Seeing double: two different homospermidine oxidases are involved in pyrrolizidine alkaloid biosynthesis in different organs of comfrey (Symphytum officinale).

Pyrrolizidine alkaloids (PAs) are toxic specialized metabolites produced in several plant species and frequently contaminate herbal teas or livestock feed. In comfrey (Symphytum officinale, Boraginaceae), they are produced in two different organs of the plant, the root and young leaves. In this study, we demonstrate that homospermidine oxidase (HSO), a copper-containing amine oxidase (CuAO) responsible for catalyzing the formation of the distinctive pyrrolizidine ring in PAs, is encoded by two individual genes. Specifically, SoCuAO1 is expressed in young leaves, while SoCuAO5 is expressed in roots. CRISPR/Cas9-mediated knockout of socuao5 resulted in hairy roots (HRs) unable to produce PAs, supporting its function as HSO in roots. Plants regenerated from socuao5 knockout HRs remained completely PA-free until the plants began to develop inflorescences, indicating the presence of another HSO that is expressed only during flower development. Stable expression of SoCuAO1 in socuao5 knockout HRs rescued the ability to produce PAs. In vitro assays of both enzymes transiently expressed in Nicotiana benthamiana confirmed their HSO activity and revealed the ability of HSO to control the stereospecific cyclization of the pyrrolizidine backbone. The observation that the first specific step of PA biosynthesis catalyzed by homospermidine synthase requires only one gene copy, while two independent paralogs are recruited for the subsequent homospermidine oxidation in different tissues of the plant, suggests a complex regulation of the pathway. This adds a new level of complexity to PA biosynthesis, a system already characterized by species-specific, tight spatio-temporal regulation, and independent evolutionary origins in multiple plant lineages.

The long road of functional recruitment-The evolution of a gene duplicate to pyrrolizidine alkaloid biosynthesis in the morning glories (Convolvulaceae).

In plants, homospermidine synthase (HSS) is a pathway-specific enzyme initiating the biosynthesis of pyrrolizidine alkaloids (PAs), which function as a chemical defense against herbivores. In PA-producing Convolvulaceae ("morning glories"), HSS originated from deoxyhypusine synthase at least >50 to 75 million years ago via a gene duplication event and subsequent functional diversification. To study the recruitment of this ancient gene duplicate to PA biosynthesis, the presence of putative hss gene copies in 11 Convolvulaceae species was analyzed. Additionally, various plant parts from seven of these species were screened for the presence of PAs. Although all of these species possess a putative hss copy, PAs could only be detected in roots of Ipomoea neei (Spreng.) O'Donell and Distimake quinquefolius (L.) A.R.Simões & Staples in this study. A precursor of PAs was detected in roots of Ipomoea alba L. Thus, despite sharing high sequence identities, the presence of an hss gene copy does not correlate with PA accumulation in particular species of Convolvulaceae. In vitro activity assays of the encoded enzymes revealed a broad spectrum of enzyme activity, further emphasizing a functional diversity of the hss gene copies. A recently identified HSS specific amino acid motif seems to be important for the loss of the ancestral protein function-the activation of the eukaryotic initiation factor 5A (eIF5A). Thus, the motif might be indicative for a change of function but allows not to predict the new function. This emphasizes the challenges in annotating functions for duplicates, even for duplicates from closely related species.

Insights into polyamine metabolism: homospermidine is double-oxidized in two discrete steps by a single copper-containing amine oxidase in pyrrolizidine alkaloid biosynthesis.

