1 Laboratoire Cycle Cellulaire, Institut de Biotechnologie des Plantes, CNRS
UMR8618, Université Paris-Sud, 91405 Orsay
Cedex, France
2 Institute of Biotechnology, University of Cambridge, Tennis Court Road,
Cambridge, CB2 1QT, UK
3 Service de Cytologie, Institut de Biotechnologie des Plantes, CNRS UMR8618,
Université Paris-Sud, 91405 Orsay Cedex,
France
* Present address: Institut des Sciences de la Vie et de la
Santé, EA 3176,
Université de Limoges, 87060 Limoges Cedex,
France
Author for correspondence (e-mail:
jasinski{at}ibp.u-psud.fr
)
Accepted 3 December 2001
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Summary |
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Key words: Arabidopsis thaliana, CDK inhibitor, Cyclin D, Endoreduplication, Plant development
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Introduction |
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In vertebrates, the existence of pathways linking development to cell cycle
control was revealed by the occurrence of developmental defects that result
from targeted mutation or overexpression of genes involved in cell cycle
function, such as cyclin D or the retinoblastoma family
(Cobrinik et al., 1996;
Sicinski et al., 1995
).
Because some of the inhibitory signals are believed to be mediated by CDK
inhibitors, it is possible that these molecules contribute to cell cycle exit
during differentiation. Consistent with this hypothesis, in double mutant mice
lacking both functional p27Kip1 and
p57Kip2, or p21Cip1 and
p57Kip2 CDK inhibitors, numerous cell types fail to
differentiate during embryonic development
(Zhang et al., 1998;
Zhang et al., 1999
). CDK
inhibitors may also play a direct role in stimulating differentiation
(Ohnuma et al., 1999
).
Although the signals that activate CKIs during differentiation remain unknown,
these CKIs clearly provide a crucial link between cell-cycle arrest and
differentiation (Myster and Duronio,
2000
). In plants, activation of division by overexpressing
AtCycD3;1 or inhibition of division by overexpressing ICK1
or KRP2 induced profound effects on plant growth and development,
providing a link between cell cycle regulation and development
(De Veylder et al., 2001
;
Riou-Khamlichi et al., 1999
;
Wang et al., 2000
).
Using a two-hybrid approach, we recently characterised the first tobacco
CKI named NtKIS1a. Its deduced polypeptide sequence displayed strong
similarity with plant CKIs and with the mammalian CDK inhibitor CIP/KIP family
(S.J., C.B. and N.G., unpublished). The similarity between these proteins
consists of a highly conserved domain similar to the CDK
interaction/inhibition domain identified in the animal CIP/KIP inhibitors
(Chen et al., 1996;
Russo et al., 1996
).
Consistent with this, we showed that NtKIS1a inhibited the kinase activity of
BY-2 cell CDK/cyclin complexes. In a two-hybrid system, NtKIS1a interacted
with both CDK and D-cyclins but not with PCNA, suggesting that it was more
closely related to p27Kip1 than to
p21Cip1 (S.J., C.B. and N.G., unpublished).
In mammals, recent work indicates that the induction of cyclin D-CDK
complexes results in a redistribution of CDK inhibitors
p27Kip1 and p21Cip1 from cyclin E-CDK2
complexes to cyclin D-CDK4/6 complexes, thereby triggering the kinase activity
of cyclin E-CDK2 (Sherr and Roberts,
1999). Thus, mammalian D-cyclins also control cell cycle
progression in a kinase independent manner, via interaction with CIP/KIP.
In this study, we examined the effects of NtKIS1a overexpression
on plant development. Gain of NtKIS1a function decreased organ size and
increased cell size. Furthermore, it blocked the endoreduplication phenomenon.
The significance of cyclin D-CKI interaction within the context of a living
Arabidopsis was studied. With this aim, we took advantage of
AtCycD3;1-overexpressing plants
(Riou-Khamlichi et al., 1999),
which had leaves curled along their proximal-distal axis and contained
numerous small and incompletely differentiated cells
(Meijer and Murray, 2001
). We
generated plants that overexpressed both AtCycD3;1 and
NtKIS1a. Analysis of these plants provided evidence for AtCycD3;1 and
NtKIS1a interaction in planta.
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Materials and Methods |
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Transformation of A. thaliana AtCycD3;1-overexpressing plants was similarly performed, except that the NtKIS1a cDNA was cloned into Bin-Hyg-TX vector. Seeds from the Agrobacterium-treated plants were selected on MS-Km containing 25 mg/l hygromycin.
