Plant Molecular Biology 16: 713-724, 1991. © 1991 Kluwer Academic Publishers. Printed in Belgium. 713 Control of transient expression of chimaeric genes by gibberellic acid and abscisic acid in protoplasts prepared from mature barley aleurone layers John V. Jacobsen and Timothy J. Close 1 CSIR O, Division of Plant Industry, Canberra, ACT 2601, Australia; 1present address: Department of Botany and Plant Sciences, University of California, Riverside, CA 92521-10124, USA Received 18 September 1990; accepted in revised form 20 December 1990 Key words: abscisic acid, aleurone protoplasts, a-amylase gene, barley, gibberellic acid, transient expres- sion Abstract Gibberellic acid (GA3) and abscisic acid (ABA) control the transcription of e-amylase genes in barley aleurone cells. This control is likely to be exerted through cis-acting hormone-responsive elements in the promoter region of the gene. In order to further define these elements, we have developed procedures for obtaining transient expression of chimaeric genes in protoplasts prepared from mature barley aleurone layers. Constructs with heterologous constitutive promoters and with heterologous and homologous GA 3- and ABA-regulated promoters were expressed specifically by these cells. This system would appear to offer great potential in gene regulation studies especially for hormonally regulated homologous genes. Functional analysis of a barley e-amylase gene has been performed using this system. A 2050 bp fragment from a high-pI e-amylase gene was fused to a reporter gene (GUS) and control of its expression was examined. Deletion analysis of this promoter fragment showed that major GA- and ABA-responsive elements occurred between 174 and 41 bp upstream from the transcription initiation site. Introduction Gibberellic acid promotes and abscisic acid inhibits the production of e-amylase in Neurone layers of barley and this response has been used extensively to study the mechanism of action of the two hormones [reviewed in 1, 10, 22]. Increased (GA3) and decreased (ABA) e-amylase production is accompanied with corresponding changes in the amounts of translatable mRNA [14, 15, 36, 38] and in the levels of mRNA tran- scripts detected by e-amylase cDNA probes [7, 9, 17, 39, 43, 44] leading to the conclusion that GA3 and ABA regulate e-amylase synthesis by causing changes in the level of its mRNA. These results do not indicate whether mRNA accumulation is the result of increased gene transcription or of stabili- zation of mRNA (or both), but run-on tran- scription experiments using nuclei isolated from barley aleurone protoplasts indicate that at least part of the increase can be explained by an increased rate of gene transcription [ 21 ]. Studies The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X54643.