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.     

Structural Requirements of Jasmonates and Mimics for Nicotine Induction inNicotiana sylvestris

Jasmonic acid (JA) is strongly implicated in the long-distance signal transduction cascade increasing nicotine synthesis in the roots of plants after leaf wounding. In order to explore the structural requirements of the inducing signal, we examined jasmonates, mimics, and a biosynthetic precursor for nicotine-inducing activity (NIA). We examine the importance of the keto group on the five-membered ring and the double bond in then-pentenyl chain by comparing the NIA of methyl jasmonate (MJ) with that of cucurbic acid, 1,3-dithiolane-MJ, 1,3-dioxolane-MJ, methyl dihydrojasmonate (DHMJ), 1,3-dioxolane-DHMJ, 1-oxo-indan-4-carboxylic acid ILE-methyl ester, and 1-hydroxyl-indan-4-carboxylic acid ILE-methyl ester. We found that: 1,3-dioxolane MJ, cucurbic acid, and 1,3-dioxolane DHMJ were less active than MJ and that the isoleucine (ILE) conjugates of 1-oxo- and l-hydroxyindanon-4-carboxylic acid had the same NIA as MJ. The activities of these indanon amino acid conjugates may be due to the structural similarity of their keto or hydroxyl groups on the five-membered ring to MJ or to the keto-enolized MJ. These results support the hypothesis that the enolization of the keto group during or prior to its interaction with the putative JA receptor is required for activity. We explore the importance of the esterification of the carboxyl functional group by comparing the NIAs of cucurbic acid and cucurbic acid methyl ester, l-oxo-indan-4-carboxylic acid, 1-oxo-indan-4-carboxylic acid methyl ester, and l-oxo-indan-4-carboxylic acid ILE-methyl ester. In all cases, the esters were more active than the free acids. We compared the NIA of MJ of different epimeric composition (8% and 20% 3R,7S-MJ); 12-oxophytodienoic acid (12-oxo-PDA) methyl ester, an important precursor of JA; and coronatine (a well-known phytotoxin and putative structural mimic of 12-oxo-PDA).We found that: (1) the epimeric composition of MJ did not affect its NIA; (2) 12-oxo-PDA methyl ester had lower NIA than MJ; and (3) coronatine significantly inhibited plant growth but did not increase nicotine biosynthesis. In summary, JA, rather than its biosynthetic precursor, 12-oxo-PDA, is likely the endogenous signal inNicotiana sylvestris, and the keto functional group on the five-membered ring and the double bond in then-pentenyl side chain are crucial components of JA for NIA.     

Damage-induced root nitrogen metabolism inNicotiana sylvestris: Testing C/N predictions for alkaloid production

Nitrogen surplus models for nicotine production induced by leaf damage predict that the observed increase in root nicotine synthesis after leaf damage results from overflow metabolism; reduced nitrogen existing in excess of growth requirements is shunted into nicotine biosynthesis. To test the nitrogen surplus model for induced nicotine production, we measured the concentrations of the majorN-containing metabolites exported from the roots and the nitrate reductase activity (NRA) of roots and shoots in damaged and undamagedNicotiana sylvestris plants. Leaf damage: (1) had no significant effect on root or shoot NRA, (2) increased nicotine export and decreased amino-acid and amide export from the roots of NO₃-fertilized plants, and (3) had no significant effect on the export of anyN-containing metabolite from the roots of NH₄-fertilized plants. These results are not consistent with the nitrogen surplus model and indicate that leaf damage has a direct influence on root alkaloid metabolism.     

Methyl jasmonate as an allelopathic agent: sagebrush inhibits germination of a neighboring tobacco,Nicotiana attenuata

