Copyright  2000 by the Genetics Society of America
Chromosome Nondisjunction and Instabilities in Tapetal Cells Are Affected by
B Chromosomes in Maize
A. Mauricio Chiavarino, Marcela Rosato, Silvia Manzanero, Guillermo Jiménez,
Mónica González-Sánchez and Marı́a J. Puertas
Departamento de Genética, Facultad de Biologı́a, Universidad Complutense, 28040 Madrid, Spain
Manuscript received December 10, 1999
Accepted for publication February 21, 2000
ABSTRACT
Abnormal mitosis occurs in maize tapetum, producing binucleate cells that later disintegrate, following
a pattern of programmed cell death. FISH allowed us to observe chromosome nondisjunction and micronucleus formation in binucleate cells, using DNA probes specific to B chromosomes (B’s), knobbed chromosomes, and the chromosome 6 (NOR) of maize. All chromosome types seem to be involved in micronucleus
formation, but the B’s form more micronuclei than do knobbed chromosomes and knobbed chromosomes
form more than do chromosomes without knobs. Micronuclei were more frequent in 1B plants and in a
genotype selected for low B transmission rate. Nondisjunction was observed in all types of FISH-labeled
chromosomes. In addition, unlabeled bridges and delayed chromatids were observed in the last telophase
before binucleate cell formation, suggesting that nondisjunction might occur in all chromosomes of the
maize complement. B nondisjunction is known to occur in the second pollen mitosis and in the endosperm,
but it was not previously reported in other tissues. This is also a new report of nondisjunction of chromosomes of the normal set (A’s) in tapetal cells. Our results support the conclusion that nondisjunction and
micronucleus formation are regular events in the process of the tapetal cell death program, but B’s strongly
increase A chromosome instability.
T
HE tapetum is the innermost cell layer that lines
each anther locule. It is in immediate contact with
the sporogenous tissue to the inside and to the middle
layer to the outside of the anther. Tapetal tissue has a
secretory role, providing nutrients required for microspore and pollen grain development (reviewed in Pacini 1997; Raghavan 1997). Particularly in Poaceae, all
pollen mother cells and pollen grains remain in physical
contact with the tapetum at every stage of development,
because a single cylinder of pollen, one cell thick, is
arranged in a locule such that each grain is in contact
with the tapetum (Kirpes et al. 1996).
The tapetum is in a dynamic state during its short
life period, lasting from primary parietal cell formation
until the dehiscent pollen stage when anther walls break
down to allow pollination. Tapetal cells begin to synthesize DNA after the microsporocytes enter meiosis (Heslop-Harrison and Mackenzie 1967). During meiosis
they undergo aberrant divisions producing bi- or multinucleate cells. Marked RNA synthesis occurs in the tapetal cells at all stages of meiosis with a peak at diplotene
(Williams and Heslop-Harrison 1979; Raghavan et
al. 1992), indicating that long before the tapetum begins
to disintegrate it acquires a complex set of specifications
in the form of mRNAs.
Corresponding author: Marı́a J. Puertas, Departamento de Genética,
Facultad de Biologı́a, Universidad Complutense, 28040 Madrid, Spain.
E-mail: [email protected]
Genetics 155: 889–897 ( June 2000)
Recent investigations have correlated the expression
of certain unique classes of mRNAs with the differentiated state of the tapetum. Yokoi et al. (1997) first demonstrated that an anther-specific promoter directed tapetum-specific expression in rice. Huang et al. (1997)
characterized a tapetum-specific transcript in Lilium longiflorum temporarily expressed during microspore development. In Brassica oleracea, pollen grains are covered
with a lipophilic pollen coat containing several forms
of oleosin. Different transcripts that originate from a
single gene whose expression is restricted to the tapetum encode the forms (Ruiter et al. 1997). In Zea mays,
the enzyme xylanase is present in the pollen coat and
the Xyl gene was found to be specifically expressed in
the tapetum after the tetrads had become individual
microspores (Bih et al. 1999). Alché et al. (1999) reported the localization of the protein Ole e I within the
pollen wall and in the tapetum. The transcripts are
present in both the microspores and the tapetum and
absent in other tissues.
