T-DNA activation tagging as a tool to isolate regulators of a metabolic pathway from a genetically non-tractable plant species

T-DNA activation tagging is a method used to generate dominant mutations in plants or plant cells by the insertion of a T-DNA which carries constituti ve enhancer elements that can cause transcriptional activation of flanking plant genes. We applied this approach to the species Catharanthus roseus (L .) G. Don (Madagascar periwinkle), in an attempt to isolate regulators of g enes that are involved in the biosynthesis of secondary metabolites of the terpenoid indole alkaloid (TIA) class. Several TIAs have pharmaceutically i nteresting activities, including the anti-tumour agents vincristine and vin blastine. The use of suspension-cultured cells enabled us to screen in a re latively easy way hundreds of thousands of T-DNA-tagged cells for resistanc e to a toxic substrate of one of the TIA biosynthetic enzymes: tryptophan d ecarboxylase. This screening yielded several interesting tagged cell lines. Further characterisation of one of the tagged cell lines led to the isolat ion of Orca3, a gene encoding an AP2/ERF-domain transcription factor that a cts as a master regulator of primary and secondary metabolism. The T-DNA ac tivation tagging results described in detail in this paper illustrate the u sefulness of this approach to isolate regulators of a complex metabolic pat hway from a genetically non-tractable plant species.