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Crosstalk in the responses to abiotic and biotic stresses in Arabidopsis: analysis of gene expression in cytochrome P450 gene superfamily by cDNA microarray.

From Arabidopsis full-length cDNA libraries, we collected ca. 7000 (7K) independent full-length cDNAs to prepare a cDNA microarray. The 7K cDNA collection contains 49 cytochrome P450 genes. In this study, expression patterns of these cytochrome P450 genes were analyzed by a full-length cDNA microarray under various treatments, such as hormones (salicylic acid, jasmonic acid, ethylene, abscisic acid), pathogen-inoculation ( Alternaria brassicicola , Alternaria alternata ), paraquat, rose bengal, UV stress (UV-C), heavy metal stress (CuSO4), mechanical wounding, drought, high salinity and low temperature. Expression of 29 cytochrome P450 genes among them was induced by various treatments. Inoculation with A. brassicicola and A. alternata as biotic stresses increased transcript levels of 12 and 5 genes in Arabidopsis plants, respectively. In addition, some of the genes were also expressed by abiotic stresses. This suggests crosstalk between abiotic and biotic stresses. The promoter sequences and cis -acting elements of each gene were studied on the basis of full-length cDNA sequences. Most cytochrome P450 genes induced by both abiotic and biotic stresses contained the recognition sites of MYB and MYC, ACGT-core sequence, TGA-box and W-box for WRKY transcription factors in their promoters. These cis -acting elements are known to participate in the regulation of plant defense. The response of each gene to multiple stresses is strictly regulated.

Abscisic Acid↗

Molecular and functional characterization of a cyclophilin with antifungal activity from Chinese cabbage.

An antifungal protein that inhibits the growth of filamentous fungal pathogens was isolated from Chinese cabbage (Brassica campestris L. ssp. pekinensis) by affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose. The N-terminal amino acid sequence of the protein was highly homologous to that of plant cyclophilins and consequently the protein was denoted as C-CyP. To understand the antifungal activity of C-CyP, we isolated a cDNA encoding its gene from a Chinese cabbage leaf cDNA library. The Chinese cabbage genome bears more than one C-CyP gene copy and C-CyP mRNA is highly expressed in all tissues except the seeds. Recombinant C-CyP catalyzed the cis-trans inter-conversion of the Ala-Pro bond of the substrate, which indicates this protein has peptidyl-prolyl cis-trans isomerase activity. It also inhibited the growth of several fungal pathogens.

Amino Acid Sequence↗

A family of glycosyl hydrolase family 45 cellulases from the pine wood nematode Bursaphelenchus xylophilus.

We have characterized a family of GHF45 cellulases from the pine wood nematode Bursaphelenchus xylophilus. The absence of such genes from other nematodes and their similarity to fungal genes suggests that they may have been acquired by horizontal gene transfer (HGT) from fungi. The cell wall degrading enzymes of other plant parasitic nematodes may have been acquired by HGT from bacteria. B. xylophilus is not directly related to other plant parasites and our data therefore suggest that horizontal transfer of cell wall degrading enzymes has played a key role in evolution of plant parasitism by nematodes on more than one occasion.

Amino Acid Sequence↗

Cloning and expression of a putative transferrin cDNA of the spruce budworm, Choristoneura fumiferana.

A spruce budworm (Choristoneura fumiferana) transferrin cDNA (CfTf) was isolated and cloned from a cDNA library that was constructed using mRNA from fifth to sixth instar larvae. CfTf cDNA encoded a predicted protein of 681 amino acids with a molecular mass of approximately 76 kDa. CfTf shared 72% and 74% identities at the amino acid level with transferrins of Manduca sexta and Bombyx mori, respectively. Like other transferrins, CfTf retains most of the N-terminal, iron-binding amino acid residues. Northern blot analyses indicated that CfTf mRNA was present at high levels after ecdysis, but that the expression level was low prior to ecdysis at the fourth-sixth instar stages. The highest level of CfTf expression was detected in the fat body. Relatively low levels of expression were detected in the epidermis and no expression was found in the midgut. Expression of CfTf mRNA could be induced by bacteria but not fungi. Expression of CfTf mRNA was suppressed by iron load.

Amino Acid Sequence↗

Effect of treatment with Trichoderma harzianum Rifai formulated in invert emulsion on postharvest decay of apple blue mold.

