Novel amphiphilic temperature responsive graft copolymers PCL-<Emphasis Type="Italic">g</Emphasis>-P(MEO<Subscript>2</Subscript>MA-<Emphasis Type="Italic">co</Emphasis>-OEGMA) via a combination of ROP and ATRP: synthesis,characterization, and sol-gel transition

A series of well-defined novel amphiphilic temperature-responsive graft copolymers containing PCL analogues P(αClεCL-co-εCL) as the hydrophobic backbone, and the hydrophilic side-chain PEG analogues P(MEO₂MA-co-OEGMA), designated as P(αClεCL-co-εCL)-g-P(MEO₂MA-co-OEGMA) have been prepared via a combination of ring-opening polymerization (ROP) and atom transfer radical polymerization (ATRP). The composition and structure of these copolymers were characterized by ¹H NMR and GPC analyses. The self-assembly behaviors of these amphiphilic graft copolymers were investigated by UV transmittance, a fluorescence probe method, dynamic light scattering (DLS) and transmission electron microscopy (TEM) analyses. The results showed that the graft copolymers exhibited the good solubility in water, and was given the low critical temperature (LCST) at 35(±1) °C, which closed to human physiological temperature. The critical micelle concentrations (CMC) of P(αClεCL-co-εCL)-g-P(MEO₂MA-co-OEGMA) in aqueous solution were investigated to be 2.0 × 10^?3, 9.1 × 10^?4 and 1.5 × 10^?3 mg·mL^?1, respectively. The copolymer could self-assemble into sphere-like aggregates in aqueous solution with diverse sizes when changing the environmental temperature. The vial inversion test demonstrated that the graft copolymers could trigger the sol-gel transition which also depended on the temperature.     

Bioengineering functional copolymers: synthesis and characterization of poly(<Emphasis Type="Italic">N</Emphasis>-isopropyl acrylamide-<Emphasis Type="Italic">co</Emphasis>-3,4-2H-dihydropyran)s

Novel bioengineering copolymers were synthesized by radical copolymerization of N-isopropylacrylamide (NIPA) and 3,4-2H-dihydropyran (DHP) with 2,2′-azobisisobutyronitrile as an initiator in acetone solution at 70 °C under nitrogen atmosphere. Structure, tacticity and compositons of the copolymers prepared in a wide range of monomer feed were confirmed by FTIR, ¹H{¹³C} NMR-DEPT and elemental analyses. The monomer reactivity ratios (r ₁ and r ₂) were detected using known two methods: r ₁ (NIPA)?=?1.25 and r ₂?=?0.035 (DHP), and r ₁ (NIPA) ?=?0.97 and r ₂?=?0.022 (DHP) by Kelen-Tüdös and Jaacks methods, respectively. It was demonstrated that the studied monomer pair has a tendency to form H-bonding beween amide/ether groups through ?NH...O< complexation which played an important role in the stereoselective chain growth, and significant decrease of allyl degradative chain transfer reactions. This phenomenon is also confirmed by the observed relatively high molecular weights of copolymers (M _v ). The synthesized water-soluble stimuli-responsive poly(NIPA-co-DHP)s exhibit thermal stability, higher glass-transition temperature, polyelectrolyte, pH- and temperature-sensitive behavior and can be attributed to the class of bioengineering functional copolymers useful for various bio- and gene-engineering, and drug delivery applications.     

Synthesis of poly(<Emphasis Type="Italic">N</Emphasis>-isopropylacrylamide-<Emphasis Type="Italic">b</Emphasis>-<Emphasis Type="Italic">N</Emphasis>-vinylcarbazole) copolymers via RAFT polymerization and its stimuli responsive morphology in aqueous media

