Name: combining raman imaging and multivariate analysis to visualize lignin, cellulose, and hemicellulose in the plant cell wall
Uploaded: 2017-06-10T14:00:00.000+00:00
Duration: 7 min 51 s
Description: Watch this Scientific Journal Video about Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall at JoVE.com

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样品制备<ol><li>从植物样品（ 例如，杨树茎）切下一个小的组织块（约3 mm x 3 mm x 5 mm）。 </li><li>将组织浸入沸腾的去离子水中30分钟。立即将其在室温（RT）下转移到去离子水中30分钟。重复此步骤，直到组织沉入容器的底部，表明组织中的空气已经被去除并且组织已经软化。 注意:对于在此步骤之前沉入底部的样品，通常重复此循环3-5次。 </li><li>在去离子水中制备20，50，70，90％（v / v）等分的聚乙二醇（PEG），以及纯PEG，并将溶液保持在65℃。 </li><li> 在一系列分级的PEG浴中孵育组织以置换水并允许PEG渗透。 注意:通常，分子量为2,000（PEG2000）的PEG用于嵌入。 <ol><li>用g处理组织如下:（a）20％PEG 1小时，（b）50％PEG 1.5小时，（c）70％PEG 2小时，（d）90％PEG 2小时，和（e）100％PEG 10小时。 </li></ol></li><li>在烤箱中预热65℃的盒子。将含有块的PEG倒入盒中，然后使用预热的镊子或针将组织块放置在所需位置。 </li><li>慢慢冷却盒子并将组织储存在室温下直至使用。 </li><li>使用锋利的剃须刀将含有目标组织的PEG块解剖成小块（约1cm×1cm×2cm），并将其安装在切片机上。 </li><li>从PEG块切割薄片（通常为3-10μm）。 </li><li>用手表玻璃中的去离子水冲洗该部分10次以从组织中去除PEG。 </li><li>将各部分用甲苯/乙醇（2:1，v / v）浸泡6小时以除去提取物。通过混合65mL去离子水，0.5mL乙酸和0.6g氯化钠来制备反应液在烧杯中举行仪式将一个部分和3 mL反应液加入到5 mL小瓶中。拧上小瓶的顶部。 </li><li>将小瓶在75℃的水浴中加热2小时以除去组织的木质素。用去离子水冲洗表面10次。 </li><li>将未处理/脱木质部分转移到玻璃显微镜载玻片上。用刷子或针头小心地打开部分。用薄纸去除额外的去离子水。 </li><li>将部分浸入D 2 O.用玻璃盖板盖住样品。用指甲油密封盖子，防止D 2 O蒸发。 </li></ol>光谱采集<ol><li>打开共焦拉曼显微镜的仪器操作软件。打开激光（波长= 532 nm），用100X显微镜物镜聚焦在晶体硅的表面，然后单击校准按钮校准仪器。 </li><li>切换仪器到光学显微镜模式并打开显微镜灯。将显微镜载玻片放在舞台上，盖子面对物镜。 </li><li>使用20X显微镜物镜查看样品，并找到感兴趣的区域。将浸油涂抹于盖玻片上并切换到浸没式显微镜物镜（60X，数值孔径NA = 1.35）。专注于样品表面。 </li><li>将仪器切换到拉曼测试模式，然后关闭显微镜灯。 </li><li>使用矩形工具选择一个映射区域。改变步长以确定获得的光谱数。 注意:注意步长（通常大于由物镜的数值孔径计算的光点直径;理论上大于1.22λ/ NA）。以下尺寸将导致过采样。 </li><li>设置最佳光谱参数，以在适当的采集时间（通常&lt;8 h）获得最佳信噪比（SNR）和光谱质量，了解样品适用性。通常，在仪器软件中输入激光（532 nm），滤光片（100％），孔（300），狭缝（100），光谱仪（1,840 cm -1 ），凹槽（1200t），物镜60X油），采集时间（2 s）。 </li><li>在数据处理之前保存光谱数据，并将其转换为通用格式（ 例如， TXT文件）。 </li></ol> 3.数据分析<ol><li>将光谱数据（TXT文件）加载到数据分析软件（ 例如 Matlab）中。在数据集上应用降噪技术来提高SNR（ 例如， Savitzsky-Golay算法或小波算法） </li><li>使用未处理的样品产生木质素和多糖的图像。对于木质素成像，由于芳环对称伸缩振动，考虑1,600cm -1附近的光谱峰。对于多糖成像（包括纤维素和半纤维素），使用光谱峰a圆2,889厘米-1 ，因为CH和CH 2伸展。 </li><li>使用脱木素样品产生纤维素和半纤维素的图像。对获取的光谱进行自我建模曲线分辨率（SMCR），以区分纤维素和半纤维素的光谱并对其分布进行成像。 </li><li>对获取的数据进行主成分分析（PCA）和聚类分析，以区分不同细胞壁层的拉曼光谱。 </li></ol>

