Journal of Cell Science 103, 831-837 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
831
Preferential mitochondrial and plastid DNA synthesis before multiple cell
divisions in Nicotiana tabacum
TAKESHI SUZUKI 1,*, SHIGEYUKI KAWANO1, ATSUSHI SAKAI1, MAKOTO FUJIE1, HARUKO KUROIWA2,
HIROMU NAKAMURA 3 and TSUNEYOSHI KUROIWA 1
1Department
of Biology, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan
of Biology, Kyouritsu Women’s Junior College University, Tokyo, Japan
3Molecular Biology Unit, Aichi Cancer Center Research Institute, Nagoya, Japan
2Department
*Author for correspondence
Summary
Organelle DNA synthesis in root meristem and cultured
cell line BY-2, both derived from Nicotiana tabacum cv.
Bright Yellow 2, was examined by immunofluorescence
microscopy of Technovit sections with antibody against
5-bromodeoxyuridine (BrdU) and co-fluorescent staining with 4 ,6-diamidino-2-phenylindole (DAPI) and
quantitative Southern hybridization. In the root meristem, the mitochondrial DNAs (mtDNAs) were synthesized in a specific region near to the quiescent center,
where a low frequency of DNA synthesis of cell nuclei
was observed. The mitochondrial nuclei (nucleoids)
changed morphologically from long ellipsoids with a
high frequency of DNA synthesis, in the region just
above the quiescent center, to granules with a low frequency of DNA synthesis, as cell distance from the quiescent center increased. Similar patterns were observed
in the cultured tobacco cell line (BY-2), in which large
amounts of preferential synthesis of DNA of both mitochondria and plastids occurred prior to cell nuclear
DNA synthesis just after stationary phase cells were
transferred to fresh medium. Granular mitochondria
which vigorously synthesized mtDNA were observed in
both lag phase and logarithmic growth phase cells. However, long, ellipsoidal mitochondria which showed a low
frequency of mtDNA synthesis were observed in stationary phase cells. Morphological changes of plastids
were more conspicuous than those of mitochondria.
After the medium was renewed, spherical plastids
became extremely elongated and string-like, for 24 h,
but were divided into small pieces after the third day.
Vigorous synthesis of plastid DNA (ptDNA) occurred
during this period of plastids elongation.
Introduction
thaliana have revealed that mitochondrial DNA synthesis
is not correlated with cell division, and morphological
changes of mitochondrial nuclei are accompanied by
changes in the rate of mitochondrial DNA synthesis
(Kuroiwa et al., 1992; Fujie et al., unpublished data). However, it is difficult to analyze the molecular mechanisms of
multiplication and distribution of these organelles in higher
plants in vivo. Using cultured cells growing under controlled conditions, rather than the tissue or organs of higher
plants in vivo, should permit such analysis at the molecular level. In tobacco cultured cells (BY-2), preferential synthesis of plastid DNA (ptDNA) and multiplication of plastids were observed following the renewal of medium
(Yasuda et al., 1988). However, no direct information has
been available on organelle DNA synthesis in mitochondria
and plastids, and especially proliferation of mitochondria in
higher plants in vitro, because it has been difficult to distinguish between the synthesis of mitochondrial DNA
Eukaryotic cells replicate by cell division preceded by DNA
synthesis. To preserve the genetic composition of self-replicating organelles in a cell, these organelles and their DNAs
must replicate in step with cell division, indicating the presence of some control mechanism between the cell nucleus
and the organelles.
Organelle DNAs of both mitochondria and plastids are
associated with specific proteins to form compact structures
in situ. These compactly organized DNA-protein complexes
are called mitochondrial nuclei and plastid nuclei. The
number, structure, distribution, and protein composition of
plastid nuclei vary considerably during plastid differentiation and development (Kuroiwa et al., 1981; Hansmann
et al., 1985; Miyamura et al., 1986; Reiss and Link, 1985;
Nemoto et al., 1988, 1990). Microautoradiographs of the
root meristem of Pelargonium zonale and Arabidopsis
Key words: organelle DNA synthesis, mitochondrial nuclei
(nucleoids), plastid nuclei (nucleoids), cell culture, antibromodeoxyuridine immunofluorescence microscopy, Nicotiana
tabacum.
