Annals of Botany 107: 1193– 1202, 2011
doi:10.1093/aob/mcr038, available online at www.aob.oxfordjournals.org
REVIEW: PART OF A SPECIAL ISSUE ON THE PLANT CELL CYCLE
Cell-cycle control as a target for calcium, hormonal and developmental
signals: the role of phosphorylation in the retinoblastoma-centred pathway
Dénes Dudits*, Edit Ábrahám, Pál Miskolczi, Ferhan Ayaydin, Metin Bilgin † and Gábor V. Horváth
†
Institute of Plant Biology, Biological Research Centre, H-6726 Szeged, Hungary
Present address: Department of Molecular and Integrative Physiology, University of Illinois, Urbana, IL 61801, USA
* For correspondence. E-mail [email protected]
Received: 21 July 2010 Returned for revision: 6 October 2010 Accepted: 7 January 2011 Published electronically: 25 March 2011
† Background During the life cycle of plants, both embryogenic and post-embryogenic growth are essentially
based on cell division and cell expansion that are under the control of inherited developmental programmes modified by hormonal and environmental stimuli. Considering either stimulation or inhibition of plant growth, the key
role of plant hormones in the modification of cell division activities or in the initiation of differentiation is well
supported by experimental data. At the same time there is only limited insight into the molecular events that
provide linkage between the regulation of cell-cycle progression and hormonal and developmental control.
Studies indicate that there are several alternative ways by which hormonal signalling networks can influence
cell division parameters and establish functional links between regulatory pathways of cell-cycle progression
and genes and protein complexes involved in organ development.
† Scope An overview is given here of key components in plant cell division control as acceptors of hormonal and
developmental signals during organ formation and growth. Selected examples are presented to highlight the
potential role of Ca2+-signalling, the complex actions of auxin and cytokinins, regulation by transcription
factors and alteration of retinoblastoma-related proteins by phosphorylation.
† Conclusions Auxins and abscisic acid can directly influence expression of cyclin, cyclin-dependent kinase
(CDK) genes and activities of CDK complexes. D-type cyclins are primary targets for cytokinins and overexpression of CyclinD3;1 can enhance auxin responses in roots. A set of auxin-activated genes (AXR1 –
ARGOS–ANT) controls cell number and organ size through modification of CyclinD3;1 gene expression. The
SHORT ROOT (SHR) and SCARECROW (SCR) transcriptional factors determine root patterning by activation
of the CYCD6;1 gene. Over-expression of the EBP1 gene ( plant homologue of the ErbB-3 epidermal growth
factor receptor-binding protein) increased biomass by auxin-dependent activation of both D- and B-type
cyclins. The direct involvement of auxin-binding protein (ABP1) in the entry into the cell cycle and the regulation of leaf size and morphology is based on the transcriptional control of D-cyclins and retinoblastomarelated protein (RBR) interacting with inhibitory E2FC transcriptional factor. The central role of RBRs in
cell-cycle progression is well documented by a variety of experimental approaches. Their function is phosphorylation-dependent and both RBR and phospho-RBR proteins are present in interphase and mitotic phase cells.
Immunolocalization studies showed the presence of phospho-RBR protein in spots of interphase nuclei or granules in mitotic prophase cells. The Ca2+-dependent phosphorylation events can be accomplished by the calciumdependent, calmodulin-independent or calmodulin-like domain protein kinases (CDPKs/CPKs) phosphorylating
the CDK inhibitor protein (KRP). Dephosphorylation of the phospho-RBR protein by PP2A phosphatase is regulated by a Ca2+-binding subunit.
Key words: Cell cycle, cyclin, cyclin-dependent kinase (CDK), CDK inhibitor protein (KRP), retinoblastomarelated protein (RBR), E2F/DP transcription factor, auxin, cytokinin, Ca2+, phosphatase (PP2A), ARGOS,
SHORT ROOT (SHR), organ size.
