L’auxine est une hormone végétale impliquée dans presque toutes les étapes du développement des plantes, de la formation de l’embryon jusqu’à la floraison, déterminant la position des organes et donc la structure de la plante. Comme pour les autres hormones, la perception de l’auxine est suivie par une transduction du signal qui produit une série de changements dans les cellules végétales dont des régulations transcriptionnelles. Cette thèse est divisée en 3 chapitres, chacun d’eux étant focalisé sur des aspects structuraux, moléculaires et évolutifs de différentes protéines impliquées dans la régulation des gènes de réponse à l’auxine.Nous avons tout d’abord centré nos études sur TOPLESS (TPL), un corépresseur qui agit au niveau de la répression des gènes de réponse à l’auxine, mais aussi dans d’autres processus végétaux compte tenu de son interaction avec de nombreux répresseurs transcriptionnels. Nous avons déterminé la structure de la partie N-terminale de TPL et compris comment TPL interagit avec différents partenaires au niveau d’un même site de liaison. Nous avons alors démontré que TPL forme un tétramère à l’aide d’une surface de tétramérisation constituée par un nouveau domaine, le domaine CRA, qui fait aussi partie du site de liaison. Les résidus impliqués dans la tétramérisation et l’interaction avec des partenaires sont très conservés depuis des centaines de millions d’années montrant ainsi l’importance du rôle de TPL depuis l’origine des plantes. Enfin, les similarités de structure entre TPL et d’autres corépresseurs qui possèdent des domaines similaires mais possédant une fonction différente montrent un bel exemple de la manière dont l’évolution joue avec des domaines protéiques pour créer de nouvelles fonctions.Nous avons ensuite étudié les préférences de liaison à l’ADN des facteurs de transcription de la réponse à l’auxine (ARF). Pour cela nous avons utilisé une combinaison d’analyses bio-informatiques de données de DAP-seq sur la liaison des ARFs sur le génome, des tests d’interaction ADN-protéine in vitro et de la modélisation de structures. Nos résultats indiquent que les différents ARFs ont des sites préférentiels de liaison sur le génome et que ces préférences sont déterminées par l’orientation et l’espacement entre motifs de liaison. Enfin, ces études suggèrent qu’en fonction du site de liaison, les ARFs pourraient se lier avec différentes conformations à l’aide de surfaces de dimérisation qui ne sont pas encore décrites. Ces résultats permettent d’expliquer comment différents ARFs coexprimés dans la même cellule peuvent fonctionner ensemble pour contribuer à une réponse transcriptionnelle à l’auxine spécifique et robuste.Finalement, nous avons remonté le temps pour positionner l’origine de la voie de signalisation de l’auxine chez les plantes. Pour cela, nous avons recherché des homologues des protéines de la voie de signalisation de l’auxine dans des algues vertes charophytes, les ancêtres les plus lointains (450 Millions d’années) des plantes. Nous avons alors trouvé un homologue des ARFs et TPL chez les premières algues multicellulaires (Chlorokybus atmophyticus). La caractérisation biochimique de l’ARF de C. atmophyticus indique qu’il partageait déjà les mêmes propriétés que les ARFs des plantes terrestres et était aussi capable d’interagir avec TPL comme certains ARFs. L’absence d’homologues du récepteur de l’auxine chez ces algues primitives indique cependant que la dépendance à l’auxine aurait été acquise plus tard avec l’apparition du système corécepteur TIR1/AFB-Aux/IAA après la divergence des charophytes vers les plantes terrestres. / Auxin is a plant hormone implicated in almost all plant developmental stages, since the embryo formation till flowering, determining the position of the organs in the plant and thus, its whole structure. As for any other hormone, auxin perception is followed by a signal transduction that finishes in a series of changes in a plant cell, including transcriptional changes. This thesis is divided in 3 chapters, each with a focus on the structural, molecular and evolutionary aspects of different proteins involved in the regulation of auxin genes response.First, we focused our studies on TOPLESS (TPL), a co-repressor implicated, not only in auxin responsive genes repression, but also in many other plant processes due to its interactions with numerous transcriptional repressors in plants. Our determination of the TPL N-terminal structure allowed us to understand that TPL can interact with different partners through the same binding site. Moreover, it revealed that TPL is a tetrameric protein, with the tetramerization interface formed by a newly identify domain, the CRA domain, that is also part of the binding site. The high residues conservation in both tetramerization interface and TPL binding site since m.y.a indicates the importance of TPL role since the origin of plants. This work also shows that the structural similarities between TPL and other co-repressor with similar domains but different function nicely exemplify how evolution plays with common features for creating new functions.Second, we studied ARF proteins, the transcription factors of the