Les flavonoïdes sont des substances naturelles connues pour leurs propriétés anti-inflammatoires, anti-cancéreuses ou anti-virales chez l'homme. Chez les végétaux, ils participent notamment à leur protection vis-à-vis d'organismes pathogènes. La voie de biosynthèse des flavonoïdes est l'une des plus étudiées chez les plantes et notamment chez la vigne : Vitis vinifera. Cependant, la ou les enzymes impliquées dans les dernières étapes de biosynthèse conduisant aux anthocyanes et aux proanthocyanidines restent, à ce jour, peu ou pas connues. L’étude que nous proposons a pour but de concevoir des sondes moléculaires d’affinité susceptibles d’interagir avec une ou plusieurs enzymes impliquées dans ces dernières étapes de biosynthèse. Ces sondes, basées sur la technologie émergeante de protéomique chimique : « Activity- and affininity Based Protein Profiling » (ABPP), ont été validées à l’aide d’une enzyme modèle : la leucoanthocyanidine dioxygénase (LDOX). Elles ont ensuite été appliquées à des extraits complexes de protéines issus de Vitis vinifera. / Flavonoids are natural substances known for their anti-inflammatory, anti-cancerous and anti-virals properties in humans. In plants, they are one of the molecules responsible for fighting pathogens. The flavonoid biosynthesis pathway as been greatly studied in plants, especially in that of the grapevine: Vitis vinifera. However, detailed studies of the exact function of the enzymes involved in the last steps of the biosynthesis of anthocyanins and proanthocyanidins remains largely lacking.The study that we propose is to synthesize molecular probes designed to specifically interact with enzymes involved in the last stages of flavonoids biosynthesis. Our probes, based on the emerging chemical proteomic technology, activity- and affinity based protein profiling (ABPP), were validated with a model enzyme: leucoanthocyanidin dioxygenase (LDOX). After which, they were used with complex protein mixtures from Vitis vinifera.

The flavonols are a varied and abundant sub-class of flavonoids that are associated with a number of essential physiological functions in plants and pharmacological activities in animals. The 2-oxoglutarate-dependant dioxygenases(2-ODDs), flavonol synthase (FLS) and flavanone 3-hydroxylase (F3H), are essential for flavonol synthesis. The primary goal of this study has been to gain a deeper understanding of the biochemistry of these enzymes in Arabidopsis. To accomplish this goal, an activity assay employing recombinant protein expression and HPLC as a detection system was developed for F3H and adapted for use with FLS. The assay was employed to establish the biochemical parameters of F3H from Arabidopsis, and to further characterize the F3H mutant allele, <i>tt6</i>(87). Enzymatic activity was demonstrated for F3H enzymes from <i>Ipomoea alba</i> (moonflower), <i>Ipomoea purpurea</i> (common morning glory), <i>Citrus sinensis</i> (sweet orange), and <i>Malus X domestica</i> (newton apple), each of which had previously been identified solely based on sequence homology. Arabidopsis contains six genes with high similarity to <i>FLS</i> from other plant species; however, all other central flavonoid pathway enzymes in Arabidopsis are encoded by single genes. The hypothesis that differential expression of FLS isozymes with varying substrate specificities is responsible for observed tissue-specific differences in flavonol accumulation was tested. Sequence analysis revealed that <i>AtFLS2, 4</i> and <i>6</i> contain premature stop codons that eliminate residues essential for enzyme activity. AtFLS1 was found to have a strong preference for dihydrokaempferol as a substrate. However, no enzyme activity was observed for AtFLS3 or AtFLS5 with a number of different substrates under a variety of reaction conditions. To identify structural elements that may contribute to the observed differences in biochemical activity, homology models for each of the isoforms were generated utilizing Arabidopsis anthocyanin synthase (ANS) as a template. A domain at the N-terminus of AtFLS1 that is missing in the other isozymes was insufficient to convey activity to an AtFLS1/5 chimera. These findings suggest a single catalytically-active form of FLS exists in Arabidopsis. The possibility that the apparently expressed but non-catalytic proteins, AtFLS2, 3, and 5, serve noncatalytic roles in flavonol production were explored by yeast 2-hybrid analysis. / Ph. D.

