IEM gustaría reconocer el Consejo Danés de Investigación (FTP y FNU) para su financiación. ERL reconoce el apoyo de una subvención de ARC DP. AB reconoce el apoyo del Centro ARC de Excelencia en Plant Cell Paredes subvención.

1. Colección de tejidos y preparación <ol><li> Recoger 100 mg de peso fresco de los tejidos vegetales (un mínimo de 10 mg de peso seco) al menos por triplicado para cada tejido de interés. Los pasos siguientes describen la preparación de material de la pared celular de los tejidos vegetativos. En el caso de los tejidos de almacenamiento, no deseado almidón es enzimáticamente eliminado antes de proceder con la extracción de los polímeros de la pared celular como se ha descrito previamente 13. </li><li> Homogeneizar las muestras a un polvo fino con nitrógeno líquido utilizando un Qiagen TissueLyser II, con 24 conjuntos de adaptador y el tubo 3 mm de tungsteno granos de carburo (30 Hz, 2 x 30 seg). Alternativamente, si solamente unas pocas muestras se procesan, un mortero y mano de mortero se utiliza. </li><li> Transfiera los homogeneizados en 10 ml tubos cónicos de plástico. </li><li> Preparar el material de la pared celular mediante el lavado de los homogeneizados en 10 ml de 80% v / v de etanol a temperatura ambiente y agitando vigorosamente durante 2 min. </li><li> Centrifugar las muestras a 3.500 xg y desechar el sobrenadante. Repetir el etanol al 70% se lava al menos tres veces o hasta que el sobrenadante es claro, en particular para los tejidos que contienen clorofila. </li><li> Realizar un lavado final con 100% de acetona y dejar los gránulos que contienen residuos insolubles en alcohol (AIR) para secar al aire durante la noche. </li><li> Tamiz las muestras de aire con un 0,4 mm de malla de 2 para conseguir un polvo fino y homogéneo y para eliminar más grande, mal partículas de tierra que en algún momento permanecen en el homogeneizado. </li><li> Pesar 10 mg de muestra de aire en microtubos, conteniendo cada una 3 mm de perlas de vidrio para ayudar al mezclado de las muestras. </li></ol> 2. Extracción de los glicanos de la pared celular <ol><li> Pectinas, y polímeros asociados con pectinas se extraen de las muestras mediante la adición de 500 l de CDTA mu M 50 (pH 7,5) a cada muestra. </li><li> Agitar brevemente los tubos para asegurar el disolvente está en contacto con el material de la muestra y, a continuación mezclar mediante el TissueLyser durante 3 horas a 8 Hz. </li><li> Centrifugar las muestras a 12.000 xg, cuidadosamente reMove los sobrenadantes y se almacena a 4 ° C. </li><li> Lavar gránulos en 1 ml de agua desionizada para diluir y eliminar cualquier disolvente restante vórtice, y se centrifuga a 13.000 x g. Repita este paso sin perturbar el precipitado y retirar todo el líquido de los tubos antes de proceder al siguiente paso. </li><li> De reticulación glicanos son secuencialmente extraída de la pared celular restante pellet con 500 l de NaOH 4 M con 0,1% w / v NaBH4, usando el mismo procedimiento descrito en los pasos 2,1 a 2,4 para la extracción CDTA. NaBH 4 se añade para reducir el aldehído (o ceto) en el extremo reductor de los polisacáridos a un alcohol evitando de este modo la base de polisacáridos descamación. </li><li> Después de la centrifugación, los sobrenadantes se retira de nuevo, se almacenaron a 4 ° C, y los sedimentos se lavaron dos veces en agua desionizada antes de proceder a la extracción siguiente. </li><li> Como una etapa opcional, los polímeros residuales, tales como la celulosa se extrajo con 500 cadoxen mu l (31% v / v 1,2-diaminanoethane con 0,78 M CdO) usando el mismo procedimiento como se describe en los pasos desde 2,1 hasta 2,4. Alternativamente, el contenido absoluto de celulosa en gránulos restantes se puede determinar usando ensayos acético / nítrico (véase la discusión). </li></ol> 3. Microarrays de impresión <ol><li> Sobrenadantes de centrífuga que contiene extrajo polímeros de la pared celular a 13.000 xg para eliminar cualquier materia en partículas. </li><li> Cargar 50 l de cada muestra en un polipropileno de 384 pocillos placa de microtitulación usando un diseño personalizado prediseñada en donde las muestras se disponen de acuerdo con el tipo de tejido y del tipo de extracción. </li><li> Diluir la muestra de polímero de la pared celular en una serie 0, 5 y 25 × dilución en serie con agua desionizada. </li><li> Los parámetros tales como la altura