, corroborant la hipòtesi de l’existència de canvis adaptatius a la Rubisco. A més, en el experiment 1 es varen trobar evidències de selecció positiva en funció de l’habitat foliar i del clima, ambdós caràcters determinants de la concentració de CO2 en el lloc de carboxilació. Aquest descobriment suposa la primera prova inequívoca de que les espècies ajusten la Rubisco en funció de les condicions ambientals que predominen. Per el contrari, en el experiment 2 els processos de selecció positiva no es varen relacionar aparentment amb el mecanisme fotosintètic CAM. En qualsevol cas, dins tots els grups estudiats varen aparèixer parells de llocs aminoacídics amb tendència a coevolucionar, la majoria ubicats a llocs importants per a la funció i estructura de la Rubisco. Es va implementar de forma pionera el model matemàtic d’arbres de decisió (DT) per a l’anàlisi de seqüències i la seva relació amb variables externes. Els resultats del DT varen corroborar en part les observacions de les anàlisis de selecció positiva, validant així l’aplicació de models alternatius per a la recerca de llocs variables. A més, dins bromèlies i orquídies, els DT varen revelar la existència de canvis aminoacídics específics, en funció de variables indicadores d’expressió de CAM (δ13C i gruixa de fulla). Dins bromèlies i orquídies es va observar variabilitat interespecífica en les constants catalítiques de la Rubisco, part de la qual es va explicar per l’existència de mecanismes de concentració de carboni dins plantes CAM. No obstant, aquesta variabilitat catalítica no es va relacionar directament amb els canvis aminoacídics detectats mitjançant els DT o sota selecció positiva. En el experiment 3, la resposta de kcat c a la temperatura va mostrar diferències entre els grups fotosintètics estudiats. Així, els valors més alts i més baixos per a l’energia d’activació (ΔHa) de kcat c es varen observar a Rhodophyta i Chlorophyta, respectivament. A les plantes terrestres, les espècies C3 d’hàbitats càlids i les espècies C4 presentaren una ΔHa de kcat c major que les plantes C3 d’hàbitats freds. Aquests resultats suggereixen que la Rubisco ha evolucionat ajustant la sensibilitat de les seves propietats cinètiques a la temperatura i indiquen una adaptació a les condicions tèrmiques locals. En el experiment 4, els paràmetres cinètics de la Rubisco (constant de Michaelis-Menten per al CO2, factor específic i kcat c ) dels 20 cultius estudiats varen mostrar variabilitat interespecífica a les tres temperatures d’assaig (15, 25 i 35 °C). La variabilitat dins la ΔHa dels paràmetres cinètics va ser significativa entre totes les espècies de cultius i tipus fotosintètics, si bé aquesta variabilitat no es va relacionar amb el clima de la regió de domesticació, possiblement degut a processos posteriors de selecció artificial. L’aplicació de dades específiques en els models matemàtics de fotosíntesi va quantificar la importància de millora de la Rubisco en els diferents cultius segons condicions canviants de temperatura i disponibilitat de CO2. Sota un rerefons evolutiu, aquesta tesi presenta informació rellevant per a processos de bioenginyeria enfocats a la millora de la Rubisco. Les anàlisis combinades de selecció positiva i coevolució resulten útils per a resoldre interaccions entre aminoàcids que haurien de tenir-se en compte a l’hora de dissenyar millors Rubiscos. A més, l’aplicació dels DT ajuda a generar relacions entre la variabilitat aminoacídica i l’ambient en que evolucionen les espècies. Per altra banda, aquesta tesi suposa l’evidència més convincent de que Rubiscos de diferents espècies no només presenten diferències en les constants catalítiques a 25 °C, sinó que també presenten diferents respostes al rang fisiològic de temperatures. Les dades aquí publicades ofereixen la possibilitat d’augmentar la precisió dels models de fotosíntesi a nivell de fulla i marquen el camí per a la millora de la Rubisco dins les espècies d’alt interès agrícola.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第9642号
農博第1270号
新制||農||846(附属図書館)
学位論文||H14||N3674(農学部図書室)
UT51-2002-G400
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 吉川 正明, 教授 伏木 亨, 教授 北畠 直文
学位規則第4条第1項該当

