Fig. 5: AHL15 overexpression-induced SE results in polyploidy by endomitosis due to chromosome mis-segregation. | Nature Communications

Fig. 5: AHL15 overexpression-induced SE results in polyploidy by endomitosis due to chromosome mis-segregation.

From: An Arabidopsis AT-hook motif nuclear protein mediates somatic embryogenesis and coinciding genome duplication

Fig. 5

a Confocal image of polyploid cells detected by CENH3-GFP-mediated centromere labeling in an embryonic structure developing on a cotyledon of a 35S:AHL15 IZE after 7 days of culture (DOC). b Percentage of polyploid nuclei (more than 11 detectable CENH3-GFP-labeled centromeres) in cotyledon cells of p35S:AHL15 IZEs after 5 or 7 DOC. Dots indicate the values of ten biological replicates per plant line, with about 200 nuclei analyzed per replicate, bar indicates the mean, and error bars the s.e.m. c, d H2B-GFP-labeled chromocenters in nuclei of cotyledon cells of p35S:AHL15 IZEs after 5 (c) or 8 (d) DOC. White arrowheads (d) indicate cells with a duplicated number of chromocenters. e–g Confocal images of H2B-GFP labeled chromocenters in endoreduplicated nuclei of wild-type root hair (e), leaf (f), or root epidermis (g) cells. h–j Confocal microscopy analysis of chromosome segregation in dividing protodermal cells in cotyledons of p35S:AHL15 IZEs after 8 DOC using the pH2B:H2B-GFP reporter. The white arrowhead indicates normal chromosome segregation during anaphase (h), the yellow arrowhead indicates mis-segregation of chromosomes during anaphase (i), and the magenta arrowhead indicates a bi-nucleated cell (j). k Confocal microscopy image of a cotyledon of a p35S:AHL15 pWOX2:NLS-YFP pAUX1:AUX1-YFP IZE. pWOX2:NLS-YFP and pAUX1:AUX1-YFP reporters were used to mark embryonic nuclei and plasma membranes, respectively. The magenta arrowhead indicates a bi-nucleated cell in an area of cells with WOX2-YFP-marked embryo cell fate. l Percentage of bi-nucleated cells, as detected by H2B-GFP labeling, in protodermal cotyledon cells of wild-type or p35S:AHL15 IZEs after 7 DOC on medium with or without 2,4-D, respectively. Dots indicate the values of ten biological replicates per plant line (two 35S:AHL15 lines were included) with about 200 nuclei analyzed per replicate, bar indicates the mean, and error bars the s.e.m. a–k Similar results were obtained from two independent experiments. Size bar indicates 6 µm. d–k Images show a merge of the transmitted light and the GFP channel (d–j), or the GFP (a, c) or YFP (k) channel alone. c–g Numbers in images indicate the frequency with which similar images from different samples were obtained.

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