832
T. Suzuki and others
(mtDNA) and ptDNA by microautoradiography. In the
present study, we were able to visualize DNA synthesis in
the nuclei of both organelles individually by indirect
immunofluorescence microscopy of Technovit sections
stained with anti-BrdU antibody.
The mechanisms of multiplication and distribution of
mitochondria and plastids are generally explained in terms
of the preservation of the genetic composition in step with
cell division. In this study, therefore, we first examined
whether the behavior of both organelles in the tobacco cultured cell line reflected that in the in vivo meristematic cells
of root apical meristem. The cytological and molecular biological analyses showed preferential organelle DNA synthesis unconnected to the DNA synthesis of the cell nucleus,
both in vivo and in vitro.
Materials and methods
Plant materials
To prepare the root meristem, seeds of tobacco cv. Bright Yellow
2 were soaked with water on two layers of filter paper at 25°C in
the light for 3 days. Tobacco cultured cells, line BY-2 derived
from Nicotiana tabacum L. cv. Bright Yellow 2, were propagated
on a gyratory shaker at 130 revs/min at 26°C in the dark as previously described (Yasuda et al., 1988; Nemoto et al., 1988). To
maintain the cultured cell line, 2 ml of stationary phase cells were
transferred weekly into 95 ml of fresh Linsmaier and Skoog (1965)
medium, as modified by Nagata et al. (1981). For sampling or
observation of the cultured cells, 2 ml of completely stationary
phase cells (9-10 days old) were transferred into the fresh medium.
The growth of the BY-2 cells was monitored by counting the numbers of cells per ml under a microscope.
Labeling cells with 5-bromodeoxyuridine (BrdU) and
indirect immunofluorescence microscopy with anti-BrdU
antibody
Three-day-old seedlings with primary roots approximately 3 mm
long were pulse-labeled for 1 h with 100 µM BrdU, a halogenated
pyrimidine analogue of thymidine, and with additional 10 µM 5fluorodeoxyuridine to enhance the incorporation of BrdU. Roots
of seedlings were fixed with 4% paraformaldehyde and embedded in Technovit 7100 resin (Kulzer and Co., GmbH, Wehrheim)
as previously described (Kuroiwa et al., 1990). Thin serial sections were cut with a glass knife on an MT-6000 XL Ultramicrotome (RMC-Eiko Co., Kawasaki), and stuck to a coverglass
with 70% ethanol. Cultured cells were pulse-labeled with BrdU
for 1 h as above at 0, 3, 6, 12, 24, 72, 120, 168 and 216 h after
the subculture. The labeled cultured cells were treated with Pectolyase Y23 and Cellulase YC (both from Seishin Pharmaceutical
Co., Tokyo) to remove cell walls (Okada et al., 1986), fixed with
Carnoy’s solution (25% glacial acetic acid in ethanol) for 30 min,
and stuck to a coverglass. The samples on coverglasses were
double-stained with 4′,6-diamidino-2-phenylindole (DAPI) and
indirect immunofluorescence stained with anti-BrdU monoclonal
antibody (Becton Dictinson Immunocytometry Systems Co., California) and fluorescein isothiocyanate (FITC)-conjugated second
antibody (goat anti-mouse Igs, Tago Inc., California) as described
previously (Munaut et al., 1990). Labeling patterns of organelles
were observed under an epi-fluorescence microscope (Olympus
BHS-RFK, Olympus Optical Co., Tokyo). Photographs were taken
at magnifications of ×50 and ×330 on 35 mm Kodak Ektachrome
400 film (Eastman Kodak Co., New York).
