Download Multiple Functions of Kip-Related Protein5

Multiple Functions of Kip-Related Protein5 Connect
Endoreduplication and Cell Elongation[C][W]
Teddy Jégu 1, David Latrasse 1, Marianne Delarue, Christelle Mazubert, Mickaël Bourge, Elodie Hudik,
Sophie Blanchet, Marie-Noëlle Soler, Céline Charon, Lieven De Veylder, Cécile Raynaud,
Catherine Bergounioux, and Moussa Benhamed*
Institut de Biologie des Plantes, Unité Mixte de Recherche 8618 Université Paris-Sud XI, 91405 Orsay, France
(T.J., D.L., M.D., C.M., E.H., S.B., C.C., C.R., C.B., M.Be.); Pôle de Biologie Cellulaire, Imagif, Centre de
Recherche de Gif, Centre National de la Recherche Scientifique, Institut Fédératif de Recherche 87, 91198
Gif-sur-Yvette cedex, France (M.Bo., M.-N.S.); Department of Plant Systems Biology, VIB, B–9052 Ghent,
Belgium (L.D.V.); and Department of Plant Biotechnology and Bioinformatics, Ghent University, B–9052
Ghent, Belgium (L.D.V.)
Despite considerable progress in our knowledge regarding the cell cycle inhibitor of the Kip-related protein (KRP) family in
plants, less is known about the coordination of endoreduplication and cell differentiation. In animals, the role of cyclindependent kinase (CDK) inhibitors as multifunctional factors coordinating cell cycle regulation and cell differentiation is well
documented and involves not only the inhibition of CDK/cyclin complexes but also other mechanisms, among them the
regulation of transcription. Interestingly, several plant KRPs have a punctuated distribution in the nucleus, suggesting that
they are associated with heterochromatin. Here, one of these chromatin-bound KRPs, KRP5, has been studied in Arabidopsis
(Arabidopsis thaliana). KRP5 is expressed in endoreduplicating cells, and loss of KRP5 function decreases endoreduplication,
indicating that KRP5 is a positive regulator of endoreduplication. This regulation relies on several mechanisms: in addition to its
role in cyclin/CDK kinase inhibition previously described, chromatin immunoprecipitation sequencing data combined with
transcript quantification provide evidence that KRP5 regulates the transcription of genes involved in cell wall organization.
Furthermore, KRP5 overexpression increases chromocenter decondensation and endoreduplication in the Arabidopsis trithoraxrelated protein5 (atxr5) atxr6 double mutant, which is deficient for the deposition of heterochromatin marks. Hence, KRP5 could
bind chromatin to coordinately control endoreduplication and chromatin structure and allow the expression of genes required
for cell elongation.
Plant development depends on meristem activity. In
meristems, stem cells generate cells that undergo an important switch during their lifetime, changing from unspecialized cells undergoing rapid division into specific
cell types that will be incorporated into primordia.
Nevertheless, differentiated plant cells do not lose their
proliferating potentialities. Dedifferentiation is commonly
associated with reentry into the cell cycle; its distinguishing feature is the withdrawal from a given differentiated state into a “stem cell”-like state that confers
pluripotentiality, a process preceding reentry into the cell
cycle (De Veylder et al., 2007). In such a context, both
stimulation and inhibition of cell proliferation are intrinsic components for plant development. Core regulators of
1
These authors contributed equally to the article.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Moussa Benhamed ([email protected]).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.112.212357
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the eukaryotic cell cycle are heterodimeric complexes
consisting of a catalytic subunit, a cyclin-dependent kinase
(CDK), and a regulatory subunit, a cyclin. The kinase
activity of CDK is regulated in several ways: by cyclin
binding (the cyclin partner being itself highly regulated),
by phosphorylation and dephosphorylation of the CDK
on evolutionarily conserved residues by specific kinases
and phosphatases, and by the binding of CDK inhibitors
(called CKIs in mammals). Plant genomes contain genes
encoding two different classes of CDK inhibitors: Siamese
related and Kip-related protein (KRP; also called Inhibitor
of Cyclin-dependent Kinase). As demonstrated by their
ability to inhibit CDK activity, plant KRP genes encode
functional CKIs. Several reports have established that
KRPs bind CDKs of the CDK A type and B type (Lui
et al., 2000; De Veylder et al., 2001; Jasinski et al., 2002;
Zhou et al., 2002), suggesting that they could all function
at the G1/S transition and at the G2/M transition. Consistently, CDK complexes containing cyclin D or A can be
inhibited by KRPs in vitro (Coelho et al., 2005), and
overexpression of KRPs can restore the growth defects
induced by D-type cyclin overexpression (Jasinski
et al., 2002; Schnittger et al., 2003; Zhou et al., 2003).
Results obtained to date suggest that all KRPs regulate
the cell cycle in a similar way. Indeed overexpression of
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KRP5 Connects Endoreduplication and Cell Elongation
various KRPs induces similar defects in plants, characterized by a stunted growth phenotype and serrated
leaf. These defects are attributable to a decrease in cell
proliferation as evidenced by reduced leaf cell number
(Wang et al., 2000; Jasinski et al., 2002). However,
KRPs may well play additional roles together with
their function in the regulation of CDK activity. This
hypothesis is supported by data obtained on CKI in
mammals; it is now clear that these proteins fulfill many
functions during development and regulate not only cell
proliferation but also cell differentiation, transcription,
and many other processes (Besson et al., 2008). In
plants, KRP overexpression does not affect cell division in the root meristem but prevents differentiated
cells from progressing into the cell cycle either for the
formation of lateral roots or for in vitro callus induction (Le Foll et al., 2008). In addition, although strong
overexpression of KRPs inhibits endoreduplication,
moderate overexpression of KRP2 promotes it (Verkest
et al., 2005). Consistently, the expression of KRPs is high
in endoreduplicating tissues such as maize (Zea mays)
endosperm or tomato (Solanum lycopersicum) fruit
(Coelho et al., 2005; Nafati et al., 2011). Together, these
results suggest that KRPs could act to maintain differentiation in addition to their role in the inhibition of cell
proliferation.
