CENH3-GFP: a visual marker for gametophytic and somatic ploidy determination in Arabidopsis thaliana. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: De Storme, Nico; Keçeli, Burcu; Zamariola, Linda; Angenon, Geert; Geelen, Danny
• Is Part Of: BMC plant biology. Volume 16:Issue 1(2016)
• Page Start: 1
• Page End: 19
• Publication Date: 2016-12
• Subjects: CENH3 -- Centromere -- Arabidopsis -- Ploidy analysis -- Meiosis -- Endomitosis; Plant molecular biology -- Periodicals; Botany -- Periodicals; 580.5
• DOI: 10.1186/s12870-015-0700-5
• Languages: English
• ISSNs: 1471-2229

Abstract

Abstract Background The 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. UsingArabidopsis thaliana as a model, we here assess the applicability of recombinantCENH3-GFP reporters for the labeling of the cell's chromocenters and for the monitoring of the gametophytic and somatic chromosome number in vivo. Results By modulating expression of aCENH3-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 thepWOX2 -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 throughp35S-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 thatCENH3 -mediated chromosome labeling provides an easy and valuable tool for the detection and characterization of endomitotic polyploidization events. Conclusions This study demonstrates that the introgression of thepWOX2 -CENH3-GFP reporter construct inArabidopsis 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.