Allelopathy of oats. II. Allelochemical effect ofl-Tryptophan and its concentration in oat root exudates

l-Tryptophan caused growth inhibition of roots and hypocotyls (or coleoptiles) of cockscomb (Amaranthus caudatus L.), lettuce (Lactuca sativa L.), cress (Lepidium sativum L.), timothy (Phleum pratense L.), rice (Oryza sativa L.), wheat (Triticum aestivum L.), and oat (Avena sativa L.), increasing the dose ofl-tryptophan increased the inhibition. The concentrations for 50% inhibition of the root growth were 0.14, 0.15, 0.21, 0.79, 0.95, 1.7, and 2.4 mM for cockscomb, cress, lettuce, timothy, rice, wheat, and oat, respectively; the concentrations for 40% inhibition of the hypocotyl (or coleoptile) growth were 0.28, 0.33, 0.43, 2.7, 4.5, 7.2, and 15 mM for cockscomb, cress, lettuce, timothy, rice, wheat and oat, respectively. The levels ofl-tryptophan in oat seedlings and in its root exudates were 29.3 mg/kg fresh wt and 0.25 mM under light conditions, and 21.1 mg/kg fresh wt and 0.18 mM under dark conditions, respectively. The presence ofl-tryptophan in the root exudates coupled with its effect on growth suggested thatl-tryptophan may play an important role in the growth inhibition of other plants in nature.     

Fatty Acid Amides,Previously Identified in Caterpillars,Found in the Cricket <Emphasis Type="Italic">Teleogryllus taiwanemma</Emphasis> and Fruit Fly <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> Larvae

Fatty acid amides (FAAs) are known elicitors that induce plants to release volatile compounds that, in turn, attract foraging parasitoids. Since the discovery of volicitin [N-(17-hydroxylinolenoyl)-l-glutamine] in the regurgitant of larval Spodoptera exigua, a series of related FAAs have been identified in several other species of lepidopteran caterpillars. We screened 13 non-lepidopteran insects for the presence of FAAs and found that these compounds were present in adults of two closely related cricket species, Teleogryllus taiwanemma and T. emma (Orthoptera: Gryllidae), and larvae of the fruit fly, Drosophila melanogaster (Diptera: Drosophilidae). When analyzed by liquid chromatography/mass spectrometry-ion trap-time-of-flight (LCMS-IT-TOF), the gut contents of both crickets had nearly identical FAA composition, the major FAAs comprising N-linolenoyl-l-glutamic acid and N-linoleoyl-l-glutamic acid. There were also two previously uncharacterized FAAs that were thought to be hydroxylated derivatives of these glutamic acid conjugates, based on their observed fragmentation patterns. In addition to these four FAAs containing glutamic acid, N-linolenoyl-l-glutamine and a small amount of volicitin were detected. In D. melanogaster, N-linolenoyl-l-glutamic acid and N-linoleoyl-l-glutamic acid were the major FAAs found in larval extracts, while hydroxylated glutamic acid conjugates, volicitin and N-linolenoyl-l-glutamine, were detected as trace components. Although these FAAs were not found in ten of the insects studied here, their identification in two additional orders of insects suggests that FAAs are more common than previously reported and may have physiological roles in a wide range of insects besides caterpillars.     

Enzymatic synthesis of N-acyl-l-amino acids in a glycerol-water system using acylase I from pig kidney

N-Medium- and long-chain acyl-l-amino acids were enzymatically synthesized from the corresponding l-amino acids and fatty acids using a reverse hydrolysis. Enzymes that are suitable for the synthetic reaction of N-acyl-l-amino acids were screened on the basis of hydrolytic activity toward N-lauroyl-l-glutamic acid as an indicator. Acylase I from pig kidney (EC 3.5.1.14) showed the highest N-acyl-l-amino acid hydrolytic activity among 57 commercially available enzymes tested. Acylase I effectively catalyzed the synthesis of N-lauroyl-l-amino acids except for N-lauroyl-l-proline and N-lauroyl-l-tyrosine in a glycerol-water system. Under the optimized reaction conditions, N-lauroyl-l-arginine and N-lauroyl-l-glutamic acid were obtained in conversions of 82 and 44%, respectively. The equilibrium constants calculated from the conversion obtained were 5.6, 15.4, 18.0, and 39.4 for the syntheses of N-lauroyl-l-glutamic acid, N_α-lauroyl-l-lysine, N-lauroyl-l-glutamine, and N-lauroyl-l-methionine, respectively. N-Acyl-l-arginines with myristic acid and palmitic acid as the fatty acid were also synthesized using acylase I.     