auxin transcriptional response, with a focus on their DNA binding preferences. For this, we used a combination of bioinformatic analyses of DAP-seq ARFs genomic binding, with in vitro DNA binding tests and structure modelling. Our results point out that different ARFs can have different preferential binding sites within the genome, with these preferences being determined by the orientation and spacing of the binding motifs. Moreover, our studies suggest that depending on the binding site, ARFs could bind with different conformations using dimerization interfaces not yet discovered. These results can explain how different ARFs co-expressed inside a plant cell can collaborate to the specificity and robustness of auxin transcriptional response by differential bindings to the genome.Finally, we travelled back in time to position the origin of auxin signalling pathway in the evolution of plants. Here we looked for protein homologues of the auxin signalling pathway in charophyte green algae, the most ancient plants ancestor (450 M years). This search retrieved an ARF and a TPL homologue in the first multicellular charophyte algae (Chlorokybus atmophyticus). The biochemical characterization of C. atmophyticus ARF indicated that it presented already the same properties of the ARFs from land plants and that it was able to interact with TPL protein, as it is the case for some ARFs. The absence of auxin receptor homologues in these primitive algae indicates however that auxin-dependency appeared with the acquisition of TIR1/AFB-Aux/IAA coreceptor system, after charophytes divergence into land plants.

Plants colonised land 460 million years ago and charophytes represent the closest living relatives of land plants. The ability to live on land may depend on the presence of certain cell wall polysaccharides such as xyloglucan, a hemicellulose exclusively found in land plants (Popper and Fry, 2003). The cell walls of charophytes are poorly characterised. The aim of this project was to use biochemical techniques to characterise the cell wall polysaccharides of charophytic algae in relation to early land plant phylogeny. Hydrolysis of Coleochaete scutata and Chara vulgaris cell walls in 2 M trifluoroacetic acid yielded predominantly GalA, Gal, Glc and Man residues and also some Ara, Xyl and traces of Fuc and Rha. In addition, hydrolysis of Chara pectin revealed an abundance of an unusual monosaccharide, 3-O-methyl-D-galactose, which was structurally identified by a series of 1-D and 2D NMR spectroscopy by COSY, TOCSY, NOESY and HSQC. 3-O-Methyl-D-galactose is more commonly found in lycophyte cell walls where its presence has been suggested to be related to lycophytes’ evolutionarily isolated position (Popper et al., 2001). The newly discovered presence of 3-O-methyl-D-galactose in charophyte pectin suggests that this polymer may be more complex than previously thought. Coleochaete and Chara hemicellulose extracts were fractionated by anion-exchange chromatography into five classes. A strongly anionic fraction from Chara hemicellulose was found to be rich in Glc, Xyl, Gal and Fuc suggestive of a xyloglucan-like polysaccharide. However, XEG was unable to produce diagnostic xyloglucan oligosaccharides in either Coleochaete or Chara hemicelluloses. Xylanase and mannanase digestion of Coleochaete and Chara hemicelluloses gave xylan- and mannan-oligosaccharides. Furthermore, lichenase digestion of Coleochaete hemicellulose yielded an unusual octasaccharide composed of approximately equimolar xylose and glucose. My work has shown that charophyte cell walls are a source of undiscovered monosaccharides and potentially novel pectic and hemicellulosic domains which may have important functions in enabling the successful colonisation of land by plants.

At maturity, spermatozoids of the green alga Chara vulgaris are biflagellated, contain little cytoplasm, and coil for approximately 2 1/4 gyres within the mother cell wall. The anterior of the cell contains an ovoid headpiece anchoring two slightly staggered basal bodies that are positioned above and directly in front of approximately 30 linearly arranged mitochondria. An elongated stellate pattern occupies the transition zone between the BBs and axonemes. Flagella emerge from the cell just in front of the nucleus and encircle the full length of the spermatozoid. The spline comprises a maximum of 38 microtubules surrounding the anterior mitochondria and gradually decreases posteriorly to a minimum of 11. The dense nucleus is narrow, cylindrical, and occupies the central revolution of the cell. Six starch-laden plastids and associated mitochondria are linearly arranged at the cell posterior. Phylogenetic analyses of charalean taxa and archegoniates based on spermatogenesis strongly support the order Charales, with Nitella as the sister group to Chara. Diagnostic features of Chara spermatozoids include absence of a lamellar strip and axonemes embedded in the cell for almost the entire length of the anterior mitochondria. Potential relationships among Charales, Coleochaetales and archegoniates are evaluated in regards to the probable course of evolution of streamlined biflagellated gametes.

