Regulation of the floral homeotic gene AGAMOUS

One of the remaining mysteries in biology is how changes in the regulation of developmental genes lead to morphological variation. Interspecies differences in gene expression may be due to evolutionary changes in cis-elements or their trans-regulators or the interplay of both. The study of such phenomena requires a good model system involving regulators linked in a common developmental pathway.; LEAFY (LFY) is central regulator of floral homeotic genes in Arabidopsis flowers and a direct activator of AGAMOUS (AG). Both LFY and AG homologs have been characterized in many higher plant species. LFY is a highly conserved transcription factor unique to the plant kingdom and AG is a member of a large MADS-box family of developmental regulators in plants. Since the functions of LFY and AG seem to be well conserved during evolution, we can take advantage of their intimate genetic relationship to determine how and to what degree these cis-trans components of the floral patterning pathway are co-evolving.; The first step of this endeavor is to better understand AG regulation. In conjunction with other supporting data, I have shown through yeast transactivation assays that a homeodomain protein WUSCHEL (WUS) and LFY can co-activate through distinct cis-regulatory regions of the AG enhancer. In order to identify additional cis-elements, I have isolated putative orthologous AG introns from 30 other angiosperms and most extensively, from within the Brassicaceae family to which Arabidopsis belongs. Through this large-scale phylogenetic footprinting approach I have discovered several highly conserved cis-elements, including two MADS-box factor binding CArG boxes, two additional LFY binding sites, an “AAGAAT” 20 bp sequence, and a pair of direct CCAAT-box repeats. The functions of all but the “AAGAAT” site have been further confirmed by reporter expression analyses.; Subsequently, I proceeded to show that the CUM1 enhancer from cucumber can partially function in Arabidopsis and that the Arabidopsis AG enhancer can also function in tomatoes. Furthermore, I discovered that the highly conserved 3′ minimal enhancers from 5 other Brassicaceae species show various degrees of reporter expression in Arabidopsis, possibly suggesting changes in the utilization or positioning of cis-elements.