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Comparative macroarray analysis of morphine containing Papaver somniferum and eight morphine free Papaver species identifies an O-methyltransferase involved in benzylisoquinoline biosynthesis

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Abstract

Benzylisoquinoline alkaloids constitute a group of about 2,500 structures and are mainly produced by plants of the order Ranunculales. But only the opium poppy, Papaver somniferum, and Papaver setigerum are able to produce morphine. In this study, we started to investigate by gene expression analysis the molecular basis for this exceptional biosynthetic ability. A sequencing project from P. somniferum seedlings was initiated using a method based on the amplified fragment length polymorphism technique that resulted in 849 UniGenes. These cDNAs were analysed on macroarrays for differential expression between morphine-containing P. somniferum plants and eight other Papaver species, which accumulate other benzylisoquinolines instead of morphine. Three cDNAs showing increased expression in P. somniferum compared to all the other Papaver species were identified. Whereas two showed no significant homology to any known protein, one putatively encoded an O-methyltransferase. Analysis of substrate specificity of the heterologously expressed protein and mass spectrometric identification of the enzymatic products identified this protein as S-adenosyl-L-methionine:(R,S)-3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (EC 2.1.1.116). Unlike other O-methyltransferases of different positional specificities implicated in benzylisoquinoline metabolism, the enzyme only accepted tetrahydroxylated tetrahydrobenzylisoquinolines as substrates; methylation was tolerated only at the 6-hydroxy position.

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Abbreviations

4′-OMT:

(R,S)-3′-Hydroxy-N-methylcoclaurine 4′-O-methyltransferase

6-OMT:

Norcoclaurine 6-O-methyltransferase

7-OMT:

Reticuline 7-O-methyltransferase

AdoMet:

S-adenosyl-L-methionine

AFLP:

Amplified fragment length polymorphism

BBE:

Berberine bridge enzyme

COR:

Codeinone reductase

Cyp80b1:

(S)-N-Methylcoclaurine 3′-hydroxylase

EST:

Expressed sequence tag

ESI-MS:

Electrospray ionization-mass spectrometry

RACE:

Rapid amplification of cDNA ends

SalAT:

Salutaridinol 7-O-acetyltransferase

TOF:

Time of flight

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Acknowledgements

We thank Silvia Wegener for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, the Deutschen Akademischen Austausch Dienst, Bonn and by the Fonds der Chemischen Industrie, Frankfurt. María Luisa Diaz-Chávez was supported by CONACYT, Mexico.

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  1. Leibniz-Institut für Pflanzenbiochemie, Weinberg 3, 06120, Halle/Saale, Germany

    Jörg Ziegler, María Luisa Diaz-Chávez, Robert Kramell, Christian Ammer & Toni M. Kutchan

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  1. Jörg Ziegler
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  2. María Luisa Diaz-Chávez
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  3. Robert Kramell
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  4. Christian Ammer
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  5. Toni M. Kutchan
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Correspondence to Jörg Ziegler.

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Ziegler, J., Diaz-Chávez, M.L., Kramell, R. et al. Comparative macroarray analysis of morphine containing Papaver somniferum and eight morphine free Papaver species identifies an O-methyltransferase involved in benzylisoquinoline biosynthesis. Planta 222, 458–471 (2005). https://doi.org/10.1007/s00425-005-1550-4

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