Abstract
Recent advances in DNA sequencing technology and analytical mass spectrometry are providing unprecedented opportunities to develop the functional genomics resources required to investigate complex biological processes in non-model plants. Opium poppy produces a wide variety of benzylisoquinoline alkaloids (BIAs), including the pharmaceutical compounds codeine, morphine, noscapine and papaverine. A functional genomics platform to identify novel BIA biosynthetic and regulatory genes in opium poppy has been established based on the differential metabolite profile of eight selected cultivars. Stem cDNA libraries from each of the eight opium poppy cultivars were subjected to 454 pyrosequencing and searchable expressed sequence tag databases were created from the assembled reads. These deep and integrated metabolite and transcript databases provide a nearly complete representation of the genetic and metabolic variances responsible for the differential occurrence of specific BIAs in each cultivar as demonstrated using the biochemically well characterized pathway from tyrosine to morphine. Similar correlations between the occurrence of specific transcripts and alkaloids effectively reveals candidate genes encoding uncharacterized biosynthetic enzymes as shown using cytochromes P450 potentially involved in the formation of papaverine and noscapine.
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Abbreviations
- BIA:
-
Benzylisoquinoline alkaloid
- CID:
-
Collision-induced dissociation
- CYP:
-
Cytochrome P450
- ESI:
-
Electrospray ionization
- EST:
-
Expressed sequence tag
- HPLC:
-
High-pressure liquid chromatography
- LC–MS/MS:
-
Liquid chromatography–tandem mass spectrometry
- MRM:
-
Multiple reaction monitoring
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Acknowledgments
PJF holds the Canada Research Chair in Plant Metabolic Processes Biotechnology. This work was sponsored by a Strategic Project Grant and a Discovery and Research Tools and Infrastructure Grant from the Natural Science and Engineering Research Council of Canada to PJF.
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11103_2012_9913_MOESM1_ESM.pdf
Chromatographic and spectral data used for identification and quantification of benzylisoquinoline alkaloids by LC–MS/MS analysis. CID spectra shown in red were taken from the references provided. All other CID spectra were determined from authentic standards (PDF 105 kb)
11103_2012_9913_MOESM2_ESM.pdf
Selected unknown compounds detected in latex extracts of eight opium poppy cultivars determined by LC–MS/MS analysis (PDF 79 kb)
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Desgagné-Penix, I., Farrow, S.C., Cram, D. et al. Integration of deep transcript and targeted metabolite profiles for eight cultivars of opium poppy. Plant Mol Biol 79, 295–313 (2012). https://doi.org/10.1007/s11103-012-9913-2
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DOI: https://doi.org/10.1007/s11103-012-9913-2