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The Plant Cell, Vol. 5, 1711-1723, December 1993 O 1993 American Society of Plant Physiologists
cdc2a Expression in Arabidopsis 1s Linked with
Competence for Cell Division
Adriana S. Hemerly,' Paulo Ferreira,' Janice de Almeida Engler,' Marc Van Montagu,a9' Gilbert Engler,b
and Dirk lnz6
a
Laboratorium voor Genetica, Universiteit Gent, K. L. Ledeganckstraat 35, 8-9000 Gent, Belgium
b Laboratoire Associe de I'lnstitut National de Ia Recherche Agronomique (France), Universiteit Gent, 8-9000 Gent, Belgium
A key regulator of the cell cycle is a highly conserved protein kinase whose catalytic subunit, p34cdc2,is encoded by
the cdc2 gene. We studied the control of the expression of the Arabidopsis cdc2a gene in cell suspensions and during
plant development. In cell cultures, arrest of the cell cycle did not significantly affect cdc2a mRNA levels, but nutrient
condltlons were important for cdc2a expression. During plant development, the pattern of cdc2a expression was strongly
correlated with the cell proliferation potential. The effects of externa1signals on cdc2a expression were analyzed. Wounding
induced expression in leaves. Lack of light altered temporal regulation of cdc2a in the apical but not root meristem of
seedlings. Differential cdc2a responses were obtained after different hormone treatments. Signals present only in intact
plants were necessary to mediate these responses. Although other control levels have yet to be analyzed, these results
suggest that the regulation of cdc2a expression may contrlbute greatly to spatial and temporal regulation of cell division
in plants. Our results also show that cdc2a expression is not always coupled with cell proliferation but always precedes
it. We propose that cdc2a expression may reflect a state of competence to divide, and that the release of other controls
1s necessary for cell division to occur,
INTRODUCTION
p34CdC2
is the catalytic subunit of a protein kinase that plays
a key role in the cell cycle control, in particular at two control
points: the Gl/S transition and the entry into mitosis (Reed,
1980; Nurse and Bissett, 1981; Reed and Wittenberg, 1990).
Cycling cells regulate cdc2 activity and expression at severa1
levels. The abundance of p34CdC2 is constant throughout the
cell cycle, whereas its rate of synthesis and kinase activity are
regulated (McGowan et al., 1990; Welch and Wang, 1992). The
activation of the mitotic kinase is characterized extensively and
involves association with cyclin and post-translational modifications (reviewed by Nurse, 1990; Maller, 1991). In contrast to
fission yeast, in which levels of cdc2 mRNA are constant during the whole cycle (Durkacz et al., 1986), the amounts of cdc2
mRNA in mammalian cells fluctuate (McGowan et al., 1990;
Welch and Wang, 1992). cdc2 transcription is activated at Gl/S
transition and decreases at late G2 or early M phase (Dalton,
1992). cdc2 expression is also regulated during exit from and
reentry into the cell cycle. Quiescent human cells have very
low levels of cdc2 mRNA and protein. As soon as the cells
reenter the cycle, the amounts of cdc2 mRNA and protein increase (Lee et al., 1988; Furakawa et al., 1990; Welch and
Wang, 1992). This form of regulation may have specific consequences for animal development because there is a positive
To whom correspondence should be addressed.
correlation between the proliferative state of tissues and the
abundance of cdc2 mRNA (Krek and Nigg, 1989; Lehner and
OFarrell, 1990).
Although the mechanisms that control the cell cycle of higher
eukaryotes seem to be largely similar, temporal and spatial
control of cell division differ for each organism following its
pattern of development. Plants, for example, have a unique
developmental plan with features not found in either animais
or fungi. Because plant cells are surrounded by a rigid cell
wall, morphogenesis is determined only by differentialcell division and expansion. Plant development is predominantly
postembryonic: during embryogenesis, the main developmental event is the establishment of the root-shoot axis. Most plant
growth occurs after germination, by iterative development at
the meristems. The primary shoot and root meristems generate cells that differentiate into many types and can continuously
produce repeating units of organs or functionally different ones.
The shoot apical meristem can switch its activity from vegetative to reproductive growth producing flowers, and roots can
be induced to make new structures like bulbs and nodules
when appropriately stimulated. Meristematic activity during
plant development can be modulated by environmental conditions, such as light, gravity, and wounding. Unlike animal
cells, most plant cells can, under appropriate conditions,
reenter the cell cycle and eventually develop into whole plants
(totipotency). An interesting point to be studied is how the
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The Plant Cell
regulation of cell cycle-controlling genes responds to the different developmental programs that each organism follows.
Particularly in plants, it is important to determine the correlation between the expression of cell cycle genes and the
totipotency of the cells.
Cell divisions in different parts of the plant have to be temporally and spatially coordinated. Although the overall growth
has an autonomous control, signals are also involved in coordinating growth throughout the plant. It is known that hormones
play a vital role in promoting or inhibiting plant growth. Very
little is known about the mechanism of action of plant-specific
mitogenic signals. Therefore, it is important to examine to what
extent the genes controlling the cell cycle are influenced by
these signals.
cdc2-homologous genes have been found in all plant species analyzed (Colasanti et al., 1991; Feiler and Jacobs, 1991;
Ferreiraetal., 1991; Hirtetal., 1991;Bergouniouxetal., 1992;
Hashimoto et al., 1992; Miao et al., 1993). We isolated a functional cdc2-homologous gene from Arabidopsis, now called
cdc2a (Ferreira et al., 1991). Recently, a second cdc2-homologous gene, cdc2b, has been found, but its function remains
to be determined (Imajuku et al., 1992). Studies of cdc2 expression by RNA gel blot analysis in maize (Colasanti et al.,
1991), rice (Hashimoto et al., 1992), petunia (Bergounioux et
al., 1992), soybean (Miao et al., 1993), and in situ hybridizations in Arabidopsis (Martinez et al., 1992) showed a correlation
between the proliferative state of the tissue and cdc2 mRNA
levels.
