Plant Cells Without Detectable Plastids are Generated in the
crumpled leaf Mutant of Arabidopsis thaliana
Yuling Chen1,2, Tomoya Asano3, Makoto T. Fujiwara4, Shigeo Yoshida5, Yasunori Machida1 and
Yasushi Yoshioka1,∗
Regular Paper
1Division
of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602 Japan
of Life Science, Hebei Normal University, Shijiazhuang 050016, PR China
3Advanced Science Research Center, Kanazawa University, 920-0934, 13-1 Takaramachi, Kanazawa, Ishikawa, Japan
4Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo,
153-8902 Japan
5Plant Science Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045 Japan
2College
Plastids are maintained in cells by proliferating prior to
cell division and being partitioned to each daughter cell
during cell division. It is unclear, however, whether cells
without plastids are generated when plastid division is
suppressed. The crumpled leaf (crl) mutant of Arabidopsis
thaliana is a plastid division mutant that displays severe
abnormalities in plastid division and plant development.
We show that the crl mutant contains cells lacking
detectable plastids; this situation probably results from an
unequal partitioning of plastids to each daughter cell. Our
results suggest that crl has a partial defect in plastid
expansion, which is suggested to be important in the
partitioning of plastids to daughter cells when plastid
division is suppressed. The absence of cells without
detectable plastids in the accumulation and replication of
chloroplasts 6 (arc6) mutant, another plastid division
mutant of A. thaliana having no significant defects in plant
morphology, suggests that the generation of cells without
detectable plastids is one of the causes of the developmental
abnormalities seen in crl plants. We also demonstrate that
plastids with trace or undetectable amounts of chlorophyll
are generated from enlarged plastids by a non-binary
fission mode of plastid replication in both crl and arc6.
Keywords: Arabidopsis thaliana • Plastid division • Plastid
partition.
Abbreviations: CaMV, cauliflower mosaic virus; CFP, cyan
fluorescent protein; DAPI, 4′,6-diamidino-2-phenylindole;
MS, Murashige and Skoog; NOS, nopaline synthase; PBS,
∗Corresponding
phosphate-buffered saline; SAM, shoot apical meristem;
RAM, root apical meristem; YFP, yellow fluorescent protein.
Introduction
Plastids originated in an endosymbiotic event between a
protoeukaryote and a photosynthetic prokaryote; they have
their own genomes and divide by binary fission (Gray 1999,
Pyke 1999, McFadden 2001, Osteryoung and Nunnari 2003).
It is generally believed that plastids do not arise de novo, but
form by division of pre-existing plastids that are passed on
from cell to daughter cell during cell division (Possingham
and Lawrence 1983). To maintain themselves in cells, plastids proliferate prior to cell division and are partitioned to
each daughter cell during cell division.
That part of the plastid division system which is derived
from plastids’ bacterial origin involves FtsZ (Osteryoung and
Vierling 1995, Osteryoung et al. 1998, Strepp et al. 1998,
Jeong et al. 2002), MinD (Colletti et al. 2000, Dinkins et al.
2001, Fujiwara et al. 2004), MinE (Itoh et al. 2001, Maple et al.
2002, Reddy et al. 2002) and SulA/GIANT CHLOROPLAST 1
(GC1) proteins (Maple et al., 2004, Raynaud et al. 2004). The
accumulation and replication of chloroplasts (arc6) gene
product is a plastid-targeted DnaJ-like protein closely related
to Ftn2, an essential cell division protein unique to cyanobacteria (Vitha et al. 2003). The arc6 mutant exhibits severe
defects in plastid division, and has only two enlarged chloroplasts per mature mesophyll cell compared with 90 chloroplasts per cell in the wild type (Robertson et al. 1995).
author: E-mail, [email protected]; Fax, +81-52-789-2966.
Plant Cell Physiol. 50(5): 956–969 (2009) doi:10.1093/pcp/pcp047, available online at www.pcp.oxfordjournals.org
© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Plant Cell Physiol. 50(5): 956–969 (2009) doi:10.1093/pcp/pcp047 © The Author 2009.
Plant cells without detectable plastids
The crumpled leaf (crl) gene product is a novel outer envelope membrane protein of plastids without an obvious transmembrane domain; it is conserved in a wide range of plants
and cyanobacteria (Asano et al. 2004a). The crl mutant also
exhibits severe defects in plastid division, and each cell contains relatively few, though enlarged, plastids (Asano et al.
2004a). Whereas most of the mutations that affect chloroplast number and size normally result in no significant
changes to the gross morphology of plants or to the plants’
fertility (Pyke and Leech 1994), the crl mutant displays
abnormalities in the orientation of the cell division plan, cell
differentiation and overall plant development (Asano et al.
2004a). The molecular mechanism by which the crl mutation affects plastid division and plant development is still
unclear. The plastid division system also contains eukaryotic
factors such as plastid division 1 (pdv1), plastid division 2
(pdv2) and accumulation and replication of chloroplast 5
(arc5) (Gao et al. 2003, Miyagishima et al. 2003). pdv1 and
pdv2 are involved in the recruitment of the ARC5 protein,
which is a eukaryotic dynamin-like protein (Gao et al. 2003,
Miyagishima et al. 2003), to the division site at a late stage of
plastid division (Miyagishima et al. 2006).