Conidia of Trichoderma harzianum were formulated in invert emulsion (water-in-oil type) at a concentration of 6.0 x 10(7) conidia/ml of the formulation. Treatments with conidia in the formulated emulsion and conidia in sterile distilled water were conducted to assess the effect on Penicillium expansum Link inoculated on apple fruits. Comparisons were made with control treatments of an uninoculated emulsion and uninoculated distilled water. The T. harzianum-containing emulsion significantly (P <or= 0.05) reduced the decay-lesion diameter compared to the T. harzianum-containing water or the uninoculated emulsion or distilled water controls (17.5, 25.7, 34.0, and 34.2 mm, respectively). Significant differences (P <or= 0.05) were also obtained between means of percent reduction in decay-lesion diameter relative to sterile distilled water control in the treatments with formulated and nonformulated conidia and control with uninoculated invert emulsion (48.8%, 24.8%, and 0.6%, respectively). Mean time period needed for sporulation of P. expansum was significantly (P <or= 0.05) increased when the treatments with formulated T. harzianum conidia were applied in comparison with the other types of treatments (14.2 versus 12.2, 9.0, and 8.8 days, respectively, for the other treatments). However, no significant differences (P <or= 0.05) were obtained in means of decay-lesion diameter or in means of time period needed for sporulation of P. expansum in the control treatments (uninoculated invert emulsion versus sterile distilled water). This indicates that the effect of the formulation ingredients on decay-lesion development of P. expansum or its sporulation was not significant. A significant (P <or= 0.05) long period of protection from P. expansum infection (up to 2 months) was also obtained when unwounded apple fruits were dipped for 30-s period in formulated T. harzianum conidia before being inoculated by P. expansum compared to the wounded fruits. This indicates the importance of the latter type of treatment in protecting apple fruits from blue mold infection for long time at postharvest stage without refrigeration.

Antibiosis↗

Malignant mimickers: chronic bacterial and fungal infections of the larynx.

Chronic infections of the larynx are notorious "copycats" of squamous cell carcinoma. Patients typically present with a historical picture and symptoms identical to those seen in a neoplastic setting: dyspnea, hoarseness, odynophagia, weight loss, and a history of tobacco and alcohol abuse. Historically, these patients were subject to an extensive resection for what was in reality a benign disease. A better understanding and awareness of these conditions has reinforced the need for a direct laryngoscopy, biopsy, and culture in the evaluation of long-lasting laryngeal lesions; this has led to more appropriate and focused treatment. The clinical mimicry of chronic laryngeal infections will be illustrated in two recent case reports, histoplasmosis and botryomycosis of the larynx, which will lead into a discussion on the differential diagnosis of bacterial and fungal laryngeal infections, their evaluation, and treatment options.

Adult↗

Organic matter transformation in the environment investigated by quantitative electron paramagnetic resonance (EPR) spectroscopy: studies on lignins.

The lignins separated from angiosperm and gymnosperm trees, peat and xylitic brown coal were investigated by quantitative EPR. Observed free radicals in lignins are sensitive to alkaline environment. Gaseous ammonia interacting with solid lignins in resonance cavity shifts quinone-hydroquinone equilibria towards formation of semiquinone anions. Complexation of copper(II) by lignins causes drastic decrease of the semiquinones in the matrices. Formation of lignin-Pb(II) complexes yielded radicals characterised by unusually low g-value (1.9999-2.0003). Monomeric structural units of the investigated lignins were recognised by pyrolysis with in situ methylation by tetramethylammonium hydroxide. Although for the natural lignins the mixture of normal semiquinone signals at g about 2.0034 and signals at g 1.9999 were observed, some monomeric components of lignins (e.g., caffeic acid, pyrogallol) gave pure lines at g = 1.9999. The bacterial oxidative biodegradation of lignin monomeric components and their Pb(II) complexes resulted in increase of the radical signals.

Bacillus subtilis↗

A basic peroxidase from wheat kernel with antifungal activity.

A basic heme-peroxidase (WP1) was purified to homogeneity from wheat (Triticum aestivum) kernels. The protein was not glycosylated and exhibited a molecular mass of 36 kDa and a pI of 8.0. The N-terminal amino acid sequence revealed a very high similarity with a wheat flour peroxidase allergen associated with baker's asthma. WPI showed indole-3-acetic acid oxidase activity in the presence of Mn2+ and phenolic cofactors. Antifungal assays performed in vitro towards phytopathogenic fungi indicated that WP1 was active in inhibiting germ tube elongation. This first report on antifungal properties of a heme-peroxidase gives experimental support to the idea that peroxidases play a defensive role against invading pathogens.

