Differential Effect of Jasmonic Acid and Abscisic Acid on
Cell Cycle Progression in Tobacco BY-2 Cells1
Agnieszka Świa̧tek, Marc Lenjou, Dirk Van Bockstaele, Dirk Inzé, and Harry Van Onckelen*
Laboratory of Plant Physiology and Biochemistry, Department of Biology, University of Antwerp,
Universiteitsplein 1, B–2610 Antwerp, Belgium (A.S., H.V.O.); Laboratory of Experimental Hematology,
University of Antwerp, Antwerp University Hospital, Wilrijkstraat 10, B–2650 Edegem, Belgium (M.L.,
D.V.B.); Vakgroep Moleculaire Genetica & Departement Plantengenetica, Vlaams Interuniversitair Instituut
voor Biotechnologie, Universiteit Gent, K.L. Ledeganckstraat 35, B–9000 Gent, Belgium (D.I.)
Environmental stress affects plant growth and development. Several plant hormones, such as salicylic acid, abscisic acid
(ABA), jasmonic acid (JA), and ethylene play a crucial role in altering plant morphology in response to stress. Developmental
regulation often has the cell cycle machinery among its targets. We analyzed the effect of JA and ABA on cell cycle
progression in synchronized tobacco (Nicotiana tabacum) BY-2 cells. Both compounds were found to prevent DNA replication, keeping the cells in the G1 stage, when applied just before the G1/S transition. However, ABA did not have any effect
on subsequent phases of the cell cycle when applied at a later stage, whereas JA effectively prevented mitosis on application
during DNA synthesis. This demonstrates that JA treatment can freeze synchronized BY-2 cells in both the G1 and G2 stages
of the cell cycle. Jasmonate administered after the S-phase was less effective in decreasing the mitotic index, suggesting that
cell sensitivity toward JA is dependent on the cell cycle phase. In cultures detained in the G2-phase, we observed a reduced
histone H1 kinase activity of kinases associated with the p13sucl protein.
In nature, plants are constantly exposed to environmental changes that force them to adapt, and only
the proper cooperation of all their organs ensures
their survival and production of healthy progeny. It
is frequently observed that stress signals not only
induce plant resistance but also affect growth rate
and cell division. For example, in wheat (Triticum
aestivum) seedlings subjected to a mild water stress,
leaf elongation is reduced in correlation with a rapid
decrease of mitotic activity in mesophyll cells of the
first leaf (Schuppler et al., 1998). Similar effects were
found in water-stressed roots of maize (Zea mays;
Sacks et al., 1997) and sunflower (Helianthus annuus;
Robertson et al., 1990), where an involvement of abscisic acid (ABA) in the inhibition of the cell cycle
was suggested. Salt stress, which can be mediated by
ABA, disrupts growth in Arabidopsis. Plants have
shortened petioles, roots, and hypocotyls and a drastic decrease in lateral root formation. At the molecular level, salt treatment strongly reduced the expression of the cyclinA2;1 and cyclinB1;1 (Burssens et al.,
2000b). Cell cycle progression can be also disturbed
by oxidative stress. When generated artificially by
the application of menadion, a source of semiquinone
1
This work was supported by Geconcenteerde Onderzoeks
Actie and by the Belgian Program on Interuniversity Poles of
Attraction (Prime Minister’s Office, Science Programming, grant
no. 15).
* Corresponding
author;
email
[email protected];
fax
32–3820 –2271.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.010592.
radicals and hydroquinones, oxidative stress disrupted DNA replication and delayed mitotic entry in
tobacco (Nicotiana tabacum) BY-2 cells and in the apical root and shoot meristems of whole tobacco plants
(Reichheld et al., 1999). In cultured parsley (Petroselinum crispum) cells, UV light has been shown to decrease expression of the histone H2A, H2B, H3, and
H4, CDKA, and cyclin B genes (Longemann et al.,
1995). Certain elements of stress signaling spatially
correlate with cell divisions in plant tissue. ANP1
and NPK1 are kinases of the mitogen-activated protein (MAP) kinase kinase kinase class, isolated from
Arabidopsis and tobacco, respectively. They mediate
response to H2O2, one of the most common reactive
oxygen species released during defense responses,
which also serves as a stress messenger (Nishihama
et al., 1997; Hirt, 2000; Kovtun et al., 2000). NPK1 is
essential for cell plate formation (Nishihama et al.,
2001). In dividing cells, they are expressed in a cell
cycle-dependent manner, suggesting their involvement in the interplay between cell cycle progression
and oxidative stress (Nakashima et al., 1998; Hirt,
2000).
Most of the external signals, however, do not exert
their effect directly on proteins that are involved in
cell division, but rather trigger their response in differentiated tissue, and in some way this response is
then coupled to the regulation of the cell cycle within
the meristems.
