Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy

Nature Biotechnology volume 22, pages 1559–1566 (2004)Cite this article

Abstract

We report on the silencing of codeinone reductase (COR) in the opium poppy, Papaver somniferum, using a chimeric hairpin RNA construct designed to silence all members of the multigene COR family through RNA interference (RNAi). After gene silencing, the precursor alkaloid (S)-reticuline—seven enzymatic steps upstream of codeinone—accumulated in transgenic plants at the expense of morphine, codeine, oripavine and thebaine. Methylated derivatives of reticuline also accumulated. Analysis verified loss of Cor gene transcript, appearance of 22-mer degradation products and reduction of enzyme activity. The surprising accumulation of (S)-reticuline suggests a feedback mechanism preventing intermediates from general benzylisoquinoline synthesis entering the morphine-specific branch. However transcript levels for seven other enzymes in the pathway, both before and after (S)-reticuline, were unaffected. This is the first report of gene silencing in transgenic opium poppy and of metabolic engineering to cause the high-yield accumulation of the nonnarcotic alkaloid reticuline.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: A summary of the biosynthetic pathway leading to morphine, showing also the methylated derivatives of reticuline; enzymes indicated are those for which cDNAs have been previously cloned and that are investigated in this study.
Figure 2: The T-DNA from the transformation vector COR 1.1/2 hpRNA designed to produce hpRNA and initiate silencing of all members of the Cor family in P. somniferum.
Figure 3: Thin layer chromatography of poppy latex alkaloids.
Figure 4: HPLC chromatograms.
Figure 5: HPLC quantification of the alkaloid contents of 18 T0 hpRNA transgenics and a set of nine independent, nontransgenic control CO58-34 plants (cntls 1–9).
Figure 6: Analysis of T1 family segregants derived from hpCOR parent 220-2-2.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Meijerink, W.J.H.J., Molina, P.E. & Abumrad, N.N. Mammalian opiate alkaloid synthesis: lessons derived from plant biochemistry. Shock 12, 165–173 (1999).

    Article  CAS  PubMed  Google Scholar 

  2. Schiff, P.L.J. Opium and its alkaloids. Amer. J. Pharm. Educ. 66, 186–194 (2002).

    Google Scholar 

  3. Caporale, L.H. Chemical Ecology—a view from the pharmaceutical industry. Proc. Natl. Acad. Sci. USA 92, 75–82 (1995).

    Article  CAS  PubMed  Google Scholar 

  4. Nielsen, B., Roe, J. & Brochmann-Hanssen, E. Oripavine—a new opium alkaloid. Planta Med. 48, 205–206 (1983).

    Article  CAS  PubMed  Google Scholar 

  5. Tetenyi, P. Opium poppy (Papaver somniferum): botany and horticulture. Hortic. Rev. 19, 373–408 (1997).

    Google Scholar 

  6. Gerardy, R. & Zenk, M.H. Formation of salutaridine from (R)-reticuline by a membrane-bound cytochrome-P-450 enzyme from Papaver somniferum. Phytochemistry 32, 79–86 (1993).

    Article  Google Scholar 

  7. Laughlin, J.C., Chung, B. & Beattie, B.M. in Poppy, The Genus Papaver (ed. Bernath, J.) 249–277 (Hardwood Academic Publishers, The Netherlands, 1998).

    Google Scholar 

  8. Dewick, P.M. in Medicinal Plant Products (ed. Dewick, P.M.) 291–400 (John Wiley & Sons, New York, 2002).

    Google Scholar 

  9. Millgate, A.G. et al. Morphine-pathway block in top1 poppies. Nature 431, 413–414 (2004).

    Article  CAS  PubMed  Google Scholar 

  10. Camacho, M.D. et al. In vitro activity of Triclisia patens and some bisbenzylisoquinoline alkaloids against Leishmania donovani and Trypanosoma brucei brucei. Phytother. Res. 16, 432–436 (2002).

    Article  CAS  Google Scholar 

  11. Rasoanaivo, P. et al. Alkaloids of Hernandia voyronii: chloroquine-potentiating activity and structure elucidation of herveline D. Planta Med. 64, 58–62 (1998).

    Article  CAS  PubMed  Google Scholar 

  12. Tshibangu, J.N., Wright, A.D. & Konig, G.M. HPLC Isolation of the anti-plasmodially active bisbenzylisoquinone alkaloids present in roots of Cissampelos mucronata. Phytochem. Analysis 14, 13–22 (2003).

    Article  CAS  Google Scholar 

  13. Angerhofer, C.K., Guinaudeau, H., Wongpanich, V., Pezzuto, J.M. & Cordell, G.A. Antiplasmodial and cytotoxic activity of natural bisbenzylisoquinoline alkaloids. J. Nat. Prod. 62, 59–66 (1999).

