Download Analysis of cell division parameters and cell cycle gene expression

Journal of Experimental Botany, Vol. 52, No. 361, pp. 1625±1633, August 2001
Analysis of cell division parameters and
cell cycle gene expression during the cultivation
of Arabidopsis thaliana cell suspensions
Caroline Richard1, Christine Granier2, Dirk InzeÂ1,3 and Lieven De Veylder1
1
Vakgroep Moleculaire Genetica, Departement Plantengenetica, Vlaams Interuniversitair Instituut voor
Biotechnologie ( VIB), Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium
2
Institut National de la Recherche Agronomique, Laboratoire d'Ecophysiologie des Plantes sous Stress
Environnementaux, 2 Place Viala, F-34060 Montpellier, France
Received 2 January 2001; Accepted 30 March 2001
Abstract
Arabidopsis thaliana cell suspension cultures were
characterized for the first time in detail in terms
of biomass accumulation, cell division rate and cell
cycle phase durations. Subsequently, this model
system was used to follow the transcription profile
of key cell cycle genes during a complete cultivation
cycle. According to the calculated changes in the
relative division rate over time, the cell cycle genes
could be classified into four groups based on their
transcriptional expression pattern. These differential
patterns of gene expression are discussed with
respect to the putative roles of the different cell cycle
genes in the division cycle. Analysis of protein levels
showed that mRNA levels did not correlate with
protein levels in all cases. Results obtained in other
systems, such as BY-2 cell suspensions or plants,
confirm that cell suspension cultures of A. thaliana
are suitable for the analysis of cell cycle regulation.
Key words: Arabidopsis thaliana, cell cycle, gene expression.
Introduction
The general mechanism of cell cycle control is evolutionarily conserved among eukaryotes. The progression
through the cell cycle phases (G1uSuG2uM) is driven by
heterodimeric SeruThr protein kinases consisting of a
catalytic subunit, the cyclin-dependent kinase (CDK) and
an activating subunit, cyclin. Some of the molecular
3
components of the cell cycle machinery originally discovered in yeast and animals have been found in higher
plants (Mironov et al., 1999). Five types of CDK-like
proteins and numerous cDNAs encoding putative cyclins
have been identi®ed in a diverse range of plant species.
The cyclins have been classi®ed in nine groups: A1,
A2, A3, B1, B2, D1, D2, D3, and D4 re¯ecting their
homologies to the mammalian cyclins A, B, and D
(Renaudin et al., 1996; De Veylder et al., 1999). The
complexes formed by association of CDKs and cyclins
can interact with other cell cycle regulators. CKS1At, a
plant homologue of p13suc1 of ®ssion yeast and the CDK
subunit (CKS) of budding yeast and vertebrates has been
isolated in Arabidopsis thaliana through a two-hybrid
screen using CDKA;1 as bait (De Veylder et al., 1997).
CKS proteins have been proposed to act as a docking
factor for positive and negative regulators of CDK
activity (Bourne et al., 1996). Also CDK inhibitors
(CKIs) have been isolated by the use of the two-hybrid
system (Wang et al., 1997; Lui et al., 2000; De Veylder
et al., 2001). In yeast and animals, CKI proteins inhibit
cell cycle progression through their association with CDK
complexes (Nakayama and Nakayama, 1998). More
recently, plant retinoblastoma protein (Rb) and E2F
homologous cDNAs have been cloned (Gutierrez, 1998).
In general, transcription of all these cell cycle genes is
strongly regulated, allowing the correct progression
through the control points of the cell cycle situated at
the late G1 phase and at the G2uM boundary. Moreover,
the cellular activity of the gene products is controlled
by their subcellular localization, phosphorylation status
and proteolytic degradation (Mironov et al., 1999).
To whom correspondence should be addressed. Fax: q32 9 264 5349. E-mail: [email protected]
ß Society for Experimental Biology 2001
1626
Richard et al.
Arabidopsis cell suspensions were used to study cell
cycle regulation. The combined analyses of cell division
rate and expression of cell cycle genes allowed the classi®cation of genes into four classes depending on their
different patterns of expression in the course of a
cultivation cycle.
Materials and methods
Arabidopsis thaliana cell suspension cultures
Cell suspension cultures of Arabidopsis thaliana (L.) Heynh.
ecotype Col-O were grown as described previously (Glab et al.,
1994) in Gamborg's B5 medium (Sigma, St Louis, MO)
containing 3% sucrose and 1 mM a-naphthaleneacetic acid
(Sigma). Cells were diluted 10-fold on a weekly basis with a
precise quantity of inoculum: 5 ml of liquid culture at day 7, the
equivalent of 1 g of fresh cells, was added to 45 ml fresh
medium, and cultivated in a 250 ml Erlenmeyer ¯ask.
