Predictions Of Optimal Defense Theory Research Articles

Age-specific allocation of glucosinolates within plant reproductive tissues

The Optimal Defense Theory (ODT) predicts that the distribution of defenses within a plant should mirror the value and vulnerability of each tissue. Although the ODT has received much experimental support, very few studies have examined defense allocation among reproductive tissues and none assessed simultaneously how these defenses evolve with age. We quantified glucosinolates in perianths, anthers and pistils at different bud maturity stages (i.e., intermediate flower buds, old flower buds and flowers) of undamaged and mechanically damaged plants of an annual brassicaceous species. The youngest leaf was used as a reference for vegetative organs, since it is predicted to be one of the most defended. In line with ODT predictions, reproductive tissues were more defended than vegetative tissues constitutively, and within the former, pistils and anthers more defended than perianths. No change in the overall defense level was found between bud maturity stages, but a significant temporal shift was observed between pistils and anthers. Contrary to ODT predictions, mechanical damage did not induce systemic defenses in leaves but only in pistils. Our results show that defense allocation in plant reproductive tissues occurs at fine spatial and temporal scales, extending the application framework of the ODT. They also demonstrate interactions between space and time in fine-scale defense allocation.

Optimal Defense Theory in an ant–plant mutualism: Extrafloral nectar as an induced defence is maximized in the most valuable plant structures

Abstract Plants allocate defences in order to decrease costs and maximize benefits against herbivores. The Optimal Defense Theory (ODT) predicts that continuously expressed (i.e. constitutive) defences are expected in structures of high value, whereas defences that are expressed or that increase their expression only after damage or upon risk of damage (i.e. induced defences) are expected in structures of low value. Although there are several studies evaluating ODT predictions, few studies have successfully tested them as a way of measuring ecological investment in extrafloral nectary (EFN)‐mediated ant–plant interactions. Here we compared extrafloral nectar production and ant attractiveness to EFNs located on vegetative versus reproductive plant structures on Qualea multiflora plants subjected to different levels of simulated herbivory. We then addressed the following predictions emerging from the ODT: (a) extrafloral nectar produced in inflorescence EFNs will have higher volumes and calories and will attract more ants than extrafloral nectar produced in leaf EFNs; (b) extrafloral nectar production (volume and calories) and ant attendance will increase after simulated herbivory in leaf EFNs but not in inflorescence EFNs; (c) higher simulated leaf herbivory will induce higher extrafloral nectar production in EFNs on leaves and (d) more attractive extrafloral nectar (higher volume and calories) will attract more ants. Extrafloral nectar volume and calorie content, as well as ant abundance, were higher in EFNs of inflorescences compared to EFNs of leaves both before and after simulated herbivory, consistent with one of our predictions. However, EFNs on both leaves and inflorescences, not on leaves only, were induced by simulated herbivory, a pattern opposite to our prediction. Plants subjected to higher levels of leaf damage produced more and higher calorie extrafloral nectar, but showed similar ant abundance. Finally, more attractive extrafloral nectar attracted more ants. Synthesis. Our results show that extrafloral nectar production before and after simulated herbivory, as well as ant recruitment, varies according to the plant structure on which EFNs are located. Our study is the first to show that ant recruitment via extrafloral nectar follows predictions from Optimal Defense Theory, and that the ant foraging patterns may be shaped by the plant part attacked and the level of damage it receives.

Inducible versus constitutive antioxidant defenses in algae along an environmental stress gradient

Optimal defense theory (ODT) predicts that antiherbivore defenses should be constitutive when plants are frequently attacked and inducible when the probability of attack is low. Like antiherbivore defenses, antioxidant defenses can be inducible or constitutive. We hypothesized the ODT predictions should apply to antioxidant defenses; thus, species inhabiting environments where oxidative stresses occur frequently should produce constitutive antioxidant defenses, whereas species in environments where stresses occur less frequently should produce inducible defenses. We tested this hypothesis by attempting to induce production of the antioxidant precursor dimethylsulfoniopropionate (DMSP) in 4 ulvoid algae species that experience different levels of environmental stress because they are zoned along a tidal gradient. The 2 lower intertidal speciesUlvaria obscuraandUlva fenestrata, which experience oxidative stresses less frequently, induced DMSP production in response to applications of the chemical oxidant hydrogen peroxide within 7 d, whereas the higher intertidal speciesUlva linzaandUlva intestinalis, which regularly experience oxidative stress, did not have increased DMSP concentrations. This study demonstrates a novel waterborne signaling mechanism for DMSP induction in marine macroalgae and provides evidence of selection for inducible antioxidant defenses in organisms experiencing less frequent environmental stresses.

