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The Plant Cell Cycle

Article  in  Annual review of plant biology · February 2003

DOI: 10.1146/annurev.arplant.54.031902.134836 · Source: PubMed

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10.1146/annurev.arplant.54.031902.134836

Annu. Rev. Plant Biol. 2003. 54:235–64

doi: 10.1146/annurev.arplant.54.031902.134836
Copyright ° c 2003 by Annual Reviews. All rights reserved
First published online as a Review in Advance on January 24, 2003

THE PLANT CELL CYCLE

Walter Dewitte and James A.H. Murray
Institute of Biotechnology, University of Cambridge, Cambridge CB2 1QT,
United Kingdom; email: [email protected], [email protected]

Key Words cell division, CDK, cyclin, cyclin-dependent kinase, Arabidopsis

■ Abstract Cell division in plants is controlled by the activity of cyclin-dependent
kinase (CDK) complexes. Although this basic mechanism is conserved with all other
eukaryotes, plants show novel features of cell-cycle control in the molecules involved
and their regulation, including novel CDKs showing strong transcriptional regulation
in mitosis. Plant development is characterized by indeterminate growth and reiteration
of organogenesis and is therefore intimately associated with cell division. This may
explain why plants have a large number of cell-cycle regulators that appear to have
overlapping and distinct functions. Here we review the recent considerable progress
in understanding how core cell-cycle regulators are involved in integrating and coor-
dinating cell division at the molecular level.

CONTENTS
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
LANDMARKS OF THE PLANT CELL CYCLE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
SYNCHRONOUS CELL SYSTEMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
GENERAL PRINCIPLES OF CELL-CYCLE CONTROL . . . . . . . . . . . . . . . . . . . . . 238
CORE REGULATORS OF THE PLANT CELL CYCLE . . . . . . . . . . . . . . . . . . . . . . 241
Cyclin-Dependent Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
CDK-Activating Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
Cyclins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
CDK Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
CDK Subunit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
Retinoblastoma Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
E2F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
CELL-CYCLE TRANSITIONS AND PROGRESSION . . . . . . . . . . . . . . . . . . . . . . . 251
From G1 Into S Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
G2 /M Transition and Mitosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
Progression Through Mitosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
Endoreduplication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
HORMONES AND THE CELL CYCLE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255

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236 DEWITTE ¥ MURRAY

INTRODUCTION
Although it seems obvious today, the concept that the cells of an organism increase
by the division of existing cells is relatively new because only in 1850 was it shown
that cells originate by the cleavage of preexisting cells, and Virchow’s aphorism
“omnis cellula e cellula” (every cell from a preexisting cell) became the foundation
of today’s theory of tissue formation (78).
During the past two decades, substantial progress has been made in our un-
derstanding of the molecular mechanisms of cell proliferation, revealing that
conserved or similar fundamental mechanisms are operational at the core of the
cell-division cycle of all eukaryotes. However, current studies of developmental
processes, coupled with the availability of whole genome sequences, have led to
the view that the mechanisms of pattern formation have evolved independently in
plants and animals (86). It is therefore to be expected that cell-cycle controls differ
between plants and other groups, not only in their details but also in the mech-
anisms by which developmental and environmental influences on cell division
interact with cell-cycle control. These plant-specific aspects of cell division are
now starting to be unravelled, and in this review we survey our current knowledge
of the molecular processes underlying cell division in plants and briefly address
potential control mechanisms of the cell cycle by plant hormones.

LANDMARKS OF THE PLANT CELL CYCLE

The mitotic cell cycle encompasses four sequential ordered phases that tempo-
rally distinguish the replication of genetic material from the segregation of du-
plicated chromosomes into two daughter cells. Lag or gap (G) phases therefore
separate the replication of the DNA (S phase) and the segregation of the chro-
mosomes (M phase, mitosis). The G1 phase (the first gap) intercedes between
the previous mitosis and the entry into the next S phase, whereas the G2 phase
separates the S phase from the subsequent M phase. Cells in G2 are therefore dis-
criminated from G1 cells by possessing a double DNA content. The gap phases
allow the operation of controls that ensure that the previous phase has been ac-
curately and fully completed, and not surprisingly the major regulatory points
in the cell cycle operate at the G1/S and G2/M boundaries, which correspond to
points of potential arrest as a consequence of evaluation of external conditions
(135a).
Cell division requires an array of complicated processes that must be executed
in a spatially and temporally controlled manner. During G1, cells must integrate
relevant signals before making the decision to initiate DNA duplication, which
implies commitment not only to S phase but also to completion of cell division.
DNA replication is initiated following the G1/S transition, and after the doubling of
the genetic material, the mechanical structures allowing separation of the chromo-
somes have to be put in place, starting with the reorganization of the cytoskeleton
early in G2 (140).
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PLANT CELL CYCLE 237

At the onset of mitosis, the interphase arrays of cortical microtubules that are
arranged tranversely with respect to the main axis of growth rearrange into a
narrow cortical ring, the preprophase band. This is unique to plants and consists of
a belt-like arrangement of microtubules and actin filaments encircling the future
division plane. The preprophase band dissolves as the mitotic spindle is built, which
segregates the chromosomes during anaphase. The microtubules then rearrange
again to form the phragmoplast, which organizes the synthesis of the new cell wall
required between the daughter cells. Phragmoplast assembly starts centrally and
expands with the growing cell wall toward the exterior of the cell (66).
Differentiating plant cells often display an alternative cycle known as endore-
duplication, characterized by an increase in the nuclear ploidy level that results
from repeated S phases with no intervening mitosis (61). In some species, such
as Arabidopsis, this produces ploidy levels up to 32C in the final stages of leaf
development (39). It is also a characteristic of certain specialized cell types, such
as trichomes and the cells within leguminous nodules that host nitrogen-fixing
bacteroids (37). In all cases, endoreduplication appears to occur only after cells
have ceased normal mitotic cycles (24, 37) and in general may be associated with
the benefit from increased DNA content to support larger cytoplasmic volumes.

