Dietrich Ober

Biosynthesis and Metabolism of Pyrrolizidine Alkaloids in Plants and Specialized Insect Herbivores

Pyrrolizidine alkaloids (PAs) encompass a typical class of plant secondary compounds. During recent years PAs have proved to be an excellent choice to exemplify various mechanistic and functional aspects of plant secondary metabolism. PAs appear to play an important role in constitutive plant chemical defense, particularly, in plant-herbivore interactions. Biochemical and physiological aspects of PA biosynthesis, allocation, and accumulation are reviewed with particular emphasis on key enzymatic steps and chemical diversification of PA backbone structures. A number of taxonomically unrelated specialized insect herbivores sequester PAs from their food plants. The biochemistry of PA sequestration in lepidopterans and leaf beetles is outlined along with the mechanisms of PA absorption, storage, pheromone biosynthesis, and insect-specific PA transformations (i.e., formation of insect alkaloids). The unique feature of PAs, that they exist in two easily interchangeable forms, the pro-toxic free base and the nontoxic N-oxide, is stressed and related to the function of PAs as defensive compounds. The first molecular evidence concerning the phylogenetic origin of PAs is presented.

Evolutionary recruitment of a flavin-dependent monooxygenase for the detoxification of host plant-acquired pyrrolizidine alkaloids in the alkaloid-defended arctiid moth Tyria jacobaeae

Larvae of Tyria jacobaeae feed solely upon the pyrrolizidine alkaloid-containing plant Senecio jacobaea. Ingested pyrrolizidine alkaloids (PAs), which are toxic to unspecialized insects and vertebrates, are efficiently N-oxidized in the hemolymph of T. jacobaeae by senecionine N-oxygenase (SNO), a flavin-dependent monooxygenase (FMO) with a high substrate specificity for PAs. Peptide microsequences obtained from purified T. jacobaeae SNO were used to clone the corresponding cDNA, which was expressed in active form in Escherichia coli. T. jacobaeae SNO possesses a signal peptide characteristic of extracellular proteins, and it belongs to a large family of mainly FMO-like sequences of mostly unknown function, including two predicted Drosophila melanogaster gene products. The data indicate that the gene for T. jacobaeae SNO, highly specific for toxic pyrrolizidine alkaloids, was recruited from a preexisting insect-specific FMO gene family of hitherto unknown function. The enzyme allows the larvae to feed on PA-containing plants and to accumulate predation-deterrent PAs in the hemolymph.

Deoxyhypusine Synthase from Tobacco cDNA ISOLATION, CHARACTERIZATION, AND BACTERIAL EXPRESSION OF AN ENZYME WITH EXTENDED SUBSTRATE SPECIFICITY

Deoxyhypusine synthase catalyzes the formation of a deoxyhypusine residue in the translation eukaryotic initiation factor 5A (eIF5A) precursor protein by transferring an aminobutyl moiety from spermidine onto a conserved lysine residue within the eIF5A polypeptide chain. This reaction commences the activation of the initiation factor in fungi and vertebrates. A mechanistically identical reaction is known in the biosynthetic pathway leading to pyrrolizidine alkaloids in plants. Deoxyhypusine synthase from tobacco was cloned and expressed in active form in Escherichia coli. It catalyzes the formation of a deoxyhypusine residue in the tobacco eIF5A substrate as shown by gas chromatography coupled with a mass spectrometer. The enzyme also accepts free putrescine as the aminobutyl acceptor, instead of lysine bound in the eIF5A polypeptide chain, yielding homospermidine. Conversely, it accepts homospermidine instead of spermidine as the aminobutyl donor, whereby the reactions with putrescine and homospermidine proceed at the same rate as those involving the authentic substrates. The conversion of deoxyhypusine synthase-catalyzed eIF5A deoxyhypusinylation pinpoints a function for spermidine in plant metabolism. Furthermore, and quite unexpectedly, the substrate spectrum of deoxyhypusine synthase hints at a biochemical basis behind the sparse and skew occurrence of both homospermidine and its pyrrolizidine derivatives across distantly related plant taxa.

