MOLECULAR CHARACTERIZATION OF THE GENE FOR CARROT CELL-WALL BETA-FRUCTOSIDASE

Carrot cell wall beta-fructosidase, previously purified and cloned, is encoded by a single, wound- and pathogen-inducible gene. The developm ental regulation of the gene was studied by determining the steady-sta te mRNA levels in different organs during carrot development: cell wal l beta-fructosidase mRNA was detected in roots and leaves of young pla nts but not during tap root development. A genomic clone was isolated and characterized. The transcription start site was determined by prim er extension analysis. Inspection of the promoter sequence (1488 bp) r evealed the presence of sequences with high homology to cis-acting ele ments for the regulation of plant genes by wounding and infection. The 5'-regulatory sequence was fused to the reporter gene beta-glucuronid ase (GUS) and tested in a transient expression assay with carrot suspe nsion cells and wounded carrot root tissue (aged disks of carrot roots ). The expression of the GUS gene in the transfected cells proved that the isolated promoter was functional. In transgenic tobacco plants co ntaining the cell wall beta-fructosidase promoter fused to GUS, the re porter gene was predominantly expressed in the shoot and root meristem s of young seedlings. No GUS expression was detected in mature tobacco plants, showing that the development-specific regulation of the cell wall beta-fructosidase promoter seen in carrot was maintained in tobac co plants. In contrast, expression of the GUS reporter gene in transge nic tobacco was not wound inducible. To analyze the functional organiz ation of the cell wall beta-fructosidase promoter, a 5'-deletion serie s was generated and tested in a transient expression assay in protopla sts of Nicotiana plumbaginifolia. Two regions containing putative sile ncer elements were identified. A comparison of these regions with know n silencer elements identified in both regions one copy of the negativ e dominant cis-acting element found in a chalcone synthase promoter of petunia.