Molecular characterization of a seed transmitted clavicipitaceous fungus occurring on dicotyledoneous plants (Convolvulaceae)

Ergoline alkaloids (syn. ergot alkaloids) are constituents of clavicipitaceous fungi (Ascomycota) and of one particular dicotyledonous plant family, the Convolvulaceae. While the biology of fungal ergoline alkaloids is rather well understood, the evolutionary and biosynthetic origin of ergoline alkaloids within the family Convolvulaceae is unknown. To investigate the possible origin of ergoline alkaloids from a plant-associated fungus, 12 endophytic fungi and one epibiotic fungus were isolated from an ergoline alkaloid-containing Convolvulaceae plant, Ipomoea asarifolia Roem. & Schult. Phylogenetic trees constructed from 18S rDNA genes as well as internal transcribed spacer (ITS) revealed that the epibiotic fungus belongs to the family Clavicipitaceae (Ascomycota) whereas none of the endophytic fungi does. In vitro and in vivo cultivation on intact plants gave no evidence that the endophytic fungi are responsible for the accumulation of ergoline alkaloids in I. asarifolia whereas the epibiotic clavicipitaceous fungus very likely is equipped with the genetic material to synthesize these compounds. This fungus resisted in vitro and in vivo cultivation and is seed transmitted. Several observations strongly indicate that this plant-associated fungus and its hitherto unidentified relatives occurring on different Convolvulaceae plants are responsible for the isolated occurrence of ergoline alkaloids in Convolvulaceae. This is the first report of an ergot alkaloid producing clavicipitaceous fungus associated with a dicotyledonous plant.Data deposition: The sequences reported in this paper have been deposited in the GenBank (accession numbers are given in the text and in Fig. 3) Dedicated to Dr. Dr. h. c. mult. Albert Hofmann, the great pioneer of ergot research, on the occasion of his 100th birthday     

Biosynthesis and accumulation of ergoline alkaloids in a mutualistic association between Ipomoea asarifolia (Convolvulaceae) and a clavicipitalean fungus

Ergoline alkaloids occur in taxonomically unrelated taxa, such as fungi, belonging to the phylum Ascomycetes and higher plants of the family Convolvulaceae. The disjointed occurrence can be explained by the observation that plant-associated epibiotic clavicipitalean fungi capable of synthesizing ergoline alkaloids colonize the adaxial leaf surface of certain Convolvulaceae plant species. The fungi are seed transmitted. Their capacity to synthesize ergoline alkaloids depends on the presence of an intact differentiated host plant (e.g. Ipomoea asarifolia or Turbina corymbosa [Convolvulaceae]). Here, we present independent proof that these fungi are equipped with genetic material responsible for ergoline alkaloid biosynthesis. The gene (dmaW) for the determinant step in ergoline alkaloid biosynthesis was shown to be part of a cluster involved in ergoline alkaloid formation. The dmaW gene was overexpressed in Saccharomyces cerevisiae, the encoded DmaW protein purified to homogeneity, and characterized. Neither the gene nor the biosynthetic capacity, however, was detectable in the intact I. asarifolia or the taxonomically related T. corymbosa host plants. Both plants, however, contained the ergoline alkaloids almost exclusively, whereas alkaloids are not detectable in the associated epibiotic fungi. This indicates that a transport system may exist translocating the alkaloids from the epibiotic fungus into the plant. The association between the fungus and the plant very likely is a symbiotum in which ergoline alkaloids play an essential role.     

Ergoline alkaloids in convolvulaceous host plants originate from epibiotic clavicipitaceous fungi of the genus Periglandula

Ergoline (i.e., ergot) alkaloids are a group of physiologically active natural products occurring in the taxonomically unrelated fungal and plant taxa, Clavicipitaceae and Convolvulaceae, respectively. The disjointed occurrence of ergoline alkaloids seems to contradict the frequent observation that identical or at least structurally related natural products occur in organisms with a common evolutionary history. This problem has now been solved by the finding that not only graminaceous but also some dicotyledonous plants belonging to the family Convolvulaceae, such as Ipomoea asarifolia and Turbina corymbosa, form close associations with ergoline alkaloid producing fungi, Periglandula ipomoeae and Periglandula turbinae. These species belong to the newly established genus Periglandula within the Clavicipitaceae. The fungus–plant associations are likely to be mutualistic symbioses.     

