The Plant Cell, Vol. 24: 4124–4134, October 2012, www.plantcell.org ã 2012 American Society of Plant Biologists. All rights reserved.
Nondisjunction in Favor of a Chromosome: The Mechanism
of Rye B Chromosome Drive during Pollen Mitosis
W
Ali M. Banaei-Moghaddam,a Veit Schubert,a Katrin Kumke,a Oda Weib,a Sonja Klemme,a Kiyotaka Nagaki,b
Jirí Macas,c Mónica González-Sánchez,d Victoria Heredia,d Diana Gómez-Revilla,d Miriam González-García,d
Juan M. Vega,d Maria J. Puertas,d and Andreas Houbena,1
a Leibniz
Institute of Plant Genetics and Crop Plant Research, 06466 Gatersleben, Germany
of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan
c Biology Centre of the Academy of Sciences of the Czech Republic, Institute of Plant Molecular Biology, Ceske Budejovice 37005,
Czech Republic
d Departamento de Genética, Facultad de Biología, Universidad Complutense, 28040 Madrid, Spain
b Institute
B chromosomes (Bs) are supernumerary components of the genome and do not confer any advantages on the organisms that
harbor them. The maintenance of Bs in natural populations is possible by their transmission at higher than Mendelian
frequencies. Although drive is the key for understanding B chromosomes, the mechanism is largely unknown. We provide
direct insights into the cellular mechanism of B chromosome drive in the male gametophyte of rye (Secale cereale). We found
that nondisjunction of Bs is accompanied by centromere activity and is likely caused by extended cohesion of the B sister
chromatids. The B centromere originated from an A centromere, which accumulated B-specific repeats and rearrangements.
Because of unequal spindle formation at the first pollen mitosis, nondisjoined B chromatids preferentially become located
toward the generative pole. The failure to resolve pericentromeric cohesion is under the control of the B-specific nondisjunction
control region. Hence, a combination of nondisjunction and unequal spindle formation at first pollen mitosis results in the
accumulation of Bs in the generative nucleus and therefore ensures their transmission at a higher than expected rate to the next
generation.
INTRODUCTION
Supernumerary or B chromosomes (Bs) are dispensable components of the genomes of numerous plant, fungi, and animal
species. Because most Bs do not confer any advantages on the
organisms that harbor them, they are thought of as parasitic or
selfish elements that persist in populations by making use of the
cellular machinery required for the inheritance and maintenance
of A chromosomes (As) (reviewed in Jones and Houben, 2003).
The maintenance of Bs in natural populations is possible by
their transmission at higher than Mendelian frequencies, and this
enables the maintenance of polymorphisms in populations
(Kimura and Kayano, 1961). The variety of mechanisms, including
segregation failure, by which B chromosomes gain advantage in
transmission are known as accumulation or drive mechanisms.
Depending on the species, B chromosome drive can be premeiotic, meiotic, or postmeiotic. In Poaceae, the drive is postmeiotic, occurring either in the first (rye [Secale cereale]), or in the
second (maize [Zea mays]) mitosis of the pollen grain (reviewed in
Jones, 1991). Drive is the key for understanding B chromosomes
and it occurs in many ways, but the molecular mechanisms remain unclear (Jones, 1991; Burt and Trivers, 2006).
1 Address
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Andreas Houben
([email protected]).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.112.105270
The behavior of rye Bs during pollen mitosis was first studied
by Hasegawa (1934), who described that the two chromatids of
the B chromosome do not separate at anaphase of first pollen
grain mitosis and in most cases are included in the generative
nucleus. In the second pollen mitosis, the generative nucleus
divides to produce two sperm nuclei. The B chromatids, which
are often nondisjoined at first pollen anaphase, segregate normally at second pollen mitosis. The irregular segregation of the
Bs during meiosis, forming univalents at metaphase I and laggards at anaphase I and II (Müntzing and Prakken, 1941), partially explains their non-Mendelian transmission, but the net
increase of B number in the progeny of 0B 3 +B crosses can
only be explained by the directed nondisjunction of the Bs to the
generative nucleus during first pollen mitosis (Müntzing, 1946a).
This mechanism has been observed in rye with up to six Bs
(Kishikawa, 1965). A similar accumulation mechanism of Bs was
reported in other species of the tribe Triticeae (i.e., Aegilops
speltoides; Mendelson and Zohary, 1972).
In rye crosses 0B 3 2B (Bs transmitted via the male) or 2B 3
0B (Bs transmitted on the female side), plants with zero, two, or
four Bs are obtained, but plants with odd numbers of Bs are only
rarely obtained in the progenies (Müntzing, 1945). Therefore,
a similar drive process must be assumed to occur in male and
female gametophytes. Indeed, Håkanson (1948) observed in the
embryo sac during first postmeiotic division an anaphase cell
with lagging B chromosomes as previously observed at pollen
anaphase.
Nondisjunction works equally well when the rye B is introduced
as an addition chromosome into hexaploid wheat (Triticum
B Chromosome Drive during Pollen Mitosis
aestivum; Lindström, 1965; Müntzing, 1970; Niwa et al., 1997;
Endo et al., 2008), hypopentaploid Triticale (Kishikawa and Suzuki,
1982), or Secale vavilovii (Puertas et al., 1985). Thus, the B controls the process of nondisjunction by itself (Matthews and Jones,
1983; Romera et al., 1991).
