Download Direct Evidence of Active and Rapid Nuclear

Direct Evidence of Active and Rapid Nuclear Degradation
Triggered by Vacuole Rupture during Programmed Cell
Death in Zinnia1
Keisuke Obara*, Hideo Kuriyama, and Hiroo Fukuda
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo,
Tokyo 113–0033, Japan
Differentiation into a tracheary element (TE) is a typical example of programmed cell death (PCD) in the developmental
processes of vascular plants. In the PCD process the TE degrades its cellular contents and becomes a hollow corpse that
serves as a water conduct. Using a zinnia (Zinnia elegans) cell culture we obtained serial observations of single living cells
undergoing TE PCD by confocal laser scanning microscopy. Vital staining was performed and the relative fluorescence
intensity was measured, revealing that the tonoplast of the swollen vacuole in TEs loses selective permeability of fluorescein
just before its physical rupture. After the vacuole ruptured the nucleus was degraded rapidly within 10 to 20 min. No
prominent chromatin condensation or nuclear fragmentation occurred in this process. Nucleoids in chloroplasts were also
degraded in a similar time course to that of the nucleus. Degradations did not occur in non-TEs forced to rupture the vacuole
by probenecid treatment. These results demonstrate that TE differentiation involves a unique type of PCD in which active
and rapid nuclear degradation is triggered by vacuole rupture.
Programmed cell death (PCD) is an active cell
death process involved in the selective elimination of
unwanted cells, and it is found throughout animal
and plant kingdoms (Ellis et al., 1991; Jones and
Dangl, 1996). The term apoptosis (Kerr et al., 1972)
usually refers to a morphological type often observed
in PCD that involves nuclear shrinkage and fragmentation, cellular shrinkage, DNA fragmentation, membrane budding, the formation of apoptotic bodies,
and digestion by macrophages (Wyllie, 1980; Kerr
and Harmon, 1991). Although none of plant PCDs
has been reported to possess all of these apoptotic
features, some plant PCD processes show a subset of
apoptotic features: Salt stress induces nuclear fragmentation and DNA degradation into oligonucleosomal fragments in barley roots (Katsuhara and Kawasaki, 1996; Katsuhara, 1997). DNA laddering is
also reported in Arabidopsis roots and maize cultured
cells during PCD induced by Man (Stein and Hansen,
1999). However, this apoptosis-like cell death does not
account for the majority of PCD in plants (Fukuda,
1998). Senescence of leaves (Smart, 1994) and ovaries
(Vercher et al., 1987) shows the features closer to necrosis than apoptosis. Differentiation into tracheary
elements (TEs), a typical example of PCD in higher
1
This work was supported in part by the Ministry of Education, Science, Sports and Culture of Japan (grant nos. 10304063,
10219201, and 10182101 to H.F.; grant nos. 10158204 and 09740587
to T.D.) and by the Japan Society for the Promotion of Science
(grant no. JSPS–RFTF96L00605 to H.F.).
* Corresponding author; e-mail [email protected];
fax 81–3–5841– 4462.
plants (Pennell and Lamb, 1997), also exhibits morphological features closer to necrosis (Fukuda, 1998).
Extensive studies about PCD during TE differentiation have been performed using the zinnia (Zinnia
elegans) culture system established by Fukuda and
Komamine (1980a). In the zinnia cell cultures, single
mesophyll cells transdifferentiate directly into TEs
without cell division (Fukuda and Komamine, 1980b).
This system is useful for studies of the processes of
PCD because differentiation occurs at a high frequency and because the sequence of events during
PCD can be followed in single cells (Chasan, 1994;
Fukuda, 1997). Isolated zinnia cells cultured with phytohormones begin to form secondary cell walls and
several hours later PCD occurs to become hollow dead
cells (Fukuda, 1997; Groover et al., 1997). A number of
ultrastructural observations of TE cell death indicated
rapid and progressive degeneration of the nucleus,
vacuole, plastids, mitochondria, and endoplasmic reticulum and finally, the removal of protoplasts, the
plasma membrane, and parts of primary walls
(O’Brien and Thimann, 1967; Srivastava and Singh,
1972; Lai and Srivastava, 1976; Esau and Charvat,
1978; Burgess and Linstead, 1984; Groover et al., 1997).