In early articles, abnormal divisions of tapetal nuclei
have been reported in a number of species (Maheswari
1950), and tapetal ontogeny and its role in pollen maturation at the ultrastructural level has been reviewed by
Echlin (1973). These investigations suggest that nuclear aberrations are an intrinsic feature of tapetal cytology. As a matter of fact, there are a large number of
articles reporting that male sterility is associated with
disturbances in the regular pathway to tapetal degenera-
890
A. M. Chiavarino et al.
tion (Lee et al. 1979; reviewed in Raghavan 1997);
and certain male sterile mutants show abnormal tapetal
development (Aarts et al. 1997; Jin et al. 1997). Recently, it has been pointed out that the tapetal degeneration is not an uncontrolled event, but Papini et al. (1999)
and Wang et al. (1999) proposed that it is a process of
programmed cell death (PCD).
In maize, tapetal cells undergo the last mitosis when
the microspores are at leptotene-zygotene stage, but cell
division is not complete since cytokinesis does not take
place and the cell becomes binucleate at pachytene. At
about the dyad stage, restitution 4n nuclei are common.
The main tapetal RNA synthesis occurs during meiotic
prophase, with a further period of accumulation in the
interval tetrad to young spores. Protein accumulation
occurs up to midmeiotic prophase; after this there is a
pause, followed by further synthesis from meiotic metaphase I to the final dissolution of the tissue (Carniel
1961; Moss and Heslop-Harrison 1967).
The present work uses fluorescence in situ hybridization (FISH) with probes specific to B chromosomes
(B’s), knobbed chromosomes, and the nucleolar organizer region (NOR), located in chromosome 6, to study
chromosome nondisjunction and micronucleus formation during the last tapetal mitosis, which gives rise to
the binucleate tapetal tissue. Both nondisjunction and
micronuclei are produced by abnormal segregation of
chromatids at anaphase. Micronuclei are produced by
lagging chromatids not included in the poles and nondisjunction is produced when the chromatids fail to
divide and both are included in the same pole. Micronuclei might also be related with nondisjunction
when the centromeres do not separate and the chromosome lags at the plate.
We use maize anthers from plants with and without
B chromosomes belonging to lines selected for high or
low B transmission rate in female 1B ⫻ male 0B crosses
(Rosato et al. 1996).
MATERIALS AND METHODS
Plants (0B and 1B) of the female high (H) and low (L) B
transmission rate lines (Rosato et al. 1996) of Pisingallo, a
native race of Zea mays from northwest Argentina (Rosato et
al. 1998), were used in the present study.
Root tips were scored for B number, and then the plants
were grown in an experimental field. Male inflorescences at
meiosis were fixed in 3:1 ethanol:acetic acid and refrigerated
at 4⬚ until analyzed. Anther squashes for determining the
convenient meiotic stage were made in 1% acetocarmine.
Binucleate tapetal cells were scored in anthers from diplotenemetaphase I using Feulgen staining. The last tapetal mitosis
was found in anthers at zygotene.
The spreading procedure was chosen to prepare nuclei for
in situ hybridization because no mechanical pressure is applied
to distribute the cells on the slides, thus largely preserving
the three-dimensional information. For cell wall digestion,
anthers were incubated in 0.3% (w/v) cytohelicase (Sepracor,
France), 0.3% (w/v) pectolyase (Sigma, St. Louis; P-3026) and
0.3% (w/v) cellulase Onozuka RS (Yakult Honsa, Tokyo) in
10 mm citrate buffer, pH 4.6, for 1 hr at 37⬚. Spread prepara-
tions of macerated anthers for in situ hybridization were made
according to Zhong et al. (1996).