Identification of host-plant chemicals stimulating oviposition by swallowtail butterfly,Papilio protenor

The ovipositional response of a Rutaceae-feeding papilionid butterfly,Papilio protenor, toCitrus host plants was evoked by the synergistic action ofl-(–)-stachydrine,d-(–)-quinic acid, (–)-synephrine, andl-(–)-proline that characterize the chemical compositions of the leaves and epicarp ofCitrus plants (C. natsudaidai andC. unshiu). The stimulatory activity of their mixture was enhanced by the addition of flavanone glycosides, naringin and hesperidin, which coexist in these plants and have previously been demonstrated to serve as oviposition stimulants. However, sugars such as sucrose, glucose, and inositols, which abound in plant tissues, exerted no effect on egg-laying by the females. On the other hand, chlorogenic acid present in the leaves of another host plant,Fagara ailanthoides, was found to act as an excellent synergist. However, there existed significant qualitative dissimilarities between the chemical compositions of the leaves ofC. unshiu andF. ailanthoides. This strongly suggests thatP. protenor is likely to utilize different categories of compounds as chemical cues in recognizing each plant as a host.     

Biochemical insight into insecticidal properties ofl-Canavanine,a higher plant protective allelochemical

l-Canavanine manifests potent insecticidal properties in a canavanine-sensitive insect such as the tobacco hornworm,Manduca sexta (L.) (Sphingidae). Investigations of the biochemical basis for the antimetabolic properties of this arginine analog reveal that it is activated and aminoacylated by arginyl tRNA synthetase and incorporated into the nascent polypeptide chain. This creates structurally aberrant, canavanine-containing proteins that can possess altered physicochemical properties. Evidence is presented in studies with the tobacco hornworm; the canavanine-adapted bruchid beetle,Caryedes brasiliensis (Bruchidae) and the weevil,Sternechus tuberculatus (Curculionidae); as well as the canavanine-resistant larvae ofHeliothis virescens [Noctuidae] to support the contention that formation of aberrant, canavanyl proteins produce deleterious biological effects and is a significant basis for canavanine's antimetabolic properties.     

High yields of ascorbyl palmitate by thermostable lipase-mediated esterification

High yields of ascorbyl palmitate (6-O-palmitoyl-l-ascorbic acid) were obtained by lipase-mediated esterification using Bacillus stearothermophilus SB 1 lipase. The final yield was greatly influenced by the initial water content of the system, quantity of enzyme, and molar ratio of palmitic acid to l-ascorbic acid. Reaction rates increased directly with temperature from 40 to 100°C. Maximum conversion (97%) was achieved after 30 min at 100°C (solvent-free), 1 h at 80°C (solvent-free), and 2 h at 60°C (solvent/hexane). The synthesis was scaled up to 1-l volume with 95% conversion using 50 mmoles of ascorbic acid and 250 mmoles of palmitic acid in hexane. Similar yields of ester were obtained in five repetitive cycles using 5 g enzyme immobilized on Accurel. The present B. stearothermophilus SB 1 lipase was a more efficient catalyst for the synthesis of ascorbyl palmitate than other commercial lipases.     

Enrichment of CLA isomers by selective esterification with l-menthol using Candida rugosa lipase