Deficient Bs (defB) lacking the heterochromatic terminal region of the long arm undergo normal disjunction at first pollen
anaphase. Therefore, it seems that the accumulation mechanism of the B by nondisjunction requires a factor located at the
end of its long arm (Müntzing, 1948; Håkanson, 1959; Endo
et al., 2008). This factor can act in trans because if a standard B
(Lima-De-Faria, 1962) or the terminal region of the long arm of
the B (Endo et al., 2008) is present in the same cell containing
a defB, nondisjunction occurs for both the standard and the
defB. Two B-specific repeat families E3900 (Blunden et al.,
1993) and D1100 (Sandery et al., 1990) reside in the long arm
terminal region. Expression analysis revealed that D1100 and
E3900 are highly transcriptionally active in anthers. In addition,
the distal heterochromatin is marked with the euchromatinspecific histone modification mark H3K4me3 (Carchilan et al.,
2007).
Here, we analyzed the fate of accumulating (standard Bs) and
nonaccumulating Bs (defBs) during microgametogenesis. This
work provides direct insights into the cellular mechanism of rye
B chromosome drive. We identified a B-specific pericentromere
organization and suggest that the failure to separate sister chromatids is caused by a B-specific pericentromere under the influence of the nondisjunction control region. In combination with
asymmetric geometry of the first pollen mitosis, the Bs accumulate preferentially in the generative nucleus, which in turn
enhances the transmission frequency of Bs to the next generation.
If the nondisjunction control region of the B is missing, normal
separation of defB chromatids occurs and A and defB centromeres intermingle in both nuclei of binucleate pollen. Hence,
no accumulation of Bs occurs in generative nuclei.
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hybridized with the rye A and B centromere-specific probe
Bilby, and the B (peri)centromere-specific probe ScCl11-1, or
the B-specific probes E3900 to discriminate between As and Bs.
In both species at anaphase, most Bs lagged behind the As
and exhibited nondisjunction. The resulting nuclei of the bicellular pollen are distinguished by contrasting chromatin configurations: The generative nucleus is more condensed, whereas
the vegetative nucleus is characterized by diffuse chromatin
(Houben et al., 2011). In 2B rye, the Bs were mostly observed
in the generative nucleus (92%). In a few cases, the Bs showed
disjunction (1.5%) or formed micronuclei near the generative
nucleus (6.5%). In 4B plants, B nondisjunction occurred because more than two B signals can be observed in bicellular
pollen. However, many pollen grains showed abnormalities
and preferential distribution of the Bs often (30%) failed (see
Supplemental Figure 2 online).
Notably, the distribution of centromeres differed between the
vegetative and generative nuclei in 0 and +B plants. In the
vegetative nuclei, the A centromeres were loosely distributed,
whereas in the generative nuclei, they were closely grouped
toward the pollen wall (Figure 1C). The A centromeres occupied
a significantly smaller area (calculated based on a maximum
intensity projection) in the generative nucleus (5.95 µm2) than in
RESULTS
A and B Centromeres Occupy Different Positions in the
Generative Nucleus of the Bicellular Pollen Grain
Bs significantly delay the progress of pollen mitosis. Anthers
from 0B, 2B, and 4B plants revealed a mitotic index of 19, 32,
and 52%, respectively (see Supplemental Figure 1 online). In 2B
plants, pollen prophases and metaphases showed a higher index than in 0B, whereas anaphases and telophases showed
a lower index. In 4B plants, the prophase index was much higher
than in 0B and 2B plants, and only a few cells were observed at
meta-, ana-, and telophase. This indicates that 2Bs delay the
first pollen prophase and metaphase, and 4B delays the first
prophase so much that only a few pollen grains are able to
follow the mitotic process normally, resulting in aborted pollen.
To obtain direct insights into the mechanism of B accumulation,
we analyzed the dynamics of chromosomes at microgametogenesis, using tissue sections to maintain the natural arrangement
of chromosomes. Pollen of +B rye (Figure 1) and of +B wheat
(Figures 2A and 2B) undergoing the first mitotic division were
Figure 1. Nondisjunction of Rye Bs.
(A) and (B) Pollen of rye +B at different stages of the first pollen mitosis in
situ hybridized with the rye centromere-specific probe Bilby (in green)
and the B centromere–specific probe ScCl11 (in red). At anaphase, as
a result of nondisjunction, the Bs were unequally distributed (A) and/or
lagged behind (B) the As and formed micronuclei. DAPI, 49,6-diamidino2-phenylindole.
(C) In the vegetative nucleus (V), the A centromeres are loosely distributed, whereas in the generative nucleus (G), the A centromeres are
closely grouped and do not intermingle with B centromeres.
Bs are arrowed. Bar = 10 µm.
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accumulation and the interior positioning of Bs in the generative
nucleus. However, this region does not control the differential
grouping of A centromeres in the generative and vegetative
nuclei.
Directed Nondisjunction of Rye Bs Is Accompanied by
Centromere Activity and Unequal Cell Division
Figure 2. Influence of the Nondisjunction Control Region on the Accumulation Process of Rye Bs.
Pollen of wheat ([A] and [B]) carrying a standard B of rye and of wheat
(C) carrying a defB of rye, missing the control region of nondisjunction at
first pollen anaphase (A) and at bicellular stage ([B] and [C]) in situ hybridized with the rye centromere-specific probe Bilby (in green), the
B-specific probe E3900 (in red), and the centromere repeat CRW2 (in
red). DAPI, 49,6-diamidino-2-phenylindole. Bs are arrowed. Bar = 10 µm.
