Chez tous les eucaryotes, la régulation de l’expression génique est primordiale pour le contrôle du cycle cellulaire. Un large éventail de gènes, incluant des régulateurs essentiels du cycle, mais aussi d’autre gènes impliqués dans la transduction du signal, la régulation hormonale et le métabolisme sont ainsi exprimé à certaines phases du cycle. Ces changements sont contrôlés à de multiples niveaux, notamment de façon transcriptionnelle et post-traductionnelle. Chez les mammifères, il est aujourd’hui évident que les micro ARNs contribuent à cette régulation en ciblant spécifiquement les transcrits d’un grand nombre de gènes régulés au cours du cycle. Cependant, nous n’avons que très peu d’informations à ce jour concernant le rôle des petits ARNs sur le contrôle de la prolifération cellulaire chez les plantes. Mes travaux de thèse ont permis de démontrer que la perte d’AGO1 affecte la prolifération cellulaire et l’activité du méristème racinaire. Nous avons également séquencé les transcrits, les petits ARNs et le dégradome à partir de cellules BY-2 synchronisées afin de déterminer le répertoire et la fonction des petit ARNs au cours du cycle cellulaire. / In all eukaryotes, regulated gene expression is key to orchestrate cell cycle progression. Not only genes encoding important core cell cycle regulators, but also genes of a variety of other factors involved in signal transduction, hormonal regulation and metabolic control are expressed at specific time points of the cell cycle. These changes in gene expression are controlled at multiple levels, including transcriptional and post-translational controls. In mammals, it became evident that microRNAs contribute to this regulation by targeting the transcripts of numerous cell cycle-regulated genes. However, in plants we still know little about the regulatory roles of small RNAs in the control of cell proliferation. During my thesis, I showed that depletion of Arabidopsis AGO1 impairs cell proliferation and root meristem activity. To further determine the repertoire and role of sRNAs in cell cycle regulation, we thus sequenced total RNAs and small RNAs, AGO1-associated small RNAs and the RNA degradome of synchronized BY2 cells at S-, G2-, M- and G1-phases of the cell cycle.

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.