Specificity in the interaction between an epibiotic clavicipitalean fungus and its convolvulaceous host in a fungus/plant symbiotum

Ipomoea asarifolia and Turbina corymbosa (Convolvulaceae) are associated with epibiotic clavicipitalean fungi responsible for the presence of ergoline alkaloids in these plants. Experimentally generated plants devoid of these fungi were inoculated with different epibiotic and endophytic fungi resulting in a necrotic or commensal situation. A symbiotum of host plant and its respective fungus was best established by integration of the fungus into the morphological differentiation of the host plant. This led us to suppose that secretory glands on the leaf surface of the host plant may play an essential role in ergoline alkaloid biosynthesis which takes place in the epibiotic fungus.Key words: ergoline alkaloids, ipomoea, turbina, convolvulaceae, claviceps, balansia, clavicipitaceae, penicillium, plant-fungus symbiotum     

Identification of ergoline alkaloids in the genus Argyreia and related genera and their chemotaxonomic implications in the convolvulaceae

The results of the identification of 21 ergoline alkaloids of 14 species of Argyreia; viz.: A. barnesii, A. capitata, A. cuneata, A. luzoninsis, A. mollis, A. maingayi, A. nervosa, A. obtusifolia, A. philippinensis, A. reticulata. A. ridleyi, A. rubicunda, A. splendens and A. sp. and 2 closely related genera; Stictocardia tiliafolia and Rivea corymbosa, by using 2-D TLC procedures are given. A brief discussion of the implications of the ergoline alkaloids and the chemotaxonomy of the Convolvulaceae is also presented.     

The Fungus Penicillium citrinum Thom 1910 VKM FW-800 Isolated from Ancient Permafrost Sediments As a Producer of the Ergot Alkaloids Agroclavine-1 and Epoxyagroclavine-1

The study of the secondary metabolites of the relict strain Penicillium citrinum VKM FW-800 isolated from ancient Arctic permafrost sediments showed that this fungus produces agroclavine-1 and epoxyagroclavine-1, which are rare ergot alkaloids with the 5R,10S configuration of the tetracyclic ergoline ring system. The production of the alkaloids by the fungus showed a biphasic behavior, being intense in the phase of active growth and slowing down in the adaptive lag phase and in the stationary growth phase. The addition of zinc ions to the incubation medium led to a fivefold increase in the yield of the alkaloids. The alkaloid-producing Penicillium fungi isolated from different regions exhibited the same tendencies of growth and alkaloid production.     

Biosynthesis of ergot alkaloids. Some new results on an old problem (Review)]

The biosynthetic pathway leading from L-tryptophan, mevalonic acid and methionine to the tetracyclic ergoline ring system of the ergot alkaloids in Claviceps species is reviewed. This pathway entails many mechanistically intriguing features. Recent studies are also discussed which reveal the stereochemical course of the isoprenylation of tryptophan and of the N-methylation of dimethylallyltryptophan (DMAT) and which shed some light on the likely steps leading from the open-chain precursors, N-methyl-DMAT to the tricyclic intermediate, chanoclavine-1. Finally, some plans are outlined to probe the evolutionary relationship of ergot alkaloid biosynthesis in fungi to that in higher plants of the family Convolvulaceae.     

