Nous avons défini le complexe tabagique selon quatre champs applicables : les pipes à fumer, le tabac, le rôle du tabac dans les rapports Autochtones-Européens et dans les échanges. Le but de ce mémoire est de mieux saisir ce que les pipes à fumer de la période historique nous dévoilent dans le contexte de l’archipel montréalais. Le dépouillement des rapports de fouille a permis de retirer d’innombrables fragments de pipes à fumer que nous retrouvons dans les contextes archéologiques de l’archipel montréalais pour la période 1642−1760. Les récits d’époque nous informent sur les habitudes reliées à la consommation du tabac ainsi que les rôles du tabac et des pipes à fumer dans les relations entre Autochtones et Européens et des processus d’échange. Des recherches sur le tabac sont venues ajouter des éléments cruciaux à ces deux sources d’information. Cette étude a été limitée à l’archipel montréalais, plus une aire de 10 km autour de celui-ci. La période étudiée est de 1642 à 1760. Nous avons constaté que les fragments des pipes à fumer se retrouvent majoritairement dans les zones de contact démontrant ainsi l’importance de ces objets dans les échanges. Ces zones de contact sont les endroits où le métissage prend place. Le tabac, que nous ne pouvons dissocier des pipes à fumer, joue aussi un rôle majeur dans les relations amérindiennes-européennes. Des dons de Nicotiana tabacum ont permis de solidifier des liens de confiance primordiaux entre les deux groupes dans les processus d’échange. Le tabac et les pipes à fumer, ont aussi subit le processus de transfert culturel, mais, le degré varie selon le type de pipes à fumer. / We defined the smoking complex according to four applicable fields: smoking pipes, tobacco, Indigenous -European relations and the role of tobacco in trade. The purpose of this research is to better understand what smoking pipes reveal to us in the context of the Montreal archipelago. The review of the excavation reports allowed us to retrieve information regarding the innumerable fragments of smoking pipes that we find in the archaeological contexts of the Montreal archipelago for the period 1642−1760. The ethnohistorical publications inform us about the habits and customs related to the use of tobacco as well as the roles that tobacco and smoking pipes played in Indigenous-European relations and exchanges. Tobacco studies have also added crucial elements to these two sources of information. This study was limited to the Montreal archipelago, plus an area of 10 km around it. The study period is from 1642 to 1760. We found that the fragments of smoking pipes are mostly found in contact zones demonstrating the cultural importance of these objects. Nicotiana tabacum strengthened bonds of trust between the two groups. These contact zones are the places where métissage takes place. Inseparable from smoking pipes, tobacco also played a major role in Amerindian-European relations. Gifts of Nicotiana tabacum favoured consolidation between the two groups and built the primordial trust necessary in exchange processes. Tobacco and smoking pipes are also part of the process of cultural transfer, but to a variable degree according to the type of smoking pipes.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23530号 / 農博第2477号 / 新制||農||1088(附属図書館) / 学位論文||R3||N5361(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 矢﨑 一史, 教授 小川 順, 教授 阪井 康能 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

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Die Biosynthese von Chlorophyll ist komplex und umfasst mehr als ein Dutzend enzymatische Schritte. Es ist nur allzu selbstverständlich, dass eine Deregulation der Chlorophyllsynthese globale Effekte auf die Zelle hat. Um diese Konsequenzen näher zu beleuchten, wurden Arabidopsis thaliana Pflanzen mit chemisch induzierter Deaktivierung von zwei Chlorophyllbiosynthesegenen (CHLH bzw. CHL27) erzeugt sowie photoautotophe Zellsuspensionskulturen von Arabidopsis thaliana hinsichtlich kurzzeitig induzierter Signalprozesse untersucht. Die Resultate verdeutlichen, dass durch Fehlregulationen innerhalb der Chlorophyllbiosynthese erzeugte reaktive Sauerstoffspezies die Transkriptionskontrolle kernkodierter Gene beeinflussen. Die Untersuchung eines enzymatischen Schrittes der Chlorophyllbiosynthese trat in dieser Arbeit in den Hauptfokus: Die Bildung des fünften, isozyklischen Ringes im Chlorophyllmolekül, katalysiert durch das bisher unzureichend erforschte Enzym Mg-Protoporphyrin-IX-monomethylester-Cyclase (Cyclase). Anhand von transgenen Cyanobakterien und Pflanzen sollte das noch unbekannte Gen