Amino Acid Sequence↗

Quantification of disease progression of several microbial pathogens on Arabidopsis thaliana using real-time fluorescence PCR.

An accurate monitoring of disease progression is important to evaluate disease susceptibility phenotypes. Over the years, Arabidopsis thaliana has become the model species to serve as a host in plant-pathogen interactions. Despite the efforts to study genetic mechanisms of host defense, little efforts are made for a thorough pathogen assessment, often still depending on symptomology. This manuscript describes the use of real-time polymerase chain reaction (PCR) to assess pathogen growth in the host Arabidopsis for a number of frequently studied pathogens. A wide range of correlations between pathogen biomass and fluorescence is demonstrated, demonstrating the theoretical sensitivity of the technique. It is also demonstrated that host DNA does not interfere with the quantification of pathogen DNA over a wide range. Finally, quantification of pathogen biomass in different plant genotypes with a varying degree of resistance shows the capability of this technique to be used for assessment of pathogen development in disease progression.

Alternaria↗

Application of classification-tree models to characterize the mycobiota of grapes on the basis of origin.

Classification-tree (CT) models are a simple and robust exploratory data analysis technique that can be used in classification, regressions and summaries of data. They distill complex ecological relationships into simplified rules and identify the species necessary for sample classification on the basis of detailed ecological inventories. The usefulness of this technique to characterize and represent differences in the grape mycobiota of distinct origins was evaluated. Grapes from four Portuguese winemaking regions were selected for a 3-year study: Alentejo, Douro, Ribatejo and Vinhos Verdes. The mycobiota of grapes was assessed with plating methods and the frequencies of isolations of the fungal taxa identified in 32 samples were used as a training dataset. The CT algorithm selected the fungal taxa and respective frequencies to classify grapes according to its region of origin. The ten-fold cross-validation technique was used for model evaluation. The success rate of the model was quantified and expressed in the number of correctly classified samples overall and into region. Furthermore, model refinement was performed using attribute selection algorithms and class redefinition. A simple tree model was generated that classified grapes into three regional origins: Douro, South (Alentejo and Ribatejo classes together) and Vinhos Verdes, on the basis of the incidence of Aspergillus niger aggregate and Penicillium thomii in samples with an accuracy of 82%. The merits and demerits of these models are discussed.

Algorithms↗

Method for the quantitative extraction of resveratrol and piceid isomers in grape berry skins. Effect of powdery mildew on the stilbene content.

A simple method for the quantitative extraction of resveratrol and its glycosides from grape berry skins has been developed. Optimal conditions for the extraction were investigated. Type of solvent, time, and temperature assayed influenced resveratrol and piceid yield. Adequate extraction was attained with ethanol/water (80:20 v/v) maintained at 60 degrees C for 30 min. Recovery (>96%) and reproducibility (6.83-15.13%) were satisfactory. After extraction, resveratrol and piceid isomers were quantified by high-performance liquid chromatography coupled to a ultraviolet-visible diode array detector. The amounts detected in 13 samples of 7 different varieties analyzed were, on average, 92.33 microg/g of dry skin for cis-piceid, 42.19 microg/g of dry skin for trans-piceid, and 24.06 microg/g of dry skin for trans-resveratrol. cis-Resveratrol was not detected in any sample. In grape berries infected by powdery mildew the contentw of these compounds were considerably increased and the degree of infection was positively related to their stilbene content.

Anticarcinogenic Agents↗

Gluconic acid, its lactones, and SO(2) binding phenomena in musts from botrytized grapes.

Intramolecular gluconic acid esterification reactions led to the formation of two lactones, gamma- and delta-gluconolactone (glucono-1,4-lactone and glucono-1,5-lactone). The presence of the first has not yet been reported in must or wine. These lactones are in equilibrium with gluconic acid, gamma- and delta-gluconolactone representing, respectively, 5.8 and 4.1% of the acid level. Correlations between must SO(2) binding power, gluconic acid, and consequently its lactones are shown. The SO(2) affinity of a mixture containing this acid and gamma- and delta-gluconolactone was determined, and gluconic acid appeared to be indirectly responsible for approximately 8% of the bindable SO(2) in musts from botrytized grapes.

Botrytis↗

Synthesis and fungistatic activity of new groups of 2,4-dihydroxythiobenzoyl derivatives against phytopathogenic fungi.

