Functional Dissections between GAMYB and Dof Transcription Factors Suggest a Role for Protein-Protein Associations in the Gibberellin-Mediated Expression of the RAmy1A Gene in the Rice Aleurone

In the germinated cereal aleurone layer, gibberellic acids (GA) induce expression of a number of genes encoding hydrolytic enzymes that participate in the mobilization of stored molecules. Previous analyses suggest that the key events controlling the GA-regulated gene expression in the aleurone are formation of active transcription machinery referred to as the GA responsive complex, followed by recruiting GAMYB. In general, bipartite promoter contexts composed of the GA-responsive element and the pyrimidine box are observed within the regulatory regions of cereal GA-responsive genes. Protein factors that recognize each promoter sequence were identified and distinct effects on the GA-mediated activation of gene expression have been also investigated; however, the connection and intercalation between two promoter motifs remain obscure. In this study, I have evaluated cooperative function of GAMYB and a pyrimidine box-binding protein OsDOF3 that influenced the promoter activity of the most predominant GA-responsive gene (RAmy1A) of rice (Oryza sativa). Transient expression of OsDOF3 in the germinated aleurone prolonged GAMYB function on the reporter expression in the absence of GA. The synergistic effect required a set of DNA bindings of two proteins on the RAmy1A promoter region. The yeast two-hybrid assay showed the physical interaction of GAMYB and OsDOF3 in yeast cells, indicating that the association of GAMYB and OsDOF3 may be a functional unit in transcription regulation. The results showed the accessory function of OsDOF3 responsible for a dosage-dependent mediation of GA signaling that leads to high-level expression of physiological target genes.