SAD: a new DOF protein from barley that activates transcription of a cathepsin B-like thiol protease gene in the aleurone of germinating seeds

Functional analysis of hydrolase gene promoters, induced by gibberellin (GA) in aleurone cells following germination, has identified a GA-responsive complex (GARC) as a tripartite element containing a pyrimidine-box motif 5'-CCTTTT-3'. We describe here the characterization of a new barley gene (Sad gene) encoding a transcription factor (SAD) of the DNA binding with One Finger (DOF) class that binds to the pyrimidine box in vitro and activates transcription of a GA-induced protease promoter in bombarded aleurone layers. RT-PCR and in situ hybridization analyses showed that the Sad transcripts accumulated in all tissues analysed, being especially abundant in the scutellum and aleurone cells upon seed germination. The SAD protein, expressed in bacteria, binds in a specific manner to two oligonucleotides containing the sequence 5'-G/CCTTTT/C-3', derived from the promoter region of the Al21 gene encoding a cathepsin B-like cysteine protease. Although the Sad transcript accumulation did not respond to external GA-incubation in aleurone cells, in transient expression experiments in co-bombarded aleurone layers, SAD trans -activated transcription from the Al21 promoter in a similar manner as did GAMYB, a MYB protein previously shown to respond to GA and to activate several hydrolase gene promoters in barley aleurone cells. In vivo interaction between the GAMYB and SAD proteins was shown in the yeast two-hybrid system, where GAMYB potentiates the SAD trans -activation capacity through interaction with its C-terminal domain.