Papaver Somniferum Cell Research Articles

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.

Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures

BackgroundPapaver somniferum (opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies.ResultsA cDNA library was prepared for opium poppy cell cultures treated with a fungal elicitor for 10 h. Using 454 GS-FLX Titanium pyrosequencing, 427,369 expressed sequence tags (ESTs) with an average length of 462 bp were generated. Assembly of these sequences yielded 93,723 unigenes, of which 23,753 were assigned Gene Ontology annotations. Transcripts encoding all known sanguinarine biosynthetic enzymes were identified in the EST database, 5 of which were represented among the 50 most abundant transcripts. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of total protein extracts from cell cultures treated with a fungal elicitor for 50 h facilitated the identification of 1,004 proteins. Proteins were fractionated by one-dimensional SDS-PAGE and digested with trypsin prior to LC-MS/MS analysis. Query of an opium poppy-specific EST database substantially enhanced peptide identification. Eight out of 10 known sanguinarine biosynthetic enzymes and many relevant primary metabolic enzymes were represented in the peptide database.ConclusionsThe integration of deep transcriptome and proteome analyses provides an effective platform to catalogue the components of secondary metabolism, and to identify genes encoding uncharacterized enzymes. The establishment of corresponding transcript and protein databases generated by next-generation technologies in a system with a well-defined metabolite profile facilitates an improved linkage between genes, enzymes, and pathway components. The proteome database represents the most relevant alkaloid-producing enzymes, compared with the much deeper and more complete transcriptome library. The transcript database contained full-length mRNAs encoding most alkaloid biosynthetic enzymes, which is a key requirement for the functional characterization of novel gene candidates.

Norcoclaurine Synthase Is a Member of the Pathogenesis-Related 10/Bet v1 Protein Family

Norcoclaurine synthase (NCS) catalyzes the first committed step in the biosynthesis of benzylisoquinoline alkaloids (BIAs). NCS from Thalictrum flavum (Tf NCS), Papaver somniferum (Ps NCS1 and Ps NCS2), and Coptis japonica (Cj PR10A) share substantial identity with pathogen-related 10 (PR10) and Bet v1 proteins, whose functions are not well understood. A distinct enzyme (Cj NCS1) with similarity to 2-oxoglutarate-dependent dioxygenases was suggested as the bona fide NCS in C. japonica. Here, we validate the exclusive role of PR10/Bet v1-type NCS enzymes in BIA metabolism. Immunolocalization of Ps NCS2 revealed its cell type-specific occurrence in phloem sieve elements, which contain all other known BIA biosynthetic enzymes. In opium poppy, NCS transcripts and proteins were abundant in root and stem, but at low levels in leaf and carpel. Silencing of NCS in opium poppy profoundly reduced alkaloid levels compared with controls. Immunoprecipitation of NCS from total protein extracts of T. flavum cells resulted in a nearly complete attenuation of NCS activity. A Ps NCS2-green fluorescent protein fusion introduced by microprojectile bombardment into opium poppy cells initially localized to the endoplasmic reticulum but subsequently sorted to the vacuole. In our hands, Cj NCS1 did not catalyze the formation of (S)-norcoclaurine from dopamine and 4-hydroxyphenylacetaldehyde.

Plant Defense Responses in Opium Poppy Cell Cultures Revealed by Liquid Chromatography-Tandem Mass Spectrometry Proteomics

Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids, including the narcotic analgesic morphine and the antimicrobial agent sanguinarine. In contrast to the plant, cell cultures of opium poppy do not accumulate alkaloids constitutively but produce sanguinarine in response to treatment with certain fungal-derived elicitors. The induction of sanguinarine biosynthesis provides a model platform to characterize the regulation of benzylisoquinoline alkaloid pathways and other defense responses. Proteome analysis of elicitor-treated opium poppy cell cultures by two-dimensional denaturing-polyacrylamide gel electrophoresis coupled with liquid chromatography-tandem mass spectrometry facilitated the identification of 219 of 340 protein spots based on peptide fragment fingerprint searches of a combination of databases. Of the 219 hits, 129 were identified through pre-existing plant proteome databases, 63 were identified by matching predicted translation products in opium poppy-expressed sequence tag databases, and the remainder shared evidence from both databases. Metabolic enzymes represented the largest category of proteins and included S-adenosylmethionine synthetase, several glycolytic, and a nearly complete set of tricarboxylic acid cycle enzymes, one alkaloid, and several other secondary metabolic enzymes. The abundance of chaperones, heat shock proteins, protein degradation factors, and pathogenesis-related proteins provided a comprehensive proteomics view on the coordination of plant defense responses. Qualitative comparison of protein abundance in control and elicitor-treated cell cultures allowed the separation of induced and constitutive or suppressed proteins. DNA microarrays were used to corroborate increases in protein abundance with a corresponding induction in cognate transcript levels.

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

BackgroundOpium 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.ResultsMetabolite fingerprinting and compound-specific profiling showed the extensive reprogramming of primary metabolic pathways in association with the induction of alkaloid biosynthesis in response to elicitor treatment. Using Chenomx NMR Suite v. 4.6, a software package capable of identifying and quantifying individual compounds based on their respective signature spectra, the levels of 42 diverse metabolites were monitored over a 100-hour time course in control and elicitor-treated opium poppy cell cultures. Overall, detectable and dynamic changes in the metabolome of elicitor-treated cells, especially in cellular pools of carbohydrates, organic acids and non-protein amino acids were detected within 5 hours after elicitor treatment. The metabolome of control cultures also showed substantial modulations 80 hours after the start of the time course, particularly in the levels of amino acids and phospholipid pathway intermediates. Specific flux modulations were detected throughout primary metabolism, including glycolysis, the tricarboxylic acid cycle, nitrogen assimilation, phospholipid/fatty acid synthesis and the shikimate pathway, all of which generate secondary metabolic precursors.ConclusionThe response of cell cultures to elicitor treatment involves the extensive reprogramming of primary and secondary metabolism, and associated cofactor biosynthetic pathways. A high-resolution map of the extensive reprogramming of primary and secondary metabolism in elicitor-treated opium poppy cell cultures is provided.

Molecular Cloning and Characterization of Tetrahydroprotoberberine cis-N-Methyltransferase, an Enzyme Involved in Alkaloid Biosynthesis in Opium Poppy

S-Adenosyl-l-methionine:tetrahydroprotoberberine cis-N-methyltransferase (EC 2.1.1.122) catalyzes the conversion of (S)-stylopine to the quaternary ammonium alkaloid, (S)-cis-N-methylstylopine, as a key step in the biosynthesis of protopine and benzophenanthridine alkaloids in plants. A full-length cDNA encoding a protein exhibiting 45 and 48% amino acid identity with coclaurine N-methyltransferase from Papaver somniferum (opium poppy) and Coptis japonica, respectively, was identified in an elicitor-treated opium poppy cell culture expressed sequence tag data base. Phylogenetic analysis showed that the protein belongs to a unique clade of enzymes that includes coclaurine N-methyltransferase, the predicated translation products of the Arabidopsis thaliana genes, At4g33110 and At4g33120, and bacterial S-adenosyl-L-methionine-dependent cyclopropane fatty acid synthases. Expression of the cDNA in Escherichia coli produced a recombinant enzyme able to convert the protoberberine alkaloids stylopine, canadine, and tetrahydropalmatine to their corresponding N-methylated derivatives. However, the protoberberine alkaloids tetrahydroxyberbine and scoulerine, and simple isoquinoline, benzylisoquinoline, and pavine alkaloids were not accepted as substrates, demonstrating the strict specificity of the enzyme. The apparent K(m) values for (R,S)-stylopine and S-adenosyl-L-methionine were 0.6 and 11.5 microm, respectively. TNMT gene transcripts and enzyme activity were detected in opium poppy seedlings and all mature plant organs and were induced in cultured opium poppy cells after treatment with a fungal elicitor. The enzyme was detected in cell cultures of other members of the Papaveraceae but not in species of related plant families that do not accumulate protopine and benzophenanthridine alkaloids.