Together, the data on cdc2 expression analyses in animals
and plants indicate that a long-term transcriptional control might
play an important role in the cdc2 regulation during development. Our study addresses this aspect of cdc2a regulation.
We demonstrate that in whole plants, cdc2a can be transcriptionally regulated by plant-mitogenic signals, such as
hormones, light, and wounding. We show that cdc2a expression is developmentally regulated and is expressed mainly in
proliferating tissues. Furthermore, we find that cdc2a expression is not always linked to cell division. In these cases, it most
probably reflects a physiological state of competence to divide.
decrease of cdc2a transcript levels was observed in cells arrested either in S phase or metaphase. In contrast, the mRNA
levels of a mitotic cyclin gene from Arabidopsis (cydAt) decreased significantly after the same treatments (Hemerly et
al., 1992) (Figure 1A).
We analyzed the effect of nutrient depletion on cdc2a expression. As shown in Figure 1B, when starved cells were
supplemented with fresh medium, cdc2a mRNA levels increased and gradually decreased upon subsequent starvation.
As seen in Figure 1C, a reduction in mRNA abundance did
not occur when cells were refreshed with medium lacking the
hormone naphthaleneacetic acid (NAA). The absence of NAA
did not arrest division after 2 days, possibly due to the presence of high levels of hormone inside the cells. On the other
hand, when cells were provided with one-tenth the normal concentration of sucrose, they stopped dividing and the amount
of cdc2a transcripts decreased significantly.
A
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HU COL.
cdc2a
cyclAt
B
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RESULTS
cdc2a Expression in Cell Cultures
Figure 1. RNA Gel Blot Analyses of cdc2a Expression in Cell
Suspensions.
To study the control of cdc2a expression at the cellular level,
we analyzed cycling cells in Arabidopsis cell cultures. Higher
levels of cdc2a mRNA were detected in actively dividing cell
suspensions compared with mRNA levels found in different
plant organs (data not shown). This suggested that there was
a positive correlation between the proliferative state of cells
and cdc2a expression. To analyze cdc2a expression in cells
arrested in the cell cycle, cell suspensions were blocked
in early S phase and metaphase using hydroxyurea and
colchicine, respectively. Figure 1A shows that only a small
(A) Cell suspensions, after growing 2 days in fresh medium, were treated
with 10 mM hydroxyurea (HU) or 0.05% colchicine (COL) for 2 days.
The RNA gel blot, with 10 ng of total RNA from treated and untreated
cells (C), was probed with cdc2a (upper blot) and cyclAt (lower blot).
(B) Starved cells (0) were diluted in complete fresh medium and aliquots were taken after 1, 2, 4, 5, and 7 days. RNA gel blot with 10 ng
of total RNA from each sample was probed with cdc2a.
(C) Cell suspensions, after growing 2 days in fresh medium, were cultured in 0.2 g LH sucrose (i SUCR) and hormone-free medium
(-HORM). Total RNA was extracted after 2 days of culture. Total RNA
from untreated cells was used as control (C). The RNA gel blot with
10 ng of total RNA was probed with cdc2a.
cdc2a Expression in Arabidopsis
Spatial and Temporal Expression of the cdc2a Gene
during Development
To study the transcriptional regulation of the cdc2a gene during plant development, the Arabidopsis cdc2a promoter was
fused with the 0-glucuronidase (gus; uidA gene from fscherichia coli) gene, introduced into Arabidopsis plants, and used
for detailed histochemical GUS analysis of gene regulation (See
Methods). The spatial and temporal expression pattern of this
chimeric gene was analyzed during development from germination to seed production, as shown in Figure 2.
In germinating seedlings, intense GUS staining was first observed in the root meristem (Figure 2A) and also later in the
shoot apical meristem. 3H-thymidine-labeling experiments
have shown that the pattern ofcdc2a expression corresponded
to the pattern of 3H-thymidineincorporation (Figure 26). Thus,
this difference in timing of the cdc2a gene expression correlated with the order of activation of the meristems.
At 8 days after germination (Figure 2C),strong GUS staining was still localized in the root and shoot apices,
corresponding to the active meristems. The expanded
cotyledons had a light vascular staining that may indicate some
cambial activity. lntense staining was found in developing first
leaves. With further growth, the expression decreased (data
not shown). Another region of GUS staining was detected in
the root-shoot junction in which the adventitious roots are initiated. In roots, GUS staining was detected all over the pericycle
and parenchyma of the vascular cylinder (Figures 2C and 2D).
The pericycle cells retain a potential to divide, because they
are responsible for the lateral root formation and root thickening. The expression in parenchyma cells might also be
correlated with a competence to divide. If so, this cell layer
might exhibit some mitotic activity contributingto the primary
root thickening at a later time or under growth conditions we
have not investigated.
Expression in the apical meristems persisted throughout the
entire course of development. Strong gos expression was detected in newly formed organs. Activity decreased as these
organs became older, concomitant with the decline in their
mitotic activity. The expression in pericycle cells was low in
older parts of roots. Fully expanded leaves had undetectable
GUS staining, although GUS activity was detected in quantitative fluorometric assays (data not shown).
As plants progress from vegetative to reproductive growth,
new organs are formed in the inflorescence, such as flower,
stem, cauline leaves, and lateral branches. In stems (Figure
2H),GUS staining was detected in the cambium and pericycle, tissues with meristematic potential, and in the cortex, where
divisions occur that contribute to the primary thickening of the
stem. GUS activity in the parenchyma cells of the protoxylem
and phloem might be an indicationof division competence or
actual mitotic activity during stem growth. All over the stem,
the intensities of GUS staining varied considerably. This was
probably associated with variations in thickening and elongation of particular regions in the stem. Developing axillary buds
1713
showed strong GUS expression correlated with their high mitotic activity (Figure 21).
