Unit of Plant Molecular Cell Biology, Institute for Biology I, RWTH Aachen University, Aachen, Germany.
Curr Protoc. 2022 Sep;2(9):e541. doi: 10.1002/cpz1.541.
Trichomes are fine outgrowths on the surface of aerial plant organs which play a role in protecting plants against water loss, UV radiation, and herbivore feeding. Throughout the years, trichomes have become a popular paradigm in biological research. For example, trichomes on rosette leaves of the reference plant Arabidopsis thaliana have been used as a model to investigate cell development, cell differentiation, and, more recently, cell wall biogenesis. State of the art -omics studies on specific cell types or tissues often require physical separation, enrichment, and purification. This, of course, also applies to leaf trichomes, and various methods have thus been proposed to separate trichomes and leaf tissue. Though most of these methods are indeed suitable for trichome isolation, they suffer in part from tedious operating procedures, low yield, poor sample purity, and reduced trichome integrity. We have thus revised a previously reported method for trichome isolation, and report here an efficient and scalable procedure for the isolation and gradient centrifugation-based purification of high-quality A. thaliana trichomes. We describe the preparation of plant material and trichome release, which is based on prolonged gentle agitation of plant seedlings in the presence of a cation-chelating agent that weakens trichome-leaf interactions. We also outline the steps for the subsequent recovery and purification of the isolated crude trichome fraction, which is based on the use of discontinuous sucrose gradient centrifugation. In addition to A. thaliana, we have found that this procedure can be applied to release and enrich glandular and non-glandular trichomes from various species, including Solanum lycopersicum and Nicotiana benthamiana. The resulting purified leaf trichomes can be subjected to different types of bioassays, including histochemistry, biochemical quantification of cell wall monosaccharides, and transcriptomics, as well as proteomic profiling. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of plant material for release and enrichment of A. thaliana trichomes Basic Protocol 2: Purification of A. thaliana trichomes by density gradient centrifugation.
表皮毛是植物地上器官表面的精细突起物，在防止植物水分流失、紫外线辐射和食草动物取食方面发挥作用。多年来，表皮毛已成为生物学研究中的一个热门范例。例如，拟南芥（Arabidopsis thaliana）莲座叶表皮毛已被用作研究细胞发育、细胞分化以及最近的细胞壁生物发生的模型。针对特定细胞类型或组织的最新“组学”研究通常需要进行物理分离、富集和纯化。这当然也适用于叶表皮毛，因此提出了各种方法来分离表皮毛和叶片组织。尽管这些方法中的大多数确实适用于表皮毛分离，但它们在部分程度上存在操作繁琐、产量低、样品纯度差和表皮毛完整性降低等问题。我们对先前报道的表皮毛分离方法进行了修订，并在此报告了一种高效且可扩展的分离和基于梯度离心的高质量拟南芥表皮毛纯化程序。我们描述了植物材料和表皮毛释放的准备过程，该过程基于在阳离子螯合剂存在下对幼苗进行长时间温和搅拌，从而削弱表皮毛与叶片的相互作用。我们还概述了后续回收和纯化分离的粗表皮毛级分的步骤，该步骤基于使用不连续蔗糖梯度离心。除了拟南芥，我们还发现该程序可用于从各种物种（包括番茄和烟草）中释放和富集腺性和非腺性表皮毛。所得纯化的叶片表皮毛可用于进行不同类型的生物测定，包括组织化学、细胞壁单糖的生化定量以及转录组学和蛋白质组学分析。© 2022 作者。 Wiley Periodicals LLC 出版的《当代协议》。 基本方案 1：用于释放和富集拟南芥表皮毛的植物材料的准备 基本方案 2：通过密度梯度离心纯化拟南芥表皮毛
