Schmidt Martin, Perera Pradeep, Schwartzberg Adam M, Adams Paul D, Schuck P James
Energy Biosciences Institute, University of California, Berkeley, USA.
J Vis Exp. 2010 Nov 1(45):2064. doi: 10.3791/2064.
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 biofuels. 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 chemicals. 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 fermentation. 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 factor 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 modifications. 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 walls. 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 wood. Analyzing the lignin Raman bands 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-μm. 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.
安全高效地满足不断增长的能源需求是一项紧迫的全球挑战。因此，旨在寻找具有成本效益和可持续解决方案的生物燃料生产研究已成为一项热门且关键的任务。木质纤维素生物质有望成为转化为液体生物燃料的主要生物质来源。然而，这些植物细胞壁材料难以进行具有成本效益和高效的降解，这对它们在生物燃料和化学品生产中的应用构成了重大障碍。特别是木质素，一种复杂且不规则的多苯基丙烷类杂聚物，在木质纤维素生物质收获后的解构过程中会产生问题。例如在生物燃料的生物质转化中，它会抑制旨在生产用于发酵的单糖的过程中的糖化作用。实际上，将植物生物质有效地用于工业目的在很大程度上取决于植物细胞壁的木质化程度。木质素的去除是一个成本高昂且具有限制作用的因素，因此为了提高细胞壁的转化率，木质素已成为植物育种和基因工程的关键目标。能够准确快速地表征植物细胞壁木质化的分析工具对于评估大量育种群体变得越来越重要。用于分离木质素等天然成分的提取程序不可避免地具有破坏性，会导致显著的化学和结构修饰。因此，分析化学原位方法是用于木质纤维素材料成分和结构表征的宝贵工具。拉曼显微镜技术依赖于单色光（如激光发出的光）的非弹性或拉曼散射，其中激光光子能量的变化与分子振动相关，并呈现出样品的固有无标记分子“指纹”。拉曼显微镜可以在样品制备最少的情况下进行无损且相对廉价的测量，从而深入了解接近天然状态的化学成分和分子结构。共聚焦拉曼显微镜的化学成像先前已用于可视化木材细胞壁中纤维素和木质素的空间分布。基于这些早期结果，我们最近采用这种方法来比较野生型和木质素缺陷型转基因毛果杨（黑杨）茎木中的木质化情况。通过分析1600至1700 cm⁻¹光谱区域内的木质素拉曼谱带，原位绘制了木质素信号强度和定位图。我们的方法可视化了木质素含量、定位和化学成分的差异。最近，我们展示了拟南芥细胞壁聚合物的拉曼成像，其横向分辨率低于微米。在此，介绍了这种方法，它能够在不染色或标记组织的情况下可视化植物细胞壁中的木质素，并比较不同组织、样品或物种中的木质化情况。
