Siamo grati a Matthew Robert Weatherhead eccellente per il servizio tecnico e John Ralph, University of Wisconsin per preziosi consigli, discussioni, e il campione di legno di pioppo. Questo lavoro è stato finanziato dal US Department of Energy (DOE) dei Grandi Laghi Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494) e dalle Scienze Chimiche, Scienze geologiche e Bioscienze Divisione, Ufficio di Basic Sciences Energy, Office of Science , US Department of Energy (nessun premio. DE-FG02-91ER20021).

1. Isolamento della parete cellulare <ol><li> Grind circa 60-70mg di aria o liofilizzato materiale vegetale con 5,5 palle mm in acciaio inox in una ml 2 tappo della provetta Sarstedt vite utilizzando un iWall, un robot macinazione e dosaggio (30 s). L&#39;alternativa non-robotica a basso throughput usando una procedura di mulini a palle (retschmill) è presentata nella Parte II, 2. </li><li> Aggiungere 1,5 ml di etanolo al 70% acquosa al materiale terra erogato, e vortice accuratamente. </li><li> Centrifugare a 10.000 rpm per 10 minuti per far sedimentare il residuo insolubile alcool. </li><li> Aspirare o decantare il sopranatante. </li><li> Aggiungere 1,5 ml di cloroformio / metanolo (1:1 v / v) per il residuo del tubo e agitare accuratamente per risospendere il pellet. </li><li> Centrifugare a 10.000 rpm per 10 minuti e aspirare o decantare il sopranatante. </li><li> Risospendere il pellet in 500 ml di acetone. </li><li> Evaporare il solvente con una corrente d&#39;aria a 35 ° C fino a secco. Se necessario campioni essiccati possono essere conservati a temperatura ambiente fino al procedimento successivo. </li><li> Per avviare la rimozione di amido dal campione risospendere il pellet in 1,5 ml di sodio 0,1 M pH 5,0 tampone acetato. </li><li> Cap i tubi Sarstedt e calore per 20 min. a 80 ° C su una piastra riscaldante. </li><li> Raffreddare la sospensione sul ghiaccio. </li><li> Aggiungere i seguenti agenti al pellet: 35 ml di 0,01% di sodio azide (NaN3), 35 amilasi microlitri (50 mg / ml H 2 O, da specie Bacillus, Sigma), 17 pullulanasi microlitri (17,8 unità da acidopullulyticus Bacillus; Sigma) . Chiudere la provetta e miscelare accuratamente. </li><li> La sospensione è incubata per una notte a 37 ° C nello shaker. Orientare i tubi in orizzontale aiutanti migliorato miscelazione. </li><li> Sospensione di calore a 100 ° C per 10 minuti su una piastra riscaldante di interrompere la digestione. </li><li> Centrifuga (10.000 rpm, 10 min) e scartare il surnatante contenente amido solubilizzato. </li><li> Lavare il rimanente a pellet per tre volte con l&#39;aggiunta di 1,5 ml di acqua, vortex, centrifugazione e decantazione delle acque di lavaggio. </li><li> Risospendere il pellet in 500 ml di acetone. </li><li> Evaporare il solvente con una corrente d&#39;aria a 35 ° C fino a secco. Potrebbe essere necessario anche per rompere il materiale nel tubo con una spatola per una migliore essiccazione. Il materiale essiccato presenta parete isolata cella (legnocellulosa). Se necessario campioni essiccati possono essere conservati a temperatura ambiente fino al procedimento successivo. </li></ol> 2. Lignina Contenuto Questo metodo si basa su un metodo riportato da Fukushima e Hatfield 3. <ol><li> Pesare 1-1,5 mg di materiale preparato parete cellulare (vedi 1) in 2 ml matraccio lasciando un tubo vuoto di uno spazio vuoto. </li><li> lavare le pareti del tubo da 250 ml di acetone per raccogliere il materiale della parete cellulare nella parte inferiore del tubo, e far evaporare l&#39;acetone molto delicato sotto il flusso d&#39;aria. </li><li> Delicatamente aggiungere 100 ml di soluzione appena fatto bromuro di acetile (25% v / v bromuro di acetile in acido acetico glaciale) lungo le pareti del tubo per evitare schizzi. </li><li> Cap volumetrico matraccio e riscaldare a 50 ° C per 2 ore </li><li> Calore per un&#39;altra ora con vortex ogni 15 minuti. </li><li> Raffreddare in ghiaccio a temperatura ambiente. </li><li> Aggiungere 400 ml di idrossido di sodio 2M e 70 ml di preparati al momento 0,5 M idrossilammina cloridrato. Vortex matracci tarati. </li><li> Riempire pallone tarato esattamente a 2,0 ml segno con acido acetico glaciale, il tappo e capovolgere più volte per mescolare. </li><li> Pipettare 200 ml di soluzione in un UV specifico 96 pozzetti e leggere in un lettore ELISA a 280 nm. </li><li> Determinare la percentuale di lignina bromuro di acetile solubile (% ABSL) utilizzando un adeguato coefficiente (pioppo = 18,21; Erbe = 17,75; Arabidopsis = 15,69) con la seguente formula: % ABSL Calc: <img src="/files/ftp_upload/1745/1745eq.jpg" alt="Figura 1" /> Moltiplicazione delle ABSL% con 10 risultati in ug / mg unità della parete cellulare Aiuta a fare almeno 3 piatto legge in media l&#39;assorbanza (abs) dal particolato può causare una lieve variazione nei valori di