This diploma thesis deals with dynamic of accumulation of non-structural carbohydrates and activity of Rubisco enzyme at elevated concentration of CO2 on beech (Fagus sylvatica L.). Three years old seedlings of beech were cultivated in minisphere with ambient (385 µmol•mol-1, variant A), and with elevated concentration CO2 (700 µmol•mol-1, variant E) for four months. In every variant the first half of plants was fertilized by nitrogen (variant N+) and the second half was control (variant N-). Plants used for experiment were at first adapted for darkness for 12 hours. Subsequently tested leaves were cut off, leafstalk including short segment of branch (approximately 1 cm) was inserted into 0.7 M solution of sucrose (variant S) or water (variant V) and exposed to radiation 200 mol•m-2•s-1 for 0, 30, 60, 120, 180 minutes. Then leaf area and fresh mass of leaf blade were established, samples were fixed in liquid nitrogen and stored in deep freezer to analysis in –70 °C. Rubisco content was determined by SDS-PAGE method, Rubisco activity spectrofotometrically and content of non-structural carbohydrates by anthrone method and HPLC method. Rubisco content was significantly lower in the N- variant than in N+ variant. Rubisco content was also significantly lower in E than in A variant, which is an evidence of down-regulation. Rubisco activity is moderately stimulated at E variant with time, but differences between variant A and E are not statistically significant. Influence of sucrose feeding to Rubisco activity was not proved. Significant differences were detected by anthrone method in non-structural carbohydrates content between variants S and V, but not between variants A and E. Statistically significant increase of sucrose content with time was detected by HPLC method at variant AS, but not at variant ES.

Thesis (Ph. D.)--Ohio State University, 2004.
Title from first page of PDF file. Document formatted into pages; contains xiii, 149 p.; also includes graphics. Includes abstract and vita. Advisor: F. Robert Tabita, Dept. of Microbiology. Includes bibliographical references (p. 144-149).

This study combined in vivo leaf gas exchange, chlorophyll fluorescence, and instantaneous carbon isotope discrimination (D) analyses with in vitro measurement of Rubisco and PEPc activity and transcript abundance of key Rubisco and PEPc regulatory enzymes, Rubisco activase (RCA) and PEPc kinase (PPCKI ), and RCA protein abundance, to provide a novel molecular perspective on the co-regulation of carboxylase activity in M. crystallinum. In C₃ M. crystallinum, Rubisco activation state was highest during the photoperiod. C3 Rubisco activity remained uniform diurnally, mirrored by the pattern of stable CO₂ assimilation rate and protein abundance of both RCA isoforms, RCA1 and RCA2. RACE-PCR was used to identify RCA2 as the larger isoform in M. crystallinum. A marked decrease in RCA2 protein occurred during CAM induction, coincident with increased PEPc protein abundance and maximal PEPc activity. Diurnal Rubisco activity was regulated independent of light intensity and electron transport rate (ETR) following CAM induction. Rubisco activation state in CAM M. crystallinum was increased during the latter half of the photoperiod (mid CAM Phase III - Phase IV), coincident with low apparent PEPc activity and the final stages of titratable acidity breakdown. Increased RCA1 protein abundance during mid-CAM Phase III was observed coincident with an increase in Rubisco activation state of approximately one third. Modulation of diurnal Rubisco activity by RCA1 represented a potential regulatory mechanism unique to the CAM mode of M. crystallinum.

In this diploma work changes of initial and total activities and content of Rubisco in beech and Norway spruce were studied. The plants were cultivated in conditions with ambient CO2 concentration (350 mol·mol–1) and elevated CO2 concentration (700 mol·mol–1). Three series of samples (at the beginning, in the middle and at the end of growing season) were taken. Initial and total Rubisco activities were measured spectrophotometrically and activation state was calculated. Rubisco content was determined by SDS–PAGE method. Rubisco activity in beech cultivated in elevated CO2 concentration decreased during the whole growing season while in beech growing in ambient CO2 concentration Rubisco activity decreased up to middle of growing season and then increased. Rubisco content in beech in ambient CO2 concentration slightly increased and in beech in elevated CO2 concentration decreased up to middle of growing season and then increased. Rubisco activities in Norway spruce both in ambient and elevated CO2 concentration decreased. Rubisco content in Norway spruce in ambient CO2 concentration decreased but in Norway spruce in elevated CO2 concentration first decreased and then increased.

