Nucleotide sequence of the tryptophan decarboxylase gene of Catharanthus roseus and expression of tdc-gusA gene fusions in Nicotiana tabacum

Abstract

The enzyme tryptophan decarboxylase (TDC; EC 4.1.1.28) converts tryptophan into tryptamine. In Catharanthus roseus and other plants capable of producing terpenoid indole alkaloids (TIAs) TDC links primary metabolism to the secondary metabolic pathway involved in the biosynthesis of these compounds. The accumulation of tdc mRNA in C. roseus cells is developmentally regulated and transcriptionally influenced by elicitors (induction) and auxins (repression). Here we report that TDC is encoded by a single copy gene in the C. roseus genome. No introns were observed upon isolation and sequencing of this gene. To study gene expression controlled by the tdc promoter, a 2 kb promoter fragment and a number of 5′ deleted promoter derivatives were joined in translational fusion to a β-d-glucuronidase reporter gene (gusA). Expression of the chimaeric constructs was monitored in stably transformed tobacco plants and in transiently transfected tobacco protoplasts. Histochemical and fluorimetric analysis of transgenic plants revealed that 1938 by of the tdc promoter (with respect to the translational start codon) give rise to GUS activity in roots, stems and leaves. No tissue or cell type specificity was noted. Promoter deletions up to nucleotide −398 directed lower levels of gusA expression but conferred the same pattern of staining for GUS activity as the −1938 construct. Further deletion of the tdc promoter up to nucleotide −232 resulted in drastically reduced GUS activity levels and loss of GUS staining in all parts of the transgenic plants. In contrast to stable transformation, the −232 tdc-gusA construct gave rise to GUS activity levels comparable to those of the −398 construct in an assay system for transient expression in protoplasts. In this system the GUS activities measured were not affected by the presence or absence of the synthetic auxin naphthalene acetic acid (NAA).