pin, recogida y tiempo de permanencia, y los pasos de lavado se encuentra en el software que controla la microarrayer. </li><li> La humedad de la cámara de impresión se controla a 60% para evitar la evaporación de la muestra. </li><li> El trabajo de impresión se inicia mediante LabNEXT software y un programa que corresponde a la disposición de microarrays. </li><li> El robot utiliza los pins de canal capilar para imprimir soluciones de la placa de muestra en 20 x 20 cm de membrana de nitrocelulosa que se une a una placa plana en la máquina. Cada punto de la matriz contiene 15 nl de solución y se imprime por triplicado. </li><li> Microarrays idénticos se imprimen uno al lado del otro en la membrana y se cortó en matrices individuales después de que el trabajo de impresión. </li><li> En cada experimento las matrices pueden ser modificados con el fin de acomodar muestras más o menos, diluciones o replicados. </li></ol> 4. Sondeo de Microarrays glicano <ol><li> Después de la impresión, bloquear los microarrays individuales en 5% w / v de leche en polvo desnatada disuelta en solución salina tamponada con fosfato (MPBS) a temperatura ambiente durante 2 horas para reducir la unión no específica. </li><li> Microarrays de sondas con anticuerpos monoclonales específicos para epítopos de la pared celular glicano durante 2 horas en MPBS al. La mayoría de antib monoclonalodies contra glucanos de la pared celular están disponibles comercialmente en tres compañías; Biosupplies ( <a href="http://www.biosupplies.com.au" target="_blank">www.biosupplies.com.au</a> ), Servicios Carbosource ( <a href="http://www.carbosource.net" target="_blank">www.carbosource.net</a> ) y PlantProbes ( <a href="http://www.plantprobes.net" target="_blank">www.plantprobes.net</a> ). </li><li> Incluir un control negativo, un microarray incubaron con MPBS al solamente y sin anticuerpo primario. </li><li> Lavar los microarrays de 3 veces en tampón fosfato salino (PBS) durante 5 minutos para eliminar la unión no específica. </li><li> Controle los microarrays con anticuerpo secundario conjugado con peroxidasa de rábano picante (HRP) en MPBS al durante 2 horas. Más anticuerpos monoclonales contra glucanos de la pared celular requiere anti-ratón o anti-rata anticuerpos secundarios. </li><li> Repetir las etapas de lavado 3 veces con tampón PBS durante 5 minutos para eliminar la unión no específica. </li><li> Desarrollar los microarrays utilizando cromógeno (3,3-diaminobencidina) o chemiluminecent (luminol) sustratos. </li></ol> 5. Cuantificación <ol><li> Después del revelado, escanear los microarrays individuales utilizando una alta resolución (1.200 dpi) Escáner de sobremesa y guardar las imágenes como negativas, de 16-bit TIFF (Figura 3). </li><li> Calcular la intensidad integral de cada mancha usando Xplore Software de procesamiento de imágenes (LabNEXT) equipado con una herramienta de cuadrícula automatizado. La intensidad de la mancha integral se deriva de la suma de los píxeles en el área de rejilla que rodean a cada punto. </li><li> Los datos de la cuadrícula para cada microarray se exporta como un archivo de texto y se puede importarse manualmente en una hoja de cálculo Excel para su análisis. Una herramienta en línea ( <a href="http://arraytemplates.charliehart.com.au/" target="_blank">http://microarray.plantcell.unimelb.edu.au/</a> ) ha sido desarrollado para traducir automáticamente y procesar los datos de cada archivo de txt. </li><li> La intensidad de la mancha integral se promedia a través de la impresión repeticiones y diluciones para obtener un lugar &quot;justo mediointensidad &quot;valor para cada muestra (Figura 2). Alternativamente, las señales de mancha correspondiente al valor de dilución sólo uno en la matriz se utilizan para cuantificar la abundancia relativa de glicano epítopo para cada muestra. </li><li> Las intensidades relativas medias al contado entre las diferentes muestras se presentan como un mapa de calor (Figura 4) utilizando el formato condicional en Excel o HeatMapper herramientas en línea ( <a href="http://bar.utoronto.ca/welcome.htm" target="_blank">http://bar.utoronto.ca/welcome.htm</a> ). Los datos para cada tipo de anticuerpo se corrige a 100 y un valor de corte de 5% se impone para eliminar la señal de fondo y los falsos positivos. </li></ol>

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No hay conflictos de interés declarado.