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作者没有什么可以披露的。

植物细胞壁是组织成几层的复合材料，包括细胞角（CC），次生壁（SW，具有S1，S2和S3层）和复合中间片（CML，中间片加上相邻的原始细胞壁），这使得在样品制备期间难以获得平坦的表面。因此，具有比木材更复杂结构的植物样品，特别是草，通常需要被固化以允许精细切片。 PEG是一种用于切割和拉曼研究的理想硬质基质，因为它可溶于水。用去离子水漂洗可以很容易地去除。用于嵌入的PEG具有不同的分子量，从1,000-20,000变化。 PEG的分子量越高，穿透能力越低10 。 D 2 O有助于降低木质素的荧光，在2,490 cm -1处有明显的峰值，但不会消除荧光干扰。二主要噪声信号与实际信号一起溢出:1）样品和背景荧光以及CCD的热波动可能导致基线漂移，2）宇宙射线可以显着影响敏感探测器，这表现在光谱作为窄带宽尖峰。 APRI方法是为了解决这些问题而开发的。 APRI包括自适应迭代重新加权的惩罚最小二乘法（airPLS）和主成分分析（PCA），以通过使用光谱特征本身来消除基线漂移和宇宙尖峰。 为了实现分布图像，应选择峰值强度/积分和全频谱线性拟合的多变量方法。前者适用于具有特定拉曼峰（ 例如木质素）的组分，而后者适用于具有强光谱重叠的组分（ 如纤维素和半纤维素）。分发通过分别在1,600cm -1和2,889cm -1附近积分特定峰，可获得木质素和多糖的对比图（图4）。然而，由于强光谱重叠，纤维素和半纤维素的图像难以通过峰积分直接产生。多元分析允许根据从有限数量的重要因素12重建原始数据的假设来对卷积信息内容进行排序。因此可以应用于区分它们的光谱并产生相应的拉曼图像。对于植物样品，提取物和木质素的存在干扰了重建纤维素和半纤维素光谱的能力。在成像数据的SMCR分析之前，必须去除这些干扰。 SMCR首先由Lawton和Sylvester引入，作为专门用于解决纯组分f的多变量技术一组光谱，没有任何追求光谱库，分析成像数据13 。光谱分类对于进一步了解植物细胞壁的结构和化学性质是重要的。这里，成像数据已经经过主成分分析（PCA）和聚类分析，以区分不同细胞壁层14的拉曼光谱。 然而，这种技术有两个限制。首先，拉曼效应较弱 - 典型的总拉曼散射截面为每分子约10 -29 cm 2 ，这使其易受强烈荧光的影响15 。改善缺陷的一种可能方法是通过尽可能薄地准备这些部分并应用基线校正算法，但荧光信号难以去除。其次，化学处理是实现细胞拉曼图像的重要手段左旋和半纤维素，并且可能增加改变原始化学成分的风险。因此，最好尽可能地除去提取物和木质素，而不会使细胞壁结构与未处理的细胞壁结构看起来太不相同。 总之，该方案适用于研究植物细胞壁内木质素，纤维素和半纤维素的分布。使用拉曼成像获得所需的信息高度依赖于操作人员在样品制备和数据分析方面的技能。良好的样品制备对于收集高质量的拉曼光谱至关重要。适当的数据分析提供了对大规模光谱的深入了解，并从图像中提取隐藏的信息。作为基本方法，该方案可用于在微观级别的化学，物理或生物处理过程中追踪主要成分的动态变化。

