Estamos muy agradecidos a Mateo Robert Weatherhead de excelente servicio técnico y John Ralph, de la Universidad de Wisconsin, por sus valiosos consejos, discusiones, y la muestra de madera de álamo. Este trabajo fue financiado por el Departamento de Energía de EE.UU. (DOE) de los Grandes Lagos del Centro de Investigación de Bioenergía (DOE BER Oficina de Ciencia-DE-FC02 07ER64494) y por las Ciencias Químicas, Geociencias y Ciencias Biológicas de la División de la Oficina de Ciencias Básicas de Energía, Oficina de Ciencia , EE.UU. Departamento de Energía (sin premio. DE-FG02-91ER20021).

1. El aislamiento de la pared celular <ol><li> Moler aproximadamente el 60-70mg de aire o de material liofilizado de plantas, con 5,5 mm bolas de acero inoxidable en un tubo de 2 ml tapa Sarstedt tornillo con un iWall, un robot de molienda y dosificación (30 s). Una alternativa no robótica de bajo rendimiento con un procedimiento de molienda (retschmill) se presenta en la Parte II 2. </li><li> Añadir 1,5 ml de etanol al 70% acuoso para el material molido dispensado, y agitar bien. </li><li> Centrifugar a 10.000 rpm durante 10 minutos para que sedimenten los residuos insolubles en alcohol. </li><li> Aspirar o decantar el sobrenadante. </li><li> Añadir 1,5 ml de cloroformo / metanol (1:1 v / v) al residuo y agitar el tubo a fondo para volver a suspender el pellet. </li><li> Centrifugar a 10.000 rpm durante 10 minutos y Aspirar o decantar el sobrenadante. </li><li> Resuspender el pellet en 500 l de acetona. </li><li> Se evapora el disolvente con una corriente de aire a 35 ° C hasta que se seque. Si es necesario muestras secas se pueden almacenar a temperatura ambiente hasta su posterior procesamiento. </li><li> Para iniciar la extracción del almidón de la muestra resuspender el precipitado en 1,5 ml de un 0,1 M de acetato de sodio pH 5,0 tampón. </li><li> Tape los tubos Sarstedt y el calor durante 20 minutos. a 80 ° C en un calentador. </li><li> Se enfría la suspensión en el hielo. </li><li> Añadir los siguientes agentes de la pastilla: 35 l de 0,01% de azida sódica (NaN3), 35 l de amilasa (50 mg / ml H 2 O, a partir de especies de Bacillus, Sigma), 17 pululanasa l (17,8 unidades de acidopullulyticus Bacillus; Sigma) . Tape el tubo y agitar bien. </li><li> La suspensión se incubó durante la noche a 37 ° C en la coctelera. La orientación de los tubos horizontales ayudantes mejor mezcla. </li><li> Suspensión de calor a 100 º C durante 10 min en un calentador de terminar la digestión. </li><li> Centrifugar (10.000 rpm, 10 min) y eliminar el sobrenadante que contiene almidón solubilizado. </li><li> Lave el resto de pellet tres veces mediante la adición de 1,5 ml de agua, agitación, centrifugación y decantación del agua de lavado. </li><li> Resuspender el pellet en 500 l de acetona. </li><li> Se evapora el disolvente con una corriente de aire a 35 ° C hasta que se seque. Puede ser necesario también para romper el material en el tubo con una espátula para un mejor secado. El material seco se presenta de la pared celular aislado (lignocelulosa). Si es necesario muestras secas se pueden almacenar a temperatura ambiente hasta su posterior procesamiento. </li></ol> 2. Contenido de lignina Este método se basa en un método reportado por Fukushima y Hatfield 3. <ol><li> Pesar 1 a 1,5 mg de material preparado de la pared celular (ver 1) en 2 ml matraz aforado de salir de un tubo vacío de un espacio en blanco. </li><li> enjuagar las paredes del tubo con 250 l de acetona para recoger el material de la pared celular en la parte inferior del tubo, y se evapora la acetona muy suave en el flujo de aire. </li><li> Suavemente añadir 100 ml de solución recién hecha bromuro de acetilo (25% v / v bromuro de acetilo en ácido acético glacial) a lo largo de las paredes del tubo para evitar salpicaduras. </li><li> Cap matraz aforado y se calienta a 50 ° C durante 2 horas </li><li> El calor durante una hora adicional con agitación cada 15 minutos. </li><li> Dejar enfriar en hielo a temperatura ambiente. </li><li> Añadir 400 l de hidróxido de sodio 2M y 70 l de recién preparada clorhidrato de 0,5 M de hidroxilamina. Vortex matraces aforados. </li><li> Rellene matraz aforado exactamente a la marca de 2,0 ml con ácido acético glacial, tapa e invierta varias veces para mezclar. </li><li> Pipetear 200 l de la solución en una placa de 96 UV específicos bien y leer en un lector de ELISA a 280 nm. </li><li> Determinar el porcentaje de lignina bromuro de acetilo soluble (% ABSL) mediante un coeficiente que corresponda (Álamo = 18,21; Pastos = 17,75; Arabidopsis = 15.69) con la siguiente fórmula: % ABSL Calc: <img src="/files/ftp_upload/1745/1745eq.jpg" alt="Figura 1" /> Multiplicación de ABSL% con 10 resultados en la unidad de la célula ug / mg pared Ayuda a hacer por lo menos tres placa se lee en un promedio de la absorbancia (ABS), ya que las partículas pueden causar una ligera