assorbenza. Nota: 0,539 centimetri rappresenta il cammino ottico, ma a seconda del piatto questo potrebbe aver bisogno di essere determinato. </li></ol> 3. Lignina Composizione Questo metodo è adottato da un metodo di recente pubblicato da Robinson e Mansfield 5. <ol><li> Trasferire circa 2 mg di materiale della parete cellulare (vedi 1.) In una provetta di vetro con tappo a vite per thioacidolysis. </li><li> preparare con cura il 2,5% trifluoruro di boro dietil etherate (BF 3), 10% ethanethiol (EtSH) soluzione. È necessario utilizzare un pallone riempito di gas azoto per spostare il volume perso in bottiglia diossano con azoto. Diossano è molto pericoloso, non prelevare campioni o apparecchiature fuori dal cofano. Volumi necessari per la preparazione della soluzione per campione: 175 microlitri diossano, 20 EtSH microlitri, 5 microlitri BF 3. </li><li> Aggiungere 200 ml di EtSH, BF 3, diossano soluzione ad ogni campione. </li><li> Purge spazio di testa flacone con gas azoto e tappo immediatamente. </li><li> Calore a 100 ° C per 4 ore con dolce mescolando ogni ora. </li><li> Reazione fine di raffreddamento in ghiaccio per 5 minuti. </li><li> Aggiungere 150 ml di bicarbonato di sodio 0.4M, vortice </li><li> Per la pulizia aggiungere 1 ml di acqua e 0,5 ml di acetato di etile, vortex e lasciare separare le fasi (acetato di etile in alto, l&#39;acqua sul fondo). </li><li> Trasferire 150 ml di strato di acetato di etile in una provetta da 2 ml Sarstedt. Assicurarsi che l&#39;acqua non viene trasferito. </li><li> Evaporare solvente da un concentratore d&#39;aria. </li><li> Aggiungere 200 microlitri e far evaporare l&#39;acetone (ripetere per un totale di due volte rimuovere l&#39;acqua in eccesso). </li><li> Per la derivatizzazione TMS aggiungere 500 ml di acetato di etile, 20 ml di piridina, e 100 ml di N, O-bis (trimetilsilil) acetamide ad ogni provetta. </li><li> incubare per 2 ore a 25 ° C. </li><li> Trasferire 100 microlitri della reazione in un GC / MS fiala e aggiungere 100 ml di acetone. </li><li> Analizzare i campioni da GC dotato di un quadrupolo-spettrometro di massa o della ionizzazione di fiamma. Un sistema HP-5MS Agilent colonna è installato (30 mm x 0,25 mm X 0,25 spessore micron). Il gradiente di temperatura seguente viene utilizzato con un ritardo di 30 minuti e un solvente 1,1 ml / min Portata: tenere iniziale a 130 ° C per 3 minuti, un 3 ° C / min fino ad un rampa di 250 ° C e mantenere per 1 minuto; permettono equilibrio alla temperatura iniziale di 130 ° C. </li><li> Picchi sono identificati con tempi di ritenzione relativi con tetracosane standard interno (opzionale) o da ioni caratteristico spettro di massa di 299 m / z, 269 m / z, monomeri e 239 m / z di S, G e H, rispettivamente (vedi fig. 2). La composizione dei componenti lignina è quantificato impostando l&#39;area totale dei picchi al 100% </li></ol> 4. Rappresentante Risultati Un esempio di analisi muro è presentato in figura 2. In questo caso fusto di pioppo (legno) è stato analizzato mediante le varie procedure descritte nella sezione protocollo. Un esempio di cromatogramma la separazione della lignina componenti dopo thioacidolysis e TMS-derivatizzazione è mostrato. Chiaramente, l&#39;abbondanza relativa di syringyl-(S), guaiacyl-(G), e p-hydroxyphenol-(H) unità può essere determinato. Il contenuto di lignina solubile bromuro di acetile si spiega da sé, ci si può aspettare tra i valori del 20-50% del peso a secco della parete. Si noti che il bromuro di acetile non solubilizzare tutti i presenti lignina nella parete, e che il grado di solubilizzazione può variare a seconda del materiale. Tuttavia, questo metodo è relativamente facile da realizzare e rapida e fornisce una eccellente approssimazione del contenuto di lignina in un materiale ligno-cellulosica. <img src="/files/ftp_upload/1745/1745fig1.jpg" alt="Figura 1" /> Figura 1:. Panoramica di analisi lignocellulosici pareti cellulari (legnocellulosa) sono isolati dal greggio materiale vegetale essiccato. Il materiale della parete viene poi pesata in aliquote e suddiviso per i vari metodi. Materiale della parete è trattato con bromuro di acetile e la lignina solubilizzato quantificato spettroscopia UV. Per la determinazione della composizione lignina, materiale della parete è sottoposto a thioacidolysis. I composti fenolici solubilizzato subire derivatizzazione TMS e possono quindi essere separati e quantificati mediante analisi GC-MS. La composizione polisaccaride matrice e cristallino protocollo contenuto di cellulosa è discusso nella Parte II, 2. <img src="/files/ftp_upload/1745/1745fig2.jpg" alt="Figura 2" /> Figura 2:. Completa analisi lignocellulosici di legno di pioppo Cippato di pioppo (Populus tremoloides) sono stati sottoposti ai protocolli descritti. Ligin composizione; H p-idrossifenil; G guaiacyl; S unità syringyl.

