In plants, flowering time is a tightly regulated process where several environmental and endogenous cues fine-tune the time of flowering. In Arabidopsis, four major genetic pathways regulate flowering time, namely photoperiod, vernalization, autonomous, and phytohormone gibberellic acid (GA) pathways. Arabidopsis is a facultative long day (LD) plant. LD promotes flowering whereas flowering is delayed in short day (SD) conditions. Here, we identified a basic-helix-loop-helix (bHLH) transcription factor called bHLH93 that is necessary to promote flowering only in SD. Also, photoperiod plays more critical roles in regulation of flowering time of bhlh93 mutant compared to GA and vernalization pathways. Thus, bHLH93 might represent a novel transcription factor absolutely required for Arabidopsis thaliana to evolve as a facultative LD plant. bhlh93 mutants also show severe adult phenotype such as shorter stature, curly and darker green leaves, and reduced fertility compared to wild type plants. These results suggest that bHLH93 controls plant stature, fertility and chlorophyll content in Arabidopsis. bHLH93 is expressed in a tissue-specific and developmental stage-dependent manner. bHLH93-YFP protein is localized in the nucleus. bHLH93 homodimerizes in yeast, and it has strong transcription activation activity in yeast. These data suggest that, like other bHLH proteins, bHLH93 may function as a transcriptional regulator in the nucleus controlling gene expression. We have identified floral repressor MAF5 as a major target of bHLH93 to promote flowering in SD. bHLH93 binds to MAF5 promoter element in vivo and in vitro. Other than MAF5, FLC and MAF1-2 are also up-regulated in bhlh93 but at a lower level than MAF5. The activation of multiple floral repressors correlates with bhlh93 flowering phenotype. Taken together, these data suggest that bHLH93 may provide selective advantage for evolution of facultative flowering behavior under varying environmental conditions for reproductive success. / text

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Les composés phénoliques de la baie, et plus particulièrement les flavonoïdes (flavonols, tanins condensés, anthocyanes), constituent un des paramètres clés contrôlant la qualité organoleptique du raisin et du vin. Ils représentent également une source de molécules antioxydantes d’intérêt grandissant pour les industries pharmacologiques, agro-alimentaires et cosmétiques. De nombreux travaux réalisés sur les plantes modèles ont démontré que la régulation de la voie de biosynthèse des flavonoïdes est essentiellement gouvernée par des facteurs de transcription de type MYB, bHLH et des protéines de type WD40. Chez la Vigne (Vitis vinifera L.), plusieurs facteurs de transcription de type MYB régulant la voie des anthocyanes et/ou des tanins condensés ont déjà été identifiés. Cependant, aucun facteur de transcription de type bHLH ou WD40 n’a encore été caractérisé. Différentes approches ont été mises en œuvre pour identifier de nouveaux régulateurs de la voie des flavonoïdes chez la Vigne. Dans un premier temps, le facteur de transcription VvMYB5b a été utilisé comme appât dans une approche de double hybride non ciblé chez la Levure (Saccharomyces cerevisiae). Dans un deuxième temps, le promoteur d’un gène codant une enzyme centrale de la voie de biosynthèse des flavonoïdes, VvDFR, a été choisi afin de développer une approche de simple hybride non ciblé chez la Levure. Enfin, une approche ciblée vers des « gènes candidats » a permis l’identification des facteurs de transcription de type bHLH VvMYC1 et VvMYCA1. Le profil d’expression de VvMYCA1 correspond à celui de l’accumulation des tanins condensés dans la pellicule, et celui de VvMYC1 corrèle avec le profil de synthèse des flavonols, des anthocyanes et des tanins condensés dans la baie de raisin, ainsi que dans les inflorescences. De plus, VvMYC1 peut interagir avec différents partenaires MYB de Vigne régulant la synthèse des anthocyanes et/ou des tanins condensés, à la fois dans la Levure mais également in planta, où l’interaction VvMYC1/VvMYBs affecte l’expression de gènes structuraux tels que l’UFGT et l’ANR. Cette interaction induit une synthèse d’anthocyanes, aussi bien en système homologue qu’en système hétérologue (Tabac et Arabidopsis). Enfin, des essais complémentaires impliquant le promoteur de VvMYC1 ont permis de démontrer que VvMYC1, en interagissant avec VvMYBPA1, peut moduler sa propre expression in vivo. / Phenolic compounds, and more specifically flavonoids (flavonols, condensed tannins and anthocyanins), are key components of the grapevine and wine quality. Because of their antioxidant activities, these compounds are of interest in pharmacological and cosmetic industries, as well as being beneficial to the human diet. Previous work on model plants showed that the flavonoid pathway was mainly regulated by the MYB and bHLH transcription factors, and WD40 proteins. In the grapevine (Vitis vinifera L.), only MYB regulators have been identified until now, and no bHLH or WD40 have been characterised. In this work, several approaches were used to identify new transcription factors involved in grapevine flavonoid biosynthesis. Firstly, the VvMYB5b protein was used as a bait in a large scale two hybrid experiment in yeast (Saccharomyces cerevisiae). Secondly, the promoter of the VvDFR gene, coding a central enzyme of the flavonoid pathway, was chosen to conduct a large scale one hybrid experiment, also in yeast. Finally, a “gene candidate” approach allowed identification of the bHLH transcription factors VvMYC1 and VvMYCA1. VvMYCA1 expression profile in berry skin and seeds correlates with condensed tannins synthesis, whereas VvMYC1 transcript accumulation in these tissues and the grapevine inflorescence correlates with condensed tannins, anthocyanins and flavonols accumulation. In yeast, VvMYC1 could physically interact with different MYB partners regulating the anthocyanin or the condensed tannins biosynthesis. This interaction was confirmed by transient promoter assays in grape cell suspensions, where co-expression of VvMYC1 with specific MYB partners activated the UFGT and ANR promoters. Likewise, this interaction induced anthocyanin accumulation in grape cells, as well as in tobacco leaves and Arabidopsis. Eventually, additional transient promoter assays revealed that VvMYC1 is involved, with VvMYBPA1, in feedback regulation of its own expression.

