Defensive function of herbivore-induced plant volatile emissions in nature.

Herbivore attack is known to increase the emission of volatiles, which attract predators to herbivore-damaged plants in the laboratory and agricultural systems. We quantified volatile emissions from Nicotiana attenuata plants growing in natural populations during attack by three species of leaf-feeding herbivores and mimicked the release of five commonly emitted volatiles individually. Three compounds (cis-3-hexen-1-ol, linalool, and cis-alpha-bergamotene) increased egg predation rates by a generalist predator; linalool and the complete blend decreased lepidopteran oviposition rates. As a consequence, a plant could reduce the number of herbivores by more than 90% by releasing volatiles. These results confirm that indirect defenses can operate in nature.

Oak leaf quality declines in response to defoliation by gypsy moth larvae.

Leaves of red oak trees that had been defoliated by gypsy moth larvae during the previous year and again during the period of the study had higher values of tanning coefficients, total phenolics, hydrolyzable and condensed tannins, dry matter contents, and toughness than did leaves of undamaged trees. These changes may influence larval growth and alter the course of further outbreaks.

Rapid changes in tree leaf chemistry induced by damage: evidence for communication between plants.

Potted poplar ramets showed increased concentrations and rates of synthesis of phenolic compounds within 52 hours of having 7 percent of their leaf area removed by tearing, as did undamaged plants sharing the same enclosure. Damaged sugar maple seedlings responded in a manner similar to that of the damaged poplars. Nearby undamaged maples had increased levels of phenolics and hydrolyzable and condensed tannin within 36 hours, but exhibited no change in rates of synthesis. An airborne cue originating in damaged tissues may stimulate biochemical changes in neighboring plants that could influence the feeding and growth of phytophagous insects.

Phenotypic, genetic and genomic consequences of natural and synthetic polyploidization of Nicotiana attenuata and Nicotiana obtusifolia.

BACKGROUND AND METHODS: Polyploidy results in genetic turmoil, much of which is associated with new phenotypes that result in speciation. Five independent lines of synthetic allotetraploid N. x obtusiata (N x o) were created from crosses between the diploid N. attenuata (Na) (male) and N. obtusifolia (No) (female) and the autotetraploids of Na (NaT) and No (NoT) were synthesized. Their genetic, genomic and phenotypic changes were then compared with those of the parental diploid species (Na and No) as well as to the natural allotetraploids, N. quadrivalvis (Nq) and N. clevelandii (Nc), which formed 1 million years ago from crosses between ancient Na and No. KEY RESULTS: DNA fingerprinting profiles (by UP-PCR) revealed that the five N x o lines shared similar but not identical profiles. Both synthetic and natural polyploidy showed a dosage effect on genome size (as measured in seeds); however, only Nq was associated with a genome upsizing. Phenotypic analysis revealed that at the cellular level, N x o lines had phenotypes intermediate of the parental phenotypes. Both allo- and autotetraploidization had a dosage effect on seed and dry biomass (except for NaT), but not on stalk height at first flower. Nc showed paternal (Na) cellular phenotypes but inherited maternal (No) biomass and seed mass, whereas Nq showed maternal (No) cellular phenotypes but inherited paternal (Na) biomass and seed mass patterns. Principal component analysis grouped Nq with N x o lines, due to similar seed mass, stalk height and genome size. These traits separated Nc, No and Na from Nq and N x o lines, whereas biomass distinguished Na from N x o and Nq lines, and NaT clustered closer to Nq and N x o lines than to Na. CONCLUSIONS: Both allo- and autotetraploidy induce considerable morphological, genetic and genomic changes, many of which are retained by at least one of the natural polyploids. It is proposed that both natural and synthetic polyploids are well suited for studying the evolution of adaptive responses.

Merging molecular and ecological approaches in plant-insect interactions.

