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.

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.

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.

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