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Genomic Analyses of Arabidopsis Flower Development: sORFs, miRNAs, and transcription factor-coding genes

Final Report Summary - GEANARAFDEV (Genomic Analyses of Arabidopsis Flower Development: sORFs, miRNAs, and transcription factor-coding genes)

The focus of the project was to identify and characterize novel components of the flower development gene regulatory network in Arabidopsis, using various genomic approaches. Extensive genetic analyses of flower development in Arabidopsis and Antirrhinum have led to the identification of key regulatory genes of the process. Most of these regulators code for transcription factors, and microarray analyses have revealed the existence of a complex transcriptional network underlying the floral development process. Yet, many components of this network likely still remain to be identified (belonging to classes of genes that have been more untractable by classic genetic and molecular biology methods, such as coding small open reading frames -sORFs-); and many protein coding genes that likely participate in the process remain functionally uncharacterised. In addition, targets of the known regulators are still largely ill-defined, so the structure of the network is also not well understood.

The specific objectives of the project were:
(1) to conduct expression profiling experiments aimed at identifying novel sORFs that may participate in flower development, and at understanding their roles;
(2) to begin to elucidate the functions of novel, uncharacterised components of the regulatory network through various reverse-genetic approaches; and
(3) to use novel genomic methods, such as ChIP-Seq, to identify target genes of known regulators, and thus help to define the structure and properties of the gene regulatory network.

The work performed since the beginning of the project encompasses the three specific objectives.

We studied the expression of novel Arabidopsis coding short Open Reading Frames during flower development, and found evidence of differential expression for many of them. Molecular characterization of the corresponding transcriptional units indicated that a fraction of those correspond to novel un-annotated genes, and analyses of their promoter sequences showed that their expression is regulated in a spatially and temporally defined manner.

To characterize the flower development gene regulatory network and to identify targets of the known regulators, we focused first on APETALA1 (AP1). AP1 is a MADS-domain transcription factor that controls the onset of Arabidopsis flower development and that, in addition, participates in the specification of sepals and petals. To understand the molecular mechanisms underlying AP1 function, we identified its target genes during floral initiation using a combination of gene expression profiling and genome-wide binding (ChIP-Seq) studies. Many of its targets encode transcriptional regulators, including known floral repressors. The latter genes are down-regulated by AP1, suggesting that it initiates floral development by abrogating the inhibitory effects of these genes. While AP1 acts predominantly as a transcriptional repressor during the earliest stages of flower development, at more advanced stages it also activates regulatory genes required for floral organ formation, indicating a dynamic mode of action. Our results further imply that AP1 orchestrates floral initiation by integrating growth, patterning and hormonal pathways. In summary, AP1 acts as a true hub in the regulatory network mediating the switch from floral induction to flower formation. This part of the project was performed in collaboration with Dr Frank Wellmer, (Smurfit Institute of Genetics - Trinity College, Dublin, Ireland) and with Prof Gerco Angenent (Plant Research International, Wageningen, The Netherlands). The results were published in Science:

Kaufmann, K., Wellmer, F., Muiño, J.M. Ferrier, T., Wuest, S.E. Kumar, V., Serrano-Mislata, A., Madueño, F., Krajewski, P., Meyerowitz, E.M. Angenent, G.C. and Riechmann, J.L. (2010) Orchestration of floral initiation by APETALA1. Science 328, 85-89.

The study of the AP1 network identified many AP1 targets whose function is currently unknown, and that we are now characterizing using the functional genomics toolkit available in Arabidopsis.

In addition, we applied the ChIP-Seq technology to another developmental regulator, TOC1 - a component of the circadian clock. The results obtained indicated that TOC1 functions as a general repressor of oscillator gene expression, thus overturning the prevailing model of the Arabidopsis circadian clock in which TOC1 was considered an activator of a subset of oscillator genes. The results were published in Science:

Huang, W., Pérez-García, P., Pokhilko, A., Millar, A.J. Antoshechkin, I., Riechmann, J.L. and Mas, P. (2012) Mapping the Core of the Arabidopsis Circadian Clock Defines the Network Structure of the Oscillator. Science 336, 75-79.