GIBBERELLIN PERCEPTION AT THE PLASMA-MEMBRANE OF AVENA-FATUA ALEURONE PROTOPLASTS

A functional assay for gibberellin (GA) receptors is described based on the induction of alpha-amylase gene expression in isolated aleurone protoplasts of Avena fatua L. by GA4 immobilised to Sepharose beads. A 17-thiol derivative of GA4, shown to be biologically active with aleurone protoplasts, has been coupled to epoxy-activated Sepharose 6B. This GA4-17-Sepharose induces high levels of alpha-amylase when incubated with isolated aleurone protoplasts, while cells of the intact aleurone layer do not respond appreciably to the immobilised GA4. In order to eliminate the possibility that GA4 may be released from the Sepharose when incubated with protoplasts, aleurone layers and isolated aleurone protoplasts have been co-incubated, and their responses to GA4, GA4-17-Sepharose and control Sepharose estimated by determining the relative amounts of alpha-amylase mRNA induced in each tissue. Evidence from these experiments is consistent with the view that GA4-17-Sepharose induces alpha-amylase gene expression in aleurone protoplasts by interacting with the protoplast surface. This indicates that GA receptors may be located at, or near, the external face of the aleurone plasma membrane.