Metabolic novelty originating from horizontal gene transfer is essential for leaf beetle survival.

Horizontal gene transfer (HGT) provides an evolutionary shortcut for recipient organisms to gain novel functions. Although reports of HGT in higher eukaryotes are rapidly accumulating, in most cases the evolutionary trajectory, metabolic integration, and ecological relevance of acquired genes remain unclear. Plant cell wall degradation by HGT-derived enzymes is widespread in herbivorous insect lineages. Pectin is an abundant polysaccharide in the walls of growing parts of plants. We investigated the significance of horizontally acquired pectin-digesting polygalacturonases (PGs) of the leaf beetle Phaedon cochleariae. Using a CRISPR/Cas9-guided gene knockout approach, we generated a triple knockout and a quadruple PG-null mutant in order to investigate the enzymatic, biological, and ecological effects. We found that pectin-digestion 1) is exclusively linked to the horizontally acquired PGs from fungi, 2) became fixed in the host genome by gene duplication leading to functional redundancy, 3) compensates for nutrient-poor diet by making the nutritious cell contents more accessible, and 4) facilitates the beetles development and survival. Our analysis highlights the selective advantage PGs provide to herbivorous insects and demonstrate the impact of HGT on the evolutionary success of leaf-feeding beetles, major contributors to species diversity.

Biosynthesis of iridoid sex pheromones in aphids.

Iridoid monoterpenes, widely distributed in plants and insects, have many ecological functions. While the biosynthesis of iridoids has been extensively studied in plants, little is known about how insects synthesize these natural products. Here, we elucidated the biosynthesis of the iridoids cis-trans-nepetalactol and cis-trans-nepetalactone in the pea aphid Acyrthosiphon pisum (Harris), where they act as sex pheromones. The exclusive production of iridoids in hind legs of sexual female aphids allowed us to identify iridoid genes by searching for genes specifically expressed in this tissue. Biochemical characterization of candidate enzymes revealed that the iridoid pathway in aphids proceeds through the same sequence of intermediates as described for plants. The six identified aphid enzymes are unrelated to their counterparts in plants, conclusively demonstrating an independent evolution of the entire iridoid pathway in plants and insects. In contrast to the plant pathway, at least three of the aphid iridoid enzymes are likely membrane bound. We demonstrated that a lipid environment facilitates the cyclization of a reactive enol intermediate to the iridoid cyclopentanoid-pyran scaffold in vitro, suggesting that membranes are an essential component of the aphid iridoid pathway. Altogether, our discovery of this complex insect metabolic pathway establishes the genetic and biochemical basis for the formation of iridoid sex pheromones in aphids, and this discovery also serves as a foundation for understanding the convergent evolution of complex metabolic pathways between kingdoms.

Occurrence and conversion of progestogens and androgens are conserved in land plants.

Progestogens and androgens have been found in many plants, but little is known about their biosynthesis and the evolution of steroidogenesis in these organisms. Here, we show that the occurrence and biosynthesis of progestogens and androgens are conserved across the viridiplantae lineage. An UHPLC-ESI-MS/MS method allowed high-throughput analysis of the occurrence and chemical conversion of progestogens and androgens in 41 species across the green plant lineage. Dehydroepiandrosterone, testosterone, and 5α-dihydrotestosterone are plants' most abundant mammalian-like steroids. Progestogens are converted into 17α-hydroxyprogesterone and 5α-pregnane-3,20-dione. Androgens are converted into testosterone and 5α-dihydrotestosterone. 17,20-Lyases, essential for converting progestogens to androgens, seem to be most effective in monocot species. Our data suggest that the occurrence of progestogens and androgens is highly conserved in plants, and their biosynthesis might favor a route using the Δ4 pathway.

Biosynthesis and antifungal activity of fungus-induced O-methylated flavonoids in maize.

