Chapter 12
Flow Cytometry and Sorting in Arabidopsis
David W. Galbraith and Guiling Sun
Abstract
Flow cytometry and sorting represents a valuable and mature experimental platform for the analysis of
cellular populations. Applications involving higher plants started to emerge around 40 years ago and are
now widely employed both to provide unique information regarding basic and applied questions in the
biosciences and to advance agricultural productivity in practical ways. Further development of this platform
is being actively pursued, and this promises additional progress in our understanding of the interactions of
cells within complex tissues and organs. Higher plants offer unique challenges in terms of flow cytometric
analysis, first since their organs and tissues are, almost without exception, three-dimensional assemblies of
different cell types held together by tough cell walls, and, second, because individual plant cells are generally
larger than those of mammals.
This chapter, which updates work last reviewed in 2014 [Galbraith DW (2014) Flow cytometry and
sorting in Arabidopsis. In: Sanchez Serrano JJ, Salinas J (eds) Arabidopsis Protocols, 3rd ed. Methods in
molecular biology, vol 1062. Humana Press, Totowa, pp 509–537], describes the application of techniques
of flow cytometry and sorting to the model plant species Arabidopsis thaliana, in particular emphasizing
(a) fluorescence labeling in vivo of specific cell types and of subcellular components, (b) analysis using both
conventional cytometers and spectral analyzers, (c) fluorescence-activated sorting of protoplasts and nuclei,
and (d) transcriptome analyses using sorted protoplasts and nuclei, focusing on population analyses at the
level of single protoplasts and nuclei. Since this is an update, details of new experimental methods are
emphasized.
Key words Flow cytometry, Sorting, Spectral analysis, Protoplasts, Fluorescent proteins, Nucleus,
Gene expression, Transcription, Next-generation sequencing, Agnostic sampling
1 Introduction
1.1 Applying Flow
Cytometry and Cell
Sorting to Vascular
Plants
The flowering plants, the Angiospermae, are a large diverse division
of the plant kingdom, which emerged approximately 140 million
years ago. They have been used in flow cytometry and cell sorting
for almost 40 years, despite the challenges provided by their predominant life form, the multicellular sporophyte, within which the
organs comprise complex interspersions of tightly connected cells
of different types [1]. Flow cytometry and sorting absolutely
requires input samples in the form of suspensions of single cells or
single particles (for example, subcellular organelles in
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_12, © Springer Science+Business Media, LLC, part of Springer Nature 2021
255
Flow Cytometry and Sorting in Arabidopsis
David W. Galbraith and Guiling Sun
Abstract
Flow cytometry and sorting represents a valuable and mature experimental platform for the analysis of
cellular populations. Applications involving higher plants started to emerge around 40 years ago and are
now widely employed both to provide unique information regarding basic and applied questions in the
biosciences and to advance agricultural productivity in practical ways. Further development of this platform
is being actively pursued, and this promises additional progress in our understanding of the interactions of
cells within complex tissues and organs. Higher plants offer unique challenges in terms of flow cytometric
analysis, first since their organs and tissues are, almost without exception, three-dimensional assemblies of
different cell types held together by tough cell walls, and, second, because individual plant cells are generally
larger than those of mammals.
This chapter, which updates work last reviewed in 2014 [Galbraith DW (2014) Flow cytometry and
sorting in Arabidopsis. In: Sanchez Serrano JJ, Salinas J (eds) Arabidopsis Protocols, 3rd ed. Methods in
molecular biology, vol 1062. Humana Press, Totowa, pp 509–537], describes the application of techniques
of flow cytometry and sorting to the model plant species Arabidopsis thaliana, in particular emphasizing
(a) fluorescence labeling in vivo of specific cell types and of subcellular components, (b) analysis using both
conventional cytometers and spectral analyzers, (c) fluorescence-activated sorting of protoplasts and nuclei,
and (d) transcriptome analyses using sorted protoplasts and nuclei, focusing on population analyses at the
level of single protoplasts and nuclei. Since this is an update, details of new experimental methods are
emphasized.
Key words Flow cytometry, Sorting, Spectral analysis, Protoplasts, Fluorescent proteins, Nucleus,
Gene expression, Transcription, Next-generation sequencing, Agnostic sampling
1 Introduction
1.1 Applying Flow
Cytometry and Cell
Sorting to Vascular
Plants
The flowering plants, the Angiospermae, are a large diverse division
of the plant kingdom, which emerged approximately 140 million
years ago. They have been used in flow cytometry and cell sorting
for almost 40 years, despite the challenges provided by their predominant life form, the multicellular sporophyte, within which the
organs comprise complex interspersions of tightly connected cells
of different types [1]. Flow cytometry and sorting absolutely
requires input samples in the form of suspensions of single cells or
single particles (for example, subcellular organelles in
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_12, © Springer Science+Business Media, LLC, part of Springer Nature 2021
255