Coniferaldehyde Research Articles

Overexpression of Artemisia annua Cinnamyl Alcohol Dehydrogenase Increases Lignin and Coumarin and Reduces Artemisinin and Other Sesquiterpenes.

Artemisia annua is the only medicinal crop that produces artemisinin for malarial treatment. Herein, we describe the cloning of a cinnamyl alcohol dehydrogenase (AaCAD) from an inbred self-pollinating (SP) A. annua cultivar and its effects on lignin and artemisinin production. A recombinant AaCAD was purified via heterogeneous expression. Enzyme assays showed that the recombinant AaCAD converted p-coumaryl, coniferyl, and sinapyl aldehydes to their corresponding alcohols, which are key intermediates involved in the biosynthesis of lignin. Km, Vmax, and Vmax/Km values were calculated for all three substrates. To characterize its function in planta, AaCAD was overexpressed in SP plants. Quantification using acetyl bromide (AcBr) showed significantly higher lignin contents in transgenics compared with wild-type (WT) plants. Moreover, GC-MS-based profiling revealed a significant increase in coumarin contents in transgenic plants. By contrast, HPLC-MS analysis showed significantly reduced artemisinin contents in transgenics compared with WT plants. Furthermore, GC-MS analysis revealed a decrease in the contents of arteannuin B and six other sesquiterpenes in transgenic plants. Confocal microscopy analysis showed the cytosolic localization of AaCAD. These data demonstrate that AaCAD plays a dual pathway function in the cytosol, in which it positively enhances lignin formation but negatively controls artemisinin formation. Based on these data, crosstalk between these two pathways mediated by AaCAD catalysis is discussed to understand the metabolic control of artemisinin biosynthesis in plants for high production.

Chemical Constituents of the Flowers of Michelia alba

Michelaine (1), (–)-anonaine, (–)-norushinsunine, (–)-ushinsunine, (–)-N-acetylanonaine, liriodenine, atherospermidine, (+)-syringaresinol, N-trans-feruloyltyramine, p-hydroxybenzaldehyde, vanillin, vanillic acid, syringic acid, coniferyl aldehyde, syringin, scopoletin, 4-acetonyl-3,5-dimethoxy-p-quinol, β-sitostenone, and stigmasta-4,22-dien-3-one were isolated from the flowers of Michelia alba (Magnoliaceae). Among them, michelaine (1) is a new 4,5-dioxoaporphine compound. The structures of these compounds were characterized and identified by spectral analyses.

Phytochemical study of the trunk bark of Citharexylum spinosum L. growing in Tunisia: Isolation and structure elucidation of iridoid glycosides

A phytochemical investigation of the trunk bark ethyl acetate extract of Citharexylum spinosum L. has led to the isolation of four previously undescribed iridoid glycosides, tunispinosides A-D, and five known phenylethanoid glycosides, verbascoside, leucosceptoside A, martynoside, isoverbascoside and plantainoside C, together with 4-hydroxy-2,6-dimethoxyphenyl 6′-O-vanilloyl-β-D-glucopyranoside, two 8,3′-neolignan glycosides, plucheosides D1-D2, coniferyl aldehyde, vanillic acid, syringic acid, ferulic acid and tyrosol. All compounds were isolated for the first time from C. spinosum. Their isolation was carried out using silica gel column and high performance liquid chromatography (HPLC). Structures were established by spectroscopic means including 1D and 2D NMR experiments, and spectrometric ESI-HRMS analysis.