variación en los valores de absorbancia. Nota: 0.539 cm representa el paso de luz, pero dependiendo de la placa de la que tenga que determinar. </li></ol> 3. Lignina Composición Este método se adoptó a partir de un método reciente publicado por Robinson y Mansfield 5. <ol><li> Transferencia de aproximadamente 2 mg de material de la pared celular (ver 1.) En un tubo de vidrio cubiertas de tornillo para tioacidolisis. </li><li> preparar cuidadosamente el 2,5% de trifluoruro de boro dietil eterato (BF 3), el 10% etanotiol (EtSH) solución. Debe utilizar un globo lleno de gas nitrógeno para desplazar el volumen perdido en la botella de dioxano con nitrógeno. El dioxano es muy peligroso, no tomar muestras o equipos fuera de la campana. Volúmenes necesarios para la preparación de la solución por muestra: 175 l dioxano, 20 EtSH l, 5 l BF 3. </li><li> Añadir 200 l de EtSH, BF 3, la solución de dioxano para cada muestra. </li><li> Purga del espacio de cabeza del vial con gas nitrógeno y la tapa inmediatamente. </li><li> El calor a 100 º C durante 4 horas con agitación suave cada hora. </li><li> Final de la reacción por enfriamiento en hielo durante 5 minutos. </li><li> Añadir 150 ml de bicarbonato de sodio 0,4 M, vortex </li><li> Para la limpieza se añade 1 ml de agua y 0,5 ml de acetato de etilo vórtice, y dejar separar las fases (acetato de etilo en la parte superior, el agua en la parte inferior). </li><li> Transferencia de 150 ml de la capa de acetato de etilo en un tubo de 2 ml Sarstedt. Asegúrese de que el agua no se transfiere. </li><li> Evaporar el solvente por un concentrador de aire. </li><li> Añadir 200 l de acetona y se evapora (repetición de un total de dos veces quitar el exceso de agua). </li><li> Para la derivación TMS añadir 500 l de acetato de etilo, 20 l de piridina, y 100 l de N, O-bis (trimetilsilil) acetamida a cada tubo. </li><li> incubar durante 2 horas a 25 ° C. </li><li> Transferencia de 100 l de la reacción en un vial de GC / MS y añadir 100 ml de acetona. </li><li> Analizar las muestras por GC equipado con un espectrómetro de masas cuadrupolo o detector de ionización de llama. Un Agilent HP-5MS columna se instala (30 mm X 0.25 mm X 0,25 m espesor de la película). El gradiente de temperatura se utiliza la siguiente con un retraso de 30 minutos disolvente y un 1,1 ml / min de caudal: retención inicial a 130 ° C durante 3 minutos, a 3 ° C / min a una rampa de 250 ° C y mantener durante 1 min, permiten el equilibrio de la temperatura inicial de 130 ° C. </li><li> Picos se identifican con tiempos de retención relativos con tetracosane patrón interno (opcional) o mediante iones característicos espectro de masas de 299 m / z, 269 m / z, y 239 monómeros m / z de S, G y H, respectivamente (ver fig. 2). La composición de los componentes de la lignina se cuantifica mediante la creación del área del pico total de 100% </li></ol> 4. Resultados representante Un ejemplo de un análisis de la pared se presenta en la Figura 2. En este caso, el álamo madre (de madera) fue analizada por los diversos procedimientos descritos en la sección de protocolo. Un cromatograma de ejemplo de la separación de la lignina, componentes después de tioacidolisis y derivación TMS-se muestra. Claramente, la abundancia relativa de syringyl-(S), guayacil (G), y p-hidroxifenol-(H) unidades se puede determinar. El contenido de lignina soluble bromuro de acetilo se explica por sí mismo, se puede esperar que los valores de entre el 20-50% del peso seco de la pared. Hay que señalar que el bromuro de acetilo no solubilizar todos los presentes la lignina en la pared, y que el grado de solubilización puede variar dependiendo del material. Sin embargo, este método es relativamente fácil de realizar y rápido y da una excelente aproximación del contenido de lignina en un material lignocelulósico. <img src="/files/ftp_upload/1745/1745fig1.jpg" alt="Figura 1" /> Figura 1:. Visión general del análisis lignocelulósicos paredes celulares (lignocelulosa) están aislados de material en bruto de plantas secas. El material de la pared se pondera en alícuotas y se divide para los ensayos de varios. Material de la pared se trata con bromuro de acetilo y la lignina solubilizada cuantificado por espectroscopia UV-. Para la determinación de la composición de la lignina, el material de la pared se somete a tioacidolisis. Los compuestos fenólicos solubilizados someterse a la derivación TMS y luego pueden ser separados y cuantificados por análisis GC-MS. La composición de polisacáridos de la matriz y el protocolo de celulosa cristalina contenido se discute en la Parte II 2. <img src="/files/ftp_upload/1745/1745fig2.jpg" alt="Figura 2" /> Figura 2:. Análisis exhaustivo lignocelulósicos de la madera de álamo, Astillas de madera de álamo (Populus tremoloides) fueron sometidos a los protocolos descritos. Ligin composición; H p-hidroxifenil; G guayacil; S syringyl unidades.

