Preparing Arabidopsis thaliana root protoplasts for cryo electron tomography

Area of Science:

• Plant Biology
• Cellular and Molecular Biology
• Microscopy

Background:

• Protoplasts are valuable tools in plant biology for studying gene expression and cellular responses.
• Existing protoplast isolation protocols have not been adapted for cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET).

Purpose of the Study:

• To develop and describe a workflow for preparing *Arabidopsis thaliana* root protoplasts suitable for cryo-electron tomography (cryo-ET).
• To enable high-resolution imaging of plant cellular structures using cryo-ET.

Main Methods:

• Protoplast isolation from *Arabidopsis thaliana* roots.
• Vitrification of protoplasts on electron microscopy (EM) grids.
• Cryo-focused ion beam (cryo-FIB) milling for sample preparation.
• Acquisition of tilt series for cryo-tomography.

Main Results:

• A comprehensive workflow for preparing plant protoplasts for cryo-ET has been established.
• The protocol facilitates the application of cryo-ET to study plant protoplasts at high resolution.
• The entire process, from plant cultivation to data acquisition, is detailed.

Conclusions:

• This protocol offers a novel approach to utilizing plant protoplasts as a model system for cryo-ET.
• The developed workflow expands the toolkit for investigating plant cell ultrastructure and dynamics.
• This method opens new avenues for high-resolution imaging in plant cell biology.