Examining the Complex Transcriptional Response of Perturbing Anthranilate Synthase in the Terpenoid Indole Alkaloid Pathway in Catharanthus Roseus

Catharanthus roseus (Madagascar periwinkle) produces two clinically important chemotherapy drugs, vinblastine and vincristine.  These drugs are produced through the terpenoid indole alkaloid (TIA) pathway.  While researchers have been studying the TIA pathway genes and regulators for the past 40 years, there are still many aspects of the pathway that are unknown.  Previous study on this system has shown that over expression of a feedback insensitive anthranilate synthase (AS) under the control of an inducible promoter increase the levels of some direct metabolites, while decrease the levels of some downstream metabolites.

Although genetic modification is specifically targeted to a particular gene AS, we hypothesized that it may cause unexpected transcriptional responses of other TIA pathway genes and regulators. In this research, RT-qPCR was applied to analyze the transcriptional response of all known TIA genes and regulators after 12, 24, and 48 hours induction of AS expression. The results show that overexpression of AS caused an increase in known terpenoid pathway gene transcripts, indole pathway gene transcripts, and some TIA pathway gene transcripts.  Notably two TIA gene transcripts were significantly down-regulated under the same conditions.  Similarly transcripts of TIA regulators were differentially expressed when AS was overexpressed.  These results explain why this AS engineered root line did not result in a higher level TIA accumulation and further indicate the complex regulation of TIA pathway after perturbing AS gene expression. Therefore, further elucidation of the TIA pathway and regulation is required to facilitate the future rational engineering of this plant for increased TIA production.