NUCLEOTIDE-SEQUENCE OF THE TRYPTOPHAN DECARBOXYLASE GENE OF CATHARANTHUS-ROSEUS AND EXPRESSION OF TDC-GUSA GENE FUSIONS IN NICOTIANA-TABACUM

The enzyme tryptophan decarboxylase (TDC; EC 4.1.1.28) converts trypto phan into tryptamine. In Catharanthus I roseus and other plants capabl e of producing terpenoid indole alkaloids (TIAs) TDC links primary met abolism to the secondary metabolic pathway involved in the biosynthesi s of these compounds. The accumulation of tdc mRNA in C. roseus cells is developmentally regulated and transcriptionally influenced by elici tors (induction) and auxins (repression). Here we report that TDC is e ncoded by a single copy gene in the C. roseus genome. No introns were observed upon isolation and sequencing of this gene. To study gene exp ression controlled by the tdc promoter, a 2 kb promoter fragment and a number of 5' deleted promoter derivatives were joined in translationa l fusion to a beta-D-glucuronidase reporter gene (gusA). Expression of the chimaeric constructs was monitored in stably transformed tobacco plants and in transiently transfected tobacco protoplasts. Histochemic al and fluorimetric analysis of transgenic plants revealed that 1938 b p of the tdc promoter (with respect to the translational start codon) give rise to GUS activity in roots, stems and leaves. No tissue or cel l type specificity was noted. Promoter deletions up to nucleotide -398 directed lower levels of gusA expression but conferred the same patte rn of staining for GUS activity as the -1938 construct. Further deleti on of the tdc promoter up to nucleotide -232 resulted in drastically r educed GUS activity levels and loss of GUS staining in all parts of th e transgenic plants. In contrast to stable transformation, the -232 td c-gusA construct gave rise to GUS activity levels comparable to those of the -398 construct in an assay system for transient expression in p rotoplasts. In this system the GUS activities measured were not affect ed by the presence or absence of the synthetic auxin naphthalene aceti c acid (NAA).