Plants produce a wide variety of natural products that can be exploited for medicinal purposes. Paclitaxel is a key anti-cancer drug originally isolated from the bark of Taxus spp. that is currently approved for use in the treatment of breast, lung and non-small cell cancers, AIDS-related Kaposi's sarcoma and coronary artery disease. Worldwide demand for paclitaxel is high and plant cell culture (PCC) is an attractive production route. Cultured cambial meristematic cells (CMCs) provide a good platform from which to increase drug production, as they possess superior growth properties on an industrial scale compared to typical dedifferentiated cell culture. Elicitors, such as methyl-jasmonate (MeJA), can up-regulate paclitaxel production in PCC, however the effect is only transient. Identification and characterisation of the key transcriptional regulators that control MeJA induced metabolic reprogramming can provide potential tools to manipulate Taxus CMC culture to produce more paclitaxel. Roche454 sequencing was employed to establish the basic transcriptomic profile of Taxus cuspidata CMCs, which was then utilized as a reference to observe the transcriptional profile of CMCs at three time points after MeJA elicitation (0.5, 2 and 12 h). Analysis of the transcriptional regulatory network identified 19 transcription factors (TFs) that were significantly up-regulated at an early time point (0.5 h) after elicitation. These TFs came from five families – AP2, MYB, NAC, bHLH and WRKY – that are well known to regulate secondary plant metabolism. An Arabidopsis thaliana transient expression assay (TEA) was employed to investigate the regulatory activity of these 19TFs against 10 paclitaxel biosynthetic promoters. The TEA screen identified 79 significant interactions with every promoter interacting with at least three TFs, which could activate or repress activity. A MYB TF was identified that could up-regulate eight out of the ten promoters tested, indicating it maybe a potential overall regulator of paclitaxel biosynthesis. In vitro electromobility shift assays established the possible binding site for this TF as an AC element, with the consensus sequence of A(A/C)C. Repressors of promoter activity were also identified, for example an AP2 TF which contains the well-established ERF associated amphiphilic repression (EAR) motif. The activity of the EAR domain was explored in vivo using a TEA assay and site directed mutagenesis mutants. Activity was lost when the mutation occurred within the domain suggesting the TF was working as an active repressor. TFs can work individually or in combination to achieve metabolic reprogramming after MeJA elicitation. One of the best characterised examples of plant combinatorial control is between particular sub classes of MYB and bHLH TFs. However investigation into possible interactions between the T. cuspidata MYB and bHLH TFs in vivo using yeast two hybrid and TEAs found few combinations that led to a significant change in regulatory activity. The regulatory activity of WRKY TFs was shown to be post-translationally controlled when the TEAs were treated with MeJA, however the mechanism by which this occurs remains to be elucidated. The interactions identified between the 19 TFs and the paclitaxel biosynthetic promoters can be exploited in the future to produce superior Taxus CMC lines with increased paclitaxel yields.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:712373 |
| Date | January 2016 |
| Creators | Howat, Susan Ann |
| Contributors | Loake, Gary ; Fry, Stephen |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/21116 |
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