Genomic DNA extraction for PCR
Leaf fragments were ground in an extraction buffer (200 mM Tris-HCl, pH
7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS). PCR, using the Promega TflI DNA
polymerase, was performed in the presence of 2% DMSO on DNA from the
isopropanol-precipitated supernatant.
RNA isolation and RT-PCR analysis
A. thaliana cauline leaves were frozen and ground in a mortar to
perform total RNA extraction using TRIzol reagent (Life Technologies). For
RT-PCR analysis, first-strand cDNA was synthesised from 5 µg of total RNA
using SuperscriptII RNase H-Reverse Transcriptase (Life Technologies)
following the manufacturer's instructions. 2.5 µl were used for PCR in a
final volume of 25 µl. PCR products were then analysed on a 1% agarose gel
and transferred onto a Hybond N+ membrane (Amersham). Hybridisations were
performed at 62°C according to Church and Gilbert's protocol
(Church and Gilbert, 1984).
NtKIS1a, AtCycD3;1 and Actin2 probes correspond to the
coding sequences.
Analyses by light and SEM microscopy
WT and 35S::NtKIS1a flowers were fixed with FAA (50% EtOH, 5% acetic acid,
10% formaldehyde), dehydrated in increasing ethanol concentrations and in
propylene oxide and embedded in araldite resin for 60 hours at 48°C. 1.5
µm sections were cut with an LKB ultrotome III ultramicrotome, coloured
with 0.5% toluidine blue and observed under a light microscope.
WT and 35S::NtKIS1a leaves of 1 cm were analysed with a scanning electron microscope (Hitachi S-3000N) under the ESED mode. Samples were slowly frozen at -12°C under partial vacuum on the Peltier stage before observation. Cell area was measured using the Optimas 6.0 software.
Flow cytometric analyses
Leaf fragments from WT, 35S::NtKIS1a and 35S::NtKIS1b plants were chopped
in Galbraith's buffer (Galbraith et al.,
1983). Mixtures were filtered and released nuclei were stained
with Dapi and analysed using a flow cytometer (Vantage, Coulter).
Histone H1 kinase assay
p9CKSHs1 beads were prepared as described
(Azzi et al., 1992). A.
thaliana plantlets (approximately 500 mg of fresh material) were ground
in a mortar in liquid nitrogen with 1 ml of extraction buffer (25 mM MOPS pH
7.2, 60 mM ß-glycerophosphate, 15 mM p-nitrophenylphosphate, 15 mM EGTA,
15 mM MgCl2, 1 mM DTT, 1 mM NaF, 1 mM NH4VO3,
1 mM phenylphosphate, 0.2 µg/ml leupeptin, 0.3 µg/ml pepstatin). The
resulting powder was slowly thawed on ice and centrifuged for 30 minutes at
18,000 g at 4°C to eliminate cell debris. Protein extract
(570 µg) was added to 10 µl of packed p9CKSHs1 beads
previously washed three times with bead buffer
(Azzi et al., 1992
) and kept
under rotation at 4°C for 1 hour 30 minutes. After a centrifugation pulse
and removal of the supernatant, beads were washed three times with bead buffer
and used for H1K assays. Samples containing the initial 10 µl packed beads
were incubated for 30 minutes at 30°C with 1
µCi[
-32P]ATP, 25 µg histone H1 (Sigma) in a final
volume of 30 µl H1K assay buffer (Azzi
et al., 1992
). The reaction was stopped by placing samples on ice.
After a centrifugation pulse, Laemmli buffer was added to 15 µl of
supernatant. Samples were analysed by 12% SDS-PAGE followed by Coomassie blue
staining to visualise histone H1 and autoradiography to detect histone H1
phosphorylation, which was further quantified with the NIH image 1.62
software.
Western blotting
After H1K assays, proteins bound to p9CKSHs1 beads were
recovered by boiling in the presence of Laemmli buffer, run on 12% SDS-PAGE
gels and transferred to a 0.1 µm nitrocellulose membrane for 1 hour in a
semi-dry system (Millipore) at 2.5 V/cm2. The membrane was further
treated as described in the ECL+Plus System protocol (Amersham). The primary
antibody was a monoclonal anti-PSTAIR antibody (Sigma), and the secondary one
was a goat anti-mouse peroxidase conjugated antibody (BioRad). Detection of
the target proteins was performed by chemoluminescence using the ECL+Plus
System (Amersham).
