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PubMed2006

Phylogenetics of Papaver and related genera based on DNA sequences from ITS nuclear ribosomal DNA and plastid trnL intron and trnL-F intergenic spacers.

BACKGROUND AND AIMS: Representatives from Papaver, Roemeria, Stylomecon and Meconopsis were studied to elucidate phylogenetic relationships between Papaver and these closely allied genera. METHODS: Two molecular data sets were used individually and combined and included sequences from the internally transcribed spacer region (ITS) of 18S-26S nuclear ribosomal DNA and the trnL intron and the trnL-trnF intergenic spacer region of plastid DNA. KEY RESULTS: Parsimony analysis demonstrated that the genus is not monophyletic unless the closely related Roemeria, Stylomecon and Meconopsis cambrica are included in a revised circumscription of Papaver. Three distinct clades are resolved in a combined ITS and trnL-F analysis. Clade 1 consists of Papaver sect. Meconella and Asian Meconopsis. Clade 2 contains a group here identified as Papaver s.s., comprising sections Carinatae, Meconidium, Oxytona, Papaver, Pilosa, Pseudopilosa and Rhoeadium. Clade 3 consists of Papaver sect. Argemonidium and Roemeria refracta. A number of diagnostic indels support these groupings. Within clade 2, sects. Papaver and Rhoeadium are either not monophyletic or lack evidence supporting their monophyly. CONCLUSIONS: The results of this molecular analysis indicate that a number of morphological characters such as valvate capsule dehiscence, dark or light filaments and sessile stigmatic discs have arisen in parallel. The phylogenetic trees are incongruent with the existing taxonomy of Papaver, and a revised classification is suggested.

DNA, Intergenic↗
PubMed2006

Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis.

Plants of the order Ranunculales, especially members of the species Papaver, accumulate a large variety of benzylisoquinoline alkaloids with about 2500 structures, but only the opium poppy (Papaver somniferum) and Papaver setigerum are able to produce the analgesic and narcotic morphine and the antitussive codeine. In this study, we investigated the molecular basis for this exceptional biosynthetic capability by comparison of alkaloid profiles with gene expression profiles between 16 different Papaver species. Out of 2000 expressed sequence tags obtained from P. somniferum, 69 show increased expression in morphinan alkaloid-containing species. One of these cDNAs, exhibiting an expression pattern very similar to previously isolated cDNAs coding for enzymes in benzylisoquinoline biosynthesis, showed the highest amino acid identity to reductases in menthol biosynthesis. After overexpression, the protein encoded by this cDNA reduced the keto group of salutaridine yielding salutaridinol, an intermediate in morphine biosynthesis. The stereoisomer 7-epi-salutaridinol was not formed. Based on its similarities to a previously purified protein from P. somniferum with respect to the high substrate specificity, molecular mass and kinetic data, the recombinant protein was identified as salutaridine reductase (SalR; EC 1.1.1.248). Unlike codeinone reductase, an enzyme acting later in the pathway that catalyses the reduction of a keto group and which belongs to the family of the aldo-keto reductases, the cDNA identified in this study as SalR belongs to the family of short chain dehydrogenases/reductases and is related to reductases in monoterpene metabolism.

Alkaloids↗
PubMed2006

[Study of Papaver somniferum cultivars identification by TD-RAPD technique].

OBJECTIVE: The feasibility of Papaver somniferum L. cultivars identification was explored by TD-RAPD technique. METHOD: Genomic DNA was extracted by improved CTAB method. One sample of species from Papaver somniferum L in xishuangbanna area. was studied by using TD-RAPD method. RESULT: We established an optimal method of extracting genomic DNA. Six primers were picked out from 10 primers. CONCLUSION: TD-RAPD could be applied to researches of molecular marker of Papaver somniferum L. TD-RAPD technique provide a method to constitute DNA database of Papaver somniferum L. and conclude the source of opium poppy.

