Direct Defense Research Articles

Reactive oxygen species as a marine grazing defense: H2O2 and wounded Ascoseira mirabilis both inhibit feeding by an amphipod grazer

Marine macroalgae can release reactive oxygen species (ROS) upon wounding and grazing. Here we address the potential role of ROS in herbivore defense. We performed feeding assays in the presence of varying concentrations of hydrogen peroxide (H2O2), a common type of ROS. H2O2 inhibited feeding by a marine amphipod grazer, Gondogeneia antarctica, over broad levels of concentration, and its potency was strongly dependent on its rate of decay in natural seawater. Because it is possible that the inhibitory levels of H2O2 are encountered in the vicinity of a sympatric macroalgal wound, we suggest that H2O2 has the potential to act as a direct anti-grazing defense in marine ecosystems. Since some sympatric macroalgae release a burst of non-H2O2 ROS, we also performed experiments to evaluate the role of these naturally-produced ROS on sympatric grazers. The presence of wounded Ascoseira mirabilis (which releases a burst of non-H2O2 ROS after wounding) during a feeding assay inhibited feeding of G. antarctica compared to the presence of intact A. mirabilis. These data are consistent with a role for ROS as a direct anti-herbivore defense in nature. However, the data are also consistent with hypotheses that involve other putative activated anti-grazing defenses.

Metabolic engineering of monoterpene biosynthesis in tomato fruits via introduction of the non-canonical substrate neryl diphosphate

Recently it was shown that monoterpenes in tomato trichomes (Solanum lycopersicum) are synthesized by phellandrene synthase 1 (PHS1) from the non-canonical substrate neryl diphosphate (NPP), the cis-isomer of geranyl diphosphate (GPP). As PHS1 accepts both NPP and GPP substrates forming different monoterpenes, it was overexpressed in tomato fruits to test if NPP is also available in a tissue highly active in carotenoid production. However, transgenic fruits overexpressing PHS1 produced only small amounts of GPP-derived PHS1 monoterpene products, indicating the absence of endogenous NPP. Therefore, NPP formation was achieved by diverting the metabolic flux from carotenoids via expression of tomato neryl diphosphate synthase 1 (NDPS1). NDPS1 transgenic fruits produced NPP-derived monoterpenes, including nerol, neral and geranial, while displaying reduced lycopene content. NDPS1 co-expression with PHS1 resulted in a monoterpene blend, including β-phellandrene, similar to that produced from NPP by PHS1 in vitro and in trichomes. Unexpectedly, PHS1×NDPS1 fruits showed recovery of lycopene levels compared to NDPS1 fruits, suggesting that redirection of metabolic flux is only partially responsible for the reduction in carotenoids. In vitro assays demonstrated that NPP serves as an inhibitor of geranylgeranyl diphosphate synthase, thus its consumption by PHS1 leads to recovery of lycopene levels. Monoterpenes produced in PHS1×NDPS1 fruits contributed to direct plant defense negatively affecting feeding behavior of the herbivore Helicoverpa zea and displaying antifungal activity against Botrytis cinerea. These results show that NPP-derived terpenoids can be produced in plant tissues; however, NPP has to be consumed to avoid negative impacts on plant metabolism.

Functionally distinct subsets of natural killer cells help shape and regulate the immune response during viral infection (INC8P.429)

Abstract Natural killer (NK) cells are innate lymphocytes that play an important role in the immune response against certain viral infections and transformed cells. Historically, NK cells have been recognized for their direct antiviral defense; however, a more complex view of NK cell function has recently emerged highlighting their ability to direct the balance between viral infection and host immunity. While the diverse effector functions of NK cells are increasingly seen as vital components of host immunity, the specific populations of NK cells that mediate these differential effects have not been identified. In this study, we provide evidence for distinct NK cell subsets that differ in their effector and immunoregulatory functions during influenza infection. We show here that during the early stages of the host immune response to viral infection, a population of effector NK cells directly contributes to viral resistance at the site of infection, while a separate helper NK subset concurrently functions to prime the adaptive immune response by expanding dendritic cells in the lymph nodes. Conversely, at the later stages of viral infection, a subset of suppressor NK cells dampens the adaptive immune response by directly regulating antigen-specific T cells. These results suggest that adoptive transfer of specific NK subsets may serve as a useful therapeutic strategy to modulate the immune response during viral infection.