Morphological observation of organelles and organelle
nucleus
The cell walls of cultured cells were removed enzymatically as
described above. Released protoplasts were fixed and stained in a
drop of 1% glutaraldehyde and 1 µg/ml of DAPI in TAN buffer
(17% (w/v) sucrose, 20 mM Tris-HCl (pH 7.65), 0.5 mM EDTA,
1.2 mM spermidine, 7 mM 2-mercaptoethanol and 0.4 mM
PMSF). The fixed protoplasts were burst by applying slight pressure to the coverslips placed on top of them. They were then
observed under an epi-fluorescence microscope equipped with a
phase-contrast objective. Photographs were taken at a magnification of ×330 on 35 mm Fuji Neopan 400 film (Fuji Photo Film
Co., Tokyo).
Quantitative analysis of organelle DNA by Southern blot
hybridization
Total DNA from tobacco cultured cells was isolated by a method
described previously (Corriveau et al., 1990). After digestion of
total DNA by HindIII, the restriction fragments were separated in
a 1% agarose gel by electrophoresis for 1.5 h at 50 V. Blotting
to nylon membrane filters and hybridization were performed by
the method of Southern (1975). For quantitative loading, preliminary Southern blot hybridization experiments were performed
with a probe of cloned nuclear-DNA fragment, containing rice
nuclear genes for both 26 S and 18 S rRNA, donated by Dr. Y.
Sano (National Institute of Genetics, Mishima). To define the
invariability of the copy number of rRNA genes during the culture, another hybridization experiment was conducted with a probe
of the psaH gene, supplied by Dr. Obokata (Hokkaido University,
Sapporo). About 5 µg of HindIII-digested total DNAs, which were
standardized to contain equal volumes of total nuclear DNA by
preliminary hybridization experiments, were loaded and then
hybridized with two probes of plastid and mitochondria. Labeling
of probes with [α-32P]dCTP was performed with a DNA labeling
kit under the conditions recommended by the manufacturer
(Amersham International PLC, Amersham). For the detection of
ptDNA, a 2.5 kb cloned fragment containing the psaA and psaB
genes of tobacco ptDNA was used as a probe. For mtDNA, a 2.0
kb cloned fragment containing the entire coding region of rice
coxI gene (Kadowaki et al., 1989) was used. The intensity of
hybridization signals was estimated by a densitometer (Densitograph AE-6900, Atto Co., Tokyo).
Results
Organelle DNA synthesis in root apical meristem
DNA synthesis in the root apex of tobacco was examined
by staining with DAPI and indirect immunofluorescence
staining with anti-BrdU antibody after pulse-labeling with
BrdU for 12 h (Fig. 1a-f). There were four types of cells
in the root apex, based on the labeling patterns of organelles
and cell nuclei. The first type, in which only organelles
were labeled with BrdU and identified by green-yellow fluorescence of FITC of second antibody to anti-BrdU antibody, was observed primarily in the neighborhood of the
quiescent center (Fig. 1c and d). The second type (both
organelles and the cell nucleus were labeled) was observed
above the quiescent center (Fig. 1c and d). The third type,
in which only the cell nucleus was labeled, was observed
in the root apical meristem above the quiescent center (Fig.
1e and f). The last type, in which neither of the organelles
nor the cell nucleus was labeled, was observed in root cap
cells (Fig. 1a and b). In the root apex of tobacco, cell
Preferential DNA synthesis in N. tabacum
nucleus DNA synthesis and cell division occurred actively
throughout the entire root meristem from about 130 µm to
400 µm from the root tip, as is typical in root meristems
of dicotyledons. In contrast, organelle DNA synthesis
occurred mainly in a specific region neighboring the quiescent center, about 130 µm from the root tip. Furthermore,
morphological changes of mitochondrial nuclei, from long
ellipsoids to granules, were observed as the distance of cells
from the quiescent center increased.