Another unresolved issue is the respective functions
of the seven members of the KRP family. Overexpression
analyses seem to indicate that all KRPs have similar
functions in planta. However, they do not inhibit the
kinase activity of the CDKA complex in vitro with the
same efficiency (Wang et al., 1997), suggesting that they
could have different behaviors. Indeed, systematic analysis of the core cell cycle interactome revealed that all
KRP proteins interact with CDKA;1 but appear to bind
preferentially to distinct cyclins (Van Leene et al., 2010);
for example, KRP5 was found to interact with CYCD2;1,
CYCD4;1, and CYCD4;2. Likewise, although all KRPs
have a nuclear localization, they can be grouped into
two categories based on their subnuclear distribution:
KRP2, KRP6, and KRP7 show a homogeneous pattern
throughout the nucleoplasm, while KRP1, KRP3, KRP4,
and KRP5 display a punctate pattern of distribution
(Bird et al., 2007). This spotted pattern has been associated with chromocenters (Jakoby et al., 2006), suggesting
a role of some KRPs in heterochromatin function.
In this work, we studied the function of one
heterochromatin-associated KRP, KRP5, in Arabidopsis
(Arabidopsis thaliana). Here, we show that KRP5 is expressed in endoreduplicating cells and that loss of
KRP5 function affects endoreduplication in dark-grown
seedlings. Although KRP5 overexpression induces the
formation of serrated leaves, as observed for other
KRPs, it does not inhibit plant growth and promotes
endoreduplication, indicating that KRP5 does not play
the same role as other KRPs in planta. The spotted nuclear
localization reported previously is due to heterochromatin
localization, and chromatin immunoprecipitation
sequencing (ChIP-seq) data combined with analyses of
mRNA level provide evidence for a role of KRP5 not only
in the inhibition of CYCD/CDK complexes but also in the
regulation of transcription. Altogether, our results suggest
that KRP5 could link cell elongation, endoreduplication,
and chromatin structure.
RESULTS
KRP5 Is Preferentially Expressed in
Endoreduplicating Cells
Plasmids developed by Deal and Henikoff (2011)
allowing the expression of a nucleus-targeted GFP under
the control of a promoter of interest were used to study
KRP5 expression pattern. The KRP5 promoter was
cloned upstream of a Nuclear Targeting Fusion protein
(NTF) tagged with GFP and used for Arabidopsis
transformation. We next used these transgenic lines
to correlate the expression of KRP5 with the distribution
of DNA content in rosette leaves, which show the
highest level of endoreduplication in Arabidopsis. DNA
content per nucleus from whole rosettes of T2 lines was
measured and analyzed by flow cytometry simultaneously with GFP fluorescence at two different stages of
development. In rosettes of 14-d-old plants, expression
of KRP5 was observed in about 20% of the cells and was
specifically enriched in endoreduplicating nuclei: 33%
of GFP-positive nuclei had a DNA content of 8C or
higher (compared with 15% in the total population; Fig.
1A). At later stages, when most leaves had reached
maturity, 84% of nuclei were labeled, suggesting that
KRP5 expression is associated with differentiation.
Furthermore, the distribution of DNA content in GFPpositive nuclei was similar to the distribution observed
in the whole population, which contains a high proportion of 4C and higher DNA contents (more than 65%),
whereas the majority of GFP-negative nuclei have a 2C
DNA content (about 65%; Supplemental Fig. S1). A
profiling of plants expressing a 35S::GFP construct was
done as a control to make sure that GFP expression did
not alter DNA content distribution and that GFP did not
accumulate preferentially in some nuclei (Supplemental
Fig. S2).
Overexpression of KRP5 Induces Endoreduplication
To examine the roles of KRP5 on plant growth and
development, Arabidopsis transgenic lines were generated in which the KRP5 complementary DNA (cDNA)
was expressed under the control of the constitutive
cauliflower mosaic virus 35S promoter (KRP5OE plants).
Their effective overexpression was monitored using
reverse transcription and quantitative PCR (qRT-PCR)
analyses (Fig. 1B). Surprisingly, KRP5OE plants did not
display reduced growth, as observed for the overexpression of other KRPs (Supplemental Fig. S3).
To analyze the effects of KRP5 overexpression on
DNA ploidy distribution, the DNA content of cauline
leaf nuclei in the wild type and 35 independent transgenic KRP5OE lines was measured by flow cytometry. In
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Jégu et al.
Figure 1. Expression of KRP5 correlates with endoreduplication. A, DNA
content distribution of GFP-positive
(GFP+) and GFP-negative (GFP2) nuclei isolated from 14-d-old rosette
leaves of Arabidopsis KRP5::NTF-GFP
plants. For each sample, more than
5,000 DAPI-stained nuclei were analyzed. The left panel shows how GFP+
nuclei were selected based on the intensity of GFP fluorescence (each nucleus is represented by a red point, and
the green box indicates a region of
high nuclei density); the right panel
shows nuclear DNA content distributions of GFP+, GFP2, and total nuclei.
The same results were obtained in two
independent experiments. B, Real-time
RT-PCR data showing the relative expression of KRP5 in wild-type Columbia (Col) and in three independent
KRP5OE lines. Average relative quantities 6 SD (n = 3) are shown for each
sample. C, DNA content distribution of
nuclei isolated from the first cauline
leaf of wild-type Columbia (Col 0) and
KRP5OE line 3. The data presented here
are representative of 35 independent
lines. D, Correlation analysis of the
DNA content with GFP fluorescence in
KRP5OE lines. The distribution shown
in gray corresponds to the DNA content of GFP2 nuclei, whereas the
green distribution corresponds to GFP+
nuclei. The dashed line represents the
distribution of the whole population.