Improvement by soil yeasts of arbuscular mycorrhizal symbiosis of soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) colonized by<Emphasis Type="Italic"> Glomus mosseae</Emphasis>

The effects of the soil yeasts Rhodotorula mucilaginosa, Cryptococcus laurentii and Saccharomyces kunashirensis on the arbuscular mycorrhizal (AM) fungus Glomus mosseae (BEG 12) was studied in vitro and in greenhouse trials. The presence of yeasts or their soluble and volatile exudates stimulated the percentage spore germination and hyphal growth of G. mosseae. Percentage root length colonized by G. mosseae and plant dry matter of soybean (Glycine max L. Merill) were increased only when the soil yeasts were inoculated prior to the AM fungus. Higher beneficial effects on AM colonization and plant dry matter were found when the soil yeasts were inoculated as an aqueous solution rather than as a thin agar slice. Although soluble and volatile exudates of yeasts benefited the AM symbiosis, their modes of action were different.This revised version was published online in May 2004 with corrections to the section of the article.     

Cloning,expression in yeast,and functional characterization of CYP71D14, a root-specific cytochrome P450 from <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Alkaloids, which are naturally occurring amines, are biosynthesized and accumulated in plant tissues such as roots, leaves, and stems. Many alkaloids have pharmacological effects on humans and animals. Cytochrome P450 (P450 or CYP) monooxygenases are known to play key roles in the biosynthesis of alkaloids in higher plants. A cDNA clone encoding a P450 protein consisting of 502 amino acids was isolated from Petunia hybrida. The deduced amino acid sequence of the cDNA clone showed a high level of similarity with the other P450 species in the CYP71D family; hence, this novel P450 was named CYP71D14. Among plant P450 species, CYP71D14 had 45.7% similarity in its amino acid sequence to CYP71D12, which is involved in the biosynthesis of the indole alkaloids vinblastine and vincristine. Expression of CYP71D14 mRNA in Petunia plants was examined by Northern blot analysis by using a full-length cDNA of CYP71D14 as a probe. CYP71D14 mRNA was expressed most abundantly in the roots. The nucleotide sequence of CYP71D14 has been submitted to the DDBJ, EMBL, and GenBank nucleotide databases under the accession number AB028462. An erratum to this article can be found at     

(Chemotaxonomic) Implications of Postharvest/Storage‐Induced Changes in Plant Volatile Profiles – the Case of Artemisia absinthium L. Essential Oil

The plant volatile profile and the essential‐oil chemical composition change during the storage of plant material. The objective of this study was to develop a mathematical model able to predict, explain, and quantify these changes. Mathematical equations, derived under the assumption that the essential oil contained within plant material could be treated as an ideal solution (Raoult's law), were applied for tracking of postharvest changes in the volatile profile of Artemisia absinthium L. (the essential oils were analyzed by GC‐FID and GC/MS). Starting from a specific chemical composition of an essential‐oil sample obtained from plant material after a short drying period (typically 5–10 d), and by using the equations derived from this model, one could easily predict evaporation‐induced changes in the volatile profile of the plant material. Based on the composition of the essential‐oil sample obtained after a given storage time t, it is possible to identify those components that were involved in chemical reactions, both as reactants and possible products. The established model even allowed the recognition of pairs of transformation, i.e., ‘daughter’ products and their ‘parent’ compounds. The obtained results highlight that the essential‐oil composition is highly dependent on the storage period of any plant material and urges caution in different types of phytochemical studies, especially chemotaxonomic ones, or practical application.     

Putrescine <Emphasis Type="Italic">N</Emphasis>-methyltransferases—a structure–function analysis