C E L L D I V I S IO N PA R A M E T E R S R E S PO N D TO
HORMONAL AND STRESS STIMULI
As discussed in several comprehensive reviews, the basic regulatory mechanisms in cell-cycle progression rely on a multicomponent system including transcriptional regulation,
protein-protein interaction, phosphorylation– dephosphorylation and protein degradation (De Veylder et al., 2007; Dudits
et al., 2007; Francis, 2007, Berckmans and De Veylder,
2009; Jurado et al., 2008). A close functional link between
plant hormones, primarily auxin, cytokinins and regulatory
proteins of cell-cycle events, has been demonstrated by different experimental approaches. The recent review by
Perrot-Rechenmann (2010) summarizes auxin actions on cell
division. Plant hormones can directly determine cell-cycle
entry and progression or can act indirectly, through a variety
of regulatory proteins involved in developmental programmes
(see Fig. 1). The auxin [2,4-dichlorophenoxy acetic acid
(2,4-D)]-stimulated entry into the S-phase is shown in Fig. 2,
where histone H4 promoter activity was increased as indicated
by the number of b-glucuronidase (GUS)-positive cells in
transgenic maize tissues. Auxin, ethylene and wounding can
also directly stimulate G2/M specific events, as reflected by
activation of the promoter of the mitotic CDKB2,1 gene in
Medicago leaves (Zhipanova et al., 2006). Treatment of
# The Author 2011. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
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1194
Dudits et al. — Cell-cycle control and signalling
KRP
AUXIN
SCR
ABP1
LIGHT
SCR,SHR
CDKB2;1+CYCD1;1
SHR
CDKA+CYCD3;1
SCFSKP2
P
CYCD6;1
EBP1
E2F/DP
RBR
P
P
RBR
E2F/A/B+DP/A/B DP/B+E2F/C
P
G1
P
RBR
S
CK
AUXIN
CDKA
CDC6/CDT1
GL2
CYCB1;1
CDKB1;1
TCP24
RBR
M
Pre-RC
ANT
P
RBR
P
G2
GEM
CYCD3;1
CYCA2;1
PP2A/Ca2+
P
CDKB2;1
CDKB2;2
+CYCD6;1
E2F/DP
G1
E2F/C
CDKB2;1
WOUNDING
ABAP1
EBP1
ETHYLENE
STM
ARGOS
KRP
P
AXR1
ETHYLENE
CPK
ABA
cullin3
AUXIN
Calcineurin
CaM
AUXIN
ACS5
Ca2+
oxidative
stress
F I G .1. Schematic overview of potential links between calcium, hormonal and developmental signals and cell cycle regulators; note that the proposed scheme is
not complete. Abbreviations: CK, cytokinin; E2F/DP, transcription factors; RBR, retinoblastoma-related protein; P, phospho-protein; CYC, cyclin; CDK, cyclindependent kinase; PP2A, phosphatase; SCR, SCARECROW; SHR, SHORT ROOT; SCF, SKP1 + CULLIN + F-box (SKP2); EBP1, plant homologue of epidermal growth factor-binding protein; SKP2, F-box protein; STM, SHOOT MERISTEMLESS; KRP, CDK inhibitor; CaM, calmodulin; CPK, calmodulin-like
domain protein kinase; ABAP1, armadillo BTB Arabidopsis protein 1; TCP24, transcription factor; CDT1, DNA replication-licensing factor; ABP1, auxinbinding protein 1; ANT, aintegumenta; ARGOS, auxin-regulated gene in organ size; AXR1, RUB1-activating enzyme; ABA, abscisic acid; GL2, GLABRA
(root hair); GEM, GL2 expression regulator; ACS5, 1-aminocyclo-propane-1-carboxil acid synthase. For detail see the text.
Control
2,4-D (1 µM)
ABA (10 µM)
Callus
tissue
Root tip
Lateral
root
F I G . 2. Auxin (2,4-D) activates while abscisic acid (ABA) depresses wheat histone H4 promoter function serving as a marker of S-phase cells in transgenic
maize tissues. The GUS (b-glucuronidase) reporter gene indicates the promoter activity by blue staining.
Dudits et al. — Cell-cycle control and signalling
Arabidopsis seedlings with auxins and cytokinins activated
both CDKA and CyclinD1;1– D2;1 genes, while KRP4 transcription was down-regulated (Cho et al., 2010).
Plant protoplast cultures offer an ideal experimental system
for studies on the hormone-dependence of cell division and the
function of cyclin-dependent kinase (CDK) complexes
(Pasternak et al., 2007). After the regeneration of the cell
wall, single alfalfa cells may enter into the division cycle synchronously, when cultured in a medium containing 2 mg L21
2,4-D as the auxin component. Cytokinin (0.5 mg L21
zeatin) was required for progression through S-phase and completion of cell division. These responses are linked to
CDKA1;1 and CDKB1;1 kinase activities. In the absence of
cytokinin, CDKA1;1 kinase protein is synthesized but lacks
histone H1 phosphorylation activity. In contrast, the other
G2/M kinase (CDKB1;1) was functional in the absence of
this hormone (Pasternak et al., 2007). The expression of
cyclin genes is activated by auxins and cytokinins, as shown
in various cell types (Nieuwland et al., 2007).
Abscisic acid (ABA) regulates plant growth and development
through its signalling network responding to stress factors (see
review by Cutler et al., 2010). Exogenously applied ABA inhibits
S-phase progression, as reflected by histone H4 promoter function in maize callus tissues (Fig. 2). Moreover, the CDK activity
in alfalfa leaves was significantly reduced by ABA even in the
presence of auxin and cytokinin (Mészáros et al., 2000).
Treatment of BY2 tobacco G1 cells with ABA inhibited the G1/
S transition after application of an aphidicolin block (Swiatek
et al., 2002). ABA is generally viewed as a growth inhibitor
having significant roles under abiotic stresses such as drought
(Skirycz and Inzé, 2010). Drought stress can essentially reduce
cell division in both roots and leaves; e.g. water limitation
decreased cell division rate in the maize root tip at the basal
region of the meristem (Sacks et al., 1997). However, in both
wheat and maize leaves, the amount of Cdc2-like protein
(CDK) was not altered by drought treatment, as shown by
western blotting with PSTAIRE antibody. In the same basal
shoot tissues, p13SUC1-bound kinase activities were 50 % lower
in extracts from the stressed compared with the non-stressed
leaves (Schuppler et al., 1998; Granier et al., 2000). Primary
roots of Arabidopsis responded to salt treatment (0.5 % NaCl)
by a reduction in the number of dividing cells and a transient
decrease in CDK activities (West et al., 2004). Activation of
CDK inhibitor genes (ICK/KRP) under stress conditions or by
ABA treatment also provides a basis for the reduction of cell division activities (Wang et al., 2008). Different stressors may differentially induce various members of the ICK/KRP gene family.