<ol>
	<li>Carroll, A., Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulosic+Biofuels.">Cellulosic Biofuels.</a> Annual Review of Plant Biology. 60, 165-165 (2009).</li><li>Foster, C. E., Martin, T., Pauly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+compositional+analysis+of+Plant+Cell+Walls+(Lignocellulosic+biomass),+Part+II:+Carbohydrates.">Comprehensive compositional analysis of Plant Cell Walls (Lignocellulosic biomass), Part II: Carbohydrates.</a> J Vis Exp. , (2010).</li><li>Fukushima, R. S., Hatfield, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+isolation+of+lignin+for+utilization+as+a+standard+to+determine+lignin+concentration+using+the+acetyl+bromide+spectrophotometric+method.">Extraction and isolation of lignin for utilization as a standard to determine lignin concentration using the acetyl bromide spectrophotometric method.</a> J. Agric. Food Chem. 49 (7), 3133-3133 (2001).</li><li>Pauly, M., Keegstra, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-wall+carbohydrates+and+their+modification+as+a+resource+for+biofuels.">Cell-wall carbohydrates and their modification as a resource for biofuels.</a> Plant J. 54 (4), 559-559 (2008).</li><li>Robinson, A. R., Mansfield, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+poplar+lignin+monomer+composition+by+a+streamlined+thioacidolysis+procedure+and+near-infrared+reflectance-based+prediction+modeling.">Rapid analysis of poplar lignin monomer composition by a streamlined thioacidolysis procedure and near-infrared reflectance-based prediction modeling.</a> Plant J. 58 (4), 706-706 (2009).</li><li>Somerville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+systems+approach+to+understanding+plant-cell+walls.">Toward a systems approach to understanding plant-cell walls.</a> Science. 306 (5705), 2206-2206 (2004).</li><li>Teeri, T. T., Brumer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery,+characterization+and+applications+of+enzymes+from+the+wood-forming+tissues+of+poplar:+Glycosyl+transferases+and+xyloglucan+endo-transglycosylases.">Discovery, characterization and applications of enzymes from the wood-forming tissues of poplar: Glycosyl transferases and xyloglucan endo-transglycosylases.</a> Biocatalysis and Biotransformation. 21, 173-173 (2003).</li></ol>

Los métodos descritos permiten una rápida evaluación cuantitativa del contenido de lignina y la composición de la biomasa de plantas lignocelulósicas. El uso del robot iWall aproximadamente 350 muestras de suelo y puede ser dispensado por día. El rendimiento de los distintos métodos de análisis por persona varía. Utilizando los protocolos descritos aquí, 30 muestras se pueden procesar por el contenido de lignina, y 15 para la composición de la lignina por día. Debido a la naturaleza cuantitativa de los cultivos de materias primas de datos óptima, variedad o genotipos pueden ser evaluados en términos de su idoneidad para la producción de biocombustibles.