In this diploma thesis, the diurnal changes of initial and total Rubisco activity and Rubisco enzyme content in beech (Fagus sylvatica) were studied under conditions of ambient (A) CO2 concentration (350 µmol.mol-1) and elevated (E) CO2 concentration (700 µmol.mol-1) during the day. Samples were taken on July 8th (from 10:00 to 21:30), on July 9th (from 04:00 to 12:00) and then on July 22th (from 04:00 to 21:30). The initial and total activity were measured spectrophotometrically and the activation level was calculated from the ratio of initial and total activities. Rubisco enzyme content was determined by SDS-PAGE method and the initial and total specific activity were calculated from the ratio of initial or total activity and Rubisco enzyme content. In our experiment no statistically significant difference was found between Rubisco activities in beeches cultivated under conditions of ambient CO2 concentration and elevated CO2 concentration, so any down-regulation of Rubisco activity did not appear under the influence of ambient CO2 concentration. Diurnal changes of Rubisco activities showed only statistically nonsignificant fluctuation. After daylight no significant increase of total Rubisco activity was observed, which demonstrates the absence or the immaterial effect of CA1P night inhibitor in beech. Intensive fluctuation of the activation level (40–90 %) proves the regulation of photosynthesis during the day via Rubisco enzyme carbamylation. Rubisco enzyme content in beech cultivated under conditions of ambient CO2 concentration was lower than that one in beech cultivated under conditions of elevated CO2 concentration. Then down-regulation of Rubisco enzyme content is presented under conditions of ambient CO2 concentration. Before daylight Rubisco enzyme content showed an indistinctive minimum on the level of statistical significance, during the day only nonrelevant fluctuation was noticed.

The graduation theses theme was to determinate changes of initial activity, total activity and Rubisco enzyme content in spinach leaves. Ascertained activities and Rubisco enzyme content were determined from extracts of spinach leaves, which were collected during two distinctively climatic different days. Both activities were determined by spectrometric method and content of enzyme Rubisco by SDS-PAGE method. The findings were compared with findings determined in earlier done works.

This thesis contains quantum chemical models and force field calculations for the RuBisCO isotope effect, the spectral characteristics of the blue-light sensor BLUF and the light harvesting complex II. The work focuses on the influence of the environment on the corresponding systems. For RuBisCO, it was found that the isotopic effect is almost unaffected by the environment. In case of the BLUF domain, an amino acid was found to be important for the UV/vis spectrum, but unaccounted for in experiments so far (Ser41). The residue was shown to be highly mobile and with a systematic influence on the spectral shift of the BLUF domain chromophore (flavin). Finally, for LHCII it was found that small changes in the geometry of a Chlorophyll b/Violaxanthin chromophore pair can have strong influences regarding the light harvesting mechanism. Especially here it was seen that the proper description of the environment can be critical. In conclusion, the environment was observed to be of often unexpected importance for the molecular properties, and it seems not possible to give a reliable estimate on the changes created by the presence of the environment.
Diese Arbeit beinhaltet quantenchemische und molekularmechanische Modelle zum Isotopeneffekt des Enzyms RuBisCO, der spektralen Charakterisierung des Blaulicht-Rezeptors BLUF und dem Lichtsammelkomplex II (LHCII). Es wurden vor allem die Einflüsse der Umgebung auf die entsprechenden Systeme untersucht. Für RuBisCO wurde gefunden, dass der Isotopeneffekt nur marginal von der Umgebung abhängt. Im Falle der BLUF Domäne wurde eine Aminosäure charakterisiert (Ser41), die bis dato experimentell noch nicht beschrieben war. Es wurde festgestellt, dass Ser41 hochmobil ist und einen systematischen Einfluss auf die spektrale Verschiebung des BLUF Chromophors (Flavin) hat. Schließlich wurde bei LHCII festgestellt, dass kleine Veränderungen in der Geometrie eines Chlorophyll b/Violaxanthin Chromophorenpaares bereits massive Einflüsse auf den Mechanismus des Lichtsammelprozesses haben können. Insbesondere hier zeigt sich, wie kritisch die genaue Beschreibung der Umgebung ist. Zusammenfassend wurde beobachtet, dass sich die Umgebung in oft unerwarteter Weise auf die molekularen Eigenschaften auswirken kann und es daher nicht möglich zu sein scheint, die entsprechenden Effekte vorher abzuschätzen.

