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Tropane alkaloids production in transgenic Hyoscyamus niger hairy root cultures over-expressing Putrescine N-methyltransferase is methyl jasmonate-dependent

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Abstract

The cDNA from Nicotiana tabacum encoding Putrescine N-methyltransferase (PMT), which catalyzes the first committed step in the biosynthesis of tropane alkaloids, has been introduced into the genome of a scopolamine-producing Hyoscyamus niger mediated by the disarmed Agrobacterium tumefaciens strain C58C1, which also carries Agrobacterium rhizogenes Ri plasmid pRiA4, and expressed under the control of the CaMV 35S promoter. Hairy root lines transformed with pmt presented fivefold higher PMT activity than the control, and the methylputrescine (MPUT) levels of the resulting engineered hairy roots increased four to fivefold compared to the control and wild-type roots, but there was no significant increase in tropane alkaloids. However, after methyl jasmonate (MeJA) treatment, a considerable increase of PMTase and endogenous H6Hase as well as an increase in scopolamine content was found either in the transgenic hairy roots or the control. The results indicate that hairy root lines over-expressing pmt have a high capacity to synthesize MPUT, whereas their ability to convert hyoscyamine into scopolamine is very limited. Exposure to MeJA strongly stimulated both polyamine and tropane biosynthesis pathways and elicitation led to more or less enhanced production simultaneously.

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Abbreviations

H6H:

Hyoscyamine-6β-hydroxylase (enzyme)

h6h :

Hyoscyamine-6β-hydroxylase (cDNA)

JA:

Jasmonic acid

MeJA:

Methyl jasmonate

MPUT:

N-methylputrescine

PCR:

Polymerase chain reaction

PMT:

Putrescine N-methyltransferase (enzyme)

pmt :

Putrescine N-methyltransferase (cDNA)

PUT:

Putrescine

RT-PCR:

Reverse transcription PCR

SD:

Spermidine

SM:

Spermine

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Acknowledgments

The authors are very grateful to Professor T. Hashimoto of the Nara Institute of Science and Technology (Kyoto, Japan) for supplying the pTVPMT plasmid carrying the tobacco pmt cDNA and to Professor M.T. Piñol of Sección de Fisiología Vegetal, Facultad de Farmacia, Universidad de Barcelona (Barcelona, Spain) for his generous gift of the plant expression vector pBMI. This research was supported by National Natural Science Foundation of China (30371746, 20572130), Shanghai Natural Science Foundation (04ZR14051, 05ZR14093), Shanghai Science and Technology Committee and Shanghai Rising-Star Program (06QB14010).

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Authors and Affiliations

  1. State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Fudan University, Shanghai, 200433, People’s Republic of China

    Lei Zhang, Zinan Wang, Yang Xia, Xiaofen Sun & Kexuan Tang

  2. Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai, 200433, People’s Republic of China

    Lei Zhang, Bin Yang, Beibei Lu, Ruxian Ding, Hanming Zhang & Wansheng Chen

  3. Laboratory of Plant Bioengineering, College of Life and Environment, Shanghai Normal University, Shanghai, 200234, People’s Republic of China

    Guoyin Kai

  4. Department of Pharmacy, Changzheng Hospital, Second Military Medical University, Shanghai, 200003, People’s Republic of China

    Wansheng Chen

  5. Plant Biotechnology Research Center, School of Agriculture and Biology, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Institute of Systems Biology, Shanghai Jiao Tong University, Shanghai, 200030, People’s Republic of China

    Kexuan Tang

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  1. Lei Zhang
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Correspondence to Wansheng Chen or Kexuan Tang.

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Zhang, L., Yang, B., Lu, B. et al. Tropane alkaloids production in transgenic Hyoscyamus niger hairy root cultures over-expressing Putrescine N-methyltransferase is methyl jasmonate-dependent. Planta 225, 887–896 (2007). https://doi.org/10.1007/s00425-006-0402-1

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