Drought or cold stresses can activate an additional CDKA1;1
inhibitor encoded by the rice EL2 gene (Peres et al., 2007).
CAL CIU M AS A C E NT R AL S I GN A L M O L E C UL E
MEDI AT I NG HO RMO NA L AN D STR E SS
EFFECTS
Stimulus-specific and dynamic alterations in the level of free
calcium in the cytosol function as a cellular signalling
control for a variety of processes, including cell division,
metabolism and gene expression (Tuteja and Mahajan,
2007). A whole set of binding proteins acting as Ca2+
sensors such as calmodulin, calmodulin-like proteins;
1195
calcineurin B-like proteins, phospholipase D, annexins, calreticulin and pistil-expressed binding protein transmit signals eliciting downstream responses. Protein kinases and phosphatases
mediate Ca2+-dependent phosphorylation events in the control
of gene expression or enzyme activities in a specific manner
(Kudla et al., 2010). In mammalian cells Ca2+ signalling is
required for cell-cycle progression and the Ca2+ signalling
apparatus is remodelled in cancer cells (Roderick and Cook,
2008). In the zygote of the brown alga Fucus serratus both
S-phase and zygotic polarization were shown to be dependent
on Ca2+ elevation in the pre-S-phase (Bothwell et al., 2008).
In plants, a transient increase in cytosolic Ca2+ can regulate
cell-cycle progression in response to abiotic stresses. Sano
et al. (2006) have demonstrated the increase of Ca2+ in BY2
tobacco cells through the application of oxidative stress
(KMnO4) or hypoosmotic treatment. These oxidative stresses
inhibited the entry of cells into mitosis and delayed the cell
cycle in a Ca2+-dependent manner. Out of several elements
of a complex signalling cascade linking cellular Ca2+ to
cell-cycle regulation, the calcium-dependent, calmodulinindependent or calmodulin-like domain protein kinases
(CDPKs/CPKs) have been proposed as active signal mediators
(Dudits et al., 1998). In the Arabidopsis genome .30 genes
encode CDPKs and members of this kinase family are activated by Ca2+ and show autophosphorylation (Bögre et al.,
1988, Cheng et al., 2002). The medicago kinase (MsCPK3)
gene responded to treatment with a high concentration of
auxin (2,4-D) known to be an inducer of asymmetric cell division and somatic embryogenesis in leaf protoplasts (Davletova
et al., 2001). In cucumber seedlings during adventitious root
formation induced by auxin (b-indole acetic acid) or nitric
oxide treatments, the CDPK enzyme was activated in a
Ca2+-dependent manner (Lanteri et al., 2006).
The link between CPK-mediated Ca2+ signals and cellcycle control was established by experiments showing the
elevated activity of an alfalfa CDK inhibitor protein
(MtKRP) after phosphorylation by the recombinant MsCPK3
protein (Pettkó-Szandtner et al., 2006). The inhibitory function
of the MtKRP protein was also demonstrated by using the
histone H1 protein and the alfalfa RBR recombinant fragment
as CDK substrate. Both MsCPK3 and MtKRP transcript levels
were increased by ABA and salt treatments, which are known
as inhibitors of cell division. The Arabidopsis KRP2 inhibitor
protein regulating the endoreduplication cycle can serve as a
substrate for mitotic CDKB1;1 kinase and this phosphorylation
can reduce KRP2 stability (Verkest et al., 2005). In the alfalfa
experimental system G2/M kinase complexes were more sensitive towards the recombinant MtKRP inhibitor than were
S-phase complexes (Pettkó-Szandtner et al., 2006).
Calmodulin, like other essential Ca2+ sensors in plants has a
primary role in abiotic and biotic stress responses as shown
by transcriptional data (Kim et al., 2009). In Arabidopsis
and tobacco cells a kinesin-like calmodulin-binding protein
(KCBP) plays a role in the formation of microtubule arrays
(Bowser and Reddy, 1997). Calcineurin B-like (CBL) proteins,
as members of the Ca2+ signalling cascade, can regulate the
biosynthesis of ethylene and polyamines (Oh et al., 2008).
In summary, the specificity of cellular responses to the
increase in the level of cytosolic Ca2+ is dependent on the
complexity of a signalling cascade aimed at well-defined
1196
Dudits et al. — Cell-cycle control and signalling
targets. In cell-cycle control, CDK activities can represent one
of these key targets that play a central role by phosphorylating
a set of regulatory proteins including cyclins, histones and
retinoblastoma-related proteins (RBRs).
