Localisation studies of cell-wall degrading enzymes in soybean

Postembedding immunoelectron microscopy has been used to investigate the diffusibility of an endo-beta-1,4-glucanase and a xylanase from A. niger in soybean. The results showed more specific localisation of the enzymes into the protein and lipid bodies of soybean cells. This was against our hypothesis that suggested that the enzymes should be localised in the cell wall.

Transcriptome analysis for discovering candidate genes involve in embryogenesis in coconut (Cocos nucifera L.) through 454 pyrosequencing

Coconut, Cocos nucifera L. is a major plantation crop, which ensures income for millions of people in the tropical region. Detailed molecular studies on zygotic embryo development would provide valuable clues for the identification of molecular markers to improve somatic embryogenesis. Since there is no ongoing genome project for this species, coconut expressed sequence tags (EST) would be an interesting technique to identify important coconut embryo specific genes as well as other functional genes in different biochemical pathways. The goal of this study was to analyse the ESTs by examining the transcriptome data of the different embryo tissue types together with one somatic tissue. Here, four cDNA libraries from immature embryo, mature embryo, microspore derived embryo and mature leaves were constructed. cDNA was sequenced by the Roche-454 GS-FLX system and assembled into 32621 putative unigenes and 155017 singletons. Of these unigenes, 18651 had significant sequence similarities to non-redundant protein database, from which 16153 were assigned to one or more gene ontology categories. Homologue genes, which are responsible for embryo development such as chitinase, beta-1,3-glucanase, ATP synthase CF0 subunit, thaumatin-like protein and metallothionein-like protein were identified among the embryo EST collection. Of the unigenes, 6694 were mapped into 139 KEGG pathways including carbohydrate metabolism, energy metabolism, lipid metabolism, amino acid metabolism and nucleotide metabolism. This collection of 454-derived EST data generated from different tissue types provides a significant resource for genome wide studies and gene discovery of coconut, a non-model species.

On the Apterous Line of the Termite Velocitermes heteropterus (Isoptera: Termitidae): Developmental Pathways and Cellulose Digestion

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Validation of reference genes for RT-qPCR normalization in common bean during biotic and abiotic stresses

Selection of reference genes is an essential consideration to increase the precision and quality of relative expression analysis by the quantitative RT-PCR method. The stability of eight expressed sequence tags was evaluated to define potential reference genes to study the differential expression of common bean target genes under biotic (incompatible interaction between common bean and fungus Colletotrichum lindemuthianum) and abiotic (drought; salinity; cold temperature) stresses. The efficiency of amplification curves and quantification cycle (C (q)) were determined using LinRegPCR software. The stability of the candidate reference genes was obtained using geNorm and NormFinder software, whereas the normalization of differential expression of target genes [beta-1,3-glucanase 1 (BG1) gene for biotic stress and dehydration responsive element binding (DREB) gene for abiotic stress] was defined by REST software. High stability was obtained for insulin degrading enzyme (IDE), actin-11 (Act11), unknown 1 (Ukn1) and unknown 2 (Ukn2) genes during biotic stress, and for SKP1/ASK-interacting protein 16 (Skip16), Act11, Tubulin beta-8 (beta-Tub8) and Unk1 genes under abiotic stresses. However, IDE and Act11 were indicated as the best combination of reference genes for biotic stress analysis, whereas the Skip16 and Act11 genes were the best combination to study abiotic stress. These genes should be useful in the normalization of gene expression by RT-PCR analysis in common bean, the most important edible legume.

Uso de Metarhizium spp. na produção de mudas pré-brotadas de cana-de-açúcar e seus efeitos na planta, em pragas e doenças

