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.     

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.     

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.     

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.     

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.     

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.     

Volatile Emissions from an Odorous Plant in Response to Herbivory and Methyl Jasmonate Exposure

Induced volatile terpenes have been commonly reported among diverse agricultural plant species, but less commonly investigated in odorous plant species. Odorous plants synthesize and constitutively store relatively large amounts of volatiles, and these may play a role in defense against herbivores. We examined the effect of herbivory and methyl jasmonate (MeJA) exposure on the release of volatile organic compounds (VOCs) in the marsh elder, Iva frutescens, which contains numerous constitutive VOCs, mainly mono- and sesquiterpenes. Our specific goal was to test for the presence of inducible VOCs in a naturally occurring plant already armed with VOCs. The abundant, native specialist leaf beetle Paria aterrima was used in herbivore induction trials. VOCs were sampled from herbivore wounded and unwounded, and from MeJA treated and untreated I. frutescens. Total VOC emissions were significantly greater in response to herbivory and MeJA treatment compared to unwounded controls. Herbivore wounding caused a substantial shift in the emission profile (42 VOCs from wounded, compared to 8 VOCs from unwounded I. frutescens), and MeJA had a similar yet less substantial influence on the emission pattern (28 VOCs from MeJA treated compared to 8 VOCs from untreated I. frutescens). Constitutive VOC emissions predominated, but some VOCs were detected only in response to herbivory and MeJA treatment, suggesting de novo synthesis. Several VOCs exhibited a delayed emission profile in contrast to the rapid release of constitutive VOCs, and principal components analysis revealed they were not associated with constitutive emissions. While I. frutescens contains many constitutive VOCs that are released immediately in response to herbivory, it also produces novel VOCs in response to feeding by the specialist P. aterrima and MeJA treatment.     

Insect-Induced Synthesis of Phytoecdysteroids in Spinach, Spinacia oleracea

Spinach (Spinacia oleracea) foliage is known to synthesize and accumulate insect molting hormones, predominantly in the form of 20-hydroxyecdysone (20E). We previously demonstrated that root 20E accumulation is increased following root damage. We designed two further experiments to address root responses to both mechanical and insect damage. In plants grown hydroponically, removal of 35% or less of the root mass did not result in changes in root 20E levels. However, removal of 70% of the root mass stimulated 6.0- and 1.5-fold increases in the root and shoot 20E concentrations, respectively. The effects of insect damage on soil-grown plants were investigated by infesting plant roots with black vine weevil (BVW: Otiorhynchus sulcatus) larvae and allowing them to feed for seven days. Decreases in root mass occurred in young plants; however, no changes were detected in mature plants. In all cases, root herbivory resulted in at least a 3.0-fold increase in root 20E concentrations. Our previous experiments implicated jasmonic acid and the analog methyl jasmonate (MJ) in signaling the damage-induced accumulation of root 20E levels. We investigated the activity of other phytohormones and growth regulators (GRs) on the 20E accumulation patterns of young plants as a means of examining the significance of jasmonates in the induction response. Hydroponic additions of MJ (0.5 M) and the synthetic auxin, 1-naphthaleneacetic acid (NAA; 0.5 M), resulted in significant increases in root 20E levels. At the concentrations tested, indole-3-acetic acid (IAA), gibberellic acid (GA₃), abscisic acid (ABA), and trans-zeatin (Z) had no effects on root 20E concentrations. However, both NAA (0.5–5.0 M) and Z (5.0 M) treatments caused increases in the root/shoot dry mass ratios, indicating shifts in resource allocation to the roots. Treatments involving ABA (5.0 M) and Z (0.5–5.0 M) caused significant increases in shoot 20E concentrations. No other hormone treatments altered shoot accumulation patterns. The mechanisms underlying the root 20E induction phenomena were investigated through the incorporation of [2-¹⁴C]mevalonic acid ([¹⁴C]MVA). Within one day, excised roots readily incorporated radioactivity into 20E from [¹⁴C]MVA. In intact plants, [¹⁴C]MVA absorbed by the roots was rapidly incorporated into root 20E pools following damage and MJ treatments. This implies that the wound-induced root 20E accumulation is the result of increased de novo 20E synthesis in the root.     