Among the messengers that may pass this type of
information are phytohormones. There is growing
evidence for both positive and negative hormonal
regulation of cell division. Exogenous application of
Plant Physiology, January 2002, Vol.
128, pp. 201–211,
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Świa̧tek et al.
cytokinins to a hormone-depleted cell culture of Arabidopsis caused accumulation of transcripts of cyclin
A1 (Burssens et al., 2000a) and cyclin D3 (cycD3;
Riou-Khamlichi et al., 2000). cycD3 transcripts also
accumulated in whole plants treated with zeatin, and
constitutive expression of cycD3 conferred callus formation in the absence of cytokinin (Riou-Khamlichi
et al., 1999). Taken together, these data suggest that
the induction of cell division by cytokinins involves
increased expression of cycD3. However, the activity
of phosphatase cdc25, which activates cyclindependent kinase (CDK) by the removal of a phosphate group from a Tyr moiety in the ATP-binding
loop, has also been shown to be cytokinin dependent
(Zhang et al., 1996). Redig et al. (1996) demonstrated
that the zeatin level fluctuates in synchronized tobacco BY-2 cells, peaking at late S and before G2/M
transition. When zeatin production was blocked, mitotic activity ceased and could only be restored by
application of exogenous zeatin (Laureys et al., 1998),
suggesting that cytokinins are essential not only for
the initiation of the cell cycle but also for the progression through it.
The information on auxin effects in the cell cycle is
much more modest. It has been shown that in a
hormone-depleted Arabidopsis cell culture, naphthylacetic acid application caused an increase in cycA2
transcript (Burssens et al., 2000a), and hormonedepleted protoplasts of petunia (Petunia hybrida) resumed cell division only when subsequently treated
with 2,4-dichlorophenoxyacetic acid (2,4-D) and benzylaminopurine but not after treatment with cytokinin alone (Trehin et al., 1998). Another class of plant
hormones, gibberellins, increased the expression of
histone H3 and a mitotic cyclin, cycOs1, in the intercalary meristem of deepwater rice (Oryza sativa). This
may explain the positive effect of gibberellins on
internodal elongation in monocotyledonous plants
(Sauter, 1997).
Certain hormones might act as messengers of negative regulation of cell division. For example, exogenous ABA reduced bromodeoxyuridine incorporation and mitotic events in root meristems of
Arabidopsis and sunflower (Robertson et al., 1990;
Leung et al., 1994). One of the possible mechanisms
underlying the ABA effect on the cell cycle is suggested in the study of Wang et al. (1998). They cloned
an Arabidopsis protein named ICK1, which showed a
homology to the cyclin-dependent kinase inhibitor
p27Kip1. ICK1 interacted with both CDKA and cycD3
and inhibited the histone H1 kinase activity of the
complex. When overexpressed in Arabidopsis, it
caused dramatic growth inhibition and decrease in
the total number of cells per plant (Wang et al., 2000).
Exogenous application of ABA up-regulated ICK1
expression, which may lead to a block of G1/S transition (Wang et al., 1998).
In our research, we investigated the possible interactions between another stress signal messenger, jas202
monic acid (JA), and cell cycle progression. JA is
widely known to inhibit root growth in Arabidopsis.
This feature has been used in isolating mutants defective in jasmonate signaling (Staswick et al., 1992;
Feys et al., 1994; Berger et al., 1996). However, the
exact mechanism of root growth inhibition is not
known. It is not directly linked with the wound and
pathogen responses generally accepted to be the
main function of jasmonates. There are several reports suggesting a role of JA in cell wall synthesis
(Koda, 1997), which might affect cell elongation. On
the other hand, it has also been reported that exogenous JA, when applied to growing soybean (Glycine
max) callus, counteracted the positive effect of cytokinins and inhibited growth, presumably due to the
inhibition of cell division (Ueda and Kato, 1982). We
investigated the effects of JA on cell cycle progression. Using a synchronized tobacco BY-2 cell line as a
model culture (Nagata et al., 1992), we compared the
effect of JA with that of ABA. We found that exogenous application of JA and ABA resulted in phasespecific disruption of the cell cycle progression in
BY-2 cells. Both ABA and JA prevented G1/S transition, but only JA, when given during the S-phase,
was capable of preventing and delaying the mitotic
entry without direct effect on DNA synthesis. This, in
turn, suggests that the jasmonate response is broader
that that of ABA and in this specific case, JA is not
acting downstream of ABA, unlike suggested elsewhere for wound response (Peña-Cortés and
Willmitzer, 1995).
RESULTS
JA Prevents Mitosis in Tobacco BY-2
BY-2 callus grown in agar-hardened medium in the
presence of 100 ␮m JA showed clear-cut reduced
growth as compared with the untreated control (Fig.