Various hydrolytic enzymes including nucleases
(Thelen and Northcote, 1989; Ye and Droste, 1996;
Aoyagi et al., 1998) and proteases (Minami and
Fukuda, 1995; Ye and Varner, 1996; Beers and Freeman, 1997) are synthesized for active degeneration of
cellular contents and are thought to accumulate in the
vacuole of TEs to sequester them from the cytoplasm.
Thus, the collapse of the vacuole is a critical irreversible step to execute the degradation of various organelles (Fukuda, 1996; Groover et al., 1997; Groover
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Obara et al.
and Jones, 1999; Kuriyama, 1999). Groover et al. (1997)
observed the process of TE differentiation using video
camera and revealed that the cessation of cytoplasmic
streaming occurs immediately after the tonoplast disruption, suggesting that TEs keep physiological activity until vacuole collapse. However, the precise kinetic
relationship between tonoplast disruption and degradation of other organelles is unknown.
In this report we present direct evidence that rapid
nuclear degradation occurs immediately after vacuole collapse.
RESULTS
Imaging of the Vacuole Rupture and the Morphological
Change of the Nucleus in Differentiating TEs
Cells at 54 h of culture were loaded with fluorescein diacetate (FDA) and SYTO16 for 1 h and 10 min,
respectively, and were observed by confocal laser
scanning microscopy (CLSM). FDA is a hydrophobic molecule that enters cells in a passive way and
becomes de-esterified in living cells to become
membrane-impermeant fluorescein. Kuriyama (1999)
reported that although fluorescein accumulates into
the vacuole of most cultured cells after 1 h of FDA
loading, it is excluded from the vacuole of highly
vacuolated TEs at the late stage of differentiation.
Therefore, to catch the moment of vacuole rupture and
analyze morphological changes in the nucleus just
before and after the vacuole rupture we chose and
observed successively highly vacuolated TEs with
green fluorescence in the cytoplasm. Figure 1 shows a
series of events occurring before and after vacuole
rupture. The central vacuole expanded greatly to a
point almost occupying the intervening space between
thickened secondary cell walls, pressing the nucleus
tightly against the plasma membrane and making it
almost flat (Fig. 1a). Seven minutes later the fluorescence disappeared from the cytoplasm and there was
no longer an obvious boundary between the cytoplasm and the vacuole (Fig. 1b). The nucleus became
spherical, probably by the liberation of vacuole pressure. The tonoplast was no longer discernable under
bright field microscopy after this event (data not
shown). Therefore, this morphological change in the
nucleus and the loss of boundary between the cyto-
Figure 1. A series of images of a TE that was undergoing vacuole rupture (a–f). This TE was stained with SYTO16 and FDA (a–e).
The red autofluorescence of the chloroplasts was merged. a, The green fluorescence of SYTO16 and fluorescein could be
observed in the nucleus and cytoplasm (7 min before vacuole rupture). The TE was highly vacuolated and its nucleus was
tightly pressed against the plasma membrane. b, Soon after vacuole rupture (0 min), the nucleus was released from the vacuolar
turgor pressure and became spherical. The heterochromatin structure could be seen just inside the nucleus. SYTO16
fluorescence in some chloroplasts appeared in this focal plane. The green fluorescence in the cytoplasm disappeared probably
because the emission spectra of fluorescein changed following vacuole rupture (see “Discussion”). c, Nuclear fluorescence
started decreasing from the central region (6 min after vacuole rupture). d, Nuclear fluorescence of SYTO16 decreased
markedly in the central region (10 min after vacuole rupture). e, The fluorescence of SYTO16 disappeared almost completely,
whereas the autofluorescence of the chloroplasts was still visible (18 min after vacuole rupture). f, The nuclear envelope could
be seen in the light image of the TE even 20 min after vacuole rupture (arrow). The bar indicates 20 ␮m.