Increasing photosynthetic efficiency remains one of the few routes left for substantial increases in crop yields. Rubisco, the enzyme responsible for fixating carbon dioxide from the atmosphere, is one of the major bottlenecks of photosynthesis. Some organisms have evolved carbon concentrating mechanisms, utilising carbonic anhydrase, to increase photosynthetic efficiency, by increasing local CO2 concentrations around Rubisco. This project aims to chemically mimic the role of carbonic anhydrase, through the use of small metal-ligand complexes, and to test their ability to perturb Rubisco's activity. In order to achieve this goal, Rubisco had to be first isolated, and a robust Rubisco assay developed, to screen the efficacy of the CA mimetics. A hydrophobic interaction chromatography protocol for the isolation of Rubisco was developed, which eliminated the requirement of salt removal before column chromatography, reducing the number of purification steps in comparison to existing methods. The rapid protocol was employed to obtain highly pure and highly active Rubisco from Spinacia oleracea and Brassica oleracea. Furthermore, the protocol revealed, for the first time, the presence of hydrophobically distinct populations of Rubisco. A fast and sensitive 384-well plate Rubisco enzymatic assay was also developed, providing a robust platform to screen and test the efficacy of the carbonic anhydrase mimetics on Rubisco's activity. A suite of existing and novel carbonic anhydrase mimetics were synthesised. A comparison of the pH-dependant CO2/HCO3- catalytic rates revealed a number of insights into the parameters that promote high interconversion rates of HCO3- to CO2: i) high electron-donating ligands, and hydrophobic ligands for 'substrate channelling' increased HCO3- dehydration rates, ii) higher pKa values for Zn-OH2, favoured the interconversion of HCO3- to CO2, iii) Zn(II) was shown to have the highest catalytic rate for HCO3- dehydration, which was attributed to the weaker binding of HCO3- to Zn(II) than to Cu(II), Co(II) and Ni(II). Additionally, this thesis provides the first proof of concept data for the use of chemical tools that could help towards mitigating the loss of Rubisco's inefficiencies. Zn(II) mimetics were shown to alter Rubisco's activity, which was attributed to a change in local CO2 concentrations, following the addition of the mimetics.

Five different forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO; IA, IB, IC, ID, II), the carboxylase of the Calvin-Benson-Bassham cycle (CBB), are utilized by plants, algae and autotrophic bacteria for carbon fixation. Discrimination against 13C by RubisCO is a major factor dictating the stable carbon isotopic composition (δ13C = {[13C/12C sample/13C/12C standard] - 1} X 1000) of biomass. To date, isotope discrimination, expressed as ε values (={[12k/13k] - 1} X 1000; 12k and 13k = rates of 12C and 13C fixation) has been measured for form IA, IB, and II RubisCOs from only a few species, with ε values ranging from 18 to 29 /. The aim of this study was to better characterize form ID and IC RubisCO enzymes, which differ substantially in primary structure from the IB enzymes present in many cyanobacteria and organisms with green plastids, by measuring isotopic discrimination and kinetic parameters (KCO2 and Vmax). Several major oceanic primary producers, including diatoms, coccolithophores, and some dinoflagellates have form ID RubisCO, while form IC RubisCO is present in many proteobacteria of ecological interest, including marine manganese-oxidizing bacteria, some nitrifying and nitrogen-fixing bacteria, and extremely metabolically versatile organisms such as Rhodobacter sphaeroides. The ε - values of the form ID RubisCO from the coccolithophore, Emiliania huxleyi and the diatom, Skeletonema costatum (respectively 11.1 / and 18.5 /) were measured along with form IC RubisCO from Rhodobacter sphaeroides and Ralstonia eutropha (respectively 22.9 / and 19.0 /). Isotopic discrimination by these form ID/IC RubisCOs is low when compared to form IA/IB RubisCOs (22-29 /). Since the measured form ID RubisCOs are less selective against 13C, oceanic carbon cycle models based on 13C values may need to be reevaluated to accommodate lower ε values of RubisCOs found in major marine algae. Additionally, with further isotopic studies, the extent to which form IC RubisCO from soil microorganisms contributes to the terrestrial carbon sink may also be determined.

