Chapter 7
Transient Expression of Fluorescent Fusion Proteins
in Arabidopsis Protoplasts
Yonglun Zeng, Changyang Ji, Youshun Lin, and Liwen Jiang
Abstract
Transient expression using protoplasts isolated from Arabidopsis suspension culture cells is a fast and useful
tool for analyzing protein subcellular localization and dynamics in plant cells. Recently, super-resolution
imaging techniques such as N-SIM (Nikon, Structured Illumination Microscopy) are widely used in cell
biology study, allowing cell biologists to obtain unattainable details and relationships of cell structures and
functions by conventional confocal imaging. To facilitate the usage of protoplasts transient expression and
super-resolution imaging for protein localization and dynamic analysis in plant cell biology research, here
we describe updated protocols of protoplasts isolation from Arabidopsis suspension culture cells and
transient expression assay for protein trafficking and localization study. Further, using GFP-tagged ERES
(Endoplasmic Reticulum Exit Site) marker proteins and RFP-tagged Golgi marker as examples, we illustrate
the major tools and methods for protein localization analysis using super-resolution imaging.
Key words Arabidopsis suspension culture cells, Protoplast, Fluorescent-tagged marker proteins,
Transient expression, Subcellular localization, Colocalization analysis, Confocal imaging, Super-resolution imaging, Structured illumination microscopy
1 Introduction
Transient expression, using protoplasts derived from suspension
culture cells, has been a key technique and reliable system for
analyzing protein subcellular localization and function with the
utilization of fluorescence reporter gene fusions in plant cell biology research [1]. In contrast to the traditional genetic and function
analysis using whole plants, which is time consuming and technically challenging, transient expression using protoplasts is a fast and
powerful method for analysis of protein function and localization.
With this technique, protein-protein interaction assay, protein subcellular localization analysis, as well as drug treatments can be
achieved within a matter of days [2–6]. Recent progress in our
understanding of plant unique sorting signals is accelerated because
deletion, substitution, and fusion experiments could be routinely
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_7, © Springer Science+Business Media, LLC, part of Springer Nature 2021
157
Transient Expression of Fluorescent Fusion Proteins
in Arabidopsis Protoplasts
Yonglun Zeng, Changyang Ji, Youshun Lin, and Liwen Jiang
Abstract
Transient expression using protoplasts isolated from Arabidopsis suspension culture cells is a fast and useful
tool for analyzing protein subcellular localization and dynamics in plant cells. Recently, super-resolution
imaging techniques such as N-SIM (Nikon, Structured Illumination Microscopy) are widely used in cell
biology study, allowing cell biologists to obtain unattainable details and relationships of cell structures and
functions by conventional confocal imaging. To facilitate the usage of protoplasts transient expression and
super-resolution imaging for protein localization and dynamic analysis in plant cell biology research, here
we describe updated protocols of protoplasts isolation from Arabidopsis suspension culture cells and
transient expression assay for protein trafficking and localization study. Further, using GFP-tagged ERES
(Endoplasmic Reticulum Exit Site) marker proteins and RFP-tagged Golgi marker as examples, we illustrate
the major tools and methods for protein localization analysis using super-resolution imaging.
Key words Arabidopsis suspension culture cells, Protoplast, Fluorescent-tagged marker proteins,
Transient expression, Subcellular localization, Colocalization analysis, Confocal imaging, Super-resolution imaging, Structured illumination microscopy
1 Introduction
Transient expression, using protoplasts derived from suspension
culture cells, has been a key technique and reliable system for
analyzing protein subcellular localization and function with the
utilization of fluorescence reporter gene fusions in plant cell biology research [1]. In contrast to the traditional genetic and function
analysis using whole plants, which is time consuming and technically challenging, transient expression using protoplasts is a fast and
powerful method for analysis of protein function and localization.
With this technique, protein-protein interaction assay, protein subcellular localization analysis, as well as drug treatments can be
achieved within a matter of days [2–6]. Recent progress in our
understanding of plant unique sorting signals is accelerated because
deletion, substitution, and fusion experiments could be routinely
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_7, © Springer Science+Business Media, LLC, part of Springer Nature 2021
157