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Plant Physiol. Vol. 125, 2001
Rapid Nuclear Degradation Triggered by Vacuole Collapse
plasm and the vacuole are considered to be markers of
tonoplast disruption. Fluorescence in the nucleus disappeared rapidly from the central part (Fig. 1, c and d)
and then from the inner edge, and it became undetectable within 20 min after vacuole rupture (Fig. 1e).
However, the nuclear envelope was still kept in such
nuclei (Fig. 1f). Moreover, the nucleus and chloroplasts did not change the location in the cell in the
period of 20 min after vacuole rupture, indicating that
these compartments may be anchored to the plasma
membrane.
Loss of Tonoplast Integrity Just before
Physical Rupture
Differentiating TEs were highly vacuolated and excluded fluorescein from the vacuole before vacuole
rupture (Fig. 1a; Groover et al., 1997; Kuriyama,
1999), indicating that the tonoplast still has selective
permeability before tonoplast rupture. By observing
a living cell successively we found a phase between
vacuole swelling and vacuole rupture. In this intermediate phase the nucleus is still pressed against the
plasma membrane, indicating that the vacuole still
keeps its osmotic pressure, whereas fluorescence in
the cytoplasm decreases markedly (Fig. 2, a and b).
Propidium iodide, which can stain the nucleus of
only cells of which the plasma membrane became
leaky, did not label the nucleus of TEs just after the
vacuole ruptured, whereas it stained the nucleus of
dead non-TEs quickly (Fig. 3). This result indicates
that the plasma membrane keeps its integrity at least
until vacuole rupture occurs. Therefore the decrease
in fluorescence in the cytoplasm prior to the vacuole
rupture does not seem to result from the diffusion of
fluorescein across the plasma membrane, but across
Figure 2. Serial fluorescence images of a TE stained with SYTO16 and FDA (a–e). Red autofluorescence of the chloroplasts
was eliminated. a, The fluorescence in the nucleus and cytoplasm was evident in the TE 7 min before vacuole rupture. The
TE was so vacuolated that its nucleus was pressed against the plasma membrane and flattened. Boundary between the
cytoplasm and vacuole was definite. b, The boundary became obscure and fluorescence intensity in the cytoplasm
decreased, whereas that in the vacuole increased (2 min before vacuole rupture). Note that the nucleus was still flattened.
c, The nucleus was released from the vacuole turgor pressure soon after vacuole rupture (0 min). d, The fluorescence
intensity in the nucleus decreased markedly in the central region (4 min after vacuole rupture). e, The fluorescence of
SYTO16 disappeared almost completely (14 min after vacuole rupture). f, Secondary wall thickening had already been
obvious even 22 min before vacuole rupture. The bar indicates 20 ␮m.
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Obara et al.
Figure 3. Images of cultured cells stained with FDA, SYTO16, and propidium iodide. Light (a, d, and g), fluorescein and
SYTO16 (b, e, and h), and propidium iodide (c, f, and i) images of cells cultured for 60 h. The TE indicated by an arrow in
a was magnified in d through f. The TE exhibited yellow fluorescence of fluorescein in the whole cell and no green
fluorescence in the cytoplasm, indicating that the TE had just undergone vacuole rupture (Kuriyama, 1999). The dead non-TE
cell indicated by an arrowhead in a was magnified in g through i. Note that propidium iodide did not stain the nucleus of
the TE just after vacuole rupture (f) although it stained the nucleus of the dead non-TE (i). Bars indicate 10 ␮m.
the tonoplast. Because fluorescein hardly diffuses
across intact membrane, at least in cultured zinnia
cells (Kuriyama, 1999), this fluorescein diffusion
across the tonoplast represents the loss of tonoplast
integrity in blocking charged molecules. Two minutes after the tonoplast lost its selective permeability,
the nucleus became almost sphere by liberation from
the vacuole pressure (Fig. 2c), indicating that the
final loss of tonoplast integrity occurs immediately
before its physical rupture.