The singer-song-writer Paul Simon sang about the '50 ways to leave your lover'; plants have at least as many ways of coping with their insect herbivores. Recent research has elucidated the mechanisms of direct and indirect plant defenses, and has provided the first proof of a protective function for indirect defenses in nature. Insect attack elicits a large transcriptional reorganization that differs from that elicited by mechanical wounding. Elicitors in herbivore oral secretions can account for herbivore-specific responses. Patterns of transcriptional changes point to the existence of central herbivore-activated regulators of metabolism.

Functional interactions in the use of direct and indirect defences in native Nicotiana plants.

Nicotiana attenuata has both direct (induced nicotine production) and indirect (induced release of mono- and sesquiterpenes) defences induced by herbivore attack; both are activated by the jasmonate cascade, albeit in different tissues (roots and shoots, respectively). The fact that both types of defences are induced suggests that their benefits are conditional. Indeed, jasmonate treatment of roots to induce nicotine production increases plant fitness correlates (lifetime viable seed production) when plants are grown in environments with herbivores, but decreases fitness when they are not. Because inducing nicotine production can make 6% of a plant's nitrogen budget unavailable for seed production, it can exact a resource-based cost. Volatile production is likely to be less costly but could make plants more 'apparent' to herbivores and thereby exact an ecological cost. Direct defences could also have ecological costs if they are sequestered by specialist herbivores and used against their enemies. Herbivory by the nicotine-tolerant herbivore Manduca sexta dramatically amplifies the increase in jasmonates and the quantity of volatiles released, but decreases the nicotine response in comparison to mechanical simulations of the wounding that larval feeding causes. The apparent switching from nicotine production to the release of volatiles may reflect incompatibilities in the use of direct and indirect defences with specialist herbivores.

Herbivore-induced allene oxide synthase transcripts and jasmonic acid in Nicotiana attenuata.

Exogenous jasmonate treatment of Nicotiana attenuata Torr. ex Wats. plants elicits durable resistance against herbivores and attack from its specialist herbivore, Manduca sexta, results in an amplification of the transient wound-induced increase in endogenous jasmonic acid levels (JA). To understand whether this "JA burst" is under transcriptional control, we cloned allene oxide synthase (AOS; EC 4.2.1.92), the enzyme that catalyzes the dehydration of 13(S)-hydroperoxy octadecatrienoic acid to an allene oxide, the first specific reaction in JA biosynthesis. An AOS cDNA coding for a 520 aa protein (58.6 kDa) with an isoelectric point of 8.74 was overexpressed in bacteria and determined to be a functional AOS. Southern blot analysis indicated the presence of more than one gene and AOS transcripts were detected in all organs, with the highest levels in stems, stem leaves and flowers. Attack by M. sexta larvae resulted in a sustained JA burst producing an endogenous JA amount 9-fold above control levels and 3-fold above maximum wound-induced levels, a response which could be mimicked by the addition of Manduca oral secretion and regurgitant to puncture wounds. M. sexta attack, wounding and regurgitant treatment transiently increased AOS transcript in the wounded leaf, but increases were not proportional to the JA response. Moreover, transcript accumulation lagged behind JA accumulation. Systemic wound-induced increases in AOS transcript, AOS activity or JA accumulation could not be detected. We conclude that increase in AOS transcript does not contribute to the initial increase in endogenous JA, but may contribute to sustaining the JA burst.

Constraints on an induced defense: the role of leaf area.