Changes in cytokinins are sufficient to alter developmental patterns of defense metabolites in Nicotiana attenuata.

Plant defense metabolites are well known to be regulated developmentally. The optimal defense (OD) theory posits that a tssue's fitness values and probability of attack should determine defense metabolite allocations. Young leaves are expected to provide a larger fitness value to the plant, and therefore their defense allocations should be higher when compared with older leaves. The mechanisms that coordinate development with defense remain unknown and frequently confound tests of the OD theory predictions. Here we demonstrate that cytokinins (CKs) modulate ontogeny-dependent defenses in Nicotiana attenuata. We found that leaf CK levels highly correlate with inducible defense expressions with high levels in young and low levels in older leaves. We genetically manipulated the developmental patterns of two different CK classes by using senescence- and chemically inducible expression of CK biosynthesis genes. Genetically modifying the levels of different CKs in leaves was sufficient to alter ontogenic patterns of defense metabolites. We conclude that the developmental regulation of growth hormones that include CKs plays central roles in connecting development with defense and therefore in establishing optimal patterns of defense allocation in plants.

Mechanisms of Optimal Defense Patterns in Nicotiana attenuata: Flowering Attenuates Herbivory-elicited Ethylene and Jasmonate SignalingF

To defend themselves against herbivore attack, plants produce secondary metabolites, which are variously inducible and constitutively deployed, presumably to optimize their fitness benefits in light of their fitness costs. Three phytohormones, jasmonates (JA) and their active forms, the JA-isoleucine (JA-Ile) and ethylene (ET), are known to play central roles in the elicitation of induced defenses, but little is known about how this mediation changes over ontogeny. The Optimal Defense Theory (ODT) predicts changes in the costs and benefits of the different types of defenses and has been usefully extrapolated to their modes of deployment. Here we studied whether the herbivore-induced accumulation of JA, JA-Ile and ET changed over ontogeny in Nicotiana attenuata, a native tobacco in which inducible defenses are particularly well studied. Herbivore-elicited ET production changed dramatically during six developmental stages, from rosette through flowering, decreasing with the elongation of the first corollas during flower development. This decrease was largely recovered within a day after flower removal by decapitation. A similar pattern was found for the herbivore-induced accumulation of JA and JA-Ile. These results are consistent with ODT predictions and suggest that the last steps in floral development control the inducibility of at least three plant hormones, optimizing defense-growth tradeoffs.

Simultaneous analysis of tissue- and genotype-specific variation in Solidago altissima (Asteraceae) rhizome terpenoids, and the polyacetylene dehydromatricaria ester

Solidago altissima is a dominant perennial of old field succession in North America with the ability to form dense, almost monospecific stands; the plant is also considered an aggressive invasive throughout Europe and Japan. S. altissima’s success is facilitated by large belowground rhizome systems that allow clonal spread and possible allelopathic suppression of competitors. A diversity of polar and nonpolar terpenes and polyacetylenes have been described from S. altissima rhizomes; however, there is little information documenting the concentration or distribution of these allelochemicals and how they relate to plant defense hypotheses. We thus developed a GC–MS method to simultaneously quantify rhizome terpenes and polyacetylenes that spanned a range of polarities and confirmed the presence of 19 terpenoids in addition to the polyacetylene dehydromatricaria ester (DME). We, (1) determined relative concentrations and variability of mono-, sesqui-, neutral diterpenes, diterpene acids and DME within a central NY population; (2) compared accumulation of these compounds in differently developed rhizome tissue; and (3) tested the alleopathic effect of DME at naturally occurring concentrations on germination and seedling growth of Asclepias syriaca, a common competitor of S. altissima. Overall, diterpene acids dominated the phytochemical profile of most genotypes (averaging 0.75% of fresh mass) with kolavenic acid being the single most abundant component. Monoterpene, sesquiterpene and acidic diterpene concentrations were all significantly greater in the actively elongating (current-year) rhizomes compared to established rhizomes and followed optimal defense theory predictions. Conversely, DME levels were lower in the current-year rhizomes than in established rhizomes. DME inhibited seed germination of A. syriaca in a dose-dependent manner. The tissue-specific accumulation of the different compounds may represent a biosynthetic and/or allocational strategy that limits the exposure of young-tissue to high concentrations of DME possibly limiting the cytotoxic effects of DME.