Rapid Degradation of the Nucleus after
Vacuole Rupture
To examine whether the disappearance of fluorescence from the nucleus stained with SYTO16 is due to
active autolysis we measured the levels of nuclear
618
fluorescence of TEs before and after vacuole rupture
(Fig. 4). Each value was shown as a relative value
against the value obtained from the nucleus of the
cells whose vacuole just ruptured. Changes in the
fluorescence in non-TEs were used as an indicator of
photobleaching. Before the tonoplast ruptured, fluorescence in TEs and non-TEs did not show substantial decrease (Fig. 4A). This tendency was observed
for at least 50 min before vacuole rupture (data not
shown). Levels of nuclear fluorescence after vacuole
rupture were measured for individual TEs every 2
min. Almost all TEs except one showed a similar
decreasing pattern of the fluorescence (Fig. 4B). This
decreasing pattern was reproduced in another 10 TEs
(data not shown). Therefore, we calculated and represented the average of TE1 to TE5 in Figure 4A. This
graph clearly indicated a sudden decrease in nuclear
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Plant Physiol. Vol. 125, 2001
Rapid Nuclear Degradation Triggered by Vacuole Collapse
Figure 4. Changes in SYTO16 fluorescence intensity in the TE and non-TE nuclei. Fluorescence intensity was determined
as the sum of pixel values per area of each organelle. The data are presented as relative values to actual pixel counts on the
0-min image. A, The pattern of changes in nuclear SYTO16 fluorescence intensity in TEs before and after vacuole rupture
were compared with that in non-TEs observed in the same visual field. The SYTO16 fluorescence intensity of the TE nuclei
decreased dramatically after vacuole rupture. B, The pattern of changes in nuclear fluorescence intensity in individual TEs
after vacuole rupture. Five TEs exhibited the similarly the rapid decrease of their nuclear fluorescence following vacuole
rupture (represented by TE 1–TE 5), although a TE degraded its nucleus relatively slowly (represented by TE 6). The values
of closed square indicates the mean of five values (TE 1–TE 5) ⫾ SE. The values of closed lozenge indicates the mean of five
non-TEs ⫾ SE.
fluorescence after vacuole rupture. Fluorescence levels decreased approximately by one-half and onequarter at 4 and 6 min after the vacuole ruptured,
respectively. In contrast, fluorescence of the nucleus
Plant Physiol. Vol. 125, 2001
in non-TEs kept high all the time during observation.
To confirm that fluorescence intensity of SYTO16
actually represents DNA contents, nuclei isolated
from cultured zinnia cells were stained with SYTO16
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Obara et al.
and were incubated with DNase I. The fluorescence
of SYTO16 was completely removed by DNase I and
the rate of decline in fluorescence intensity after
DNase treatment was similar to that of nuclei stained
with 4⬘-6-diamidino-2-phenylindole (DAPI; Fig. 5).
These results indicate that the rapid decrease in
Figure 5. Images of DNA degradation in isolated nuclei. Differential interference contrast (a–c) and fluorescence (d–l)
images of isolated nuclei are shown. Nuclei were stained with SYTO16 (d, e, g, h, j, and k) or DAPI (f, i, and l). Nuclei were
treated with DNase I for 0 min (b, c, e, and f), 5 min (h and i), and 15 min (k and l). Images of the nucleus 0 min (a and d),
5 min (g), and 15 min (j) after mock treatment are also shown. Note that SYTO16 fluorescence after DNase I treatment
declined similarly to DAPI fluorescence, a marker of DNA content, (e, f, h, i, k, and l), whereas SYTO16 fluorescence
remained intensely without DNase I treatment (d, g, and j). The bar indicates 5 ␮m.
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Plant Physiol. Vol. 125, 2001
Rapid Nuclear Degradation Triggered by Vacuole Collapse
nuclear fluorescence in TEs results from active autolysis of DNA, but not from other factors such as
photobleaching.