Functional Analysis of an Uridine Diphosphate Glycosyltransferase Involved in the Biosynthesis of Polyphenolic Glucoside in Tea Plants (Camellia sinensis)

Polyphenols are one of the largest groups of compounds that confer benefits to the health of plants and humans. Flavonol glycosides are a major ingredient of polyphenols in Camellia sinensis. Flavonol-3-O-glycosides are characteristic astringent taste compounds in tea infusion. A polyphenolic glycosyltransferase (CsUGT72AM1) belonging to cluster IIIb was isolated from the tea plant. The full-length cDNA of CsUGT72AM1 is 1416 bp. It encodes 472 amino acids with a calculated molecular mass of 50.92 kDa and an isoelectric point of 5.21. The recombinant CsUGT72AM1 protein was expressed in Escherichia coli and exhibited catalytic activity toward multiple flavonoids and coniferyl aldehyde. The enzyme assay indicated that rCsUGT72AM1 could perform glycosidation of flavonols or coniferyl aldehyde in vitro to form 3-O-glucoside or 4-O-glucoside, respectively. Interestingly, this enzyme also had activities and performed multisite glycosidation toward flavanones. The consistent products were confirmed to be naringenin-7-O-glucoside and -4'-O-glucoside by the nuclear magnetism assay. In addition, in the enzyme assay with cyanidin as the substrate, the results suggested that the glycosylated activity of CsUGT72AM1 was remarkably inhibited by a high concentration of anthocyanins. The above results indicate that CsUGT72AM1 may be involved in the metabolism of flavonol, flavanone, anthocyanin, and lignin.

Photocatalytic Degradation of β-O-4 Lignin Model Compound by In2S3 Nanoparticles Under Visible Light Irradiation

Cleaving the abundant β-O-4 linkages in lignin is a key issue for producing value-added products by controlled lignin depolymerization. Herein, hydrothermally synthesized In2S3 nanoparticles were primarily used to photodegrade guaiacylglycerol-β-guaiacyl ether, a β-O-4 lignin model compound, under visible light irradiation. The as-synthesized In2S3 nanoparticles are found to be typical β-In2S3 nanocrystals of cubic phase and composed of large plate-like particles and small granular particles by using X-ray diffraction technique and field-emission scanning electron microscopy. The bandgap energy of the In2S3 nanoparticles is estimated to be 1.78 eV using an UV-visible diffuse reflectance spectroscopy. The photodegradation and structure variation of lignin model compound were evaluated by the variation of its UV-vis absorption spectrum, Fourier transform infrared spectrum, and X-ray photoelectron spectroscopy, while its degradation products were identified by using the gas chromatography-mass spectrometry. The results show that the as-synthesized In2S3 nanoparticles can photocatalytically break the β-O-4 linkage and oxidize the hydroxyl/methoxyl groups of lignin model compound under visible light irradiation although the lignin model compound is photo-resistant even under UV irradiation. The photodegradation products of lignin model compound consist of various aromatic monomers including value-added acetovanillone, vanillin, and coniferyl aldehyde. A possible pathway is proposed for photodegrading lignin model compound in the presence of the as-synthesized In2S3 nanoparticles under visible light irradiation.

Comparison of tolerance of four bacterial nanocellulose-producing strains to lignocellulose-derived inhibitors

BackgroundThrough pretreatment and enzymatic saccharification lignocellulosic biomass has great potential as a low-cost feedstock for production of bacterial nanocellulose (BNC), a high value-added microbial product, but inhibitors formed during pretreatment remain challenging. In this study, the tolerance to lignocellulose-derived inhibitors of three new BNC-producing strains were compared to that of Komagataeibacter xylinus ATCC 23770. Inhibitors studied included furan aldehydes (furfural and 5-hydroxymethylfurfural) and phenolic compounds (coniferyl aldehyde and vanillin). The performance of the four strains in the presence and absence of the inhibitors was assessed using static cultures, and their capability to convert inhibitors by oxidation and reduction was analyzed.ResultsAlthough two of the new strains were more sensitive than ATCC 23770 to furan aldehydes, one of the new strains showed superior resistance to both furan aldehydes and phenols, and also displayed high volumetric BNC yield (up to 14.78 ± 0.43 g/L) and high BNC yield on consumed sugar (0.59 ± 0.02 g/g). The inhibitors were oxidized and/or reduced by the strains to be less toxic. The four strains exhibited strong similarities with regard to predominant bioconversion products from the inhibitors, but displayed different capacity to convert the inhibitors, which may be related to the differences in inhibitor tolerance.ConclusionsThis investigation provides information on different performance of four BNC-producing strains in the presence of lignocellulose-derived inhibitors. The results will be of benefit to the selection of more suitable strains for utilization of lignocellulosics in the process of BNC-production.