This research is the outcome of investigations of the ability of Nitella congesta, a charophyte, to hyperaccumulate metal contaminants, as well as contribute to the sustainable development of a chain of lakes derived from the sand mine voids at Capel 250km south of Perth, Western Australia. Studies were conducted to ascertain the taxonomy of Nitella congesta as well as its life cycle pattern in relation to the hydrological regime of the lakes of the wetlands. It was observed that a decrease in the availability of water in the lakes particularly on the onset of summer, initiated the production of fruiting bodies whiles prolonged availability of water ensured a prolonged vegetative growth. / Aquatic plants play an important role in the structuring of freshwater communities. Freshwater macrophytes such as Nitella congesta have been reported to serve as food source as well as provide refuge and shelter for macroinvertebrates. Thus the presence of freshwater macrophytes in one way or the other has a direct impact on the species abundance and diversity of macroinvertebrates that use them as their habitat. A study of the impact of Nitella congesta as a suitable macrophyte on diversity and abundance of macroinvertebrates showed a positive outcome. It was observed that species richness and diversity were high in Nitella congesta dominated sites of the lakes. Experimental outcome showed that Nitella congesta is a hyperaccumulator of metals. Both the mucilage and the thallus displayed concentrations of few metals. / The disappearance of submerged macrophytes such as charophytes in shallow lakes is a major problem caused by eutrophication. There has been an approved proposal to discharge treated waste water with a phosphorus concentration of about 4,000μg/L into the lakes of the Capel Wetlands Centre. This necessitated a study of the impact of eutrophication on the establishment of Nitella congesta as a functional macrophyte for the enhancement of the ecological structure of the wetlands. / Results showed that though eutrophication will initially increase the primary productivity of the lakes of the wetlands, the eventual consequence will be the loss of Nitella congesta as a functional macrophyte in the wetlands. / At the last stage of the study, a consistent decrease in pH readings of the lakes and lack of successful germination of Nitella congesta as a result of prolonged drought and exposure of the lake sediment was observed. This necessitated a thorough study of the impact of climate change on the establishment of Nitella congesta in the wetlands. In conclusion, it was observed that Nitella congesta could serve as a suitable tool for the rehabilitation of the wetlands.

Aquesta tesi té com a objectiu fonamental la caracterització taxonòmica, biostratigràfica i paleoecològica de les associacions de caròfits de les fàcies lacustres i transicionals del Garumnià basal (Cretaci superior) de les Conques de Vallcebre, Coll de Nargó, Tremp i Àger. L’altre objectiu d’aquesta tesi és la caracterització tafonòmica i paleoecològica de les restes fòssils de plantes vasculars productores dels lignits garumnians. La flora de caròfits del Garumnià basal està constituïda per girogonits de Feistiella malladae, Peckichara cancellata, P. sertulata, Microchara cristata, M. parazensis, Platychara caudata, P. turbinata i Dughiella obtusa, i utricles de Clavator brachycerus. Pel que fa als òrgans vegetatius les calcàries garumnianes son molt riques en tal•lus de grans dimensions de Charaxis sp. associats a Peckichara sp., i de tal•lus més gràcils de Clavatoraxis microcharophorus Villalba-Breva et Martín-Closas 2011, portadors de Microchara sp. També és freqüent la presència de Munieria grambastii associada a vegades a Clavator brachycerus. Des del punt de vista biostratigràfic, les associacions de caròfits de les conques d’Àger i Vallcebre pertanyen a la biozona de caròfits Peckichara cancellata (Campanià superior - Maastrichtià inferior basal), mentre que les associacions de caròfits de les conques de Coll de Nargó i Tremp pertanyen a la biozona Septorella ultima (Maastrichtià inferior). En base a l’estudi estratigràfic i sedimentològic i l’anàlisi de microfàcies s’ha determinat que la sedimentació a la Conca d’Àger està formada majoritàriament per calcàries lacustres organitzades