Wir sind dankbar, dass Matthew Robert Weatherhead für einen exzellenten technischen Service und John Ralph, University of Wisconsin für wertvolle Ratschläge, Diskussionen und die Pappel Probe. Diese Arbeit wurde vom US Department of Energy (DOE) Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494) und von der Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science finanziert , US Department of Energy (Preis-Nr. DE-FG02-91ER20021).

1. Zellwand Isolation <ol><li> Grind etwa 60-70mg von Luft-oder gefriergetrocknete Pflanzenmaterial mit 5,5 mm Edelstahl-Kugeln in einem 2 ml Sarstedt Schraubverschluss Röhrchen mit einem iWall, ein Mahlen und Abfüllen Roboter (30 s). Eine Alternative nicht-Roboter-low-Durchsatz-Verfahren unter Anwendung einer Kugelmühle (retschmill) ist in Teil II 2 dargestellt. </li><li> Fügen Sie 1,5 ml 70% wässrigem Ethanol auf die verzichtet Mahlgut und gründlich vortexen. </li><li> Zentrifuge bei 10.000 rpm für 10 min zur Pelletierung der Alkohol unlöslichen Rückstand. </li><li> Verwerfen Sie den Überstand. </li><li> Fügen Sie 1,5 ml Chloroform / Methanol (1:1 v / v) Lösung des Rückstandes und schütteln Rohr gründlich, um das Pellet zu resuspendieren. </li><li> Zentrifuge bei 10.000 rpm für 10 min und Verwerfen des Überstandes. </li><li> Pellet in 500 ul Aceton. </li><li> Das Lösungsmittel wird mit einem Luftstrom bei 35 ° C, bis es trocken. Bei Bedarf getrockneten Proben können bei Raumtemperatur bis zur weiteren Verarbeitung gespeichert werden. </li><li> Zur Einleitung der Entfernung von Stärke aus der Probe das Pellet in 1,5 ml einer 0,1 M Natriumacetat-Puffer pH 5,0. </li><li> Cap der Sarstedtröhrchen und Wärme für 20 min. bei 80 ° C in einem Heizblock. </li><li> Kühlen Sie die Suspension auf Eis. </li><li> Fügen Sie die folgenden Substanzen, um das Pellet: 35 ul von 0,01% Natriumazid (NaN 3), 35 ul Amylase (50 ug / mL H 2 O, von Bacillus-Arten, Sigma), 17 ul Pullulanase (17,8 Einheiten aus Bacillus acidopullulyticus; Sigma) . Cap der Röhre und gründlich vortexen. </li><li> Die Suspension wird über Nacht bei 37 ° C in den Shaker. Die Ausrichtung der Rohre horizontal Helfer eine bessere Durchmischung. </li><li> Hitze Suspension bei 100 ° C für 10 min in einem Heizblock, um die Verdauung zu kündigen. </li><li> Zentrifuge (10.000 rpm, 10 min) und Überstand verwerfen, die solubilisierten Stärke. </li><li> Waschen Sie die verbleibende Pellet dreimal durch Zugabe von 1,5 ml Wasser, Vortexen, Zentrifugieren und Dekantieren des Waschwassers. </li><li> Pellet in 500 ul Aceton. </li><li> Das Lösungsmittel wird mit einem Luftstrom bei 35 ° C, bis es trocken. Es kann notwendig sein auch zu brechen das Material in der Röhre mit einem Spatel für eine bessere Trocknung. Das getrocknete Material präsentiert isoliert Zellwand (Lignocellulose). Bei Bedarf getrockneten Proben können bei Raumtemperatur bis zur weiteren Verarbeitung gespeichert werden. </li></ol> 2. Ligningehalt Diese Methode basiert auf einem gemeldeten Methode Fukushima und Hatfield 3. <ol><li> Wiegen Sie 1 bis 1,5 mg