CoMPP es un método rápido y sensible para perfilar la composición glicanos de cientos de plantas derivadas de las muestras en cuestión de días. Este método complementa las plataformas de glicano ya disponibles bacterianas o de mamíferos de matriz para cribado de alto rendimiento de las interacciones de hidratos de carbono con glicanos de proteínas de unión tales como lectinas, receptores, y anticuerpos 16. Con una gran diversidad de sondas disponibles para la detección de glucanos de la pared celula...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> 3 mm de carburo de tungsteno bolas </td><td> Qiagen </td><td> 69997 </td><td></td></tr><tr><td> Colección de microtubos (1,2 mm) </td><td> Qiagen </td><td> 19560 </td><td> 1,5 ml tubos de microcentrífuga también se puede utilizar </td></tr><tr><td> Qiagen TissueLyser II </td><td> Qiagen </td><td> 85300 </td><td></td></tr><tr><td> 3 perlas de vidrio mm </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 cadmio </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 nitrocelulosa (0,22 micras de tamaño de poro) </td><td> GE Water &amp; Process Technologies </td><td> EP2HY00010 </td><td> diferentes membranas de tamaño de poro son adecuados para diferentes tipos de anclaje </td></tr><tr><td> Xact II microarrayer robot </td><td> Labnext </td><td> 001A </td><td> la Xact II robot fue equipado con una costumbre 20 x 20 cm de placa cerámica a la que se une la membrana de nitrocelulosa </td></tr><tr><td> Xtend pins RM microarrays </td><td> Labnext </td><td> 0037-350 </td><td> pins deben ser adecuados para manchas en membranas </td></tr><tr><td> Placas de microtitulación de 384 pocillos (polipropileno) </td><td> Greiner </td><td> 781207 </td><td></td></tr><tr><td> Anti-glicanos anticuerpos monoclonales </td><td> Sondas vegetales / CarboSource / Biosupplies </td><td></td><td> Sitios Web; 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) y Biosupplies ( <a href="http://www.biosupplies.com.au" target="_blank">www.biosupplies.com.au</a> ). </td></tr><tr><td> IgG anti-rata (molécula completa) - anticuerpo peroxidasa producida en cabra. </td><td> Sigma </td><td> A9037 </td><td> el tipo de anticuerpos secundarios depende del anticuerpo primario utilizado (por ejemplo, criado en rata, ratón de cabra, etc,). </td></tr><tr><td> SIGMA RÁPIDO 3,3 &#39;-diaminobenzidina tabletas </td><td> Sigma </td><td> D4293 </td><td> el tipo de desarrollo de reactivo depende de los anticuerpos secundarios utilizados y el método de detección (colourmetric, o chemiluminecent). </td></tr><tr><td> SuperSignal West Pico Substrato quimioluminiscente </td><td> Thermoscientific </td><td> 34080 </td><td> véase anteriormente </td></tr><tr><td> Xplore Software de procesamiento de imágenes </td><td> LabNext </td><td> 008 </td><td> muchos tipos de software con herramientas automáticas de grillado disponiblespara medir el valor de píxel de manchas de microarrays. </td></tr><tr><td> Polisacáridos de plantas </td><td> Sigma / Megazyme </td><td></td><td></td></tr></tbody></table>

