The regulation of <i>ZCT1</i>, a transcriptional repressor of monoterpenoid indole alkaloid biosynthetic genes in <i>Catharanthus roseus</i>

Mortensen, Samuel; Weaver, Jessica D.; Sathitloetsakun, Suphinya; Cole, Lauren F.; Rizvi, Noreen F.; Cram, Erin J.; Lee‐Parsons, Carolyn W. T.

doi:10.1002/pld3.193

Cited by 12 publications

(10 citation statements)

References 83 publications

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“…C. roseus hairy roots were generated as described in Rizvi et al. ( Mortensen et al, 2019 ) with the modification made in Mortensen et al ( Mortensen et al, 2019 ). The infection of plants with H. arabidopsidis and trypan blue staining were conducted as previously described ( Stuttmann et al., 2011 ).…”

Section: Methodsmentioning

confidence: 99%

High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns

Grützner

Martin

Horn

et al. 2021

Plant Communications

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The recent discovery of the mode of action of the CRISPR/Cas9 system has provided biologists with a useful tool for generating site-specific mutations in genes of interest. In plants, site-targeted mutations are usually obtained by the stable transformation of a Cas9 expression construct into the plant genome. The efficiency of introducing mutations in genes of interest can vary considerably depending on the specific features of the constructs, including the source and nature of the promoters and terminators used for the expression of the Cas9 gene and the guide RNA, and the sequence of the Cas9 nuclease itself. To optimize the efficiency of the Cas9 nuclease in generating mutations in target genes in Arabidopsis thaliana , we investigated several features of its nucleotide and/or amino acid sequence, including the codon usage, the number of nuclear localization signals (NLSs), and the presence or absence of introns. We found that the Cas9 gene codon usage had some effect on its activity and that two NLSs worked better than one. However, the highest efficiency of the constructs was achieved by the addition of 13 introns into the Cas9 coding sequence, which dramatically improved the editing efficiency of the constructs. None of the primary transformants obtained with a Cas9 gene lacking introns displayed a knockout mutant phenotype, whereas between 70% and 100% of the primary transformants generated with the intronized Cas9 gene displayed mutant phenotypes. The intronized Cas9 gene was also found to be effective in other plants such as Nicotiana benthamiana and Catharanthus roseus .

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Section: Methodsmentioning

confidence: 99%

High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns

Grützner

Martin

Horn

et al. 2021

Plant Communications

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120

133

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“…The ZCT1, ZCT2 and ZCT3 transcriptional regulators have been shown to act as negative regulators of genes involved in TIA metabolism and regulation of TIA metabolism (Pauw et al, 2004;Chebbi et al, 2014;Mortensen et al, 2019a;Mortensen et al, 2019b).…”

Section: Resultsmentioning

confidence: 99%

“…ZCT1 and ZCT2, but not ZCT3, also repress expression of HDS, part of the methyl erythritol pathway (Chebbi et al, 2014). ZCT1 has also been shown to repress its own promoter (Mortensen et al, 2019b).…”

Section: Introductionmentioning

confidence: 99%

Repression of ZCT1, ZCT2 and ZCT3 affects expression of terpenoid indole alkaloid biosynthetic and regulatory genes

Gibson

2021

PeerJ

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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.

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“…A MAP kinase cascade modulates the TIA biosynthetic pathway by activating its downstream target AP2/ERF TF genes (Paul et al, 2017). In addition, the zinc-finger TF ZCT1 acts as a transcriptional repressor in the TIA biosynthetic pathway (Mortensen et al, 2019). In Ophiorrhiza pumila, OpWRKY2 and OpWRKY3 were identified as two positive regulators in the biosynthesis of camptothecin (Wang et al, 2019a;Hao et al, 2021).…”

Section: Transcription Factors Involved In the Biosynthesis Of Alkaloidsmentioning

confidence: 99%

Environmental and Genetic Factors Involved in Plant Protection-Associated Secondary Metabolite Biosynthesis Pathways

Zhan

Chen

et al. 2022

Front. Plant Sci.

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Plant specialized metabolites (PSMs) play essential roles in the adaptation to harsh environments and function in plant defense responses. PSMs act as key components of defense-related signaling pathways and trigger the extensive expression of defense-related genes. In addition, PSMs serve as antioxidants, participating in the scavenging of rapidly rising reactive oxygen species, and as chelators, participating in the chelation of toxins under stress conditions. PSMs include nitrogen-containing chemical compounds, terpenoids/isoprenoids, and phenolics. Each category of secondary metabolites has a specific biosynthetic pathway, including precursors, intermediates, and end products. The basic biosynthetic pathways of representative PSMs are summarized, providing potential target enzymes of stress-mediated regulation and responses. Multiple metabolic pathways share the same origin, and the common enzymes are frequently to be the targets of metabolic regulation. Most biosynthetic pathways are controlled by different environmental and genetic factors. Here, we summarized the effects of environmental factors, including abiotic and biotic stresses, on PSM biosynthesis in various plants. We also discuss the positive and negative transcription factors involved in various PSM biosynthetic pathways. The potential target genes of the stress-related transcription factors were also summarized. We further found that the downstream targets of these Transcription factors (TFs) are frequently enriched in the synthesis pathway of precursors, suggesting an effective role of precursors in enhancing of terminal products. The present review provides valuable insights regarding screening targets and regulators involved in PSM-mediated plant protection in non-model plants.

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The regulation of ZCT1, a transcriptional repressor of monoterpenoid indole alkaloid biosynthetic genes in Catharanthus roseus

Cited by 12 publications

References 83 publications

High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns

High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple introns

Repression of ZCT1, ZCT2 and ZCT3 affects expression of terpenoid indole alkaloid biosynthetic and regulatory genes

Environmental and Genetic Factors Involved in Plant Protection-Associated Secondary Metabolite Biosynthesis Pathways

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