en centenars de seqüències de somització, que van des de fàcies lacustres profundes, passant per ambients marginals, ben il•luminats dominats per herbeis de caràcies, fins a fàcies de vora de llac dominades per herbeis de clavatoràcies. A les conques de Vallcebre, Coll de Nargó i Tremp, la sedimentació del Garumnià basal està organitzada en diversos cicles, els quals comencen amb dipòsits salabrosos formats per margues amb mol•luscs eurihalins a la base, seguits per lignits i calcàries d’ambients salabrosos dominades per porocaràcies, i calcàries d’aigua dolça amb caràcies i clavatoràcies. Les diferències en el registre sedimentari i la flora de caròfits mostren que les conques sud pirinenques presentaven una polaritat de fàcies sud-nord durant l’inici de la sedimentació continental del Cretaci superior, i que aquesta continentalització va començar al sud i desprès va continuar al nord, d’est a oest. L’estudi de la macroflora del Garumnià basal de les conques de Vallcebre, Coll de Nargó i Tremp indica que la comunitat vegetal dels aiguamolls del Maastrichtià inferior estava dominada per la conífera queirolepidiàcia Frenelopsis, que va ser la planta productora de la matèria orgànica precursora del lignit. Associada a aquestes coníferes hi havia la palmera Sabalites longirhachis, i en alguns casos altres angiospermes d’aiguamoll, representades per les seves llavors, especialment Bergacarpon viladricii Marmi, Gomez, Villalba-Breva et Martín-Closas 2012. Altres plantes vasculars significatives encara que menys abundants eren les falgueres, cicadals i pandanals. / This thesis aims to characterize the taxonomy, biostratigraphy and palaeoecology of charophyte assemblages of lacustrine and transitional facies from the basal Garumnian (Late Cretaceous) of the Vallcebre, Coll de Nargó, Tremp and Àger basins. The other objective of this study is the taphonomic and palaeoecological characterization of the precursory vascular plants of the Garumnian lignite. The charophyte flora of the basal Garumnian consists of gyrogonites of Feistiella malladae, Peckichara cancellata, P. sertulata, Microchara cristata, M. parazensis, Platychara caudata, P. turbinata and Dughiella obtusa, and utricles of Clavator brachycerus. Regarding the vegetative organs, Garumnian limestones are rich in large thalli of Charaxis sp. associated to Peckichara sp., and smaller thalli of Clavatoraxis microcharophorus Villalba-Breva et Martín-Closas 2011, with attached Microchara sp. Also Munieria grambastii thalli are abundant, associated sometimes with Clavator brachycerus. From a biostratigraphic point of view, charophyte assemblages of Vallcebre and Àger basins belong to the Peckichara cancellata biozone (Late Campanian – Earliest Maastrichtian), while charophyte assemblages of Coll de Nargó and Tremp basins belong to the Septorella ultima biozone (Early Maastrichtian). Based on the sedimentological and stratigraphic studies and microfacies analyses, the sedimentation in the Àger Basin is organised into hundreds of small order sedimentary cycles corresponding to lacustrine infilling sequences, ranging from deep lacustrine facies, through marginal environments dominated by characean meadows, to lakeshore facies dominated by clavatorecean meadows. In the Vallcebre, Coll de Nargó and Tremp basins, the deposits of the basal Garumnian are organized into small number of cycles that show a transition from brackish deposits formed by marls with euryhaline molluscs, lignites and limestones with porocharaceans, to lacustrine limestones with characeans and clavatoraceans. The differences in the sedimentary record and in the charophyte flora show that the Southern Pyrenean Basin displayed a south to north shift of facies during the onset of the Upper Cretaceous continental sedimentation and that this continentalisation began in the south and then continued to north, from east to west. The study of megafossil plant remains from the basal Garumnian of the Vallcebre, Coll de Nargó and Tremp basins indicates that the cheirolepidiacean conifer Frenelopsis was the main component of the wetlands vegetation associated with Sabalites longirhachis palms, and in some cases with other angiosperms, such as the plant bearing Bergacarpon viladricii Marmi, Gomez, Villalba-Breva et Martín-Closas 2012, probably a commelinid.