Nuclear factors GT-1 and 3AF1 interact with multiple sequences within the promoter of the Tdc gene from Madagascar periwinkle: GT-1 is involved in UV light-induced expression

Abstract

Plant secondary metabolites of the terpenoid indole alkaloid (TIA) class comprise several compounds with pharmaceutical applications. A key step in the TIA biosynthetic pathway is catalysed by the enzyme tryptophan decarboxylase (TDC), which channels the primary metabolite tryptophan into TIA metabolism. In Catharanthus roseus (Madagascar periwinkle), the Tdc gene is expressed throughout plant development. Moreover, Tdc gene expression is induced by external stress signals, such as fungal elicitor and UV light. In a previous study of Tdc promoter architecture in transgenic tobacco it was shown that the −538 to −112 region is a quantitative determinant for the expression level in different plant organs. Within this sequence one particular region (−160 to −99) was identified as the major contributor to basal expression and another region (−99 to −37) was shown to be required for induction by fungal elicitor. Here, the in vitro binding of nuclear factors to the −572 to −37 region is described. In extracts from tobacco and C. roseus, two binding activities were detected that could be identified as the previously described nuclear factors GT-1 and 3AF1, based on their mobility and binding characteristics. Both factors appeared to interact with multiple regions in the Tdc promoter. Mutagenesis of GT-1 binding sites in the Tdc promoter did not affect the basal or elicitor-induced expression levels. However, induction of the Tdc promoter constructs by UV light was significantly lower, thereby demonstrating a functional role for GT-1 in the induction of Tdc expression by UV light.