SYNCHRONOUS CELL SYSTEMS

Within the shoot apices of intact plants cell division is essentially asynchronous
and there is little or no coordination of division timing between cells (35). Although
certain cellular aspects, including the intracellular localization of proteins, can be
studied in individual cells and therefore do not require consistent timing of division
between cells, the biochemical and molecular analysis of cell division is predicated
on the availability of cell systems that allow populations of cells to be synchronized.
Although a certain degree of synchrony can be achieved in intact seedlings or plant
tissues by the use of inhibitor/block release [e.g., the work of Sala et al. (111)],
the most suitable systems for detailed analysis are in vitro suspension cultures of
plant cells, which can be grown in shake flasks (42). In such systems, cell division
is removed from any developmental context. Ideally, all cells progress through
the cell cycle at the same rate from the same initial starting point, and various
procedures have been developed for different cultures that allow an approximation
of this ideal. These procedures involve some method of accumulating cells at a
specific point in the cycle, followed by the reactivation of cell-cycle progression.
The major approaches are the use of reversible inhibitors of specific cell-cycle
processes to block progression through the cycle, followed by release of the syn-
chronized cells, and the removal and resupply of specific nutrients or other factors
that may be combined with cycles of subculturing. The application of inhibitors to
cell-cycle studies has been reviewed recently (96); the most important agents are
the DNA-polymerase inhibitor aphidicolin, which blocks cell-cycle progression
in early S phase, and the antimicrotubule herbicides such as propyzamide, which
block cells in early mitosis (89).
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238 DEWITTE ¥ MURRAY

A number of cultures capable of various degrees of synchronization have been

developed in species including Acer (65), Catharanthus roseus (3), alfalfa (64a),
Nicotiana plumbaginifolia (146), and soybean (75). However, the gold standard
of synchronizable cultures, providing not only the best levels of synchronization
in plants but also in all higher eukaryotes, is the tobacco Bright Yellow-2 (BY-2)
cell line developed by Nagata and coworkers (89), which has become a major
factor in the progress of understanding plant cell division. This culture is readily
synchronizable with aphidicolin to yield cells that retain a degree of synchrony for
more than one complete cycle, and mitotic synchrony can be further enhanced by
adding an additional block/release step with propyzamide.
Cultures of the key plant species Arabidopsis are recalcitrant to the development
of effective synchrony, but we have recently selected cultures that offer sufficient
levels of synchrony for molecular and biochemical analysis, using both aphidicolin
and nutrient-induced synchrony (80, 81).

GENERAL PRINCIPLES OF CELL-CYCLE CONTROL

The eukaryotic cell cycle is regulated at multiple points, but all or most of these
involve the activation of a special class of serine-threonine protein kinases, which
are functionally defined as requiring binding for activity to a regulatory protein
known as a cyclin and are therefore named cyclin-dependent kinases (CDKs).
Yeasts have a single CDK responsible for cell-cycle control that possesses the
canonical sequence PSTAIRE (single amino-acid code) within its cyclin-binding
domain. This CDK is conserved in all eukaryotes and is hence often referred to by
its fission yeast name of cdc2. However, in higher eukaryotes there are multiple
additional CDKs that have roles at different points in the cell cycle; these CDKs
are not conserved between animals and plants and have variant sequences in their
cyclin-binding domain. In animals the nomenclature CDK1 to CDK7 has been
adopted (95), whereas in plants the lack of direct equivalents (except between
CDK1 and CDKA) has led to the adoption of an alphabetical suffix [CDKA to
CDKE (62)], which replaces an earlier confused nomenclature based on cdc2.
The first cyclins were identified in sea urchin eggs as proteins whose levels
fluctuate during the cell cycle and were so named because of this transitory and
cyclical appearance (33). They provide the primary mechanism for control of CDK
activity because the CDK subunit is inactive unless bound to an appropriate cyclin
(Figure 1). Cyclins are a diverse group of proteins with low overall homology that
share a large, rather poorly conserved region responsible for their interaction with
the CDK; this region is referred to as the cyclin core. The cyclin core stretches about
250 amino acid residues and is organized in two folds of five helices. The first fold
is the cyclin box and comprises ∼100 amino acid residues (93), representing the
region of highest conservation, although it contains only five absolutely invariant
positions. The crystal structure reveals the cyclin box as the face of interaction
with the cognate CDK (60).
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PLANT CELL CYCLE 239

Figure 1 Cyclin-dependent kinase (CDK) activity is regulated at multiple levels.

Monomeric CDK lacks activity until it is complexed with cyclins (CYC) and acti-
vated by phosphorylation by CDK-activating kinase (CAK). Its activity can therefore
be regulated by the abundance of the CDK or cyclin components through controlled
transcription, translation, intracellular localization, or regulated destruction, or by the
regulated activity of CAK. In addition, activity can be inhibited by phosphorylation
by WEE1 kinases or the binding of inhibitor proteins [Kip-related proteins (KRP)].
Inhibitors may block the assembly of CDK/cyclin complexes or inhibit the kinase activ-
ity of assembled dimers. CDK subunit (CKS) proteins scaffold interactions with target
substrates.

Cyclins fall into many classes that share homology and, at least to some degree,
conserved function between animals and plants. A classification based on sequence
organization indicates that five types of cyclins exist in plants (A, B, C, D, and H
types) (105, 134). A-type cyclins generally appear at the beginning of S phase, are
involved in S-phase progression, and are destroyed around the G2/M transition.
B-type cyclins appear during G2, control G2/M and mitotic transitions, and are
destroyed as cells enter anaphase. D-type cyclins control progression through G1
and into S phase and differ from A and B types by generally not displaying a cyclical
expression or abundance; their presence appears to depend on extracellular signals
that stimulate or maintain division. If such signals are removed, levels of D-type
cyclins decline rapidly, resulting in cells remaining blocked in G1. In animals E-
type cyclins are strongly regulated at the G1/S boundary, but a direct equivalent
has not been observed to date in plant cells.
The levels of cyclins are generally determined by highly regulated transcription
as well as by specific protein-turnover mechanisms. A- and B-type cyclins possess
a “destruction box” that targets their timely removal by the anaphase-promoting
complex during early-to-mid mitosis. D-type cyclins are conjugated to ubiquitin
by an SCF complex and then subjected to proteasome degradation.
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240 DEWITTE ¥ MURRAY

