Identification of Dof proteins with implication in the gibberellin-regulated expression of a peptidase gene following the germination of rice grains.

Type III carboxypeptidase (CPD3) is one of the hydrolytic enzymes whose expression is up-regulated by gibberellins (GA) in the aleurones of germinated cereal grains. A number of pyrimidine boxes and a sequence resembling the gibberellic acid response element (GARE) are observed in the region upstream of the transcription initiation site of the CPD3 gene, showing a characteristic of cereal GA-responsive genes. Transient gene expression assays in germinated rice aleurone demonstrated that the CPD3 promoter was able to confer hormonally responses on the expression of the reporter gene. By southwestern screening, several cDNAs encoding the Dof class proteins were isolated from a rice aleurone library. Each mRNA accumulation for five novel members of Dof proteins (OsDof1--5) occurs with a different time course and in a tissue-specific manner following the germination of grains. Of these, the expression of the OsDof3 gene is abundant in aleurones where it precedes that of the CPD3 gene, implying that this is an early response gene of GA. The OsDof3 protein, expressed in Escherichia coli, selectively bound AAAG motifs of the pyrimidine boxes through the DNA-binding activity of its Dof domain. Co-expression experiments in aleurones suggested that the OsDof3 protein should play a regulatory role in the expression of the CPD3 gene under the control of GA.