IEM gostaria de reconhecer o dinamarquês Research Council (FTP e FNU) para financiamento. ERL reconhece o apoio de uma DP ARC subvenção. AB agradece o apoio do Centro de Excelência em ARC Usina concessão paredes celulares.

1. Coleção de tecidos e Preparação <ol><li> Recolher 100 mg de peso fresco de tecidos de planta (um mínimo de 10 mg de peso seco) em pelo menos em triplicado para cada tecido de interesse. Os passos que se seguem descrevem a preparação de material de parede celular a partir de tecidos vegetativos. No caso de tecidos de armazenamento, não-desejado amido é enzimaticamente removida antes de se proceder à extracção de polímeros da parede celular, como descrito anteriormente 13. </li><li> Homogeneizar a amostra até um pó fino com azoto líquido utilizando um Qiagen TissueLyser II, com 24 conjuntos de tubos de adaptador e 3 mm grânulos de carboneto de tungsténio (30 Hz, 2 x 30 s). Em alternativa, se apenas alguns exemplos estão a ser tratados, de um almofariz e pilão é usado. </li><li> Transfira os homogeneizados em 10 ml de plástico tubos cónicos. </li><li> Preparar o material da parede celular, por lavagem dos homogeneizados em 10 ml de etanol a 80% v / v à temperatura ambiente e agitação em vórtex vigorosamente durante 2 min. </li><li> Centrifugue as amostras a 3500 xg e elimine o sobrenadante. Repita o etanol a 70%, pelo menos, lava três vezes ou até que o sobrenadante é claro, em particular para os tecidos que contêm clorofila. </li><li> Realizar uma lavagem final com acetona a 100% e deixar os grânulos contendo os resíduos de álcool insolúvel (AIR) secar ao ar durante a noite. </li><li> Sieve as amostras de ar com uma malha de 0,4 mm 2 para conseguir um pó fino, homogéneo e para remover maior, mal partículas do solo, que por vezes ficam no homogenato. </li><li> Pesar 10 mg de amostra de ar em microtubos contendo, cada um, de 3 mm de contas de vidro para auxiliar a homogeneização das amostras. </li></ol> 2. Extracção de Glicanos parede celular <ol><li> Pectinas, e polímeros relacionados com as pectinas são extraídas as amostras pela adição de 500 ul de 50 mM CDTA (pH 7,5) para cada amostra. </li><li> Brevemente os tubos de vórtice para garantir o solvente está em contacto com o material da amostra e, em seguida misturar com o TissueLyser durante 3 horas a 8 Hz. </li><li> Centrifugue as amostras a 12.000 xg, cuidadosamente remover-se os sobrenadantes e armazenar a 4 ° C. </li><li> Lavar peletes em 1 ml de água desionizada para diluir e remover qualquer remanescente de vórtice, solvente e centrifuga-se a 13.000 x g. Repetir esta etapa, sem perturbar o sedimento e remover todo o líquido a partir dos tubos, antes de prosseguir para o passo seguinte. </li><li> Reticulantes glicanos são sequencialmente extraída da parede celular de pellet restante com 500 ul de NaOH 4M com 0,1% w / v NaBH4, usando o mesmo processo descrito nos passos 2,1-2,4 para a extracção CDTA. NaBH4 é adicionada para reduzir o aldeído (ou cetona) grupo na extremidade redutora dos polissacáridos para uma base de álcool impedindo descamação de polissacarídeos. </li><li> Após centrifugação, os sobrenadantes são novamente removidos, armazenados a 4 ° C, e as pelotas lavadas duas vezes em água desionizada antes de se proceder à extracção seguinte. </li><li> Como passo opcional, os polímeros residuais, tais como a celulose são extraídos com 500 uL cadoxen (31% v / v 1,2-diaminoethane com 0,78 M CdO), utilizando o mesmo procedimento tal como descrito nos passos 2,1-2,4. Alternativamente, o teor de celulose total em peletes restantes podem ser determinadas usando ensaios acético / nítrico (ver discussão). </li></ol> 3. Microarrays impressão <ol><li> Sobrenadantes de centrífuga contendo polímeros extraída da parede celular a 13000 xg para remover qualquer matéria particulada. </li><li> Carregar 50 ul de cada amostra para um polipropileno de 384 poços de placas de microtitulação utilizando uma disposição de pré-concebido costume em que as amostras são organizados de acordo com o tipo de tecido e do tipo de extracção. </li><li> Dilui-se a parede celular da amostra de polímero em um 0, 5 e 25 da série de diluição em série × com água desionizada. </li><li> Parâmetros