Kinetic analysis of the cell suspension cultures
Changes in biomass over time were followed daily for 15 d after
the dilution of a cell suspension culture. Relative growth
rate (RGR; mg produced mg 1 and d 1) was calculated as the
slope (at time j) of the relationship between the logarithm of
the biomass (B) and time:
RGRj ˆ ‰d(lnB)udtŠj
(1)
Cell numbers were determined daily after dilution of the cell
suspension culture. One millilitre of cell suspension was treated
with 1 ml of enzymatic solution (2% cellulase, 0.1% pectinolyase in 0.66 M sorbitol) for 20 min at 37 8C. Cell density was
then determined with a haemocytometer. Measurements were
performed in triplicate on each of two separate samples from
the same culture. Repetition on ®ve different cultures was
performed.
Dead and living cells were distinguished with ¯uorescein
diacetate staining. Cell suspension cultures (200 ml) were
incubated for 2 min in 0.1% ¯uorescein diacetate. The proportion of dead cells (%dead) in the cell suspension culture was
estimated by counting dead and living cells under a ¯uorescent
microscope. The number of dead cells (Ndead) in the culture
was then calculated as:
Ndead ˆ %dead 3 Ntotal u100
(2)
1
Relative cell division rate (RDRj; cells produced cell and d 1)
at time j was calculated by local linear regression taking into
account the total number of cells (Ntotal) at times j 1, j,
and jq1:
RDRj ˆ ‰d(lnNtotal )udtŠj
(3)
RDR was also calculated by considering the number of living
cells instead of the total number of cells.
Assuming that all cells divide at similar rates, the doubling
time, i.e. the time required for a population of cells at time j to
double in number, can be considered as a correct estimate of the
duration of cell cycle (tcycle, j) (Green and Bauer, 1977; Beemster
et al., 1996). From equation (3), it follows that:
tcycle; j ˆ ln(2)uRDRj
(4)
Nuclei isolation, ¯ow cytometry and duration of
cell cycle phases
Cells were ®rst treated with an enzyme solution (2% cellulase,
0.1% pectinolyase in 0.66 M sorbitol) for 20 min at 37 8C. After
two washes with B5 medium, cells were resuspended in
Galbraith buffer (Galbraith et al., 1983) and ®ltered through a
30 mm nylon mesh. The obtained nuclei were stained with the
¯uorescent dye 49,6-diamidino-2-phenylindole (5 mg ml 1) and
subjected to ¯ow cytometric analysis. Fluorescence intensity of
at least 10 000 nuclei was measured using a BRYTE HS ¯ow
cytometer (Biorad, Hercules, CA, USA).
Duration of cell cycle phases can be determined by combining
calculation of cell doubling time with ¯ow cytometry data
(Webster and MacLeod, 1980; Tardieu and Granier, 2000).
The M phase was not considered in the calculation because
the nuclear envelope is absent during M phase. Because the
duration of the M phase has been shown to be short both in
meristems (NougareÁde and Rembur, 1985) and cell suspensions (Nagata et al., 1992; Francis et al., 1995), its exclusion
does not modify the conclusions on the duration of the other
phases.
RNA extraction and reverse transcription PCR
Reverse transcription (RT)-PCR was used because levels of
expression of most cell cycle genes are too low to be detected
with classical Northern hybridization. Total RNA was extracted
with Trizol (GibcouBRL, Gaithersburg, MD, USA) according
to the manufacturer's instructions.
Two independent protocols were used for cDNA preparation, the Superscript Preampli®cation System (GibcouBRL) and
the kit universal riboclone cDNA synthesis system (Promega,
Madison, WI, USA). Gene expression obtained with these two
kits was compared as an additional control. The Superscript Preampli®cation System (GibcouBRL) was used for
®rst-strand cDNA synthesis with oligo(dT) primer solution
on a 3 mg RNA template. The universal riboclone cDNA
synthesis system (Promega) used mRNA prepared with
magnetic particules Dynabeads oligo(dT) 25 (Dynal, Oslo,
Norway) from 75 mg of total RNA. First-strand synthesis
of the cDNA was driven on half the volume of the mRNA
obtained (approximately 0.5 mg). The quantity of cDNA
obtained was estimated under UV by comparing the sample
with a standard dilution of plasmid DNA labelled with ethidium
bromide. The primer combinations used for the different
cell cycle genes are presented in Table 1.
Five nanograms of cDNA from the kit universal riboclone
cDNA synthesis system (Promega) or 1 ml of the 20 ml of
cDNA product with the Superscript Preampli®cation System
(GibcouBRL) were submitted to the RT-PCR with 300 ng of
each gene-speci®c primer, 160 mM of dNTP, 10 ml of PCR
buffer, and 0.8 ml of Taq polymerase (Amersham Pharmacia
Biotech, Little Chalfont, UK) in a total volume of 100 ml.
The PCR was performed with one denaturation cycle of 4 min
at 94 8C and 15 or 20 cycles of denaturation for 45 s at 94 8C,
primers annealing for 45 s at 55 8C, and elongation for 45 s at
72 8C. PCRs were done under conditions preserving linearity
between the RNA input and the amount of PCR product.