DNA Primers↗
PubMed2001

Molecular characterization of the salutaridinol 7-O-acetyltransferase involved in morphine biosynthesis in opium poppy Papaver somniferum.

Salutaridinol 7-O-acetyltransferase (EC ) catalyzes the conversion of the phenanthrene alkaloid salutaridinol to salutaridinol-7-O-acetate, the immediate precursor of thebaine along the morphine biosynthetic pathway. We have isolated a cDNA clone that corresponds to the internal amino acid sequences of the native enzyme purified from a cell suspension culture of opium poppy Papaver somniferum. The recombinant enzyme acetylated the 7-hydroxyl moiety of salutaridinol in the presence of acetyl-CoA. The apparent K(m) value for salutaridinol was determined to be 9 microm and 54 microm for acetyl-CoA. The gene transcript was detected in extracts from Papaver orientale and Papaver bracteatum in addition to P. somniferum. Genomic DNA gel blot analysis indicated that there is likely a single copy of this gene in the P. somniferum genome. The amino acid sequence of salutaridinol 7-O-acetyltransferase is most similar (37% identity) to that of deacetylvindoline acetyltransferase of Catharanthus roseus. Salutaridinol 7-O-acetyltransferase is the second enzyme specific to morphine biosynthesis for which we have isolated a cDNA. Taken together with the other cDNAs cloned encoding norcoclaurine 6-O-methyltransferase, (S)-N-methylcoclaurine 3'-hydroxylase, the cytochrome P-450 reductase, and codeinone reductase, significant progress has been made toward accumulating genes of this pathway to enable the end goal of a biotechnological production of morphinan alkaloids.

Acetyltransferases↗
PubMed2006

Effects of Papaver rhoeas extract on the acquisition and expression of morphine-induced behavioral sensitization in mice.

In the present study, the effects of a water-alcohol extract of Papaver rhoeas on the acquisition and expression of morphine-induced behavioral sensitization in mice were investigated. The subcutaneous (s.c.) administration of morphine (50 mg/kg) induced locomotor activity in animals, whereas the drug did not show an effect at a dose of 5 mg/kg. On the other hand, intraperitoneal (i.p.) administration of the plant extract (25, 50 and 100 mg/kg) did not show any effect. The locomotor behavioral response was enhanced in mice pretreated with morphine (5 mg/kg, daily x 3 days) alone, indicating that sensitization had developed. Extract (25, 50 and 100 mg/kg, i.p.) administration, 30 min before each of the three daily doses of morphine decreased the development of sensitization. Moreover, intraperitoneal administration of the plant extract (25, 50 and 100 mg/kg) 30 min before the test reduced the expression of morphine-induced behavioral sensitization. The results indicate that administration of the extract of Papaver rhoeas reduced the acquisition and expression of morphine-induced behavioral sensitization in mice.

Animals↗
PubMed2004

Effects of Papaver rhoeas extract on the expression and development of morphine-dependence in mice.

The problem of drug dependence still remains unresolved. In the present study, the effects of water-alcohol extract of Papaver rhoeas on the expression and acquisition of naloxone-induced jumping and diarrhea in morphine-dependent mice were investigated. Administration of three daily doses of morphine (12.5, 25 and 50 mg/kg) for three days in order to develop dependence to morphine caused a significant and dose-dependent increase in the number of jumping and diarrhea when the animals were challenged with naloxone (4 mg/kg). On the other hand, administration with the plant extract (25, 50 and 100 mg/kg) did not show any effect. Injection of extract (25, 50 and 100 mg/kg) 30 min before the naloxone administration in morphine-dependent mice decreased the number of jumping and diarrhea. Administration of extract (25, 50 and 100 mg/kg) 30 min before morphine injection increased the number of jumping but decreased the diarrhea. It could be concluded that the extract of Papaver rhoeas can ameliorate the withdrawal syndrome in morphine-dependent mice. Therefore, the extract might be useful to treatment of withdrawal signs in opioid addicts.

