From the Department of Plant Sciences, The Weizmann Institute of Science, Rehovot 76100, Israel
Received for publication, February 26, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cellular dedifferentiation is the major process
underlying totipotency, regeneration, and formation of new stem cell
lineages in multicellular organisms. In animals it is often associated with carcinogenesis. Here, we used tobacco protoplasts (plant cells
devoid of cell wall) to study changes in chromatin structure in the
course of dedifferentiation of mesophyll cells. Using flow cytometry
and micrococcal nuclease analyses, we identified two phases of
chromatin decondensation prior to entry of cells into S phase. The
first phase takes place in the course of protoplast isolation,
following treatment with cell wall degrading enzymes, whereas the
second occurs only after protoplasts are induced with phytohormones to
re-enter the cell cycle. In the absence of hormonal application,
protoplasts undergo cycles of chromatin condensation/decondensation and
die. The ubiquitin proteolytic system was found indispensable for
protoplast progression into S phase, being required for the second but
not the first phase of chromatin decondensation. The emerging model
suggests that cellular dedifferentiation proceeds by two functionally
distinct phases of chromatin decondensation: the first is a transitory
phase that confers competence for cell fate switch, which is followed,
under appropriate conditions, by a second
proteasome-dependent phase representing a commitment for
the mitotic cycle. These findings might have implications for a
wide range of dedifferentiation-driven cellular processes in higher eukaryotes.
A high proportion of mature plant cells retain characteristic
features of totipotent stem cells, i.e. they have the
capability to dedifferentiate, re-enter the cell cycle, and
proliferate, eventually giving rise to the various organs that make up
a new plant (1). In multicellular organisms, cellular dedifferentiation is the major process enabling the regeneration of complex tissues and
organs as well as the establishment of new stem cell lineages; in
animals it is often associated with carcinogenesis. The molecular mechanism(s) underlying re-entry of differentiated animal cells into
the cell cycle and reactivation of DNA synthesis have been intensively
studied. Yet, little is known about the early events that accompany
cellular dedifferentiation, i.e. the establishment of
competence for cell fate switch and the determination of cell fate.
The tobacco protoplast system provides an outstanding experimental tool
for the study of the biochemical and molecular basis for cellular
dedifferentiation. The fully differentiated, non-dividing mesophyll
cells of tobacco leaves can be separated from their original tissue by
cell wall-degrading enzymes. This treatment results in the formation of
a large population of protoplasts (plant cells devoid of cell wall)
that, following treatment with phytohormones (auxin and cytokinin) can
re-enter the cell cycle and proliferate (2-5). This system
demonstrates a unique attribute of plant cells, totipotency,
i.e. the capability for cloning in plants (1).
Dedifferentiation of mature tobacco cells was shown to be accompanied
by a sharp increase in ubiquitin gene expression (6), the biochemical
tag that marks proteins for degradation by the ubiquitin proteasome
system (7). Elevation in ubiquitin gene expression probably represents
a critical point in cellular reorganization, namely, selective
destruction of proteins involved in maintaining the previous function
of a cell and a concomitant activation of proteins that are essential
for cell proliferation (8-11). Genetic studies in
Arabidopsis demonstrated that some of the factors involved in auxin response are components of the cell cycle-regulated
ubiquitin-protein-ligase complex (E3), known as SCF (Skp1-Cdc53-F-box
protein) (Ref. 12 and references therein). The SCF complex is required
for the ubiquitination of specific phosphorylated substrates
during G1 to S transition (9-11).
In the course of cellular dedifferentiation, mature cells undergo
remarkable changes in their pattern of gene expression, switching from
a program that drives the specific function of a given somatic cell to
a new one that directs cell multiplication. How do differentiated cells
remodel their gene expression program in a rapid and orderly manner?
Conceivably, chromatin reorganization could serve that purpose. Indeed,
it has been suggested that one of the first stages in cellular
dedifferentiation is the unfolding of chromatin supercoiling (13). We
currently study mechanism(s) underlying dedifferentiation of mature
plant cells, focusing on changes in chromatin structure. Using
FACS1 and MNase analyses, we
found that differentiated tobacco mesophyll cells undergo two distinct
phases of chromatin decondensation prior to entry into S phase. We also
report the involvement of the ubiquitin proteasome system in chromatin
decondensation and entry of cells into S phase.