Next we examined whether the rapid nuclear digestion after vacuole rupture is TE-specific or if it
occurs even in non-TE when the vacuole of non-TE is
forced to rupture. Probenecid, which is known to
inhibit organic anion transport (Cole et al., 1990;
Oparka et al., 1991; Wright and Oparka, 1994), not
only accelerated vacuole rupture in TEs, but also
induced vacuole rupture in non-TEs, although in
these, it occurred asynchronously after a long delay
(Kuriyama, 1999). Therefore, cells were incubated
with 100 ␮m probenecid for 12 h and were loaded
with SYTO16. Non-TEs in which the vacuole had
ruptured were recognized by the presence of balloonlike structures composed of fragmented tonoplast
replacing the central vacuole. It was difficult to catch
the moment of the probenecid-induced vacuole rupture of non-TEs because the timing of vacuole rupture after probenecid treatment varied widely among
non-TEs. Therefore, non-TEs that had undergone
vacuole rupture, but exhibited relatively intense nuclear SYTO16 fluorescence were chosen and observed
successively to examine the changes in the intensity
of nuclear fluorescence because such non-TEs were
expected to be cells that had lost the vacuole very
recently. The intensity of nuclear fluorescence of nonTEs with the vacuole did not change significantly for
30 min (Fig. 6). Although the nuclear fluorescence
intensity of four non-TEs of the five that had undergone vacuole rupture decreased during the 30-min
period, the decrease was much slower than that of
TEs after vacuole rupture. This result suggests that
active nuclear digestion does not occur in non-TEs
after vacuole rupture and that changes in physiological conditions in the cytoplasm caused by vacuole
rupture such as pH and Ca2⫹ concentration are not
the reason for the observed decline in nuclear
fluorescence.
Rapid Degradation of Nucleic Acids Compared with
Chlorophyll in Chloroplasts after Vacuole Rupture
DNA in chloroplasts and mitochondria forms a
complex “nucleoid” with proteins (Kuroiwa et al.,
1982, 1998). To examine whether degradation of
nucleoids in mitochondria and chloroplasts occurs
similarly to nuclear degradation or not, cells were
loaded with SYTO16 and observed successively with
focusing on the surface layer of thin cytoplasm.
Nucleoids in mitochondria and chloroplasts in TEs
exhibited strong green fluorescence before vacuole
rupture, indicating that nucleic acids in these organelles are not degraded markedly before the vacuole ruptures (Fig. 7a). We confirmed the observation
by Groover et al. (1997) that cytoplasmic streaming
continued until the vacuole ruptured.
After vacuole rupture the nucleoids in chloroplasts
appeared to be degraded rapidly, which is similar to
the degradation of the nuclear chromatin (Fig. 7, b, c,
and d). Fluorescence of nucleic acids in chloroplasts
and the nucleus decreased simultaneously by onehalf within approximately 6 min and to undetectable
level within 16 min after vacuole rupture (Fig. 8). In
contrast, chlorophyll fluorescence did not decrease
detectably even at 16 min after vacuole rupture (Fig.
8). Moreover, the appearance of chloroplasts detected
by autofluorescence did not change even at the time
when nucleic acids in them were mostly degraded
Figure 6. Changes in the intensity of nuclear SYTO16 fluorescence in non-TEs that have undergone probenecid-induced
vacuole rupture. Because the timing of probenecid-induced vacuole rupture in non-TEs varied widely, non-TEs that had
already undergone vacuole rupture and still exhibit intense nuclear SYTO16 fluorescence were chosen and analyzed
(represented by non-TE 1–TE 5). The value of closed square indicates the mean of five living non-TEs that had the vacuole.
Error bars represent SE.
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Figure 7. Fluorescence images of a TE that was undergoing vacuole rupture. The autofluorescence of the several chloroplasts was in focus. a, Nucleic acids in the nucleus, chloroplasts, and mitochondria could be observed in a TE 5 min before
vacuole rupture. The nucleus was pressed against the plasma membrane by the large central vacuole. b, The nucleus was
released from pressure soon after vacuole rupture (0 min). c, SYTO16 fluorescence in some chloroplasts disappeared within
4 min after vacuole rupture. d, Nucleic acids in the nucleus and chloroplasts were almost completely degraded, whereas
chloroplast autofluorescence was still visible. The bar indicates 20 ␮m.
