Biosynthesis of poppy isoquinoline alkaloids in nature and in vitro culture. 2. Bracteum poppy (Papaver bracteatum Lindl.)

Literature data and the data of the author's investigations on production of isoquinoline alkaloids by Papaver bracteatum Lindl. have been analyzed. Information on the methods of regulation and cell localization of morphine and sanguinarine biosynthesis is presented. The works studying differentiation processes in tissue cultures of bracteum poppy and relationship thereof with thebaine biosynthesis have been analyzed. Possible mechanism determining the induction of somatic embryos development and thebaine biosynthesis in the culture in vitro are proposed.     

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.     

Benzylisoquinoline alkaloid biosynthesis in opium poppy

Opium poppy (Papaver somniferum) is one of the world’s oldest medicinal plants and remains the only commercial source for the narcotic analgesics morphine, codeine and semi-synthetic derivatives such as oxycodone and naltrexone. The plant also produces several other benzylisoquinoline alkaloids with potent pharmacological properties including the vasodilator papaverine, the cough suppressant and potential anticancer drug noscapine and the antimicrobial agent sanguinarine. Opium poppy has served as a model system to investigate the biosynthesis of benzylisoquinoline alkaloids in plants. The application of biochemical and functional genomics has resulted in a recent surge in the discovery of biosynthetic genes involved in the formation of major benzylisoquinoline alkaloids in opium poppy. The availability of extensive biochemical genetic tools and information pertaining to benzylisoquinoline alkaloid metabolism is facilitating the study of a wide range of phenomena including the structural biology of novel catalysts, the genomic organization of biosynthetic genes, the cellular and sub-cellular localization of biosynthetic enzymes and a variety of biotechnological applications. In this review, we highlight recent developments and summarize the frontiers of knowledge regarding the biochemistry, cellular biology and biotechnology of benzylisoquinoline alkaloid biosynthesis in opium poppy.     

Antisense RNA-mediated suppression of benzophenanthridine alkaloid biosynthesis in transgenic cell cultures of California poppy

California poppy (Eschscholzia californica Cham.) cell cultures produce several benzophenanthridine alkaloids, such as sanguinarine, chelirubine, and macarpine, with potent pharmacological activity. Antisense constructs of genes encoding two enzymes involved in benzophenanthridine alkaloid biosynthesis, the berberine bridge enzyme (BBE) and N-methylcoclaurine 3'-hydroxylase (CYP80B1), were introduced separately into California poppy cell cultures. Transformed cell lines expressing antisense BBE or antisense CYP80B1 constructs and displaying low levels of BBE or CYP80B1 mRNAs, respectively, showed reduced accumulation of benzophenanthridine alkaloids compared with control cultures transformed with a beta-glucuronidase gene. Pathway intermediates were not detected in any of the transformed cell lines. The suppression of benzophenanthridine alkaloid biosynthesis using BBE or CYP80B1 antisense RNA constructs also reduced the growth rate of the cultures. Two-dimensional (1)H-nuclear magnetic resonance and in vivo (15)N-nuclear magnetic resonance spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic cell lines. However, transformed cells with reduced benzophenanthridine alkaloid levels contained larger cellular pools of several amino acids including alanine, leucine, phenylalanine, threonine, and valine compared with controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was less than 2-fold higher in antisense-suppressed cells relative to controls. These results show that alterations in the metabolic flux through benzophenanthridine alkaloid biosynthesis can affect the regulation of amino acid pools. These data provide new insight into the metabolic engineering of benzophenanthridine alkaloid pathways.     