(A) and (B) At anaphase, due to nondisjunction, standard Bs lag behind
the As (A) and are placed into the generative nucleus (b).
(C) If the nondisjunction control region is missing, normal segregation of
Bs occurs irrespective of the differential grouping of A centromeres in the
generative (G) and vegetative (V) nuclei.
(D) Pollen at bicellular stage of 0B rye probed with Bilby.
the vegetative nucleus (14.55 µm2; n = 12, t test P < 0.0003).
Interestingly, centromeres of As and Bs did not intermingle and
Bs often occupied a different position toward the location of the
cell plate.
To test whether the nondisjunction control region of the B is
required for the contrasting distribution of As and Bs, we analyzed the wheat-rye B addition line BS-2 (Endo et al., 2008),
which is carrying a defB with the nondisjunction control region
missing. Here, a random segregation of defBs was found at first
pollen mitosis, and the centromeres of As and Bs were interspersed (Figure 2C). Like in rye, the centromeres in the vegetative nucleus were loosely distributed, whereas in the generative
nucleus, the centromeres were associated. The same distribution
of centromeres was found in pollen of 0B plants (Figure 2D). Hence,
the nondisjunction control region of the B is required for the
To test whether the absence of centromere activity causes
nondisjunction of Bs, we assayed the centromeric histone H3
variant CENH3 at microgametogenesis. CENH3 was selected
because in mammals (Howman et al., 2000) and Hordeum hybrids (Sanei et al., 2011) its loss results in the failure of chromosome segregation. In parallel, the dynamics of microtubule
fibers was visualized to test whether in rye and wheat the first
pollen mitosis represents an asymmetric cell division, as previously reported for other plants (reviewed in Tanaka, 1997; Borg
et al., 2009).
All centromeres of As and Bs were equally labeled by CENH3
(Figure 3; see Supplemental Figure 3 online). At first pollen
prophase, all chromosomes together with the surrounding tubulin spindle apparatus were located toward the pollen cortex
(see Supplemental Figure 3A online). Next, an asymmetric anaphase spindle was formed. The peripheral generative spindle
bundle is blunt and the pole is nearly in contact with the pollen
cortex. By contrast, the interior spindle bundle is long and pointed
(Figure 3A; see Supplemental Figure 3B online). These are typical
features of an asymmetrical cell division (Borg et al., 2009). At late
anaphase, in rye +B (Figure 3B) and wheat +B (Figure 3D), we
identified CENH3 signals of the same intensity as segregated
As, corresponding with the position of lagging Bs (Figures 1A
and 2B). This suggests that centromere inactivity is not the causal
reason for the lack of separation of B chromatids at first pollen
mitosis. It is more likely that the cohesion between B chromatids
is higher than the microtubule traction force required for the
separation of chromatids.
Microtubules reorganize into the bipolar phragmoplast array
between reforming nuclei at subsequent telophase; consequently,
a curved cell plate is formed separating the generative and vegetative cells (see Supplemental Figure 3C online). In both species,
centromeres of As in the generative nuclei had a smaller distance from each other than in the vegetative nuclei (Figures 3C
and 3E). A small number of CENH3 signals were observed toward
the interior of the generative nucleus localized within the same
region where B centromeres were detected by fluorescence in
situ hybridization (FISH) (Figures 1C and 2B). Likely, the observed
asymmetrical microtubule spindle organization is causing a preferential accumulation of joined B chromatids in the generative
nucleus. At the second pollen mitosis, the spindle organization
was symmetric and both sperm nuclei were equally marked by
CENH3 (see Supplemental Figure 3D online).
The Composition of Centromere Repeats Differs between
As and Bs
The centromeric sequences of rye A and B chromosomes were
compared because of the different segregation behavior of both
types of chromosomes. Therefore, different parts of Ty3/Gypsy-
B Chromosome Drive during Pollen Mitosis
Figure 3. B Centromeres Are Active during Pollen Mitosis.
Distribution of CENH3 (in red) and of a-tubulin (in green) at different
stages of the first pollen mitosis of rye +B ([A] to [C]) and wheat +B ([D]
and [E]). DAPI, 49,6-diamidino-2-phenylindole. Apparent B positions are
arrowed. Bar = 10 µm.
(A) Formation of an asymmetric anaphase spindle. The peripheral generative spindle (G) is blunt and the interior spindle (V) is long and pointed.
(B) At late anaphase, CENH3 signals of lagging chromosomes are
arrowed.
(C) Binucleate pollen with a decondensed vegetative (V) and a condensed generative nucleus (G). Note the differential grouping of CENH3
signals.
(D) Anaphase of wheat +B exhibiting CENH3 signals of lagging chromosomes (arrowed).
(E) In binucleate pollen, the vegetative (V) and the generative nucleus
show ungrouped and clustered CENH3 signals, respectively. In merge
images, 49,6-diamidino-2-phenylindole is shown in blue.
type centromeric retrotransposons like the rye-specific element
Bilby (Francki, 2001) and the sequences originally identified in
wheat centromeric sequences 6C6-3/-4 (Zhang et al., 2004),
192-bp repeat (Ito et al., 2004), CCS1 (Aragón-Alcaide et al.,
1996), and CRW2 (Liu et al., 2008), were in silico compared with
454-sequence reads (Martis et al., 2012) from flow-sorted As
and Bs as well as from genomic DNA of 0B and +4B rye plants.
