Este trabalho foi financiado pela Fundação Alemã de Ciência (DFG) ea Iniciativa de Excelência dos governos federal alemão e do Estado.

1. Reticulação de material vegetal <ol><li> Colheita 1-2g de folhas de Arabidopsis (6 a 10 centímetros de diâmetro de roseta) em um tubo plástico de 50 ml e encher-se para 40mL com tampão de reticulação. </li><li> Certifique-se de folhas de ficar imersa, por exemplo, encher um direito adaptado esponja do filtro acima da superfície buffer. Em seguida, coloque os tubos em um dessecador. </li><li> Vácuo infiltrado por 10min. A cada tubo adicionar 2,5 ml de glicina 2M para parar de reticulação, e inverter os tubos para justificar a dispersão igual de glicina. </li><li> Vácuo infiltrado por outro 5min e descarte de fluidos, enquanto o corte de folhas em uma peneira. </li><li> Lave as folhas duas vezes com 1L de água em um copo de vidro, em seguida, deixa secar bem com papel toalha. </li><li> Coleta de folhas em um tubo de plástico fresco e congelar em nitrogênio líquido. Loja deixa a -80 ° C até o isolamento da cromatina. </li></ol> 2. Isolamento de cromatina <ol><li> Folhas foram moídas com argamassa e pistilo que foram mantidas em nitrogênio líquido até o uso. </li><li> Transferência de pó para um tubo plástico de 50 mL e suspendê-tampão de extração 30mL # 1. </li><li> Incubar por 15 min a 4 ° C em um agitador vertical. </li><li> Filtrado de suspensão por quatro camadas de miracloth em um tubo plástico de 50 mL fresco. </li><li> Rotação por 20min em 2800 xg, a 4 ° C. </li><li> Remover o sobrenadante e pellet suspender com um pincel em tampão de extração 1mL # 2. Primeiro adicionar um pequeno volume de tampão # 2, então suspender o pellet com um pincel, em seguida, adicione o restante do buffer. </li><li> Transferência de suspensão para um tubo de microcentrífuga 1,5 ml e girar para 10min a 12.000 xg, a 4 ° C. </li><li> Enquanto isso, prepare-microcentrífuga 2mL tubos contendo tampão de extração 1,5 ml # 3. </li><li> Remover o sobrenadante do tubo girou (passo 2.7) e suspender sedimento em 300 ul de tampão de extração n º 3. Primeiro adicionar um pequeno volume de tampão # 3, suspender pellet com um pincel, e depois adicionar o buffer restante. </li><li> Pipeta com cuidado o pellet suspenso (passo 2.9) em cima do tampão de extração 1,5 mL # 3 preparada no passo 2.8. Certifique-se que as duas fases não se misturam. Rotação por 1h a 16.000 xg, a 4 ° C. </li><li> Remover o sobrenadante e pellet suspender cromatina com um pincel em 300 ul de tampão de sonicação. </li><li> Sonicate cromatina com um sonotrodo, ou em um banho ultra-sônico, para um tamanho de DNA de aproximadamente 400pb. Quando sonicando, fazer cromatina certeza não vai ser aquecido acima de aproximadamente 30 ° C para proteger sensíveis ao calor ligações cruzadas. A duração da sonicação precisa ser determinado experimentalmente, porque varia significativamente entre os dispositivos sonicação diferentes. Para orientação, as definições para dois dispositivos diferentes são fornecidos: </li></ol> Para sonicação com uma marca Bandelin sonotrodo expor cromatina em tubo de microcentrífuga de 0,6 ml cinco vezes a 20 explosões com configurações de 25% de energia de ciclo, = 50. Ao fazê-lo amostra, fresco após cada 20 rajadas º em banho de gelo. Para sonicação com um banho de ultra-som Bioruptor Diagenode, preencha banho com água e gelo e sonicate dez vezes por 30 segundos em tubos de microcentrífuga 1,5 ml com ajuste de &#39;alto&#39;. Depois a cada 30 segundos, as amostras de fresco por mais 30 