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Occasional co-localization of CENH3-GFP foci during microsporogenesis. Representative images of pWOX2-CENH3-GFP fluorescence in uninuclear stage Arabidopsis thaliana microspores. Most microspores clearly exhibit five distinct centromeric GFP foci (A and B), reflecting the haploid chromosome number in diploid Arabidopsis thaliana (2x = 10). However, in some microspores, a lower number of centromeric GFP foci is observed due to the spatial co-localization or Z-axis based projection overlap of two or more GFP foci (C and D; see arrows). Scale bar, 5 μm. (DOC 151 kb)

Sequences of primers used in this study. (DOC 25Â kb)

Occasional co-localization of CENH3-GFP foci during microsporogenesis. Representative images of pWOX2-CENH3-GFP fluorescence in uninuclear stage Arabidopsis thaliana microspores. Most microspores clearly exhibit five distinct centromeric GFP foci (A and B), reflecting the haploid chromosome number in diploid Arabidopsis thaliana (2x = 10). However, in some microspores, a lower number of centromeric GFP foci is observed due to the spatial co-localization or Z-axis based projection overlap of two or more GFP foci (C and D; see arrows). Scale bar, 5 μm. (DOC 151 kb)

DAPI-stained chromosome spreads of 3x Arabidopsis male meiocytes. DAPI-stained chromosome spreads of somatic (A) and male meiotic (B, C and D) nuclei of triploid Arabidopsis thaliana plants (3x = 15). The somatic nucleus exhibits 15 distinct chromosomes, reflecting the triploid somatic chromosome number. Male meiotic chromosome spreads reveal the formation of five trivalents at diakinesis (B) and metaphase I (C), that inherently lead to an unbalanced segregation of homologous chromosomes at anaphase I (D; 8–7 chromosome segregation). Scale bar, 10 μm. (DOC 206 kb)

Expression of pLAT52-CENH3-GFP in mature pollen grains. CENH3-GFP expression driven by the pollen-specific LAT52 promoter in mature Arabidopsis thaliana pollen grains exhibits a strong fluorescent signal in the cytoplasm and the vegetative nucleus. Scale bar, 20 μm. (DOC 294 kb)

CENH3-GFP does not complement cenh3 −/− seed lethality. Genotypic analysis of F2 progeny resulting from several F1 cenh3-1/CENH3 plants that harbor the pWOX2-CENH3-GFP transgene reveals an absence of homozygous cenh3 −/− plants, indicating that cenh3 −/− seed fertility is not restored by the incorporation of the pWOX2 transcribed CENH3-GFP fusion protein. (DOC 25 kb)

Centromeric localization of CENH3 GFP-tailswap in different tissues. Representative images of the expression and subcellular localization of CENH3 GFP-tailswap in different tissues of Arabidopsis thaliana; including a young developing petal (A), a mature petal (B), unicellular stage microspores (C) and a bicellular stage microspore (E). Corresponding bright field images of the developing microspores are also presented (D and F). Scale bars, 20 μm. (DOC 241 kb)

Cenh3-1/CENH3 plants harboring pWOX2-CENH3-GFP show seed abortion. Representative pictures of siliques of Arabidopsis thaliana wild type (A), cenh3-1/CENH3 (B), and cenh3-1/CENH3 plants hemizygous for the pWOX2-CENH3-GFP transgene (C). Heterozygous cenh3-1/CENH3 plants harboring pWOX2-CENH3-GFP show a similar frequency of seed abortion as seen in the control cenh3-1/CENH3 plants, supporting the notion that pWOX2-CENH3-GFP does not complement cenh3 −/− seed abortion. (DOC 425 kb)

Frequency distribution of pWOX2-CENH3-GFP signals in nuclei of late uninuclear and binuclear megaspores isolated from triploid Arabidopsis thaliana plants. (DOC 54Â kb)

