Phytohormones and volatile organic compounds, like geosmin, in the ectomycorrhiza of Tricholoma vaccinum and Norway spruce (Picea abies).

The ectomycorrhizospheric habitat contains a diverse pool of organisms, including the host plant, mycorrhizal fungi, and other rhizospheric microorganisms. Different signaling molecules may influence the ectomycorrhizal symbiosis. Here, we investigated the potential of the basidiomycete Tricholoma vaccinum to produce communication molecules for the interaction with its coniferous host, Norway spruce (Picea abies). We focused on the production of volatile organic compounds and phytohormones in axenic T. vaccinum cultures, identified the potential biosynthesis genes, and investigated their expression by RNA-Seq analyses. T. vaccinum released volatiles not usually associated with fungi, like limonene and ß-barbatene, and geosmin. Using stable isotope labeling, the biosynthesis of geosmin was elucidated. The geosmin biosynthesis gene ges1 of T. vaccinum was identified, and up-regulation was scored during mycorrhiza, while a different regulation was seen with mycorrhizosphere bacteria. The fungus also released the volatile phytohormone ethylene and excreted salicylic and abscisic acid as well as jasmonates into the medium. The tree excreted the auxin, indole-3-acetic acid, and its biosynthesis intermediate, indole-3-acetamide, as well as salicylic acid with its root exudates. These compounds could be shown for the first time in exudates as well as in soil of a natural ectomycorrhizospheric habitat. The effects of phytohormones present in the mycorrhizosphere on hyphal branching of T. vaccinum were assessed. Salicylic and abscisic acid changed hyphal branching in a concentration-dependent manner. Since extensive branching is important for mycorrhiza establishment, a well-balanced level of mycorrhizospheric phytohormones is necessary. The regulation thus can be expected to contribute to an interkingdom language.

First Feasibility Analysis of Ballistocardiography on a Passenger Flight.

The project SCARAB2 (Scalable, Robust and Adaptive on Board Ballistocardiography) aims to use Ballistocardiography (BCG) to monitor flight passengers. In order to show that recorded BCG data from flights give evaluable information even in the noisy environment of an airplane, we monitored a heart-healthy passenger using BCG. Furthermore, we show that there can be a conclusion to heart activities from the recorded ballistocardiogram by comparing the data to a concurrently recorded electrocardiogram (ECG).

Functional variation in a key defense gene structures herbivore communities and alters plant performance.

Plant genetic diversity structures animal communities and affects plant population productivity. However, few studies have investigated which traits are involved and the mechanisms mediating these effects. We studied the consequences of varying the expression of a single biosynthetic gene in jasmonate (JA) defense hormones, which are essential for defense against herbivores but constrain plant growth, in experimental mesocosm populations of wild tobacco (Nicotiana attenuata) plants under attack from three native herbivores. Empoasca leafhoppers preferentially attack JA-deficient N. attenuata plants in nature, and the specialist Tupiocoris notatus mirids avoid Empoasca-damaged plants. However, in experimental mesocosm populations having equal numbers of wild-type (WT) and JA-deficient plants that are silenced in the expression of the biosynthetic gene lipoxygenase 3 (LOX3), Empoasca sp. attacked both genotypes. Empoasca sp. damage, rather than JA, determined T. notatus damage, which was reduced in mixed populations. The growth of specialist Manduca sexta larvae was reduced on WT vs. asLOX3 monocultures, but differed in mixtures depending on caterpillar density. However, seed capsule number remained similar for WT and asLOX3 plants in mixtures, not in monocultures, in two experimental scenarios reflecting high and low caterpillar attack. At high caterpillar density, WT plants growing in mixtures produced more seed capsules than those growing in monocultures while seed production of asLOX3 plants did not differ by population type. However, at low caterpillar density, asLOX3 plants growing in mixed populations produced more seed capsules than those growing in monoculture, while seed capsule production did not differ for WT by population type. Thus, mixed populations had a more stable output of seed capsules under the two scenarios. This may result from a balance between JA-mediated herbivore defense and plant competitive ability in mixed populations.

