Stomata: gatekeepers of uptake and defense signaling by green leaf volatiles in maize.

Plants adapt to balance growth-defense tradeoffs in response to both biotic and abiotic stresses. Green leaf volatiles (GLVs) are released after biotic and abiotic stresses and function as damage-associated signals in plants. Although, GLVs enter plants primarily through stomata, the role of stomatal regulation on the kinetics of GLV uptake remains largely unknown. Here, we illustrate the effect of stomatal closure on the timing and magnitude of GLV uptake. We closed stomata by either exposing maize (Zea mays) plants to darkness or applying abscisic acid, a phytohormone that closes the stomata in light. Then, we exposed maize seedlings to (Z)-3-hexen-1-ol and compared its dynamic uptake under different stomatal conditions. Additionally, we used (E)-3-hexen-1-ol, an isomer of (Z)-3-hexen-1-ol not made by maize, to exclude the role of internal GLVs in our assays. We demonstrate that closed stomata effectively prevent GLV entry into exposed plants, even at high concentrations. Furthermore, our findings indicate that reduced GLV uptake impairs GLV-driven induction of biosynthesis of sesquiterpenes, a group of GLV-inducible secondary metabolites, with or without herbivory. These results elucidate how stomata regulate the perception of GLV signals, thereby dramatically changing the plant responses to herbivory, particularly under water stress or dark conditions.

Complex and Beautiful: Unraveling the Intricate Communication Systems Among Plants and Insects.

My research focuses on elucidating the chemical communication systems linking plants, herbivores, and natural enemies. My interests in integrating chemistry and agriculture led to my graduate studies in the emerging field of chemical ecology. My thesis research resulted in the identification, synthesis, and application of boll weevil sex pheromones. My research group subsequently developed chemical lures for more than 20 species of pest insects. I then shifted my focus to some of the first studies of the chemical signals produced by plants being attacked by herbivores. When insects feed, elicitors in the insects' oral secretions, such as volicitin, a fatty acid-amino acid conjugate elicitor, stimulate plants to release volatile organic compounds. Parasitoid wasps learn to associate these species-specific volatiles with their herbivore hosts. These volatiles also prime nearby plants to activate a faster and higher defense response upon attack. Throughout my career, I have collaborated with scientists from diverse disciplines to tackle fundamental questions in chemical ecology and create innovative solutions for insect management. Our collaborative research has fundamentally changed and improved our understanding of the ongoing coevolution of plants, their herbivores, and the natural enemies that attack those herbivores.

The dual function of elicitors and effectors from insects: reviewing the 'arms race' against plant defenses.

KEY MESSAGE: This review provides an overview, analysis, and reflection on insect elicitors and effectors (particularly from oral secretions) in the context of the 'arms race' with host plants. Following injury by an insect herbivore, plants rapidly activate induced defenses that may directly or indirectly affect the insect. Such defense pathways are influenced by a multitude of factors; however, cues from the insect's oral secretions are perhaps the most well studied mediators of such plant responses. The relationship between plants and their insect herbivores is often termed an 'evolutionary arms race' of strategies for each organism to either overcome defenses or to avoid attack. However, these compounds that can elicit a plant defense response that is detrimental to the insect may also benefit the physiology or metabolism of an insect species. Indeed, several insect elicitors of plant defenses (such as the fatty acid-amino acid conjugate, volicitin) are known to enhance an insect's ability to obtain nutritionally important compounds from plant tissue. Here we re-examine the well-known elicitors and effectors from chewing insects to demonstrate not only our incomplete understanding of the specific biochemical and molecular cascades involved in these interactions but also to consider the role of these compounds for the insect species itself. Finally, this overview discusses opportunities for research in the field of plant-insect interactions by utilizing tools such as genomics and proteomics to integrate the future study of these interactions through ecological, physiological, and evolutionary disciplines.

Herbivorous Caterpillars and the Green Leaf Volatile (GLV) Quandary.

