Name: sequencing of plant wall heteroxylans using enzymic, chemical (methylation) and physical (mass spectrometry, nuclear magnetic resonance) techniques
Uploaded: 2016-03-24T14:00:00.000+00:00
Duration: 11 min 49 s
Description: Watch this Scientific Journal Video about Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques at JoVE.com

This project was supported by funds from Commonwealth Scientific and Research Organisation Flagship Collaborative Research Program, provided to the High Fibre Grains Cluster via the Food Futures Flagship. AB also acknowledges the support of an Australia Research Council (ARC) grant to the ARC Centre of Excellence in Plant Cell Walls (CE110001007).

W-溶胶AXS 1.还原性末端的标签（RE）糖残基与2-氨基苯甲酰胺（2AB） <ol><li>在65℃孵育的W-溶胶AXS用在1M的NaBH 3 CN（氰基硼氢化钠）（pH5.5）中进行2小时的存在2AB（0.2M）到多糖主链链的还原末端转化成它们的荧光衍生物。 注意:以下步骤应在通风橱作为加入NaBH 3 CN释放有毒的氰化物气体，当它是在与水接触来进行。 <ol><li>称出的NaBH 3 CN（62.8毫克）和溶解于水（1毫升）的微量离心管（1.5ml）中以制备1M的加入NaBH 3 CN溶液。在1M的NaBH 3 CN溶液（1ml）中加热在65溶解2AB试剂（27.2毫克） &#160; ℃，调节反应混合物（0.2M 2AB，1M的NaBH 3 CN）与10％乙酸的pH 5.5的pH值。 </li><li>添加反应混合物中加入200μl（0.2M 2AB，1M的NaBH 3 CN）至W-所以升AXS（1毫克）在有盖的玻璃管，混合使用涡旋混合器。孵育在65 2小时&#160;在通风橱°以下。酷悬浮RT，并添加4卷。无水乙醇。 </li><li>放置悬浮于冷藏（4℃）O / N，以沉淀多糖。 </li><li>离心机（1500 XG，10分钟，RT），除去上清液。用无水乙醇（4次），丙酮（1x）和甲醇（1×）广泛洗涤沉淀，每次洗涤之间离心。在40°CO / N真空干燥。 注意:彻底清洗也消除残留2AB。 </li></ol></li></ol> 2.来自2 AB标记的代低聚木糖的W-溶胶AXS <ol><li>溶解2AB在微量离心管的乙酸钠缓冲液500微升（100毫米，pH值5）（1.5毫升）标记的W-溶胶AXS（1毫克）。加入4单位木聚糖内切的（GH 11，[M1]），在37孵育&#160; °下16小时。 </li><li>通过加热反应mixt破坏酶的活性URE在沸水浴10分钟。冷却该悬浮液至室温并转移到玻璃管用帽。