Novel functions and interactors for Brassinosteroid receptors In Arabidopsis thaliana = Noves funcions i interactors pels receptors de Brassinosteroids d’Arabidopsis thaliana

The general objective of this PhD thesis is to investigate the mechanistic bases for plant hormones Brassinosteroids (BRs) action in plant vascular development. To this end, we have used a multidisciplinary approach by combining mathematical modeling with exhaustive analysis of the BRs mutants vascular phenotypes. Furthermore, high throughput proteomic techniques with accurate confocal microscopy to identify new components of the BR signaling pathway with cellular resolution have been performed. Our work describes for the first time the composition of BRI1 and BRL3 receptor protein complexes in Arabidopsis. These results represent a conceptual advance for the molecular understanding of BR signaling pathway in plant development, as disclose several key points beyond the current state of the art. Despite their cellular specificity and their role as functional BR receptors the study of BRL receptors has been neglected and no reports have been published on them. Several barriers may have hampered the study of BRL pathways in the plant. Despite BRL being functional BR receptors, obstacles in: (i) the visualization of vascular inner tissues, (ii) the biological variability that exists within the shoot stem vasculature where BRL proteins are localised, and (iii) the lack of an apparent phenotype for the brl mutants, altogether might have hampered the study of these receptors. This PhD thesis challenges the study of the functional role of BRL receptors by using multidisciplinary approaches ranging from systems biology to quantitative biochemistry and high-resolution microscopy. The purification and identification of BRL3-receptor protein complex has permitted to assign a specific role for BRL3 receptor in planta, independently from the BRI1 main receptor (also reported in this study). To better understand the function of the BRL receptors in planta and to investigate novel BR specific functions in distinct cell-types, we have carried different approaches: 1. Investigate the contribution of BRs to the vascular development in the shoot of Arabidopsis plants by comprehensive phenotypic analysis of vascular-patterning defects in BR-signaling and synthesis mutants. 2. Biochemical characterization of BRI1 and BRL3 signalosome complexes by MS- analysis. 3. Functional characterization of BRL3 signalosome in plant growth and development by genetic analysis of candidate interactors identified by MS analysis. First, a role for BRs in regulating vascular development in the shoot inflorescence stem of Arabidopsis has been demonstrated. Based on our experimental results, mathematical modeling (in collaboration with Physicists at the group of Dr. Ibañes, UB) was mutually beneficial to jointly establish the role of auxin in plant vascular patterning. Our initial experimental approach accounted for exhaustive analysis of BRs mutants while did not concern auxin hormone action. Through computational and modeling predictions, it was found that BRs alone were not sufficient to create the VB patterning, suggesting the requirement of auxin transport for the VB formation. Thus, our results unveil coordinated roles for auxin and BRs in establishing the vascular patterning within Arabidopsis shoots. Second, experimental evidence for two different BR perception complexes, BRI1 and BRL3 complexes, operating at distinct and specific cell types is provided. Several BRI1 interactors and downstream signaling components have been identified by extensive proteomic studies while no evidence for the composition of the native BRI1 receptor complex has been reported. Additionally, BRL receptors have been described to be active BR receptors that play specific roles in Arabidopsis yet no study on BRL receptor complexes composition nor downstream signaling components has been shown. Here we disclose the composition of both BRI1 and BRL3 receptor complexes providing novel candidate interactors and components to the BR signaling field. Of note, identification of the BRL3 receptor complex composition in combination with high-resolution microscopy and genetic analysis reveals a novel function for BAK1 and BRL1 proteins through interaction with BRL3 in BR-mediated root development. Collectively, these results highlight the power of using multidisciplinary approaches for the study of signaling pathways within specific cellular domains in the plant.
Aquesta tesis doctoral té com a objectiu principal investigar noves funcions de les hormones vegetals esteroides, Brassinoesteroides (BRs), en el desenvolupament del teixit vascular de la planta. Per tal d’assolir aquest objectiu hem utilitzat una aproximació multidisciplinària, combinant models matemàtics i computacionals amb anàlisis exhaustius dels fenotips vasculars de plantes mutants en BRs, en la planta model Arabidopsis thaliana . A més, hem combinat tècniques de proteòmica amb microscopia confocal, amb l’objectiu d’identificar nous components de senyalització de BRs amb una resolució cel•lular. Els nostres resultats indiquen l’inici d’una ruta alternativa a la visió clàssica de la ruta de senyalització dels BRs descrita fins al moment. Tot i que els principals components del senyal ja han estat identificats pel seu homòleg més pròxim, el receptor BRI1, els complexes de BRL1 i BRL3 juntament amb els candidats co-receptors així com els components de la ruta de senyalització encara no s’han descrit. Per tal d’entendre millor la funció d’aquests receptors de BRs i d’investigar funcions de BRs específiques pels diferents tipus cel•lulars, vam plantejar diferents aproximacions: 1. Estudi de la contribució dels Brassinoesteroides en la formació del patró vascular de la tija d’Arabidopsis 2. Identificació de nous components de senyalització del receptor BRL3, involucrats en la regulació del desenvolupament vascular. 3. Caracterització funcional del signalosoma de BRL3 en el creixement i en el desenvolupament de la planta. Aquest estudi demostra que el transport polar de l’auxina i la senyalització de BRs determinen el patró radial dels feixos vasculars de la tija principal de les plantes d’Arabidopsis. Les nostes dades experimentals juntament amb l’elaboració d’un nou model matemàtic ens va permetre entendre com s’elabora el patró radial de la tija d’Arabidopsis, i els resultats es van publicar a la revista PNAS (Ibañes et al., 2009). A més, el nostre estudi demostra la formació de complexes receptors de BRs específics de tipus cel•lular, els quals participen en diferents activitats cel•lulars durant el creixement i desenvolupament de l’arrel. També revela funcions noves i específiques per a la senyalització de BRs a través dels receptor BRL3 en el nínxol de cèl•lules mare de l'arrel d'Arabidopsis.