Kyoto University (京都大学)
0048
新制・課程博士
博士(工学)
甲第13793号
工博第2897号
新制||工||1428(附属図書館)
26009
UT51-2008-C709
京都大学大学院工学研究科合成・生物化学専攻
(主査)教授 今中 忠行, 教授 青山 安宏, 教授 濵地 格
学位規則第4条第1項該当

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第13901号
農博第1716号
新制||農||956(附属図書館)
学位論文||H20||N4368(農学部図書室)
UT51-2008-C817
京都大学大学院農学研究科食品生物科学専攻
(主査)教授 吉川 正明, 教授 井上 國世, 教授 入江 一浩
学位規則第4条第1項該当

The binding of carboxy-arabinitol bisphosphate (CABP), carboxy-arabinitol 1-phosphate (CA1P), carboxy-ribitol bisphosphate (CRBP), to carbamylated sites or xylulose bisphosphate (XuBP) and ribulose bisphosphate (RuBP) to decarbamylated sites on ribulose bisphosphate carboxylase/oxygenase (rubisco) exhibits negative cooperativity. The binding of ligands to decarbamylated sites was highly pH-dependent between 7.5 and 8.5. Lower pH enhanced binding affinity. The binding of ligands to carbamylated sites was pH-independent. A binding model for negative cooperative interactions among catalytic sites is proposed based on the observations and the crystallographic structure of rubisco. Fully activated, purified rubisco slowly loses its activity during in vitro catalysis after exposure to RuBP. This time-dependent kinetics is termed as "fallover". Two different fallover patterns were demonstrated during CO₂ fixation, one with little loss of activator CO₂ at pH 8.5 and the second with loss of activator CO₂ at pH 7.5. The two inhibitors being produced during fallover were isolated by high performance anion exchange chromatography (HPAE) and identified as XuBP and 3-ketoarabinitol 1,5-bisphosphate by pulsed amperometric detection (PAD) either directly or after reduction by NaBH₄. Because of the weak binding affinity of XuBP to catalytic sites of rubisco at pH 8.5, there was little loss of activator CO₂ during fallover. 3-keto-arabinitol-P₂ which binds to carbamylated rubisco sites became the major inhibitor at that pH. However, at pH 7.5, the binding of XuBP to decarbamylated sites can cause a shift in equilibrium with loss of carbamylated sites even in the presence of excess CO₂ and Mg²⁺, resulting in the loss of activator CO₂ during fallover. XuBP was isolated and identified from celery leaves by HPAE-PAD. A new method was developed for the determination of the substrate specificity of rubisco. It is based on the specific ¹⁴C-labeling of 3-phosphoglycerate (PGA) from the carboxylase reaction and its dilution from the oxygenase reaction. Therefore, the ratio of carboxylation to oxygenation can be measured directly by determining the specific radioactivity of PGA produced from both reactions with HPAE-PAD separation of the total PGA and scintillation counting of ¹⁴C-labeled PGA.