Yeast two-hybrid assays
Yeast two-hybrid assays were performed according to the protocol described
in the Matchmaker Two-Hybrid system (Clontech).
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Results |
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The T2 progeny of four T1 lines (one displaying a strong, one displaying a medium, and two displaying no significant or a weak phenotype) was analysed. According to the number of T2 plants displaying a serrated leaf phenotype, three cases were observed. In the first case, which described the progeny of a T1 strong line, 12 plants out of 12 grown, presented the serrated phenotype with a gradient in the depth of the teeth. Among these 12 plants, 5 displayed a more severe phenotype than the T1 parents, were affected in flower morphology and were sterile. In the second case, which described the progeny of a T1 medium line, 8 plants out of 12 were affected in leaf morphology but remained fertile. Finally, in the third case, which described the progeny of a weak T1 line, only 1 plant out of 12 T2 presented serrated leaves and was fertile (Fig. 1C). The number of T2 plants that displayed a serrated phenotype was assumed to reflect the disjunction of the transgene insertions present in the T1 parents. Interestingly, we observed stronger phenotypes when there were more T2 plants affected (Fig. 1C). Consequently, it was suggested that the strength of the phenotype was to some extent related to the number of transgene insertions.
Closer inspection of the extreme flower phenotype showed a conservation of all the whorls with, however, a reduced growth of petals and stamens (Fig. 1A,f,g). To understand the reasons for the sterility, flower transversal sections of WT and 35S::NtKIS1a were compared (Fig. 2). In 35S::NtKIS1a anthers, pollen grains were formed. Nevertheless, they showed a strong heterogeneity in size. Furthermore, the anthers had a disorganised structure and failed to dehisce (Fig. 2b,d). The gynoecium structure was also affected (Fig. 2f). Its organisation in three layers, exo, meso and endocarp, was not clearly observed compared with the WT (Fig. 2e). Moreover, most of the cells were enlarged and their shape seemed to be modified. Additionally, the transmitting tract, a tissue specialised in directing pollen tube growth, was nearly absent in 35S::NtKIS1a. The ovules displayed a disorganised structure but were apparently not affected in number.
|
35S::NtKIS1a plants show reduced CDK kinase activity
The CDK kinase activity was analysed in two 35S::NtKIS1a lines, one strong
and one medium. Results shown in Fig.
3 demonstrated that the CDK kinase activity measured in these
lines was significantly decreased compared with WT. Moreover, since 60%
inhibition was observed in strong line and 37% in the medium one, it suggested
that the CDK kinase activity inhibition was correlated to the strength of the
phenotype.
|
Gain of NtKIS1a function in Arabidopsis plants decreases
organ size and increases cell size
The decrease in plant and organ size observed in the 35S::NtKIS1a lines
could reflect an alteration in cell size and/or cell number. To investigate
this last point at the cellular level, we examined the size of cells in
35S::NtKIS1a petals and leaves in comparison with the wildtype
(Fig. 4). In WT petals, the
shape and size of the cells were different along the organ and defined three
regions: basal (III), median (II) and distal (I)
(Fig. 4A,B). In the basal and
median regions, the cells had a lengthened shape, basal cells being larger
than median cells, whereas in the distal region, cells had a round shape and a
small size. Interestingly, in the 35S::NtKIS1a, the cells had a constant
lengthened shape all along the petal. The distal cells were still smaller than
the basal cells but had lost their round shape. Moreover, the petal margin was
serrated (Fig. 4B). Comparison
of cells from WT and 35S::NtKIS1a demonstrated that the cell size at all three
regions was significantly increased in the outer epidermis of 35S::NtKIS1a
petals (Fig. 4C). Since
35S::NtKIS1a petals were smaller, it suggested that petals had fewer cells per
organ than the WT.
|
The size of leaf lower epidermis cells from two 35S::NtKIS1a lines was analysed using scanning electron microscopy and the cell area was further quantified using the optimas 6.0 software. The results show that the cells were significantly enlarged in NtKIS1a-overexpressing plants compared with a WT plant (Fig. 4D). In WT, cell area varied from 180 to 3516 µm2 and three categories of cell area can be described: area less than 300 µm2; area between 300 and 3000 µm2; and area greater than 3000 µm2 (Fig. 4E). In the 35S::NtKIS1a line displaying a medium phenotype, cells with an area less than 300 µm2 were never observed. Additionally, in the two remaining groups, the average cell area was increased. In the 35S::NtKIS1a line displaying a strong phenotype, only cells with an area greater than 3000 µm2 were observed, on average 6.3-fold higher than the corresponding cell area in the WT (Fig. 4E).