vorbereitet Zellwandmaterial (siehe 1) in 2 ml-Messkolben der Ausfahrt aus einer Tube leer für einen Rohling. </li><li> Spülen Rohrwandungen mit 250 ul Aceton an die Zellwand Material auf den Boden des Röhrchens zu sammeln und verdampft das Aceton sehr sanft unter Luftstrom. </li><li> Vorsichtig mit 100 ul frisch gemacht Acetylbromid-Lösung (25% v / v Acetylbromid in Eisessig) entlang der Rohrwände um Spritzer zu vermeiden. </li><li> Cap-Messkolben und Wärme bei 50 ° C für 2 Stunden </li><li> Wärme für eine zusätzliche Stunde mit Vortexen alle 15 Minuten. </li><li> Cool auf Eis auf Raumtemperatur. </li><li> Add 400 ul 2M Natronlauge und 70 ul frisch zubereitete 0,5 M Hydroxylaminhydrochlorid. Vortex-Messkolben. </li><li> Füllen Sie Messkolben genau auf die 2,0 ml Marke mit Eisessig, verschließen und mehrmals zu mischen. </li><li> Je 200 ul der Lösung in eine UV spezifischen 96-Well-Platte und las in einem ELISA-Reader bei 280nm. </li><li> Bestimmen Sie den Prozentsatz der Acetylbromid Lignin (% ABSL) unter Verwendung eines geeigneten Koeffizienten (Pappel = 18.21; Grasses = 17,75; Arabidopsis = 15,69) mit folgender Formel: % ABSL Calc: <img src="/files/ftp_upload/1745/1745eq.jpg" alt="Abbildung 1" /> Multiplikation% ABSL mit 10 Treffern in der ug / mg Zellwand-Einheit Es hilft zu tun mindestens 3 Platte liest die Absorption (abs), da Partikel können eine leichte Variation in Extinktionswerte Ursache Durchschnitt. Hinweis: 0,539 cm stellt die Schichtdicke, sondern in Abhängigkeit von der Platte könnte dies müssen bestimmt werden. </li></ol> 3. Ligninzusammensetzung Diese Methode ist aus einer neueren Methode von Robinson und Mansfield 5 veröffentlicht hat. <ol><li> Etwa 2 mg der Zellwand-Material (siehe 1.) In einer Schraube verschlossen Glasrohr für thioacidolysis. </li><li> sorgfältig vorbereiten 2,5% Bortrifluoriddiethyletherat (BF 3), 10% Ethanthiol (EtSH)-Lösung. Sie müssen einen Luftballon mit Stickstoff gefüllt, um die verlorenen Volumens im Dioxan Flasche mit Stickstoff zu verdrängen. Dioxan ist sehr gefährlich, nicht nehmen Proben oder Geräte aus der Haube. 20 pl EtSH;; 5 pl BF 3 175 ul Dioxan: Volumes für die Vorbereitung der Lösung pro Probe benötigt. </li><li> Add 200 ul EtSH, BF 3, Dioxanlösung zu jeder Probe. </li><li> Purge Vial Kopfraum mit Stickstoffgas und sofort wieder aufsetzen. </li><li> Hitze auf 100 ° C für 4 Stunden unter leichtem Mischen jede Stunde. </li><li> End Reaktion durch Kühlen auf Eis für 5 Minuten. </li><li> Add 150 ul 0,4 M Natriumbicarbonat, Wirbel </li><li> Für den Clean-up 1 ml Wasser und 0,5 ml Ethylacetat, Wirbel und lassen Phasen zu trennen (Ethylacetat auf, Wasser auf der Unterseite). </li><li> Transfer 150 ul des Ethylacetatschicht in ein 2 ml Sarstedt Röhrchen. Achten Sie darauf, kein Wasser übertragen wird. </li><li> Dampft Lösungsmittel durch einen Konzentrator mit Luft. </li><li> Geben Sie 200 ul Aceton und verdampft (Wiederholung für insgesamt zwei Mal zu entfernen überschüssiges Wasser). </li><li> Für die TMS-Derivatisierung add 500 ul Ethylacetat, 20 ul