Maize Stem Response to Long-Term Attack by Sesamia nonagrioides.

Plants defend themselves against herbivores by activating a plethora of genetic and biochemical mechanisms aimed at reducing plant damage and insect survival. The short-term plant response to insect attack is well understood, but less is known about the maintenance of this response over time. We performed transcriptomic and metabolomics analyses in order to identify genes and metabolites involved in the long-term response of maize to attack by the corn borer Sesamina nonagrioides. To determine the role of elicitors present in caterpillar secretions, we also evaluated the response of maize stem challenged with insect regurgitates. The integrative analysis of the omics results revealed that the long-term response in maize is characterized by repression of the primary metabolism and a strong redox response, mainly mediated by germin-like proteins to produce anti-nutritive and toxic compounds that reduce insect viability, and with the glutathione-ascorbate cycle being crucial to minimize the adverse effects of reactive oxygen species (ROS) on the plant. Our results suggest that different defense mechanisms are involved in the long-term response compared to those reported during the early response. We also observed a marginal effect of the caterpillar regurgitates on the long-term defensive response.

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.

High-throughput quantification of more than 100 primary- and secondary-metabolites, and phytohormones by a single solid-phase extraction based sample preparation with analysis by UHPLC-HESI-MS/MS.

BACKGROUND: Plant metabolites are commonly functionally classified, as defense- or growth-related phytohormones, primary and specialized metabolites, and so forth. Analytical procedures for the quantifications of these metabolites are challenging because the metabolites can vary over several orders of magnitude in concentrations in the same tissues and have very different chemical characteristics. Plants clearly adjust their metabolism to respond to their prevailing circumstances in very sophisticated ways that blur the boundaries among these functional or chemically defined classifications. But if plant biologists want to better understand the processes that are important for a plant's adaptation to its environment, procedures are needed that can provide simultaneous quantifications of the large range of metabolites that have the potential to play central roles in these adjustments in a cost and time effective way and with a low sample consumption. RESULTS: Here we present a method that combines well-established methods for the targeted analysis of phytohormones, including jasmonates, salicylic acid, abscisic acid, gibberellins, auxins and cytokinins, and extends it to the analysis of inducible and constitutive defense compounds, as well as the primary metabolites involved in the biosynthesis of specialized metabolites and responsible for nutritional quality (e.g., sugars and amino acids). The method is based on a single extraction of 10-100 mg of tissue and allows a broad quantitative screening of metabolites optimized by their chemical characteristics and concentrations, thereby providing a high throughput analysis unbiased by the putative functional attributes of the metabolites. The tissues of Nicotiana attenuata which accumulate high levels of nicotine and diterpene glycosides, provide a challenging matrix that thwarts quantitative analysis; the analysis of various tissues of this plant are used to illustrate the robustness of the procedure. CONCLUSIONS: The method described has the potential to unravel various, until now overlooked interactions among different sectors of plant metabolism in a high throughput manner. Additionally, the method could be particularly beneficial as screening method in forward genetic approaches, as well as for the investigation of plants from natural populations that likely differ in metabolic traits.

Olive fruits infested with olive fly larvae respond with an ethylene burst and the emission of specific volatiles.

Olive fly (Bactrocera oleae R.) is the most harmful insect pest of olive (Olea europaea L.) which strongly affects fruits and oil production. Despite the expanding economic importance of olive cultivation, up to now, only limited information on plant responses to B. oleae is available. Here, we demonstrate that olive fruits respond to B. oleae attack by producing changes in an array of different defensive compounds including phytohormones, volatile organic compounds (VOCs), and defense proteins. Bactrocera oleae-infested fruits induced a strong ethylene burst and transcript levels of several putative ethylene-responsive transcription factors became significantly upregulated. Moreover, infested fruits induced significant changes in the levels of 12-oxo-phytodienoic acid and C12 derivatives of the hydroperoxide lyase. The emission of VOCs was also changed quantitatively and qualitatively in insect-damaged fruits, indicating that B. oleae larval feeding can specifically affect the volatile blend of fruits. Finally, we show that larval infestation maintained high levels of trypsin protease inhibitors in ripe fruits, probably by affecting post-transcriptional mechanisms. Our results provide novel and important information to understand the response of the olive fruit to B. oleae attack; information that can shed light onto potential new strategies to combat this pest.