Several herbivorous caterpillars contain effectors in their oral secretions that alter the emission of green leaf volatiles (GLVs) produced by the plants upon which the caterpillars are feeding. These effectors include an isomerase, a fatty acid dehydratase (FHD), and a heat-stable hexenal trapping (HALT) molecule. GLVs serve as signaling compounds in plant-insect interactions and inter-and intra-plant communication. However, it is not known whether these GLV-altering effectors are common among herbivorous caterpillars, or the evolutionary context of these effectors in relation to GLV emission by host plants in response to feeding damage. Here, we examined the distribution and activity of the isomerase, FHD, and HALT effectors across 10 species spanning 7 lepidopteran families. Six of the 10 species possessed all three effectors in their oral secretions. Activity from the HALT and FHD effectors was observed in all examined caterpillar species, while activity from the isomerase effector varied in some species and was absent in others. There was no discernable pattern in effector activity based on evolutionary divergence, since individual species within a family did not possess similar mechanisms to alter GLV emission. These data, demonstrating the GLV-altering effectors acting at different steps in the GLV biosynthetic pathway and present in the examined caterpillar species at different combinations with different activities, highlight the importance of these effectors in changing the emission of these compounds during caterpillar herbivory. Understanding the prevalence and roles of GLV-altering effectors and GLV emission itself will open new research areas in the dynamics of plant-insect interactions.

Herbivore-specific plant volatiles prime neighboring plants for nonspecific defense responses.

Plants produce species-specific herbivore-induced plant volatiles (HIPVs) after damage. We tested the hypothesis that herbivore-specific HIPVs prime neighboring plants to induce defenses specific to the priming herbivore. Since Manduca sexta (specialist) and Heliothis virescens (generalist) herbivory induced unique HIPV profiles in Nicotiana benthamiana, we used these HIPVs to prime receiver plants for defense responses to simulated herbivory (mechanical wounding and herbivore regurgitant application). Jasmonic acid (JA) accumulations and emitted volatile profiles were monitored as representative defense responses since JA is the major plant hormone involved in wound and defense signaling and HIPVs have been implicated as signals in tritrophic interactions. Herbivore species-specific HIPVs primed neighboring plants, which produced 2 to 4 times more volatiles and JA after simulated herbivory when compared to similarly treated constitutive volatile-exposed plants. However, HIPV-exposed plants accumulated similar amounts of volatiles and JA independent of the combination of priming or challenging herbivore. Furthermore, volatile profiles emitted by primed plants depended only on the challenging herbivore species but not on the species-specific HIPV profile of damaged emitter plants. This suggests that feeding by either herbivore species primed neighboring plants for increased HIPV emissions specific to the subsequently attacking herbivore and is probably controlled by JA.

Elevated carbon dioxide reduces emission of herbivore-induced volatiles in Zea mays.

Terpene volatiles produced by sweet corn (Zea mays) upon infestation with pests such as beet armyworm (Spodoptera exigua) function as part of an indirect defence mechanism by attracting parasitoid wasps; yet little is known about the impact of climate change on this form of plant defence. To investigate how a central component of climate change affects indirect defence, we measured herbivore-induced volatile emissions in plants grown under elevated carbon dioxide (CO2 ). We found that S. exigua infested or elicitor-treated Z. mays grown at elevated CO2 had decreased emission of its major sesquiterpene, (E)-ß-caryophyllene and two homoterpenes, (3E)-4,8-dimethyl-1,3,7-nonatriene and (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene. In contrast, inside the leaves, elicitor-induced (E)-ß-caryophyllene hyper-accumulated at elevated CO2 , while levels of homoterpenes were unaffected. Furthermore, gene expression analysis revealed that the induction of terpene synthase genes following treatment was lower in plants grown at elevated CO2 . Our data indicate that elevated CO2 leads both to a repression of volatile synthesis at the transcriptional level and to limitation of volatile release through effects of CO2 on stomatal conductance. These findings suggest that elevated CO2 may alter the ability of Z. mays to utilize volatile terpenes to mediate indirect defenses.

