Comparative macroarray analysis of morphine containing Papaver somniferum and eight morphine free Papaver species identifies an O-methyltransferase involved in benzylisoquinoline biosynthesis

Benzylisoquinoline alkaloids constitute a group of about 2,500 structures and are mainly produced by plants of the order Ranunculales. But only the opium poppy, Papaver somniferum, and Papaver setigerum are able to produce morphine. In this study, we started to investigate by gene expression analysis the molecular basis for this exceptional biosynthetic ability. A sequencing project from P. somniferum seedlings was initiated using a method based on the amplified fragment length polymorphism technique that resulted in 849 UniGenes. These cDNAs were analysed on macroarrays for differential expression between morphine-containing P. somniferum plants and eight other Papaver species, which accumulate other benzylisoquinolines instead of morphine. Three cDNAs showing increased expression in P. somniferum compared to all the other Papaver species were identified. Whereas two showed no significant homology to any known protein, one putatively encoded an O-methyltransferase. Analysis of substrate specificity of the heterologously expressed protein and mass spectrometric identification of the enzymatic products identified this protein as S-adenosyl-L-methionine:(R,S)-3-hydroxy-N-methylcoclaurine 4-O-methyltransferase (EC 2.1.1.116). Unlike other O-methyltransferases of different positional specificities implicated in benzylisoquinoline metabolism, the enzyme only accepted tetrahydroxylated tetrahydrobenzylisoquinolines as substrates; methylation was tolerated only at the 6-hydroxy position.     

Host range of Peronospora somniferi and Pe. meconopsidis within selected members of the Papaveraceae under controlled conditions

Opium poppy, belonging to the family Papaveraceae, is grown for its alkaloid compounds of pharmaceutical value. Downy mildew caused by Peronospora somniferi and Pe. meconopsidis, substantially impacts crop production. The present study was conducted to identify the host range of Pe. somniferi and Pe. meconopsidis within selected weed and ornamental members of the Papaveraceae family. Nine Papaver spp., Meconopsis cambrica and a nonhost control (tomato, Solanum lycopersicum) were challenged with both pathogens under controlled glasshouse or laboratory conditions using infested soil or foliar applied sporangia as inocula. Peronospora somniferi and Pe. meconopsidis induced disease symptoms, including sporulation, in at least one trial for all tested species except for Pa. atlanticum and tomato. Species-specific PCR testing of foliage of challenged plants confirmed infections by both pathogen species of symptomatic plants, identifying these as hosts. Positive PCR tests were also obtained from Pa. atlanticum plants for both pathogens. However, in the absence of pathogen sporulation structures as further evidence of infection, the host status of Pa. atlanticum remains inconclusive. Testing of seeds collected from Pe. somniferi and Pe. meconopsidis infected plants of Pa. somniferum, Pa. dubium, Pa. rhoeas and Pa. nudicaule showed presence of both pathogens, indicating likely ability for seed transmission in these species. We identified new hosts of these pathogens and discuss potential implications of these alternative hosts in pathogen survival, dissemination and epidemic initiation.     

Papaver bracteatum,potential commercial source of codeine

Papaver bracteatum, native to Iran and southern Russia, has been grown successfully in many countries. Research in the northwest United States has confirmed the potential for its commercial production as a source of the alkaloid thebaine. Potential for the chemical conversion of thebaine into codeine, one of man’s most widely used alkaloidal medicinal agents, is reviewed. Economic and social advantages of growing this species over opium poppy (Papaver somniferum) are discussed. The value of the seed oil for cooking and industrial use is considered.     

A cultivated poppy (Papaver sp.) invades wild habitats of Papaver fauriei in the mountain area of Rishiri Island, Japan

Many tiny yellow poppies are grown in the town area of Rishiri Island, Japan. Because the phenotype of this cultivated poppy is similar to that of Papaver fauriei, which is endemic to Mt. Rishiri, Rishiri Island, residents on the island call the cultivated poppy “P. fauriei” although the origin of the cultivated poppy is uncertain. To estimate the origin of the cultivated poppy, its internal transcribed spacer (ITS) sequence was compared with those found in the Far East wild poppies P. fauriei, P. alboroseum, P. miyabeanum and P. nudicaule. Although the ITS sequence of the cultivated poppy was not identical to those found in the wild species, it was most similar to that of P. miyabeanum, indicating that the cultivated poppy is not P. fauriei. However, cultivated poppy seeds have been sown several times over a period of at least 20 years in wild P. fauriei habitats on Mt. Rishiri in the hope of aiding the recovery of P. fauriei populations in wild habitats. Poppy plants in the wild habitats where such seeds have been sown showed the same ITS sequences as those of the cultivated poppy, indicating that the cultivated poppy is established in these wild habitats. This is a case of a nonindigenous species being introduced to wild habitats through human actions.     