(Fig. 7d). These data indicate a rapid and preferential
degradation of nucleic acids in chloroplasts compared with chlorophyll. We could not analyze the
kinetics of mitochondrial nucleoids degradation because mitochondria were not stationary.
DISCUSSION
Plant PCD does not involve phagocytosis by adjacent cells, which is often observed in animal apoptosis (Greenberg, 1996). In the case of TE PCD, differentiating TEs synthesize newly hydrolytic enzymes
and degenerate cellular contents without the assistance of neighboring cells. Our serial observation of
SYTO16-stained nuclei provided direct evidence that
nuclear degradation occurs only after the disruption
of the tonoplast (Fig. 4A). Likewise, in the PCD process during aerenchyma formation in maize (Campbell and Drew, 1983) and senescence of unpolinated
ovaries (Vercher et al., 1987), the tonoplast disruption
is suggested to play a critical role in autolysis. In
animal PCD there is a type of PCD that is suggested
to involve the secretion of hydrolases from lysozomes leading to the loss of cytoplasm (Clarke, 1990).
Jones (2000) proposed that plant PCD in which vacuole collapse plays a critical role in autolysis may be
similar to the lysozomal PCD in animal.
Once the vacuole ruptured, nucleic acids in the nucleus and chloroplasts were degraded rapidly to undetectable levels within approximately 15 min (Figs. 4
Figure 8. Changes in the intensity of nuclear SYTO16, chloroplast SYTO16, and chlorophyll fluorescence following vacuole
rupture. The data are presented as relative values to actual pixel counts per area on the 0-min image. Error bars represent SE.
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Plant Physiol. Vol. 125, 2001
Rapid Nuclear Degradation Triggered by Vacuole Collapse
and 8). However, chlorophyll was not degraded and
remained intensely fluorescence after SYTO16 fluorescence was undetectable (Fig. 8). Active and rapid digestion of chloroplastic DNA compared with chlorophyll is also observed in chloroplast of male origin in
mated Chlamydomonas reinhardtii (Kuroiwa et al., 1982;
Nishimura et al., 1999). These results suggest that
plants possess a mechanism of digesting unneeded or
unwanted nucleic acids actively, rapidly, and selectively. On the other hand, digestion of chloroplast
DNA during senescence of rice coleoptiles proceeds
gradually and spans days. In this senescence digestion
of membrane structure, proteins, and DNA in chloroplasts commences before tonoplast disruption (Inada
et al., 1998). In leaf senescence the hydrolysis of the
nucleus occurs at a very late stage (Noodén and Guiamet, 1996), which is preceded by breakdown of membrane structure and proteins in the chloroplast (Bate et
al., 1990; Bleecker and Patterson, 1997). Therefore,
there seems to be different PCD mechanisms in plants,
as typically shown in TE PCD and leaf senescence
(Fukuda, 2000).
Rapid digestion of nucleic acids after vacuole rapture in differentiating TEs suggests the strong activity of nuclease(s) released from the vacuole. It has
been reported that some nucleases accumulated specifically in differentiating TEs (Thelen and Northcote, 1989; Ye and Droste, 1996; Aoyagi et al., 1998).
Of these nucleases, ZEN1 (a 43 kD-nuclease) is only
the nuclease that can degrade double-strand DNA
(Thelen and Northcote, 1989; Aoyagi et al., 1998).
This enzyme, which is thought to accumulate in the
vacuole, is synthesized just before autolysis of TEs
(Aoyagi et al., 1998). Therefore, this nuclease may be
responsible for the active degradation of nucleic
acids in the nucleus and chloroplasts.
After vacuole rupture the nucleus was degraded
from the central region (Fig. 1, b, c, and d). The
inner-edge region of the nucleus, which exhibits
chromatin condensation, was digested a little later
than the central region (Fig. 1, b, c, and d). This delay
may be due to the difficulty for the nuclease(s) to
attack DNA in these condensed region, heterochromatin. The nucleus kept its spherical form even after
the nucleic acids were degraded (Fig. 1f), indicating
the absence of nuclear fragmentation in PCD during
TE differentiation, unlike apoptosis, and suggesting
the presence of the mechanism keeping the nuclear
shape. The process of nuclear degradation is summarized in Figure 9.