Sanguinarine biosynthesis is associated with the endoplasmic reticulum in cultured opium poppy cells after elicitor treatment

Three key benzylisoquinoline alkaloid biosynthetic enzymes, (S)-N-methylcoclaurine-3'-hydroxylase (CYP80B1), berberine bridge enzyme (BBE), and codeinone reductase (COR), were localized in cultured opium poppy (Papaver somniferum) cells by sucrose density gradient fractionation and immunogold labeling. CYP80B1 catalyzes the second to last step in the formation of (S)-reticuline, the last common intermediate in sanguinarine and morphine biosynthesis. BBE converts (S)-reticuline to (S)-scoulerine as the first committed step in sanguinarine biosynthesis, and COR catalyzes the penultimate step in the branch pathway leading to morphine. Sanguinarine is an antimicrobial alkaloid that accumulates in the vacuoles of cultured opium poppy cells in response to elicitor treatment, whereas the narcotic analgesic morphine, which is abundant in opium poppy plants, is not produced in cultured cells. CYP80B1 and BBE were rapidly induced to high levels in response to elicitor treatment. By contrast, COR levels were constitutive in the cell cultures, but remained low and were not induced by addition of the elicitor. Western blots performed on protein homogenates from elicitor-treated cells fractionated on a sucrose density gradient showed the cosedimentation of CYP80B1, BBE, and sanguinarine with calreticulin, and COR with glutathione S-transferase. Calreticulin and glutathione S-transferase are markers for the endoplasmic reticulum (ER) and the cytosol, respectively. In response to elicitor treatment, large dilated vesicles rapidly developed from the lamellar ER of control cells and fused with the central vacuole. Immunogold localization supported the association of CYP80B1 and BBE with ER vesicles, and COR with the cytosol in elicitor-treated cells. Our results show that benzylisoquinoline biosynthesis and transport to the vacuole are associated with the ER, which undergoes major ultrastructural modification in response to the elicitor treatment of cultured opium poppy cells.     

Alkaloid biosynthesis in Papaver sp. cells in culture and during organogenesis

In vitro cell cultures of two Papaver species, P. somniferum and P. bracteatum initiated from mature seeds were screened for their ability to produce alkaloids. Protocols for callus induction, somatic embryogenesis and organogenesis were established. The alkaloid contents were analysed by high-performance-liquid chromatography, thin-layer chromatography and spectrophotometric assays. Undifferentiated callus produced small amounts of sanguinarine, which increased with the degree of tissue differentiation. Embryogenic calli were maintained in culture for more than 2 years, retaining a high regeneration capability. Thin-layer chromatography analysis revealed variations in alkaloid spectrum between parallel cell lines. The morphinan alkaloid, thebaine, was found to be accumulated exclusively in morphogenous strains of P. bracteatum, and morphine was the major alkaloid in the spectrum of P. somniferum dedifferentiated callus. Regenerant plants synthesized thebaine and sanguinarine at the same level as juvenile plants grown from P. bracteatum seeds. We revealed differences in the ability to produce different types of alkaloids: seed-derived plants were able to accumulate thebaine while undifferentiated primary cell cultures produced only sanguinarine. The production of either sanguinarine and morphinan alkaloids are found in regenerants showing that both metabolic pathways were active in young plantlets.     

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.     

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.     

Molecular characterization of berberine bridge enzyme genes from opium poppy.

In Papaver somniferum (opium poppy) and related species, (S)-reticuline serves as a branch-point intermediate in the biosynthesis of numerous isoquinoline alkaloids. The berberine bridge enzyme (BBE) ([S]-reticuline:oxygen oxidoreductase [methylene bridge forming], EC 1.5.3.9) catalyzes the stereospecific conversion of the N-methyl moiety of (S)-reticuline into the berberine bridge carbon of (S)-scoulerine and represents the first committed step in the pathway leading to the antimicrobial alkaloid sanguinarine. Three unique genomic clones (bbe1, bbe2, and bbe3) similar to a BBE cDNA from Eschscholtzia californica (California poppy) were isolated from opium poppy. Two clones (bbe2 and bbe3) contained frame-shift mutations of which bbe2 was identified as a putative, nonexpressed pseudogene by RNA blot hybridization using a gene-specific probe and by the lack of transient expression of a chimeric gene fusion between the bbe2 5' flanking region and a beta-glucuronidase reporter gene. Similarly, bbe1 was shown to be expressed in opium poppy plants and cultured cells. Genomic DNA blot-hybridization data were consistent with a limited number of bbe homologs. RNA blot hybridization showed that bbe genes are expressed in roots and stems of mature plants and in seedlings within 3 d after germination. Rapid and transient BBE mRNA accumulation also occurred after treatment with a fungal elicitor or with methyl jasmonate. However, sanguinarine was found only in roots, seedlings, and fungal elicitor-treated cell cultures.     