Direct and indirect chemical defences against insects in a multitrophic framework

Plant secondary metabolites play an important role in mediating interactions with insect herbivores and their natural enemies. Metabolites stored in plant tissues are usually investigated in relation to herbivore behaviour and performance (direct defence), whereas volatile metabolites are often studied in relation to natural enemy attraction (indirect defence). However, so-called direct and indirect defences may also affect the behaviour and performance of the herbivore's natural enemies and the natural enemy's prey or hosts, respectively. This suggests that the distinction between these defence strategies may not be as black and white as is often portrayed in the literature. The ecological costs associated with direct and indirect chemical defence are often poorly understood. Chemical defence traits are often studied in two-species interactions in highly simplified experiments. However, in nature, plants and insects are often engaged in mutualistic interactions with microbes that may also affect plant secondary chemistry. Moreover, plants are challenged by threats above- and belowground and herbivory may have consequences for plant-insect multitrophic interactions in the alternative compartment mediated by changes in plant secondary chemistry. These additional associations further increase the complexity of interaction networks. Consequently, the effect of a putative defence trait may be under- or overestimated when other interactions are not considered.

Differential allocation and deployment of direct and indirect defences by Vicia sepium along elevation gradients

Summary Dissecting drivers of plant defence investment remains central for understanding the assemblage of communities across different habitats. There is increasing evidence that direct defence strategies against herbivores, including secondary metabolites production, differ along ecological gradients in response to variation in biotic and abiotic conditions. In contrast, intraspecific variation in indirect defences remains unexplored. Here, we investigated variation in herbivory rate, resistance to herbivores and indirect defences in ant‐attracting Vicia species along the elevation gradient of the Alps. Specifically, we compared volatile organic compounds (VOCs) and ant attraction in high‐ and low‐elevation ecotypes. Consistent with adaptation to the lower herbivory conditions that we detected at higher elevations in the field, high‐elevation plants were visited by fewer ants and were more susceptible to herbivore attack. In parallel, constitutive volatile organic compound production and subsequent ant attraction were lower in the high‐elevation ecotypes. We observed an elevation‐driven trade‐off between constitutive and inducible production of VOCs and ant attraction along the environmental cline. At higher elevations, inducible defences increased, while constitutive defence decreased, suggesting that the high‐elevation ecotypes compensate for lower indirect constitutive defences only after herbivore attack. Synthesis. Overall, direct and indirect defences of plants vary along elevation gradients. Our findings show that plant allocation to defences are subject to trade‐offs depending on local conditions, and point to a feedback mechanism linking local herbivore pressure, predator abundance and the defence investment of plants.

Modulation of flavonoid metabolites in Arabidopsis thaliana through overexpression of the MYB75 transcription factor: role of kaempferol-3,7-dirhamnoside in resistance to the specialist insect herbivore Pieris brassicae

Anthocyanins and flavonols are secondary metabolites that can function in plant defence against herbivores. In Arabidopsis thaliana, anthocyanin and flavonol biosynthesis are regulated by MYB transcription factors. Overexpression of MYB75 (oxMYB75) in Arabidopsis results in increasing anthocyanin and flavonol levels which enhances plant resistance to generalist caterpillars. However, how these metabolites affect specialist herbivores has remained unknown. Performance of a specialist aphid (Brevicoryne brassicae) was unaffected after feeding on oxMYB75 plants, whereas a specialist caterpillar (Pieris brassicae) gained significantly higher body mass when feeding on this plant. An increase in anthocyanin and total flavonol glycoside levels correlated negatively with the body mass of caterpillars fed on oxMYB75 plants. However, a significant reduction of kaempferol-3,7-dirhamnoside (KRR) corresponded to an increased susceptibility of oxMYB75 plants to caterpillar feeding. Pieris brassicae caterpillars also grew less on an artificial diet containing KRR or on oxMYB75 plants that were exogenously treated with KRR, supporting KRR's function in direct defence against this specialist caterpillar. The results show that enhancing the activity of the anthocyanin pathway in oxMYB75 plants results in re-channelling of quercetin/kaempferol metabolites which has a negative effect on the accumulation of KRR, a novel defensive metabolite against a specialist caterpillar.

