Efficient Isolation and Purification of High‐Quality Arabidopsis thaliana Trichomes

AbstractTrichomes 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 trichomesBasic Protocol 2: Purification of A. thaliana trichomes by density gradient centrifugation