The differentiation of plant vascular tissue is regulated by plant hormones and transcription factors. One of the key plant hormones involved in this process is auxin. Auxin signals are mediated by auxin response factor transcription factors (ARFs). These transcription factors are involved in the perception of auxin signals and the subsequent activation or deactivation of suites of downstream genes. Based on its mutant phenotype, one of the most interesting members of this family is the ARF MONOPTEROS (MP). This thesis investigates the role played by MP in secondary vascular differentiation, as well as taking a look at other molecular aspects of secondary vascular differentiation, with a focus on the model plants Arabidopsis thaliana and poplar (Populus trichocarpa and hybrid poplar). A dexamethasone inducible RNAi silencing strategy was developed, and transgenic Arabidopsis lines produced. When silencing was induced in these lines from germination, a phenotype closely resembling the mp mutant was observed. When MP silencing was induced in bolting stems, early senescence, as well as a dramatic reduction in interfascicular fibre production was observed, and these stems were thinner and less rigid than empty vector controls. RNA from these stems was isolated and used in a global transcript profiling microarray experiment. This experiment showed that several auxin-related genes, as well as several transcription factors, were differentially regulated in response to MP silencing. Because Arabidopsis is not a typical woody plant, further investigation into the role played by MP in wood formation was done using the model tree poplar. A BLAST search of a poplar xylem EST database identified a single promising partial sequence. Based on this sequence information, a poplar MP homolog was isolated and named PopMP1. The full-length sequence of this gene demonstrated remarkable structural conservation when compared with that of Arabidopsis. Subsequent complete sequencing of the poplar genome revealed a second copy of the MP gene in poplar and named PopMP2. Expression profiling across a range of tissues suggests that subfunctionalization has occurred between the two copies. Overexpression transgenic lines for PoptrMP1 were developed. AtHB8 is known to be regulated by MP in Arabidopsis, and a poplar HB8 homolog was upregulated in the transgenic lines. However, no obvious physical phenotype in these lines was apparent. To investigate the transcriptome-wide changes associated with initiation of cambium formation in poplar stems, a global transcript profiling experiment was performed. Out of 15400 genes tested, 2320 met an arbitrary cutoff of >1.3 fold and p-value <0.05 and were labeled differentially expressed (DE). These included several transcription factors and showed remarkable similarity to analogous data from Arabidopsis. The conclusions drawn from this thesis support the hypothesis that MP plays roles in later development, and do not rule out the possibility that MP is directly involved in wood development. The data reported also offer a large number of candidate for further investigation into the genetic control of wood development.

The differentiation of plant vascular tissue is regulated by plant hormones and transcription factors. One of the key plant hormones involved in this process is auxin. Auxin signals are mediated by auxin response factor transcription factors (ARFs). These transcription factors are involved in the perception of auxin signals and the subsequent activation or deactivation of suites of downstream genes. Based on its mutant phenotype, one of the most interesting members of this family is the ARF MONOPTEROS (MP). This thesis investigates the role played by MP in secondary vascular differentiation, as well as taking a look at other molecular aspects of secondary vascular differentiation, with a focus on the model plants Arabidopsis thaliana and poplar (Populus trichocarpa and hybrid poplar). A dexamethasone inducible RNAi silencing strategy was developed, and transgenic Arabidopsis lines produced. When silencing was induced in these lines from germination, a phenotype closely resembling the mp mutant was observed. When MP silencing was induced in bolting stems, early senescence, as well as a dramatic reduction in interfascicular fibre production was observed, and these stems were thinner and less rigid than empty vector controls. RNA from these stems was isolated and used in a global transcript profiling microarray experiment. This experiment showed that several auxin-related genes, as well as several transcription factors, were differentially regulated in response to MP silencing. Because Arabidopsis is not a typical woody plant, further investigation into the role played by MP in wood formation was done using the model tree poplar. A BLAST search of a poplar xylem EST database identified a single promising partial sequence. Based on this sequence information, a poplar MP homolog was isolated and named PopMP1. The full-length sequence of this gene demonstrated remarkable structural conservation when compared with that of Arabidopsis. Subsequent complete sequencing of the poplar genome revealed a second copy of the MP gene in poplar and named PopMP2. Expression profiling across a range of tissues suggests that subfunctionalization has occurred between the two copies. Overexpression transgenic lines for PoptrMP1 were developed. AtHB8 is known to be regulated by MP in Arabidopsis, and a poplar HB8 homolog was upregulated in the transgenic lines. However, no obvious physical phenotype in these lines was apparent. To investigate the transcriptome-wide changes associated with initiation of cambium formation in poplar stems, a global transcript profiling experiment was performed. Out of 15400 genes tested, 2320 met an arbitrary cutoff of >1.3 fold and p-value <0.05 and were labeled differentially expressed (DE). These included several transcription factors and showed remarkable similarity to analogous data from Arabidopsis. The conclusions drawn from this thesis support the hypothesis that MP plays roles in later development, and do not rule out the possibility that MP is directly involved in wood development. The data reported also offer a large number of candidate for further investigation into the genetic control of wood development.

