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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.
Bücher und Buchkapitel
Papaver species are known to produce a large variety of benzylisoquinoline alkaloids, with each species exhibiting a specific alkaloid profile, but only a few genes involved in the biosynthesis or regulation of these complex pathways are known so far. Here we used a genomic approach to discover genes responsible for the determination of a specific alkaloid profile. A stem expressed sequence tag database of ~1100 unique genes from Papaver somniferum was created. Gene expression analysis of these sequences in P. bracteatum, P. somniferum and P. somniferum ‘Noscapine’ exhibited 39 cDNAs showing differential expression coincident with morphine accumulation.