A wheat alpha-Amy2 promoter is regulated by gibberellin in transformed oat aleurone protoplasts

Gibberellin (GA(3))-responsive aleurone protoplasts isolated from Avena sativa have been successfully used as a transient expression system to analyse promoter fusions between the wheat alpha-amylase gene alpha-Amy2/54 and the reporter gene GUS. Following PEG-mediated uptake of plasmid DNA, transient expression directed by the alpha-Amy2/54 promoter was found to be regulated in the same way as the endogenous oat alpha-amylase genes. Expression was thus dependent on the inclusion of GA(3) in the protoplast incubation media, could not be detected before a lag phase of 2 days following transformation and was inhibited by simultaneous addition of abscisic acid (ABA) with GA(3) to the media. In contrast, expression from the CaMV 35S promoter in the same system was not affected by GA(3) or ABA and could be detected 1 day after transformation. Introduction of a further three different promoters into the aleurone protoplasts confirmed that GA(3) specifically controlled transient expression from the alpha-Amy2/54 promoter only. Promoter deletions of the alpha-Amy2/54: GUS fusion demonstrated that sequences within 300 bp of the start of transcription of the gene were sufficient to direct high-level expression that was regulated by GA(3) and ABA.