Morphological changes of mitochondrial nuclei and plastid
nuclei in cultured cell line
The above results suggest that organelle DNA synthesis is
not related to cell division. However, it is difficult to
analyze further the behavior of organelles during cell division at the molecular level by such observations in vivo.
Cultured cells are more suitable for analysis biochemically
or molecular biologically than the tissue or organs of higher
plants in vivo. The tobacco cultured cells of BY-2 multiplied about 50-fold in a week when 2 ml of suspension of
9-day-old cell clusters was transferred to fresh medium
(Fig. 2). The mitotic indices from 48 to 120 h after the
transfer were 4-5%. The 4-day period from about 24 h to
120 h after the transfer was determined to be the logarithmic growth phase and the mean duplication time was estimated to be about 18.8 h. The period beyond 120 h was
determined to be the stationary phase. Both organelles and
their nuclei were examined morphologically by phase-contrast microscopy and DAPI-fluorescence microscopy at 0,
24, 72, 120, 168 and 216 h after the transfer (Fig. 3). These
morphological changes are illustrated in Fig. 7, below. At
the time of transfer, spherical plastids and elongated mitochondria were observed but their fluorescence was quite
weak (Fig. 3a, b, c and d). Within 24 h after the transfer,
plastids were extremely elongated and string-like. The
number of pt-nuclei apparently increased to the maximum
and then fluorescence became stronger (Fig. 3e, f, g and h).
Small granular mitochondria with relatively intensely fluorescent nuclei were also observed during this time. After
Fig. 2. Growth curve and mitotic indices of tobacco cultured cell
line BY-2.
833
the third day, the number and fluorescence intensity of ptnuclei decreased gradually, and elongated plastids were
divided into small pieces (Fig. 3i-x). However, the number
and fluorescence intensity of mt-nuclei increased until the
third day and then decreased slowly. After 120 h, mitochondria were growing longer gradually (Fig. 3m-r).
Changes of DNA content of organelles during cell culture
The above results indicate that morphological changes of
organelles occur during the proliferation of cultured cells
and in the in vivo root meristem. In the root meristem, the
synthesis of mtDNA occurred only in mitochondria with
long ellipsoidal mt-nuclei just above the quiescent center.
Since morphological changes of both organelles and
organelle nuclei occurred along with the proliferation of
cells in the culture, the DNA content of organelles must
also change during these processes. To confirm the above
hypothesis, total DNAs were isolated from cultured cells at
0, 12, 24, 48, 72, 120, 168, 216, 264 and 312 h after the
transfer, and analyzed by quantitative Southern hybridization (Figs 4 and 5). HindIII digestions of total DNAs,
which contained equal amounts of nuclear DNA determined
by hybridization signals from a nuclear probe of rRNA
genes (7.0 kb bands in Fig. 4a), were loaded and then
hybridized with two probes specific for ptDNA and
mtDNA. The possibility that the copy number of rRNA
genes varied during the culture was eliminated by an additional hybridization experiment with a probe of nuclear
single-copy gene psaH (data not shown). The probe specific
for ptDNA hybridized into two DNA fragments consisting
of a major 3.6 kb band and a minor 2.3 kb band (middle
and lower bands in Fig. 4a). The probe specific for mtDNA
hybridized into a DNA fragment of 4.1 kb (Fig. 4b). The
hybridization signal intensities of both organelles changed
over the time course of the culture as compared to the standardized hybridization signals of cell nuclear DNA (upper
bands in Fig. 4a). Hybridization signal intensities, which
indicated the relative amount of organelle DNAs per cell,
increased suddenly at 12 h after the transfer, reached a peak
between 24 and 72 h, and then gradually subsided (Fig. 5).
These results suggest that the amount of organelle DNA
per cell increased suddenly, 3.2- and 4.4-fold in plastids
and mitochondria, respectively, due to preferential
organelle DNA synthesis without cell division within 24 h
after the transfer, and then decreased gradually according
to the distribution of organelles by cell division.