P L A N T R E T I N O B L A S TO M A - R E L AT E D
P ROT E I N S A S P H O S P H O - P ROT E I N S I N T H E
CO NTROL O F T HE C ELL DI VI SI ON C YC L E
The RBRs, as structural and functional counterparts of the
mammalian tumour suppressor pRb proteins in higher plants,
have divergent roles in the regulation of the division cycle
and development (Gutierrez, 1998; Durfee et al., 2000;
Wildwater et al., 2005; Wyrzykowska et al., 2006;
Sablowski, 2007; Costa and Gutierrez-Marcos, 2008; Paz
Sanchez et al., 2008; Chen et al., 2009; Sabelli and Larkins,
2009). RBR cDNA clones have been identified from both
dicot and monocot plant species. While dicot plants have
only a single gene, monocot cereal species carry at least two
distinct genes with characteristic expression patterns
(Lendvai et al., 2007; Miskolczi et al., 2007). Microarray
analysis showed an elevated expression of the Arabidopsis
RBR1 gene in young roots, stems and light-grown seedlings
(de Almeida et al., 2009). In the mammalian cell division
cycle retinoblastoma proteins ( pRbs) act as a ‘pocket
domain’ protein regulating G1- to S-phase transition through
phosphorylation-dependent interaction with the E2F family
transcription factors either repressing or activating genes
required for cell-cycle check point transition. The Rb – E2F
complexes are involved in several basic cellular events such
as carcinogenesis, apoptosis and cell differentiation (reviewed
by Poznic, 2009).
Also in plants, the RBR functions are controlled by phosphorylation and protein – protein interactions. Similarly to
human pRBs, plant RBR proteins are composed of an aminoterminal region, A and B domains in the pocket region and a
C-terminal domain. These proteins have several potential
CDK phosphorylation sites (Durfee et al., 2000; Boniotti
and Gutierrez, 2001; Miskolczi et al., 2007). Interactions
between CDKs and D-type cyclins are required for the formation of active kinase complexes that can phosphorylate
RBR proteins, as shown by Nakagami et al. (1999). These
plant cyclins may have the LxCxE motif that mediates the
binding of a variety of proteins to RBR proteins (Huntley
et al., 1998; Soni et al., 1995). The NtRBR1 protein of
tobacco was phosphorylated by the CYCD3;3/CDKA
complex and this kinase activity was detected in G1 and
S-phase cell extracts from synchronized tobacco BY-2 cells
(Nakagami et al., 2002).
The cell-cycle phase-dependent phosphorylation of plant
RBRs relies on various kinase complexes and determines differential RBR functions. In synchronized alfalfa cells, western blotting of total protein extracts detected the 115-kDa full-size
MsRBR protein in all cell-cycle phases with limited variation
(Ábrahám et al., 2011). Immunoblotting the same protein
samples with anti-phospho-pRb antibodies indicated a low
phospho-MsRBR protein level in the control culture with G1
cells. This was elevated in samples with S-phase cells. Protein
amounts cross-reacting with anti-phospho-pRb peptide antibodies were found to be high in extracts isolated from cell
populations representing a significant frequency of G2/
M-phase cells. Subsequently the phospho-MsRBR protein was
reduced in samples containing G1 cells. In these studies, both
the MedsaCDKA1;1 PSTAIRE motif and the mitotic
MedsaCDKB2;1 kinases phosphorylated the His-tagged
C-terminal fragment of the MsRBR protein produced in vitro.
Kawamura et al. (2006) generated antibodies against the
C-terminal region of tobacco NtRBR1 protein and different
phospho-serine peptides containing sequences from NtRBR1.
The NtRBR1 protein was phosphorylated by both CDKA and
CDKB kinases immunoprecipitated from actively growing
cells. Antibodies, recognizing specific phospho-serine residues,
cross-reacted differentially with the NtRBR1 protein in various
phases of the cell cycle. Hirano et al. (2008) demonstrated by
pull-down assay that the non-phosphorylated AtRBR1 protein
could bind to the E2Fa protein, whereas the hyperphosphorylated form did not interact with the E2Fa protein.
These findings are in agreement with the general model of G1/
S-phase transition control, where the hypophosphorylated Rb
proteins act as transcriptional repressor by inhibition of E2F
functions in the regulation of the expression of
S-phase-specific genes (Poznic, 2009).
The presence of phospho-MsRBR proteins in interphase and
mitotic cells was also demonstrated by immunolocalization
experiments (Ábrahám et al., 2011). These studies showed
intensive staining of the interphase nuclei and the signals
were concentrated into spots. As shown by Fig. 3, in mitosis
the prophase cells have labelled granules. These structures
cannot be recognized in later phases such as prometaphase,
metaphase, anaphase and telophase. These observations emphasize the presence of phospho-RBR protein in the G2 phase and
mitosis in plant cells. The functional significance of pRB
tumour suppressor and Rbf1 proteins has been demonstrated
in mammalian and Drosophila G2/M cells (Nair et al., 2009;
Lavoie, 2008). Analysis of the MsRBR protein in cells from cultures at different growing phases revealed an increase in both
forms of this protein during exponential growth. Linking division activity to the presence of the MsRBR proteins was also
supported by hormone starvation experiments. The prolonged
withdrawal of synthetic auxin and kinetin stopped the growth
of A2 alfalfa cultures and the MsRBR protein could not be
detected in these cells (Ábrahám et al., 2011). The cell divisiondependent presence of plant RBR proteins could also be
F I G . 3. Immunodetection of phospho-MsRBR protein in prophase alfalfa
cells as nuclear granules by antibodies produced against the human
phospho-pRb peptide (green labelling). These nuclear granules cannot be
recognized in later mitotic phases as shown by anaphase cells. The red staining
of DNA was carried out by DAPI (diamidino-2-phenylindole).
