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Chromatin Ring Formation at Plant Centromeres

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Chromatin Ring Formation at Plant Centromeres

Veit Schubert et al. Front Plant Sci.

Abstract

We observed the formation of chromatin ring structures at centromeres of somatic rye and Arabidopsis chromosomes. To test whether this behavior is present also in other plant species and tissues we analyzed Arabidopsis, rye, wheat, Aegilops and barley centromeres during cell divisions and in interphase nuclei by immunostaining and FISH. Furthermore, structured illumination microscopy (super-resolution) was applied to investigate the ultrastructure of centromere chromatin beyond the classical refraction limit of light. It became obvious, that a ring formation at centromeres may appear during mitosis, meiosis and in interphase nuclei in all species analyzed. However, varying centromere structures, as ring formations or globular organized chromatin fibers, were identified in different tissues of one and the same species. In addition, we found that a chromatin ring formation may also be caused by subtelomeric repeats in barley. Thus, we conclude that the formation of chromatin rings may appear in different plant species and tissues, but that it is not specific for centromere function. Based on our findings we established a model describing the ultrastructure of plant centromeres and discuss it in comparison to previous models proposed for animals and plants.

Keywords: CENH3; centromere organization; interphase nucleus; meiosis; mitosis; repetitive DNA; super-resolution microscopy.

Figures

Figure 1
Figure 1
Centromeric chromatin (pAL) ring formation in differentiated 2–16C leaf nuclei of A. thaliana. In addition to globular reticulate chromatin structures (triangles), ring and half-ring formation (asterisks) appears at all ploidy levels, especially around chromocenters (arrows). In 16C nuclei due to loss of cohesion centromeres may become separated but they keep their globular structure as demonstrated by the 17 signals in the right nucleus. Bar size in inset = 0.5 μm.
Figure 2
Figure 2
CENH3 chromatin ring formation at centromeres of rye during mitosis, meiosis and in interphase nuclei. (A) The 14 CENH3 labeled centromeres visible as rings in interphase become separated at somatic metaphase which segregate at their chromatids during anaphase. The main ring structures may be formed by several subrings (asterisk). (B) CENH3 structures during meiosis in pollen mother cells and somatic anther cells. After centromere alignment in early prophase I homologous centromeres coalesce and form ring structures which are split at the end of prophase I. The single rings composed by the two sister centromers present in metaphase I start to separate again (triangle) and in interkinesis clearly two rings are visible at each centromere. They are required to separate the sister chromatids in anaphase II. In prophase I centromeres may also associate (asterisk). At metaphase I and anaphase I bivalents the CENH3 chromatin ring structures are characterized by a cap/crown-like shape comprising extensions where spindle fibers may attach (arrows; see also Supplementary Movies 7–12). Somatic interphase nuclei (two of them in tapetum cells, and single ones in other cells) from anthere tissues show Rabl orientation (Rabl, 1885). Also in these nuclei CENH3 structures may coalesce and form ring structures. Bar size in inset = 0.5 μm.
Figure 3
Figure 3
CENH3-positive centromere structures during meiosis of hexaploid wheat. In prophase I the four centromeres of paired sister chromatids of both homologs are fused and form ring-like structures which become separated in metaphase I where only two sister chromatids are fused and spindle fibers attach (right).
Figure 4
Figure 4
Centromere chromatin substructures in embryo (A), spike (B), and endosperm (C) tissues of Ae. speltoides. CENH3 chromatin ring formation appears pronounced in embryo interphase nuclei (A1−2), but not at metaphase chromosomes (A3). The centromeric CRW2 repeat shows mainly a globular organization formed by chromatin fibers present also for CENH3 chromatin in spike tissue during the cell cycle (B1−4). Due to a high degree of chromatin condensation the CENH3 chromatin ring formation is less clearly visible in endosperm nuclei (C). Bar size in inset = 0.5 μm.
Figure 5
Figure 5
Ultrastructural organization of centromeres and subtelomeric regions at metaphase chromosomes and in interphase nuclei of barley. Anti-CENH3 and centromere-specific BAC7-repeats were used as centromeric probes. Repeat HvT01 detects the subtelomeric regions. In interphase nuclei CENH3 chromatin may compose highly condensed globular structures embedded in ring chromatin containing centromeric repeats (A1). Alternatively, CENH3 chromatin may show ring structures and the centromere repeats (BAC7) may be organized by chromatin fibers in a less condensed globular manner (A2−3). In addition, both CENH3 and centromeric BAC7 repeats may form rings (A4). (B1−3) While the centromeric repeats (BAC7) establish reticulate chromatin substructures at centromeres during the somatic cell cycle at centromeres, subtelomeric repeats (HvT01) may form ring-like structures at the subtelomeres in interphase (B1), prometaphase (B2), and metaphase (B3). Bar size in insets = 0.5 μm.
Figure 6
Figure 6
Models of plant centromere chromatin organization at interphase, somatic metaphase, and metaphase I chromosomes. In interphase CENH3-containing chromatin (red) may form ring-like (top) or globular (bottom) structures embedded in CENH3-negative heterochromatin (dark blue). At somatic metaphase chromosomes (side view of a single chromatid) the CENH3 chromatin forms rings (top) or globular/pad-like (bottom) structures where spindle fibers attach. The CENH3 chromatin is surrounded by pericentromeric heterochromatin (dark blue). The spindle fibers attach mainly to the pericentromeric flanks of the primary constriction to transfer pulling forces from the microtubules to the more stable chromosome arms (see also Wanner et al., 2015). At metaphase I bivalents, single fused CENH3 chromatin rings composed by the two sister centromers show a crown- like shape (bottom homolog) due to the pulling forces of the microtubules. They become separated again (top homolog) during the transition to anaphase I.

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