Organelle DNA synthesis in a cultured cell line
Although the above results indicate that organelle DNA is
synthesized immediately following the renewal of medium,
they do not directly show that preferential organelle DNA
synthesis occurs prior to cell nucleus DNA synthesis during
culture. To confirm the synthesis of organelle DNA prior
to that of cell nuclei, cultured cells pulse-labeled with BrdU
were examined by immunofluorescence microscopy with
antibody against BrdU. Preferential synthesis of organelle
DNA following medium renewal was observed in individual cells of BY-2 by indirect immunofluorescence staining
with anti-BrdU antibody as well as in the root meristem.
The cells were classified into four types according to the
labeling patterns of organelles and cell nuclei (Fig. 6). In
834
T. Suzuki and others
Fig. 3. Epi-fluorescence (a, c, e, g, i, k, m, o, q, s, u and w), and phase-contrast (b, d, f, h, j, l, n, p, r, t, v and x) images of mitochondria,
plastids and cell nuclei in BY-2. Cells harvested at 0 (a, b, c and d), 24 (e, f, g and h), 72 (i, j, k and l), 120 (m, n, o and p), 168 (q, r, s and
t) and 216 (u, v, w and x) h after transfer into fresh medium were treated with cellulolytic enzymes to remove cell walls, and observed
under an epi-fluorescence microscope after staining with DAPI. Small pictures (right two files: bar, 10 µm) show parts of the cytoplasm
of the large pictures (left two files: bar, 10 µm) at higher magnification. Small arrowheads and large arrowheads show mitochondria and
plastids, respectively.
Preferential DNA synthesis in N. tabacum
835
Table 1. Change in the proportion of four different cell
types classified by their labeling patterns
Frequency of labeling pattern (%)
Labeling
pattern
N −O−
N−O+
N +O+
N +O−
Fig. 4. Quantitative Southern blot hybridization patterns of
tobacco total DNAs extracted according to the time course after
transfer. HindIII-digested total DNAs were measured in contrast
with the hybridization signals of cell nuclear DNA (7.0 kb bands
in a). The probe specific for plastid DNA hybridized to two DNA
fragments of a major 3.6 kb band and a minor 2.3 kb band (a), and
the probe specific for mitochondrial DNA hybridized to a DNA
fragment with 4.1 kb (b).
the cells of the first type, neither organelles nor the cell
nucleus was labeled (N−O−; Fig. 6a and b). In the second
type of cells, only organelles were labeled (N−O+; Fig. 6c
and d). Both organelles and the cell nucleus were labeled
in the third type of cells (N+O+; Fig. 6e and f). Only the
cell nucleus was labeled in the last type of cells (N+O−;
Fig. 6g and h). The proportions of these four different types
of cultured cells were followed during culture (Table 1),
and the changes in frequencies of labeled organelles and
cell nuclei are shown in Fig. 7. At the time of the transfer,
99% of the cells were type of N−O−, and only 1% of the
cells were N−O+. The frequency of N−O+ type cells
increased immediately, reached peaks of 56-63% after 6 to
72 h, and decreased suddenly at 120 h after the transfer.
N+O+ cells were observed after 6 h and reached a maximum frequency of 40% at 72 h after the transfer. Only a
few N+O− cells were observed after 24 h, and their fre-
Fig. 5. Relative changes in the DNA levels of both mitochondria
(d) and plastids (s) per cell nucleus in tobacco BY-2 cells during
the culture. Relative values for organelle DNA amounts were
determined by measuring the intensities of hybridization signals
with a densitometer.
0
3
6
99
1
0
0
68
32
0
0
36
60
5
0
Time after transfer (h)
12
24
72
120
12
59
29
0
5
63
30
3
0
56
40
4
35
17
24
24
168
216
76
3
10
11
98
2
0
0
quency reached a peak of 24% at 120 h after the transfer.