During flower and silique development, the intensity and localization of GUS staining varied (Figures 2E to 2G).In different
stages of flower buds (previously determined by Smyth et al.,
1990), GUS staining was observed in actively growing organs.
In stage 6 to 7 flowers, GUS activity was detected in the stamen and gynoecium primordia (Figure 2J). In stage 9 to 10
flowers, GUS staining was stronger in fast growing petals and
the gynoecium (Figure 2K). In fully grown flowers, GUS staining was found within the gynoecium, mainly in the ovules where
integumental cells are actively dividing during embryo sac development (Figures 2E and 2L). After fertilization, the flower
undergoes changes leading to the formation of a silique, where
the embryo develops. As the silique grew, GUS staining was
detected throughout the silique wall, septum, funiculus, and
developing embryos (Figures 2F and 2M). No GUS staining
was detected in mature siliques (Figure 2G). It is important
to point out that we detected a reduction of GUS activity in
the same tissues, over short periods, that did not result from
protein dilution during cell growth. This indicates that the GUS
protein is not as stable as has been thought (Jefferson et al.,
1987), at least in dividing cells. The GUS protein might have
a faster turnover in these cells, possibly because of higher levels of proteases.
To ensure that the cdc2a promoter region fused to GUS contained all the regulatory sequences necessary to drive the
correct pattern of cdc2a expression, whole-mount in situ
hybridizationswere performed in Arabidopsis seedlings using
cdc2a as a probe. The pattern of expression in roots and apical meristem confirmed the results obtained with histochemical
GUS analysis (Figures 2N to 2s).
Environmental Control of cdc2a Expression
Because environmentalfactors can trigger changes in cell division and expansion patterns, it seemed likely that cdc2a
expression could be affected by them. Light constitutes one
of the most distinctive influences of the environment on plant
development. We investigated the effect of light on cdc2a
expression during germination. In etiolated seedlings, root development is not significantly affected; correspondingly, cdc2a
had the same pattern of expression there as in light-grown
plants (data not shown). In the aerial part of the plant, the absence of light altered the temporal but not the spatial regulation
of cdc2a expression. Four-day-old seedlings germinated under light showed GUS activity in the apical meristem. In
contrast, dark-grown seedlings showed no GUS activity in this
region until they were 7 days old, as shown in Figure 36. Because this meristem did not divide in the darkness, as
demonstrated by the absence of 3H-thymidineincorporation
(data not shown), the activation of cdc2a expression might
correlate with the acquisitionof a competence to divide rather
than with cell division.
1714
The Plant Cell
Figure 2. cdc2a Expression in Arabidopsis.
ccfc2a Expression in Arabidopsis
1715
induction of cdc2a expression caused by wounding might be
correlated with increased division competence in cells around
the wounded area.
^PH^^
I
Figure 3. Histochemical Localization of GUS Activity in 7-Day-Old
Arabidopsis Seedlings.
(A) Apical meristem of a light-grown plant.
(B) Apical meristem of a dark-grown plant.
Wound healing in plants can trigger proliferation of parenchyma cells to form a protective layer. In GUS analysis of
wounded leaves, we found cdc2a expression around damaged
surfaces. To exclude artifacts, such as incomplete penetration
of substrate, some leaves were cut and fixed with acetone after 30 min, whereas others were fixed immediately. Only leaves
fixed 30 min after wounding showed induction of cdc2 expression around the damaged surface, as shown in Figure 4B. The
intensity and kinetics of the response varied within and between plants. Because significant cell proliferation in the
wounded area was not seen in Arabidopsis leaves, the
Hormonal Control of cdc2a Expression in Intact Plants
Besides the intrinsic cellular control, hormones also regulate
cell division in plants, although the precise mechanism by
which this control is mediated is unknown. To investigate if
hormones could have an effect on transcriptional regulation
of the cdc2a gene, we used roots of transgenic Arabidopsis
plants containing the cdc2 promoter-gus fusion as a test system. To correlate induction or repression of cdc2a gene
expression with DMA synthesis, roots were treated simultaneously with hormones and 3H-thymidine. After histochemical
GUS detection, embedded roots were sectioned and autoradiography was performed for detection of 3H-thymidine
incorporation (see Methods).
Figure 5 shows the GUS activity in roots of intact plants
treated with different hormones. After a 72-hr treatment with
different cytokinins (benzyl-6-aminopurine, 6-[Y,Y-dimethylallylamino]purine, and kinetin), the roots showed increased
GUS activity in the pericycle and parenchyma cells of the vascular cylinder (Figure 5H); this induction was only seen in the
upper part of the main root. GUS staining in this region was
almost undetectable in untreated roots (Figure 5G). 3H-thymidine labeling in cells with induced GUS activity (Figure 51)
and increased cell number after longer hormone incubation
Figure 2. (continued).
Histochemical localization of GUS activity in transgenic Arabidopsis plants is shown in (A) to (M).
(A) Three-day-old seedling.
(B) Longitudinal section through a 3-day-old root. Black grains correspond with localization of 3H-thymidine labeling.
(C) Eight-day-old seedling.
(D) Cross-section through an 8-day-old root.
(E) Mature flower.
(F) Developing silique.
(G) Mature silique.
(H) Cross-section through an inflorescence stem.
(I) Longitudinal section through an axillary bud.
(J) Longitudinal section through a stage 6 to 7 flower bud.
(K) Oblique section through a stage 9 to 10 flower bud.
(L) Longitudinal section through the ovary shown in (E).
(M) Longitudinal section through the silique shown in (F).
Whole-mount in situ hybridizations of Arabidopsis seedlings are shown in (N) to (S).
(N) Apical meristem of a 6-day-old seedling hybridized with a digoxigenin-labeled cdcZa antisense probe. Black stain represents RNR/RNA hybrids
detected by gold-labeled antibodies.