The partitioning of plastids to each daughter cell has been
suggested to be stochastic, with a tendency toward equality
when a plant has a certain number of plastids per cell (Birky
1983). Involvement of actin-dependent repositioning of
plastids in the perinuclear region in unbiased partitioning
has been reported in cultured tobacco mesophyll protoplasts that contain a number of chloroplasts (Sheahan et al.
2004). Yet it is not clear how plastids are maintained in the
plastid division mutants whose cells contain only a few plastids each. A mathematical analysis of a physical model of
organelle partition suggested that the probability of equal
partitioning of plastids increases steeply as the ratio of total
organelle volume to total cell volume increases (Birky 1983).
This analysis indicated that the probability of an event in
which one daughter cell receives no organelles is not negligible when the number of organelles is <10, while the probability of such an event decreases almost linearly with
increasing organelle volume ratio (Birky 1983). Therefore,
organelle volume could have an important effect on plastid
partitioning in cells containing only a few plastids. Indeed, it
has been reported that the ratio of plan chloroplast area to
plan cell area is constant in mesophyll cells of Arabidopsis
thaliana, regardless of chloroplast number (Pyke and Leech
1992, Pyke and Leech 1994). In other words, when chloroplast division is inhibited, chloroplasts enlarge so that the
volume they occupy in each cell remains constant. It has also
been demonstrated that the average size of arc6 plastids is
double that of wild-type plastids in shoot apical meristem
and leaf primordia (Robertson et al. 1995).
Whereas plant cells (excluding male gametes) that have
lost plastids have only very rarely been found (Sears 1980),
stomatal guard cells lacking chloroplasts have been reported
in arc6 (Robertson et al. 1995). It is questionable, however,
whether those stomatal guard cells may actually contain
plastids without chlorophyll, given that the presence of plastids without chlorophyll in stomatal guard cells has been
reported in suffulta, a tomato plastid division mutant (Forth
and Pyke 2006). Therefore, it is controversial as to whether
the loss of plastids occurs in the sporophyte phase.
Here, we demonstrate the presence of cells lacking detectable plastids in crl but not arc6 by visualizing plastids using a
plastid-targeted yellow fluorescent protein (YFP) and by
staining organelle DNA with DAPI (4′,6-diamidino-2phenylindole). This situation probably results from an
unequal partitioning of plastids to each daughter cell during
cell division. Although it is generally assumed that cells lacking plastids would not be viable, our results suggest that
such cells are viable. The existence of cells without detectable plastids in crl plants, which exhibit significant abnormalities of overall plant morphology, but not in arc6 plants,
which do not (Pyke et al. 1994), suggests that the generation
of cells without detectable plastids is at least one of the
causes of the morphological abnormalities seen in crl
plants.
Results
Cotyledons of mature embryos contained cells
without detectable plastids in crl
All cells in cotyledons of mature wild-type embryos contained plastids with chlorophyll (Fig. 1A). When we examined cells in cotyledons of mature crl embryos using
epifluorescence microscopy, however, we detected no chlorophyll fluorescence in almost half of the cells. The average
number of chloroplasts per cell in crl embryos was decreased
to 2.1 compared with 17.0 in wild-type embryos (Table 1).
We looked for the presence of plastids in the cells without
chloroplasts in cotyledons of mature embryos of the crl and
wild-type plants that expressed TPFtsZ1-1:YFP, a plastid-targeted YFP, using confocal microscopy. No cells without chloroplast were found in the wild-type plants expressing
TPFtsZ1-1:YFP (Fig. 1A). Among the crl plants expressing the
protein, in contrast, no YFP signal was detected in 14% of
the cells (Fig. 1A, G).
Because it was possible that TPFtsZ1-1:YFP was not expressed
or that TPFtsZ1-1:YFP was not imported into plastids in the
cells without a YFP signal, we confirmed the absence of plastids by staining plastid DNA with DAPI and observing the
cells through confocal microscopy. For this analysis, we used
the cotyledons of mature embryos that expressed both
TPFtsZ1-1:YFP and a mitochondria-localized DsRed (mtDsRed)
so that plastid DNA could be distinguished from mitochondrial DNA. As shown in Fig. 2C and Supplementary Movie 3,
no plastid DNA was detected in the cells without the YFP
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Fig. 1 Frequency of cells containing undetectable
plastids. Cells from plants expressing TPFtsZ1-1:YFP
were examined as described in Materials and
Methods. (A) Cells from cotyledons of mature
embryos (n = 97 for the wild type, n = 104 for crl,
n = 98 for arc6). (B) Guard cells in the cotyledons
of 13- and 14-day-old plants (n = 91 for the wild
type, n = 56 for crl, n = 54 for arc6). (C) Mesophyll
cells from the rosette leaves of 18-day-old plants
(n = 105 for the wild type, n = 95 for crl, n = 103 for
arc6). (D–G) Representative confocal images of
each type of cell from cotyledons of mature crl
embryos. Images showing chlorophyll fluorescence
are overlaid with the corresponding images
showing YFP fluorescence. The corresponding
Nomarski images (DIC) are shown in the righthand panels. The following stacks of optical serial
sections along the z-axis are merged: a 27-image
stack of a 0.43 µm interval for (D), a 34-image
stack of a 0.43 µm interval for (E), a 25-image stack
of a 0.43 µm interval for (F) and a 27-image stack
of a 0.43 µm interval for (G). Chl, cells containing
chloroplasts; Chl + P, cells containing chloroplasts
and plastids with trace or undetectable chlorophyll
fluorescence; P, cells containing plastids with trace
or undetectable chlorophyll fluorescence; N.D.,
cells containing no detectable plastids. Scale
bars = 10 µm.