The organization of metabolism is an essential feature of cellular biochemistry. Metabolism does not occur as a linear assembly of freely diffusing enzymes, but as a complex web in which multiple interactions are possible. Because of the crowded environment of the cell, there must be structured and ordered mechanisms that control metabolic pathways. The following work will examine two metabolic pathways, one that is ubiquitous among living organisms and another that is entirely unique to plants, and examine the organization of each in an attempt to further define mechanisms that are fundamental features of metabolic control. One study offers some of the first characterizations of genes involved in [Fe-S] cluster assembly in Arabidopsis. The other explores the mechanisms that control localization of an enzyme that is part of the well-characterized flavonoid biosynthetic pathway. These two distinct pathways serve as unique models for genetic and biochemical studies that contribute to our overall understanding of plant metabolism. / Ph. D.

Transcription factors (TFs) are ubiquitous gene expression regulators and play essential roles in almost all biological processes. This Ph.D. project is primarily focused on the functional characterisation of MYB112 - a member of the R2R3-MYB TF family from the model plant Arabidopsis thaliana. This gene was selected due to its increased expression during senescence based on previous qRT-PCR expression profiling experiments of 1880 TFs in Arabidopsis leaves at three developmental stages (15 mm leaf, 30 mm leaf and 20% yellowing leaf). MYB112 promoter GUS fusion lines were generated to further investigate the expression pattern of MYB112. Employing transgenic approaches in combination with metabolomics and transcriptomics we demonstrate that MYB112 exerts a major role in regulation of plant flavonoid metabolism. We report enhanced and impaired anthocyanin accumulation in MYB112 overexpressors and MYB112-deficient mutants, respectively. Expression profiling reveals that MYB112 acts as a positive regulator of the transcription factor PAP1 leading to increased anthocyanin biosynthesis, and as a negative regulator of MYB12 and MYB111, which both control flavonol biosynthesis. We also identify MYB112 early responsive genes using a combination of several approaches. These include gene expression profiling (Affymetrix ATH1 micro-arrays and qRT-PCR) and transactivation assays in leaf mesophyll cell protoplasts. We show that MYB112 binds to an 8-bp DNA fragment containing the core sequence (A/T/G)(A/C)CC(A/T)(A/G/T)(A/C)(T/C). By electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation coupled to qPCR (ChIP-qPCR) we demonstrate that MYB112 binds in vitro and in vivo to MYB7 and MYB32 promoters revealing them as direct downstream target genes. MYB TFs were previously reported to play an important role in controlling flavonoid biosynthesis in plants. Many factors acting upstream of the anthocyanin biosynthesis pathway show enhanced expression levels during nitrogen limitation, or elevated sucrose content. In addition to the mentioned conditions, other environmental parameters including salinity or high light stress may trigger anthocyanin accumulation. In contrast to several other MYB TFs affecting anthocyanin biosynthesis pathway genes, MYB112 expression is not controlled by nitrogen limitation, or carbon excess, but rather is stimulated by salinity and high light stress. Thus, MYB112 constitutes a previously uncharacterised regulatory factor that modifies anthocyanin accumulation under conditions of abiotic stress. / Transkriptionsfaktoren (TFs) sind ubiquitäre Regulatoren der Genexpression und spielen eine essentielle Rolle in nahezu allen biologischen Prozessen. Diese Doktorarbeit hat vor allem die funktionelle Charakterisierung von MYB112 zum Thema, einem Mitglied der R2R3-MYB-TF-Familie aus der