Characterisation of novel target promoters for the dexamethasone-inducible/tetracycline-repressible regulator TGV using luciferase and isopentenyl transferase as sensitive reporter genes

Abstract.

The chimeric transcriptional activator TGV mediates dexamethasone (dx)-inducible and tetracycline (tc)-repressible transgene expression in tobacco (dx-on/tc-off system). The expression profiles of four different synthetic target promoters, comprising multiple TGV binding sites upstream of a core promoter, were characterised using the sensitive luciferase assay. Induction factors of over 1000 were measured in roots and leaves of over 30% of the transgenic plants, irrespective of the promoter used. Promoters P_TF and P_Tax, which carry the –48 to +1 region of the Cauliflower Mosaic Virus 35S promoter, showed higher expression levels in both the uninduced and induced states than P_Top10 and P_TFM, which harbour several point mutations in this region. Moreover, P_Tax expressed higher background activities than P_TF, indicating that the sequence of the synthetic regulatory region can influence background levels. The usefulness of the dx-on/tc-off system for experiments addressing gene function was demonstrated by using it to control the expression of isopentenyl transferase. This enzyme catalyses the rate-limiting step in cytokinin biosynthesis and causes phenotypic effects even at low expression levels. Only dx-induced transgenic plants displayed phenotypic alterations indicative for increased cytokinin synthesis (e.g. outgrowth of lateral buds). Simultaneous treatment of selected buds with the anti-inducer tc suppressed bud growth. This result suggests that cytokinins cannot serve as mobile signals to elicit the release of apical dominance in tissues compromised for enhanced cytokinin synthesis.