Le dépérissement du pin d'Alep (Pinus halepensis Mill. ) constaté en conditions naturelles a initié le développement de recherches concernant l'effet de stress abiotiques sur cette espèce. L'étude présentée dans le cadre de ée travail de thèse s'inscrit dans ce contexte, évaluant l'effet de l'ozone, de la sécheresse et du stress salin sur le métabolisme carboné du pin d'Alep. L'ozone entraîne l'apparition de nécroses, liées à une diminution de la teneur en chlorophylles. Une diminution de l'activité de la rubisco, enzyme de fixation du carbone, et une augmentation d'enzymes impliquées dans les voies cataboliques telles la phosphoenolpyruvate, carboxylase (pEPe), la pyruvate kinase (PK), les enzymes maliques (EM-NAD et EM-NADP) sont par ailleurs démontrées dans les aiguilles des pins soumis au polluant. La plante semble ainsi mettre en place une stratégie visant à la production de composés utilisables dans les processus de réparation et/ou de défense vis à vis des radicaux induits par l'ozone. La sécheresse et le stress salin permettraient une protection de la plante contre les radicaux générés par le polluant, vraisemblablement par une fermeture des stomates limitant ainsi la pénétration de l'ozone dans les aiguilles. La baisse d'activité rubisco sous ozone nous a suscité l'intérêt d'une étude de la régulation de cette enzyme, et d'une enzymele associée, la rubisco activase, à un niveau moléculaire. A ces fins, le clonage de fragments d'ADNc codant pour la grande sous unité de la rubisco et pour la rubisco activase a été réalisé, permettant ainsi une étude phylogénétique des deux protéines. Une diminution de la quantité de protéine et de transcrits de la grande sous unité de la rubisco et de la rubisco activase a été démontré sous ozone, suggérant une potentielle corégulation des deux protéines dans ces conditions. Bien que les effets de la sécheresse soient similaires, aucune corrélation entre les deux protéines n'a pu être déterminée. Lors de la combinaison des deux contraintes, une différence de réponse des transcrits des deux protéines est mise en évidence. Pour la première fois, la rubisco activase est mise en évidence et clonée partiellement chez un conifère et sa régulation sous contrainte étudiée
The decline of aleppo pine forests (Pinus halepensis M. ) observed in natural conditions have prompted research concerning the effects of abiotic stresses on this species. For this purpose, the effects of ozone, water stress and salt stress on aleppo pine's carbon metabolism were carried out in this study. Ozone did induce chlorotic mottles, linked to a decrease in chlorophyll content. A decline in total rubiseo activity, and an increase in the activities of several enzymes associated to the catabolic pathway such as pyruvate kinase (PK), phosphoenolpyruvate carboxylase (PEPc) and malic enzymes (EM-NAD and EM-NADP) were shown in needles of pines submitted to ozone. These results tend to show that an increase in compounds potentially used in detoxifying repair processes is occuring under ozone stress. Water and salt stresses would limit the deleterious effects of ozone on plant metabolism, presumably by inducing stomatal closure which would limit the pollutant input into the leaves. Studies on the regulation of rubisco and rubisco aetivase, were undertaken at a molecular level in needles of pines submitted to ozone and/or drought. For this purpose, the cloning of cDNA fragments coding for rubisco large subunit (LSU) and for rubisco activase (RCA) was realized, allowing a phylogenetic analysis of both proteins. A decrease of LSU and RCA protein quantities, associated to a transcriptional regulation, was shown in response to ozone,> suggesting a potential coregulation of both proteins in those particular conditions. Even though the effects of drought on the large subunit of rubisco and on rubisco activase were similar, no correlation between the effects of this stress on the two proteins could be drawn. When ozone was combined to drought, a difference in the regulation of rubisco large subunit and of rubisco activase was shown. Under those conditions, rubisco large subunit was under a transcriptional control whereas rubisco activase was under a post transcriptionnal regulation. For the very first time in this study, a conifer' s rubisco activase is partially cloned and its regulation studied in reponse to abiotic constraints

