Estudio del mecanismo de señalización por giberelinas en el endospermo de semillas de arabidopsis thaliana

Abstract

ABSTRACT Seeds are fundamental organs for the survival and dispersal of plants and its germination is a critical process that determines crop yield. The endosperm has an important role in the control of germination, providing nutrients to the embryo. In addition, this tissue senses environmental conditions and integrates this information into signals that are transmitted to the embryo to control its growth. Gibberellins (GAs) have a fundamental role in germination, promoting the elongation of the embryo and the weakening of the endosperm cell walls, essential events in this proccess. Although our group had prevously characterised a regulatory module controlling embryo growth during germination, the molecular mechanisms regulating GA-responses and cell expansion in endosperm, as well as its relationship with the embryo, were unknown. The first objective of this thesis is the identification of new regulators involved in endosperm-specific GA signaling during seed germination, by using two complementary approaches based on phylogenetic analysis coupled with the screening of a library of transcription factors and in silico analysis of whole genome expression data during germination. The second part of this work focuses on determining the relevance of endosperm-specific GA signaling in seed germination. Phylogenetic analysis of the marker gene EXPA2, wich is induced by GAs specifically in the endosperm during germination, allowed the identification of a functional element conserved in other species of bassicaceae (CS-EXPA2). We demostrate that CS-EXPA2 contains sequences recognized by the transcription factors NAC25 and NAC1L, both expressed in the endosperm, and indispensable for the transactivation and inducibility of the gen EXPA2 by GAs. NAC25 and NAC1L are able to form homo and heterodimers in planta and NAC25 interacts with the DELLA protein RGL2, a repressor of the GAs signaling mechanism. Such interaction avoids the transactivaction capacity of NAC25 on EXPA2. Functional analysis of the loss of function of NAC25 and NAC1L indicates that they are activators of transcription, mainly of genes encoding cell wall remodeling enzymes, and that its regulation is essential for cell elongation in the endosperm and subsequent germination. In silico analysis of complete genome expression data, allowed the identification of four additional transcription factors expressed in the endosperm during germination (TF1-TF4) as potential regulators of EXPA2. In this thesis we show that TF1 and TF2 are transcriptional activators of EXPA2 in planta, and that both are able to homodimerize and interact with DELLA proteins. In addition, TF1 interacts with NAC25. Although the functional analysis of their loss of function did not allow them to be assigned a relevant role under standard germination conditions, molecular evidences indicate the possible existence of combinatorial control events that could be revealed in the absence of gene redundancy and/or in the presence of specific environmental conditions. Finally, we demonstrate that EXPA2 expression depends on a early signal coming from the embryo and we show evidences suggesting that a corrdinated expansion between embryo and endosperm is required to protect embryo's radicle integrity during germination. Taken as a whole, our results show a fundamental role of NAC25, NAC1L and RGL2 in GA-mediated signalling in the endosperm, driving endosperm cell expansion and controlling seed to seedling transition.