Direct Type I IFN but Not MDA5/TLR3 Activation of Dendritic Cells Is Required for Maturation and Metabolic Shift to Glycolysis after Poly IC Stimulation

Author SummaryImmune responses are orchestrated by a specialized cell type called dendritic cells (DCs). In order to achieve durable and robust immunity, DCs need to undergo an intricate differentiation process known as maturation. This process is poorly understood at the moment. Poly IC, which mimics viral RNA and is an agonist for the viral pattern recognition receptors (PRRs) that signal “danger” to the immune system, can induce full maturation of DCs and it has been shown that this requires type I IFN signaling. In this study we set out to examine the specific signals provided by direct PRR or IFN stimulation that are required for DC maturation. We found to our surprise that type I IFN can regulate almost all steps of the DC's maturation process without requiring direct PRR involvement. We also show that type I IFN regulates several metabolic switches essential for preservation of DC integrity: it stimulates the expression of the hypoxia-inducible factor 1α (Hif1α), which controls the metabolic shift from oxidative phosphorylation (used by resting cells to generate energy) to aerobic glycolysis, a less efficient but faster energy-producing process. This metabolic shift was required to meet increased energy demands of activated DCs and to prevent their premature death, thus sustaining an immune response to viral infection.

Review of recent advances on the production and eco-physiological roles of green leaf volatiles

We review advances in understanding the production, eco-physiological roles, and related regulatory mechanism of green leaf volatiles (GLVs). GLVs are six-carbon aldehydes, alcohols and esters derived from the hydroperoxide lyase branch of the octadecanoic acid pathway in plants. Besides leaves, other tissues such as root, stem, fruit, and seed are also reported to be capable of producing the GLVs regulated at the levels of transcription and post-transcription in plants. GLVs composition and amounts are influenced by the developmental stage and growth environment such as seasonal change in a given plant. In addition, the formation of GLVs can be induced by biotic stress such as insect attack, inoculation of pathogenic bacteria and inhabitation of beneficial fungi, as well as abiotic ones such as nitrogen deficiency. Although GLVs are primarily recognized as important contributors to unique flavors, they also play important roles in direct and indirect defense responses. They not only inhibit the reproduction of bacteria, fungi and insects, but also induce the synthesis of various defensive compounds. They can also prime different signal induction pathways. The potential roles that GLVs played in the quality formation of Chinese herbs, especially genuine ones, are discussed based on their wide distribution and multi-layered defensive roles in plants. It is necessary and urgent to carry out related research on GLVs in Chinese herbs for their sustainable and green development.

Maize Developmental Stage Affects Indirect and Direct Defense Expression

Herbivores elicit a complex indirect and direct defense response in plants, but little is known about how defenses are expressed throughout a plant's life cycle. Here we report the characterization of direct and indirect defense profiles for two vegetative stages of maize, v1 and v3. Indirect defense expression was evaluated by measuring plant headspace volatiles in response to artificial herbivory and caterpillar feeding. To assess direct defense expression, transcripts of four proteinase inhibitors (PI) and larval performance were measured. Artificial herbivory significantly elevated total induced volatiles in v1 and v3 compared with controls, but this induction was marginal in v1 when compared with v3. Both maize stages produced similar total amounts of volatiles, accounting for fresh weight, of different compositions during caterpillar feeding. However, on a per-plant basis, v1 produced less volatiles compared with v3. In contrast, jasmonic acid levels, an indicator of an antiherbivore defense response, increased similarly in both stages. During caterpillar feeding, both developmental stages contained higher levels of PI transcripts compared with controls. However, v1 plants contained more cystatin-like PI transcripts and total larval mass was reduced compared with v3 plants. These results suggest that in maize, direct and indirect defenses against insect herbivory differ between seedling and juvenile plants.

Reactive oxygen species and the Antarctic macroalgal wound response.