Folivory results in both leaf damage and the loss of photosynthetic capacity. Leaf damage activates the production of induced defenses, but diminished photosynthetic capacity resulting from lost leaf area may impair a plant's ability to respond defensively. Because damage-induced nicotine production in Nicotiana sylvestris (Solanaceae) is an energy-demanding, active process, we predicted that the loss of leaf area would constrain this plant's ability to produce an induced nicotine pool. We examined our prediction in an experiment which combined leaf puncture and removal protocols, quantified induced nicotine pools on a whole-plant basis, and accounted for losses in the nicotine pool due to removed leaves and lost growth potential. In contrast to our prediction, leaf removal did little to diminish the growth-corrected estimates of the induced nicotine pool in plants with sufficient damage "cue"; only when plants had lost 88% or more of their leaf area did the induced nicotine pool decline significantly. These results demonstrate that the induced defense is relatively insensitive to current photosynthetic capacity. In contrast to the size of the induced nicotine pool, the concentration of nicotine in the remaining shoot tissues continued to rise as puncture damage increased over all defoliation levels tested. The mechanisms responsible for inducible nicotine production may have evolved as a means of providing shoot tissues with protection that is proportional to the amount of damage incurred while keeping production costs constant for the remaining plant parts.

Induced responses in Nicotiana attenuata affect behavior and growth of the specialist herbivore Manduca sexta.

Many plants employ induced responses against generalist herbivores. Specialist herbivores, however, may employ several mechanisms to overcome the negative effects of induced plant defenses. Here we test how the behavior and development of specialist Manduca sexta larvae are affected by induced responses in their natural host plant Nicotiana attenuata. On a spatial scale relevant to both the plant and the herbivore, we first determined how methyl jasmonate (MeJA)-induced responses, such as increased nicotine production, affect the tendency of larvae to leave induced plants. When larvae were allowed to move between two plants planted in one pot, they left an MeJA-treated plant faster than a control plant. When both plants in the pot were MeJA-treated, the larvae developed more slowly than when both plants were uninduced, or when the larvae had the opportunity to move to an uninduced neighbor. The sooner larvae moved from an MeJA-treated plant to an untreated neighbor, the larger the body mass they attained. This demonstrates that M. sexta larvae can compensate behaviorally for the deleterious effects of induced plant responses. These effects were observed in plants grown under both low and high N supply rates, though the effects were more pronounced under high N. To examine the consequences of the timing and the direction of the host plant switching behavior for larval development, neonate larvae were fed leaves excised from induced and uninduced plants. Larvae confined to MeJA-treated leaves had higher mortality rates and grew slower than larvae fed only control leaves. This demonstrates that MeJA-induced responses decrease growth and development of specialist herbivores that do not have the behavioral option of moving to an uninduced plant. The sooner the larvae were switched to MeJA-treated leaves, the slower their development compared to larvae fed only uninduced leaves. In contrast, the sooner larvae fed MeJA-treated leaves were switched to control leaves, the faster they developed. Again the effects of MeJA treatment were stronger in plants grown under high N supply. We propose that induced plants growing in close competition with an uninduced conspecific may offset the fitness costs of these induced responses and perhaps obtain a fitness benefit by motivating herbivores to move to their neighboring competitors.

Ecophysiological comparison of direct and indirect defenses in Nicotiana attenuata.

After herbivore attack, plants launch a suite of direct and indirect defense responses that must be coordinated if plants are to realize a fitness benefit from these responses. Here we characterize the volatile emissions in the native tobacco plant, Nicotiana attenuata Torr. ex Wats., that are elicited by tobacco hornworm (Manduca sexta L.) attack and are known to function as attractants for parasitoids. To provide the first ecophysiological comparison of examples of both types of defense in the same species, we characterize the elicitation and signaling mechanisms, the resources required, and the potential costs and benefits of the volatile release and compare these traits with those of the well-described induced direct defense in this species, nicotine production. The release of (E)-ß-ocimene, cis-α-bergamotene and linalool is dramatically induced within 24 h by application of methyl jasmonate (MeJA), caterpillar feeding, and the treatment of mechanical wounds with larval oral secretions (OS), but not by mechanical damage alone. Plants from different geographic locations produce volatile blends that differ in composition. The most consistently released component from all genotypes, cis-α-berga-motene, is positively related to the amount of MeJA and the level of wounding if OS are applied to the wounds. The volatile release is strongly light dependent, dropping to undetectable quantities during dark periods, even when temperatures are elevated to match those of the light period. Inhibitors of wound-induced jasmonate accumulation (salicylates and auxins), which are known to inhibit wound-induced nicotine production, do not inhibit the release of volatiles. By individually inducing different leaf positions with OS and, on other plants, excising them after induction, we demonstrate that the emission is largely a systemic, whole-plant response, which is maximally triggered when the second fully expanded leaf is induced. We conclude that while both are whole-plant, systemic responses that utilize recently acquired resources for their production and are activated by the jasmonate cascade, the elicitation of the volatile release exhibits greater tissue sensitivity and utilizes additional signaling components than does nicotine production. In contrast to the large investment of fitness-limiting resources required for induced nicotine production or the resources used in benzyl acetone release from flowers for pollinator attraction, the resource requirements for the volatile release are minor. Hence the argument that the volatile release incurs comparatively large physiological costs cannot be supported in this system.