Southern blot and non-radioactive hybridization
After electrophoresis, PCR products were blotted on HybondNq membranes (Amersham Pharmacia Biotech) as described
(Southern, 1975). Fluorescein-labelled probes speci®c for the
Cell cycle gene expression in Arabidopsis
1627
Table 1. Gene-speci®c primers used for reverse transcription PCR analysis
Gene
Accession
Primers
CYCA2;1
Z31589
CYCB1;1
X62279
CYCB2;1
AJ297936
CYCD1;1
X83369
CYCD2;1
X83370
CYCD3;1
X83371
CYCD4;1
AJ131636
CDKA;1
X57839
CDKB1;1
X57840
ICK1
U94772
ICK2
AJ251851
KRP3
AJ301554
KRP4
AJ301555
CKS1At
AJ000016
E2Fb
AJ294533
59-GCTCCAGCTACTTGGTGTCACTTG-39
59-CCGCTGAAGCGGCAATTAGGGATGG-39
59-CGAGACGCCCCCACTACTTAGACTT-39
59-CGGGTTTAGCTCGAATCGGACATGC-39
59-GCTGAAGCCCTCAGTTCCAAGTGCT-39
59-CCTGTCGCCGCCCTCTGATGCAGAC-39
59-CCGGTGTGTTTTCCGGTGAGTCAAC-39
59-CCTTGGTCCTCTACTTTCACTCCCC-39
59-GGCGGCGGATTTACGAACGAGATTG-39
59-GCCCGCCTTCCAAAGAGCTCTCTCT-39
59-GCCACCGTCTCCTCCTCTCTGTAAT-39
59-GCCCATGGCAGATGCAAAATCGGCT-39
59-GAACACTCGAGTGTAATGGCAGAGG-39
59-CATCATACTAGTTATAATAATGTAAG-39
59-GCCACTCTCATAGGGTTCTCCATCG-39
59-GGCATGCCTCCAAGATCCTTGAAGT-39
59-GGGTCTTGGTCGTTTTACTGTT-39
59-CCAAGACGATGACAACAGATACAGC-39
59-GCAGCTACGGAGCCGGAGAATTGT-39
59-TCTCCTTCTCGAAATCGAAATTGTACT-39
59-CGGCTCGAGGAGAACCACAAACACGC-39
59-CGAAACTAGTTAATTACCTCAAGGAAG-39
59-GATCCCGGGCGATATCAGCGTCATGG-39
59-GATCCCGGGTTAGTCTGTTAACTCC-39
59-GGCGGATCCGTTTCTAATCATCTACCTTCGTCC-39
59-GAATCCATGGGGTACATAAG-39
59-CTCATAGAATTCTTGCATATAAC-39
59-GAGAGACATATGGGTCAGATCC-39
59-GGGAATTCCTGAAGCGTAAGACAGATTTGGTAAACC-39
59-GGGGATCCTCAGCTACCTGTAGGTGATCTCGTAGC-39
different cell cycle genes were prepared with the GeneImages
random prime labelling module (Amersham Pharmacia Biotech)
and the GeneImages CDP-star detection module allowed the
probe detection (Amersham Pharmacia Biotech). The signals
were revealed by autoradiography after they were exposed for
a few minutes to a Kodak ®lm.
Western blot analysis
Protein extracts were prepared from A. thaliana cell suspensions
in homogenization buffer (HB) containing 50 mM TRIS-HCl
(pH 7.2), 60 mM b-glycerophosphate, 15 mM nitrophenyl
phosphate, 15 mM EGTA, 15 mM MgCl2, 2 mM dithiothreitol, 0.1 mM vanadate, 50 mM NaF, 20 mg ml 1 leupeptin,
20 mg ml 1 aprotein, 20 mg ml 1 soybean trypsin inhibitor,
100 mM benzamidine, 1 mM phenylmethylsulphonyl¯uoride,
and 0.1% Triton X-100. Protein extract (30 mg) was denaturated
for 10 min at 95 8C in SDS loading buffer. Proteins were
separated on a 12.5% SDS-PAGE gel and immunoblotted on a
nitrocellulose membrane (Hybond-Cq; Amersham Pharmacia
Biotech). Filters were blocked overnight with 2% milk powder
in phosphate-buffered saline containing 0.1% Tween-20 (PBST),
washed ®ve times with PBST, and probed for 2 h with antiCDKA;1 (1u5000), anti-CDKB1;1 (1u2000), and anti-CKS1At
(1u5000) diluted in blocking solution. The blots were rinsed
®ve times with PBST and incubated for 1 h with anti-rabbit
horseradish peroxidase conjugated antibodies (Amersham
Pharmacia Biotech) diluted in the blocking solution. The
membranes were washed ®ve times with PBST, and signals
were developed using a chemiluminescent detection kit (NEN
Life Science Products Inc., Boston, MA, USA).