Protoplast and Nuclei Preparation--
Protoplasts were isolated
from sterile leaves of Nicotiana tabacum ("Samsun NN")
essentially as described (14). Freshly prepared protoplasts were
washed and incubated at 22 °C in VKM medium containing 0.5 µg/ml
6'-benzylaminopurine (BAP) and 2 µg/ml Bromodeoxyuridine (BrdUrd) Labeling--
Protoplasts reactivated
for S phase (72 h after their preparation) were pulse-labeled with 10 µM BrdUrd (Sigma) for 30 min, after which nuclei were
isolated, stained with PI, and analyzed by FACS. Nuclei were then
sorted, and DNA was prepared (from about 50,000 nuclei) as described
below, resolved on 0.8% agarose gel, transferred onto nitrocellulose
membrane, and probed with anti-BrdUrd (Becton Dickinson).
Decondensation Assay--
Equal amounts of nuclei (determined by
pack volume and relative density) prepared from protoplasts or from
tobacco leaves were first permeabilized by incubation in Hamilton
buffer containing 0.15% Triton X-100 (15), washed and resuspended in
600 µl of nuclei digestion buffer (50 mM Tris-HCl, pH
8.0, 0.3 M sucrose, 5 mM MgCl2, 1.5 mM NaCl, 0.1 mM CaCl2, and 5 mM RNA Preparation and Northern Blot Analysis--
Total RNA was
prepared from protoplasts by using the Tri-Reagent kit (Sigma)
according to the manufacturer's protocol. Equal amounts of RNA were
resolved on 1.4% agarose formaldehyde gel, transferred to a BioTrace
pure nitrocellulose membrane (Gelman Sciences), and hybridized with
32P-labeled Arabidopsis Skp1 antisense RNA.
Arabidopsis Skp1 was obtained by polymerase chain reaction
using PWO DNA polymerase (Roche Molecular Biochemicals) and the
following primers: Skp1-sense, 5'-GTCGACGAATTCGAGCTCTCACTCGAGTCATTCAAAAGCCCATTGATTCTCC-3'
flanked with the EcoRI and BamHI restriction
sites, and Skp1-antisense primer,
5'-GTCGACGAATTCGAGCTCTCACTCGAGTCATTCAAAAGCCCATTGATTCTCC-3' flanked with
HincII and EcoRI sites. As a template we used the BAC clone T4L20 (GenBankTM/EBI accession number AL023094),
kindly provided by the Arabidopsis Biological Resource
Center (ABRC). The amplified DNA fragment was digested with
BamHI and EcoRI, subcloned into the same sites of
pBluescript SK, and sequenced to confirm identity. To prepare antisense
RNA probes, the plasmid DNA was linearized with BamHI and
subjected to in vitro transcription using the Riboprobe
System (Promega) and T7 RNA polymerase according to the manufacturer's protocol.
First Phase Chromatin Decondensation Occurs in Freshly Prepared
Protoplasts--
Tobacco leaves were treated with cell wall-degrading
enzymes to produce a large population of protoplasts. We compared the FACS histograms of PI-stained nuclei isolated from freshly prepared protoplasts (t-0 protoplasts) versus nuclei prepared from
leaves. Both t-0 protoplasts and leaf nuclei displayed a single peak
corresponding to G0/G1 content of DNA (Fig.
1, A and B).
