Concomitant occurrence of pyrrolizidine and quinolizidine alkaloids in the hemiparasite Osyris alba L. (Santalaceae)

The investigation of the alkaloid extracts of the hemiparasitic plant Osyris alba, collected from three different localities in southern France, revealed the concomitant presence of both pyrrolizidine (PA) and quinolizidine (QA) alkaloids in the samples from two of these localities. The sample from the third locality contained only PAs. The eight QAs identified were sparteine, N-methylcytisine, cytisine, methyl-12-cytisine acetate, hydroxy-N-methylcytisine, N-acetylcytisine, lupanine, and anagyrine. Of the eleven detected PAs, eight were identified as chysin A, chysin B, 1-carboxypyrrolizidine-7-olide, senecionine, integerrimine, retrorsine, senecivernine and a new alkaloid janfestine (7R-hydroxychysin A or 1R-carbomethoxy-7R-hydroxypyrrolizidine). PAs were mainly present as their N-oxides This is, to our knowledge, the first report demonstrating the simultaneous presence of two classes of alkaloids, quinolizidine and pyrrolizidine alkaloids, in a single parasitic plant. As these alkaloids do not occur in the same host plant, the results indicate that Osyris must have tapped more than one host plant concomitantly. Since both quinolizidine and pyrrolizidine alkaloids serve as defence compounds against herbivores, affecting different molecular targets, the simultaneous acquisition of the two types of alkaloids by a single plant could provide a novel mode of defence of hemiparasites against herbivores.     

Attract and deter: a dual role for pyrrolizidine alkaloids in plant–insect interactions

Pyrrolizidine alkaloids (PAs) are the major defense compounds of plants in the Senecio genus. Here I will review the effects of PAs in Senecio on the preference and performance of specialist and generalist insect herbivores. Specialist herbivores have evolved adaptation to PAs in their host plant. They can use the alkaloids as cue to find their host plant and often they sequester PAs for their own defense against predators. Generalists, on the other hand, can be deterred by PAs. PAs can also affect survival of generalist herbivores. Usually generalist insects avoid feeding on young Senecio leaves, which contain a high concentration of alkaloids. Structurally related PAs can differ in their effects on insect herbivores, some are more toxic than others. The differences in effects of PAs on specialist and generalists could lead to opposing selection on PAs, which may maintain the genetic diversity in PA concentration and composition in Senecio species.     

Evolutionary recruitment of a flavin-dependent monooxygenase for stabilization of sequestered pyrrolizidine alkaloids in arctiids

Pyrrolizidine alkaloids are secondary metabolites that are produced by certain plants as a chemical defense against herbivores. They represent a promising system to study the evolution of pathways in plant secondary metabolism. Recently, a specific gene of this pathway has been shown to have originated by duplication of a gene involved in primary metabolism followed by diversification and optimization for its specific function in the defense machinery of these plants. Furthermore, pyrrolizidine alkaloids are one of the best-studied examples of a plant defense system that has been recruited by several insect lineages for their own chemical defense. In each case, this recruitment requires sophisticated mechanisms of adaptations, e.g., efficient excretion, transport, suppression of toxification, or detoxification. In this review, we briefly summarize detoxification mechanism known for pyrrolizidine alkaloids and focus on pyrrolizidine alkaloid N-oxidation as one of the mechanisms allowing insects to accumulate the sequestered toxins in an inactivated protoxic form. Recent research into the evolution of pyrrolizidine alkaloid N-oxygenases of adapted arctiid moths (Lepidoptera) has shown that this enzyme originated by the duplication of a gene encoding a flavin-dependent monooxygenase of unknown function early in the arctiid lineage. The available data suggest several similarities in the molecular evolution of this adaptation strategy of insects to the mechanisms described previously for the evolution of the respective pathway in plants.     

