Developmental regulation of benzylisoquinoline alkaloid biosynthesis in opium poppy plants and tissue cultures

Summary Opium poppy (Papaver somniferum L.) contains a number of pharmaceutically important alkaloids of the benzylisoquinoline type including morphine, codeine, papaverine, and sanguinarine. Although these alkaloids accumulate to high concentrations in various organs of the intact plant, only the phytoalexin sanguinarine has been found at significant levels in opium poppy cell cultures. Moreover, even sanguinarine biosynthesis is not constitutive in poppy cell suspension cultures, but is typically induced only after treatment with a funga-derived elicitor. The absence of appreciable quantities of alkaloids in dedifferentiated opium poppy cell cultures suggests that benzylisoquinoline alkaloid biosynthesis is developmentally regulated and requires the differentiation of specific tissues. In the 40 yr since opium poppy tissues were first culturedin vitro, a number of reports on the redifferentiation of roots and buds from callus have appeared. A requirement for the presence of specialized laticifer cells has been suggested before certain alkaloids, such as morphine and codeine, can accumulate. Laticifers represent a complex internal secretory system in about 15 plant families and appear to have multiple evolutionary origins. Opium poppy laticifers differentiate from procambial cells and undergo articulation and anastomosis to form a continuous network of elements associated with the phloem throughout much of the intact plant. Latex is the combined cytoplasm of fused laticifer vessels, and contains numerous large alkaloid vesicles in which latex-associated poppy alkaloids are sequestered. The formation of alkaloid vesicles, the subcellular compartmentation of alkaloid biosynthesis, and the tissue-specific localization and control of these processes are important unresolved problems in plant cell biology. Alkaloid biosynthesis in opium poppy is an excellent model system to investigate the developmental regulation and cell biology of complex metabolic pathways, and the relationship between metabolic regulation and cell-type specific differentiation. In this review, we summarize the literature on the roles of cellular differentiation and plant development in alkaloid biosynthesis in opium poppy plants and tissue cultures.     

Opium poppy and Madagascar periwinkle: model non-model systems to investigate alkaloid biosynthesis in plants

Alkaloids represent a large and diverse group of compounds that are related by the occurrence of a nitrogen atom within a heterocyclic backbone. Unlike other types of secondary metabolites, the various structural categories of alkaloids are unrelated in terms of biosynthesis and evolution. Although the biology of each group is unique, common patterns have become apparent. Opium poppy ( Papaver somniferum ), which produces several benzylisoquinoline alkaloids, and Madagascar periwinkle ( Catharanthus roseus ), which accumulates an array of monoterpenoid indole alkaloids, have emerged as the premier organisms used to study plant alkaloid metabolism. The status of these species as model systems results from decades of research on the chemistry, enzymology and molecular biology responsible for the biosynthesis of valuable pharmaceutical alkaloids. Opium poppy remains the only commercial source for morphine, codeine and semi-synthetic analgesics, such as oxycodone, derived from thebaine. Catharanthus roseus is the only source for the anti-cancer drugs vinblastine and vincristine. Impressive collections of cDNAs encoding biosynthetic enzymes and regulatory proteins involved in the formation of benzylisoquinoline and monoterpenoid indole alkaloids are now available, and the rate of gene discovery has accelerated with the application of genomics. Such tools have allowed the establishment of models that describe the complex cell biology of alkaloid metabolism in these important medicinal plants. A suite of biotechnological resources, including genetic transformation protocols, has allowed the application of metabolic engineering to modify the alkaloid content of these and related species. An overview of recent progress on benzylisoquinoline and monoterpenoid indole alkaloid biosynthesis in opium poppy and C. roseus is presented.     

Quantitative <Superscript>1</Superscript>H NMR metabolomics reveals extensive metabolic reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures

Background  

Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a model system to study plant alkaloid metabolism. The plant is cultivated as the only commercial source of the narcotic analgesics morphine and codeine, but also produces many other alkaloids including the antimicrobial agent sanguinarine. Modulations in plant secondary metabolism as a result of environmental perturbations are often associated with the altered regulation of other metabolic pathways. As a key component of our functional genomics platform for opium poppy we have used proton nuclear magnetic resonance (¹H NMR) metabolomics to investigate the interplay between primary and secondary metabolism in cultured opium poppy cells treated with a fungal elicitor.     