Commercially available preparations of CLA are composed of almost equal amounts of 9-cis,11-trans (9c,11t)-CLA and 10-trans,12-cis (10t,12c)-CLA. Each isomer was fractionated and enriched, for availability as a food supplement, by a process comprising selective esterification with l-menthol by Candida rugosa lipase, distillation, and n-hexane extraction. The first selective esterification of CLA isomers was conducted with an equimolar amount of l-menthol of 30°C. The oil phase of the reaction mixture was fractionated into an l-menthyl ester fraction (9c,11t-CLA rich) and an FFA fraction (10t,12c-CLA rich) by distillation. The FFA fraction was esterified again with an equimolar amount of l-menthol to enrich 10t,12c-CLA. The 10t,12c-CLA preparation was obtained as the resulting FFA fraction by distillation. 10t,12c-CLA was enriched to 91% with 40% recovery. To enrich 9c,11t-CLA, the l-menthyl ester fraction in the first esterification was chemically hydrolyzed, and the resulting FFA were esterified again with an equimolar amount of l-menthol. The 9c, 11t-CLA preparation was obtained by chemical hydrolysis of the resulting l-methyl ester fraction, followed by n-hexane extraction. 9c,11t-CLA was enriched to 94% with 42% recovery. This effective process for purification of CLA isomers using l-methol is applicable to the production of food supplements.     

A novel ∈-lysine acylase from <Emphasis Type="Italic">Streptomyces mobaraensis</Emphasis> for synthesis of <Emphasis Type="Italic">N∈</Emphasis>-acyl-<Emphasis Type="SmallCaps">l</Emphasis>-lysines

A novel ∈-lysine acylase (N ₆-acyl-l-lysine amidohydrolase; EC 3.5.1.17) was isolated from Streptomyces mobaraensis and purified to homogeneity by SDS-PAGE from the culture broth. The purified enzyme was monomeric, with a molecular mass of approximately 60 kDa. The enzyme was inactivated by the presence of 1,10-phenanthroline and activated in the presence of Co²⁺ and Zn²⁺. The enzyme showed a pH optimum of 8.0 and was stable at temperatures of up to 50°C for 1 h at pH 8.0. The enzyme specifically catalyzed the hydrolysis of the amide bond of various N∈-acyl-l-lysines. Furthermore, the enzyme efficiently catalyzed the synthesis of N∈-acyl-l-lysines with fatty and aromatic acyl groups in an aqueous buffer. In the syntheses of N∈-decanoyl-l-lysine, N∈-lauroyl-l-lysine, and N∈-myristoyl-l-lysine, the product precipitated and the yield was 90% or higher using 10 mM FA and 0.5 M l-lysine as the substrate.     

Allelopathy of oats. I. Assessment of allelopathic potential of extract of oat shoots and identification of an allelochemical

The allelopathic potential of oat (Avena sativa L.) extracts was investigated under laboratory conditions. The ethyl ether-, acetone-, and water-soluble fractions obtained from the extract of oat shoots inhibited the germination and growth of roots and hypocotyls of lettuce (Lactuca sativa L.). The inhibitory activity of the water-soluble fraction was maximum, followed by that of ethyl ether-soluble and acetone-soluble fraction. An active principle of the water-soluble fraction was isolated and its structure was determined by spectral data asl-tryptophan.l-Tryptophan inhibited the growth of hypocotyls and roots of lettuce seedlings at concentrations greater than 0.03 and 0.1 mM, respectively. These results suggested thatl-tryptophan may be an allelochemical which affects the growth or germination of different plant species.     

Behavioral responses of crayfish (Orconectes virilis andOrconectes rusticus) to chemical feeding stimulants

We conducted two experiments to assess how chemical stimuli affect feeding behavior, grooming, and walking in the crayfishesOrconectes virilis andOrconectes rusticus. In the first experiment,O. virilis was tested with 29 amino acids; in the second experiment,0. rusticus was tested with 12 amino acids, 13 additional single compounds, and two six-compound mixtures. InO. virilis, the following amino acids, in order of potency, elicited feeding movements:l-isoleucine, glycine, hydroxy-l-proline,l-glutamate,l-valine, and B-alanine. Grooming increased in response tol-phenylalanine,l-tryptophan,l-tyrosine,l-leucine,l-methionine, and D-aspartate. InO. rusticus, both mixtures and the following single compounds, in order of potency, elicited feeding movements: cellobiose, sucrose, glycine, maltose, glycogen, nicotinic acid methyl ester, putrescine, andl-glutamate. Grooming increased in response to putrescine only, and walking increased in response to glycogen only. The responsiveness of these crayfishes to a wide variety of chemicals may reflect the omnivorous foraging habits of these crustaceans.     