Catalytic Oxidation of Lignins into the Aromatic Aldehydes: General Process Trends and Development Prospects.

This review discusses principal patterns that govern the processes of lignins’ catalytic oxidation into vanillin (3-methoxy-4-hydroxybenzaldehyde) and syringaldehyde (3,5-dimethoxy-4-hydroxybenzaldehyde). It examines the influence of lignin and oxidant nature, temperature, mass transfer, and of other factors on the yield of the aldehydes and the process selectivity. The review reveals that properly organized processes of catalytic oxidation of various lignins are only insignificantly (10–15%) inferior to oxidation by nitrobenzene in terms of yield and selectivity in vanillin and syringaldehyde. Very high consumption of oxygen (and consequentially, of alkali) in the process—over 10 mol per mol of obtained vanillin—is highlighted as an unresolved and unexplored problem: scientific literature reveals almost no studies devoted to the possibilities of decreasing the consumption of oxygen and alkali. Different hypotheses about the mechanism of lignin oxidation into the aromatic aldehydes are discussed, and the mechanism comprising the steps of single-electron oxidation of phenolate anions, and ending with retroaldol reaction of a substituted coniferyl aldehyde was pointed out as the most convincing one. The possibility and development prospects of single-stage oxidative processing of wood into the aromatic aldehydes and cellulose are analyzed.

Profiling of Saccharomyces cerevisiae transcription factors for engineering the resistance of yeast to lignocellulose-derived inhibitors in biomass conversion

BackgroundYeast transcription factors (TFs) involved in the regulation of multidrug resistance (MDR) were investigated in experiments with deletion mutants, transformants overexpressing synthetic genes encoding TFs, and toxic concentrations of lignocellulose-derived substances added to cultures as complex mixtures or as specific compounds, viz. coniferyl aldehyde, 5-hydroxymethylfurfural, and furfural.ResultsIn the presence of complex mixtures of toxic substances from spruce wood, transformants overexpressing YAP1 and STB5, TFs involved in oxidative stress response, exhibited enhanced relative growth rates amounting to 4.589 ± 0.261 and 1.455 ± 0.185, respectively. Other TFs identified as important for resistance included DAL81, GZF3, LEU3, PUT3, and WAR1. Potential overlapping functions of YAP1 and STB5 were investigated in experiments with permutations of deletions and overexpression of the two genes. YAP1 complemented STB5 with respect to resistance to 5-hydroxymethylfurfural, but had a distinct role with regard to resistance to coniferyl aldehyde as deletion of YAP1 rendered the cell incapable of resisting coniferyl aldehyde even if STB5 was overexpressed.ConclusionsWe have investigated 30 deletion mutants and eight transformants overexpressing MDR transcription factors with regard to the roles the transcription factors play in the resistance to toxic concentrations of lignocellulose-derived substances. This work provides an overview of the involvement of thirty transcription factors in the resistance to lignocellulose-derived substances, shows distinct and complementary roles played by YAP1 and STB5, and offers directions for the engineering of robust yeast strains for fermentation processes based on lignocellulosic feedstocks.