ycf54 hinsichtlich seiner physiologischen Funktion in dem Cyclase-Enzymschritt analysiert werden. Das Fehlen von Ycf54 in Synechocystis sp. PCC6803 bzw. des homologen LCAA-Proteins in Nicotiana tabacum und Arabidopsis thaliana führt zu starken Cyclase-Substrat-Akkumulationen, verringerten Chlorophyllgehalten und reduzierten Ycf59- bzw. CHL27-Proteingehalten. Ein Mangel von Ycf54/LCAA beeinträchtigt daher die Funktionalität des Cyclase-Komplexes und scheint sich zudem interessanterweise auch auf die Stabilität photosynthetischer Antennenkomplexe auszuwirken. Mittels Pulldown-Assays konnte für Arabidopsis thaliana die direkte physikalische Interaktion zwischen LCAA und CHL27 bestätigt werden. Darüber hinaus sind erste Hinweise für die Ferredoxin-NADP-Reduktase als potenziellen Interaktionspartner gezeigt. / Synthesis of chlorophyll is a complex metabolic process and encompasses more than a dozen enzymatic reactions. It is self-evident that a deregulation of chlorophyll biosynthesis evokes global cellular impacts. To elucidate these consequences Arabidopsis thaliana plants with chemically inducible deactivation of two chlorophyll biosynthesis genes (CHLH and CHL27, respectively) were generated and photoautotrophic cell suspension cultures of Arabidopsis thaliana were used for short induced signal processes. The results illustrate that reactive oxygen species provoked by a deregulated chlorophyll synthesis affect the control of transcription of nuclear genes. The investigation of one enzymatic step of chlorophyll biosynthesis was placed as main focus: The formation of the isocyclic ring of the chlorophyll molecule catalyzed by the Mg protoporphyrin IX monomethyl ester cyclase (short: cyclase), an enzyme which is not fully investigated so far. The still unknown hypothetical chloroplast open reading frame (ycf) ycf54 should be analyzed concerning it’s physiological function in the enzymatic step of the cyclase using transgenic cyanobacteria and plants. Lack of Ycf54 in Synechocystis sp. PCC6803 and the homologous LCAA protein in Nicotiana tabacum and Arabidopsis thaliana, respectively, leads to chlorophyll deficiency, a strong accumulation of the cyclase substrate and reduced protein contents of Ycf59 and CHL27, respectively. A deficit of Ycf54/LCAA impairs the functionality of the cyclase complex and also might compromise the stability of photosynthetic antenna complexes. Using pull-down assays a direct physical interaction between LCAA and CHL27 could be confirmed. Additionally, first evidences for ferredoxin NADP reductase as a potential interaction partner was given.

The supply of precursors from primary metabolism is often overlooked when engineering secondary metabolism for increased product yields. This is because precursor supply may be assumed to be non-limiting, and it is considered difficult to engineer primary metabolism, because control of carbon flow (flux) is generally distributed among most enzymes of the pathway. The aim of this thesis was to increase the production of sterols, part of the isoprenoid class of secondary metabolites, in tobacco (Nicotiana tabacum) Bright Yellow 2 (BY-2) cell cultures. This was achieved by genetically engineering increased activity of mitochondrial citrate synthase, an enzyme of the tricarboxylic acid (TCA) cycle that is involved in the provision of cytosolic acetyl coenzyme A, the primary metabolite precursor to sterols. Metabolic flux analysis revealed that citrate synthase exerts significant control over cyclic TCA cycle flux in BY-2 cells and suggested that increasing the activity of downstream enzymes within secondary metabolism could lead to a further redirection of TCA-cycle-derived precursors into sterol biosynthesis. Attempts were made to achieve this by genetically engineering increased activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), a key enzyme of secondary metabolism involved in sterol biosynthesis. Consistent with previous research, transgenic lines had increased sterol levels. However, the high sterol phenotype was unstable, and attempts to co-express HMGR and citrate synthase genes were unsuccessful. The thesis demonstrates that increasing the provision of precursors to secondary metabolites can result in increased yields of those secondary metabolites but suggests that in most cases the activity of enzymes within secondary metabolism has a greater effect on those yields. It also reveals that single enzymes can exert significant control of flux within primary metabolism, although the control exerted by specific enzymes probably changes with the demands placed on metabolism.