Fungal infection of grasses, including rice (Oryza sativa), sorghum (Sorghum bicolor), and barley (Hordeum vulgare), induces the formation and accumulation of flavonoid phytoalexins. In maize (Zea mays), however, investigators have emphasized benzoxazinoid and terpenoid phytoalexins, and comparatively little is known about flavonoid induction in response to pathogens. Here, we examined fungus-elicited flavonoid metabolism in maize and identified key biosynthetic enzymes involved in the formation of O-methylflavonoids. The predominant end products were identified as two tautomers of a 2-hydroxynaringenin-derived compound termed xilonenin, which significantly inhibited the growth of two maize pathogens, Fusarium graminearum and Fusarium verticillioides. Among the biosynthetic enzymes identified were two O-methyltransferases (OMTs), flavonoid OMT 2 (FOMT2), and FOMT4, which demonstrated distinct regiospecificity on a broad spectrum of flavonoid classes. In addition, a cytochrome P450 monooxygenase (CYP) in the CYP93G subfamily was found to serve as a flavanone 2-hydroxylase providing the substrate for FOMT2-catalyzed formation of xilonenin. In summary, maize produces a diverse blend of O-methylflavonoids with antifungal activity upon attack by a broad range of fungi.

The Ca2+ sensor proteins CML37 and CML42 antagonistically regulate plant stress responses by altering phytohormone signals.

KEY MESSAGE: Calmodulin-like-proteins (CML) belong to a family of calcium-sensing proteins that are unique for plants and involved in many different developmental and stress-related reactions. In defense against herbivory, some pathogens and drought, CML37 acts as a positive and CML42 as a negative regulator, respectively. We provide evidence that both CMLs act antagonistically in the regulation of induced defense responses. A double knock-out line, cml37 x cml42, thus shows wild-type phenotypes upon all kind of stresses we used. A transient increase in the cytosolic calcium concentration is one of the first reactions that can be measured in plant cells upon abiotic as well as biotic stress treatments. These calcium signals are sensed by calcium binding proteins such as calmodulin-like proteins (CMLs), which transduce the sensed information into appropriate stress responses by interacting with downstream target proteins. In previous studies, CML37 has been shown to positively regulate the plants' defense against both the insect herbivore Spodoptera littoralis and the response to drought stress. In contrast, CML42 is known to negatively regulate those two stress responses. Here, we provide evidence that these two CMLs act antagonistically in the regulation of induced responses directed against drought and herbivory stress as well as in the defense against the necrotrophic pathogen Alternaria brassicicola. Both CMLs shape the plant reactions by altering the phytohormone signaling. Consequently, the phytohormone-regulated production of defensive compounds like glucosinolates is also antagonistically mediated by both CMLs. The finding that CML37 and CML42 have antagonistic roles in diverse stress-related responses suggests that these calcium sensor proteins represent important tools for the plant to balance and fine-tune the signaling and downstream reactions upon environmental stress.

Species-Specific and Distance-Dependent Dispersive Behaviour of Forisomes in Different Legume Species.

Forisomes are giant fusiform protein complexes composed of sieve element occlusion (SEO) protein monomers, exclusively found in sieve elements (SEs) of legumes. Forisomes block the phloem mass flow by a Ca2+-induced conformational change (swelling and rounding). We studied the forisome reactivity in four different legume species-Medicago sativa, Pisum sativum, Trifolium pratense and Vicia faba. Depending on the species, we found direct relationships between SE diameter, forisome surface area and distance from the leaf tip, all indicative of a developmentally tuned regulation of SE diameter and forisome size. Heat-induced forisome dispersion occurred later with increasing distance from the stimulus site. T. pratense and V. faba dispersion occurred faster for forisomes with a smaller surface area. Near the stimulus site, electro potential waves (EPWs)-overlapping action (APs), and variation potentials (VPs)-were linked with high full-dispersion rates of forisomes. Distance-associated reduction of forisome reactivity was assigned to the disintegration of EPWs into APs, VPs and system potentials (SPs). Overall, APs and SPs alone were unable to induce forisome dispersion and only VPs above a critical threshold were capable of inducing forisome reactions.

Glucosinolate Abundance and Composition in Brassicaceae Influence Sequestration in a Specialist Flea Beetle.