Polyamines are important metabolites in plant development and abiotic and biotic stress responses. Copper-containing amine oxidases (CuAOs) are involved in the regulation of polyamine levels in the cell. CuAOs oxidize primary amines to their respective aldehydes and hydrogen peroxide. In plants, aldehydes are intermediates in various biosynthetic pathways of alkaloids. CuAOs are thought to oxidize polyamines at only one of the primary amino groups, a process frequently resulting in monocyclic structures. These oxidases have been postulated to be involved in pyrrolizidine alkaloid (PA) biosynthesis. Here, we describe the identification and characterization of homospermidine oxidase (HSO), a CuAO of Heliotropium indicum (Indian heliotrope), involved in PA biosynthesis. Virus-induced gene silencing of HSO in H. indicum leads to significantly reduced PA levels. By in vitro enzyme assays after transient in planta expression, we show that this enzyme prefers Hspd over other amines. Nuclear magnetic resonance spectroscopy and mass spectrometry analyses of the reaction products demonstrate that HSO oxidizes both primary amino groups of homospermidine (Hspd) to form a bicyclic structure, 1-formylpyrrolizidine. Using tracer feeding, we have further revealed that 1-formylpyrrolizidine is an intermediate in the biosynthesis of PAs. Our study therefore establishes that HSO, a canonical CuAO, catalyzes the second step of PA biosynthesis and provides evidence for an undescribed and unusual mechanism involving two discrete steps of oxidation that might also be involved in the biosynthesis of complex structures in other alkaloidal pathways.

CRISPR/Cas9-Mediated Genome Editing in Comfrey (Symphytum officinale) Hairy Roots Results in the Complete Eradication of Pyrrolizidine Alkaloids.

Comfrey (Symphytum officinale) is a medicinal plant with anti-inflammatory, analgesic, and proliferative properties. However, its pharmaceutical application is hampered by the co-occurrence of toxic pyrrolizidine alkaloids (PAs) in its tissues. Using a CRISPR/Cas9-based approach, we introduced detrimental mutations into the hss gene encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis. The resulting hairy root (HR) lines were analyzed for the type of gene-editing effect that they exhibited and for their homospermidine and PA content. Inactivation of only one of the two hss alleles resulted in HRs with significantly reduced levels of homospermidine and PAs, whereas no alkaloids were detectable in HRs with two inactivated hss alleles. PAs were detectable once again after the HSS-deficient HRs were fed homospermidine confirming that the inability of these roots to produce PAs was only attributable to the inactivated HSS and not to any unidentified off-target effect of the CRISPR/Cas9 approach. Further analyses showed that PA-free HRs possessed, at least in traces, detectable amounts of homospermidine, and that the PA patterns of manipulated HRs were different from those of control lines. These observations are discussed with regard to the potential use of such a CRISPR/Cas9-mediated approach for the economical exploitation of in vitro systems in a medicinal plant and for further studies of PA biosynthesis in non-model plants.

Development of an activity assay for characterizing deoxyhypusine synthase and its diverse reaction products.

Deoxyhypusine synthase transfers an aminobutyl moiety from spermidine to the eukaryotic translation initiation factor 5A (eIF5A) in the first step of eIF5A activation. This exclusive post-translational modification is conserved in all eukaryotes. Activated eIF5A has been shown to be essential for cell proliferation and viability. Recent reports have linked the activation of eIF5A to several human diseases. Deoxyhypusine synthase, which is encoded by a single gene copy in most eukaryotes, was duplicated in several plant lineages during evolution, the copies being repeatedly recruited to pyrrolizidine alkaloid biosynthesis. However, the function of many of these duplicates is unknown. Notably, deoxyhypusine synthase is highly promiscuous and can catalyze various reactions, often of unknown biological relevance. To facilitate in-depth biochemical studies of this enzyme, we report here the development of a simple and robust in vitro enzyme assay. It involves precolumn derivatization of the polyamines taking part in the reaction and avoids the need for the previously used radioactively labeled tracers. The derivatized polyamines are quantified after high-performance liquid chromatography coupled to diode array and fluorescence detectors. By performing kinetic analyses of deoxyhypusine synthase and its paralog from the pyrrolizidine alkaloid-producing plant Senecio vernalis, we demonstrate that the assay unequivocally differentiates the paralogous enzymes. Furthermore, it detects and quantifies, in a single assay, the side reactions that occur in parallel to the main reaction. The presented assay thus provides a detailed biochemical characterization of deoxyhypusine synthase and its paralogs.