Results
Growth characteristics of an Arabidopsis thaliana
cell suspension culture
A freshly diluted Arabidopsis culture was analysed daily
for 15 d. Biomass increased linearly over time for approximately 10 d after cell dilution. Afterwards, it reached a
plateau (Fig. 1a). RGR was high during the ®rst 2 d after
cell dilution, decreased at day 3 to 0.2 mg mg 1 d 1 and
stabilized (0.3 mg mg 1 d 1) between day 4 and
day 6 (Fig. 1b). At day 7, RGR decreased and was
approximately null from day 10 on.
Cell number increased in the suspension from day 1 to
day 7 (Fig. 2a). Cell numbers remained high between days
7 and 11, but afterwards decreased over time. This
decrease in cell numbers coincided with an increase in cell
death (Fig. 2a). The percentage of cell death was low and
relatively constant during the ®rst 7 d after cell dilution
(approximately 3% of total cell number), but increased
linearly afterwards. On day 14, the proportion of dead
cells was 38% of the total cell number. Because cell death
was very low during the cell division period, RDRs
calculated by considering either the total cell number or
living cells only were approximately the same (Fig. 2b).
Only RDRs calculated with the total number of cells
will be considered hereafter. The RDR increased 2-fold
1628
Richard et al.
Fig. 1. Temporal changes of biomass (a) and RGR (b) in Arabidopsis
thaliana cell suspension culture. Vertical bars, interval of con®dence at
the 0.05 probability level (n ˆ 5).
Fig. 3. Temporal change in the cell cycle duration calculated on the
total number of cells in the Arabidopsis thaliana cell suspension culture.
(b) Temporal change in percentage of nuclei in G1 (solid diamond),
G2 (open diamond), and S (solid inverted triangle). (c), Temporal change
in the durations of G1, G2, and S (symbols as in b).
increased over time, whereas that in G2 decreased. On the
®rst day after dilution, durations of the S and G1 phases
were approximately the same (11.4 h and 10.9 h, respectively) and that of G2 was close to 26 h at this time.
Durations of G2 and S decreased between days 1 and 2
and reached their lowest value on day 2 (10 h and 2.5 h,
respectively). Duration of all the phases increased with
time after day 2. The increase in cell cycle duration
observed between days 2 to 5 was essentially due to the
more pronounced increase in duration of the G1 phase.
Transcriptional regulation of cell cycle genes
Fig. 2. (a) Temporal change in total cell number (solid circle) and
number of dead cells (solid triangle) in the Arabidopsis thaliana cell
suspension culture. Vertical bars, interval of con®dence at the 0.05
probability level (n ˆ 5). (b) Temporal change in the RDR calculated
either by considering the total number of cells (solid circle) or the living
cells (open circle).
between day 1 and 2 and, thereafter, decreased over time
to become negligible around day 8. The shortest cell
doubling time (24.5 h) was observed on day 2 (Fig. 3a).
Cell doubling time increased slowly between day 2 and
day 5 and reached 53 h on day 5, after which it increased
dramatically and reached 250 h on day 6.
The percentage of cells in G1 was always higher than
in G2 or S (Fig. 3b). The percentage of cells in G1 and S
The expression of various cell cycle genes was analysed at
four time points after initial dilutions of the culture: day 1
(characterized by a high RDR), day 5 (speci®c for the
exponential accumulation of biomass and decrease in cell
division rate), day 8 (entry into stationary phase, total
cessation of cell division), and day 12 (stationary phase,
linear increase in cell death).
Expression of all studied cyclin genes and CDKB1;1
decreased with time during the culture cultivation
while expression of CDKA;1 remained constant
The cyclin genes CYCA2;1, CYCB1;1, CYCB2;1, and
CDKB1;1 displayed decreasing levels of mRNA during
a culture cycle (Fig. 4). CYCB1;1 and CYCB2;1 show a
very close expression pattern and their corresponding
mRNA levels decreased rapidly compared to CYCA2;1.
Cell cycle gene expression in Arabidopsis
1629
Fig. 4. Transcriptional regulation of cell cycle genes during the cultivation of Arabidopsis thaliana cell suspensions. Four time points corresponding to
contrasted values of RDR have been retained. RNA speci®c of these days was submitted to semi-quantitative RT-PCR. The hybridization bands were
quanti®ed by densitometry. The intensity of spots was compared with the strongest signal (100).
In contrast to A- and B-type cyclins, the steady-state
levels of the D-type cyclins (CYCD2;1, CYCD3;1, and
CYCD4;1) decreased in a shorter time interval from day 1
to day 5. The mRNA levels of the CDKA;1 gene remained
constant during the cultivation period, whereas those of
CDKB1;1 decreased but to a minor extent compared to
cyclins. From day 1 to day 12 it was reduced only by a
factor of 2.