Defensive properties of pyrrolizidine alkaloids against microorganisms

The understanding of the selection factors that drive chemical diversification of secondary metabolites of constitutive defence systems in plants, such as pyrrolizidine alkaloids (PAs), is still incomplete. Historically, plants always have been confronted with microorganisms. Long before herbivores existed on this planet, plants had to cope with microbial pathogens. Therefore, plant pathogenic microorganisms may have played an important role in the early evolution of the secondary metabolite diversity. In this review, we discuss the impact that plant-produced PAs have on plant-associated microorganisms. The objective of the review is to present the current knowledge on PAs with respect to anti-microbial activities, adaptation and detoxification by microorganisms, pathogenic fungi, root protection and PA induction. Many in vitro experiments showed effects of PAs on microorganisms. These results point to the potential of microorganisms to be important for the evolution of PAs. However, only a few in vivo studies have been published and support the results of the in vitro studies. In conclusion, the topics pointed out in this review need further exploration by carrying out ecological experiments and field studies.     

Cellular distribution of alkaloids and their translocation via phloem and xylem: the importance of compartment pH

The physico‐chemical background of alkaloid allocation within plants is outlined and discussed exemplarily for pyrrolizidine alkaloids (PAs) and nicotine. The trigger for this discourse is the finding that, for example, PAs, which are taken up from the soil, are translocated in the xylem, whereas – when genuinely present in plants – they are allocated as N‐oxides via phloem. Special emphasis is put on the impact of different pH values in certain compartments, as this entails significant changes in the relative lipophilic character of alkaloids: tertiary alkaloids diffuse readily through biomembranes, while the corresponding protonated alkaloids are retained in acidic compartments, i.e. vacuoles or xylem. Therefore, this phenomenon, well known as the ‘ion trap mechanism’, is also relevant for long‐distance transport of alkaloids. Any efficient allocation of typical tertiary alkaloids within the phloem can thus be excluded. In contrast, due to their strongly increased hydrophilic properties, alkaloid‐N‐oxides or quarternary alkaloids cannot diffuse through biomembranes and, consequently, would be retained in the acidic xylem during translocation. The major aim of this paper is to sharpen the mind for the chemical peculiarities of alkaloids and to consider them adequately in forthcoming investigations on allocation of alkaloids.     

The genotype dependent presence of pyrrolizidine alkaloids as tertiary amine in Jacobaea vulgaris

Secondary metabolites such as pyrrolizidine alkaloids (PAs) play a crucial part in plant defense. PAs can occur in plants in two forms: tertiary amine (free base) and N-oxide. PA extraction and detection are of great importance for the understanding of the role of PAs as plant defense compounds, as the tertiary PA form is known for its stronger influence on several generalist insects, whereas the N-oxide form is claimed to be less deterrent. We measured PA N-oxides and their reduced tertiary amines by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We show that the occurrence of tertiary PAs is not an artifact of the extraction and detection method. We found up to 50% of tertiary PAs in shoots of Jacobine - chemotype plants of Jacobaea vulgaris. Jacobine and its derivatives (jacoline, jaconine, jacozine and dehydrojaconine) may occur for more than 20% in reduced form in the shoots and more than 10% in the roots. For 22 PAs detected in F(2) hybrids (J. vulgaris × Jacobaea aquatica), we calculate the tertiary amine percentage (TA%=the tertiary amine concentration/(tertiary amine concentration+the corresponding N-oxide concentration) × 100). We found that the TA% for various PAs was genotype-dependent. Furthermore, TA% for the different PAs were correlated and the highest correlations occurred between PAs which share high structural similarity.     

Local adaptation in oviposition choice of a specialist herbivore: The cinnabar moth

Specialist herbivores feed on a restricted number of related plant species and may suffer food shortage if overexploitation leads to periodic defoliation of their food plants. The density, size and quality of food plants are important factors that determine the host plant choice of specialist herbivores. To explore how all these factors influence their oviposition behaviour, we used the cinnabar moth Tyria jacobaeae and the hybrids of a cross between Jacobaea vulgaris and J. aquatica as a study system. While defoliation by the cinnabar moth is common in the coastal area of The Netherlands, it is relatively rare in inland ragwort population. Ragworts contain pyrrolizidine alkaloids (PAs) and those that are found in coastal areas are rich in jacobine-like PAs while those that occur inland are rich in erucifoline-like PAs. We tested how the oviposition preference was influenced by plant size, nitrogen and water content and PA composition. We used cinnabar moth populations from a regularly defoliated area, Meijendel, and Bertogne, a rarely defoliated area. Our results revealed no effects of nitrogen or water content on oviposition preference. Moths from both populations laid larger egg batches on the plants rich in jacobine-like PAs. Moths from Meijendel preferred larger plants and spread their eggs over more egg batches that were, on average, smaller than those of Bertogne moths. These results suggest that Meijendel moths adopted a oviposition strategy to cope with potential defoliation.     

Exogenous application of plant defense hormones alters the effects of live soils on plant performance

The overall effect of a live soil inoculum collected from nature on plant biomass is often negative. One hypothesis to explain this phenomenon is that the overall net pathogenic effect of soil microbial communities reduces plant performance. Induced plant defenses triggered by the application of the plant hormones jasmonic acid (JA) and salicylic acid (SA) may help to mitigate this pathogenic effect of live soil. However, little is known about how such hormonal application to the plant affects the soil and how this, in turn, impacts plant growth. We grew four plant species in sterilized and inoculated live soil and exposed their leaves to two hormonal treatments (JA and SA). Two species (Jacobaea vulgaris and Cirsium vulgare) were negatively affected by soil inoculation. In these two species foliar application of SA increased biomass in live soil but not in sterilized soil. Two other species (Trifolium repens and Daucus carota) were not affected by soil inoculum and for these two species foliar application of SA reduced plant biomass in both the sterilized and live soil. Application of JA reduced plant biomass in both soils for all species. We subsequently carried out a multiple generation experiment for one of the plant species, J. vulgaris. In each generation, the live soil was a mixture of 10% soil from the previous generation and 90% sterilized soil and the same hormonal treatments were applied. The negative effects of live soil on plant biomass were similar in all four generations, and this negative effect was mitigated by the application of SA. Our research suggests that the application of SA can mitigate the negative effects of live soil on plant growth. Although the inoculum of soil containing a natural live soil microbial community had a strong negative effect on the growth of J. vulgaris, we found no evidence for an increase or decrease in negative plant-soil feedback in either the control or the SA treated plants. Also plant performance did not decrease consistently with succeeding generations.     

Competition Increases Sensitivity of Wheat (Triticum aestivum) to Biotic Plant-Soil Feedback

Plant-soil feedback (PSF) and plant competition play an important role in structuring vegetation composition, but their interaction remains unclear. Recent studies suggest that competing plants could dilute pathogenic effects, whereas the standing view is that competition may increase the sensitivity of the focal plant to PSF. In agro-ecosystems each of these two options would yield contrasting outcomes: reduced versus enhanced effects of weeds on crop biomass production. To test the effect of competition on sensitivity to PSF, we grew Triticum aestivum (Common wheat) with and without competition from a weed community composed of Vicia villosa, Chenopodium album and Myosotis arvensis. Plants were grown in sterilized soil, with or without living field inoculum from 4 farms in the UK. In the conditioning phase, field inocula had both positive and negative effects on T. aestivum shoot biomass, depending on farm. In the feedback phase the differences between shoot biomass in T. aestivum monoculture on non-inoculated and inoculated soils had mostly disappeared. However, T. aestivum plants growing in mixtures in the feedback phase were larger on non-inoculated soil than on inoculated soil. Hence, T. aestivum was more sensitive to competition when the field soil biota was present. This was supported by the statistically significant negative correlation between shoot biomass of weeds and T. aestivum, which was absent on sterilized soil. In conclusion, competition in cereal crop-weed systems appears to increase cereal crop sensitivity to soil biota.     