signal, though it was detected in both the crl cells with
enlarged plastids and wild-type cells with ordinary plastids
(Fig. 2A, B, Supplementary Movies 1, 2). Mitochondrial
DNA was detected in both wild-type and crl plants as
small dots that overlaid with mtDsRed signal (Fig. 2A–C,
958
Supplementary Movies 1–3). Faint background signals that
overlapped with the YFP signals in the DsRed channel were
derived from chlorophyll fluorescence (Fig. 2A, B). These
results indicate that the cotyledons of mature crl embryos
contain cells without detectable plastids. The presence of
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Plant cells without detectable plastids
Table 1 Average numbers of plastids per cell
Plant phenotype
Cotyledon in embryosa
Stomatal guard cellb
Mesophyll cellc
Pavement celld
Wild type
17.0 ± 2.0
3.5 ± 0.3
41.7 ± 6.8
6.5 ± 0.6
crl
2.1 ± 0.4
0.57 ± 0.14
2.8 ± 0.7
2.7 ± 0.9
arc6
2.9 ± 0.4
1.34 ± 0.23
1.1 ± 0.1
3.1 ± 0.1
Only the plastids having detectable chlorophyll fluorescence were counted for stomatal guard cells, mesophyll cells and cells in cotyledon in embryos.
aCotyledons of mature embryos were used for the analysis (n = 97 for the wild type, n = 108 for crl, n = 98 for arc6).
bCotyledons of 13- and 14-day-old plants were used for the analysis (n = 92 for the wild type, n = 56 for crl, n = 55 for arc6).
cThe first and second leaves of 18-day-old plants were used for the analysis (n = 50 for the wild type, n = 92 for crl, n = 103 for arc6).
dThe first and second leaves of 18-day-old plants expressing TP
FtsZ1-1:YFP were used for the analysis (n = 103 for the wild type, n = 102 for crl, n = 93 for arc6).
Fig. 2 Cells without detectable plastids existed in crl embryos. Confocal images of cells from cotyledons of wild type (WT) and crl embryos after
DAPI staining are shown. Each plant expresses the plastid-targeted YFP (TPFtsZ1-1:YFP) and the mitochondria-targeted DsRed (mtDsRed). Stacks
of 22, 23 and 21 images of a 0.41 µm interval along the z-axis are merged in (A), (B) and (C), respectively. The absence of detectable plastid DNA
in the cells in (C) was confirmed through examination of each optical section. Each optical section of (A), (B) and (C) is shown in Supplementary
Movies 1, 2 and 3, respectively. Images showing DAPI fluorescence and the same images showing DsRed fluorescence are overlaid with the same
images showing YFP fluorescence. The corresponding Nomarski images (DIC) are shown in the right-hand panels. Scale bars = 5 µm.
nuclear and mitochondrial DNA, and the localization of
mtDsRed in mitochondria in the YFP-negative cells, indicate
that the cells without detectable plastids were alive and that
they retained at least some physiological activity (Fig. 2C).
In both wild-type and crl cells, some mitochondria had no
detectable DAPI signals. This indicates that some mitochondria contained undetectable amounts of DNA.
Among cells from mature embryos of arc6 plants, which
we observed using epifluorescence microscopy, the average
number of chloroplasts per cell was reduced to 2.9 (Table 1),
and chlorophyll fluorescence was not detected in 5% of the
cells (‘P’ in Fig. 1A). Our confocal analysis of the cotyledons
of mature arc6 embryos expressing TPFtsZ1-1:YFP revealed
that all of the cells we examined contained plastids (Fig. 1A).
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This result indicates that the reduction in the number of
plastids per cell was insufficient for the generation of cells
without detectable plastids.
The cells without detectable plastids were generated
by an unequal allocation of plastids during cell
division
An analysis of the dividing cells of torpedo-stage crl embryos
expressing TPFtsZ1-1:YFP revealed the presence of dividing
cells in which plastids were segregating to only one of the
two daughter cells (Fig. 3B). Among dividing cells in torpedostage wild-type embryos, in contrast, plastids were segregating to the two daughter cells almost equally (Fig. 3A).
These results suggest that cells without detectable plastids
are generated by the unequal allocation of a crl cell’s plastids
to each of its daughter cells. We also observed dividing cells
in which only stromules were segregating to one of the two
daughter cells (Fig. 3C). The daughter cells that received
only stromules would probably appear to have no plastids
after the degradation of the stromule fragments. In torpedostage crl embryos (n = 43), the frequency of dividing cells
that generated one daughter cell without detectable plastids
was 4.6%, and the frequency of dividing cells that generated
one daughter cell containing only stromules was also 4.6%.
Investigation of the dividing cells in arc6 embryos expressing TPFtsZ1-1:YFP revealed that a few enlarged plastids
expanded to take up the volume of the entire cell (Fig. 3E).
Such expanded plastids are probably dissected by cell plates
during division and allocated to daughter cells. In contrast to
those in arc6 embryos, the enlarged plastids in crl embryos
were relatively compact: in many cases they existed only in a
small part of each cell (Fig. 3B, C). To quantify the degree of
plastid expansion in each genotype, we measured the ratio
of plan plastid area to plan cell area in dividing cells as
described in the Materials and Methods section, below. As
shown in Fig. 4, the ratio of plan plastid area to plan cell area
in crl cells was approximately 80% of that in wild-type cells,
whereas that in arc6 cells was approximately 110% of that in
wild-type cells. In addition, some crl cells were observed to
have ratios <0.2 (Fig. 4). These results suggest that plastids in
crl embryos do not expand enough to prevent the occasional
loss of plastids during cell division.