The Conjugated Double Bond of Coniferyl Aldehyde Is Essential for Heat Shock Factor 1 Mediated Cytotoprotection

Coniferyl aldehyde (1) is previously reported as a potent inducer of heat shock factor 1 (HSF1). Here, we further examined the active pharmacophore of 1 for activation of HSF1 using the derivatives coniferyl alcohol (2), 4-hydroxy-3-methoxyphenylpropanal (3), and 4-hydroxy-3-methoxyphenylpropanol (4). Both 1 and 2 resulted in increased survival days after a lethal radiation (IR) dose. The decrease in bone marrow (BM) cellularity and Ki67-positive BM cells by IR was also significantly restored by 1 or 2 in mice. These results suggested that the vinyl moiety of 1 and 2 is necessary for inducing HSF1, which may be useful for developing small molecules for cytoprotection of normal cells against damage by cytotoxic drugs and radiation.

Supercritical carbon dioxide extraction of fast pyrolysis oil from softwood

Supercritical carbon dioxide (scCO2) was studied for separation of bio-oil applying different pressures (100, 200, 300bar), temperatures (60, 80°C), carriers (SiO2, activated carbon), and times (1–6h). The maximum extraction yield (13–14wt%) was obtained at 300bar, 60°C with SiO2 as carrier. The composition of the scCO2 extracts was determined by GC–MS/FID. The concentration progress of 32 typical compounds in the extracts at 200bar was monitored in detail. At 200bar pressure, the contents in the scCO2 extracts of aldehydes, furanones, coniferyl alcohol, coniferyl aldehyde, and vanillin increased with extraction time. Acids, ketones and most of the guaiacols decreased while phenols and pyrans remained the same. More acids, ketones, and furaldehydes could be obtained at a lower temperature and more aromatic compounds could be extracted when SiO2 as the carrier was employed. The yields of individual components were mainly depending on the duration of extraction.

The Structure-Antioxidant Activity Relationship of Ferulates.

The antioxidant activity of ferulic acid (1), iso-ferulic acid (2), coniferyl aldehyde (3), methyl ferulate (4), and ethyl ferulate (5) were investigated using 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and ferric-reducing antioxidant power (FRAP) assays and autoxidation of triacylglycerols of commercially available sunflower oil (TGSO). The compounds tested for ability to scavenge ABTS radical cations was in the order of ferulic acid > coniferyl aldehyde ≈ iso-ferulic acid > ethyl ferulate ≈ methyl ferulate. The results of the FRAP assay for ferulic acid, iso-ferulic acid, and coniferyl aldehyde were similar to and higher than those of methyl ferulate and ethyl ferulate. In the lipid system, iso-ferulic acid showed weak antioxidant activity. The other ferulates exhibited much stronger, yet similar, activities.

Effects of decomposed products from Japanese cedar hydrolyzates on acetic acid fermentation by Clostridium thermocellum and Moorella thermoacetica (C. thermoaceticum)

Abstract Japanese cedar (Cryptomeria japonica) was treated with hot-compressed water and as decomposed products, the following compounds were recovered: furfural, 5-hydroxymethyl furfural, levoglucosan, lactic acid, glycolic acid, coniferyl alcohol, coniferyl aldehyde and vanillin. The impacts and fermentability of these compounds were studied on acetic acid fermentation by the co-culture of Clostridium thermocellum and Moorella thermoacetica. It was found that furfural, 5-HMF and lignin-derived products strongly limited acetic acid production by free cells. Importantly, co-immobilized C. thermocellum and M. thermoacetica expressed increased tolerance towards the decomposed products and successfully provided acetic acid corresponding to 93% of the theoretical maximum from Japanese cedar hydrolyzates.

