Progress toward the isolation of "hidden" cell populations of dark-growing organs

The key to the evolutionary success of multicellularity, which arose independently in plants and animals, is the different functions that highly specialized cell types can perform. Individual genes, pathways, and metabolites can have diverse or even opposing roles depending on the tissue context. However, to study specific cell types is challenging. This limitation can be overcome by cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS). This approach makes possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and RNA-Seq analysis.

In our study, in order to discriminate between viable protoplasts and debris, we have combined the use of different markers: cFDA for cell viability and DAPI for loss of cell membrane integrity to generate a suitable gate for protoplasting. The main motivation of our work was to implement a protoplasting and FACS protocol in order to performance transcriptomics analyses of cell types. We used the D-Root device to cultivate the seedling with roots in darkness, conditions similar to nature. In addition, our protocols were optimized to use the minimum number/amount of enzymes, but also to extract hidden cell type populations, which are not easily accessible because of their location inside mature root tissues.

In conclusion, the success of this work consists of isolating individual cells from scarce and non-accessible tissues grown in darkness, in the D-Root device and analyzing them in detail in order to determine if they are all the same or there is already a specialization.