<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>

I metodi descritti permettono una rapida valutazione quantitativa del contenuto di lignina e la composizione della biomassa vegetale lignocellulosiche. Utilizzando il robot iWall circa 350 campioni di terreno e può essere erogato al giorno. La velocità dei vari metodi analitici per persona varia. Utilizzando i protocolli descritti qui, a 30 campioni possono essere trattati per contenuto di lignina, e 15 per la composizione della lignina al giorno. A causa della natura quantitativa della materia prima ottimale dei dati raccolti, varietà o genotipi può essere valutata in termini di idoneità per la produzione di biocarburanti.

<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 necessità di fonti rinnovabili, carbon neutral, e sostenibile di materie prime per l&#39;industria e la società è diventata una delle questioni più pressanti per il 21 ° secolo. Questo ha riacceso interesse per l&#39;uso di prodotti vegetali come materie prime industriali per la produzione di combustibili liquidi per il trasporto<sup&gt; 1</sup&gt; E altri prodotti come materiali biocomposite<sup&gt; 7</sup&gt;. Biomassa vegetale rimane una delle più grandi riserve non sfruttate del pianeta<sup&gt; 4</sup&gt;. È principalmente costituito da pareti cellulari che sono composti da polimeri ricchi di energia compresa la cellulosa, emicellulosa vari (polisaccaridi della matrice, e la lignina polifenoli<sup&gt; 6</sup&gt; E quindi talvolta chiamate legnocellulosa. Tuttavia, le pareti delle cellule vegetali si sono evoluti per essere recalcitranti alla degradazione come pareti fornire resistenza alla trazione di cellule e le piante intere, allontanare gli agenti patogeni, e permettere all&#39;acqua di essere trasportati in tutta la pianta, nel caso di alberi fino a più del 100 m sopra livello del suolo. Grazie alle varie funzioni di mura, vi è una immensa diversità strutturale tra le mura di specie vegetali diverse e tipi di cellule all&#39;interno di una singola pianta<sup&gt; 4</sup&gt;. Quindi, a seconda di ciò che le specie coltivate, la varietà delle colture, o tessuto vegetale viene utilizzato per una bioraffineria, le fasi di lavorazione per la depolimerizzazione da processi chimici / enzimatici e successiva fermentazione degli zuccheri vari biocarburanti liquidi devono essere adeguate e ottimizzati. Questo fatto rafforza l&#39;esigenza di una caratterizzazione approfondita delle materie prime da biomassa vegetale. Qui descriviamo una metodologia completa di analisi che consente la determinazione della composizione del legnocellulosa ed è suscettibile di un mezzo di analisi high-throughput. In questa prima parte ci si concentra sull&#39;analisi della lignina polifenoli (Figura 1). Il metodo inizia con la preparazione di materiale destarched parete cellulare. Il legnocellulosa risultanti sono poi divisi per determinare il contenuto di lignina solubilizzazione acetylbromide<sup&gt; 3</sup&gt;, E la sua composizione lignina in termini di syringyl, unità guaiacyl-e p-idrossifenil<sup&gt; 5</sup&gt;. Il protocollo per analizzare i carboidrati nella biomassa lignocellulosica compreso il contenuto di cellulosa e polisaccaridi composizione della matrice è discusso nella 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

