One of the reasons that methods of transcript profiling based
on flow sorting are becoming particularly popular is that robust
methods have emerged for amplification of cDNAs produced from
very small amounts of RNA, even down to the levels of polyA
+
RNA contained within a single protoplast (see, for example, [109–
117] and references therein), or a single nucleus [118]. This means
that the sort process itself is no longer a limiting factor in transcriptomics. For the comprehensive analysis of cellular proteomes,
metabolomes, and complements of small RNAs in sorted cells/
protoplasts, first reports indicated a requirement for much larger
numbers (1.5 Â 10
5 [18] and 10
6 [77, 119]) for metabolite experiments, and 10
6 for proteomics and small RNA experiments
[67, 69]). Larger numbers of sorted nuclei (5 Â 10
4 ) were also
found necessary for chromatin profiling [120]. Recently, DNA
binding sites of transcription factors have been measured for mammalian samples containing 1000 cells and histone modification
landscapes from samples of 100 cells [120]. Further technical
advances, based on recent results obtained with mammalian systems [121], predict the numbers required for these measurements
will continue to drop, ultimately perhaps to the level of individual
cells/nuclei.
Development of methods of flow sorting that accommodate
expression of multiple FPs has progressed more slowly. Flow analysis and sorting of protoplasts based on two-color (GFP and RFP)
expression was one early example [72]. However, based on the
previous discussion, it should now be possible to consider employing many more FPs (i.e., combinations of up to six different FPs
[59]), for the study of achlorophyllous plant organs. For the study
of leaves, stems, and other aerial organs, due to the predominance
of chlorophyll autofluorescence, flow cytometric analysis of expression of multiple FPs will likely be limited to four different FPs, i.e.,
variants of BFP, CFP, either GFP or YFP, and one of the orange/
red FPs emitting fluorescence below around 620 nm. Examples of
work in this direction include the production [122] of “Kaleidocell” Arabidopsis plants, in which nuclei, plastids, mitochondria,
and plasma membranes were separately tagged with cyan, red,
yellow, and green fluorescent proteins. Unfortunately, the majority
of the transgene fluorescence disappears about 2~3 weeks after seed
germination, perhaps due to gene silencing (Kato, pers. commun.)
since homozygotes carry eight expression cassettes of very similar
sequence. Hirakawa and Matsunaga [123] describe ameliorating
silencing effects by reducing transgene similarities. It is clear that a
systematic evaluation of long-term expression stabilities of multiple
FPs in plants is needed, and flow cytometry, particularly Spectral
Analysis, is the method of choice for this quantitation (see Fig. 3).
Analysis of protoplasts by flow cytometry at various stages in
culture following molecular manipulations (transfection, drug
treatments, etc.) can also be desirable. For example, CRISPR
Flow Cytometry and Sorting in Arabidopsis
265
on flow sorting are becoming particularly popular is that robust
methods have emerged for amplification of cDNAs produced from
very small amounts of RNA, even down to the levels of polyA
+
RNA contained within a single protoplast (see, for example, [109–
117] and references therein), or a single nucleus [118]. This means
that the sort process itself is no longer a limiting factor in transcriptomics. For the comprehensive analysis of cellular proteomes,
metabolomes, and complements of small RNAs in sorted cells/
protoplasts, first reports indicated a requirement for much larger
numbers (1.5 Â 10
5 [18] and 10
6 [77, 119]) for metabolite experiments, and 10
6 for proteomics and small RNA experiments
[67, 69]). Larger numbers of sorted nuclei (5 Â 10
4 ) were also
found necessary for chromatin profiling [120]. Recently, DNA
binding sites of transcription factors have been measured for mammalian samples containing 1000 cells and histone modification
landscapes from samples of 100 cells [120]. Further technical
advances, based on recent results obtained with mammalian systems [121], predict the numbers required for these measurements
will continue to drop, ultimately perhaps to the level of individual
cells/nuclei.
Development of methods of flow sorting that accommodate
expression of multiple FPs has progressed more slowly. Flow analysis and sorting of protoplasts based on two-color (GFP and RFP)
expression was one early example [72]. However, based on the
previous discussion, it should now be possible to consider employing many more FPs (i.e., combinations of up to six different FPs
[59]), for the study of achlorophyllous plant organs. For the study
of leaves, stems, and other aerial organs, due to the predominance
of chlorophyll autofluorescence, flow cytometric analysis of expression of multiple FPs will likely be limited to four different FPs, i.e.,
variants of BFP, CFP, either GFP or YFP, and one of the orange/
red FPs emitting fluorescence below around 620 nm. Examples of
work in this direction include the production [122] of “Kaleidocell” Arabidopsis plants, in which nuclei, plastids, mitochondria,
and plasma membranes were separately tagged with cyan, red,
yellow, and green fluorescent proteins. Unfortunately, the majority
of the transgene fluorescence disappears about 2~3 weeks after seed
germination, perhaps due to gene silencing (Kato, pers. commun.)
since homozygotes carry eight expression cassettes of very similar
sequence. Hirakawa and Matsunaga [123] describe ameliorating
silencing effects by reducing transgene similarities. It is clear that a
systematic evaluation of long-term expression stabilities of multiple
FPs in plants is needed, and flow cytometry, particularly Spectral
Analysis, is the method of choice for this quantitation (see Fig. 3).
Analysis of protoplasts by flow cytometry at various stages in
culture following molecular manipulations (transfection, drug
treatments, etc.) can also be desirable. For example, CRISPR
Flow Cytometry and Sorting in Arabidopsis
265