Here, we report the successful synthesis of series of stimuli responsive amphiphilic diblock copolymers (SRABCs) poly(N-isopropylacrylamide-b-N-vinylcarbazole) [poly(NIPAAm-b-NVK)] through reversible addition fragmentation chain transfer (RAFT) polymerization. Copolymers with fixed hydrophilic [poly(NIPAAm)] block length and variable (with three different) hydrophobic [poly(NVK)] block lengths were synthesized and the block length ratio was confirmed from their molecular weight data. The self-assembly nature of synthesized block copolymers was confirmed by determining critical micelle concentration (CMC). Self-assembled block copolymers showed rice-grain like morphology for copolymers having equivalent hydrophobic/hydrophilic chain length but in case of block copolymers having smaller and bigger hydrophobic chain length with respect to hydrophilic chain length displayed vesicular morphology. The thermo and pH responsiveness of the block copolymers was found to be influenced by variation in length and chemical composition of the blocks. Due to their thermo and pH responsiveness resulted self-assembled structures underwent morphology transitions from vesicular and rice grain like to micellar structure in aqueous medium. The probable applications of the studied stimuli responsive amphiphilic diblock copolymers can be found in the nanotechnology and biotechnology are indicated.

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Graphical abstract Synthesis, self-assembly and stimuli responsiveness of poly(NIPAAm-b-NVK) copolymers.

     

Aqueous radical polymerization of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylacrylamide redox-initiated by aerobically catalytic oxidation of water-soluble tertiary amines

Catalytic oxidation of water-soluble tertiary amines by complexes of Cu^II, Fe^III and Co^II was utilized to initiate radical polymerization of N,N-dimethylacrylamide (DMAAm) in aqueous solution at 70–80 °C. The oxidation of tertiary amines by Cu^II was studied by proton nuclear magnetic resonance spectroscopy and online ultraviolet–visible spectrophotometry. The polymerization kinetics was monitored by gas chromatography, and molecular weight of the PDMAAm was measured by gel-permeation chromatography coupled with multi-angle laser light scattering. Oxidation of tertiary amines occurs predominantly via formation of C_alpha·radicals to initiate polymerization of electron-deficient monomers and N-dealkylation, and redox equilibrium between Cu^I/L and Cu^II/L is established at a faster rate in aqueous media. Fe^III and Cu^II complexes are efficient catalysts as each catalyst molecule could generate above 10 propagating radicals in 5 h, while Co^II complex might involve in oxidation of tertiary amines in non-radical pathway, leading to a low catalytic efficiency. Water-soluble tertiary amines such as N,N-dialkylethanolamine (alkyl = methyl, ethyl etc.) are reducing agents of a higher activity in aqueous media than those primary or secondary analogues. Our strategy renders it possible to prepare polymer of alpha-amino functionality via one-pot process from commercially available commodity reagents under practical conditions with negligible catalyst residue.     

(1<Emphasis Type="Italic">S</Emphasis>,3<Emphasis Type="Italic">R</Emphasis>)-<Emphasis Type="Italic">cis</Emphasis>-Chrysanthemyl Tiglate: Sex Pheromone of the Striped Mealybug, <Emphasis Type="Italic">Ferrisia virgata</Emphasis>

Derivatives of 2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylic acid (chrysanthemic acid) are classic natural pyrethroids discovered in pyrethrum plants and show insecticidal activity. Chrysanthemic acid, with two asymmetric carbons, has four possible stereoisomers, and most natural pyrethroids have the (1R,3R)-trans configuration. Interestingly, chrysanthemic acid–related structures are also found in insect sex pheromones; carboxylic esters of (1R,3R)-trans-(2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropyl)methanol (chrysanthemyl alcohol) have been reported from two mealybug species. In the present study, another ester of chrysanthemyl alcohol was discovered from the striped mealybug, Ferrisia virgata (Cockerell), as its pheromone. By means of gas chromatography–mass spectrometry, nuclear magnetic resonance spectrometry, and high-performance liquid chromatography analyses using a chiral stationary phase column and authentic standards, the pheromone was identified as (1S,3R)-(?)-cis-chrysanthemyl tiglate. The (1S,3R)-enantiomer strongly attracted adult males in a greenhouse trapping bioassay, whereas the other enantiomers showed only weak activity. The cis configuration of the chrysanthemic acid–related structure appears to be relatively scarce in nature, and this is the first example reported from arthropods.     