<table><tbody><tr><td>Hydroxylamine Hydrochloride</td><td>Sigma-Aldrich</td><td>255580</td><td></td></tr><tr><td>Acetyl Bromide</td><td>Aldrich</td><td>135968</td><td></td></tr><tr><td>Ethanethiol</td><td>Sigma-Aldrich</td><td>E3708</td><td></td></tr><tr><td>Borontrifluoride diethyl etherate</td><td>Fluka</td><td>15719</td><td></td></tr><tr><td>N,O,-Bis(trimethylsilyl) acetimide</td><td>Fluka</td><td>15241</td><td></td></tr><tr><td>Dioxane</td><td>Sigma-Aldrich</td><td>296309</td><td></td></tr><tr><td>Spectromax Plus 384</td><td>Molecular Devices</td><td>Plus384</td><td></td></tr><tr><td>GC-MS</td><td>Agilent Technologies</td><td>6890 GC/5975B MSD</td><td>(lignin composition)</td></tr><tr><td>5.5mm Stainless Steel Balls</td><td>Salem Ball Company</td><td>(N/A)</td><td></td></tr><tr><td>96 well plate heat spreader</td><td>Biocision</td><td>Coolsink 96F</td><td></td></tr><tr><td>Heating block</td><td>Techne</td><td>Dri-block DB-3D</td><td></td></tr><tr><td>Sample concentrator</td><td>Techne</td><td>FSC400D</td><td></td></tr></tbody></table>

comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) part i: lignin

La necesidad de renovables, neutral de carbono y sostenible de materias primas para la industria y la sociedad se ha convertido en uno de los problemas más acuciantes para el siglo 21. Esto ha reavivado el interés en el uso de productos vegetales como materia prima industrial para la producción de combustibles líquidos para el transporte<sup&gt; 1</sup&gt; Y otros productos tales como materiales biocompuesto<sup&gt; 7</sup&gt;. La biomasa vegetal es uno de los más grandes reservas sin explotar en el planeta<sup&gt; 4</sup&gt;. Se está compuesta mayormente por las paredes celulares que están compuestos de polímeros ricos en energía como celulosa, hemicelulosa diferentes (polisacáridos de la matriz, y la lignina de polifenoles<sup&gt; 6</sup&gt; Y por lo tanto a veces se denomina lignocelulosa. Sin embargo, las paredes celulares vegetales han evolucionado para ser recalcitrantes a la degradación de las paredes ofrecen resistencia a la tracción a las células y las plantas enteras, y evitar agentes patógenos, y permitir que el agua se transporta por toda la planta, en el caso de árboles de hasta más de 100 m por encima de a nivel del suelo. Debido a las diversas funciones de las paredes, hay una inmensa diversidad estructural dentro de las paredes de diferentes especies de plantas y tipos de células dentro de una sola planta<sup&gt; 4</sup&gt;. Por lo tanto, dependiendo de lo que las especies de cultivo, las variedades de cultivo, o tejido de la planta se utiliza para la bio-refinerías, los pasos del proceso de despolimerización por procesos químicos / enzimáticos, y posterior fermentación de los azúcares distintos a los biocombustibles líquidos deben ser ajustadas y optimizadas. Este hecho se basa la necesidad de una caracterización exhaustiva de materias primas de biomasa de las plantas. A continuación se describe una metodología completa de análisis que permite la determinación de la composición de la lignocelulosa y es susceptible a un medio de análisis de alto rendimiento. En esta primera parte nos centramos en el análisis de la lignina en polifenoles (Figura 1). El método comienza con la preparación de material de desalmidonadas pared celular. La lignocelulosa resultantes se dividen para determinar su contenido de lignina por solubilización acetylbromide<sup&gt; 3</sup&gt;, Y su lignina composición en términos de sus unidades syringyl, guayacil-y p-hidroxifenil<sup&gt; 5</sup&gt;. El protocolo para el análisis de los hidratos de carbono en la biomasa lignocelulósica incluyendo el contenido de celulosa y la composición de polisacáridos de la matriz se discute en la Parte II<sup&gt; 2</sup&gt;.