No crystal structure of a plant CDK has been reported, but all eukaryotic CDKs
share substantial structural similarity, characterized by a bilobal structure. The
catalytic cleft, with ATP- and substrate-binding sites, lies between the N-terminal
and C-terminal lobes, and cyclin binding stabilizes the catalytic site within the
otherwise rather flexible structure [reviewed by Joubès et al. (62)]. However, access
to the active site remains restricted by a loop of the CDK known as the “T-loop.”
Phosphorylation of a conserved threonine residue within the T-loop induces a
conformational change that allows proper binding of the substrate and access to the
γ -phosphate group of bound ATP. Phosphorylation of Thr160, or the functionally
equivalent residue, is therefore essential for CDK activity and is carried out by a
CDK-activating kinase (CAK). Two forms of CAK are known, one form classified
as CDKF (131, 134), that can substitute for the CAK mutation in yeast, as well
as heterodimeric complexes consisting of CDK7 and cyclin H (animals) or their
homologues CDKD/CYCH in Arabidopsis.
Further regulation of CDK activity occurs by inhibitory phosphorylation of
amino-terminal residues around threonine-14 and tyrosine-15, which is catalyzed
by the WEE1 kinase, recently reported in maize and Arabidopsis (122, 127, 134).
In yeast and mammals activation of CDK activity occurs through a phosphatase
known as CDC25. Although homology searches do not reveal a direct homologue
in plants, it is likely that a functional equivalent is present.
Thus, the activity of the basic CDK-cyclin module is potentially controlled not
only through the levels of cyclins and CDK, owing to regulated transcription, trans-
lation, protein turnover, and/or sequestration and intracellular localization, but also
by activating (CAK-mediated) and inhibitory (WEE1-mediated) phosphorylation.
However, further mechanisms of modulation exist through the action of inhibitor
and scaffolding proteins. CDK inhibitors have key roles in controlling cell-cycle
progression, and there is limited sequence conservation between the animal class
of CKI, known as Kip, and plant CDK inhibitors. Plant proteins are known as ICK
(inhibitors of CDK) or KRP (Kip-related proteins) and appear to bind both CDK
and cyclin subunits (136). CDK subunit (CKS) proteins related to budding yeast
p16suc1 also exist in plants and animals. These proteins can act as inhibitors and
activators of CDK activity and appear to have a role in scaffolding the interaction
of the CDK complex with substrates (7).
CDK activity results in the covalent addition of a phosphate group to a substrate
protein at a serine or threonine residue, which modifies the substrate’s properties. A
well-known example of a substrate that is phosphorylated at a specific cell-cycle
phase is the extensive phosphorylation of histone H1 in mitotic chromosomes;
histones are therefore a commonly used substrate for CDK/cyclin phosphoryla-
tion assays. Other known CDK targets include components of the cytoskeleton
and the retinoblastoma (Rb) protein, whose phosphorylation controls the G1/S
transition. In other cases, CDK phosphorylation results in the destruction of spe-
cific cell-cycle components because recognition of targets for ubiquitin-mediated
proteolysis by F-box proteins normally requires previous phosphorylation. In
this way, the cell cycle can be given direction because the activation of CDK
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PLANT CELL CYCLE 241

activity results in the destruction of components needed for the previous phase
(65a).

CORE REGULATORS OF THE PLANT CELL CYCLE

Cyclin-Dependent Kinases
The sequencing of the complete Arabidopsis genome has allowed the full comple-
ment of a plant’s cell cycle genes to be defined. In Arabidopsis there appear to be
four types of CDK and two types of CAK (62, 134).
The functions of CDKA and CDKB are the best documented. In Arabidopsis
there is a single gene for CDKA; it carries the PSTAIRE amino acid hallmark, and
as in other plants, the abundance of CDKA mRNA does not fluctuate significantly
throughout the cell cycle (36, 81, 114, 123). CDKA kinase activity is upregulated
during G1 phase and is high during S phase. Results in tobacco BY-2 cells suggest
that activity remains high throughout G2 until the G2/M transition (104, 123),
whereas in Arabidopsis cells there appears to be a reduction in activity during G2
and a second peak at the G2/M transition (81).
The expression of A-type CDK in plants is confined to tissues in which the cells
have retained the capacity to divide (36, 114). However, CDKA activity within cells
is not regulated at the level of its expression because constitutive overexpression
does not provoke a phenotype (49). Rather its activity is regulated posttransla-
tionally, and constitutive overexpression of a dominant-negative mutant form of
Arabidopsis CDKA protein, which does not possess kinase activity, causes reduced
cell number in transgenic tobacco plants (49). These and other results suggest that
CDKA activity is rate limiting and essential for plant cell division, and although
some of its cyclin partners are known, it is not clear with which of these CDKA
performs its essential role(s).
Immunolocalization experiments, and more recently the use of a CDKA-green
fluorescent protein (GFP) fusion in tracking and cosedimentation experiments,
provided detailed information on the intercellullar localization of CDKA during the
cell cycle (15, 125, 141). During interphase CDKA-GFP is detected in the nucleus
and the cytoplasm with the nuclear CDKA-GFP tightly associated with chromatin.
During late G2 CDKA-GFP is briefly associated with the cortical portion of the
preprophase band. During prophase it is detected in the condensing chromatin,
and during prometaphase it is recruited to the metaphase spindle, a result also
confirmed in cosedimentation experiments. At anaphase CDKA is localized to
the central region of the spindle; during telophase the CDKA accumulates on the
midline of the phragmoplast. At later stages of telophase CDKA reassociates with
the chromatin (141). This close involvement of CDKA with the rearrangements of
the microtubules that accompany mitosis suggests it has a key role in controlling
microtubule dynamics and hence also mitotic progression (126).
B-type CDKs are unique to plants, and the Arabidopsis CDKBs are subdivided
in two subgroups, each with two members (134). The expression of both CDKB1
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242 DEWITTE ¥ MURRAY

