Article Figures & Data
Figures
- Figure 1.
Root-Tip Metaphases with Graphically Presented Chromosomal Types.
(A) and (C) to (I) Double-target FISH with 45S (green) and 5S rDNA (red, arrowed) as probes.
(A) to (E) Bivalent forming species.
(A) and (B) Oe. elata ssp hookeri strain hookeri de Vries.
(B) DAPI gray-scale image (left) of the metaphase plate shown in (A) and its graphical interpretation (right); arrowheads indicate secondary constrictions.
(C) Oe. elata ssp hookeri strain johansen Standard.
(D) Oe. grandiflora strain grandiflora Tuscaloosa.
(E) Oe. glazioviana strain blandina de Vries.
(F) to (I) Species that exhibit rings at meiosis.
(F) Oe. biennis strain suaveolens Grado.
(G) Oe. biennis strain suaveolens Standard.
(H) Oe. villosa ssp villosa strain bauri Standard.
(I) Oe. glazioviana strain r/r-lamarckiana Sweden.
I to VII, a to n, chromosome types distinguished on the basis of statistical calculations, i.e., their lengths and arm ratios are means from Supplemental Tables 2 and 3. Note that this designation does not imply chromosome homology/homoeology between strain or species. Single asterisks, chromosomal markers distinguished by their morphology and arrangement of rDNA loci; double asterisks, chromosomal markers based exclusively on morphology; arrowhead in I indicates the centromere of the chromosome “g” of Oe. glazioviana strain r/r-lamarckiana Sweden. Bars = 5 μm.
- Figure 2.
The Organization of Chromatin States on Chromosomes and within Nuclei.
(A) Oe. elata ssp hookeri strain hookeri de Vries. Giemsa C-banding; arrowheads indicate C-band-positive NOR-heterochromatin.
(B) Oe. elata ssp hookeri strain hookeri de Vries. Sequential C-banding/DAPI (top) and FISH with 45S rDNA (green) and 5S rDNA (red, arrowed) probes (bottom) on early metaphase chromosomes. Note that the subtelomeric regions are still slightly diffuse; arrowheads indicate NOR-heterochromatin.
(C) Oe. villosa ssp villosa strain bauri Standard. C-banding/DAPI on late metaphase chromosomes; arrowheads indicate NOR-heterochromatin.
(D) Oe. biennis strain suaveolens Grado. Immunodetection of H3K9me2 (top, red) and DAPI-stained chromosomes (bottom).
(E) and (F) Oe. elata ssp hookeri strain johansen Standard. On metaphase chromosomes, euchromatin indexed by H3K4me2 (E) or H3K27me3 (F) occupies small distal regions, that is, satellites and segments comparable in size to satellites (merged images, red signals). Arrowheads indicate satellites; arrows indicate regions where NOR-heterochromatin is located.
(G) Oe. biennis strain suaveolens Standard. A fragment of DAPI-stained early prophase from root tip, disturbed by strong squashing. The relative position of centromere-containing early-condensing chromatin blocks (not marked), NOR-heterochromatin blocks (arrows, three of them are marked), and terminal heterochromatin (arrowheads, only two of them are marked) is clearly visible.
(H) to (J) Oe. elata ssp hookeri strain johansen Standard. Distribution of H3K4me2 (H) and H3K27me3 ([I] and [J]) on prophase chromosomes ([H] and [I]) or early metaphase chromosome (J). Until late metaphase, the late-condensing chromatin in Oenothera is not fully condensed. Top, DAPI images; bottom, immunosignals (red) merged with DAPI (blue). Arrowheads indicate DAPI-positive terminal heterochromatin.
(K) and (L) Oe. glazioviana strain r/r-lamarckiana Sweden. Immunodetection of H3K27me2 (red, merged images).
(K) Metaphase plate with H3K27me2-enriched chromosome termini.
(L) DAPI-bright terminal heterochromatin (DAPI staining, blue, top) strictly colocalizes with H3K27me2 immunosignals (red, merged, bottom).
(M) Oe. biennis strain suaveolens Grado. A metaphase chromosome (left, DAPI staining) with secondary constriction and its H3K27me2 pattern (right). Arrowhead indicates the satellite; arrow indicate region where NOR-heterochromatin is located.
(N) Oe. elata ssp hookeri strain hookeri de Vries. Nuclei stained with DAPI (left and right) or CMA3 (middle) with facultative chromocenters and minute blocks of terminal heterochromatin (arrowed in [M], left).
(O) Oe. biennis strain suaveolens Grado. Nucleus with facultative chromocenters (DAPI staining, top) that are negative for H3K4me2 (red, bottom).
(P) Oe. elata ssp hookeri strain johansen Standard. In the nuclei of mature parenchyma, facultative chromocenters are lacking (DAPI staining, top). DAPI-bright terminal heterochromatin is also detectable as spots enriched in H3K27me2 (red, bottom).
(Q) Oe. elata ssp hookeri strain hookeri de Vries. In nuclei from root epidermis, chromocenters are partially decondensed.
(R) Graphical interpretation of how the main chromatin states are organized in cycling nuclei (top), noncycling nuclei deprived of the facultative chromocenters (middle), and noncycling nuclei with facultative chromocenters partially decondensed (bottom). Polarization of nuclear architecture and NOR-heterochromatin were not included in the graphical interpretations.
Bars = 5 μm.
- Figure 3.
Arrangement and Condensation State of Chromatin Domains in Cycling Nuclei.