A close pattern of expression for all the studied CKI
and CKS genes
The maximum of mRNA levels for the CKI genes (ICK1,
ICK2, KRP3, and KRP4) was reached during the
exponential accumulation in biomass, while the cells
actively divided (Fig. 4). The mRNA levels of ICK1 and
KRP3 were up to 6-fold higher on day 5 than on day 8,
while cell division rate was close to 0. However, ICK2 and
KRP4 were still expressed in the early stationary phase.
CKS gene expression increased with time to reach a
maximum value around day 5, and decreased afterwards
(Figs 4, 5).
E2Fb was expressed only at the beginning and
end of the culture
The E2Fb gene presented a particular pattern of mRNA
accumulation. Expression was detected at the beginning
of the cultivation at day 1. A 7-fold decrease from day
1 to day 5 was recorded. Remarkably, expression could
1630
Richard et al.
Fig. 5. CDKA;1, CDKB1;1, and CKS1At RNA and protein pro®les
during 10 d of cultivation of Arabidopsis thaliana suspension cells.
RNA samples and protein extracts were prepared on the indicated
days and used for RT-PCR using CDKA;1-, CDKB1;1-, and CKSspeci®c primers and probes, and Western blotting with CDKA;1-,
CDKB1;1-, and CKS-speci®c antibodies, respectively.
again be observed when cells were in late stationary phase
(day 12) (Fig. 4).
Translational regulation of cell cycle gene expression
Currently, only a few speci®c antibodies against plant cell
cycle proteins are available. However, the results obtained
demonstrate that mRNA levels and protein levels did not
correlate in all cases.
The levels of CDKA;1 mRNA and proteins remained
constant during the whole cultivation (Fig. 5). For
CDKB1;1, mRNA and protein levels showed a similar
decreasing accumulation pattern. The above correlations
between amounts of transcripts and proteins were not
observed for CKS1At. The CKS1At mRNA levels
increased until days 5±6 and remained high until late
stationary phase. In contrast, on Western blotting a
constant CKS1At signal was observed from day 1 to
day 4, then the CKS1At protein amounts decreased
to become undetectable after day 7.
Discussion
Growth characteristics of Arabidopsis
cell suspension culture
Three days after dilution, the relative increase in biomass
of the cell suspension culture is stabilized indicating that
with time changes in biomass follow an exponential
progression. After this phase, RGR decreases with time.
As in plant leaves (Granier and Tardieu, 1998), in cell
suspensions, cell division and increase in biomass occur
®rst simultaneously and then cell division stops a few days
before the arrest of biomass increase.
RDR is never constant, indicating that temporal
changes in cell numbers are never exponential. After
day 2, the cell cycle duration increases with time as
documented in BY-2 cell suspension (Francis et al., 1995).
The peak of RDR observed at day 2 correlates perfectly
with the measured temporal changes in CDK activity of
proliferating Arabidopsis cells suspensions grown under
similar conditions (Stals et al., 2000).
As noticed for the tobacco BY-2 cell line (Francis et al.,
1995), the calculations lead to the conclusion that,
with time, the increase in cell cycle duration is due
to a progressive lengthening of the G1 phase without
any signi®cant changes on the duration of S and G2.
Lengthening of the G1 phase has also been reported in
plants under different situations: spatial gradient in RDR
in dicotyledonous leaves (Granier and Tardieu, 1998),
effects of temperature (Wimber, 1966), sucrose starvation
(Van't Hof et al., 1973), toxic metals on roots (Powell
et al., 1986), and water de®cit or reduction in incident
light on leaves (Granier and Tardieu, 1999a, b).
Different behaviours of CDKA;1 and CDKB1;1:
evidence for a post-translational regulation of CDKA;1
Expression of CDKA;1 and CDKB1;1 genes correlates
with their protein level. Whereas the CDKA;1 mRNA and
protein levels remain constant with time, the CDKB1;1
protein and mRNA levels gradually decrease in parallel
with a reduction in cell division rate. Transcript and
protein levels of CDKA;1 do not correlate with cell
division rate or with the CDKA;1 kinase activities as
quanti®ed by the af®nity of this protein to p9CKShs1 beads
and in vitro by phosphorylation of histone H1 (Stals et al.,
2000). The differences in activity and in abundance of
CDKA;1 indicate a post-translational regulation of this
kinase, as shown in wheat (Schuppler et al., 1998) and
maize leaves (Granier et al., 2000).
Levels of cyclin gene expression correlate with
cell division rate
The expression of cyclin genes decreases with time,
following the reduction in cell division rate. However,
two different patterns emerge: A- and B-type cyclins are
weakly produced when RDR is negligible (on day 8).