Experimental Evidence for Three Pheromone Races of the Scarab Beetle <Emphasis Type="Italic">Phyllophaga anxia</Emphasis> (LeConte)

This study offers experimental evidence for the existence of three pheromone races of the northern genitalic form of Phyllophaga anxia: one race in which females produce and males respond mainly to l-valine methyl ester, a second producing and responding to l-isoleucine methyl ester, and a third producing and responding to an intermediate range of blends of the two compounds. At Franklinville, NY, pheromone gland contents of females were analyzed using coupled gas chromatography–electroantennogram detection. Two types of females were found, one that produced greater than 99% l-valine methyl ester and another that produced greater than 99% l-isoleucine methyl ester. Capture–mark–release–recapture field tests with males at Franklinville established that most males were recaptured in traps baited with the same blends with which they were originally captured. The populations characterized at Franklinville, NY, have also been found at numerous locations from eastern Canada and the northeast and north central USA, sometimes in allopatry and sometimes in sympatry. At a site in Carver, MA, P. anxia males responded to blends of the methyl esters of l-valine and l-isoleucine, and Carver females produced blends similar to those to which the males responded. Populations responding to blends have been identified only from southeastern Massachusetts and Rhode Island. At a field site near Waterloo, NY, the addition of small proportions of l-isoleucine methyl ester to lures containing l-valine methyl ester did not affect trap captures, but higher proportions of l-isoleucine methyl ester were inhibitory, decreasing trap captures.     

<Emphasis Type="SmallCaps">l</Emphasis>-DOPA Increases Lignification Associated with <Emphasis Type="BoldItalic">Glycine max</Emphasis> Root Growth-Inhibition

l-3,4-dihydroxyphenylalanine (l-DOPA), an allelochemical exuded from the roots of velvet bean [Mucuna pruriens (L.) DC. var. utilis], presents a highly inhibitory action to plant growth. The effects of l-DOPA on phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and peroxidase (POD, EC 1.11.1.7) activities, and phenolic compound and lignin content in soybean [Glycine max (L.) Merr.] roots were investigated to determine the possible phytotoxic mechanism. Three-day-old seedlings were cultivated in half-strength Hoagland nutrient solution (pH 6.0), without or with 0.1 to 1.0 mM l-DOPA in a growth chamber (25°C, 12-hr light to 12-hr darkness photoperiod, irradiance of 280 μmol m⁻² s⁻¹) for 24 hr. In general, the length, fresh weight, and dry weight of the roots decreased, whereas PAL and POD activities and phenolic compound and lignin content increased after l-DOPA treatments. Results showed the susceptibility of soybean to l-DOPA and reinforce the role of this nonprotein amino acid as a strong allelochemical. The present findings also suggest that l-DOPA-induced inhibition in soybean roots may be because of a cell wall stiffening process related to the formation of cross-linking between cell wall polymers linked to lignin production.     

Continuous production of acyl <Emphasis Type="SmallCaps">l</Emphasis>-ascorbates using a packed-bed reactor with immobilized lipase

Saturated acyl (6-O-caproyl, lauroyl, and myristoyl) and unsaturated acyl (6-O-oleoyl, linoleoyl, and arachidonoyl) l-ascorbates were continuously synthesized at 50°C using a system where a column packed with ascorbic acid powder and a packed-bed reactor with an immobilized lipase from Candida antarctica were connected in series. A productivity of 1.6–1.9 kg/L reactor·d was achieved for at least 11 d. The surface tension of the caproyl or lauroyl l-ascorbate in aqueous solution was measured at various temperatures and pH to estimate the critical micelle concentration (CMC) of the acyl l-ascorbate. The CMC values were independent of temperature but dependent on the pH. The value of the caproyl ascorbate increased with an increase in pH.     

A Feeding Stimulant for Manduca sexta from Solanum surattenses

The acceptance of Solanum surattenses as a host plant for the larvae of Manduca sexta was explained by the presence of feeding stimulants in foliage. Bioassay-guided fractionation of plant extracts resulted in the isolation of a highly active compound (1), which was identified as a furostan derivative {26-O-β-d-glucopyranosyl-(25R)-furosta-5-ene-3-β-yl-O-α-l-rhamnopyranosyl-(1″-2′)-O-α-l-rhamnopyranosyl-(1′″-3″)-O-β-d-glucopyranoside}. This compound has the same steroidal core substructure as that in a stimulant (indioside D) previously identified from potato foliage. However, the sugar substituents attached to the core are different.     