Reduction of Pyrrolizidine Alkaloid Levels in Comfrey (Symphytum officinale) Hairy Roots by RNAi Silencing of Homospermidine Synthase.

Comfrey is a medicinal plant, extracts of which are traditionally used for the treatment of painful inflammatory muscle and joint problems, because the plant contains allantoin and rosmarinic acid. However, its medicinal use is limited because of its toxic pyrrolizidine alkaloid (PA) content. PAs encompass more than 400 different compounds that have been identified from various plant lineages. To date, only the first pathway-specific enzyme, homospermidine synthase (HSS), has been characterized. HSS catalyzes the formation of homospermidine, which is exclusively incorporated into PAs. HSS has been recruited several times independently in various plant lineages during evolution by duplication of the gene encoding deoxyhypusine synthase (DHS), an enzyme of primary metabolism. Here, we describe the establishment of RNAi knockdown hairy root mutants of HSS in Symphytum officinale. A knockdown of HSS by 60 - 80% resulted in a significant reduction of homospermidine by ~ 86% and of the major PA components 7-acetylintermedine N-oxide and 3-acetylmyoscorpine N-oxide by approximately 60%. The correlation of reduced transcript levels of HSS with reduced levels of homospermidine and PAs provides in planta support for HSS being the central enzyme in PA biosynthesis. Furthermore, the generation of PA-depleted hairy roots might be a cost-efficient way for reducing toxic by-products that limit the medicinal applicability of S. officinale extracts.

Specific Distribution of Pyrrolizidine Alkaloids in Floral Parts of Comfrey (Symphytum officinale) and its Implications for Flower Ecology.

Pyrrolizidine alkaloids (PAs) are a typical class of plant secondary metabolites that are constitutively produced as part of the plant's chemical defense. While roots are a well-established site of pyrrolizidine alkaloid biosynthesis, comfrey plants (Symphytum officinale; Boraginaceae) have been shown to additionally activate alkaloid production in specialized leaves and accumulate PAs in flowers during a short developmental stage in inflorescence development. To gain a better understanding of the accumulation and role of PAs in comfrey flowers and fruits, we have dissected and analyzed their tissues for PA content and patterns. PAs are almost exclusively accumulated in the ovaries, while petals, sepals, and pollen hardly contain PAs. High levels of PAs are detectable in the fruit, but the elaiosome was shown to be PA free. The absence of 7-acetyllycopsamine in floral parts while present in leaves and roots suggests that the additional site of PA biosynthesis provides the pool of PAs for translocation to floral structures. Our data suggest that PA accumulation has to be understood as a highly dynamic system resulting from a combination of efficient transport and additional sites of synthesis that are only temporarily active. Our findings are further discussed in the context of the ecological roles of PAs in comfrey flowers.

Crystal structure of pyrrolizidine alkaloid N-oxygenase from the grasshopper Zonocerus variegatus.

The high-resolution crystal structure of the flavin-dependent monooxygenase (FMO) from the African locust Zonocerus variegatus is presented and the kinetics of structure-based protein variants are discussed. Z. variegatus expresses three flavin-dependent monooxygenase (ZvFMO) isoforms which contribute to a counterstrategy against pyrrolizidine alkaloids (PAs). PAs are protoxic compounds produced by some angiosperm lineages as a chemical defence against herbivores. N-Oxygenation of PAs and the accumulation of PA N-oxides within their haemolymph result in two evolutionary advantages for these insects: (i) they circumvent the defence mechanism of their food plants and (ii) they can use PA N-oxides to protect themselves against predators, which cannot cope with the toxic PAs. Despite a high degree of sequence identity and a similar substrate spectrum, the three ZvFMO isoforms differ greatly in enzyme activity. Here, the crystal structure of the Z. variegatus PA N-oxygenase (ZvPNO), the most active ZvFMO isoform, is reported at 1.6 Šresolution together with kinetic studies of a second isoform, ZvFMOa. This is the first available crystal structure of an FMO from class B (of six different FMO subclasses, A-F) within the family of flavin-dependent monooxygenases that originates from a more highly developed organism than yeast. Despite the differences in sequence between family members, their overall structure is very similar. This indicates the need for high conservation of the three-dimensional structure for this type of reaction throughout all kingdoms of life. Nevertheless, this structure provides the closest relative to the human enzyme that is currently available for modelling studies. Of note, the crystal structure of ZvPNO reveals a unique dimeric arrangement as well as small conformational changes within the active site that have not been observed before. A newly observed kink within helix α8 close to the substrate-binding path might indicate a potential mechanism for product release. The data show that even single amino-acid exchanges in the substrate-entry path, rather than the binding site, have a significant impact on the specific enzyme activity of the isoforms.