como altura do pino, coleta e tempo de espera, e de lavagem são definidas no software que controla a microarrayer. </li><li> A humidade da câmara de impressão é controlada em 60% para evitar a evaporação da amostra. </li><li> O trabalho de impressão é iniciado usando LabNEXT software e de um programa, que corresponde ao esquema de microarray. </li><li> O robô utiliza pinos do canal capilar para imprimir as soluções da amostra sobre a placa de 20 x 20 centímetros membrana de nitrocelulose, que é anexada a uma placa plana na máquina. Cada ponto na matriz contém 15 nl de uma solução e é impressa em triplicado. </li><li> Microarrays idênticos são impressas lado a lado sobre a membrana e corte em matrizes individuais, após o trabalho de impressão está completa. </li><li> Em cada experimento, as matrizes podem ser modificados de modo a acomodar mais ou menos amostras, diluições ou repetições. </li></ol> 4. Sondagem de Microarrays glicano <ol><li> Após a impressão, bloquear os microarrays individuais em 5% w / v de leite em pó desnatado dissolvido em solução salina tamponada com fosfato (MPBS) à temperatura ambiente durante 2 horas para reduzir a ligação não específica. </li><li> Microarrays de sonda com anticorpos monoclonais específicos para a célula-parede glicano epítopos durante 2 h em MPBS. A maioria dos Antib monoclonalodies glicanos contra paredes celulares estão comercialmente disponíveis a partir de três empresas; Biosupplies ( <a href="http://www.biosupplies.com.au" target="_blank">www.biosupplies.com.au</a> ), Carbosource Serviços ( <a href="http://www.carbosource.net" target="_blank">www.carbosource.net</a> ) e (PlantProbes <a href="http://www.plantprobes.net" target="_blank">www.plantprobes.net</a> ). </li><li> Incluir um controlo negativo, um microarray incubadas com MPBS e não apenas o anticorpo primário. </li><li> Lavar os microarrays 3 vezes em tampão fosfato salino (PBS) durante 5 minutos para remover ligação não específica. </li><li> Sondar os microarrays com anticorpo secundário conjugado com peroxidase de rábano (HRP) em MPBS durante 2 horas. A maioria dos anticorpos monoclonais contra glicanos da parede celular exigem anti-rato ou anti-ratazana de anticorpos secundários. </li><li> Repita os passos de lavagem 3 vezes com tampão PBS, durante 5 minutos para remover ligação não específica. </li><li> Desenvolver os microarrays utilizando cromogênico (3,3-diaminobenzidina) ou chemiluminecent (luminol) substratos. </li></ol> 5. Quantificação <ol><li> Após o desenvolvimento, digitalizar os microarrays individuais usando uma alta resolução (1200 dpi) scanner de desktop e salvar as imagens negativas, como arquivos TIFF de 16 bits (Figura 3). </li><li> Calcular a intensidade integral de cada mancha usando Xplore Software Image Processing (LabNEXT) equipado com uma ferramenta automatizada de grade. A intensidade local integrante é derivado a partir da soma de pixels na área da grelha em torno de cada ponto. </li><li> Os dados de grade para cada microarray é exportado como um arquivo txt e pode ser importado manualmente em uma planilha do Excel para análise. Uma ferramenta online ( <a href="http://arraytemplates.charliehart.com.au/" target="_blank">http://microarray.plantcell.unimelb.edu.au/</a> ) foi desenvolvido para traduzir automaticamente e processar dados de arquivos txt individuais. </li><li> A intensidade de manchas integral é na média entre impressão repetições e diluições para obter um ponto &quot;médiavalor de intensidade &quot;para cada amostra (Figura 2). Alternativamente, os sinais de ponto correspondente a um único valor de diluição em série são utilizados para quantificar a abundância relativa de epítopo de glicano para cada amostra. </li><li> As intensidades relativas local médias entre diferentes amostras são apresentados como um mapa de calor (Figura 4), ​​utilizando ferramentas de formatação condicional no excel ou online heatmapper ( <a href="http://bar.utoronto.ca/welcome.htm" target="_blank">http://bar.utoronto.ca/welcome.htm</a> ). Os dados para cada tipo de anticorpo é corrigida para 100 e um valor de corte de 5% é aplicada para remover o sinal de fundo e falsos positivos. </li></ol>