However, nuclei prepared from t-0 protoplasts (Fig. 1B, G1
nuclei) reproducibly showed an upward shift in fluorescence
intensity compared with leaf nuclei (Fig. 1A, G0 nuclei). To
verify that these changes in PI fluorescence reflected intrinsic
differences in the PI binding capabilities of chromatin, both
populations of nuclei were mixed and incubated on ice for 15-30 min
prior to FACS analysis. Fig. 1C shows that even after nuclei
mixing, each population retained its position in the FACS histogram
supporting an intrinsic difference in PI binding capabilities of
chromatin. Because PI intercalates between the strands of DNA without
sequence specificity, its fluorescence intensity is directly
proportional to the level of accessible DNA, hence reflecting the
condensation state of chromatin. We next applied a biochemical test,
namely the MNase assay, to test whether the observed increment in
fluorescence intensity of protoplasts nuclei reflected an increase in
DNA accessibility to PI conferred by DNA decondensation (17). To this
end, permeabilized nuclei from leaves or from t-0 protoplasts were
treated with MNase for various time periods, and protected DNA
fragments were resolved by agarose gel electrophoresis. As shown in
Fig. 1D, digestion by MNase resulted in a typical
nucleosomal ladder visualized by ethidium bromide staining, where each
band represents DNA protected by one or several nucleosomes. Chromatin
of t-0 protoplasts showed a higer sensitivity to MNase than chromatin
from leaf nuclei (Fig. 1D, compare lane 6 with
lane 11). This verified that chromatin in nuclei of t-0
protoplasts is at a relatively decondensed configuration compared with
chromatin in leaf nuclei. By analyzing the effect of leaf and
protoplast nuclear protein extracts on the activity of MNase, we
excluded the possibility that the reduced sensitivity of leaf chromatin
to MNase resulted from the presence of nuclease inhibitors, otherwise
absent from protoplasts nuclei (data not shown). Taken together, we
suggest that the removal of the cell wall and the separation of
differentiated mesophyll cells from their original tissue induce a
stress response leading to conformational changes in chromatin.
Progression of Protoplasts into S Phase Is Accompanied by a Second
Phase of Chromatin Decondensation--
Following preparation,
protoplasts re-enter the cell cycle and reactivate DNA synthesis only
upon application of the phytohormones auxin and cytokinin. In the
absence of phytohormones protoplasts undergo cycles of chromatin
condensation/decondensation and die (Fig.
2). FACS analysis showed that within
24 h chromatin became condensed (Fig. 2C), shifting
from the G1 to G0 position (Fig. 2,
G1 and G0), which is typical for leaf nuclei
(Fig. 2A). The chromatin is then gradually decondensed, and
sub-G1 population, which is typical for cells undergoing
apoptotic cell death (18), starts to accumulate (Fig. 2, D
and E), eventually leading to protoplast death (not shown).
In the presence of phytohormones, protoplasts re-enter the cell cycle
(Fig. 3); division patterns were first
evident at 72 h after hormonal application (Fig. 3D, right panel). FACS analysis showed that most freshly
prepared protoplasts (t-0 protoplasts, Fig. 3A) as well as
those incubated with hormones for 24 h (Fig. 3B) had
DNA content corresponding to the G1 phase. By 48 h
(Fig. 3C), about 10% of protoplasts approached S phase, and
by 72 h (Fig. 3D) 30-40% were in S and G2
phases of the cell cycle. An extra G1 peak (referred to as
supra-G1; Fig. 3, *G1), revealing an increase in
PI fluorescence intensity, was reproducibly evident at 72 h after
protoplast preparation (Fig. 3D). In contrast, cycling
tobacco cells showed monophasic G1 (see Fig.
4C), suggesting that the
occurrence of a supra-G1 peak is unique to differentiated
cells re-entering the cell cycle. When protoplasts, however, were
incubated for a longer duration (96 h), almost no original
G1 nuclei were apparent; the majority of nuclei shifted to
the supra-G1 position (Fig. 3E). This pattern suggests that the supra-G1 peak represents a stage at which
nuclei are competent to re-enter S phase, and, therefore, may be
equivalent to G1 nuclei of cycling cells. To examine this
possibility, we established a cell suspension culture of N. tabacum (cycling cells) and compared the FACS histograms of nuclei
prepared from this culture with that of N. tabacum
protoplasts. Fig. 4 shows that G1 nuclei of cycling cells
had a higher fluorescence intensity than nuclei of t-0 protoplasts
(Fig. 4, compare A with C), coinciding with the
position of the supra-G1 nuclei (Fig. 4, compare
B with C). Each population of nuclei retained its
position in the histogram after mixing (data not shown). These results
indicate that the supra-G1 peak represents chromatin
relaxation rather than increased DNA content. This conclusion is
strengthened by the finding that treatment of protoplasts with HU (a
blocker of DNA replication) resulted in accumulation of both
G1 and supra-G1 nuclei (Fig. 5A, G1 and *G1), and by the
absence of BrdUrd labeling from supra-G1 nuclei (Fig. 5,
B and C). These results verify that the increased fluorescence intensity of PI-stained nuclei (supra-G1)
prior to entry of cells into S phase resulted from chromatin
decondensation, apparently an essential event for replication factors
to approach and be assembled onto origins to initiate DNA
replication.