The frequency pattern of all tested sequences was similar in the
compared sets of 454 reads of As and Bs (see Supplemental
Figure 4 online). All A (peri)centromere-located sequences were
also identified at the B chromosome of rye. The in silico results
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were confirmed by PCR using primers specific for the tested
centromeric sequences (see Supplemental Table 1 online) and
genomic DNA of 0B and +4B rye, 0B and +2B wheat, and DNA
of sorted rye Bs (see Supplemental Figure 5 online). Amplicons
of expected size were obtained and no major sequence differences (GenBank accession numbers JQ963501 to JQ963591)
were identified between As and Bs.
It has been reported for several species that the transcriptional activity of repetitive DNA sequences located in the centromeric heterochromatin is crucial for proper function of the
centromere (Ekwall, 2004; Morey and Avner, 2004; BouzinbaSegard et al., 2006). To test the activity of A and B centromeric
repeats, we analyzed the transcripts of root, leaf, and anther
tissues of rye with and without Bs (see Supplemental Figure 6
online). Each repeat, even different parts of the same repeat
(e.g., ScCl11-1, ScCl11-2, and ScCl11-3 or Sc6C6-3 and
Sc6C6-4) displayed its unique expression pattern. Except for
Sc192 bp, ScCl11-1, and ScCl11-2, all centromeric transcripts
were detectable. The transcription level was highest in anthers.
Bs did obviously not influence the expression of centromeric
repeats.
The centromere localization of all A and B centromere- and of
the B centromere-specific sequences was analyzed by in situ
hybridization and fluorescence wide-field microscopy in somatic
metaphases (Figures 4A and 4B; see Supplemental Figure 7
online). Notably, Bs revealed more extended Bilby and ScCCS1
signals along the centromeres than the As (Figures 4A and 4B).
Using structural illumination microscopy (SIM) to achieve a resolution below the limit of light microscopy, the differential distribution of the rye-specific centromere sequence Bilby became
even more obvious. All As displayed distinct ring-like hybridization signals. By contrast, the centromere of the Bs revealed
an extended and diffuse distribution of Bilby signals (Figure 4C).
To confirm this finding, the volumes of A- and B-located Bilby
signals were comparatively quantified. Compared with As, the
volumes of B-localized Bilby signals occupied a 3.4-fold larger
volume in interphase (n = 57 As and 12 Bs, P < 0.001) and a 2.8fold larger volume in metaphase chromosomes (n = 157 As and
12 Bs, P < 0.001).
The reduced condensation level of pachytene chromosomes
enabled a quantitative length comparison of the higher order
organization of centromeric repeats between As and Bs. In B
centromeres, Bilby repeats are interrupted by insertion of the
ScCl11 B-specific centromeric repeats (Figure 4D). The middle
part of the centromeric region of the B was observed to be
narrower than the rest, whereas this narrower region was not
observed in the As. To avoid possible differences in length
measurements due to colors used for probe detection, both
probes were pairwise detected with Cy3 and fluorescein. The
mean length of the centromeric region was shorter in green Bilby
(10.33 µm) than in red ScCl11 (13.83 µm), (t = 24.302, P value =
0.0002; n = 14). However, the difference was not significant
when the probes were detected with the opposite colors (mean
red Bilby length = 13.13 µm, mean green ScCl11 length = 14.62
µm; t = 21.465, P value = 0.15; n = 15). In 10 pachytene cells
(five of each type of probe detection), the seven A centromeres
could be individually observed. The mean length of the Bilby
signal on the As was significantly shorter than the mean length
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of Bs is more extended and Bs have accumulated additional
sequences, like ScCl11 and mitochondrion-derived sequences.
This suggests that the centromere of the B originated from an
A centromere that underwent subsequent sequence accumulations and rearrangements. The B-specific extension of the
pericentromere seems to be involved in the formation of a
B-specific pericentric heterochromatin.
B Centromere–Specific Sequences Do Not Interact with the
Centromere-Specific Histone Variant CENH3
After the identification of a specific composition of the B centromere, we asked whether the B-located sequences ScCl11
and mitochondrial DNA interact with CENH3-containing nucleosomes and therefore contribute to an active B centromere.
Chromatin immunoprecipitation (ChIP) was performed using
an anti-Os-CENH3 antibody and chromatin from +B rye. No
significant interaction was found between CENH3 and ScCl11 or
mitochondrial sequences (Figure 5A). By contrast, a significant
CENH3 interaction was found for Bilby. Other Ty3/Gypsy-type
sequences, including Sc192 bp and ScCCS1, are targeted by
CENH3 too, although at lower levels. This indicates that different
parts of the rye centromeric retrotransposon differently bind to
CENH3, as previously shown in maize (Zhong et al., 2002)
and rice (Oryza sativa; Nagaki et al., 2005). Hence, the rye B
centromere–specific sequences identified so far are not components of the active B centromere. Consistently, after indirect
immunostaining of somatic chromosomes, no obvious differences were found between the CENH3 signals of As and Bs
(Figure 5B).
Figure 4. The Organization of Rye A and B (Peri)centromeric Regions
Differs.
(A) to (D) Mitotic metaphase chromosomes of rye + Bs (arrowed) labeled
with the A and B centromere-located sequences Bilby ([A] and [C]) as
well as with the B-specific repeats ScCl11 ([A] and [C]) and E3900 (B).
FISH results were analyzed by fluorescence wide-field microscopy (WF)
([A], [B], and [D]) and by SIM (C). In contrast with As, the centromere of
the Bs (arrowed) revealed an extended and diffuse distribution of Bilby
signals. A centromeres displayed distinct ring-like hybridization signals.