As shown in Fig. 3D, DAPI staining of the dividing cells in
crl embryos expressing TPFtsZ1-1:YFP also revealed the presence of dividing cells without detectable plastids. The frequency of such dividing cells was low, but this result suggests
that cells without detectable plastids are able to enter the M
phase at least once.
Cells without detectable plastids were present in crl
plants after germination
We examined cells in the expanding rosette leaves of 12day-old plants expressing TPFtsZ1-1:YFP using confocal
960
microscopy. In both crl and arc6 plants, the average number
of plastids per cell was decreased in the mesophyll and pavement cells of rosette leaves (Table 1). No YFP signal was
detected in 16% of the crl cells (n = 301), whereas all of the
wild-type (n = 244) and arc6 (n = 303) cells we examined had
YFP signals. Most of the cells without detectable plastids in
the expanding rosette leaves of crl plants were either stomatal guard cells or epidermal cells (data not shown). We
also observed that about 18% of pavement cells in the
expanded leaves of crl plants did not contain detectable
plastids (data not shown). These observations are consistent
with the absence of cells without detectable plastids in the
mesophyll of expanded leaves of crl plants (Fig. 1C). Our
confocal analysis of cells from crl plants expressing both
TPFtsZ1-1:YFP and mtDsRed after DAPI staining demonstrated
the existence of YFP-negative cells having no detectable
plastid DNA (Fig. 5C, D, arrowheads, Supplementary
Movies 6, 7). In YFP-positive cells under the same conditions, we detected plastid DNA and mitochondrial DNA in
both wild-type and crl cells (Fig. 5A–D, 5C, D, arrows, Supplementary Movies 4–7). Thus cells without detectable
plastids were present in crl leaves that developed after
germination.
Plastids without chlorophyll proliferated in crl and
arc6 embryo cells
Our confocal analysis of the cotyledons of mature embryos
of crl plants expressing TPFtsZ1-1:YFP also revealed that
approximately 50% of the cells (the sum of the frequencies
of ‘C + P’ and ‘P’ in Figs. 1A, E, F and 6B) had plastids with
undetectable or weak chlorophyll fluorescence (hereafter
referred to as plastids without chlorophyll). Far more of the
wild-type cells contained plastids with chlorophyll fluorescence (Figs. 1A, 6A), but 5% had both chloroplasts and plastids without chlorophyll (‘C + P’ in Fig. 1A). In cotyledons of
mature arc6 embryos expressing TPFtsZ1-1:YFP, we observed
cells containing plastids without chlorophyll at a frequency
of approximately 70% (the sum of the frequencies of ‘C + P’
and ‘P’ in Figs. 1A and 6C). Whole-mount immunolocalization of Tic110, a core component of the translocons at the
inner envelope membranes of plastids (Inaba et al. 2005),
revealed that Tic110 localized on the plastids without chlorophyll, confirming that YFP localized in plastids (Supplementary Fig. S1A–C). DAPI staining of cells from cotyledons
of mature crl embryos revealed that most plastids contained
detectable amounts of DNA (Fig. 6E). This result suggests
that most plastids without chlorophyll contain DNA. Yet we
also observed plastids in crl embryos without detectable
amounts of DNA (Fig. 6F, arrowheads). Therefore, some
plastids without chlorophyll might not contain detectable
amounts of DNA.
Collectively, our results indicate that plastids without
chlorophyll are generated in both crl and arc6 cells, and
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Plant cells without detectable plastids
Fig. 3 An unequal segregation of plastids to each daughter cell occurred during cell division in crl. Confocal images of the cells of embryos at the
torpedo stage from the wild-type plant (A), the crl plant (B–D) and the arc6 plant (E) expressing TPFtsZ1-1:YFP. Images showing DAPI fluorescence
are overlaid with the corresponding image showing TPFtsZ1-1:YFP fluorescence. The cell in (E) contained an enlarged plastid. The following stacks
of optical serial sections along the z-axis are merged: a 27-image stack of a 0.39 µm interval for (A) and (C), a 24-image stack of a 0.39 µm interval
for (B) and (D), and a 16-image stack of a 0.39 µm interval for (E). The corresponding Nomarski images (DIC) are shown in the right-hand panels.
Scale bars = 5 µm.
consequently that neither crl nor arc6 is required for this
manner of plastid proliferation. As cells without detectable
plastids exist in cotyledons of mature crl embryos, our results
also indicate that this manner of plastid proliferation was
not sufficient to prevent the generation of cells without
detectable plastids.
Plastids containing trace or undetectable amounts
of chlorophyll existed in crl and arc6 guard cells and
mesophyll cells
The average number of chloroplasts per guard cell was
decreased both in crl and in arc6 (Table 1). An analysis of the
Plant Cell Physiol. 50(5): 956–969 (2009) doi:10.1093/pcp/pcp047 © The Author 2009.
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Fig. S1E). Enlarged plastids in arc6 cells were also observed
giving rise to YFP-containing protrusions with trace or undetectable amounts of chlorophyll fluorescence (Fig. 7E).