添加4卷。无水乙醇，并将该悬浮液在冷藏（4℃）O / N，以沉淀任何未消化的多糖。 </li><li>离心（1500 xg离心10分钟，室温）以分离未消化多糖（片）和木聚糖内切产生低聚木糖（上清液）。倒出上清到一个干净的玻璃管，并把它变成一个温水浴（40℃）。 </li><li>蒸发在氮气流下乙醇到终点体积（〜500微升）。冻结在-80℃的上清液4小时，并在一个冷冻干燥器回收低聚木糖干燥冷冻上清液。 </li></ol> 3.从KOH-溶胶AXS和2AB标签低聚木糖的生成<ol><li>对待切木聚糖酶KOH-溶胶AXS（GH 11，[M1]）以上（部分2.1-2.4）所描述的生成低聚木糖。 </li><li>治疗endoxylanase产生低聚木糖从KOH溶胶AXS以上（部分1.1.1-1.1.2）描述2AB反应混合物（0.2M 2AB，1M硼氢化钠3 CN）。 </li><li>倒出上清到一个干净的玻璃管，并把它变成一个温水浴（40℃）。蒸发在氮气流下乙醇到终点体积（〜500微升）。冻结在-80℃的上清液4小时，并在一个冷冻干燥器回收低聚木糖干燥冷冻上清液。 </li></ol> 4. MALDI-TOF-MS <ol><li> MALDI基质溶液的制备<ol><li>加的2，5- dihydroxbenzoic苯甲酸（DHB）的小舀到含有0.1％甲酸的一管（MALDI-基质溶液）50％乙腈（500毫升）中。混合使用一个旋涡，如果迅速溶解，加DHB的另一个小瓢。注:MALDI-矩阵的理想解决方案浓度为10微克/微升-1。 </li></ol></li><li> MALDI-目标板的制作<ol><li>存款aqueo我们寡糖（本机）的样品（5-10微克）（W-溶胶和/或KOH-SOL）到MALDI-目标板。使用单独的尖端添加0.3微升的MALDI基质溶液和通过上下抽吸混合。使混合物在RT干燥。 注意:正确干燥的样品应当包括向着光点的中心指向长针形晶体。如果存款是粘性和/或油污样品既可以过于集中或由盐，这些沉积物是不可能产生良好的光谱和样本应进一步纯化。 </li><li>引入目标板进入MS源和正（+ IVE）离子模式。调整加速器电压到离子源1 19.0千伏和离子源2 16.3千伏调整激光功率，以大于70％。选择在MALDI-靶板样品当场单击开始，开始激光发射。 注意:目标的所有区域不会产生信号。特定的点只会给一个信号，这或者是由于一些激光发射采样/基质混合物或晶体的特性的枯竭。 <ol><li>移动激光到目标的不同区域采集和平均在大约200随机光谱以获得满意的信号 - 噪声。 </li></ol></li></ol></li></ol> 5. ESI-QTOF-MS <ol><li>采用纳米HPLC加上使用RP电喷雾电离（ESI）飞行（QTOF）MS仪器在线色谱分离的四极时分析切木聚糖酶生成低聚糖（本机）C-18柱（75微米×150毫米; 3.5微米珠尺寸）。 <ol><li>所述木聚糖内切产生的含水寡糖混合物转移到小瓶中，并放入HPLC自动采样器。编程的5-80％在乙腈中，分别在流动相的0.1％（体积/体积）的水甲酸和0.1％（V / V）甲酸的梯度洗脱，经60分钟。 </li><li>调节流速，以0.2微升/分钟。调整正离子模式中的扫描范围帘气体10，GS1 4，源温度100℃，离子喷雾电压2300伏，和去聚类电位50:300-1,600 米/ z和的0.5次/秒使用ESI源条件如下一扫描速率的五，运行LC色谱程序和洗脱低聚糖。所得到的总离子色谱（TIC）是由软件自动保存。 </li><li>打开保存的TIC，并选择提取色谱图。在命令行中键入预期的群众（如 271，403，535，667，799 931 M / Z）。点击进入扫描色谱图。处理，根据制造商的说明14使用软件:ESI-QTOF MS色谱数据的选择离子扫描。 </li></ol></li></ol> 6. ESI- 质谱 <ol><li>插入1〜2微升每-O-甲基化的寡糖在50％乙腈样品放入使用注射器一纳米喷雾尖端（如由Pettolino 等人 1所记载的甲基化）。修剪使用玻璃切割器的纳米喷嘴以适合附连到MS的离散纳米喷雾支架。 </li><li>根据预期的质量范围（200-1,500 米/ z）和帘气体至10，离子喷雾电压1,900 V和极性到正集的质量。 </li><li>按下获取按钮以打开相关的窗口中，输入数据的文件名 ​​，以获得总离子扫描（ESI-MS 1）。然后按STOP按钮。 </li><li>从产品的离子更改扫描类型片段的关注峰。输入感兴趣的质量（ 如885 M / Z）产生碎片，并调整质量范围（200-900 M / Z）。按下获取按钮和调整碰撞能量（在化合物标签向上和/或向下），以实现母离子的整个碎片（885米/ z）和获得的片段离子扫描（ESI-MS 2）。 </li><li>输入感兴趣（ 如711 M / Z）片段离子的质量，并调整质量范围（200-720 米/ z）表示 。按采样按键的d调整碰撞能量，以达到前体离子的整个碎片（710 米/ z）和获得的片段离子扫描（ESI-MS 3）。 </li></ol> 7. H 1 NMR光谱<ol><li>溶解寡糖木聚糖内切生成混合物（KOH-溶胶，天然形式）（〜500微克）在D 2 O中（1.0毫升，99.9％）在一个塑料试管（15毫升）中。冻结在-80℃的悬浮液4小时，在冷冻干燥机以回收低聚木糖干燥冰冻的悬浮液中。 </li><li>为了与D 2 O.充分交流的H 2 o重复两次7.1 </li><li>溶解干燥的寡糖在D 2 O中（0.6ml毫升，99.9％），并添加丙酮的0.5微升（在D 2 O中的5％）作为内标。转移氘化寡糖进入NMR样品管中。 </li><li>持由顶部核磁共振含有管样品，并在塑料离心器插入样品管。放置在采样深度尺微调。脓H或拉该样品管以调节样品的深度，以保证样品的顶部和底部深度计的中心线是相等的。 </li><li>除去深度规和插入所述试样附连到配备有冷冻探针的600MHz的NMR波谱仪自动采样器。 </li><li>登录和开放式光谱仪控制软件。输入样品文件名。打开现有的数据集，然后使用"EDC"命令将其保存到一个新的名称。键入样品的位置一批在自动进样器，然后按"ENTER"。 注:按"ENTER"键会轻轻滴样品管的磁铁孔中，其中，将被定位在探针的顶部。 </li><li>通过在命令行中输入"edte"设置所需的样品温度。等待样品温度继续下一步骤之前达到所需的值。进入"锁定"在命令行，并选择适当的溶剂（D 2 O）。等待＆＃34;锁定完成"的消息显示在窗口的底部。 </li><li>键入"ATMM"在命令行，然后单击"优化"的ATMM菜单栏的顶部。选择启动调谐和探针匹配所选通道（在此情况下1H）。 </li><li>在为垫补过程中，轻微的调整对磁场做出直到均匀磁场通过在命令行中输入"ZG"，进入周围的sample.Acquire实现了信号命令行中键入"topshim"。 </li><li>分析根据制造商的使用在2.225 ppm的参考丙酮的内标1小时的化学位移说明使用软件15光谱。 </li></ol>