segundos. <ol start="13"><li> Spin-sonicado amostra para 5min a 16.000 g a 4 ° C para precipitar o material não dissolvido. O sobrenadante pode ser usado diretamente para imunoprecipitação. Alternativamente, as amostras podem ser de choque congelado em nitrogênio líquido e armazenadas a -80 ° C até à sua utilização. </li></ol> 3. Imunoprecipitação <ol><li> Prepare os tubos de 2 mL de microcentrífuga contendo proteína-A 40μL. Agarose e 1.8mL de anticorpos de ligação de tampão Adicionar 200μL da preparação da cromatina (passo 2.13). </li><li> Incubar os tubos por 1h em um agitador vertical. </li><li> Descarte de proteína-A agarose contas e usar o sobrenadante para imunoprecipitação. </li><li> Remover uma alíquota de 40μL para determinar a concentração de cromatina (&#39;input&#39;). </li><li> Adicionar 30μL de proteína-A. Agarose ea quantidade de anticorpos específicos de modificação que está indicado na tabela abaixo de reagentes e equipamentos específicos para suspensão da cromatina 400μL sonicado </li><li> Incubar por 2.5h ou durante a noite em um agitador vertical a 4 ° C. </li><li> Spin down contas de 2min em 440 x g. </li><li> Lavar talão pellet com 900μL de cada tampão de lavagem (veja a lista abaixo). Para cada buffer, contas incubar no buffer de 10min em um agitador vertical a 4 ° C, em seguida, girar para a 2min a 440 xg, elimine o sobrenadante e adicionar o próximo buffer. <ol><li> Tampão de lavagem pouco sal </li><li> Tampão de lavagem de alta sal </li><li> LiCl tampão de lavagem </li><li> TE tampão de lavagem </li></ol></li><li> Após a última lavagem, remover completamente qualquer solução remanescente. </li></ol> 4. De crosslinking de histonas e DNA, e purificação de DNA precipitado <ol><li> Adicionar 100μL de reticulação de-buffer para o pellet agarose e amostra &#39;input&#39; do 40μl a partir do passo 3.4, mix de 5min em um misturador vórtice, as amostras de spin, e incubate a 65 ° C durante a noite. </li><li> Purificar DNA com DNA comercial ou kit de purificação de PCR. </li><li> Tipicamente 5μl de uma diluição de 1:05 do eluído finais são suficientes para executar convencionais lugar específico quantitativa em tempo real de RT-PCR (RT-qPCR). </li></ol> 5. Tabela de buffers <table border="1"><tbody><tr><td colspan="2"> Buffer de reticulação </td></tr><tr><td> Butirato de sódio </td><td> 10mM </td></tr><tr><td> Sacarose </td><td> 400mm </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10mM </td></tr><tr><td> β-mercaptoetanol </td><td> 5mM </td></tr><tr><td> Formaldeído </td><td> 3% (v / v) </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Tampão de extração # 1 </td></tr><tr><td> Butirato de sódio </td><td> 10mM </td></tr><tr><td> Sacarose </td><td> 400mm </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10mM </td></tr><tr><td> β-mercaptoetanol </td><td> 5mM </td></tr><tr><td> Inibidor de Protease completo </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Tampão de extração # 2 </td></tr><tr><td> Butirato de sódio </td><td> 10mM </td></tr><tr><td> Sacarose </td><td> 250mm </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10mM </td></tr><tr><td> β-mercaptoetanol </td><td> 5mM </td></tr><tr><td> MgCl 2 </td><td> 10mM </td></tr><tr><td> Triton X-100 </td><td> 1% (w / v) </td></tr><tr><td> Inibidor de Protease completo </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Tampão de extração # 3 </td></tr><tr><td> Butirato de sódio </td><td> 10mM </td></tr><tr><td> Sacarose </td><td> 1.7M </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 10mM </td></tr><tr><td> β-mercaptoetanol </td><td> 5mM </td></tr><tr><td> MgCl 2 </td><td> 2mM </td></tr><tr><td> Triton X-100 </td><td> 0,15% (w / v) </td></tr><tr><td> Inibidor de Protease completo </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Tampão de sonicação </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 25mM </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 5mM </td></tr><tr><td> SDS </td><td> 0,5% (w / v) </td></tr><tr><td> Inibidor de Protease completo </td><td> 1x </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Tampão de ligação do anticorpo </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 50mM </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 1mM </td></tr><tr><td> NaCl </td><td> 150mm </td></tr><tr><td> Triton X-100 </td><td> 0,1% (w / v) </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Tampão de lavagem pouco sal </td></tr><tr><td> NaCl </td><td> 150mm </td></tr><tr><td> SDS </td><td> 0,1% (w / v) </td></tr><tr><td> Triton X-100 </td><td> 1% (w / v) </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 2mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 20mM </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> Tampão de lavagem de alta sal </td></tr><tr><td> NaCl </td><td> 500mM </td></tr><tr><td> SDS </td><td> 0,1% (w / v) </td></tr><tr><td> Triton X-100 </td><td> 1% (w / v) </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 2mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 20mM </td></tr></tbody></table><table border="1"><tbody><tr><td colspan="2"> LiCl tampão de lavagem </td></tr><tr><td> Cloreto de lítio </td><td> 250mm </td></tr><tr><td> Nonidet-P40 </td><td> 1% (v / v) </td></tr><tr><td> Desoxicolato de sódio </td><td> 1% (w / v) </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 1mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 20mM </td></tr></tbody></table><table border="1"><tbody><tr><td> TE tampão de lavagem </td></tr><tr><td> EDTA-NaOH, pH 8,0 </td><td> 10mM </td></tr><tr><td> Tris-HCl, pH 8,0 </td><td> 1mM </td></tr></tbody></table><table border="1"><tbody><tr><td> Tampão Decrosslinking </td></tr><tr><td> Tris-HCl, pH 6,8 </td><td> 62,5 mm </td></tr><tr><td> NaCl </td><td> 200mM </td></tr><tr><td> SDS </td><td> 2% (w / v) </td></tr><tr><td> TDT </td><td> 10mM </td></tr></tbody></table> 6. Resultados representativos: Expressão do gene Arabidopsis thaliana defesa PR2 codifica uma β-1,3-glucanase com atividade antimicrobiana 12, foi induzido por benzothiadiazole (BTH), um análogo sintético do ácido salicílico hormônio vegetal 3. Figura 1 demonstra que a ativação de PR2 expressão é associada a um aumento na acetilação de resíduos de lisina na histona H3 e H4 do promotor PR2. Como a densidade diminui nucleosome durante a ativação do gene 13, os valores de acetilação das histonas foram normalizados para o sinal obtido com um anticorpo para uma região invariante de 3 histonas. <img src="/files/ftp_upload/3096/3096fig1.jpg" alt="Figura 1"/> Figura 1 BTH induz acetilação das histonas no promotor PR2 em Arabidopsis. As plantas foram tratadas com 100μM BTH ou um controle de pó molhável. Após 72h, foram coletadas folhas e cromatina foi isolado. O sinal obtido após precipitação com acetilação anticorpos específicos (como indicado na figura) foi normalizada para o sinal obtido por precipitação com um anticorpo para um domínio invariante de histona H3. Cromatina precipitado foi quantificada por RT-qPCR. Dados são padronizados para níveis de modificação das histonas na ausência de tratamento BTH.