Taken together, our results indicate that plastids without
chlorophyll were generated from enlarged plastids in guard
cells and mesophyll cells in both crl and arc6 plants.
Our results also indicate that arc6 stomatal guard cells
having no chloroplasts containing plastids without chlorophyll (Fig. 1B).
Discussion
Fig. 4 The ratio of plan plastid area to plan cell area in dividing cells
was decreased in crl. The ratio of plan plastid area and plan cell area in
dividing cells in torpedo-stage embryos of the wild type (Col-0), crl and
arc6 was measured as described in Materials and Methods (n = 17 for
the wild type, n = 20 for crl, n = 20 for arc6). The ratio for each dividing
cell is indicated as a filled circle. The averages of these ratios (0.48 for
the wild type, 0.35 for crl and 0.49 for arc6) are indicated as horizontal
lines.
guard cells in the cotyledons of crl plants expressing TPFtsZ1-1:YFP also revealed that 18% of these guard cells contained
no detectable plastids and that approximately 35% of them
contained plastids without chlorophyll (Fig. 1B, the sum of
the frequencies of ‘C + P’ and ‘P’, Fig. 7B). Plastids without
chlorophyll were also detected in the wild type, but at a
lower frequency (approximately 10%, the sum of the frequencies of ‘C + P’ and ‘P’ in Fig. 1B). The localization of YFP
to plastids in guard cells was confirmed by whole-mount
immunolocalization of Tic110 (Supplementary Fig. S1D, F).
We observed the enlarged plastids in crl cells giving rise to
YFP-containing protrusions with trace or undetectable
amounts of chlorophyll fluorescence, by a process of budding and fragmentation (Fig. 7D). Thus, the plastids without
chlorophyll in the crl guard cells were probably generated
from enlarged chloroplasts. Plastids without chlorophyll
were also found in crl rosette leaf mesophyll cells containing
chloroplasts at a frequency of 27% (Figs. 1C, 7G).
Guard cells of cotyledons and mesophyll cells of rosette
leaves in arc6 plants expressing TPFtsZ1-1:YFP contained plastids without chlorophyll, at approximate frequencies of 70%
(the sum of the frequencies of ‘C+ P’ and ‘P’ in Figs. 1B and
7C) and 10% (Figs. 1C, 7H), respectively. Localization of
Tic110 on the YFP signals confirmed the plastid localization
of TPFtsZ1-1:YFP in the arc6 guard cells (Supplementary
962
We demonstrated that crl plants expressing TPFtsZ1-1:YFP
contained cells without detectable plastids in the cotyledons
of their mature embryos and in their rosette leaves. The frequency of cells without detectable plastids was high: approximately 15% in both organs (Fig. 1A, B). DAPI staining of
YFP-negative cells expressing TPFtsZ1-1:YFP and mtDsRed
indicated that these cells did not contain plastids with
detectable DNA (Figs. 2, 5). These results strongly suggest
that crl plants contain cells that have no plastids. As an alternative explanation, it is also possible that cells in which plastids are not detectable actually contain aberrant plastids
which do not import proteins and which contain small or
undetectable amounts of plastid DNA. Even if such aberrant
plastids exist, they must be non-functional because protein
import into plastids is necessary for them to function. Therefore, crl plants probably contain cells either lacking plastids
or containing only non-functional plastids.
Our data indicated that the cells without detectable plastids were viable rather than lethal (Figs. 2C, 5C, D). Plastids
are believed to be essential for plant cell viability because
they are the site of many plant metabolic pathways that are
crucial for plant growth, development and metabolism. For
example, plastids are a major site of lipid and amino acid
synthesis (Galili 1995, Ohlrogge and Browse 1995, Neuhaus
and Emes 2000, Tetlow et al. 2004). The precursors for the
hormones gibberellin (Kasahara et al. 2002), cytokinin
(Kasahara et al. 2004) and abscisic acid (Milborrow and Lee
1998, Hirai et al. 2000, Kasahara et al. 2004), as well as those
for carotenoids (DellaPenna and Pogson 2006) and chlorophylls (Tanaka and Tanaka 2007), are synthesized in plastids.
Jasmonates, lipid-derived plant hormones, are also synthesized in plastids (Wasternack 2007). In addition, several steps
of pyrimidine and purine metabolic pathways take place in
plastids (Zrenner et al. 2006). In order for cells lacking plastids to be viable, metabolites that are ordinarily synthesized
in plastids must be supplied from neighboring cells to those
without detectable plastids.
Our analysis of dividing cells in embryos indicated that
the cells without plastids in crl plants were probably generated by the aberrant segregation of plastids during cell division. As we indicated for the dividing cells in arc6 embryos,
Plant Cell Physiol. 50(5): 956–969 (2009) doi:10.1093/pcp/pcp047 © The Author 2009.