Putrescine N-methyltransferase (PMT) is a key enzyme of plant secondary metabolism at the start of the specific biosynthesis of nicotine, of tropane alkaloids, and of calystegines that are glycosidase inhibitors with nortropane structure. PMT is assumed to have developed from spermidine synthases (SPDS) participating in ubiquitous polyamine metabolism. In this study decisive differences between both enzyme families are elucidated. PMT sequences were known from four Solanaceae genera only, therefore additional eight PMT cDNA sequences were cloned from five Solanaceae and a Convolvulaceae. The encoded polypeptides displayed between 76% and 97% identity and typical amino acids different from plant spermidine synthase protein sequences. Heterologous expression of all enzymes proved catalytic activity exclusively as PMT and K _cat values between 0.16 s⁻¹ and 0.39 s⁻¹. The active site of PMT was initially inferred from a protein structure of spermidine synthase obtained by protein crystallisation. Those amino acids of the active site that were continuously different between PMTs and SPDS were mutated in one of the PMT sequences with the idea of changing PMT activity into spermidine synthase. Mutagenesis of active site residues unexpectedly resulted in a complete loss of catalytic activity. A protein model of PMT was based on the crystal structure of SPDS and suggests that overall protein folds are comparable. The respective cosubstrates S-adenosylmethionine and decarboxylated S-adenosylmethionine, however, appear to bind differentially to the active sites of both enzymes, and the substrate putrescine adopts a different position.     

Screening for Ergot Alkaloid Producers among Microscopic Fungi by Means of the Polymerase Chain Reaction

The potential of the polymerase chain reaction for the detection of ergot alkaloid producers among microscopic fungi of the generaPenicilliumand Clavicepswas evaluated. Twenty-three strains of various species of fungi with a previously studied capacity for alkaloid production were used. The internal fragment of the gene encoding 4-dimethylallyltryptophan synthase, the enzyme catalyzing the first step in the biosynthesis of ergot alkaloids, was amplified using degenerate primers. This approach revealed an about 1.2-kb specific DNA fragment in micromycetes synthesizing ergot alkaloids with complete tetracyclic ergoline system. Microorganisms that produce alkaloids with modified C or D ergoline rings, as well as -cyclopiazonic acid, did not yield the PCR fragment of the expected size. This fragment was also not found in fungi incapable of ergot alkaloid production.     

A phylogeny inferred from large ribosomal subunit (26S) rDNA sequences suggests that Cuscuta is a derived member of Convolvulaceae

Cuscuta is a parasitic angiosperm that has been considered alternatively either as a genus within Convolvulaceae or as a monogeneric family in its own right. Although typically placed in the Solanales,Cuscuta has also been positioned within the Polemoniales. Extreme reduction of morphological and anatomical characters, as well as chloroplast genome reductions and rearrangements, has made the phylogenetic placement ofCuscuta uncertain. Analysis of 26S rDNA sequences suggests thatCuscuta is a derived member of Convolvulaceae. Molecular results are discussed in relation to the morphological and anatomical characters of autotrophic members of Convolvulaceae.     

Alkaloid profile and antimicrobial activity of <Emphasis Type="Italic">Lupinus angustifolius</Emphasis> L. alkaloid extract

Purpose of the present study was to evaluate alkaloid profile of the aerial parts of Lupinus angustifolius growing in Turkey by capillary gas chromatography-mass spectrometry (GC-MS). Fifteen alkaloids were identified by capillary GC-MS. 13α-Hydroxylupanine (50.78%) and lupanine (23.55%) were determined as the main alkaloids in the aerial parts of L. angustifolius. Ammodendrine, isoangustifoline, tetrahydrorhombifoline, angustifoline, α-isolupanine, 5,6-dehydrolupanine, 11,12-dehydrolupanine, 13α-acetoxylupanine, 13α-isovaleroyloxylupanine, 13α-valeroyloxylupanine, 13α-tigloyloxylupanine, 13α-cis-cinnamoyloxylupanine and 13α-cis-cinnamoyloxy-17-oxolupanine were identified as the minor alkaloids of the plant. Furthermore, antibacterial and antifungal activities of L. angustifolius alkaloid extract were tested against standard strains of the following bacteria; Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus as well as the fungi; Candida albicans and C. krusei. The alkaloid extract showed significant activity on B. subtilis, S. aureus and P. aeruginosa while it was weakly active on E. coli. On the other hand, the extract possessed moderate activity against C. albicans and C. krusei.     

