Interphase chromosome arrangement in Arabidopsis thaliana is similar in differentiated and meristematic tissues and shows a transient mirror symmetry after nuclear division

Abstract

Whole-mount fluorescence in situ hybridization (FISH) was applied to Arabidopsis thaliana seedlings to determine the three-dimensional (3D) interphase chromosome territory (CT) arrangement and heterochromatin location within the positional context of entire tissues or in particular cell types of morphologically well-preserved seedlings. The interphase chromosome arrangement was found to be similar between all inspected meristematic and differentiated root and shoot cells, indicating a lack of a gross reorganization during differentiation. The predominantly random CT arrangement (except for a more frequent association of the homologous chromosomes bearing a nucleolus organizer) and the peripheric location of centromeric heterochromatin were as previously observed for flow-sorted nuclei, but centromeres tend to fuse more often in nonendoreduplicating cells and NORs in differentiated cells. After mitosis, sister nuclei revealed a symmetric arrangement of homologous CTs waning with the progress of the cell cycle or in the course of differentiation. Thus, the interphase chromosome arrangement in A. thaliana nuclei seems to be constrained mainly by morphological features such as nuclear shape, presence or absence of a nucleolus organizer on chromosomes, nucleolar volume, and/or endopolyploidy level.

Figure 1.—

Chromosome territory organization in differentiated and meristematic A. thaliana tissues. (A–F) Differently labeled probes for the top (red) and bottom (green) arm of chromosome AT2 were hybridized to A. thaliana seedlings. Possible arrangements of homologous arm territories are shown for part of a differentiated primary leaf (A, with premature trichomes), for a pair of guard cells (B, sister cells s1 and s2), for part of a differentiated primary root (C, front view), for a root hair (D), and for shoot- and root-tip meristematic cells (E and F, respectively). (G) Association of homologous or heterologous chromosome territories for AT3 (yellow), AT4 (green), and AT5 (red) was analyzed in meristematic root-tip nuclei. Bars, 5 μm.

Figure 2.—

Interphase arrangement of the major heterochromatic blocks in differentiated and meristematic A. thaliana tissues. As shown for cortex and epidermal cells (A, spherical- shaped, and B, spindle-shaped nucleus), for guard cells (C), for root meristematic cells (D), and for root hairs (E), 45S rDNA signals (green) are in most nuclei associated with a single nucleolus and in meristematic cells (D) are more dispersed than in differentiated cells. Centromeric 180-bp repeats (red) are preferentially localized at the nuclear periphery (visible in 3D) in all tested cell types. 5S rDNA (yellow) is associated with the flanking centromeres. In root-hair nuclei (E), 5S rDNA FISH signals appear to be particularly elongated (right, with higher magnification as insert). Bars, 5 μm.

Figure 3.—

Numbers of centromeric, 45S rDNA, and 5S rDNA FISH signals in several differentiated and meristematic cell types from leaves, stems, and roots of entire A. thaliana seedlings. (A) Number of centromeric FISH signals. Centromeric 180-bp repeat FISH signals reveal a lower tendency to fuse in endoreduplicated cells (cortex and epidermal cells, vascular cells from leaf, stem, and root as well as root hairs) than in nonendoreduplicating cell types (guard cells, shoot-tip, root-tip, and secondary root-tip meristems). (B) Number of 45S rDNA FISH signals. Mitotically active cells of the shoot tip, root tip, and secondary root tip revealed the largest nucleoli, and 45S rDNA signals were less often associated than in differentiated cells. (C) Number of 5S rDNA FISH signals. Compared to centromeric signals, the number of 5S rDNA signals was more homogenous among the different cell types. 5S rDNA signals show a lower association frequency between heterologous loci.

Figure 4.—

Transient mirror symmetry between sister nuclei in A. thaliana primary root meristem through nuclear division. Differently labeled probes for the top (T, red) and bottom (B, green) arm of chromosome AT2 and 10 adjacent BACs from AT3 top arm (yellow) were hybridized to the A. thaliana root tip. (A1) In the primary root meristem (DAPI staining), initial cells (i) undergo a longitudinal division followed by a transverse division of the upper sister cell, yielding a pair of sister cells (s1 and s2). (A2) CT arrangement of a pair of sister cells (s1 and s2) magnified. Superimposed maximum intensity projections of 24 serial sections (Z-interval = 0.2 μm) reveal a mirror-symmetric positional arrangement of the homologous AT2 arm territories and the 10 adjacent BACs of AT3. (A3) Observing this arrangement from front (left), top (center), and side (right) views of sister cells s1 and s2, the symmetry appears to be imperfect as to the shape and the strict positioning of the homologous regions. (B) Differences increased when the pair s3 and s4 were compared with its closest neighbor pair s5 and s6. (C1–C4) The mirror symmetry between sister nuclei could be related to the symmetry observed between the two sets of chromatids during a different stage of mitotic division: metaphase (C1), early anaphase (C2), late anaphase (C3), and telophase (C4). Bars, 5 μm.