The ratio of auxin to cytokinin determines de novo organogenesis in plants. Relatively little is known about the effect of genetically altered auxin signaling on in vitro organogenesis. Here, callusogenesis, shoot, and root formation were studied in loss- (LOF) and gain-of-function (GOF) alleles in two phylogenetically related Auxin Response Factors (ARFs), MONOPTEROS (MP/ARF5) and NON-PHOTOTROPHIC HYPOCOTYL 4 (NPH4/ARF7). Reduced MP activity greatly diminished shoot regeneration, and partially diminished callusogenesis and root formation. LOF in NPH4 strongly decreased callusogenesis, and mildly decreased shoot and root regeneration in particular categories of explants. By contrast, organogenesis responses were strongly increased in aerial explants carrying the GOF transgene dMP. Thus, both MP and NPH4 seem to act as positive regulators of certain organogenesis processes and the GOF dMP transgene may be of interest for stimulating organogenesis in plant species with poor regeneration properties. Also, organogenesis in vitro may reveal unknown developmental ARF functions.

The ratio of auxin to cytokinin determines de novo organogenesis in plants. Relatively little is known about the effect of genetically altered auxin signaling on in vitro organogenesis. Here, callusogenesis, shoot, and root formation were studied in loss- (LOF) and gain-of-function (GOF) alleles in two phylogenetically related Auxin Response Factors (ARFs), MONOPTEROS (MP/ARF5) and NON-PHOTOTROPHIC HYPOCOTYL 4 (NPH4/ARF7). Reduced MP activity greatly diminished shoot regeneration, and partially diminished callusogenesis and root formation. LOF in NPH4 strongly decreased callusogenesis, and mildly decreased shoot and root regeneration in particular categories of explants. By contrast, organogenesis responses were strongly increased in aerial explants carrying the GOF transgene dMP. Thus, both MP and NPH4 seem to act as positive regulators of certain organogenesis processes and the GOF dMP transgene may be of interest for stimulating organogenesis in plant species with poor regeneration properties. Also, organogenesis in vitro may reveal unknown developmental ARF functions.

L'auxine est une hormone végétale qui coordonne plusieurs processus de développement des plantes à travers la régulation d'un ensemble spécifique de gènes. Les Auxin Response Factors (ARF) sont des régulateurs transcriptionnels qui modulent l'expression de gènes de réponse à l’auxine. Des données récentes montrent que les membres de la famille des ARF sont impliqués dans la régulation du développement des fruits de la nouaison à la maturation. L'objectif principal de la thèse est d’étudier la part qui revient aux ARF dans le contrôle du développement et de la maturation des fruits et d’en comprendre les mécanismes d’action. L’analyse des données d’expression disponibles dans les bases de données a révélé que, parmi tous les ARF de tomates, SlARF2 affiche le plu haut niveau d'expression dans le fruit avec un profil distinctif d’expression associé à la maturation. Nous avons alors entrepris la caractérisation fonctionnelle de SlARF2 afin d’explorer son rôle dans le développement et la maturation des fruits. Deux paralogues, SlARF2A et SlARF2B, ont été identifiés dans le génome de la tomate. Nous avons montré que l’expression de SlARF2A dans le fruit est régulée par l'éthylène tandis que celle de SlARF2B est induite par l'auxine. La sous-expression de SlARF2A, comme celle de SlARF2B, entraine un retard de maturation alors que l’inhibition simultanée des deux paralogues conduit à une inhibition plus sévère de la maturation suggérant une redondance fonctionnelle entre les deux paralogues lors de la maturation des fruits. Les fruits présentant une sous-expression des gènes SlARF2 produisent de faibles quantités d'éthylène, montrent une faible accumulation de pigments et une plus grande fermeté. Le traitement avec de l'éthylène exogène ne peut pas inverser les phénotypes de défaut de maturation suggérant que SlARF2 pourrait agir en aval de la voie de signalisation de l'éthylène. L'expression des gènes clés de biosynthèse et de signalisation de l'éthylène est fortement perturbée dans les lignées sous-exprimant SlARF2 et les gènes majeurs qui contrôlent le processus de maturation (RIN, CNR, NOR, TAGL1) sont sensiblement sous-régulés. Les données suggèrent que SlARF2 est essentiel pour la maturation des fruits et qu’il pourrait agir au croisement des voies de signalisation de l'auxine et de l'éthylène. Dans le but de mieux comprendre les mécanismes moléculaires par lesquels les ARF régulent l'expression des gènes de réponse à l'auxine, nous avons étudié l'interaction des SlARFs avec des partenaires protéiques ciblés, principalement les co-répresseurs de type Aux/IAA et Topless (TPL) décrits comme les acteurs clés dans la répression des gènes dépendant de la signalisation auxinique. Une fois les gènes codant pour les membres de la famille TPL de tomate isolés, une approche double hybride dans la levure a permis