and CDKB2 has been demonstrated and documented (80, 81, 98, 114, 123). Both
subgroups exhibit cell-cycle regulation of their expression, a feature that is unique
to plant CDKBs and has not been observed for any type of CDKs in other
eukaryotes.
The CDKB1 subgroup normally has the hallmark sequence PPTALRE and is
expressed from the onset of S phase until mitosis in a number of species examined
including Antirrhinum (cdc2c) (36), alfalfa (cdc2MsD) (76), tobacco (123), and
Arabidopsis (81, 114). The CDKB2 type is normally characterized by PPTTLRE
and is expressed only in G2-M cells where examined in Antirrhinum (cdc2d ) (36),
Arabidopsis (81), and alfalfa (cdc2MsF ) (76).
It is presumed that CDKB1 and CDKB2 kinase activity is limiting for the G2/M
transition. Constitutive overexpression of a dominant-negative mutant of a CDKB
in tobacco increased the proportion of cells with a 4C content, indicating that
cell cycle progression is inhibited some time prior to nuclear membrane degra-
dation (98). By in situ hybridization, CDKB expression is confined to actively
dividing tissues and is dispersed in a patchy pattern in these tissues, indicative of
a cell-phase-specific accumulation. Its expression is excluded from endoredupli-
cating tissues (58, 63). Furthermore, double-labeling experiments in Antirrhinum
revealed the accumulation of CDKB1 transcripts in cells in S, G2, and M phase
and of CDKB2 transcripts in cells in G2 and M (36). Activity peaks at the G2/M
transition (123). Immunolocalization of alfalfa CDKB2;1 (cdc2MsF ) revealed
colocalization with microtubular structures such as the preprophase band, pre-
prophase spindle, metaphase spindle, and phragmoplast (83). CDKs may also
function outside the cell cycle, as downregulation of Arabidopsis CDKB1;1 re-
duced the growth of etiolated seedlings primarily by inhibiting the elongation rates,
independently of cell division or endoreduplication (145).
C-type CDKs are most closely related to the cholinesterase-related cell-division
controller (CHED) kinases of mammalian cells; they share the PITAIRE motif with
these proteins, and no function in the cell cycle has yet been demonstrated (73a).
In mammals, CHED kinases are suspected to be involved with megakaryocyte
differentiation in hematopoiesis (71). Tomato CDKC failed to interact with A,
B, or D cyclins, and although it is preferentially expressed in dividing tissues, its
expression pattern does not indicate a cell-phase-specific regulation (63). In alfalfa
the expression of CDKC is also constant throughout the cell cycle (76). However,
no loss-of-function data are yet available to definitely rule out a cell-cycle function
of this type of CDK.
The SPTAIRE CDK, found in alfalfa, was classified as an E-type CDK (62),
and on the basis of homology a related sequence was detected in Arabidopsis
(134). Their possible function in the cell cycle is still unclear. CDKE maintains an
unchanged basal level throughout the cell cycle (76).

CDK-Activating Kinases
The first plant CAK, R2, was identified in rice (45, 112, 144) and served as a model
to identify three related sequences in the Arabidopsis genome that were classified
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PLANT CELL CYCLE 243

into the group of D-type CDKs (134). Additionally, a further, unrelated Arabidop-
sis CAK was isolated in a yeast complementation assay, and the kinase activity
of immunoprecipitates with anti-CAK antibody toward CDKA was confirmed.
Unlike monomeric yeast CAK, the Arabidopsis CAK did not display activity as
a monomer, suggesting the need for a posttranslational modification or the asso-
ciation with a regulatory subunit for activity (131). Because the sequence of this
cak1At was unrelated to CDKD and the rice CAK R2, apart from its conserved
kinase domain, it was recently classified in a distinct group, CDKF (134). It there-
fore appears that there are two classes of CAK in Arabidopsis. Rice CAK R2
not only has kinase activity toward CDK but also phosphorylates the C-terminal
domain of RNA polymerase II. R2 is preferentially expressed during S phase,
and the kinase activity associated with R2 using the carboxy-terminal domain as
a substrate peaks during S phase (34). Unlike rice R2, the Arabidopsis CDKF;1
complexes cannot phosphorylate the carboxy-terminal domain, but specifically
phosphorylate CDKs (131). Inducible cosuppression of Arabidopsis CDKF using
either sense or antisense constructs led to a decrease of CDK activity and caused a
premature differentiation in root cells (132). The use of the specific CDK inhibitor
roscovitine also caused differentiation, but other blockers of the cell cycle such as
aphidicolin did not. This suggests that continued CDK activity (rather than cell
division itself) is directly important in maintaining the undifferentiated state of
meristematic cells.

Cyclins
A-TYPE CYCLINS A-type and B-type cyclins could be isolated in a variety of plant
species by polymerase chain reaction (PCR)-based cloning strategies, benefiting
from the relatively conserved cyclin box (46, 48). Although early analyses (based
on a limited numbers of sequences) suggested ambiguities in the assignation of
cyclins between the A and B subgroups, later, more detailed studies showed that
animal and plant A- and B-type cyclins show clear relationships in their overall se-
quence and the nature of their destruction box (105, 106). Levels of A-type cyclins,
like B-type cyclins, are tightly controlled by cell-cycle-dependent proteolysis, and
this is conferred by the N-terminal destruction box (41).
A-type cyclins are subdivided into three different subclasses: CYCA1, CYCA2,
and CYCA3 (12, 105). In Arabidopsis 10 sequences encoding A-type cyclins have
been identified; four of these are predicted by bioinformatics, and the expression
of the other six has been confirmed (134). In most plants screened, members of all
three subclasses of A-type cyclins have been found.
Expression analysis in various models indicated that A-type cyclins are ex-
pressed before B-type cyclins in the plant cell cycle, beginning around the onset of
S phase (38, 56, 70, 82, 103, 116). In tobacco BY2 cells, sequential expression of
different A-type cyclins was analyzed during the cell cycle. The expression of two
A3-type cyclins was upregulated at the G1/S transition, whereas a gene belonging
to the A1-type was induced at mid-S phase. These data suggested that the members
of the different subclasses have different biological functions (103).
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244 DEWITTE ¥ MURRAY