Ammonia Released by <Emphasis Type="Italic">Streptomyces aburaviensis</Emphasis> Induces Droplet Formation in <Emphasis Type="Italic">Streptomyces violaceoruber</Emphasis>

Streptomyces violaceoruber grown in co-culture with Streptomyces aburaviensis produces an about 17-fold higher volume of droplets on its aerial mycelium than in single-culture. Physical separation of the Streptomyces strains by either a plastic barrier or by a dialysis membrane, which allowed communication only by the exchange of volatile compounds or diffusible compounds in the medium, respectively, still resulted in enhanced droplet formation. The application of molecular sieves to bioassays resulted in the attenuation of the droplet-inducing effect of S. aburaviensis indicating the absorption of the compound. ¹H-NMR analysis of molecular-sieve extracts and the selective indophenol-blue reaction revealed that the volatile droplet-inducing compound is ammonia. The external supply of ammonia in biologically relevant concentrations of ≥8 mM enhanced droplet formation in S. violaceoruber in a similar way to S. aburaviensis. Ammonia appears to trigger droplet production in many Streptomyces strains because four out of six Streptomyces strains exposed to ammonia exhibited induced droplet production.     

Ring-opening polymerization of <Emphasis Type="SmallCaps">l</Emphasis>-lactide catalyzed by a novel molybdenum-based catalytic system

As a transparent material that can be completely biodegradable, poly(l-lactide) (PL-LA) has recently received considerable attention. In this study, it our first efforts to fabricate l-lactide (L-LA) by a novel molybdenum-based catalytic system consisting of molybdenum pentachloride (MoCl₅) as the main catalyst and m-cresol substituted alkyl aluminum Al(OPhCH₃)(i-Bu)₂ as the co-catalyst. The effects of different types of phosphorus ligands, Al:Mo molar ratios, catalyst contents,catalyst components (separate catalysis of m-cresol aluminum and cocatalysis of Al/Mo system) and polymerization temperature were investigated. The T_g and T_m of the resulting poly(l-lactide) (PL-LA) were characterized by differential scanning calorimetry (DSC), and the molecular weight and molecular weight distribution were determined by gel permeation chromatography (GPC). The GPC results showed that the molecular weight of the PL-LA was higher than that 10⁴ g/mol and the molecular weight distribution was narrow. The structures of PL-LA was detected by ¹H NMR spectroscopy (¹H NMR) and X-ray diffraction (XRD) validation, which demonstrated that a moalr ratio of Mo/Al/l-lactide?=?1:30:1000 showed the higher conversion rate and molecular weight. In comparison to the separate catalysis of m-cresol aluminum, the molecular weight of PL-LA prepared by the cocatalysis of Al/Mo system was slightly improved, and the molecular chains were relatively regular and the crystallinity was higher.     

<Emphasis Type="Italic">m</Emphasis>-Cresol-substituted triethyl aluminum/MoCl<Subscript>5</Subscript>/TBP catalytic system for coordination polymerization of butadiene

Coordination polymerization of butadiene was initiated by a catalyst system consisting of tributyl phosphate (TBP) as ligand, molybdenum pentachloride as primary catalyst and triethyl aluminum substituted by m-cresol as co-catalyst. The effects of the substitution of m-cresol on the activity of the catalyst system, molecular weight and molecular weight distribution, intrinsic viscosity and microstructures of the resulting polymers were investigated in details. The molecular weight and molecular weight distribution of the polymerization products were determined by GPC. The microstructure of the polymerization products was characterized by FTIR, ¹³C NMR and DSC techniques. The experimental results indicated that the polymerization activity of the reaction system and the molecular weight of the polymerization products gradually increased with the increase of the substitution content of m-cresol, namely, Al(OPhCH₃)₂Et?>?Al(OPhCH₃)Et₂?>?Al(OPhCH₃)_0.5Et_2.5>AlEt₃. The 1,2-structure contents of the polymerization products could be adjusted between 89 and 91% through the control of the substitution of m-cresol, and there was minute quantities of crystalline structures in the resulting polymers due to the increasing content of the syndiotactic 1,2-polybutadiene. In a word, the existence and increase of steric hindrance of m-cresol made it easier for polymerization products to form interdisciplinary 1,2-structure.     