1). Subsequently, we examined the effect of JA on the
cell cycle progression. Duration of the specific phases
of the cell cycle progression as shown in Figure 2A
was estimated by means of flow cytometry, abundance of histone H4 mRNA, thymidine incorporation, and mitotic index (Nagata et al., 1992; Laureys
et al., 1998; Ehsan et al., 1999; Reichheld et al., 1999).
After aphidicolin release, at the beginning of the
Figure 1. JA inhibits growth of BY-2 callus when applied to the agar
medium: A, Control; B, 100 ␮M JA.
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Plant Physiol. Vol. 128, 2002
Jasmonic Acid Inhibits Cell Cycle
delay of the occurrence of the mitotic peak. Similar
results were obtained when methyl jasmonate was
used (data not shown). Fluorescein diacetate staining
revealed that cells remained viable during the experiments and 24 h afterward. Data presented in Figure
2B were confirmed by multiple repetitions of this
experiment.
To examine whether the DNA synthesis was affected by JA treatment, the aphidicolin-released culture was divided in two subcultures: One of them
was treated with 200 ␮m JA, and the other one was
kept as control. DNA synthesis in both cultures was
monitored by flow cytometry and thymidine incorporation (Figs. 3 and 4). The thymidine incorporation protocol used was a pulse-labeling method,
which reflected the rate and frequency of replication. Thymidine incorporation did not differ between JA treated and control culture (Fig. 3). Flow
cytometry results (Fig. 4) revealed that both cultures
formed comparable G2 populations (about 70% of
all cells). However, the transition from 4C to 2C
DNA content occurred only in the control culture
between the 6th and 8th hour after aphidicolin
release.
Mitotic Block Depends on the Application Time
As DNA synthesis appeared to be undisturbed by
jasmonate application, we examined further the time
requirement of JA application on the inhibition and
delay of mitosis. The aphidicolin-synchronized culture was divided into five subcultures: control and
four others to which 100 ␮m of JA was applied at 0,
Figure 2. A, Scheme of aphidicolin-based synchronization protocol.
B, The effect of various concentrations of ABA and JA on mitotic
activity in synchronized BY-2 cells. Both hormones were applied
immediately after aphidicolin release.
experiment, the main population of cells was at the
early S-phase. Such cultures were divided into four
subcultures, to which hormones were applied. We
compared the effect of (⫾)JA and (⫾)ABA on the
mitotic ratio. Although the ABA-treated culture
showed little, if any, response (Fig. 2B), JA treatment
decreased the mitotic index in a concentrationdependent manner (Fig. 2B). Besides a moderate reduction, 10 ␮m JA concentration caused a 1- to 2-h
Plant Physiol. Vol. 128, 2002
Figure 3. DNA replication during treatment with a JA concentration,
which effectively prevents mitosis. Thymidine incorporation by the
synchronized culture treated immediately after aphidicolin release
compared with untreated control. White symbols correspond to thymidine incorporation and black symbols to mitotic index.
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Świa̧tek et al.
Effect of Jasmonate and ABA on G1/S Transition
Using aphidicolin-released cultures limited our observation to S, G2, and mitotic stages of the cell cycle
because by the time cells reached another division
cycle, they lost synchrony to such an extent that
obtained results became difficult to interpret.
Prior knowledge that ABA acts negatively on the
cell cycle progression at the level of the G1/S transition led us to compare the effect of ABA and JA at
this point. To focus on G1/S transition, the
aphidicolin-synchronized culture was subsequently
treated with propyzamide, an agent that inhibits tubulin polymerization and therefore prevents spindle
assembly and chromosome separation. Propyzamide
was removed when most of the cells were arrested in
metaphase. The scheme in Figure 6 represents the
approximate timing of the cell cycle phases within
the experimental setup. Using this method, a highly
synchronized culture (⬎90% of the cells) at M/G1
transition can be obtained. In a first set of experiments, we compared the effect of ABA and JA on
DNA synthesis and mitosis. The propyzamidereleased culture was divided into five subcultures:
One was treated with 200 ␮m ABA 2 h after the
release, which corresponded to early G1-phase; a
second one was supplied with ABA at 7 h, when cells
were already involved in DNA synthesis; a third one
was treated at 2 h with 100 ␮m JA; a fourth one was
supplied with JA at 7 h; and the fifth one was treated
with an equal volume of methanol and kept as a
control. At appropriate time points, samples were
taken for mitotic index, thymidine incorporation, and
flow cytometric analysis (Figs. 7 and 8). The control
culture reached S-phase between 6 and 11 h and its
mitotic peak 16 h after propyzamide release. Both of
the cultures that were treated with 200 ␮m ABA or
100 ␮m JA during S-phase (7 h after the release)
Figure 4. Flow cytometric analysis of the synchronized culture
treated in the same manner as in Figure 3. We took samples before,
after the aphidicolin release and later, every 2 h from jasmonatetreated and reference cultures.