Radioactive Tracer Feeding Experiments and Product Analysis to Determine the Biosynthetic Capability of Comfrey (Symphytum officinale) Leaves for Pyrrolizidine Alkaloids.

This protocol delivers a method to determine the biosynthetic capability of comfrey leaves for pyrrolizidine alkaloids independently from other organs like roots or flowers. The protocol applies and combines radioactive tracer experiments with standard and modern techniques like thin layer chromatography (TLC), solid-phase extraction (SPE), high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS).

Variability of Pyrrolizidine Alkaloid Occurrence in Species of the Grass Subfamily Pooideae (Poaceae).

Pyrrolizidine alkaloids (PAs) are a class of secondary metabolites found in various unrelated angiosperm lineages including cool-season grasses (Poaceae, subfamily Pooideae). Thesinine conjugates, saturated forms of PA that are regarded as non-toxic, have been described to occur in the two grass species Lolium perenne and Festuca arundinacea (Poaceae, subfamily Pooideae). In a wider screen, we tested various species of the Pooideae lineage, grown under controlled conditions, for their ability to produce thesinine conjugates or related structures. Using an LC-MS based targeted metabolomics approach we were able to show that PA biosynthesis in grasses is limited to a group of very closely related Pooideae species that produce a limited diversity of PA structures. High variability in PA levels was observed even between individuals of the same species. These individual accumulation patterns are discussed with respect to a possible function and evolution of this type of alkaloid.

Identification of a Second Site of Pyrrolizidine Alkaloid Biosynthesis in Comfrey to Boost Plant Defense in Floral Stage.

Pyrrolizidine alkaloids (PAs) are toxic secondary metabolites that are found in several distantly related families of the angiosperms. The first specific step in PA biosynthesis is catalyzed by homospermidine synthase (HSS), which has been recruited several times independently by duplication of the gene encoding deoxyhypusine synthase, an enzyme involved in the posttranslational activation of the eukaryotic initiation factor 5A. HSS shows highly diverse spatiotemporal gene expression in various PA-producing species. In comfrey (Symphytum officinale; Boraginaceae), PAs are reported to be synthesized in the roots, with HSS being localized in cells of the root endodermis. Here, we show that comfrey plants activate a second site of HSS expression when inflorescences start to develop. HSS has been localized in the bundle sheath cells of specific leaves. Tracer feeding experiments have confirmed that these young leaves express not only HSS but the whole PA biosynthetic route. This second site of PA biosynthesis results in drastically increased PA levels within the inflorescences. The boost of PA biosynthesis is proposed to guarantee optimal protection especially of the reproductive structures.

Comprehensive Structural Characterization of the Bacterial Homospermidine Synthase-an Essential Enzyme of the Polyamine Metabolism.