Mechanisms and time course of menthol-induced cutaneous vasodilation.

Menthol is a vasoactive compound that is widely used in topical analgesic agents. Menthol induces cutaneous vasodilation, however the underlying mechanisms are unknown. Determining the rates of appearance and clearance of menthol in the skin is important for optimizing topical treatment formulation and dosing. The purpose of this study was to determine the mechanisms contributing to menthol-mediated cutaneous vasodilation and to establish a time course for menthol appearance/clearance in the skin. Ten young (23±1years, 5 males 5 females) subjects participated in two protocols. In study 1, four intradermal microdialysis fibers were perfused with increasing doses of menthol (0.1-500mM) and inhibitors for nitric oxide (NO), endothelium derived hyperpolarizing factors (EDHFs), and sensory nerves. Skin blood flow was measured with laser Doppler flowmetry and normalized to %CVCmax. In study 2, two intradermal microdialysis fibers were perfused with lactated Ringer's solution. 0.017mL·cm-2 of a 4% menthol gel was placed over each fiber. 5µL samples of dialysate from the microdialysis fibers were collected every 30min and analyzed for the presence of menthol with high performance gas chromatography/mass spectrometry. Skin blood flow (laser speckle contrast imaging) and subjective ratings of menthol sensation were simultaneously obtained with dialysate samples. In study 1, menthol induced cutaneous vasodilation at all doses ≥100mM (all p<0.05). However, inhibition of either NO, EDHFs, or sensory nerves fully inhibited menthol-mediated vasodilation (all p>0.05). In study 2, significant menthol was detected in dialysate 30min post menthol application (0.89ng, p=0.0002). Relative to baseline, cutaneous vasodilation was elevated from minutes 15-45 and ratings of menthol sensation were elevated from minute 5-60 post menthol application (all p<0.05). Menthol induces cutaneous vasodilation in the skin through multiple vasodilator pathways, including NO, EDHF, and sensory nerves. Topical menthol is detectable in the skin within 30min and is cleared by 60min. Skin blood flow and perceptual measures follow a similar time course as menthol appearance/clearance.

Variation in the Volatile Profiles of Black and Manchurian Ash in Relation to Emerald Ash Borer Oviposition Preferences.

Emerald ash borer (EAB; Agrilus planipennis) is a devastating pest of ash (Fraxinus spp.) in its invaded range in North America. Its coevolved Asian hosts are more resistant and less preferred for oviposition than susceptible North American species. We compared EAB oviposition preferences and bark and canopy volatile organic compound (VOC) emissions of resistant Manchurian ash and susceptible black ash, and examined relationships between VOC profiles and oviposition. In the field, black ash was highly preferred for oviposition while no eggs were laid on Manchurian ash, and we found clear differences in the VOC profiles of Manchurian and black ash. We detected 78 compounds emitted from these species, including 16 compounds that elicited EAB antennal activity in prior studies. Four compounds were unique to black and 11 to Manchurian ash. Emission rates of 14 canopy and 19 bark volatiles varied among the two species, including four previously reported as antennally active. Specifically, 7-epi-sesquithujene (bark) emissions were greater from black ash, while ß-caryophyllene (canopy), linalool (bark), and α-cubebene (bark) were emitted at higher rates by Manchurian ash. No relationships were found between the emission rate of any single compound or group of compounds (e.g. monoterpenes) suggesting that preference may be based on complex profile combinations. This is the first study to directly compare VOCs of black and Manchurian ash as well as the first to examine bark- and canopy-specific VOCs. The unique bark and canopy VOC profiles of these two species implicates potentially important variation in VOCs between a closely related resistant and susceptible species that provides a foundation for future studies of host preferences of EAB.

A small, differentially regulated family of farnesyl diphosphate synthases in maize (Zea mays) provides farnesyl diphosphate for the biosynthesis of herbivore-induced sesquiterpenes.