Artemisia tridentata ssp. tridentata is the dominant and defining shrub in the Great Basin Desert, with well-documented allelopathic tendencies that have generally been ascribed to its most abundantly released secondary metabolites. However, as a minor component, sagebrush releases a highly biologically active substance, methyl jasmonate (MeJA), which is known to function as both a germination inhibitor and promoter in laboratory studies. Nicotiana attenuata is a tobacco species native to the Great Basin Desert and grows in newly burned juniper–sagebrush habitats for 2–3 yr following a fire. With a combination of field and laboratory studies, we examined the role of MeJA release from sagebrush by both air and water transport in inhibiting N. attenuata seed germination. We demonstrated that sagebrush interacts allelopathically with the seed bank of N. attenuata through its release of MeJA. In the field, seeds buried 0–40 cm from sagebrush plants for 4 months in net bags had significantly reduced germination compared to seeds buried similarly but protected in plastic bags. Moreover, germination on soils collected from underneath sagebrush plants was reduced by 60% compared to seeds placed on soils collected between sagebrush plants or outside of the sagebrush population. Exposure to A. tridentata seeds and seedlings did not affect N. attenuata germination, suggesting that established sagebrush plants only influence the tobacco's seed bank. In the laboratory, exposure of seeds to sagebrush emissions resulted in germination delays of up to 6 d. Exposure to volatile and aqueous MeJA also inhibited germination of N. attenuata seeds at quantities that are released naturally by sagebrush: 3.5 g/hr and 1.12 g/seed cup (56 ng/seed), respectively. A. tridentata seeds were significantly more resistant to MeJA, being inhibited at 336 g MeJA (16.8 g/seed), 300 times greater than the level of aqueous MeJA required to inhibit N. attenuata seeds. MeJA inhibited N. attenuata germination regardless of the seed's dormancy state and the specific epimer (trans- or cis-) of MeJA. Germination on sagebrush chaff that had been heated to reduce MeJA content was negatively correlated with the amount of MeJA remaining in the chaff. Germination of a nondormant, conspecific tobacco, N. trigonophylla, which grows in the same area but is not associated with fire, is less sensitive than N. attenuata to the extracts of sagebrush litter, but similarly sensitive to MeJA. Additionally, four of five other tobacco species that are not known to be associated with sagebrush are less sensitive to MeJA, suggesting an evolved sensitivity to MeJA. To determine the proportion of germination inhibition of a sagebrush extract that could be attributed to MeJA, we serially diluted sagebrush extracts with water and restored the quantity of MeJA of the original extract by adding appropriate quantities of synthetic MeJA; 16–60% of the inhibitory activity of the original extract could be attributed to the MeJA. We conclude that MeJA release from sagebrush plays an allelopathic role for N. attenuata seed banks, but other unidentified compounds are also involved.     

Effects of octadecanoid metabolites and inhibitors on induced nicotine accumulation inNicotiana sylvestris

We examined the effects of inhibitors of the octadecanoid pathway (n-propyl gallate, acetosalicylic acid, salicylhydroxamic acid, methyl salicylate, and antipyrine) on wound- and jasmonate-induced nicotine accumulation and compared the nicotine-inducing ability of exogeneous additions of linolenic acid (18:3) and its methyl ester, linoleic acid (18:2), abscisic acid, traumatic acid, and methyl dihydrojasmonate to the nicotine-inducing ability of exogenous additions of methyl jasmonate (MJ). The first four of these inhibitors significantly reduced wound-induced nicotine accumulation when applied in a lanolin paste to wounded tissues immediately after wounding at concentrations of 89–90µg/plant. When methyl salicylate and propyl gallate were mixed individually with MJ, neither inhibited MJ-induced nicotine synthesis, which suggests that the inhibitors block jasmonate synthesis or release from stored pools and not its effects. Linolenic acid or its methyl ester applied to undamaged plants or damaged plants (to either damaged or undamaged leaves) or to the roots of hydroponically growing plants did not induce nicotine accumulation or increase nicotine accumulation above levels found in damaged plants. Similarly, traumatic acid, linoleic acid, and abscisic acid did not induce nicotine accumulations. Methyl dihydrojasmonate, which is biosynthetically derived from linoleic acid, had 12–56% of the nicotine-inducing acitivity of MJ when added to the roots of hydroponically grown plants. The signal transduction pathway mediating wound-induced nicotine production therefore shares many features of the pathway eliciting wound-induced proteinase inhibitor production but differs in not being regulated at the lipase step in jasmonic acid production and not being responsive to abscisic acid.     