Efficient production of <Emphasis Type="SmallCaps">d</Emphasis>-1,2,4-butanetriol from <Emphasis Type="SmallCaps">d</Emphasis>-xylose by engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> whole-cell biocatalysts

We have developed a whole-cell bioconversion system for the production of d-1,2,4-butanetriol (BT) from renewable biomass. A plasmid pETduet-xylB-yjhG-T7-adhP-T7-mdlC was constructed and transformed to Escherichia coli BL21(DE3) to obtain the whole cells of E. coli BL21-XYMA capable of bioconversion d-xylose to BT. Then, the factors including carbon sources, nitrogen sources, metal ions, and culture conditions (pH, temperature, IPTG) were identified, and their effects on the whole-cell activity for BT production were investigated. To obtain the highest whole-cell activity, the optimal cultivation parameters are: 15 g·L^–1 yeast extract, 5 g·L^–1 sucrose, 3 g·L^–1 KH₂PO₄, 5 g·L^–1 NaCl, 3 g·L^–1 NH₄Cl, 0.25 g·L^–1 MgSO₄·7H₂O and 1 mL·L^–1 the mixture of trace elements. With the optimized whole cells of E. coli BL21-XYMA, 60 g·L^–1 of xylose was converted to 28 g·L^–1 BT with a molar yield of 66 %, which is higher than those reported in the biotechnological system.

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Synthesis and characterization of maleylated cellulose-<Emphasis Type="Italic">g</Emphasis>-polyacrylamide hydrogel using TiO<Subscript>2</Subscript> nanoparticles under sunlight

Graft polymerization onto the cellulose is one way to produce semisynthetic copolymers and semiconductors were hardly used as initiators. Maleylated cellulose (MC) with different degree of carboxyl groups was synthesized and degree of carboxyl groups was determined using titration method. Then the graft copolymers of acrylamide (AM) on MC were synthesized by titanium dioxide semiconductor photoinitiator in aqueous suspension under sunlight. The effect of different parameters, such as the degree of carboxyl groups, degassing of atmosphere, reactor type, light source, MC/AM ratio, and initiator concentration, was evaluated in the synthesis of graft copolymers. MC with a high degree of carboxyl groups about 2.8 mmol g^?1 was selected for graft photopolymerization. Maximum monomer conversion (55%) for Maleylated cellulose-g-polyacrylamide (MC-g-PAM) was achieved with 0.5 mg TiO₂, MC/AM = 0.056, argon atmosphere, sunlight source, and double quartz tube reactor. The maximum amount of equilibrium swelling (41 g g^?1) was achieved for MC-g-PAM with 34% monomer conversion. The resulting graft copolymers were characterized by FT-IR, SEM, and TGA. Synthesis of MC-g-PAM using TiO₂ nanoparticles (NPs) as the initiator was done successfully that shows the TiO₂ NPs are useable in graft polymerization of acrylamide monomers onto the MC under sunlight.     

Sexual Deception in the <Emphasis Type="Italic">Eucera</Emphasis>-Pollinated <Emphasis Type="Italic">Ophrys leochroma</Emphasis>: A Chemical Intermediate between Wasp- and <Emphasis Type="Italic">Andrena-</Emphasis>Pollinated Species

Ophrys flowers mimic sex pheromones of attractive females of their pollinators and attract males, which attempt to copulate with the flower and thereby pollinate it. Virgin females and orchid flowers are known to use the same chemical compounds in order to attract males. The composition of the sex pheromone and its floral analogue, however, vary between pollinator genera. Wasp-pollinated Ophrys species attract their pollinators by using polar hydroxy acids, whereas Andrena-pollinated species use a mixture of non-polar hydrocarbons. The phylogeny of Ophrys shows that its evolution was marked by episodes of rapid diversification coinciding with shifts to different pollinator groups: from wasps to Eucera and consequently to Andrena and other bees. To gain further insights, we studied pollinator attraction in O. leochroma in the context of intra- and inter-generic pollinator shifts, radiation, and diversification in the genus Ophrys. Our model species, O. leochroma, is pollinated by Eucera kullenbergi males and lies in the phylogeny between the wasp and Andrena-pollinated species; therefore, it is a remarkable point to understand pollinator shifts. We collected surface extracts of attractive E. kullenbergi females and labellum extracts of O. leochroma and analyzed them by using gas chromatography with electroantennographic detection (GC-EAD) and gas chromatography coupled with mass spectrometry (GC-MS). We also performed field bioassays. Our results show that O. leochroma mimics the sex pheromone of its pollinator’s female by using aldehydes, alcohols, fatty acids, and non-polar compounds (hydrocarbons). Therefore, in terms of the chemistry of pollinator attraction, Eucera-pollinated Ophrys species might represent an intermediate stage between wasp- and Andrena-pollinated orchid species.     