Dudits et al. — Cell-cycle control and signalling
concluded from additional experiments. Only the nonphosphorylated AtRBR1 protein was detected in stationaryphase MM2d Arabidopsis cells. Transfer into fresh medium
resulted in the elevation of detectable amounts of AtRBR1
protein and after 8 h its phosphorylated form was detected by
western blotting (Hirano et al., 2008). During the growth of
Arabidopsis roots the auxin-binding protein 1 (ABP1) is essential for the G1/S transition through interaction with the cyclinD/
RBR pathway (Tromas et al., 2009).
The direct involvement of plant RBRs in the control of cell
division, endoreduplication and differentiation was shown by
reduction of the expression of NtRBR1 gene through
virus-induced gene silencing in tobacco plants (Park et al.,
2005). The transcription of E2F and S-phase genes such as ribonucleotide reductase (RNR), proliferating cell nuclear antigen
(PCNA), mini chromosome maintenance (MCM), histone H1
and replication origin activation protein (CDC6) was significantly up-regulated in infected tissues of these plants. Leaf
cells were observed with enlarged nuclei representing higher
ploidy levels. Endoreduplication was also enhanced in transgenic plants over-expressing the E2Fa-DP genes (De Veylder
et al., 2002; Kosugi and Ohashi, 2003). In cultured
Arabidopsis cells (MM2d cell line) the RNAi-induced downregulation of AtRBR1 increased G2-phase cells (Hirano et al.,
2008). Dependence of the activation of S-phase entry and cell
proliferation on RBR mRNA levels can be also demonstrated in
monocot cells. Fig. 4 presents an increase in the frequency of
S-phase cells incorporating 5-ethynyl-2′ -deoxyuridine (EdU)
and biomass production in rice callus culture after antisense
transformation of the OsRBR1 gene. A recent publication
(Kotogány et al., 2010) describes the use of EdU labelling of
S-phase cells in plant tissues.
P H O S P H ATA S E P P 2 A R E G U L ATO RY S U B U N I T,
A P HO SPHO PROTEIN W IT H CALC IU M -B IN DI NG
M OT I V E S I N T E R ACT S W I T H P L A N T R B R
P ROT E I N S
Post-translational modifications of regulatory proteins by both
kinases and phosphatases are essential in controlling molecular
Relative
transcript
level OsRBR;1
1197
and physiological responses. Protein phosphatases regulate
practically every step of the mitotic cycle (reviewed by
Bollen et al., 2009). PP1, PP2A, PP4 and the dual specificity
phosphatases Cdc25 and Cdc14 are involved in regulatory processes. During mitosis, PP1 and PP2A control mitotic kinases
(Cdk1, Nek2A, Plk1, Aurora A), and dephosphorylate specific
pools of mitotic kinase substrates. Upon mitotic exit, they
function as guides to promote the controlled removal of
mitotic phosphorylations. In animal cells, PP1c interacts with
pRB and directly dephosphorylates the protein (Vietri et al.,
2006; Kiss et al., 2008). In addition, PP2A was reported to
be implicated in the dephosphorylation of RBRs, particularly
upon oxidative stress (Cicchillitti et al., 2003). A PP2A regulatory subunit (PR70) was shown to associate with pRb and
mediate its dephosphorylation (Magenta et al., 2008). In chondrocytes, FGF promoted the dephosphorylation of p107 by
inducing an association between PP2A and p107 (Kolupaeva
et al., 2008). Although phosphorylation of RBRs by CDKs
in plants is well documented (Nakagami et al., 1999), up till
now not a single report has been published on plant phosphatases that are responsible for the dephosphorylation of
phospho-RBRs.
In yeast two-hybrid screens, using full-length MsRBR1
protein of alfalfa as bait, a Medicago truncatula interactor
was identified, which showed a significant level of similarity
to PP2A protein phosphatase B′ regulatory subunits (39 %
identity and 57 % homology with murine PR59 PP2A regulatory subunit). Further pairwise assays revealed that this protein
discriminated between the rice RBR proteins in interaction
strength. Like the alfalfa RBR protein, OsRBR1 showed
strong association with the PP2A regulatory subunit, whereas
OsRBR2 failed to interact with this prey (Lendvai et al.,
2007). Similar selectivity was reported for mammalian
pocket domain proteins. Murine RBs were shown to interact
with the PR59 PP2A B′ regulatory subunit in a differential
manner; the p107 protein was the only one to show a strong
association but pRB did not (Voorhoeve et al., 1999). The
PR59-associated PP2A complex dephosphorylated p107 in
vivo, and over-expression of the phosphatase regulatory
subunit in U2OS cells resulted in inhibition of cell-cycle
Control
Over-expression
line 80
Antisense
line 89
1
2·35
0·49
2·25
–
4·80
Frequency of
S-phase cells
Cell mass
in suspension
culture g per 50 mL
F I G . 4. The transcript level from the rice retinoblastoma-related gene (RBR) influences the number of S-phase cells and biomass in transgenic rice cell cultures.