In contrast, D-type cyclins are not expressed while the
cell division rate is null. In whole plants, CYCA2;1 is
expressed in dividing cells, but also in speci®c nonactively dividing cells, such as parenchyma and root
pericycle (Burssens et al., 2000). CYCB1;1 expression is
linked to active cell division because CYCB1;1 transcripts
are exclusively detected in newly forming organs and
tissues (Ferreira et al., 1994). Such differential behaviour
of A- and B-type cyclins is not visible in this model
system.
In plants, D-type cyclins are involved in a direct
response to nutritional signals and plant hormones
(Soni et al., 1995; Fuerst et al., 1996; De Veylder et al.,
Cell cycle gene expression in Arabidopsis
1999). In Arabidopsis cell suspensions, the expression level
of CYCD2;1 and CYCD4;1 is signi®cantly reduced after
growth in media depleted of hormones and sucrose and,
after re-addition of sucrose, the initial transcript levels
are rapidly reached. CYCD3;1 transcript levels depend
strongly on cytokinin and are induced at the G1-to-S
transition after this phytohormone is added again. These
observations imply a function for the D-type cyclins as
part of the cellular machinery that integrates diverse
signals impinging on the cell cycle (Fuerst et al., 1996).
The absence, or very low abundance, of D-type cyclins
after day 5 of subculturing suggests that, at this stage,
essential mitogenic signals are depleted in the medium.
Unfortunately, no speci®c antibodies against cyclins
of A. thaliana are available to follow the cyclin evolution at the protein level. Nevertheless, the amount of
CYCB2;1 that is co-puri®ed with CDKA;1 on p9CKShs1
beads is low 4 d after subculturing, while the associated
kinase activity decreases by 50% (Stals et al., 2000). The
decreasing amount of CYCB2;1 proteins correlate with
decreasing transcript levels. Although preliminary, this
®nding suggests that in plants, as in yeast, cyclin transcript levels re¯ect the levels of the corresponding proteins
(Nasmyth, 1993).
1631
protein. Alternatively, the translation of the mRNA could
be repressed. Because the CKS proteins are essential for
cell division, down-regulation of the CKS1At protein
level may be one of the mechanisms that arrests the cell
cycle under unfavourable conditions.
E2Fb, a particular pattern of expression
The E2Fb transcript accumulation pattern can be correlated with an expression in non-cycling cells or a
G1 phase-speci®c expression, as shown by ¯ow cytometry.
Indeed after day 6, the population of nuclei in G1
continues to increase to reach 80% at day 12 (data not
shown). These hypotheses are also consistent with a dual
role of the animal E2F in S phase entry and apoptosis
(Lavia and Jansen-DuÈrr, 1999). Further studies need to
be done to analyse in detail these possibilities. However,
the pattern observed for this gene is different from that
of D-type cyclins, also involved in G1-to-S transition.
This difference could be explained by a different sensitivity to mitogens present in the culture medium.
Hormones or sucrose do not clearly affect E2Fb expression, in contrast to D-type cyclins that are sensitive to
mitogens (Soni et al., 1995; Fuerst et al., 1996; De Veylder
et al., 1999; Riou-Khamlichi et al., 1999; C Richard,
unpublished data).
CKIs and CKS1At, a close pattern of regulation
In cell suspension cultures, the CDK inhibitors, ICK1 and
KRP3, are strongly expressed during the exponential
growth phase in which cells divide actively, indicating
a role at the G1 and G2 checkpoints. Transcriptional
activation of CKIs could be necessary to control the
correct progression through the different phases of the
cell cycle. Recently, animal CKIs were shown to play a
role as adaptor proteins that help to assemble CDKucyclin
complexes (LaBaer et al., 1997; Cheng et al., 1999). A
similar function might be true for ICK1 and KRP3.
In contrast, ICK2 and KRP4 are expressed when cell
division rate is high, but also when division stops
and when cell weight increases due to cell expansion
only. In animals, CKIs have been postulated to play
a distinct role in differentiation. Cell suspensions are
not really appropriate to investigate the role of CKIs
in such a mechanism. In whole plants, CKI genes are
involved in the exit of mitosis and are strongly expressed
in differentiated tissues that have stopped dividing
(L De Veylder, unpublished data).
Like for ICK2 and KRP4, transcription of CKS1At
is maintained at high levels even after the cessation of
cell division. However, no correlation between protein
and mRNA levels has been recorded. An increase of
the transcript level until days 5±6 is noticed while the
amount of protein decreases over the same period, suggesting that an active degradation mechanism is switched
on that suppresses the accumulation of the CKS1At
Conclusions
An A. thaliana cell suspension culture, of which the main
cell cycle parameters were determined, was used as a
model system for studying the transcriptional regulation
of key cell cycle genes during a cultivation cycle. The
genes examined can be classi®ed into four groups according to their transcript pro®le: (i) a gene with a constant
level of expression independently of variations in cell
division rate (CDKA;1); (ii) genes with a decreasing
expression parallel to decreasing cell division rate during
cultivation (A-, B-, and D-type cyclins; CDKB1;1);