The Ubiquitin Proteasome System Is Required for the Second Phase of
Chromatin Decondensation and Reactivation of S Phase--
The
expression of various ubiquitin genes is enhanced in the course of
dedifferentiation of mature tobacco cells (6). To investigate the
involvement of the ubiquitin pathway in dedifferentiation, we tested
the progression of protoplasts into S phase in the presence of MG132, a
specific inhibitor of the ubiquitin proteasome system. MG132 had no
effect on the occurrence of the first phase chromatin decondensation
(data not shown). Freshly prepared protoplasts induced to re-enter the
cell cycle were incubated with MG132 at various time points (0, 24, and
48 h), and their progression into S phase was monitored by FACS
72 h after preparation. Addition of MG132 at times 0 and 24 h
completely abolished progression of protoplasts into S phase (Fig.
6, A and B). When
MG132 was added after 48 h (the time at which about 10% of the
protoplasts approached S phase (see Fig. 3C)), the FACS
histogram showed two peaks corresponding to G1 and
G2 content of DNA; no or very small amount of S phase
protoplasts could be detected (Fig. 6C). Notably, treatment
of protoplasts with MG132 diminished the supra-G1 peak (Fig. 6, compare C with E). In contrast, the
protease inhibitor leupeptin, used as a control in these experiments,
enhanced protoplast progression into S phase (Fig. 6D).
These findings demonstrate the indispensability of the ubiquitin
proteolytic system for the second phase chromatin decondensation and
for protoplasts entry into S phase; progression from S to
G2 phase is proteasome-independent. Consistent with the
requirement for the proteasome system, Skp1 mRNA, a component of
the cell cycle-regulated E3 ubiquitin ligase (SCF), is absent in
leaves, but its level is markedly increased in protoplasts progressing
into S phase (Fig. 6F).
The results presented here demonstrate that chromatin
decondensation is an integral part in cellular dedifferentiation.
Tobacco mesophyll cells re-entering the cell cycle displayed two
successive phases of increased PI fluorescence intensity of nuclei
prior to reactivation of S phase. These phases reflect increased
accessibility of DNA to PI conferred by chromatin relaxation rather
than DNA synthesis. The first phase (transition from G0 to
G1), which occurs in the course of protoplast isolation, is
ubiquitin-independent and does not require exogenous application of
auxin and cytokinin. The second phase (transition from G1
to supra-G1) is auxin/cytokinin-dependent and
occurs just before entry of cells into S phase. In agreement with these
findings, an extra G1 peak, not attributed to DNA synthesis but rather to conformational changes in chromatin, has been recorded by
FACS in dedifferentiating red blood cells of frog (13). Also, two
phases of chromatin decondensation, prior to reactivation of DNA
synthesis, were found in chicken erythrocyte nuclei incubated in
Xenopus egg extract (17). The mechanism underlying chromatin remodeling of somatic nuclei transplanted into Xenopus egg
extract has recently been shown to require the activity of the
chromatin-remodeling nucleosomal adenosine triphosphatase (ATPase) ISWI
(19), a member of the SWI2/SNF2 superfamily. The ISWI is a subunit of
several distinct nucleosome remodeling complexes that increase the
accessibility of DNA in chromatin (20). Taken together, it appears that
chromatin decondensation is a common mechanism underlying cellular
dedifferentiation both in plants and animals. We propose that the first
phase of chromatin decondensation represents a transitory phase that is necessary for activation of genes whose products are required for the
establishment of competence for cell fate switch (Fig. 7). The ensuing cell fate determination
may be governed by growth factors and environmental signals. In plant