The highly conserved bacterial homospermidine synthase (HSS) is a key enzyme of the polyamine metabolism of many proteobacteria including pathogenic strains such as Legionella pneumophila and Pseudomonas aeruginosa; The unique usage of NAD(H) as a prosthetic group is a common feature of bacterial HSS, eukaryotic HSS and deoxyhypusine synthase (DHS). The structure of the bacterial enzyme does not possess a lysine residue in the active center and thus does not form an enzyme-substrate Schiff base intermediate as observed for the DHS. In contrast to the DHS the active site is not formed by the interface of two subunits but resides within one subunit of the bacterial HSS. Crystal structures of Blastochloris viridis HSS (BvHSS) reveal two distinct substrate binding sites, one of which is highly specific for putrescine. BvHSS features a side pocket in the direct vicinity of the active site formed by conserved amino acids and a potential substrate discrimination, guiding, and sensing mechanism. The proposed reaction steps for the catalysis of BvHSS emphasize cation-π interaction through a conserved Trp residue as a key stabilizer of high energetic transition states.

Paralogue Interference Affects the Dynamics after Gene Duplication.

Proteins tend to form homomeric complexes of identical subunits, which act as functional units. By definition, the subunits are encoded from a single genetic locus. When such a gene is duplicated, the gene products are suggested initially to cross-interact when coexpressed, thus resulting in the phenomenon of paralogue interference. In this opinion article, we explore how paralogue interference can shape the fate of a duplicated gene. One important outcome is a prolonged time window in which both copies remain under selection increasing the chance to accumulate mutations and to develop new properties. Thereby, paralogue interference can mediate the coevolution of duplicates and here we illustrate the potential of this phenomenon in light of recent new studies.

Single cell subtractive transcriptomics for identification of cell-specifically expressed candidate genes of pyrrolizidine alkaloid biosynthesis.

Progress has recently been made in the elucidation of pathways of secondary metabolism. However, because of its diversity, genetic information concerning biosynthetic details is still missing for many natural products. This is also the case for the biosynthesis of pyrrolizidine alkaloids. To close this gap, we tested strategies using tissues that express this pathway in comparison to tissues in which this pathway is not expressed. As many pathways of secondary metabolism are known to be induced by jasmonates, the pyrrolizidine alkaloid-producing species Heliotropium indicum, Symphytum officinale, and Cynoglossum officinale of the Boraginales order were treated with methyl jasmonate. An effect on pyrrolizidine alkaloid levels and on transcript levels of homospermidine synthase, the first specific enzyme of pyrrolizidine alkaloid biosynthesis, was not detectable. Therefore, a method was developed by making use of the often observed cell-specific production of secondary compounds. H. indicum produces pyrrolizidine alkaloids exclusively in the shoot. Homospermidine synthase is expressed only in the cells of the lower leaf epidermis and the epidermis of the stem. Suggesting that the whole pathway of pyrrolizidine alkaloid biosynthesis might be localized in these cells, we have isolated single cells of the upper and lower epidermis by laser-capture microdissection. The resulting cDNA preparations have been used in a subtractive transcriptomic approach. Quantitative real-time polymerase chain reaction has shown that the resulting library is significantly enriched for homospermidine-synthase-coding transcripts providing a valuable source for the identification of further genes involved in pyrrolizidine alkaloid biosynthesis.

New aspect of plant-rhizobia interaction: alkaloid biosynthesis in Crotalaria depends on nodulation.

Infection of legume hosts by rhizobial bacteria results in the formation of a specialized organ, the nodule, in which atmospheric nitrogen is reduced to ammonia. Nodulation requires the reprogramming of the plant cell, allowing the microsymbiont to enter the plant tissue in a highly controlled manner. We have found that, in Crotalaria (Fabaceae), this reprogramming is associated with the biosynthesis of pyrrolizidine alkaloids (PAs). These compounds are part of the plant's chemical defense against herbivores and cannot be regarded as being functionally involved in the symbiosis. PAs in Crotalaria are detectable only when the plants form nodules after infection with their rhizobial partner. The identification of a plant-derived sequence encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis, suggests that the plant and not the microbiont is the producer of PAs. Transcripts of HSS are detectable exclusively in the nodules, the tissue with the highest concentration of PAs, indicating that PA biosynthesis is restricted to the nodules and that the nodules are the source from which the alkaloids are transported to the above ground parts of the plant. The link between nodulation and the biosynthesis of nitrogen-containing alkaloids in Crotalaria highlights a further facet of the effect of symbiosis with rhizobia on the ecologically important trait of the plant's chemical defense.

