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.     

Wound-induced changes in root and shoot jasmonic acid pools correlate with induced nicotine synthesis inNicotiana sylvestris spegazzini and comes

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 from¹⁵NO₃ 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.     

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.     

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.     

Alkaloidal responses to damage inNicotiana native to North America

We performed field tests of alkaloid induction inNicotiana attenuata plants growing in southwestern Utah with mimicry of the two major types of damage inflicted by invertebrate and vertebrate herbivores: leaf damage and stalk removal, respectively. In undamaged plants, seasonal increases in leaf nicotine content occurred at a rate of 0.046% leaf dry mass/day. Leaf damage doubled the accumulation rate to 0.086–0.138% leaf dry mass/day, while stalk removal resulted in a quadrupling of the accumulation rate to 0.206% leaf dry mass/day. These damage-induced increases in nicotine accumulation are significantly larger than between-plant and phenological variations. Leaf damage to the nornicotine-(N. repanda andN. trigonophylla) and anabasine-accumulating (N. glauca)Nicotiana species native to North America resulted in 1.5- to 5-fold increases in their principal leaf alkaloid pools. We conclude that alkaloid induction is not limited to nicotine-accumulatingNicotiana species and that herbivores feeding on previously damaged plants are likely to encounter tissues with alkaloid titers significantly higher than those of undamaged plants.     

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.     

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.     

Cost-cutting of nitrogen source for economical production of cellulolytic enzymes by Trichoderma inhamatum KSJ1

For saccharifying food wastes, cellulolytic enzymes were produced using Trichoderma inhamatum KSJ1 in modified Mandel’s medium. In a previous study, 0.1% bacto peptone in Mandel’s medium was established as the best organic nitrogen source for the production of cellulolytic enzymes using strain KSJ1. However, economically, peptone was too expensive. Therefore, soybean, yeast and Chunggookjang (fermented soybean paste) were substituted for peptone in this research. Also, yeast or ground soybean hydrolyzed by sulfuric acid or from a culture broth of Bacillus alcalophilus, a strain producing protease, was added to the medium as the nitrogen source to the production of cellulolytic enzyme. In the cultivation using 0.5% yeast hydrolyzed with a culture solution of B. alcalophilus as the nitrogen source, the activities of FPase and amylase were 0.20 and 2.17 U/mL in a 100 mL flask, compared to 0.35 and 1.24 U/mL with the 0.1% peptone as control, respectively. In a 10 L jar fermenter, the activities of FPase and amylase were improved to 0.40 and 4.82 U/mL in the cultivation, respectively, using 0.5% yeast hydrolyzed with the culture broth, compared with 0.38 and 3.79 U/mL, respectively, for the 0.1% peptone as control. Therefore, hydrolyzed yeast was established as an available nitrogen source for the industrial scale production of cellulolytic enzymes by strain KSJ1, resulting in a 52.3% cost reduction in the production of cellulolytic enzyme by substitution of the expensive nitrogen sources.     

Catalytic reduction of NH4NO3 by NO: Effects of solid acids and implications for low temperature DeNOx processes

Ammonium nitrate is thermally stable below 250 °C and could potentially deactivate low temperature NO_x reduction catalysts by blocking active sites. It is shown that NO reduces neat NH₄NO₃ above its 170 °C melting point, while acidic solids catalyze this reaction even at temperatures below 100 °C. NO₂, a product of the reduction, can dimerize and then dissociate in molten NH₄NO₃ to NO⁺ + NO₃⁻, and may be stabilized within the melt as either an adduct or as HNO₂ formed from the hydrolysis of NO⁺ or N₂O₄. The other product of reduction, NH₄NO₂, readily decomposes at ≤100 °C to N₂ and H₂O, the desired end products of DeNO_x catalysis. A mechanism for the acid catalyzed reduction of NH₄NO₃ by NO is proposed, with HNO₃ as an intermediate. These findings indicate that the use of acidic catalysts or promoters in DeNO_x systems could help mitigate catalyst deactivation at low operating temperatures (<150 °C).     

Continuous production of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate): Effects of C/N ratio and dilution rate on HB/HV ratio

Ralstonia eutropha was cultivated in a continuous stirred fermenter with various C/N ratios (20, 30, and 40), dilution rates, and organic salt substrates (sodium propionate or sodium valerate) to explore the microbial growth and the poly(3HB-co-3HV) accumulation. When sodium propionate was used as the secondary carbon source, the HB/HV molar ratio at various C/N ratios and dilution rates did not change appreciably (approximately 90: 10). The highest poly(3HB-co-3HV) content in biomass (41.8%) and poly(3HB-co-3HV) productivity (0.100 g/(L·h)) occurred under the condition with a C/N ratio of 20 and dilution rate of 0.06 h⁻¹. When sodium valerate was used as the secondary carbon source, the productivity of poly(3HB-co-3HV) increased with increasing dilution rate for the C/N ratio of 30 and 40. The average HB/HV molar ratio ranged from 48: 52 to 78: 32. The feeding of sodium valerate promoted the accumulation of HV better than feeding sodium propionate did. This study shows that a potential strategy of manipulating by both C/N ratio and dilution rate could be used to control the HV unit fraction in poly(3HB-co-3HV) in a continuous cultivation.     