    Article  CAS  PubMed  Google Scholar 

  14. Chen, Q., Peng, W.L., Qi, S.J. & Xu, A.L. Apoptosis of human highly metastatic lung cancer cell line 95-D induced by acutiaporberine, a novel bisalkaloid derived from Thalictrum acutifolium. Planta Med. 68, 550–553 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. Seifert, F., Todorov, D.K., Hutter, K.J. & Zeller, W.J. Cell cycle effects of thaliblastine. J. Cancer Res. Clin. Oncology 122, 707–710 (1996).

    Article  CAS  Google Scholar 

  16. Nakaoji, K., Nayeshiro, H. & Tanahashi, T. Norreticuline and reticuline as possible new agents for hair growth acceleration. Biol. Pharm. Bull. 20, 586–588 (1997).

    Article  CAS  PubMed  Google Scholar 

  17. Brochmann-Hanssen, E. A second pathway for the terminal steps in the biosynthesis of morphine. Planta Med. 50, 343–345 (1984).

    Article  CAS  PubMed  Google Scholar 

  18. Kutchan, T.M. in The Alkaloids vol. 50 (ed. Cordell, G.) 253–316, (Academic Press, San Diego, 1998).

    Google Scholar 

  19. Facchini, P.J. & Park, S.U. Developmental and inducible accumulation of gene transcripts involved in alkaloid biosynthesis in opium poppy. Phytochemistry 64, 177–186 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Ounaroon, A., Decker, G., Schmidt, J., Lottspeich, F. & Kutchan, T.M. (R,S)-Reticuline 7-O-methyltransferase and (R,S)-norcoclaurine 6-O-methyltransferase of Papaver somniferum—cDNA cloning and characterization of methyl transfer enzymes of alkaloid biosynthesis in opium poppy. Plant J. 36, 808–819 (2003).

    Article  CAS  PubMed  Google Scholar 

  21. Unterlinner, B., Lenz, R. & Kutchan, T.M. Molecular cloning and functional expression of codeinone reductase: the penultimate enzyme in morphine biosynthesis in the opium poppy Papaver somniferum. Plant J. 18, 465–475 (1999).

    Article  CAS  PubMed  Google Scholar 

  22. Grothe, T., Lenz, R. & Kutchan, T.M. Molecular characterization of the salutaridinol 7-O-acetyltransferase involved in morphine biosynthesis in opium poppy Papaver somniferum. J. Biol. Chem. 276, 30717–30723 (2001).

    Article  CAS  PubMed  Google Scholar 

  23. Bird, D.A., Franceschi, V.R. & Facchini, P.J. A tale of three cell types: alkaloid biosynthesis is localized to sieve elements in opium poppy. Plant Cell 15, 2626–2635 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Weid, M., Ziegler, J. & Kutchan, T.M. The roles of latex and the vascular bundle in morphine biosynthesis in the opium poppy. Papaver somniferum. Proc. Natl. Acad. Sci. USA 101, 13957–13962 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Wesley, S.V. et al. Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J. 27, 581–590 (2001).

    Article  CAS  PubMed  Google Scholar 

  26. Wang, M.B. & Waterhouse, P.M. Application of gene silencing in plants. Curr. Opin. Plant Biol. 5, 146–150 (2002).

    Article  CAS  PubMed  Google Scholar 

  27. Helliwell, C.A., Wesley, S.V., Wielopolska, A.J. & Waterhouse, P.M. High-throughput vectors for efficient gene silencing in plants. Funct. Plant Biol. 29, 1217–1225 (2002).

    Article  CAS  Google Scholar 

  28. Dittrich, H. & Kutchan, T.M. Molecular cloning, expression, and induction of berberine bridge enzyme, an enzyme essential to the formation of benzophenanthridine alkaloids in the response of plants to pathogenic attack. Proc. Natl. Acad. Sci. USA 88, 9969–9973 (1991).

    Article  CAS  PubMed  Google Scholar 

  29. Bird, D.A. & Facchini, P.J. Berberine bridge enzyme, a key branch-point enzyme in benzylisoquinoline alkaloid biosynthesis, contains a vacuolar sorting determinant. Planta 213, 888–897 (2001).

    Article  CAS  PubMed  Google Scholar 

  30. Pauli, H.H. & Kutchan, T.M. Molecular cloning and functional heterologous expression of two alleles encoding (S)-N-methylcoclaurine 3′-hydroxylase (CYP80B1), a new methyl jasmonate-inducible cytochrome P-450-dependent mono-oxygenase of benzylisoquinoline alkaloid biosynthesis. Plant J. 13, 793–801 (1998).