Enzymatic synthesis of high-purity structured lipids with caprylic acid at 1,3-positions and polyunsaturated fatty acid at 2-position

We attempted to synthesize high-purity structured triacylglycerols (TAG) with caprylic acid (CA) at the 1,3-positions and a polyunsaturated fatty acid (PUFA) at the 2-position by a two-step enzymatic method. The first step was synthesis of TAG of PUFA (TriP), and the second step was acidolysis of TriP with CA. Candida antarctica lipase was effective for the first reaction. When a reaction medium of PUFA/glycerol (3∶1, mol/mol) and 5% immobilized Candida lipase was mixed for 24 h at 40°C and 15 mm Hg, syntheses of TAG of γ-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids reached 89, 89, 88, and 83%, respectively. In these reactions, the lipase could be used for at least 10 cycles without significant loss of activity. In the second step, the resulting trieicosapentaenoin was acidolyzed at 30°C for 48h with 15 mol parts CA using 7% of immobilized Rhizopus delemar lipase. The CA content in the acylglycerol fraction reached 40 mol%. To increase the content further, the acylglycerols were extracted from the reaction mixture with n-hexane and were allowed to react again with CA under conditions similar to those of the first acidolysis. After three successive acidolysis reactions, the CA content reached 66 mol%. The content of dicapryloyl-eicosapentaenoyl-glycerol reached 86 wt% of acylglycerols, and the ratio of 1,3-dicapryloyl-2-eicosapentaenoyl-glycerol to 1(3),2-dicapryloyl-3(1)-eicosapentaenoyl-glycerol was 98∶2 (w/w). In this reaction, the lipase could be used for at least 20 cycles without significant loss of activity. Repeated acidolysis of the other TriP with CA under similar conditions synthesized 1,3-dicapryloyl-2-γ-linolenoyl-glycerol, 1,3-dicapryloyl-2-arachidonoyl-glycerol, and 1,3-dicapryloyl-2-docosahexaenoyl-glycerol in yields of 58, 87, and 19 wt%, respectively.     

No Evidence for the Induction of Brown Algal Chemical Defense by the Phytohormones Jasmonic Acid and Methyl Jasmonate

Induced chemical defense reactions are widespread in marine brown algae. Despite the evidence that the biosynthesis of defense metabolites can be up-regulated upon herbivory, we do not know how this regulation of biosynthetic pathways to secondary metabolites is achieved in brown algae. In higher plants, the phytohormone jasmonic acid (JA) is crucial for the mediation of induced chemical defenses, and several findings of this metabolite from marine sources have been reported. We tested the hypothesis that JA or related metabolites play a role in induced brown algal defense. Quantification of oxylipins with a detection limit around 20 ng g⁻¹ algal tissue did not reveal the presence of JA in the seven examined brown algal species Dictyota dichotoma, Colpomenia peregrina, Ectocarpus fasciculatus, Fucus vesiculosus, Himanthalia elongata, Saccharina latissima (formerly Laminaria saccharina), and Sargassum muticum. Moreover, treatment with ecologically relevant concentrations of JA and methyl jasmonate did not lead to a significant change in the profile of medium- and non-polar metabolites of the tested algae. Only when high concentrations of ≥500 μg ml⁻¹ medium of the phytohormones were applied that a metabolic response which could be attributed to unspecific stress was observed. Bioassays with D. dichotoma that focused on medium- and non-polar compounds confirmed the lack of a biological role of JA and methyl jasmonate in the induction of algal induced chemical defenses. The phytohormone-treated samples did not exhibit any increased defense potential towards the amphipod Ampithoe longimana and the isopod Paracerceis caudata. JA and related phytohormones, known to be active in higher plants, thus appear to play no role in brown algae for induction of the defense chemicals studied here.     

