ALBERT

Notes: Abstract Plants respond to herbivore attack with a bewildering array of responses, broadly categorized as direct and indirect defenses, and tolerance. Plant-herbivore interactions are played out on spatial scales that include the cellular responses, well-studied in plant-pathogen interactions, as well as responses that function at whole-plant and community levels. The plant's wound response plays a central role but is frequently altered by insect-specific elicitors, giving plants the potential to optimize their defenses. In this review, we emphasize studies that advance the molecular understanding of elicited direct and indirect defenses and include verifications with insect bioassays. Large-scale transcriptional changes accompany insect-induced resistance, which is organized into specific temporal and spatial patterns and points to the existence of herbivore-specific trans-activating elements orchestrating the responses. Such organizational elements could help elucidate the molecular control over the diversity of responses elicited by herbivore attack.

Notes: The oxylipin pathway mediates wound- and herbivore-induced defense reactions in Nicotiana attenuata as evidenced by a transient jasmonic acid (JA)-burst that precedes these defense responses. The fate of this induced JA-burst remains unknown. Two derivatives of JA, its methylester, methyl jasmonate (MeJA) and cis-jasmone (cisJ), are thought to be a means of disposing of JA through volatilization at the plant surface. In N. attenuata, the headspace quantities of these compounds did not change over 3 days, although levels of MeJA and cisJ increased 100- and 70-fold, respectively, in surface extracts of attacked leaves after feeding of Manduca sexta larvae or application of larval regurgitant to mechanical wounds. Inhibition of the wound-induced increase in JA with indole-3-acetic acid (IAA) revealed an association between the JA accumulation and subsequent increases in MeJA and cisJ. Induced systemic increases of MeJA were not of local origin and therefore do not contribute to the inactivation of the JA-burst in the wounded leaf. The total amount of MeJA and cisJ produced could only account for 9% of the JA-burst elicited by herbivore attack and therefore their production do not represent major disposal pathways of JA in N. attenuata.

Notes: Abstract The induced defenses of plants are, by definition, examples of adaptive phenotypic plasticity; however, they have rarely been considered as such due largely to the difficulty of defining the character states of these dynamic chemical responses. Nicotine functions as an inducible defense in Nicotiana sylvestris and is one of the few induced defenses which is understood on a whole-plant basis. Nicotine biosynthesis is restricted to the roots and increases after wounding or herbivore attack to the shoot. Newly synthesized nicotine is transported throughout the plant; induced plants are better protected from subsequent herbivore attack in laboratory trials. Root nicotine production is activated by an endogenously-produced signal, jasmonic acid (JA), which is synthesized in response to leaf damage and either directly through transport or indirectly via another, as yet unknown signal, increases JA pools and nicotine biosynthesis in the roots. The addition of JA or its methyl ester (MJ) to plants stimulates nicotine production in the same way that leaf damage does. In this study I use MJ to elicit the induced defense without wounding and describe a new character state for this induced defense: the allometrically determined setpoint. Plants were induced with 45 μg of MJ, once or in different combinations of 2 or 3 times during a 21-day period of vegetative growth, allowed 7 days between elicitations for the response to relax, and harvested during one of 4 harvests in order to quantify biomass and whole-plant nicotine pools. For plants induced only once, the largest induced change in whole-plant nicotine pools occurred during the second induction period, when elicitation increased nicotine pools by 5.99 mg, approximately 87% more than either the first or third induction periods. However, plants induced once had the same whole-plant nicotine pools at the end of the experiment regardless of whether they were induced during the highly responsive second period or during the less responsive first and second induction periods. Sequential elicitations consistently increased nicotine pools, but after 3 inductions, the nicotine pools were not significantly higher than those in plants which had been induced twice; plants can only increase their nicotine pools in response to 2 inductions when grown under these conditions. These results suggest that this plant has allometrically-determined setpoints for this induced defense which are determined by the number of times a plant's wound-signal cascade is stimulated, rather than by the quantity of nicotine synthesized during an induction episode.

Notes: Abstract The functional significance of herbivore-induced plant traits known to directly or indirectly influence herbivore performance remains largely untested under field conditions due to the difficulty of uncoupling the response to herbivory from the act of herbivory. The signals that activate many of the induced responses in plants are endogenously produced in response to wounding, unlike many of the predator-induced responses found in aquatic invertebrates (which are activated by exogenous cues derived from predators). Jasmonates, endogenously-produced damage signals, activate diverse wound-induced responses in plants including induced nicotine production in Nicotiana sylvestris. The results presented here are from two experiments which illustrate the use of jasmonates to uncouple induced nicotine production in Nicotiana attenuata (Torrey ex. Watson) from wounding. The exogenous addition of methyl jasmonate (MJ) in small quantities (11 μg for a 1.4 g dry mass plant) to roots of hydroponically-grown plants induces de novo nicotine synthesis and increases whole-plant nicotine concentrations just as wounding does. The MJ-induced changes were proportional to the quantity of MJ given. Moreover, the effects of MJ were additive to the effects of damage. Applications of MJ to shoots were less effective. Root treatments also worked with plants growing in a field plot. The application of MJ represents a promising tool for examining the functional significance of induced nicotine responses in plants growing in their native environments.