The horseradish flea beetle Phyllotreta armoraciae exclusively feeds on Brassicaceae, which contain glucosinolates as characteristic defense compounds. Although glucosinolates are usually degraded by plant enzymes (myrosinases) to toxic isothiocyanates after ingestion, P. armoraciae beetles sequester glucosinolates. Between and within brassicaceous plants, the glucosinolate content and composition can differ drastically. But how do these factors influence sequestration in P. armoraciae? To address this question, we performed a five-day feeding experiment with three Arabidopsis thaliana lines that differ four-fold in glucosinolate content and the composition of aliphatic and indolic glucosinolates. We quantified the amounts of ingested, sequestered, and excreted glucosinolates, and analyzed the changes in glucosinolate levels and composition in beetles before and after feeding on Arabidopsis. P. armoraciae accumulated almost all ingested glucosinolate types. However, some glucosinolates were accumulated more efficiently than others, and selected glucosinolates were modified by the beetles. The uptake of new glucosinolates correlated with a decrease in the level of stored glucosinolates so that the total glucosinolate content remained stable at around 35 nmol/mg beetle fresh weight. Beetles excreted previously stored as well as ingested glucosinolates from Arabidopsis, which suggests that P. armoraciae regulate their endogenous glucosinolate level by excretion. The metabolic fate of ingested glucosinolates, i.e. the proportions of sequestered and excreted glucosinolates, depended on glucosinolate type, content, and composition in the food plant. Overall, P. armoraciae sequestered and excreted up to 41% and 31% of the total ingested aliphatic and indolic glucosinolates from Arabidopsis, respectively. In summary, we show that glucosinolate variability in Brassicaceae influences the composition but not the level of sequestered glucosinolates in P. armoraciae beetles.

Biosynthetic and Functional Color-Scent Associations in Flowers of Papaver nudicaule and Their Impact on Pollinators.

Despite increasing evidence for biosynthetic connections between flower pigments and volatile compounds, examples of such relationships in polymorphic plant species remains limited. Herein, color-scent associations in flowers from Papaver nudicaule (Papaveraceae) have been investigated. The spectral reflectance and scent composition of flowers of four color cultivars was determined. We found that pigments and volatiles occur in specific combinations in flowers of P. nudicaule. The presence of indole in the bouquets is strongly associated with the occurrence of yellow pigments called nudicaulins, for which indole is one of the final biosynthetic precursors. Whereas yellow flowers emit an excess of indole, orange flowers consume it during nudicaulin production and lack the substance in their bouquet. By using the honeybee, Apis mellifera, evaluations were made on how color and scent affect the discrimination of these flowers by pollinators. Honeybees were able to discriminate artificial odor mixtures resembling those of the natural flower odors. Bees trained with stimuli combining colors and odors showed an improved discrimination performance. The results indicate that the indole moiety of nudicaulins and emitted indole might be products of the same biochemical pathway. We propose that conserved pathways account for the evolution of color-scent associations in P. nudicaule and that these associations positively affect flower constancy of pollinators.

Phyllotreta striolata flea beetles use host plant defense compounds to create their own glucosinolate-myrosinase system.

The ability of a specialized herbivore to overcome the chemical defense of a particular plant taxon not only makes it accessible as a food source but may also provide metabolites to be exploited for communication or chemical defense. Phyllotreta flea beetles are adapted to crucifer plants (Brassicales) that are defended by the glucosinolate-myrosinase system, the so-called "mustard-oil bomb." Tissue damage caused by insect feeding brings glucosinolates into contact with the plant enzyme myrosinase, which hydrolyzes them to form toxic compounds, such as isothiocyanates. However, we previously observed that Phyllotreta striolata beetles themselves produce volatile glucosinolate hydrolysis products. Here, we show that P. striolata adults selectively accumulate glucosinolates from their food plants to up to 1.75% of their body weight and express their own myrosinase. By combining proteomics and transcriptomics, a gene responsible for myrosinase activity in P. striolata was identified. The major substrates of the heterologously expressed myrosinase were aliphatic glucosinolates, which were hydrolyzed with at least fourfold higher efficiency than aromatic and indolic glucosinolates, and ß-O-glucosides. The identified beetle myrosinase belongs to the glycoside hydrolase family 1 and has up to 76% sequence similarity to other ß-glucosidases. Phylogenetic analyses suggest species-specific diversification of this gene family in insects and an independent evolution of the beetle myrosinase from other insect ß-glucosidases.