Opium poppy: blueprint for an alkaloid factory

Opium poppy (Papaver somniferum) produces a large number of benzylisoquinoline alkaloids, including the narcotic analgesics morphine and codeine, and has emerged as one of the most versatile model systems to study alkaloid metabolism in plants. As summarized in this review, we have taken a holistic strategy—involving biochemical, cellular, molecular genetic, genomic, and metabolomic approaches—to draft a blueprint of the fundamental biological platforms required for an opium poppy cell to function as an alkaloid factory. The capacity to synthesize and store alkaloids requires the cooperation of three phloem cell types—companion cells, sieve elements, and laticifers—in the plant, but also occurs in dedifferentiated cell cultures. We have assembled an opium poppy expressed sequence tag (EST) database based on the attempted sequencing of more than 30,000 cDNAs from elicitor-treated cell culture, stem, and root libraries. Approximately 23,000 of the elicitor-induced cell culture and stem ESTs are represented on a DNA microarray, which has been used to examine changes in transcript profile in cultured cells in response to elicitor treatment, and in plants with different alkaloid profiles. Fourier transform-ion cyclotron resonance mass spectrometry and proton nuclear magnetic resonance mass spectroscopy are being used to detect corresponding differences in metabolite profiles. Several new genes involved in the biosynthesis and regulation of alkaloid pathways in opium poppy have been identified using genomic tools. A biological blueprint for alkaloid production coupled with the emergence of reliable transformation protocols has created an unprecedented opportunity to alter the chemical profile of the world’s most valuable medicinal plant.

Gene transcript and metabolite profiling of elicitor-induced opium poppy cell cultures reveals the coordinate regulation of primary and secondary metabolism

Elicitor-induced sanguinarine accumulation in opium poppy (Papaver somniferum) cell cultures provides a responsive model system to profile modulations in gene transcripts and metabolites related to alkaloid biosynthesis. An annotated expressed sequence tag (EST) database was assembled from 10,224 random clones isolated from an elicitor-treated opium poppy cell culture cDNA library. The most abundant ESTs encoded defense proteins, and enzymes involved in alkaloid metabolism and S-adenosylmethionine-dependent methyl transfer. ESTs corresponding to 40 enzymes involved in the conversion of sucrose to sanguinarine were identified. A corresponding DNA microarray was probed with RNA from cell cultures collected at various time-points after elicitor treatment, and compared with RNA from control cells. Several diverse transcript populations were coordinately induced, with alkaloid biosynthetic enzyme and defense protein transcripts displaying the most rapid and substantial increases. In addition to all known sanguinarine biosynthetic gene transcripts, mRNAs encoding several upstream primary metabolic enzymes were coordinately induced. Fourier transform-ion cyclotron resonance-mass spectrometry was used to characterize the metabolite profiles of control and elicitor-treated cell cultures. Principle component analysis revealed a significant and dynamic separation in the metabolome, represented by 992 independent detected analytes, in response to elicitor treatment. Identified metabolites included sanguinarine, dihydrosanguinarine, and the methoxylated derivatives dihydrochelirubine and chelirubine, and the alkaloid pathway intermediates N-methylcoclaurine, N-methylstylopine, and protopine. Some of the detected analytes showed temporal changes in abundance consistent with modulations in the profiles of alkaloid biosynthetic gene transcripts.

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.

Berberine bridge enzyme, a key branch-point enzyme in benzylisoquinoline alkaloid biosynthesis, contains a vacuolar sorting determinant.

In opium poppy (Papaver somniferum L.), (S)-reticuline is the last common intermediate in sanguinarine and morphine biosynthesis. Sanguinarine accumulates in the vacuole of cultured opium poppy cells in response to treatment with fungal elicitors. The first committed step in sanguinarine biosynthesis is catalyzed by the berberine bridge enzyme (BBE), which converts (S)-reticuline to (S)-scoulerine. An N-terminal signal peptide and novel vacuolar sorting determinant were identified and characterized in BBE. In vitro translation of BBE mRNA in the presence of canine pancreatic microsomes produced a glycosylated, proteolysis-resistant protein, confirming the existence of a signal peptide. Transcripts encoding a BBE N-terminal deletion series fused to beta-glucuronidase or green fluorescent protein (GFP) were also translated in the presence of canine microsomes, and introduced into cultured opium poppy cells via microprojectile bombardment. The signal peptide was restricted to the first 25 amino acids and shown to initially target BBE to the endoplasmic reticulum. Fusion of 50 N-terminal residues from BBE to GFP resulted in the localization of the reporter to the vacuole. GFP was also sorted to the vacuole when fused to a heterologous N-terminal signal peptide followed by BBE amino acids 26-50. The BBE vacuolar sorting determinant was further localized between residues 26 and 41 by deletion analysis. The final subcellular destination of BBE is consistent with the vacuolar sequestration of sanguinarine. However, the vacuolar pH is below the functional range for BBE, suggesting that the enzyme is active only prior to its entry into the vacuole.

Distribution of morphinan and benzo[ c]phenanthridine alkaloid gene transcript accumulation in Papaver somniferum

The opium poppy Papaver somniferum L. produces the antimicrobial benzo[ c]phenanthridine alkaloid sanguinarine and the narcotic analgesic morphinan alkaloid morphine. Transcripts of three genes of alkaloid biosynthesis in P. somniferum in developing seedlings, mature plants and plant cell suspension culture were monitored for temporal/spatial or for methyl jasmonate-induced accumulation by RNA gel blot analysis. These genes encoded ( S)- N-methylcoclaurine 3′-hydroxylase (CYP80B1) that is common to morphine and sanguinarine biosynthesis, the berberine bridge enzyme (BBE) that lies on the pathway to sanguinarine, and codeinone reductase (COR) the penultimate enzyme of morphine biosynthesis. In developing P. somniferum seedlings, the morphine precursor thebaine was present throughout the first twenty days of germination. In contrast, sanguinarine was present in detectable quantities only after day five after germination and continued to increase at least until day twenty. Accumulation of cyp80b1, bbe1 and cor1 gene transcripts paralleled these differences. In the mature poppy plant, cyp80b1, bbe1 and cor1 gene transcripts were detected in the root, the stem, the leaf lamina and the leaf mid rib. Only cyp80b1 and cor1, however, were found in the flower bud and the capsule. Consistent with the fact that sanguinarine accumulation, but not that of morphine, can be induced in opium poppy cell suspension culture by addition of methyl jasmonate to the culture medium, cyp80b1 and bbe1, but not cor1 transcript accumulated in response to elicitor treatment.

Polyamine Content and Somatic Embryogenesis in Papaver somniferum Cells Transformed with sam-1 Gene

Summary Papaver somniferum calluses transformed with the sam-1 gene from Arabidopsis thaliana, which encodes a SAM-synthetase, were subcultured over a 4-year period. The stability of the expression as well as the level of SAM synthetase activity were evaluated in 5 transgenic cell lines (STSI, STSII, STSIII, STSIV, STSV) and in the control. All transgenic cell lines exhibited a level of SAM-synthetase activity higher than that of the control. The enhancement of the enzyme activity has been confirmed by Northern blot analyses. The level of polyamines (putrescine, spermidine, spermine and 1,3-diaminopropane) was also evaluated in cell lines cultured either in Linsmaier and Skoog medium containing growth regulators (LS) or on a hormone free medium (LSHF). Cell lines cultured in LS medium showed a variable yield of the polyamine content. Putrescine was the major polyamine. After transfer to a hormone free medium, all of the cell lines, except STSIV, were able to form embryo-like structures. In this condition, the polyamine level decreases about 4-fold, spermidine being the most abundant.

Analysis of promoters from tyrosine/dihydroxyphenylalanine decarboxylase and berberine bridge enzyme genes involved in benzylisoquinoline alkaloid biosynthesis in opium poppy.