Role of Microniches in Protecting Introduced Rhizobium leguminosarum biovar trifolii against Competition and Predation in Soil

The importance of microniches for the survival of introduced Rhizobium leguminosarum biovar trifolii cells was studied in sterilized and recolonized sterilized loamy sand and silt loam. The recolonized soils contained several species of soil microorganisms but were free of protozoa. Part of these soil samples was inoculated with the flagellate Bodo saltans, precultured on rhizobial cells. The introduced organisms were enumerated in different soil fractions by washing the soil, using a standardized washing procedure. With this method, free organisms and organisms associated with soil particles or aggregates >50 μm were separated. The total number of rhizobia was influenced slightly (silt loam) or not at all (loamy sand) by the recolonization with microorganisms or by the addition of flagellates alone. However, when both flagellates and microorganisms were present, numbers of rhizobia decreased drastically. This decrease was more than the sum of both effects separately. Nevertheless, populations of rhizobia were still higher than in natural soil. In the presence of flagellates, higher percentages of rhizobia and other microorganisms were associated with soil particles or aggregates >50 μm than in the absence of flagellates. In recolonized soils, however, the percentages of particle-associated rhizobia were lower than in soils not recolonized previous to inoculation. Thus, the presence of other microorganisms hindered rhizobial colonization of sites where they are normally associated with soil particles or aggregates.     

Testing chemical defence based on pyrrolizidine alkaloids

Pyrrolizidine alkaloids are considered the primary defence mechanism in aposematic ithomiine butterflies and arctiid moths. Despite evidence that pyrrolizidine alkaloids are effective against some invertebrate predators, proof for a protective function of pyrrolizidine alkaloids against vertebrate predators is fragmented. The present work shows that the pyrrolizidine alkaloid monocrot-aline is unpalatable to the pileated finch,Coryphospingus pileatusand that the unpalatability is learned through association with a specific colour pattern (blue stripes). In a series of trials, using mealworms as model prey, birds rejected those to which pyrrolizidine alkaloid solution had been applied topically but accepted prey devoid of the alkaloid. Subsequent offerings of prey with pyrrolizidine alkaloid and a painted blue-striped pattern led to consistent rejections by the experimental birds. Birds were then offered blue-striped painted larvae without pyrrolizidine alkaloids (‘mimics’), which were rejected at levels similar to the previous trial. The predators learned to recognize the prey as unpalatable items based on their experience in the previous encounters. These results provide evidence for the protective capacity of the pyrrolizidine alkaloid against a vertebrate predator and supports the role of these chemicals in aposematism in the Lepidoptera.     

Preference for high concentrations of plant pyrrolizidine alkaloids in the specialist arctiid moth Utetheisa ornatrix depends on previous experience

Secondary metabolites are one the most pervasive defensive mechanisms in plants. Many specialist herbivores have evolved adaptations to overcome these defensive compounds. Some herbivores can even take advantage of these compounds by sequestering them for protection and/or mate attraction. One of the most studied specialist insects that sequesters secondary metabolites is the arctiid moth Utetheisa ornatrix. This species sequesters pyrrolizidine alkaloids (PAs) from its host plant, the legume Crotalaria spp. The sequestered PAs are used as a predator repellent and as a mating pheromone. We used this species to test larval preference for different concentrations of PAs. We purified PAs from plant material and added them at different concentrations to an artificial diet. Larvae of U. ornatrix previously feeding on low and high PA concentration artificial diets were allowed to choose between two new artificial diets with different PA concentrations. The amount of PAs sequestered and larval preference were dependent on their previous exposure to low or high PA content in the diet. Larvae that were pretreated with a low PA diet significantly consumed more diet with the high PA concentration, while larvae that were pretreated with a high PA diet showed no discrimination between future feeding of different PA concentration diets. We discuss our results using mechanistic and evolutionary approaches. Finally, we discuss how these results have important implications on the evolution of plant herbivore interactions and how specialist herbivores may decrease the levels of chemical defenses on plant populations.     

