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

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 from(15)NO3 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.

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.

Amino acids occur in most floral nectars but their role in pollinator attraction is relatively unstudied. Nectars of butterfly-pollinated flower tend to have higher concentrations of amino acids than do flowers pollinated by bees and many other animals, suggesting that amino acids are important attractants of butterflies to flowers. In order to determine whether amino acids are important in attracting butterflies and bees, we tested the preference of cabbage white butterflies (Pieris rapae) and honey bees (Apis mellifera) by allowing them to feed from artificial flowers containing sugar-only or sugar-amino acid mimics ofLantana camara nectar. Honey bees and female cabbage white butterflies consumed more sugar-amino acid nectar than sugar-only nectar. In addition, female cabbage white butterflies visited artificial flowers containing sugar-amino acid nectars more frequently than flowers containing sugar-only nectars; honey bees spent more time consuming the sugar-amino acid nectar. Male cabbage white butterflies did not discriminate between the two nectars. These results support the hypothesis that the amino acids of nectar contribute to pollinator attraction and/or feeding.