Elicitation of sanguinarine production in two-phase cultures of Eschscholtzia californica

Large increases in sanguinarine production were observed in suspension cultured Eschscholtzia californica cells treated with elicitors prepared from yeast extract, Colletotrichum lindemuthianum and Verticillum dahliae. Compounded silicone fluid, an ideal accumulation phase for the two-phase culture of E. californica, accumulated a large amount of sanguinarine produced in a specific manner and increased sanguinarine production. Elicitation in combination with two-phase culture additionally increased net sanguinarine production, as well as the sanguinarine concentration in the accumulation phase.     

Ultrastructural changes associated with the accumulation and secretion of sanguinarine in Papaver bracteatum suspension cultures treated with fungal elicitor

Suspension cultures of Papaver bracteatum Arya II Lindl., grown without hormone in the presence of conidial extracts of Verticillium dahliae Kleb., accumulate millimolar quantities of the benzophenanthridine alkaloid, sanguinarine. Under the fluorescence microscope, the elicitor-treated cells display an orange-yellow fluorescence characteristic of sanguinarine, primarily near the periphery of the cells. Electron-microscopic inspection showed the presence of slightly dilated endoplasmic reticulum and of electron-dense protuberances on the tonoplast of large central vacuoles. These osmiophilic aggregates lining the tonoplast bud into spherical bodies, appear to become detached from the membrane and are released into the vacuole. Upon subcellular fractionation of elicited cells on Renografin step gradients, sanguinarine was found to be distributed in all bands but with 86% concentrated in the gradient pellet. Analysis of the pellet by electron microscopy showed that it contained electron-dense fragments similar to the osmiophilic bodies observed on the tonoplast of intact elicited cells. In elicited cell cultures, most of the sanguinarine was recovered from medium in a 100·g sedimenting, cell-free, particulate fraction accounting for as much as 85% of the media sanguinarine and 62% of the total sanguinarine. The sanguinarine-rich 100·g media pellet was determined to be two-thirds protein, one-third RNA and was essentially devoid of phenolics, phospholipid and DNA. The pellet consisted of electrondense material and cytoplasmic remnants resembling those found in the Renografin pellet and tonoplast aggregates of intact cells. When placed under hypotonic conditions or extracted with aqueous buffer, pH 3–11, the pellet did not release sanguinarine. These observations provide evidence for storage of sanguinarine at electron-dense deposits which occur on the tonoplast and as freely floating bodies in vacuoles.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - EM electron microscopy - ER endoplasmic reticulum - HPLC high-pressure liquid chromatography - MRST Murashige and Skoog's revised tobacco medium     

Stimulation of Sanguinarine Production by Combined Fungal Elicitation and Hormonal Deprivation in Cell Suspension Cultures of Papaver bracteatum

Fungal elicitor preparations from either homogenized mycelia of Dendryphion penicillatum (Cda.) Fr., a specific pathogen of Papaver species, or conidia of Verticillium dahliae Kleb., a general pathogen, were added to 14-day-old suspension cultures of Papaver bracteatum. Plant tissue cultures were grown either in the presence or absence of 0.1 milligram of 2,4-dichlorophenoxyacetic acid per liter and 0.5 milligram of 6-benzylam-inopurine per liter. Dendryphion extracts elicited an accumulation of the benzophenanthridine alkaloid, sanguinarine, which was not greatly influenced by hormone deprivation. Millimolar concentrations of dopamine were detected under all conditions. Thebaine was found when cells were cultured in hormone-free media, but it was not elicitor dose dependent. Verticillium-elicited cultures accumulated sanguinarine in an elicitor-dose-dependent manner only under conditions of hormonal deprivation, resulting in an elevation of sanguinarine levels 5- to 500-fold greater than controls (2-10% dry weight). Most of the sanguinarine accumulated in the medium (23 milligrams per liter), with 85% of the alkaloid associated with a 100g sedimenting fraction that, upon light microscopic inspection, proved to be devoid of cells. In bioassays, sanguinarine showed significant biological activity at concentrations as low as 5 to 10 micrograms per milliliter against three general plant pathogens, Verticillium dahliae, Botrytis cinerea Pers. ex Fr., and Rhizoctonia solani Kuehn. Dendryphion was less affected by sanguinarine addition and displayed an ability to metabolize the alkaloid as evidenced by its loss from the media, subsequent accumulation in the mycelia, and ultimate disappearance over a 48-hour period. By comparison, dopamine and thebaine were less toxic to the general plant pathogens.     