A mixed diet of toxic plants enables increased feeding and anti-predator defense by an insect herbivore

Some insect herbivores sequester plant secondary metabolites (PSMs) for their own defense, raising the interesting possibility that grazing herbivores are defended by combinations of PSMs from different plant species. In this study, we tested the hypothesis that the grazing caterpillar, Grammia incorrupta, deters the ant, Aphaenogaster cockerelli, by eating a mixture of plants containing iridoid glycosides (IGs) and those containing pyrrolizidine alkaloids (PAs), and that this deterrence is greater than that attained by eating either plant alone. This hypothesis was tested against the non-mutually exclusive hypothesis that mixing plants containing PAs with those containing IGs improves growth performance. Caterpillar survival and growth were measured on three experimental diets: a PA plant, an IG plant, and a mixture of the two. We measured the degree of deterrence associated with these, and an additional experimental diet devoid of PSMs at naturally occurring A. cockerelli nests. Caterpillars fed both plants gained more mass than those fed either plant alone, but took longer to develop. These differences were not caused by diet-based variation in growth efficiency, but by eating more food when offered the mixed-plant diet relative to single-plant diets. The mixed diet was shown to provide deterrence to ants, whereas caterpillars fed single-plant diets were not significantly more deterrent than caterpillars that had eaten the PSM-free diet. We hypothesize that enhanced defense results from increased food consumption in response to multiple plant species, perhaps leading to greater PSM sequestration. Through this mechanism, bottom-up and top-down effects may mutually reinforce the grazing dietary strategy.     

Evolution of pyrrolizidine alkaloids in Phalaenopsis orchids and other monocotyledons: Identification of deoxyhypusine synthase, homospermidine synthase and related pseudogenes

In order to study the evolution of pathways of plant secondary metabolism, we use the biosynthesis of pyrrolizidine alkaloids (PAs) as a model system. PAs are regarded as part of the plant’s constitutive defense against herbivores. Homospermidine synthase (HSS) is the first specific enzyme of PA biosynthesis. The gene encoding HSS has been recruited from the gene encoding deoxyhypusine synthase (DHS) from primary metabolism at least four times independently during angiosperm evolution. One of these recruitments occurred within the monocot lineage. We have used the PA-producing orchid Phalaenopsis to identify the cDNAs encoding HSS, DHS and the substrate protein for DHS, i.e., the precursor of the eukaryotic initiation factor 5A. A cDNA identified from maize was unequivocally characterized as DHS. From our study of Phalaenopsis, several pseudogenes emerged, of which one was shown to be a “processed pseudogene”, and others to be transcribed. Sequence comparison of the HSS- and DHS-encoding sequences from this investigation with those of monocot species taken from the databases suggest that HSS and probably the ability to produce PAs is an old feature within the monocot lineage. This result is discussed with respect to the recent discovery of structural related PAs within grasses.     

Metabolic profiling in Echium plantagineum: presence of bioactive pyrrolizidine alkaloids and napthoquinones from accessions across southeastern Australia

Geographically distinct populations of Paterson’s curse (Echium plantagineum L., Boragineacea), found near roadsides across New South Wales and Victoria, Australia were surveyed along 3 distinct longitudinal transects in spring of 2011 for presence of pyrrolizidine alkaloids and naphthoquinones in sampled plants. Composite samples of shoots and roots were collected from each of 45 sites; shoot extracts were subjected to solid phase extraction and LC-ESI/MS for determination of pyrrolizidine alkaloids (PAs) and related N-oxides (PANOs), while root periderm extracts were analysed for naphthoquinone content spectrophotometrically and by LC-ESI/MS. Metabolic profiling of 12 possible PAs and PANOs showed their consistent appearance in all shoot extracts, with lepthamine N-oxide, echimidine-N oxide and echumine N-oxide predominant. The three major PANOs were significantly higher in northern sampling locations than those further south. Root extracts contained shikonin and several related naphthoquinones, as well as two of the major PANOs found in the leaves. Naphthoquinones were highest in the northwest corner of the sampled region. The patterns of abundance of secondary metabolites in E. plantagienum suggest that climate change might result in greater production of defensive compounds by E. plantagineum, making this weed increasingly toxic to livestock.     