Watch this Scientific Journal Video about Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin at JoVE.com

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

La biomasa vegetal es uno de los principales de carbono-neutral recurso renovable que podría ser utilizado para la producción de biocombustibles. La biomasa vegetal se compone principalmente de las paredes celulares, un material compuesto estructuralmente complejos denomina lignocelulosa. Aquí se describe un protocolo para un análisis exhaustivo del contenido y la composición de la lignina polifenólicos.

Análisis Integral de la composición de las paredes celulares vegetales (biomasa lignocelulósica) Parte I: La lignina

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st ...

comprehensive-compositional-analysis-plant-cell-walls-lignocellulosic

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JoVE Journal

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Cell Biology

Biology

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part I: Lignin

Unit 1

Plant Cell Structure and Organization

SCIENTISTS IN ACTION

32.1K vistas.  Michigan State University (MSU). La biomasa vegetal es uno de los principales de carbono-neutral recurso renovable que podría ser utilizado para la producción de biocombustibles. La biomasa vegetal se compone principalmente de las paredes celulares, un material compuesto estructuralmente complejos denomina lignocelulosa. Aquí se describe un protocolo para un análisis exhaustivo del contenido y la composición de la lignina polifenólicos.

Video: Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part I: Lignin

Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The analysis does not usually require extensive sample preparation; structural and chemical information can be obtained without labeling. However, each Raman image contains thousands of spectra; this raises difficulties when extracting hidden information, especially for components with similar chemical structures. This work introduces a multivariate analysis to address this issue. The protocol establishes a general method to visualize the main components, including lignin, cellulose, and hemicellulose within the plant cell wall. In this protocol, procedures for sample preparation, spectral acquisition, and data processing are described. It is highly dependent upon operator skill at sample preparation and data analysis. By using this approach, a Raman investigation can be performed by a non-specialist user to acquire high-quality data and meaningful results for plant cell wall analysis.

This protocol aims to present a general method to visualize lignin, cellulose, and hemicellulose in plant cell walls using Raman imaging and multivariate analysis.

Combinación de imágenes Raman y Análisis Multivariante para visualizar Lignina, Celulosa y Hemicelulosa en el Muro de la Célula Vegetal

The application of Raman imaging to plant biomass is increasing because it can offer spatial and compositional information on aqueous solutions. The ...

Investigación

Bioquímica

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

La lignina Down-regulación de<em&gt; Zea mays</em&gt; Vía dsRNAi y Análisis de lignina Klason

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

Bioingeniería

Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital structural and protective role in the development and life of higher plants. Although the intricate mechanisms regulating lignification processes in vivo strongly impact the industrial valorization of many plant-derived products, the scientific community still has a long way to go to decipher them. In a simple three-step workflow, the dual labeling protocol presented herein enables bioimaging studies of actively lignifying zones of plant tissues. The first step consists in the metabolic incorporation of two independent chemical reporters, surrogates of the two native monolignols that give rise to lignin H- and G-units. After incorporation into growing lignin polymers, each reporter is then specifically labeled with its own fluorescent probe via a sequential combination of bioorthogonal SPAAC/CuAAC click reactions. Combined with lignin autofluorescence, this approach leads to the generation of three-color localization maps of lignin within plant cell walls by confocal fluorescence microscopy and provides precise spatial information on the presence or absence of active lignification machinery at the scale of plant tissues, cells and different cell wall layers.

BLISS, a dual labeling protocol for studying lignification dynamics, was developed. Using synthetic monolignol reporters and a sequential combination of SPAAC and CuAAC bioorthogonal click reactions, this methodology paves the way to in-depth analysis of the factors that regulate the biogenesis of lignins in planta.

Visualización dinámica de lignificación en plantas con la química Click: doble etiquetado es felicidad!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital ...

Química

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid (TGA)

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls and is an aromatic heteropolymer primarily composed of three monolignols with significant industrial importance. Lignin plays an important role in plant growth and development, protects&#160;from biotic and abiotic stresses, and in the quality of animal fodder, the wood, and industrial lignin products. Accurate estimation of lignin content is essential for both fundamental understanding of the lignin biosynthesis and industrial applications of biomass. The thioglycolic acid (TGA) method is a highly reliable method of estimating the total lignin content in the plant biomass. This method estimates the lignin content by forming thioethers with the benzyl alcohol groups of lignin, which are soluble in alkaline conditions and insoluble in acidic conditions. The total lignin content is estimated using a standard curve generated from commercial bamboo lignin.