DAPI, 49,6-diamidino-2-phenylindole.
(D) Pachytene chromosomes of rye + 2B (arrowed) with the A and B
centromere-located sequences Bilby and with the B-specific repeat
ScCl11. The Bilby signal on the Bs is significantly larger that the signal on
the As.
of the Bilby signal on the B, independent of whether Bilby was
detected in green (5.31 µm in the As versus 9.73 µm in the Bs of
the same cells) or in red (7.79 µm in the As versus 13.46 µm in
the Bs of the same cells). Hence, the Bilby signal of the Bs is
significantly larger than the signal on the As irrespective of the
color used for detection (t = 4.933; P value = 0.0001; n = 10).
We conclude that all A centromere sequences are also present in the centromeres of the Bs. However, the contrasting
distribution of Bilby signals suggests that the higher sequence
organization of A and B centromeres differs. The pericentromere
Figure 5. Not All Sequences of the Rye B (Peri)centromere Interact with
the Centromere-Specific Histone Variant CENH3.
(A) ChIP analysis of (peri)centromeric sequences of rye using anti-OsCENH3. The B-specific repeat E3900 and rDNA were used as negative
control. The columns and error bars represent the average percentage of
immunoprecipitation (IP%) and SD from five independent experiments.
**P < 0.01 and ***P < 0.001 in the Tukey’s honestly significant difference
test.
(B) Immunostaining of a haploid mitotic metaphase cell of rye +2Bs
(arrowed) with anti-Os-CENH3 (in red) as control. Bar = 10 µm.
B Chromosome Drive during Pollen Mitosis
DISCUSSION
We provide direct insight into the cellular and molecular basis
of the non-Mendelian accumulation mechanism of a selfish
rye chromosome. Comparative analysis of the centromere repeat composition revealed differences between A and B chromosomes of rye. Centromere sequences of As are also shared
by the B centromere. In addition, in the Bs, these sequences
are intermingled with and extended by ScCl11 repeats and
mitochondrion-derived DNA. This indicates that the centromere of the B originated from an A chromosome and afterwards new sequences accumulated in the B centromere.
However, ScCl11 and mitochondrial sequences are not part
of the CENH3-containing chromatin but rather represent B-specific
elements of the pericentromere. A comparable composition
was also reported for the maize B centromere. Unlike maize A
chromosomes, in which the centromeres are composed of a
CentC satellite and centromere-specific retrotransposons, the
centromere of the maize B chromosome exhibits a different
sequence composition and organization. CentC and centromerespecific retrotransposons were disrupted by maize B-specific
centromeric sequences (Jin et al., 2005). The B-specific accumulation of repeats in the pericentromere is likely involved in the
formation of pericentric heterochromatin, which is known to play
a role in chromosome segregation (Yamagishi et al., 2008). In
particular, it has been suggested that heterochromatin is required
in sister chromatid cohesion. In fission yeast, repeats flanking the
kinetochore are essential for cohesion (Bernard et al., 2001).
Unequal Cell Division: the Key for the Preferential Drive of
Bs in the Generative Nucleus
An important clue comes from our finding that the microtubule
spindle is asymmetrical during first pollen mitosis in rye and
wheat. The asymmetry of this division plays a critical role in the
determination and subsequent fate of the two unequal daughter
cells, the vegetative and the generative (Twell, 2011). As a result
of the asymmetric cell division and of the subsequent formation
of contrasting interphase structures, the centromeres of the
As are clustered in the more condensed generative nucleus,
whereas in the decondensed vegetative nucleus, the centromeres are distributed over a much larger area. Notably, the A
and B centromeres do not intermingle in the generative nuclei.
Likely, the distinct interphase position of the Bs is a consequence
of B nondisjunction because defBs, lacking the nondisjunction
control region, grouped together with the centromeres of the As.
By contrast, the Bs exhibiting nondisjunction lagged behind the
separated A chromatids during anaphase and were preferentially
located toward the position of the former mitotic equatorial plate.
Considering the asymmetric geometry of the spindle, it is
probable that, as proposed by (Jones, 1991), inclusion of Bs in
the generative nucleus is caused by the fact that the equatorial
plate is closer to the generative pole and lagging Bs are passively included in the generative nuclei as the nuclear membrane
is formed. Alternatively, the Bs may preferentially accumulate in
the generative nucleus due to a higher tension force on the B
centromere toward the generative pole. It has been suggested
that asymmetric spindle positioning in single-cell-stage of
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Caenorhabditis elegans embryos is caused by an initial stochastic different net pulling force on the two microtubule asters.
The increase of these unequal forces through an unknown
mechanism will cause different positioning of the nucleus/
centrosomal complex, which has similar initial positions relative to
anterior-posterior cortical polarity cues (Grill et al., 2001; Siller
and Doe, 2009). Whether a comparable mechanisms exists in
plants remains to be determined. However, it seems that asymmetrical spindles are also a key component of the premeiotic drive
in, for example, the Asteraceae Crepis capillaris (Rutishauser and
Rothlisberger, 1966) and of the meiotic drive of Bs in, for example,
the grasshopper Myrmeleotettix maculatus (Hewitt, 1976). Hence,
the asymmetry of the mitotic spindle seems to be a major component of the B accumulation mechanisms.
Why Does Nondisjunction of Bs Mainly Occur
in Gametophytes?