The following repetitive DNA sequences were used in FISH
as probes:
1. pZmBs, a clone containing the maize B chromosome-specific sequence (Alfenito and Birchler 1993), kindly provided by J. A. Birchler (Columbia, MO). This probe labels
the B centromeric regions and, in especially good slides,
a small B telomeric region is also labeled.
2. pZm4-21, a clone containing the maize 180-bp knob repeat
(Peacock et al. 1981), kindly provided by J. A. Birchler.
3. pTa71, a clone containing the rDNA gene unit, the 5.8S,
18S, and 28S genes and the intergenic spacer from Triticum
aestivum (Gerlach and Bedbrook 1979).
The probes were labeled by nick translation with biotin16-dUTP (Boehringer Mannheim, Mannheim, Germany;
1093070000) or digoxigenin-11-dUTP (Boehringer Mannheim 1093088000), using a nick translation kit (Boehringer
Mannheim 976776).
Slides were incubated with RNase A (1 ␮g/ml, Sigma) in
2⫻ SSC for 1 hr at 37⬚; 1⫻ SSC is 0.15 m NaCl, 0.015 m sodium
citrate. Subsequently the slides were rinsed three times for 5
min in 2⫻ SSC, fixed in 4% paraformaldehyde (Sigma) in
1⫻ SSC for 10 min at room temperature, washed in 2⫻ SSC,
and then sequentially dehydrated in an ethanol series of 70,
95, and 100%, 3 min each, and air-dried. Prior to hybridization, the chromosome preparations were denatured in 70%
(v/v) formamide in 2⫻ SSC at 62⬚ for 1 min, dehydrated
through an ice-cold ethanol series, and air-dried.
The hybridization mixture, containing 2 ng/ml of a specific
repetitive probe, was denatured by boiling for 10 min,
quenched on ice for 7 min, and added to the denatured slides.
Hybridization was performed overnight at 37⬚. Posthybridization washes of the slides were done with 2⫻ SSC at room
temperature and 1⫻ SSC at 37⬚, both for 30 min. Biotinlabeled probe detection was performed with avidin conjugated
to Cy3 (Amersham PA 43000). Digoxigenin-labeled probe indirect detection was performed with mouse anti-digoxigenin
(Boehringer Mannheim 1333062). The secondary antibody
was anti-mouse conjugated to fluorescein isothiocyanate
(Boehringer Mannheim 124616). After detection, the slides
were washed in detection buffer (4⫻ SSC, 0.2% Tween 20)
and counterstained with 4⬘,6-diamidino-2-phenylindole (DAPI;
Boehringer Mannheim 236276). Slides were mounted in Vectashield (Vector, Burlingame, CA; H 1000).
Hybridization signals were photographed, using an epifluorescence Olympus microscope, on Fujicolor Professional 400
NPH film and the negatives were scanned at 1350 dpi with a
Nikon film scanner. The images were optimized for best contrast and brightness by means of commercial image-processing
software.
A sample of root tips was studied for control. The roots
were not subjected to any pretreatment so that we could observe normal mitotic anaphases and telophases. FISH in the
root tips was done using the same probes and procedure as
described above for the anthers.
RESULTS
Binucleate tapetal cells were scored with standard
Feulgen staining for the presence of micronuclei in 0B
and 1B plants of the L and H lines, using at least six
anthers per individual. Table 1 shows that the frequency
of micronuclei in 1B plants was 12.63% in the L line,
whereas only 3.92% of the tapetal cells showed micronuclei in the H line. A two-way ANOVA showed that
Nondisjunction in the Tapetum
891
TABLE 1
Binucleate tapetal cells with and without micronucleus in 1B and 0B plants of the L and H lines
No. of cells
B transmission line
L
4 individuals
L
2 individuals
H
4 individuals
H
2 individuals
B number
Without Mn
With Mn
Total
1B
9850
(87.37)
1775
(98.17)
6081
(96.08)
1583
(99.00)
1424
(12.63)
33
(1.83)
248
(3.92)
16
(1.00)
11,274
0B
1B
0B
1,808
6,329
1,599
Percentages are shown in parentheses. Mn, micronucleus.
