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Raquel Martin Arevalillo

Functional, structural and evolutionary aspects of transcriptional response to auxin

Published on 27 November 2017

Thesis presented on November 27, 2017

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 identified 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 signaling pathway in the evolution of plants. Here we looked for protein homologues of the auxin signaling 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.