The data presented here are representative of two independent experiments.
the wild type, endoreduplication occurs in cauline
leaves, but it is not as high as in rosette leaves. In all
KRP5OE lines, increases in the 8C and 16C populations
was observed, correlated with a decrease in the proportion of nuclei with 2C DNA content, illustrating
an activation of the endoreduplication cycle (Fig. 1C;
Supplemental Table S1). These results suggest that
KRP5 is involved in the activation of endoreduplication. To confirm this result, we asked whether the
accumulation of KRP5 in the nucleus was associated
with higher DNA content. Flow cytometry allowed
us to monitor DNA content and GFP fluorescence
simultaneously. KRP5-GFP was detected almost exclusively in endoreduplicated nuclei with an 8C and
higher DNA content and in some nuclei with a 4C
DNA content, which could correspond to a first
round of endoreduplication (Fig. 1D). This observation supports the hypothesis that KRP5 stimulates
endoreduplication.
KRP5 Promotes Endoreduplication in Etiolated Seedlings
To provide further evidence for a role of KRP5 as a
positive regulator of endoreduplication, we asked
whether the loss of KRP5 may result in a reduction of
endoreduplication. A search in Arabidopsis transfer
DNA (T-DNA) insertion mutant collections was performed. An Arabidopsis insertion line (SK_27217) was
obtained from the SALK collection of T-DNA insertion
lines (Robinson et al., 2009; Fig. 2A). The genotype of
this krp5 mutant, which contains an insertion in the
first exon, was confirmed by PCR. qRT-PCR analysis
was used to assess the effect of the T-DNA mutation
on the KRP5 transcript in homozygous mutant plants.
This analysis showed that the expression of the KRP5
gene in the krp5 mutant corresponds to 10% of that in
the wild-type Columbia plants (Fig. 2B), indicating
that the mutation is not a full knockout but severely
affects the expression of the KRP5 gene.
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KRP5 Connects Endoreduplication and Cell Elongation
Figure 2. KRP5 mutation induces a decrease of endoreduplication and
cell elongation in etiolated seedlings. A, Schematic diagram of the
Arabidopsis KRP5 gene. Introns are indicated with a single line, exons
with dark gray boxes, and untranslated regions with light gray boxes.
The position of the T-DNA is indicated by the dashed arrow, and the
positions of primers used for genotyping are indicated by arrows with
numbers. The results of a PCR experiment performed for genotyping
with the indicated primer combination are shown on the right. B, qRTPCR quantification of KRP5 expression in the wild type (Col 0) and the
krp5 mutant. C, qRT-PCR quantification of KRP5 expression in the wild
type (Col 0) grown in the light (16 h of light) or in darkness. D, DNA
content distribution of nuclei isolated from 14-d-old seedlings of the
wild type (WT; blue), KRP5OE (black), krp5 mutants (green), and krp5
mutants retransformed with the 35S:KRP5-GFP construct (krp5
KRP5OE; red) germinated in the dark condition. Data are averages 6 SD
from three independent experiments. E, Cortical cell size distribution
of etiolated hypocotyls from wild-type (Col; white), krp5 (gray), and
KRP5OE (OE; black) plants. For each sample, a total of 55 cells were
measured.
We did not observe any difference in the DNA
content of nuclei isolated from mature leaves (data not
shown). We reasoned that KRPs may have partially
redundant functions and that expression analysis may
help us to identify in which particular cellular context
KRP5 could have a prominent role in the regulation of
endoreduplication. A survey of publicly available gene
expression data (https://www.genevestigator.com/gu/)
revealed that KRP5 is highly expressed in dark-grown
seedlings. To confirm this, we compared KRP5 expression
in dark- and light-grown seedlings. We observed an
8-fold induction of KRP5 expression in dark versus
light conditions (Fig. 2C). Accordingly, in etiolated
seedlings, we observed that the level of endoreduplication correlated with GFP labeling in the KRP5::
NTF-GFP lines (Supplemental Fig. S4). Hypocotyl
growth in etiolated seedlings is mediated only by cell
elongation and is associated with increased endoreduplication (Traas et al., 1998; Sugimoto-Shirasu and
Roberts, 2003). Together with the increased expression
of KRP5 in the dark, this suggests that KRP5 could
regulate endoreduplication during etiolation. To test
this, the DNA content of nuclei isolated from etiolated
7-d-old plantlets of the wild type and the krp5 mutant
was measured by flow cytometry. Etiolated krp5 mutants displayed a moderate but reproducible decrease
in the 16C population (Student’s t test, P = 0.0049),
providing evidence for an inhibition of the endoreduplication cycle in the mutant context (Fig. 2D;
Supplemental Table S2). This result is consistent with
the opposite effect of KRP5 overexpression observed in
the same condition (Fig. 2D; Supplemental Table S2).
This reduction of the proportion of 16C nuclei was
reversed by the introduction of the 35S::KRP5-GFP
construct in the mutant background, demonstrating
that reintroduction of the KRP5 cDNA complements
the mutant phenotype (Fig. 2D; Supplemental Table
S2). We also checked the level of endoreduplication in
krp5 mutants grown under the light condition, and no
difference was observed compared with the wild type
(Supplemental Fig. S5).