The central nervous system is formed from epiblast precursor cells through Neurulation. Neural induction can be studied in its main aspects in vitro. However, the process is poorly understood, especially in regard to when and how a cell becomes specified, and then committed, to be a neural cell. It is, on the other hand, well established that neural formation requires absence or, inhibition of the BMP signalling both in vivo and in vitro. ID1 is a direct target of BMP signalling with major influence on in vitro neural differentiation. A cDNA library screen, looking for transcription factors negatively regulated by ID1, reported TWIST1, along with only two other proteins. Twist1 expression is upregulated during in vitro neural differentiation. Furthermore, targeted deletion of Twist1 has dramatic consequences on anterior neural development. Twist1 knock-out mice fail to form the closed neural tube in the prospective brain, followed by exencephaly and, early embryonic death. In this thesis I investigate the influence on in vitro neural differentiation of a TWIST1 constitutively active form, insensitive to ID1 inhibition. I report that this transcriptionally active TWIST1 accelerates neural differentiation, in vitro and, biases it, towards dorsal phenotypes. I provide, for the first time, evidence for Twist1 expression in the neural tissue, observed weakly in a restricted domain, temporally and spatially, in the dorsal part of the neural tube. I propose a new model for TWIST1 influence at this level. I also investigate how TWIST1 actions depend on levels of expression and dimer choice. I found that, TWIST1 can exert its neural modulating actions only at low levels, as high levels divert a cell fate towards non-neural lineages.

Spiders are able to spin a variety of silk types for various purposes, each with their own unique properties. The mechanical properties of spider silk out-perform the mechanical properties of many man-made materials we use today, including tensile steel, KevlarTM, and nylon. To further understand the proteins the silks are made of and how they are synthesized in the silk glands, transcriptional and proteomic analysis was conducted. Transcriptional regulation of silk genes was investigated to determine how and why several silk proteins are transcribed into mRNA products together in the same gland. The tubuliform gland is one of the major contributors of egg case silk production. The mRNA of major ampullate spidroins 1 and 2 (MaSp1, MaSp2) and tubuliform spidroin 1 (TuSp1) is found in the tubuliform glands, but not all are translated into proteins for egg case silk purposes. To understand why not all of the transcribed mRNA products are not being translated into proteins, the promoter sequences of MaSp1, MaSp2, and TuSp1 were aligned and found to contain an E-Box site. Several constructs containing the cDNA of the promoter sequences and cDNA of bHLH transcription factors were built to test transcriptional regulation of MaSp1, MaSp2, and TuSp1. Proteomic analysis of egg case silk and the tubuliform glands was also conducted to identify further proteins synthesized in the tubuliform glands and to determine which of these proteins are ultimately incorporated into the egg case silk fibers by MS/MS analysis. Multiple silk proteins were identified within the tubuliform glands and incorporated into the egg case fibers, suggesting silks are composite fibers of multiple spidroins.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18667号 / 生博第320号 / 新制||生||43(附属図書館) / 31581 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 佐藤 文彦, 教授 河内 孝之, 教授 荒木 崇 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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