Our data clearly show that vacuole rupture is a
trigger of nuclear degradation in TEs. Then what is the
mechanism of vacuole rupture? As shown in a previous paper (Kuriyama, 1999), the vacuole of differentiating TE swells before rupturing in differentiating
TEs. We found that the fluorescence of fluorescein in
the cytoplasm of differentiating TEs diminished rapidly just before the physical rupture of the vacuole;
that is, the tonoplast kept pressing the nucleus against
the plasma membrane (Fig. 2b). This rapid decrease in
the fluorescence was shown to result from alteration
of tonoplast permeability (Fig. 3). As regards the permeability of the tonoplast, the following three possibilities are proposed: (a) Fluorescein diffused across
the tonoplast; (b) proton was released from the vacuole into the cytoplasm; as a result, cytoplasmic pH
Figure 9. Model for the description of autolytic process during TE differentiation. a, TEs are highly vacuolated before vacuole
rupture. Cytoplasmic streaming continues to occur until this stage (Groover et al., 1997). b, The loss of tonoplast function
occurs. However, the vacuole is not so immediately fragmented that it still presses the nucleus against the plasma
membrane. c, Later, the nucleus becomes spherical. Nucleases (Thelen and Northcote, 1989; Ye and Droste, 1996; Aoyagi
et al., 1998) attack nucleic acids. Cys proteases (Minami and Fukuda, 1995; Ye and Varner, 1996; Beers and Freeman 1997)
degrade heterochromatin structure (data not shown) that can be seen just inside the nucleus. d, Degradation proceeds. e,
DNA digestion precedes the breakdown of whole nuclear and chloroplast structure, which are still attached to the plasma
membrane. f, TEs lose their contents and become mature. The wall at the tip becomes porous (Burgess and Linstead, 1984).
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Obara et al.
changed from neutral to acidic, and then green fluorescence from fluorescein decreased greatly; and (c) 1
and 2 occurred simultaneously. At present we do not
know which is correct. In any case, however, it is sure
that selective permeability of the tonoplast changes at
this stage. Although the permeability-change of the
tonoplast prior to its rupture is beginning to be revealed, a key mechanism leading to vacuole rupture
remains an enigma.
MATERIALS AND METHODS
25 mW for 0.4 s. Before vacuole rupture images were taken
at intervals of 5 or 7 min to avoid too much exposure of the
laser beam to cells. Immediately after the vacuole rupture
the interval was changed to 2 min. In each image, brightness of pixels in the region drawn around the nucleus of
TEs and non-TEs was measured and normalized by the
value measured in the image representing the moment of
vacuole collapse in individual cells. Fluorescence from the
nucleic acids in chloroplasts and auto-fluorescence of chlorophyll were also measured and normalized by the same
way.
Plant Material and Culture
The first leaves of 14-d-old seedlings of zinnia (Zinnia
elegans cv Canary Bird [Takii Shubyo, Kyoto]) were used for
the isolation of mesophyll cells in suspension culture according to the method of Fukuda and Komamine (1980a). All
experiments were performed with cells cultured in inductive D medium that contained 0.1 mg/L ␣-naphthylacetic
acid and 0.2 mg/L benzyladenine as phytohormones.
Fluorescence Staining
For CLSM, FDA (Aldrich Chemical, Milwaukee, WI) was
added to the medium to final concentration of 0.1 ␮g mL⫺1.
After 1 h of incubation with rotation, SYTO16 (Molecular
Probes, Eugene, OR) was added to the medium at final
concentration of 1 ␮m and was incubated for 10 min.
SYTO16 is a vital fluorescence dye that is membranepermeable and stains DNA and RNA (Luther and Kamentsky, 1996). When the integrity of the plasma membrane was examined, propidium iodide (Wako Pure
Chemicals, Osaka), which stains DNA and RNA, but is
membrane-impermeable, was added to the medium to final
concentration of 1.5 ␮g mL⫺1. All staining and incubation
was performed at 27°C.