Plant cells without detectable plastids
Fig. 5 Cells without detectable plastids existed in crl rosette leaves. Confocal images of cells from expanding wild-type (WT) and crl rosette leaves
after DAPI staining are shown. Each plant expresses TPFtsZ1-1:YFP and mtDsRed. The following stacks of optical serial sections along the z-axis are
merged: a 21-image stack of a 0.41 µm interval for (A), a 20-image stack of a 0.41 µm interval for (B), a 31-image stack of a 0.41 µm interval for
(C) and a 25-image stack of a 0.41 µm interval for (D). The absence of detectable plastid DNA in the cells in (C) and (D) was confirmed by
observing each optical section. Each optical section of (A), (B), (C) and (D) is shown in Supplementary Movies 4, 5, 6 and 7, respectively. Images
showing DAPI fluorescence and the corresponding images showing DsRed fluorescence are overlaid with the corresponding images showing YFP
fluorescence. The corresponding Nomarski images (DIC) are shown in the right panels. The shapes of the cells are indicated by dashed lines in
(C) and (D). Arrowheads indicate the cells without detectable plastid DNA. Arrows indicate enlarged plastids containing DNA. All cells in these
images were taken from the expanding rosette leaves of 12-day-old plants. Scale bars = 5 µm.
the expansion of plastids to take up the entire volume of the
cell probably ensures the even allocation of plastids to both
daughter cells during cell division when the division of plastids is suppressed (Figs. 3E, 4). Our results are consistent
with Birky’s mathematical analysis of a physical model of
plastid partition (Birky 1983). It has been reported that the
ratio of plan chloroplast area to plan cells area is constant in
mesophyll cells, regardless of chloroplast number (Pyke and
Leech 1992, Pyke and Leech 1994). The same mechanism
probably functions in dividing cells and ensures the allocation of plastids to both daughter cells when the number of
plastids is decreased. The proliferation and expansion of plastids prior to the onset of cell division may be an important
step in plastid allocation to daughter cells. Plastids in crl,
however, may be partially defective in their ability to expand,
and may for this reason be lost from cells during cell division.
A deficiency in crl at a locus which encodes an outer envelope membrane protein may cause plastids to lose their
membrane integrity and prevent their expansion. Such a
deficiency in membrane integrity may also prevent the constriction of the plastid division ring, leading to the suppression of plastid division. The existence of dividing cells lacking
detectable plastids in crl embryos (Fig. 3D) suggests that
plastids are not essential for plant cell division, and that a
plant cell may have no cell cycle checkpoint to prevent the
generation of cells without plastids.
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Fig. 6 Plastids without chlorophyll were generated in crl and arc6 embryo cells. Confocal images of cells from cotyledons of mature embryos
expressing TPFtsZ1-1:YFP. Wild-type (WT) (A, D), crl (B, E, F) and arc6 (C) cells are shown. Images showing chlorophyll fluorescence are overlaid with
the corresponding images showing YFP fluorescence in (A–C). Images of DAPI fluorescence are overlaid with the corresponding images showing
TPFtsZ1-1:YFP fluorescence in (D–F). The corresponding Nomarski images (DIC) are shown in the right-hand panels. The following stacks of optical
serial sections along the z-axis are merged: a 30-image stack of a 0.44 µm interval for (A), a 32-image stack of a 0.43 µm interval for (B), a 30-image
stack of a 0.46 µm interval for (C), a 30-image stack of a 0.39 µm interval for (D), a 25-image stack of a 0.39 µm interval for (E) and a 24-image stack
of a 0.39 µm interval for (F). Arrowheads in (F) indicate plastids having no distinct plastid DNA. Scale bars = 10 µm (A–C), 5 µm (D–F).
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Plant Cell Physiol. 50(5): 956–969 (2009) doi:10.1093/pcp/pcp047 © The Author 2009.
Plant cells without detectable plastids
Fig. 7 Plastids without chlorophyll were generated in crl and arc6 stomatal guard cells and mesophyll cells. (A–E) Confocal images of the guard
cells from cotyledons of the crl mutant, the arc6 mutant and the wild type (WT) expressing TPFtsZ1-1:YFP. Images showing chlorophyll fluorescence
and the corresponding images showing YFP fluorescence are overlaid with the corresponding Nomarski image (DIC). (A) Wild-type stomatal
guard cells containing chloroplasts. (B) crl stomatal guard cells with plastids containing undetectable amounts of chlorophyll. (C) arc6 stomatal
guard cells with plastids containing undetectable amounts of chlorophyll. (D, E) Enlarged chloroplasts giving rise to protrusions in crl (D) and
arc6 (E). (F–H) Confocal images of mesophyll cells from the first or second leaves of (F) wild-type (WT), (G) crl and (H) arc6 plants expressing
TPFtsZ1-1:YFP. Images showing YFP fluorescence are overlaid with the corresponding images of chlorophyll fluorescence. The corresponding
Nomarski images (DIC) are shown in the right-hand panels. Arrows in (G) and (H) indicate plastids with trace or undetectable amounts
of chlorophyll. Images were taken from 13- and 14-day-old plants that had undergone neither fixation nor incubation in EDTA (A–E) or from
18-day-old plants (F–H). Scale bars = 5 µm (A–C), 2 µm (D, E), 20 µm (F–H).
In crl mesophyll cells, we found no cells without detectable plastids (Fig. 1C). This might be because the size of the
chloroplasts of crl mesophyll cells was large enough to ensure
plastid partitioning to daughter cells. It is also possible that
mesophyll cells without detectable plastids were not classified as mesophyll cells in our analysis; it was, in fact, difficult
to identify cells without detectable plastids and/or those
containing only plastids without chlorophyll as mesophyll
cells because of the presence of epidermal cells whose shape
is similar to that of mesophyll cells in spongy tissue and palisade tissues.