Down-regulation of ZmEXPB6 (Zea mays ß-expansin 6) protein is correlated with salt-mediated growth reduction in the leaves of Z. mays L.

The salt-sensitive crop Zea mays L. shows a rapid leaf growth reduction upon NaCl stress. There is increasing evidence that salinity impairs the ability of the cell walls to expand, ultimately inhibiting growth. Wall-loosening is a prerequisite for cell wall expansion, a process that is under the control of cell wall-located expansin proteins. In this study the abundance of those proteins was analyzed against salt stress using gel-based two-dimensional proteomics and two-dimensional Western blotting. Results show that ZmEXPB6 (Z. mays ß-expansin 6) protein is lacking in growth-inhibited leaves of salt-stressed maize. Of note, the exogenous application of heterologously expressed and metal-chelate-affinity chromatography-purified ZmEXPB6 on growth-reduced leaves that lack native ZmEXPB6 under NaCl stress partially restored leaf growth. In vitro assays on frozen-thawed leaf sections revealed that recombinant ZmEXPB6 acts on the capacity of the walls to extend. Our results identify expansins as a factor that partially restores leaf growth of maize in saline environments.

Evolution of homospermidine synthase in the convolvulaceae: a story of gene duplication, gene loss, and periods of various selection pressures.

Homospermidine synthase (HSS), the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, is known to have its origin in the duplication of a gene encoding deoxyhypusine synthase. To study the processes that followed this gene duplication event and gave rise to HSS, we identified sequences encoding HSS and deoxyhypusine synthase from various species of the Convolvulaceae. We show that HSS evolved only once in this lineage. This duplication event was followed by several losses of a functional gene copy attributable to gene loss or pseudogenization. Statistical analyses of sequence data suggest that, in those lineages in which the gene copy was successfully recruited as HSS, the gene duplication event was followed by phases of various selection pressures, including purifying selection, relaxed functional constraints, and possibly positive Darwinian selection. Site-specific mutagenesis experiments have confirmed that the substitution of sites predicted to be under positive Darwinian selection is sufficient to convert a deoxyhypusine synthase into a HSS. In addition, analyses of transcript levels have shown that HSS and deoxyhypusine synthase have also diverged with respect to their regulation. The impact of protein-protein interaction on the evolution of HSS is discussed with respect to current models of enzyme evolution.

Distinct cell-specific expression of homospermidine synthase involved in pyrrolizidine alkaloid biosynthesis in three species of the boraginales.

Homospermidine synthase (HSS) is the first specific enzyme in pyrrolizidine alkaloid (PA) biosynthesis, a pathway involved in the plant's chemical defense. HSS has been shown to be recruited repeatedly by duplication of a gene involved in primary metabolism. Within the lineage of the Boraginales, only one gene duplication event gave rise to HSS. Here, we demonstrate that the tissue-specific expression of HSS in three boraginaceous species, Heliotropium indicum, Symphytum officinale, and Cynoglossum officinale, is unique with respect to plant organ, tissue, and cell type. Within H. indicum, HSS is expressed exclusively in nonspecialized cells of the lower epidermis of young leaves and shoots. In S. officinale, HSS expression has been detected in the cells of the root endodermis and in leaves directly underneath developing inflorescences. In young roots of C. officinale, HSS is detected only in cells of the endodermis, but in a later developmental stage, additionally in the pericycle. The individual expression patterns are compared with those within the Senecioneae lineage (Asteraceae), where HSS expression is reproducibly found in specific cells of the endodermis and the adjacent cortex parenchyma of the roots. The individual expression patterns within the Boraginales species are discussed as being a requirement for the successful recruitment of HSS after gene duplication. The diversity of HSS expression within this lineage adds a further facet to the already diverse patterns of expression that have been observed for HSS in other PA-producing plant lineages, making this PA-specific enzyme one of the most diverse expressed proteins described in the literature.

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.

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.