Plant biomass is the most abundant renewable resource on Earth; is mainly composed of lignin, cellulose, and hemicellulose; and is considered an attractive source of bioenergy and bio-based chemicals1. Unfortunately, it can resist degradation and confer hydrolytic stability or structural robustness to the plant cell wall. Such resistance is attributable to the accessible surface area, biomass particle size, degree of polymerization, cellulose crystallinity, and protective lignin2. A comprehensive understanding of the structural and chemical nature of the plant cell wall is thus significant from the viewpoint of plant biology and chemistry, as well as from that of commercial utilization. Commonly used wet chemistry analyses, such as chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy, only provide average compositional data of the measured sample. Furthermore, these methods are invasive and destroy the original structure of the plant tissue3. 
The Raman imaging technique is a powerful tool for the nondestructive visualization of spatially resolved chemical information4. It uses a laser light to cause inelastic scattering with a photon and relies on changes in polarizability arising from the molecular vibrations. In this case, water causes weak Raman scattering, which makes this approach suitable for in situ investigations of biological samples5. The application of the Raman imaging technique to the plant cell wall can elucidate the structure and composition of plant cell walls in their native state, with the resolution on the scale of the single cell and even of the cell wall layers6. A typical Raman imaging analysis of a plant cell wall generally consists of three steps: 1) sample preparation, 2) spectral acquisition, and 3) data processing. 
Although one of the major advantages of Raman imaging is the ability to achieve label-free and non-destructive spectra with minimal sample preparation, physical sample sectioning is still necessary to expose the surface of interest. This process should be performed carefully to obtain a flat surface, since the technique depends on maintaining optical focus7. Spectral acquisition requires a balance between image quality and extensive acquisition times8. Data processing aims to effectively extract the chemical information from the image data, especially for the components with similar chemical structures, such as cellulose and hemicellulose. Due to the strong spectral overlap, the exact spectra are difficult to discern. In this case, multivariate analysis is a straightforward approach to effectively uncover the hiding structural and chemical information9. This work presents a general protocol describing the use of Raman imaging to visualize the main components in plant cell walls, including lignin, cellulose, and hemicellulose.

<table><tbody><tr><td>Microtome</td><td>Thermo Scientific</td><td>Microm HM430</td><td></td></tr><tr><td>Confocal Raman microscope</td><td>Horiba Jobin Yvon</td><td>Xplora</td><td></td></tr><tr><td>Oven</td><td>Shanghai ZHICHENG</td><td>ZXFD-A5040</td><td></td></tr></tbody></table>

combining raman imaging and multivariate analysis to visualize lignin, cellulose, and hemicellulose in the plant cell wall

拉曼成像对植物生物量的应用正在增加，因为它可以提供水溶液的空间和组成信息。分析通常不需要大量样品制备;结构和化学信息可以在没有标记的情况下获得。然而，每个拉曼图像包含数千个光谱;这在提取隐藏信息时尤其是具有类似化学结构的组件提出了困难。这项工作引入了一个多变量分析来解决这个问题。该方案建立了可视化植物细胞壁内主要成分（包括木质素，纤维素和半纤维素）的一般方法。在该协议中，描述了样品制备，光谱采集和数据处理的过程。它在样品制备和数据分析方面高度依赖操作员的技能。通过使用这种方法，可以由非专业用户执行拉曼调查以获得high质量数据和植物细胞壁分析的有意义的结果。

图1给出了用于植物细胞壁的拉曼成像的典型的微拉曼系统的概述。例如，杨树（ Populus nigra L.）的原始拉曼光谱具有显着的基线漂移和峰值（ 图2a ）。在执行拉曼成像数据集（APRI）的自动预处理方法之后，这两个光谱污染物被成功地去除（ 图2b ）。 图3中显示了杨树的典型拉曼光谱，其波段分配列于表1 。木质素的拉曼图像通过归结于芳环结构的1,550-1,650cm -1的光谱区域的积分而产生（ 图4a ）。 图4d显示了多糖的拉曼图像，通过在2,889cm -1处整合峰来实现。为了成像纤维素和半纤维素，对脱木素样品的拉曼成像数据进行SMCR。相应的光谱和图像如图5所示 。 图6显示了PCA和聚类分析实现的结果。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55910/55910fig1.jpg" /> 图1:植物细胞壁拉曼成像示意图。横截面样品（杨树为例）用微拉曼系统测量，该微拉曼系统将光学显微镜与电荷耦合器件（CCD）检测器耦合到拉曼高分辨率光谱仪。在明场图像中，植物细胞壁被组织成细胞角（CC），复合中层（CML）和次生壁（SW）。通常，单峰产生拉曼图像整合或强度。在原始数据中找到两个光谱污染物（ 即基线漂移和宇宙峰值）。 <a href="http://ecsource.jove.com/files/ftp_upload/55910/55910fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55910/55910fig2.jpg" /> 图2: APRI预处理之前（ a ）和之后（ b ）的拉曼光谱。两个光谱污染物被成功地去除。 <a href="http://ecsource.jove.com/files/ftp_upload/55910/55910fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55910/55910fig3.jpg" /> 图3:杨树细胞壁的典型拉曼光谱。 </str典型的拉曼光谱来自次级壁。频带分配如表1所示。 <a href="http://ecsource.jove.com/files/ftp_upload/55910/55910fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55910/55910fig4.jpg" /> 图4:杨树细胞壁内的木质素（ a ）和多糖（ b ）的拉曼图像。木质素图像通过将峰值整合在1,600cm -1附近而产生 。多糖图像通过将峰值约2,889cm -1积分而产生。 <a href="http://ecsource.jove.com/files/ftp_upload/55910/55910fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" class ="xfigimg"src ="/ files / ftp_upload / 55910 / 55910fig5.jpg"/&gt; 图5:杨树细胞壁中纤维素（ a ）和半纤维素（ b ）的拉曼图像。对脱木素样品的拉曼成像数据进行SMCR。脱木质素有助于暴露纤维素和半纤维素的光谱特征。纤维素主要集中在SW中，而半纤维素的分布在整个白杨细胞壁中几乎是均匀的。 <a href="http://ecsource.jove.com/files/ftp_upload/55910/55910fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" class="xfigimg" src="/files/ftp_upload/55910/55910fig6.jpg" /> 图6:用于自动识别杨树细胞壁中不同细胞壁层的PCA和聚类分析结果。通过使用PCA和聚类分析，将光谱分为对应于细胞腔，CC，CML和SW的四个部分。这些层的平均光谱如下。结果表明，木质素沿着CML和CC浓缩，而多糖主要集中在SW中。 <a href="http://ecsource.jove.com/files/ftp_upload/55910/55910fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>波数（cm -1 ） </td><td>组件</td><td>分配</td></tr><tr><td> 1095 </td><td>纤维素，半纤维素</td><td>重原子CC和CO伸缩振动</td></tr><tr><td> 1123 </td><td>纤维素，半纤维素</td><td>重原子CC和CO伸缩振动</td></tr><tr><td> 1163 </td><td>纤维素，半纤维素/ TD&gt; <td>重原子CC和CO伸缩振动加HCC和HCO弯曲振动</td></tr><tr><td> 1275 </td><td>木质素</td><td>芳基OH和芳基O-CH3的芳基-O;愈创木酚环C = O组</td></tr><tr><td> 1331 </td><td>木质素，纤维素，半纤维素</td><td> HCC和HCO弯曲振动</td></tr><tr><td> 1378 </td><td>纤维素，半纤维素</td><td> HCC，HCO和HOC弯曲振动</td></tr><tr><td> 1460 </td><td>木质素，纤维素，半纤维素</td><td> HCH和HOC弯曲振动</td></tr><tr><td> 1603 </td><td>木质素</td><td>芳环伸缩振动，对称振动</td></tr><tr><td> 1656 </td><td>木质素</td><td>环共轭C = C的松柏醇的伸缩振动; C = O甲醛的伸缩振动</td></tr><tr><td> 2889 </td><td> C，H </td><td> CH和CH 2伸缩振动</td></tr><tr><td&gt; 2940 </td><td> L，C，H </td><td> CH伸缩振动在OCH 3不对称振动</td></tr></tbody></table> 表1: 拉曼峰位置和带分配

Watch this Scientific Journal Video about Combining Raman Imaging and Multivariate Analysis to Visualize Lignin, Cellulose, and Hemicellulose in the Plant Cell Wall at JoVE.com

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

该方案旨在提出使用拉曼成像和多变量分析在植物细胞壁中可视化木质素，纤维素和半纤维素的一般方法。

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 ...

combining-raman-imaging-multivariate-analysis-to-visualize-lignin

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.8K 次观看.  Beijing Forestry University. 该方案旨在提出使用拉曼成像和多变量分析在植物细胞壁中可视化木质素，纤维素和半纤维素的一般方法。 

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

High-throughput Screening of Recalcitrance Variations in Lignocellulosic Biomass: Total Lignin, Lignin Monomers, and Enzymatic Sugar Release

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the use of non-renewable energy sources. In order to identify which plants, out of a diverse pool, have the desired chemical traits for downstream applications, attributes, such as cellulose and lignin content, or monomeric sugar release following an enzymatic saccharification, must be compared. The experimental and data analysis protocols of the standard methods of analysis can be time-consuming, thereby limiting the number of samples that can be measured. High-throughput (HTP) methods alleviate the shortcomings of the standard methods, and permit the rapid screening of available samples to isolate those possessing the desired traits. This study illustrates the HTP sugar release and pyrolysis-molecular beam mass spectrometry pipelines employed at the National Renewable Energy Lab. These pipelines have enabled the efficient assessment of thousands of plants while decreasing experimental time and costs through reductions in labor and consumables.

Plant cell wall structure and chemistry traits are evaluated to identify ideal feedstocks for biofuels and bio-materials. Standard methods have limitations when applied to large data sets. These high-throughput pretreatment, enzyme saccharification, and pyrolysis-molecular beam mass spectrometry methods compare large numbers of biomass samples with decreased experimental time and cost.

总木质素，木质素单体，和酶促糖推出:顽拗变奏曲在木质纤维素生物质的高通量筛选

The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the ...

科研

环境科学

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 ...

生物化学

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

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

基于原生自荧光的sFLIM测量植物部分荧光探针和木质素之间的相互作用

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

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

Ultrafast Lignin Extraction from Unusual Mediterranean Lignocellulosic Residues

Pretreatment is still the most expensive step in lignocellulosic biorefinery processes. It must be made cost-effective by minimizing chemical requirements as well as power and heat consumption and by using environment-friendly solvents. Deep eutectic solvents (DESs) are key, green, and low-cost solvents in sustainable biorefineries. They are transparent mixtures characterized by low freezing points resulting from at least one hydrogen bond donor and one hydrogen bond acceptor. Although DESs are promising solvents, it is necessary to combine them with an economic heating technology, such as microwave irradiation, for competitive profitability. Microwave irradiation is a promising strategy to shorten the heating time and boost fractionation because it can rapidly attain the appropriate temperature. The aim of this study was to develop a one-step, rapid method for biomass fractionation and lignin extraction using a low-cost and biodegradable solvent. 
In this study, a microwave-assisted DES pretreatment was conducted for 60 s at 800 W, using three kinds of DESs. The DES mixtures were facilely prepared from choline chloride (ChCl) and three hydrogen-bond donors (HBDs): a monocarboxylic acid (lactic acid), a dicarboxylic acid (oxalic acid), and urea. This pretreatment was used for biomass fractionation and lignin recovery from marine residues (Posidonia leaves and aegagropile), agri-food byproducts (almond shells and olive pomace), forest residues (pinecones), and perennial lignocellulosic grasses (Stipa tenacissima). Further analyses were conducted to determine the yield, purity, and molecular weight distribution of the recovered lignin. In addition, the effect of DESs on the chemical functional groups in the extracted lignin was determined by Fourier-transform infrared (FTIR) spectroscopy. The results indicate that the ChCl-oxalic acid mixture affords the highest lignin purity and the lowest yield. The present study demonstrates that the DES-microwave process is an ultrafast, efficient, and cost-competitive technology for lignocellulosic biomass fractionation.

Deep eutectic solvent-based, microwave-assisted pretreatment is a green, fast, and efficient process for lignocellulosic fractionation and high-purity lignin recovery.

超高速利格宁从不寻常的地中海木质细胞残留物中提取

Pretreatment is still the most expensive step in lignocellulosic biorefinery processes. It must be made cost-effective by minimizing chemical requirements ...

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.

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

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 ...