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

Abstract

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 constitutive 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 genes that are involved in the biosynthesis of secondary metabolites of the terpenoid indole alkaloid (TIA) class. Several TIAs have pharmaceutically interesting activities, including the anti-tumour agents vincristine and vinblastine. The use of suspension-cultured cells enabled us to screen in a relatively easy way hundreds of thousands of T-DNA-tagged cells for resistance to a toxic substrate of one of the TIA biosynthetic enzymes: tryptophan decarboxylase. This screening yielded several interesting tagged cell lines. Further characterisation of one of the tagged cell lines led to the isolation of Orca3, a gene encoding an AP2/ERF-domain transcription factor that acts as a master regulator of primary and secondary metabolism. The T-DNA activation tagging results described in detail in this paper illustrate the usefulness of this approach to isolate regulators of a complex metabolic pathway from a genetically non-tractable plant species.