Skip to main content
SpringerLink
Log in

Genetically engineered hairy root cultures of Hyoscyamus senecionis and H. muticus: ploidy as a promising parameter in the metabolic engineering of tropane alkaloids

  • Original Article
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Key message

Tetraploidy improves overexpression of h6h and scopolamine production of H. muticus, while in H. senecionis, pmt overexpression and elicitation can be used as effective methods for increasing tropane alkaloids.

Abstract

The effects of metabolic engineering in a polyploid context were studied by overexpression of h6h in the tetraploid hairy root cultures of H. muticus. Flow cytometry analysis indicated genetic stability in the majority of the clones, while only a few clones showed genetic instability. Among all the diploid and tetraploid clones, the highest level of h6h transgene expression and scopolamine accumulation was interestingly observed in the tetraploid clones of H. muticus. Therefore, metabolic engineering of the tropane biosynthetic pathway in polyploids is suggested as a potential system for increasing the production of tropane alkaloids. Transgenic hairy root cultures of Hyoscyamus senecionis were also established. While overexpression of pmt in H. senecionis was correlated with a sharp increase in hyoscyamine production, the h6h-overexpressing clones were not able to accumulate higher levels of scopolamine than the leaves of intact plants. Applying methyl jasmonate was followed by a sharp increase in the expression of pmt and a drop in the expression of tropinone reductase II (trII) which consequently resulted in the higher biosynthesis of hyoscyamine and total alkaloids in H. senecionis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

BSTFA:

N,O-Bis (trimethylsilyl)trifluoroacetamide

GC:

Gas chromatography

GC–MS:

Gas chromatography–mass spectrometry

MeJa:

Methyl jasmonate

MS:

Murashige and Skoog

PMT:

Putrescine N-methyltransferase

TMCS:

Trimethylchlorosilane

TRI:

Tropinone reductase I

TRII:

Tropinone reductase II

TA:

Tropane alkaloids

References

  • Arroo R, Woolley J, Oksman-Caldentey K-M (2007) Henbane, Belladonna, Datura and Duboisia. In: Pua E, Davey M (eds) Biotechnology in agriculture and forestry, Transgenic Crops VI, vol 61. Springer, Berlin, pp 189–204

    Google Scholar 

  • Biondi S, Fornale S, Oksman-Caldentey KM, Eeva M, Agostani S, Bagni N (2000) Jasmonates induce over-accumulation of methylputrescine and conjugated polyamines in Hyoscyamus muticus L. root cultures. Plant Cell Rep 19:691–697

    Article  CAS  Google Scholar 

  • Cardillo AB, Otalvaro AAM, Busto VD, Talou JR, Velasquez LME, Giulietti AM (2010) Scopolamine, anisodamine and hyoscyamine production by Brugmansia candida hairy root cultures in bioreactors. Process Biochem 45:1577–1581

    Article  CAS  Google Scholar 

  • Dehghan E, Häkkinen S, Oksman-Caldentey K-M, Shahriari Ahmadi F (2012) Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.). Plant Cell Tissue Organ Cult 110:35–44

    Article  CAS  Google Scholar 

  • Dehghan E, Shahriari Ahmadi F, Ghotbi Ravandi E, Reed DW, Covello PS, Bahrami AR (2013) An atypical pattern of accumulation of scopolamine and other tropane alkaloids and expression of alkaloid pathway genes in Hyoscyamus senecionis. Plant Physiol Biochem 70:188–194

    Article  CAS  PubMed  Google Scholar 

  • Hashimoto T, Yun D, Yamada Y (1993) Production of tropane alkaloids in genetically engineered root cultures. Phytochemistry 32:713–718

    Article  CAS  Google Scholar 

  • Jouhikainen K, Lindgren L, Jokelainen T, Hiltunen R, Teeri TH, Oksman-Caldentey K-M (1999) Enhancement of scopolamine production in Hyoscyamus muticus L. hairy root cultures by genetic engineering. Planta 208:545–551

    Article  CAS  Google Scholar 

  • Kai G et al (2007) Molecular cloning and characterization of a new cDNA encoding hyoscyamine 6-beta-hydroxylase from roots of Anisodus acutangulus. J Biochem Mol Biol 40:715–722