Because there is strong evidence that the level of
ploidy has some impact on cell size determination
(Sugimoto-Shirasu and Roberts, 2003), cortical cell
sizes of etiolated hypocotyls were compared between
the wild type, KRP5 OE , and the krp5 mutant. As
shown in Figure 2E, cell size distribution indicated
that, globally, cortical cells are longer in the hypocotyls
of KRP5 OE compared with the wild type, whereas
these cells are shorter in krp5 mutants. This observation is consistent with differences in the ploidy levels
obtained for each genotype. We also measured the
total hypocotyl length of the wild type, KRP5OE, and
the krp5 mutant, and no significant differences were
observed (Supplemental Fig. S6), indicating either that
the differences in cell size are not important enough to
affect the global organ size or that cell number is altered
in these plants. Taken together, our results point to a
role of KRP5 as a positive regulator of endoreduplication, raising the question of the molecular mechanisms
underlying this function.
KRP5 Can Antagonize CYCD3;1 Function
The primary role of KRPs is to inhibit CDK/cyclin
complexes. Therefore, we asked whether KRP5 could
promote endoreduplication via CDK/cyclin inactivation. As stated above, KRPs preferentially bind D-type
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Figure 3. KRP5 can antagonize CYCD3;1 function. Phenotype (A) and DNA content distribution
(B) in nuclei of rosette leaves isolated from the
wild type (WT; blue), AtCYCD3;1 overexpressors
(CycD3;1OE; red), and double transformants
CycD3;1OE/KRP5OE (green). The data shown in B
are representative of three independent experiments. For each sample, a minimum of 5,000
DAPI-stained nuclei were analyzed.
cyclins. The synthesis of D-type cyclins is initiated
during G1 and drives the G1/S-phase transition. Constitutive overexpression of CYCD3;1 in Arabidopsis
plants leads to dramatic changes in plant morphology.
CYCD3;1-overexpressing plants also show an increase
in the proportion of cells with a 4C DNA content, and
CYCD3s have been identified as negative regulators of
endoreduplication (Dewitte et al., 2007). To determine
whether KRP5 could inhibit CDK/CYCD3;1 activity
and rescue the CYCD3;1 overexpression phenotype, we
transformed CYCD3;1-overexpressing lines with the
35S:KRP5-GFP construct. The presence of the KRP5
transgene was monitored via qRT-PCR analyses and
GFP visualization. Interestingly, the phenotype of the
CYCD3OE/KRP5OE cooverexpression plants was similar
to that of the wild type (Fig. 3A), suggesting that these
two proteins have antagonistic activities in planta.
Furthermore, flow cytometry analysis revealed that
CYCD3OE/KRP5OE plants displayed wild-type levels
of endoreduplication in cauline leaves, demonstrating
that KRP5 overexpression overcomes the effect of
CYCD3;1 overexpression (Fig. 3B; Supplemental Fig. S7;
Supplemental Table S3). Hence, KRP5 could promote
endoreduplication by inhibiting CYCD3. However, other
mechanisms may also contribute to the KRP5-dependent
activation of endoreduplication. For example, in mammals, p21, a KIP protein, regulates differentiation by directly modulating gene expression (Devgan et al., 2005).
Because KRP5 has a punctate distribution in nuclei, we
investigated its ability to bind chromatin.
KRP5 Colocalizes with Heterochromatin Marks and Binds
Different Types of Genes
To examine the subnuclear distribution of KRP5,
we performed immunodetection experiments using
an antibody raised against GFP and with an H3K9me2
antibody, which is a cytological mark for visible
heterochromatin. Immunostaining showed that the
KRP5-GFP signal was located inside and outside
chromocenters (Fig. 4A), as reported previously for
other KRPs (Jakoby et al., 2006; Bird et al., 2007). These
observations prove that KRP5 localizes into euchromatic
and heterochromatic regions, suggesting that it could
play a role in chromatin structure.
To identify KRP5-binding sites, we performed ChIPseq on chromatin isolated from Arabidopsis plantlets.
The MACS (for model-based analysis of ChIP-seq) algorithm was used to determine which loci are significantly
enriched for KRP5-GFP (Fig. 4B). The MACS algorithm is
designed to detect transcription factor-binding events,
which typically produce short peaks with strong signal
intensity (Zhang et al., 2008). We identified around 1,090
KRP5-bound genomic loci (Supplemental Table S4).
First, we analyzed the KRP5 peak distance to the transcriptional start site (TSS); we showed that KRP5 binding
was not centered on the TSS and that the Gaussian curve
displays its maximum at 1,000 bp downstream of the
TSS (Fig. 4C).
To annotate the KRP5-binding sites, we used the
Genomic Position Annotation Tool (Krebs et al., 2008).
Of these loci, 5% were identified as pseudogenes, 1%
as tRNA, 69% as transposable elements, and 25% as
protein-coding genes (Fig. 4D). These data were consistent with the dual subnuclear localization of KRP5
in chromocenters, which are known to be full of
transposable elements, and in euchromatin. To analyze
the Gene Ontology (GO) of the 25% protein-coding
KRP5 target genes, we used an integrated Web-based
GO analysis toolkit, AgriGO, and generated a hierarchical
tree graph of overrepresented GO terms in the biological
process category, showing a significant enrichment in
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KRP5 Connects Endoreduplication and Cell Elongation
Figure 4. KRP5 colocalizes with heterochromatin
marks and binds different types of genes. A, Immunofluorescence detection of KRP5-GFP and
H3K9me2 in an isolated nucleus of a KRP5OE
transgenic plant. The same distribution was observed in all GFP-labeled nuclei. B, Distribution
of KRP5-binding sites on chromosome 2. KRP5binding sites appear as cyan peaks, genes are
shown in blue, and transposons are shown in
black. C, Distribution of KRP5 peaks around the
TSS. KRP5 preferentially binds to regions situated
downstream of the TSS. D, Annotation of KRP5binding genes. A total of 74% of KRP5-binding
sites correspond to transposable elements and
pseudogenes, whereas 25% correspond to protein-coding genes. Among those, the hierarchical
tree graph on the right shows a significant enrichment in “plant-type cell wall organization.”