cells, auxin and cytokinin induce dedifferentiation and re-entry into
the cell cycle, auxin by itself may induce redifferentiation (21),
whereas in the absence of phytohormones, cells die.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
naphthalene acetic acid
(NAA). In certain assays, protoplasts or leaves were incubated in the
presence of 100 µM of the ubiquitin proteasome inhibitor
MG132 (N-carbobenzyloxy (N-CBZ)-Leu-Leu-Leu-al, Sigma), or the protease inhibitor leupeptin (acetyl-Leu-Leu-Arg-al, Sigma), or 5 mM hydroxyurea (Sigma). All FACS measurements
were performed on isolated nuclei to avoid interference of plastid and
mitochondrial DNAs. Nuclei were isolated in a Hamilton buffer essentially as described (15, 16), washed twice with FACS buffer (10 mM MES, 0.2 M sucrose, 0.01% Triton X-100, 2.5 mM EDTA, 2.5 mM dithiothreitol (16)) to remove
soluble contaminants, and passed through two layers each of 150- and
100-µm filters to remove cell debris. Nuclei were precipitated
(1000 × g, 7 min, 4 °C), resuspended in FACS buffer
supplemented with 50 µg/ml DNase-free RNase A (Roche Molecular
Biochemicals) and 50 µg/ml PI (Sigma), viewed under the microscope,
and subjected to FACS analysis using FACSort (Becton Dickinson). The
position of PI fluorescence intensity for G0/G1
nuclei has been changed from one experiment to another as a result of
alteration in the amplifier gains for FL-2, which was necessary to
accommodate fluorescence intensity of both
G0/G1 and G2 nuclei.
-mercaptoethanol (15)). A sample (80 µl) was
removed for untreated control. MNase (1000 units/ml) was then added and
at various time points, samples (80 µl) were taken, mixed with 350 µl of stop solution (2 mg/ml proteinase K (Roche Molecular
Biochemicals), 10 mM NaCl, 1 mM MgCl2, 10 mM Tris-HCl, pH 7.5, and 2% SDS,
(17)), and incubated overnight at 37 °C. To prepare DNA, each sample
was added 140 µl of 5 M potassium acetate, mixed well,
incubated on ice for 15 min, and centrifuged (15 min, 12,000 × g, 4 °C). The supernatant was collected, extracted once
with phenol/chloroform/isoamyl alcohol (25:24:1), and DNA was
precipitated by adding 1 ml of 100% ethanol, followed by
centrifugation. The DNA pellet was resuspended in 30 µl of TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), treated with RNase A (20 µg/ml, 25 min at room temperature), and nuclease
digestion products were resolved on 1.6% agarose gels and stained with
ethidium bromide.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (53K):
[in a new window]
Fig. 1.
First phase chromatin decondensation revealed
in freshly prepared protoplasts. Nuclei were prepared from tobacco
leaves (A) or from freshly prepared protoplasts
(B), stained with PI, and subjected to FACS analysis. Note
the higher PI fluorescence intensity in protoplast nuclei
(G1) compared with leaf nuclei (G0).
C, FACS histogram of a mixture of both types of nuclei.
D, chromatin from freshly prepared protoplasts shows
increased sensitivity to MNase. MNase (1000 units/ml) was applied for
the indicated time periods (min) to nuclei prepared from tobacco leaves
(lanes 2-6) or from freshly isolated protoplasts
(lanes 7-11). DNA was extracted and separated by 1.6%
agarose gel, followed by ethidium bromide staining. None
(lanes 2 and 7) is DNA prepared from untreated
nuclei. Positions of DNA size markers (M, lane 1)
are indicated in base pairs (bp) on the left.
View larger version (17K):
[in a new window]
Fig. 2.
Protoplasts undergo cell death in the absence
of phytohormones. Protoplasts grown in the absence of hormones
( H) were sampled at time 0, 24, 48, and 72 h
(B-E, respectively). Nuclei were prepared, stained with PI,
and subjected to FACS analysis. The FACS histogram of leaf nuclei
(A) is shown. The positions of G0 and
G1 nuclei are marked by dashed lines and
sub G1 is indicated by an arrow.