1, 2, or 4 h after aphidicolin release. The control
culture treated with a corresponding volume of
methanol reached a mitotic peak of 50% at 6 h after
release (Fig. 5). In the culture treated with JA at 0 h,
a mitotic peak of only 16% was observed 9 h after
aphidicolin release. JA treatment at late S-phase (2 h)
was less effective, and application just before G2/M
transition (4 h) resulted in a mitotic peak at the same
time as in the control yet slightly reduced in height.
This experiment revealed that the effect of JA depended on the stage of the cell cycle in which cells
were treated.
204
Figure 5. The effect of application time of 100 ␮M JA on the frequency of mitosis in synchronized BY-2 cells. After aphidicolin
release, the culture was divided into four subcultures. JA was added
immediately, 2 or 4 h after the release. One subculture was kept
untreated as control.
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Plant Physiol. Vol. 128, 2002
Jasmonic Acid Inhibits Cell Cycle
Figure 6. Scheme of synchronization protocol with subsequent block and release of aphidicolin and propyzamide.
showed similar DNA synthesis profiles as the control
(Fig. 7). Flow cytometry showed the formation of a
G2 population (4C DNA content) in both cultures
(Fig. 8). The mitotic index was slightly reduced in the
ABA-treated culture, with a mitotic peak at 17 h. A
stronger reduction of mitotic index occurred in the
JA-treated culture, consistent with previous results
(Fig. 2B) showing that it was JA and not ABA that
interfered with G2/M transition. In contrast, cultures
supplied with either 100 ␮m JA or 200 ␮m ABA at the
2nd h, which was before G1/S transition, displayed
similar strongly reduced thymidine incorporation.
Corresponding flow cytometric data showed only a
minor amount of JA- or ABA-treated cells with 4C
DNA content at the 10th h of the experiment when
compared with the control (Fig. 8). Taken together,
these data strongly support the idea of a negative
regulation of G1/S transition by both ABA and JA.
There was also a difference in mitotic index between
ABA- and JA-treated cultures. Although both hormones similarly decreased thymidine incorporation
when applied before G1/S transition, JA treated-
cultures had almost no mitotic events, whereas cell
divisions were still observed after ABA treatment. As
shown above, JA prevents DNA synthesis when applied at G1, 2 h after propyzamide release. To pinpoint more precisely the possible targets affected by
the presence of jasmonate, we applied JA at various
points between propyzamide release and initiation of
DNA synthesis (Fig. 9). The propyzamide-released
culture was divided into six subcultures to which 100
␮m JA was applied 1, 2, 3, 4, 5, or 6 h after release,
and one culture was left untreated as the control. As
in previous experiments, JA applied before G1/S
transition, i.e. before the 6th h after propyzamide
release, caused a strong and similar decrease in thymidine incorporation. For sake of clarity, only data
for the 2nd, 5th, and 6th h were presented in Figure
9. When applied at the 6th h, when most of the cells
were at the beginning of S-phase, JA had only a
moderate effect on DNA synthesis. These data
pointed to a very specific target on which JA acts
during G1/S transition.
Histone H1 Kinase Activity in Jasmonate-Treated Cells
Figure 7. The effect of 200 ␮M ABA and 100 ␮M JA applied at
different stages of the cell cycle on thymidine incorporation (represented by white symbols) and mitotic index (black symbols) in synchronized BY-2 cells. Hormone was applied at G1 (2 h after the
release) or early S-phase (7 h after the release) and culture was
sampled for thymidine incorporation, mitotic index, and flow cytometry (see Fig. 8). The results are compared with an untreated reference culture.
Plant Physiol. Vol. 128, 2002
There was an intriguing discrepancy between the
application time (0 up to 2 h after aphidicolin release,
which corresponds to S-phase) when jasmonate treatment has its maximal effect on G2/M transition (Fig.
5) and the apparent undisturbed progression of replication (Figs. 3 and 4). This excluded the possibility
of toxicity of the high concentrations of JA used. On
the other hand, it raised the question of which of the
elements of the cell cycle machinery were affected
and how.
We measured the activities of CDKs, the core protein kinases of the cell cycle, upon separation from
other kinases by affinity purification with p13agarose. The histone H1 kinase activity in the cycling
control culture remained high, with a transient slight
increase in the activity at 6 h (Fig. 10), just before the
mitotic peak, which occurred at 7 h (data not shown).
