LARSON, An Arabidopsis Homeobox Gene in Flower Development

University Of Maryland, College Park, College Park MD

Investigators

Abstract

0212847 Liu The long-term objective is to reveal the mechanisms determining how cells in a multicellular organism assume their developmental fates. Using Arabidopsis flower as a model, the focus of this proposal is to reveal the regulatory mechanism for the spatial and temporal-specific expression of AGAMOUS (AG), a gene for stamen and carpel identity specification. AG mRNA is normally expressed in the inner two whorls of a flower and is absent from the outer two whorls. LEUNIG (LUG), a putative transcriptional co-repressor with sequence similarity to the yeast co-repressor TUP, was previously shown to play a crucial role in repressing AG expression in the outer two whorls. Since LUG does not encode a DNA binding domain, how LUG is recruited to the AG cis-regulatory element is unknown. The newly identified LARSON (LSN) gene is an excellent candidate for being a DNA-binding partner of LUG. lsn-1 mutation enhances the defects of lug mutation in AG regulation. Further, preliminary data indicated that LSN encodes a homeobox protein, can bind to the AG cis-regulatory elements, and can interact with LUG in yeast. In addition, as LUG mRNA is expressed in all floral whorls, the outer whorl-specific repression activity of LUG could be conferred by interaction with an outer-whorl-specific factor. Could LSN hold the crucial role in providing the spatial and temporal specificity to the LUG co-repressor? The proposed experiments are aimed at testing these hypotheses. First, the molecular identity of LSN will be confirmed by transformation rescue of the lsn-1 mutant phenotype. Several reverse genetic methods will be employed to identify additional alleles of lsn. Second, the RNA and protein expression profile of LSN will be determined by in situ hybridization and immuno-localization or LSN-GFP. Third, by expressing LSN cDNA in ectopic whorls (where LUG is expressed), the ectopic co-repressor activity will be tested. Fourth, to establish that LSN directly regulates AG, the electrophoretic mobility shift assay will be used to identify the binding sites of LSN within AG cis-regulatory elements. Further, LSN-VP16 chimeric protein expressed in transgenic plants will be used to establish the functional relevance of LSN binding to AG in vivo. Finally, physical interactions between LSN and LUG proteins will be tested using a co-immunoprecipitation assay. Other LSN-interacting proteins will be identified by a yeast two-hybrid screen. The proposed study will elucidate the basic mechanism of transcriptional repression in higher plant development and provide excellent training opportunities for undergraduate and graduate students.