Seed Dormancy in Acer: An assessment of the Rôle of the Structures Covering the Embryo

Cells of a seven year old strain of Papaver somniferum L. when cultured for 2 weeks and incubated with substances known to elicit the formation of phytoalexins, responded by turning reddish brown within 6 h and accumulating sanguinarine. Morphinan alkaloids were not detected. Media (100 ml) containing 1 ml of Botrytis spec. preparations raised the level of sanguinarine in the cells 26 times over the maximum level found in controls. Over a culture period of 79 h the cells achieved a sanguinarine concentration of 2.9% of dry weight. Media (100 ml) with 1 ml of Rhodotorula rubra preparation, 15 mg arachidonic acid, 1 mg actinomycin, 0.5 ml of Helminthosporium gramineum, Sclerotinia sclerotiorum, or 5 ml Colletotrichum gloeosporoides preparation elicited a considerable, but relatively weaker response. Sanguinarine accumulation was also found to occur in the medium and reached a concentration of 43% of total sanguinarine per culture when cells were cultured in 100 ml medium with 5 ml Colletotrichum preparation for 24 h. Young poppy cell cultures initiated 9 months ago responded to the presence of Botrytis material as did 7-year-old cultures.     

Alkaloid synthesis is coupled to shoot morphogenesis in Argemone mexicana L. (Papaveraceae) in vitro cultures

During the induction process of an in vitro callus culture of Argemone mexicana L. (Papaveraceae), the levels of two benzylisoquinoline alkaloids known as berberine and sanguinarine displayed opposing trends. While the berberine levels steadily decreased from the initial explant stage up to the early proliferation of unorganized parenchymatous cell masses, the sanguinarine content increased. Once the callus culture was established, sanguinarine was the primary alkaloid present and berberine could no longer be detected. However, upon shoot regeneration, the berberine accumulation recovered, but sanguinarine was found in the newly formed leafy tissue. After root formation, sanguinarine was relocated to this organ, whereas berberine was evenly distributed between both tissues. Explants from stem internodes did not form callus, and berberine—plus sanguinarine—containing axillary shoots emerged from lateral buds in the induction medium. In contrast to callus-derived shoots, no root formation was observed. Therefore, alkaloid synthesis in A. mexicana in vitro cultures is related to the level of tissue organization in different ways, and while berberine accumulation seems to require the presence of differentiated organs, this is not the case for sanguinarine. Moreover, leafy parts of rootless shoots acquired the capacity to accumulate sanguinarine, which is usually absent in aerial tissues of mature plants. However, when these shoots were rooted, sanguinarine was mainly located in the newly formed roots, while berberine was detected in the shoots at similar levels found in the roots.

     

N-Acylethanolamines in signal transduction of elicitor perception. Attenuation Of alkalinization response and activation of defense gene expression