In vitro efficacy of <Emphasis Type="Italic">Hyptis suaveolens</Emphasis> L. (Poit.) essential oil on growth and morphogenesis of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">gladioli</Emphasis> (Massey) Snyder &; Hansen

Hyptis suaveolens L. (Poit.) essential oil was tested in vitro on the growth and morphogenesis of Fusarium oxysporum f.sp. gladioli (Massey) Snyder & Hansen, which causes Fusarium corm rot and yellows in various susceptible cultivars of gladiolus. The fungitoxicity of the oil was measured by percentage radial growth inhibition using the poisoned food technique (PF) and volatile activity assay (VA). The mycelial growth of the test fungus was completely inhibited at 0.998 and 0.748 μg ml⁻¹ concentration of oil in PF and VA, respectively. Essential oil was found to be fungicidal in nature at 1.247 and 0.998 μg ml⁻¹ concentration of oil in PF and VA, respectively. Determination of conidial germination in the presence of oil was also carried out and it was found that the oil exhibited 100% inhibition of conidial germination at 0.450 μg ml⁻¹ concentration. The effect of essential oil on the yield of mycelial weight was observed and it was found that at 0.873 μg ml⁻¹ concentration no mycelium was recorded and 100% inhibition was observed. The fungitoxicity of oil did not change even on exposure to 100°C temperature or to autoclaving, and the oil also retained its fungicidal nature even after storage of 24 months. The main changes observed under light microscopy after oil treatment were a decrease and loss of conidiation and anomalies in the hyphae such as a decrease in the diameter of hyphae and granulation of cytoplasm. The treatment of the oil also showed highly reduced cytoplasm in the hyphae, showing clear retraction of the cytoplasm from the hyphae and ultimately in some areas hyphae without cytoplasm were also found. GC-MS studies of the essential oil revealed that the oil consisted of 24 compounds with 1,8-cineole as major component accounting for 44.4% of the total constituents.     

Lumi-ergometrine – structural identification and occurrence in sclerotia

The fungus Claviceps purpurea grows on grasses and cereal grains and produces six predominant ergot alkaloids. These toxic substances undergo different transformation reactions during storage and cereal processing. One of these reactions is the addition of water to a double bond in the ergoline skeleton. Since light is required for this process, the substances formed were named lumi-ergot alkaloids. From these, a new asymmetric carbon and consequently two epimers with different polarities are formed. For investigations of lumi-ergot alkaloids, ergometrine was used exemplarily as it represents one of the six ergot alkaloids predominantly formed by Claviceps purpurea. The main reaction product, the less polar compound of the two lumi-ergometrine epimers, was separated by HPLC and unambiguously identified as 10-(S)-lumi-ergometrine using X-ray structural analysis. A HPLC-MS/MS method was developed for the detection of this substance in sclerotia extracts. Using this method, the existence of both epimeric forms of lumi-ergometrine could be proved in the sclerotia. This is the first time that the existence of a lumi-transformation product of ergot alkaloids was proved in naturally grown samples.     

Host plant selection by a monophagous herbivore is not mediated by quantitative changes in unique plant chemistry: Agonopterix alstroemeriana and Conium maculatum

Host plant selection by ovipositing females is a key process determining the success of phytophagous insects. In oligophagous lepidopterans, host-specific plant secondary chemicals are expected to be dominant factors governing oviposition behavior; distinctive compounds can serve as high-contrast signals that clearly differentiate confamilial hosts from non-hosts increasing the accuracy of host quality evaluation. Agonopterix alstroemeriana (Clerk) (Lepidoptera: Oecophoridae) and Conium maculatum L. (Apiaceae) form an extremely specialized plant-herbivore system, with A. alstroemeriana monophagous on C. maculatum, a plant with few other insect herbivores at least in part due to its virtually unique capacity among plants to produce piperidine alkaloids. Here we have studied the response of A. alstroemeriana oviposition to unique host plant secondary metabolites, piperidine alkaloids, and widespread compounds, mono- and sesquiterpenes, in a concentration-dependent fashion. Rates of oviposition were negatively correlated with Z-ocimene concentrations. To confirm the deterrent properties of this monoterpene for A. alstroemeriana oviposition, we conducted a choice experiment using artificially damaged C. maculatum plants, with higher emission of volatiles, and undamaged control plants. Damaged plants were less preferred as oviposition sites compared to the controls. The lack of association between oviposition and piperidine alkaloids, defenses unique to Conium species, suggests that quantitative changes of these species-specific chemicals do not play a predominant role in host selection by the monophagous A. alstroemeriana.     