Sex ratio of mirid populations shifts in response to hostplant co-infestation or altered cytokinin signaling .

Herbivore species sharing a host plant often compete. In this study, we show that host plant-mediated interaction between two insect herbivores - a generalist and a specialist - results in a sex ratio shift of the specialist's offspring. We studied demographic parameters of the specialist Tupiocoris notatus (Hemiptera: Miridae) when co-infesting the host plant Nicotiana attenuata (Solanaceae) with the generalist leafhopper Empoasca sp. (Hemiptera: Cicadellidae). We show that the usually female-biased sex ratio of T. notatus shifts toward a higher male proportion in the offspring on plants co-infested by Empoasca sp. This sex ratio change did not occur after oviposition, nor is it due differential mortality of female and male nymphs. Based on pyrosequencing and PCR of bacterial 16S rRNA amplicons, we concluded that sex ratio shifts were unlikely to be due to infection with Wolbachia or other known sex ratio-distorting endosymbionts. Finally, we used transgenic lines of N. attenuata to evaluate if the sex ratio shift could be mediated by changes in general or specialized host plant metabolites. We found that the sex ratio shift occurred on plants deficient in two cytokinin receptors (irCHK2/3). Thus, cytokinin-regulated traits can alter the offspring sex ratio of the specialist T. notatus.

Hoverfly preference for high honeydew amounts creates enemy-free space for aphids colonizing novel host plants.

The existence of an enemy-free space can play an important role in aphid host race formation processes, but little is known about the mechanisms that create an area of low predation pressure on particular host plants. In this paper, we identify a mechanism generating lower predation pressure that promotes the maintenance of the different host races of the pea aphid (Acyrthosiphon pisum) complex, a well-studied model for ecological speciation. The pea aphid consists of at least 15 genetically distinct host races which are native to specific host plants of the legume family, but can all develop on the universal host plant Vicia faba. Previous work showed that hoverfly (Episyrphus balteatus) oviposition preferences contribute to the enemy-free space that helps to maintain the different pea aphid host races, and that higher amounts of honeydew are more attractive to ovipositing hoverflies. Here we demonstrated that aphid honeydew is produced in large amounts when aphid reproduction rate was highest, and is an important oviposition cue for hoverflies under field conditions. However, on less suitable host plants, where honeydew production is reduced, pea aphids enjoy lower predation rates. A reduction in enemy pressure can mitigate the performance disadvantages of aphids colonizing a novel host and probably plays an important role in pea aphid host race formation.

Enemy-free space promotes maintenance of host races in an aphid species.

The enormous biodiversity of herbivorous insects may arise from ecological speciation via continuous host-plant switches. Whether such switches are successful depends on the trade-off between different selection pressures that act on herbivores. Decreased herbivore performance due to suboptimal nutrition might be compensated for by a reduced natural enemy pressure. As a consequence, an "enemy-free space" on a certain plant might facilitate host-plant switches and maintain biotypes. To test this hypothesis, we used the pea aphid (Acyrthosiphon pisum) complex, which consists of at least 11 genetically distinct host races that are native to specific legume host plants but can all develop on the universal host plant Vicia faba. Three A. pisum host races native to Trifolium pratense, Pisum sativum, and Medicago sativa were investigated in experiments on their respective host plants and on the universal host plant V. faba. We found that hoverflies preferred to oviposit on P. sativum and the universal host V. faba. Since feeding by hoverfly larvae suppressed aphid population growth on these host plants, the native hosts M. sativa and T. pratense provided enemy-free space for the respective A. pisum races. Mobile predators, such as ants and ladybird beetles, preferred Pisum race aphids on V. faba over P. sativum. Thus, all three of the native host plants studied supply enemy-free space for A. pisum compared to the universal host V. faba. Reducing encounters between aphid races on V. faba would reduce gene flow among them and could contribute to maintaining the host races.