Wound-induced changes in root and shoot jasmonic acid pools correlate with induced nicotine synthesis inNicotiana sylvestris spegazzini and comes

Leaf damage by herbivores inNicotiana sylvestris Spegazzini and Comes (Solanaceae) produces a damage signal that dramatically increasesde novo nicotine synthesis in the roots. The increased synthesis leads to increases in whole-plant nicotine pools, which in turn make plants more resistant to further herbivore attack. Because signal production and the response to the signal occur in widely separated tissues, the speed with which different damage signals exit a damaged leaf can be studied. We propose that electrical damage signals should exit a leaf faster (less than 60 min) than chemical damage signals. Excision of a leaf induces a smaller increase in nicotine production than does puncture damage, so we examined our proposition by excising previously punctured leaves at 1, 60, and 960 min after leaf puncture and quantifying the induced whole-plant nicotine pools six days later when the induced nicotine production had reached a maximum. Significant induced nicotine production occurred only if punctured leaves were excised more than 1 hr after puncture, which is consistent with the characteristics of a slow-moving chemical signal rather than a fast-moving electrical signal. We explore the nature of the chemical signal and demonstrate that additions of 90µg or more of methyl jasmonate (MJ) in an aqueous solution to the roots of hydroponically grown plants inducede novo nicotine synthesis from¹⁵NO₃ in a manner similar to that induced by leaf damage. We examine the hypothesis that jasmonic acid (JA) functions in the transfer of the damage signal from shoot to root. Using GC-MS techniques to quantify whole-plant JA pools, we demonstrate that leaf damage rapidly (<0.5 hr) increases shoot JA pools and, more slowly (<2 hr), root JA pools. JA levels subsequently decay to levels found in undamaged plants within 24 hr and 10 hr for shoots and roots, respectively. The addition of sufficient quantities (186µg) of MJ in a lanolin paste to leaves from hydroponically grown plants significantly increased endogenous root JA pools and increasedde novo nicotine synthesis in these plants. However, the addition of 93µg or less of MJ did not significantly increase endogenous root JA pools and did not significantly affectde novo nicotine synthesis. We propose that wounding increases shoot JA pools, which either directly through transport or indirectly through a systemin-like signal increase root JA pools, which, in turn, stimulate root nicotine synthesis and increase whole-plant nicotine pools.     

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.     

Up in smoke: I. Smoke-derived germination cues for postfire annual,Nicotiana attenuata torr. Ex. Watson

Some postfire annuals with dormant seeds use heat or chemical cues from charred wood to synchronize their germination with the postfire environment. We report that wood smoke and polar extracts of wood smoke, but not the ash of burned wood, contain potent cue(s) that stimulate germination in the postfire annual plant,Nicotiana attenuata. We examined the responses of seeds from six populations of plants from southwest Utah to extracts of smoke and found the proportion of viable seeds that germinated in the presence of smoke cues to vary between populations but to be consistent between generations. With the most dormant genotypes, we examine three mechanisms by which smoke-derived chemical cues may stimulate germination (chemical scarification of the seed coat and nutritive- and signal-mediated stimulation of germination) and report that the response is consistent with the signal-mediated mechanism. The germination cue(s) found in smoke are produced by the burning of hay, hardwood branches, leaves, and, to a lesser degree, cellulose. Moreover, the cues are found in the common food condiment, liquid smoke, and we find no significant differences between brands. With a bioassay-driven fractionation of liquid smoke, we identified 71 compounds in active fractions by GC-MS and AA spectrometry. However, when these compounds were tested in pure form or in combinations that mimicked the composition of active fractions over a range of concentrations, they failed to stimulate germination to the same degree that smoke fractions did. Moreover, enzymatic oxidation of some of these compounds also failed to stimulate germination. In addition, we tested 43 additional compounds also reported from smoke, 85 compounds that were structurally similar to those reported from smoke and 34 compounds reported to influence germination in other species. Of the 233 compounds tested, 16 proved to inhibit germination at the concentrations tested, and none reproduced the activity of wood smoke. By thermally desorbing smoke produced by cellulose combustions that was trapped on Chromosorb 101, we demonstrate that the cue is desorbed between 125 and 150°C. We estimate that the germination cues are active at concentrations of less than 1 pg/seed and, due to their chromatographic behavior, infer that a number of different chemical structures are active. In separate experiments, we demonstrate that cues remain active for at least 53 days in soil under greenhouse conditions and that the application of aqucous extracts of smoke to soil containing seeds results in dramatic increases in germination of artificial seed banks. Hence, although the chemical nature of the germination cue remains elusive, the stability of the germination cues, their water-solubility, and their activity in low concentrations suggest that these cues could serve as powerful tools for the examination of dormant seed banks and the selective factors thought to be important in the evolution of postfire plant communities.     