“plant type cell wall organization” (Fig. 4D). This result suggests a role of KRP5 in the regulation of genes
involved in plant cell wall organization and in heterochromatin structure, raising the question of the mechanisms allowing its recruitment on chromatin.
To identify chromatin-related proteins that may facilitate KRP5 binding on chromatin, we searched for
interacting partners. AtBAF60, a subunit of the putative
SWI/SNF chromatin-remodeling complex, was previously characterized by Tandem Affinity Purification of
Tagged Proteins experiment as a KRP5 partner (Van
Leene et al., 2010). To confirm that KRP5 proteins can
interact with AtBAF60, we used bimolecular fluorescence complementation in protoplasts isolated from BY-2
cell suspensions (Walter et al., 2004). BY-2 cell protoplasts were transformed with constructs encoding a
fusion between KRP5 and the N-terminal region of
yellow fluorescent protein (YFP; KRP5-YFPN) and
between the whole AtBAF60 amino acid sequence and
the C-terminal region of YFP (AtBAF60-YFPC). As shown
in Supplemental Figure S8, YFP fluorescence could be
detected in the nucleus of protoplasts cotransformed with
KRP5-YFPN and AtBAF60-YFPC, demonstrating that
KRP5 can interact with AtBAF60 in plant cells. These
results suggest that KRP5 could bind chromatin and
control transcription through the action of chromatinremodeling complexes.
KRP5 Binding Correlates with Transcription Activation
To determine if a correlation exists between
chromatin-bound KRP5 and a transcriptional activation
or repression, we first selected a set of KRP5-binding
genes: CDC20.1, a gene involved in endoreduplication
regulation; AtGRP9, AtRX2, EXT10, and EXT2, which
are involved in cell wall structure; and Copia78, a
transposable element. All of these target genes were
confirmed by chromatin immunoprecipitation (ChIP)PCR (Supplemental Fig. S9). Then, the expression
levels of these genes were quantified by qRT-PCR,
showing a correlation between the presence of KRP5
and an activation of the transcription (Fig. 5). These
results suggest that KRP5 could act directly to promote
gene transcription, unraveling a new mechanism by
which KRP5 may control endoreduplication or cell
differentiation. However, 69% of KRP5-binding sites
do not correspond to euchromatin but to heterochromatin regions, which raises the question of the role of
KRP5 at these sites.
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KRP5 Overexpression Induces Chromocenter
Decondensation and Further Endoreduplication
in Mutants Deficient for the Deposition of
Heterochromatin Marks
Previous work showed that ARABIDOPSIS
TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATXR6,
two redundant genes encoding histone methyltransferases, play a critical role in maintaining heterochromatin
condensation and gene silencing in Arabidopsis. Moreover, these proteins can act to suppress DNA rereplication
in heterochromatic regions of the Arabidopsis genome
(Jacob et al., 2010). Because we found KRP5 to bind to
heterochromatin, we asked whether this association
could also participate in the control of endoreduplication.
To test this, we transformed the atxr5 atxr6 double mutant with the 35S:KRP5-GFP construct. Then, we measured the DNA content of the first cauline leaf nuclei in
the atxr5 atxr6 double mutant, KRP5OE, and in five
independent atxr5 atxr6/KRP5OE lines by flow cytometry. We observed that the proportion of 16C nuclei was
increased when KRP5 was overexpressed in the atxr5
atxr6 double mutant context compared with KRP5OE
alone (Fig. 6A; Supplemental Table S1).
To confirm that the increase in endoreduplication
could be correlated with a destabilization of the chromatin structure, we performed cytological observation
of leaf nuclei. As shown in Figure 6B, overexpression
of KRP5 in the atxr5 atxr6 double mutant context
Figure 5. KRP5 binding correlates
with transcription activation. Quantification of the expression level is
shown by qRT-PCR of six KRP5binding genes in the wild type (Col)
and in a KRP5 overexpressor (KRP5OE).
Each panel shows the position of
KRP5 peaks (cyan), a schematic
representation of the target gene
(dark blue; in which exons are
represented by boxes and introns
by lines), and the quantification of
expression. Error bars represent SD
values from at least three repetitions. [See online article for color
version of this figure.]
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KRP5 Connects Endoreduplication and Cell Elongation
dramatically decreased the heterochromatin compaction. Twenty-one percent of KRP5OE atxr5/atxr6 nuclei
presented this specific chromatin phenotype; comparatively, 0% of wild-type, KRP5OE, and atxr5 atxr6 nuclei
showed this strong phenotype (n = 100). Altogether, our
results suggested that condensation of heterochromatin
functions as a barrier to endoreduplication and that
KRP5 can promote heterochromatin decondensation
when the deposition of H3K27me1 is inhibited.
DISCUSSION
One principle of eukaryotic DNA replication is that
the genome is duplicated only once each time a cell
divides. However, in response to a variety of physiological signals or developmental cues, cells can exit from
the classical mitotic cell cycle to start endoreduplication.
This corresponds to a modified cell cycle lacking mitosis, using several regulators. As a general rule, this
process is associated with cell elongation and differentiation. Until now, it has been unclear how a dividing
cell exits its division program and enters the endocycle.
Here, we show that KRP5 is highly expressed in
endoreduplicating cells, that it is required to fully induce endocycles in etiolated seedlings, and that its
overexpression stimulates endoreduplication. Furthermore, posttranscriptional regulations seem to restrict KRP5 accumulation to endoreduplicating cells,
since we observed that the KRP5-GFP fusion protein,
Figure 6. KRP5 acts in heterochromatin condensation. A, DNA content distribution of nuclei
isolated from the first cauline leaf of the wild type
(WT; blue), the atxr5 atxr6 double mutant (green),
and KRP5OE (black) and atxr5 atxr6 KRP5OE (red)
lines. Endoreduplication caused by KRP5 overexpression was much increased in the atxr5 atxr6
background compared with the wild-type background. Results are averages 6 SD calculated from
five different measures for each line. For each
sample, a minimum of 5,000 DAPI-stained nuclei
were analyzed. B, Immunofluorescence detection
of KRP5-GFP in isolated nuclei of wild-type Columbia, double mutant atxr5 atxr6, KRP5OE, and
atxr5 atxr6/KRP5OE.