d’établir des cartes exhaustives d'interactions protéine-protéine entre les membres des ARFs et des Aux/IAA d’une part et les ARFs et les TPL d’autre part. L'étude a révélé que les Aux/IAA interagissent préférentiellement avec les SlARF activateurs et qu’à l’inverse les Sl-TPL interagissent uniquement avec les SlARF répresseurs. Les données favorisent l'hypothèse que les ARF activateurs recrutent les Sl-TPL via leur interaction avec les Aux/IAA, tandis que les ARF répresseurs peuvent interagir directement avec les Sl-TPL. Les études d’interactions ont permis également d’identifier de nouveaux partenaires comme les protéines VRN5 et LHP1, composantes des complexes Polycomb PRC impliqués dans la repression par voie épigénétique de la transcription par modification de l'état de méthylation des histones. Au total, le travail de thèse apporte un nouvel éclairage sur le rôle et les mécanismes d'action des ARF et identifie SlARF2 comme un nouvel élément du réseau de régulation contrôlant le processus de maturation des fruits chez la tomate. / The plant hormone auxin coordinates plant development through the regulation of a specific set of auxin-regulated genes and Auxin Response Factors (ARFs) are transcriptional regulators modulating the expression of auxin-response genes. Recent data demonstrated that members of this gene family are able to regulate fruit set and fruit ripening. ARFs are known to act in concert with Aux/IAA to control auxin-dependent transcriptional activity of target genes. However, little is known about other partners of ARFs. The main objective of the thesis research project was to gain more insight on the involvement of ARFs in fruit development and ripening and to uncover their interaction with other protein partners beside Aux/IAAs. Mining the tomato expression databases publicly available revealed that among all tomato ARFs, SlARF2 displays the highest expression levels in fruit with a marked ripening-associated pattern of expression. This prompted us to uncover the physiological significance of SlARF2 and in particular to investigate its role in fruit development and ripening. Two paralogs, SlARF2A and SlARF2B, were identified in the tomato genome and transactivation assay in a single cell system revealed that the two SlARF2 proteins are nuclear localized and act as repressors of auxin-responsive genes. In fruit tissues, SlARF2A is ethylene-regulated while SlARF2B is auxin-induced. Knock-down of SlARF2A or SlARF2B results in altered ripening with spiky fruit phenotype, whereas simultaneous down-regulation of SlARF2A and SlARF2B leads to more severe ripening inhibition suggesting a functional redundancy among the two SlARF2 paralogs during fruit ripening. Double knock-down fruits produce less climacteric ethylene and show delayed pigment accumulation and higher firmness. Exogenous ethylene treatment cannot reverse the ripening defect phenotypes suggesting that SlARF2 may act downstream of ethylene signaling. The expression of key ethylene biosynthesis and signaling genes is dramatically disturbed in SlARF2 down-regulated fruit and major regulators of the ripening process, like RIN, CNR, NOR, TAGL1, are under-expressed. The data support the notion that SlARF2 is instrumental to fruit ripening and may act at the crossroads of auxin and ethylene signaling. Altogether, while ethylene is known as a key hormone of climacteric fruit ripening, the ripening phenotypes associated with SlARF2 down-regulation bring unprecedented evidence supporting the role of auxin in the control of this developmental process. To further extend our knowledge of the molecular mechanism by which ARFs regulate the expression of auxin-responsive genes we sought to investigate interactions SlARF and putative partners, mainly Aux/IAAs and Topless co-reppressors (TPLs) reported to be key players in gene repression dependent on auxin signaling. To this end, genes encoding all members of the tomato TPL family were isolated and using a yeast-two-hybrid approach comprehensive protein-protein interaction maps were constructed. The study revealed that Aux/IAA interact preferentially with activator SlARFs while Sl-TPLs interact only with repressor SlARFs. The data support the hypothesis that activator ARFs recruit Sl-TPLs co-repressors via Aux/IAAs as intermediates, while repressor ARFs can physically interact with Sl-TPLs. Further investigation indicated that SlARFs and Sl-TPLs can interact with polycomb complex PRC1 PRC2 components, VRN5 and LHP1, known to be essential players of epigenetic repression of gene transcription through the modification of histones methylation status. These data establish a potential link between ARFs and epigenetic regulation and thereby open new and original perspectives in understanding the mode of action of ARFs. Altogether, the thesis work provides new insight on the role of ARFs and their underlying action mechanisms, and defines SlARF2 as a new component of the regulatory network controlling the ripening process in tomato.