ALD5, PAD1, ATF1 and ATF2 facilitate the catabolism of coniferyl aldehyde, ferulic acid and p-coumaric acid in Saccharomyces cerevisiae

The ability of Saccharomyces cerevisiae to catabolize phenolic compounds remains to be fully elucidated. Conversion of coniferyl aldehyde, ferulic acid and p-coumaric acid by S. cerevisiae under aerobic conditions was previously reported. A conversion pathway was also proposed. In the present study, possible enzymes involved in the reported conversion were investigated. Aldehyde dehydrogenase Ald5, phenylacrylic acid decarboxylase Pad1, and alcohol acetyltransferases Atf1 and Atf2, were hypothesised to be involved. Corresponding genes for the four enzymes were overexpressed in a S. cerevisiae strain named APT_1. The ability of APT_1 to tolerate and convert the three phenolic compounds was tested. APT_1 was also compared to strains B_CALD heterologously expressing coniferyl aldehyde dehydrogenase from Pseudomonas, and an ald5Δ strain, all previously reported. APT_1 exhibited the fastest conversion of coniferyl aldehyde, ferulic acid and p-coumaric acid. Using the intermediates and conversion products of each compound, the catabolic route of coniferyl aldehyde, ferulic acid and p-coumaric acid in S. cerevisiae was studied in greater detail.

Toxicity of Plant Secondary Metabolites Modulating Detoxification Genes Expression for Natural Red Palm Weevil Pesticide Development.

This study aimed to explore the larvicidal and growth-inhibiting activities, and underlying detoxification mechanism of red palm weevil against phenylpropanoids, an important class of plant secondary metabolites. Toxicity of α-asarone, eugenol, isoeugenol, methyl eugenol, methyl isoeugenol, coumarin, coumarin 6, coniferyl aldehyde, diniconazole, ethyl cinnamate, and rosmarinic acid was evaluated by incorporation into the artificial diet. All of the phenylpropanoids exhibited dose- and time-dependent insecticidal activity. Among all the tested phenylpropanoids, coumarin exhibited the highest toxicity by revealing the least LD50 value (0.672 g/L). In addition, the most toxic compound (coumarin) observed in the current study, deteriorated the growth resulting tremendous reduction (78.39%) in efficacy of conversion of digested food (ECD), and (ECI) efficacy of conversion of ingested food (70.04%) of tenth-instar red palm weevil larvae. The energy-deficient red palm weevil larvae through their intrinsic abilities showed enhanced response to their digestibility resulting 27.78% increase in approximate digestibility (AD) compared to control larvae. The detoxification response of Rhynchophorus ferrugineus larvae determined by the quantitative expression of cytochrome P450, esterases, and glutathione S-transferase revealed enhanced expression among moderately toxic and ineffective compounds. These genes especially cytochrome P450 and GST detoxify the target compounds by enhancing their solubility that leads rapid excretion and degradation resulting low toxicity towards red palm weevil larvae. On the other hand, the most toxic (coumarin) silenced the genes involved in the red palm weevil detoxification mechanism. Based on the toxicity, growth retarding, and masking detoxification activities, coumarin could be a useful future natural red palm weevil-controlling agent.

Quercus humboldtii(Colombian oak): Characterisation of wood phenolic composition with respect to traditional oak wood used in oenology

The use of new oak barrels and the demand for oak wood in oenology is increasing. Thus, it is necessary to search for new wood sources to supply the current demand in cooperage. The aim of this work was to study the composition of ellagitannins and low molecular weight phenolic compounds (LMWP) by HPLC-DAD of green wood Quercus humboldtii Bonpl. (Colombian), compared to the species typically used in cooperage: Quercus sessiliflora Salisb. (French and Romanian) and Quercus alba L. (USA). In Colombian oak wood, the same LMWP and ellagitannins as in the traditional species were identified. The most abundant LMWP was ellagic acid, and its concentration was lower in Q. humboldtii and Q. alba than in Q. sessiliflora , so these two presented a lower content of phenolic acids. As regards phenolic aldehydes, Colombian oak wood only showed significant differences in the content of coniferyl and sinapic aldehydes in relation to French oak. Finally, the total ellagitannin content of Q. humboldtii was lower than that of European oaks and similar to that of American oak ( Q. alba ). None of the ellagitannins studied presented significant differences compared to Q. alba . Therefore, Q. humboldtii wood was more similar in terms of phenolic composition to Q. alba than to Q. sessiliflora .