In a recent study of N-acylphosphatidylethanolamine (NAPE) metabolism in elicitor-treated tobacco (Nicotiana tabacum L.) cells, we identified a rapid release and accumulation of medium-chain N-acylethanolamines (NAEs) (e.g. N-myristoylethanolamine or NAE 14:0) and a compensatory decrease in cellular NAPE (K.D. Chapman, S. Tripathy, B. Venables, A.D. Desouza [1998] Plant Physiol 116: 1163–1168). In the present study, we extend this observation and report a 10- to 50-fold increase in NAE 14:0 content in leaves of tobacco (cv Xanthi) plants treated with xylanase or cryptogein elicitors. Exogenously supplied synthetic NAE species affected characteristic elicitor-induced and short- and long-term defense responses in cell suspensions of tobacco and long-term defense responses in leaves of intact tobacco plants. In general, synthetic NAEs inhibited elicitor-induced medium alkalinization by tobacco cells in a time- and concentration-dependent manner. Exogenous NAE 14:0 induced expression of phenylalanine ammonia lyase in a manner similar to fungal elicitors in both cell suspensions and leaves of tobacco. NAE 14:0, but not myristic acid, activated phenylalanine ammonia lyase expression at submicromolar concentrations, well within the range of NAE 14:0 levels measured in elicitor-treated plants. Collectively, these results suggest that NAPE metabolism, specifically, the accumulation of NAE 14:0, are part of a signal transduction pathway that modulates cellular defense responses following the perception of fungal elicitors.     

Involvement of lipoxygenase in elicitor-stimulated sanguinarine accumulation in Papaver somniferum suspension cultures

The involvement of lipoxygenase (LOX, EC 1.13.11.12) in elicitor-induced opium poppy defense response was investigated. Papaver somniferum L. suspension cultures were treated with abiotic elicitor methyl jasmonate (MJ), fungal elicitor (Botrytis cinerea homogenate) and phenidone (specific inhibitor of LOX) to determine the involvement of this enzyme in production of sanguinarine, the major secondary metabolite of opium poppy cultures. P. somniferum suspension cultures responded to elicitor treatment with strong and transient increase of LOX activity followed by sanguinarine accumulation. LOX activity increased in elicited cultures, reaching 9.8 times of the initial value at 10 h after MJ application and 2.9 times after B. cinerea application. Sanguinarine accumulated to maximal levels of 169.5 ± 12.5 μg g^?1 dry cell weight in MJ-elicited cultures and 288.0 ± 10.0 μg g^?1 dry cell weight in B. cinerea-elicited cultures. The treatment of cells with phenidone before elicitor addition, significantly reduced sanguinarine production. The relative molecular weight of P. somniferum LOX (83 kDa) was estimated by using immunobloting and its pH optimum was shown to be pH 6.5.     

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.     

Involvement of 3-hydroxy-3-methylglutaryl coenzyme a reductase in the regulation of sesquiterpenoid phytoalexin synthesis in potato

The importance of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in the regulation of sesquiterpenoid phytoalexin accumulation in potato (Solanum tuberosum L. cv Kennebec) was examined. Wounding of potato tubers produced a large temporary increase in HMG-CoA reductase activity of the microsomal and organelle fractions. Treatment of wounded tuber tissue with the sesquiterpenoid phytoalexin elicitor arachidonic acid further increased and prolonged the HMG-CoA reductase activity in the microsomal but not the organelle fraction. Incubation of elicitor-treated tuber tissue in white light reduced organelle and microsomal HMG-CoA reductase activity to 50% and 10%, respectively, of the activity of tissues held in darkness. Constant light also reduced overall phytoalexin accumulation 58% by greatly reducing levels of lubimin. Rishitin accumulation was not significantly altered by light. Application of nanomolar amounts of mevinolin, a highly specific inhibitor of HMG-CoA reductase, to elicitor-treated tuber tissue produced a large decline in lubimin accumulation and did not markedly alter rishitin accumulation. These results indicate that HMG-CoA reductase has a role in the complex regulation of sesquiterpenoid phytoalexin accumulation in potato.     

Alkaloidal content of argentine Argemone

The alkaloid content of different Argentine Argemone has been determined. Two varieties of A. subfusiformis subsp. subfusiformis Ownb. and A. subfusiformis subsp. subinermis Ownb. yielded a similar ratio and content of the following alkaloids: protopine, allocrytopine, berberine, sanguinarine, and chelerythrine. The A. subfusiformis taxa showed a markedly high sanguinarine content in roots as opposed to aerial parts. A. polyanthemos (Fedde) Ownb. showed a different ratio between alkaloids but a qualitative similar result. N-Norchelerythrine was isolated from A. polyanthemos. The chemotaxonomic value of the alkaloid analyses is discussed.     