Synthesis and swelling behavior of metal-chelating superabsorbent hydrogels based on sodium alginate-<Emphasis Type="Italic">g</Emphasis>-poly(AMPS-<Emphasis Type="Italic">co</Emphasis>-AA-<Emphasis Type="Italic">co</Emphasis>-AM) obtained under microwave irradiation

A metal-chelating superabsorbent hydrogel based on poly(2-acrylamido-2-methylpropanesulfonic acid-co-acrylic acid-co-acrylamide) grafted onto sodium alginate backbone, NaAlg-g-poly(AMPS-co-AA-co-AM) is prepared under microwave irradiation. The Taguchi method is used for the optimization of synthetic parameters of the hydrogel based on water absorbency. The Taguchi L₉ (3⁴) orthogonal array is chosen for experimental design. Mass concentrations of crosslinker MBA \(C_{\text{MBA}}\) initiator KPS \(C_{\text{KPS}}\), sodium alginate \(C_{\text{NaAlg}}\) and mass ratio of monomers \(C_{\text{AM/AA/AMPS}}\) are chosen as four factors. The analysis of variance of the test results indicates the following optimal conditions: 0.8 g L^?1 of MBA, 0.9 g L^?1 of KPS, 8 g L^?1 of NaAlg and \(R_{\text{AM/AA/AMPS}}\) equals to 1:1.1:1.1. The maximum water absorbency of the optimized final hydrogel is found to be 822 g g^?1. The relative thermal stability of the optimized hydrogel in comparison with sodium alginate is demonstrated via thermogravimetric analysis. The prepared hydrogel is characterized by FTIR spectroscopy and scanning electron microscopy. The influence of the environmental parameters on water absorbency such as the pH and the ionic force is also investigated. The optimized hydrogel is used as adsorbent for hazardous heavy metal ions Pb(II), Cd(II), Ni(II) and Cu(II) and their competitive adsorption is also discussed. Isotherm of adsorption and effect of pH, adsorption dosage and recyclability are investigated. The results show that the maximum adsorption capacities of lead and cadmium ions on the hydrogel are 628.93 and 456.62 mg g^?1, respectively. The adsorption is well described by Langmuir isotherm model. The hydrogel is also utilized for the loading of potassium nitrate as an active agrochemical agent and the release of this active agent has also been investigated.     

Complex-radical copolymerization of <Emphasis Type="Italic">N-</Emphasis>vinyl pyrrolidone with isostructural analogs of maleic anhydride

Radical copolymerizations of N-vinyl-2-pyrrolidone (VP) with isostructural analogs of maleic anhydride (MA), such as citraconic anhydride (CA) and N-substituted maleimides [maleimide (MI), N-ethylmaleimide (EMI) and N-phenylmaleimide (PhMI)] were studied. Compositions of copolymers synthesized in a wide range of monomer feed ratios were determined by alkali titration (for anhydride copolymers), FTIR and ¹H NMR spectroscopy using 1495 and 630 cm^-1 (for VP-MI), 1289 and 1225 cm^-1 (for VP-EMI) and 1050 and 3067 cm^-1 (for VP-PhMI) analytical bands and integral areas of CH₂ (pyrrolidone ring) and CH (MI), CH₃ (EMI) and CH= (benzene ring in PhMI) groups, respectively. Electron-donor VP monomer was found to have substantially different reactivities in the radical copolymerization with MA, CA and N-substituted (H, C₂H₅ and phenyl) malemides as electron-acceptor comonomers. Effects of H-bonding and N→O=C coordination on the monomer reactivity ratios were evaluated. Tendency to alternation of the monomer pairs increases in the order of VP–MA > VP–CA > VP-MI > VP-PhMI > VP-EMI. Structure-thermal property-relationship for the synthesized copolymers was also studied.     