A Wheat Cinnamyl Alcohol Dehydrogenase TaCAD12 Contributes to Host Resistance to the Sharp Eyespot Disease.

Sharp eyespot, caused mainly by the necrotrophic fungus Rhizoctonia cerealis, is a destructive disease in hexaploid wheat (Triticum aestivum L.). In Arabidopsis, certain cinnamyl alcohol dehydrogenases (CADs) have been implicated in monolignol biosynthesis and in defense response to bacterial pathogen infection. However, little is known about CADs in wheat defense responses to necrotrophic or soil-borne pathogens. In this study, we isolate a wheat CAD gene TaCAD12 in response to R. cerealis infection through microarray-based comparative transcriptomics, and study the enzyme activity and defense role of TaCAD12 in wheat. The transcriptional levels of TaCAD12 in sharp eyespot-resistant wheat lines were significantly higher compared with those in susceptible wheat lines. The sequence and phylogenetic analyses revealed that TaCAD12 belongs to IV group in CAD family. The biochemical assay proved that TaCAD12 protein is an authentic CAD enzyme and possesses catalytic efficiencies toward both coniferyl aldehyde and sinapyl aldehyde. Knock-down of TaCAD12 transcript significantly repressed resistance of the gene-silenced wheat plants to sharp eyespot caused by R. cerealis, whereas TaCAD12 overexpression markedly enhanced resistance of the transgenic wheat lines to sharp eyespot. Furthermore, certain defense genes (Defensin, PR10, PR17c, and Chitinase1) and monolignol biosynthesis-related genes (TaCAD1, TaCCR, and TaCOMT1) were up-regulated in the TaCAD12-overexpressing wheat plants but down-regulated in TaCAD12-silencing plants. These results suggest that TaCAD12 positively contributes to resistance against sharp eyespot through regulation of the expression of certain defense genes and monolignol biosynthesis-related genes in wheat.

Detoxification of furanic and phenolic lignocellulose derived inhibitors of yeast using laccase immobilized on bacterial cellulosic nanofibers

Biotransformation of lignocellulose by microbial fermentation is usually preceded by thermo-chemical pretreatments followed by enzymatic hydrolysis of cellulose. Derivatives formed during the pretreatment of the lignocellulosic biomass inhibit enzymatic hydrolysis as well as microbial fermentation. Pretreated lignocellulose hydrolysate contains many derivatives of either furanic or phenolic inhibitory derivatives. In the present study, laccase was used to detoxify three different types of lignocellulosic derivatives that are highly toxic to microbial fermentation due to their low hydrophilic nature, namely furfural, acetosyringone, and coniferyl aldehyde. A minimal inhibitory concentration (MIC) test was carried out with Saccharomyces cerevisiae. The MIC of furfural, acetosyringone, and coniferyl aldehyde was 12mM, 24mM, and 1.5mM, respectively. Laccase was immobilized on to cellulose nanofiber produced by Gluconacetobacter xylinus. Immobilized laccase showed a better pH and thermal stability than free laccase. Reuse of immobilized laccase retains 85% of its enzyme activity after 16 recycles. Immobilized laccase completely degraded the three lignocellulose inhibitory derivatives after 36h of incubation at 40°C. Finally, the degradation was confirmed by ultraviolet visible spectroscopy (UV–VIS spectrum), high performance liquid chromatography and liquid chromatography mass spectrometry. Interestingly, it was found that the effect of enzymatic degradation depends on the structural variation of the lignocellulosic derivatives as laccase alone detoxified the furfural and coniferyl aldehyde, whereas a redox mediator HOBt was needed for the detoxification of ketone based lignin derivative acetosyringone.