HERBIVORY MEDIATES GRASS–ENDOPHYTE RELATIONSHIPS

Endophytic fungi are plant symbionts living asymptomatically within plant tissues. Neotyphodium spp., which are asexual vertically transmitted systemic fungal endophytes of cool-season grasses, are predicted to be plant mutualists. These endophytes increase host plant resistance to environmental stresses and/or increase the production of alkaloid-based herbivore deterrents. The ubiquity of this defense mutualism is unclear, and a variety of alternative mechanisms may explain the observed variation in infection rates, levels of deterrence, and the maintenance of asexual endophytes in grass populations. We found that grass-endophyte interactions are variable and ordered along an herbivory gradient in an undisturbed subarctic alpine ecosystem. Native grass populations in grazed sites had significantly greater frequency of Neotyphodium infection compared to ungrazed sites. Tillers from grazed sites had significantly higher hyphal densities compared to ungrazed sites. The ability of grass-Neotyphodium constituents to deter vertebrate herbivory in natural systems is thought to be rare. In grazed meadows, we showed that endophyte infection resulted in the deterrence of grazing by native vertebrate herbivores. However, the same herbivores did not distinguish between infected and uninfected grass harvested from ungrazed areas. These results demonstrate that the relationship between vertically transmitted endophytes and grasses in the alpine tundra vary greatly within populations. This may be based in part on defense mutualism and is consistent, under varying levels of herbivory, with the predictions of optimal defense theory.

Testing the optimal defense theory and the growth-differentiation balance hypothesis in Arabidopsis thaliana

Two prominent theories proposed to explain patterns of chemical defense expression in plants are the optimal defense theory (ODT) and the growth-differentiation balance hypothesis (GDBH). The ODT predicts that plant parts with high fitness value will be highly defended, and the GDBH predicts that slow growing plant parts will have more resources available for defense and thus will have higher defense levels than faster growing tissues. We examined growth rate, fitness value, and defense protein levels in leaves of a wild and lab ecotype of Arabidopsis thaliana to address whether patterns of defense protein expression in this plant conform to predictions of either the ODT or the GDBH. We divided leaves of A. thaliana into six leaf classes based on three developmental stages: vegetative, bolting, and flowering; with two leaf ages at each stage: young and old. We assessed the fitness value of leaves by determining the impact of the removal of each leaf class on total seed production and germination rates. Although A. thaliana was highly tolerant to defoliation, young leaves were more valuable than old in general, and young leaves on bolting plants were the most valuable leaf class in particular. Young leaves on vegetative plants grew fastest in both ecotypes, while old leaves on bolting and flowering plants grew slowest. Finally, defense levels were assessed in each leaf class by quantifying the constitutive and inducible expression of four defense-related proteins. Expression of guaiacol peroxidase and chitinase activity conformed largely to GDBH predictions. Expression of trypsin inhibitor and polyphenoloxidase activity varied by leaf class and treatment, but conformed to neither GDBH nor ODT predictions.

Is induction response negatively correlated with constitutive resistance in black mustard?

Black mustard, Brassica nigra, is highly variable in both constitutive resistance and induction response following damage by herbivores. A focal population from Ithaca, New York, was used to test the following two predictions of optimal defense theory: (1) that allocation to resistance will reduce plant performance in the absence of herbivores; and (2) that induction response will be negatively correlated with constitutive resistance. The experiment consisted of a half-sib mating design with 47 paternal families and four dams per sire, fully crossed with a damage treatment consisting of 25% leaf removal by the cabbage white butterfly, Pieris rapae, when plants had four leaves. Leaf trichome density, sinigrin concentration, and glucobrassicin concentration were 38%, 19%, and 16% higher, respectively, for the seventh leaf of damaged plants. Paternal families did not vary significantly in their induction response. Narrow-sense heritabilities were h(S)(2) = 0.51, 0.76, and 0.50 for constitutive leaf trichome density, sinigrin concentration, and glucobrassicin concentration, respectively. Positive genetic correlations were found between glucobrassicin concentration and days to first flower, suggesting a genetic cost of resistance. Induction responses were negatively correlated with constitutive allocation for leaf trichome density and sinigrin concentration. The results were therefore consistent with optimal defense theory, offering modest evidence for both predictions.

