Rapid analysis of an osmotic stress responsive promoter by transient expression in intact wheat embryos

Expression of the wheat ‘Em’ genes in embryos is strongly induced both by abscisic acid and by subjection to osmotic stress. We have examined the basis of this induction in an homologous system by con structing fusions between the promoter sequences of a cloned ‘Em’ gene and the GUS reporter gene. The ABA-and stress-mediated expression of these constructs has been assayed following delivery to intact wheat embryos by particle bombardment. Staining of bombarded embryos with the chromogenic substrate X-gluc enabled a simple and rapid visual identification of promoter activity by scoring the numbers of stained spots. Although not rigorously quantitative, a general correspondence between number of expression events (spots) and promoter activity could be inferred when the results obtained with bombarded embryos were compared with those obtained by the fluorlmetnc measurement of GUS activity in cereal aleurone protoplasts transfected with the same constructs. Analysis of a series of 5'-deletions of the ‘Em’ promoters indicated that the stress-responsive cis-acting regulatory elements comprise the same sequences as those responsible for ABA-mediated gene expression.