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Não há conflitos de interesse declarados.

Etapas mais críticas no protocolo são a reticulação de DNA e histonas, o tipo ea quantidade de material foliar, moagem do material, ea sonicação de cromatina. Para uma discussão mais detalhada dessas questões, ver refs. 9 e 10. Sonicação e crosslinking: É importante para interromper cromatina durante a sonicação a fragmentos de cerca de 400pb. Sonicação exaustiva irá destruir cromatina interrompendo DNA e histonas, enquanto que sonicação insuficiente vai deixar fragmentos de cromatina longo. Estes afetam a especificidade do método, porque modificações das histonas distal do locus testados não podem ser discriminados de alterações local. Eficiência sonicação é melhor testado através da recolha de cromatina 20μL de amostras de DNA antes e depois sonicação, de reticulação e histonas, isolando o DNA e determinar o comprimento do DNA cortado por eletroforese em gel de agarose. RNAse deve ser adicionado às amostras antes de eletroforese, porque a preparação da cromatina ainda contém grandes quantidades de RNA nesta fase de purificação que pode interferir com a visualização de DNA fragmentado. As amostras são úteis para imunoprecipitação da cromatina se o DNA de cromatina não-cortado é maior do que 10kbp, enquanto que o DNA de cromatina sheared forma uma mancha com intensidade máxima do sinal em torno de 400pb de DNA. Rotineiramente usamos 3% (v / v) de formaldeído para crosslinking cromatina em folhas intactas, mas 1% (v / v) de formaldeído pode ser suficiente na maioria dos casos. Em nossas mãos, as concentrações de formaldeído baixo permitem menor tempo de sonicação e sinais de fundo em reações consequente PCR. No entanto, quantidades insuficientes de formaldeído muitas vezes resultam em baixa reprodutibilidade do sinal de PCR entre os experimentos independentes. Isso pode ser devido a penetração insuficiente de folhas com formaldeído em baixas concentrações. Certifique-se de trocar o seu estoque de formaldeído com freqüência como o composto tende a polimerizar com o tempo. As soluções de formaldeído são estáveis ​​apenas para cerca de um mês, e este período é prolongado por quase estabilização metanol. É aconselhável a análise de diferentes concentrações de formaldeído durante a configuração de condições experimentais. Quantidade de material vegetal e de moagem: É importante para se infiltrar pequenas quantidades de material foliar em um grande volume de tampão para evitar que as folhas grudem umas nas outras. Folha furando muitas vezes resulta em infiltração insuficiente de formaldeído. Além disso, o material vegetal muito vai bloquear os poros do tecido miracloth. A eficiência de moagem depende consideravelmente usando um almofariz e um pilão, com o ajuste perfeito. Rotineiramente moer com nitrogênio líquido refrigerado pilão na ausência de nitrogênio líquido adicional de três vezes por 50 segundos até que o material é moído para um pó fino.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> Proteína-A-Agarose </td><td> Roche </td><td> 11 134 515 001 </td><td> depende da classe de anticorpos </td></tr><tr><td> Proteína-G-Agarose </td><td> Roche </td><td> 11 243 233 001 </td><td> depende da classe de anticorpos </td></tr><tr><td> Anti-hyperacetyl-H4 </td><td> Millipore </td><td> 06-946 </td><td> usamos 5μL </td></tr><tr><td> Anti-acetil-H4K5 </td><td> Millipore </td><td> 07-327 </td><td> usamos 5μL </td></tr><tr><td> Anti-acetil-H4K8 </td><td> Millipore </td><td> 07-328 </td><td> usamos 5μL </td></tr><tr><td> Anti-acetil-H4K12 </td><td> Millipore </td><td> 07-595 </td><td> usamos 5μL </td></tr><tr><td> Anti-acetil-H4K16 </td><td> Millipore </td><td> 07-329 </td><td> usamos 5μL </td></tr><tr><td> Anti-acetil-H4K18 </td><td> Millipore </td><td> 07-354 </td><td> usamos 1μL </td></tr><tr><td> Anti-acetil-H3K9 </td><td> Millipore </td><td> 07-352 </td><td> usamos 5μL </td></tr><tr><td> Anti-acetil-H3K14 </td><td> Millipore </td><td> 07-353 </td><td> usamos 1μL </td></tr><tr><td> Anti-trimetil-H3K4 </td><td> Diagenode </td><td> PAB-003-50 </td><td> usamos 5μL </td></tr><tr><td> Anti-dimetil-H3K4 </td><td> Millipore </td><td> 07-030 </td><td> usamos 5μL </td></tr><tr><td> Anti-monometil-H3K4 </td><td> Abcam </td><td> ab8895 </td><td> 2,5-10μL </td></tr><tr><td> Anti-H3 </td><td> Abcam </td><td> ab1791 </td><td> usamos 1μL para verificar a densidade das histonas </td></tr><tr><td> Bioruptor </td><td> Diagenode </td><td> UCD TO-200 </td><td></td></tr><tr><td> Sonotrodo MS72 </td><td> Bandelin </td><td></td><td></td></tr><tr><td> Miracloth </td><td> Calbiochem </td><td> 475855 </td><td></td></tr><tr><td> Inibidor de Protease completo </td><td> Roche </td><td> 11836145001 </td><td></td></tr></tbody></table>