there are nonsignificant differences between individuals
within lines, but there are significant differences between lines (F ⫽ 28.85; P ⬍ 0.000001) and between 0B
and 1B plants (F ⫽ 20.08; P ⬍ 0.000001). The interaction
is also significant (F ⫽ 24.14; P ⬍ 0.000001), indicating
that the B dose differently affects the H or L genotype.
Table 1 shows that micronuclei are mainly formed in
the L line, but not necessarily corresponding to the B’s,
because there are micronuclei in 0B plants.
To test the frequency of micronuclei corresponding
to the B’s, we carried out FISH with the pZmBs probe,
specifically labeling 1B plants of both lines. The results
are summarized in Table 2. In this table, cells with and
without micronuclei have to be considered separately,
because the cells with micronuclei were preferentially
scored to study the distribution of the B label. In most
cells, B labels were normally distributed (Figure 1A),
but in a number of cases, two B labels were found in
the same nucleus of the binucleate cell, indicating that
B nondisjunction had occurred in the preceding mitosis
(Figure 1B). A contingency ␹2 test showed that there
are significant differences between lines (␹2 ⫽ 13.08,
1 d.f., P ⫽ 0.0003), indicating that the frequency of B
nondisjunction in the tapetal cells is higher in the L
line.
Surprisingly, only 16.47% of the binucleate cells of
the L line with micronuclei showed the B label in the
micronucleus (Figure 1C), indicating that most micronuclei do not correspond to the B and that A chromosomes (A’s) are also unstable in tapetal mitosis (Figure 1, D and E). Nevertheless, the B’s seem to be more
unstable, because if all chromosomes were equally unstable the expected frequency of B micronuclei would
be 1/21 ⫽ 4.76%, because 21 chromosomes are present;
but a frequency of 16.47% is found (Table 2).
A contingency ␹2 test was made to test if B nondisjunction was related to the formation of A chromosome
micronuclei, resulting in significant differences (␹2 ⫽
5.05, 1 d.f., P ⬍ 0.025). The deviation is such that the
number of cells with B nondisjunction plus A micronuclei is higher than expected and, conversely, the
number of cells with B normal disjunction plus A micronuclei is lower than expected. In the H line the
number of cells with micronuclei is very low, and no
calculations can be made.
To determine the nature of the A chromosomes forming micronuclei we used FISH with the pZmBs and the
pZm4-21 probes, specific to the maize B’s and to the
heterochromatic knobs, respectively.
At root tip mitotic metaphase, it was determined that
this maize race is polymorphic for the heterochromatic
knobs, with five large and at least three small knobs. In
TABLE 2
Types of binucleate tapetal cells observed with the pZmBs probe in 1B plants
Types of cells
B
transmission
line
Normal
B nondisjunction
Unlabeled Mn
B nondisjunction and Mn
Labeled Mn
L
8 individuals
H
3 individuals
517
(63.51)
179
(76.17)
297
(36.49)
56
(23.83)
79
(44.89)
68
(38.64)
1
(50.00)
29
(16.47)
1
(50.00)
Without Mn
With Mn
Percentages are shown in parentheses. Mn, micronucleus.
892
A. M. Chiavarino et al.
Figure 1.—(A–E) Localization of the B-specific probe (red) and the knob-specific probe (green) in binucleate tapetal cells.
(A) Normal disjunction of the B’s and unequal knob distribution. (B) B nondisjunction and unequal knob distribution. The
labels corresponding to the centromere and telomere of the two B chromatids are visible side by side. (C) B in the micronucleus.