(O) Apical meristem as shown in (N) but hybridized with the cdc2a sense probe.
(P) to (R) Roots at different stages hybridized with the cdc2a antisense probe. Dark-brown stain represents RNA/RNA hybrids detected by alkaline
phosphatase-labeled antibodies.
(S) Root tip hybridized with the cdc2a sense probe.
Micrographs in (D) and (H) to (M) were taken using dark-field optics. In (B), (D), and (H) to (S), bars = 100 urn. AM, apical meristem; AR anther
primordium; C, cortex; CZ, cambial zone; E, endodermis; Em, embryo; F, funiculus; G, gynoecium; LP, leaf primordium; O, ovule; P, petal; Pe,
pericycle; Ph, phloem; S, septum; St, stamen; SW, silique wall; VC, vascular cylinder; X, xylem; XP, xylem parenchyma.
1716
The Plant Cell
Figure 4. Histochemical Localization of GUS Activity in Leaves of Transgenic Arabidopsis Plants after Wounding.
(A) GUS assay performed on a leaf fixed with acetone immediately
after wounding.
(B) GUS assay performed on a leaf fixed with acetone 30 min after
wounding.
Arrows indicate wounded regions.
inhibited. It remains to be determined whether these cells completed normal rounds of mitosis.
When roots were treated with 10~6 M IAA and 10~6 M
benzyl-6-aminopurine simultaneously, the pattern of GUS staining was similar to that obtained with cytokinin alone. No
induction of GUS activity was observed in excised roots treated
only with cytokinins but was observed when both auxin and
cytokinins were present.
Treatment with 10~6 to 10~8 M abscisic acid (ABA) for only
1 day completely inhibited GUS activity in the lateral root tips
and decreased the activity over the vascular cylinder of the
entire root (Figure 5B). The expression in the main root tip was
unaffected. Root tips where cdc2a expression was inhibited
showed no 3H-thymidine incorporation, indicating an arrest of
cell division. An inhibitory effect of cell division in roots by ABA
has been described previously (Phillips, 1971; Newton, 1977).
In treatments with 10~5 to 10~7 M ethephon, 1-aminocyclopropane-1-carboxylate, or gibberellin A3, the pattern of cdc2a
expression was not affected (data not shown).
Hormonal Control of cdc2a Expression in Protoplasts:
Dedifferentiation and Reinitiation of Cell Division
showed that there was a correlation of cdc2a expression with
cell division.
Treatment with 10~6 M indole-3-acetic acid (IAA) for 72 hr
induced lateral root formation and resulted in an intriguing
expression pattern: most of the root tips that exhibited homogeneous GUS activity in untreated plants showed a three-zone
pattern. One zone exhibiting reduced staining was intercalated
between two strongly stained zones (Figure 5C). Roots treated
with 10~6 M NAA showed the same GUS activity pattern (data
not shown). This pattern became visible after 2 days and disappeared after 4 days in auxin-containing medium (data not
shown). Similar experiments with other plants transformed with
different promoter-gus fusions that drive expression in root
tips (including the 35S cauliflower mosaic virus promoter) never
showed this three-zone pattern. Because cell sizes in auxintreated and untreated roots are the same, a possible artifact
due to GUS dilution in the inhibition zone can be excluded.
Interestingly, excised roots treated with any of these hormones
did not exhibit the three-zone pattern of GUS staining. Whereas
IAA and NAA have comparable effects, roots treated with
10~6 M 2,4-D behaved differently. Localized cell division in
roots is stimulated by 2,4-D, triggering the formation of periodic calluslike thickenings in place of lateral roots. These
undifferentiated tissues showed strong uniform GUS staining
(data not shown). However, when a lower concentration of 2,4-D
was used (10~7 M), the three-zone pattern was detected in
structures resembling lateral roots (Figure 5D). 3H-thymidinelabeling experiments have shown a similar pattern of nucleotide incorporation in the root tips of auxin-treated and untreated
roots (Figures 5E and 5F). These data show that DNA synthesis occurred in the region in which cdc2a expression was
A particular phenomenon of most plant cells is their capacity
to dedifferentiate and reinitiate division under appropriate conditions. In contrast to Arabidopsis, tobacco protoplasts can be
easily regenerated and have proven particularly suitable for
studies of the hormonal requirements for cell division. Therefore, cdc2a expression was analyzed in tobacco leaf protoplasts
derived from transgenic plants containing the Arabidopsis
cdc2a promoter fused to gus. The overall developmental regulation of the cdc2a promoter-gus chimeric gene in tobacco
was found to be comparable to that in Arabidopsis (data not
shown). DNA synthesis was monitored by the rate of 3Hthymidine incorporation into protoplasts.
As shown in Figure 6, quantitative fluorometric GUS measurements revealed low levels of GUS activity in freshly
prepared tobacco mesophyll protoplasts. When protoplasts
were cultivated in medium containing appropriate concentrations of auxin and cytokinin to stimulate cell division, induction
of GUS activity was detected. Protoplasts cultivated with auxin
alone exhibited only a small increase in GUS activity. In contrast, cytokinins alone could induce GUS activity without
stimulating significant DNA synthesis and cell division. In protoplasts cultivated with ABA, GUS activity decreased. These
data show that an induction of cdc2a expression occurs in protoplasts during their dedifferentiation and reinitiation of cell
division. Moreover, cdc2a expression can be induced by hormones in protoplasts without cell division.
The DNA content profile of nuclei of freshly isolated mesophyll protoplasts of tobacco was monitored by flow cytometry.
It showed the presence of 95% of 2C nuclei (G1 phase) (data
not shown). Because most of the protoplast population is in
G1, they have to undergo DNA synthesis before the first mitosis. To study whether the activation of cdc2a transcription in
cdc2a Expression in Arabidopsis
the dedifferentiation process is coupled to entry into S phase,
protoplasts were treated with hydroxyurea. The results showed
that blockage in early S phase had no significant effect on GUS
activity (Figure 6). These data indicate that in protoplast culture, where quiescent cells are reinduced to divide, the
activation of cdc2a transcription occurs at or before G1/S transition and is not dependent on completion of DMA synthesis
or progression through the cell cycle.