Tissue-specific induction of herbivore resistance: seaweed response to amphipod grazing.

Tissues within individual plants can vary greatly in the strength of their constitutive (i.e., permanent) and induced resistance to herbivores. Optimal defense theory predicts that defenses should be allocated among tissues in proportion to the value of the tissues to the plant and the tissue-specific risk of attack by grazers. We examined the relationship between tissue value and defense in the highly-differentiated brown seaweed Sargassum filipendula. Tissues within S. filipendula varied widely in palatability to the herbivorous amphipod Ampithoe longimana, with younger tissues preferred over older tissues and blades preferred over stipes. Old stipes (at the base of the plant), which linked the other tissues to the seafloor and were thus the most valuable tissue to the plant, were defended constitutively and resisted amphipod grazing by virtue of their toughness rather than via deterrent chemistry. Induction of resistance as a result of amphipod grazing occurred only in the top stipes, which contain the meristematic tissue responsible for future growth. Induction in the top stipes was not due to toughness or other structural properties, as the unpalatability persisted when top stipes were dried, ground to a fine powder, and reconstituted into an agar matrix. This suggests that the induced resistance to grazing resulted from an increase in chemical defenses. The demonstration of constitutive or induced defenses in only the more valuable tissues of the seaweed is consistent with predictions of optimal defense theory. Our finding of induction due to mesograzer (amphipod) feeding is also consistent with the notion that it is these small, more sedentary, herbivores that are most likely to induce defenses in seaweeds.

IS INDUCTION RESPONSE NEGATIVELY CORRELATED WITH CONSTITUTIVE RESISTANCE IN BLACK MUSTARD?

Black mustard, Brassica nigra, is highly variable in both constitutive resistance and induction response following damage by herbivores. A focal population from Ithaca, New York, was used to test the following two predictions of optimal defense theory: (1) that allocation to resistance will reduce plant performance in the absence of herbivores; and (2) that induction response will be negatively correlated with constitutive resistance. The experiment consisted of a half-sib mating design with 47 paternal families and four dams per sire, fully crossed with a damage treatment consisting of 25% leaf removal by the cabbage white butterfly, Pieris rapae, when plants had four leaves. Leaf trichome density, sinigrin concentration, and glucobrassicin concentration were 38%, 19%, and 16% higher, respectively, for the seventh leaf of damaged plants. Paternal families did not vary significantly in their induction response. Narrow-sense heritabilities were h2s = 0.51, 0.76, and 0.50 for constitutive leaf trichome density, sinigrin concentration, and glucobrassicin concentration, respectively. Positive genetic correlations were found between glucobrassicin concentration and days to first flower, suggesting a genetic cost of resistance. Induction responses were negatively correlated with constitutive allocation for leaf trichome density and sinigrin concentration. The results were therefore consistent with optimal defense theory, offering modest evidence for both predictions.

Changes in the glucosinolate-myrosinase defense system in Brassica juncea cotyledons during seedling development.

Optimal defense theory (ODT) predicts that plant defenses will be allocated to plant organs and tissues in proportion to their relative fitness values and susceptibilities to attack. This study was designed to test ODT predictions on the myrosinase-glucosinolate defense system in Brassica juncea by examining the relationships between the fitness value of B. juncea cotyledons and the levels and effectiveness of cotyledon defenses. Specifically, we estimated fitness value of cotyledons during plant development by measuring plant growth and seed production after cotyledon damage or removal at successive seedling ages. Cotyledon removal within five days of emergence had a significant impact on growth and seed production, but cotyledon removal at later stages did not. Consistent with ODT, glucosinolate and myrosinase levels in cotyledons also declined with seedling age, as did relative defenses against a generalist herbivore, Spodoptera eridania, as estimated by bioassay. Declines in glucosinolates were as predicted by a passive, allometric dilution model based on cotyledon expansion. Declines in myrosinase activity were significantly more gradual than predicted by allometric dilution, suggesting active retention of myrosinase activity as young cotyledons expand.