Preparation and Characteristics of Polyphenylsilsesquioxane-<Emphasis Type="Italic">b</Emphasis>-Polyurethane Copolymer as a Dielectric Material

Polyphenylsilsesquioxane-b-polyurethane copolymers were prepared in the presence of dibutyltin dilaurate catalyst by copolymerization of polyphenylsilsesquioxane (PPSQ) with a α,ω-diisocyanate group terminated polyurethane (DPU) prepolymer of various molecular weights. Additionally, DPU prepolymers were prepared by the reaction of isophorone diisocyanate and various polycaprolactones (PCL, M _w : 400, 830, 1250). The introduction of air-filled pores in the copolymer matrix was accomplished by removing the DPU segment in an electric furnace equipped with a N₂ gas injection port. The thermogravimetric properties of the products were measured in order to identify the thermal decomposition temperature of the DPU prepolymer and to ascertain the thermal stability of the copolymer matrix. Furthermore, the atomic force microscopy (AFM) and scanning electron microscopy (SEM) were measured to obtain more detailed information on the surface of copolymer film. With the results of the AFM analysis of the materials prepared with the DPU prepolymers of lower molecular weight, it was possible to identify well-dispersed air-filled pores in the copolymer matrix film. In particular, the dielectric constant drastically decreased with the introduction of the air-filled pores.     

Interspecific Cross-Attraction between the South American Cerambycid Beetles <Emphasis Type="Italic">Cotyclytus curvatus</Emphasis> and <Emphasis Type="Italic">Megacyllene acuta</Emphasis> is Averted by Minor Pheromone Components

During field screening trials conducted in Brazil in 2015, adults of both sexes of the cerambycid beetles Cotyclytus curvatus (Germar) and Megacyllene acuta (Germar) (subfamily Cerambycinae, tribe Clytini) were significantly attracted to racemic 3-hydroxyhexan-2-one and racemic 2-methylbutan-1-ol, chemicals which previously have been identified as male-produced aggregation-sex pheromones of a number of cerambycid species endemic to other continents. Subsequent analyses of samples of beetle-produced volatiles revealed that males of C. curvatus sex-specifically produce only (R)-3-hydroxyhexan-2-one, whereas males of M. acuta produce the same compound along with lesser amounts of (2S,3S)-2,3-hexanediol and (S)-2-methylbutan-1-ol. Follow-up field trials showed that both sexes of both species were attracted to synthetic reconstructions of their respective pheromones, confirming that males produce aggregation-sex pheromones. The minor pheromone components of M. acuta, (S)-2-methylbutan-1-ol and (2S,3S)-2,3-hexanediol, synergized attraction of that species, but antagonized attraction of C. curvatus to (R)-3-hydroxyhexan-2-one. Beetles of other cerambycine species also were attracted in significant numbers, including Chrysoprasis linearis Bates, Cotyclytus dorsalis (Laporte & Gory), and Megacyllene falsa (Chevrolat). Our results provide further evidence that 3-hydroxyhexan-2-one is a major component of attractant pheromones of numerous cerambycine species world-wide. Our results also highlight our increasing understanding of the crucial role of minor pheromone components in imparting species specificity to cerambycid pheromone blends, as is known to occur in numerous species in other insect families.     

Improvement of bacterial cellulose production in <Emphasis Type="Italic">Acetobacter xylinum</Emphasis> using byproduct produced by <Emphasis Type="Italic">Gluconacetobacter hansenii</Emphasis>

A single sugar α-linked glucuronic acid based oligosaccharide (SSGO) is water soluble oligosaccharides (WSOS) obtained by Gluconacetobacter hansenii PJK (KCTC 10505BP) as a byproduct during bacterial cellulose (BC) production. In this study, SSGO was used for the improvement of BC production by the vinegar bacterium, Acetobacter xylinum, which produces heteropolysaccharides as a byproduct. The addition of 1.0% SSGO to the chemically defined medium (CDM) resulted in an 89.3% increase in BC production by A. xylinum after 15 days of cultivation under static condition, and a 52.3% increase in BC production by G. hansenii. Both the dry cell weight and live cell density of A. xylinum increased 50% with the addition of 1.0% SSGO. SSGO successfully improved BC production by A. xylinum.     