Morphological and molecular analyses support the existence of host-specific Peronospora species infecting Chenopodium

About 20 species of Peronospora have been reported to cause downy mildew on Chenopodium, but, particularly in plant pathology literature, only one species, P. farinosa, is considered to be involved. We performed sequence analysis of the ITS rDNA to reveal the phylogenetic relationships of Peronospora specimens from five species of Chenopodium, viz. C. album, C. ambrosioides, C. bonus-henricus, C. hybridum, and C. polyspermum. The five clades corresponded to particular Chenopodium species, and showed a high level of sequence divergence. Differences in the morphology of the conidia and ultimate branchlets also supported the separation of the five groups at the host species level. These results suggest that the names P. variabilis, P. boni-henrici, P. chenopodii, and P. chenopodii-polyspermi should be used for the four downy mildew pathogens specific to C. album, C. bonus-henricus, C. hybridum, and C. polyspermum, respectively. The Peronospora on C. ambrosioides was found to be an independent species.     

Ability of the endangered species Papaver fauriei to produce hybrids with a cultivated poppy (Papaver sp.)

Papaver fauriei is an endemic and endangered species that grows only on the gravelly alpine slopes of Mt. Rishiri, Japan. Cultivated poppy (Papaver sp.), the species name of which is unknown, has been introduced to the natural habitats of P. fauriei through human activities. Because the appearance and internal transcribed spacer (ITS) sequences of these two poppies are highly similar, it is of concern that they could produce hybrids in their natural habitats. Thus, first, the ability of these two poppies to produce hybrids was analyzed by artificial fertilization in this study. A large number of seeds were produced by reciprocal crosses between P. fauriei and the cultivated poppy, comparable with the number of seeds obtained by self‐ or cross‐fertilization of P. fauriei or the cultivated poppy. In addition, high germination was observed for seeds obtained from crosses between the two poppies, and deleterious phenotypes, such as albinism and dwarfism, were not detected in the F₁ generation. These results indicate that after pollination, there is no reproductive isolation between the two poppies. Second, we sequenced the internal transcribed spacer (ITS) region of 240 poppy individuals collected from the gravelly alpine slopes of Mt. Rishiri, and 66 showed the sequence of P. fauriei, whereas 174 showed the sequence of the cultivated poppy. However, the ITS sequence that confirms hybridism between the two poppies was not detected in these individuals, indicating that hybridization of P. fauriei and the cultivated poppy rarely occurs under natural conditions. Unknown mechanism(s) appear to prevent cross‐pollination between the two poppies.     

Re-consideration of Peronospora farinosa infecting Spinacia oleracea as distinct species, Peronospora effusa

Downy mildew is probably the most widespread and potentially destructive global disease of spinach (Spinacia oleracea). The causal agent of downy mildew disease on various plants of Chenopodiaceae, including spinach, is regarded as a single species, Peronospora farinosa. In the present study, the ITS rDNA sequence and morphological data demonstrated that P. farinosa from S. oleracea is distinct from downy mildew of other chenopodiaceous hosts. Fifty-eight spinach specimens were collected or loaned from 17 countries of Asia, Europe, Oceania, North and South America, which all formed a distinct monophyletic group. No intercontinental genetic variation of the ITS rDNA within Peronospora accessions causing spinach downy mildew disease was found. Phylogenetic trees supported recognition of Peronospora from spinach as a separate species. Microscopic examination also revealed morphological differences between Peronospora specimens from Spinacia and P. farinosa s. lat. specimens from Atriplex, Bassia, Beta, and Chenopodium. Consequently, the name Peronospora effusa should be reinstated for the downy mildew fungus found on spinach. Here, a specimen of the original collections of Peronospora effusa is designated as lectotype.     

Purification and Characterization of Calmodulins from Papaver somniferum and Euphorbia lathyris

Calmodulins (CaM) were isolated and characterized from two well-known latex producing plants, Papaver somniferum and Euphorbia lathyris. The molecular weights of both were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 17,000 comparable to that of bovine brain CaM. Amino acid compositions also compared similarly with those of known CaMs, with regard to the presence of trimethyllysine and the ratio of phenylalanine to tyrosine. The Cornish-Bowden equation (SΔn) revealed strong statistical correlations of P. somniferum and E. lathyris CaM with those of other plants and animals, although their amino acid compositions were not identical. Both plant CaM stimulated CaM dependent cAMP phosphodiesterase: for Papaver somniferum the K_a was found to be 1.09 nanomolar and for Euphorbia lathyris, 2.01 nanomolar.     