How scent and nectar influence floral antagonists and mutualists.

Many plants attract and reward pollinators with floral scents and nectar, respectively, but these traits can also incur fitness costs as they also attract herbivores. This dilemma, common to most flowering plants, could be solved by not producing nectar and/or scent, thereby cheating pollinators. Both nectar and scent are highly variable in native populations of coyote tobacco, Nicotiana attenuata, with some producing no nectar at all, uncorrelated with the tobacco's main floral attractant, benzylacetone. By silencing benzylacetone biosynthesis and nectar production in all combinations by RNAi, we experimentally uncouple these floral rewards/attractrants and measure their costs/benefits in the plant's native habitat and experimental tents. Both scent and nectar increase outcrossing rates for three, separately tested, pollinators and both traits increase oviposition by a hawkmoth herbivore, with nectar being more influential than scent. These results underscore that it makes little sense to study floral traits as if they only mediated pollination services.

Application of Silicone Tubing for Robust, Simple, High-throughput, and Time-resolved Analysis of Plant Volatiles in Field Experiments.

Plant volatiles (PVs) mediate manifold interactions between plants and their biotic and abiotic environments (Dicke and Baldwin, 2010; Holopainen and Gershenzon, 2010). An understanding of the physiological and ecological functions of PVs must therefore be based on measurements of PV emissions under natural conditions. Yet sampling PVs in natural environments is difficult, limited by the need to transport, maintain, and power instruments, or else to employ expensive sorbent devices in replicate. Thus PVs are usually measured in the artificial environments of laboratories or climate chambers. However, polydimethysiloxane (PDMS), a sorbent commonly used for PV sampling (Van Pinxteren et al., 2010; Seethapathy and Górecki, 2012), is available as silicone tubing (ST) for as little as 0.60 €/m (versus 100-550 € apiece for standard PDMS sorbent devices). Small (mm-cm) ST pieces can be placed in any experimental setting and used for headspace sampling with little manipulation of the organism or headspace. ST pieces have absorption kinetics and capacities sufficient to sample plant headspaces on a timescale of minutes to hours, producing biologically meaningful "snapshots" of PV blends. When combined with thermal desorption (TD)-GC-MS analysis - a 40-year-old and widely available technology - ST pieces yield reproducible, sensitive, spatiotemporally resolved, quantitative data from headspace samples taken in natural environments (Kallenbach et al., 2014).

A robust, simple, high-throughput technique for time-resolved plant volatile analysis in field experiments.

Plant volatiles (PVs) mediate interactions between plants and arthropods, microbes and other plants, and are involved in responses to abiotic stress. PV emissions are therefore influenced by many environmental factors, including herbivore damage, microbial invasion, and cues from neighboring plants, and also light regime, temperature, humidity and nutrient availability. Thus, an understanding of the physiological and ecological functions of PVs must be based on measurements reflecting PV emissions under natural conditions. However, PVs are usually sampled in the artificial environments of laboratories or climate chambers. Sampling of PVs in natural environments is difficult, being limited by the need to transport, maintain and provide power to instruments, or use expensive sorbent devices in replicate. Ideally, PVs should be measured in natural settings with high replication, spatio-temporal resolution and sensitivity, and modest costs. Polydimethylsiloxane (PDMS), a sorbent commonly used for PV sampling, is available as silicone tubing for as little as 0.60 € m(-1) (versus 100-550 € each for standard PDMS sorbent devices). Small pieces of silicone tubing (STs) of various lengths from millimeters to centimeters may be added to any experimental setting and used for headspace sampling, with little manipulation of the organism or headspace. STs have sufficiently fast absorption kinetics and large capacity to sample plant headspaces over a timescale of minutes to hours, and thus can produce biologically meaningful 'snapshots' of PV blends. When combined with thermal desorption coupled to GC-MS (a 40-year-old widely available technology), use of STs yields reproducible, sensitive, spatio-temporally resolved quantitative data from headspace samples taken in natural environments.