Repeated Evolution of the Pyrrolizidine Alkaloid–Mediated Defense System in Separate Angiosperm Lineages

Species of several unrelated families within the angiosperms are able to constitutively produce pyrrolizidine alkaloids as a defense against herbivores. In pyrrolizidine alkaloid (PA) biosynthesis, homospermidine synthase (HSS) catalyzes the first specific step. HSS was recruited during angiosperm evolution from deoxyhypusine synthase (DHS), an enzyme involved in the posttranslational activation of eukaryotic initiation factor 5A. Phylogenetic analysis of 23 cDNA sequences coding for HSS and DHS of various angiosperm species revealed at least four independent recruitments of HSS from DHS: one within the Boraginaceae, one within the monocots, and two within the Asteraceae family. Furthermore, sequence analyses indicated elevated substitution rates within HSS-coding sequences after each gene duplication, with an increased level of nonsynonymous mutations. However, the contradiction between the polyphyletic origin of the first enzyme in PA biosynthesis and the structural identity of the final biosynthetic PA products needs clarification.

Cell-specific expression of homospermidine synthase, the entry enzyme of the pyrrolizidine alkaloid pathway in Senecio vernalis, in comparison with its ancestor, deoxyhypusine synthase.

Pyrrolizidine alkaloids (PAs) are constitutive plant defense compounds with a sporadic taxonomic occurrence. The first committed step in PA biosynthesis is catalyzed by homospermidine synthase (HSS). Recent evidence confirmed that HSS evolved by gene duplication from deoxyhypusine synthase (DHS), an enzyme involved in the posttranslational activation of the eukaryotic translation initiation factor 5A. To better understand the evolutionary relationship between these two enzymes, which are involved in completely different biological processes, we studied their tissue-specific expression. RNA-blot analysis, reverse transcriptase-PCR, and immunolocalization techniques demonstrated that DHS is constitutively expressed in shoots and roots of Senecio vernalis (Asteraceae), whereas HSS expression is root specific and restricted to distinct groups of endodermis and neighboring cortex cells located opposite to the phloem. All efforts to detect DHS by immunolocalization failed, but studies with promoter-β-glucuronidase fusions confirmed a general expression pattern, at least in young seedlings of tobacco (Nicotiana tabacum). The expression pattern for HSS differs completely from its ancestor DHS due to the adaptation of HSS to the specific requirements of PA biosynthesis.

Pyrrolizidine alkaloid biosynthesis, evolution of a pathway in plant secondary metabolism.

The system of pyrrolizidine alkaloids has proven to be a powerful system for studying the evolution of a biosynthetic pathway in plant secondary metabolism. Pyrrolizidine alkaloids are typical plant secondary products produced by the plant as a defense against herbivores. The first specific enzyme, homospermidine synthase, has been shown to have evolved by duplication of the gene encoding deoxyhypusine synthase, which is involved in primary metabolism. Despite the identical function of homospermidine synthase for pyrrolizidine alkaloid biosynthesis in the various plant lineages, this gene duplication has occurred several times independently during angiosperm evolution. After duplication, these gene copies diverged with respect to gene function and regulation. In the diverse plant lineages producing pyrrolizidine alkaloids, homospermidine synthase has been shown to be expressed in a variety of tissues, suggesting that the regulatory elements were recruited individually after the duplication of the structural gene. The molecular, kinetic, and expression data of this system are discussed with respect to current models of gene and pathway evolution.

Polyphyletic Origin of Pyrrolizidine Alkaloids within the Asteraceae. Evidence from Differential Tissue Expression of Homospermidine Synthase

The evolution of pathways within plant secondary metabolism has been studied by using the pyrrolizidine alkaloids (PAs) as a model system. PAs are constitutively produced by plants as a defense against herbivores. The occurrence of PAs is restricted to certain unrelated families within the angiosperms. Homospermidine synthase (HSS), the first specific enzyme in the biosynthesis of the necine base moiety of PAs, was originally recruited from deoxyhypusine synthase, an enzyme involved in the posttranslational activation of the eukaryotic initiation factor 5A. Recently, this gene recruitment has been shown to have occurred several times independently within the angiosperms and even twice within the Asteraceae. Here, we demonstrate that, within these two PA-producing tribes of the Asteraceae, namely Senecioneae and Eupatorieae, HSS is expressed differently despite catalyzing the same step in PA biosynthesis. Within Eupatorium cannabinum, HSS is expressed uniformly in all cells of the root cortex parenchyma, but not within the endodermis and exodermis. Within Senecio vernalis, HSS expression has been previously identified in groups of specialized cells of the endodermis and the adjacent root cortex parenchyma. This expression pattern was confirmed for Senecio jacobaea as well. Furthermore, the expression of HSS in E. cannabinum is dependent on the development of the plant, suggesting a close linkage to plant growth.

