Preparing Arabidopsis thaliana root protoplasts for cryo electron tomography

Frontiers in Plant Science

Plant Science

Hugo Germain¹  Ingrid Berenice Sanchez Carrillo¹  Teura Barff¹  Patrick C. Hoffmann²  Martin Beck³ 

[1] Department of Chemistry, Biochemistry, and Physics, Université du Québec à Trois-Rivières, Trois-Rivières, QC, Canada;Department of Molecular Sociology, Max-Planck-Institute for Biophysics, Frankfurt, Germany;Department of Molecular Sociology, Max-Planck-Institute for Biophysics, Frankfurt, Germany;Institute of Biochemistry, Goethe University Frankfurt, Frankfurt, Germany;

关键词: structural biology;    Arabidopsis thaliana;    protoplasts;    cryo-EM;    cryo-ET;   

DOI  :  10.3389/fpls.2023.1261180

 received in 2023-07-19, accepted in 2023-09-04,  发布年份 2023

来源: Frontiers

【 摘 要 】

The use of protoplasts in plant biology has become a convenient tool for the application of transient gene expression. This model system has allowed the study of plant responses to biotic and abiotic stresses, protein location and trafficking, cell wall dynamics, and single-cell transcriptomics, among others. Although well-established protocols for isolating protoplasts from different plant tissues are available, they have never been used for studying plant cells using cryo electron microscopy (cryo-EM) and cryo electron tomography (cryo-ET). Here we describe a workflow to prepare root protoplasts from Arabidopsis thaliana plants for cryo-ET. The process includes protoplast isolation and vitrification on EM grids, and cryo-focused ion beam milling (cryo-FIB), with the aim of tilt series acquisition. The whole workflow, from growing the plants to the acquisition of the tilt series, may take a few months. Our protocol provides a novel application to use plant protoplasts as a tool for cryo-ET.

【 授权许可】

Unknown   
Copyright © 2023 Sanchez Carrillo, Hoffmann, Barff, Beck and Germain