The Nicotiana attenuata†GLA1 lipase controls the accumulation of Phytophthora parasitica-induced oxylipins and defensive secondary metabolites.

Nicotiana attenuata plants silenced in the expression of GLYCEROLIPASE A1 (ir-gla1 plants) are compromised in the herbivore- and wound-induced accumulation of jasmonic acid (JA). However, these plants accumulate wild-type (WT) levels of JA and divinyl-ethers during Phytophthora parasitica infection. By profiling oxylipin-enriched fractions with targeted and untargeted liquid chromatography-tandem time-of-flight mass spectrometry approaches, we demonstrate that the accumulation of 9-hydroxy-10E,12Z-octadecadienoic acid (9-OH-18:2) and additional C18 and C19 oxylipins is reduced by ca. 20-fold in P. parasitica-infected ir-gla1 leaves compared with WT. This reduced accumulation of oxylipins was accompanied by a reduced accumulation of unsaturated free fatty acids and specific lysolipid species. Untargeted metabolic profiling of total leaf extracts showed that 87 metabolites accumulated differentially in leaves of P. parasitica-infected ir-gla1 plants with glycerolipids, hydroxylated-diterpene glycosides and phenylpropanoid derivatives accounting together for ca. 20% of these 87 metabolites. Thus, P. parasitica-induced oxylipins may participate in the regulation of metabolic changes during infection. Together, the results demonstrate that GLA1 plays a distinct role in the production of oxylipins during biotic stress responses, supplying substrates for 9-OH-18:2 and additional C18 and C19 oxylipin formation during P. parasitica infection, whereas supplying substrates for the biogenesis of JA during herbivory and mechanical wounding.

Leaf-herbivore attack reduces carbon reserves and regrowth from the roots via jasmonate and auxin signaling.

Herbivore attack leads to resource conflicts between plant defensive strategies. Photoassimilates are required for defensive compounds and carbon storage below ground and may therefore be depleted or enriched in the roots of herbivore-defoliated plants. The potential role of belowground tissues as mediators of induced tolerance-defense trade-offs is unknown. We evaluated signaling and carbohydrate dynamics in the roots of Nicotiana attenuata following Manduca sexta attack. Experimental and natural genetic variability was exploited to link the observed metabolite patterns to plant tolerance and resistance. Leaf-herbivore attack decreased sugar and starch concentrations in the roots and reduced regrowth from the rootstock and flower production in the glasshouse and the field. Leaf-derived jasmonates were identified as major regulators of this root-mediated resource-based trade-off: lower jasmonate levels were associated with decreased defense, increased carbohydrate levels and improved regrowth from the rootstock. Application and transport inhibition experiments, in combination with silencing of the sucrose non-fermenting (SNF) -related kinase GAL83, indicated that auxins may act as additional signals that regulate regrowth patterns. In conclusion, our study shows that the ability to mobilize defenses has a hidden resource-based cost below ground that constrains defoliation tolerance. Jasmonate- and auxin-dependent mechanisms may lead to divergent defensive plant strategies against herbivores in nature.

Progressive 35S promoter methylation increases rapidly during vegetative development in transgenic Nicotiana attenuata plants.

