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High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.
One phytatray of approximately 1,500 one-week-old PSCR::GFP seedlings yielded about 60,000 protoplasts (as measured by hemacytometer). 2.6% of 65,000 FACS-processed events were defined as being GFP-positive and were sorted (Figure 4b).
Eight plates of approximately 1,500 four-day-old PWOX5::GFP seedlings each (12,000 total) yielded about 30,000,000 protoplasts (as measured by hemacytometer). 0.063% of 16,000,000 FACS-processed events were defined as being GFP-positive and were sorted (Figure 4c).
10,000 sorted events are typically used for RNA extraction and can yield from 20 to 140 ng total RNA (Figure 5).
Protoplasts can, in principle, be derived from a variety of plant tissues, optimizing favorable conditions will greatly enhance RNA quality and quantity. Both the protoplasting solution and the elective incubation buffer used will influence this aspect.
Many different fluorescent proteins can be used, depending on the capabilities of the FACS used, e.g. GFP, RFP, YFP, CFP or their many variants and derivatives. The expression of the markers could be driven not just by cell type-specific promoters or enhancer traps, as shown here, but also by your favorite promoter, e.g. a hormone responsive synthetic reporter gene5. Dual color FACS of plant protoplasts is also feasible. This can, for example, be applied when performing a transient transformation assay using a vector with a fluorescent positive selection marker5.
Protoplasts can also be sorted for the analysis of genomic DNA, proteins or metabolites6. Keeping protoplasts alive after sorting is challenging due to their fragile nature, using a larger (130 μm) nozzle and lower sheath pressure and sorting them into an optimized incubation buffer will enhance post-sort protoplast viability.
This work was supported by the National Science Foundation (grant no. DBI 0519984) and the National Institutes of Health (grant no. 5R01GM078279).
The authors have nothing to disclose
Bastiaan O. R. Bargmann, Center for Genomics and Systems Biology, Department of Biology, New York University.
Kenneth D. Birnbaum, Center for Genomics and Systems Biology, Department of Biology, New York University.