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Jégu et al.
although it was expressed under the control of a constitutive promoter, accumulated in endoreduplicated
nuclei. This result is in apparent contradiction to previously reported KRP overexpression studies (De Veylder
et al., 2001; Jasinski et al., 2002; Zhou et al., 2002;
Schnittger et al., 2003) but is in agreement with the
observed stimulation of the endoreduplication cycle in
weak KRP2OE plants (Verkest et al., 2005). In addition,
although KRP5 overexpression does not affect overall
plant size, some results reported here suggest that
KRP5 can negatively influence cell proliferation.
Indeed, the smaller cell size observed in the krp5 hypocotyl did not correlate with a reduction of hypocotyl length. Likewise, the bigger cells observed in
KRP5 OE plants did not affect the overall organ length.
Together, these observations suggest that KRP5, like
other KRPs, can inhibit cell proliferation, but maybe
with a lower efficiency. Whether these contrasting
outcomes of KRP overexpression reflect specificities of
functions remains to be fully established, but these
observations, together with the recent finding that
KRPs appear to bind preferentially to different cyclins
(Van Leene et al., 2010), hint at a previously unsuspected specialization of KRPs in plants.
The data reported here suggest that KRP5 can promote endocycles via at least three different modes of
action. The first mechanism is the inhibition of CDK/
cyclin D3 complexes, as evidenced by the ability of KRP5
overexpression to overcome the effects of CYCD3;1
overexpression in planta. CYCD3;1 expression rises as
cells reenter the cell cycle, and its ectopic expression in
cell cultures results in the accumulation of cells in G2
(Menges et al., 2006). Plants overexpressing CYCD3;1
exhibit ectopic divisions in leaves (Dewitte et al., 2003).
Furthermore, cellular expansion and endoreduplication are inhibited in CYCD3;1OE plants, whereas the
cycd3 triple mutant has a reduced number of cells and
increased endoreduplication levels (Dewitte et al.,
2007), pointing to a role of CYCD3 as a negative regulator of endoreduplication. Hence, the ability of KRP5
to inhibit the activity of cyclin D3 could represent a
mechanism to promote endoreduplication.
In addition to its role in the regulation of CDK/
cyclin activity, our results point to a role of KRP5 in the
regulation of gene expression, highlighting another
mechanism by which it may stimulate the switch from
proliferation to endocycles. Indeed, our results demonstrate that KRP5 binds to various loci on chromatin,
possibly via an interaction with the chromatin-remodeling
factor AtBAF60, to modulate gene expression. Such a
mechanism has been reported in mammalian cells,
where the cell cycle inhibitor p16 interacts with the SWI/
SNF complex Brg1 (Becker et al., 2009). Among KRP5
targets, we identified CDC20 in our ChIP-seq experiment.
CDC20 is an activator of APC/C, a multisubunit protein
complex acting in the degradation process of mitotic
cyclins (Marrocco et al., 2010). Overexpression of CDC20.1
promotes endoreduplication in the uvi4 mutant, which is
deficient for an inhibitor of APC/C (Iwata et al., 2011).
Hence, KRP5-dependent induction of CDC20 expression
may contribute to promote endocycles. Furthermore,
the observation that genes associated with plant cell wall
organization and with cell elongation (Esmon et al., 2006;
Sánchez-Rodríguez et al., 2010) are significantly overrepresented among KRP5 target genes and induced by
KRP5 overexpression suggests that KRP5 may regulate
simultaneously endoreduplication and cell elongation. In
most cases, endoreduplication is associated with cell
elongation in plants, as observed in wild-type leaf
epidermal cells and etiolated hypocotyls (Traas et al.,
1998; Sugimoto-Shirasu and Roberts, 2003), even though
cell growth and nuclear DNA content can be uncoupled
in some instances (Beemster et al., 2002; Jasinski et al.,
2002). Although we cannot rule out the possibility that
increased expression of genes related to endocycles or
cell elongation is an indirect effect of KRP5 overexpression, the concomitant observation of KRP5
binding to these loci strongly suggests that KRP5 directly regulates their expression. This role of KRP5 as a
link between cell cycle regulation and the control of
cell growth and differentiation is consistent with the
situation observed in animals. Indeed, KRPs are putative homologs of mammalian CKI such as p21 and
p27 (Inzé and De Veylder, 2006), which have been
demonstrated to participate in a number of other
specific protein-protein interactions beyond their association with CDK/cyclin complexes. For example,
p21 has the potential to physically associate with
transcription factors and coactivators and to modulate
their function (Dotto, 2000). In particular, p21 can bind
to the coactivator p300 and modulate transcription,
depending on the nature of the core promoter (Snowden
et al., 2000). Recently, Devgan and colleagues have
shown that in keratinocytes, p21 functions as a transcriptional regulator that associates physically with the
promoter of the Wnt4 gene (Ferrándiz et al., 2012).
More generally, it has been demonstrated that p21 can
interact with several transcription factors such as CBP, C/
EBPa, E2Fs, Myc, Nrf2, STAT3, and others (for review,
Figure 7. Model summarizing the role of KRP5 as a multifunctional
protein connecting the transcriptional regulation and/or activity of key
genes of the cell cycle, the modulation of the heterochromatin structure, and plant cell wall organization. [See online article for color
version of this figure.]