View larger version (60K):
[in a new window]
Fig. 3.
Protoplasts approaching S phase display a
second phase chromatin decondensation. Protoplasts were induced to
re-enter the cell cycle by auxin and cytokinin. At the indicated time
points after hormonal induction (t-0 to t-96),
nuclei were isolated, and progression of protoplasts into S phase was
monitored by FACS analysis (A-E). The appearance of
protoplasts is shown (right panels, size
bars = 20 µm); cell division patterns are seen at 72 and
96 h. Note the additional G1 peak designated
supra-G1 (*G1) at D, a characteristic
feature of protoplasts re-entering the cell cycle. To compare changes
in fluorescence intensity in both G1 and G2
nuclei, we changed the amplifier gains for FL-2 to put the
G1 peak at about channel 360.
View larger version (22K):
[in a new window]
Fig. 4.
Supra-G1 nuclei converge with the
G1 nuclei of cycling cells. Nuclei were prepared from
t-0 protoplasts (A), t-72 protoplasts (B), and
from N. tabacum cycling cells (C) and subjected
to FACS analysis. To compare changes in fluorescence intensity in both
G1 and G2 nuclei, we changed the amplifier
gains for FL-2 to place the G1 peak at about channel
300.
View larger version (23K):
[in a new window]
Fig. 5.
The supra-G1 peak does not
reflect initiation of DNA synthesis. A, FACS histogram
of HU-treated protoplasts. HU (5 mM) was applied after
36 h and progression into S phase was analyzed 72 h after
protoplast preparation. The FACS histogram shows
accumulation of G1 and supra-G1 (*G1) nuclei.
B, the FACS histogram of BrdUrd-labeled t-72 protoplasts.
The sorted fractions of supra-G1 (*G1) and S
phase nuclei are indicated. C, detection of BrdUrd-labeled
DNA. DNA was extracted from sorted nuclei and separated on a 0.8%
agarose gel containing ethidium bromide (EB, upper
panel). DNA was Southern blotted and subjected to Western analysis
using BrdUrd antibody ( BrdU, lower panel).
M indicates the BamHI- and
EcoRI-digested lambda DNA marker (the slow migrating
fragment is shown).
View larger version (26K):
[in a new window]
Fig. 6.
The second phase chromatin decondensation and
re-entry into the cell cycle require the activity of the ubiquitin
proteasome system. MG132 (100 µM) was added to
protoplasts at the indicated time points (0, 24, and 48 h) and
progression into S phase was determined by FACS 72 h after
preparation (A-C). D, FACS histogram of
protoplasts treated with 100 µM protease inhibitor
leupeptin applied at t-0. E, untreated protoplasts. The
positions of G1 (G1) and supra-G1
(*G1) are indicated. F, the mRNA level of
Skp1 increases during protoplasts progression into S phase. Northern
blot analysis of total RNAs (upper panel) isolated from
tobacco leaves (L) or from protoplasts at the indicated
times (0, 24, and 48 h). The amount of RNA loaded in each lane was
estimated by the relative ethidium bromide staining of ribosomal RNAs
(rRNAs, lower panel).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 7.
A model proposing the distinction between
competence for cell fate switch and cell fate determination. A
differentiated plant cell responds to removal of cell wall (cellulase)
by decondensing its chromatin (1st phase), an event marking
a transitory phase, i.e. competence for cell fate switch.
This phase of chromatin decondensation appears to be essential, though
not sufficient, for re-entry of cells into S phase. At this stage,
additional signals determine cell fate: auxin and cytokinin induce
chromatin decondensation (2nd phase), followed by
reactivation of S phase and proliferation, eventually giving rise to
production of new plants. Auxin by itself may induce redifferentiation
(19), whereas in the absence of hormonal application, cells die.
The occurrence of the supra-G1 peak appears to be a unique feature of differentiated cells re-entering the cell cycle not found in cycling cells. The accumulation of protoplasts at the supra-G1, just before entry into S phase, indicates that the transition rate from G1 to supra-G1 is faster than from supra-G1 to S phase. This progression pattern may mark a check-point control known as the restriction point in animals or START in yeast, a point of commitment to the mitotic cell cycle (22).
The stepwise manner by which chromatin decondensation occurs points to various levels of heterochromatin compaction, i.e. heterochromatin may not be a homogeneous but rather a heterogeneous structure displaying different degrees of chromatin condensation. This assumption is supported by recent findings demonstrating variations in chromatin (de)condensation within heterochromatin segments in meiotic chromosomes of Arabidopsis (23). We propose that the first phase of chromatin decondensation occurs in a less condensed heterochromatin, often called facultative heterochromatin, which is likely to be located at the boundary region between eu- and heterochromatin. This border chromatin might have an important biological significance in cellular plasticity, i.e. the interplay between differentiation, proliferation, and cell death.
The role played by the ubiquitin system in regulating cell cycle progression has been well documented in animals (10-11) and recently described also in plants (24). By using a specific inhibitor to the proteasome system we demonstrated its role in driving protoplasts into S phase. The fact that treatment of protoplasts with the proteasome inhibitor MG132 eliminated the supra-G1 and, consequently, entry into S phase supports the notion that DNA decondensation is essential for reactivation of DNA synthesis (25). Furthermore, our results suggest that the activity of the ubiquitin proteolytic system plays a role, either directly or indirectly, in DNA relaxation, thereby enabling the assembly of replication proteins onto origins to initiate DNA replication.
It is long standing that auxin and cytokinin are required to induce
cell division in plant tissue culture (26) and are indispensable for
the progression of protoplasts into S phase (3-5). Auxin and cytokinin
presumably evoke signaling pathway(s) leading to a commitment for the
mitotic cell cycle and reactivation of DNA replication (4, 27, 28).
Notably, several mutations that confer resistance to auxin were found
to encode components of the cell cycle-regulated ubiquitin ligase
complex SCF (12) required for G1-S transition (10, 11).
This implies that auxin exerts its effect, at least in part, by
inducing an orderly regulated protein destruction via the
ubiquitin proteasome system. Given that the ubiquitin proteolytic
system is required for redifferentiation of Zinnia mesophyll
cells into tracheary elements (29), we suggest that the ubiquitin
proteolytic system is not only involved in cell cycle progression but
also in regulating cell fate determination.
![]() |
ACKNOWLEDGEMENT |
---|
We thank D. Aviv for invaluable help with protoplast preparations, A. Eshel and S. Lev-Yadun for helpful discussion of results, D. Ginzberg for providing BrdUrd antibody, and G. Katzenelson and D. Dolev for general assistance. We also thank A. Sharp and E. Ariel for helping with the FACS analysis and the Arabidopsis Biological Resource Center (The Ohio State University) for providing BAC clones.
![]() |
FOOTNOTES |
---|
* This research was supported by a grant from the Israel Science Foundation (ISF).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Plant Sciences, The
Weizmann Inst. of Science, Rehovot 76100, Israel. Tel.: 972-8-934-3505; Fax: 972-8-934-4181; E-mail: gideon.grafi@weizmann.ac.il.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M101756200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
FACS, fluorescence-activated cell sorter;
BAP, 6'-benzylaminopurine;
NAA, naphthalene acetic acid;
PI, propidium iodide;
HU, hydroxyurea;
MNase, micrococcal nuclease;
MES, 4-morpholineethanesulfonic acid;
SCF, Skp1-Cdc53-F-box protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Takebe, I., Labib, G., and Melchers, G. (1971) Naturwissenchaften 58, 318-320 |
2. | Nagata, T., and Takebe, I. (1970) Planta 92, 301-308 |
3. | Galun, E. (1981) Annu. Rev. Plant Physiol. 32, 237-266 |
4. | Trehin, C., Planchais, S., Glab, N., Perennes, C., Tregear, J., and Bergounioux, C. (1998) Planta 206, 215-224[CrossRef][Medline] [Order article via Infotrieve] |
5. | Carle, S. A., Bates, G. W., and Shannon, T. A. (1998) J. Plant Growth Regul. 17, 221-230[Medline] [Order article via Infotrieve] |
6. | Jamet, E., Durr, A., Parmentier, Y., Criqui, M. C., and Fleck, J. (1990) Cell Differ. Dev. 29, 37-46[Medline] [Order article via Infotrieve] |
7. |
Hershko, A.
(1988)
J. Biol. Chem.
263,
15237-15240 |
8. | Durr, A., Jamet, E., Criqui, M. C., Genschik, P., Parmentier, Y., Marbach, J., Plesse, B., Lett, M. C., Vernet, T., and Fleck, J. (1993) Biochimie (Paris) 75, 539-545[Medline] [Order article via Infotrieve] |
9. | Hershko, A. (1997) Curr. Opin. Cell Biol. 9, 788-799[CrossRef][Medline] [Order article via Infotrieve] |
10. | Peters, J-M. (1998) Curr. Opin. Cell Biol. 10, 759-768[CrossRef][Medline] [Order article via Infotrieve] |
11. | Krek, W. (1998) Curr. Opin. Genet. Dev. 8, 36-42[CrossRef][Medline] [Order article via Infotrieve] |
12. | Gray, W. M., and Estelle, M. (2000) Trends Biochem. Sci. 25, 133-138[CrossRef][Medline] [Order article via Infotrieve] |
13. | Chiabrera, A., Hinsenkamp, M., Pilla, A. A., Ryaby, J., Ponta, D., Belmont, A., Beltrame, F., Grattarola, M., and Nicolini, C. (1979) J. Histochem. Cytochem. 27, 375-381[Abstract] |
14. | Zelcer, A., and Galun, E. (1976) Plant Sci. Lett. 7, 331-336 |
15. | Van Blokland, R., ten Lohuis, M., and Meyer, P. (1997) Mol. Gen. Genet. 257, 1-13[CrossRef][Medline] [Order article via Infotrieve] |
16. | Saxena, P. K., Fowke, L. C., and King, J. (1985) Protoplasma 128, 184-189 |
17. | Blank, T., Trendelenburg, M., and Kleinschmidt, J. A. (1992) Exp. Cell Res. 202, 224-232[Medline] [Order article via Infotrieve] |
18. | Ormerod, M. G., Collins, M. K., Rodriguez-Tarduchy, G., and Robertson, D. (1992) J. Immunol. Methods 153, 57-65[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Kikyo, N.,
Wade, P. A.,
Guschin, D.,
Ge, H.,
and Wolffe, A. P.
(2000)
Science
289,
2360-2362 |
20. | Varga-Weisz, P. D., and Becker, P. B. (1998) Curr. Opin. Cell Biol. 10, 346-353[CrossRef][Medline] [Order article via Infotrieve] |
21. | Vissenberg, K., Quelo, A-H., van Gestel, K., Olyslaegers, G., and Verbelen, J-P. (2000) Cell Biol. Int. 24, 343-349[CrossRef][Medline] [Order article via Infotrieve] |
22. | Zetterberg, A., Larsson, O., and Wiman, K. G. (1995) Curr. Opin. Cell Biol. 7, 835-842[CrossRef][Medline] [Order article via Infotrieve] |
23. | Fransz, P. F, Armstrong, S., de Jong, J. H., Parnell, L. D., van Drunen, C., Dean, C., Zabel, P., Bisseling, T., and Jones, G. H. (2000) Cell 100, 367-376[Medline] [Order article via Infotrieve] |
24. |
Genschik, P.,
Criqui, M. C.,
Parmentier, Y.,
Derevier, A.,
and Fleck, J.
(1998)
Plant Cell
10,
2063-2076 |
25. | Hanks, S. K., and Rao, P. N. (1980) J. Cell Biol. 87, 285-291[Abstract] |
26. | Skoog, F., and Miller, C. O. (1957) Symp. Soc. Exp. Biol. 11, 118-131 |
27. | Meskiene, I., and Hirt, H. (2000) Plant Mol. Biol. 42, 791-806[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Riou-Khamlichi, C.,
Huntley, R.,
Jacqmard, A.,
and Murray, J. A.
(1999)
Science
283,
1541-1544 |
29. |
Woffenden, B. J.,
Freeman, T. B.,
and Beers, E. P.
(1998)
Plant Physiol.
118,
419-430 |