Structure of the genes encoding Hordeum vulgare (1----3,1----4)-beta-glucanase isoenzymes I and II and functional analysis of their promoters in barley aleurone protoplasts

Abstract

Barley (1----3,1----4)-beta-glucanase isoenzyme II is synthesized in the aleurone cells during germination and secreted into the endosperm for hydrolysis of the cell walls. Its synthesis is stimulated by gibberellic acid (GA3) and repressed by abscisic acid. The gene for isoenzyme I is expressed in the aleurone, scutellum and prominently in young leaves. Close functional relatedness between the two enzymes is attested by 92% identity at the level of the amino acid sequence. The structural genes for the two enzymes each contain a large intron of 2505 bp and 2952 bp, respectively, in the codon for amino acid 25 of the 28-residue signal peptide. During evolution, homologous regions of the two introns have changed position and orientation. Furthermore, a large palindromic sequence of 327 bp in the 5' end of the intron is present only in the gene for isoenzyme II. In transient expression assays using barley aleurone protoplasts and chloramphenicol acetyl transferase as reporter the promoter of the isoenzyme I gene showed no response to GA3. However, removal of a unique 151 bp region extending from positions -402 to -552 upstream of the TATA box permitted low levels of GA3-induced expression of the reporter gene, suggesting a silencer function for this domain. High levels of GA3-responsive expression were obtained in aleurone protoplasts using the promoter of the gene encoding isoenzyme II. Truncation of this promoter revealed that sequences located within 253 bp upstream from the TATA box are sufficient to direct GA3-stimulated expression. Using the homologous barley aleurone protoplast transfection assay, it was possible to reproduce the in vivo expression characteristics of the genes for the barley (1----3,1----4)-beta-glucanase isoenzymes I and II with reporter gene constructs.