An alfalfa CYCA3 (CycMs3) was expressed prior to DNA synthesis upon cell-
cycle reentry of differentiated cells and could complement a G1-cyclin-deficient
yeast, suggesting a possible role in cell-cycle reentry or the G1/S transition (82).
Although expression timing is consistent with this interpretation, the ability to
rescue G1-cyclin-deficient yeast cells should not be regarded as a criterion for
cyclins involved in G/S control because B-type cyclins, which are not expressed
during G1, can share this potential (20).
Localized induction of CYCA3;2 expression stimulated cell division in pri-
mordia and young organs in tobacco, suggesting that in certain situations CYCA
activity can be rate limiting for cell division. Intriguingly, this transient local in-
duction in cell division in primordia led to a reduction in cell number in the cognate
region of the mature leaf (142), suggesting that control of cell division in localized
domains is important for proper morphogenesis.
A detailed study of the expression kinetics of A2-type cyclins in partially syn-
chronized cell suspension cultures revealed that transcript levels peaked during G2,
dropped during M phase, and then rose again during G1 and S. CYCA2 protein lev-
els dropped during G1, rose during S, and then remained at a constant level during
G2 and M phase. However, the CYCA2-associated kinase activity was biphasic;
it transiently peaked during mid-S phase, then dropped to a background level and
reached its maximum level at the G2/M transition (110).
Intriguingly, CYCA2 was shown to be able to interact with the maize retinoblas-
toma protein in a yeast two-hybrid experiment. This interaction requires an intact
cyclin-box, indicating that a yeast CDK probably forms a complex with CYCA2,
and this heterodimer interacts with the Rb instead of directly with the CYCA2
(110).
A detailed expression analysis of Arabidopsis CYCA2;1 expression in the root
pericycle indicated that it was associated with the pericycle cells at the xylem poles,
which arrest in G2, and was not detected with the pericycle cells at the phloem poles,
which remain in the G1 phase of the cell cycle (5). Although a role for CYCA-
kinase activity in the G1/S transition certainly cannot be excluded at this point,
particularly in the case of CYCA3, it seems from the maxima of analyzed CYCA-
associated kinase activities during both S and G2, and their expression pattern in
tissues, that they are predominantly involved in S and G2-phase progression rather
then in G1 progression.
Details on subcellular localization of CYCAs during the cell cycle are starting
to emerge, but we are still far from a complete picture. During S phase a CYCA3; 1-
GFP fusion protein was restricted to the nucleus and nucleolus; in cells undergoing
mitosis no CYCA3;1-GFP was detected (18). Similarly, in alfalfa CYCA2 was de-
tected in the nucleus until prophase but was undetectable during mitosis (110). Im-
munolocalization experiments in Zea mays indicated that CYCA1 becomes nuclear
after prophase and binds to microtubular arrays during mitosis (84, 85). Generally,
these data are consistent with distinct roles for the various classes of A-type cyclins.

B-TYPE CYCLINS Among the first cell-cycle genes cloned in plants were three
B-type cyclins, two from carrot and soybean (46) and one from Arabidopsis (48).
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PLANT CELL CYCLE 245

Apart from their sequence differences, B-type cyclins distinguish themselves from
A-type cyclins by their somewhat later expression pattern during the cell cycle.
All identified B-type cyclins were subdivided into two subclasses, CYCB1 and
CYCB2 (105), but recently the presence of a B-type cyclin gene in the Arabidopsis
genome, which encodes for a B-type-cyclin-like protein without the typical B-type
destruction box, was predicted and assigned to a third class, CYCB3 (134). There
are two genes encoding for both CYCB1 and CYCB2 cyclins in Arabidopsis.
Microinjection of tobacco CYCB1;1 mRNA into Xenopus oocytes overcame
their natural G2/M arrest, as do mammalian cyclins (99). Ectopic expression of
the CYCB1;2, but not of the CYCB1;1, cyclin in normally endoreduplicating tri-
chomes forces the G2/M transition and causes the trichome cell to convert what
would normally be endoreduplication cycles into cell divisions producing multi-
cellular trichomes (113), indicating that plant CYCB1 are indeed functionally
involved in the G2/M transition.The mechanism involved in G2-M-specific tran-
scriptional regulation has been the topic of extensive studies. In synchronized
cell suspensions and plants, CYCB transcripts are absent until S phase, rise dur-
ing G2, reach a maximum during G2 and early M phase, and decline rapidly as
M phase progresses (5, 51, 81, 99, 129). The use of a β-glucuronidase (GUS) re-
porter fused to the full-length CYCB1;1 promoter indicated that the levels of GUS
mRNA mimicked the expression kinetics of the CYCB1;1 in cell systems, showing
that the kinetics of B-type cyclin mRNA accumulation are conferred by transcrip-
tional regulation (56, 117, 129). Deletion analysis of the Catharanthus CYCB1;1
promoter in combination with the luciferase reporter gene allowed the identifica-
tion of a nine-base-pair element containing a central core pentamer with homology
to Myb-binding sites that is responsible for the M-phase-specific regulation. Fur-
thermore, this MSA (M-phase-specific activator) element was conserved in the
promoters of several M-phase-regulated genes (57, 130). Three cDNAs encod-
ing Myb proteins (NtMybA1, NtMybA2, and NtMybAB) were isolated from a
tobacco BY-2 cDNA library using MSA elements from Catharanthus and tobacco
as bait in a one-hybrid screen (55). Furthermore, NtMybA1 and A2 activated the
MSA-containing promoters, whereas NtMybB inhibited their activity. It there-
fore appears that Myb factors are conserved MSA-interaction factors and regulate
M-phase-specific transcription. However, different factors may regulate other
CYCB genes, as two interacting proteins, which were found to be a putative Myb-
like factor with a Myb-DNA binding domain and a protein that contains a Myc-type
dimerization domain and leucine zipper, were identified as binding the Arabidopsis
CYCB1;1 promoter (97).
As mentioned above, levels of B-type cyclins are regulated not only by tran-
scriptional control but also by proteolysis, and CYCB destruction by the anaphase
promoting complex marks the transition from metaphase into anaphase in ani-
mals, and possibly in plants (41). CYCB protein levels are therefore high only
during a narrow window of the cell cycle; tobacco CYCB1;1 protein levels rose
at the end of S phase and dropped early in the M phase in a synchronized tobacco
BY2-culture (17). Unlike the situation in vertebrate and yeast cells, in which over-
expression of a nondegradable form of cyclin B blocks mitosis, in BY-2 cells
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246 DEWITTE ¥ MURRAY

nondegradable CYCB1;1 did not profoundly perturb progression through mitosis,

nor did it increase CDK kinase activity, but CYCB1;1 negatively affected cy-
tokinesis, resulting in a low frequency of multinucleate cells (18). This indicates
that additional mechanisms regulate CDK and CYCB1 activity (17, 41) and that
CYCB1 destruction is not a prerequisite for the completion of mitosis.
In G2 tobacco, CYCB1;1 is either nuclear or cytoplasmic. In prophase,
CYCB1;1-GFP fusion protein was found on the condensing chromosomes, and no
colocalization with mitotic spindles was observed for this cyclin. As expected, in
anaphase and telophase the fusion protein was undetectable because it is destroyed
at the metaphase/anaphase boundary (18).
The expression of B-type cyclins in plants is mainly associated with cycling
cells (5, 58, 114). Fusion of GUS to the promoter and the coding region for the
first 50 amino acid residues of Arabidopsis CYCB1;1, a region that includes the
destruction box, produces a GUS fusion protein that is expressed only in G2/M
cells and is destroyed as cells pass through mitosis (16). This marker is therefore an
excellent indicator of mitotic activity in plant tissues. However, the situation at the
protein level is more complex, as in maize root tips only the CYCB1;2 protein was
completely absent from nondividing tissues; CYCB1;1, CYCB2;1, and CYCA1;1
disappeared from the cytoplasm but persisted in the nuclei of elongating cells at a
lower level (85).
Targeted constitutive overexpression of Arabidopsis CYCB1;1 to division-com-
petent cells promoted cell division in root meristems and increased root growth,
indicating that this CYCB1 might be rate limiting for division in these cells (29),
whereas ectopic expression of CYCB1;1 failed to overcome the G2/M block in
endoreduplicating cells (113). However, the mechanism behind these phenomena
is unclear and may be linked with the specific ability of some root cells to arrest
in G2 (5).

