NSF Award
9108232
The research in this proposal is designed to examine stigma/pollen interactions in the self-fertile crucifer Arabidopsis thaliana. The molecular work will initially focus on the members of a multigene family in Arabidopsis that are homologous to the well characterized S-multigene family in Brassica. In the self-incompatible crucifer, Brassica, members of the S-multigene family produce protein products functioning in the stigma/pollen interactions of pollination, including the cell-cell recognition and rejection of self pollen, which prevents inbreeding. Since Arabidopsis is self-fertile, it does not have functional self-incompatibility genes but does carry S- homologous regions that may be equivalent to the members of the Brassica S-multigene family involved in the pollination response. Their analysis in Arabidopsis system will elucidate aspects of the pollination process at the molecular level. Specifically, Arabidopsis S-homologous genomic clones and a PCR-generated fragment already isolated using Brassica S-gene sequences are being characterized by restriction mapping and sequencing to develop Arabidopsis S-homologous and gene-specific probes. With these probes expression and organization of the S-multigene family in the Arabidopsis genome will be determined. Expression of an S-related sequence has already been demonstrated by RNA blot analysis. Additional S-related transcripts will be isolated from a cDNA library prepared from flower polyA+ RNA. Their temporal and spatial expression will be analyzed by in situ hybridization and GUS reporter gene expression regulated by the S-related gene(s)' promoter. The available molecular genetics of Arabidopsis (RFLP maps and contiguous cosmid and YAC libraries) will facilitate characterization of the genomic organization of the S-related multigene family. The ease of Arabidopsis transformation via Agrobacterium will facilitate study of the function of the S-related gene(s) by gene inactivation using antisense constructs. Ligand blotting will be used to determine if the S-related proteins bind specifically to any pollen proteins.