Treatment with 200 ␮m JA resulted in a decrease of
the H1 kinase activity detectable as soon as 2 h after
aphidicolin release (Fig. 10), whereas the level of the
CDKA protein remained unaffected, as detected by
western blot using anti-PSTAIRE antibody (data not
shown).
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Świa̧tek et al.
Figure 8. The effect of 200 ␮M ABA and 100 ␮M JA applied at different stages of the cell cycle. Hormone was applied at G1
(2 h after the propyzamide release) or early S-phase (7 h after the release), and culture was sampled for thymidine
incorporation, mitotic index (see Fig. 7), and flow cytometry. The central graph represents the G1 DNA content in the culture
2 h after the propyzamide was removed. Surrounding graphs represent DNA content in the treated samples isolated 8 h later,
which corresponds to the late S-phase in the untreated culture (top left).
DISCUSSION
In experiments reported by Ueda and Kato (1982)
on the soybean callus, growth inhibition caused by
jasmonate was attributed to the interference with
action of the exogenous cytokinins, whose presence
was essential for the survival of the culture. BY-2
cells synthesize sufficient endogenous cytokinins to
sustain growth independently of exogenous supply
(Nagata et al., 1992). In BY-2 callus, however, we also
observed growth inhibition by JA. Detailed analysis
of the cell cycle progression in synchronized suspension culture revealed that G1/S and G2/M transitions were blocked. JA has been reported to change
the ratio between cytokinin ribosides and free bases
and to decrease the level of zeatin in potato (Solanum
tuberosum; Dermastia et al., 1994). In BY-2 cells, zeatin
levels peak sharply before G2/M transition (Redig et
al., 1996), and its exogenous supply can rescue G2
arrest caused by the inhibition of cytokinin production (Laureys et al., 1998). However, a block of G2/M
transition caused by JA could not be recovered by
zeatin treatment (data not shown). This means that
the effect of JA was not directly linked with cytokinin
action, and we have to seek its targets elsewhere.
We decided to compare the effects of JA on cell
cycle progression with those of ABA, which were
206
already documented in Arabidopsis (Leung et al.,
1994; Wang et al., 1998). Moreover, ABA was proposed to be a primary factor in wound response,
which triggers JA synthesis (Peña-Cortés and
Willmitzer, 1995). ABA has been previously reported
to diminish the number of mitotic events and thymidine incorporation in root meristems of sunflower
(Robertson et al., 1990) and to reduce bromodeoxyuridine incorporation into Arabidopsis root meristems (Leung et al., 1994).
Our results demonstrate that exogenous ABA application inhibits G1/S transition in synchronized
BY-2 cells, which is in agreement with previous literature data indicating ICK1 among targets of ABA
action (Wang et al., 1998). This protein is a potent
inhibitor of histone H1 kinase activity of the p13suc1associated kinases. It can interact with both CDKA1
and cycD3, and its expression is induced by ABA
(Wang et al., 1998).
We also found that ABA had no effect on further
cell cycle progression when applied during S-phase.
In contrast with ABA, JA was also able to hinder
G2/M transitions. The effect of JA on G2/M transitions was remarkably more pronounced when applied during S-phase than when applied at the late
G2-phase. This suggests that either the JA sensor
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Plant Physiol. Vol. 128, 2002
Jasmonic Acid Inhibits Cell Cycle
Figure 9. The effect of application time of JA on the initiation of DNA
synthesis. The culture, prepared by combined aphidicolin/propyzamide block, was divided into subcultures to which JA was added at
a given time point. The subcultures were sampled for thymidine
incorporation (white symbols) and mitotic index (black symbols).
mechanisms are most sensitive during S-phase or
that there is delay between signal and response. The
decrease in CDK activity detectable after 2 h of treatment suggests that the response is fast and occurs
already in late S-phase. However, what is really triggered by JA in the cell remains an enigma.
There is evidence for the regulation of gene expression by jasmonates in BY-2 cells. For example, cathepsin D inhibitor from potato is positively regulated by JA in potato plants, and a cathepsin D
promoter-␤-glucuronidase fusion remains jasmonate
inducible when expressed in BY-2 cells (Ishikawa et
al., 1994). This suggests that the signaling and transcription machinery necessary to confer jasmonate
responsiveness of a gene promoter is present in BY-2.
JA treatment was reported to change the protein
expression pattern in nonsynchronized BY-2, and
several cDNAs were identified by differential screening (Imanishi et al., 1998). Another interesting feature
of the effect of jasmonate on BY-2 cells is the intriguing sensitivity of the microtubular network of
S-phase cells (Abe et al., 1990). Synchronized BY-2
cells respond with a total disruption of microtubular
network when JA is applied but only when the cells
are engaged in DNA synthesis. The microtubules
seem to restore at the later stages of the cell cycle.