MAIN CONCLUSION: Of the three functional FPPS identified in maize, fpps3 is induced by herbivory to produce FDP important for the formation of the volatile sesquiterpenes of plant defense. Sesquiterpenes are not only crucial for the growth and development of a plant but also for its interaction with the environment. The biosynthesis of sesquiterpenes proceeds over farnesyl diphosphate (FDP), which is either used as a substrate for protein prenylation, converted to squalene, or to volatile sesquiterpenes. To elucidate the regulation of sesquiterpene biosynthesis in maize, we identified and characterized the farnesyl diphosphate synthase (FPPS) gene family which consists of three genes. Synteny analysis indicates that fpps2 and fpps3 originate from a genome duplication in an ancient tetraploid ancestor. The three FPPSs encode active enzymes that produce predominantly FDP from the isopentenyl diphosphate and dimethylallyl diphosphate substrates. Only fpps1 and fpps3 are induced by elicitor treatment, but induced fpps1 levels are much lower and only increased to the amounts of fpps3 levels in intact leaves. Elicitor-induced fpps3 levels in leaves increase to more than 15-fold of background levels. In undamaged roots, transcript levels of fpps1 are higher than those of fpps3, but only fpps3 transcripts are induced in response to herbivory by Diabrotica virgifera virgifera. A kinetic of transcript abundance in response to herbivory in leaves provided further evidence that the regulation of fpps3 corresponds to that of tps23, a terpene synthase, that converts FDP to the volatile (E)-ß-caryophyllene. Our study indicates that the differential expression of fpps1 and fpps3 provides maize with FDP for both primary metabolism and terpene-based defenses. The expression of fpps3 seems to coincide with the herbivore-induced emission of volatile sesquiterpenes that were demonstrated to be important defense signals.

N-(18-hydroxylinolenoyl)-L-glutamine: a newly discovered analog of volicitin in Manduca sexta and its elicitor activity in plants.

Plants attacked by insect herbivores release a blend of volatile organic compounds (VOCs) that serve as chemical cues for host location by parasitic wasps, natural enemies of the herbivores. Volicitin, N-(17-hydroxylinolenoyl)-L-glutamine, is one of the most active VOC elicitors found in herbivore regurgitants. Our previous study revealed that hydroxylation on the 17th position of the linolenic acid moiety of N-linolenoyl-L-glutamine increases by more than three times the elicitor activity in corn plants. Here, we identified N-(18-hydroxylinolenoyl)-L-glutamine (18OH-volicitin) from larval gut contents of tobacco hornworm (THW), Manduca sexta. Eggplant and tobacco, two solanaceous host plants of THW larvae, and corn, a non-host plant, responded differently to this new elicitor. Eggplant and tobacco seedlings emitted twice the amount of VOCs when 18OH-volicitin was applied to damaged leaf surfaces compared to N-linolenoyl-L-glutamine, while both these fatty acid amino acid conjugates (FACs) elicited a similar response in corn seedlings. In both solanaceous plants, there was no significant difference in the elicitor activity of 17OH- and 18OH-volicitin. Interestingly, other lepidopteran species that have 17OH-type volicitin also attack solanaceous plants. These data suggest that plants have developed herbivory-detection systems customized to their herbivorous enemies.

Volatiles from intact and Lygus-damaged Erigeron annuus (L.) Pers. are highly attractive to ovipositing Lygus and its parasitoid Peristenus relictus Ruthe.

Trap cropping and biological control can provide a sustainable means of controlling insect pests. Insects in the genus Lygus (Hemiptera: Miridae) are major pests on cotton and horticultural crops throughout the United States, and pesticide resistance within Lygus populations necessitates more sustainable long-term management techniques. Here, we explore behavioral responses of Lygus bugs (L. rubrosignatus Knight) and an introduced parasitoid, Peristenus relictus (Hymenoptera: Braconidae), to a common field edge plant, Erigeron annuus, which has the potential to serve as a trap host. Erigeron annuus is attractive to Lygus in the field, with Lygus preferentially moving to Erigeron patches compared to more abundant cotton plants. To determine the role of odor cues in mediating this attraction, we collected volatiles from E. annuus with and without Lygus damage, and then tested the attractiveness of these volatiles vs. those of cotton to Lygus females and female P. relictus wasps using Y-tube and wind tunnel bioassays. We found that undamaged E. annuus emits high concentrations of a complex volatile blend (60+ compounds), with novel compounds induced and constitutive compounds up-regulated in response to damage. Additionally, both female Lygus bugs and female P. relictus wasps are highly attracted to E. annuus volatiles over those of cotton in almost every combination of damage treatments. Our results suggest that Erigeron annuus would be an effective trap plant to control Lygus in cotton, since it is highly attractive to both the pest and its natural enemy.