Rosette leaves of 35S::NtKIS1a plants display a decreased DNA
content
Most Arabidopsis organs consist of cells with polyploid nuclei
attributable to endoreduplication, which increases cell size
(Bergounioux et al., 1992;
Koornneef, 1994
). Since an
increase in size was revealed in 35S::NtKIS1a plant cells, the ploidy level in
different organs of 35S::NtKIS1a, 35S::NtKIS1b and WT plants was compared by
flow cytometric analysis. In rosette leaf cells of WT plants, the maximal
endoreduplication level reached 64C, the proportion of cells with a high DNA
content increased with leaf aging, as already observed (S.C. Brown, personal
communication). Conversely, the distribution pattern of DNA content in cauline
leaves was modified and reached a 2C and 4C distribution in flower buds.
Surprisingly, in 35S::NtKIS1a plants displaying a strong phenotype, cells of
all the tested organs, even the rosette leaves, displayed a 2C and 4C DNA
content distribution (Fig. 5).
This result demonstrated that the endoreduplication phenomenon was absent in
these 35S::NtKIS1a plants. Moreover, in these plants, the proportion of 4C DNA
content cells was very low resulting in an overwhelming 2C DNA content cell
population.
|
Overexpression of NtKIS1a rescues normal development in
AtCycD3;1-overexpressing plants
The flow cytometric analysis performed on NtKIS1a-overexpressing
Arabidopsis plants suggested that NtKIS1a prevents endoreduplication
and may influence S phase progression. Therefore, to gain more insight into
the various pathways in which NtKIS1a could be involved we first hypothesised
that it might interact with D-cyclin/CDK complexes. Indeed, we previously
showed that NtKIS1a interacts with tobacco D-cyclins in a yeast two-hybrid
system (S.J., C.B. and N.G., unpublished). Additionally, the
Arabidopsis D-cyclin AtCycD3;1 represented one of the preys obtained
in a two-hybrid screen using NtKIS1a as a bait (S.J., C.B. and N.G.,
unpublished). This interaction was further confirmed
(Fig. 6A), demonstrating that
these two proteins were interacting partners in a two-hybrid system. In
Arabidopsis, AtCycD3;1 overexpression resulted in the formation of
abnormal plants that contained numerous small, incompletely differentiated
cells (Meijer and Murray,
2001). Moreover, these plants showed disorganised meristem, late
flowering and delayed senescence
(Riou-Khamlichi et al.,
1999
).
|
To determine whether NtKIS1a could inhibit the CDK/cyclin D3;1 activity and thus rescue the AtCycD3;1-overexpression phenotype, NtKIS1a and AtCycD3;1 were combined in the context of a living Arabidopsis plant by two different methods. First, we performed hand-pollination of AtCycD3;1 pistils with NtKIS1a-overexpressing pollen. The presence of both AtCycD3;1 and NtKIS1a transgenes was controlled in F1 plants by PCR analysis (not shown) and their effective overexpression was checked by RT-PCR (Fig. 6B, left). Interestingly, the phenotype of the AtCycD3;1xNtKIS1a plants was similar to that of the WT, making clear differences to those observed in both AtCycD3;1 or NtKIS1a-overexpressing plants (Fig. 6C, top). Indeed, both serration and/or undulation and curling of leaves, associated with NtKIS1a and AtCycD3;1 overexpression, respectively, disappeared in the crossed plants. Closer inspection of the restored phenotype was performed by scanning electron microscopy of leaf lower epidermis cells. Cell area measurement revealed that the three groups, previously described for WT plants (Fig. 4), were also represented in the AtCycD3;1xNtKIS1a plants (Fig. 6D). Indeed, in the crossed plants, cells with an area greater than 300 µm2 reappeared compared with 35S::AtCycD3;1 plants and, reciprocally, cells with an area less than 3000 µm2 reappeared compared with 35S::NtKIS1a plants (Fig. 4E; Fig. 6D). These results suggested an in vivo functional co-operation between these two cell cycle regulators.