Our data demonstrate undisturbed DNA synthesis
despite the lack of organized microtubular structures. This can be easily understood because propyzamide or oryzaline treatment, which also disrupts
the microtubular network completely, manifests its
effect only at metaphase, when the lack of mitotic
spindle stops the cycle, leaving the condensed chromosomes scattered in the cytoplasm. Thus, the lack
of microtubular network during S-phase is not sufficient to prevent DNA synthesis or mitotic entry, and
Plant Physiol. Vol. 128, 2002
other mechanisms must be involved in the cell cycle
arrest caused by jasmonates. León et al. (1998) demonstrated that JA-induced gene expression in Arabidopsis was inhibited by (2,5-di-tert-butyl)-1,4hydroqinone, which mobilizes Ca2⫹ from internal
stores. The phosphatase inhibitor okadaic acid had a
similar effect, whereas a kinase inhibitor, staurosporine, positively regulated the expression of the genes
normally induced by JA, thus indicating the need for
protein phosphatase acting downstream of JA (Rojo
et al., 1998). Because plant MAP kinases mediate
various aspects of the stress responses, the hypothesis that jasmonate signaling might negatively crossreact with a MAP kinase pathway, which is positively controlled by phosphorylation, seems very
tempting. However, two tobacco kinases (which are
involved in wound response) related to mitotic alfalfa (Medicago sativa) kinase MMK3 (Bögre et al.,
1999), salicylic acid-induced protein kinase, and
wound-induced protein kinase are not influenced by
JA (Kumar and Klessig, 2000). The only kinase in
tobacco for which there was reported control by jasmonates is WAPK; expression of this kinase is induced by JA but its sequence is not related to MAP
kinases (Lee et al., 1998).
It has been clear that JA inhibits plant growth,
because plant extracted jasmonates were shown to
inhibit sheath elongation in rice seedlings and hypocotyl and root elongation in lettuce (Lactuca sativa)
seedlings (Yamane et al., 1981). Plant growth may be
inhibited by the disruption of either the meristem
activity or cell expansion in the elongation zone. Our
results seem to favor the first possibility, because we
Figure 10. Histone H1 kinase activity in jasmonates-treated synchronized BY-2 cells. The aphidicolin-released culture was divided into
two subcultures, one immediately treated with 200 ␮M JA to suppress
mitosis completely, the other left untreated. A, Histone H1 phosphorylation by p13 affinity purified protein from treated and untreated
cultures, sampled as indicated. B, Quantification of the data presented in A.
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Świa̧tek et al.
observe the disturbance of both the G1/S and G2/M
transitions after JA treatment. The down-regulation
of CDK activity after jasmonate treatment of
aphidicolin-released cells suggests the possibility
that JA targets the cell cycle machinery as a part of a
stress response.
Inhibition of root elongation in Arabidopsis was
exploited to isolate jasmonate-insensitive mutants
jar1 (Staswick et al., 1992), jin1 and jin 4 (Berger et al.,
1996), and coi1 (Feys et al., 1994), all of them nonallelic. Only the COI1 gene has been identified (Xie et
al., 1998), revealing a sequence with a F-box motif
and a Leu-rich repeat that are characteristic for the
component of Skp1/Cdc53(cullin)/F-box protein
complex, which is involved in other organisms in the
targeting for proteolysis cell cycle components, such
as yeast (Saccharomyces cerevisiae) Cln1 and Cln2 cyclins, human cyclin E, and mouse (Mus musculus)
p27KIP1 CDK inhibitor (Kipreos and Pagano, 2000).
Also, plant B-type cyclins, which accumulate before mitotic entry, undergo proteolysis in a ubiquitindependent manner to permit anaphase and exit from
mitosis (Genschik et al., 1998).
The activity of the CDK decreased after JA treatment, whereas the level of the PSTAIRE protein remained unaffected. This suggests that decreased
CDK activity could be related to cyclin availability.
However, to our knowledge, there is no evidence of
any involvement of Coi protein in cell cycle regulation, nor has its expression been demonstrated in
BY-2 cells.
A second level of the control of CDKA activity
involves the phosphorylation and subsequent removal of the phosphate group from the Tyr residue
by cdc25. The Tyr phosphatase activity of the enzyme
specific for the CDK is positively regulated by cytokinins (Zhang et al., 1996). In human cells, cdc25 is
also involved in the response to UV-mediated DNA
damage in a p53-independent pathway, where DNA
damage induces S-phase arrest via activation of Chk1
kinase, which phosphorylates cdc25A and targets it
for proteasome degradation (Mailand et al., 2000). In
plant cells, there is growing evidence for the role of
jasmonates in UV response (Conconi et al., 1996).