1717
DISCUSSION
cdc2a Expression Has Long-Term Control That Is
Developmental^ Regulated
The activation of the mitotic kinase, involving post-translational
modifications of p34cdc2, is the best-characterized level of
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Figure 5. Histochemical Localization of GUS Activity in Roots of Transgenic Arabidopsis Plants after Hormone Treatments.
(A) Untreated root.
(B) Root treated with 10~6 M ABA for 3 days.
(C) Root treated with 10~6 M IAA for 3 days.
(D) Root treated with 10~7 M 2,4-D for 3 days.
(E) Longitudinal section through the untreated root shown in (A) labeled with 3H-thymidine. Black grains correspond with the localization of 3Hthymidine labeling.
(F) Longitudinal section through the root shown in (C), labeled with 3H-thymidine. Black grains correspond with the localization of 3H-thymidine
labeling.
(G) Upper part of the main root of a 4-week-old plant not treated with hormones.
(H) Same part of the root as shown in (G), but treated with 1CT6 M benzyl-6-aminopurine.
(I) Cross-section through the root shown in (G) labeled with 3H-thymidine.
(J) Cross-section through the same part of the root shown in (H) labeled with 3H-thyrnidine. White grains correspond with the localization 3Hthymidine labeling.
Micrographs in (E) and (F) were taken using bright-field optics; micrographs in (I) and (J) were taken using dark-field optics. VC, vascular cylinder.
Bars = 100 urn.
1718
The Plant Cell
............ ....................................
.............
.............
.............
o
FI
C
NM+2.4-0 N M
BAP
EAP
KIN
ABA
HU
HU+
NM+BAP
Flgure 6. Measurement of GUS Activity and 3H-ThymidineIncorpo-
ration in Transgenic Tobacco Leaf Protoplasts.
Freshly isolated protoplasts (FI) were cultured 3 days in medium containing 3H-thymidineand iO-6 M of the indicated hormones, except
for the NAA+BAP treatment (NAA, 5 x 10" M; benzyl-6-aminopurine
[BAP], 10" M). C, protoplastscultured in medium without hormones;
HU, 10 mM hydroxyurea; KIN, kinetin; MU, 4"thylumbelliferone. The
mean values of GUS activity represent four replicates of three independent experiments. The mean values of 3H-thymidine incorporation
represent two replicates of two independent experiments. Standard
errors are shown.
regulation of the cdc2 gene and appears to impose a shortterm control over p34cdc2in cycling cells. It has been suggested that there must also be a long-term control of cdc2 gene
expression that occurs in processes such as cell differentiation (Lee et al., 1988; Krek and Nigg, 1989; Dalton, 1992). This
long-term control would be important during plant growth, in
which cell divisions and differentiation in meristems continuously give rise to new organs. Our data show that cdc2a is
transcriptionally regulated during plant development and is
expressed mainly in dividing cells. As cells are differentiated,
cdc2a transcription substantially decreases. We must consider
that our results could have been influenced by gus (uidA)mRNA
stability and GUS enzyme stability/activity. However, our analyses of the transcriptional regulation of the cdc2a gene during
development correlated well with previous results obtained by
in situ hybridizations, in which steady state mRNA levels were
analyzed (Martinez et al., 1992).
We have found no specificity of cdc2a expression for any
of the plant meristems and dividing cells, which suggests that
cdc2a probably participates in the spatial and temporal regulation of cell divisions throughout the plant. Although Cdc2a
protein levels cannot be extrapolated from our studies and still
have to be analyzed in Arabidopsis tissues, they might be regulated, at least in part, by transcriptional control. Positive
correlations between amount of Cdc2-like proteins and cell
proliferation were reported in wheat (John et al., 1990) and
carrot (Gorst et al., 1991).
cdc2a Expression 1s Linked with Competence for Cell
Dlvision
Although there was a positive correlation between cdc2a
mRNA levels and the proliferative state of cells in our studies,
it is clear that cdc2a expression was not restricted to dividing
cells. Similar observations were made recently in Arabidopsis (Martinez et al., 1992) and soybean (Miao et al., 1993). We
found several cases in which cdc2a expression increased but
was not followed by cell division. The observed variations in
cdc2a mRNA levels could correspond with a degree of division competence (see below).
In contrast to what was observed in Drosophila(Lehner and
OFarrell, 1990) and chicken (Krek and Nigg, 1989), in which
no cdc2 mRNA was found in differentiated adult tissues, we
did observe cdc2a expression in some nondividing, differentiated tissues. The different patterns of expression between
plants and animals might reflect the unique features in each
developmental program (Colasanti et al., 1991; Mia0 et al.,
1993). In contrast to animals, few dicotyledonous plant cells
are terminally differentiated. Many tissues retain cells able to
dedifferentiate if given the proper developmental cues. Our
data suggest that fully differentiated plant cells do not switch
off cdc2a expression. Thus, these cells potentially retain a pool
of the essential kinase that can be used if necessary.
Because the molecular mechanism that controls cell division when plants respond to an environmental stimulus is not
known, we have investigated the effect of light and wounding
on cdc2a expression. In the absence of light, cells of the apical meristem do not divide, but we observed in most cases
an induction of cdc2a expression. lnduction of cdc2a expression in the apical meristem during the normal developmental
program might be oneof several necessary steps leading ultimately to cell division. Actual cell division might depend on
environmental cues, such as light.