Over-expression of the rice RBR1 gene reduces while down-regulation of this gene increases the frequency of DNA-synthesizing S-phase cells labelled with
5-ethyl-2′ -deoxyuridine (EdU) as shown in yellow and green. The over-expressing culture (line 80) could not be cultured for a prolonged time.
1198
Dudits et al. — Cell-cycle control and signalling
progression and accumulation of the cells in G1 phase
(Voorhoeve et al., 1999). This analogy has also supported
the suggestion that plant RBRs belonging to the RBR1 or
RBR2 subfamilies have different roles in the regulation of
plant cell division and differentiation. Previous results have
also suggested that PP2A has an important cell division regulatory role, since its activity contributes to the control of
mitotic kinases and microtubule organization in alfalfa
(Ayaydin et al., 2000).
Orthologues of all PP2A subunits have been described in
plants. The PP2A catalytic subunits are localized to various
cell compartments and play diverse functions ranging from
metabolism to cell-cycle control. In Arabidopsis, the catalytic
subunit isoforms of PP2A are encoded by five genes, each of
which appears to be expressed in all tissues, albeit at different
levels (Arino et al., 1993; Casamayor et al., 1994;
Pérez-Callejón et al., 1998). Database searches in the
rice genome revealed the presence of five PP2A catalytic subunits (Os02g0217600, Os03g0167700, Os3g0805300,
Os06g0574500 and Os10g0410600) and a single PP2A A
regulatory subunit (Os09g0249700). Since the active holoenzyme in general consists of one catalytic, one A- and one
B-type regulatory subunit, the correct characterization of this
RBR-dephosphorylating plant PP2A phosphatase holoenzyme
requires the identification of all PP2A subunits.
Sequence analysis of the OsRBR1-interacting OsPP2A B′
regulatory protein (encoded by the Os10g0476600 gene)
revealed that it contains EF-hand domains potentially regulating its function by Ca2+-binding. Several examples in the literature have already demonstrated such regulation;
experiments showed that the PR70 member of the PPP2R3
family of human regulatory subunits targets protein phosphatase 2A to Cdc6 (Davis et al., 2008). Two functional
EF-hand calcium-binding motifs mediate the calciumenhanced interaction of PR70 with PP2A. Another report
suggested that Ca2+ binding to EF-hand 1 of the B′ /PR70
subunit was able to further increase PP2A affinity for certain
substrates like Thr-75-DARPP32 protein, or to alter the orientation of phospho-serine or -threonine in the active site of the
catalytic subunit (Ahn et al., 2007). In the yeast two-hybrid
experiments it was possible to demonstrate that the interaction
between the OsRBR1 and OsPP2A B′ proteins needs an intact
pocket domain of the RBR and the presence of the EF-hand
domains on the regulatory subunit (P. Yu et al., BRC
Szeged, Hungary, unpubl. res.). Such a finding may support
the hypothesis that the dephosphorylation of plant RBR proteins might be influenced by the increase in intracellular
Ca2+; thus it could respond to extracellular stimuli, e.g.
environmental stress factors.
A N OV ERV IE W OF POT E NTI AL L IN KS
B E T W E E N H O R M O N A L A N D DE V E LO P M E N TA L
S IG N A L L I N G A N D CE L L - C Y C L E R E G U L ATOR S
Over the past 20 years, starting with the report on the cloning
of the Cdc2 gene from pea (Feiler and Jacobs, 1990), biochemical and genetic approaches involving recombinant
DNA have led to a substantial increase in understanding the
functions of the key cell-cycle regulators and have provided
good evidence that their role is integrated into the
developmental programme of plants in a complex way (de
Jager et al., 2005; Maughan et al., 2006; De Veylder et al.,
2007; Busov et al., 2008). However, the picture is far from
complete. Figure 1 summarizes the previously discussed
data, demonstrating selected cases where plant hormones interact with regulatory components of cell-cycle control. These
growth regulators can directly influence events mediated by
cyclin, CDK, KRP, E2F/DP and RBR. It is possible to see
the pivotal role of auxins that can also be realized through
pathways regulating organ formation or plant structure. This
summary outlines the links with Ca2+ signalling. The proposed scheme emphasizes the presence of the RBR protein
and its phosphorylated form in the G2/M cell-cycle phase
and cites examples for interplay between regulators of plant
structure and cell proliferation or differentiation.
Studies in root hair development offer an excellent example
for a joint component in the regulation of DNA replication and
epidermal cell fate (Caro et al., 2007). Root hair initiation
from epidermal cells occurs in trichoblast progenitors that do
not express the homeobox transcription factor GL2
(GLABRA2). Activity of this gene is controlled by the GEM
(GL2expression modulator) protein interacting with a DNA
replication licensing factor, CDT1. Over-expression of GEM
represses GL2 and results in ectopic root hair formation,
while GEM acts as a repressor of cell division. The armadillo
BTB Arabidopsis protein 1 (ABAP1) has been described as a
regulator of cell-cycle progression in leaves by integrating
plant developmental signals with DNA replication and transcription controls (Masuda et al., 2008). The 2- and 5-fold
reduction in ABAP1 levels in Arabidopsis plants caused an
increase in the expression of AtCDT1 genes, resulting in stimulation of rosette and leaf growth. ABAP1 is a nuclear G1/early
S-specific protein that interacts with the transcription factor
AtTCP24 and this complex binds to promoters of AtCDT1
genes. The authors suggest that ABAP1 acts as a member of
a negative feedback loop to control DNA replication during
leaf development.