D-TYPE CYCLINS D-type cyclins were originally identified by their capacity to

complement yeast strains defective in G1 cyclins (19, 120). They were defined as
D-type cyclins on the basis of a low sequence homology to animal D-type cyclins
and the presence of the conserved LxCxE motif, which is responsible for their
interaction with retinoblastoma-related (Rb or RBR) proteins (1, 53). They have
been identified in a variety of plant species [see review by Meijer & Murray (79)];
in Arabidopsis, ten D-type cyclins sequences have been identified, and these can
be classified according to Vandepoele et al. (134) into seven subclasses. Using
this classificiation of Arabidopsis D-type cyclins, the CYCD3 subclass has three
members, the CYCD4 family two, and the other groups all have a single member.
The CYCD4 subgroup has relatively high homology with CYCD2;1 (CYCD4;1
and CYCD4;2 have 60% and 54% similarity with CYCD2;1, respectively), sim-
ilar to the homology shared by other subgroup members (CYCD3;1 shares 61%
similarity with CYCD3;2 and 57 % with CYCD3;3), and it has been proposed
that the CYCD4 cyclins should be regarded as members of the CYCD2 subgroup
(54, 94).
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PLANT CELL CYCLE 247

D-type cyclins share conserved structural features with mammalian D-types

in addition to the cyclin box, such as the Rb-interaction motif and in most cases
putative PEST (proline, glutamine, serine and threonine rich) sequences involved
in protein degradation. The Rb-interaction domain consists of an LxCxE (single-
letter code, where x represents any residue) amino acid motif that is conserved
in both animals and plants and is located near the N terminus of the protein. For
CYCD2;1 and CYCD3;1, Rb binding has been demonstrated in vitro and depends
on an intact LxCxE motif (1, 53). Two cyclin sequences, CYCD4;2 and CYCD6;1,
coding for a CYCD-related protein without a canonical LxCxE motif, have been
predicted, and CYCD5;1 has a slightly divergent motif. The biological role of these
proteins is still unclear. In Arabidopsis both CYCD2;1 and CYCD3;1 form active
complexes with CDKA but not with CDKB1;1 (47). Interaction with other possible
CDK partners has not been probed yet, although alfalfa CYCD4 associated with
the alfalfa CDKB2 in yeast (83).
Expression of D-type cyclins is low in stationary BY-2 cell cultures (121), al-
though in an Arabidopsis cell culture CYCD2;1 and CYCD3;1 transcripts were
constant throughout the growth cycle from subculturing to stationary phase. How-
ever, when the culture medium was adjusted so that sugar levels were depleted
by the time stationary phase was reached, CYCD3;1 transcripts dropped signifi-
cantly. These results show that CYCD2;1 and CYCD3;1 expression is not limited
to actively dividing cells but rather is dependent on medium composition and
hence extrinsic signals. Moreover, together with other evidence, it shows that both
CYCD2;1 and CYCD3;1 expression is dependent on sugar availability and is in-
duced in response to sugar supply (108). Induction of both CYCD2;1 and CYCD3;1
by sugars occurred without progression through G1 or de novo protein synthesis,
as shown by its insensitivity to cycloheximide, a protein synthesis inhibitor that
also blocks progression through G1 phase in Arabidopsis (38). This induction is,
however, highly sensitive to inhibitors of protein phosphatases used at low con-
centrations, and the response profiles of CYCD2;1 and CYCD3;1 induction are
different to two phosphatase inhibitors, indicative of the involvement of different
phosphatases or pathways (108).
Further analysis showed that although CYCD2;1 and CYCD3;1 transcript levels
drop on sucrose removal, total levels of CYCD2;1 protein remain fairly constant,
whereas CYCD3;1 protein levels follow the transcript kinetics (47). CYCD3;1
therefore parallels the classic response of mammalian D-type cyclins to exter-
nal mitogenic signals (118). However, the kinase activity associated with both
CYCD2;1 and CYCD3;1 dropped upon sucrose removal. Immunoprecipitation re-
vealed that in stationary phase CYCD2;1 is not bound to CDKA but is sequestered
in an unknown complex that removes CYCD2;1-associated kinase activity without
affecting the total protein level (47).
Human D-type cyclins are degraded by ubiquitin-mediated proteolyis that in-
volves phosphorylation of the threonine-286 residue of the protein. Threonine-286
or its equivalent is located within the hydrophilic PEST domain and is conserved
in D-type cyclins of various organisms including plants, and therefore a similar
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248 DEWITTE ¥ MURRAY