Table 1 and Fig. 7 clearly show that the DNA synthesis in
organelles preferentially occurred ahead of that in cell
nuclei. The frequency of labeled organelles suddenly
increased from 1 to 93% within only 24 h and immediately
decreased after 72 h. Although the frequency of labeled
organelles stayed within the range of 93-96%, the intensity
of that labeling slightly decreased after 24 h. On the other
hand, the frequency of labeled cell nuclei increased 5-33%
from 6 to 24 h after that of organelles and gradually
increased to 48% at 120 h after the transfer, when the frequencies of labeled organelles and cell nuclei gradually
decreased. These results clearly show that organelle DNAs
are synthesized preferentially ahead of cell nuclear DNA
just after the renewal of medium.
Discussion
In the present study, mtDNAs of root meristem cells were
found to be synthesized in a specific region neighboring the
quiescent center. In this region, DNA synthesis of cell
nuclei is observed at low frequency by fluorescence staining with DAPI and indirect immunofluorescence staining
with anti-BrdU antibody and FITC-conjugated second antibody in a thin section of BrdU-labeled root meristem of
tobacco embedded in Technovit 7100 resin. Essentially the
same phenomenon of preferential synthesis of organelle
Fig. 7. DNA synthesis and morphological changes of organelles
and cell nucleus during cell culture. Upper drawings indicate
morphological changes of plastid (pt) and mitochondrion (mt).
Black dots show organelle nuclei and open circles show starch
grains. Filled areas under the drawings indicate the activity of
DNA synthesis of organelles (pt & mt) and cell nucleus (N).
Thickness of areas reflects percentage of cells labeled with BrdU.
836
T. Suzuki and others
DNA is found in the root meristem of Pelargonium zonale
and Arabidopsis thaliana (Kuroiwa et al., 1992; Fujie et al.,
unpublished data). These results emphasize that DNA synthesis of organelles is unconnected to cell division. Moreover, in tobacco cultured cells, quantitative Southern blot
analysis and immunofluorescence microscopy with antiBrdU antibody show that large amounts of preferential
mitochondrial and plastid DNA synthesis occur prior to cell
nuclear DNA synthesis just after stationary phase cells are
transferred to fresh medium (Fig. 7). Organelle DNA synthesis of in vitro cultured cells resembles that of in vivo
root meristematic cells. However, the patterns of morphological changes of both mitochondria and plastids seem to
depend upon the different growth conditions. In tobacco
root meristem, the mitochondrial nuclei changed from long
ellipsoids with a high frequency of DNA synthesis to granules with a low frequency of DNA synthesis as the distance
from the quiescent center increased (Fig. 1). The same pattern was also observed in the root meristem of P. zonale
(Kuroiwa et al., 1992). However, in the cultured cell line
BY-2, elongated, ellipsoidal mitochondria were observed
only in stationary phase cells 120 h following transfer,
instead of during the logarithmic growth phase of mtDNA
(Figs 3 and 7). mtDNA synthesis was not observed in these
long ellipsoidal mitochondria. On the other hand, the granular mitochondria present in the logarithmic phase, vigorously synthesized mtDNA. The frequency of mtDNA synthesis increased gradually until 72 h after the transfer and
then decreased slowly. The mitochondria in the cultured
cells do not completely resemble those in the root meristem and are more similar to those in more differentiated
meristematic cells. The morphological difference between
the mitochondria in the cultured cells and those in the root
meristem might aid in understanding the relationship
between cell differentiation and morphological changes of
mitochondria.