Host suitability affects odor association in Cotesia marginiventris: implications in generalist parasitoid host-finding.

Insect herbivores often induce plant volatile compounds that can attract natural enemies. Cotesia marginiventris (Hymenoptera: Braconidae) is a generalist parasitoid wasp of noctuid caterpillars and is highly attracted to Spodoptera exigua-induced plant volatiles. The plasticity of C. marginiventris associative learning to volatile blends of various stimuli, such as host presence, also has been shown, but little is known about how this generalist parasitoid distinguishes between host species of varying suitability. Spodoptera exigua is an excellent host that yields high parasitoid emergence, while Trichoplusia ni serves as a sub-optimal host species due to high pre-imaginal wasp mortality. We have found that S. exigua and T. ni induce different volatile blends while feeding on cotton. Here, wind tunnel flight assays were used to determine the importance of differentially induced volatiles in host-finding by C. marginiventris. We found that, while this generalist parasitoid wasp can distinguish between the two discrete volatile blends when presented concurrently, a positive oviposition experience on the preferred host species (S. exigua) is more important than host-specific volatile cues in eliciting flight behavior towards plants damaged by either host species. Furthermore, wasps with oviposition experience on both host species did not exhibit a deterioration in positive flight behavior, suggesting that oviposition in the sub-optimal host species (T. ni) does not cause aversive odor association.

Volatile profile differences and the associated Sirex noctilio activity in two host tree species in the Northeastern United States.

Sirex noctilio females are known to be attracted to stem sections of stressed pine trees for oviposition. The volatile profiles and attractiveness of Eastern white pine (Pinus strobus) and two chemotypes of Scots pine (P. sylvestris) were compared after stem injection with herbicide. In general, trap captures on herbicide-treated trees were higher than on controls. The high-carene chemotype of Scots pine captured the highest numbers of females, followed by the low-carene chemotype, and finally the Eastern white pine. Herbicide-treated trees of both species emitted larger quantities of volatiles than the controls. The herbicide treatment induced higher volatile emission rates in the Scots pine chemotypes than in white pine, although there was no difference between the two chemotypes. However, qualitative differences were found between the volatile profiles of the two species as well as between the two Scots pine chemotypes, which could account for the differential attractiveness of the species and chemotypes tested.

Male-produced pheromone in the European woodwasp, Sirex noctilio.

A male-produced pheromone that attracts both males and females was identified for the European woodwasp, Sirex noctilio, a serious pest of pine trees. Males displayed excitatory behaviors when placed in groups, and were attracted to the odors from males that were 2-5-d-old, but not to odors from males that were 0-1-d-old. An unsaturated short-chain alcohol, (Z)-3-decen-1-ol, was discovered in samples collected on SuperQ filters over groups of males and identified by using micro-derivatization reactions and gas chromatography coupled with mass spectrometry (GC-MS). The compound was not detected in volatile samples from females. Gas chromatography coupled electroantennographic detection (GC-EAD) of antennae from males exposed to male headspace odors produced strong antennal responses to the main peak of (Z)-3-decen-1-ol, as well as to an unknown minor component that had a similar retention time. Antennae from both males and females responded to synthetic (Z)-3-decen-1-ol. Several different synthetic candidates for the GC-EAD active minor components were selected based on GC-MS and GC-EAD responses to male headspace collections. These synthetic compounds were tested for antennal activity using GC-EAD, and those that produced strong responses were blended with the major component and tested for male attraction in the Y-tube olfactometer at different concentrations and ratios. Males tested in the Y-tube olfactometer were attracted to a synthetic blend of (Z)-3-decen-1-ol and (Z)-4-decen-1-ol at a ratio of 100:1. Whereas the addition of some suspected minor compounds reduced attraction, the addition of a third compound found in male emanations that produced strong male antennal responses, (E,E)-2,4-decadienal (at a ratio of 100:1:1), resulted in attraction of both males (Y-tube and wind tunnel) and females (wind tunnel).

Phytohormone-based activity mapping of insect herbivore-produced elicitors.

In response to insect attack, many plants exhibit dynamic biochemical changes, resulting in the induced production of direct and indirect defenses. Elicitors present in herbivore oral secretions are believed to positively regulate many inducible plant defenses; however, little is known about the specificity of elicitor recognition in plants. To investigate the phylogenic distribution of elicitor activity, we tested representatives from three different elicitor classes on the time course of defense-related phytohormone production, including ethylene (E), jasmonic acid (JA), and salicylic acid, in a range of plant species spanning angiosperm diversity. All families examined responded to at least one elicitor class with significant increases in E and JA production within 1 to 2 h after treatment, yet elicitation activity among species was highly idiosyncratic. The fatty-acid amino acid conjugate volicitin exhibited the widest range of phytohormone and volatile inducing activity, which spanned maize (Zea mays), soybean (Glycine max), and eggplant (Solanum melongena). In contrast, the activity of inceptin-related peptides, originally described in cowpea (Vigna unguiculata), was limited even within the Fabaceae. Similarly, caeliferin A16:0, a disulfooxy fatty acid from grasshoppers, was the only elicitor with demonstrable activity in Arabidopsis thaliana. Although precise mechanisms remain unknown, the unpredictable nature of elicitor activity between plant species supports the existence of specific receptor-ligand interactions mediating recognition. Despite the lack of an ideal plant model for studying the action of numerous elicitors, E and JA exist as highly conserved and readily quantifiable markers for future discoveries in this field.

Pea aphids, Acyrthosiphon pisum, suppress induced plant volatiles in broad bean, Vicia faba.

Plants defend themselves against herbivory through several means, including the production of airborne volatile organic compounds (VOCs). These VOCs benefit plants by attracting natural enemies of their herbivores. The pea aphid, Acyrthosiphon pisum, is able to feed on its host plant, Vicia faba, without inducing detectable changes in plant VOC emission. Levels of VOCs emission are not significantly different between control plants and those fed upon by aphids for up to 5 days. Using a second herbivore, the beet armyworm caterpillar, Spodoptera exigua, we demonstrate that several expected caterpillar-induced VOCs are reduced when co-infested with pea aphids, thus demonstrating that pea aphids have the ability to inhibit the release of certain VOCs. This study shows, for the first time, that aphids not only avoid triggering plant volatile emission, but also can actively inhibit herbivore-induced volatiles.

Active role of fatty acid amino acid conjugates in nitrogen metabolism in Spodoptera litura larvae.

Since the first fatty acid amino acid conjugate (FAC) was isolated from regurgitant of Spodoptera exigua larvae in 1997 [volicitin: N-(17-hydroxylinolenoyl)-L-glutamine], their role as elicitors of induced responses in plants has been well documented. However, studies of the biosyntheses and the physiological role of FACs in the insect have been minimal. By using (14)C-labeled glutamine, glutamic acid, and linolenic acid in feeding studies of Spodoptera litura larvae, combined with tissue analyses, we found glutamine in the midgut cells to be a major source for biosynthesis of FACs. Furthermore, 20% of the glutamine moiety of FACs was derived from glutamic acid and ammonia through enzymatic reaction of glutamine synthetase (GS). To determine whether FACs improve GS productivity, we studied nitrogen assimilation efficiency of S. litura larvae fed on artificial diets containing (15)NH(4)Cl and glutamic acid. When the diet was enriched with linolenic acid, the nitrogen assimilation efficiency improved from 40% to >60%. In the lumen, the biosynthesized FACs are hydrolyzed to fatty acids and glutamine, which are reabsorbed into tissues and hemolymph. These results strongly suggested that FACs play an active role in nitrogen assimilation in Lepidoptera larva and that glutamine containing FACs in the gut lumen may function as a form of storage of glutamine, a key compound of nitrogen metabolism.

Plant-insect dialogs: complex interactions at the plant-insect interface.

Although five different classes of insect herbivore-produced elicitors of plant volatiles have been identified, this is only a part of the complex, chemically mediated interactions between insect herbivores and their host plants. The defensive reactions of the plant, following physical injury by the herbivore, are influenced by a multitude of factors including, but not necessarily limited to, the elicitors and numerous other herbivore-associated molecules, as well as microbes on the plant surface that may alter plant defensive pathways. Ultimately, a thorough and accurate understanding of the chemical ecology of insect-plant interactions will require a more holistic approach, taking into consideration the ecological and physiological context in which a plant perceives and responds to herbivore-associated signals.

Plants on constant alert: elevated levels of jasmonic acid and jasmonate-induced transcripts in caterpillar-resistant maize.

This study was conducted to determine if constitutive levels of jasmonic acid (JA) and other octadecanoid compounds were elevated prior to herbivory in a maize genotype with documented resistance to fall armyworm (Spodoptera frugiperda) and other lepidopteran pests. The resistant inbred Mp708 had approximately 3-fold higher levels of jasmonic acid (JA) prior to herbivore feeding than the susceptible inbred Tx601. Constitutive levels of cis-12-oxo-phytodienoic acid (OPDA) also were higher in Mp708 than Tx601. In addition, the constitutive expression of JA-inducible genes, including those in the JA biosynthetic pathway, was higher in Mp708 than Tx601. In response to herbivory, Mp708 generated comparatively higher levels of hydrogen peroxide, and had a greater abundance of NADPH oxidase transcripts before and after caterpillar feeding. Before herbivore feeding, low levels of transcripts encoding the maize insect resistance cysteine protease (Mir1-CP) and the Mir1-CP protein were detected consistently. Thus, Mp708 appears to have a portion of its defense pathway primed, which results in constitutive defenses and the ability to mount a stronger defense when caterpillars attack. Although the molecular mechanisms that regulate the constitutive accumulation of JA in Mp708 are unknown, it might account for its enhanced resistance to lepidopteran pests. This genotype could be valuable in studying the signaling pathways that maize uses to response to insect herbivores.

Fatty acid-amino acid conjugates diversification in lepidopteran caterpillars.

Fatty acid amino acid conjugates (FACs) have been found in noctuid as well as sphingid caterpillar oral secretions; in particular, volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] and its biochemical precursor, N-linolenoyl-L-glutamine, are known elicitors of induced volatile emissions in corn plants. These induced volatiles, in turn, attract natural enemies of the caterpillars. In a previous study, we showed that N-linolenoyl-L-glutamine in larval Spodoptera litura plays an important role in nitrogen assimilation which might be an explanation for caterpillars synthesizing FACs despite an increased risk of attracting natural enemies. However, the presence of FACs in lepidopteran species outside these families of agricultural interest is not well known. We conducted FAC screening of 29 lepidopteran species, and found them in 19 of these species. Thus, FACs are commonly synthesized through a broad range of lepidopteran caterpillars. Since all FAC-containing species had N-linolenoyl-L-glutamine and/or N-linoleoyl-L-glutamine in common, and the evolutionarily earliest species among them had only these two FACs, these glutamine conjugates might be the evolutionarily older FACs. Furthermore, some species had glutamic acid conjugates, and some had hydroxylated FACs. Comparing the diversity of FACs with lepidopteran phylogeny indicates that glutamic acid conjugates can be synthesized by relatively primitive species, while hydroxylation of fatty acids is limited mostly to larger and more developed macrolepidopteran species.