Plant–Plant Signaling: Application of trans- or cis-Methyl Jasmonate Equivalent to Sagebrush Releases Does Not Elicit Direct Defenses in Native Tobacco

Nicotiana attenuata plants growing in close proximity to damaged sagebrush (Artemisia tridentata ssp. tridentata) suffer less herbivory than plants near undamaged sagebrush. Sagebrush constitutively releases methyl jasmonate (MeJA), a compound that when applied directly to N. attenuata, elicits herbivore resistance and the direct defense traits [protease inhibitors (PIs), nicotine]. Damage increases the release of volatile MeJA, primarily in the cis epimer, suggesting that cis-MeJA may mediate this apparent interplant signaling. We characterized sagebrush's MeJA plume before and after damage in nature and in the laboratory, and compared the activity of trans- and cis-MeJA in inducing PIs, nicotine, and Manduca sexta resistance in N. attenuata. We used both lanolin applications and aqueous sprays that mimic natural exposures, and we determined the amount of volatilized MeJA required to elicit a nicotine response in open-grown plants. Wounding rapidly and transiently increased cis-MeJA emissions from damaged parts (but not systemically), and the released plume did not rapidly dissipate in nature. cis-MeJA was not consistently more active than trans-MeJA, and the order of exposure (trans- then cis-) did not influence activity. We conclude that volatile MeJA, either trans- or cis-, when applied at levels consistent with those released by sagebrush does not elicit direct defenses in N. attenuata.     

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.     

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.     

Constraints to herbivore-induced systemic responses: bidirectional signaling along orthostichies in Nicotiana attenuata

We investigated the impact of leaf vascular connections on systemically transmitted herbivore-induced gene expression in Nicotiana attenuata. Although systemic signaling is clearly associated with the plant vascular system, few studies consider vascular architecture when measuring systemically induced defenses. N. attenuata is a plant with dispersed phyllotaxis approximating 3/8 in the rosette stage of growth. We mimicked Manduca sexta herbivory by introducing larval regurgitant to wounds produced with a standardized continuous mechanical wounding and investigated mRNA accumulation of genes. Herbivory in N. attenuata induces the expression of genes coding for a proteinase inhibitor protein (PI), threonine deaminase (TD, EC 4.3.1.19), a luminal-binding protein (BiP), and an -dioxygenase (-DOX). We measured the systemic response of sink leaves when orthostichous (growing at an angular distance of 0 degrees) source leaves were treated, and vice versa, and compared it to the systemic response of leaves growing at the maximum angular distance of 180 degrees. Vascular architecture clearly controlled the intensity of systemic mRNA accumulation within the 4-hr time frame of the experiment. In addition, we found signal translocation to be bidirectional, travelling from source to sink as well as from sink to source leaves, which argues against a phloem-based assimilate-linked signal identity.     

Jasmonate-Induced Responses of Nicotiana sylvestris Results in Fitness Costs Due to Impaired Competitive Ability for Nitrogen

