Cell type-specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines

To investigate the relationship between developmental events and gene expression, cell-specific resolution of gene activity is critical. Such high-resolution data have been difficult to obtain at a genomic level because cells first need to be isolated, and then sufficient amounts of mRNA must be collected, or subsequently amplified, for a large-scale profiling analysis. Genomics methods have tremendous potential to infer developmental circuits and, in combination with genetic tools, to discover the unknown downstream targets of known developmental regulators. We have developed a method that can be used to isolate up to hundreds of thousands of plant cells of a specific cell type, with very high purity, which can then be used for microarray analysis. The method makes use of reporter lines expressing green fluorescent protein (GFP) in histologically defined cell types, of which large collections are now available (Table 1). The GFP Line of interest is bulked and the tissue is collected and rapidly converted into protoplasts. GFP-positive cells are then isolated using a fluorescence-activated cell sorter (FACS). Total RNA is isolated, labeled using standard procedures and applied to microarrays (Fig. 1). The technique has been used to generate expression profiles of cell types and tissues in the Arabidopsis thaliana root, although it can be used for any tissue whose cell walls can be readily digested. The protocol presented here has been optimized for roots.