Modellpflanze Arabidopsis thaliana. Ausgesucht wurde das Gen aufgrund seiner erhöhten Expression in seneszenten Blättern, basierend auf vorangegangenen qRT-PCR Expressions-Profiling Experimenten für 1880 TFs in Arabidopsis Blättern aus drei Entwicklungsstadien (15 mm Blatt, 30 mm Blatt und 20 % vergilbtes Blatt). MYB112-Promotor-GUS-Fusionslinien wurden generiert um das Expressionsmuster von MYB112 detailliert zu untersuchen. Unter Zuhilfenahme transgener Ansätze in Kombination mit Metabolomics und Transcriptomics können wir zeigen, dass MYB112 eine wichtige Rolle in der Regulation des pflanzlichen Flavonoid-Metabolismus spielt. In MYB112 Überexpressoren und MYB112-defizienten Mutanten kommt es zu erhöhter bzw. verminderter Anthocyanin-Akkumulation. Expressions-Profiling zeigt, dass MYB112 einerseits als ein positiver Regulator des Transkriptionsfaktors PAP1 fungiert, was zu einer erhöhten Anthocyanin-Biosynthese führt, andererseits als negativer Regulator von MYB12 und MYB111 auftritt, welche beide die Flavonol-Biosynthese kontrollieren. Wir haben früh auf MYB112 reagierende Gene durch eine Kombination verschiedener Ansätze identifiziert. Diese umfassen Genexpressions-Profiling (Affymetrix ATH1 Microarrays und qRT-PCR) und Transaktivierungs-Experimente in Mesophyll-Protoplasten aus Blättern. Wir zeigen, dass MYB112 an eine 8-bp DNA-Fragment, welches die Kernsequenz (A/T/G)(A/C)CC(A/T)(A/G/T)(A/C)(T/C) aufweist. Mit Hilfe von Electrophoretic Mobility Shift Assay (EMSA) und Chromatin-Immunopräzipitation gekoppelt mit qPCR (ChIP-qPCR) zeigen wir, dass MYB112 in vitro und in vivo an die Promotoren von MYB7 und MYB32 bindet was sie damit als direkte Zielgene von MYB112 identifiziert. Es wurde bereits gezeigt, dass MYB TFs eine wichtige Rolle bei der Kontrolle der Flavonoid-Biosynthese in Pflanzen haben. Viele Faktoren, die oberhalb des Anthocyanin-Biosyntheseweges agieren, werden bei Stickstofflimitierung oder erhöhter Saccharose-Konzentration auch verstärkt exprimiert. Außer den erwähnten Bedingungen können auch andere Umweltparameter, wie z. B. erhöhter Salzgehalt und Starklicht zu erhöhter Expression führen. Im Gegensatz jedoch zu einigen anderen MYB TFs, die einen Einfluss auf Gene des Anthocyanin-Biosyntheseweges ausüben, ist die Expression von MYB112 nicht durch Stickstoff-Limitierung oder Kohlenstoffüberfluss kontrolliert, sondern wird durch erhöhten Salzgehalt sowie Starklicht stimuliert. Somit ist MYB112 ein neuer Regulator, der eine Anthocyanin-Akkumulation unter abiotischen Stressbedingungen kontrolliert.

Highbush blueberry (Vaccinium corymbosum L.) has one of the highest antioxidant capacities and flavonoid concentrations of any fruit or vegetable, and regular consumption of blueberries has been connected to a wide range of health benefits. A diversity of flavonoids (flavonols, anthocyanins, proanthocyanidins) are likely responsible for many of the health benefits, and these compounds also significantly contribute to the organoleptic properties of ripe blueberries. Despite the potential importance of these flavonoids in diet, there has been little investigation into the molecular genetics of blueberry flavonoid biosynthesis. Therefore, I developed a real-time quantitative PCR protocol to monitor expression of flavonoid genes throughout development and ripening. Following evaluation of five reference genes, expression profiling of biosynthetic genes revealed that flavonoid synthesis is tightly controlled at the transcriptional level in a biphasic developmental pattern. These results are discussed in relation to flavonoid metabolite accumulation profiles, which were produced as part of a collaboration. Finally, in conjunction with a second group of collaborating scientists, some promising preliminary evidence is provided suggesting that the hormone abscisic acid might have a role in regulating ripening initiation in blueberry. / Graduate