La enzima Rubisco es uno de los sitemas biológicos más importantes. Por un lado controla la fotosíntesis y por otro cataliza la reacción que inicia el proceso de la fotorrespiración. Este comportamiento dual y aparentemente contradictorio de la enzima la ha hecho objeto de numerosos estudios. En la Tesis que se presenta hemos aplicado las técnicas propias de la Química Teórica para estudiar su comportamiento. En primer lugar, nuestros resultados indican que la razón para el comportamiento bifuncional de la enzima radica en la geometría tensionada que tiene el sustrato en el interior del centro activo, que hace que tanto el estado electrónico triplete como el singlete se encuentren accesibles y participen en los procesos químicos. En segundo lugar, hemos proporcionado una explicación al fenómeno de autoinactivación de la enzima observado experimentalmente. En tercer lugar, hemos obtenido las estructuras de transición correspondientes a las reacciones de enolización, carboxilación y oxigenación catalizadas por la enzima, caracterizando su acción catalítica y obtieniendo claves para interpretar su comportamiento: el sustrato se encuentra preparado para experimentar adición de CO2 o de O2, y el hecho de que ocurra una u otra reacción sólo depende de la molécula de gas que alcance el centro activo. De esto se desprende que el proceso de oxigenación es inevitable, una vez enolizado el sustrato, y está indisolublemente ligado al de carboxilación.