Our results have also shown an induction of cdc2a expression upon wounding. Although leaf cells have low levels of
cdc2a mRNA and are competent to divide under appropriate
conditions, it is well known that in tissue culture, the induction
of cell proliferation occurs mainly in the wounded regions of
leaves. We can speculate that in the wounded area, cells acquire a higher degree of competence to divide. Higher induction
of cdc2a expression after wounding normally occurs in young
leaves but other factors, probably related to the growth conditions, seem to affect the expression. It is known that the wound
response is influenced by carbon dioxide, temperature, light,
and nutrient conditions (Green and Ryan, 1973). The response
of plant cells is also dependent on the age of the tissue and
is less pronounced in older tissues (Kahl, 1982).
Our studies on Arabidopsis cell suspensions have shown
that in cells blocked in early S phase or metaphase of the cell
cycle, the amount of cdc2a mRNA remained approximately
constant. In contrast, the mRNA levels of the Arabidopsis mitotic cyclin cyc7At decreased dramatically. The different
responses of the two genes after treatment with hydroxyurea
cdc2a Expression in Arabidopsis
and colchicine might indicate the existence of different modes
of regulation. Whereas the cyclin gene may be tightly controlled
by completion of preceding biochemical events in the cell cycle, the correlation between cdc2a expression and proliferative
competence indicate that this gene is controlled by more longterm developmental programs acting throughout the life of
differentiated cells.
The concept that cdc2 expression in noncycling plant cells
is correlated with a degree of competence to divide parallels,
to a certain extent, what has been seen in mammalian cell
cultures. There have been several examples of a decrease in
cdc2 transcript levels in mammalian cells as they become
quiescent; after serum stimulation, the amount of transcripts
increases when cells reenter the cell cycle (Lee et al., 1988;
Stein et al., 1991;Welch and Wang, 1992).Quiescent cells that
retain the competence to divide have low levels of cdc2 mRNA
(Stein et al., 1991). In contrast, senescent human cells that
fail to reenter the cell cycle have no detectable amount of cdc2
mRNA (Stein et al., 1991).Plant cells cannot be cultured in
an arrested state. Although cell starvation is remotely comparable, the cells die after an extensive period of culture. Analyses
of cdc2a expression as starvation progressed showed that the
amount of cdc2a transcripts gradually decreased and rose
again as cells reinitiated division.
Totipotent nondividing plant cells, at least superficially, can
be compared to quiescent animal cells. They are not cycling,
but upon stimulus, they can reenter the cell cycle. We have
shown that leaf mesophyll cells have low levels of cdc2a expression. When their protoplasts are stimulated to divide, there
is an induction of cdc2a expression. Our results also indicate
that cdc2a expression can be induced by hormones without
the occurrence of cell division. In this case, cdc2a expression
might be an early step in a cascade of events necessary for
future cell division.
Undifferentiated quiescent plant cells can also be found in
dormant meristems. Upon stimulation, these become active
again. Analyses of transgenic tobacco plants, containing the
Arabidopsis cdc2a promoter-gus fusion, showed GUS staining in dormant axillary buds (data not shown). This expression
may reflect the competence of dormant meristematic cells to
restart division.
cdc2a Expression Can Se Regulated Transcriptionally
by Hormones
Plant hormones are known regulators of cell division, and so
we analyzed their effects on cdc2a expression. In roots of intact plants, hormones couid trigger an induction or inhibition
of the cdc2a expression in different tissues according to the
hormone applied. In several cases, induced cdc2a expression
occurred concomitantly with cell division. This correlation
was also observed in alfalfa cell cultures (Hirt et al., 1991),
Arabidopsis roots (Martinez et al., 1992), and soybean roas
(Miao et al., 1993).However, as discussed above, in protoplast
1719
analysis we found situations in which the hormonal induction
of cdc2a expression was not followed by cell division.
The hormone effects observed on cdc2 expression are probably due not only to the exogenous hormones, but also to their
interactionswith endogenous growth factors in specific tissues.
The auxin effect on root tips and the induction of cdc2a expression by cytokinins, for example, were not seen in excised
tissues that might be rapidly depleted of interna1 hormone
stores (Theologis and Ray, 1982;Alliotte et al., 1989).In intact
plants, the upper part of the main root might contain high levels of auxin due to transport of this hormone. Because cdc2a
expression was induced in the vascular cylinder in excised
roots treated with both auxins and cytokinins, the simultaneous presence of auxins and cytokinins are possibly necessary
for the induction of cdc2a expression in this region. Further
investigationof which signals are present internally and needed
for these cdc2a responses could help to reveal the types of
hormonal control over cell division.
The three-zone pattern observed in root tips of intact plants
after auxin treatment is another example of how cdc2a expression can apparently be uncoupled from cell division. In this
case, the transcription of the cdc2a gene was inhibited in cells
in which DNA synthesis was occurring, as deduced from the
3H-thymidine incorporation. A similar GUS staining pattern
was described for the histone H4 gene from Arabidopsis in
some root tips of plants not treated with hormones (Atanassova
et al., 1992).We are now investigatingwhether the cells where
cdc2a expression was inhibited are undergoing mitosis. There
is a possibility that these cells, prior to auxin treatment, contained sufficient CdcPa protein to allow cell division to proceed.
Down-regulation of cdc2a expression could also be correlated
with an irregular cell cycle. An inhibitory effect of IAA in root
meristems was observed in broad bean, where roots treated
with the hormone show a reduced mitotic index and an extended mitotic cycle (Macleod and Davidson, 1966). However,
why this inhibition is localized in such a sharply defined zone
of the root tip remains to be explained. It is also possible that
endoreduplication is occurring in the inhibition zone, and
CdcPa would not be required for GllS transition.
Based on our data, Figure 7 presents a highly simplified
model for the regulation of cdc2a expression during plant development. We should emphasize that this model cannot be
extrapolated to explain the regulation of cdc2a activity, which
is mainly posttranslational. We propose that in dividing cells,
high levels of cdc2a mRNA are found. When cells stop dividing and start to differentiate, cdc2a mRNA levels decrease.