Tyrosine/dihydroxyphenylalanine decarboxylase (TYDC) and the berberine bridge enzyme (BBE) represent the entry point and a key branch point, respectively, in the biosynthesis of benzylisoquinoline alkaloids in select species of the Papaveraceae and Fumariaceae. Genomic clones for tydc7 and bbe1 from opium poppy (Papaver somniferum L.) were isolated. Deletion analysis of tydc7 and bbe1 5'-flanking regions revealed the location of putative regulatory domains necessary for expression of the beta-glucuronidase (gus) reporter gene in a transient assay system based on the microprojectile bombardment of cultured opium poppy cells. A 105-nucleotide region between -393 and -287 of the tydc7 5'-flanking region, and a 155-nucleotide region between -355 and -200 of the bbe1 5'-flanking region, were found to be essential for promoter activity. RNA gel blot analysis showed that tydc7 and bbe1 expression is induced in cultured opium poppy cells in response to wounding or treatment with a pathogen-derived elicitor. Time-courses for the induction of tydc7 and bbe1 mRNAs in wounded cells were nearly identical to those for GUS activity in cells bombarded with select promoter-gus constructs when the -393 to -287 region of tydc7, or the -355 to -200 region of bbe1, was present. Our data suggest that the wound signal caused by the entry of DNA-coated microcarriers into opium poppy cells was sufficient to induce tydc7 and bbe1 promoter activity, and that wound-responsive regulatory elements are located within domains identified by deletion analysis.

Cryopreservation does not affect the expression of a foreign sam gene in transgenic Papaver somniferum cells

Transgenic cell lines of Papaver somniferum have been obtained via Agrobacterium tumefaciens. Papaver somniferum is known to be genetically unstable in in vitro culture conditions. Cryopreservation at ultra-low temperature is an appropriate strategy for long-term preservation of germplasm. We have studied the effects of osmotic stress due to cryoprotectants during pretreatment and of storage at –196 °C on the stability and the expression of the foreign sam1 gene from Arabidopsis thaliana. We established that the integrity, transcription of the transgene and enzymatic activity of its product were not affected by cryopreservation in liquid nitrogen

Temporal Correlation of Tyramine Metabolism with Alkaloid and Amide Biosynthesis in Elicited Opium Poppy Cell Cultures : In Honour of Professor G. H. Neil Towers 75th Birthday

Aromatic amines are involved in the biosynthesis of many plant secondary metabolites, including benzylisoquinoline alkaloids and hydroxycinnamic acid amides. Incorporation of tyramine and dopamine into both the antibiotic alkaloid sanguinarine and hydroxycinnamic acid amides was shown to occur in elicitor-treated opium poppy cell cultures. A temporal correlation was demonstrated between the biosynthesis of alkaloids and amides, the accumulation of tyrosine\\dopa decarboxylase (TYDC) and berberine bridge enzyme (BBE) mRNAs, and the induction of tyramine hydroxycinnamoyl transferase (THT) activity. The induction of TYDC mRNAs occurred most rapidly, whereas the subsequent activation of BBE mRNAs and THT activity correlated with the synthesis and accumulation of sanguinarine and amides, respectively. In pulse-labeling experiments, a maximum of 1.2 and 0.6% of exogenous 14C-tyramine and 14C-dopamine, respectively, was incorporated into sanguinarine after elicitor treatment of cell cultures. In contrast, a maximum of 24 and 35% of exogenous 14C-tyramine and 14C-dopamine, respectively, was incorporated into an acid-insoluble fraction of the cell wall after elicitor treatment. Insolubilized radioactivity was released from cell walls after hydrolytic extraction and recovered mostly as hydroxycinnamic acid amides and free amines. The elicitor-induced incorporation of 14C-amines into both sanguinarine and cell walls decreased during the cell culture growth cycle. The prolonged induction of THT activity in elicited cultures, coupled with high and constitutive peroxidase activity, suggests that the observed decrease in cell wall labeling 5 h after the addition of elicitor was due to hydroxycinnamoyl-CoA or H2O2 limitation.

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

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.