Oxidation kinetics of linoleic acid in the presence of saturated acyl <Emphasis Type="SmallCaps">l</Emphasis>-ascorbate

The oxidation processes of linoleic acid (LA) in the presence of l-ascorbic acid or saturated acyl l-ascorbate additives were measured at various temperatures and molar ratios of the additive to LA. Higher oxidative stability of LA was observed at higher additive levels for all additives. The addition of the ascorbates lengthened the induction period for the oxidation of LA. An autocatalytic kinetic rate equation was used to model the oxidation processes of LA mixed with the ascorbates, and the dependence of the rate constant, k, on acyl-chain carbon number was determined. At any temperature, the use of ascorbate additives decreased the k value for LA, and there was a slight tendency for k values to decrease with increasing acyl-chain length. The apparent activation energy, E _a, and the frequency factor, k ₀, for the rate constant were determined from Arrhenius plots. The calculated E _a and k ₀ values also decreased with increasing ascorbate acyl-chain length.     

Involvement of reactive oxygen species generated from melanin synthesis pathway in phytotoxicty of <Emphasis Type="SmallCaps">L</Emphasis>-Dopa

l-DOPA is an active allelochemical that inhibits plant growth. To determine whether the phytotoxicity is due to the reactive oxygen species generated during its oxidation to melanin, oxidative damage, melanin accumulation, and the effect of antioxidants on its phytotoxicity were examined in l-DOPA-tolerant (barnyard grass) and -susceptible (lettuce) plants, and in suspension-cultured carrot cells. l-DOPA suppressed root elongation in lettuce compared to barnyard grass. Levels of melanin and thiobarbituric acid reactive substances (TBARS) increased remarkably in l-DOPA-treated lettuce roots, but not in barnyard grass. l-DOPA also suppressed carrot cell growth to 60% of the control at 1 mM. Melanin content in 1 mM l-DOPA-treated carrot cells increased continuously; however, ascorbic acid and -tocopherol suppressed accumulation. When melanin formation was inhibited by ascorbic acid and -tocopherol, growth of l-DOPA-treated cells was restored. TBARS levels were higher in 1 mM l-DOPA-treated carrot cells than in untreated control cells 2 d after treatment, but not at 4 or 6 d. Ascorbic acid and -tocopherol suppressed the production of lipid peroxide during the initial 2 d. These results suggest that the phytotoxicity of l-DOPA is due to oxidative stress caused by reactive oxygen species from the melanin synthesis pathway.     

Biocatalytic synthesis of chiral intermediates for antiviral and antihypertensive drugs

The chiral intermediate (1S,2R) [3-chloro-2-hydroxy-1-(phenylmethyl)propyl] carbamic acid, 1,1-dimethylethyl ester 2a was prepared for the total synthesis of a human immunodeficiency virus protease inhibitor, BMS-186318. The stereoselective reduction of (1S) [3-chloro-2-oxo-1(phenylmethyl)propyl] carbamic acid, 1,1-dimethylethyl ester 1 was carried out using microbial cultures, among which Streptomyces nodosus SC 13149 efficiently reduced 1 to 2a. A reaction yield of 80%, enantiomeric excess (e.e.) of 99.8%, and diastereomeric purity of 99% were obtained for chiral alcohol 2a. Chiral l-6-hydroxy norleucine 3, an intermediate in the synthesis of antihypertensive drug, was prepared by reductive amination of 2-keto-6-hydroxyhexanoic acid 4 using beef liver glutamate dehydrogenase. The cofactor NADH required for this reaction was regenerated using glucose dehydrogenase from Bacillus sp. A reaction yield of 80% and e.e. of 99.5% were obtained for l-6-hydroxynorleucine 3. To avoid the lengthy chemical synthesis of the ketoacid, a second route was developed in which racemic 6-hydroxynorleucine [readily available from hydrolysis of 5-(4-hydroxybutyl) hydantoin 5] was treated with d-amino acid oxidase from Trigonopsis variabilis to selectively convert the d-isomer of racemic 6-hydroxynorleucine to 2-keto-6-hydroxyhexanoic acid 4 and l-6-hydroxynorleucine 3. Subsequently, the 2-keto-6-hydroxyhexanoic acid 4 was converted to l-6-hydroxynorleucine by reductive amination using glutamate dehydrogenase. A reaction yield of 98% and an e.e. of 99.5% were obtained.     