detection of histone modifications in plant leaves

Estrutura da cromatina é importante para a regulação da expressão gênica em eucariotos. Neste processo, a remodelação da cromatina, a metilação do DNA e modificações covalentes nas caudas amino-terminal de histonas H3 e H4 desempenham papéis essenciais 1-2. H3 e H4 modificações das histonas incluem metilação de lisina e arginina, acetilação da lisina e fosforilação de resíduos de serina 1-2. Essas modificações estão associadas tanto com a ativação do gene, a repressão, ou um estado preparado de gene que suporta a ativação mais rápida e robusta de expressão após a percepção de sinais apropriados (micróbio-padrões moleculares associados, luz, hormônios, etc) 3-7. Aqui, apresentamos um método para a detecção confiável e sensível de modificações específicas da cromatina em genes de plantas selecionadas. A técnica é baseada na reticulação de (modificado) histonas e DNA com formaldeído 8,9, extração e sonicação da cromatina, cromatina imunoprecipitação (CHIP) com a modificação de anticorpos específicos 9,10, de reticulação de histona-DNA complexos, e gene-específicos em tempo real, PCR quantitativo. A abordagem tem se mostrado útil para detectar modificações específicas histonas associadas C 4 fotossíntese em 5,11 milho e imunidade sistêmica em 3 Arabidopsis.

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Detection of Histone Modifications in Plant Leaves

Uma abordagem confiável e útil para detectar modificações das histonas em genes específicos de plantas é descrito. A abordagem combina cromatina imunoprecipitação (chip) e real-time PCR quantitativo. Ela permite a detecção de modificações das histonas em genes específicos, com um papel em diversos processos fisiológicos.

Detecção de histona Modificações nas folhas das plantas

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

detection-of-histone-modifications-in-plant-leaves

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24.7K visualizações.  RWTH Aachen University. Uma abordagem confiável e útil para detectar modificações das histonas em genes específicos de plantas é descrito. A abordagem combina cromatina imunoprecipitação (chip) e real-time PCR quantitativo. Ela permite a detecção de modificações das histonas em genes específicos, com um papel em diversos processos fisiológicos.

Vídeo: Detection of Histone Modifications in Plant Leaves

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

Detecção de modificações pós-translacionais sobre Native Nucleossomos Intact por ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Biologia

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

Detecção de interações proteína na planta através de um gateway de Complementação de fluorescência Compatível Bimolecular (BiFC) Sistema

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Profiling glicana de Polímeros da planta da parede celular usando Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Identificação de modificações pós-translacionais de Complexos de proteína vegetal

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

A técnica de infiltração-centrifugação para Extração de apoplásticas Fluid das folhas da planta Usando<em&gt; Phaseolus vulgaris</em&gt; Como exemplo

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Quantificação de alta resolução Celulose cristalina Acumulação em<em&gt; Arabidopsis</em&gt; Raízes para monitorar celular específico do tecido da parede Modificações

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

De alto rendimento CRISPR Vector construção e caracterização de DNA Modificações pela geração de raízes de tomateiro Cabeludo

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Fluxo de trabalho completo para Análise de histona modificações pós-traducionais Usando Bottom-up Espectrometria de Massa: De histona Extração de Análise de Dados

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

Identificação de novos reguladores de transpiração vegetal por triagem de imagens térmicas em larga escala em Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

Global Level Quantification of Histone Post-Translational Modifications in a 3D Cell Culture Model of Hepatic Tissue

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid tissues due to unnaturally rapid cell replication. This is particularly challenging when modeling mature chromatin, as fast replicating cells are frequently involved in DNA replication and have a heterogeneous polyploid population. Presented below is a workflow for modeling, treating, and analyzing quiescent chromatin modifications using a three-dimensional (3D) cell culture system. Using this protocol, hepatocellular carcinoma cell lines are grown as reproducible 3D spheroids in an incubator providing active nutrient diffusion and low shearing forces. Treatment with sodium butyrate and sodium succinate induced an increase in histone acetylation and succinylation, respectively. Increases in levels of histone acetylation and succinylation are associated with a more open chromatin state. Spheroids are then collected for isolation of cell nuclei, from which histone proteins are extracted for the analysis of their post-translational modifications. Histone analysis is performed via liquid chromatography coupled online with tandem mass spectrometry, followed by an in-house computational pipeline. Finally, examples of data representation to investigate the frequency and occurrence of combinatorial histone marks are shown.

This protocol outlines how a three-dimensional cell culture system can be used to model, treat, and analyze chromatin modifications in a near-physiological state.

Quantificação de nível global de modificações pós-translacionais histone em um modelo de cultura celular 3D de tecido hepático

Flat cultures of mammalian cells are a widely used in vitro approach for understanding cell physiology, but this system is limited in modeling solid ...