Shifting Nicotiana attenuata's diurnal rhythm does not alter its resistance to the specialist herbivore Manduca sexta.

Arabidopsis thaliana plants are less resistant to attack by the generalist lepidopteran herbivore Trichoplusia ni when plants and herbivores are entrained to opposite, versus identical diurnal cycles and tested under constant conditions. This effect is associated with circadian fluctuations in levels of jasmonic acid, the transcription factor MYC2, and glucosinolate contents in leaves. We tested whether a similar effect could be observed in a different plant-herbivore system: the wild tobacco Nicotiana attenuata and its co-evolved specialist herbivore, Manduca sexta. We measured larval growth on plants under both constant and diurnal conditions following identical or opposite entrainment, profiled the metabolome of attacked leaf tissue, quantified specific metabolites known to reduce M. sexta growth, and monitored M. sexta feeding activity under all experimental conditions. Entrainment did not consistently affect M. sexta growth or plant defense induction. However, both were reduced under constant dark conditions, as was M. sexta feeding activity. Our data indicate that the response induced by M. sexta in N. attenuata is robust to diurnal cues and independent of plant or herbivore entrainment. We propose that while the patterns of constitutive or general damage-induced defense may undergo circadian fluctuation, the orchestration of specific induced responses is more complex.

Feeding on Leaves of the Glucosinolate Transporter Mutant gtr1gtr2 Reduces Fitness of Myzus persicae.

As aphids are a pest on various crops worldwide, a better understanding of the interaction between aphids and plant host defenses is required. The green peach aphid (Myzus persicae) feeds on a variety of plant species, including the model plant Arabidopsis thaliana (Arabidopsis), in which glucosinolates function as a major part of the chemical defense. Several studies have shown that glucosinolates play a role in interactions between Arabidopsis and the green peach aphid. In this work, we used a recently identified Arabidopsis glucosinolate transporter mutant (gtr1gtr2 dKO), with altered glucosinolate content in the vasculature, to investigate the role of defense compound transport in aphid infestation. By monitoring aphid performance on caged leaves and analyzing glucosinolates in leaf tissue and phloem sap, as well as inside aphids, we examined if a change in spatial distribution of glucosinolates within a leaf influences aphid performance. Based on reduced glucosinolate content in the phloem sap of the transporter mutant, we hypothesized that aphids would perform better on gtr1gtr2 dKO leaves compared to WT. Unexpectedly, aphids performed poorly on gtr1gtr2 dKO leaves. Our data suggest that higher glucosinolate content in tissues surrounding the phloem of the double transporter mutant may play a role in reducing aphid performance on this genotype.

Pea Aphid (Acyrthosiphon pisum) Host Races Reduce Heat-Induced Forisome Dispersion in Vicia faba and Trifolium pratense.

Although phloem-feeding insects such as aphids can cause significant damage to plants, relatively little is known about early plant defenses against these insects. As a first line of defense, legumes can stop the phloem mass flow through a conformational change in phloem proteins known as forisomes in response to Ca2+ influx. However, specialized phloem-feeding insects might be able to suppress the conformational change of forisomes and thereby prevent sieve element occlusion. To investigate this possibility, we triggered forisome dispersion through application of a local heat stimulus to the leaf tips of pea (Pisum sativum), clover (Trifolium pratense) and broad bean (Vicia faba) plants infested with different pea aphid (Acyrthosiphon pisum) host races and monitored forisome responses. Pea aphids were able to suppress forisome dispersion, but this depended on the infesting aphid host race, the plant species, and the age of the plant. Differences in the ability of aphids to suppress forisome dispersion may be explained by differences in the composition and quantity of the aphid saliva injected into the plant. Various mechanisms of how pea aphids might suppress forisome dispersion are discussed.

The first step in the biosynthesis of cocaine in Erythroxylum coca: the characterization of arginine and ornithine decarboxylases.