BACKGROUND: Genetically modified plants are widely used in agriculture and increasingly in ecological research to enable the selective manipulation of plant traits in the field. Despite their broad usage, many aspects of unwanted transgene silencing throughout plant development are still poorly understood. A transgene can be epigenetically silenced by a process called RNA directed DNA methylation (RdDM), which can be seen as a heritable loss of gene expression. The spontaneous nature of transgene silencing has been widely reported, but patterns of acquirement remain still unclear. RESULTS: Transgenic wild tobacco plants (Nicotiana attenuata) expressing heterologous genes coding for antimicrobial peptides displayed an erratic and variable occurrence of transgene silencing. We focused on three independently transformed lines (PNA 1.2, PNA 10.1 and ICE 4.4) as they rapidly lost the expression of the resistance marker and down-regulated transgene expression by more than 200 fold after only one plant generation. Bisulfite sequencing indicated hypermethylation within the 35S and NOS promoters of these lines. To shed light on the progress of methylation establishment, we successively sampled leaf tissues from different stages during plant development and found a rapid increase in 35S promoter methylation during vegetative growth (up to 77% absolute increase within 45 days of growth). The levels of de novo methylation were inherited by the offspring without any visible discontinuation. A secondary callus regeneration step could interfere with the establishment of gene silencing and we found successfully restored transgene expression in the offspring of several regenerants. CONCLUSIONS: The unpredictability of the gene silencing process requires a thorough selection and early detection of unstable plant lines. De novo methylation of the transgenes was acquired solely during vegetative development and did not require a generational change for its establishment or enhancement. A secondary callus regeneration step provides a convenient way to rescue transgene expression without causing undesirable morphological effects, which is essential for experiments that use transformed plants in the analysis of ecologically important traits.

Feeding-induced rearrangement of green leaf volatiles reduces moth oviposition.

The ability to decrypt volatile plant signals is essential if herbivorous insects are to optimize their choice of host plants for their offspring. Green leaf volatiles (GLVs) constitute a widespread group of defensive plant volatiles that convey a herbivory-specific message via their isomeric composition: feeding of the tobacco hornworm Manduca sexta converts (Z)-3- to (E)-2-GLVs thereby attracting predatory insects. Here we show that this isomer-coded message is monitored by ovipositing M. sexta females. We detected the isomeric shift in the host plant Datura wrightii and performed functional imaging in the primary olfactory center of M. sexta females with GLV structural isomers. We identified two isomer-specific regions responding to either (Z)-3- or (E)-2-hexenyl acetate. Field experiments demonstrated that ovipositing Manduca moths preferred (Z)-3-perfumed D. wrightii over (E)-2-perfumed plants. These results show that (E)-2-GLVs and/or specific (Z)-3/(E)-2-ratios provide information regarding host plant attack by conspecifics that ovipositing hawkmoths use for host plant selection. DOI:http://dx.doi.org/10.7554/eLife.00421.001.

Jasmonate signaling in the field, part I: elicited changes in jasmonate pools of transgenic Nicotiana attenuata populations.

Nicotiana attenuata, a wild tobacco species native of the southwestern USA that grows in the immediate postfire environment, is one of the important host plants for herbivore populations recolonizing recently burned habitats in the Great Basin Desert. Based on more than 20 years of field research on this eco-genomics model system established in our group, we have developed a genetic and analytical toolbox that allows us to assess the importance of particular genes and metabolites for the survival of this plant in its native habitat. This toolbox has been extensively applied to study the activation of jasmonate signaling after the attack of different herbivore species. Here, we provide detailed guidelines for the analysis, under field conditions, of induced changes in jasmonate pools during insect herbivory. The procedures range from selection and field release of well-characterized transgenic lines for testing the physiological consequences of manipulating jasmonate biogenesis, metabolism, or perception to the metabolic elicitation of chewing herbivore attack and the quantification of the resulting changes in jasmonate fluxes.

Jasmonate signaling in the field, part II: insect-guided characterization of genetic variations in jasmonate-dependent defenses of transgenic and natural Nicotiana attenuata populations.

