Regulació espai-temporal de la via de senyalització de brasinosteroides al ninxol de cél·lules mare
- Autores: Nadja Bosch Moreno
- Directores de la Tesis: Ana Isabel Caño Delgado (dir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2021
- Idioma: catalán
- Tribunal Calificador de la Tesis: Jordi Moreno Romero (presid.), María Coca López (secret.), Teresa Altabella Artigas (voc.)
- Programa de doctorado: Programa de Doctorado en Biología y Biotecnología Vegetal por la Universidad Autónoma de Barcelona
- Materias:
- Enlaces
- Tesis en acceso abierto en: TDX
- Resumen
The present PhD thesis dissertation describes the configuration of BRAVO protein complex in Arabidopsis stem cell niche, while demonstrates that BRAVO-WOX5-BES1 are part of a main regulator network that comprises BRAVO-WOX5 heterodimers, and together contribute to cell specific regulation of BR-controlled quiescence in root stem cell niches.
The current data provide new insights into the QC division organization in plant roots. It was investigated the composition of BRAVO protein complex from Arabidopsis primary root in vivo by IP and LC-MS/MS techniques. Giving the low expression of native BRAVO, we conducted an exhaustive screening for BRAVO interactors in Yeast to increase the sensitivity of our approach. The analyses revealed that BRAVO, a member of the R2R3 MYB family, interact with a homeobox superfamily protein. The work further demonstrate that BRAVO interacts with WOX5, BES1 and TPL. This result was confirmed by molecular techniques in vitro by Y2H assays and in vivo using FRET-FLIM and BiFC in Nicotiana benthamiana leaves. Our data provides evidences of BRAVO directly interaction with WOX5, and at the same time both could be part of the BES1/TPL transcriptional complex at the SCN trough the BR signalling cascade. We display that the interaction of BES1-TPL is essential for the QC division in root SCN. Increasing BR levels induce QC division through a fine mechanism which is accurately controlled by BRAVO. The results establish that TPL regulates QC cell division through BES1-mediated suppression of BRAVO, and in response to BRs, the last step seems to be the promotion of the QC division. By a genetical and a mathematical analysis, we revealed that BRAVO and WOX5 interaction is essential for stem cell fate. Of particular interest is the fact that BRAVO and WOX5 reinforce each other at the root stem cell niche. This was surprising, since WOX5 levels are oppositely regulated by BRs than in BRAVO. The exhaustive analysis of the expression pattern of both genes in all the simple and double KO mutants, support that BRAVO is required to maintain normal WOX5 levels in the QC. In addition our data are coherent with the fact that WOX5 can induce BRAVO expression but only in the BRAVO native domain. Consistent with our hypothesis, a mathematical model predicted that WOX5 transcriptionally represses itself and activates BRAVO expression, taking in account the heterodimers and complex formation. In this scenario, the model interactions indicate that BRAVO is unable to activate WOX5 expression outside of its domain, in agreement with the results of the BRAVO overexpression line. We show a regulatory network of our interactions predicted by the mathematical model. We added the protein fold changes predicted by this model when changing the BR concentrations due to the BR signalling cascade in different situations and we observed better correlation of BRAVO and WOX5 protein concentrations when one of them are absent. But not when both are out. The exact reasons for these differences are not clear. This mechanism could be a compensation mechanism. And finally, in the last chapter, we delve into evolution with the aim to comprehend the primitive organisation and function of our present root SCN.