Os fungos do gênero Metarhizium são entomopatogênicos e ainda apresentam relações endofíticas e podem viver saprofiticamente no solo. A associação desses microrganismos com insetos é bem conhecida, mas as interações diretas com as plantas ainda são incipientes. Objetivou-se com este estudo, determinar a capacidade de colonização endofítica em raízes de cana-de-açúcar (Saccharum spp.) de M. robertsii, M. anisopliae e três linhagens brasileiras recém-descobertas, bem como revelar o potencial destes fungos como antagonistas a dois fungos fitopatogênicos responsáveis pela podridão-vermelha (Fusarium moniliforme e Colletotrichum falcatum) e no controle de duas importantes pragas, a broca da cana-de-açúcar, Diatraea saccharalis e o nematóide-das-galhas, Meloidogyne javanica. Foram utilizados 24 isolados de Metarhizium spp. nos experimentos de promoção de crescimento de plantas após a inoculação em gemas de cana-de-açúcar em condições de casa-de-vegetação e campo. O efeito da inoculação do fungo em mudas de cana-de-açúcar foi investigado quanto a mortalidade e redução no desenvolvimento de D. saccharalis e redução nas populações de M. javanica. Experimentos de inibição in vitro em cultivo pareado foram conduzidos com os fungos F. moniliforme e C. falcatum e Trichoderma harzianum. Além disto, foram realizados testes qualitativos \"in vitro\" para avaliar a capacidade desses fungos em produzir β-1,3-Glucanase e Sideróforos. A inoculação de Metarhizium de todas as espécies testadas promoveu o crescimento de parte aérea, peso úmido e seco de raízes em relação ao controle (água). Enquanto o tratamento de gemas rotineiramente usado na usina onde se realizou a produção de mudas pré-brotadas com aplicação do fungicida Comet® + o fertilizante Biozyme TF resultou em pegamento de 32% e 59,6%, apenas com o uso de isolados de Metarhizium spp. estes valores foram de até 50% e 86,4%, nos dois experimentos em campo, respectivamente. As mortalidades observadas em lagartas de D. saccharalis que se alimentaram das plantas inoculadas com isolados de Metarhizium spp. atingiram valores de até 80%, sendo a mortalidade confirmada pela esporulação do fungo de até 55%. As lagartas sobreviventes apresentaram menor peso do que aquelas do controle não tratado. A inoculação de Metarhizium spp. em mudas de cana-de-açúcar conferiu proteção a M. javanica, resultando em uma densidade de massa de ovos por grama de raiz nas mudas inoculadas com o fungo até 59,8% menor no primeiro experimento e 64,1% menor no segundo em relação as mudas sem inoculação. Os ensaios de antagonismo mostraram que, todos os isolados de Metarhizium spp. apresentaram alguma forma de inibição tanto para F. moniliforme como para C. falcatum. Dos 24 isolados testados, 20,8% produziram a enzima hidrolítica, β-1,3-glucanase, o que pode estar associado a capacidade de inibição dos fungos fitopatogênicos. Constatou-se que 8 (33,3%) dos isolados produziram sideróforos e sugere que esses fungos apresentam mecanismos de disponibilização do ferro. O conhecimento gerado com este estudo poderá subsidiar novas estratégias de utilização de Metarhizium spp. em campo na produção de MPB de cana-de-açúcar.

Taxtomina A e Piriformospora indica no controle de Phytophthora nicotianae em citros e Phytophthora plurivora em faia (Fagus sylvatica)

Espécies de Phytophthora tem se destacado ao longo da história devido ao seu potencial destrutivo, se iniciando com a devastadora P. infestans na Irlanda e se estende até os dias de hoje com P. nicotianae em citros e P. plurivora em faia. Uma característica importante deste grupo de patógenos é que as medidas de controle da doença se baseiam na prevenção da entrada do patógeno na área visto que, uma vez instalado, o produtor precisa conviver com o mesmo, pois não se dispõem de métodos efetivos de controle. Neste sentido, a busca por métodos de controle torna-se primordial. O endofítico radicular Piriformospora indica, tem-se destacado em vários patossistemas devido a sua habilidade de induzir resistência contra patógenos, aumentar a tolerância à estresses abióticos e promover o crescimento de plantas. Taxtomina A, produzida por Streptomyces scabies, é capaz de ativar mecanismos de defesa de plantas, os quais são efetivos contra agentes patogênicos. Objetivou-se com este trabalho avaliar o efeito de P. indica e da taxtomina A sobre P. nicotianae em citros e P. plurivora em faia. Ambos foram avaliados quanto ao seu efeito direto sobre os patógenos em questão. O indutor de defesa vegetal Bion® foi utilizado em alguns ensaios para fins de comparação. Plântulas de citros e faia foram tratadas com concentrações crescentes de taxtomina e parâmetros fisiológicos, bioquímicos e de controle da doença foram avaliados. Taxtomina A não apresenta efeito direto sobre os patógenos avaliados. Os dados de incidência da doença em plântulas de faia tratadas com taxtomina A nas concentrações de 10, 25, 50 e 100 μg se mostraram consistentes com a quantidade de DNA do patógeno no sistema radicular, demonstrando que, aparentemente, a toxina induziu suscetibilidade nas plântulas de faia. Em citros, para os parâmetros fisiológicos e bioquímicos avaliados, em linhas gerais, a taxtomina A nas concentrações de 50 e 100 μg demonstrou potencial de aplicação no patossistema citros - P. nicotianae. Quando avaliada a mortalidade de plantas inoculadas com o patógeno e tratadas com taxtomina, bem como, quando quantificado o DNA do oomiceto no sistema radicular, as referidas concentrações também apresentaram os melhores desempenhos. Plântulas das mesmas espécies foram submetidas a inoculação com P. indica, sendo avaliados os efeitos na promoção de crescimento, na atividade de enzimas e de genes relacionados ao processo de defesa, bem como, no controle da doença. Não foi observado efeito direto do endofítico radicular sobre os patógenos avaliados. Quando plântulas de citros foram inoculadas com P. indica e depois com P. nicotianae, não foi observada promoção de crescimento e controle da doença. As análises histológicas e moleculares demonstraram a presença do endofítico no sistema radicular de plântulas de citros e faia. Análises bioquímicas revelaram apenas aumentos pontuais no teor de proteínas e na atividade da β-1,3-glucanase e da peroxidase no tratamento com P. indica + P. nicotianae. Os genes PR-1.4, PR-1.8, PR-β-glucosidase e Hsp70 foram induzidos em plântulas inoculadas com P. indica e com o patógeno, bem como no tratamento com Bion® e patógeno, porém em menor magnitude. O endofítico P. indica ativa o sistema de defesa de plântulas de citros, no entanto, os mecanismos ativados não são efetivos para o controle da doença na interação citros - P. nicotianae.