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KRP5 Connects Endoreduplication and Cell Elongation
see Dotto, 2000; Perkins, 2002; Chen et al., 2009). p21
has been described to act both as a repressor and as a
coactivator of the expression of genes (Poole et al.,
2004; Fritah et al., 2005; Sestáková et al., 2010). Thus,
the transcription function of p21 could be conserved in
its Arabidopsis homolog KRP5 and provide a molecular
link between the onset of endoreduplication and cell
growth or differentiation.
Finally, KRP5 may modulate nuclear DNA content
by acting on heterochromatin structure. Indeed, 75% of
KRP5 targets are in heterochromatin regions, suggesting that KRP5 may modulate chromatin organization.
Many lines of evidence link chromatin structure and
DNA replication: replication timing requires specific
chromatin marks (Casas-Delucchi et al., 2012), and
recently, the chromatin factor CAF-1 has been implicated not only in the control of meristem structure but
also in the control of genome replication at several
stages of development (Exner et al., 2006). Another example is that mutations in the genes encoding the two
homologous H3K27 monomethyltransferases ATXR5
and ATXR6 lead to the rereplication of specific genomic
portions. These data indicate that ATXR5 and ATXR6
are components of a pathway required to suppress
rereplication in Arabidopsis and, more importantly,
link chromatin structure to rereplication (Jacob et al.,
2010). The synergistic phenotype of the KRP5OE atxr5/
atxr6 plants reported here suggests that in a context
where heterochromatin is less condensed, endoreduplication is increased, possibly because heterochromatin
could function as a barrier against endoreduplication.
The ability of KRP5 to bind to heterochromatin regions
and to stimulate the expression of transposable elements
suggests that it may participate in the formation of a
more accessible chromatin structure for the replication
complexes, thereby creating a favorable chromatin
context for endoreduplication. In agreement with this
hypothesis, we found that KRP5 overexpression severely
enhances the chromocenter decondensation phenotype
of atxr5 atxr6 mutants.
In summary, we show that KRP5 is a multifunctional
protein that connects cell elongation and endoreduplication through the combination of at least three modes of
action: inhibition of CDK/cyclin complexes, transcriptional regulation of genes involved in endoreduplication,
and cell wall synthesis and modulation of heterochromatin
structure (Fig. 7). Loss-of-function analysis revealed only
very mild defects caused by KRP5 deficiency, pointing to
high redundancy between KRPs and suggesting that other
members of the family can substitute for KRP5 in most cell
types and at most developmental stages. Future work will
help to clarify whether other KRPs are also multifunctional
and what their distinctive roles are in planta.
MATERIALS AND METHODS
Plant Material and Growth Conditions
Arabidopsis (Arabidopsis thaliana) plants were grown in chambers at 20°C
on soil or on sterile one-half-strength Murashige and Skoog medium and 7.5%
agar in long-day (16 h of light) conditions. Seeds were surface sterilized by
treatment with Bayrochlore (Bayrol) for 20 min, washed, and imbibed in
sterile water for 2 to 4 d at 4°C to obtain homogeneous germination.
The T-DNA insertion line krp5 (SK_27217) from the SALK collection of
T-DNA insertion lines was obtained from the Arabidopsis Biological Resource
Center. The double mutant atxr5 atxr6 was obtained from Scott D. Michaels.
RNA Extraction and Real-Time Quantitative PCR Analysis
Total RNA were extracted from seedlings with the RNeasy MiniPrep kit
(Qiagen) according to the manufacturer’s instructions. First-strand cDNA was
synthesized from 2 mg of total RNA using Improm-II reverse transcriptase
(A3802; Promega) according to the manufacturer’s instructions. A 1/25th
fraction of the synthesized cDNA was mixed with 100 nM of each primer
and LightCycler 480 Sybr Green I master mix (Roche Applied Science) for
quantitative PCR analysis. Products were amplified and fluorescent signals
were acquired with a LightCycler 480 detection system. The specificity of
amplification products was determined by melting curves. AtPTF2 was used
as an internal control for signal normalization. Exor4 relative quantification
software (Roche Applied Science) automatically calculates relative expression
levels of the selected genes with algorithms based on the comparative
threshold cycle method. Data were from duplicates of at least two biological
replicates. The sequences of primers can be found in Supplemental Table S5.
Promoter Cloning
The 1,617 bp upstream of the ATG was amplified with Pfu DNA polymerase
(primers used are listed in Supplemental Table S4) and cloned in pGEM-T
Easy vector. After sequencing, a BamHI/SpeI fragment was cloned in the intact
system developed by Deal and Henikoff (2011).
Confocal Imaging
Transformed BY-2 protoplasts were directly imaged on a TCS-SP2 upright
microscope (Leica Microsystems). To observe YFP, excitation was at 514 nm,
and the spectral detector was set between 520 and 550 nm. Transmitted light
was also collected. All images were acquired with similar gain adjustments.
ChIP and ChIP-seq Analysis
ChIP assays were performed on 10-d-old in vitro seedlings using anti-GFP
(Santa Cruz) or IgG control (Millipore) antibodies using a procedure adapted
from Gendrel et al. (2005). Briefly, after plant material fixation in 1% (v/v)
formaldehyde, tissues were homogenized and nuclei were isolated and lysed.
Cross-linked chromatin was sonicated using a water bath Bioruptor UCD-200
(Diagenode; 30-s-on/30-s-off pulses, at high intensity for 24 min). Protein/
DNA complexes were immunoprecipitated with antibodies overnight at 4°C
with gentle shaking and incubated for 1 h at 4°C with 50 mL of Dynabeads
Protein A (Invitrogen; 100-02D). Immunoprecipitated DNA was then recovered using the IPure kit (Diagenode) and analyzed by quantitative real-time
PCR. An aliquot of untreated sonicated chromatin was processed in parallel
and used as the total input DNA control. The sequences of primers can be
found in Supplemental Table S5.