Terminally differentiated cells have no cdc2a expression. In
fully differentiated cells, which remain totipotent, low levels of
cdc2a mRNA are found (e.g., leaf mesophyll cells). lnduction
of cdc2a expression (by signals like hormone or wounding)
might precede or induce competence for cell division. Competent cells would then have high levels of cdc2a mRNA (root
pericycle cells and wounded leaf cells). Meristematic cells can
also stop dividing without differentiating (dormancy). They
would be competent to divide and contain high levels of cdc2a
1720
The Plant Cell
o
we have found, except that we have sequenced 1.5 kb further in the
5' end to obtain the promoter.
n d i fd,fterentiotion
f e ; ; ; i a t e terminally
d
Construction of Chimerlc cdc2a Promoter-p-Glucuronldase
Gene
I
I
cell
activation
of p 3 4 C d C 2
"\
cyclin
cdc25
?
-
signals
signals
Figure 7. Schematic Representationof a HighlySimplified Modelfor
the Regulation of cdc2a Expression during Plant Development.
Black and stippledsquares representcells with high levels and moderate levels of cdc20, respectively; the white square representscells with
no detectable cdc2a mRNA.
mRNA. Activation of ~
3followed
4 by cell
~ division
~
might
~
depend on further signals (e.g., hormone and light). A regulatory
step could be the expression of other cell cycle genes and/or
regulation of ~ 3 4 leve1
~ ~ and
~ 2post-translational modifications. Moreover, a cascade of signals and regulatory events
must be present in each stage of the proposed model. Their
study would be of great importance in understanding the mechanisms that control cell division during plant development.
METHODS
A region of 1.7 kb of Arabidopsis genomic DNA containing the promoter region and the leader of cdc2a gene was amplified by PCR as
described by Saiki et al. (1985), except that it was performed in 30
cycles of 94OC for 1 min, 51oC for 1 min, and 72OC for 3 min with a
final extension of 10 min. The oligonucleotides used as primers were
5'-AAGTTTTGTCGACATATATAT-3'and 5'-AACCTGATCCATGGATTCCTG-3: each containing the underlined Sal1 and Ncol sites, respectively. The amplified fragment was digested with Sal1 and Ncol and
ligated into these sites in the pGUSl vector (Peleman et al., 1989),
upstream of the P-glucuronidase(gus; uidA from Escherichiacoli) coding sequence. The cdc2a promoter-gus-3' octopine synthase (ocs)
cassette was excised by digestionwith Sal1and Smal and ligated into
the Sal1 and Scal restriction sites of the binary vector pGSV4 (D.
HBrouart, M. Van Montagu, and D. InzB, manuscriptsubmitted), generating the plasmid pVPC2AGUS. A control without the promoter was
also constructed by transferringonly the gus-3'ocs cassette into the
vector pGSV4,creating the plasmidpVPGUS. The constructswere mobilized into Agrobacterium tumefaciens C58C1RifR(pGV2260)(Deblaere
et al., 1985) usinga triparental matingsystem (Van Haute et al., 1983).
lhnsgenic Plants
Arabidopsis ecotype C24 plants transformed with pVPC2AGUS and
pVPGUSwere oMtned using the rodtransformation method(Valvekens
et al., 1988). Nicotiana tabacum cv Petit Havana(SRl) was also transformed with the same constructs using the leaf disc protocol (Horsch
et al., 1985). For each construct, 10 independenttransgenic plant lines
were analyzed in the R2 generation. All pVPGUS transformants
showed no GUS activity both in histochemical and fluorometric assays. There was no qualitative difference in gus expression in the
pVPC2AGUStransformants. One representativetransgenic line, containing one copy of the pVPC2AGUS construct, was chosen for
subsequent GUS analysis.
lsolatlon of the cdc2a Gene
Histochemlcal GUS Assays
Polymerasechain reaction (PCR) was used to amplify a fragment of
genomic DNA homologousto the cell cycle-related gene cdc2. PCR
was performed essentially as described by Ferreira et al. (1991), except that 100 ng of total DNA from Arabidopsis thaliana ecotype C24
was used as template. One clone, identical in the coding region of
the previously isolated cdc2a cDNA, was used to screen a genomic
library (X Charon 35) prepared with total DNA from Arabidopsis ecotype Columbia(kindgift of DianeJofuku, University of California, Santa
Cruz, CA) and severa1positivecandidates were isolated.After restriction mapping and hybridizations with different parts of the cDNA,
subclonescoveringa region of ~5 kb were generated in pUC18 and
sequenced by the method of Sanger et al. (1977). Seven introns were
mapped within the cdc2a coding region and one in the 5' untranslated region. During the course of this work, lmajuku et al. (1992)
reported a cdc2a genomic nucleotidesequence identical to the one
Histochemical assays for gus expression were performedas described
by Jefferson et al. (1987) with some modifications. Organs of transgenic plants, or entire seedlings, were prefixedwith cold 90% acetone
for 1 hr at -204c, washed twice with 100 mMsodium phosphate buffer,
pH 7.4, and immersed in the enzymatic reaction mixture containing
0.5 mg mL-l 5-bromo-4-chloro-3-indolyl p-D-glucuronic acid (X-gluc;
Biosynth, Staad, Switzerland), O 5 mM potassiumferricyanide, and O 5
mM potassiumferrocyanideas catalysts in 100 mMsodium phosphate
buffer, pH 7.4. The reaction was conducted at 37% in the dark for a
period of 1 hr to overnight, depending on how quickly the substrate
penetrated in the given tissue. Control experiments with untreatedmaterial showed that the pretreatment with acetone allowed a better
penetration of the substrate without altering GUS activity and localization (G. Engler, manuscript in preparation).
cdc2a Expression in Arabidopsis
Thin sections of GUS-stainedmaterial were prepared according to
Peleman et al. (1989).3H-thymidineincorporation was detected in this
material by microautoradiography.
