A G-box element from the Catharanthus roseus strictosidine synthase (Str) gene promoter confers seed-specific expression in transgenic tobacco plants

The enzyme encoded by the strictosidine synthase (Str) gene from Catharanthus roseus catalyses a key step in the biosynthesis of the pharmaceutically important terpenoid indole alkaloids. Str cDNA and genomic clones have already been isolated, allowing us to study the regulation of Stp gene expression. Here we focus on the role of a putative cis-acting element, CA-CGTG, in the Str promoter. This sequence is known as a G-box, and functions as a transcription-regulating sequence in a number of other promoters. By means of electrophoretic mobility shift assays it was demonstrated that the Str G-box is capable of interacting with nuclear factors in tobacco and with the cloned tobacco G-box-binding factor TAF-1. Disruption of the Str G-box sequence by two single-nucleotide mutations prevented binding of factors, thereby demonstrating the specificity of the observed interactions. Functional analysis in transgenic tobacco plants demonstrated that these mutations also reduced the transcriptional activity of constructs containing tetramers of the Str G-box sequence. Expression directed by a tetramer of the Str G-box fused to a truncated promoter containing only a TATA box was confined to seeds and was found to increase during seed maturation. Thus, the Str G-box tetramer is able to direct seed-specific expression independently of other regulatory sequences. G-box-directed expression in leaves required the presence of an enhancer region from the cauliflower mosaic virus (CaMV) 35S promoter. The results indicate that the G-box needs to interact with other elements to drive expression in leaf, and that it can by itself confer seed-specific expression as a multimer. The fact that only some of the G-boxes found in different promoters serve as seed-specific elements indicates that sequences flanking the G-box determine the transcriptional activity in different tissues. Based on sequence comparisons we propose that the nucleotides at positions -4, -3, -2 and/or +4 are important in determining seed-specific expression.