The discovery that some of the nondisjunction control region–
specific repeats produce noncoding RNA predominantly in
anthers (Carchilan et al., 2007) suggests an intriguing possibility,
namely, that the nondisjunction of Bs occurs because the
control region provides RNA to maintain the cohesion in key
regions of B sister chromatids. One might imagine that the
failure in mitotic segregation reflects a failure to properly resolve
the pericentromeric heterochromatin during first pollen mitosis.
The cell cycle type-specific segregation failure of Bs triggers the
question: In which aspect the first pollen mitosis differs from
other mitotic events in other cell types? We argue that either
a haploid tissue type–specific expression of nondisjunction
controlling transcripts (Carchilan et al., 2007) and/or the formation of a contrasting chromatin composition during first
pollen mitosis (Houben et al., 2011) ensure cell type–specific
accumulation.
Although no similarity between D1100/E3900 and B centromere–located sequences has been found, it is notable that some
similarity exists at the amino acid level between a part of the
E3900 sequence (which encodes a partial reading frame for the
gag protein of an long terminal repeat retrotransposon, most
closely related to a highly conserved Ty3/gypsy family) and
conserved centromeric sequences of CentC and osrch3 in
maize and rice, respectively (Langdon et al., 2000). However,
sequence similarity between noncoding RNA and the target
region seems not to be a functional requirement. For instance,
dosage compensation in both flies and mammals requires noncoding RNAs, which spread in cis to coat the X chromosome. The
regions of the Xist RNA that are required for the localization on the
inactivated X have no obvious sequence similarity (Wutz et al.,
2002). Therefore, it seems possible that B-derived noncoding
RNAs act as guide molecules to direct protein complexes to
specific genomic loci, such as the B pericentromere. Whether
the B transcripts act directly or indirectly in the nondisjunction
process of Bs remains to be answered. However, recent identification of a number of rye B-located genic sequences (Martis et al.,
2012) provides a basis to speculate about a role of protein-coding
genes in nondisjunction control as alternative possibility. For
instance, a specific interaction of proteins and pericentromeric
sequences has been shown in murine fibroblastic cells infected
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by a virus strain (Mansuroglu et al., 2010). Here, the nonstructural protein encoded by this virus interacts specifically with
clusters of the pericentromeric g-satellite sequence of the host
genome. This interaction induces sister chromatid cohesion and
segregation defects.
As proposed by Masonbrink and Birchler (2010), nondisjunction of maize Bs in the sporophyte and the gametophyte are
probably related, but the gametophyte may be a more tolerant
environment for B nondisjunction. In maize, B nondisjunction
also occurs during tapetal programmed cell death (Chiavarino
et al., 2000; González-Sánchez et al., 2004), and in the endosperm (Alfenito and Birchler, 1990) and in plants containing
multiple Bs, the nondisjunction rate in root meristems is increased (Masonbrink and Birchler, 2010). By contrast, in a number
of species, nondisjunction of Bs occurs regularly in somatic tissue
(listed in Jones and Rees, 1982). However, as the drive mechanism of Bs is effective in gametophytes only, abnormal segregation of Bs in somatic tissue will not affect the accumulation of Bs.
It is tempting to speculate that the extended cohesion between
sister chromatids of Bs is also part of their unique behavior during
meiosis if occurring as a univalent. Unlike A chromosomes, univalent Bs of many species (e.g., Poa alpina [Müntzing, 1946b],
Anthoxanthum aristatum [Östergren, 1947], Godetia nutans
[Hakansson, 1945], and rye [Manzanero et al., 2000]), split sister
chromatids much less frequently at anaphase I.
The Drive Mechanism of the B Chromosome of Rye: A Model
Figure 6. Model to Explain the Drive Mechanism of the Rye B Chromosome at Micro Gametogenesis.
(A) to (E) Pollen grain with 7As +1 standard B chromosome.
(F) to (J) In the case that the nondisjunction control region of the B is
missing (defB), no accumulation of the defB occurs.
(A) and (E) At first pollen mitosis, metaphase chromosomes are located
toward the pollen cortex and an asymmetric spindle is formed. The peripheral generative (G) spindle bundle is blunt and the interior spindle
bundle toward the vegetative pole (V) is longer and pointed.
(B) In contrast with the separated A sister chromatids, the majority of Bs
stay in between both spindle poles. The failure to resolve the pericentromeric cohesion of the B is under the control of the B-specific nondisjunction control element. Likely, the B-specific centromere organization
is involved in the formation of a B-specific pericentric heterochromatin.
Despite centromere activity, the cohesion between B chromatids in key
regions is probably stronger than the microtubule traction force required for
the separation of the chromatids.
(C) The placement of Bs toward the generative nucleus is probably realized because the equatorial plate is closer to the generative pole and
lagging Bs are included in the generative nucleus as the nuclear membrane is formed; alternatively, a higher spindle tension force toward the
generative pole might preferentially pull the Bs to the generative nucleus.
Unlike As, nondisjoined Bs accumulate preferentially at a position close
to the phragmoplast.
(D) and (E) During the second pollen mitosis, the generative nucleus
divides to produce two sperm nuclei (S), each with an unreduced number
of Bs.
(F) to (J) If the nondisjunction control region of the B is missing, normal
separation of defB chromatids occurs and A and defB centromeres intermingle (H) in both nuclei of binucleate pollen.