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA

Here, we present a modified TGA method for estimation of lignin content in herbaceous plant biomass. This method estimates the lignin content by forming specific thioether bonds with lignin and presents an advantage over the Klason method, as it requires a relatively small sample for lignin content estimation.

Estimación del contenido de lignina de biomasa vegetal utilizando ácido tioglicólico (TGA)

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls ...

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates

The need for renewable, carbon neutral, and sustainable raw materials for industry and society has become one of the most pressing issues for the 21st century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation2 and other products such as biocomposite materials6. Plant biomass remains one of the greatest untapped reserves on the planet4. It is mostly comprised of cell walls that are composed of energy rich polymers including cellulose, various hemicelluloses, and the polyphenol lignin5 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls contribute extensively to the strength and structural integrity of the entire plant. Despite its necessary rigidity, the cell wall is a highly dynamic entity that is metabolically active and plays crucial roles in numerous cell activities such as plant growth and differentiation5. Due to the various functions of walls, there is an immense structural diversity within the walls of different plant species and cell types within a single plant4. Hence, depending of what crop species, crop variety, or plant tissue is used for a biorefinery, the processing steps for depolymerisation by chemical/enzymatic processes and subsequent fermentation of the various sugars to liquid biofuels need to be adjusted and optimized. This fact underpins the need for a thorough characterization of plant biomass feedstocks. Here we describe a comprehensive analytical methodology that enables the determination of the composition of lignocellulosics and is amenable to a medium to high-throughput analysis (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its monosaccharide composition of the hemicelluloses and other matrix polysaccharides1, and its content of crystalline cellulose7. The protocol for analyzing the lignin components in lignocellulosic biomass is discussed in Part I3.

Comprehensive Compositional Analysis of Plant Cell Walls Lignocellulosic biomass Part II: Carbohydrates

Plant biomass is a major carbon-neutral renewable resource that could be used for the production of biofuels. Plant biomass consists mainly of cell walls, a structurally complex composite material termed lignocellulosics. Here we describe a protocol for a comprehensive analysis of the content and composition of wall derived carbohydrates.

Análisis Integral de la composición de las paredes celulares vegetales (biomasa lignocelulósica) Parte II: Los hidratos de carbono

Biología

Quantitative 31P NMR Analysis of Lignins and Tannins

The development of sustainable biorefinery products is confronted, among others, with the challenge of lignin and tannin valorization. These abundant, renewable aromatic biopolymers have not been widely exploited due to their inherent structural complexity and high degrees of variability and species diversity. The lack of a defined primary structure for these polyphenols is further compounded with complex chemical alterations induced during processing, eventually imparting a large variety of structural features of extreme significance for any further utilization efforts.
Consequently, a protocol for the rapid, simple, and unequivocal identification and quantification of the various functional groups present in natural polyphenols, is a fundamental prerequisite for understanding and accordingly tailor their reactivity and eventual utility.
Quantitative 31P NMR offers the opportunity to rapidly and reliably identify unsubstituted, o-mono substituted, and o-disubstituted phenols, aliphatic OHs, and carboxylic acid moieties in lignins and tannins with broad application potential.
The methodology consists of an in situ quantitative lignin or tannin labeling procedure using a suitable 31P containing probe, followed by the acquisition of a quantitative 31P NMR spectrum in the presence of an internal standard. The high natural abundance of the 31P nucleus allows for small amounts of the sample (~30 mg) and short NMR acquisition times (~30-120 min) with well-resolved 31P signals that are highly dependent on the surrounding chemical environment of the labeled OH groups.

Quantitative 31P NMR Analysis of Lignins and Tannins

31P NMR is a powerful tool for the structural elucidation of polyphenols. This fast, easy, precise, quantitative, and highly reproducible analytical procedure, that allows for the quantification and differentiation of the different types of hydroxy, phenolic, and carboxylic groups in lignins and tannins has now become a routine analytical tool.

Análisis cuantitativo de RMN de 31P de ligninas y taninos

The development of sustainable biorefinery products is confronted, among others, with the challenge of lignin and tannin valorization. These abundant, ...

