Abstract
BackgroundThe in vivo determination of the cell-specific chromosome number provides a valuable tool in several aspects of plant research. However, current techniques to determine the endosystemic ploidy level do not allow non-destructive, cell-specific chromosome quantification. Particularly in the gametophytic cell lineages, which are physically encapsulated in the reproductive organ structures, direct in vivo ploidy determination has been proven very challenging. Using Arabidopsis thaliana as a model, we here assess the applicability of recombinant CENH3-GFP reporters for the labeling of the cell’s chromocenters and for the monitoring of the gametophytic and somatic chromosome number in vivo.ResultsBy modulating expression of a CENH3-GFP reporter cassette using different promoters, we isolated two reporter lines that allow for a clear and highly specific labeling of centromeric chromosome regions in somatic and gametophytic cells respectively. Using polyploid plant series and reproductive mutants, we demonstrate that the pWOX2-CENH3-GFP recombinant fusion protein allows for the determination of the gametophytic chromosome number in both male and female gametophytic cells, and additionally labels centromeric regions in early embryo development. Somatic centromere labeling through p35S-CENH3-GFP shows a maximum of ten centromeric dots in young dividing tissues, reflecting the diploid chromosome number (2x = 10), and reveals a progressive decrease in GFP foci frequency throughout plant development. Moreover, using chemical and genetic induction of endomitosis, we demonstrate that CENH3-mediated chromosome labeling provides an easy and valuable tool for the detection and characterization of endomitotic polyploidization events.ConclusionsThis study demonstrates that the introgression of the pWOX2-CENH3-GFP reporter construct in Arabidopsis thaliana provides an easy and reliable methodology for determining the chromosome number in developing male and female gametes, and during early embryo development. Somatically expressed CENH3-GFP reporters, on the other hand, constitute a valuable tool to quickly determine the basic somatic ploidy level in young seedlings at the individual cell level and to detect and to quantify endomitotic polyploidization events in a non-destructive, microscopy-based manner.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0700-5) contains supplementary material, which is available to authorized users.
Highlights
The in vivo determination of the cell-specific chromosome number provides a valuable tool in several aspects of plant research
DNA flow cytometry cannot be used for the ploidy analysis of cell types which are embedded in surrounding tissue
We demonstrate that CENH3-GFP confers distinct labeling of centromeres in several tissue types, including developing gametes, and show that this allows for a tissue- or cell-specific chromosome quantification
Summary
The in vivo determination of the cell-specific chromosome number provides a valuable tool in several aspects of plant research. Current techniques to determine the endosystemic ploidy level do not allow non-destructive, cell-specific chromosome quantification. Several techniques have been developed that allow for the determination of organ- or plant-specific ploidy levels; including DNA flow cytometry [10,11,12], chromosome spreading and fluorescent in situ hybridization (FISH) [13,14,15]. None of these methodologies enables in vivo chromosome quantification on a single-cell level. DNA flow cytometry cannot be used for the ploidy analysis of cell types which are embedded in surrounding tissue (e.g., vascular cell layers, egg cells, early embryonic cells)
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