FUNCTIONAL ANALYSIS OF TRANSCRIPTION FACTORS INVOLVED IN REPRODUCTIVE MERISTEM IDENTITY IN ARABIDOPSIS THALIANA

Flowering is the most important process in a plant’s life as it is the essential step for its reproduction. Flower development starts with a tightly regulated process called the floral transition in which different regulatory pathways, which are regulated by environmental and internal signals, culminate in the transition from vegetative to reproductive growth. Subsequently, flowers develop instead of leaves and the formation of these flowers is controlled by complex regulatory pathways. In model organism Arabidopsis thaliana there are at least five different pathways that regulate the floral transition to guarantee that it occurs under the best possible conditions. The signals derived from these pathways are than integrated at the level of the floral pathway integrators which are LEAFY (LFY), FLOWERING LOCUS T (FT), SOPPRESSOR OF OVEREXPRESSION OF CO (SOC1). These genes are responsible for the switch from the shoot apical meristem (SAM) to the inflorescence meristem (IM) and are involved in the activation of the floral meristem identity (FMI) genes: LFY, LATE MERISTEM IDENTITY1 (LMI1), APETALA1 (AP1), CAULIFLOWER (CAL), SHORT VEGETATIVE PHASE (SVP) and AGAMOUS-LIKE 24 (AGL24). In fact, after the floral transition, the inflorescence meristem (IM) starts to produce floral meristems from its flanks. These meristems remain undifferentiated until stage 3 of flower development, thanks to the action of the FMI genes; afterwards, when some of these genes become repressed, the floral organs start to differentiate. In these first undifferentiated stages, the floral meristem grows and produces enough cells to support the subsequent differentiation of all the floral organs. Many of the genes involved in these two processes, floral transition and floral meristem determination, are MADS-box transcription factors. The MADS-box family is one of the best-characterized gene families in Arabidopsis and the its members represent key regulators of developmental processes. MADS-box factors are combinatorial proteins that act via multimerization and interact with other regulatory proteins in complexes to regulate the transcription of target genes. The aim of this thesis is the analysis of the genetic interactions of MADS-box transcription factors playing key roles during the floral transition and early stages of flower development. The floral pathways integrator SOC1 is a MADS-box gene that integrates at least four pathways that control flowering (photoperiod, vernalization, autonomous and gibberellin pathways), giving rise to the activation of the floral meristem identity genes (Parcy, 2005). In chapter 2, , we show that AGAMOUS-LIKE 42 (AGL42), AGAMOUS-LIKE 71 (AGL71) and AGAMOUS-LIKE 72 (AGL72) that are phylogenetically related to SOC1, are also involved in the floral transition of both the shoot apical meristem and axillary meristems and moreover, are involved in the gibberellin pathway. The soc1 agl42 ami::agl71-72 mutant shows an aerial rosettes bearing nodes phenotype. Our findings suggest that the SOC1-like genes are involved in the floral transition especially in the axillary meristem and the GA pathway is the main player controlling flowering in these axillary meristems both under short day and long day conditions. Furthermore SOC1 is able to directly control the expression of AGL42, AGL71 and AGL72 to maintain a proper expression level of SOC1-like genes. In chapter 3, the interactions between the floral meristem identity genes SVP, AGL24, AP1, CAL, which are all MADS-box transcription factors, and LFY is described. The lfy mutant shows partial reversions of flowers in inflorescence shoot-like structures and this phenotype is enhanced in the lfy ap1 double mutant. Here we show that combining the lfy mutant with agl24, svp single or agl4 svp double mutant enhances the lfy phenotype and that the agl24 svp lfy triple mutant phenocopies the ap1 lfy double mutant. Analysis of the molecular interactions between LFY and AGL24 and SVP showed that LFY is, together with AP1, a repressor of AGL24 and SVP whereas AGL24 and SVP positively regulate AP1 and LFY by direct binding to their regulatory regions. Since all genes are important to establish floral meristem identity this regulatory loop is probably important to maintain the correct relative expression levels of these genes. In chapter 4, we focalize our attention on SVP, a MADS-box gene involved in floral repression, before the floral transition, and in floral meristem (FM) identity determination, after the floral transition. An interesting feature of SVP is that it is the only Floral Meristem Identity Gene identified so far that is expressed exclusively in the undifferentiated FM. To date some transcription factors that are able to bind the SVP genomic region have already been identified by ChIP experiments, but it is still not clear how this gene is regulated. To understand this as a first step we are interested in the identification of the SVP minimal promoter region that fully comprises all its regulatory elements. We use, for our studies, lines that contain different SVP promoter fragments, that are cloned as transcriptional or translational fusions to the uidA gene, that encodes the beta-glucuronidase enzyme. This studies show that at least two regions are necessary for normal SVP expression: a 1 Kb fragment, located from 3000 to 2000 bp upstream of the start codon, and the first intron. In fact constructs lacking one of these two regions aren’t able to express GUS in the flower primordia. In conclusion this work contributes to get a better understanding of what exactly happens during the floral transition and, afterwards, in undifferentiated flower meristems.