Notes: Abstract The morphological and chemical characteristics of flowers which attract pollinators present a dilemma for plants; advertising may increase the “apparency” of plants to their predators and some pollinators are also predators. We explore how a self-compatible disturbance species,Nicotiana attenuata, copes with this potential dilemma by examining the changes in emission of chemicals from flowers in response to pollination and herbivory. We propose that chemical changes induced by herbivory and pollination reflect the function of the chemicals in the plant. The emission of a single compound, benzyl acetone (BA, 4-phenyl-2-butanone), by flowers increases dramatically (50x) in the evening, peaking just after dark —a pattern of emission characteristic of moth-pollinated flowers. Pools of BA were found only in the outer lip of the corolla where pollinators come in contact with the flower, and diurnal changes in the size of the corolla pool closely paralleled the amount emitted by intact flowers throughout the day, as determined by headspace sampling. Pollination dramatically decreases both the pools of BA in the corolla and its emission from flowers. Similarly, nicotine, a broadly biocidal defense metabolite and an induced defense in vegetative and reproductive tissues, is also found in the headspace of flowers and is principally localized in the basal parts of the corolla below the attachment of the filaments and the nectar reward. Moreover, the dynamics of the corolla pools of BA and nicotine throughout the day are consistent with their roles in advertisement and defense, respectively. The corolla pools of nicotine are stable throughout the day except during the period of peak BA production and emission when nicotine pools decrease significantly. The coordinated increase in BA emission and decline in nicotine pools are not inexorably linked, because herbivory or mechanical damage to corolla tissue rapidly increases corolla nicotine pools without affecting the increase in BA pools. Similarly, leaf damage results in a slower, systemic increase in corolla nicotine pools during reproductive growth but again does not affect BA pools. Excised flowers emitted BA in a manner similar to that of intact flowers, and excision of a majority of flowers from a plant did not alter the BA emission patterns of the remaining flowers. We conclude that althoughN. attenuata's defensive and advertisement chemistries respond synchronously to some environmental stimuli, the flowers' chemical responses to pollinators and herbivoresare distinct and the differences reflect their ecological roles. We propose that the cost-benefit framework of the optimal defense and apparency theories can be fruitfully applied to the allocation of defense metabolites and floral volatiles that function in pollinator attraction, and that this framework can be readily tested by manipulating the patterns of the emissions of plants in the field.

Notes: Abstract 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 15N-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.

Notes: Abstract 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 15N 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−1) 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 15N-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 of 15N 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 plants in the high-nutrient soil from a 1-year-old burn in a piñyon-juniper forest (the plants' natural habitat) and in low-nutrient soil from an adjacent unburned area, and induced nicotine production in half of the plants with a 500 μg root MJ treatment. Plants grown in burned soils had an estimated lifetime fitness that was on average 2.8-fold greater than that of plants grown in unburned soils. MJ treatment reduced fitness estimates by 43% and 71% in the burned and unburned soils, respectively. We conclude that while hydroponic culture allows one to rigorously quantitate nitrogen allocation to growth, reproduction and defense, the allocation patterns of plants grown in hydroponic culture differ from those of plants grown in soil. Under hydroponic conditions, plants have low reproductive allocations and reproductive-defense tradeoffs are not detected. Reproductive-defense tradeoffs are readily discernible in soil-grown plants, but under these growing conditions, the nitrogen-basis for the tradeoff is difficult to quantify.

Notes: Abstract. 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, 2-13C]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 long-distance signal-transduction pathway.

Notes: Abstract. Jasmonic acid (JA) is part of a long-distance signal-transduction pathway that effects increases in de-novo nicotine synthesis in the roots of Nicotiana sylvestris Speg et Comes (Solanaceae) after leaf wounding. Elevated nicotine synthesis increases whole-plant nicotine pools and makes plants more resistant to herbivores. Leaf wounding rapidly increases JA pools in damaged leaves, and after a 90-min delay, root JA pools also increase. The systemic response in the roots could result from either: (i) the direct transport of JA from wounded leaves, or (ii) JA synthesis or its release from conjugates in roots in response to a second, systemic signal. We synthesized [2-14C]JA, and applied it to a single leaf in a quantity (189 μg) known to elicit both a whole-plant nicotine and root JA response equivalent to that found in plants subjected to leaf wounding. We quantified radioactive material in JA, and in metabolites both more and less polar than JA, from treated and untreated leaves and roots of plants in eight harvests after JA application. [2-14C]Jasmonic acid was transported from treated leaves to roots at rates and in quantities equivalent to the wound-induced changes in endogenous JA pools. The [2-14C]JA that had been transported to the roots declined at the same rate as endogenous JA pools in the roots of plants after leaf wounding. Most of the labeled material applied to leaves was metabolized or otherwise immobilized at the application site, and the levels of [2-14C]JA in untreated leaves did not increase over time. We measured the free JA pools before and after four different hydrolytic extractions of root and shoot tissues to estimate the size of the potential JA conjugate pools, and found them to be 10% or less of the free JA pool. We conclude that the direct transport of wound-induced JA from leaves to roots can account for the systemic increase in root JA pools after leaf wounding, and that metabolism into less polar structures determines the duration of this systemic increase. However, the conclusive falsification of this hypothesis will require the suppression of all other signalling pathways which could have shoot-to-root transport kinetics similar to that of endogenous JA.