(iii) genes with a peak in transcript level when the relative
division rate is maximum (ICK1, ICK2, KRP3, KRP4,
CKS1At); and (iv) a gene expressed only at the start and
the end of the cultivation cycle, when cell division was
high and arrested, respectively (E2Fb). Although protein
levels do not always correlate with the transcript pro®le,
as demonstrated here for the CKS1At gene, the observed
differences in temporal expression of the examined cell
cycle genes could correspond with differential functions
of their gene products in the cell cycle. Thus, the
A. thaliana culture system is useful for cell cycle studies.
Acknowledgements
The authors wish to thank Dr Isabelle Landrieu and Dr James
Dat for critical reading of the manuscript, Martine De Cock for
help in preparing it, and Rebecca Verbanck and Stijn Debruyne
1632
Richard et al.
for illustrations. This work was supported by grants from
Interuniversity Poles of Attraction Programme (Belgian State,
Prime Minister's Of®ceÐFederal Of®ce for Scienti®c, Technical
and Cultural Affairs; P4u15). CR and CG are indebted to
the BiopoÃle veÂgeÂtal d'Amiens (France) and the MinisteÁre des
Affaires EtrangeÁres (France) for a PhD fellowship and a
Lavoisier post-doctoral fellowship, respectively.
References
Beemster GTS, Masle J, Williamson RE, Farquhar GD. 1996.
Effects of soil resistance to root penetration on leaf expansion in wheat (Triticum aestivum L.): kinematic analysis
of leaf elongation. Journal of Experimental Botany 47,
1663±1678.
Bourne Y, Watson MH, Hickey MJ, Holmes W, Rocque W,
Reed SI, Tainer JA. 1996. Crystal structure and mutational
analysis of the human CDK2 kinase complex with cell cycleregulatory protein CksHs1. Cell 84, 863±874.
Burssens S, de Almeida Engler J, Beeckman T, Richard C,
Shaul O, Ferreira P, Van Montagu M, Inze D. 2000. The
developmental expression of the Arabidopsis thaliana CycA2;1
gene. Planta 211, 623±631.
Cheng M, Olivier P, Diehl JA, Fero M, Roussel MF,
Roberts JM, Sherr CJ. 1999. The p21Cip1 and p27Kip1 CDK
`inhibitors' are essential activators of cyclin D-dependent
kinases in murine ®broblasts. The EMBO Journal 18,
1571±1583.
De Veylder L, Beeckman T, Beemster GTS, Krols L, Terras F,
Landrieu I, Van Der Schueren E, Maes S, Nandts M, Inze D.
2001. Functional analysis of cyclin-dependent kinase inhibitors of Arabidopsis. The Plant Cell 13, (in press).
De Veylder L, de Almeida Engler J, Burssens S, Manevski A,
Lescure B, Van Montagu M, Engler G, Inze D. 1999. A new
D-type cyclin of Arabidopsis thaliana expressed during lateral
root primordia formation. Planta 208, 453±462.
De Veylder L, Segers G, Glab N, Casteels P, Van Montagu M,
Inze D. 1997. The Arabidopsis Cks1At protein binds to the
cyclin-dependent kinases CDKA;1 and CDKB1;1. FEBS
Letters 412, 446±452.
Ferreira PCG, Hemerly AS, de Almeida Engler J,
Van Montagu M, Engler G, Inze D. 1994. Developmental
expression of the Arabidopsis cyclin gene cyc1At. The
Plant Cell 6, 1763±1774.
Francis D, Davies MS, Braybrook C, James NC, Herbert RJ.
1995. An effect of zinc on M-phase and G1 of the plant cell
cycle in the synchronous TBY-2 tobacco cell suspension.
Journal of Experimental Botany 46, 1887±1894.
Fuerst RAUA, Soni R, Murray JAH, Lindsey K. 1996.
Modulation of cyclin transcript levels in cultured cells of
Arabidopsis thaliana. Plant Physiology 112, 1023±1033.
Galbraith DW, Harkins KR, Maddox JM, Ayres NM,
Sharma DP, Firoozabady E. 1983. Rapid ¯ow cytometric
analysis of the cell cycle in intact plant tissues. Science 220,
1049±1051.
Glab N, Labidi B, Qin L-X, Trehin C, Bergounioux C, Meijer L.
1994. Olomoucine, an inhibitor of the cdc2ucdk2 kinases
activity, blocks plant cells at the G1 to S and G2 to M cell
cycle transitions. FEBS Letters 353, 207±211.
Granier C, Tardieu F. 1998. Spatial and temporal analyses of
expansion and cell cycle in sun¯ower leaves. A common
pattern of development for all zones of a leaf and different
leaves of a plant. Plant Physiology 116, 991±1001.
Granier C, Tardieu F. 1999a. Water de®cit and spatial pattern of
leaf development. Variability in responses can be simulated
using a simple model of leaf development. Plant Physiology
119, 609±619.