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CoMPP é um método rápido e sensível para perfilar a composição de glicano de centenas de plantas derivadas de amostras em uma questão de dias. Este método complementa os já disponíveis bacterianas ou de mamíferos plataformas de matriz de glicano para rastreio de alto rendimento de interacções de hidratos de carbono com glicano de proteínas de ligação, tais como lectinas, receptores e anticorpos 16. Com uma grande diversidade de testes disponíveis para a detecção de glicanos da parede celula...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> 3 milímetros de tungstênio contas Carbide </td><td> Qiagen </td><td> 69997 </td><td></td></tr><tr><td> Microtubos de coleta (1,2 mm) </td><td> Qiagen </td><td> 19560 </td><td> 1,5 ml tubos de microcentrífuga também pode ser usado </td></tr><tr><td> Qiagen TissueLyser II </td><td> Qiagen </td><td> 85300 </td><td></td></tr><tr><td> 3 milímetros pérolas de vidro </td><td> Sigma Aldrich </td><td> Z143928 </td><td></td></tr><tr><td> CDTA </td><td> Sigma Aldrich </td><td> 34588 </td><td></td></tr><tr><td> Óxido de cádmio </td><td> Sigma Aldrich </td><td> 202894 </td><td></td></tr><tr><td> 1,2-diaminoetano </td><td> Sigma Aldrich </td><td> 03550 </td><td></td></<html> tr&gt; <tr><td> Membrana de nitrocelulose (tamanho de poro 0,22 um) </td><td> GE Water &amp; Process Technologies- </td><td> EP2HY00010 </td><td> diferentes dos poros das membranas de tamanho são adequados para diferentes tipos de pinos </td></tr><tr><td> Xact II microarrayer robô </td><td> Labnext </td><td> 001A </td><td> o robot Xact II foi equipado com uma placa de 20 x 20 cm personalizado cerâmica à qual a membrana de nitrocelulose é anexado </td></tr><tr><td> Xtend pinos microarray RM </td><td> Labnext </td><td> 0037-350 </td><td> pinos devem ser adequados para detectar a membranas </td></tr><tr><td> 384 poços de microtitulação (polipropileno) </td><td> Greiner </td><td> 781207 </td><td></td></tr><tr><td> Anti-glicano anticorpos monoclonais </td><td> Sondas de plantas / CarboSource / Biosupplies </td><td></td><td> Sites; PlantProbes ( <a href="http://www.plantprobes.net" target="_blank">www.plantprobes.net</a> ), Carbosource (ww.carbosource.net &quot;target =&quot; _blank &quot;&gt; www.carbosource.net) e Biosupplies ( <a href="http://www.biosupplies.com.au" target="_blank">www.biosupplies.com.au</a> ). </td></tr><tr><td> Anti-IgG de rato (molécula inteira) - Peroxidase anticorpo produzido em cabra. </td><td> Sigma </td><td> A9037 </td><td> o tipo de anticorpos secundários depende do anticorpo primário utilizado (por exemplo, criados em etc rato, de cabra, mouse). </td></tr><tr><td> SIGMA RÁPIDO 3,3 &#39;-diaminobenzidina-comprimidos </td><td> Sigma </td><td> D4293 </td><td> o tipo de desenvolvimento de reagente depende dos anticorpos secundários utilizados e o método de detecção (colourmetric ou chemiluminecent). </td></tr><tr><td> SuperSignal Substrato West Pico quimioluminescente </td><td> Thermoscientific </td><td> 34080 </td><td> veja acima </td></tr><tr><td> Xplore Software Processamento de Imagem </td><td> LabNext </td><td> 008 </td><td> tipos de software com muitas ferramentas automáticas gridagem estão disponíveispara medir o valor de pixel de pontos de microarrays. </td></tr><tr><td> Polissacarídeos de plantas </td><td> Sigma / Megazyme </td><td></td><td></td></tr></tbody></table>