La Ribulosa 1,5-bisfosfato carboxilasa oxigenasa (Rubisco) cataliza el primer paso en la asimilación fotosintética de carbono a través del ciclo de Calvin y es, por tanto, la principal vía de entrada del CO2 de la atmósfera en la biosfera. La estructura del enzima en organismos eucariotas es un hexadecámero compuesto por 8 subunidades grandes (de 51-58KDa, de codificación cloroplástica) y 8 subunidades pequeñas (de 12-18KDa, de codificación nuclear). La Rubisco desempeña una importante función no catalítica como reserva de nitrógeno, azufre y carbono, al degradarse de forma rápida y selectiva en condiciones de senescencia natural o inducida por estrés. Una de las respuestas más generalizadas ante diferentes tipos de estrés en distintos organismos es la oxidación de grupos tioles de la Rubisco, que se ha relacionado in vitro e in vivo con la inactivación y susceptibilización proteolítica del enzima. Con estos antecedentes, en la presente tesis se ha profundizado en el conocimiento de los mecanismos que regulan la actividad y la disponibilidad proteolítica de la Rubisco a través del estado de oxidación de residuos de cisteína del propio enzima. Dentro de este tema, se estudiaron los siguientes aspectos concretos:1. Patrón diferencial de proteolisis de Rubisco reducida y oxidada.La utilización de proteasas de baja especificidad de secuencia como sondas estructurales reveló que la accesibilidad del sitio de corte tras la Lys 18 de la subunidad grande de la Rubisco de diferentes especies no depende del estado redox del enzima. Por otro lado, la oxidación de las cisteínas conservadas de Rubisco induce un cambio conformacional progresivo que favorece la exposición del lazo Ser61-Thr68 de la subunidad grande a la acción de proteasas exógenas. El corte proteolítico en este lazo, situado en la interfase entre las dos subunidades grandes que componen cada uno de los dímeros del holoenzima, promueve el desensamblaje y la digestión completa (no restringida) del enzima.La proteolisis de la Rubisco por subtilisina puede modelizarse mediante un sistema de tres ecuaciones diferenciales acopladas, que incluye cuatro constantes cinéticas de primer orden: k1, para el procesamiento tras la Lys 18, k2, para el corte en el lazo Ser61-Thr68 de una subunidad grande intacta, k2´, para el corte en el mismo lazo de una subunidad grande previamente procesada tras la Lys 18 y k3, para el desensamblaje y la consecuente proteolisis no restringida. El modelo describe correctamente la cinética de proteolisis y puede utilizarse como herramienta para detectar cambios conformacionales y comportamientos anómalos en enzimas mutantes.2. Obtención de mutantes de Rubisco por sustitución de residuos conservados de cisteína.Los procedimientos de transformación cloroplástica y nuclear de C. reinhardtii han permitido la obtención de mutantes fotosintéticamente activos de cisteínas conservadas de la subunidad grande (C84S, C247S, C284S, C427S, C449S, C459S y C449S/C459S) y de la subunidad pequeña (C41S, C83S) de la Rubisco. La futura caracterización de estos mutantes puede ayudar a desvelar el papel concreto que cada uno de estos residuos pueda desempeñar en el control redox del enzima.3. Estudio del papel del par Cys 449-459 en la modulación redox de la Rubisco.De entre los mutantes obtenidos, se escogieron los correspondientes a los residuos de cisteína 449 y 459 de la subunidad grande de la Rubisco para una caracterización más detallada. El residuo de cisteína 459, con un alto grado de conservación entre las secuencias de plantas superiores, algas verdes y cianobacterias, se encuentra en C. reinhardtii a una distancia de la cisteína 449 compatible con la formación de un puente disulfuro. La sustitución de los residuos de cisteína 449 y 459 no provoca cambios apreciables en el centro activo del enzima en estado reducido aunque supone una ligera desestabilización de la estructura nativa, percibible sólo a temperaturas muy superiores a la fisiológica. Los residuos de cisteína 449 y 459 de la subunidad grande de la Rubisco son necesarios para inactivar completamente el enzima a potenciales redox elevados y favorecen de forma redundante la exposición del lazo Ser61-Thr68 a la acción de proteasas exógenas in vitro en condiciones oxidantes.Las cisteínas 449 y 459 tienen un papel aparentemente redundante de forma que su relevancia funcional sólo se pone de manifiesto in vivo al sustituir ambos residuos. Así, el doble mutante, a diferencia de los mutantes simples, presenta un mayor contenido de Rubisco, proteínas totales y pigmentos fotosintéticos por célula durante la fase estacionaria de crecimiento. El estrés salino se ha descrito como un desencadenante de estrés oxidativo en el interior del cloroplasto, y se escogió como modelo para el estudio del papel redox de las cisteínas 449 y 459 in vivo, en condiciones que inducen la degradación de Rubisco. La sustitución simultánea de los residuos de cisteína 449 y 459 intensifica la degradación de Rubisco y los procesos de inactivación, polimerización en agregados de alto peso molecular y asociación de la proteína con las membranas, relacionados con el catabolismo del enzima. Se presenta una discusión sobre los posibles mecanismos por los cuales las cisteínas 449 y 459 pueden estar implicadas en estos procesos in vivo, en base a los datos bibliográficos de los que se dispone hasta el momento.
In the present work, it has been intended to further characterize the mechanisms that regulate the activity and the proteolytic availability of Rubisco through the redox state of its cysteine residues. The following aspects were studied:1. Differential proteolytic pattern upon Rubisco oxidation.The oxidation of conserved cysteine residues induces a progressive conformational change that favours the exposure of the Ser61-Thr68 loop of the large subunit to exogenous proteases. A proteolytic cut in this loop promotes disassembly and the complete digestion of the enzyme. A mathemathical model with three coupled differential equations is proposed. This model fits properly the observed kinetics of proteolytic fragmentation and can be used as a tool to detect conformational changes in mutant enzymes. 2. Site-directed mutagenesis of conserved cysteine residues of Rubisco in Chlamydomonas reinhardtii. In order to test the contribution of each of the conserved cysteines to redox regulation, photosynthetically active Rubisco mutants corresponding to large subunit (C84S, C247S, C284S, C427S, C449S, C459S, C449S/C459S) and small subunit (C41S, C83S) conserved cysteines residues were obtained.3. Study of the role of the Cys449-459 pair in Rubisco redox modulation through analyis of the phenotype of the C449S, C459S and C449S/C459S mutants.Cysteine residues 449 and 459 of the large subunit are involved in full inactivation of the enzyme at strong oxidizing conditions, and favour the exposure of loop Ser61-Thr68 to in vitro proteolytic attack. Cysteines 449 and 459 have a redundant role in vivo, in such a way that their physiological role becomes evident only after substitution of both residues. The double mutant accumulates a higher biomass per cell and shows a more intense response to saline stress in processes (degradation, aggregation, association to membranes and inactivation) related to the catabolism of the enzyme.