The barley cystatin gene (Icy) is regulated by DOF transcription factors in aleurone cells upon germination.

A genomic clone (Icy gene) encoding a barley cystatin has been characterized. The gene contains one intron interrupting its ORF that encodes a protein of 107 amino acid residues. A DNA fragment of -1058 bp upstream of the ATG translation initiation codon has been sequenced and several promoter deletions fused to the beta-glucuronidase (GUS; uidA gene) reporter gene obtained. Transient expression assays in different barley tissues have indicated that a -631 bp promoter fragment was sufficient for full activity. In bombarded barley aleurone layers the GUS-driven expression by this promoter is repressed by GA(3) incubation, as is the accumulation of the Icy transcripts detected. A spatial and temporal location of cystatin transcripts by in situ hybridization techniques indicates that this gene is ubiquitously expressed and its transcripts are particularly abundant in leaves and roots, and in seeds, both during development and upon germination. Two DOF transcription factors, SAD and BPBF, previously described as involved in gene expression regulation in seeds, interact specifically in vitro with oligonucleotides containing DOF binding-sites derived from the Icy gene promoter. Transient expression experiments in co-bombarded aleurone layers demonstrate that BPBF strongly represses transcription of the native Icy promoter, even when co-transfected with SAD that behaves as an activator in this in vivo system.