Equal knob distribution. (D) Normal disjunction of the B’s. Knob in the micronucleus. (E) B nondisjunction. Equal knob
distribution. Unlabeled micronucleus. (F and G) Localization of the B-specific probe (red) and the specific probe for chromosome
6 (green) in binucleate tapetal cells. (F) Normal distribution of the B and nondisjunction of chromosome 6. (G) Nondisjunction
of both the B and chromosome 6. (H and I) Localization of the B-specific probe (red) and the chromosome 6-specific probe
(green) in the last tapetal telophase before binucleate cell formation. (H) B nondisjunction, chromosome 6 label on the bridge.
(I) B nondisjunction, normal disjunction of chromosome 6, unlabeled delayed chromatid and bridge. ( J–L) Localization of the
knob-specific probe (green) in the last tapetal telophase. ( J) Knob in a delayed chromatid. (K) Unlabeled delayed chromatid.
(L) Knob on the bridge.
particular, chromosome 6 shows one small knob on the
short arm and a DAPI⫹ interstitial band on the long
arm. The NOR is located on the short arm. The large
number of knobs makes it difficult to count the number
of knobs per nucleus. Even so, unequal knob distribu-
tion between the two nuclei of the binucleate cell was
evident in a number of cells (Figure 1, A and B). This
indicates that not only the B, but also the knob carrying
chromosomes undergo nondisjunction in the tapetal
mitosis.
Nondisjunction in the Tapetum
893
TABLE 3
Types of binucleate tapetal cells without micronucleus observed with the pZmBs
and the pZm4-21 probes in 1B plants
Types of cells
B transmission line
Normal
Normal for the B’s,
unequal knob
distribution
L
4 individuals
H
2 individuals
87
(34.94)
71
(51.45)
63
(25.30)
33
(23.91)
B nondisjunction,
equal knob
distribution
B nondisjunction,
unequal knob
distribution
54
(21.69)
15
(10.87)
45
(18.07)
19
(13.77)
Percentages are shown in parentheses.
Also in this case we scored cells with and without
micronuclei separately. Table 3 shows the types of cells
without micronuclei observed with both probes in 1B
plants of both lines. All four possible combinations were
found: normal disjunction of B’s and knobs, B disjunction and unequal distribution of knobs, B nondisjunction and equal distribution of knobs, and B nondisjunction and unequal distribution of knobs. A contingency ␹2 test was made between the observed and the
expected distributions assuming independent assortment, resulting in nonsignificant differences for the L
line (␹2 ⫽ 0.29, 1 d.f., P ⫽ 0.59), whereas in the H line
the distributions significantly differ (␹2 ⫽ 6.36, 1 d.f.,
P ⫽ 0.01).
B nondisjunction frequency is 0.3976, and the frequency of knob unequal distribution is 0.4337 in the L
line. In the H line B nondisjunction frequency is 0.2464
and the frequency of knob unequal distribution is
0.3768 (Table 3). Since there is only one B and there
are eight chromosomes with knobs, it seems that the B
tends to undergo nondisjunction with higher frequency
than knobbed A chromosomes. B nondisjunction frequency is significantly higher in the L line (␹2 ⫽ 9.0,
1 d.f., P ⬍ 0.003), but the frequency of unequal distribution of knobs is similar in both lines (␹2 ⫽ 1.19, 1 d.f.,
P ⫽ 0.28).
The double FISH allowed us to determine that three
types of micronuclei can be formed: B micronuclei,
knob micronuclei, and micronuclei without label corresponding to A chromosomes or chromosome fragments
lacking knobs (Figure 1, C, D, and E, respectively). In
the L line we found 42 binucleate cells with one micronucleus, 6 showing B label (14.28%), 25 showing knob
label (59.52%), and 11 without label (26.19%). As there
is 1 B chromosome and there are 8 knobbed chromosomes and 12 chromosomes without knobs, the probability of forming micronuclei decreases in the order B ⬎
knobbed chromosome ⬎ chromosome without knob.