After immunoprecipitation of the chromatin, ChIP-seq libraries were generated and sequenced. In this work, we used the anti-GFP antibody for ChIP-seq,
and input chromatin was used as a control to minimize biologically irrelevant
experimental bias. Alignment was performed using Bowtie (Langmead et al.,
2009) version 0.12.7 on the Arabidopsis genome (The Arabidopsis Information
Resource 10). Default Bowtie parameters were used except as follows: -best
-strata (used to get the best mapping position with the minimum of mismatch).
Peak calling was performed with MACS (Zhang et al., 2008) and gene annotation with the Genomic Position Annotation Tool (Krebs et al., 2008).
Immunofluorescence
Seedlings were fixed in 4% paraformaldehyde in PHEM (60 mM PIPES, 25
mM HEPES, 10 mM EGTA, and 2 mM MgCl2, pH 6.9) during 1 h at room
temperature (apply vacuum for 20 min to facilitate uptake of the fixation solution). Seedlings were washed 5 min in PHEM and 5 min in phosphate
buffered saline (PBS), pH 6.9, and chopped on a petri dish in PBS supplemented with 0.1% (w/v) Triton. The mixture was filtered (50 mm) and centrifugated 10 min at 2,000g. The supernatant was carefully removed, and the
Plant Physiol. Vol. 161, 2013
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Jégu et al.
pellet was washed once with PBS, gently resuspended in 20 mL of PBS, and a
drop was placed on a poly-Lys slide and air dried. Slides were rehydrated with
PBS and permeabilized two times by 10 min of incubation in PBS plus 0.1% (v/v)
Tween 20 (PBST). Slides were placed in a moist chamber and incubated overnight
at 4°C with primary antibody anti-H3K9me2 (Millipore; 07-441) or anti-GFP
(Santa Cruz) in PBST supplemented with bovine serum albumin (3%, w/v).
Slides were washed five times for 10 min each in PBST (at room temperature)
and incubated 1 h at room temperature in the dark with the secondary antibody
(Alexa Fluor 594 goat anti-rabbit; A11037; Invitrogen) diluted 1:400 (v/v) in PBST
and 3% bovine serum albumin. Slides were washed five times for 10 min each in
PBST. Slides were mounted with a drop of Vectashield with 49,6-diamino-2phenylindole (DAPI) and observed as described for pollen mitosis analysis
with the suitable cube fluorescence filters (Band Pass [BP] 340–380, Dichroic
Splitter [DS] 400, BP 450–490 for DAPI; BP 570–590, DS 595, BP 605–655 for A594).
Flow Cytometry
For flow cytometric nuclei analysis, tissues were chopped with a razor blade in
1 mL of Galbraith buffer supplemented with 1% polyvinylpyrrolidone 10,000, 5
mM metabisulfite, and 5 mg mL21 RNase from a stock solution at 50 units mg21.
Propidium iodide was added to the filtered supernatants at 50 mg mL 21.
Endoreduplication levels of 5,000 to 10,000 stained nuclei were determined
using a Cyflow SL flow cytometer (Partec) with a 532-nm solid-state laser
(100 mW) excitation and emission collected after a 590-nm long-pass filter.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Nuclear DNA content distribution of GFP+ and
GFP2 nuclei isolated from rosette leaves of Arabidopsis KRP5::NTFGFP plants.
Supplemental Figure S2. DNA content distributions of nuclei isolated
from leaves of 35S::GFP plants.
Supplemental Figure S3. Phenotype of the KRP5-overexpressing line.
Supplemental Figure S4. DNA content distributions of nuclei isolated
from KRP5::NTF-GFP hypocotyls of 7-d-old dark-grown seedlings.
Supplemental Figure S5. DNA content distributions of nuclei isolated
from light-grown wild-type plants, krp5 mutants, and KRP5OE.
Supplemental Figure S6. Average hypocotyl lengths of etiolated seedlings
of krp5, wild-type, and KRP5OE plants.
Supplemental Figure S7. DNA content distribution in nuclei of rosette
leaves isolated from the wild type, AtCYCD3;1 overexpressors, and
three different double transformant CycD3;1OE/KRP5OE lines.
Supplemental Figure S8. Interaction between KRP5 and AtBAF60 visualized by bimolecular fluorescence complementation.
Supplemental Figure S9. ChIP from KRP5OE transgenic plants was performed using anti-GFP or anti-IgG antibody.
Supplemental Table S1. Quantification of nuclear DNA content distribution in the first cauline leaf of wild-type, KRP5OE, atxr5 atxr6, and
KRP5OE atxr5 atxr6 plants.
Supplemental Table S2. Quantification of nuclear DNA content distribution in hypocotyls of 7-d-old dark-grown seedlings of the wild type,
krp5, krp5 KRP5OE, and KRP5OE.
Supplemental Table S3. Quantification of nuclear DNA content distribution in the wild type, CYCD3;1OE, and KRP5OE CYCD3;1OE from 14-d-old
growing plantlets.
Supplemental Table S4. List of KRP5 target genes.
Supplemental Table S5. Primer sequences.
ACKNOWLEDGMENTS
We are very grateful to Steven Henikoff (Fred Hutchinson Cancer Research
Center) for providing INTACT vector and ACT2p::BirA seeds, to James A.H.
Murray (Cardiff School of Biosciences) and Catherine Riou (Limoges University) for providing 35S::CYCD3;1, to Scott D. Michaels (Department of Biology,
Indiana University) for the atrx5 atrx6 double mutant, and to François Parcy
(Commissariat à l’Energie Atomique) for providing pBiFP vectors for bimolecular fluorescence complementation. We thank Gilles Sante (Orsay University) for taking excellent care of our plants and Séverine Domenichini (Orsay
University) for her expert help with microscopy.
Received December 6, 2012; accepted February 1, 2013; published February 20,
2013.
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