    Article  CAS  PubMed  Google Scholar 

  31. Park, S.U., Yu, M. & Facchini, P.J. Antisense RNA-mediated suppression of benzophenanthridine alkaloid biosynthesis in transgenic cell cultures of California poppy. Plant Physiol. 128, 696–706 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Park, S.U., Yu, M. & Facchini, P.J. Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids. Plant Mol. Biol. 51, 153–164 (2003).

    Article  CAS  PubMed  Google Scholar 

  33. Bock, A., Wanner, G. & Zenk, M.H. Immunocytological localization of two enzymes involved in berberine biosynthesis. Planta 216, 57–63 (2002).

    Article  CAS  PubMed  Google Scholar 

  34. Deus-Neumann, B. & Zenk, M.H. Accumulation of alkaloids in plant vacuoles does not involve an ion-trap mechanism. Planta 167, 44–53 (1986).

    Article  CAS  PubMed  Google Scholar 

  35. Burbulis, I.E. & Winkel-Shirley, B. Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway. Proc. Natl. Acad. Sci. USA 96, 12929–12934 (1999).

    Article  CAS  PubMed  Google Scholar 

  36. Panicot, M. et al. A polyamine metabolon involving aminopropyl transferase complexes in Arabidopsis. Plant Cell 14, 2539–2551 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. He, X.Z. & Dixon, R.A. Genetic manipulation of isoflavone 7-O-methyltransferase enhances biosynthesis of 4′-O-methylated isoflavonoid phytoalexins and disease resistance in alfalfa. Plant Cell 12, 1689–1702 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Facchini, P.J., Bird, D.A., MacLeod, B.P., Park, S.-U. & Samanani, N. in Integrative Phytochemistry: From Ethnobotany to Molecular Ecology (ed. Romeo, J.T.) 143–180, (Pergamon, Amsterdam, 2003).

    Book  Google Scholar 

  39. Schunmann, P.H.D. et al. A suite of novel promoters and terminators for plant biotechnology. Funct. Plant Biol. 30, 443–452 (2003).

    Article  CAS  Google Scholar 

  40. Schunmann, P.H.D., Surin, B. & Waterhouse, P.M. A suite of novel promoters and terminators for plant biotechnology—II. The pPLEX series for use in monocots. Funct. Plant Biol. 30, 453–460 (2003).

    Article  CAS  Google Scholar 

  41. Lazo, G.R., Stein, P.A. & Ludwig, R.A. A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology 9, 963–967 (1991).

    Article  CAS  PubMed  Google Scholar 

  42. Chitty, J.A., Allen, R.S., Fist, A.J. & Larkin, P.J. Genetic transformation in commercial Tasmanian cultivars of opium poppy, Papaver somniferum, and movement of transgenic pollen in the field. Funct. Plant Biol. 30, 1045–1058 (2003).

    Article  CAS  Google Scholar 

  43. Lenz, R. & Zenk, M.H. Purification and properties of codeinone reductase (NADPH) from Papaver somniferum cell cultures and differentiated plants. Eur. J. Biochem. 233, 132–139 (1995).

    Article  CAS  PubMed  Google Scholar 

  44. Hirata, K., Poeaknapo, C., Schmidt, J. & Zenk, M.H. 1,2-dehydroreticuline synthase, the branch point enzyme opening the morphinan biosynthetic pathway. Phytochemistry 65, 1039–1046 (2004).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank Neil Smith and Peter Waterhouse for helpful discussions and 22-mer detection; Pat Hallam, Peta Dolan and Noel Davies for HPLC and LC/MS work; Stephen Pyne and Alison Ung for chiral determinations; Toni Kutchan, Meinhart Zenk and Susanne Frick for helpful discussions.

Author information

Authors and Affiliations

  1. CSIRO Plant Industry, PO Box 1600, Canberra, 2601, Australian Capital Territory, Australia

    Robert S Allen, Anthony G Millgate, Julie A Chitty, Jennifer Thisleton & Philip J Larkin

  2. Tasmanian Alkaloids Pty Ltd, PO Box 130, Westbury, 7303, Tasmania, Australia

    James A C Miller & Anthony J Fist

  3. Johnson & Johnson Research Pty Ltd, Locked Bag 4555, Strawberry Hills, 2012, New South Wales, Australia

    Wayne L Gerlach

Authors
  1. Robert S Allen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anthony G Millgate
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Julie A Chitty
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jennifer Thisleton
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. James A C Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anthony J Fist
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Wayne L Gerlach
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Philip J Larkin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Philip J Larkin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

RT-PCR analysis of eight T1 family segregants from hpCOR parent 220-2-2; these are the same individuals as shown in Figure 6. (PDF 341 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Allen, R., Millgate, A., Chitty, J. et al. RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy. Nat Biotechnol 22, 1559–1566 (2004). https://doi.org/10.1038/nbt1033

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1033

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing