Pradhan Mitra Prajakta, Loqué Dominique
Feedstocks Division, Joint Bioenergy Institute; Physical Biosciences Division, Lawrence Berkeley National Laboratory.
Feedstocks Division, Joint Bioenergy Institute; Physical Biosciences Division, Lawrence Berkeley National Laboratory;
J Vis Exp. 2014 May 13(87):51381. doi: 10.3791/51381.
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 (40-50%), hemicellulose (25-30%), and lignin (20-30%). 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 Mäule 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.
拟南芥是一种常用于理解和操控植物各种细胞过程的模式生物，并且它已被广泛应用于次生细胞壁形成的研究中。次生细胞壁的沉积发生在初生细胞壁形成之后，这一过程仅由诸如形成导管和纤维组织的那些特化细胞来执行。大多数次生细胞壁由纤维素（40 - 50%）、半纤维素（25 - 30%）和木质素（20 - 30%）组成。已经分离出了几个影响次生细胞壁生物合成的突变体，并且相应的突变体可能会或可能不会表现出明显的生化成分变化或可见表型，因为这些突变可能会被补偿反应所掩盖。历史上一直使用染色程序来在细胞层面显示差异。这些方法完全是视觉分析手段；然而，它们在快速和关键分析中的作用非常重要。刚果红和荧光增白剂是用于检测多糖的染料，而马勒试剂和间苯三酚通常用于确定木质素的差异，甲苯胺蓝O用于对多糖和木质素进行差异染色。切片、染色和成像这些看似简单的技术对初学者来说可能是一项挑战。本研究以拟南芥模型的样品制备为起点，详细介绍了多种染色方法的方案，这些方案可轻松用于观察植物次生细胞壁中的细胞和组织结构。