The biofuel market is dominated by ethanol and biodiesel derived from cellulosic and lipid-based biomass crops. This is largely due to the relatively low costs and reliability of production. At present, production of non-food plant-derived oils for biofuel production in the U.S. is minimal. A research team from the Commonwealth Scientific and Industrial Research Organization (CSIRO), an independent Australian federal government research institution, has developed an efficient transgenic system to engineer oil production in tobacco leaves. This novel system is comprised of multiple transgenes that direct the endogenous metabolic flux of oil precursors towards triacylglycerol (TAG) production. Additional genes were incorporated to store and protect the accumulated oil in vegetative tissues. Preliminary greenhouse tests by the CSIRO research group indicated an oil content of > 30% by dry weight (DW) in tobacco leaf lamina. Here we evaluated two transgenic lines against a non-transgenic control in 2017 and 2018 in greenhouse and field production systems. The 2017 pilot study showed that the high leaf-oil tobacco line was viable and will grow in the field in Kentucky. Chemical analyses revealed significantly higher oil content compared to the non-transgenic control despite several logistical setbacks. These promising discoveries prompted the deployment of additional transgenic line assessments and further data validation in 2018. Line evaluations in 2018 revealed that the LEC2:WRI1:DGAT:OLE transgenic line had the highest leaf oil content (≥ 19.3% DW-1) compared to both the WRI1:DGAT:OLE transgenic line (≤ 5.6% DW-1) and non-transgenic control (≤ 2.1% DW-1). The results of this research will contribute to the successful development of transgenic tobacco lines engineered to accumulate high concentrations of TAG in the leaves.

Plants do not display an adaptive immune system but express an efficient innate immune system defending them by inducing sophisticated multilevel defense responses against different potential pathogens. Indeed, plant immune responses are triggered upon the detection of many common pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs) through specific pattern-recognition receptors (PRRs) and/or pathogen- or plant-derived signal molecules called elicitors. Cryptogein (Cry) and oligogalacturonides (OGs) are well known elicitors of defense reactions and their recognition induce a Ca2+-dependent signaling pathway: Ca2+ influx and subsequent free cytosolic [Ca2+] ([Ca2+]cyt) variations are earliest steps to trigger downstream plant defense signaling. Here we have demonstrated that elicitor-induced Ca2+ signaling in tobacco also takes place in mitochondria and chloroplasts. Pharmacological studies indicated that IP3-channels play an important role in the regulation of Ca2+ signaling in mitochondria and chloroplasts. Mitochondrial respiration and energy dissipation mechanisms in chloroplasts are partly controlled by [Ca2+] in these organelles. Moreover, using pharmacological and genetic approaches, our data demonstrated that glutamate receptors homologs (GLRs) participate in OGs-mediated Ca2+ signaling in Arabidopsis. GLRs partly control OGs-induced nitric oxide (NO) production, reactive oxygen species (ROS) production and expression of defense-related genes. Importantly, plants treated with GLRs antagonists exhibited compromised resistance to necrotrophic fungal pathogen, Botrytis cinerea and biotrophic oomycete, Hyaloperonospora arabidopsidis. Analysis of Atglr single mutants revealed the important contribution of AtGLR3.3 in resistance against H. arabidopsidis. Moreover, striking similarities in gene expression levels were observed after OGs elicitation/H. arabidopsidis infection. Finally, transcriptomic analysis demonstrated that about 60 % of the total OGs-modulated genes modified their expression in GLRs-dependent manner. These GLRs-dependent genes belong to different functional categories including the category "responses to biotic stresses". Taken together, these data provide strong evidences of 1) elicitor-induced Ca2+ signaling in mitochondria and chloroplasts in tobacco and 2) the regulation of elicitor/pathogen mediated plant defense signaling pathways through GLRs in Arabidopsis thaliana

