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.

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.

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.