Cells showing the three types of micronuclei were found
in all eight possible combinations between B disjunction/nondisjunction and equal/unequal knob distribu-
tion. The small number of cells in some classes did not
allow determining if the eight classes appeared with
random assortment.
In the H line only five cells with a micronucleus were
found, two with B label and three with knob label, indicating again that all chromosomes form fewer micronuclei in this line.
Table 4 shows the types of binucleate tapetal cells
observed in 0B and 1B plants using the pTa71 probe,
specific to chromosome 6, which is the only maize chromosome carrying the nucleolar organizing region, located on the short arm. Cells with nondisjunction of one
or both chromosome 6’s were observed. Nonsignificant
differences were found between the number of cells
undergoing nondisjunction of chromosome 6 in the L
and H lines, irrespective of the presence of B’s (␹2 ⫽
0.23, 1 d.f., P ⫽ 0.63), but nondisjunction of chromosome 6 was more frequent in 1B than in 0B plants (␹2 ⫽
28.4, 1 d.f., P ⫽ 0.00001). Cells with micronuclei were
observed only in the L line indicating again that this
line is prone to micronucleus formation.
The behavior of the B chromosome and chromosome
6 was studied carrying out FISH with the pZmBs and
the pTa71 probes. The types of binucleate cells without
micronuclei are shown in Table 5 and Figure 1, F and
G. A contingency ␹2 test showed that nondisjunction of
the B chromosome and nondisjunction of chromosome
6 do not occur as independent events in the L line
(␹2 ⫽12.02, 1 d.f., P ⫽ 0.0005). The deviation is such
that more cells are observed than expected when both
chromosomes undergo normal disjunction or both
chromosomes undergo nondisjunction. Conversely,
fewer cells are observed than expected when only one
chromosome undergoes nondisjunction. However, the
contingency ␹2 test showed that nondisjunction of the
B chromosome and nondisjunction of chromosome 6
occur as independent events in the H line (␹2 ⫽ 2.26,
1 d.f., P ⫽ 0.09). The frequency of nondisjunction of
chromosome 6 is 0.1784 in the L line and 0.1172 in the
H line.
In the L line, 3 cells showed a micronucleus with the
894
A. M. Chiavarino et al.
TABLE 4
Types of binucleate tapetal cells observed with the pTa71 probe, which labels the NOR
in chromosome 6, in 0B and 1B plants
Types of cells
Without Mn
B transmission
line
L
5 individuals
L
2 individuals
H
4 individuals
H
2 individuals
B
number
0B
1B
0B
1B
Normal
Nondisjunction of
one chromosome 6
435
(94.36)
161
(83.85)
365
(94.32)
96
(84.95)
26
(5.64)
30
(15.63)
22
(5.68)
17
(15.05)
With Mn
Nondisjunction of
both chromosome 6’s
1
(0.52)
Chromosome
6 in Mn
Unlabeled
Mn
1
2
1
Percentages are shown in parentheses. Mn, micronucleus.
B label (13.64%), 5 showed the chromosome 6 label
(22.73%), and 14 showed no label (63.63%). In the H
line only 3 cells with a micronucleus were found.
The same probes already described were used for
FISH in four anthers of 1B plants of the L line, where
pollen mother cells were at the zygotene stage, to observe the last tapetal mitosis, just before the binucleate
cell formation. Bridges and delayed chromatids were
commonly observed either labeled or unlabeled (Figure
1, H–L). The delayed unlabeled chromatids indicate
that A chromosomes without heterochromatic knobs
may also suffer an abnormal mitosis.
To compare the tapetal mitosis with cell division in
the root meristem, 100 telophases of root tip cells were
studied as controls in two 1B plants of the L and two
of the H line. All labels studied were normally distributed in 100% of the cases, indicating that nondisjunction or any type of chromosome instability does not
occur either for the B or for the A chromosomes in any
of the lines.