Second, the AtCycD3;1-overexpressing line was transformed with the
NtKIS1a-containing transgene, resulting in double overexpressing
plants. The presence of the NtKIS1a transgene was controlled on
genomic DNA in the Kanamycin-resistant (KmR) T1 plantlets
(AtCycD3;1+NtKIS1a) and its effective expression was monitored through RT-PCR
analyses (Fig. 6B, right). As
previously observed for crossed plants, double transformants displayed a
WT-like phenotype (Fig. 6C,
bottom). At the level of leaf lower epidermis cell area, the AtCycD3;1+NtKIS1a
plants displayed a distribution similar to that found in crossed plants (not
shown). Furthermore, the double transformed plants had an advanced flowering
time compared with the flowering time of the 35S::AtCycD3;1 plants
(Riou-Khamlichi et al., 1999)
(not shown).
Since a wildtype-related phenotype was recovered through the combination of NtKIS1a and AtCycD3;1 by two independent manners, it strongly suggested that these two proteins, in addition to being two-hybrid interacting partners, co-operated in planta to restore a pseudo wildtype balance of their opposite activities towards cell cycle progression.
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Discussion |
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It was previously shown that depth of toothing or lobing could be affected
by alteration of gibberellin levels
(Chandra Sekhar and Sawhney,
1991). Investigation of such a relationship between NtKIS1a and
gibberellins should be helpful to understand how NtKIS1a
overexpression impaired leaf morphogenesis. Interestingly, there was a
gradient in the strength of the serrated phenotype with deeper teeth in the
strongest phenotype. Gradual modifications were observed in the T1 generation
between normal plants, which were fertile and displayed only weakly serrated
leaves, and the tiny extremely serrated plants displaying sterile flowers with
short petals and non dehiscent anthers. It is important to note that abnormal
flowers were never observed without strongly serrated leaves. It suggested
that affecting cell division in petals required higher level of CKIs activity
than needed for modifying leaf shape and size. Indeed, the analysis of the
transgene disjunction demonstrated that the extreme phenotype affecting both
leaves and flowers was only found in the strong T1 lines, where all the T2
progeny presented a serrated phenotype
(Fig. 1C). It suggested that
only the plants containing the highest number of transgene copies were
affected in flowers. Remarkably, in 35S::NtKIS1a plants, all organs were
formed with the right identity. Therefore, despite NtKIS1a being
expressed under the constitutive 35S promoter, it did not interfere with
identity acquisition of meristems nor organs. NtKIS1a would preferentially act
at terminal differentiation of the plant organ cells since only the
morphogenesis of organs was affected.
Cell size and endoreduplication
There is a large body of evidence indicating that the final size of a cell
is linked to its DNA content (Traas et
al., 1998). In 35S::NtKIS1a rosette leaves, cells displayed a 2C
and 4C DNA content distribution (Fig.
5), demonstrating that the endoreduplication phenomenon, known to
occur in the WT rosette, was affected. Interestingly, in the 35S::NtKIS1a
plants, which displayed a strong serrated phenotype, endoreduplication was
completely prevented (Fig. 5),
whereas this block was only partial in less affected plants (medium and weak
lines, not shown). This result demonstrated that the NtKIS1a CDK inhibitor
interfered with the endoreduplication phenomenon in Arabidopsis when
overexpressed. However, these results did not provide information towards the
involvement of NtKIS1a in endoreduplication in tobacco. Surprisingly, the
endoreduplication block observed in NtKIS1a-overexpressing
Arabidopsis was accompanied with a phenomenal cell enlargement:
6.3-fold in the strong lines in which the endoreduplication block was complete
(Figs 4,
5). It demonstrated that, in
35S::NtKIS1a Arabidopsis rosette leaves, endoreduplication and cell
size could be uncoupled.
Endoreduplication and differentiation
Endoreduplication is the major mechanism leading to somatic
polyploidisation in plants and has been described in many specific cell types
that are highly specialised (Joubes and
Chevalier, 2000). This phenomenon represents a growing field of
interest in plant biology as it characterises the switch between cell
proliferation and cell differentiation during developmental steps.
Endoreduplication shares several characteristics with the mitotic cycle. In
particular, the endoreduplicative cycle appears to be under control of the
same CDK/cyclin complexes. However, both cycles are mutually exclusive and
higher eukaryotes have developed strategies that ensure an inhibition of
endoreduplication during mitosis and vice versa
(Larkins et al., 2001
). A
variety of biological processes, especially cell differentiation, have been
proposed to involve endoreduplication
(Joubes and Chevalier, 2000
).