Granier C, Tardieu F. 1999b. Leaf expansion and cell division
are affected by reducing absorbed light before but not after
the decline in cell division rate in the sun¯ower leaf. Plant,
Cell and Environment 22, 1365±1376.
Granier C, Inze D, Tardieu F. 2000. Spatial distribution of cell
division rate can be deduced from that of p34cdc2 kinase
activity in maize leaves grown at contrasting temperatures
and soil water conditions. Plant Physiology 124, 1393±1402.
Green PB, Bauer K. 1977. Analysing the changing cell cycle.
Journal of Theoretical Biology 68, 299±315.
Gutierrez C. 1998. The retinoblastoma pathway in plant cell
cycle and development. Current Opinion in Plant Biology
1, 492±497.
LaBaer J, Garrett MD, Stevenson LF, Slingerland JM,
Sandhu C, Chou HS, Fattaey A, Harlow E. 1997. New
functional activities of the p21 family of CDK inhibitors.
Genes and Development 11, 847±862.
Lavia P, Jansen-DuÈrr P. 1999. E2F target genes and cell-cycle
checkpoint control. BioEssays 21, 221±230.
Lui H, Wang H, DeLong C, Fowke LC, Crosby WL, Fobert PR.
2000. The Arabidopsis Cdc2a-interacting protein ICK2 is
structurally related to ICK1 and is a potent inhibitor of
cyclin-dependent kinase activity in vitro. The Plant Journal
21, 379±385.
Mironov V, De Veylder L, Van Montagu M, Inze D. 1999.
Cyclin-dependent kinases and cell division in higher
plantsÐthe nexus. The Plant Cell 11, 509±521.
Nagata T, Nemoto Y, Hasezawa S. 1992. Tobacco BY-2 cell line
as the `HeLa' cell in the cell biology of higher plants.
International Review of Cytology 132, 1±30.
Nakayama K-i, Nakayama K. 1998. CipuKip cyclin-dependent
kinase inhibitors: brakes of the cell cycle engine during
development. BioEssays 20, 1020±1029.
Nasmyth K. 1993. Control of the yeast cell cycle by the Cdc28
protein kinase. Current Opinion in Cell Biology 5, 166±179.
NougareÁde A, Rembur J. 1985. Le point veÂgeÂtatif en tant que
modeÁle pour l'eÂtude du cycle cellulaire et de ses points de
controÃle. Bulletin de la SocieÂte botanique de France, ActualiteÂs
botaniques 132, 9±34.
Powell MJ, Davies MS, Francis D. 1986. The in¯uence of zinc
on the cell-cycle in the root meristem of a zinc-tolerant and
nontolerant cultivar of Festuca rubra L. The New Phytologist
102, 419±428.
Renaudin J-P, Doonan JH, Freeman D, et al. 1996. Plant cyclins:
a uni®ed nomenclature for plant A-, B- and D-type cyclins
based on sequence organization. Plant Molecular Biology 32,
1003±1018.
Riou-Khamlichi C, Huntley R, Jacqmard A, Murray JAH. 1999.
Cytokinin activation of Arabidopsis cell division through a
D-type cyclin. Science 283, 1541±1544.
Schuppler U, He P-H, John PCL, Munns R. 1998. Effect of
water stress on cell division and cell-division-cycle 2-like
cell-cycle kinase activity in wheat leaves. Plant Physiology
117, 667±678.
Soni R, Carmichael JP, Shah ZH, Murray JAH. 1995. A
family of cyclin D homologs from plants differentially
controlled by growth regulators and containing the conserved
retinoblastoma protein interaction motif. The Plant Cell 7,
85±103.
Southern EM. 1975. Detection of speci®c sequences among
DNA fragments separated by gel electrophoresis. Journal of
Molecular Biology 98, 503±517.
Cell cycle gene expression in Arabidopsis
Stals H, Casteels P, Van Montagu M, Inze D. 2000. Regulation
of cyclin-dependent kinases in Arabidopsis thaliana. Plant
Molecular Biology 43, 583±593.
Tardieu F, Granier C. 2000. Quantitative analysis of cell division
in leaves: methods, developmental patterns and effects of
environmental conditions. Plant Molecular Biology 43,
555±567.
Van't Hof J, Hoppin DP, Yagi S. 1973. Cell arrest in G1 and G2
of the mitotic cycle of Vicia faba root meristems. American
Journal of Botany 60, 889±895.
1633
Wang H, Fowke LC, Crosby WL. 1997. A plant cyclindependent kinase inhibitor gene. Nature 386, 451±452.
Webster PL, MacLeod RD. 1980. Characteristics of root
apical meristem cell population kinetics: a review of analyses
and concepts. Environmental and Experimental Botany 20,
335±358.
Wimber DE. 1966. Duration of the nuclear cycle in
Tradescantia root tips at three temperatures as
measured with H3-thymidine. American Journal of Botany
42, 296±301.