DISCUSSION
The use of FISH with DNA probes specific to B chromosomes, knobbed chromosomes, and chromosome 6
(NOR) has allowed us to detect that these particular
chromosomes, and probably all maize chromosomes,
undergo a peculiar behavior in binucleate tapetal cells.
Nondisjunction of A chromosomes and micronuclei
occur in tapetal cells of plants with the normal 0B chromosome complement, indicating that these aberrations
are regular events in the process of anther maturation,
although they occur at low frequency. However, the
presence of 1B chromosome increases the frequency
of both events, whereas micronucleus frequency in 1B
plants is particularly increased in the low B transmission
rate line. This indicates that both the B’s and the genotype influence A chromosome instability.
Micronucleus formation seems to be more a controlled than a random event. First, not all chromosomes
were found forming micronuclei with the same frequency, but the B’s form more micronuclei than do the
knobbed A’s, and the knobbed A’s form more micronuclei
than do the A’s lacking heterochromatic knobs. Second,
binucleate cells with two micronuclei were not found,
and they should have been observed according to the
frequency of cells with one micronucleus. For example,
in 1B plants of the L line the frequency of binucleate
cells with one micronucleus is 0.1263 (Table 1). The
random probability of forming two micronuclei is
(0.1263)2 ⫽ 0.0159; therefore (0.0159 ⫻ 11,274) ⫽
179.84 cells with two micronuclei are expected, but
none were observed.
Alternatively, it is possible that two micronuclei were
not observed because both fused in a single restitution
micronucleus; however, the probability that this occurred in all cases seems to be negligible.
Nondisjunction of B chromosomes is known to occur
typically at the second pollen mitosis (Randolph 1941;
Roman 1947; Carlson 1986). It has been recently reported to occur also at the first pollen mitosis (Rusche
et al. 1997) and in the endosperm (Alfenito and
Birchler 1990), but it was not previously reported in
other tissues. The B-specific probe allowed us to describe this phenomenon in tapetal cells. Similarly, the
other specific probes detected nondisjunction of the
knobbed A chromosomes including chromosome 6
(NOR). This is also a new report of A chromosome
nondisjunction in tapetal cells.
It should be noted that B nondisjunction is more
frequent than nondisjunction of a particular knobbed
chromosome, assuming that all knobbed chromosomes
have the same probability to undergo nondisjunction.
1
(0.32)
1
(0.27)
36
(9.72)
15
(4.84)
99
(26.76)
85
(27.42)
Percentages are shown in parentheses.
205
(55.41)
191
(61.61)
L
4 individuals
H
4 individuals
29
(7.84)
18
(5.81)
Normal
B transmission line
Normal for the B’s,
nondisjunction of
chromosome 6
B nondisjunction,
normal for
chromosome 6
One chromosome
6 and B
nondisjunction
Normal for
the B’s,
nondisjunction
of both
chromosome 6’s
Types of cells
Types of binucleate tapetal cells without micronucleus observed with the pZmBs and the pTa71 probes in 1B plants
TABLE 5
B nondisjunction,
nondisjunction
of both
chromosome 6’s
Nondisjunction in the Tapetum
895
But the most remarkable result is that nondisjunction
of A chromosomes is significantly increased from 5 to
15% by the presence of B chromosomes (Table 4), although it is not influenced by the genotype H or L.
Our hypothesis is that there is a basal level of a transacting substance producing nondisjunction in tapetal
cells, whose concentration and/or effect is increased
threefold by B chromosomes. The results shown in Tables 3 and 5 support this hypothesis because B and
knobbed chromosome nondisjunction or B’s and chromosome 6 nondisjunction are not independent events.
The observed number of binucleate cells where both
chromosomes behave normally or both undergo nondisjunction is higher than expected. In addition, we have
observed more cells with B nondisjunction and A micronucleus than expected if these were independent events
(Table 2), indicating again that B chromosomes induce
a general instability in the A’s.
