In an important recent paper Kristensen et al. address a question of fundamental importance in plant biotechnology: how are metabolic pathways affected upon introduction of a transgene? Analysis of the transcriptome and metabolome of Arabidopsis thaliana engineered to produce the cyanogenic glucoside dhurrin demonstrated that plants can be tailored in a rational manner with marginal inadvertent effects.

The formation and storage of plant natural products such as phenylpropanoids, terpenoids and alkaloids are dynamic and complex processes that involve multiple subcellular compartments and cell types. Evidence is emerging to show that consecutive enzymes of phenylpropanoid and flavonoid biosynthesis are organized into macromolecular complexes that can be associated with endomembranes, that monoterpenoid biosynthetic enzymes are exclusively localized to highly specialized glandular trichome secretory cells and that complex monoterpenoid indole- and morphinan alkaloids require a combination of phloem parenchyma, laticifers and epidermal cells for their synthesis and storage. Highly ordered, protein-mediated processes that involve intra- and intercellular translocation need be considered when attempting to understand how a plant can regulate the formation and accumulation of complex but well-defined natural product profiles.

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In the present study morphinan, tetrahydrobenzylisoquinoline, benzo[c]phenanthridine, and phthalideisoquinoline alkaloids were determined qualitatively and quantitatively by HPLC and LC-MS analysis in tissues of the Tasmanian Papaver somniferum L. elite cultivar C048-6-14-64. The data were compared with the results from the low-morphine cultivar “Marianne”. In the elite cultivar, 91.2% of the latex alkaloids consist of the three pharmaceutically most valuable alkaloids: morphine, codeine, and thebaine. In the root system, the major alkaloids are sanguinarine/10-hydroxysanguinarine and dihydrosanguinarine/10-hydroxydihydrosanguinarine. In the stems and leaves of C048-6-14-64, the same alkaloids were measured as in the latex. In the stems, a gradient in relative total alkaloid content from the top downward toward the roots was observed. The concentration of morphine was decreasing toward the roots, whereas an increasing gradient from the upper to the lower stem parts was detected for codeine. The relative total alkaloid concentration in leaves remained constant; no gradient was observed. The cultivar “Marianne” displayed a shifted pattern of alkaloid accumulation and reduced levels of total alkaloid. In the condiment cultivar, 80.5% of the alkaloids of the latex consisted of the two phthalideisoquinoline alkaloids narcotoline and noscapine. Only 18.8% of the relative total alkaloid content were morphinan alkaloids. In contrast to the narcotic cultivar, in which the benzo[c]phenanthridines in roots dominated over the morphinan and tetrahydrobenzylisoquinoline alkaloids, the concentration of benzo[c]phenanthridines in “Marianne” was similar to that of morphinan and tetrahydrobenzylisoquinoline alkaloids. These data suggest a differential alkaloid regulation in each cultivar of P. somniferum.

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.

The process of catalytic dephosphorylation of geranylgeranyl diphosphate (GGPP) to give geranylgeraniol (GGOH) in Croton stellatopilosus leaves was examined by in vivo chloroplast feedings with [1-3H]GGPP and [1-3H]GGMP and in vitro enzyme-catalyzed reactions. The results strongly suggest that the formation of GGOH from GGPP proceeds in the chloroplasts via two successive monodephosphorylation reactions. Hence, we name the enzyme geranylgeranyl diphosphate phosphatase rather than geranylgeranyl diphosphatase based on its catalytic mechanism.

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.

Isoquinoline alkaloids are a large class of compounds derived from the amino acid L-tyrosine containing many physiologically active members. Among the isoquinoline alkaloids, morphine is one of the pharmaceutically important members that is still derived from the plant that produces it, the opium poppy Papaver somniferum. P. somniferum produces over 80 alkaloids derived from L-tyrosine. We have isolated cDNAs encoding several enzymes of tetrahydrobenzylisoquinoline-derived alkaloid biosynthesis from this plant. The first enzyme in the biosynthetic pathway for which we have isolated a cDNA is norcoclaurine 6-O-methyltransferase. The next is the cytochrome P-450-dependent monooxygenase (S)-N-methylcoclaurine 3’-hydroxylase. These enzymes are common to the morphine, noscapine and sanguinarine biosynthetic pathways. Specific to the sanguinarine pathway is the berberine bridge enzyme that oxidatively cyclizes the N-methyl moiety of (S)-reticuline to the bridge carbon C-8 of (S)-scoulerine. Finally, specific to morphine biosynthesis are salutaridinol 7-O-acetyltransferase and codeinone reductase the penultimate enzyme of the morphine pathway that reduces codeinone to codeine. Given the number of cDNAs specific to various alkaloid biosynthetic pathways that we now have, attempts at metabolic engineering of P. somniferum can be made. We describe herein details of the isolation and biochemical characterization of a cDNA encoding the P. somniferum O-methyltransferase OMTPS3.

Opium poppy (Papaver somniferum L.) produces a large variety of isoquinoline alkaloids. The aim of this investigation is to understand the regulation of biosynthesis and the ecological function of the alkaloids in the plant. Agrobacterium-mediated transformations of opium poppy were used to introduce the berberine bridge enzyme cDNA bbe 1 in the antisense orientation into seedling explants. After induction of callus on an appropriate medium, embryos were developed via somatic embryogenesis. After the embryos were developed into plantlets with leaves and roots they were transferred to soil. In this way, forty-nine phenotypically normal T0 plants were produced. Forty-six plants produced viable seeds and were used to produce T1 plants. These plants were then analyzed for the presence of the bbe 1 transgene and for the content of alkaloid in latex and root. Selected plants showed a differential alkaloid pattern in latex compared to the wild type. In this paper, the results of a plant with an altered alkaloid profile, heritable at least to the T2 generation, is presented. This represents the first example of metabolic engineering of the alkaloid pathways in opium poppy.