The introduction of genetically modified plants into natural habitats represents a valuable means to determine organismic level functions of a gene and its effects on a plant's interaction with other organisms. Nicotiana attenuata, a wild tobacco species native of the southwestern USA that grows in the immediate postfire environment, is one of the important host plants for herbivore populations recolonizing recently burned habitats in the Great Basin Desert. Here, we provide detailed guidelines for the analysis, under field conditions, of jasmonate-dependent defense and its impact on the plant's native herbivore community. The procedures are based on the field release of transgenic lines silenced for jasmonate biogenesis, metabolism, or perception to conduct association studies between defense trait expression (secondary metabolite and trypsin proteinase inhibitor accumulation) and insect infestations. Additionally, because some insects have evolved mechanisms to "eavesdrop" on jasmonate signaling when selecting their host plants, we describe how leafhoppers of the species Empoasca, which selectively colonize jasmonate-deficient plants, can be used as "bloodhounds" for identifying natural variations in jasmonate signaling among natural N. attenuata populations.

Empoasca leafhoppers attack wild tobacco plants in a jasmonate-dependent manner and identify jasmonate mutants in natural populations.

Choice of host plants by phytophagous insects is essential for their survival and reproduction. This choice involves complex behavioral responses to a variety of physical and chemical characteristics of potential plants for feeding. For insects of the order Hemiptera, these behavioral responses involve a series of steps including labial dabbing and probing using their piercing mouthparts. These initial probing and feeding attempts also elicit a rapid accumulation of phytohormones, such as jasmonic acid (JA), and the induced defense metabolites they mediate. When Nicotiana attenuata plants are rendered JA deficient by silencing the initial committed step of the JA biosynthesis pathway, they are severely attacked in nature by hemipteran leafhoppers of the genus Empoasca. By producing N. attenuata plants silenced in multiple steps of JA biosynthesis and perception and in the biosynthesis of the plant's three major classes of JA-inducible insecticidal defenses, we demonstrate that the choice of plants for feeding by Empoasca leafhoppers in both nature and the glasshouse is independent of the accumulation of major insecticidal molecules. Moreover, this choice is independent of the presence of Candidatus Phytoplasma spp. and is not associated with detectable changes in plant volatiles but instead depends on the plant's capacity to mediate JA signaling. We exploited this trait and used Empoasca leafhoppers to reveal genetic variation in JA accumulation and signaling hidden in N. attenuata natural populations.

C12 derivatives of the hydroperoxide lyase pathway are produced by product recycling through lipoxygenase-2 in Nicotiana attenuata leaves.

In response to diverse stresses, the hydroperoxide lyase (HPL) pathway produces C(6) aldehydes and 12-oxo-(9Z )-dodecenoic acid ((9Z )-traumatin). Since the original characterization of (10E )-traumatin and traumatic acid, little has been added to our knowledge of the metabolism and fluxes associated with the conversion of (9Z )-traumatin into diverse products in response to wounding and herbivory. A liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method was developed to quantify C(12) derivatives of the HPL pathway and to determine their metabolism after wounding and simulated herbivory in Nicotiana attenuata leaves. Ninety-eight per cent of the (9Z )-traumatin produced was converted to 9-hydroxy-(10E )-traumatin (9-OH-traumatin); two-thirds by product recycling through lipoxygenase-2 (NaLOX2) activity and one-third by nonenzymatic oxidation. Thirty-eight per cent of the de novo produced 9-OH-traumatin was conjugated to glutathione, consistent with this oxylipin being a reactive electrophile species. 12-OH-(9Z )-dodecenoic and dodecenedioic acids also showed rapid increases after wounding and simulated herbivory and a role for C(12) derivatives as signals in these processes was consistent with their ability to elicit substantial changes in gene expression. These results underscore the importance of metabolite reflux through LOX2, an insight which creates new opportunities for a functional understanding of C(12) derivatives of the HPL pathway in the regulation of stress responses.

Rapid modification of the insect elicitor N-linolenoyl-glutamate via a lipoxygenase-mediated mechanism on Nicotiana attenuata leaves.