Some of the genes induced by jasmonates are common for UV and pathogen response, such as chalcone
synthase, Phe ammonia lyase in parsley and Arabidopsis (Longemann et al., 1995; Long and Jenkins,
1998), and polyphenol oxidase, Leu aminopeptidase,
Thr deaminase, and proteinase inhibitors in tomato
(Lycopersicon esculentum) plants (Conconi et al., 1996).
UV exposure cannot induce mRNA expression of
those genes in the tomato JL-5 mutant, defective in
jasmonate synthesis, suggesting the requirement for
octadecanoid compounds for a proper response
(Conconi et al., 1996). On the other hand, the levels of
neither 12-oxophytodienoic acid nor JA increase after
UV exposure of tomato plants (Stratmann et al.,
2000), which might imply that the actual cross-talk
208
between jasmonate signaling and UV response is
downstream of jasmonates.
UV and fungal elicitor exposure of a cell culture of
parsley decreases the expression of many genes essential for the cell cycle, such as histones H2A, H2B,
H3, H4, CDKA, and cyclin B1;1 (Longemann et al.,
1995), leading to the inhibition of growth. If there is
cross-talk between those two pathways, one might
expect JA to exert a negative effect on cell proliferation by triggering similar check point mechanisms as
UV light. We then can assume a physiological role for
the cell cycle block caused by jasmonates as being a
distress signal, which slows the vegetative growth
during defense responses.
MATERIALS AND METHODS
Cell Culture and Synchronization
BY-2 cells were maintained as described by Nagata et al.
(1992) with modifications: The culture was refreshed
weekly by transfer of 0.5 mL of a 7-d-old culture into 50 mL
of fresh Murashige and Skoog medium (Duchefa, Haarlem,
The Netherlands) pH 5.8, containing 3% (w/v) Suc (Duchefa), 0.2 g L⫺1 KH2PO4 (Merck, Darmstadt, Germany), 10
mg L⫺1 myo-inositol (Sigma, Bornem, Belgium), 1 mg L⫺1
thiamin hydrochloride (Sigma), and 0.2 mg L⫺1 2,4-D
(Serva, Hiedelberg, Germany), referred to hereafter as “medium.” The culture was kept at 27°C at constant darkness
and 130 rpm. For maintenance on petri dishes, medium
was additionally supplied with 1% (w/v) agar (Sigma).
The synchronization protocol was based on the method of
Nagata et al. (1992) as illustrated in Figure 2A. The stationary culture was transferred in a proportion 14:100 to the
fresh medium, supplied with 5 mg L⫺1 aphidicolin (ICN
Biomedicals, Asse, Belgium). After 24 h of incubation, cells
were released by extensive washing with fresh medium
without 2,4-D and thiamine, (4 L per 100 mL of blocked
culture). Afterward, cells were transferred into fresh medium and divided equally into an appropriate number of
subcultures, which were subsequently treated with methanol solutions of (⫾)JA, (⫾)methyl jasmonate (Apex Organics, Honiton, Devon, UK), or (⫾)ABA (Sigma) in various concentrations, prepared to obtain a 1:1000 dilution
with the volume of experimental culture. An appropriate
volume of methanol was added to the control culture. For
a double block, an additional step was included. After
aphidicolin release 1.54 mg L⫺1 propyzamide was added
and after approximately 6 h, when at least 60% of the cells
were either in prophase or in metaphase, the culture was
released by washing and cells were transferred to the fresh
medium.
Thymidine Incorporation
DNA synthesis was monitored in 1-mL samples by pulse
labeling with 1 ␮Ci of [methyl-3H]thymidine (Amersham
Pharmacia Biotech Benelux, Roosendaal, The Netherlands)
for 30 min at 28°C on a rotary shaker. Labeled cells were
collected by centrifugation 5 min at 2,000 rpm and imme-
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Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 128, 2002
Jasmonic Acid Inhibits Cell Cycle
diately frozen in liquid N2. Total DNA and protein was
extracted by grinding and precipitated with 10% (w/v)
trichloracetic acid, containing 10 mm thymidine (Sigma).
The pellet was washed with 5% (w/v) trichloracetic acid,
70% (v/v) ethanol, and acetone, and suspended in 0.2 n
NaOH. Protein content was measured with the Bradford
reagent (Bio-Rad Laboratories, Hercules, CA), using bovine
serum albumin as standard. The remaining sample was
hydrolyzed overnight in 37°C, and incorporated radioactivity was measured by scintillation counting. Upon
quench correction, total DNA synthesis was expressed as
Bq per ␮g of protein in the sample.