Transformation of Papaver somniferum cell suspension cultures with sam1 from A. thaliana results in cell lines of different S‐adenosyl‐L‐methionine synthetase activity

We have developed a procedure for opium poppy (Papaver somniferum) transformation using Agrobacterium tumefaciens‐mediated gene delivery. Hypocotyl‐derived cell suspension cultures of P. somniferum were cocultivated with A. tumefaciens strain GV3101(pMP90) harbouring either the binary vector pTHW136 or the binary vector pO35SSAM. The former contained the uidA reporter gene and the nptII selectable gene, encoding the enzymes β‐glucuronidase and neomycin phosphotransferase II, respectively. The latter contained the sam1 gene encoding the enzyme S‐adenosyl‐L‐methionine (SAM) synthetase and the nptII gene. Putatively transformed cell lines were selected on media supplemented with paromomycin. Integration of the foreign genes was confirmed by Southern blot analysis with and without PCR amplification prior to hybridization. SAM synthetase activity was measured in extracts of 5 transformed cell lines. One of them expressed a significant overactivity while two others had a lower activity than the control cell line, leading us to question the possible partial cosuppression of both the resident and the foreign sam genes. To our knowledge, this is the first report of Agrobacterium tumefaciens‐mediated transformation of Papaver somniferum cell suspension cultures.

Transformation of Papaver somniferum cell suspension cultures with sam1 from A. thaliana results in cell lines of different S-adenosyl-L-methionine synthetase activity

Transformation of Papaver somniferum cell suspension cultures with sam1 from A. thaliana results in cell lines of different S-adenosyl-L-methionine synthetase activity

Uncoupled defense gene expression and antimicrobial alkaloid accumulation in elicited opium poppy cell cultures.

Treatment of opium poppy (Papaver somniferum L.) cell cultures with autoclaved mycelial homogenates of Botrytis sp. resulted in the accumulation of sanguinarine. Elicitor treatment also caused a rapid and transient induction in the activity of tyrosine/dopa decarboxylase (TYDC, EC 4.1.1.25), which catalyzes the conversion of L-tyrosine and L-dopa to tyramine and dopamine, respectively, the first steps in sanguinarine biosynthesis. TYDC genes were differentially expressed in response to elicitor treatment. TYDC1-like mRNA levels were induced rapidly but declined to near baseline levels within 5 h. In contrast, TYDC2-like transcript levels increased more slowly but were sustained for an extended period. Induction of TYDC mRNAs preceded that of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) mRNAs. An elicitor preparation from Pythium aphanidermatum was less effective in the induction of TYDC mRNA levels and alkaloid accumulation; however, both elicitors equally induced accumulation of PAL transcripts. In contrast, treatment with methyl jasmonate resulted in an induction of TYDC but not PAL mRNAs. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and the protein kinase inhibitor staurosporine partially blocked the fungal elicitor-induced accumulation of sanguinarine. However, only staurosporine and okadaic acid, an inhibitor of protein phosphatases 1 and 2A, blocked the induction of TYDC1-like transcript levels, but they did not block the induction of TYDC2-like or PAL transcript levels. These data suggest that activation mechanisms for PAL, TYDC, and some later sanguinarine biosynthetic enzymes are uncoupled.

Production of sanguinarine by elicited plant cell culture III. Immobilized bioreactor cultures

Papaver somniferum cells were surface-immobilized and grown efficiently and maintained highly viable in 6-1 modified airlift bioreactors. These immobilized cultures were elicited with chitin in the presence of XAD-7 adsorbent polymeric resin, in parallel with control suspension cultures carried out in flasks, for sanguinarine production. Results showed production levels of the larger cultures operated under apparently non limiting conditions of up to 80 mg l −1 or 0.52% dw after 600 h, of which 70–80% was found in the resin. These productions were 3- to 14-times lower than obtained from control cultures. This poor performance of immobilized cultures is explained by dissolved oxygen limitations within the biofilm as well as by various other restrictive factors associated to the culture system used in relation to the elicitation process, including the inherent physiological heterogeneity of the plant cell biofilm and the insoluble nature of the elicitor used which may have hindered its penetration of the immobilized biomass.