Scopolamine in <Emphasis Type="Italic">Brugmansia Suaveolens</Emphasis> (Solanaceae): Defense,Allocation, Costs,and Induced Response

Brugmansia suaveolens (Solanaceae) contains tropane alkaloids (TAs), which can act as chemical defenses. Selective pressures might modulate the allocation of alkaloids within the plant, as postulated by optimal-defense theory. By tracing scopolamine, the most abundant TA in this species, we found that scopolamine in an artificial diet, in concentrations similar to those in leaves of B. suaveolens, increased mortality and prolonged developmental time of the larvae of the generalist noctuid moth Spodoptera frugiperda. A diet of undamaged leaves of B. suaveolens also showed a large negative effect on the growth of larvae of S. frugiperda compared to a diet of leaves of Ricinus communis, a species that did not have negative effects on this moth; more valuable plant parts, such as young leaves, flowers, and unripe fruits with seeds, have higher scopolamine concentrations than other tissues; leaves of B. suaveolens increase their content of scopolamine after artificial damage. The highest induction was found 24 hr after the damage, and after that, scopolamine content decreased to constitutive levels. This increase represented a cost, because in another experiment, a treatment with methyl jasmonate, an elicitor hormone, increased scopolamine production 9.5-fold and decreased leaf growth 2.3-fold; a diet of artificially damaged leaves of B. suaveolens showed a negative effect on the growth of larvae of S. furgiperda compared to undamaged leaves, suggesting that damage by herbivores induces resistance. Our data are in line with the optimal-defense theory, but experiments in the field with herbivores that share an evolutionary history with B. suaveolens must be undertaken to understand the dynamics of TA allocation in response to herbivory.     

Exogenous jasmonates simulate insect wounding in tomato plants (Lycopersicon esculentum) in the laboratory and field

Wounding increases the levels and activities of several defense-related proteins in the foliage of the tomato plant,Lycopersicon esculentum Mill. Evidence indicates that two of these responses, the systemic increases in polyphenol oxidase and proteinase inhibitors, are regulated by an octadecanoid-based signalling pathway which includes the wound hormone, jasmonic acid. It is not known whether other responses to wounding are also regulated by this same signalling pathway. In this paper, we show that application of jasmonates (jasmonic acid or its volatile derivative, methyl jasmonate) in low concentrations to foliage of young tomato plants induced, in a dose-dependent manner, the same protein responses-polyphenol oxidase, proteinase inhibitors, lipoxygenase, and peroxidase-as doesHelicoverpa zea Boddie feeding. Application of jasmonic acid to a single leaflet of four-leaf tomato plants induced these four proteins in a spatial pattern nearly identical to that produced by localized feeding ofH. zea. Exogenous jasmonic acid also decreased suitability of foliage for the beet armyworm,Spodoptera exigua Hubner in the laboratory. Based on these results, we conducted an experiment to measure the effects of jasmonic acid spray under field conditions. We provide the first evidence that jasmonic acid spray on field plants induces production of chemical defenses above the levels found in unsprayed controls. Exogenous jasmonic acid sprayed on plants in agricultural plots increased levels of polyphenol oxidase and proteinase inhibitors. Because application of jasmonic acid induces these defensive compounds at low concentrations in a manner similar to natural wounding, it may prove to be a useful tool for stimulating plant resistance to insects in the field.     

The Effect of Exogenous Jasmonic Acid on Induced Resistance and Productivity in Amaranth (Amaranthus hypochondriacus) Is Influenced by Environmental Conditions

Amaranthus hypochondriacus is a C4 pseudocereal crop capable of producing reasonable grain yields in adverse environmental conditions that limit cereal performance. It accumulates trypsin inhibitors and alpha-amylase inhibitors in seeds and leaves that are considered to act as insect feeding deterrents. Foliar trypsin and alpha-amylase inhibitors also accumulate by treatment with exogenous jasmonic acid (JA) in controlled laboratory conditions. Three field experiments were performed in successive years to test if two nonphytotoxic dosages of JA were capable of inducing inhibitor activity in A. hypochondriacus in agronomical settings, and if this induced response reduced insect herbivory and insect abundance in foliage and seed heads. The performance of JA-treated plants was compared to insecticide-treated plants and untreated controls. The effect of exogenous JA on the foliar levels of six additional putatively defence proteins was also evaluated. Possible adverse effects of JA induction on productivity were evaluated by measuring grain yield, seed protein content, and germination efficiency. The results present a complex pattern and were not consistent from year to year. To some extent, the yearly variability observed could have been consequence of growth under drought versus nondrought conditions. In a drought year, JA-treated plants had lower levels of insect herbivory-derived damage in apical leaves and panicle than control plants, whereas in nondrought years, there was an inconsistent effect on aphids, with no effect on lepidopteran larvae. JA treatments reduced the size of the insect community in seed heads. The effect varied with year. Exogenous JA did not adversely affect productivity, and in the absence of drought stress, the higher dosage enhanced grain yield. Induction of defensive proteins by JA, although sporadic, was more effective in nondrought conditions. The patterns of foliar protein accumulation observed suggest that they may be part of a constitutive, rather than inducible, chemical defense mechanism that is developmentally regulated and critically dependent on the environment. The results emphasize the difficulties that are often encountered when evaluating the performance of chemical elicitors of induced resistance in field settings.     