Methyl jasmonate induced gene expression in wheat delays symptom development by the crown rot pathogen Fusarium pseudograminearum

The necrotrophic fungal pathogen Fusarium pseudograminearum (F. pseudograminearum) causes crown rot disease (CR) in wheat. This host-pathogen interaction has not been studied previously at the molecular level. In this study. using real-time quantitative PCR, the expression of 26 selected wheat genes was examined 1, 2 and 4 days after inoculation of wheat seedlings of the CR susceptible cultivar Kennedy and the partially field-resistant cultivar Sunco. Reproducible induction of eight defence genes consisting of PR1.1, PR2 (beta,1-3 glucanase), PR3 (chitinase), PR4 (wheativin), PR5 (thaumatin-like protein). TaPERO (peroxidase), PR10 and TaGLP2a (germin-like) was observed. These genes were induced in both cultivars, however. some genes were induced more rapidly in Sunco than in Kennedy. MJ treatment also induced the above pathogen responsive defence genes in both cultivars while benzo(1,2,3)thiadiazole-7-carbothionic acid S-methyl ester (BTH) treatment weakly induced them in Kennedy only. Similarly. treatment with MJ before inoculation significantly delayed the development of necrotic symptoms for 2 weeks in both wheat cultivars, while BTH pre-treatments delayed symptom development in Kennedy only. The chemically induced protection, therefore, correlated with induction of the F. pseudograminearum-responsive genes. These results support the emerging role of jasmonate signalling in defence against necrotrophic fungal pathogens in monocots and future manipulation of this pathway may improve CR resistance in wheat. (c) 2006 Elsevier Ltd. All rights reserved.

Transgenic barley expressing a protein-engineered, thermostable (1,3-1,4)-beta-glucanase during germination.

The codon usage of a hybrid bacterial gene encoding a thermostable (1,3-1,4)-beta-glucanase was modified to match that of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene. Both the modified and unmodified bacterial genes were fused to a DNA segment encoding the barley high-pI alpha-amylase signal peptide downstream of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene promoter. When introduced into barley aleurone protoplasts, the bacterial gene with adapted codon usage directed synthesis of heat stable (1,3-1,4)-beta-glucanase, whereas activity of the heterologous enzyme was not detectable when protoplasts were transfected with the unmodified gene. In a different expression plasmid, the codon modified bacterial gene was cloned downstream of the barley high-pI alpha-amylase gene promoter and signal peptide coding region. This expression cassette was introduced into immature barley embryos together with plasmids carrying the bar and the uidA genes. Green, fertile plants were regenerated and approximately 75% of grains harvested from primary transformants synthesized thermostable (1,3-1,4)-beta-glucanase during germination. All three trans genes were detected in 17 progenies from a homozygous T1 plant.

Evaluation of the beta-glucanolytic enzyme complex of Trichoderma harzianum Rifai for the production of gluco-oligosaccharide fragments by enzymatic hydrolysis of 1,3;1,6-beta-D-glucans

Botryosphaeran, a new exopolysaccharide from the endophytic fungus Botryosphaeria rhodina MAMB-05, and algal laminarin were hydrolyzed by partially-fractionated enzymes of the beta-glucanolytic complex from Trichoderma harzianum Rifai. beta-Glucanase fractions (F-I and F-II) separated by gel permeation chromatography presented different modes of attack on botryosphaeran and laminarin. Botryosphaeran was hydrolyzed to the extent of 66% (F-I) and 98% (F-II) within 30 min, and its main hydrolysis products were gluco-oligosaccharides of DP >= 4, with lesser amounts of glucose, di- and tri-saccharides. The action of enzyme fractions I and II on laminarin resulted in 15% conversion to glucose, while the percentage of saccharification was radically different (70% for F-I and 25% for F-II). The different product arrays within the polysaccharide hydrolysates can be explained by the difference in the enzymes' specificities within each enzyme fraction, and the molecular structures of the polysaccharides and their complexity.

