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

Expression of the wheat ‘Em’ genes in embryos isstrongly induced both by abscisic acid and by subjection toosmotic stress. We have examined the basis of this inductionin an homologous system by con structing fusions between thepromoter sequences of a cloned ‘Em’ gene and theGUS reporter gene. The ABA-and stress-mediated expression ofthese constructs has been assayed following delivery to intactwheat embryos by particle bombardment. Staining of bombardedembryos with the chromogenic substrate X-gluc enabled a simpleand rapid visual identification of promoter activity by scoringthe 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 resultsobtained with bombarded embryos were compared with those obtainedby the fluorlmetnc measurement of GUS activity in cereal aleuroneprotoplasts transfected with the same constructs. Analysis ofa series of 5'-deletions of the ‘Em’ promoters indicatedthat the stress-responsive cis-acting regulatory elements comprisethe same sequences as those responsible for ABA-mediated geneexpression. Key words: Abscisic acid, osmotic stress, Em genes, wheat embryos, transient expression