我们非常感谢马修罗伯特韦瑟优质的技术服务和约翰拉尔夫，威斯康星大学的宝贵意见，讨论和杨木样本。这项工作的美国能源部（DOE）的大湖生物能源研究中心（美国能源部的BER科学DE - FC02 - 07ER64494办事处）和化学科学，地球科学和生物科学部，基础能源科学办公室，科学办公室资助美国能源部（无奖。DE - FG02 - 91ER20021）。

1。细胞墙隔离<ol><li>大约60 - 70mg的空气或冷冻干燥植物材料研磨5.5毫米，在2毫升Sarstedt螺丝帽使用一个iWall管不锈钢球，研磨和配药机器人（30秒）。另一种非机械手的低吞吐量的程序，使用一个球，磨（retschmill）是在第二部分2。 </li><li> 1.5毫升的70％的水溶液乙醇添加到免除地面材料，旋涡彻底。 </li><li>以10,000 rpm离心10分钟沉淀不溶于酒精残留。 </li><li>吸或倒出上清液。 </li><li>加入1.5毫升氯仿/甲醇（1:1 V / V）的解决方案去渣，彻底动摇管悬浮沉淀。 </li><li>离心10,000 RPM为10分钟，并吸出或倒出上清液。 </li><li> 500μL丙酮中的悬浮颗粒。 </li><li>蒸发与空气流在35 ° C，待干溶剂。 如果需要干燥的样品可存放在室温，直到进一步的处理。 </li><li>要启动从样本中去除淀粉颗粒悬浮在一个0.1 M的醋酸钠缓冲液的pH值5.0分中评1.5毫升。 </li><li>第20分钟Sarstedt管和热。在80 ° C在加热块。 </li><li>清凉冰暂停。 </li><li>添加以下代理颗粒：35微升0.01％叠氮化钠（NaN3的），35μL淀粉酶（50μg/ mL的H 2 O; 芽孢杆菌的物种，Sigma公司）; 17μL普鲁兰（17.8 acidopullulyticus芽孢杆菌;单位西格玛） 。第管和涡彻底。 </li><li>悬挂在37℃摇床中的C孵育过夜。定向管水平助手改善混合。 </li><li>热悬浮在100 ° C下10分钟，在加热块终止消化。 </li><li>离心（10,000 RPM，10分钟），弃上清液含溶解淀粉。 </li><li>洗净，加入1.5毫升的水，振荡，离心，洗涤水调迁，其余颗粒三次。 </li><li> 500μL丙酮中的悬浮颗粒。 </li><li>蒸发与空气流在35 ° C，待干溶剂。这可能是必要的，也打破了管具有更好的干燥锅铲的材料。 干燥后的材料呈现孤立的细胞壁（lignocellulosics）。如果需要干燥的样品可存放在室温，直到进一步的处理。 </li></ol> 2。木质素含量这种方法是基于一个福岛和Hatfield 3方法。 <ol><li>称取1 - 1.5毫克的细胞壁物质准备（1）到2 ml容量瓶中留下一管空一个空白。 </li><li> 250丙酮液收集管底部的细胞壁物质冲洗管壁，丙酮蒸发下的气流非常温柔。 </li><li>轻轻地沿管壁添加100μL新鲜的乙酰溴铵溶液（25％V / V冰醋酸中的乙酰溴），防止飞溅。 </li><li>第容量瓶和热在50℃为2小时彗星</li><li>热与涡旋每15分钟增加一小时。 </li><li>在冰上冷至室温。 </li><li>新增400个2M的氢氧化钠和70μL新鲜配制的0.5 M的盐酸羟胺液。涡容量瓶。 </li><li>完全填充容量瓶用冰醋酸，第2.0毫升标志和颠倒几次混合。 </li><li>吸取200μL的解决方案的紫外线具体的96孔板，在酶标仪在280nm处读取。 </li><li>乙酰溴溶解木质素（％ABSL）的比例，使用适当的系数（白杨= 18.21;草= 17.75;拟南芥= 15.69），用下面的公式确定： ABSL％Calc工作： <img src="/files/ftp_upload/1745/1745eq.jpg" alt="图1" /> 10微克/毫克细胞壁单位乘法％ABSL 它可以帮助做至少有3盘读取的平均吸光度（ABS），因为微粒可导致吸光度值略有变化。注：0.539厘米的光程，但根据板，这可能需要确定。 </li></ol> 3。木质素组成这种方法是通过从罗宾逊和曼斯菲尔德5最近出版了一方法。 <ol><li>转移到一个螺丝thioacidolysis上限玻璃管约2毫克细胞壁物质（见1）。。 </li><li>精心准备二乙基乙醚2.5％，三氟化硼（BF 3），10％的乙硫醇（EtSH）解决方案。您必须使用一个气球充满氮气，以取代二恶烷在用氮气瓶的丢失量。二恶烷是非常危险的，不拿样品或设备的引擎盖。需要为每个样品溶液的制备：175μL的二恶烷; 20μLEtSH; 5μL高炉3卷。 </li><li>加入200μLEtSH，BF 3，每个样品中的二恶烷的解决方案。 </li><li>样品瓶顶空与氮气和第立即清除。 </li><li>热在100 ° C时轻轻搅拌每隔一小时4小时。 </li><li>冰上冷却5分钟结束反应。 </li><li> 0.4M碳酸氢钠加入150μL，涡旋</li><li>对于清理中添加1毫升的水和0.5毫升乙酸乙酯，旋涡，让独立的阶段（顶部，底部水醋酸乙酯）。 </li><li>转移到2毫升Sarstedt管150μL的醋酸乙酯层。确保没有水转。 </li><li>溶剂蒸发与空气浓缩。 </li><li>添加200μL丙酮和蒸发（共两次重复删除多余的水分）。 </li><li>对于TMS衍生添加醋酸乙酯500μL，20μL吡啶，N，O -双（三甲基硅基）乙酰胺，以每管100μL。 </li><li>孵育2小时25 ° C。 </li><li>转移100μL成小瓶的GC / MS的反应，并添加100μL丙酮。 </li><li>配备一个四极质谱仪或火焰离子化检测器的气相色谱法分析样品。安捷伦HP - 5MS柱的安装（X 0.25毫米X 0.25微米薄膜厚度30毫米）。以下的温度梯度是用溶剂延迟了30分钟和一个1.1毫升/分钟流速：初始保持在130 ° C，3分钟3℃/ min的坡道至250℃，保持1分钟;平衡到初始温度130 ° C </li><li>米/ 269米/ Z，Z，和239米/ Z，S，G和H单体，分别为（见图相对保留使用四烷内标（可选）倍或299频谱特征质量离子峰确定。 2）。木质素组件的组成是通过设置总峰面积的100％的量化</li></ol> 4。代表性的成果墙上分析的一个例子是在图2。在这种情况下，杨树干（木）协议部分所述的各种程序进行了分析。木质素元件分离后thioacidolysis和TMS衍生的一个例子色谱图。显然，紫丁香的相对丰度（S），愈创木，（G）和P - hydroxyphenol - （H）的单位可确定。乙酰溴溶于木质素的内容是不言自明，可以壁干重的20-50％之间的预期值。应该注意的乙酰溴不溶解在墙上的木质素目前，增溶程度可以有所不同，视物。然而，这种方法是比较容易开展和快速的，并给出了在一个木质纤维材料的木质素含量的优秀近似值。 <img src="/files/ftp_upload/1745/1745fig1.jpg" alt="图1" /> 图1：木质纤维分析概述。细胞壁（lignocellulosics）原油干植物材料隔离。墙体材料，然后加权成等份，并细分为各种检测。墙体材料是乙酰溴和溶解的木质素由紫外线光谱量化处理。木质素成分的测定，墙体材料是受到thioacidolysis。溶酚醛树脂进行TMS衍生化和GC - MS分析，然后可以分离和量化。矩阵多糖的组成和结晶纤维素含量协议是讨论在第二部分 2 。 <img src="/files/ftp_upload/1745/1745fig2.jpg" alt="图2" /> 图2：杨树（ 胡杨tremoloides） 杨树木材的木质纤维综合分析木片所描述的协议。 Ligin组成，H P -羟基苯基，G愈创木，S紫丁香单位。

<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>

描述的方法，使木质素含量和木质纤维植物的生物量组成的快速定量评估。使用iWall机器人约350个样本，每天可地面和免除。每人的各种分析方法的吞吐量不同。使用这里描述的协议，30个样本可以处理的木质素含量，并每天为木质素组成15。由于数据的最佳原料作物的定量性质，品种或基因型，可用于生产生物燃料的适用性评估。

<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

可再生，碳中性的，和可持续发展的原材料行业和社会的需要，已成为面向21世纪的最紧迫的问题之一。这再度引发了在植物产品作为工业原料使用液体燃料的运输生产的兴趣<sup&gt; 1</sup&gt;和其他产品，如生物复合材料<sup&gt; 7</sup&gt;。植物生物量的仍然是这个星球上最大的未开发储量<sup&gt; 4</sup&gt;。它主要是由细胞组成的墙壁，能源丰富的聚合物包括纤维素，半纤维素（矩阵多糖，各种多酚木质素<sup&gt; 6</sup&gt;，因此有时被称为lignocellulosics。然而，植物细胞壁的演变顽抗退化如墙壁提供抗拉强度细胞和整个植物，抵御病原体，并让水在整个工厂运输，在树木的情况下，超过100米以上地面水平。由于墙的各种功能，内有不同的植物种类和细胞类型的墙壁上一个巨大的结构在一个单一的植物多样性<sup&gt; 4</sup&gt;。因此，根据什么样的作物品种，作物品种，或植物组织是一个生物炼制，加工为解聚/酶的化学过程和随后的各种糖发酵的液体生物燃料的步骤需要进行调整和优化。这一事实的基础需要一个彻底的植物生物质原料分析。在这里，我们描述了一个全面的分析方法，使lignocellulosics组成的决心，是适合中期，以高通量分析。在第一部分中，我们专注于多酚木质素的分析（图1）。方法开始准备destarched细胞壁物质。造成lignocellulosics分裂acetylbromide增溶，以确定其木质素含量<sup&gt; 3</sup&gt;，其在紫丁香，愈创木和P -羟基苯单位的木质素组成<sup&gt; 5</sup&gt;。在木质纤维素生物质的碳水化合物，包括纤维素含量和基质多糖成分分析的协议是在第二部分讨论<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

植物生物量是一个主要的碳中性的可再生资源，可用于生产生物燃料的使用。植物生物量主要由细胞壁，一个结构复杂的复合材料，被称为lignocellulosics。在这里，我们描述为一个全面的内容和组成的多酚类木质素分析协议。

综合植物细胞壁成分分析（木质纤维素生物质）第一部分：木质素

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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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 次观看.  Michigan State University (MSU). 植物生物量是一个主要的碳中性的可再生资源，可用于生产生物燃料的使用。植物生物量主要由细胞壁，一个结构复杂的复合材料，被称为lignocellulosics。在这里，我们描述为一个全面的内容和组成的多酚类木质素分析协议。

视频: 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.

结合拉曼成像和多变量分析可视化植物细胞壁中的木质素，纤维素和半纤维素

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

科研

生物化学

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.

木质素下调<em&gt;玉米</em&gt;通过dsRNAi和Klason木质素分析

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

生物工程

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.

点击化学可视化植物木动力学: 双标记是幸福!

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

化学

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.

使用硫二甘酸（TGA）估计植物生物质木质素含量

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.

综合植物细胞壁成分分析（木质纤维素生物质）第二部分：碳水化合物

生物学

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.

利格宁和单宁的定量 31P NMR 分析

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.

无标签原位成像在植物细胞壁的木质化

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.

糖基谱研究植物细胞壁聚合物，用微阵列

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.

组织化学染色<em&gt;拟南芥</em&gt;次生壁元素

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.

植物表皮脂质聚酯单体的分离及成分分析

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

综合植物细胞壁成分分析（木质纤维素生物质）第一部分：木质素

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