(J) Consequently, no accumulation of the defBs occurs. In addition, the
nondisjunction control region of the B is required for the accumulation
On the basis of the above-mentioned observations, we propose
a model of how the accumulation mechanism works for the B
chromosome of rye (Figure 6). At all mitotic stages of microgametogenesis, the centromeres of As and Bs are active. However, sister chromatid cohesion differs between As and Bs at first
pollen mitosis, and the B-specific pericentromeric repeats are
involved in the formation of pericentric heterochromatin. The failure to resolve the pericentromeric cohesion is under the control of
the B-specific nondisjunction control element. Due to unequal
spindle formation, joined B chromatids become preferentially
located toward the generative pole. As a consequence of a
missing or delayed transport of Bs at anaphase, the B centromeres locate opposite to the clustered A centromeres in the
resulting generative nucleus. In the second pollen mitosis, the
generative nucleus divides to produce two sperm nuclei, each
with an unreduced number of Bs. Hence, a combination of nondisjunction and of unequal spindle formation at first pollen mitosis
results in the directed accumulation of Bs to the generative
nucleus and therefore ensures their transmission at a higher than
Mendelian rate to the next generation.
As in rye, the accumulation mechanism in maize Bs requires
a factor located on the end of the long arm of the B that can act
in trans (Roman, 1947; Carlson, 1978; Lamb et al., 2006), and
the B centromeric heterochromatin, irrespective of centromere
function, is required for efficient nondisjunction (Carlson, 2006;
and for the interior position of Bs in generative nuclei. However, this
region does not control the differential grouping of centromeres of A
chromosomes in generative and vegetative nuclei.
B Chromosome Drive during Pollen Mitosis
Han et al., 2007). As the Bs of rye and maize originated independently, comparable drive mechanisms in both species
evolved in parallel. Although much of repetitive DNA evolution
is governed by neutral evolutionary processes (Charlesworth
et al., 1994), we propose that some B-located repeats, like those
located in the Ab10 maize chromosome involved in neocentromere
meiotic drive (Mroczek et al., 2006), the satellites involved in segregation distortion of Drosophila melanogaster (Frank, 2000)
or in centromere-associated drive in female meiosis (Fishman
and Saunders, 2008) are functionally involved in the regulation
of chromosome segregation to ensure the maintenance of Bs
in natural populations.
METHODS
Plant Material and Plant Cultivation
Plants with Bs from the self-fertile inbred line 7415 of rye (Secale cereale)
(Jimenez et al., 1994) and from hexaploid wheat (Triticum aestivum) cv
Lindström with added standard rye Bs (Lindström, 1965) and hexaploid
wheat cv Chinese Spring with added truncated rye Bs (line BS-2) (Endo
et al., 2008) were grown at the same temperature, humidity, and light
conditions (16 h light, 22°C day/16°C night).
Genomic DNA and RNA Extraction, PCR, and RT-PCR
Genomic DNA was extracted from leaf tissue of rye and wheat with and
without B chromosomes via the DNeasy plant mini kit (Qiagen) according
to the manufacturer’s instructions. Rye Bs were flow-sorted and reamplified as described by Kubaláková et al. (2003). Total RNA was isolated
from roots, leaves, and anthers (staged between meiosis and development
of mature pollen by microscopy) using the Trizol method (Chomczynski and
Sacchi, 1987). The quality of RNA was checked on denaturing agarose gel,
and possible DNA contamination was removed by treatment of total RNA
with DNase (Ambion TURBO DNase; Invitrogen). Absence of DNA contamination was confirmed by PCR using primers specific for Bilby (Francki,
2001) or CRW2 repeats (Liu et al., 2008) (see Supplemental Table 1 online).
cDNA was synthesized with 2.5 µg of DNase-treated total RNA using the
RevertAid H Minus first-strand cDNA synthesis kit (Fermentas) according to
the manufacturer’s instructions. PCR and RT-PCR reaction was performed
in a 25-mL reaction containing 100 ng of genomic DNA or cDNA, respectively,1 mM each primer (see Supplemental Table 1 online), buffer,
deoxynucleotide triphosphates, and 1 unit of Taq polymerase (Qiagen).
Thirty-five amplification cycles (45 s at 95°C, 1 min at corresponding
annealing temperature [see Supplemental Table 1 online], and 1 min at 72°C)
were run. Primers specific for the constitutively expressed GAPDH gene
were used for RT-PCR control.
Generation and Analysis of Rye Centromeric Sequences
The cereal centromeric sequences Bilby (Francki, 2001), 6C6-3/-4 (Zhang
et al., 2004), 192-bp repeat (Ito et al., 2004), CCS1 (Aragón-Alcaide et al.,
1996), and CRW2 (Liu et al., 2008) were compared with rye B-derived
sequence 454 reads (European Bioinformatics Institute–European Nucleotide Archive accession number ERP001061) by BLAST to identify
similar B-located sequences for primer design (see Supplemental Table 1
online). In addition, their frequencies on A and B chromosomes were
determined by PROFREP algorithm (Macas et al., 2007). PCR products from
+B rye DNA were purified and used as FISH and/or dot blot hybridization
probes. PCR products obtained from genomic rye 0B DNA and sorted rye B
chromosomes were cloned and sequenced. Sequences were deposited in
the GenBank database under accession numbers JQ963501 to JQ963591.