    CAS  PubMed  Google Scholar 

  • Kai G, Yang S, Zhang Y, Luo X, Fu X, Zhang A, Xiao J (2012) Effects of different elicitors on yield of tropane alkaloids in hairy roots of Anisodus acutangulus. Mol Biol Rep 39:1721–1729

    Article  CAS  PubMed  Google Scholar 

  • Kang SM, Jung HY, Kang YM, Yun DJ, Bahk JD, Yang JK, Choi MS (2004) Effects of methyl jasmonate and salicylic acid on the production of tropane alkaloids and the expression of PMT and H6H in adventitious root cultures of Scopolia parviflora. Plant Sci 166:745–751

    Article  CAS  Google Scholar 

  • Khatamsaz M (1998) Flora of Iran, vol 24. Research Institute of Forests and Rangelands, Iran

    Google Scholar 

  • Kushwaha AK, Sangwan NS, Trivedi PK, Negi AS, Misra L, Sangwan RS (2013) Tropine forming tropinone reductase gene from Withania somnifera (Ashwagandha): biochemical characteristics of the recombinant enzyme and novel physiological overtones of tissue-wide gene expression patterns. PLoS One 8:e74777

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li R, Reed DW, Liu EW, Nowak J, Pelcher LE, Page JE, Covello PS (2006) Functional genomic analysis of alkaloid biosynthesis in Hyoscyamus niger reveals a cytochrome P450 involved in littorine rearrangement. Chem Biol 13:513–520

    Article  CAS  PubMed  Google Scholar 

  • Mateus L, Cherkaoui S, Christen P, Oksman-Caldentey KM (2000) Simultaneous determination of scopolamine, hyoscyamine and littorine in plants and different hairy root clones of Hyoscyamus muticus by micellar electrokinetic chromatography. Phytochemistry 54:517–523

    Article  CAS  PubMed  Google Scholar 

  • Moyano E, Fornalé S, Palazón J, Cusidó RM, Bagni N, Piñol MT (2002) Alkaloid production in Duboisia hybrid hairy root cultures overexpressing the pmt gene. Phytochemistry 59:697–702

    Article  CAS  PubMed  Google Scholar 

  • Moyano E, Jouhikainen K, Tammela P, Palazon J, Cusido RM, Piñol MT, Teeri TH, Oksman-Caldentey K-M (2003) Effect of pmt gene overexpression on tropane alkaloid production in transformed root cultures of Datura metel and Hyoscyamus muticus. J Exp Bot 54:203–211

    Article  CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Ochatt S, Jacas L, Patat-Ochatt EM, Djenanne S (2013) Flow cytometric analysis and molecular characterization of Agrobacterium tumefaciens-mediated transformants of Medicago truncatula. Plant Cell Tissue Organ Cult 113:237–244

    Article  CAS  Google Scholar 

  • Oksman-Caldentey K-M, Arroo R (2000) Regulation of tropane alkaloid metabolism in plants and plant cell cultures. In: Verpoorte R, Alfermann A (eds) Metabolic engineering of plant secondary metabolism. Kluwer, Dortrecht, pp 253–281

    Chapter  Google Scholar 

  • Oksman-Caldentey K-M, Vuorela H, Isenegger M, Strauss A, Hiltunen R (1987) Selection for high tropane alkaloid content in Hyoscyamus muticus plants. Plant Breed 99:318–326

    Article  CAS  Google Scholar 

  • Palazón J, Moyano E, Cusidó RM, Bonfill M, Oksman-Caldentey KM, Piñol MT (2003) Alkaloid production in Duboisia hybrid hairy roots and plants overexpressing the h6h gene. Plant Sci 165:1289–1295

    Article  Google Scholar 

  • Portsteffen A, Dräger B, Nahrstedt A (1994) The reduction of tropinone in Datura stramonium root cultures by two specific reductases. Phytochemistry 37:391–400

    Article  CAS  PubMed  Google Scholar 

  • Rischer H, Häkkinen ST, Ritala A, Seppänen-Laakso T, Miralpeix B, Capell T, Christou P, Oksman- Caldentey K-M (2013) Plant cells as pharmaceutical factories. Curr Pharm Design 19:5640–5660