Biomassa vegetale è uno dei principali carbon-neutral risorsa rinnovabile che potrebbero essere utilizzati per la produzione di biocarburanti. Biomassa vegetale è costituita principalmente delle pareti delle cellule, un materiale composito chiamato strutturalmente complesso legnocellulosa. Qui si descrive un protocollo per un&#39;analisi completa del contenuto e la composizione della lignina polifenoliche.

Completa l&#39;analisi della composizione delle pareti cellulari delle piante (biomassa lignocellulosica) Parte I: 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 ...

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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 Views.  Michigan State University (MSU). Biomassa vegetale è uno dei principali carbon-neutral risorsa rinnovabile che potrebbero essere utilizzati per la produzione di biocarburanti. Biomassa vegetale è costituita principalmente delle pareti delle cellule, un materiale composito chiamato strutturalmente complesso legnocellulosa. Qui si descrive un protocollo per un'analisi completa del contenuto e la composizione della lignina polifenoliche.

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.

Combinazione di Raman Imaging e analisi multivariata per la visualizzazione di lignina, cellulosa e emicellulosa nella parete cellulare delle piante

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

Ricerca

Biochimica

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-regulation dei<em&gt; Zea mays</em&gt; Via dsRNAi e Klason lignina analisi

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

Bioingegneria

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.

Visualizzazione di lignificazione Dynamics in piante con chimica clicca: doppia etichettatura è beatitudine!

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

Chimica

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.

Stima del contenuto di Lignina da biomassa vegetale mediante acido tioglicolico (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.

Completa l&#39;analisi della composizione delle pareti cellulari delle piante (biomassa lignocellulosica) Parte II: Carboidrati

Biologia

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.

Analisi NMR quantitativa 31P di Lignine e Tannini

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.

Senza etichetta In situ</emImaging> di lignificazione in pareti delle cellule vegetali

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.

Profiling Glycan dei polimeri cellula vegetale da parete usando i 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.

Colorazione istochimica di<em&gt; Arabidopsis thaliana</em&gt; Cellula secondaria Elementi da parete

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.

Isolamento e analisi della composizione delle piante cuticola lipidi poliestere monomeri

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

Completa l'analisi della composizione delle pareti cellulari delle piante (biomassa lignocellulosica) Parte I: lignina

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Capitoli in questo video

Combinazione di Raman Imaging e analisi multivariata per la visualizzazione di lignina, cellulosa e emicellulosa nella parete cellulare delle piante

La lignina down-regulation dei

Visualizzazione di lignificazione Dynamics in piante con chimica clicca: doppia etichettatura è beatitudine!

Stima del contenuto di Lignina da biomassa vegetale mediante acido tioglicolico (TGA)

Completa l'analisi della composizione delle pareti cellulari delle piante (biomassa lignocellulosica) Parte II: Carboidrati

Analisi NMR quantitativa ³¹P di Lignine e Tannini

Senza etichetta In situ di lignificazione in pareti delle cellule vegetali

Profiling Glycan dei polimeri cellula vegetale da parete usando i microarrays

Colorazione istochimica di

Isolamento e analisi della composizione delle piante cuticola lipidi poliestere monomeri