Label-free in situ Imaging of Lignification in Plant Cell Walls

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find cost-effective and sustainable solutions has become a topical and critical task. Lignocellulosic biomass is poised to become the primary source of biomass for the conversion to liquid biofuels1-6. However, the recalcitrance of these plant cell wall materials to cost-effective and efficient degradation presents a major impediment for their use in the production of biofuels and chemicals4. In particular, lignin, a complex and irregular poly-phenylpropanoid heteropolymer, becomes problematic to the postharvest deconstruction of lignocellulosic biomass. For example in biomass conversion for biofuels, it inhibits saccharification in processes aimed at producing simple sugars for fermentation7. The effective use of plant biomass for industrial purposes is in fact largely dependent on the extent to which the plant cell wall is lignified. The removal of lignin is a costly and limiting factor8 and lignin has therefore become a key plant breeding and genetic engineering target in order to improve cell wall conversion.
Analytical tools that permit the accurate rapid characterization of lignification of plant cell walls become increasingly important for evaluating a large number of breeding populations. Extractive procedures for the isolation of native components such as lignin are inevitably destructive, bringing about significant chemical and structural modifications9-11. Analytical chemical in situ methods are thus invaluable tools for the compositional and structural characterization of lignocellulosic materials. Raman microscopy is a technique that relies on inelastic or Raman scattering of monochromatic light, like that from a laser, where the shift in energy of the laser photons is related to molecular vibrations and presents an intrinsic label-free molecular "fingerprint" of the sample. Raman microscopy can afford non-destructive and comparatively inexpensive measurements with minimal sample preparation, giving insights into chemical composition and molecular structure in a close to native state. Chemical imaging by confocal Raman microscopy has been previously used for the visualization of the spatial distribution of cellulose and lignin in wood cell walls12-14. Based on these earlier results, we have recently adopted this method to compare lignification in wild type and lignin-deficient transgenic Populus trichocarpa (black cottonwood) stem wood15. Analyzing the lignin Raman bands16,17 in the spectral region between 1,600 and 1,700 cm-1, lignin signal intensity and localization were mapped in situ. Our approach visualized differences in lignin content, localization, and chemical composition. Most recently, we demonstrated Raman imaging of cell wall polymers in Arabidopsis thaliana with lateral resolution that is sub-&mu;m18. Here, this method is presented affording visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species without staining or labeling of the tissues.

Label-free in situ Imaging of Lignification in Plant Cell Walls

A method based on confocal Raman microscopy is presented that affords label-free visualization of lignin in plant cell walls and comparison of lignification in different tissues, samples or species.

Sin etiquetas In situ Imágenes de lignificación de las paredes celulares vegetales

Meeting growing energy demands safely and efficiently is a pressing global challenge. Therefore, research into biofuels production that seeks to find ...

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.

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 ...

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 ...

Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester of glycerol and modified fatty acids that constitutes the framework of the cuticle that covers aerial plant surfaces. Suberin is a related lipid polyester that is deposited on the cell walls of certain tissues, including the root endodermis and the periderm of tubers, tree bark and roots. These lipid polymers are highly variable in composition among plant species, and often differ among tissues within a single species. Here, we describe a detailed protocol to study the monomer composition of cutin in Arabidopsis thaliana leaves by sodium methoxide (NaOMe)-catalyzed depolymerisation, derivatization, and subsequent gas chromatography-mass spectrometry (GC/MS) analysis. This method can be used to investigate the monomers of insoluble polyesters isolated from whole delipidated plant tissues bearing either cutin or suberin. The method can by applied not only to characterize the composition of lipid polymers in species not previously analyzed, but also as an analytical tool in forward and reverse genetic approaches to assess candidate gene function.

Lipid polyesters constitute the structural components of two cell wall modifications, the plant cuticle and suberin-containing diffusion barriers. In this video, we describe a method to depolymerize cutin from whole delipidated leaves. The method can be applied to investigating mutants compromised in either cutin or suberin biosynthesis.

Aislamiento y composicional Análisis de Planta cutícula lípidos poliéster Monómeros

Terrestrial plants produce extracellular aliphatic biopolyesters that modify cell walls of specific tissues. Epidermal cells synthesize cutin, a polyester ...

Análisis Integral de la composición de las paredes celulares vegetales (biomasa lignocelulósica) Parte I: La lignina

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