The biosynthesis of papaverine proceeds via (S)-reticuline

Papaverine is one of the earliest opium alkaloids for which a biosynthetic hypothesis was developed on theoretical grounds. Norlaudanosoline (=tetrahydropapaveroline) was claimed as the immediate precursor alkaloid for a multitude of nitrogen containing plant metabolites. This tetrahydroxylated compound was proposed to be fully O-methylated. The resulting tetrahydropapaverine should then aromatize to papaverine. In view of experimental data, this pathway has to be revised. Precursor administration to 8-day-old seedlings of Papaver followed by direct examination of the metabolic fate of the stable-isotope-labeled precursors in the total plant extract, without further purification of the metabolites, led to elucidation of the papaverine pathway in vivo. The central and earliest benzylisoquinoline alkaloid is not the tetraoxygenated norlaudanosoline, but instead the trihydroxylated norcoclaurine that is further converted into (S)-reticuline, the established precursor for poppy alkaloids. The papaverine pathway is opened by the methylation of (S)-reticuline to generate (S)-laudanine. A second methylation at the 3′ position of laudanine leads to laudanosine, both known alkaloids from the opium poppy. Subsequent N-demethylation of laudanosine yields the known precursor of papaverine: tetrahydropapaverine. Inspection of the subsequent aromatization reaction established the presence of an intermediate, 1,2-dihydropapaverine, which has been characterized. The final step to papaverine is dehydrogenation of the 1,2-bond, yielding the target compound papaverine. We conclusively show herein that the previously claimed norreticuline does not play a role in the biosynthesis of papaverine.     

Metabolic engineering with a morphine biosynthetic P450 in opium poppy surpasses breeding

Morphine biosynthesis was genetically engineered in an industrial elite line of the opium poppy (Papaver somniferum L.), to modify the production of alkaloids in plants. The cytochrome P-450-dependent monooxygenase (S)-N-methylcoclaurine 3'-hydroxylase (CYP80B3) lies on the pathway to the benzylisoquinoline alkaloid branch point intermediate (S)-reticuline. Overexpression of cyp80b3 cDNA resulted in an up to 450% increase in the amount of total alkaloid in latex. This increase occurred either without changing the ratio of the individual alkaloids, or together with an overall increase in the ratio of morphine. Correspondingly, antisense-cyp80b3 cDNA expressed in opium poppy caused a reduction of total alkaloid in latex up to 84%, suggesting that the observed phenotypes were dependent on the presence of the transgene. This study found compelling evidence, that cyp80b3 is a key regulation step in morphine biosynthesis and provides practical means to genetically engineer valuable secondary metabolites in this important medicinal plant.     

Isolation and partial characterization of norcoclaurine synthase, the first committed step in benzylisoquinoline alkaloid biosynthesis, from opium poppy

Norcoclaurine synthase (NCS) catalyzes the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) to yield norcoclaurine, the common precursor to all benzylisoquinoline alkaloids produced in plants. In opium poppy (Papaver somniferum L.), NCS activity was detected in germinating seeds, young seedlings, and all mature plant organs, especially stems and roots. However, the highest levels of activity were found in cell-suspension cultures treated with a fungal elicitor. NCS activity was induced more than 20-fold over an 80-h period in response to elicitor treatment. Compared to opium poppy. basal NCS activity was 3-and 5-fold higher in benzylisoquinoline alkaloid-producing cell cultures of Eschscholzia californica and Thalictrum flavum ssp. glaucum, respectively. In contrast, NCS activity was not detected in cultured cells of Nicotiana tabacum and Catharanthus roseus, which do not produce benzylisoquinoline alkaloids. NCS displayed maximum activity between pH 6.5 and 7.0, and a broad temperature optimum between 42 and 55 degrees C. Enzyme activity was not affected by Ca2+ or Mg2+, and was not inhibited by a variety of benzylisoquinoline alkaloids. NCS showed hyperbolic saturation kinetics for 4-HPAA, with an apparent Km of 1.0 mM. However, the enzyme exhibited sigmoidal saturation kinetics for dopamine with a Hill coefficient of 1.84. NCS enzymes from E. californica and T. flavum displayed similar properties. These data indicate that NCS exhibits positive cooperativity between substrate-binding sites. Enzymes of this type catalyze regulatory, or rate-limiting, steps in metabolism, suggesting that NCS plays a role in controlling the rate of pathway flux in benzylisoquinoline alkaloid biosynthesis.