We present the first evidence for a fitness cost of an inducible response that is detectable in a nitrogen (N) currency. Nicotine is an induced defense in Nicotiana sylvestris that can utilize 5–8% of the plant's total N, an investment that apparently cannot be recouped by metabolism. Induced nicotine production is endogenously regulated by jasmonic acid (JA), and we treated leaves with the methyl ester of this wound hormone (MeJA) in quantities (0, 25, 250 g) known to elicit changes in endogenous JA and subsequent nicotine responses comparable to those elicited by mechanical wounding and herbivory in this species. We grew plants in competition chambers (CCs) in which three same-sized plants could compete for a communal but fixed pool of ¹⁵NO₃ to quantify the outcome of competition for this fitness-limiting resource that is used both in defense and seed production. Competition profoundly increased all measures of growth and reproductive performance measured per milligram of N acquired. While plants acquired all the N supplied to them in the hydroponic solution, plants grown in CCs (as compared those grown in individual chambers—ICs) retained more of this N and produced more biomass, had larger nicotine contents, allocated less of their N to nicotine, produced larger floral stalks with more flowers, aborted fewer flowers, matured more capsules, and produced a greater mass of seed. Plants grown in ICs produced heavier seed, but this difference did not translate into a difference in seed viability.MeJA treatment increased nicotine concentrations in proportion to the amount applied and significantly reduced growth (13–23%) and reproductive (31–44%) performance for plants grown with uninduced competitors, reflecting a large opportunity cost of induction. The effects of MeJA treatment on growth and reproduction were significantly less pronounced for plants grown in ICs. MeJA treatment significantly reduced the ability of plants to compete for [¹⁵N]KNO₃ (reducing uptake by 9.5% and 23.7% for 25- and 250-g MeJA-treated plants, respectively); no reductions in N acquisition were found in IC grown plants treated with MeJA. This impairment of competitive ability could account for 41–47% of the jasmonate-induced reductions in biomass by the day 15 harvest and 12–20% of the reductions in seed set and, in addition, created by "opportunity benefit" for neighboring uninduced plants, which grew larger, aborted fewer flowers, and matured more seed (a 28% increase) than did uninduced plants competing with similarly uninduced plants.Competition dramatically increased plant growth and reproductive performance, and MeJA treatment of these high-performing plants significantly reduced their competitive ability, which translated into opportunity costs for induced plants and opportunity benefits for neighboring uninduced plants. Induced plants minimized these fitness costs by reducing their use of recently acquired N for nicotine biosynthesis when growing with competitors. MeJA treatments also altered stalk length, flower production, flower abortion, and allocation to seed mass. In spite of all this plasticity, induced responses incur large fitness costs, costs that could be in part attributed to reductions in competitive ability for N. We conclude that inducibility functions to minimize these costs.     

Calcium chloride as a major component contributing to aphid resistance ofNicotiana benthamiana

Substances with antiaphid activity were extracted from the leaf surface of aphid-resistantN. benthamiana and purified. Sugar esters and diacyl glycerols were isolated from the chloroform extract, but only diacyl glycerols showed significant activity. However, the major activity was found in the water extract rather than in the chloroform extract. From the water extract calcium chloride was isolated as the most abundant active substance. Only calcium chloride showed significant activity among several calcium salts and chlorides of several metals that are abundant in plants. Calcium contents per unit area of leaf surface ofN. benthamiana and aphid-resistantN. gossei were almost equal to each other and 10–100 times higher than that of aphid-susceptibleN. tabacum.     

Differential activity and degradation of plant volatile elicitors in regurgitant of tobacco hornworm (Manduca sexta) larvae

Plants respond to insect herbivory by emitting volatile compounds that attract natural enemies of the herbivores. Biosynthesis of many of these volatiles in plants is induced by herbivore-produced compounds. Components of tobacco hornworm (THW) regurgitant were investigated for their efficacy as elicitors of corn seedling volatiles. Two components that elicited the strongest release of volatiles were isolated and identified as N-linolenoyl-L-glutamine (18:3-GLN) and N-linolenoyl-L-glutamic acid (18:3-GLU). The approximately 10 times more active 18:3-GLN, which also is found in the regurgitant of several other Lepidopteran larvae, was rapidly degraded when THW regurgitant was left at room temperature, while 18:3-GLU degraded at a much slower rate. Different dietary sources of THW and tobacco bud worm larvae, including both host and nonhost plants, did not affect the amino acid composition of the fatty acid–amino acid conjugates in the regurgitant.     

Mite predator responses to prey and predator-emitted stimuli

We found that the searching behavior of two acarine predators,Amblyseius fallacis andPhytoseiulus macropilis, for prey,Tetranychus urticae, is affected by the following stimuli: (1) prey silk and associated feces, whose combined physical and chemical properties elicit reduction in the rate of predator movements and longer halts; (2) kairomone extracted from prey silk and associated feces, which, upon contact, elicits frequent predator return to prey-inhabited locales; and (3) predator-emitted marking pheromone, which elicits shorter duration of search in presearched prey locales. We also found that treatment of filter paper with prey kairomone or silk enhanced predator location of prey eggs, leading us to speculate that application of synthetic prey kairomone could be useful in pest management programs.     