Secondary metabolism in plants produces numerous compounds with wide-ranging activities, including the antineoplastic compound taxol and related taxanes. The biotechnological production of taxol has so far been based on empirical studies. The aim of the present work has been to study how the factors that enhance taxane production affect the metabolic profiles and gene expression in productive cells. As a consequence of this work, new potential candidates have been obtained for unknown taxane biosynthetic genes, some bottle-neck steps of taxane biosynthesis (in vitro and in silico) have been identified and a master regulator, not only for taxane biosynthesis, but also for other secondary metabolism routes, has been characterized. Coronatine, a powerful and less harmful elicitor than methyl jasmonate, has been successfully assayed and found to increase taxane production. In the different studies of this work, the expression level of genes that participate in taxol biosynthesis has been determined, clarifying their involvement in the production of this anti-cancer agent. / El metabolismo secundario de las plantas produce numerosos compuestos con un amplio rango de actividades, entre los que se encuentra el compuesto antineoplásico taxol y los taxanos relacionados, la producción biotecnológica del cual se basa en estudios empíricos. El objetivo de este trabajo ha sido estudiar como los factores que incrementan la producción de taxanos afectan los perfiles metabólicos y la expresión génica en los cultivos. De esta manera se han identificado nuevos genes candidatos que codifican para los genes desconocidos de la biosíntesis, algunos pasos limitantes de ésta y se ha caracterizado un regulador relacionado con el metabolismo secundario. Se ha ensayado la coronatina, un elicitor más eficiente para mejorar la producción de taxanos y menos dañino que el jasmonato de metilo. En los diferentes ensayos de este trabajo han sido determinados los niveles de expresión de genes que participan en la biosíntesis de taxol, ayudando a comprender su papel en la producción de este anticancerígeno.

Proteins of DREPP family (20-25 kDa, syn. PCaP1 in Arabidopsis thaliana) first appeared in ferns and we have shown that several independent duplications of DREPP protein occurred during evolution of large families (Poaceae, Brassicaceae, Solanaceae and Asteraceae) and in group Coniferophyta. Secondary losses of one paralogue occurred in subfamilies Pooideae and Solanoideae.We have also detected two large-scale modification of DREEP protein in Asparagales and Brassicaceae (this divergent paralogue was previously described as MAP18 protein). We have examined colinearity of chromosome fragments in vicinity both PCaP1 and MAP18 paralogues in Arabidopsis thaliana and we hypothesize that MAP18 gene arose during genome duplication on the origin of Brassicaceae family. DREPP protein was previously identified in detergent-resistant membrane microdomain fraction and a myristyl anchor was shown to be necessary for their membrane localization. Membrane association was shown to be modified by the interaction of unique N-terminal domain with PtdInsPs, which was inhibited by binding of Ca-calmodulin (Nagasaki et al., 2008). The mutation of Gly2 by Ala in the myristilation site, or C-terminal GFP-fusion (GFP-DREPP), affect membrane association in Arabidopsis thaliana (Nagasaki et al., 2008). Several DREPP paralogues in...

