Abstract
Terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus is a complex and highly regulated process. Understanding the biochemistry and regulation of the TIA pathway is of particular interest as it may allow the engineering of plants to accumulate higher levels of pharmaceutically important alkaloids. Toward this end, we generated a transgenic C. roseus hairy root line that overexpresses the CrBPF1 transcriptional activator under the control of a β-estradiol inducible promoter. CrBPF1 is a MYB-like protein that was previously postulated to help regulate the expression of the TIA biosynthetic gene STR. However, the role of CrBPF1 in regulation of the TIA and related pathways had not been previously characterized. In this study, transcriptional profiling revealed that overexpression of CrBPF1 results in increased transcript levels for genes from both the indole and terpenoid biosynthetic pathways that provide precursors for TIA biosynthesis, as well as for genes in the TIA biosynthetic pathway. In addition, overexpression of CrBPF1 causes increases in the transcript levels for 11 out of 13 genes postulated to act as transcriptional regulators of genes from the TIA and TIA feeder pathways. Interestingly, overexpression of CrBPF1 causes increased transcript levels for both TIA transcriptional activators and repressors. Despite the fact that CrBPF1 overexpression affects transcript levels of a large percentage of TIA biosynthetic and regulatory genes, CrBPF1 overexpression has only very modest effects on the levels of the TIA metabolites analyzed. This finding may be due, at least in part, to the up-regulation of both transcriptional activators and repressors in response to CrBPF1 overexpression, suggesting that CrBPF1 may serve as a “fine-tune” regulator for TIA biosynthesis, acting to help regulate the timing and amplitude of TIA gene expression.
Highlights
Generation of Transgenic Hairy Roots Expressing CrBPF1 under the Control of a β-Estradiol Inducible Promoter CrBPF1 was identified by screening for proteins that bind the STR promoter
Thirteen transcriptional regulators have been postulated to act in regulation of the Terpenoid indole alkaloid (TIA) pathway, the effects of most of these transcriptional regulators on expression of the majority of the known TIA biosynthetic and regulatory genes have not yet been determined
Terpenoid indole alkaloid production is dependent on the synthesis of precursors by both the indole and terpenoid pathways, the combination of these precursors by STR and subsequent reactions carried out by different branches of the TIA pathway
Summary
Transcriptional activators and repressors play important roles in regulating TIA biosynthesis Expression of both OctadecanoidResponsive Catharanthus AP2-domain protein 2 (ORCA2) and ORCA3 increases rapidly upon fungal elicitation (Menke et al, 1999; van der Fits and Memelink, 2000). ORCA2 and ORCA3 are AP2-domain transcription factors that activate STR expression through a jasmonate signal transduction pathway by binding to the jasmonate and elicitor-responsive element (JERE) in the STR promoter (Menke et al, 1999; van der Fits and Memelink, 2001). CrBPF1 promotes STR transcription through a signal transduction pathway that is responsive to elicitors but not jasmonate and acts downstream of protein phosphorylation and calcium influx (van der Fits et al, 2000). The results of these transcriptional and metabolic profiling experiments have revealed the role of CrBPF1 in regulation of TIA metabolism
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