Animals↗
PubMed2005

Effects of Papaver rhoeas extract on the acquisition and expression of morphine-induced conditioned place preference in mice.

In the present study, the effects of water-alcohol extract of Papaver rhoeas on the acquisition and expression of morphine-induced conditioned place preference (CPP) in mice were investigated. Subcutaneous (s.c.) administration of morphine (1, 10 and 20 mg/kg) produced place preference. On the other hand, intraperitoneal (i.p.) administration of the plant extract (25, 50 and 100 mg/kg) did not show any effect. Injection of extract (25, 50 and 100 mg/kg, i.p.) 30 min before the morphine administration decreased the acquisition of morphine CPP. Administration of the plant extract (25, 50 and 100 mg/kg, i.p.) 30 min before the test did not change the expression of morphine-induced CPP. It could be concluded that Papaver rhoeas reduced the acquisition but not the expression of morphine-induced conditioned place preference.

Animals↗
PubMed2004

Cytomechanical properties of papaver pollen tubes are altered after self-incompatibility challenge.

Self-incompatibility (SI) in Papaver rhoeas triggers a ligand-mediated signal transduction cascade, resulting in the inhibition of incompatible pollen tube growth. Using a cytomechanical approach we have demonstrated that dramatic changes to the mechanical properties of incompatible pollen tubes are stimulated by SI induction. Microindentation revealed that SI resulted in a reduction of cellular stiffness and an increase in cytoplasmic viscosity. Whereas the former cellular response is likely to be the result of a drop in cellular turgor, we hypothesize that the latter is caused by as yet unidentified cross-linking events. F-actin rearrangements, a characteristic phenomenon for SI challenge in Papaver, displayed a spatiotemporal gradient along the pollen tube; this suggests that signal propagation occurs in a basipetal direction. However, unexpectedly, local application of SI inducing S-protein did not reveal any evidence for localized signal perception in the apical or subapical regions of the pollen tube. To our knowledge this represents the first mechanospatial approach to study signal propagation and cellular responses in a well-characterized plant cell system. Our data provide the first evidence for mechanical changes induced in the cytoplasm of a plant cell stimulated by a defined ligand.

Actins↗
PubMed2000

Evidence for DNA fragmentation triggered in the self-incompatibility response in pollen of Papaver rhoeas.

Studies of the molecular and biochemical basis of self-incompatibility (SI) in Papaver rhoeas have revealed much about the signalling pathways triggered in pollen early in this response. The aim of the current investigation was to begin to study downstream events in order to elucidate some of the later cellular responses involved in the SI response and identification of the mechanisms controlling the irreversible inhibition of pollen tube growth. We have used the FragEL assay to investigate if there is any evidence for DNA fragmentation stimulated in pollen of P. rhoeas in an S-specific manner. Our data clearly demonstrate that S proteins are responsible for triggering this, specifically in incompatible, and not compatible, pollen. DNA fragmentation was first detected in incompatible pollen tubes 4 h after challenge with S proteins, and continued to increase for a further 10 h. This provides the first evidence, to our knowledge, that this phenomenon is associated with the SI response. We also demonstrate that mastoparan, which increases [Ca2+]i, also triggers DNA fragmentation in these pollen tubes, thereby implicating an involvement of Ca2+ signalling in this process. Together, our data represent a significant breakthrough in understanding of the SI response in Papaver pollen.

Calcium Signaling↗
PubMed2003

Genomic organization of the Papaver rhoeas self-incompatibility S(1) locus.