Inducible <Emphasis Type="Italic">De Novo</Emphasis> Biosynthesis of Isoflavonoids in Soybean Leaves by <Emphasis Type="Italic">Spodoptera litura</Emphasis> Derived Elicitors: Tracer Techniques Aided by High Resolution LCMS

Isoflavonoids are a characteristic family of natural products in legumes known to mediate a range of plant-biotic interactions. For example, in soybean (Glycine max: Fabaceae) multiple isoflavones are induced and accumulate in leaves following attack by Spodoptera litura (Lepidoptera: Noctuidae) larvae. To quantitatively examine patterns of activated de novo biosynthesis, soybean (Var. Enrei) leaves were treated with a combination of plant defense elicitors present in S. litura gut content extracts and L-α-[¹³C₉, ¹⁵N]phenylalanine as a traceable isoflavonoid precursor. Combined treatments promoted significant increases in ¹³C-labeled isoflavone aglycones (daidzein, formononetin, and genistein), ¹³C-labeled isoflavone 7-O-glucosides (daidzin, ononin, and genistin), and ¹³C-labeled isoflavone 7-O-(6″-O-malonyl-β-glucosides) (malonyldaidzin, malonylononin, and malonylgenistin). In contrast levels of ¹³C-labeled flavones and flavonol (4′,7-dihydroxyflavone, kaempferol, and apigenin) were not significantly altered. Curiously, application of fatty acid-amino acid conjugate (FAC) elicitors present in S. litura gut contents, namely N-linolenoyl-L-glutamine and N-linoleoyl-L-glutamine, both promoted the induced accumulation of isoflavone 7-O-glucosides and isoflavone 7-O-(6″-O-malonyl-β-glucosides), but not isoflavone aglycones in the leaves. These results demonstrate that at least two separate reactions are involved in elicitor-induced soybean leaf responses to the S. litura gut contents: one is the de novo biosynthesis of isoflavone conjugates induced by FACs, and the other is the hydrolysis of the isoflavone conjugates to yield isoflavone aglycones. Gut content extracts alone displayed no hydrolytic activity. The quantitative analysis of isoflavone de novo biosynthesis, with respect to both aglycones and conjugates, affords a useful bioassay system for the discovery of additional plant defense elicitor(s) in S. litura gut contents that specifically promote hydrolysis of isoflavone conjugates.     

The Role of the Glucosinolate-Myrosinase System in Mediating Greater Resistance of <Emphasis Type="Italic">Barbarea verna</Emphasis> than <Emphasis Type="Italic">B. vulgaris</Emphasis> to <Emphasis Type="Italic">Mamestra brassicae</Emphasis> Larvae