Pyridin, und 100 ul N, O-Bis (trimethylsilyl) acetamid in jedes Röhrchen. </li><li> inkubieren für 2 Stunden bei 25 ° C. </li><li> Transfer-100 ul der Reaktion in ein GC / MS Fläschchen und 100 ul Aceton. </li><li> Analysieren Sie die Proben durch GC mit einem Quadrupol-Massenspektrometer oder Flammenionisationsdetektor ausgestattet. Ein Agilent HP-5ms-Säule installiert ist (30 mm x 0,25 mm X 0,25 um Filmdicke). Die folgenden Temperaturgefälle ist mit einem 30 min Lösungsmittel Verzögerung und einem 1,1 ml / min Durchfluss verwendet: Initial halten bei 130 ° C für 3 min; 3 ° C / min Rampe zu einer 250 ° C und für 1 min halten; ermöglichen Equilibrierung auf die ursprüngliche Temperatur von 130 ° C. </li><li> Peaks werden von relativen Retentionszeiten mit Tetracosan interner Standard (optional) oder durch charakteristische Massenspektrum Ionen von 299 identifizierten m / z, 269 m / z und 239 m / z für S, G, H und Monomere sind (siehe Abb.. 2). Die Zusammensetzung des Lignin-Komponenten wird durch die Einstellung der gesamten Peakfläche um 100% quantifiziert </li></ol> 4. Repräsentative Ergebnisse Ein Beispiel für eine Wand-Analyse ist in Abbildung 2 dargestellt. In diesem Fall Pappel Stamm (Holz) wurde durch die verschiedenen Verfahren in das Protokoll Abschnitt beschrieben analysiert. Ein Beispiel-Chromatogramm der Trennung von Lignin-Komponenten nach thioacidolysis und TMS-Derivatisierung wird angezeigt. Offensichtlich ist die relative Häufigkeit von Syringyl-(S), Guaiacyl-(G) und p-hydroxyphenol-(H)-Einheiten bestimmt werden kann. Für den Inhalt der Acetylbromid Lignin ist selbsterklärend, kann man erwarten, Werte zwischen 20-50% der Wand Trockengewicht. Man sollte beachten, dass Acetylbromid nicht lösen alle das Lignin in der Wand, und dass der Grad der Solubilisierung kann variieren je nach Material. Allerdings ist dieses Verfahren relativ einfach durchzuführen und eine schnelle und gibt einen hervorragenden Angleichung der Ligningehalt in einer Lignocellulose-Material. <img src="/files/ftp_upload/1745/1745fig1.jpg" alt="Abbildung 1" /> Abbildung 1:. Übersicht von Lignocellulose-Analyse Zellwände (Lignocellulose) werden aus Rohöl getrocknetes Pflanzenmaterial isoliert. Die Wand wird anschließend in Aliquots gewichtet und unterteilt für die verschiedenen Tests. Das Wandmaterial ist mit Acetylbromid und das gelöste Lignin durch UV-Spektroskopie quantifiziert behandelt. Für die Bestimmung der Ligninzusammensetzung ist Wandmaterial thioacidolysis unterzogen. Die solubilisierten Phenole unterziehen TMS-Derivatisierung und kann dann getrennt und quantifiziert werden durch GC-MS-Analyse. Die Matrix Polysaccharid Zusammensetzung und kristalline Cellulose-Gehalt Protokoll wird in Teil II 2 diskutiert. <img src="/files/ftp_upload/1745/1745fig2.jpg" alt="Abbildung 2" /> Abbildung 2:. Umfassende lignocellulosischen Analyse von Pappelholz Hackschnitzel aus Pappel (Populus tremoloides) wurden zu den beschriebenen Protokollen unterzogen. Ligin Zusammensetzung; H p-hydroxyphenyl; G Guaiacyl; S Syringyleinheiten.