OPTIMAL DEFENSE THEORY PREDICTS THE ONTOGENY OF AN INDUCED NICOTINE DEFENSE

Optimal Defense (OD) theory predicts that the within-plant allocation of secondary metabolites that function as defenses will be positively correlated with the fitness value of particular plant parts. Here, we experimentally examine this prediction by ex- ploiting our understanding of the mechanisms of wound-induced nicotine production in Nicotiana sylvestris (Solanaceae) to manipulate the patterns of nicotine allocation and to determine their fitness consequences. In two perturbation experiments conducted over three stages of ontogeny (rosette, elongation, and flowering), we wounded or removed leaves of different ages (young, mature, or old) and determined the effects on nicotine allocation (whole-plant and within-plant) and fitness (lifetime viable seed production). OD theory predicts that, as leaves age and their fitness value decreases, the allocation of defense to particular leaves and the fitness consequences of their removal should be positively cor- related. We found that (1) leaf removal results in a significant decrease in seed mass at the elongation stage, but not at the rosette or flowering stages; (2) the relative value of leaves decreases from young and mature to old leaves; (3) leaf damage significantly increases the whole-plant nicotine contents of rosette-stage plants, but not of elongation- or flowering- stage plants, and after damage, younger leaves are more heavily defended than older leaves at the elongation and flowering stages; and (4) regardless of ontogenetic stage, plants distribute nicotine among leaves in accordance with their relative fitness value, thus sup- porting OD theory predictions. Leaf value increases after N fertilization at the flowering stage, but is not changed if adjacent leaves are removed at earlier growth stages. Moreover, plants are capable of sending their root-synthesized nicotine to specific leaves after damage; at the elongation stage, the new and young leaves receive greater proportional allocations of nicotine than other leaves. The cessation of significant whole-plant nicotine inductions at later stages in ontogeny is not due to the root's decreased ability to respond to the plant's wound signal, jasmonic acid (JA) with increased nicotine biosynthesis, but rather to the decline in a leaf's sensitivity to wounding and in its ability to export JA from the leaves to roots. Ontogeny has profound effects on the use of this induced defense. Plants mount systemic nicotine inductions at the rosette stage that rely on large increases in de novo nicotine synthesis, switch to the selective targeting of nicotine to the new and young leaves at the elongation stage without large increases in de novo nicotine synthesis, and allocate to reproductive structures, but not leaves, at the flowering stage. These changes are consistent with the predictions of OD theory.

Optimal Defense Theory Predicts the Ontogeny of an Induced Nicotine Defense

Optimal Defense (OD) theory predicts that the within-plant allocation of secondary metabolites that function as defenses will be positively correlated with the fitness value of particular plant parts. Here, we experimentally examine this prediction by exploiting our understanding of the mechanisms of wound-induced nicotine production in Nicotiana sylvestris (Solanaceae) to manipulate the patterns of nicotine allocation and to determine their fitness consequences. In two perturbation experiments conducted over three stages of ontogeny (rosette, elongation, and flowering), we wounded or removed leaves of different ages (young, mature, or old) and determined the effects on nicotine allocation (whole-plant and within-plant) and fitness (lifetime viable seed production). OD theory predicts that, as leaves age and their fitness value decreases, the allocation of defense to particular leaves and the fitness consequences of their removal should be positively correlated. We found that (1) leaf removal results in a significant decrease in seed mass at the elongation stage, but not at the rosette or flowering stages; (2) the relative value of leaves decreases from young and mature to old leaves; (3) leaf damage significantly increases the whole-plant nicotine contents of rosette-stage plants, but not of elongation- or flowering-stage plants, and after damage, younger leaves are more heavily defended than older leaves at the elongation and flowering stages; and (4) regardless of ontogenetic stage, plants distribute nicotine among leaves in accordance with their relative fitness value, thus supporting OD theory predictions. Leaf value increases after N fertilization at the flowering stage, but is not changed if adjacent leaves are removed at earlier growth stages. Moreover, plants are capable of sending their root-synthesized nicotine to specific leaves after damage; at the elongation stage, the new and young leaves receive greater proportional allocations of nicotine than other leaves. The cessation of significant whole-plant nicotine inductions at later stages in ontogeny is not due to the root's decreased ability to respond to the plant's wound signal, jasmonic acid (JA) with increased nicotine biosynthesis, but rather to the decline in a leaf's sensitivity to wounding and in its ability to export JA from the leaves to roots. Ontogeny has profound effects on the use of this induced defense. Plants mount systemic nicotine inductions at the rosette stage that rely on large increases in de novo nicotine synthesis, switch to the selective targeting of nicotine to the new and young leaves at the elongation stage without large increases in de novo nicotine synthesis, and allocate to reproductive structures, but not leaves, at the flowering stage. These changes are consistent with the predictions of OD theory.

