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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 ?-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.

SUBMITTER: Li CY

PROVIDER: S-EPMC4589645 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

Li Chun Yao CY Leopold Alex L AL Sander Guy W GW Shanks Jacqueline V JV Zhao Le L Gibson Susan I SI

Frontiers in plant science 20151001

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 ...[more]

PMID: 26483828

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Project description:Terpenoid indole alkaloids (TIAs) include several valuable pharmaceuticals. As Catharanthus roseus remains the primary source of these TIA pharmaceuticals, several research groups have devoted substantial efforts to increase production of these compounds by C. roseus. Efforts to increase TIA production by overexpressing positive regulators of TIA biosynthetic genes have met with limited success. This limited success might be due to the fact that overexpression of several positive TIA regulators turns on expression of negative regulators of TIA biosynthetic genes. Consequently, a more effective approach for increasing expression of TIA biosynthetic genes might be to decrease expression of negative regulators of TIA biosynthetic genes. Towards this end, an RNAi construct was generated that expresses a hairpin RNA carrying nucleotide fragments from three negative transcriptional regulators of TIA genes, ZCT1, ZCT2 and ZCT3, under the control of a beta-estradiol inducible promoter. Transgenic C. roseus hairy root lines carrying this ZCT RNAi construct exhibit significant reductions in transcript levels of all three ZCT genes. Surprisingly, out of eight TIA biosynthetic genes analyzed, seven (CPR, LAMT, TDC, STR, 16OMT, D4H and DAT) exhibited decreased rather than increased transcript levels in response to reductions in ZCT transcript levels. The lone exception was T19H, which exhibited the expected negative correlation in transcript levels with transcript levels of all three ZCT genes. A possible explanation for the T19H expression pattern being the opposite of the expression patterns of the other TIA biosynthetic genes tested is that T19H shunts metabolites away from vindoline production whereas the products of the other genes tested shunt metabolites towards vindoline metabolism. Consequently, both increased expression of T19H and decreased expression of one or more of the other seven genes tested would be expected to have similar effects on flux through the TIA pathway. As T19H expression is lower in the ZCT RNAi hairy root lines than in the control hairy root line, the ZCTs could act directly to inhibit expression of T19H. In contrast, ZCT regulation of the other seven TIA biosynthetic genes tested is likely to occur indirectly, possibly by the ZCTs turning off expression of a negative transcriptional regulator of some TIA genes. In fact, transcript levels of a negative TIA transcriptional regulator, GBF1, exhibited a strong, and statistically significant, negative correlation with transcript levels of ZCT1, ZCT2 and ZCT3. Together, these findings suggest that the ZCTs repress expression of some TIA biosynthetic genes, but increase expression of other TIA biosynthetic genes, possibly by turning down expression of GBF1.

Dataset Information

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

Publications

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

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