Response of a leaf beetle to two food plants,only one of which provides a sequestrable defensive chemical

Oreina elongata is a chemically defended leaf beetle. If its food plant contains pyrrolizidine alkaloids, all life stages of the beetle sequester them. However, one of the two known host-plant genera does not contain these alkaloids. In this paper we compare the adult feeding preference and larval performance of two populations, one feeding on Adenostyles alliariae (which contains alkaloids) and one on Cirsium spinosissimum (devoid of alkaloids). Adults of the population living on C. spinosissimum preferred the alkaloid-containing A. alliariae, while adults of the population feeding on A. alliariae showed no preference for either plant. On the other hand, larval growth of both populations is better on C. spinosissimum, without alkaloids. This is especially so in the population that never naturally encounters pyrrolizidine alkaloids; the population living on A. alliariae is apparently better adapted to its host's secondary compounds. The data are discussed in terms of cost of defense and trade-offs between growth and defense.     

Purification,cloning, functional expression and characterization of perakine reductase: the first example from the AKR enzyme family,extending the alkaloidal network of the plant <Emphasis Type="Italic">Rauvolfia</Emphasis>

Perakine reductase (PR) catalyzes an NADPH-dependent step in a side-branch of the 10-step biosynthetic pathway of the alkaloid ajmaline. The enzyme was cloned by a “reverse-genetic” approach from cell suspension cultures of the plant Rauvolfia serpentina (Apocynaceae) and functionally expressed in Escherichia coli as the N-terminal His₆-tagged protein. PR displays a broad substrate acceptance, converting 16 out of 28 tested compounds with reducible carbonyl function which belong to three substrate groups: benzaldehyde, cinnamic aldehyde derivatives and monoterpenoid indole alkaloids. The enzyme has an extraordinary selectivity in the group of alkaloids. Sequence alignments define PR as a new member of the aldo-keto reductase (AKR) super family, exhibiting the conserved catalytic tetrad Asp52, Tyr57, Lys84, His126. Site-directed mutagenesis of each of these functional residues to an alanine residue results in >97.8% loss of enzyme activity, in compounds of each substrate group. PR represents the first example of the large AKR-family which is involved in the biosynthesis of plant monoterpenoid indole alkaloids. In addition to a new esterase, PR significantly extends the Rauvolfia alkaloid network to the novel group of peraksine alkaloids. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequences reported in this article have been submitted to the Gene Bank under Accession No: AY766462.     

Implementation of functional genomics for gene discovery in alkaloid producing plants

Two centuries after the discovery of the first alkaloids, many enzymes involved in plant alkaloid biosynthesis have been identified. Nevertheless, the biosynthetic pathways for most of the plant alkaloids still remain incompletely characterised and understanding the regulatory mechanisms controlling the onset and flux of alkaloid biosynthesis is virtually inexistent. This information is however crucial to allow modelling of metabolic networks and predictive metabolic engineering. In the postgenomics era, new functional genomics tools, enabling comprehensive investigations of biological systems, are continuously emerging and are now gradually being implemented in the field of plant secondary metabolism as well. Here we discuss the advances these promising new technologies have already brought and may still bring with regard to the dissection of plant alkaloid biosynthesis. Encouraging results were obtained in alkaloid producing species such as Papaver somniferum, Catharanthus roseus and Nicotiana tabacum. Therefore we anticipate that functional genomics and the knowledge it brings along, will eventually allow a better exploitation of the plant biosynthetic machinery.