mechanism for the regulation of D-type cyclin levels might be operational in plants
(90, 94).
Although both CYCD2;1 and CYCD3;1 expression is sugar regulated, CYCD3;1
expression shows strong responses to plant hormones, whereas CYCD2;1 does not
(94). CYCD3;1 is rapidly super-induced by cytokinin within one hour of treatment
at physiological concentrations, not only in cultured cells (107, 120) but also in
whole seedlings, as detected by RNA gel blots and in situ hybridization. CYCD3;1
expression is also elevated in the high cytokinin mutant pt/amp1 (13, 107).
However, the hormone response of CYCD3;1 is not limited to its super-induction
by cytokinin, as it is also upregulated by auxin, gibberelins, and brassinosteroids
(52, 94). The response to cytokinins, like its sugar response, is phosphatase medi-
ated and does not appear to require de novo protein synthesis (107), in contrast to
the induction by epi-brassinolide, which does involve de novo protein biosynthe-
sis but no regulation by phosphorylation (52). This suggests that the brassinolide
regulation of CYCD3;1 requires transcription and/or translation of upstream reg-
ulators. Abscisic and jasmonic acids reduce the levels of CYCD3;1 transcripts
in Arabidopsis cells, consistent with the effects of these hormones in blocking
cell-cycle progression in BY-2 cells (94, 128).
In Antirrhinum seedlings, CYCD1, CYCD3;1 (CYCD3a), and CYCD3;2
(CYCD3b) respond to sucrose. Interestingly, only CYCD1 and CYCD3;1 expres-
sion is significantly upregulated by cytokinin. Furthermore, only CYCD1 displays
a mild response to auxin (40).
CYCD3;1 might have an additional role besides integrating sucrose and hor-
mone signaling. Toxin-induced cell-cycle arrest in early S phase by aphidicolin
or in G1 by low levels of cycloheximide causes a strong reduction in CYCD3;1
transcript levels (38, 81), indicating that CYCD3;1 expression may also assess
correct cell-cycle progression.
In tissues, CYCD expression seems to be associated with proliferating tissues
and is excluded from differentiated tissues. CYCD4;1 expression is associated
with developing lateral roots, embryogenesis, and vascular tissues (25). CYCD3;1
transcripts accumulated in proliferating shoot tissues such as the meristematic,
young leaf, and developing vascular tissues (107, 28). Furthermore, D-type cy-
clins of the same class can be differentially expressed in dividing tissues during
development. In Antirrhinum, CYCD3;2 (CYCD3b) is expressed throughout the
vegetative meristem, whereas the cytokinin-inducible CYCD3;1 (CYCD3a) tran-
scripts are confined to early stages in the development of organ primordia. In
Antirrhinum floral meristems, CYCD3;1 expression was detected in the periphery
of the inflorescence meristem, in young floral meristems, and in the tips of older
floral organs. CYCD3;2 transcripts, however, were abundant throughout the inflo-
rescence meristem and floral meristems, being apparently expressed in all dividing
cells. CYCD1 was also expressed throughout the inflorescence (40).
CYCDs may also provide a key link between developmental controls and cell
proliferation. AINTEGUMENTA encodes a transcription factor of the AP2 domain
family and was identified as a gene that regulates cell division in ovules and flow-
ers (31). Ectopic overexpression of AINTEGUMENTA in Arabidopsis increases
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PLANT CELL CYCLE 249

the size of leaves by extending the period of cell proliferation and increasing leaf
cell number and also causes increased expression of CYCD3;1 (87). Overexpres-
sion of CYCD3;1 also dramatically increased leaf cell number but did not affect
the expression of AINTEGUMENTA (28), suggesting that AINTEGUMENTA may
regulate cell number in organ development by controlling CYCD3 expression.

H-TYPE CYCLINS Genes encoding H-type cyclins have been identified in Ara-
bidopsis, rice, and poplar (134, 143). In rice and poplar high transcript levels are
associated with dividing cells. Rice cyclin H interacts specifically with the CAK
R2 but not with other CDKs, and increases the kinase activities of R2 and enhances
the rescue by R2 of a CAK mutation in budding yeast. These observations indicate
that cyclin H is the regulatory subunit of CAK (143). Further information on the
regulation of CYCH and CAK itself is likely to show whether these proteins have
a regulatory role in the plant cell cycle.

CDK Inhibitors
Binding of CDK inhibitors to CDK/cyclin complexes modulates the kinase ac-
tivity. In Arabidopsis the seven genes identified that encode proteins with limited
homology to the animal Kip/Cip family of CDK inhibitors are referred to as Kip-
related proteins (KRPs) or ICKs (inhibitor of CDK) (24, 74, 137). Apart from the
Kip-related region, the overall homology of most KRPs is rather low. The bind-
ing of some of these KRPs to CDKA has been reported independently by two
research groups, and a consensus result from their experiments was the binding
of ICK1, ICK2, and ICK7 to CDKA and the absence of CDKA interaction with
ICK5 (24, 148). Also, no KRP was found capable of binding CDKB in a two-
hybrid system in these studies. All KRP/ICK proteins interacted with CYCD3;1,
CYCD2;1, and CYCD1;1 (148), and four interacted with CYCD4;1 (24). No KRP
interacted with CYCB1;1 (24, 148). Inhibition of CDKA activity has been demon-
strated in vitro for both ICK1/KRP1 and KRP2 (139). In addition, an in vivo assay
demonstrated the ability of ICK1 to delay the progression from nuclear envelope
breakdown to metaphase in Tradescantia stamen hairs (14). Together these results
suggest that some or all KRP proteins act to inhibit formation of CYCD-CDK
complexes and to inhibit the kinase activity of preformed CDKA complexes,
but there is no direct evidence that KRP binding blocks CDK-cyclin complex
formation.
The inhibitory effect of KRP proteins on CDKA activity has been confirmed as
also affecting cell-cycle progression in transgenic plants. The targeted overexpres-
sion of KRP1 resulted in petals with a reduced cell number (148), and constitu-
tive overexpression of KRP1, KRP2, and KRP4 resulted in smaller serrated leaves
(24, 148). Aberrant floral morphology was found upon the overexpression of KRP1
and KRP4 (148). Constitutive overexpression of KRP2 reduced cell numbers in
leaves, which was correlated with and hence partially compensated by an increase
in cell size. Leaf development is characterized by largely distinct phases in which
first cell division and later cellular differentiation and accompanying expansion are
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250 DEWITTE ¥ MURRAY

the predominant processes (30). In KRP2-overexpressing plants cell division was

inhibited in the first phase, but the timing of the switch between division and cel-
lular differentiation/expansion was unaffected, suggesting no direct role for KRPs
in integrating proliferation and cellular differentiation (24).
KRP transcripts are differentially distributed in Arabidopsis plants (24, 139),
and in synchronized Arabidopsis expression systems their transcripts are differ-
entially regulated. During cell-cycle reentry of sucrose-starved cells, KRP2 is
transiently expressed during the G1 phase, but its transcripts subsequently disap-
pear. KRP1/ICK1 transcripts are present in sucrose-starved cells, and its tran-
scripts then decline but increase again at the ensuing mitosis, whereas KRP3
is limited to mitotic cells (81). KRP1/ICK1 is also strongly upregulated by ab-
scisic acid treatment (138). These results suggest different roles for the KRP
proteins during the cell cycle. KRP2 may be specifically involved in cell-cycle
reentry, and KRP1/ICK1 in maintaining cells in stationary phase and abscisic acid
responses.