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大多数基质相细胞壁多糖似乎已经无规取代的主链（有两个糖基和非糖基残基），根据植物物种，发育阶段和组织类型3是高度可变的。由于多糖是次级基因产物的序列没有模板产生的，因此没有单一的分析方法，例如存在对于核酸和蛋白质，它们的测序。纯化的特定键的水解酶的可用性提供了一个有力的工具来降解多糖寡糖然后，可以是色谱分馏，和结合的化学和物理技术使用时完全测?...

Heteroxylans是属于禾本科的主壁和所有被子植物3-6的次生壁的主要非纤维素多糖多糖家族。木聚糖主链中它们的类型和与糖基（葡糖醛酸（中，GlcA），阿拉伯糖（阿拉伯糖））和非糖基（O-乙酰基，阿魏酸），这取决于组织类型，发育阶段和物种7个残基的取代的图案不同。 从小麦壁（ 普通小麦 ）胚乳组成主要由阿拉伯木聚糖（AXS）（70％）和（1→3）（1→4）-β-D-葡聚糖（20％）与少量的纤维素和heteromannans的（2％各）8。木聚糖主链可以是各种未取代的和主要的单取代的（主要是O-2位和在较小程度上O-3的位置）和二 - 取代的（O-2和O-3位）用α-L-阿拉˚F残留9。还原性末端（RE）异质从双子叶植物的木聚糖（例如， 拟南芥 ）10和裸子植物（例如，云杉（ 云杉 ））11包含一个特性四糖的糖基序列; -β-D-木糖对 - （1→3）-α-L-鼠李糖对 - （1→2）-α-D-半乳糖p A-（1→4）-D-的Xyl 页 。理解heteroxylan生物合成和功能（生物和工业），重要的是要充分序列木聚糖主链理解的种类和取代的模式以及还原端（RE）的序列。 用于还原端（RE）和heteroxylans的内部区域的糖基序列（多个）的结构特征的特定的技术在本手稿中描述。该技术依赖于荧光团标记（用2氨基苯甲酰胺（2AB））之前，酶（木聚糖内切）水解heteroxylan链的还原端（RE）。这种方法，特别是用于对RE测序，是首先由纽约实验室10,12-13报道，但现在扩展为包括内部区域测序，并且是已建立的技术的组合是同样适用于独立的隔离的源的所有heteroxylans。这种方法也可以使用（如果有的话），相应的内切水解酶应用于其他类多糖。 在本研究中，如所述脱浆小麦胚乳细胞壁中分离作为不溶醇残基（空气）中，依次用水（W-溶胶FR）萃取，含有1％的NaBH 4（KOH-溶胶FR）1M KOH在Ratnayake 等人。（2014）2。从两个W-和KOH溶胶FRS释放寡糖然后甲基化并且两个天然和甲基化寡糖的详细的结构分析使用MALDI-TOF-MS，ESI-QTOF-MS-加上HPLC用的组合进行使用RP C-18柱在线色谱分离和ESI-MSñ。木聚糖内切消化的KOH溶胶AXS还特征在于核磁共振（NMR）。

<table><tbody><tr><td>2 aminobenzamide (2AB)</td><td>Sigma-Aldrich (www.sigmaaldrich.com)</td><td>A89804</td><td></td></tr><tr><td>sodium borohydride (NaBH&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich (www.sigmaaldrich.com)</td><td>247677</td><td>Hazardous, handle with care</td></tr><tr><td>sodium cyanoborohydride (NaBH&lt;sub&gt;3&lt;/sub&gt;CN)</td><td>Sigma-Aldrich (www.sigmaaldrich.com)</td><td>156159</td><td>Hazardous, handle with care</td></tr><tr><td>endo-1,4-&amp;beta;-Xylanase M1 (from Trichoderma viride) (120101a)</td><td>Megazyme (www.megazyme.com)</td><td>E-XYTR1</td><td></td></tr><tr><td>Deuterium Oxide (D&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich (www.sigmaaldrich.com)</td><td>151882</td><td></td></tr><tr><td>Freeze dryer (CHRIST-ALPHA 1-4 LD plus)</td><td></td><td></td><td></td></tr><tr><td>RP C18 Zorbax eclipse plus column&amp;nbsp;</td><td>Agilent&amp;nbsp;</td><td></td><td>(2.1&amp;times;100 mm; 1.8 &amp;micro;m bead size)&amp;nbsp;</td></tr><tr><td>MicroFlex MALDI-TOF MS&amp;nbsp;&amp;nbsp; (Model - MicroFlex LR)</td><td>(Bruker Daltonics, Germany)</td><td></td><td></td></tr><tr><td>(ESI) -(QTOF) MS&amp;nbsp;&amp;nbsp; (Model # 6520)</td><td>(Agilent, Palo Alto, CA )</td><td></td><td></td></tr><tr><td>ESI-MS&lt;sup&gt;n &lt;/sup&gt;&amp;nbsp;- ion-trap&amp;nbsp; (Model # 1100 HCT)</td><td>(Agilent, Palo Alto, CA).</td><td></td><td></td></tr><tr><td>Bruker Avance III 600 MHz -NMR</td><td>Bruker Daltonics, Germany</td><td></td><td></td></tr><tr><td>Topspin (version 3.0)-Biospin- software&amp;nbsp;</td><td>Bruker&amp;nbsp;</td><td></td><td></td></tr><tr><td>GC-MS (Model # 7890B)</td><td>Agilent&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

sequencing of plant wall heteroxylans using enzymic, chemical (methylation) and physical (mass spectrometry, nuclear magnetic resonance) techniques

这个协议描述了用于还原末端（RE）和heteroxylans的内部区域的糖基序列（多个）的表征的具体的技术。脱浆小麦胚乳细胞壁中分离作为不溶醇残基（AIR）1，并依次用水（W-溶胶星期五）萃取和含有1M的KOH 1％的NaBH 4（KOH溶胶星期五），为通过Ratnayake 等描述人 （2014）2。两种不同的方法（ 见图1中的摘要）被采用。在第一，完整的W-溶胶AXS用2AB处理来标记原始RE主链糖残基，然后用木聚糖内切处理，以生成2AB标记的RE和内部区域分别还原的低聚糖，的混合物。在第二种方法中，所述KOH溶胶星期五与内木聚糖酶水解成第一生成其随后用2AB标记的寡糖的混合物。该酶解释放（（UN）的标签）从两个低聚糖W-和KOH溶胶FRS然后甲基化并且两个天然和甲基化寡糖的详细的结构分析使用MALDI-TOF-MS，RP-HPLC-ESI-QTOF-MS和ESI-MS n的组合被执行。木聚糖内切消化的KOH溶胶AXS的特征还在于用核磁共振（NMR）也提供了关于异头配置信息。这些技术可以应用于其他类使用适当的内切水解酶多糖。

的木聚糖内切消化2AB标记的W-溶胶AXS生成2AB标记的RE寡糖的混合物和一系列从木聚糖链（ 图1中的内部区域衍生的未标记的（无2AB标签）寡糖;从Ratnayake 等人 。 2）。然后将一系列的色谱方法被用来分馏异构体的复杂混合物。最后，MS技术被用于识别异构结构，然后由质谱技术测序。这里，我们提出的代表，而不是该方法的全面的，例子。...

Watch this Scientific Journal Video about Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques at JoVE.com

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

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Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

Principles of Nuclear Magnetic Resonance

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7.6K 次观看.  University of Melbourne. This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR). 

视频: Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

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

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharide fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.

A protocol for the structural analysis of polysaccharides by gel electrophoresis (PACE), using mannan as an example, is described.

植物甘露聚糖细胞壁多糖的结构表征

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

Preparation of Fungal and Plant Materials for Structural Elucidation Using Dynamic Nuclear Polarization Solid-State NMR

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR and sensitivity-enhanced DNP experiments. The sample processing procedure of plant biomass is also detailed. This method allows the measurement of a series of 1D and 2D 13C-13C/15N correlations spectra, which enables high-resolution structural elucidation of complex biomaterials in their native state, with minimal perturbation. The isotope-labeling can be examined by quantifying the intensity in 1D spectra and the polarization transfer efficiency in 2D correlation spectra. The success of dynamic nuclear polarization (DNP) sample preparation can be evaluated by the sensitivity enhancement factor. Further experiments examining the structural aspects of the polysaccharides and proteins will lead to a model of the three-dimensional architecture. These methods can be modified and adapted to investigate a wide range of carbohydrate-rich materials, including the natural cell walls of plants, fungi, algae and bacteria, as well as synthesized or designed carbohydrate polymers and their complex with other molecules.

A protocol for preparing 13C,15N-labeled fungal and plant samples for multidimensional solid-state NMR spectroscopy and dynamic nuclear polarization (DNP) investigations is presented.

动态核极化固态核磁共振法制备结构澄清用真菌和植物材料

This protocol shows how uniformly 13C, 15N-labeled fungal materials can be produced and how these soft materials should be proceeded for solid-state NMR ...

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

Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part 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 century. This has rekindled interest in the use of plant products as industrial raw materials for the production of liquid fuels for transportation1 and other products such as biocomposite materials7. 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 (matrix polysaccharides, and the polyphenol lignin6 and thus sometimes termed lignocellulosics. However, plant cell walls have evolved to be recalcitrant to degradation as walls provide tensile strength to cells and the entire plants, ward off pathogens, and allow water to be transported throughout the plant; in the case of trees up to more the 100 m above ground level. 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 depolymerization 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. In this first part we focus on the analysis of the polyphenol lignin (Figure 1). The method starts of with preparing destarched cell wall material. The resulting lignocellulosics are then split up to determine its lignin content by acetylbromide solubilization3, and its lignin composition in terms of its syringyl, guaiacyl- and p-hydroxyphenyl units5. The protocol for analyzing the carbohydrates in lignocellulosic biomass including cellulose content and matrix polysaccharide composition is discussed in Part II2.

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

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 the polyphenolic lignin.

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 ...

生物学

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

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

胞外多糖的OLIGO质谱分析（OLIMP）

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

RWA 侵染小麦品种中细胞壁相关酶的表征

Elucidating &#946;-1,3-Glucanase and Peroxidase Physicochemical Properties of Wheat Cell Wall Defense Mechanism Against Diuraphis noxia Infestation

Wheat plants infested by Russian wheat aphids (RWA) induce a cascade of defense responses, including the hypersensitive responses (HR) and induction of pathogenesis-related (PR) proteins, such as &#946;-1,3-glucanase and peroxidase (POD). This study aims to characterize the physicochemical properties of cell wall-associated POD and &#946;-1,3-glucanase and determine their synergism on the cell wall modification during RWASA2-wheat interaction. The susceptible Tugela, moderately resistant Tugela-Dn1, and resistant Tugela-Dn5 cultivars were pregerminated and planted under greenhouse conditions, fertilized 14 days after planting, and irrigated every 3 days. The plants were infested with 20 parthenogenetic individuals of the same RWASA2 clone at the 3-leaf stage, and leaves were harvested at 1 to 14 days post-infestation. The Intercellular wash fluid (IWF) was extracted using vacuum filtration and stored at -20 &#176;C. Leaf residues were crushed into powder and used for cell wall components. POD activity and characterization were determined using 5 mM guaiacol substrate and H2O2, monitoring change in absorbance at 470 nm. &#946;-1,3-glucanase activity, pH, and temperature optimum conditions were demonstrated by measuring the total reducing sugars in the hydrolysate with DNS reagent using &#946;-1,3-glucan and &#946;-1,3-1,4-glucan substrates, measuring the absorbance at 540 nm, and using glucose standard curve. The pH optimum was determined between pH 4 to 9, temperature optimum between 25 and 50 &#176;C, and thermal stability between 30 &#176;C and 70 &#176;C. &#946;-1,3-glucanase substrate specificity was determined at 25 &#176;C and 40 &#176;C using curdlan and barley &#946;-1,3-1,4-glucan substrates. Additionally, the &#946;-1,3-glucanase mode of action was determined using laminaribiose to laminaripentaose. The oligosaccharide hydrolysis product patterns were qualitatively analyzed with thin-layer chromatography (TLC) and quantitatively analyzed with HPLC. The method presented in this study demonstrates a robust approach for infesting wheat with RWA, extracting peroxidase and &#946;-1,3-glucanase from the cell wall region and their comprehensive biochemical characterization.

作者聚焦：在小麦-RWA 相互作用中整合 #946;-葡聚糖酶和过氧化物酶的生化功能

The present protocol describes procedures used to study and characterize cell wall-related enzymes, mainly &#946;-1,3-glucanase and peroxidase, in wheat plants. Their activity levels increase during wheat-RWA interaction and are involved in the plant defense response through cell wall reinforcement, which deters aphid feeding.

阐明小麦细胞 壁防御机制 的 #946;-1,3-葡聚糖酶和过氧化物酶的物理化学特性

Wheat plants infested by Russian wheat aphids (RWA) induce a cascade of defense responses, including the hypersensitive responses (HR) and induction of ...

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

摘要

探索更多视频

此视频中的章节

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

植物甘露聚糖细胞壁多糖的结构表征

动态核极化固态核磁共振法制备结构澄清用真菌和植物材料

点击化学可视化植物木动力学: 双标记是幸福!

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

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

胞外多糖的OLIGO质谱分析（OLIMP）

阐明小麦细胞壁防御机制的 #946;-1,3-葡聚糖酶和过氧化物酶的物理化学特性

阐明小麦细胞壁防御机制的 #946;-1,3-葡聚糖酶和过氧化物酶的物理化学特性

阐明小麦细胞壁防御机制的 #946;-1,3-葡聚糖酶和过氧化物酶的物理化学特性

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

摘要

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此视频中的章节

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