4131
FISH and Indirect Immunostaining
Preparations of semithin pollen sections and of root tip meristems as well
as subsequent FISH or indirect immunostaining were performed according to Houben et al. (2011). FISH on pachytene chromosomes was
performed as described (González-García et al., 2006). FISH probes were
labeled with ChromaTide Texas Red-12-dUTP, Alexa Fluor 488-5-dUTP
(Invitrogen), or Cy5-dUTP (GE Healthcare Life Sciences) by nick translation. The primary antibodies, mouse anti-a tubulin (Sigma-Aldrich;
catalog number T 9026; diluted 1:100), and rabbit anti-Os-CENH3 (Nagaki
et al., 2004) were used for indirect immunostaining. The anti-Os-CENH3
antibody is able to recognize CENH3 of all tested grass species, including
rye and common wheat. Imaging was performed using an Olympus BX61
microscope and an ORCA-ER charge-coupled device camera (Hamamatsu). Deconvolution microscopy was employed to remove out-of-focus
information. All images were collected in gray scale and pseudocolored
with Adobe Photoshop 6. Maximum intensity projections were processed
with the program AnalySIS (Soft Imaging System).
Quantification of Fluorescence Signals
and Super-Resolution Microscopy
Quantification of FISH signals was performed with an epifluorescence
microscope (Zeiss Axiophot) using a 3100/1.45 Zeiss a plan-fluar objective and a three-chip Sony color camera (DXC-950P). The microscope
was integrated into a Digital Optical 3D Microscope system (Confovis) to
measure the signals along x, y, and z axes as a basis to calculate the signal
volumes. ImageJ software (Collins, 2007) was used to determine the
length of centromeric regions of pachytene chromosomes. To measure
the areas occupied by CENH3 signals within the vegetative and generative nuclei, maximum intensity projections from image stacks acquired
by the Olympus BX61 microscope and processed with SIS software
(Olympus) were used. To achieve an optical resolution of ;100 nm, we
applied SIM using a 363/1.40 objective of an Elyra super-resolution
microscope system (Zeiss).
ChIP Assay
ChIP was performed as described by Nagaki et al. (2003) using young rye
leaves with Bs and the anti-Os-CENH3 antibody (Nagaki et al., 2004).
ChIP without antibody served as a mock treatment. Briefly, after incubation with the antibody, the chromatin solution was separated into
supernatant (Sup) and pellet (Pel) fractions. DNA was blotted on Hybond
N+ membranes (GE) and used for dot-blot hybridization with rye (peri)
centromere-localized sequences (Bilby, Sc6C6-4, Sc192 bp, ScCCS1,
ScCRW2, ScCl11-2, and mitochondrial DNA [barley (Hordeum vulgare)
BAC MmHB 0205G01]) and noncentromeric repeats (E3900 and 45S
rDNA) (pTa71; Gerlach and Bedbrook, 1979). The probes were radiolabeled by [a-32P]dCTP using the DecaLabel DNA labeling kit (Fermentas)
and hybridized to the ChIP DNA on the membranes. The signals were
captured by a phosphor imager (Fujifilm FLA-5100) and quantified by TINA
2.09 software. In each case, the percentage of immunoprecipitation
[defined as Pel/(Pel + Sup)] of the mock experiments was subtracted from
the percentage of immunoprecipitation of the antibody to CENH3 treatments (IP% = the percentage of immunoprecipitation [CENH3] 2 the
percentage of immunoprecipitation [Mock]). Each experiment was repeated
in five independent reactions. The data were analyzed statistically by
pairwise comparison of the IP% of rye centromere-localized sequences
and the IP% of rDNA using the Tukey’s honestly significant difference test.
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers JQ963501 to JQ963591.
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Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Bs Significantly Delay the Progress of Pollen
Mitosis.
Supplemental Figure 2. Pollen of Rye +4B Plants at Bicellular Stage.
Supplemental Figure 3. Distribution of CENH3 and of a-Tubulin at
Pollen Mitosis of Rye +B.
Supplemental Figure 4. Sequence Comparison of A and B Centromere-Located Repeats.
Supplemental Figure 5. PCR Amplification of the Centromeric
Repeats of Plants with and without Bs.
Supplemental Figure 6. Transcription Analysis of the (Peri)centromeric Repeats of Rye with and without Bs.
Supplemental Figure 7. Distribution of Centromeric Sequences along
A and B Chromosomes of Rye.
Supplemental Table 1. Primer Sequences and PCR Conditions.
ACKNOWLEDGMENTS
We thank Takashi Endo for providing the wheat-rye B addition line BS-2
and Pavel Neumann, Ingo Schubert, and R. Neil Jones for fruitful
discussions. We also thank Karla Meier for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (HO 1779/10-1/14-1) and by Spanish Grants BFU 2006-10921,
CCG10-UCM/GEN-5559, and AGL 2011-28542. M.G.-G. was supported
by a grant from the Ministry of Education of Spain.
AUTHOR CONTRIBUTIONS
A.H. designed the research. A.M.B.-M, V.S., K.K., O.W., S.K., K.N., J.M.,
M.G.-S., V.H., D.G.-R., M.G.-G., J.M.V., and M.J.P. performed research.
A.M.B.-M., M.J.P., and A.H. wrote the article.
Received September 17, 2012; revised September 17, 2012; accepted
September 30, 2012; published October 26, 2012.
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Pollen Mitosis
Ali M. Banaei-Moghaddam, Veit Schubert, Katrin Kumke, Oda Weiβ, Sonja Klemme, Kiyotaka Nagaki,
Jirí Macas, Mónica González-Sánchez, Victoria Heredia, Diana Gómez-Revilla, Miriam
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Plant Cell 2012;24;4124-4134; originally published online October 26, 2012;
DOI 10.1105/tpc.112.105270
This information is current as of June 17, 2017
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References
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