glycan profiling of plant cell wall polymers using microarrays

Paredes das células de planta são matrizes complexas de glicanos heterogéneos, que desempenham um papel importante na fisiologia e desenvolvimento de plantas e fornecer as matérias-primas para as sociedades humanas (por exemplo, as indústrias de madeira, papel, têxteis, e biocombustíveis) 1,2. No entanto, compreender a biossíntese e função destes componentes permanece um desafio. Glicanos de parede celular são quimicamente e conformacionalmente diversificada, devido à complexidade dos blocos de construção, os resíduos glicosilo. Estas ligações formam em posições múltiplas e diferem na estrutura do anel, a configuração isomérica ou anomérico, e, além disso, são substituídas por uma matriz de não-açúcar resíduos. Composição varia de glicano na célula diferente e / ou tipos de tecidos, ou mesmo sub-domínios de uma única célula de parede 3. Além disso, a sua composição é também modificado durante o desenvolvimento 1, ou em resposta a estímulos ambientais 4. Em excesso de 2.000 genes têm paredes celulares de plantas são matrizes complexas de glicanos heterogéneas foram previstos para ser envolvido na biossíntese de glicanos parede celular e modificação em 5 Arabidopsis. No entanto, relativamente poucos dos genes biossintéticos foram funcionalmente caracterizados 4,5. Genética reversa são difíceis porque os genes expressos diferencialmente são muitas vezes, muitas vezes em níveis baixos, entre os tipos de células 6. Além disso, estudos mutantes são muitas vezes dificultada pela redundância gene ou mecanismos compensatórios para garantir o funcionamento adequado parede celular é mantida 7. Assim, novas abordagens são necessárias para rapidamente caracterizar a diversidade de estruturas de glicano e facilitar abordagens genômica funcional para a compreensão a síntese da parede celular e modificação. Os anticorpos monoclonais (mAb) 8,9 surgiram como uma ferramenta importante para a determinação da estrutura de glicano e distribuição nas plantas. Estes reconhecem distepítopos INCT presentes dentro das principais classes de glicanos da parede celular vegetal, incluindo pectinas, xiloglucanos, xilanos, mananos, glucanos e Arabinogalactanos. Recentemente o seu uso tem sido estendido para experiências em grande escala de triagem para determinar a abundância relativa de glicanos em uma ampla gama de tipos de tecido da planta e, simultaneamente, 9,10,11. Apresentamos aqui um microarray-based método de rastreio de glicano chamado Comprehensive Polymer Microarray Profiling (CoMPP) (Figuras 1 &amp; 2) 10,11, que permite múltiplas amostras (100 s) a ser rastreada utilizando uma plataforma de microarray miniaturizado com o reagente reduzida e os volumes de amostra. Os sinais no local de microarray pode ser formalmente quantificadas para dar semi-quantitativas de dados sobre a ocorrência de glicano epítopo. Esta abordagem é bem adequado para o rastreamento de alterações glicano em sistemas biológicos complexos 12 e proporcionando uma visão global da composição da parede celular particularmente quando o conhecimento préviof este não está disponível.

A abundância relativa de glicanos em seis tipos de tecidos (filamentos das anteras, pólen, ovários, pétalas, sépalas e estigma) de flores Nicotiana alata foi determinada utilizando CoMPP. A Figura 3A mostra um microarray representativa que foi sondado com mAb específico para JIM5 parcialmente (baixo) homogalaturonano methylesterified (HG), um epitopo que ocorre em polissacarídeos pécticos 14. O epitopo JIM5 é detectada em extractos de CDTA de todos os tecidos florais no entanto é mais alta ...

Watch this Scientific Journal Video about Glycan Profiling of Plant Cell Wall Polymers using Microarrays at JoVE.com

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Uma técnica chamada<strong&gt; C</strong&gt; Omprehensive<strong&gt; M</strong&gt; Icroarray<strong&gt; P</strong&gt; Profiling olymer (CoMPP) para a caracterização de glicanos da parede celular vegetal é descrito. Este método combina a especificidade dos anticorpos monoclonais dirigidos para epitopos definidos glicano-com uma plataforma de microarray em miniatura analítico permitindo rastreio de ocorrência de glicano em uma ampla variedade de contextos biológicos.

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 ...

glycan-profiling-of-plant-cell-wall-polymers-using-microarrays

Research

JoVE Journal

Biology

14.5K visualizações.  University of Melbourne. Uma técnica chamada C Omprehensive M Icroarray P Profiling olymer (CoMPP) para a caracterização de glicanos da parede celular vegetal é descrito. Este método combina a especificidade dos anticorpos monoclonais dirigidos para epitopos definidos glicano-com uma plataforma de microarray em miniatura analítico permitindo rastreio de ocorrência de glicano em uma ampla variedade de contextos biológicos...

Vídeo: Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Bacterial Gene Expression Analysis Using Microarrays

Análise de Expressão Gênica Utilizando bacteriana Microarrays

Biologia

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism 2, 3. Auxin activates a H+ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins 4, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins 5. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.
 To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 &deg;C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.

Medição Usina Extensão parede celular (Creep) induzida pelo pH ácido e por Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Investigando Tecido e órgãos específicos Responses Fitocromo usando FACS assistida Célula tipo Profiling expressão específica no Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

Análise do genoma de Aminoacylation (carregamento) Níveis de tRNA Usando Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

Oligo Profiling Massa (OLIMP) de polissacarídeos extracelulares

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

Profiling Proteome Redox tiol Usando Isótopos Espectrometria de Massa Tagging

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Coloração histoquímica de<em&gt; Arabidopsis thaliana</em&gt; Celular Secundária parede Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...

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 ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

assistida por laser Microdissecção (LAM) como ferramenta para transcricional Profiling de tipos de células individuais

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Um método fácil para a planta polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...