Identification and cloning of related self-incompatibility S-genes in Papaver rhoeas and Papaver nudicaule

 The primary goal of this study was to identify, clone and analyse new S-gene sequences in order to provide a basis for identifying amino acid residues that confer S-allele specificity. Three new putative S-alleles from Papaver rhoeas and Papaver nudicaule were identified using immunological and PCR methods. cDNAs encoding full-length open reading frames of the P. rhoeas S ₈ and P. nudicaule Sn ₁ genes were isolated. Nucleotide sequencing of these cDNAs, together with the partial S ₇ sequence obtained by PCR, was used to derive the corresponding amino acid sequences. It is of interest that the P. nudicaule Sn₁ sequence, which is the first S-allele isolated from another species of Papaver, shares a closer sequence identity to the P. rhoeas S₃ amino acid sequence than S₃ does to S₁ from P. rhoeas. The identity of the S₈ allele was confirmed by expressing the coding region in Escherichia coli and demonstrating that the recombinant protein, designated S₈e, specifically inhibited S ₈ pollen in an in vitro bioassay. Information from sequence analysis of the S₈, Sn₁ and partial S₇ amino acid sequences revealed important information about Papaver S-proteins. It confirmed previous observations based on only two S-alleles, that whilst exhibiting a high degree of amino acid sequence polymorphism ranging from 51.3% to 63.7%, these molecules probably share very similar secondary structures. These studies also revealed that, in contrast to the S-proteins from the Solanaceae and Brassica, amino acid sequence variation is not found in hypervariable blocks, but instead, is found throughout the S-proteins, interspersed with numerous short strictly conserved segments. Received: 16 March 1998 / Revision accepted: 19 May 1998     

Taxonomy and phylogenetic position of <Emphasis Type="Italic">Phyllactinia takamatsui</Emphasis>, a newly described powdery mildew on cotoneaster,based on molecular and morphological data

The newly recognised powdery mildew species Phyllactinia takamatsui on Cotoneaster nummularius (Rosaceae) is described and illustrated. This species, collected in Kerman Province, Iran, is well characterised by its conidial morphology and rDNA ITS sequences clearly different from allied species. Conidia are broadly ellipsoid to subcylindrical, i.e. they are not clavate-spathuliform as in most Phyllactinia species. The rDNA ITS sequence analysis showed that this species is closely allied to other species described on hosts belonging to Rosaceae, such as Ph. mali and Ph. pyri-serotinae. The ITS sequence of P. takamatsui was 92 to 94 % similar to that of the closest known relatives. The new species is described in detail, illustrated and compared with other similar taxa.     

3属4种罂粟科植物幼苗初生维管系统的结构

罂粟科属间演化关系一直存在争议。本文以罂粟科罂粟属的虞美人（Papaver rhoeas）和东方罂粟（Papaver orientale）、白屈菜属的白屈菜（Chelidonium majus）以及紫堇属的地丁草（Corydalis bungeana）为材料,应用整体透明和石蜡连续切片的方法,在明场显微镜和偏光显微镜下对其幼苗期的结构及发育进行了观察。结果表明,3属植物幼苗子叶节区下部存在的单中柱类型各异：罂粟属为外始式二原型圆形单中柱,白屈菜属为外始式二原型近圆形单中柱,紫堇属为外始式二原型“S”形单中柱。3属植物的子叶脉序也存在区别：虞美人和东方罂粟具单一叶脉,无叶隙;白屈菜为环结曲行羽状脉;地丁草为基出完全顶聚脉。根据上述幼苗初生维管系统的结构,认为3属中罂粟属较为原始,白屈菜属较为进化,紫堇属更为进化。     

Pseudoidium javanicum,a new species of powdery mildew on Acalypha spp. from Indonesia

Pseudoidium javanicum is proposed as a new species based on analyses of 28S, ITS and IGS rDNA sequences, and morphological data. This new species was found on Acalypha wilkesiana var. marginata, A. argentea, and A. cristata collected from Cibodas Botanical Garden, Bogor (West Java Province, Indonesia). Our analyses showed that all these specimens have identical rDNA sequences and similar morphological characteristics. They form a distinct clade separated from other species of Erysiphaceae. Pseudoidium javanicum differs from Erysiphe acalyphae by having shorter conidiophores and foot cells 1–3 times as long as the 0–2 following cells. The conidial size of Ps. javanicum is also smaller than that of E. jatrophae.     

