Rapid progress has been made in the past few years towards understanding the molecular and genetic mechanisms controlling flower development. These studies have identified early-acting genes that specify the identity of floral meristems, and later-acting genes that determine the fate of floral organ primordia. One family of genes that has emerged as playing a major role in flower development are the MADS-box genes, which encode putative transcription factors. Indeed, five different Arabidopsis MADS-box genes, APETALA1, CAULIFLOWER, AGAMOUS, APETALA3, and PISTILLATA, have been characterized in terms of their roles in specifying both floral meristem and organ identity. Furthermore, MADS-box genes have documented roles in regulating gene expression in diverse organisms, including humans, Drosophila, and yeast. The focus of this proposal is on the further characterization of three recently published MADS-box genes (AGL4-6), on nine additional genes that were recently isolated (AGL8-9 and AGL11-17), and on the isolation and characterization of additional members of this important gene family in Arabidopsis. To identify new MADS-box genes, PCR amplifications and low stringency hybridizations will be used. Full length cDNA and genomic clones will be isolated and sequenced, and deduced protein products will be compared to those already identified to look at evolutionary relationships. RFLP mapping will determine if the cloned gene maps near a previously identified mutation. RNA blotting and in situ hybridizations in wild type and mutant plants will define the temporal and spatial pattern of gene expression, and provide insights into the regulatory interactions between these and other genetic loci. Transgenic plants that express sense and antisense RNAs will be generated by Agrobacterium-mediated transformation, followed by targeted EMS mutant screens to identify candidate mutations in cloned loci. Preliminary results from these experiments support the hypothe sis that MADS-box genes play important and diverse roles in plant development. Characterization of MADS-box genes will provide insights into the roles and evolution of this important gene family in Arabidopsis, and will contribute to our understanding of related genes in distantly related plants and other organisms.