Whereas both crl and arc6 have severe defects in plastid
division, only crl shows significant defects in gross plant morphology (Robertson et al. 1995, Asano et al. 2004a). We have
demonstrated that crl shows developmental abnormalities
in embryos and in leaves (Asano et al. 2004a), in both of
which we have also found cells without detectable plastids
(Figs. 2C; 5C, D). The existence of cells without detectable
plastids in crl but not in arc6 suggests that the existence of
cells without detectable plastids is at least one of the causes
of crl’s morphological defects. A number of studies have
indicated the importance of plastid integrity in organ development (Chatterjee et al. 1996, Keddie et al. 1996, Mandel
et al. 1996, Uwer et al. 1998, Albert et al. 1999, Wang et al. 2000,
Apuya et al. 2001, Despres et al. 2001, Apuya et al. 2002,
Kuroda and Maliga 2003, Asano et al. 2004b, Raynaud et al.
2005, Hust and Gutensohn 2006, Garton et al. 2007). Plastids
have also been shown to be necessary for organ development in a cell-autonomous manner through the analysis of
variegated mutants. For example, in the immutans mutant
of Arabidopsis, sectors containing functional or dysfunctional plastids exist next to each other; white sectors contain
dysfunctional plastids and poorly developed palisade layers
(Aluru et al. 2001). Thus, crl cells without detectable plastids
Plant Cell Physiol. 50(5): 956–969 (2009) doi:10.1093/pcp/pcp047 © The Author 2009.
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may not be differentiated properly because of their lack of
plastids, and may thus lose the ability to undergo organized
cell division. A patchy distribution of improperly differentiated cells in the developing organs of a crl plant may affect
organ morphology.
An alternative explanation for the defects in crl’s gross
plant morphology is that a defect in cell cycle progression
may occur in cells without detectable plastids. It has been
shown that defects in cell cycle progression disturb the pattern of cell division and differentiation during plant development (Suzuki et al. 2004, Jenik et al. 2005, Suzuki et al. 2005,
Inagaki et al. 2006). An example of this can be seen in the
tebichi (teb) mutant of A. thaliana (Inagaki et al. 2006). The
teb mutant has defects in the pattern of cell division and in
the overall structures of shoot apical meristems (SAMs),
root apical meristem (RAMs) and embryos, as well as in the
morphology of leaves, stems and roots (Inagaki et al. 2006).
A possible mechanism by which a defect in cell cycle progression could lead to a defect in cell differentiation has been
proposed: stochastic delay of cell cycle progression in the
meristem may cause improper cell organization, which
affects the positional information that is essential for proper
cell differentiation (Inagaki et al. 2006). In crl, a short supply
of metabolites could cause a delay in cell cycle progression in
cells without detectable plastids; in this case, the existence of
such cells, which would have less mitotic activity in SAMs,
RAMs and embryos, could cause distortion in the pattern of
cell organization, which in turn would affect the positional
information.
It has been reported that enlarged chloroplasts in the
tomato suffulta (su) mutant give rise to a wild-type-like population of chromoplasts in ripe fruit through a process of
plastid budding and fragmentation (Forth and Pyke 2006).
Plastid-derived protrusions lacking chlorophyll in guard cells
have also been reported in su (Forth and Pyke 2006). Our
results indicate that similar plastid-derived protrusions lacking chlorophyll were also generated from enlarged chloroplasts in crl and arc6 guard cells (Fig. 7D, E). Thus, enlarged
chloroplasts in crl and arc6 probably undergo a similar process of plastid proliferation, not only in guard cells, but also
in mesophyll cells and in cells of the cotyledon primordia of
embryos. Our results also indicate that neither ARC6 nor
CRL is required for this mode of plastid division. Moreover,
the FtsZ ring does not form a ring structure in arc6 chloroplasts (Vitha et al. 2003), so this mode of plastid division
must not require the formation of an FtsZ ring. This mode of
plastid division was not observed in wild-type cells (Fig. 1),
confirming that it can only occur when the binary fission of
plastids is perturbed, as has been suggested with regard to
the tomato su mutant (Forth and Pyke 2006). Finally, our
DAPI staining experiment revealed the existence of plastids
without detectable plastid DNA in crl (Fig. 6F). Such plastids
were also found in arc6 embryos, but not in wild-type
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embryos (data not shown). Thus plastid DNA might not
always be transmitted to the daughter plastids in the budding and fragmentation mode of plastid proliferation.
Materials and Methods
Plant materials and growth conditions
The arc6 mutant line (CS286) was obtained from the Arabidopsis Biological Resource Center (ABRC) and backcrossed
with Col-0 three times before analysis. Plants were grown at
22°C under long-day conditions (16 h of light, 8 h of dark) or
continuous light conditions on Murashige and Skoog (MS)
plates or soil. For growth on plates, the plants were grown on
MS growth medium containing 0.5% (w/v) gellan gum
(Wako Pure Chemical Industries, Osaka, Japan) and 2%
(w/v) sucrose. The mtDsRed-expressing plants were generated by a genetic cross with a mtDsRed-expressing line,
which was a gift from S. Arimura. The mtDsRed has the presequence of the mitochondrial ATPase δ′ subunit of A. thaliana (Sakamoto and Wintz 1996) at the N-terminus of the
DsRed monomer (Clontech Laboratories, Inc., Mountain
View, CA, USA) and transcribed by the cauliflower mosaic
virus 35S RNA (CaMV 35S) promoter.