Determination of feeding preference of Formosan subterranean termite (Coptotermes formosanus Shiraki) for some amino acid additives

A choice feeding test using 21 amino acids was conducted to determine the feeding preference of Formosan subterranean termite,Coptotermes formosanus Shiraki, in the laboratory. Significantly more filter paper treated withd-aspartic acid orl-glutamic acid was consumed by Formosan subterranean termites than was control filter paper treated with water. In two-choice feeding tests, termites consumed significantly more filter paper treated withd-aspartic acid orl-aspartic acid than paper treated with water. Addingl-proline,l-lysine, orl-isoleucine to filter paper significantly increased consumption compared with control filter paper in no-choice tests. The use of amino acid additives in termite baits is briefly discussed.     

Mechanism of Selective Phytotoxicity of l-3,4-Dihydroxyphenylalanine (l-Dopa) in Barnyardglass and Lettuce

l-3,4-Dihydroxyphenylalanine (l-dopa) is one of the few allelochemicals in which the phytotoxic action mechanism has been studied. Excess exogenous l-dopa suppresses root elongation in some plant species, and the inhibitory action is species-selective. The main factor of phytotoxicity of l-dopa is considered to be oxidative damage by reactive oxygen species (ROS) and/or free radical species (FRS). This study was performed to elucidate the mechanism of species-selective phytotoxicity. The involvement of ROS/FRS and polyphenol oxidase (PPO) in species-selective phytotoxicity was examined with barnyardgrass (Echinochloa crus-galli L.) and lettuce (Lactuca sativa L.), tolerant and susceptible species, respectively. Lipid peroxidation and melanin accumulation correlated with growth inhibition by L-dopa. Antioxidants, ascorbic acid and α-tocopherol, decreased lipid peroxidation and melanin accumulation and rescued lettuce root from growth inhibition. The oxidation of L-dopa by PPO was much greater in lettuce than in barnyardgrass. From these results, the phytotoxicity of L-dopa is considered due to the oxidative damage caused by ROS/FRS generated from the melanin synthesis pathway. PPO activity might be involved in the mechanism of species-selective phytotoxicity between barnyardgrass and lettuce.     

Role of Catechol Structure in the Adsorption and Transformation Reactions of <Emphasis Type="SmallCaps">l</Emphasis>-D<Emphasis Type="SmallCaps">opa</Emphasis> in Soils

3-(3′,4′-Dihydroxyphenyl)-l-alanine (l-DOPA), which is synthesized in velvet bean (Mucuna pruriens), inhibits plant growth. The concentration of l-DOPA in soil is reduced by adsorption and transformation reactions, which can result in the reduction of its plant-growth-inhibitory activity. To determine which part of the l-DOPA structure is involved in the adsorption and soil transformation reactions, we compared the kinetics of l-DOPA disappearance in a volcanic ash soil with that of l-phenylalanine (3-phenyl-l-alanine) and l-tyrosine (3-(4′-hydroxyphenyl)-l-alanine), compounds that are similar in structure to l-DOPA but do not have a catechol (o-dihydroxybenzene) moiety. l-Phenylalanine and l-tyrosine were not adsorbed and transformed in the soil at equilibrium pH values between 4 and 7. These results suggest that the adsorption and transformation reactions of l-DOPA in the soil involve the catechol moiety and not the amino and carboxylic acid groups, which are common to all three compounds. Like l-DOPA, (+)-catechin, another allelochemical that contains a catechol moiety, underwent adsorption and soil transformation reactions. Thus, we concluded that the concentrations of allelochemicals bearing a catechol moiety in soils will decrease rapidly owing to adsorption and transformation reactions, and this decrease will be faster in soils with a high pH value or high adsorption ability. Owing to this decrease in concentration, allelopathic phenomena may not occur.