Communication between plants: induced resistance in wild tobacco plants following clipping of neighboring sagebrush.

The possibility of communication between plants was proposed nearly 20 years ago, although previous demonstrations have suffered from methodological problems and have not been widely accepted. Here we report the first rigorous, experimental evidence demonstrating that undamaged plants respond to cues released by neighbors to induce higher levels of resistance against herbivores in nature. Sagebrush plants that were clipped in the field released a pulse of an epimer of methyl jasmonate that has been shown to be a volatile signal capable of inducing resistance in wild tobacco. Wild tobacco plants with clipped sagebrush neighbors had increased levels of the putative defensive oxidative enzyme, polyphenol oxidase, relative to control tobacco plants with unclipped sagebrush neighbors. Tobacco plants near clipped sagebrush experienced greatly reduced levels of leaf damage by grasshoppers and cutworms during three field seasons compared to unclipped controls. This result was not caused by an altered light regime experienced by tobacco near clipped neighbors. Barriers to soil contact between tobacco and sagebrush did not reduce the difference in leaf damage although barriers that blocked air contact negated the effect.

Rapid HPLC screening of jasmonate-induced increases in tobacco alkaloids, phenolics, and diterpene glycosides in Nicotiana attenuata.

A rapid, HPLC-based screening procedure for the main classes of secondary metabolites in Nicotiana attenuata leaves (alkaloids, phenolics, and diterpene glycosides) is reported. In a single step, leaves are extracted in aqueous acidified (0.5% acetic acid) methanol, and the extracted compounds are separated by reversed-phase HPLC with an acidic water/acetonitrile gradient in <30 min. The utility of the method in quantifying changes in the secondary metabolites after methyl jasmonate treatment of the plants, a treatment known to elicit resistance to herbivores in nature, is illustrated. Methyl jasmonate treatment elicited dramatic increases in some secondary metabolites (caffeoylputrescine, nicotine, and diterpene glycosides increased 12.5-, 1.4-, and 1.9-fold, respectively) but left others, such as rutin, unchanged. Such broad-based analytical screens will help characterize environmental and genetic changes in secondary metabolite profiles.

Mechanism of damage-induced alkaloid production in wild tobacco.

Greenhouse-grown tobacco plants of the speciesNicotiana sylvestris (Solanaceae) subjected to leaf damage show a fourfold increase in the alkaloid content of their undamaged leaves. This increase in nicotine and nornicotine concentrations begins 19 hr after the end of the damage regime, reaches a maximum at nine days, and wanes to control levels 14 days after the start of leaf damage. The increase in leaf alkaloid content in damaged plants is largely due to a 10-fold increase in the alkaloid concentration of the xylem fluid entering leaves, which, in turn, suggests that increased synthesis of alkaloids is occurring in the roots. This research distinguishes between positive and negative cues affecting the change in xylem fluid alkaloid concentrations. A negative cue, such as auxin, when lost or diminished as a result of leaf damage could signal the alkaloidal response. Indeed, exogenous applications of auxin to damaged leaves inhibit the alkaloidal response. However, attempts to block endogenous auxin transport by steam girdling or applying an auxin transport inhibitor fail to mimic the effect of leaf damage on leaf alkaloid concentrations. The damage cue appears to be a positive cue that is related to the timing and the amount of leaf damage rather than to the amount of leaf mass lost. Moreover, when performed proximally to leaf damage, steam girdling truncates the alkaloidal response. This induced alkaloidal response appears to be triggered by a phloem-borne cue that allows the plant to distinguish between different types of leaf damage. The physiological and ecological consequences of the mechanism of this damage-induced alkaloidal response are further explored.