BACKGROUND: Some plants distinguish mechanical wounding from herbivore attack by recognizing specific constituents of larval oral secretions (OS) which are introduced into plant wounds during feeding. Fatty acid-amino acid conjugates (FACs) are major constituents of Manduca sexta OS and strong elicitors of herbivore-induced defense responses in Nicotiana attenuata plants. RESULTS: The metabolism of one of the major FACs in M. sexta OS, N-linolenoyl-glutamic acid (18:3-Glu), was analyzed on N. attenuata wounded leaf surfaces. Between 50 to 70% of the 18:3-Glu in the OS or of synthetic 18:3-Glu were metabolized within 30 seconds of application to leaf wounds. This heat-labile process did not result in free alpha-linolenic acid (18:3) and glutamate but in the biogenesis of metabolites both more and less polar than 18:3-Glu. Identification of the major modified forms of this FAC showed that they corresponded to 13-hydroxy-18:3-Glu, 13-hydroperoxy-18:3-Glu and 13-oxo-13:2-Glu. The formation of these metabolites occurred on the wounded leaf surface and it was dependent on lipoxygenase (LOX) activity; plants silenced in the expression of NaLOX2 and NaLOX3 genes showed more than 50% reduced rates of 18:3-Glu conversion and accumulated smaller amounts of the oxygenated derivatives compared to wild-type plants. Similar to 18:3-Glu, 13-oxo-13:2-Glu activated the enhanced accumulation of jasmonic acid (JA) in N. attenuata leaves whereas 13-hydroxy-18:3-Glu did not. Moreover, compared to 18:3-Glu elicitation, 13-oxo-13:2-Glu induced the differential emission of two monoterpene volatiles (beta-pinene and an unidentified monoterpene) in irlox2 plants. CONCLUSIONS: The metabolism of one of the major elicitors of herbivore-specific responses in N. attenuata plants, 18:3-Glu, results in the formation of oxidized forms of this FAC by a LOX-dependent mechanism. One of these derivatives, 13-oxo-13:2-Glu, is an active elicitor of JA biosynthesis and differential monoterpene emission.

Nicotiana attenuata SIPK, WIPK, NPR1, and fatty acid-amino acid conjugates participate in the induction of jasmonic acid biosynthesis by affecting early enzymatic steps in the pathway.

Wounding and herbivore attack elicit the rapid (within minutes) accumulation of jasmonic acid (JA) that results from the activation of previously synthesized biosynthetic enzymes. Recently, several regulatory factors that affect JA production have been identified; however, how these regulators affect JA biosynthesis remains at present unknown. Here we demonstrate that Nicotiana attenuata salicylate-induced protein kinase (SIPK), wound-induced protein kinase (WIPK), nonexpressor of PR-1 (NPR1), and the insect elicitor N-linolenoyl-glutamate [corrected] (18:3-Glu) participate in mechanisms affecting early enzymatic steps of the JA biosynthesis pathway. Plants silenced in the expression of SIPK and NPR1 were affected in the initial accumulation of 13-hydroperoxy-linolenic acid (13-OOH-18:3) after wounding and 18:3-Glu elicitation by mechanisms independent of changes in 13-lipoxygenase activity. Moreover, 18:3-Glu elicited an enhanced and rapid accumulation of 13-OOH-18:3 that depended partially on SIPK and NPR1 but was independent of increased 13-lipoxygenase activity. Together, the results suggested that substrate supply for JA production was altered by 18:3-Glu elicitation and SIPK- and NPR1-mediated mechanisms. Consistent with a regulation at the level of substrate supply, we demonstrated by virus-induced gene silencing that a wound-repressed plastidial glycerolipase (NaGLA1) plays an essential role in the induction of de novo JA biosynthesis. In contrast to SIPK and NPR1, mechanisms mediated by WIPK did not affect the production of 13-OOH-18:3 but were critical to control the conversion of this precursor into 12-oxo-phytodienoic acid. These differences could be partially accounted for by reduced allene oxide synthase activity in WIPK-silenced plants.