Despite the long history of cocaine use among humans and its social and economic significance today, little information is available about the biochemical and molecular aspects of cocaine biosynthesis in coca (Erythroxylum coca) in comparison to what is known about the formation of other pharmacologically-important tropane alkaloids in species of the Solanaceae. In this work, we investigated the site of cocaine biosynthesis in E. coca and the nature of the first step. The two principal tropane alkaloids of E. coca, cocaine and cinnamoyl cocaine, were present in highest concentrations in buds and rolled leaves. These are also the organs in which the rate of alkaloid biosynthesis was the highest based on the incorporation of ¹³CO2. In contrast, tropane alkaloids in the Solanaceae are biosynthesized in the roots and translocated to the leaves. A collection of EST sequences from a cDNA library made from young E. coca leaves was employed to search for genes encoding the first step in tropane alkaloid biosynthesis. Full-length cDNA clones were identified encoding two candidate enzymes, ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), and the enzymatic activities of the corresponding proteins confirmed by heterologous expression in E. coli and complementation of a yeast mutant. The transcript levels of both ODC and ADC genes were highest in buds and rolled leaves and lower in other organs. The levels of both ornithine and arginine themselves showed a similar pattern, so it was not possible to assign a preferential role in cocaine biosynthesis to one of these proteins.

The organ-specific expression of terpene synthase genes contributes to the terpene hydrocarbon composition of chamomile essential oils.

BACKGROUND: The essential oil of chamomile, one of the oldest and agronomically most important medicinal plant species in Europe, has significant antiphlogistic, spasmolytic and antimicrobial activities. It is rich in chamazulene, a pharmaceutically active compound spontaneously formed during steam distillation from the sesquiterpene lactone matricine. Chamomile oil also contains sesquiterpene alcohols and hydrocarbons which are produced by the action of terpene synthases (TPS), the key enzymes in constructing terpene carbon skeletons. RESULTS: Here, we present the identification and characterization of five TPS enzymes contributing to terpene biosynthesis in chamomile (Matricaria recutita). Four of these enzymes were exclusively expressed in above-ground organs and produced the common terpene hydrocarbons (-)-(E)-ß-caryophyllene (MrTPS1), (+)-germacrene A (MrTPS3), (E)-ß-ocimene (MrTPS4) and (-)-germacrene D (MrTPS5). A fifth TPS, the multiproduct enzyme MrTPS2, was mainly expressed in roots and formed several Asteraceae-specific tricyclic sesquiterpenes with (-)-α-isocomene being the major product. The TPS transcript accumulation patterns in different organs of chamomile were consistent with the abundance of the corresponding TPS products isolated from these organs suggesting that the spatial regulation of TPS gene expression qualitatively contribute to terpene composition. CONCLUSIONS: The terpene synthases characterized in this study are involved in the organ-specific formation of essential oils in chamomile. While the products of MrTPS1, MrTPS2, MrTPS4 and MrTPS5 accumulate in the oils without further chemical alterations, (+)-germacrene A produced by MrTPS3 accumulates only in trace amounts, indicating that it is converted into another compound like matricine. Thus, MrTPS3, but also the other TPS genes, are good markers for further breeding of chamomile cultivars rich in pharmaceutically active essential oils.

Bark Beetle Attack History Does Not Influence the Induction of Terpene and Phenolic Defenses in Mature Norway Spruce (Picea abies) Trees by the Bark Beetle-Associated Fungus Endoconidiophora polonica.