Damage-induced alkaloids in tobacco: Pot-bound plants are not inducible.

Field-grown wild tobacco plants (Nicotiana sylvestris) were subjected to a defoliation regime designed to mimic the rate and amount of leaf mass removed by one tobacco hornworm per plant. Undamaged leaves on these plants undergo a dramatic (457% for leaf position 5, 410% for leaf position 8) increase in total leaf alkaloids compared to same-age and positioned control leaves on undamaged control plants. However, potted greenhouse-grown plants fail to exhibit the same damage-induced increase in alkaloid content. The greenhouse environment differs from the field environment in factors known to influence leaf alkaloid content, particularly soil N, P, K, near-UV radiation, and relative humidity. However, altering these environmental factors does not make potted plants able to increase their leaf alkaloid levels in response to defoliation. Transplanting plants into larger pots with more soil does allow the plants to respond to defoliation. Thirty days after transplanting, the plants are again unresponsive to damage, probably as a result of becoming "pot-bound." This result suggests a mechanism for the induction response, specifically that leaf damage triggers synthesis of these alkaloids in the roots, and offers a potentially valuable experimental tool for the study of induced-plant defenses in tobacco and other plants that synthesize alkaloids in their root tissues.

Jasmonate-induced responses are costly but benefit plants under attack in native populations.

Herbivore attack is widely known to reduce food quality and to increase chemical defenses and other traits responsible for herbivore resistance. Inducible defenses are commonly thought to allow plants to forgo the costs of defense when not needed; however, neither their defensive function (increasing a plant's fitness) nor their cost-savings function have been demonstrated in nature. The root-produced toxin nicotine increases after herbivore attack in the native, postfire annual Nicotiana attenuata and is internally activated by the wound hormone, jasmonic acid. I treated the roots of plants with the methyl ester of this hormone (MeJA) to elicit a response in one member of each of 745 matched pairs of plants growing in native populations with different probabilities of attack from herbivores, and measured the lifetime production of viable seed. In populations with intermediate rates of attack, induced plants were attacked less often by herbivores and survived to produce more seed than did their uninduced counterparts. Previous induction did not significantly increase the fitness of plants suffering high rates of attack. However, if plants had not been attacked, induced plants produced less seed than did their uninduced counterparts. Jasmonate-induced responses function as defenses but are costly, and inducibility allows this species to forgo these costs when the defenses are unnecessary.

Herbivory simulations in ecological research.

Because of the experimental advantages that they offer, mechanical simulations of grazing are more commonly used than true herbivory in ecological studies of the impact of herbivory on plants. However, few studies have explicitly compared plant responses to herbivory and to mechanical simulations. Most such comparisons report differences in plant responses to mechanical versus true herbivory, even though the amounts and types of tissue removed were similar. Moreover, studies that also attempted to mimic the timing of leaf damage report differences in plant responses to the different damage modes. Because a plant's response to herbivory is complex and is activated by more than merely the removal of tissue, exact mechanical simulations may prove difficult.

Up in smoke: II. Germination ofNicotiana attenuata in response to smoke-derived cues and nutrients in burned and unburned soils.