Differential tissue distribution of metabolites in Jacobaea vulgaris,Jacobaea aquatica and their crosses

Plants are attacked by many different herbivores. Some will consume whole leaves or roots, while others will attack specific types of tissue. Thus, insight into the metabolite profiles of different types of leaf tissues is necessary to understand plant resistance against herbivores. Jacobaea vulgaris, J. aquatica and three genotypes of their crossings were used to study the variation in metabolomic profiles between epidermis and mesophyll tissues. Extracts of epidermis and mesophyll tissues were obtained using carborundum abrasion (CA). Subsequently, ¹H nuclear magnetic resonance (NMR) spectroscopy and multivariate data analyses were applied to compare the metabolome profiles. Orthogonal partial least-squares-discriminant analysis (OPLS-DA) resulted in a clear separation of epidermis and mesophyll extracts. The epidermis contained significantly higher amounts of jacaranone and phenylpropanoids, specifically chlorogenic (5-O-CQA) and feruloyl quinic (FQA) acids compared to the mesophyll. In contrast, the mesophyll showed significantly higher concentrations of pyrrolizidine alkaloids (PAs), specifically jacobine and jaconine. The tissue specific distribution of these compounds was constant over all genotypes tested. Phenylpropanoids, 5-O-CQA and FQA, as well as PAs are known for their inhibitory effect on herbivores, especially against thrips. Thrips feeding commences with the penetration of the epidermis, followed by ingestion of sub-epidermal or mesophyll. Thrips thus may have to encounter phenylpropanoids in the epidermis as the first line of defence, before encountering the PAs as the ultimate defence in the mesophyll. The finding of tissue specific defense may have a major impact on studies of plant resistance. We cannot judge resistance using analyses of a whole roots, leafs or flowers. In such a whole-organism approach, the levels of potential defense compounds are far below the real ones encountered in tissues involved in the first line of defense. Instead, it is of great importance to study the defence compounds in the specific tissue to which the herbivore is confined.     

Effects of soil organisms on aboveground multitrophic interactions are consistent between plant genotypes mediating the interaction

Belowground communities can affect interactions between plants and aboveground insect communities. Such belowground–aboveground interactions are known to depend on the composition of belowground communities, as well as on the plant species that mediates these interactions. However, it is largely unknown whether the effect of belowground communities on aboveground plant–insect interactions also depends on genotypic variation within the plant species that mediates the interaction. To assess whether the outcome of belowground–aboveground interactions can be affected by plant genotype, we selected two white cabbage cultivars [Brassica oleracea L. var. capitata (Brassicaceae)]. From previous studies, it is known that these cultivars differ in their chemistry and belowground and aboveground multitrophic interactions. Belowground, we inoculated soils of the cultivars with either nematodes or microorganisms and included a sterilized soil as a control treatment. Aboveground, we quantified aphid [Brevicoryne brassicae (L.) (Hemiptera: Aphididae)] population development and parasitoid [Diaeretiella rapae (McIntosh) (Hymenoptera: Braconidae)] fitness parameters. The cultivar that sustained highest aphid numbers also had the best parasitoid performance. Soil treatment affected aphid population sizes: microorganisms increased aphid population growth. Soil treatments did not affect parasitoid performance. Cultivars differed in their amino acid concentration, leaf relative growth rate, and root, shoot, and phloem glucosinolate composition but showed similar responses of these traits to soil treatments. Consistent with this observation, no interactions were found between cultivar and soil treatment for aphid population growth or parasitoid performance. Overall, the aboveground community was more affected by cultivar, which was associated with glucosinolate profiles, than by soil community.     

Plant–soil feedback effects on plant quality and performance of an aboveground herbivore interact with fertilisation

Plant–soil feedback (PSF) effects on plant performance can be influenced by the availability of nutrients in the soil. Recent studies have shown that PSF effects can also change aboveground plant–insect interactions via soil‐mediated changes in plant quality, but whether this is influenced by soil nutrient availability is unknown. We examined how fertilisation influences PSF effects on aboveground plant‐aphid interactions in ragwort Jacobaea vulgaris. We grew J. vulgaris in soil conditioned by conspecific plants and in unconditioned soil at two levels of fertilisation and measured soil fungal communities, plant biomass, concentrations of primary (amino acids) and secondary (pyrrolizidine alkaloids; PAs) metabolites in phloem exudates, performance of the specialist aphid Aphis jacobaeae and sequestration of PAs by the aphid. We observed a strong interaction between soil conditioning and fertilisation on amino acid and PA concentrations in phloem exudates of J. vulgaris and on aphid performance, with opposite effects of soil conditioning at the two fertilisation levels. Plant biomass was reduced by soil conditioning and increased by fertilisation. Aphids contained high PA concentrations, converted N‐oxides into tertiary amines and preferentially sequestered certain PA compounds, but PA sequestration was not affected by any of the treatments. We conclude that effects of PSF and fertilisation on plant chemistry and aphid performance are interdependent. Our study highlights the need to consider the importance of abiotic soil conditions on the outcome of PSF effects on aboveground plant–insect interactions.     

Pyrrolizidine alkaloids in human diet.

Pyrrolizidine alkaloids are the leading plant toxins associated with disease in humans and animals. Upon ingestion, metabolic activation in liver converts the parent compounds into highly reactive electrophiles capable of reacting with cellular macromolecules forming adducts which may initiate acute or chronic toxicity. The pyrrolizidine alkaloids present a serious health risk to human populations that may be exposed to them through contamination of foodstuffs or when plants containing them are consumed as medicinal herbs. Some pyrrolizidine alkaloids (PA) adducts are persistent in animal tissue and the metabolites may be re-released and cause damage long after the initial period of ingestion. PAs are also known to act as teratogens and abortifacients. Chronic ingestion of plants containing PAs has also led to cancer in experimental animals and metabolites of several PAs have been shown to be mutagenic in the Salmonella typhimurium/mammalian microsome system. However, no clinical association has yet been found between human cancer and exposure to PAs. Based on the extensive reports on the outcome of human exposure available in the literature, we conclude that while humans face the risk of veno-occlusive disease and childhood cirrhosis PAs are not carcinogenic to humans.     

Independent recruitment of a flavin-dependent monooxygenase for safe accumulation of sequestered pyrrolizidine alkaloids in grasshoppers and moths

Several insect lineages have developed diverse strategies to sequester toxic pyrrolizidine alkaloids from food-plants for their own defense. Here, we show that in two highly divergent insect taxa, the hemimetabolous grasshoppers and the holometabolous butterflies, an almost identical strategy evolved independently for safe accumulation of pyrrolizidine alkaloids. This strategy involves a pyrrolizidine alkaloid N-oxygenase that transfers the pyrrolizidine alkaloids to their respective N-oxide, enabling the insects to avoid high concentrations of toxic pyrrolizidine alkaloids in the hemolymph. We have identified a pyrrolizidine alkaloid N-oxygenase, which is a flavin-dependent monooxygenase, of the grasshopper Zonocerus variegatus. After heterologous expression in E. coli, this enzyme shows high specificity for pyrrolizidine alkaloids of various structural types and for the tropane alkaloid atropine as substrates, a property that has been described previously for a pyrrolizidine alkaloid N-oxygenase of the arctiid moth Grammia geneura. Phylogenetic analyses of insect flavin-dependent monooxygenase sequences suggest that independent gene duplication events preceded the establishment of this specific enzyme in the lineages of the grasshoppers and of arctiid moths. Two further flavin-dependent monooxygenase sequences have been identified from Z. variegatus sharing amino acid identities of approximately 78% to the pyrrolizidine alkaloid N-oxygenase. After heterologous expression, both enzymes are also able to catalyze the N-oxygenation of pyrrolizidine alkaloids, albeit with a 400-fold lower specific activity. With respect to the high sequence identity between the three Z. variegatus sequences this ability to N-oxygenize pyrrolizidine alkaloids is interpreted as a relict of a former bifunctional ancestor gene of which one of the gene copies optimized this activity for the specific adaptation to pyrrolizidine alkaloid containing food plants.