Genetic diversity and host range of powdery mildews on Papaveraceae

Because of the strong morphological similarity of the powdery mildew fungi that infect papaveraceous hosts, a total of 39 samples were studied to reveal the phylogeny and host range of these fungi. ITS and 28S sequence analyses revealed that the isolates identified earlier as Erysiphe cruciferarum on papaveraceous hosts represent distinct lineages and differ from that of E. cruciferarum sensu stricto on brassicaceous hosts. The taxonomic status of the anamorph infecting Eschscholzia californica was revised, and therefore, a new species name, Erysiphe eschscholziae, is proposed. The taxonomic position of the Pseudoidium anamorphs infecting Glaucium flavum, Meconopsis cambrica, Papaver dubium, and Stylophorum diphyllum remain unclear. This study revealed that Erysiphe macleayae exhibits a specific host range different from that of E. cruciferarum, the common pathogen of papaveraceous hosts. Although E. macleayae occurred naturally on Macleaya cordata, Macleaya microcarpa, M. cambrica, and Chelidonium majus only, our inoculation tests revealed that the fungus was capable of infecting Argemone grandiflora, Glaucium corniculatum, Papaver rhoeas, and Papaver somniferum, indicating that these plant species may also be taken into account as potential hosts. Erysiphe cruciferarum originating from P. somniferum was not able to infect A. grandiflora, C. majus, E. californica, M. cordata, and P. rhoeas. The emergence of E. macleayae on M. microcarpa is reported here for the first time from the Czech Republic and Slovakia. The appearance of chasmothecia of E. macleayae on C. majus in Slovakia was reported, as well. Erysiphe cruciferarum was identified on G. corniculatum and reported here for the first time from Slovakia.     

Bound morphine and codeine in the capsule of Papaver somniferum

The polysaccharide fraction of the capsule of Papaver somniferum contained bound morphine and codeine. The alkaloids appear to be bound to the polymer by two different types of linkage.     

Taxonomy and phylogeny of Puccinia lagenophorae: a study using rDNA sequence data, morphological and host range features

Puccinia lagenophorae is a rust fungus originating from Australasia which has spread throughout the world. A phylogenetic analysis of taxa related to this species was performed using rDNA (LSU, ITS) sequence data. The analyses revealed a well-supported cluster including all specimens of P. lagenophorae. By evaluating morphological and sequence data, the species is taxonomically re-defined and a list of synonyms is provided. Puccinia distincta on Bellis perennis, a species recently separated from P. lagenophorae, P. saccardoi, a species on the Goodeniaceae, and P. byliana, a species so far only known from South Africa, are reduced to synonymy in P. lagenophorae, as are several other species. Our analysis indicates that P. lagenophorae is likely not derived from the northern hemisphere species P. obscura, but from a species from Australia host-alternating between Asteraceae (aecial host) and Cyperaceae/Juncaceae (telial host). Another related species, P. stylidii (on Stylidium sp., Stylidiaceae) may have been derived from the same parental species as P. lagenophorae. From ontogenetical and morphological studies, the presence of pycnia could not be confirmed in the life cycle of this species, and the width of the pedicel of teliospores at the point of attachment was found to be highly variable and not a taxonomic character. The number of known host species is approximately 150, including 41 new host plants recorded herein.     

Phylogenetic and genetic studies in Papaver section Oxytona: cytogenetics,isozyme analysis and chloroplast DNA variation

Summary Chromosome behaviour at meiosis, isozyme studies and analysis of the chloroplast DNA restriction fragments were used to assess the phylogenetic relations among the three Papaver species of the section Oxytona. The multivalents observed in diplotene — diakinesis stages of meiosis of the hexaploid P. pseudo-orientale and its tetraploid hybrid with P. bracteatum indicate the autopolyploid nature of this section. Further evidence supporting this conclusion was obtained from isozyme analysis. The same number of isozymes was expressed in all the species regardless of their ploidy level. Inheritance studies conducted with Pgi, Dia and Acp allozymes demonstrated, for the first time, the transfer and expression of genetic material among these species. The differences found in the chloroplast DNA restriction fragments of the Oxytona species and of P. somniferum indicate intensive evolution of the chloroplast DNA in the genus Papaver. The similarity of the chloroplast DNA restriction patterns and of the isozymes in P. orientale and P. pseudo-orientale suggested that P. orientale was the female parent in the cross generating P. pseudo-orientale and that the latter species is of recent origin.Contribution no. 2199 —E, 1987 series from the Agricultural Research Organization, The Volcani center, Bet Dagan 50 250, Israel