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.     

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.     

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.     

Effect of triacontanol on photosynthesis,alkaloid content and growth in opium poppy (Papaver Somniferum L.)

The influence of different foliar applications of Triacontanol (Tria.) on growth, CO₂ exchange, capsule development and alkaloid accumulation in opium poppy was studied in glasshouse conditions. Plant height, capsule number and weight, morphine content, CO₂ exchange rate, total chlorophyll and fresh and dry weight of the shoot were significantly maximum at 0.01 mg/l Tria. At the highest concentration (4 mg/l) total chlorophyll, CO₂ exchange rate and plant height were significantly inhibited. Thebaine and codeine contents remained unaffected at all the concentrations. The concentration of Fe, Mn, Cu in shoots were maximum at .01 and Zn at 0.1 mg/l Tria. Increase in shoot weight, leaf area ratio and chlorophyll content were significantly correlated with morphine content.CIMAP Communication No. 839.     

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.     

Identification and characterization of latex-specific proteins in opium poppy

Latex from the opium poppy, Papaver somniferum L., was analyzed by polyacrylamide gel electrophoresis (PAGE). Two latex-specific bands were identified in protein samples of poppy latex using one-dimensional native PAGE. Second dimension analysis with SDS-PAGE indicates that these proteins have a relative molecular weight of approximately 20 kilodaltons. We have termed these polypeptides the major latex proteins (MLPs). Polyclonal antibodies prepared against the MLPs were used to probe protein gel blots of latex and poppy tissues known to lack laticifers. Laticifer-free tissues showed no reaction with anti-MLP immunoglobulin G indicating that MLPs are found only in poppy latex. MLP distribution was also examined in mature opium poppy tissues by immunocytochemistry. Laticifers were differentially labeled by fluorescein isothiocyanate secondary labeling of anti-MLP immunoglobulin G and could easily be identified in both transverse and longitudinal section. Fractionation studies of isolated latex showed that MLPs are concentrated in the latex cytosol and not in alkaloidal vesicles. Analysis of latex proteins by conventional two-dimensional electrophoresis indicates that the two MLP bands are composed of several distinct polypeptides with similar relative molecular weights. The pIs of these molecules range from 6.0 to 3.5. The role(s) of MLPs in laticifer metabolism has not been determined.