Reactive oxygen species (ROS) are commonly produced by algal, vascular plant, and animal cells involved in the innate immune response as cellular signals promoting defense and healing and/or as a direct defense against invading pathogens. The production of reactive species in macroalgae upon injury, however, is largely uncharacterized. In this study, we surveyed 13 species of macroalgae from the Western Antarctic Peninsula and show that the release of strong oxidants is common after macroalgal wounding. Most species released strong oxidants within 1min of wounding and/or showed cellular accumulation of strong oxidants over an hour post-wounding. Exogenous catalase was used to show that hydrogen peroxide was a component of immediate oxidant release in one of five species, but was not responsible for the entire oxidative wound response as is common in vascular plants. The other component(s) of the oxidant cocktail released upon wounding are unknown. We were unable to detect protein nitration in extracts of four oxidant-producing species flash frozen 30s after wounding, but a role for reactive nitrogen species such as peroxynitrite cannot be completely ruled out. Two species showed evidence for the production of a catalase-activated oxidant, a mechanism previously known only from the laboratory and from the synthetic drug isoniazid used to kill the human pathogen Mycobacterium tuberculosis. The rhodophyte Palmaria decipiens, which released strong oxidants after wounding, also produced strong oxidants upon grazing by a sympatric amphipod, suggesting that oxidants are involved in the response to grazing.

Two herbivore-induced cytochrome P450 enzymes CYP79D6 and CYP79D7 catalyze the formation of volatile aldoximes involved in poplar defense.

Aldoximes are known as floral and vegetative plant volatiles but also as biosynthetic intermediates for other plant defense compounds. While the cytochrome P450 monooxygenases (CYP) from the CYP79 family forming aldoximes as biosynthetic intermediates have been intensively studied, little is known about the enzymology of volatile aldoxime formation. We characterized two P450 enzymes, CYP79D6v3 and CYP79D7v2, which are involved in herbivore-induced aldoxime formation in western balsam poplar (Populus trichocarpa). Heterologous expression in Saccharomyces cerevisiae revealed that both enzymes produce a mixture of different aldoximes. Knockdown lines of CYP79D6/7 in gray poplar (Populus × canescens) exhibited a decreased emission of aldoximes, nitriles, and alcohols, emphasizing that the CYP79s catalyze the first step in the formation of a complex volatile blend. Aldoxime emission was found to be restricted to herbivore-damaged leaves and is closely correlated with CYP79D6 and CYP79D7 gene expression. The semi-volatile phenylacetaldoxime decreased survival and weight gain of gypsy moth (Lymantria dispar) caterpillars, suggesting that aldoximes may be involved in direct defense. The wide distribution of volatile aldoximes throughout the plant kingdom and the presence of CYP79 genes in all sequenced genomes of angiosperms suggest that volatile formation mediated by CYP79s is a general phenomenon in the plant kingdom.

Histopathological findings on Carassius auratus hepatopancreas upon exposure to acrylamide: correlation with genotoxicity and metabolic alterations

Acrylamide is an amide used in several industrial applications making it easily discharged to aquatic ecosystems. The toxicity of acrylamide to aquatic organisms is scarcely known, although previous studies with murine models provided evidence for deleterious effects. To assess the effects of acrylamide to freshwater fish, goldfish (Carassius auratus L.) were exposed to several concentrations of waterborne acrylamide and analysed for genotoxic damage, alterations to detoxifying enzymes and histopathology. Results revealed a dose-dependent increase in total DNA strand breakage, the formation of erythrocytic nuclear abnormalities and in the levels of hepatic cytochrome P4501A (CYP1A) and glutathione S-transferase (GST) activity. In addition, acrylamide induced more histopathological changes to pancreatic acini than to the hepatic parenchyma, regardless of exposure concentration, whereas hepatic tissue only endured significant alterations at higher concentrations of exposure. Thus, results confirm the genotoxic potential of acrylamide to fish and its ability to induce CYP1A, probably as a direct primary defence mechanism. This strongly suggests the substance's pro-mutagenic potential in fish, similarly to what is known for rodents. However, the deleterious effects observed in the pancreatic acini, more severe than in the liver, could indicate a specific, albeit unknown toxic mechanism of acrylamide to fish that overran the organism's metabolic defences against a chemical agent rather than causing a general systemic failure.