The self-incompatibility (SI) response in Papaver rhoeas depends upon the cognate interaction between a pollen-expressed receptor and a stigmatically expressed ligand. The genes encoding these components are situated within the S-locus. In order for SI to be maintained, the genes encoded by the S-locus must be co-inherited with no recombination between them. Several hypotheses, including sequence heterogeneity and chromosomal position, have been put forward to explain the maintenance of the S-locus in the SI systems of the Brassicaceae and the Solanaceae. A region of the Papaver rhoeas genome encompassing part of the self-incompatibility S(1) locus has been cloned and sequenced. The clone contains the gene encoding the stigmatic component of the response, but does not contain a putative pollen S-gene. The sequence surrounding the S(1) gene contains several diverse repetitive DNA elements. As such, the P. rhoeas S-locus bears similarities to the S-loci of other SI systems. An attempt to localize the P. rhoeas S-locus using fluorescence in situ hybridization (FISH) has also been made. The potential relevance of the findings to mechanisms of recombination suppression is discussed.

Amino Acid Sequence↗
PubMed2005

A rapid and reliable solid-phase extraction method for high-performance liquid chromatographic analysis of opium alkaloids from papaver plants.

A rapid and reliable solid-phase extraction method for HPLC analysis of opium alkaloids from Papaver plants was established. Fifty mg of dried and powdered plant sample was extracted with 5 ml of 5% acetic acid for 30 min under sonication. After centrifugation, 3 ml of the supernatant was loaded on a reversed-phase cation-exchange solid-phase extraction cartridge. After seriate washings with 0.1 M hydrochloric acid and methanol, alkaloids were eluted with a mixture of 28% ammonia and methanol (1:19). The eluate was concentrated under nitrogen stream at 40 degrees C and the residue was dissolved in 50% aqueous methanol for high performance liquid chromatographic analysis. With this solid-phase extraction method, the recovery of morphine, codeine, oripavine, thebaine, papaverine, noscapine and sanguinarine was from 99.94 to 112.18% when the standard alkaloids were added to the plant samples. Opium alkaloids of a variety of genus Papaver plants cultivated in a field and phytotron were analyzed by this method.

Alkaloids↗
PubMed1999

[Determination of thebaine in various tissues of papaver bracteatum by gas chromatography].

Papaver bracteatum is an important source of opiates because of its high thebaine content. An efficient and convenient method of determining thebaine, not only in the root, but in the more highly pigmented capsule, stem and leaf, is urgently required. We decided to examine these in detail on a wide variety of plant material and to choose an assay method which is convenient and gives reproducible results. A simple method for the quantitative extraction and analysis of thebaine in the different tissues (capsule, root, leaf and stem) of papaver bracteatum has been established. The powdered materials were extracted with methanol under ultrasonic condition. The procedure of extraction is simple, rapid and reliable. The assay method gave satisfactory reproducibility for a wide range of plant materials (coefficient of variation 2.9% to 5.4%). The linear range of response of thebaine was from 5.0 to 500.0 mg/L. The minimum limit of detection was 2 ng. The operating time of the assay is about 20 min. The presence of other alkaloids did not interfere with the assay reported here in.

Chromatography, Gas↗
PubMed2005

[Determination of papaverine in seeds of Papaver somniferum L. and soup of chafing dish by high performance liquid chromatography-fluorescence detection].

A method of reversed-phase high performance liquid chromatography coupled with fluorescence detection was developed for the separation and determination of papaverine in seeds of Papaver somniferum L. and soup of chafing dish. The liquid chromatographic experiment was carried out under the following conditions: the detector was a fluorescence with excitation wavelength at 285 nm and emission wavelength at 355 nm. An RP-C18 column (250 mm x 4.6 mm i.d., 5 microm) was used. The mobile phase was a mixture of methanol and 0.02 mol/L ammonium acetate (70: 30, v/v). The flow rate was 0.8 mL/min. A linear range was obtained from 0.1 ng to 0.1 microg with a good correlation. The lowest detection limit was 0.02 ng. The average recovery of seeds for Papaver somniferum L. was 99.0% - 100.8%. The method is rapid, accurate, sensitive and suitable for the separation and determination of papaverine in foods.

Chromatography, High Pressure Liquid↗