However, the role and the control of the endocycle are poorly characterised in
plants. Interestingly, since two plant CKIs [NtKIS1a (this paper) and
KRP2 (De Veylder et al.,
2001
)] prevented endoreduplication when overexpressed, it
suggested that CKIs could be potent regulators of this phenomenon in
Arabidopsis. Similarly, in Caenorhabditis elegans, it was
suggested that the CDK inhibitor CKI-1 might play a role in endoreduplication,
since it was expressed in differentiated intestinal cells that underwent
endoreduplicative cell cycles (Hong et
al., 1998
). In mammals, p57Kip2 is involved in
the transition to the endocycle in trophoblast giant cells during terminal
differentiation and interestingly, ectopic expression of a stabilised
p57Kip2 mutant protein blocked endoreduplication
(Hattori et al., 2000
).
Furthermore, the mutation of the CDK and cyclin binding domains of
p57Kip2 abrogated its inhibitory activity on
endoreduplication when overexpressed
(Hattori et al., 2000
).
Consistent with this, endoreduplication occurred in plants overexpressing
NtKIS1b (Fig. 5),
which lacked the C-terminal end, necessary for both CDK and cyclin interaction
(S.J., C.B. and N.G., unpublished).
NtKIS1a and cyclin D3;1 interaction
In mammals, D-cyclins are thought to drive cell cycle progression by
associating with their CDK partners (CDK4 and CDK6) and by guiding these
kinases to their cellular substrates
(Sherr, 1995). In addition to
their well-documented CDK-dependent role, D-cyclins play a kinase independent
function by sequestering cell cycle inhibitors p27Kip1 and
p21Cip1, thereby triggering the kinase activity of cyclin
E-CDK2 (Sherr and Roberts,
1999
). Moreover, in the context of a living mouse, the
significance of cyclin D1-p27Kip1 interaction was
demonstrated, since deletion of p27Kip1 rescues
developmental abnormalities of cyclin D1-deficient mice
(Geng et al., 2001
;
Tong and Pollard, 2001
).
NtKIS1a was shown to interact with tobacco and Arabidopsis D-cyclins
(S.J., C.B. and N.G., unpublished) (Fig.
6A), known as positive regulators of the cell cycle machinery
(De Veylder et al., 1999
;
Riou-Khamlichi et al., 1999
;
Riou-Khamlichi et al., 2000
).
In order to address the significance of cyclin D-NtKIS1a interaction in the
context of a living Arabidopsis, AtCycD3;1
(Riou-Khamlichi et al., 1999
)
and NtKIS1a were overexpressed simultaneously in plants in two ways.
Our phenotypic analyses revealed that overexpression of NtKIS1a
together with AtCycD3;1 restored normal plant development. Regarding
the leaf phenotype, both the serration and/or undulation observed in NtKIS1a
lines and the curling of AtCycD3;1 line disappeared in double overexpressing
plants (Fig. 6C). This leaf
phenotype restoration was also confirmed at the cellular level, since in
double overexpressing plants, cell areas could be described by three
categories such as in WT (Fig.
4E; Fig. 6D).
Furthermore, several other traits were addressed in the double overexpressing
plants (cell shape, plant and organ size, flowering time; not shown) and all
converged on the WT phenotype restoration. Consequently, one interpretation
would be that the enhanced inhibitory activity brought by NtKIS1a
overexpression, would allow compensation of the hyperproliferative properties
associated with AtCycD3;1 overexpression. As already mentioned above
for animals, D-cyclins control the activity of CDK/cyclin E, responsible for S
phase entry, via titration of CIP/KIP
(Sherr and Roberts, 1999
).
Although E-cyclins have not yet been found in plants, a member of the D-cyclin
family (cyclin
2) has some characteristics in common with E-cyclins
(Soni et al., 1995
). Thus, a
second interpretation could be that NtKIS1a would be sequestrated by
AtCycD3;1, allowing the activation of putative E-cyclins and then
re-activation of mitotic activity. To summarise, our findings indicate that
NtKIS1a and AtCycD3;1 function to antagonise each other and this represents
the first demonstration in planta of co-operation between a cell cycle
inhibitor, NtKIS1a, and a cell cycle activator, AtCycD3;1.
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Acknowledgments |
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