Year of Publication

2011

Degree Name

Doctor of Philosophy (PhD)

Document Type

Dissertation

College

Agriculture

Department

Plant Physiology

First Advisor

Dr. Ling Yuan

Abstract

Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are an important source of natural or semi-synthetic anticancer drugs. Biosynthesis of TIAs is tissue-specific and induced by certain phytohormones and fungal elicitors, indicating the involvement of a complex transcriptional control network. However, the transcriptional regulation of the TIA pathway is poorly understood. This study reports the isolation and characterization of the G10H promoter and two WRKY transcription factors regulating TIA biosynthesis.

Geraniol 10-hydroxylase (G10H) controls the first committed step in the biosynthesis of terpenoid indole alkaloids (TIA). The C. roseus G10H promoter sequence was isolated by a PCR-based genome walking method. Sequence analysis revealed that the G10H promoter contains several potential eukaryotic regulatory elements involved in regulation of gene expression. For functional characterization, fusion constructs of G10H promoter fragments with the GUS reporter gene were generated and expression was analyzed in a tobacco protoplast transient expression assay. Gain-of-function experiments revealed the presence of three potential transcriptional enhancers located in regions between -191 and -147, -266 and -188, and -318 and -266, respectively. The G10H promoter was capable of conferring stable GUS expression in transgenic tobacco plants and C. roseus hairy roots. In transgenic tobacco seedlings, GUS expression was tissue-specific, restricted to the leaf and actively growing cells around the root tip. GUS expression was not detected in the hypocotyls, root cap and older developing areas of the root. The GUS expression in both transgenic C. roseus hairy roots and tobacco seedlings were responsive to fungal elicitors and methyljasmonate. Compared to other known promoters of TIA pathway genes, the G10H promoter contains unique binding sites for several transcription factors, suggesting that the G10H promoter may be regulated by a different transcriptional cascade.

The majority of TIA pathway gene promoters contain typical W-box elements, which are frequently found to be the binding sites of WRKY transcription factors. CrWRKY1 and CrWRKY2 transcription factors were isolated using a degenerate PCR method. The C. roseus WRKY transcription factor, CrWRKY1 is preferentially expressed in roots and induced by phytohormones, jasmonate, gibberellic acid and ethylene. Overexpression of CrWRKY1 in C. roseus hairy roots up-regulated several key TIA pathway genes, especially tryptophan decarboxylase (TDC), as well as transcriptional repressors ZCT1, ZCT2 and ZCT3. In contrast, CrWRKY1 overexpression repressed the transcriptional activators ORCA2, ORCA3 and CrMYC2. Overexpression of a dominant-repressive form of CrWRKY1, created by fusing the SRDX-repressor domain to CrWRKY1, resulted in down-regulation of TDC and ZCTs but up-regulation of ORCA3 and CrMYC2. CrWRKY1 bound to the W-box elements of the TDC promoter in electrophoretic mobility shift, yeast one-hybrid and C. roseus protoplast assays. In CrWRKY1 hairy roots, up-regulation of TDC increased TDC activity, tryptamine concentration and resistance to 4-methyl tryptophan inhibition. Compared to control roots, CrWRKY1 hairy roots accumulated up to 3-fold higher levels of serpentine. The preferential expression of CrWRKY1 in roots and its interaction with transcription factors, including ORCA3, CrMYC2 and ZCTs, may play a key role in determining the root-specific accumulation of serpentine in C. roseus plants.

CrWRKY2 is induced by methyljasmonate induction. In plant, CrWRKY2 expression is mainly found in young leaves and the stem. The stable transformation of CrWRKY2 in C. roseus hairy roots up-regulated many pathway genes, especially the genes in vindoline biosynthesis. The accumulation of vindoline was observed in CrWRKY2 hairy roots.

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