Synthesis of novel beta-aryl-beta-(methylthio)acroleins via Vilsmeier-Haack protocol as potential 1,3-dielectrophilic three-carbon building blocks

A new general route for the synthesis of novel beta-aryl-beta-(methylthio)acroleins, a class of stable potential 1,3-dielectrophilic synthons, has been reported. The overall protocol involves treatment of either beta-chloroacroleins or their precursor iminium salts (generated in situ from the corresponding active methylene ketones under Vilsmeier-Haack reaction conditions) with S,S-dimethyldithiocarbonates (DDC)/aqueous KOH in either a one-pot or two-step process. The dimethyldithiocarbonate (DDC)/30% aqueous KOH has been shown to be an excellent source of methylthiolate anion. (C) 2014 Elsevier Ltd. All rights reserved.

Comparative analysis of allantoin quercetin, and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid in Nitraria tangutorum Bobr. Seed by HPLC-APCI-MS and CE

This paper describes the simultaneous determination of allantoin, quercetin, and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (MTCCA) in Nitraria tangutorum Bobr seed by HPLC-APCI-MS and CE (capillary electrophoresis) methods. The final optimized chromatographic conditions were investigated in a reversed-phase Eclipse XDB-C8 column (150 x 4.6 mm, 5 mu m). A seventeen-minute gradient elution, (A: aqueous acetonitrile 20% (v/v); B: aqueous acetonitrile 60% (v/v); C: pure acetonitrile 100%) at a flow rate of 1.0 mL/min was selected for the separation of three natural products with diode array detection (DAD) at 220 nm. A CE experiment was carried out in a fused silica capillary with 32 mmol/L boric acid (pH 10), 32 mmol/L SDS and acetonitrile (10.0%, v/v). The applied potential and temperature was, respectively, set at 19 kV and 25 degrees C. After development, the validation was performed in parallel for HPLC and CE, with the same standards and sample to avoid differences due to the manipulation. The validation parameters of both techniques were adequate for the intended purpose.

beta-1,2,3-Triazolyl-Nucleosides as Nicotinamide Riboside Mimics

The synthesis of a series of pyridine- and piperidine-substituted 1,2,3-triazolides linked to a riboside moiety is described. The presence of a triazolide substituent on the pyridine moiety permitted the facile reduction of the latter under mild hydrogenation conditions. These analogues were modelled as to define their similarity to nicotinamide riboside and quantify their ability to bind NAD-dependent protein deacetylases.

Differential roles of integrins alpha(2)beta(1), and alpha(IIb)beta(3) in collagen and CRP-induced platelet activation

Collagen and collagen-related peptide (CRP) activate platelets by interacting with glycoprotein (GP)VI. In addition, collagen binds to integrin alpha(2)beta(1) and possibly to other receptors. In this study, we have compared the role of integrins alpha(2)beta(1) and alpha(IIb)beta(3) in platelet activation induced by collagen and CRP. Inhibitors of ADP and thromboxane A(2) (TxA(2)) substantially attenuated collagen-induced platelet aggregation and dense granule release, whereas CRP-induced responses were only partially inhibited. Under these conditions, a proportion of platelets adhered to the collagen fibres resulting in dense granule release and alpha(IIb)beta(3) activation. This adhesion was substantially mediated by alpha(2)beta(1). The alpha(IIb)beta(3) antagonist lotrafiban potentiated CRP-induced dense granule release, suggesting that alpha(IIb)beta(3) outside-in signalling may attenuate GPVI signals. By contrast, lotrafiban inhibited collagen-induced dense granule release. These results emphasise the differential roles of alpha(2)beta(1) and alpha(IIb)beta(3) in platelet activation induced by collagen and CRP. Further, they show that although ADP and TxA(2) greatly facilitate collagen-induced platelet activation, collagen can induce full activation of those platelets to which it binds in the absence of these mediators, via a mechanism that is dependent on adhesion to alpha(2)beta(1).

Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-beta-D-glucan in endosperm of wheat

(1,3;1,4)-beta-d-Glucan (beta-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative beta-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total beta-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable beta-glucan was also reduced by about 50% in the transgenic lines, and the M(r) distribution of the fraction was decreased from an average of 79 to 85 x 10(4) g/mol in the controls and 36 to 57 x 10(4) g/mol in the transgenics. Immunolocalization of beta-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a beta-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.