Silencing nature's narcotics: metabolic engineering of the opium poppy

The opium poppy, Papaver somniferum L., and its narcotic and analgesic alkaloids, have an ancient history of use (and abuse) by humankind. A recent article by Allen and co-workers describes the metabolic engineering of morphine biosynthesis to block morphine formation and accumulate a potentially valuable pathway intermediate, (S)-reticuline. This work highlights the potential for modifying the production of pharmaceuticals in plants, but also raises questions about the complex regulation of biosynthetic pathways.     

Subcellular localization of sanguinarine biosynthetic enzymes in cultured opium poppy cells

Benzylisoquinoline alkaloids are a large and structurally diverse group of natural plant products that includes many compounds with potent biological activities, including the antimicrobial agent sanguinarine. The putative subcellular localization of the sanguinarine pathway was determined using in-frame N-terminal fusions between cDNAs encoding nine consecutive biosynthetic enzymes and the gene encoding the green fluorescent protein (GFP). Expression constructs were introduced into cultured opium poppy cells by particle bombardment, and the localization of fusion proteins was visualized using epifluorescence microscopy. GFP fusions with two O-methyltransferases and two N-methyltransferases in the sanguinarine pathway all produced non-targeted fluorescence in the cytosol and nucleus. Interspersed between these soluble proteins are five membrane-bound cytochromes P450. Corresponding cDNAs are available for three P450s, all of which produced fluorescence when fused to GFP in association with the endoplasmic reticulum (ER). Two enzymes with suggested or known N-terminal signal peptides were initially associated with the ER, but were subsequently transported to the central vacuole suggesting their occurrence in the ER lumen. The alternating localization of these biosynthetic enzymes to three subcellular compartments indicates extensive trafficking of pathway intermediates across the endomembranes and suggests a key role for compartmentalization in the regulation of sanguinarine metabolism.     

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.     

Self-regulation of phytoalexin production: a non-biosynthetic enzyme controls alkaloid biosynthesis in cultured cells of Eschscholzia californica

Benzophenanthridine alkaloids are strong antimicrobials of Papaveraceae and attractive lead compounds for drug development. The cytotoxicity of these compounds requires the producing plant to limit the pathogen-triggered burst of biosynthesis. Cells of Eschscholzia californica excrete early benzophenanthridines to the cell wall, followed by re-uptake and reduction in the cytoplasm by the detoxifying enzyme sanguinarine reductase. We now discovered that this enzyme is a core component of self-control in alkaloid production. RNAi-based silencing of sanguinarine reductase gave rise to mutants that either show a complete stop of elicitor-triggered alkaloid production or a burst of biosynthesis that severalfold surpasses the wild type level. These unexpected phenotypes reflect impacts of substrate or product of sanguinarine reductase: the substrate, sanguinarine, inhibits phospholipase A2 at the plasma membrane, an initial component of the signal path towards expression of biosynthetic enzymes. The product, dihydrosanguinarine, inhibits enzymes of early biosynthesis, prior to reticuline formation. By tuning these steady states, sanguinarine reductase adjusts the capacity of alkaloid biosynthesis: a minimum activity is sufficient to prevent the blockade of the induction pathway by sanguinarine, while the full activity of the same enzyme causes a limitation of the biosynthetic flow via dihydrosanguinarine.