The morphological changes of plastids in the cultured
cells were more obvious than those of mitochondria (Figs
3 and 7). These changes were not observed in the root
meristem. The spherical plastids became extremely
elongated and string-like during the first 24 h after the
renewal of medium. The elongated plastids broke into small
pieces after the third day (Fig. 7). A similar observation
was reported in BY-2 cells subcultured weekly (Yasuda et
al., 1988). In this case, the plastid division occurred
between 24 and 48 h after the subculture. This is also supported by the observation that the surface area of a dumbbell-shaped plastid is reduced to half of its original value
during this time. However, the two culture systems differ
in that the extremely elongated string-shaped plastids were
not observed in BY-2 subcultured weekly. The stringshaped plastid seems to be observed only when completely
stationary phase cells are transferred to fresh medium as a
9-day subculture. Miyamura et al. (1990) observed ellipsoid and elongated sausage-shaped plastids, in which several spherical and cup-shaped pt-nuclei were located side
by side, in the leaf base of Triticum aestivum. They
suggested that the division of plastids occurs in this area.
From the results presented here, plastid elongation seems
to occur along with the preferential synthesis of ptDNA.
This preferential synthesis may be in preparation for sev-
eral plastid divisions following multiple cell divisions without organelle DNA synthesis.
The preferential synthesis of organelle DNA prior to that
of cell nuclear DNA, both in vivo and in vitro, suggests
that organelle DNA is synthesized and stored in large
amounts to prepare for the following multiple cell divisions
without organelle DNA synthesis. This behavior of
organelles was observed in both in vivo and in vitro systems. In the in vitro culture system, the preferential synthesis of organelle DNA is apparently induced by the stimulation of medium renewal. Moreover, the multiplication
and differentiation of organelles can also be controlled artificially by modifying the medium. For example, when BY2 cultured stationary phase cells were transferred into
medium with additional benzyladenine, the proplastids were
converted to amyloplasts and this was accompanied by
changes in the transcriptional activities of plastid genes
(Sakai et al., 1992). A culture system like BY-2 offers a
good in vitro experimental system in which to study the
molecular mechanism of replication, multiplication and distribution of organelles.
This work was supported by a grant-in-aid (to T.K.) for Special Research on Priority Areas (project no. 03256105, Molecular
Structure of Chromosomes) from the Ministry of Education, Science and Culture, Japan, and a grant pioneering research project
in biotechnology from the Ministry of Agriculture, Forestry and
Fisheries, Japan. We are grateful to Dr. Y. Sano (Nagoya University), Dr. K. Kadowaki (National Institute of Agrobiological
Resources) and Dr. J. Obokata (Hokkaido University) for providing us with clones of 26 S and 18 S rRNAs, and coxI and psaH
genes, respectively.
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(Received 29 June 1992 - Accepted 3 August 1992)
Fig. 6. Epi-fluorescence images with DAPI (a, c, e and g), and immunofluorescence images with anti-BrdU antibody (b, d, f and h) of four
different types in BY-2 cells. Cells were labeled with BrdU for 1 h at 0, 3, 6, 12, 24, 72, 120, 168 and 216 h after the transfer. Cells were
classified into four types, according to their labeling pattern: (a and b) organelles and cell nucleus (N) not labeled (N−O−); (c and d) only
organelles labeled (N−O+); (e and f) both organelles and cell nucleus labeled (N+O+); (g and h) only cell nucleus labeled (N+O−).
Arrowhead and arrow show a labeled mitochondrion and a labeled plastid, respectively. Bar, 10 µm.
Fig. 1. Epi-fluorescence images with DAPI (a, c and e), and immunofluorescence images with anti-BrdU (b, d and f) of root meristem of
tobacco labeled with BrdU for 12 h. Green-yellow fluorescent signals of organelles appear heavier on the proximal portion of the
meristem than on the distal portion of the meristem from the quiescent center (a and b). Organelle DNA synthesis occurs primarily in the
cells close to the quiescent center (c and d). Very little organelle DNA synthesis occurs in the distal part of central cylinder cells (e and f).
Arrows indicate the cell nuclei of neighboring cells of the quiescent center. Large arrowheads show mitochondrial nuclei with DNA
synthesis. Small arrowheads show mitochondrial nuclei without DNA synthesis. Bars, 50 µm (a and b); 10 µm (c, d, e and f).