Ce travail a pour objectif de tester des solutions viables et durables pour la phytoremédiation de sols contaminés en éléments traces. Il inclut à la fois des données sur l'évaluation initiale et résiduelle des risques (biomonitoring) et sur des solutions de phytomanagement à long terme utilisant des plantes et microorganismes associés, en particulier la phytoextraction aidée couplant l’épuisement du pool labile de contaminants du sol, la production de matière première végétale et la restauration de services écosystémiques. La phytotoxicité du Cu, le rôle améliorant des amendements organiques et minéraux et la tolérance des plantes ont été examinés utilisant des essais biologiques ainsi qu’une technique de dilution du sol. L'utilité d’une lignée de mutant de tournesol (Helianthus annuus) et d’une lignée parental de tabac (Nicotiana tabacum) pour le biomonitoring de sols contaminés en Cu a été investiguée. Les paramètres biochimiques qui sont en relation avec le statut antioxidant des plantes et leurs réponses moléculaires à l’excès de Cu ont en général montré une plus grande sensibilité que les traits morphologiques. Le tabac est plus tolérant au Cu que le tournesol. Des bactéries endophytes issues de différentes sources, notamment des graines d’une population métallicole d’une graminée (Agrostis capillaris) peuvent stimuler la croissance du tournesol et du tabac aux expositions en Cu modérément en excès. Pour ces plantes annuelles, accumulatrices secondaires du Cu et à phénotype d’exclusion, l'augmentation de la capacité de phytoextraction de Cu par les parties aériennes s’effectue principalement par l’accroissement de la biomasse aérienne, plutôt que par celui de sa concentration en Cu. Par conséquent, une attention a été prêtée aux pratiques agricoles dans les essais en parcelles sur site. Plusieurs options d’amélioration ont été examinées in situ: l'application d’amendements du sol, l'utilisation de lignées de mutants et de variants somaclonaux, la rotation de cultures et des cultivars, la bioaugmentation, la fertilisation, l’irrigation, etc. Deux lignées de mutants et des cultivars commerciaux de tournesol ainsi qu’une lignée parentale de tabac ont montré un fort potentiel pour la phytoextraction du Cu, accompagné d’une production significative de graines et/ou autre biomasse valorisable. Les solutions de restauration écologique basées sur la phytoextraction, utilisant des plantes annuelles accumulatrices secondaires de Cu permettraient (1) la décontamination progressive des sols contaminés en Cu au cours des rotations culturales, (2) un retour financier lié à la valorisation de la biomasse végétale, et (3) la restauration de services écosystémiques. / This work aimed at assessing sustainable phytoremediation options for trace element-contaminated soils. It includes both the assessment of initial and residual risks (biomonitoring) and long-term sustainable decontamination options using plants and associated microbes, especially aided phytoextraction with the secondary purposes of producing plant-based feedstock and restoring ecosystem services. Copper phytotoxicity, the improving role of soil conditioners, and plant tolerance were tested using a bioassay as well as a fading technique. The usefulness of a mutant line of sunflower (Helianthus annuus) and a motherline of tobacco (Nicotiana tabacum) for the biomonitoring of Cu-contaminated soils was investigated. Biochemical parameters in relation to antioxidant status of plants and molecular responses to Cu excess generally showed a greater sensitivity than morphologic ones. Tobacco has a higher Cu tolerance than sunflower. Endophytic bacteria from various sources, notably from the seeds of metallicolous populations of grasses (Agrostis capillaris) can promote the growth of sunflower and tobacco exposed to Cu excess. For annual Cu-secondary accumulator plants with an excluder phenotype, increase in shoot Cu removal occurred primarily through increase in shoot biomass, rather than in shoot Cu concentration. Therefore, attention in field trials was paid to agricultural practices. Various improving options were tested in situ: application of soil amendments, the use of mutant lines and somaclonal variation, cultivars and crop rotation, bioaugmentation, fertilization, irrigation, etc. Two mutant lines and some commercial cultivars of sunflower as well as the motherline of tobacco showed a high potential for Cu phytoextraction as well as for plant-based feedstock. Ecological restoration options for Cu-contaminated soils based on phytoextraction using annual Cu-secondary accumulator plants with a high shoot biomass would (1) result in the progressive decontamination of Cu-contaminated soils during crop rotations, (2) provide a financial return through biomass valorization, and (3) promote ecosystem services.