Optimisation of SFE method on-line coupled to FT-IR spectroscopy for the real-time monitoring of the extraction of tagitinin C in T. diversifolia

The monitoring in real time of dynamic extractions of tagitinin C from Tithonia diversifolia leaves was carried out with a home made high-pressure fiber optic cell which coupled a supercritical fluid extractor with carbon dioxide as the extraction medium and a FT-IR spectrometer equipped with a mercury cadmium telluride detector (MCT). The shape of extraction curves obtained during the monitoring was used to decide when to stop the supercritical fluid extraction (SFE).No significant density dependence of the molar absorption coefficient or wavenumber of the CO stretching vibration (ν_CO) of tagitinin C at 1668 cm⁻¹ was noticed.The physical characteristics of SCCO₂ governing the extraction yield of the active component from leaves were optimized by means of a central composite design (CCD). The studied variables were temperature (40, 60 and 80 °C) and pressure (8.0, 14.0 and 20.0 MPa) of the supercritical fluid.The composition profile of T. diversifolia extracts obtained by SFE was investigated in the range from 3400 to 2600 cm⁻¹ according to the pressure and temperature conditions of SCCO₂. The qualitative approach of the extracts composition was accomplished through the CH stretching vibrations (ν_CH) of components.     

Fe/N/C non-precious catalysts for PEM fuel cells: Influence of the structural parameters of pristine commercial carbon blacks on their activity for oxygen reduction

Fifteen commercial SRCC furnace carbon blacks of various grades, ranging from N1 to N9, were used as carbon supports in the preparation of Fe/N/C type electrocatalysts for the oxygen reduction reaction (ORR) in PEM fuel cell conditions. All catalysts were prepared by loading the various carbon grades with 0.2 wt.% Fe as iron acetate and heat-treating the resulting material at 950 °C in pure NH₃. This reaction provides the nitrogen content and the microporosity necessary to synthesize and host the Fe/N/C catalytic sites that perform ORR. The maximum catalytic activity (V_pr max) for each carbon grade was determined by optimizing pyrolysis time. The aim of this study is to determine which structural characteristics of the pristine carbon black are important for maximizing catalytic activity. Three structural parameters that influenced the catalytic site density on the carbon support were identified. They are: (i) the average particle diameter of the pristine carbon black, d_particle, available from BET area measurements; (ii) the amount of disordered phase which is proportional to W_D, the width at half maximum of the D peak in the Raman spectrum of the pristine carbon; and (iii) the mean size of the graphene layers characterizing the graphitic crystallites in the carbon black, L_a. The latter is available by Rietveld analysis of the XRD spectra of the pristine carbons. The best catalytic activities are obtained for the smallest d_particle, the largest W_D, and the largest L_a. Optimizing these three parameters maximizes the fraction of the pristine carbon black that becomes microporous upon reaction with NH₃ and, therefore, enables the formation of Fe/N/C catalytic sites. A FeN₂₊₂/C structure bridging two adjacent graphitic crystallites is proposed as a potential model for most of the catalytic sites present in such Fe/N/C type catalysts.     

Evidence for the accessory glands as the site of production of the oviposition attractant and/or stimulant ofLutzomyia longipalpis (Diptera: Psychodidae)

The phlebotomine sandflyLutzomyia longipalpis Lutz and Neiva, the vector of visceral leishmaniasis in South America, has recently been shown to produce an oviposition semiochemical. In the present study it was found that a nonpolar extract of eggs was attractive and/or stimulatory to ovipositing females. A Chromatographic investigation indicated the presence of similar compounds in accessory glands and egg extracts. Extract of accessory gland was also found to elicit a positive oviposition response. It is concluded that the pheromone is produced in the accessory glands and is secreted onto the eggs during oviposition.     

Polyaniline supported CuI: An efficient catalyst for C–N bond formation by N-arylation of N(H)-heterocycles and benzyl amines with aryl halides and arylboronic acids, and aza-Michael reactions of amines with activated alkenes

Polyaniline supported CuI catalyst was prepared, characterized and effectively used in the N-arylation of N(H)-heterocycles and benzylamines with aryl halides and arylboronic acids and in aza-Michael reaction of amines with activated alkenes. The catalyst was recovered by simple filtration and reused for several cycles with consistent activity.