Rhoades et al. (1967) and Rhoades and Dempsey
(1973) carried out elegant genetic experiments where
they followed the pattern of loss for specific marker
genes affecting aleurone color or endosperm development. They demonstrated that B chromosomes caused
elimination of chromatin from knob-bearing members
of the A set in aleurone cells. They hypothesized that the
B’s suppressed the replication of the heterochromatic
knobs at the second microspore mitosis thus producing
dissimilar sperm cells.
The results reported in the present article indicate
that A chromosome instability induced by B chromosomes in the tapetum is, in all probability, a phenomenon related to knob elimination in the aleurone. Interestingly, both the tapetum and the aleurone are tissues
playing nutritive roles essential to the pollen and embryo, respectively. Both tissues degenerate after their
nutritive role is accomplished. Aleurone cells form a
secretory tissue that releases hydrolases to digest the
endosperm and nourish the embryo; they are unnecessary for postembryonic development and die as soon as
germination is complete following PCD (Kuo et al. 1996;
Wang et al. 1996; Pennell and Lamb 1997).
There are a large number of articles demonstrating
that tapetal aberrant mitosis, ploidy changes, and degeneracy are essential events for the normal maturation of
pollen grains. However, only recently it has been
pointed out that the process of tapetal degeneracy is
actually a process of PCD (Papini et al. 1999; Wang et
al. 1999) with the cellular remnants necessary for pollen
development acting as secretion products.
Our results support the view that chromosome instability is a regular event occurring during tapetal degeneracy, which might be one of the first steps in the PCD
process, first, because the aberrations occur as controlled events in normal 0B plants and are influenced
by the genotype and, second, because of the similarity
between our observations and those of Rhoades et al.
(1967) in the aleurone.
B chromosomes are stable in somatic tissues (Alfen-
896
A. M. Chiavarino et al.
ito and Birchler 1990), but in the archesporial mitosis
they become unstable, increasing A chromosome instabilities. It is possible that the onset of PCD produces B
nondisjunction, which, irrespective of its influence on A
chromosomes, is an essential feature of B chromosomes
when it occurs at pollen grain mitosis, because it is
necessary for their own transmission and persistence in
populations.
B nondisjunction, and chromatin elimination from
knobbed A chromosomes induced by the B’s, has been
related to the suppression of heterochromatin replication at second pollen division (Rhoades and Dempsey
1972, 1973), although direct evidence of nondisjunction
induced by lack of replication has never been obtained.
On the contrary, Alfenito and Birchler (1990), using
markers on B-A translocations, reported replicative nondisjunction of B chromosomes in the endosperm.
Our results support the view that nondisjunction does
not result from faulty replication of chromosomes or
chromosome segments, because the number of fluorescent labels in tapetal telophase or binucleate cells always
correspond to that expected if all chromosomes were
fully replicated (Figure 1). Particularly, in the case of B
chromosomes, replicative nondisjunction seems evident
since the B-specific probe labels both chromosome
ends. Micronuclei carrying fluorescent labels also seem
to correspond to full chromatids and not to chromosome fragments, because unlabeled chromatin is always
present in addition to the label in the micronucleus.
Similarly, abnormal segregation at tapetal telophase
produced delayed chromatids and not small fragments.
Tapetal tissue has an important role in developing
anthers of angiosperms. In a further work we will analyze
whether the abnormalities in the tapetum due to the
B’s have any influence on pollen viability. It is interesting that the L line, which tends to lose the B’s, is that
producing more abnormalities. This may influence the
B polymorphism at the population level since those
plants transmitting more B’s seem to induce less instability in the normal A chromosomes.
This work was supported by grant PB 98-0678 of the DGICYT of
Spain. Mauricio Chiavarino is a postdoctoral grant holder of the
Fundación Antorchas (Argentina) and Marcela Rosato is a postdoctoral grant holder of the CONICET (Argentina).
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Communicating editor: J. A. Birchler