Abstract
Background: The terpenoid indole alkaloid (TIA) pathway leads to the production of pharmaceutically important drugs, such as the anticancer compounds vinblastine and vincristine. Unfortunately, these drugs are produced in trace amounts, causing them to be very costly. To increase production of these drugs, an improved understanding of the TIA regulatory pathway is needed. Towards this end, transgenic Catharanthus roseus hairy roots that overexpress the ORCA2 TIA transcriptional activator were generated and characterized.Results: Transcriptional profiling experiments revealed that overexpression of ORCA2 results in altered expression of key genes from the indole and terpenoid pathways, which produce precursors for the TIA pathway, and from the TIA pathway itself. In addition, metabolite-profiling experiments revealed that overexpression of ORCA2 significantly affects the levels of several TIA metabolites. ORCA2 overexpression also causes significant increases in transcript levels of several TIA regulators, including TIA transcriptional repressors.Conclusions: Results presented here indicate that ORCA2 plays a critical role in regulation of TIA metabolism. ORCA2 regulates expression of key genes from both feeder pathways, as well as the genes (STR and SGD) encoding the enzymes that catalyze the first two steps in TIA biosynthesis. ORCA2 may play an especially important role in regulation of the downstream branches of the TIA pathway, as it regulates four out of five genes characterized from this part of the pathway. Regulation of TIA transcriptional repressors by ORCA2 may provide a mechanism whereby increases in TIA metabolite levels in response to external stimuli are transient and limited in magnitude.
| Original language | English (US) |
|---|---|
| Article number | 155 |
| Journal | BMC plant biology |
| Volume | 13 |
| Issue number | 1 |
| DOIs | |
| State | Published - Oct 8 2013 |
Bibliographical note
Funding Information:The authors would like to thank Dr. Christie A.M. Peebles at Colorado State University for advice on production of C. roseus transgenic hairy root lines, Dr. Kenneth Beckman and Ms. Trianna Full at the Biomedical Genomics Center at the University of Minnesota for helping with quantitative RT-PCR analysis, Dr. Sarah O’Connor from the John Innes Centre for the gift of strictosidine, Dr. Ann Perera and the W. M. Keck Metabolomics Research Laboratory for assistance with the LC-MS analysis and Syngenta AG for providing the alcohol-inducible expression system. Financial support for conducting the research was provided by NSF CBET-0729753 (JVS) and NSF CBET-0729625 (SIG) and for preparing the manuscript by NSF CBET-1064903 (SIG). The sponsor did not play a role in the collection, analysis or interpretation of the data and was not involved in the writing of the report or the decision to submit the article for publication.
Keywords
- Catharanthus roseus
- Hairy root cultures
- ORCA2
- Terpenoid indole alkaloids