Electrochemical synthesis and characterization of poly(2,2’-dithiodianiline) thin films loaded with copper microparticles. Application to the amperometric analysis of γ-aminobutyric acid

Summary Modified electrodes with poly(2,2’-dithiodianiline),PDTDA, were prepared from the corresponding monomer by cyclic voltammetry between 0.0 V and 1.5-1.6 V on gold or stainless steel respectively. The potentiodynamic method proved to bring about good quality and adherent thin films. The wide potential window shown by this modified electrode allowed attempting the insertion of copper in the polymeric matrix using several strategies. The response of this modified electrode copper was checked in the presence of γ-aminobutyric acid (GABA). Copper loaded on PDTDA/Au electrodes showed the highest sensitivity along with a stable and reproducible response for the detection of GABA. The surface morphology and composition analysis by SEM and XPS shows that copper is deposited in the polymeric matrix as uniformly scattered microparticles. The surface of these particles is mainly composed of Cu(I) species.     

Analysis ofcis- andtrans-fatty acid isomers in hydrogenated and refined vegetable oils by capillary gas-liquid chromatography

For quantitation ofcis- andtrans-fatty acid isomers, infrared (IR) spectroscopy, gas-liquid chromatography (GLC) on highly polar stationary phases or the combination (GLC-IR) may be used. IR offers the advantage of simplicity and speed, but the lower determination limit of 5% and the lack of detailed information limit its use. Detailed fatty acid information, required for, e.g., food-labeling purposes, can only be obtained with GLC methods. Most of the GLC methods are optimized for partially hydrogenated samples. AOCS Official Method Ce 1c-89 prescribes a single, highly polar stationary phase, SP2340, but underestimates the amount oftrans isomers due to 18∶1 positional isomer overlap. The combined GLC-IR method may circumvent this problem but at the cost of time, effort, and precision.Trans isomers in refined (deodorized or stripped) oils are different in type and levels from isomers in partially hydrogenated oils; theirtrans isomers are mono-trans trienoic and dienoic isomers, occurring at levels up to about 1–3%. GLC conditions for hydrogenated samples are often not suitable for refined oils because of overlap problems, but this time in the 18∶3 region. Through careful selection of stationary phase and temperature program optimization (Drylab^®GC), we have developed a single method that is suitable for hydrogenated, as well as refined, processed oils. The accuracy was checked withcis andtrans fatty acid fractions isolated by silverion exchange high-performance liquid chromatography. Thetrans values obtained with the optimized method are in good agreement with the results obtained for the isolated fractions. We propose that recommended methods describe GLC conditions in terms of separation criteria rather than recommending only a fixed combination of stationary phase and temperature program.     

Direct synthesis of poly(l-lactic acid) in supercritical carbon dioxide with dicyclohexyldimethylcarbodiimide and 4-dimethylaminopyridine

The direct synthesis of poly(l-lactic acid) (PLLA) from an l-lactic acid oligomer has been performed in supercritical carbon dioxide (scCO₂) using an esterification promoting agent, dicyclohexyldimethylcarbodiimide (DCC), and 4-dimethylaminopyridine (DMAP) as a catalyst. PLLA within M_n of 13,500 g/mol was synthesised in 90% yield at 3500 psi and 80 °C after 24 h. The molecular weight distribution of the products was narrower than PLLA prepared with melt-solid phase polymerisation under conventional conditions. Both DCC and DMAP showed high solubility in scCO₂ (DCC: 7.6 wt% (1.63×10⁻² mol/mol CO₂) at 80 °C, 3385 psi, DMAP: 4.5 wt% (1.62×10⁻²mol/mol CO₂) at 80 °C, 3386 psi) and supercritical fluid extraction was found to be effective at removing excess DMAP and DCC after the polymerisation was complete. We show that DCC and DMAP are effective esterification promoting reagents with further applications for condensation polymerisations in scCO₂.