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