Plasmid construction
The coding sequence of cyan fluorescent protein (CFP) in a
construct expressing CFP fused with the transit peptide of
AtFtsZ1-1 (90 amino acids) by the CaMV 35S promoter
(Fujiwara et al. 2004) was exchanged with that of YFP (EYFP,
Clontech Laboratories, Inc.). A chimeric gene comprising the
CaMV 35S promoter region, the AtFtsZ1-1 transit peptide
sequence, the YFP gene and the NOS terminator sequence
was ligated into the cloning site of the binary vector
pSMAB704 (provided by Dr. H. Ichikawa) to yield pSMABZ1TP-YFP. pSMAB-Z1TP-YFP was transformed into A. thaliana ecotype Columbia by the Agrobacterium-mediated
floral dip method (Clough and Bent 1998). A total of 331
transgenic Arabidopsis seedlings were selected on bialaphoscontaining MS plates, and one transgenic line, FL6-4, which
showed high and stable expression of the plastid-targeted
YFP (designated as TPFtsZ1-1:YFP), was further characterized
for microscopic observations.
Plastid analysis
Samples for plastid analysis were prepared by the method
described by Pyke and Leech (1991) with slight modifications. Samples were fixed with 2% (w/v) paraformaldehyde
in phosphate-buffered saline (PBS; 8.1 mM Na2HPO4, 1.5 mM
KH2PO4, 137 mM NaCl, 2.7 mM KCl) for 0.5–1 h. Samples
were then incubated at 65°C for 1 h or at 4°C overnight in
0.1 M Na2EDTA (pH 9.0). Chloroplast and plastid numbers
were determined in pavement cells, mesophyll cells and cells
from the cotyledons of mature embryos in plants expressing
Plant Cell Physiol. 50(5): 956–969 (2009) doi:10.1093/pcp/pcp047 © The Author 2009.
Plant cells without detectable plastids
TPftsZ1-1:YFP. Cotyledons of mature embryos were dissected
under a stereomicroscope before analysis. Chloroplasts and
plastids in guard cells in the cotyledons of plants expressing
TPftsZ1-1:YFP were counted without fixation. For observation
of DNA, specimens were treated as described above, and
then stained with 1 µg ml−1 DAPI in TAN buffer (Nemoto
et al. 1988).
Confocal microscopy
Chlorophyll fluorescence, YFP fluorescence, DsRed fluorescence and Alexa Fluor 546 fluorescence were observed with
an LSM 510 confocal microscope (Carl Zeiss, Inc.,
Oberkochlen, Germany) and an FV-1000 confocal microscope (Olympus Co., Tokyo, Japan). The excitation wavelength for DAPI was 405 nm, and emission was collected at
425–475 nm. The excitation wavelength for YFP was 488 nm,
and emission was collected at 510–530 nm for LSM510 and
at 500–530 nm for FV-1000. The excitation wavelength
for chlorophyll was 488 nm, and emission was collected at
659–798 nm for LSM510 and at 655–755 nm for FV-1000.
The excitation wavelength for DsRed was 543 nm, and emission was collected at 560–660 nm. The excitation wavelength
for Alexa Fluor 546 was 545 nm, and emission was collected
at 563–595 nm.
Analysis of plan plastid area and plan cell area
The plan area of plastids in dividing cells and that of the cells
themselves were determined as follows: torpedo-stage
embryos expressing TPFtsZ1-1:YFP were dissected from ovules
under a stereomicroscope and then fixed with 2% (w/v)
paraformaldehyde in PBS and treated in 0.1 M Na2EDTA (pH
9.0) as described above. Cells having condensed chromosomes were scanned in three dimensions with an FV-1000
confocal microscope. Image stacks along the z-axis were
merged, and areas of plastids and those of cells were measured using FV10-ASW software (Olympus Co., Tokyo,
Japan). The ratio was obtained by dividing the area of the
plastids in each cell by the area of the cell itself.
Whole-mount immunolocalization
Whole-mount immunolocalization of Tic110 was performed as described by Friml et al. (2003) with slight modifications as follows: cell walls were digested with 2% cellulase
R-10 (Yakult, Tokyo, Japan)/0.02% pectolyase Y-23 (Kyowa
Chemical Products, Osaka, Japan) in 0.4 M mannitol, 1 mM
CaCl2 and 0.2% bovine serum albumin (BSA; pH 5.5) for
30–60 min at 25°C. Antiserum against Arabidopsis Tic110
(1 : 500; Inaba et al. 2005) and Alexa Fluor 546 goat anti-rabbit IgG (1 : 1,000; Invitrogen Corp., Carlsbad, CA, USA) were
used for Tic110 detection. Alexa Fluor 546 fluorescence was
observed with an LSM510 confocal microscope (Carl Zeiss,
Inc., Oberkochlen, Germany).
Supplementary data
Supplementary data are available at PCP online.
Funding
The Ministry of Education, Culture, Sports, Science and
Technology (No. 17051012 to Y.Y., No. 19060003 to Y.M. and
Y.Y.); the Japan Society for the Promotion of Science (No.
18570037 to Y.Y.).
Acknowledgments
We thank S. Arimura (University of Tokyo) for providing the
seeds of the mtDsRed plant, H. Ichikawa (NIAS) for the
pSMAB704 vector, the Arabidopsis Biological Research
Center (USA) for the seeds of the arc6 mutant (CS286), and
Danny J. Schnell (University of Massachusetts at Amherst)
for providing antiserum against Arabidopsis Tic110.
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(Received February 7, 2009; Accepted March 21, 2009)
Plant Cell Physiol. 50(5): 956–969 (2009) doi:10.1093/pcp/pcp047 © The Author 2009.
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