Alterations in the Helicoverpa armigera midgut digestive physiology after ingestion of pigeon pea inducible leucine aminopeptidase.

Jasmonate inducible plant leucine aminopeptidase (LAP) is proposed to serve as direct defense in the insect midgut. However, exact functions of inducible plant LAPs in the insect midgut remain to be estimated. In the present investigation, we report the direct defensive role of pigeon pea inducible LAP in the midgut of Helicoverpa armigera (Lepidoptera: Noctuidae) and responses of midgut soluble aminopeptidases and serine proteinases upon LAP ingestion. Larval growth and survival was significantly reduced on the diets supplemented with pigeon pea LAP. Aminopeptidase activities in larvae remain unaltered in presence or absence of inducible LAP in the diet. On the contrary, serine proteinase activities were significantly decreased in the larvae reared on pigeon pea LAP containing diet as compared to larvae fed on diet without LAP. Our data suggest that pigeon pea inducible LAP is responsible for the degradation of midgut serine proteinases upon ingestion. Reduction in the aminopeptidase activity with LpNA in the H. armigera larvae was compensated with an induction of aminopeptidase activity with ApNA. Our findings could be helpful to further dissect the roles of plant inducible LAPs in the direct plant defense against herbivory.

An unconventional antimicrobial protein histone from freshwater prawn Macrobrachium rosenbergii: Analysis of immune properties

In this study, we have reported the first histone characterized at molecular level from freshwater prawn Macrobrachium rosenbergii (MrHis). A full length cDNA of MrHis (751 base pairs) was identified from an established M. rosenbergii cDNA library using GS-FLX™ technique. It encodes 137 amino acid residues with a calculated molecular mass of 15 kDa and an isoelectric point of 10.5. MrHis peptide contains a histone H2A signature between 21 and 27 amino acids. Homologous analysis showed that MrHis had a significant sequence identity (99%) with other known histone H2A groups especially from Penaeus monodon. Phylogenetic analysis of MrHis showed a strong relationship with other amino acid sequences from histone H2A arthropod groups. Further phylogenetic analysis showed that the MrHis belongs to histone H2A superfamily and H2A1A sub-family. Secondary structure of MrHis showed that the protein contains 50.36% α-helical region and 49.64% coils. The 3D model of MrHis was predicted by I-Tasser program and the model was evaluated for quality analysis including C-score analysis, Ramachandran plot analysis and RMSD analysis. The surface view analysis of MrHis showed the active domain at the N terminal. The antimicrobial property of MrHis protein was confirmed by the helical structure and the total hydrophobic surface along with its net charge. The MFE of the predicted RNA structure of MrHis is −128.62 kcal/mol, shows its mRNA stability. Schiffer–Edmundson helical wheel analysis of the N-terminal of MrHis showed a perfect amphipathic nature of the peptide. Significantly (P < 0.05) highest gene expression was noticed in the hemocyte and is induced with viral (WSBV and MrNV) and bacteria (A eromonas hydrophila and Vibrio harveyi) infections. The coding sequence of recombinant MrHis protein was expressed in a pMAL vector and purified to study the antimicrobial properties. The recombinant product showed antimicrobial activity against both Gram negative and Gram positive bacteria. In this study, the recombinant MrHis protein displayed antimicrobial activity in its entirety. Hence, it is possible to suggest that the activity may be due to the direct defense role of histone or its N-terminal antimicrobial property. However, this remains to be verified by detailed investigations.

The HERBIVORE ELICITOR-REGULATED1 gene enhances abscisic acid levels and defenses against herbivores in Nicotiana attenuata plants.

