Transcriptional control of stem cell specification in the Arabidopsis embryo: defining cell type-specific transcriptomes

Early plant embryogenesis condenses the fundamental processes underlying plant development into a short sequence of predictable steps. The main tissues, as well as stem cell niches (meristems) for their post-embryonic maintenance, are specified through genetic control networks. These stem cell niches, in which new cells are continuously produced and organized into organs, contain morphologically undifferentiated and pluripotent stem cells as well as organizer cells that prevent differentiation of adjoining stem cells. In past years, much has been learned about the control of embryonic patterning, and several key regulators have been identified. However, much of the genetic networks underlying the establishment of (stem) cell and tissue identity, critical for us to fully understand plant development, remains unresolved. A key question is how cell fates are instructed by unique cellular transcriptomes.

Figure 1. Schematic figure of detailing the concept behind the INTACT method. A transgenic nuclear tagging factor (NTF) containing GFP and a biotinylation site is co-expressed in the cell type of interest (A, green) together with a biotin ligase (BirA, blue) in the Arabidopsis embryo. After homogenization, biotin-tagged nuclei are purified using streptavidin-coated beads (B, dark green).
Figure 1. Schematic figure of detailing the concept behind the INTACT method. A transgenic nuclear tagging factor (NTF) containing GFP and a biotinylation site is co-expressed in the cell type of interest (A, green) together with a biotin ligase (BirA, blue) in the Arabidopsis embryo. After homogenization, biotin-tagged nuclei are purified using streptavidin-coated beads (B, dark green).

Our (Joakim Palovaara, Shunsuke Sagia, Jos Wendrich) project is to utilize a newly developed method called INTACT to determine the transcriptome of the stem cells, organizer cell and their precursors of the future root apical meristem in the early Arabidopsis embryo. INTACT is a two-component transgenic labelling system (Fig. 1), where a nuclear lamina-localized GFP protein that contains an in vivo biotinylation site (Nuclear Tagging Factor or NTF) is expressed with cell type-specific promoters (Fig. 2a-c). In the same plant, a biotin ligase (BirA) is also transgenically expressed. In those cells that express both NTF and BirA, the enzyme biotinylates the NTF protein and biotin-tagged nuclei can be efficiently isolated from crude nuclear preparations using streptavidin-coated beads for transcriptome profiling (Figs. 1, 2d). With this system, we will be able to compose a gene expression atlas of each cell-type during stem cell specification and identify putative key regulators of this event. A reverse genetics approach will be applied in order to functionally investigate these regulators.

Figure 2. Confocal images of suspensor-specific (A), hypophysis-specific (B), and vascular-specific (C) NTF expression in the Arabidopsis embryo. (D) Biotin-tagged nucleus (red) bound to streptavidin-coated beads. GFP fluorescence in (A-C) is in green while membranes are counterstained with FM4-64 (red).
Figure 2. Confocal images of suspensor-specific (A), hypophysis-specific (B), and vascular-specific (C) NTF expression in the Arabidopsis embryo. (D) Biotin-tagged nucleus (red) bound to streptavidin-coated beads. GFP fluorescence in (A-C) is in green while membranes are counterstained with FM4-64 (red).

Techniques used: INTACT, RNA isolation, qRT-PCR, microarray, RNA-seq, (confocal) microscopy.

See also: Transcriptomics approaches in the early Arabidopsis embryo Trends in Plants Sciences 2013