After fertilization of an egg cell, a series of cell divisions is
accompanied by early specialization of cells and, in late
globular-stage embryos, the primary shoot and root meristems
are laid down as the main centres of cell division and starting
points for the formation of differentiated cells. The transition
from the meristematic zone to the elongation and the differentiation zone either in the root tip or the basal region of leaf
depends on the co-ordination between proliferation and differentiation processes. This cell fate determination is under the
influence of plant hormones such as auxin, brassinosteroids,
cytokinins and gibberellin. In addition ABA, ethylene and jasmonic acid are considered as stress-responsive regulators.
Wolters and Jürgens (2009) reviewed the essential proteins
and corresponding genes involved in hormone actions and outlined hormonal regulation in meristem functions and in starting differentiation.
The interplay between the basic cell-cycle machinery and
auxin could be clearly demonstrated in the regulation of
lateral root initiation. Auxin accumulation in the pericycle
cells primes lateral root formation through the induction of
cell division (see review by Péret et al., 2009). D-type
cyclins represent rate-limiting factors in G1 – S phase transition
during cell-cycle progression. The loss-of-function mutation in
Dudits et al. — Cell-cycle control and signalling
Arabidopsis CYCD4;1 caused a reduction in the number of
enlarged pericycle cells (Nieuwland et al., 2009). In the
absence of this kind of cyclin D, lateral root density was
lowered and this defect could be restored by auxin treatment.
Over-expression of another D-type cyclin (CYCD3;1) resulted
in enhanced auxin response and increased the lateral root
density in the presence of 0.1 or 1 mM naphthalene acetic
acid (De Smet et al., 2010). The activation of cell division is
an essential but not sufficient prerequisite for lateral root
initiation. This was clearly demonstrated by over-expression
of the CYCD3;1 gene in the solarity root1 (srl1) mutant
(Vanneste et al., 2005). In this mutant background, cell division was induced without lateral root initiation. Based on
microarray transcript profiling data, the authors list a number
of genes involved in cell cycle, auxin signalling, transport,
conjugation and biosynthesis genes among the lateral root
initiation genes. Primary auxin-responsive genes out of cellcycle genes such as CYCA2;4 and CDKB2;1 genes have promoters with auxin-responsive regulatory elements. The heterodimeric transcription factor E2FA/DPA is one of the key
activators of S-phase entry. Lateral root density was found to
be reduced in transgenic Arabidopsis plants over-expressing
these transcriptional factors (De Smet et al., 2010). In contrast,
auxin-induced lateral root formation was increased in this line.
The authors concluded that enhancement of the division
capacity in pericycle cells results in the formation of new
lateral roots in an auxin-dependent manner. The SHORT
ROOT (SHR) and SCARECROW (SCR) transcription factor
network controls root patterning through identification of
asymmetric cell division. The recent microarray analysis of
cell-type-specific transcriptional effects has identified
CDKB2;1, CDKB2;2 and CYCD6;1 as key downstream
targets for SHR/SCR as regulators of the cell-cycle machinery
(Sozzani et al., 2010). These studies provide experimental
support for a direct molecular link between these key developmental regulators and a cell-cycle gene.
Beside auxins, ethylene can directly modulate root growth
by modifying cell division. As an example, this can be concluded from studies on the CULLIN3 knockdown mutant of
Arabidopsis (Thomann et al., 2009). The CULLIN3-based ubiquitin protein ligase determines the stability of ACS5, a
member of the 1-aminocyclo-propane-1-carboxylic acid
synthases (ACS) that catalyses a rate-limiting step in ethylene
biosynthesis. The lack of the CUL3A/B function can contribute to the stabilization of the ACS5 protein and the induction
of ethylene. This change resulted in a reduction in the root
meristem size and cell number. The authors suggest premature
exit of cells from the meristem and transition to cell expansion.
The functionality of shoot apical meristem (SAM) is derived
from co-ordinated actions of several plant hormones with a
pivotal role conscribed to auxins, cytokinins and gibberellins
(see review by Vernoux et al., 2010). Products of the homeobox
genes WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM)
control SAM organization by specifying stem cell niche or
allowing the proliferation of meristem cells (Lenhard et al.,
2002). In transgenic Arabidopsis plants with ectopic expression
of the STM gene (AINTEGUMENTA promoter/STM), the
CycB1;1::CDBGUS reporter gene as a mitotic marker indicated
the promotion of cell division in leaf primordial cells. Wu et al.
(2005) characterized the STIMPY (STIP) homeobox gene,
1199
which is also required for the growth of SAM, as manifesting
the WUS function. The expression of histone H4, as an
S-phase-specific marker was not detected in the apical region
of mutant stip seedlings. The authors reported that sucrose treatment could restore the stip mutant phenotype.