Nicotiana attenuata plants can distinguish the damage caused by herbivore feeding from other types of damage by perceiving herbivore-associated elicitors, such as the fatty acid-amino acid conjugates (FACs) in oral secretions (OS) of Manduca sexta larvae, which are introduced into wounds during feeding. However, the transduction of FAC signals into downstream plant defense responses is still not well established. We identified a novel FAC-regulated protein in N. attenuata (NaHER1; for herbivore elicitor regulated) and show that it is an indispensable part of the OS signal transduction pathway. N. attenuata plants silenced in the expression of NaHER1 by RNA interference (irHER1) were unable to amplify their defenses beyond basal, wound-induced levels in response to OS elicitation. M. sexta larvae performed 2-fold better when reared on irHER1 plants, which released less volatile organic compounds (indirect defense) and had strongly reduced levels of several direct defense metabolites, including trypsin proteinase inhibitors, 17-hydroxygeranyllinallool diterpene glycosides, and caffeoylputrescine, after real and/or simulated herbivore attack. In parallel to impaired jasmonate signaling and metabolism, irHER1 plants were more drought sensitive and showed reduced levels of abscisic acid (ABA) in the leaves, suggesting that silencing of NaHER1 interfered with ABA metabolism. Because treatment of irHER1 plants with ABA results in both the accumulation of significantly more ABA catabolites and the complete restoration of normal wild-type levels of OS-induced defense metabolites, we conclude that NaHER1 acts as a natural suppressor of ABA catabolism after herbivore attack, which, in turn, activates the full defense profile and resistance against herbivores.

Leaf drop affects herbivory in oaks

Leaf phenology is important to herbivores, but the timing and extent of leaf drop has not played an important role in our understanding of herbivore interactions with deciduous plants. Using phylogenetic general least squares regression, we compared the phenology of leaves of 55 oak species in a common garden with the abundance of leaf miners on those trees. Mine abundance was highest on trees with an intermediate leaf retention index, i.e. trees that lost most, but not all, of their leaves for 2-3 months. The leaves of more evergreen species were more heavily sclerotized, and sclerotized leaves accumulated fewer mines in the summer. Leaves of more deciduous species also accumulated fewer mines in the summer, and this was consistent with the idea that trees reduce overwintering herbivores by shedding leaves. Trees with a later leaf set and slower leaf maturation accumulated fewer herbivores. We propose that both leaf drop and early leaf phenology strongly affect herbivore abundance and select for differences in plant defense. Leaf drop may allow trees to dispose of their herbivores so that the herbivores must recolonize in spring, but trees with the longest leaf retention also have the greatest direct defenses against herbivores.

Chrysanthemum expressing a linalool synthase gene ‘smells good’, but ‘tastes bad’ to western flower thrips

Herbivore-induced plant volatiles are often involved in direct and indirect plant defence against herbivores. Linalool is a common floral scent and found to be released from leaves by many plants after herbivore attack. In this study, a linalool/nerolidol synthase, FaNES1, was overexpressed in the plastids of chrysanthemum plants (Chrysanthemum morifolium). The volatiles of FaNES1 chrysanthemum leaves were strongly dominated by linalool, but they also emitted small amount of the C11-homoterpene, (3E)-4,8-dimethyl-1,3,7-nonatriene, a derivative of nerolidol. Four nonvolatile linalool glycosides in methanolic extracts were found to be significantly increased in the leaves of FaNES1 plants compared to wild-type plants. They were putatively identified by LC-MS-MS as two linalool-malonyl-hexoses, a linalool-pentose-hexose and a glycoside of hydroxy-linalool. A leaf-disc dual-choice assay with western flower thrips (WFT, Frankliniella occidentalis) showed, initially during the first 15 min of WFT release, that FaNES1 plants were significantly preferred. This gradually reversed into significant preference for the control, however, at 20-28 h after WFT release. The initial preference was shown to be based on the linalool odour of FaNES1 plants by olfactory dual-choice assays using paper discs emitting pure linalool at similar rates as leaf discs. The reversal of preference into deterrence could be explained by the initial nonvolatile composition of the FaNES1 plants, as methanolic extracts were less preferred by WFT. Considering the common occurrence of linalool and its glycosides in plant tissues, it suggests that plants may balance attractive fragrance with 'poor taste' using the same precursor compound.

