Transcriptional regulation of taxol™ biosynthesis in taxus cuspidate procambium cells
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This thesis presents an investigation into the transcriptional regulation of TaxolTM biosynthsis in Taxus cuspidata cell suspension cultures. The potent anticancer drug TaxolTM has been shown to be successful in the treatment of breast, lung and ovarian cancer and the acquired immunodeficiency syndrome (AIDS) related Kaposi’s sarcoma. Produced by all species of yew, TaxolTM belongs to the class of taxane diterpenoids and is of huge pharmaceutical importance. The plant material utilised in this thesis is a cell suspension culture initiated from isolated procambium cells of T. cuspidata. The latter is a meristematic tissue giving rise to the conductive tissue of plants. This un-differentiated cell suspension culture exhibits an increased and stable production of TaxolTM in response to the plant hormone elicitor methyljasmonate, limited cell aggregation and fast growth when compared to a cell suspension culture initiated from differentiated cells (somatic) of T. cuspidata. In order to assess the stem cell characteristics of the employed procambium cell suspension culture, the transcriptome of T. cuspidata was sequenced utilising Roche/ 454 and Illumina/ Solexa NlaIII tag sequencing technoloxiv gies. Statistical analysis uncovered differential expression profiles of 563 genes present within the procambium cell derived transcriptome by comparison with the somatic cell derived transcriptome. Gene ontology analysis of the latter identified that genes associated with response to stress and defence response were upregulated in the differentially expressed portion within the procambium cell suspension culture. This is consistent with the characteristics of animal stem cells which exhibit robust defence strategies to environmental stress. Furthermore PHLOEM INTERCALATED WITH XYLEM (PXY ) and TRACHEARY ELEMENT DIFFERENTIATION 2 (TED2), which are essential for ordered procambium cell division and differentiation into trachaery elements respectively in A. thaliana and Z. elegans, are up-regulated in the T. cuspidata procambium cell suspension culture. Further T. cuspidata homologues of the jasmonate signalling components JASMONATE ZINC FINGER LIKE ZIM DOMAIN 2 (JAZ2) and JAZ3 were identified among up-regulated transcripts in response to jasmonate treatment in both the procambium and the somatic cell line. Blast analysis identified 211 transcription factors within the APETELA 2 (AP2), BASIC-HELIX-LOOPHELIX (bHLH), WRKY, MYB and BASIC-LEUCIN-ZIPPER (bZIP) families. Further characterisation established 21 transcription factors which are significantly up-regulated in response to jasmonate treatment and show a higher expression level in procambium cells. These provide promising targets for further functional characterisation to elucidate their involvement within TaxolTM biosynthesis. In order to investigate transcriptional regulation of the TaxolTM structural genes, a 513 bp fragment corresponding to the TAXADIENE SYNTHASE (TASY ) promoter was cloned by genome walking. In-silico analysis of the TASY and 3’-N-DEBENZOYLTAXOL N-BENZOYLTRANSFERASE (DBTNBT) promoter resulted in the identification of methyljasmonate and pathogen-responsive elements which may significantly contribute to jasmonate mediated accumulation of TaxolTM. Analysis of a chimeric promoter construct driving the reporter gene β-GLUCURONIDASE (GUS) in N. benthamiana confirmed jasmonate-responsiveness of the TASY promoter. Finally, comparison of the expression level of genes coding for potentially rate-limiting enzymes within the TaxolTM pathway established a significantly increased expression of BACCATIN II PHENYLPROPANOYLTRANSFERASE (BAPT) in response to jasmonate treatment within the procambium cell suspension culture. Furthermore transcripts of TASY, PHENYLALANINE AMINOMUTASE (PAM) and DBTNBT show an overall higher expression and prolonged transcript accumulation in procambium compared to somatic cells. In this thesis jasmonate-signalling components, jasmonate-responsive transcription factors and differential gene expression profiles of TaxolTM structural genes were identified which, may contribute to an increased TaxolTM production in the utilised procambium cell suspension culture. Furthermore the T. cuspidata procambium cell suspension culture was found to have an increased level of stress- and defence-response reflected by differential gene expression profiles and content of phenolic compounds and TaxolTM.