Leaf initiation on the flanks of the SAM and early development rely on the division of cells in regions with increased
auxin concentration (Scarpella et al., 2010). The direct involvement of an auxin-binding protein (ABP1) in the alteration of
cell-cycle gene expression and leaf size/morphology was
demonstrated by transient reduction of this protein (Braun
et al., 2008). Conditional repression of ABP1 activity in
Arabidopsis leaves caused a reduction in cell size in leaves
and these cellular changes are linked to lowered transcript
levels of cyclin D genes (CYCD3;1, CYCD6;1). The E2FC
and RBR genes were activated in the same tissues. The
authors concluded that the ABP1 protein is required for
entry to the division cycle that was shown in other experimental systems such as tobacco BY2 cultured cells (David et al.,
2007). Proteolysis of cell-cycle control proteins such as
mitotic cyclins is dependent on ubiquitination that can be
carried out by E3 ligases as components of the anaphasepromoting complexes (APCs). Serralbo et al. (2006) characterized the Arabidopsis HOBBIT (HBT) gene encoding a homologue of the CDC27 protein as a subunit of APC. Analysis of
leaves of Arabidopsis plants lacking the HBT functions
revealed that both cell division and later elongation were
impaired. Importantly, the authors observed the rescue of division in epidermal cells by the underlying T mesophyll cells.
The role of HOBBIT function in the regulation of cell division
and elongation can differ in roots and leaves. HBT reduction
leads to a decrease in endoreduplication in roots, whereas
leaf clones showed decreased mitotic activity.
Horváth et al. (2006) described significant alterations in
organ size depending on down- or up-regulation of the
potato EBP1 gene both in potato and Arabidopsis plants.
This plant gene shows functional and structural homology to
human EBP1, the ErbB-3 epidermal growth factor receptorbinding protein. The EBP1 function depends on auxin and,
in over-expressing potato plants, CYCD3;1 expression in the
meristem increased in comparison with the wild type. The
expression level of the G2/M-specific CDKB1;1 gene was
found 2 – 3 times higher in young leaves. In transgenic
Arabidopsis plants the endogenous RBR1 protein level was
negatively regulated by EBP1 protein. Figure 1 presents a
cascade of auxin-responsive proteins (AXR1, ARGOS,
AINTEGUMENTA) that can modify plant organ size by elevating the expression of the CYCD3;1 gene (Hu et al.,
2003). AXR1 is a component of the RUB1-activating
enzyme, which is involved in an early step of auxin signalling
(Leyser, 2006). Over-expression of ARGOS (auxin-regulated
gene involved in organ size) genes increased leaf size by production of more cells. In these 35S-ARGOS Arabidopsis plants
the expression of ANT and CYCD3;1 genes was enhanced in
tissues. The ANT gene encodes a transcription factor of the
AP2 domain family and its ectopic expression resulted in an
increase in cell numbers and enlarged organs (Mizukami and
Fischer, 2000). The authors proposed a role for ANT in maintenance of the meristematic competence of cells through
stimulation of cyclin D3 gene expression.
1200
Dudits et al. — Cell-cycle control and signalling
CO NCL UD IN G R E M A R K S
Despite impressive progress in the discovery of plant genes
and encoded proteins functioning as key elements of the cellcycle machinery, there is a need for a deeper knowledge of
how cell division contributes to organ growth and the completion of the developmental programme under normal or suboptimal environmental conditions. The plant-specific nature of
regulation during cell-cycle progression can be clearly recognized in the mode of action of plant hormones regulating the
expression of several division control genes, including
cyclins, CDK and CDK inhibitor protein genes (KRP).
Hormonal modification of cell division activity can be partially linked to Ca2+-sensitive molecular pathways based on
post-translational modifications such as protein phosphorylation or dephosphorylation. In the future, extensive studies
on the role of Ca2+ signalling in cell-cycle events will be
expected to substantially improve our understanding of how
cell division is dependent on developmental and environmental factors. The central significance of RBRs not only in
the regulation of cell division, but also in plant organ development is now widely demonstrated in various experimental
systems. RBR functions are regulated by phosphorylation,
therefore studies on the roles of CDKs and phosphatases will
highlight novel pathways in cell-cycle control. The interactions
between E2F transcription factors and RBRs are considered
basic molecular regulatory processes in G1 – S phase transition.
Detection of the alfalfa RBR protein and its phosphorylated
form in G2 and mitotic cells extends the present models and
supports future efforts to clarify the functional role of plant
RBRs during mitosis. The present overview has cited several
examples for the influence of developmental genes such as
SHORT ROOT (SHR), SCARECROW (SCR), SHOOT
MERISTEMLESS (STM), AINTEGUMENTA (ANT) and auxinregulated gene in organ size (ARGOS) that modulate cell division activity through cell-cycle control elements. As presented
in Fig. 1, the elements of the cell-cycle control machinery primarily serve as acceptors of signals reflecting the hormonal or
metabolic status of cells and participate in the determination of
actual cell division activity according to the developmental
programme.
AC KN OW LED GEMEN T S
Several unpublished results cited in this review originate from
a research project supported by a grant from OTKA
(Hungarian Scientific Research Grant number NK-69227).
Edit Ábrahám was supported by the János Bolyai Research
Fellowship of the Hungarian Academy of Sciences. The
authors thank to Mátyás Cserháti for critically reading this
manuscript.
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