Nicotiana attenuata is a native tobacco that is commonly found usually one growing season after fires in the blackbrush, sagebrush and pinyon-juniper forests of the Great Basin desert of North America. This plant also occurs in isolated dry washes and roadsides for many consecutive seasons. Postfire annuals are thought to synchronize their germination from the seed bank with the postfire environment in response to increases in (1) fire-related cues or (2) nutrient supply rates resulting from the mineralization of nutrients by fire, or (3) the removal of allelochemicals produced by the dominant vegetation occupying the sites before the burn or the microbial community associated with the dominant vegetation. We examine the effect of these three changes on the germination ofN. attenuata seed from artificial seed banks made with burned and unburned soil taken in 1993 from under four dominant shrub species (Coleogyne ramosissima, Yucca baccata, Lycium andersonnii, Purshia tridentata) of an area that burned in 1992 and from two dry washes in whichN. attenuata populations have persisted since at least 1988. We utilize our recent discovery that aqueous extracts of wood smoke contain potent germination cue(s) for this species and the established observation that nitrate stimulates germination in manyNicotiana species. In two experiments, we added smoke-derived germination cues and nutrients separately and in combinations to the artificial seed banks, measured germination rates, and inferred the effect of burning by the response of the seed banks to these additions. Germination rates of seed in burned soil were consistently higher than those in unburned soil collected from under all species tested; concentrations of nitrate, P, Mn, and Ca were also higher in burned than unburned soils. Because the addition of more cue and nitrate to burned soil increased germination rates, these soil components may not be at concentrations sufficient to saturate the germination response one year after a fire. The germination of seeds in soil collected from beneath unburnedYucca bacatta plants increased to the same level as that found under burned plants of the same species with the addition of cue and nitrate. Similarly, unburned bitterbrush soil attained the same germination potential with the addition of cue and a complete nutrient solution as burned bitterbrush soil. We conclude that the effect of fire on the germination potential of bitterbrush and yucca soil is due to increases in germination cue and nutrients. However, since the addition of cue and nutrients to the unburned soils under blackbrush and wolfberry shrubs did not elevate the germination potential of these soils to that found in burned soils, we conclude that these species alter the soil so as to inhibit germination and burning reverses this alteration. The presence of persistentN. attenuata populations in washes could not be attributed to particular chemical characteristics of these soils. Additions of cue dramatically increased germination potential of these soils, whereas the addition of nitrate did not. The concentrations of most mineral nutrients resembled those found in the unburned sites with the exception of Mn, Cu, and Zn, which were higher. However, these cations do not influence germination rates. Treatment of soil taken from these washes with cue resulted in significant increases in germination ofN. attenuata seeds in the natural seed bank compared to water-treated controls, demonstrating that the plants growing in washes also produce dormant seeds that require the smoke cue for germination.

Plasticity in allocation of nicotine to reproductive parts inNicotiana attenuata.

Although little is known about the patterns of chemical defense allocation in reproductive tissues, optimal defense theory predicts a high constitutive allocation due to the tissues' high fitness value. To examine this prediction, we quantified the short- and long-term changes in the nicotine pools of reproductive tissues in response to both floral and leaf damage. Recently opened flowers (stage 5 capsules) do not alter their nicotine pools within a day in response to herbivory byManduca sexta larvae or mechanical damage to the corolla. Similarly, leaf damage during both vegetative and reproductive growth does not influence the nicotine pools of the first three stage-5 capsules produced. However, the nicotine pools of capsules produced later in reproductive growth were significantly larger (1.2- to 1.9-fold) on plants with leaf damage. These differences in floral nicotine pools were a result of both increases in nicotine pools of capsules on damaged plants and decreases in the nicotine pools of capsules on undamaged plants during reproductive growth. Leaf damage did not affect the rate of capsule maturation or the mass of stage-5 capsules at any time during reproductive growth. An allometric analysis of nicotine pools and biomass of reproductive parts in all stages of development from damaged and undamaged plants demonstrates that damaged plants allocated a significantly larger quantity of nicotine to reproductive parts in all stages of development than did undamaged plants. Given that nicotine is thought to be synthesized in the roots and transported to leaves and reproductive parts, nicotine could be allocated to reproductive parts in proportion to the number of developing capsules on a plant. We excised the first 27 stage-5 capsules on plants with and without leaf damage, with the expectation that plants with fewer capsules would allocate a larger amount of nicotine to the remaining capsules. In contrast to the prediction of this passive allocation model, floral excision did not affect nicotine pools on plants with or without leaf damage. These results demonstrate that the allocation of nicotine to reproductive parts is more strongly influenced by damage to vegetative rather than reproductive tissues. Reproductive parts are constitutively defended over the short term, but the set points for defense allocation are apparently increased by damage to vegetative tissues during reproductive growth. The decrease in allocation of nicotine to reproductive parts in undamaged plants during reproductive growth suggests an optimization of resource allocation as plants realize their potential fitness.