Genetic, morphological and cyanogen content evaluation of a new collection of Caribbean Lima bean (Phaseolus lunatus L.) landraces

Lima bean is a species cultivated broadly in the Americas and has been cultivated in the Caribbean for at least 500 years. In order to determine the genetic structure and diversity of Lima bean from the Caribbean, 50 landraces from Haiti, the Dominican Republic and Puerto Rico were collected and analyzed using 24 SSR markers. All landraces in this Caribbean collection were found to be of Middle-American descent. The genetic diversity (HE) was highest in the landraces from Puerto Rico and lowest in Haitian landraces. The observed proportion of heterozygotes (HO) was higher in the Haitian landraces and lowest in Puerto Rican landraces. Unweighted pair-group method with arithmetic averaging analysis showed that the landraces clustered into 3 clusters with all Haitian landraces grouping in one cluster. We also assessed the agro-morphological characteristics of the collection as well as the content of cyanogenic glucoside, linamarin, in leaves and dry seeds. Lima bean, which is a model crop for indirect plant defenses against herbivory, also possess linamarin as a source of direct plant defense. Upon tissue damage, linamarin is converted to toxic hydrogen cyanide. In our collection 44.6 % of the landraces had average seed HCN content ≤200 ppm which is the permitted level for Lima bean seed in the US. Our results also identified the landraces in this collection which have high linamarin levels in the leaves while having low levels in the seeds. Such landraces have the desirable combination of traits and will be the focus of our future plans for agronomic trait improvement though breeding.

Elevated carbon dioxide impairs the performance of a specialized parasitoid of an aphid host feeding on Brassica plants

Elevated concentrations of atmospheric carbon dioxide (CO2), a consequence of anthropogenic global change, may profoundly interfere with tritrophic interactions. Such effects with a focus on parasitoids as natural antagonists of herbivores have rarely been investigated. In particular, studies on effects of secondary metabolites induced by crop plant acclimation to elevated CO2 on higher trophic levels were yet missing. We used the system composed of Brassica plants, the aphid Brevicoryne brassicae and the endoparasitoid Diaeretiella rapae, which is specialized on aphids feeding on brassicacean plants, to compare effects of elevated CO2 (800ppm) versus ambient CO2 (400ppm). Plants were exposed to the CO2 concentrations for up to 10weeks, aphids for 2–3 generations, and parasitoids for 1 generation, to allow for acclimation. Concomitant bioassays with herbivore-infested plants and parasitoids showed a significantly lower proportion of hosts parasitized under elevated compared to ambient CO2 after a 10-week plant exposure. Parasitoid progeny emerged earlier but offspring adults were shorter lived. Plant glucosinolate concentrations were higher under elevated compared to ambient CO2, whereas, contrary to expectation, aphid glucosinolate concentrations were significantly lower. Likewise aphid body mass remained approximately 20% lower under elevated compared to ambient CO2. Thus, elevated CO2 seems to have enhanced plant direct defense by an increase of natural plant defense compounds, however, it led to a decrease in indirect defense, likely due to the reduced host size. Our results point, for the first time, to a conflict between bottom-up and top-down control under elevated CO2.

Formation of the unusual semivolatile diterpene rhizathalene by the Arabidopsis class I terpene synthase TPS08 in the root stele is involved in defense against belowground herbivory.

Secondary metabolites are major constituents of plant defense against herbivore attack. Relatively little is known about the cell type-specific formation and antiherbivore activities of secondary compounds in roots despite the substantial impact of root herbivory on plant performance and fitness. Here, we describe the constitutive formation of semivolatile diterpenes called rhizathalenes by the class I terpene synthase (TPS) 08 in roots of Arabidopsis thaliana. The primary enzymatic product of TPS08, rhizathalene A, which is produced from the substrate all-trans geranylgeranyl diphosphate, represents a so far unidentified class of tricyclic diterpene carbon skeletons with an unusual tricyclic spiro-hydrindane structure. Protein targeting and administration of stable isotope precursors indicate that rhizathalenes are biosynthesized in root leucoplasts. TPS08 expression is largely localized to the root stele, suggesting a centric and gradual release of its diterpene products into the peripheral root cell layers. We demonstrate that roots of Arabidopsis tps08 mutant plants, grown aeroponically and in potting substrate, are more susceptible to herbivory by the opportunistic root herbivore fungus gnat (Bradysia spp) and suffer substantial removal of peripheral tissue at larval feeding sites. Our work provides evidence for the in vivo role of semivolatile diterpene metabolites as local antifeedants in belowground direct defense against root-feeding insects.