A radiation of Psylliodes flea beetles on Brassicaceae is associated with the evolution of specific detoxification enzymes.

Flea beetles of the genus Psylliodes have evolved specialized interactions with plant species belonging to several distantly related families, mainly Brassicaceae, Solanaceae, and Fagaceae. This diverse host use indicates that Psylliodes flea beetles are able to cope with different chemical defense metabolites, including glucosinolates, the characteristic defense metabolites of Brassicaceae. Here we investigated the evolution of host use and the emergence of a glucosinolate-specific detoxification mechanism in Psylliodes flea beetles. In phylogenetic analyses, Psylliodes species clustered into four major clades, three of which contained mainly species specialized on either Brassicaceae, Solanaceae, or Fagaceae. Most members of the fourth clade have broader host use, including Brassicaceae and Poaceae as major host plant families. Ancestral state reconstructions suggest that Psylliodes flea beetles were initially associated with Brassicaceae and then either shifted to Solanaceae or Fagaceae, or expanded their host repertoire to Poaceae. Despite a putative ancestral association with Brassicaceae, we found evidence that the evolution of glucosinolate-specific detoxification enzymes coincides with the radiation of Psylliodes on Brassicaceae, suggesting that these are not required for using Brassicaceae as hosts but could improve the efficiency of host use by specialized Psylliodes species.

Hijacking the Mustard-Oil Bomb: How a Glucosinolate-Sequestering Flea Beetle Copes With Plant Myrosinases.

Myrosinase enzymes play a key role in the chemical defense of plants of the order Brassicales. Upon herbivory, myrosinases hydrolyze the ß-S-linked glucose moiety of glucosinolates, the characteristic secondary metabolites of brassicaceous plants, which leads to the formation of different toxic hydrolysis products. The specialist flea beetle, Phyllotreta armoraciae, is capable of accumulating high levels of glucosinolates in the body and can thus at least partially avoid plant myrosinase activity. In feeding experiments with the myrosinase-deficient Arabidopsis thaliana tgg1 × tgg2 (tgg) mutant and the corresponding Arabidopsis Col-0 wild type, we investigated the influence of plant myrosinase activity on the metabolic fate of ingested glucosinolates in adult P. armoraciae beetles. Arabidopsis myrosinases hydrolyzed a fraction of ingested glucosinolates and thereby reduced the glucosinolate sequestration rate by up to 50% in adult beetles. These results show that P. armoraciae cannot fully prevent glucosinolate hydrolysis; however, the exposure of adult beetles to glucosinolate hydrolysis products had no impact on the beetle's energy budget under our experimental conditions. To understand how P. armoraciae can partially prevent glucosinolate hydrolysis, we analyzed the short-term fate of ingested glucosinolates and found them to be rapidly absorbed from the gut. In addition, we determined the fate of ingested Arabidopsis myrosinase enzymes in P. armoraciae. Although we detected Arabidopsis myrosinase protein in the feces, we found only traces of myrosinase activity, suggesting that P. armoraciae can inactivate plant myrosinases in the gut. Based on our findings, we propose that the ability to tolerate plant myrosinase activity and a fast glucosinolate uptake mechanism represent key adaptations of P. armoraciae to their brassicaceous host plants.

The phytopathogenic fungus Sclerotinia sclerotiorum detoxifies plant glucosinolate hydrolysis products via an isothiocyanate hydrolase.

Brassicales plants produce glucosinolates and myrosinases that generate toxic isothiocyanates conferring broad resistance against pathogens and herbivorous insects. Nevertheless, some cosmopolitan fungal pathogens, such as the necrotrophic white mold Sclerotinia sclerotiorum, are able to infect many plant hosts including glucosinolate producers. Here, we show that S. sclerotiorum infection activates the glucosinolate-myrosinase system, and isothiocyanates contribute to resistance against this fungus. S. sclerotiorum metabolizes isothiocyanates via two independent pathways: conjugation to glutathione and, more effectively, hydrolysis to amines. The latter pathway features an isothiocyanate hydrolase that is homologous to a previously characterized bacterial enzyme, and converts isothiocyanate into products that are not toxic to the fungus. The isothiocyanate hydrolase promotes fungal growth in the presence of the toxins, and contributes to the virulence of S. sclerotiorum on glucosinolate-producing plants.

One Pathway Is Not Enough: The Cabbage Stem Flea Beetle Psylliodes chrysocephala Uses Multiple Strategies to Overcome the Glucosinolate-Myrosinase Defense in Its Host Plants.

The cabbage stem flea beetle (Psylliodes chrysocephala) is a key pest of oilseed rape in Europe, and is specialized to feed on Brassicaceae plants armed with the glucosinolate-myrosinase defense system. Upon tissue damage, the ß-thioglucosidase enzyme myrosinase hydrolyzes glucosinolates (GLS) to form toxic isothiocyanates (ITCs) which deter non-adapted herbivores. Here, we show that P. chrysocephala selectively sequester GLS from their host plants and store these throughout their life cycle. In addition, P. chrysocephala metabolize GLS to desulfo-GLS, which implies the evolution of GLS sulfatase activity in this specialist. To assess whether P. chrysocephala can largely prevent GLS hydrolysis in ingested plant tissue by sequestration and desulfation, we analyzed the metabolic fate of 4-methylsulfinylbutyl (4MSOB) GLS in adults. Surprisingly, intact and desulfo-GLS together accounted for the metabolic fate of only 26% of the total ingested GLS in P. chrysocephala, indicating that most ingested GLS are nevertheless activated by the plant myrosinase. The presence of 4MSOB-ITC and the corresponding nitrile in feces extracts confirmed the activation of ingested GLS, but the detected amounts of unmetabolized ITCs were low. P. chrysocephala partially detoxifies ITCs by conjugation with glutathione via the conserved mercapturic acid pathway. In addition to known products of the mercapturic acid pathway, we identified two previously unknown cyclic metabolites derived from the cysteine-conjugate of 4MSOB-ITC. In summary, the cabbage stem flea beetle avoids ITC formation by specialized strategies, but also relies on and extends the conserved mercapturic acid pathway to prevent toxicity of formed ITCs.

Tissue-Specific Emission of (E)-α-Bergamotene Helps Resolve the Dilemma When Pollinators Are Also Herbivores.

More than 87% of flowering plant species are animal-pollinated [1] and produce floral scents and other signals to attract pollinators. These floral cues may however also attract antagonistic visitors, including herbivores [2]. The dilemma is exacerbated when adult insects pollinate the same plant that their larvae consume. It remains largely unclear how plants maximize their fitness under these circumstances. Here we show that in the night-flowering wild tobacco Nicotiana attenuata, the emission of a sesquiterpene, (E)-α-bergamotene, in flowers increases adult Manduca sexta moth-mediated pollination success, while the same compound in leaves is known to mediate indirect defense against M. sexta larvae [3, 4]. Forward and reverse genetic analyses demonstrated that both herbivory-induced and floral (E)-α-bergamotene are regulated by the expression of a monoterpene-synthase-derived sesquiterpene synthase (NaTPS38). The expression pattern of NaTPS38 also accounts for variation in (E)-α-bergamotene emission among natural accessions. These results highlight that differential expression of a single gene that results in tissue-specific emission of one compound contributes to resolving the dilemma for plants when their pollinators are also herbivores. Furthermore, this study provides genetic evidence that pollinators and herbivores interactively shape the evolution of floral signals and plant defense.

Gene coexpression analysis reveals complex metabolism of the monoterpene alcohol linalool in Arabidopsis flowers.

The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (-)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined.