1 Institute of Microbiology and Genetics, University of Vienna, Vienna
Biocenter, Dr Bohrgasse 9, A-1030 Vienna, Austria
2 Centro de Investigaciones Biológicas CSIC, Velázquez 144,
E-28006-Madrid, Spain
3 Institute of Microbiology, Academy of Sciences of the Czech Republic,
Vídeská 1083, 142 20 Prague 4, Czech Republic
4 School of Biological Sciences, Royal Holloway, University of London, Egham
TW20 0EX, UK
* Author for correspondence (e-mail: l.bogre{at}rhul.ac.uk)
Accepted 31 October 2002
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Summary |
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Key words: Cyclin B, Mitosis, Checkpoint, Plant development
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Introduction |
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The first functional evidence that Cdks are pivotal for mitotic control in
plants came from experiments in which microinjection of an active Cdk complex
induced entry into mitosis (Hush et al.,
1996). The plant functional homologue of the animal Cdk1 is named
CdkA and has a protein kinase activity that peaks at the G1-S and the G2-M
transitions (Bögre et al.,
1997
). Treatment of cells with roscovitine, a drug that most
specifically inhibits CdkA, blocks cell cycle progression both in G1 and G2
phases, further indicating multiple roles for plant CdkA during G1 and G2
phases (Binarova et al., 1998
).
Another group of plant Cdks is divided into two subgroups, CdkB1 and CdkB2,
and exists only in plants (Joubes et al.,
2000
). The tightly regulated expression and activity of B-type
Cdks, as well as the block in G2 phase by the expression of a
dominant-negative mutant form of CdkB1, suggest a specific function during
G2-phase and mitosis (Magyar et al.,
1997
; Porceddu et al.,
1999
; Porceddu et al.,
2001
).
There are three A-type and two B-type cyclin groups in plants
(Renaudin et al., 1998). In
synchronized BY2 cells cyclin A3 is expressed during an earlier time window,
from the G1-S transition until onset of mitosis compared with cyclin A1 and
A2, which are expressed during mid S phase until midmitosis
(Reichheld et al., 1996
).
Cyclin A2-associated kinase activity peaks both in S phase and mitosis in
synchronized alfalfa cells (Roudier et
al., 2000
). In animals a single A-type cyclin associates with
different Cdks during S phase and mitosis
(Resnitzky et al., 1995
;
den Elzen and Pines, 2001
),
while in plants two A-type cyclins, the tobacco cyclin A1 and the alfalfa
cyclin A2, were found only associated with CdkA both in vitro
(Nakagami et al., 1999
), and
in the yeast two-bybrid system (Roudier et
al., 2000
).
The expression of the B1- and B2-type plant cyclins is tightly controlled
and restricted to late G2 and M phases
(Hirt et al., 1992;
Fobert et al., 1994
). A
tobacco Cyclin B1-GFP fusion was found to be distributed between the cytoplasm
and nucleus in interphase and to associate with the chromatin during prophase
and metaphase until it is degraded at the onset of anaphase
(Criqui et al., 2000
;
Criqui et al., 2001
). These
authors also found that ectopically expressed cyclin B1 did not alter mitotic
progression in cultured cells, while expression of cyclin B1 in
Arabidopsis was shown to stimulate root formation by producing more
cells as building blocks for tissue enlargement
(Doerner et al., 1996
).
Evidence for specialized functions of closely related plant B-type cyclins
came from the finding that ectopic expression of the Arabidopsis cyclin
B1;2, but not of cyclin B1;1 inhibited endoreduplication and
induced cell divisions, resulting in multicellular trichomes
(Schnittger et al., 2002
).
Recently, we presented evidence for the existence of a topoisomerase
II-dependent checkpoint in plant cells by showing that the ectopic expression
of Cyclin B2 alone is able to override the checkpoint and induce cells to
enter into mitosis without delay
(Gimenez-Abian et al.,
2002
).
Plant B2-type cyclins are known to be expressed from late G2 phase to
mitosis (Hirt et al., 1992;
Ferreira et al., 1994
) but the
role and localization of their protein has not been studied. We show that the
cyclin B2 protein is localized to the nucleus and its expression is restricted
to a short time window during early prophase by its proteolysis. We found that
the induction of ectopic cyclin B2 expression during G2-phase drives cells to
enter mitosis earlier and allows caffeine and okadaic acid to overcome a
replication checkpoint block induced by hydroxyurea. Thus, ectopic cyclin B2
expression interferes with the control of progression from G2 to M phase,
which probably causes the developmental abnormalities observed in transgenic
plants.
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Materials and Methods |
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Synchronization and cell cycle analysis
Synchronization was carried out by diluting a 7-day-old culture 1:5 and
adding 10 mg/l aphidicolin (Sigma) 8 hours later for 16 hours. After removal
of the aphidicolin by washing cells five times with medium, chlortetracycline
(Sigma) was added at a concentration of 0.1 mg/l to induce cycB2-expression.
Drugs were added 8 hours after aphidicolin removal in G2 phase. The following
drug concentrations were used in these experiments: 10 µM amiprophos
methyl, 50 µM taxol, 10 µM propyzamide, 100 µM roscovitine (a gift
from M. Strnad, Olomouc, Czech Republic), 100 µM MG 132 (Calbiochem), 5
µM lactacystin (Affinity, UK) and 1 µM epoxomicin (Affiniti, UK). To
follow cell cycle progression, flow cytometric analysis was performed as
described (Bögre et al.,
1997) using a PAS2 flow cytometer (Partec, Münster, Germany).
For mitotic index, cells were fixed in a 3:1 (v/v) ethanol/acetic acid
mixture, then washed with 70% (v/v) ethanol. The DNA was stained with 1
µg/ml DAPI and observed by epifluorescence microscopy.
BrdU incorporation and labeling
A stationary phase cell culture (7-day-old culture) was diluted 1:10 with
fresh medium, and bromo-deoxyuridine labeling reagent was added immediately at
the dilution as recommended in the cell proliferation kit (RPN20) (Amersham
Pharmacia Biotech). Cells were grown for 15 hours and samples were taken every
3 hours, fixed and processed for immunostaining for incorporated
bromo-deoxyuridine (BrdU) using monoclonal antibody to BrdU (Amersham
Pharmacia Biotech) or Cycb2-HA using HA.11 monoclonal antibody (BabCO,
Richmond, California). For each timepoint, 1000 cells were analyzed for BrdU
or HA-staining.
Immunostaining and microscopy
For indirect immunofluorescence, cells were fixed and stained as previously
described (Bögre et al.,
1997). The HA.11 monoclonal antibody (BabCO, Richmond, California)
was used in a 1:1000 dilution and with the secondary anti-mouse Cy3-conjugated
antibody (Sigma) at a dilution of 1:200. DNA was stained with 1 µg/ml DAPI
in PBS. Microtubule staining was performed using an anti
-tubulin mouse
monoclonal antibody DM1A (Sigma) at a dilution of 1:200, and anti mouse
FITC-conjugated secondary antibody (Sigma). For GFP observation, a drop of
cell suspension was transferred on a slide, carefully covered with a
coverslip, and observed with an upright fluorescence microscope (Axioplan 2;
Zeiss, Jena, Germany) equipped with a GFP filter (HQ480/20X; HQ510/20M; AF
Analysentechnik, Jena, Germany). Typical exposure times were in a range of few
seconds. Images were taken using a cooled charge-coupled device
black-and-white digital camera (SPOT-2; Diagnostic Instruments, Burroughs,
Michigan) and Metaview imaging software (Diagnostic Instruments, Burroughs,
Michigan).
RNA and protein blotting, CDK activity measurements
Northern blots were performed as described
(Hirt et al., 1992). Proteins
on western blots were detected by using anti-rabbit PSTAIRE antibody
(Bögre et al., 1997
), the
HA.11 monoclonal antibody (BabCO, Richmond, California) or the polyclonal
rabbit anti N-terminal cyclin B1;1 antibody (kindly provided by Pascal
Genschik, Strasbourg, France). Cdk activities were measured in the samples
after immunoprecipitation with the HA.11 monoclonal antibody or after binding
to p13suc1, as described previously
(Bögre et al., 1997
).
Regeneration from tobacco leaf disks
Leaf disks with 1 cm diameter were excised from sterile plants and placed
on MS medium (Sigma) with 0.5/0.1 (rooting), 0.1/0.5 (shooting) and 0.5/0.5
(callusing) naphtalene acetic acid (NAA)/6-benzylaminopurine (BAP)-containing
media with and without 0.1 mg/l Cl-tetracycline for 3 weeks, when roots on 10
inocula for each treatments were counted and plates were photographed.
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Results |
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To facilitate the studies of cell cycle progression, we generated cell cultures from the CycB2-HA and CycB2-GFP transgenic plants, which displayed tight transcriptional regulation by Cl-tetracycline. In cultured cells derived from line CycB2-HA-2 the cycB2-HA transcript was clearly detectable 10 minutes after adding Cl-tetracycline, and reached its maximal level of expression within 1 hour (Fig. 1B). We established the maximal induction of CycB2-HA protein at 0.01 mg/ml Cl-tetracycline (Fig. 1C). Correspondingly, in vitro kinase assays after immunoprecipitation using an anti HA antibody revealed that the CycB2-HA protein associated with and activated a histone H1 kinase (Fig. 1D), indicating that the cyclin moiety in these fusion proteins retained its normal function to bind and activate Cdk. The level of cycB2-HA mRNA expression was slightly lower than that of the endogenous mitotic cyclin Nt;tCycB1;1 (Fig. 2B). In view of the lack of a cyclin B2-specific antibody, we could not determine the endogenous cyclin B2 levels, but we found that the CycB2-HA-associated histone H1 kinase activity reached only around 20% of the total cellular Cdk activity purified from these cells by binding to p13suc1 (Fig. 1D). Thus, it is likely that the cyclin B2 protein was not overproduced, but its expression was temporally unscheduled.
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Ectopically expressed cyclin B2 accelerates entry into mitosis
To assess the consequences of altered cyclin B2 expression on cell cycle
regulation, we synchronized the CycB2-HA-2 cell culture by aphidicolin
treatment, which blocks cell cycle progression specifically during S phase.
Following removal of the inhibitor, we induced the expression of CycB2-HA at
various time points during the cell cycle. We found that ectopic expression of
CycB2-HA during either S or early G2 phase did not initiate mitosis. However,
cells entered mitosis 2 hours earlier compared with control cells when
expression of CycB2-HA was ectopically induced at mid G2 phase
(Fig. 2). Several lines of
evidence supported the finding of this advanced entry into mitosis by 2 hours.
First, the mitotic index peaked at 10 hours in cells ectopically expressing
CycB2-HA, instead of 12 hours in control cells
(Fig. 2A). Second, monitoring
cell cycle progression by flow cytometry revealed that after cells had passed
through mitosis, the percentage of cells with a G1 DNA increased 2 hours
earlier in CycB2-HA expressing cells (data not shown). Third, we followed the
mRNA expression of the tobacco cyclin B1 and of histone H4,
as stage-specific markers for mitosis and S phase, respectively. Mirroring the
results of mitotic index above, the peak of the endogenous cyclin B1
expression also appeared 2 hours earlier in tetracycline-treated cells
(Fig. 2B).
Because microtubule structures are highly specific landmarks for the
different stages of mitosis, we used these features to further refine and
confirm the above interpretation. The pre-prophase band (PPB) appears between
late G2 phase and late prophase, the spindle structures during mitosis, and
the phragmoplast marks the cytokinetic plate during late anaphase and
telophase (Staiger and Lloyd,
1991). We quantified the relative proportion of PPB, spindle and
phragmoplast visualized by tubulin immunostaining in synchronized cells
expressing cycB2-HA. Disassembly of the PPB, which occurred 2 hours earlier
than in control cells, was the most pronounced effect of ectopic CycB2-HA
expression (Fig. 2C).
Ectopic cyclin B2 expression overrides the hydroxyurea-induced
S-phase checkpoint only in the presence of caffeine or okadaic acid
Blocking DNA synthesis arrests cells before mitosis, and blocks the
expression of mitotic A- and B-type cyclins in plants
(Renaudin et al., 1998).
First, we tested whether ectopic cyclin B2 expression could induce
S-phase-arrested cells to enter into mitosis. The induction of CycB2-HA
expression in asynchronously dividing cells by treatment with tetracycline for
16 hours slightly increased the mitotic index
(Fig. 3A, lanes 1 and 7) and
this effect was abolished when cells were treated at the same time with HU
(Fig. 3A, lanes 4 and 10).
Thus, cyclin B2 alone is unable to override the HU-activated checkpoint block
and we found a similar result in aphidicolin treated cells.
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Drugs such as caffeine or okadaic acid are known to cancel the
DNA-replication checkpoint in yeast and animal cells, but were found to be
ineffective in plant cells (Amino and
Nagata, 1996; Pelayo et al.,
2001
). A reason for this could be that in plants mitotic cyclins
are not expressed in S-phase-arrested cells. Thus, to gain further evidence
that CycB2 is a potent inducer for mitosis, we tested whether its ectopic
expression could help caffeine or okadaic acid to override the S-phase block.
In an asynchronously growing control culture the mitotic index was determined
to be 7% and the addition of caffeine or okadaic acid for 1 hour did not
modify the percentage of mitotic cells. The addition of hydroxyurea (HU) for
16 hours, which blocks cells in S-phase, decreased the mitotic index (MI) and
this low MI was not increased by a subsequent 1 hour treatment with caffeine
or okadaic acid (Fig. 3A, left
panel). Thus, caffeine or okadaic acid were unable to override the HU-induced
S-phase block, which is in agreement with the results found before in onion
root cells (Amino and Nagata,
1996
; Pelayo et al.,
2001
). However, when caffeine or okadaic acid were added to the
HU-blocked cells expressing CycB2-HA, the mitotic indices were raised to a
level of around 50 and 25%, respectively, when compared with control cells
without HU. Caffeine was slightly more effective than okadaic acid in
stimulating mitosis in HU blocked cells, together with ectopic CycB2-HA
expression (Fig. 3A, right
panel). Thus, CycB2-HA expression allowed caffeine or okadaic acid to override
replication checkpoints in S phase.
To address whether caffeine and okadaic acid stimulated cells to enter into mitosis by elevating the CycB2-HA-associated Cdk activity in HU-blocked cells, we immunopurified the CycB2-HA-associated kinase complex with an antibody directed against the HA-tag and measured its histone H1-kinase activity. In the absence of tetracycline, no CycB2-HA protein or CycB2-HA-associated Cdk activity were found, while the Cdk amounts, as detected by the PSTAIRE antibody, were constant in these extracts (Fig. 3B, left panel). In the presence of tetracycline, CycB2-HA protein expression was increased by equal amounts after all treatments, showing that the tetracycline-induction of CycB2-HA expression was similar in all the treatments. We found that the CycB2-HA-associated Cdk activity in cells treated with tetracycline was increased after treatment with caffeine and okadaic acid. The addition of HU abolished the CycB2-HA-associated Cdk activity. In agreement with its ability to induce mitosis in HU-blocked cells, caffeine and okadaic acid elevated the activity of CycB2-associated Cdk in HU-treated cells (Fig. 3B, right panel). Okadaic acid was again less effective in raising the Cdk activity than was caffeine. Thus, we conclude that the activity of CycB2-associated Cdk is inhibited in the presence of HU, and this inhibition is cancelled by treatment with caffeine and okadaic acid, which supports our findings that caffeine or okadaic acid together with CycB2 expression override the replication-checkpoint in plant cells.
Cyclin B2 degradation is initiated in between pre-prophase and
prophase and persists until mid-G1 phase
To learn more about how cyclin B2 promotes the G2-M transition, we
determined its localization in cultured cells by indirect immunofluorescence
using an anti HA-antibody (Fig.
4). In interphase cells CycB2-HA was localized to the nucleus. In
late G2-phase cells, which display a pre-prophase band, CycB2-HA was still
present within the nucleus, but it was absent in pro-metaphase cells with
condensed chromatin and visible spindle microtubules
(Fig. 4A, PM). This suggests
that CycB2-HA is degraded as cells progress from pre-prophase to
pro-metaphase. No CycB2-HA staining was observed in cells in meta-, ana- or in
telophase, and it was missing in around 40% of interphase cells
(Fig. 4A).
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To confirm the localization of the cyclin B2 protein in live cells, we used
a cell line expressing a cyclin B2-GFP fusion. Similar to the CycB2-HA protein
visualized by immunostaining with the HA antibody, CycB2-GFP was also found
within the nucleus in interphase cells and it was never found in mitotic cells
(Fig. 4B, left panel). We were
unable to follow the degradation of CycB2-GFP by time-lapse microscopy,
because UV irradiation of cells during late G2-phase blocked cell cycle
progression, presumably by activating a prophase checkpoint, similar to that
described in animal cells (Rieder and
Cole, 1998).
In an asynchronously growing culture around 60% of interphase cells
displayed CycB2-HA or CycB2-GFP staining within the nucleus after treatment
with tetracycline. To define more precisely at what stage of interphase cyclin
B2 was stable, we blocked cell cycle progression at the G1-S transition by
treatment with aphidicolin. Both CycB2-HA and CycB2-GFP signals were
detectable in around 90% of aphidicolin-blocked cells. After release from the
block, a high proportion of cells (around 90%) remained positive for nuclear
CycB2-GFP or CycB2-HA signals, which decreased when cells progressed through
mitosis (data not shown). Thus, CycB2-HA and CycB2-GFP were stable during S
and G2 phases, and became unstable as cells entered mitosis and G1 phase. To
map the time window within G1 during which CycB2-HA was stable, we observed
stationary phase cells arrested with a G1 DNA content. In these cells we never
found CycB2 protein expression, indicating that it is unstable or not
translated (Fig. 4C). Adding
back fresh medium reinitiated the cell cycle and cells reached S phase within
0 to 15 hours, which was shown by monitoring incorporation of BrdU. CycB2-HA
protein reappeared at around 6-9 hours in a large proportion of cells
(Fig. 4C). Thus, there is a
transition point within G1 when cyclin B2 becomes stabilized, similar to
B-type cyclins of yeast and animal cells
(Amon et al., 1993;
Brandeis and Hunt, 1996
).
We analyzed the timing of the initiation of CycB2-HA degradation by using
drugs that inhibited cell cycle progression at specific time points.
Inhibition of Cdk activity by adding roscovitine to synchronized cells during
mid G2 phase (6 hours after the release from aphidicolin treatment) blocks
cells at late G2 phase and G2-M interface, characterized by a fully developed
prophase spindle and condensed chromatin surrounded by a persistent nuclear
envelope (Binarova et al.,
1998). Inducing such a G2-arrest kept CycB2-HA intact within the
nucleus, while in control cells CycB2-HA became degraded after prophase
(Fig. 5A). Thus, the
degradation of cyclin B2 requires either an active Cdk or nuclear envelope
breakdown.
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Cyclin B2 destruction during mitosis is not affected by the spindle
checkpoint
Activation of the spindle checkpoint by treatment of cells with microtubule
disrupting drugs leads to a metaphase arrest and stabilization of cyclin B1
(Criqui et al., 2000). To
study whether the spindle checkpoint might influence cyclin B2 degradation in
plants, we treated the synchronized CycB2-HA-2 cells in late G2-phase with the
microtubule stabilizing drug taxol, as well as with the microtubule disrupting
drug amiprophos methyl (APM), and followed CycB2-HA localization with
immunostaining. Both APM and taxol arrested a large proportion of cells in
metaphase, without any detectable CycB2-HA-derived signal
(Fig. 5B,C).
To confirm that CycB2 is unstable in metaphase-arrested cells, and that the
signal is not masked and therefore undetectable by immunofluorescence, we used
the CycB2-GFP cell line and followed the CycB2-GFP fluorescence in living
cells during activation of the spindle checkpoint by depolymerizing
microtubules with propyzamide (Fig.
6A). Again, we found that the CycB2-GFP-derived fluorescence was
clearly present in propyzamide-treated interphase cells, but it was missing in
metaphase-arrested cells (Fig.
6A, right panel, arrow). When the same treatment was applied to a
cell line expressing a cyclin B1-GFP fusion, we found that the GFP signal was
strongly present in association with chromosomes in metaphase-arrested cells
(Fig. 6A, left panel) as it was
reported previously (Criqui et al.,
2000; Criqui et al.,
2001
).
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To further ascertain that the CycB2-HA protein is indeed decreased when the
spindle checkpoint has been activated and that cyclin B1 is stabilized in
these cells, we analyzed protein extracts of synchronized cells by
immunoblotting using the anti HA-antibody for detection of CycB2-HA protein
and the anti cyclin B1;1 antibody for detection of endogenous cyclin B1
(Criqui et al., 2000). In
control cells, both the expression of CycB2-HA and cyclin B1 increased during
early mitosis, reaching the maximum at 9 hours after aphidicolin removal, from
which point both cyclin levels declined rapidly
(Fig. 7). Treatment with
propyzamide at 7 hours during G2 phase did not affect the levels of CycB2-HA
protein, and it decreased at 15 hours to a similar level as in the control
cells (Fig. 7A). Contrary to
this, the endogenous cyclin B1 protein level remained high in
propyzamide-treated cells (Fig.
7B). This strongly suggests, that cyclin B1, but not cyclin B2 is
stabilized by the spindle-checkpoint in plants.
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Treatment with the proteosome inhibitor MG 132 in the presence of
ectopic expression of Cyclin B2 abrogates the metaphase alignment of
chromosomes
Degradation of A- and B-type cyclins is blocked by proteosome inhibitors
such as MG 132, epoxomicin or lactacystin, both in plant and in animal cells
and they arrest cells with over-condensed metaphase chromosomes
(Genschik et al., 1998).
Surprisingly, we found that the CycB2-HA derived signal was barely detectable
in MG 132-treated cells (Fig.
5C).
To confirm that proteosome inhibitors differently affect B-type cyclin levels in vivo, we treated CycB1-GFP- and CycB2-GFP-expressing cells with MG 132, as well as with two other inhibitors: epoxomicin and lactacystin. Similar to our findings by immunolocalization in MG 132-treated cells, CycB2-GFP was barely detectable, while CycB1-GFP was strongly present and localized to the condensed chromosomes in cells which were arrested in metaphase after treatment with epoxomicin (Fig. 6B), MG 132 or lactacystin (data not shown).
Immunodetection of the CycB2-HA and the endogenous cyclin B1 protein levels further substantiated the above results. We treated cells before mitosis, at 7 hours after aphidicoline release with epoxomicin, lactacystin or MG 132, and prepared extracts from cells at 15 hours for immunoblotting. We found that CycB2-HA protein levels were unaffected by the proteosome inhibitors, and were as low in the treated cells as in the control cells (Fig. 7A), while the cyclin B1 protein levels increased (Fig. 7B).
In contrast to wild-type cells, we found that CycB2-HA-expressing cells arrested in the presence of MG 132 at a stage when chromosomes were not aligned along the metaphase plate (Fig. 5C). In untreated cells, which ectopically expressed CycB2-HA, we observed a slight increase in the percentage of metaphase cells compared with control cells, but we never found abnormalities in chromosome alignment. This indicates that increased levels of cyclin B2 during prophase might interfere with chromosome alignment at the metaphase plate. No such abnormalities were observed in control cells or CycB1-overexpressing cells treated with the proteosome inhibitors.
Ectopic expression of cyclin B2 interferes with root development
We initially observed, that constitutive expression of CycB2 severely
retards plant growth with most pronounced effects on root development. To
investigate how ectopic expression of CycB2 could interfere with shoot and
root regeneration, we used a transgenic line with Cl-tetracycline inducible
cycB2-HA expression. Detached leaves from transgenic plants were placed on
Cl-tetracycline-containing medium, in combination with different ratios of
cytokinin and auxin. It is well established that high ratios of cytokinin to
auxin favor shoot regeneration, while the reverse would privilege root growth
(Sugiyama, 1999). Our results
show that ectopic expression of CycB2 specifically blocked root development in
a medium containing high auxin to cytokinin ratios, conditions in which root
development is normally encouraged (Fig.
8A). However, the initiation of shoot development in these
transgenic plants was not detectably modified on high ratios of cytokinin to
auxin containing medium (data not shown).
|
To determine whether the inhibition of root development can be traced to an altered cell cycle progression, we measured the distribution of cells with a G1 and G2 DNA content. Most cells in leaves without hormone treatment have G1 DNA content, irrespective of tetracycline-induced cycB2-HA expression (data not shown). Control experiments, in which leaves were incubated on auxin-containing medium but without induction of cycB2-HA expression, showed that after 3 days the cell cycle was initiated in a proportion of cells (as judged from BrdU incorporation for S-phase and observing mitotic figures in leaf pieces) and 30% of the cells displayed a G2 DNA content. By contrast, in leaves that were incubated with auxin while the expression of cycB2-HA was induced, only about 15% of the cells displayed G2 DNA content after 3 days (Fig. 8B), while similar percentages of S-phase and mitosis, as compared with control samples, were observed. The reduced proportion of cells with G2 DNA content indicates that, as in the previous experiments with cultured cells, the G2-phase is accelerated upon ectopic expression of cycB2 in the presence of auxin.
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Discussion |
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The plant cyclin B2 appears to play a similar role to cyclin A in animal
cells during entry into mitosis. In animals, microinjection of exogenous
cyclin A into G2 cells rapidly induces chromosome condensation, while
inhibition of cyclin A-Cdk-complex by p21cip1/waf1 prevents G2
cells from entering prophase and induces early prophase cells to return back
to interphase (Furuno et al.,
1999). Irradiation-induced DNA damage identifies a late G2 control
point in animal cells, from where the chromosomes return to an uncondensed
interphase stage (Rieder and Cole,
1998
). An analogous G2-checkpoint was observed in onion cells,
where chromosome condensation is reversed by inhibiting protein synthesis
before mid-prophase (Gracia-Herdugo et al., 1974). This reversible
condensation phase coincides with the timing of cyclin A-associated Cdk1
activation in animal cells (Clute and
Pines, 1999
), and our findings suggest that it might be a cyclin
B2-associated Cdk kinase in plant cells. Although animal cyclin A is expressed
during a broad time window and has a role both in S-phase and during entry
into mitosis (Rosenblatt et al.,
1992
), plant cyclin B2 expression is confined to late G2-phase and
early mitosis. The existence of several subgroups of plant A- and B-type
cyclins, which are specifically expressed during S, G2 or mitotic phases,
suggests a high degree of specialization in cyclin function in plants
(Renaudin et al., 1998
).
The PPB is a microtubule structure formed in late G2-phase, and it
disassembles as cells enter mitosis in late prophase
(Utrilla et al., 1993).
Further evidence that ectopic cyclin B2 expression affects the G2 to M phase
transition is given by the lower percentage of PPBs in these cells. This
indicates that either the time window during which the PPB is present becomes
shorter or a cyclin B2-associated Cdk activity directly induces the
disassembly of the PPB. It has been shown that microinjections of an active
Cdk complex into plant cells can cause the disassembly of PPB and nuclear
envelope breakdown (Hush et al.,
1996
).
Ectopic Cyclin B2 expression could not initiate mitosis in S or early G2
phase. In yeast and animals, entry into mitosis is regulated by the
phosphorylation of the Thr14 and Tyr15 inhibitory sites
of CDK (Walworth, 2001). The
existence of phosphorylation-mediated Cdk-regulatory pathways during plant
mitosis is indicated by the presence of a wee1-kinase homolog in
maize and Arabidopsis, which inactivates Cdks through its
phosphorylation at the conserved Tyr15 residue
(Sun et al., 1999
).
Surprisingly, no apparent homologs of the cdc25 phosphatase have been
found in the fully sequenced genome of Arabidopsis, but
dephosphorylation of Tyr15 is tightly regulated in response to
cytokinin or during stress treatment
(Reichheld et al., 1999
).
Expression of S. pombe cdc25 in tobacco can induce cells to resume
cell divisions that have been arrested in G2 phase by cytokinin starvation
(John, 1996
;
Zhang et al., 1996
).
Constitutive expression of cdc25 in tobacco plants leads to a number
of developmental abnormalities, such as precocious flowering, aberrant leaves
and flowers, and a smaller size of dividing cells in lateral roots compared
with control plants (Bell et al.,
1993
).
There is a conserved DNA-synthesis checkpoint-signaling pathway in yeast
and animal cells that regulates CDK activity through the phosphorylation of
the Thr14 and Tyr15 inhibitory sites. Caffeine is a drug
that inhibits checkpoint function in animal cells
(Schlegel and Pardee, 1986;
Andreassen and Margolis, 1992
;
Moser et al., 2000
), such as
the replication checkpoint during S phase
(Schlegel and Pardee, 1986
)
and the DNA damage checkpoint in G2 phase
(Blasina et al., 1999
). In
plants, caffeine is able to override the G2 DNA damage checkpoint
(Hartley-Asp et al., 1980
;
González-Fernández et al.,
1985
) but it is unable to cancel replication checkpoints in S
phase (Amino and Nagata, 1996
;
Pelayo et al., 2001
). Okadaic
acid, a protein phosphatase 2A inhibitor, induces premature mitosis in animal
cells in a similar way to caffeine (Ghosh
et al., 1996
). Correspondingly, endothal, another PP2A-specific
phosphatase inhibitor, was found to prematurely activate a plant mitotic CDK
resulting in microtubule and chromosome condensation abnormalities
(Ayaydin et al., 2000
).
Caffeine was found to releases the checkpoint-induced block in G2 phase by
inhibiting the ATM kinase, which works upstream of the two checkpoint kinases:
Chk1 and Chk2 (Blasina et al.,
1999
; Brondello et al.,
1999
). In a current model the Chk1 kinase directly inhibits the
Cdc25 phosphatase and thus stops activation of CDK by keeping it in a
phosphorylated state at the Thr14 and Tyr15 inhibitory
sites. The animal target of caffeine, the ATM kinase, has been conserved in
plants (Garcia et al., 2000
),
but little is understood about its function during checkpoint pathways in
plants.
We show that, in hydroxyurea-treated cells, cyclin B2 expression stimulated
the entry into mitosis only when the replication checkpoint pathway was
compromised by caffeine or okadaic acid. Moreover, under these conditions, the
CycB2-associated Cdk activity was also induced. This suggests that at least
one of the targets of the replication checkpoint in plants is the
CycB2-associated Cdk. In animal cells, the nuclear-localized cyclin A
stimulates entry into mitosis (Furuno et
al., 1999), and cyclin B1 is able to abolish the replication
checkpoint in response to caffeine when experimentally altered to localize to
the nucleus (Tam et al., 1995
;
Toyoshima et al., 1998
). The
plant cyclin B2 is nuclear, and during a period of S phase, we found the
localization of CycB2 to be speckled within the nucleus (M. W.,
unpublished).
In animal cells, A-type cyclins are degraded in pro-metaphase, while B-type
cyclins undergo proteolysis in metaphase. Degradation of both cyclins requires
the anaphase-promoting complex (APC). Although the APC is activated already
during prometaphase, destruction of cyclin B in animal cells appears to be
selectively inhibited by the spindle assembly checkpoint, resulting in
stabilization of cyclin B until all chromosomes are aligned along the
metaphase plate (Geley et al.,
2001). Both cyclin types contain the conserved destruction box
motif recognized by the APC, but in A-type cyclins, this motif is not
sufficient to trigger proteolysis during prophase. Rather, prophase
destruction of cyclin A is mediated by an extended destruction box including
multiple overlapping elements (Kaspar et
al., 2001
). Plant cyclin A and B degradation during mitosis is
also dependent on their conserved destruction box motif and likely to be
mediated by the APC (Genschik et al.,
1998
). Similar to animal cyclins, plant cyclin A is undetectable
in metaphase (Roudier et al.,
2000
), while plant cyclin B1 is present in metaphase and its
degradation is inhibited upon activation of the spindle assembly checkpoint
(Criqui et al., 2000
). The
plant cyc B2;2 genes are grouped together with B-type cyclins, based
on similarities within the cyclin box, but the arrangement of the N-terminal
destruction box motifs of the plant cyclin B2 sequence better
resembles that of animal A-type cyclins which have multiple destruction box
elements. Our studies indicate that the plant cyclin B2 protein uses a similar
degradation mechanism as animal A-type cyclins and that it is degraded during
late prophase, again similar to animal cyclin A. Treatment of cells with the
proteosome inhibitors MG 132, epoxomicin or lactacystin arrested cells in
metaphase, as expected, but surprisingly, cyclin B2 protein was undetectable
by indirect immunofluorescence, suggesting that it is degraded in spite of the
presence of the inhibitors in these cells. To exclude the possibility that the
cyclin B2 was undetectable due to technical difficulties, such as masking the
epitope, we repeated these experiments by treating cells expressing CycB1-GFP
or CycB2-GFP with the same proteosome inhibitors and followed GFP fluorescence
in living cells. Furthermore, we directly compared the protein level of
CycB2-HA with that of endogenous cyclin B1 in proteosome-inhibitor-treated
cells by immunoblotting. Both experiments led to the same conclusion: the
degradation of cyclin B2 is hardly affected by treatments with proteosome
inhibitors, while cyclin B1 destruction is readily blocked and cells are
arrested in metaphase. These results indicate that cyclin B2 is either not
targeted by the proteosome at pro-metaphase and proteolysed by some other
mechanism or its destruction is more effective than that of other substrates,
including cyclin B1. Recently, it was shown that cyclin A and cyclin F are
degraded by proteolysis independent of proteosome in animal cells
(Welm et al., 2002
;
Fung et al., 2002
).
Collectively, the data showing that cyclin B2 is only present until
pro-metaphase promote our conclusion that plant cyclin B2 functions during
early mitosis.
The expression of cyclin B2 to a level not significantly higher than that
of endogenous cyclin somewhat delayed metaphase, without affecting subsequent
chromosome alignment along the metaphase plate. Compromising proteolysis in
these cells by treatment with proteosome inhibitors resulted in an
accumulation of cells with chromosomes that were not correctly aligned at the
metaphase plate. By contrast, MG 132-treatment of wild-type cells arrests
cells in metaphase with normal spindle and chromosome alignment
(Genschik et al., 1998). This
shows that Cyclin B2 ectopic expression, together with impaired proteolysis
synergistically affects chromosome alignment during metaphase. A similar
effect has been reported in animal cells upon overexpression of cyclin A
during late G2-phase and mitosis (den
Elzen and Pines, 2001
). This again suggests that the plant cyclin
B2 has analogous functions to animal cyclin A in regulating mitosis.
Proper chromosome alignment at the metaphase plate is controlled by a
complex anchor of many proteins associated with chromosomes mainly at their
kinetochore/centomeric region. Kinetochore localization of phosphoepitopes
recognized by the MPM2 antibody suggests that the function of these proteins
is regulated by mitosis-specific phosphorylation in plants
(Binarova et al., 1993). In
Drosophila, Cyclin A inhibits chromosome disjunction by acting
synergistically with other proteins involved in this process
(Sigrist et al., 1995
;
Parry and O'Farrell, 2001
).
Among these are the Drosophila securin Pim, which stabilizes sister
chromosome cohesion until it is degraded at the metaphase to anaphase
transition (Leismann et al.,
2000
), and the Cdk inhibitor Rux, which interacts genetically and
physically with Cyclin A and inhibits its in vitro kinase activity
(Foley et al., 1999
).
The increased number of metaphase cells observed upon ectopic cyclin B2 expression suggests that in plants B2-type cyclins might have a similar role to cyclin A in animal cells, delaying the progression through metaphase.
We found that cyclin B2 was non-detectable in stationary phase cells, which
are arrested in G1, and begins to accumulate before cells enter S phase. This
confirms the results obtained in yeast and animal cells
(Amon et al., 1994;
Brandeis and Hunt, 1996
;
Huang et al., 2001
) and shows
that APC activity is switched off during late G1 to S phase in plants. In
yeast and animal cells, APC activity is required during G1 phase to prevent
expression of proteins that may interfere with cellular polarization events or
lead to premature DNA replication or disturb spindle assembly. Before mitosis,
the APC must be turned off to allow mitotic substrates to accumulate.
Inactivation of the APC has been linked to mid-G1, which is corresponding to
the start in yeast, and is suggested to depend on raising G1 cyclin activity
(Amon et al., 1994
). Recently,
it was shown that during a normal cell cycle the APC is inactivated in a
graded manner, and that its complete inactivation occurs in S phase and
requires S phase cyclins (Huang et al.,
2001
).
We found that ectopic expression of cyclin B2 in leaf segments interferes
with root regeneration induced by auxin. This re-differentiation process is
initiated in a single differentiated cell within the vascular bundle and was
mapped by hormone shift-experiments to a relatively short time window, about
24 hours, during which auxin induces the cell to re-enter cell division and
re-polarize (Attfield and Evans,
1991). The majority of leaf cells enter the cell cycle from the G1
phase, and a 24 hour period might coincide with the time, when a proportion of
cells are in G2-phase. Shortening of G2-phase may disrupt some events, such as
cell polarization, which is required for the determination of the root fate.
Timing of cell cycle phases was shown to be an important factor in determining
polarized growth in yeast (Lew and Reed,
1993
). In plants, the timing of the presence of the PPB, which is
particularly altered upon ectopic CycB2-HA expression, could be an important
factor for the orientation of cell division. In line with this is a recent
report which implicates Cdk1 as an important factor in regulating the
orientation of asymmetric cell divisions in Drosophila
(Tio et al., 2001
). Our
results suggest that enhancing the expression of one of the key cell cycle
regulators, a cyclin B2 gene, is sufficient to prevent formation of a root
meristem under conditions when it is normally formed.
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