Rapid Activation of Phenylpropanoid Metabolism in Elicitor-Treated Hybrid Poplar (Populus trichocarpa Torr. & Gray x Populus deltoides Marsh) Suspension-Cultured Cells

Elicitor induction of phenylpropanoid metabolism was investigated in suspension-cultured cells of the fast-growing poplar hybrid (Populus trichocarpa Torr. & Gray × Populus deltoides Marsh) H11-11. Treatment of cells with polygalacturonic acid lyase or two fungal elicitors resulted in rapid and transient increases in extractable l-phenylalanine ammonia lyase and 4-coumarate:coenzyme A ligase enzyme activities. The substrate specificity of the inducible 4-coumarate:coenzyme A ligase enzyme activity appeared to differ from substrate specificity of 4-coumarate:coenzyme A ligase enzyme activity in untreated control cells. Large and transient increases in the accumulation of l-phenylalanine ammonia-lyase and 4-coumarate:coenzyme A ligase mRNAs preceded the increases in enzyme activities and were detectable by 30 minutes after the start of elicitor treatment. Chalcone synthase, cinnamyl alcohol dehydrogenase, and coniferin β-glucosidase enzyme activities were unaffected by the elicitors, but a large and transient increase in β-glucosidase activity capable of hydrolyzing 4-nitrophenyl-β-glucoside was observed. Subsequent to increases in l-phenylalanine ammonialyase and 4-coumarate:coenzyme A ligase enzyme activities, cell wall-bound thioglycolic acid-extractable compounds accumulated in elicitor-treated cultures, and these cells exhibited strong staining with phloroglucinol, suggesting the accumulation of wall-bound phenolic compounds.     

Sanguinarine suppresses IgE induced inflammatory responses through inhibition of type II PtdIns 4-kinase(s)

The effects of sanguinarine on IgE mediated early signaling mechanisms leading to inflammatory mediators release were investigated. Pretreatment of RBL 2H3 cells with sanguinarine inhibited IgE induced activation of type II PtdIns 4-kinase activity. Concomitant with type II PtdIns 4-kinase inhibition, sanguinarine also inhibited IgE induced degranulation and β hexosaminidase release in RBL 2H3 cells. In vitro assays showed sanguinarine inhibited type II PtdIns 4-kinase activity in a dose dependent fashion with no effect on PtdIns 3-kinase activity. Fluorescence spectroscopic studies suggested that sanguinarine binds to type II PtdIns 4-kinases α and β isoforms with a K_d of 2.4 and 1.8 μM, respectively. Kinetic studies showed that sanguinarine competes with PtdIns binding site of type II PtdIns 4-kinase β. These results suggest that the anti-inflammatory effects of sanguinarine on PtdIns 3-kinase signaling pathway are more likely an indirect effect and emphasize the importance of the cross talk between type II PtdIns 4-kinases and PtdIns 3-kinases.     

Increased N-acylphosphatidylethanolamine biosynthesis in elicitor-treated tobacco cells