(A) and (B) F1 hybrid between Oe. elata ssp hookeri strain johansen Standard and Oe. grandiflora strain grandiflora Tuscaloosa. Premeiotic interphase with chromocenters well developed (A) or completely decondensed (B). The chromocenters in premeiotic interphase are more compact than their counterparts from the root meristem. This difference is most likely due to the fact that root meristems, compared with other tissues, were subjected to longer enzymatic treatment and more harshly squashed (to obtain well-spread and flattened metaphase plates).
(C) to (G) Oe. glazioviana strain r/r-lamarckiana Sweden. C-banding/DAPI with standard ([C], [F], and [G]) or prolonged ([D] and [E]) hot saline incubation performed on root-tip nuclei. Facultative chromocenters are still visible in (C) and (F) (some of them marked with arrowheads); however, in (D) and (E) (left image), they could not be differentiated and only terminal heterochromatin and NOR-heterochromatin were clearly detectable. This was due the difference between constitutive and facultative heterochromatin in response to the degradative action of the prolonged incubation in hot SSC. This difference probably reflects structural differences between the two chromatin fractions. The nucleus in (E) was after C-banding/DAPI with prolonged SSC incubation, subjected to double-target FISH with 45S rDNA (middle, five signals) and 5S rDNA (right, two signals) probes. Arrows in (E) indicate NOR-heterochromatin blocks that reside on chromosomes possessing 5S rDNA sites; arrowheads point to the remaining NOR-heterochromatin blocks. The rest of the heterochromatin blocks represent terminal heterochromatin that shows a clear tendency for fusion.
(F) and (G) Nuclei with a clear Rabl arrangement. Arrowheads indicate arbitrarily indicated centromere-containing chromosomal parts; arrows indicate terminal heterochromatin blocks.
(F) Interphase nucleus from root meristem.
(G) Mitotic prophase.
(H) F1 hybrid between Oe. elata ssp hookeri strain johansen Standard and Oe. grandiflora strain grandiflora Tuscaloosa. Meiotic prophase. Centromere-containing condensed chromosome regions are more clustered than they are in somatic prophase.
Bars = 5 μm.
- Figure 4.
Facultative Chromocenters, Translocation Breakpoints, and Their Consequences.
(A) A given facultative chromocenter enters prophase as already condensed centromere-containing chromosomal segment (early condensing chromatin). Euchromatin in prophase is late condensing (late-condensing chromatin) and decreasingly diffuse until middle/late metaphase. It is the change in condensation state of euchromatin that is critical for a total chromosome length during progression of the prophase stage. For simplicity, the illustrated cycling nucleus possesses only seven facultative chromocenters. Only two prophase chromosomes are shown (in boxes). C-banding-positive terminal heterochromatin, C-banding-positive NOR-heterochromatin, and NORs are also shown.
(B) As a consequence of the organization shown in (A), prophase chromosomes can be drawn as a simplified model for a general discussion on translocations in Oenothera. For this illustrative purpose, the term “early-condensing chromatin” and “facultative heterochromatin/facultative chromocenter,” although reflecting different stages (prophase versus interphase), designate the same region, i.e., middle chromosome region. 1 to 3, Schematically depicted three different reciprocal translocations. Breakpoints are indicated by triangles, and translocating segments are within dashed ovals. Translocation 1 does not change chromosome morphology since it involves breakpoints occurring at the junction between middle chromosome regions and euchromatin (yellow triangles). Translocation 2 involves breakpoints within the decondensed (active) 45S rDNA (NOR) and leads to multiplication and diversification of 45S rDNA sites. Translocation 3, involving breakpoints occurring within middle chromosome regions (red triangles), causes significant alterations in chromosome morphology.
(C) Cycling nucleus with the idealized Rabl arrangement. The breakpoint regions (junctions between facultative heterochromatin and euchromatin) are located at the same distance from the centromere pole. Thus, such an organization should facilitate physical interactions between breakpoint sites, allowing regular exchanging of euchromatic segments in Oenothera. For simplicity, only three interphase chromosomes were depicted.
- Figure 5.
Evolution of PTH Triggered by Bivalents with Nonhomologous Middle Regions.
(A) Two structurally heterozygous bivalents, 1a2/1b2 and 3a4/3b4, segregating independently (top panel). The presence of such bivalents, however, reduces fertility to 50% (bottom panel). Out of the four possible gametes, two of them (1a2/3a4 and 1b2/3b4) will miss one of the middle regions and thus are genetically unbalanced and lethal. Only the remaining two (1a2/3b4 and 1b2/3a4) contain both middle regions.
(B) Half of the progeny of the fertile gametes from (A) possesses structurally heterozygous bivalents (1a2/1b2 + 3a4/3b4), and the other half is structurally homozygous but splits into two categories: (1a2/1a2 + 3b4/3b4) and (1b2/1b2 + 3a4/3a4).
(C) The two types of homozygous progeny from (B) differ by their chromosomes. Upon hybridization (top panel), they will produce offspring with structurally heterozygous bivalents (1a2/1b2 and 3b4/3a4) again (bottom panel). Structurally heterozygous bivalents can therefore persist in a population.
(D) Since the plants with two structurally heterozygous bivalents have their fertility reduced to 50% (and only half of the viable offspring is true breeding), there is a selection for the fixation of such meiotic configurations in which nonhomologous middle regions are alternately arranged (top panel). Such an arrangement produces only two types of fully fertile gametes (bottom panel) and the offspring is uniform over generations.
(E) and (F) When more than two heterozygous bivalents are present, the fertility is more severely reduced and larger meiotic rings arise as a consequence. For example, three structurally heterozygous bivalents ([E], left panel) reduce gamete fertility to 25% ([E], right panel). However, the alternate arrangement of the chromosomes in a ring ([F], left panel) still produces 100% of the viable gametes ([F], right panel).
For convenience, only chromosomes involved in translocations are shown, and rings are represented as chains.
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