Whole-Mount In Sltu Hybridlzatlon
Whole-mount in situ hybridizationsof Arabidopsis seedlings were performed as described previously (Ludevid et al., 1992) and modified
according to J. de Almeida Engler, M. Van Montagu, and G. Engler
(manuscript in preparation).Anti-digoxigenin alkaline phosphatase Fab
fragment (Boehringer Mannheim, Germany) and 0.8-nm colloidal
gold-conjugatedanti-digoxigeninantibodies (Boehringer Mannheim)
were used as markers.
Hormone Treatments
Four-week-old plants growing in sterile conditions in germination
medium (Valvekens et al., 1988)were transferredto a sterile semisolid
medium containing Murashige and Skoog salts (Flow Laboratories,
McLean, VA), 0.3%agar, and the given hormone. A range of hormone
concentrations (from 10-* to lO+ M) and incubation times (from 20
hr to 6 days) were tested. Unless otherwise indicated, all hormones
were applied in aconcentration of 10-6 M for 72 hr. 3H-thymidinelabeling was done in the last 20 hr of treatment, after which the plantswere
transferred to fresh medium containing 2 pCi mL-l of methyVHthymidine (47 Ci mmol-I; Amersham, Aylesbury, U.K.). Histochemical GUS analysis was performed as described above.
1721
radiolabeled RNA probe(RiboprobeGemini IICore System; Promega)
at 65OC,in 50% formamide, 5 x SSPE (1 x SSPE is 3.6 M NaCI, 0.2 M
NaP04,pH 7.7,0.02M Na2-EDTA),5 x Denhardh solution (1 x Den0.02%BSA), 0.25%nonfat
hardt‘s solution is 0.020/0 Ficoll, 0.02%WP,
dry milk, 0.5% SDS, and 20 pg mL-I denatured salmon sperm, and
then washed at 68OC twice in 2 x SSC (1 x SSC is 150 mM NaCI,
15 mM N*-citrate, pH 7.0),1% SDS, twice in 1 x SSC, 1% SDS, and
once in 0.1 x SSC, 0.1% SDS.
Protoplast Analyses
Protoplastswere isolatedfrom in vitro-cultured tobacw plants (Boerjan
et al., 1992). Protoplasts (2 x 105 mL”) were incubated in 2 mL of
culture medium in multiwell tissue culture plates (No. 3047; Falcon,
Plymouth,U.K.). Theculture mediacontainedthe appropriate hormones
and 2 pCi mL-l of methyL3Hthymidine.The protoplasts were cultured
for 72 hr, at 25OC, 5000 lux, and 16 hr of light. For quantitative GUS
measurements, 1 mL of protoplastculture was mixed vigorously with
500 pL of twofold concentrated GUS buffer (Jefferson et al., 1987) and
centrifuged for 5 min at 14,000 rpm in an Eppendorf centrifuge; the
supernatant was used for GUS and protein analyses. Determination
of protein concentration was performed using a protein assay kit (Bradford; Bio-Rad).Quantitative analyses of GUS activity were conducted
with 6 pg of total protein using the fluorometric assay (Breyne et al.,
1993). 3H-thymidineincorporation was measured in the other 1-mL
protoplast culture as described by Zelcer and Galun (1976).
ACKNOWLEDGMENTS
Cell Culture
Cell suspension cultures of Arabidopsis ecotype Columbia (Axelos et
al., 1992) were a gift from Bernard Lescure (CNRS-INRA, Toulouse,
France). The cells were maintained in Gamborg’s 85 medium (Flow
Laboratories), containing 20 g L-’ sucrose and 1 pM naphthaleneacetic acid (NAA). The cultures were kept in continuous light at 26OC
rotatingat 100 rpm. Every7 days, one-fifthof each culture was diluted
with 4 volumes of fresh medium.
For the starvationexperiments, one-thirdof a 7-day-oldculture was
diluted in 2 volumes of fresh medium. Aliquots were taken every day,
and the cell growth was monitored by 3H-thymidineincorporationand
by cell numbers. In the experiments in which cells were directly cultured in low-sucrose (0.2 g L-l) and hormone-free media, one-third of
a 2-day-old culture was collected by filtration, washed severa1times,
and incubated overnight with the appropriate medium. To removeany
carry-over nutrients, the suspensions were filtered again and reincubated in fresh medium.
We thank Dr. Catherine Bergouniouxfor the flow cytometry analyses;
Drs. DianeJofuku and Bernard Lescurefor their kind gifts; Drs. Allan
Caplan and Tom Gerats for critical readingof the manuscript;Raimundo
Villarroel for nucleotide sequencing; Chris Genetellofortechnical assistance; Martine De Cock for preparationof the manuscript; and Karel
Spruyt and Vera Vermaerckefor photcgraphsand drawings.This work
was supported by grantsfrom the Belgian Programme on Interuniversity Poles of Attraction (Prime Minister‘s Office, Science Policy
Programming, No. 38), and ‘Vlaams Actieprogramma Biotechnologie”
(ETC 002). A.S.H. and P.F. are indebted to the Coordenaçãode Aperfeiçoamento de Pessoal de Nível Superior (CAPES 1387/89-12) and
Conselho Nacionalde DesenvolvimentoCientífico e Tecnoldgico(CNPq
204081/82-2) for predoctoral fellowships, respectively. G.E. and D.I.
are research engineer and research director of the lnstitut National
de Ia Recherche Agronomique (France), respectively.
Received August 4, 1993; accepted October 5, 1993.
RNA Analyses
Total RNA was isolatedas described by Logemann et al. (1987).RNA
samples were separated on 1.2%agarose-formaldehyde gel and transferred to nylon filters (Hybond-N, Amersham). To confirm that equal
amounts of RNA were loaded in each lane, RNAs were visualized in
the formaldehydegels by adding 1 pL of ethidium bromide(stock concentration of O5 mg mL-I) in each sample. Filters were hybridized with
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