Expression pattern of pWOX2-CENH3-GFP in early megasporogenesis. Representative images of pWOX2-CENH3-GFP fluorescence in early stage megaspores of Arabidopsis thaliana, displaying either the haploid number of five centromeric GFP foci in uninuclear megaspores (A, B and C) or ten GFP signals in binuclear megaspores (D; nuclei are spatially overlapping). Scale bar, 5 μm. (DOC 291 kb)

Centromeric localization of CENH3 GFP-tailswap in different tissues. Representative images of the expression and subcellular localization of CENH3 GFP-tailswap in different tissues of Arabidopsis thaliana; including a young developing petal (A), a mature petal (B), unicellular stage microspores (C) and a bicellular stage microspore (E). Corresponding bright field images of the developing microspores are also presented (D and F). Scale bars, 20 μm. (DOC 241 kb)

CENH3-GFP co-localizes with dense DAPI stained chromocenters. Expression of p35S-CENH3-GFP in DAPI stained guard and epidermal cells reveals that the CENH3-GFP signals co-localize with dense DAPI stained chromocenters, indicating that the CENH3-GFP fusion protein is targeted to the centromeric chromosome region. Scale bar, 5 μm. (DOC 311 kb)

CENH3-GFP co-localizes with dense DAPI stained chromocenters. Expression of p35S-CENH3-GFP in DAPI stained guard and epidermal cells reveals that the CENH3-GFP signals co-localize with dense DAPI stained chromocenters, indicating that the CENH3-GFP fusion protein is targeted to the centromeric chromosome region. Scale bar, 5 μm. (DOC 311 kb)

Cenh3-1/CENH3 plants harboring pWOX2-CENH3-GFP show seed abortion. Representative pictures of siliques of Arabidopsis thaliana wild type (A), cenh3-1/CENH3 (B), and cenh3-1/CENH3 plants hemizygous for the pWOX2-CENH3-GFP transgene (C). Heterozygous cenh3-1/CENH3 plants harboring pWOX2-CENH3-GFP show a similar frequency of seed abortion as seen in the control cenh3-1/CENH3 plants, supporting the notion that pWOX2-CENH3-GFP does not complement cenh3 −/− seed abortion. (DOC 425 kb)

DAPI-stained chromosome spreads of 3x Arabidopsis male meiocytes. DAPI-stained chromosome spreads of somatic (A) and male meiotic (B, C and D) nuclei of triploid Arabidopsis thaliana plants (3x = 15). The somatic nucleus exhibits 15 distinct chromosomes, reflecting the triploid somatic chromosome number. Male meiotic chromosome spreads reveal the formation of five trivalents at diakinesis (B) and metaphase I (C), that inherently lead to an unbalanced segregation of homologous chromosomes at anaphase I (D; 8–7 chromosome segregation). Scale bar, 10 μm. (DOC 206 kb)

CENH3-GFP does not complement cenh3 −/− seed lethality. Genotypic analysis of F2 progeny resulting from several F1 cenh3-1/CENH3 plants that harbor the pWOX2-CENH3-GFP transgene reveals an absence of homozygous cenh3 −/− plants, indicating that cenh3 −/− seed fertility is not restored by the incorporation of the pWOX2 transcribed CENH3-GFP fusion protein. (DOC 25 kb)

Expression pattern of pWOX2-CENH3-GFP in early megasporogenesis. Representative images of pWOX2-CENH3-GFP fluorescence in early stage megaspores of Arabidopsis thaliana, displaying either the haploid number of five centromeric GFP foci in uninuclear megaspores (A, B and C) or ten GFP signals in binuclear megaspores (D; nuclei are spatially overlapping). Scale bar, 5 μm. (DOC 291 kb)

Ploidy distribution of young and mature petals in Arabidopsis thaliana. Histograms representing the DNA ploidy distribution of nuclei isolated from a young, developing petal at flower stage 5â 6 (A) and a mature petal at anthesis (B) of a diploid Arabidopsis thaliana plant. (DOC 67Â kb)

Frequency distribution of pWOX2-CENH3-GFP signals in nuclei of late uninuclear and binuclear megaspores isolated from triploid Arabidopsis thaliana plants. (DOC 54Â kb)