PHLOROTANNIN ALLOCATION AMONG TISSUES OF NORTHEASTERN PACIFIC KELPS AND ROCKWEEDS

Optimal defense theory (ODT) predicts antiherbivore defensive compounds will be allocated so that the most valuable or most susceptible tissues will be best defended. The growth–differentiation balance hypothesis (GDBH) predicts that defense allocation will be a result of trade‐offs between growth and defense. Thus, these two theories predict opposite allocation patterns with respect to “valuable,” actively growing meristematic and reproductive tissues. ODT predicts that meristems and reproductive tissues should have higher defense levels than nonmeristematic vegetative tissues; the GDBH predicts the defense levels of meristems and reproductive tissues will be lower than vegetative tissues. We examined allocation patterns of phlorotannins in 21 species of kelps (Order Laminariales) and rockweeds (Order Fucales) from nine sites on the west coast of the United States to determine if allocation patterns better matched the predictions of ODT or the GDBH and to look for differences in allocation patterns among sites. Within‐species differences in phlorotannin levels occurred in 10 of the 21 species examined. Meristems of both kelps and rockweeds had higher phlorotannin levels than nonmeristematic vegetative tissues, consistent with ODT. Phlorotannin levels in reproductive tissues of kelps were higher than vegetative tissues, but levels in reproductive tissues of rockweeds were lower than vegetative tissues, indicating that allocation strategies may follow taxonomic lines. Allocation patterns differed among sites in four of the 16 species collected from more than one site. Differences in allocation patterns among sites were usually changes in the ratios of phlorotannins in well‐defended compared to poorly defended tissues, rather than changes in which tissues were well defended or poorly defended. We concluded that environmental variability can have large effects on the concentration of phlorotannins in algae but has limited effects on allocation patterns among tissues.

The Allometry of Nitrogen to Growth and an Inducible Defense under Nitrogen‐Limited Growth

As plants become nitrogen—limited in their growth, carbon/nutrient (C/N) theory predicts a decreasing allocation of nitrogen to nitrogen—intensive defense compounds, while optimal defense (OD) theory predicts an increasing allocation to defense; CN ratio of the plant does not change, while OD theory does. We compare C/N and OD theory predictions for the inducible nicotine production of a native tobacco, Nicotiana sylvestris. We define allocation as the slope of the ln—ln allometric relationship between whole—plant nicotine and nicotine—free biomass in undamaged and damaged plants, harvested every other day in eight consecutive harvests. Relative growth, nitrogen, and nitrogen in nicotine accumulation rates (RGRs, RNARs, and RNicARs) were calculated to estimate the instantaneous investment of nitrogen in nicotine and the growth of plant tissues. Nicotine accumulation continues during nitrogen stress, resulting in a 3.29—fold increase in the nicotine pools of undamaged plants, and is accelerated in response to leaf damage, resulting in 4.06 fold increase in the nicotine pools of damaged plants. Nitrogen pool size did not change after day 2, and both undamaged and damaged plants allocated an increasing proportion of their nitrogen pool to nicotine throughout the experiment. Damage increased the allocation of nitrogen to nicotine; the slope of the ln nicotine vs ln nicotine—free biomass regression for damaged plants was significantly greater (16.9%; P < .041) than the slope for undamaged plants as measured during the period of damage—induced nicotine accumulation (days 2—10 damage). Allocation of nitrogen to nicotine accumulating is clearly not a passive function of the nitrogen in excess of growth requirements, and damage increases this allocation. Trade—offs between biomass and nicotine production over the course of the experiment between damaged and undamaged plants were not apparent. However, the short—term estimates indicate otherwise. On day 2, there were a significant decrease in RGR, with a correlated increase in RNicAR in response to damage, suggesting that there was a short—term trade—off between allocation to growth and nicotine accumulation or other induced processes. By the end of the experiment, each damaged plant had accumulated 0.595 mg N in nicotine above that accumulated by undamaged plants, and additional investment representing 1.94% of the total nitrogen pool. However 2 d after damage, damaged plants allocated 0.131 mg more N to nicotine than undamaged plants, representing a short—term investment of 17.7% of the nitrogen potentially made available by the decrease in RGR (.0740 mg). Plants invest substantial portions of their nitrogen in an induced defense, but our estimates do not include the nitrogen costs of production, storage, transport, detoxification, or the indirect cost of this induced defense.