Twenty-six compounds, derivatives of amides, hydrazines, hydrazides, hydrazones, and semicarbazides, with a 2,4-dihydroxythiobenzoyl moiety, were synthesized from sulfinyl-bis(2,4-dihydroxythiobenzoyl). The compositions and chemical structures of these compounds were confirmed by IR, (1)H NMR, EI-MS, and elemental analysis. Antifungal properties of chemicals under in vitro conditions against five phytopathogenic fungi were estimated. In vivo studies against Erisiphe graminis were also carried out. The compounds N-substituted with an 2,4-dihydroxythiobenzamide group proved to be the most active. N-2-(1-Cinnamylbenzene ester)-2,4-dihydroxythiobenzamide, under in vitro conditions, showed activity at the level of 80-100% development of most pathogens at a concentration of 20 microg/mL and partially at a concentration of 200 microg/mL. For compounds with -HN-NH- or -NH-N= moiety, weak or no fungistatic properties were found at the concentrations studied.

Alternaria↗

Characterization of European wine glycerol: stable carbon isotope approach.

Glycerol of about 170 European wines was analyzed using gas chromatography-combustion-isotope ratio mass spectrometry technique. (13)C/(12)C isotopic ratio measurements were performed to characterize glycerol's delta(13)C values of genuine wine samples from European Union wine-producing countries. Glycerol was also successfully dosed using an internal reference, 1,5-pentanediol.

Botrytis↗

Protection of originality of Tokaji Aszú: amines and organic acids in botrytized wines by high-performance liquid chromatography.

Amine and organic acid composition of Aszú wines from the Tokaj region of Hungary, nonbotrytized Hungarian wines from different regions, and foreign botrytized wines were analyzed by high-performance liquid chromatography. Hungarian and foreign wines (36 Hungarian and seven foreign botrytized wines) were compared in different ways by calculation of ratios of given amine compounds, analyses of variance, principal component, and discriminant analysis. In wines, putrescine and in some samples 3-methyl-butylamine and/or phenyl ethylamine were found in remarkable concentrations, while in botrytized wines four other amines were verified in high concentration. Good separation between Aszú and foreign botrytized wines was found by calculation of the amine component's ratio. The first two principal components of the principal component analysis accounted for 77 and 84% of the total variance in the data of amines and acids, respectively. The component scores of samples grouped according to Aszú, foreign botrytized, and nonbotrytized wines. Linear discriminant analysis was used for differentiation of Aszú, foreign botrytized, and normal wines. Using nine amines and two acids as variables, the correct classification was 97.6%. On the basis of results, an objective evaluation method can be elaborated for quality control in order to protect the authenticity and origin of wine specialties made from botrytized grapes.

Acids↗

Odor-active alcohols from the fungal transformation of alpha-farnesene.

Submerged microbial cultures were screened for their potential to oxifunctionalize alpha-farnesene. The major oxidation product in all transforming cultures, 3,7,11-trimethyldodeca-1,3(E),5(E)10-tetraen-7-ol, showed a pleasant citrus-like odor and peak concentrations of 170 mg L-1. An Aspergillus niger isolate from mango generated another two terpene alcohols identified as diastereomeric menth-1-en-3-[2-methyl-1,3-butadienyl]-8-ol, a new natural compound with an apricot-like odor. The regiospecifity of the oxygen attack with concurrent lack of stereoselectivity suggested that the initial step of the bioconversion resembled the chemical autoxidation starting with the generation of an intermediate resonance-stabilized carbon-centered radical or carbocation.

Alcohols↗

Role of carbonyl compounds in SO(2) binding phenomena in musts and wines from botrytized grapes.

Carbonyl compounds play an important role in musts from botrytized grapes. Some of them, such as glyoxal and methylglyoxal, may explain a considerable part of bindable SO(2). Others, such as 2- and 5-oxogluconic acids, produced by gluconic acid oxidation in proportions respectively from 2.5 per 1 play an interesting role as SO(2) binding indicator. Finally, the levels of some compounds such as dihydroxyacetone, 5-oxofructose, and delta-gluconolactone in balance with gluconic acid are well correlated with SO(2) binding powers and also explain a large part of the bindable SO(2) in musts. During alcoholic fermentation, only dihydroxyacetone among these three compounds is metabolized by yeast. Thus, two compounds present in grapes, delta-gluconolactone and 5-oxofructose, with three yeast SO(2)-binding byproducts, ethanal, pyruvic, and 2-oxoglutaric acids, explain much of the SO(2) binding power in wines from botrytized grapes.

Botrytis↗