Previous studies with tobacco (Nicotiana tabacum L.) cell suspensions indicated that elicitation of defense response (production of phytoalexins) with xylanase (1,4-β-D-xylanxylanohydrolase: EC 3.2.1.8) resulted in a dramatic acylation of phytosterols (Moreau et al. 1994). N-acylphosphatidylethanolamine (NAPE), an acylated derivative of phosphatidylethanolamine (PE), was recently demonstrated to be synthesized in vivo in plant tissues (Chapman and Moore 1993a). Here we report that acylation of PE was increased in elicitor-treated cells. NAPE levels increased 3-fold (from 1.6 to 4.8 mol% of total phospholipids) after a 2-h treatment of cell suspensions with xylanase (1 δg ml^?1). Specific activity of NAPE synthase increased in parallel with NAPE levels. Levels of NAPE and NAPE synthase activity declined during the period of 2–4 h after elicitation while levels of acylated sterolglycosides (ASG) continued to increase. Radiolabeling studies with [2^?14C]-ethanolamine confirmed that three times as much NAPE was synthesized in elicitor-treated cells compared to that in unelicited cells. Patterns of incorporation of [1-¹⁴C]-palmitic acid into membrane phospholipids in elicitor-treated cells suggested that increased acylation of lipids may be a result of changes in the acyl-coenzyme A pool. Treatment of cells with purified ethylene biosynthesis-inducing xylanase (EIX; 1 δg ml^?1 cells) resulted in increased levels of NAPE synthase activity comparable to those observed with the commercial preparations of xylanase. Boiled xylanase did not elicit an increase in the specific activity of NAPE synthase. Collectively our results demonstrate that the accumulation of NAPE in tobacco cells is attributable to increased activity of NAPE synthase. This suggests that NAPE may be specifically synthesized to play a protective role in membranes of plant cells as has been suggested for membranes of damaged animal cells.     

Sanguinarine induces apoptosis of human osteosarcoma cells through the extrinsic and intrinsic pathways

The quaternary benzo[c]phenanthridine alkaloid sanguinarine inhibits the proliferation of cancerous cells from different origins, including lung, breast, pancreatic and colon, but nothing is known of its effects on osteosarcoma, a primary malignant bone tumour. We have found that sanguinarine alters the morphology and reduces the viability of MG-63 and SaOS-2 human osteosarcoma cell lines in concentration- and time-dependent manner. Incubation with 1 μmol/L sanguinarine for 4 and 24 h killed more efficiently MG-63 cells than SaOS-2 cells, while incubation with 5 μmol/L sanguinarine killed almost 100% of both cell populations within 24 h. This treatment also changed the mitochondrial membrane potential in both MG-63 and SaOS-2 cells within 1 h, caused chromatin condensation and the formation of apoptotic bodies. It activated multicaspases, and increased the activities of caspase-8 and caspase-9 in both MG-63 and SaOS-2 cells. These data highlight sanguinarine as a novel potential agent for bone cancer therapy.     

Purification and characterization of an inducible sesquiterpene cyclase from elicitor-treated tobacco cell suspension cultures

An elicitor-inducible sesquiterpene cyclase, which catalyzes the conversion of farnesyl diphosphate to 5-epi-aristolochene (IM Whitehead, DR Threlfall, DF Ewing [1989] Phytochemistry 28:775-779) and representing a committed step in the phytoalexin biosynthetic pathway in tobacco, was purified by a combination of hydrophobic interaction, anion exchange, hydroxylapatite, and chromatofocusing chromatography. From 2 kilograms of elicited tobacco (Nicotiana tabacum) cells, approximately 500 micrograms of cyclase protein was purified, representing greater than a 130-fold increase in the specific activity of the enzyme and a 4% recovery of the starting activity. The purified enzyme resolved as two major polypeptides of 60 and 62 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biochemical characterization of the enzyme activity included an absolute requirement for magnesium, an isoelectric point of 4.5 to 4.9, and a K_m for farnesyl diphosphate of 2 to 5 micromolar. The purified cyclase protein was used to generate mouse polyclonal antibodies which efficiently inhibited cyclase activity in an in vitro assay. Electrophoresis of extracts from elicitor-treated cells or purified cyclase enzyme on native polyacrylamide gels separated the cyclase enzyme into four polypeptides as shown by immunoblot analysis using poly- and monoclonal antibodies. Proportionate cyclase enzyme activity comigrated with those polypeptides. No cyclase polypeptides were detectable in extracts of control cells by immunoblot analysis. However, immunoblot analysis of proteins from elicitor-treated cells using four independent monoclonal antibody lines and the polyclonal antibodies detected the same polypeptides, regardless of whether the proteins were separated by native or SDS-PAGE. The results suggest an induction of multiple cyclase polypeptides in elicitor-treated cells resulting from either the expression of multiple genes or multiple post-translational processing events.