Coniferaldehyde inhibits LPS-induced apoptosis through the PKC α/β II/Nrf-2/HO-1 dependent pathway in RAW264.7 macrophage cells

Coniferaldehyde (CA) exerts anti-inflammatory properties by inducing heme oxygenase-1 (HO-1). To define the regulation mechanism by which CA induces a cytoprotective function and HO-1 expression, the up-stream regulations involved in the activation of nuclear transcription factor-erythroid 2-related factor (Nrf)-2/HO-1 pathway were investigated. CA dramatically increased the Nrf-2 nuclear translocation and HO-1 expression. Lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, and cell death were down-regulated by CA, which were reversed by inhibition of HO-1 activity. Furthermore, CA specifically enhanced the phosphorylation of protein kinase C (PKC) α/β II. Selective inhibition of PKC α/β II using Go6976 or siRNA abolished the CA-induced Nrf-2/HO-1 signaling, and consequently suppressed the cytoprotective activity of CA on the LPS-induced cell death. Together, our results elucidate the regulatory mechanism of PKC α/β II as the upstream molecule of Nrf-2 required for HO-1 expression during CA-induced anti-inflammatory cytoprotective function in LPS stimulated macrophages.

Degradation and polymerization of monolignols by Abortiporus biennis, and induction of its degradation with a reducing agent.

This study was carried out to better understand the characteristic modification mechanisms of monolignols by enzyme system of Abortiporus biennis and to induce the degradation of monolignols. Degradation and polymerization of monolignols were simultaneously induced by A. biennis. Whole cells of A. biennis degraded coniferyl alcohol to vanillin and coniferyl aldehyde, and degraded sinapyl alcohol to 2,6-dimethoxybenzene- 1,4-diol, with the production of dimers. The molecular weight of monolignols treated with A. biennis increased drastically. The activities of lignin degrading enzymes were monitored for 24 h to determine whether there was any correlation between monolignol biomodification and ligninolytic enzymes. We concluded that complex enzyme systems were involved in the degradation and polymerization of monolignols. To degrade monolignols, ascorbic acid was added to the culture medium as a reducing agent. In the presence of ascorbic acid, the molecular weight was less increased in the case of coniferyl alcohol, while that of sinapyl alcohol was similar to that of the control. Furthermore, the addition of ascorbic acid led to the production of various degraded compounds: syringaldehyde and acid compounds. Accordingly, these results demonstrated that ascorbic acid prevented the rapid polymerization of monolignols, thus stabilizing radicals generated by enzymes of A. biennis. Thereafter, A. biennis catalyzed the oxidation of stable monolignols. As a result, ascorbic acid facilitated predominantly monolignols degradation by A. biennis through the stabilization of radicals. These findings showed outstanding ability of A. biennis to modify the lignin compounds rapidly and usefully.

Male tarsi specific odorant-binding proteins in the diving beetle Cybister japonicus sharp.

Odorant binding proteins (OBPs) play critical roles in chemical communication of insects, as they recognize and transport environmental chemical signals to receptors. The diving beetle Cybister japonicus Sharp shows a remarkable sexual dimorphism. The foreleg tarsi of males are equipped with large suction cups, believed to help holding the female during underwater courtship and mating. Here, we identified two OBPs highly and specifically expressed in male tarsi, suggesting important functions of these structures in chemical communication. The first protein, CjapOBP1, exhibits the 6 conserved cysteines motif of classic OBPs, while the second, CjapOBP2, contains only four cysteines and can be assigned to the sub-class of C-minus OBPs. Both proteins were expressed in a bacterial system and the purified recombinant proteins were used to for antibodies preparation. Western Blot analysis showed that CjapOBP1 is predominantly expressed in male tarsi and could be also detected in antennae and palpi of both sexes, while CjapOBP2, besides male tarsi, is also present in testis. Ligand-binding experiments showed a good binding affinity between CjapOBP1, CjapOBP2 and citral and coniferyl aldehyde, respectively. These results support a possible function of these two OBPs in the male foreleg tarsi of diving beetles in chemical communication.