glycan profiling of plant cell wall polymers using microarrays

Paredes celulares de las plantas son matrices complejas de los glicanos heterogéneos, que desempeñan un papel importante en la fisiología y el desarrollo de las plantas y proporcionar las materias primas para las sociedades humanas (por ejemplo, industrias de la madera, papel, textiles y de biocombustibles) 1,2. Sin embargo, la comprensión de la biosíntesis y función de estos componentes sigue siendo un reto. Glucanos de la pared celular son químicamente y conformacionalmente diversa debido a la complejidad de sus bloques de construcción, los residuos de glicosilo. Estos forman uniones en las posiciones múltiples y difieren en estructura de anillo, configuración isomérica o anomérico, y además, están sustituidos con una matriz de no azúcar residuos. Glycan composición varía en células diferentes y / o tipos de tejidos o incluso sub-dominios de una sola célula de la pared 3. Además, su composición también se modifica durante el desarrollo 1, o en respuesta a señales ambientales 4. En exproceso de 2.000 genes tienen paredes celulares de las plantas son matrices complejas de los glicanos heterogéneos ha previsto que participen en la biosíntesis de la pared celular y la modificación de glicanos en Arabidopsis 5. Sin embargo, relativamente pocos de los genes biosintéticos se han caracterizado funcionalmente 4,5. Ida y enfoques de genética son difíciles debido a que los genes son a menudo expresados ​​diferencialmente, a menudo a niveles bajos, entre los tipos de células 6. Además, los estudios mutantes son a menudo obstaculizado por la redundancia de genes o mecanismos compensatorios para asegurar el funcionamiento adecuado de la pared celular se mantiene 7. Así, nuevos enfoques son necesarios para caracterizar rápidamente la diversa gama de estructuras de glucanos y para facilitar enfoques de la genómica funcional para la biosíntesis de la pared celular y la comprensión modificación. Los anticuerpos monoclonales (mAbs) 8,9 han surgido como una herramienta importante para determinar la estructura de glicano y distribución en las plantas. En ellos se reconoce distINCT presentes dentro de las principales categorías de glicanos la pared celular vegetal, incluidas las pectinas, xiloglucanos, xilanos, mananos, glucanos y arabinogalactanos epítopos. Recientemente su uso se ha extendido a los experimentos de cribado a gran escala para determinar la abundancia relativa de los glicanos en una amplia gama de plantas y tipos de tejidos simultáneamente 9,10,11. Aquí presentamos un método basado en micromatrices glicano exploración llamada integral Microarray de perfiles de polímero (CoMPP) (Figuras 1 y 2) 10,11 que permite múltiples muestras (100 segundos) para ser seleccionados utilizando una plataforma de microarrays miniaturizado con reactivo reducida y volúmenes de muestra. Las señales de punto del microarray puede ser formalmente cuantificados para dar semi-cuantitativos de los datos sobre la ocurrencia glycan epítopos. Este método es muy adecuado para el seguimiento de los cambios en glicanos complejos sistemas biológicos 12 y que proporciona una visión global de la composición de la pared celular en particular cuando el conocimiento previo of este no estará disponible.

La abundancia relativa de los glicanos en seis tipos de tejido (filamentos de anteras, polen, ovarios, pétalos, sépalos y el estigma) de Nicotiana alata flores se determinó usando CoMPP. La Figura 3A muestra un microarray representante que se ha sondeado con mAb específico para JIM5 parcialmente (bajo) methylesterified homogalacturonan (HG), un epítopo que se produce en polisacáridos pécticos 14. El epítopo JIM5 se detecta en extractos CDTA de todos los tejidos de las flores sin embargo es m?...

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

Una técnica llamada<strong&gt; C</strong&gt; Omprehensive<strong&gt; M</strong&gt; Icroarray<strong&gt; P</strongProfiling&gt; olymer (CoMPP) para la caracterización de los glicanos la pared celular vegetal se describe. Este método combina la especificidad de los anticuerpos monoclonales dirigidos a epítopos definidos glicano con una miniatura plataforma de microarrays analítico que permite detección de la ocurrencia de glicanos en una amplia gama de contextos biológicos.

Perfiles de Polímeros Glycan la pared celular vegetal utilizando 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 vistas.  University of Melbourne. Una técnica llamada C Omprehensive M Icroarray P olymer (CoMPP) para la caracterización de los glicanos la pared celular vegetal se describe. Este método combina la especificidad de los anticuerpos monoclonales dirigidos a epítopos definidos glicano con una miniatura plataforma de microarrays analítico que permite detección de la ocurrencia de glicanos en una amplia gama de contextos biol?...

Video: Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Bacterial Gene Expression Analysis Using Microarrays

Gen bacteriano y Análisis de Expresión El uso de microarrays

Investigación

Biología

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.

Celda de medición Planta de Extensión de la pared (creep) inducida por el pH ácido y por Alfa-expansina

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.

La investigación de los tejidos y órganos específicos de las respuestas fitocromo usando FACS asistida por células de tipo Perfiles de expresión específica en 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.

Todo el genoma análisis de Aminoacilación (de carga) Niveles de tRNA utilizando 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 perfiles Misa (OLIMP) de polisacáridos 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.

Creación de perfiles proteoma redox tiol Uso del etiquetado de espectrometría de masas

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.

La tinción histoquímica de<em&gt; Arabidopsis thaliana</em&gt; Celda Secundaria Elements Pared

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.

Alta resolución Cuantificación de celulosa cristalina acumulación en<em&gt; Arabidopsis</em&gt; Las raíces para monitorear la célula específica de tejido de pared Modificaciones

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.

Asistida por láser microdisección (LAM) como herramienta para la transcripción de perfiles de distintos tipos de células

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.

Un método fácil para la planta de perfiles de polisomas

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