CrBPF1 overexpression alters transcript levels of terpenoid indole alkaloid biosynthetic and regulatory genes

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.

The basic helix‐loop‐helix transcription factor BIS2 is essential for monoterpenoid indole alkaloid production in the medicinal plant Catharanthus roseus

“…Ultimately, coordinate regulation of the complete set of MIA biosynthesis genes will require a more complex combination of transcriptional activators and repressors. Indeed, several other TFs have been reported as positive or negative regulators of MIA genes, including G-box-binding factors (GBFs), the MYB TF box P-binding factor-1 (BPF1), WRKY1, AT-hook TFs and EAR-domain containing zinc finger TFs Sib eril et al, 2001;Pauw et al, 2004;Vom Endt et al, 2007;Suttipanta et al, 2011;Li et al, 2015).…”

Transcriptional Regulation and Transport of Terpenoid Indole Alkaloid in Catharanthus roseus: Exploration of New Research Directions

“…As for regulatory genes, overexpressing CrBPF1 increased the transcripts of ORCA3 , CrMYC1 , CrMYC2 , BIS1 , GBF2 and ZCTs , but little affected the expressions of ORCA2 and CrWRKY1 . Although overexpression of CrBPF1 could not obviously increase the accumulation of TIAs and even caused the decrease of serpentine, it could extensively regulate TIA pathway genes and increase the expression of TIA transcriptional repressors, which may be a good direction to the research of TIA biosynthesis [ 44 ].…”

Exploring the production of high-value compounds in plant Catharanthus roseus hairy roots and yeast Yarrowia lipolytica

“…Many previous C. roseus engineering studies showed that the modification of TIA genes usually led to complex transcriptional changes in other pathway genes and transcription factors [16,17,33]. Similarly, the complicated mRNA changes in the TIA pathway regulators were triggered after inducing two vindoline pathway genes T16H and 16OMT.…”

“…and Str genes were enhanced after induction by JA and fungal elicitation, which suggests that CrMYC1 activates the expression of the Str gene [32]. CrMYC2 is postulated to act upstream of ORCA2 and ORCA3, activating their transcription [33].…”

“…CrMYC1 [32] and CrMYC2 [33] are basic helix-loop-helix transcription factors. Both of these two transcription factors are induced by JA and fungal elicitation.…”