Terpenes and phenolics are important constitutive and inducible conifer defenses against bark beetles and their associated fungi. In this study, the inducible defenses of mature Norway spruce (Picea abies) trees with different histories of attack by the spruce bark beetle, Ips typographus were tested by inoculation with the I. typographus-associated fungus Endoconidiophora polonica. We compared trees that had been under previous attack with those under current attack and those that had no record of attack. After fungal inoculation, the concentrations of mono-, sesqui-, and diterpenes in bark increased 3- to 9-fold. For the phenolics, the flavan-3-ols, catechin, and gallocatechin, increased significantly by 2- and 5-fold, respectively, while other flavonoids and stilbenes did not. The magnitudes of these inductions were not influenced by prior bark beetle attack history for all the major compounds and compound classes measured. Before fungal inoculation, the total amounts of monoterpenes, diterpenes, and phenolics (constitutive defenses) were greater in trees that had been previously attacked compared to those under current attack, possibly a result of previous induction. The transcript levels of many genes involved in terpene formation (isoprenyl diphosphate synthases and terpene synthases) and phenolic formation (chalcone synthases) were significantly enhanced by fungal inoculation suggesting de novo biosynthesis. Similar inductions were found for the enzymatic activity of isoprenyl diphosphate synthases and the concentration of their prenyl diphosphate products after fungal inoculation. Quantification of defense hormones revealed a significant induction of the jasmonate pathway, but not the salicylic acid pathway after fungal inoculation. Our data highlight the coordinated induction of terpenes and phenolics in spruce upon infection by E. polonica, a fungal associate of the bark beetle I. typographus, but provide no evidence for the priming of these defense responses by prior beetle attack.

Constitutive emission of the aphid alarm pheromone, (E)-ß-farnesene, from plants does not serve as a direct defense against aphids.

BACKGROUND: The sesquiterpene, (E)-ß-farnesene (EBF), is the principal component of the alarm pheromone of many aphid species. Released when aphids are attacked by enemies, EBF leads aphids to undertake predator avoidance behaviors and to produce more winged offspring that can leave the plant. Many plants also release EBF as a volatile, and so it has been proposed that this compound could act to defend plants against aphid infestation by 1) deterring aphids from settling, 2) reducing aphid performance due to frequent interruption of feeding and 3) inducing the production of more winged offspring. Here we tested the costs and benefits of EBF as a defense against the green peach aphid, Myzus persicae, using transgenic Arabidopsis thaliana lines engineered to continuously emit EBF. RESULTS: No metabolic costs of EBF synthesis could be detected in these plants as they showed no differences in growth or seed production from wild-type controls under two fertilizer regimes. Likewise, no evidence was found for the ability of EBF to directly defend the plant against aphids. EBF emission did not significantly repel winged or wingless morphs from settling on plants. Nor did EBF reduce aphid performance, measured as reproduction, or lead to an increase in the proportion of winged offspring. CONCLUSIONS: The lack of any defensive effect of EBF in this study might be due to the fact that natural enemy attack on individual aphids leads to a pulsed emission, but the transgenic lines tested continuously produce EBF to which aphids may become habituated. Thus our results provide no support for the hypothesis that plant emission of the aphid alarm pheromone EBF is a direct defense against aphids. However, there is scattered evidence elsewhere in the literature suggesting that EBF emission might serve as an indirect defense by attracting aphid predators.

Pectin Digestion in Herbivorous Beetles: Impact of Pseudoenzymes Exceeds That of Their Active Counterparts.

Many protein families harbor pseudoenzymes that have lost the catalytic function of their enzymatically active counterparts. Assigning alternative function and importance to these proteins is challenging. Because the evolution toward pseudoenzymes is driven by gene duplication, they often accumulate in multigene families. Plant cell wall-degrading enzymes (PCWDEs) are prominent examples of expanded gene families. The pectolytic glycoside hydrolase family 28 (GH28) allows herbivorous insects to break down the PCW polysaccharide pectin. GH28 in the Phytophaga clade of beetles contains many active enzymes but also many inactive counterparts. Using functional characterization, gene silencing, global transcriptome analyses, and recordings of life history traits, we found that not only catalytically active but also inactive GH28 proteins are part of the same pectin-digesting pathway. The robustness and plasticity of this pathway and thus its importance for the beetle is supported by extremely high steady-state expression levels and counter-regulatory mechanisms. Unexpectedly, the impact of pseudoenzymes on the pectin-digesting pathway in Phytophaga beetles exceeds even the influence of their active counterparts, such as a lowered efficiency of food-to-energy conversion and a prolongation of the developmental period.