Gene duplications and the time thereafter - examples from plant secondary metabolism.

Gene duplications are regarded as one of the central mechanisms for the origin of new genes. Recent studies in plant secondary metabolism have provided several examples of genes that originated by duplication with successive diversification. In this review, the mechanisms of gene duplication are explained and several models discussed that suggest the way that gene duplicates develop into genes with new functions. Signatures of gene duplication and diversification processes are discussed using the biosynthesis of benzoxazinones and of pyrrolizidine alkaloids as examples.

The evolution of pyrrolizidine alkaloid biosynthesis and diversity in the Senecioneae

Pyrrolizidine alkaloids are characteristic secondary metabolites of the Asteraceae and some other plant families. They are especially numerous and diverse in the tribe Senecioneae and form a powerful defense mechanism against herbivores. Studies into the evolution of pyrrolizidine alkaloid biosynthesis using Senecio species have identified homospermidine synthase as the enzyme responsible for the synthesis of the first specific intermediate. These studies further indicated that the homospermidine synthase-encoding gene was recruited following gene duplication of deoxyhypusine synthase and that this occurred independently in several different angiosperm lineages. A review of published pyrrolizidine alkaloid data shows that the Senecioneae are characterized by a large qualitative and quantitative variation in pyrrolizidine alkaloid profiles and that these data demonstrate little phylogenetic signal. This suggests that although the first steps of this pathway are highly conserved, the diversification of secondarily derived pyrrolizidine alkaloids is extremely plastic.

Flavin-dependent monooxygenases as a detoxification mechanism in insects: new insights from the arctiids (lepidoptera).

Insects experience a wide array of chemical pressures from plant allelochemicals and pesticides and have developed several effective counterstrategies to cope with such toxins. Among these, cytochrome P450 monooxygenases are crucial in plant-insect interactions. Flavin-dependent monooxygenases (FMOs) seem not to play a central role in xenobiotic detoxification in insects, in contrast to mammals. However, the previously identified senecionine N-oxygenase of the arctiid moth Tyria jacobaeae (Lepidoptera) indicates that FMOs have been recruited during the adaptation of this insect to plants that accumulate toxic pyrrolizidine alkaloids. Identification of related FMO-like sequences of various arctiids and other Lepidoptera and their combination with expressed sequence tag (EST) data and sequences emerging from the Bombyx mori genome project show that FMOs in Lepidoptera form a gene family with three members (FMO1 to FMO3). Phylogenetic analyses suggest that FMO3 is only distantly related to lepidopteran FMO1 and FMO2 that originated from a more recent gene duplication event. Within the FMO1 gene cluster, an additional gene duplication early in the arctiid lineage provided the basis for the evolution of the highly specific biochemical, physiological, and behavioral adaptations of these butterflies to pyrrolizidine-alkaloid-producing plants. The genes encoding pyrrolizidine-alkaloid-N-oxygenizing enzymes (PNOs) are transcribed in the fat body and the head of the larvae. An N-terminal signal peptide mediates the transport of the soluble proteins into the hemolymph where PNOs efficiently convert pro-toxic pyrrolizidine alkaloids into their non-toxic N-oxide derivatives. Heterologous expression of a PNO of the generalist arctiid Grammia geneura produced an N-oxygenizing enzyme that shows noticeably expanded substrate specificity compared with the related enzyme of the specialist Tyria jacobaeae. The data about the evolution of FMOs within lepidopteran insects and the functional characterization of a further member of this enzyme family shed light on this almost uncharacterized detoxification system in insects.