Alkaloidal responses to damage inNicotiana native to North America.

We performed field tests of alkaloid induction inNicotiana attenuata plants growing in southwestern Utah with mimicry of the two major types of damage inflicted by invertebrate and vertebrate herbivores: leaf damage and stalk removal, respectively. In undamaged plants, seasonal increases in leaf nicotine content occurred at a rate of 0.046% leaf dry mass/day. Leaf damage doubled the accumulation rate to 0.086-0.138% leaf dry mass/day, while stalk removal resulted in a quadrupling of the accumulation rate to 0.206% leaf dry mass/day. These damage-induced increases in nicotine accumulation are significantly larger than between-plant and phenological variations. Leaf damage to the nornicotine-(N. repanda andN. trigonophylla) and anabasine-accumulating (N. glauca)Nicotiana species native to North America resulted in 1.5- to 5-fold increases in their principal leaf alkaloid pools. We conclude that alkaloid induction is not limited to nicotine-accumulatingNicotiana species and that herbivores feeding on previously damaged plants are likely to encounter tissues with alkaloid titers significantly higher than those of undamaged plants.

Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. IV. Insect-Induced ethylene reduces jasmonate-induced nicotine accumulation by regulating putrescine N-methyltransferase transcripts.

Attack by the specialist herbivore, Manduca sexta, on its native host Nicotiana attenuata Torr. ex Wats. produces a dramatic ethylene release, a jasmonate burst, and a suppression of the nicotine accumulation that results from careful simulations of the herbivore's damage. Methyl-jasmonate (MeJA) treatment induces nicotine biosynthesis. However, this induction can be suppressed by ethylene as pretreatment of plants with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene receptors, restores the full MeJA-induced nicotine response in herbivore attacked plants (J. Kahl, D.H. Siemens, R.J. Aerts, R. Gäbler, F. Kühnemann, C.A. Preston, I.T. Baldwin [2000] Planta 210: 336-342). To understand whether this herbivore-induced signal cross-talk occurs at the level of transcript accumulation, we cloned the putrescine methyltransferase genes (NaPMT1 and NaPMT2) of N. attenuata, which are thought to represent the rate limiting step in nicotine biosynthesis, and measured transcript accumulations by northern analysis after various jasmonate, 1-MCP, ethephon, and herbivory treatments. Transcripts of both root putrescine N-methyltransferase (PMT) genes and nicotine accumulation increased dramatically within 10 h of shoot MeJA treatment and immediately after root treatments. Root ethephon treatments suppressed this response, which could be reversed by 1-MCP pretreatment. Moreover, 1-MCP pretreatment dramatically amplified the transcript accumulation resulting from both wounding and M. sexta herbivory. We conclude that attack from this nicotine-tolerant specialist insect causes N. attenuata to produce ethylene, which directly suppresses the nitrogen-intensive biosynthesis of nicotine.