    Article  CAS  Google Scholar 

  • Saghai-Maroof MA, Soliman K, Gorgensen RA, Allard RW (1984) DNA spacer length polymorphism in Barley: Mendelian inheritance, chromosomal location and population genetics. Proc Natl Acad Sci USA 81:8014–8018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sato F, Hashimoto T, Haciya A, Tamura K, Choi K-B, Morishige T, Fujimoto H, Yamada Y (2001) Metabolic engineering of plant alkaloid biosynthesis. Proc Natl Acad Sci USA 98:367–372

    Article  CAS  PubMed  Google Scholar 

  • Sevon N, Oksman-Caldentey K-M (2002) Agrobacterium rhizogenes mediated transformation: root cultures as a source of alkaloids. Planta Med 68(10):859–868

    Article  CAS  PubMed  Google Scholar 

  • Wang X, Chen M, Yang C, Liu X, Zhang L, Lan X, Tang K, Liao Z (2011) Enhancing the scopolamine production in transgenic plants of Atropa belladonna by overexpressing pmt and h6h genes. Physiol Plant 143:309–315

    Article  CAS  PubMed  Google Scholar 

  • Yamada Y, Hashimoto T, Endo T (1990) Biochemistry of alkaloid production in vitro. In: Charlwood B, Rhodes MJC (eds) Secondary products from plant tissue culture. Clarendon Press, Oxford, pp 201–225

    Google Scholar 

  • Zárate R, Jaber-Vazdekis N, Medina B, Ravelo Á (2006) Tailoring tropane alkaloid accumulation in transgenic hairy roots of Atropa baetica by over-expressing the gene encoding hyoscyamine 6β-hydroxylase. Biotechnol Lett 28:1271–1277

    Article  PubMed  Google Scholar 

  • Zhang L et al (2004) Engineering tropane biosynthetic pathway in Hyoscyamus niger hairy root cultures. Proc Natl Acad Sci USA 101:6786–6791

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang L, Yang B, Lu B, Kai G, Wang Z, Xia Y, Ding R, Zhang H, Sun X, Chen W, Tang K (2007) Tropane alkaloids production in transgenic Hyoscyamus niger hairy root cultures over-expressing putrescine N-methyltransferase is methyl jasmonate-dependent. Planta 225:887–896

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This research was financially supported by the Deputy of Research Affairs, Ferdowsi University of Mashhad, Iran.

Author information

Author notes

  1. Esmaeil Dehghan

    Present address: Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Room Y4.306D, Dallas, TX, 75390, USA

Authors and Affiliations

  1. Department of Plant Biotechnology, Faculty of Agriculture, Ferdowsi University of Mashhad, P.O. Box 9177948978, Mashhad, Iran

    Esmaeil Dehghan, Zeinab Hasanpour & Farajollah Shahriari Ahmadi

  2. Department of Biochemistry, UT Southwestern Medical Center, Dallas, TX, 75390, USA

    Esmaeil Dehghan

  3. National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK, S7N 0W9, Canada

    Darwin W. Reed & Patrick S. Covello

  4. Unitat de Fisiologia Vegetal, Facultat de Farmacia, Universitat de Barcelona, 08028, Barcelona, Spain

    Javier Palazon

  5. VTT Technical Research Centre of Finland Ltd., P.O. Box 1000, 02044 VTT, Espoo, Finland

    Kirsi-Marja Oksman-Caldentey

Authors
  1. Esmaeil Dehghan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Darwin W. Reed
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Patrick S. Covello
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zeinab Hasanpour
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Javier Palazon
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kirsi-Marja Oksman-Caldentey
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Farajollah Shahriari Ahmadi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Esmaeil Dehghan.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Communicated by Maike Petersen.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 2016 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dehghan, E., Reed, D.W., Covello, P.S. et al. Genetically engineered hairy root cultures of Hyoscyamus senecionis and H. muticus: ploidy as a promising parameter in the metabolic engineering of tropane alkaloids. Plant Cell Rep 36, 1615–1626 (2017). https://doi.org/10.1007/s00299-017-2178-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-017-2178-0

Keywords

Search

Navigation