<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>

Die beschriebenen Methoden ermöglichen eine schnelle quantitative Beurteilung der Ligningehalt und Zusammensetzung von Lignocellulose-Biomasse-Anlage. Mit dem iWall Roboter ca. 350 Proben können Boden und abgegeben werden pro Tag. Der Durchsatz der verschiedenen analytischen Methoden pro Person variiert. Mit der beschriebenen Protokolle hier aus können 30 Proben für Ligningehalt verarbeitet werden, und 15 für Ligninzusammensetzung pro Tag. Durch die quantitative Natur der Daten optimal Rohstoff Getreide können Sorte oder Genotypen in Bezug auf ihre Eignung für die Produktion von Biokraftstoffen bewertet werden.

<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

Der Bedarf an erneuerbaren, klimaneutralen und nachwachsenden Rohstoffen für die Industrie und die Gesellschaft hat sich zu einem der drängendsten Probleme des 21. Jahrhunderts. Dies hat das Interesse an der Verwendung von pflanzlichen Erzeugnissen als industrielle Rohstoffe für die Produktion von flüssigen Kraftstoffen für den Transport neu entfacht<sup&gt; 1</sup&gt; Und andere Produkte wie Biokomposit Materialien<sup&gt; 7</sup&gt;. Pflanzlicher Biomasse bleibt eines der größten unerschlossenen Reserven auf dem Planeten<sup&gt; 4</sup&gt;. Es ist vor allem der Zellwände, die von energiereichen Polymeren, einschließlich Cellulose, Hemicellulosen verschiedenen (Matrix Polysaccharide und das Polyphenol Lignin sind zusammen<sup&gt; 6</sup&gt; Und damit manchmal Lignocellulose bezeichnet. Allerdings haben pflanzlichen Zellwände entwickelt, um widerspenstig, um den Abbau als Wände Zugfestigkeit bereitzustellen, um Zellen und die gesamte Pflanzen, Pathogene abzuwehren, und damit das Wasser in der Pflanze transportiert werden; im Fall von Bäumen bis zu mehr die 100 m über Erdgeschoss. Aufgrund der vielfältigen Funktionen von Wänden, gibt es eine immense strukturelle Vielfalt innerhalb der Mauern der verschiedenen Pflanzenarten und Zelltypen innerhalb einer einzigen Anlage<sup&gt; 4</sup&gt;. Daher, je von dem, was Pflanzenarten und Abwechslung, oder Pflanzengewebe ist für eine Bioraffinerie verwendet wird, müssen die Verarbeitungsschritte für die Depolymerisation durch chemische / enzymatische Prozesse und die anschließende Vergärung der verschiedenen Zuckerarten auf flüssige Biokraftstoffe angepasst und optimiert werden. Diese Tatsache unterstreicht die Notwendigkeit, für eine gründliche Charakterisierung pflanzlicher Biomassen. Hier beschreiben wir eine umfassende analytische Methode, die Bestimmung der Zusammensetzung von Lignocellulose ermöglicht und ist offen für eine mittel-bis High-Throughput-Analyse. In diesem ersten Teil haben wir auf die Analyse der Polyphenol Lignin (Abbildung 1) zu konzentrieren. Das Verfahren beginnt mit der Vorbereitung der Stärke befreite Zellwandmaterial. Die daraus resultierende Lignocellulose werden dann bis zu seinem Ligningehalt von acetylbromide Solubilisierung bestimmen split<sup&gt; 3</sup&gt;, Und seine Ligninzusammensetzung in Bezug auf seine Syringyl, Guaiacyl-und p-hydroxyphenyl Einheiten<sup&gt; 5</sup&gt;. Das Protokoll für die Analyse der Kohlenhydrate in Lignozellulose einschließlich Cellulose-Gehalt und Matrix Polysaccharidzusammensetzung ist in Teil II diskutiert<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

Pflanzlicher Biomasse ist ein wichtiger klimaneutrale erneuerbare Ressource, die für die Herstellung von Biokraftstoffen verwendet werden könnten. Pflanzliche Biomasse besteht im Wesentlichen aus Zellwände, eine strukturell komplexen Verbundwerkstoff bezeichnet Lignocellulose. Hier beschreiben wir ein Protokoll für eine umfassende Analyse des Inhalts und der Zusammensetzung der Polyphenol-Lignin.

Umfassende Analyse der Zusammensetzung von pflanzlichen Zellwänden (Lignozellulose) Teil I: Lignin

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 ...

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

Unit 1

Plant Cell Structure and Organization

SCIENTISTS IN ACTION

32.1K Aufrufe.  Michigan State University (MSU). Pflanzlicher Biomasse ist ein wichtiger klimaneutrale erneuerbare Ressource, die für die Herstellung von Biokraftstoffen verwendet werden könnten. Pflanzliche Biomasse besteht im Wesentlichen aus Zellwände, eine strukturell komplexen Verbundwerkstoff bezeichnet Lignocellulose. Hier beschreiben wir ein Protokoll für eine umfassende Analyse des Inhalts und der Zusammensetzung der Polyphenol-Lignin.

Serie: 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.

Kombinieren von Raman Imaging und multivariater Analyse zur Visualisierung von Lignin, Cellulose und Hemicellulose in der Pflanzenzellwand

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

Forschung

Biochemie

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.

Lignin Herunterregulierung<em&gt; Zea mays</em&gt; Über dsRNAi und Klason Lignin Analyse

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

Biotechnik

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.

Visualisierung von Lignification Dynamik in Anlagen mit Klicken Sie auf Chemie: doppelte Kennzeichnung ist Glückseligkeit!

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

Chemie

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.

Abschätzung des Ligningehalts von pflanzlicher Biomasse mit Thioglykolsäure (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.

Umfassende Analyse der Zusammensetzung von pflanzlichen Zellwänden (Lignozellulose) Teil II: Kohlenhydrate

Biologie

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.

Quantitative 31P NMR Analyse von Ligninen und Tanninen

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.

Label-freie In situ Imaging der Verholzung in pflanzlichen Zellwänden

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.

Glycan Profiling of Plant Cell Wall Polymers mit 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.

Histochemische Färbung von<em&gt; Arabidopsis thaliana</em&gt; Sekundärzellwand-Elemente

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.

Isolierung und Analyse der Zusammensetzung von Pflanzen Cuticle Lipid Polyester Monomere

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

Umfassende Analyse der Zusammensetzung von pflanzlichen Zellwänden (Lignozellulose) Teil I: Lignin

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