Protein Extraction and Histone H1 Kinase Assays
Kinase activity was measured according to Reichheld et
al. (1999). Protein extracts were prepared by grinding cells
in liquid N2 with mortar and pestle to a fine powder. Frozen
powder was suspended in extraction buffer, containing 60
mm ␤-glycerolophosphate, 15 mm p-nitrophenylphosphate,
25 mm Tris-HCl, pH 7.5, 15 mm ethyleneglycol-bis-(-aminoethyl ether)-N,N⬘-tetraacetic acid (EGTA), 15 mm MgCl2, 2
mm dithiothreitol, 1 mm NaVO3, 50 mm NaF, 20 mg L⫺1
antipain, 20 mg L⫺1 aproteine, 20 mg L⫺1 soybean (Glycine
max) trypsine inhibitor, 100 ␮m benzamidine, 1 mm phenylmethylsulfonyl fluoride, and 0.1% (v/v) Nonidet P-40
and centrifuged at 4°C, 13,000 rpm for 5 min. An amount of
supernatant, corresponding to 100 ␮g of protein, was adjusted with extraction buffer to equal volume where necessary and incubated at 4°C for 2 h with p13suc1-agarose
beads (Oncogene, San Diego). After three times of washing
with extraction buffer, beads were incubated for 20 min in
the reaction buffer containing 50 mm Tris-HCl, pH 7.8, 15
mm MgCl2, 5 mm EDTA, 2 mm dithiothreitol, 2 mg L⫺1 of
recombinant cAMP-dependent kinase inhibitor (Sigma),
0.6 g L⫺1 of histone H1 (Sigma), 10 ␮m ATP, and 15 mCi
L⫺1 of [␥-32P]ATP (Amersham Pharmacia Biotech).
Adding 5⫻ Laemmli loading buffer stopped reaction,
and histone was separated from ATP by SDS-PAGE in
17.5% (w/v) acrylamide gel. Radioactivity of the bands
was visualized and quantified with PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Microscopy and Flow Cytometry
Fluorescein diacetate 5 g L⫺1 (Serva) and 4⬘,6-diaminophenylindole were used for viability staining and DNA
visualization, respectively. For mitotic index analysis, cells
were sampled during the experiment, 0.5 mL per sample,
and fixed in solution ethanol/acetic acid 3:1 (v/v). Cells
were washed in phosphate-buffered saline, stained with
4⬘,6-diamino-phenylindole, and counted (Nikon fluorescent microscope, Nikon Europe, Badhoevedorp, The Netherlands). Five hundred cells were counted per slide, and
stages from early prophase until anaphase were considered
as mitotic. Two-milliliter samples were taken for flow cytometry. Cells were washed with 0.66 m sorbitol in Murashige and Skoog medium, pH 5.8, and the cell wall was
removed by treatment for 1 h at 37°C with 0.1% (w/v)
Plant Physiol. Vol. 128, 2002
pectolyase and 2% (w/v) cellulase (Sigma) dissolved in
0.66 m sorbitol in Murashige and Skoog medium. Protoplasts were washed twice with buffer containing 45 g L⫺1
mannitol and 18 g L⫺1 Glc in Murashige and Skoog medium, pH 5.8, and sedimented by centrifugation at 1,500g
for 5 min. Nuclei were released from the pellet in 250 ␮L of
Galbraith buffer (45 mm MgCl2, 30 mm sodium citrate, 20
mm 3-(N-morpholino)-propanesulfonic acid [MOPS], 1%
[v/v] Triton X-100, pH 7.0) and fixed with 5 ␮L of 37%
(v/v) formaldehyde (Sigma). DNA staining was performed
according to the method of Vindelov et al. (1983). Fixed
samples were washed with phosphate-buffered saline and
filtered through a 20-␮m nylon membrane. One-hundred
microliters of filtrate was treated with 200 ␮L of solution A
containing 3.4 mm trisodium citrate, 0.1% (v/v) Nonidet
P-40, 0.5 mm Tris, pH 7.6, and 1.5 mm spermine tetrahydrochloride (Sigma; stock solution), to which 30 mg L⫺1
trypsine was added. After 10 min of incubation at room
temperature, 150 ␮L of solution B containing the stock
solution to which 0.5 g L⫺1 trypsine inhibitor and 100 mg
L⫺1 of ribonuclease A were added and the sample was
incubated for another 10 min. Finally, the nuclei were
stained with solution C containing the stock solution to
which 0.4 g L⫺1 propidium iodide (Sigma) and 1.2 g L⫺1
spermine tetrahydrochloride were added, and the sample
was incubated for 1 h at 4°C and analyzed on FACScan
(Becton-Dickinson, San Jose, CA) analytical flow
cytometer.
ACKNOWLEDGMENTS
The authors thank Dr. Lieven de Veylder for kindly
sharing his expertise in kinase assays and Prof. Herman
Slegers and his coworkers for help with radioactivity work,
especially Bert Grobben for PhosphorImager analysis.
Received July 3, 2001; returned for revision August 31,
2001; accepted October 8, 2001.
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