We investigated the influences of two structurally similar glucosinolates, phenethylglucosinolate (gluconasturtiin, NAS) and its (S)-2-hydroxyl derivative glucobarbarin (BAR), as well as their hydrolysis products on larvae of the generalist Mamestra brassicae (Lepidoptera: Noctuidae). Previous results suggested a higher defensive activity of BAR than NAS based on resistance toward M. brassicae larvae of natural plant genotypes of Barbarea vulgaris R. Br. (Brassicaceae) dominated by BAR. In the present study, the hypothesis of a higher defensive activity of BAR than NAS was tested by comparing two Barbarea species similarly dominated either by BAR or by NAS and by testing effects of isolated BAR and NAS on larval survival and feeding preferences. Larvae reared on leaf disks of B. verna (Mill.) Asch. had a lower survival than those reared on B. vulgaris P- and G-chemotypes. Leaves of B. verna were dominated by NAS, whereas B. vulgaris chemotypes were dominated by BAR or its epimer. In addition, B. verna leaves showed a threefold higher activity of the glucosinolate-activating myrosinase enzymes. The main product of NAS from breakdown by endogenous enzymes including myrosinases (“autolysis”) in B. verna leaves was phenethyl isothiocyanate, while the main products of BAR in autolyzed B. vulgaris leaves were a cyclized isothiocyanate product, namely an oxazolidine-2-thione, and a downstream metabolite, an oxazolidin-2-one. The glucosinolates BAR and NAS were isolated and offered to larvae on disks of cabbage. Both glucosinolates exerted similar negative effects on larval survival but effects of NAS tended to be more detrimental. Low concentrations of BAR, but not of NAS, stimulated larval feeding, whereas high BAR concentrations acted deterrent. NAS only tended to be deterrent at the highest concentration, but the difference was not significant. Recoveries of NAS and BAR on cabbage leaf disks were similar, and when hydrolyzed by mechanical leaf damage, the same isothiocyanate-type products as in Barbarea plants were formed with further conversion of BAR to cyclic products, (R)-5-phenyloxazolidine-2-thione [(R)-barbarin] and (R)-5-phenyloxazolidin-2-one [(R)-resedine]. We conclude that a previously proposed generally higher defensive activity of BAR than NAS to M. brassicae larvae could not be confirmed. Indeed, the higher resistance of NAS-containing B. verna plants may be due to a combined effect of rather high concentrations of NAS and a relatively high myrosinase activity or other plant traits not investigated yet.     

Modifying the Alkylglucosinolate Profile in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Alters the Tritrophic Interaction with the Herbivore <Emphasis Type="Italic">Brevicoryne brassicae</Emphasis> and Parasitoid <Emphasis Type="Italic">Diaeretiella rapae</Emphasis>

Arabidopsis thaliana was used as an experimental model plant to investigate a tritrophic interaction between the plant, a specialist aphid herbivore, Brevicoryne brassicae, and its natural enemy, the parasitoid Diaeretiella rapae. The A. thaliana ecotype Col-5 was transformed with a functional 2-oxoglutarate dependent dioxygenase (BniGSL-ALK) that converts 3-methylsulfinylpropylglucosinolate and 4-methylsulfinylbutylglucosinolate to 2-propenylglucosinolate and 3-butenylglucosinolate, respectively. This transformation results in a change in the glucosinolate hydrolysis profile where 3-butenylisothiocyanate, 2-propenylisothiocyanate and 5-vinyloxazolidine-2-thione are produced in contrast to the wild-type plant where 4-methylsulfinylbutylisothiocyanate is the main product. Performance of B. brassicae was affected negatively by transforming Col-5 with BniGSL-ALK in terms of mean relative growth rates. In a series of behavioral bioassays, naïve D. rapae females were able to discriminate between B. brassicae infested and uninfested Col-5 plants transformed with BniGSL-ALK, with parasitoids showing a preference for B. brassicae infested plants. By contrast, naïve D. rapae females were unable to discriminate between aphid infested and uninfested Col-5 plants. Subsequent air entrainments of B. brassicae infested Col-5 plants transformed with BniGSL-ALK further confirmed the presence of 3-butenylisothiocyanate in the headspace. By contrast, no glucosinolate hydrolysis products were recorded from similarly infested Col-5 plants.     

Development of Cre-<Emphasis Type="Italic">lox</Emphasis> based multiple knockout system in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> R1

The extremophilic bacterium Deinococcus radiodurans R1 has been considered as an attractive microorganism due to its remarkable tolerance to various external stresses. Considering the nature of D. radiodurans R1, it has potential as a platform microorganism for industrial applications, including biorefinery and bioremediation process. However, D. radiodurans R1 is well known for its hard genetic manipulation. Thus, much effort has been made to develop efficient genetic engineering tools for making D. radiodurans R1 suitable for industrial platform microorganism. Although a plasmid-based single gene knockout method has been reported, development of multiple gene knockout system has not yet been reported. Here we report, for the first time, Cre-lox based rapid and efficient multiple knockout method for metabolic engineering of D. radiodurans R1. Also, deletion of dr0053 gene was successfully achieved within seven days to make biofilm overproducing strain.