Jasmonic acid (JA) is thought to be part of a signal-transduction pathway which dramatically increases de-novo nicotine synthesis in the roots and increases whole-plant (WP) nicotine pools in response to the wounding of the leaves in Nicotiana sylvestrisSpegazzini and Comes (Solanaceae). We report the synthesis of a doubly labeled JA ([1, 2-13 C]JA) and use it as an internal standard to quantify by gas chromatography-mass spectrometry the changes in root and shoot JA pools in plants subjected to differing amounts of standardized leaf wounding. Wounding increased JA pools 10-fold locally in damaged leaves within 90 min and systemically in the roots (3.5-fold) 180 min after wounding. If JA functions as an intermediary between stimulus and response, quantitative relationships among the stimulus, JA, and the response should exist. To examine these relationships, we varied the number of punctures in four leaves and quantified both the resulting JA in damaged leaves after 90 min and the resulting WP nicotine concentration after 5 d. We found statistically significant, positive relationships among number of leaf punctures, endogenous JA, and WP nicotine accumulation. We used two inhibitors of wound-induced nicotine production, methyl salicylate and indole-3-acetic acid, to manipulate the relationships between wound-induced changes in JA and WP nicotine accumulation. Since wounding and the response to wounding occur in widely separated tissues, we applied inhibitors to different plant parts to examine their effects on the local and systemic components of this response. In all experiments, inhibition of the wound-induced increase in leaf JA 90 min after wounding was associated with the inhibition of the nicotine response 5 d after wounding. We conclude that wound-induced increases in leaf JA are an important component of this longdistance signal-transduction pathway.Abbreviations: JA = jasmonic acid; WP = whole plant; MSA = methyl salicylate

Resource-based tradeoffs in the allocation of a limiting resource are commonly invoked to explain negative correlations between growth and defense in plants, but critical examinations of these tradeoffs are lacking. To rigorously quantify tradeoffs in a common currency, we grew Nicotiana attenuata plants in individual hydroponic chambers, induced nicotine production by treating roots with methyl jasmonate (MJ) and standardized leaf puncturing, and used N to determine whether nitrogen-based tradeoffs among nicotine production, growth, and seed production could be detected. Plants were treated with a range of MJ quantities (5, 45 or 250 μg plant) to effect a physiologically realistic range of changes in endogenous jasmonic acid levels and increases in nicotine production and accumulation; MJ treatments were applied to the roots to target JA-induced nicotine production, since nicotine biosynthesis is restricted to the roots. Leaf puncturing and 5 μg MJ treatments increased de novo nicotine synthesis and whole-plant (WP) nicotine pools by 93 and 66%, while 250 μg MJ treatments increased these values 3.1 and 2.5-fold. At these high rates of nicotine production, plants incorporated 5.7% of current nitrogen uptake and 6.0% of their WP nitrogen pools into nicotine. The N-labeled nicotine pools were stable or increased for the duration of vegetative growth, indicating that the N-nicotine was not metabolized and re-used for growth. Plants with elevated nicotine production grew more slowly and the differences in plant biomass gain between MJ-treated plants and controls were linearly related to the differences in nicotine accumulation. Despite the reductions in rosette-stage growth associated with nicotine production, estimates of lifetime fitness (cumulative lifetime seed production, mass/seed, seed viability) were not affected by any treatment. Only two treatments (leaf puncturing and 250 μg MJ) increased the allocations ofN acquired at the time of induction to seed production. On average, plants used only 14.9% of their WP nitrogen pool for seed production, indicating that either the nitrogen requirements for seed production or the reproductive effort of these hydroponically-grown plants are low. To determine if seed production is strongly influenced by the amount of vegetative biomass attained before reproduction, the experiment was repeated with plants that had 44% of their leaf area (or 29% of their WP biomass) removed before MJ treatments with a removal technique that minimized the nicotine response. MJ treatments of these plants dramatically increased nicotine production and accumulation, but these plants also suffered no measurable fitness consequences from either the leaf removal or MJ treatments. We conclude that when N. attenuata plants are grown in these individual hydroponic chambers, their allocation to reproduction is sufficiently buffered to obscure the large increases in nitrogen allocations to an inducible defense. To determine whether soil-grown plants are similarly buffered, we grew two genotypes of plan...

Nicotiana attenuata is a post-fire annual that utilizes jasmonate-inducible nicotine production as an inducible chemical defense which, in turn, can utilize 6% of a plant' s nitrogen budget and be costly to seed production. We characterize the nitrogen pools of burned soils in the plant' s native environment (piñyon-juniper woodlands) and examine how variation in nitrogen source and supply rate influence the patterns of allocation to growth and inducible and constitutive nicotine production. Available soil nitrogen increases dramatically (40-fold) immediately after a fire and consists principally of ammonia which is subsequently oxidized to nitrate during post-fire succession. We simulate these changes in nitrogen availability in hydroponic culture and use allometric techniques to characterize changes in allocation. In two experiments, we alter (1) nitrate supply rates 8-fold and (2) the ratio of ammonia:nitrate under consistent nitrogen supply rates. In both experiments, we increase the allocation to nicotine by treating roots with methyl jasmonate (MJ), the methyl ester of the plant' s internal wound signal, jasmonic acid, which increases nicotine production in the roots after shoot herbivory. MJ treatments decrease whole plant (WP) growth, increase root:shoot ratio, and increase WP nicotine pools in all nitrogen environments. Overall, source and supply rate of nitrogen have no effect on either the constitutive or induced allometric relationships of nicotine accumulation and growth. This remarkable homeostasis in allocation patterns contradicts a key prediction of carbon nutrient (C/N) theory. With N-pulse-chase techniques, we demonstrate that plants preferentially utilize ammonia for nicotine production over nitrate when both nitrogen sources are available. This preferential use of ammonia may allow  N. attenuata to reduce the biochemical costs of producing nicotine in the post-fire environment.

SVMM.^HYIn Nicotiana .n'hestris Spegazzini and Comes {Solanaceae), we examined the relationships among wounding, endogenous leaf jasmonic acid (JA) pools, and whole-plant (\VP) nicotine accumulation ovet a range of woundmg intensities and spatial distributions, in order to explore optimal defence (OD) theory-predictions. We quantitatively wounded one or four leaves and then quantified: (1) JA in damaged and undamaged leaves 90 min after wounding; (2) WP nicotine concentration after 5 d (the times when JA and nicotine attain the largest wound-induced concentrations). We find: (1) statistically significant, positive relationships on a !eaf-by-leaf basis among tbe number of leaf punctures, endogenous leaf JA, and WP nicotine accumulation; (2) tbat young, undamaged leaves have a bigber concentration of JA tban do older, undamaged leaves, and produce a greater amount of JA per puncture tban older leaves, but tbat all leaves bave tbe same JA content (ng JA per leaf); and (3) that a damaged leaf produces less JA when other leaves in tbe canopy are wounded than when it is the only wounded leaf in tbe canopy, but tbat when it is tbe only wounded ieaf, tbe pbylotactically adjacent, undamaged leaves do not mcrease their JA concentrations. Tbe observation tbat younger leaves produce more JA per puncture tban do older leaves is consistent with OD theory predictions. The observation that a small amount of damage localized to a single leaf is as effective as a larger amount of damage dispersed across the canopy in increasing leaf JA and WP nicotine accumulation shows the plant's ability to differentiate between dispersed and localized damage. Because the quantity of JA in a wounded leaf 90 min after wounding is a reliable indicator of the WP nicotine response to wounding, this trait provides insight into how plants integrate information about environmental insults and tailor tbeir defence responses.

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