CLSM
After stained cells were transferred onto poly-d-Lyscoated dishes (Glass Bottom Microwell Dishes, MatTek
Corporation, Ashland, MA), they were placed on the inverted platform of a confocal laser scanning microscope
(Meridian Instruments Far East, Tokyo). Dyes were excited
using a 488-nm line from an argon laser, and detection was
performed through a band-pass filter allowing the recording of green fluorescence from 515 to 545 nm. A long pass
filter above 630 nm was also used when the red autofluorescence of the chlorophyll was to be overlaid.
Measurement of Fluorescence
To quantify degradation of the nucleus, cells were
loaded only with SYTO16 to prevent the fluorescence of
FDA from overlapping because they have similar fluorescence emission spectra. Time-lapse images were taken for
TEs and non-TEs in the same field and analyzed using
INSIGHT-IQ system (Meridian Instruments Far East). In
each image cells were equally exposed to the laser beam at
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Isolation of Nuclei
The zinnia cells that had been cultured for 18 h were
collected on nylon meshes (pore size of 10 ␮m) and washed
in a mannitol solution composed of 0.7 m mannitol and
MES (5 mm 2-morpholinoethanesulfonic acid), pH 5.5.
These cells were resuspended in an enzyme solution containing 1% (w/v) cellulase onozuka RS (Yakult, Tokyo),
0.1% (w/v) pectolyase Y-23 (Seishin Pharmaceutical, Tokyo ), 0.6 m mannitol, 5 mm MgCl2, and 20 mm MES, pH
5.5, and were incubated at 27°C for 1 h to produce protoplasts. Protoplasts were collected by centrifuge at 200g for
1 min and resuspended in a solution containing 24% (w/v)
Suc and 5 mm MES, pH 5.5. The mannitol solution was
overlaid onto the resuspension. After centrifugation at 200g
for 5 min, the middle layer containing purified protoplasts
was collected. Purified protoplasts were washed in mannitol solution. Isolation of nuclei from purified protoplasts
was performed according to the method of Willmitzer and
Wagner (1981) with some modifications. The standard
buffer used here was composed of 0.25 m Suc, 10 mm NaCl,
5 mm EDTA, 0.15 mm spermine, 0.5 mm spermidine, 20 mm
2-mercaptoethanol, 0.2 mm phenylmethylsulfonyl fluoride,
0.1% (w/v) bovine serum albumin, and 10 mm MES, pH
6.0. Purified protoplasts were agitated in the standard
buffer supplemented with 1% (w/v) Triton X-100 and were
centrifuged at 1,000g for 10 min. The precipitate was suspended in the standard buffer supplemented with 0.1%
(w/v) Triton X-100 and 70% (v/v) percoll (Pharmacia Biotech, Piscataway, NJ), and was overlaid with the standard
buffer supplemented with 0.1% (w/v) Triton X-100 and
25% (v/v) percoll. After centrifugation at 600g for 20 min
the interphase was collected and diluted with the standard
buffer. The subsequent centrifugation at 1,000g for 10 min
gave a pellet of nuclei.
DNA Degradation in Isolated Nuclei
Isolated nuclei were resuspended in a DNase reaction
buffer containing 15 mm NaCl, 0.15 mm spermine, 0.5 mm
spermidine, 10% (w/v) Suc, 10 mm MnSO4, and MES, pH
6.0, and were incubated with a dye (1 ␮m SYTO16 or 2
␮g/mL DAPI) and 70U/mL DNase I. Fluorescence images
of each sample were taken immediately (0 min), and at 5
and 15 min after the addition of DNase I or reaction buffer
under an epifluorescence microscope (model BX-50-FLA,
Olympus, Tokyo) equipped with charge-coupled device
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Copyright © 2001 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 125, 2001
Rapid Nuclear Degradation Triggered by Vacuole Collapse
camera (HC-2500, Fuji Photo Film, Tokyo). Exposure of each
image was 0.1 s. All experiments were performed at 25°C.
Received July 24, 2000; returned for revision August 31,
2000; accepted November 2, 2000.
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