Behavioral and electrophysiological responses of natural enemies to synomones from tea shoots and kairomones from tea aphids,Toxoptera aurantii

Olfactometer bioassays and electrophysiological studies showed that the lacewing, Chrysopa sinica, the aphid parasitoid, Aphidius sp., and the coccinellid, Coccinella septempunctata, all responded to volatiles from tea aphids, Toxoptera aurantii, to hexane or ether rinses of tea aphid cuticles, and to synomones released by aphid-damaged tea shoots, as well as to the tea shoot–aphid complex. Each natural enemy spent more time searching on a filter paper treated with tea aphid honeydew than on a blank control filter paper. The interaction between synomones from aphid-damaged shoots and kairomones from tea aphids enhanced the responses to the plant–host complex. There was a significant, logistic dose–response relationship between the number of natural enemies responding and the odor stimulus concentration. Volatile components from the plant–host complex, obtained by air entrainment, were identified by their mass spectra and retention times and confirmed by comparison with standard samples. These were (Z)-3-hexen-1-ol, benzaldehyde, (E)-2-hexenal, (Z)-3-hexenyl acetate, ocimene, linalool, geraniol, indole, and (E)-2-hexenoic acid. The main components in a hexane rinse from tea aphid cuticle were benzaldehyde, undecane, 2,5-hexanedione, 2,5-dihydrothiophene, linalool, 4-methyl-octane, and eicosane, whereas the main components from an ether rinse were (E)-2-hexenoic acid, heptadecane, pentadecane, eicosane, tetratetracontane, and nonadecane. Benzaldehyde elicited the strongest responses from natural enemies in the olfactometer and the largest electroantennogram (EAG) responses. While the amount of odor was small, Coccinella septempunctata was slightly more sensitive than Chrysopa sinica and Aphidius sp. An increase in doses of benzaldehyde, (E)-2-hexenal, and (Z)-3-hexenyl acetate caused the EAG responses of each natural enemy to decrease. When the doses of (Z)-3-hexen-1-ol, linalool, and geranoil increased, EAGs of Chrysopa sinica and Aphidius sp. increased, but EAGs of Coccinella septempunctata decreased. When the dose of indole increased, EAGs of Coccinella septempunctata decreased, but those of Aphidius sp. increased. This study demonstrates that tea shoot–aphid complexes emit volatile synomones, while the odors from tea aphids, aphid cuticle extracts, and tea aphid honeydew contain kairomones, to which the natural enemies show a logistic dose–response.     

Behavioral responses ofLittoraria irrorata (SAY) to water-borne odors

Behavioral responses of the gastropod molluscLittoraria (=Littorina)irrorata indicate that it can discriminate among environmental odors. Snails were assayed for responses to 11 odors from plants and animals potentially representing food, shelter, location in the environment, and predators. Crushed conspecifics were included as an alarm odor. Except for odor of crushed conspecifics, all odor sources were water-borne from living intact organisms. Behavioral responses were categorized as no response, positive response, or negative response. For some analyses, negative responses were subdivided into withdrawing and turning responses. Snails responded positively to several plant odors. They did not respond to odors of intact conspecifics, fiddler crabs, or grass shrimp. They responded negatively to odors of a plant found at the upper limit of their minimal habitat, predatory blue crabs, crushed conspecifics, predatory gastropods, and ribbed mussels. Odors of blue crabs on different diets affect the type of negative response the snails display.     

Phenolic biosynthesis,leaf damage,and insect herbivory in birch (Betula pendula)

The effect of both caterpillar herbivory and artificial damage on phenylalanine ammonia lysase (PAL) activity of birch foliage was measured, using an intact cell assay. After artificial damage there was a small increase in PAL activity in damaged leaves but no change in adjacent undamaged ones. Insect grazing produced a larger increase in PAL activity, and the enzyme activity was also increased in adjacent undamaged leaves. Artificial damage increased the phenolic levels of the damaged leaves. Insect grazing caused a larger, longer-lasting increase in phenolic levels and also elevated phenolic levels in undamaged leaves. The possible role of these wound-induced biochemical changes in birch is discussed.     

Behavioral and cardiac responses of the rabbit,Oryctolagus cuniculus,to chemical fractions from anal gland

Unrestrained rabbits, 2–3 months old and nestlings, were exposed to the odors of the acid-neutral, acid, neutral, and basic fractions of the anal gland of male rabbits. The acid-neutral, acid, and neutral fractions elicited the strongest response in the form of avoidance. The heart rates of the nestlings were monitored using a radio telemetry technique. Statistically significant lowering of the heart rate and an increase in variability occurred on exposure to the fractions. The extent of the changes varied consistently in relation to a given odor, being greatest for the acid and neutral fractions. The results indicate the usefulness of heart-rate monitoring as a tool in the study of the perception of odors by animals.