CDK Subunit
CDK subunit (CKS) proteins are proposed to act as docking factors that can influ-
ence the interactions between the kinase complex and the substrates. Interaction
of Arabidopsis CKS1 and CDKA and CDKB has been demonstrated (24, 148).
The expression of CKS1 (26) is associated with dividing cells as well as with
endoreduplicating cells (58). Recently, a second homologue in Arabidopsis has
been identified by genomic analyis (134).

Retinoblastoma Protein
A single gene for an Rb-related protein exists in Arabidopsis, but in maize there are
two genes (RBR1 and RBR2) as well as differentially spliced transcripts, although
their functional significance is unknown (1). Plant Rb proteins are nuclear and are
characterized by many potential CDK phosphorylation sites (1, 22, 91). In animals
and plants Rb proteins interact with several proteins including the E2F family of
transcription factors, CYCD, and RbAp48-like proteins (1, 22, 53). In maize the
RBR genes are expressed in all tissues, but the strongest expression is associated
with the shoot apex (1). When Rb protein abundance was examined in developing
maize leaves, a gradient along the leaf lamina was found, with the highest levels
associated with differentiating cells exiting the cell cycle at the distal end of the
leaf and lowest levels associated with the proliferative zone at the leaf base (53).
The Arabidopsis homologue of the Rbp48-related Rb-interacting protein MSI1
has recently been shown to be a component of the CAF chromatin assembly factor
complex together with the proteins encoded by the FAS1 and FAS2 genes (64b),
whose loss leads to fasciation of the shoot apical meristem and inability to maintain
the expression of the meristem identity gene WUSCHEL. These results suggest
links between Rb’s role in cell-cycle control and cellular differentiation.
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PLANT CELL CYCLE 251

E2F
E2F binds DNA as heterodimers with dimerisation partner (DP) proteins, and
homologues of E2F and DP proteins are found in plants and interact with Rb
(2, 21, 43, 69, 77, 100, 115). In Arabidopsis a family of three E2F protein [E2Fa,
E2Fb, and E2Fc, according to the latest nomenclature (134)], two DP (a and
b) proteins, and three DP-E2F-like genes [DEL (also known as ELP), with mixed
sequence characteristics] have been described (21). E2Fa (also known as E2F3) and
E2Fb (E2F1) transactivate gene expression, whereas E2Fc (E2F2) appears to have
weak or no transactivation potential (21, 69, 77, 109). E2Fa and E2Fb translocate
to the nucleus and transactivate an E2F reporter gene when coexpressed with DPa
but not with DPb (69). The DEL proteins do not interact with DP or E2F proteins
but can bind to E2F sites in a monomeric form and repress the transcription of
genes under E2F promotor control (68). The E2F/DP transcription factors can
trigger genes involved in DNA replication. Rice OsE2F1 binds to the putative E2F
binding site in the promoter of proliferating cell nuclear antigen, a gene involved
in DNA replication, and confers transactivation to an E2F-reporter gene (67); cell-
cycle regulation of the tobacco ribonucleotide reductase gene is controlled by E2F
elements (10, 11).
Combined overexpression of E2Fa/DPa transcription factors in Arabidopsis
stimulated the expression of S-phase-specific genes such as DNA polymerase α,
ORC, MCM, and CDC6 (23) and stimulated the entry into S phase of fully dif-
ferentiated leaf cells (109, 23). In leaves this led to either ectopic divisions or
increased rounds of endoreduplication; the choice between these two alternatives
is proposed to be controlled by the presence or absence of a putative mitotis-
promoting factor (23). In trichomes, CYCB1;2 can provide this role (113), suggest-
ing that this cyclin or a regulator of its expression or activity might provide such an
activity.

CELL-CYCLE TRANSITIONS AND PROGRESSION

From G1 Into S Phase

A model for control of the G1/S transition has been proposed in which D-type
cyclins are primary mediators of the G1/S transition and hence have a major re-
sponsibility for stimulating the mitotic cell cycle (88) (Figure 2). Transcription of
D-type cyclins is activated by extracellular signals and leads to the formation of
active CDKA-CYCD complexes. These phosphorylate and hence inactivate the Rb
protein so that it loses its E2F association and can no longer block the activation of
E2F-regulated genes. E2F is then able to activate transcription of genes involved
in S phase and other growth and cell-cycle processes (10, 23).
The role of multiple CYCDs during G1/S control is not yet understood, nor is
it known whether Rb is their only important target. Do different CYCD kinases
phosphorylate different positions within Rb or do they act sequentially during
progression through G1 into S phase, or in different cell types?
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252 DEWITTE ¥ MURRAY

Figure 2 Model for the control of G1/S transition. CYCD3 transcription is induced
in response to sucrose and hormones and forms active complexes with CDKA. In
nondividing cells, or cells in G1, genes with E2F sites are held transcriptionally inactive
because bound E2F recruits retinoblastoma (Rb) to these promoters. The rising levels
of CYCD kinase activity phosphorylate Rb, causing it to lose E2F association. E2F-
regulated genes including CDC6, CYCD3;1, E2Fc, and CDKB1;1 are thereby activated.
The control incorporates positive and negative feedback loops because CYCD3;1 is
an E2F target, as is the E2Fc, which lacks transactivation potential and is therefore a
competitive inhibitor of E2F-regulated activity.

The role of the multiple E2F genes is also not fully resolved, although it seems
likely that E2Fc is involved in a negative-feedback loop. As already mentioned,
E2F2c appears to be a competitive inhibitor of E2F-activated gene expression
owing to its lack of an effective activation domain, and it contains an E2F-binding
site in its promoter. Increasing levels of E2F activation activity would therefore
lead to increased competitive inhibition of E2F-directed expression. This model
has been supported by the finding that when CYCD3;1 is overexpressed, resulting
in Rb inactivation, strongly increased levels of E2Fc are detected (28).

G2/M Transition and Mitosis

The G2/M transition is controlled by the level of CDK-CYCB kinase activity, and
inhibition of CDKs arrests cells in G2 and stabilizes the preprophase band (6). Dur-
ing G2 levels of transcripts encoding CYCB increase and probably associate with
both CDKA and CDKB1. Further regulation of CDK activity by both KRP proteins
and inhibitory phophorylation by WEE1 kinases is likely. Phosphatases therefore
probably play an important role in M-phase onset, although a direct homologue of
CDC25 phosphatases that activate CDKs in animals and yeast has not been found.
However, because inhibition of serine/threonine-specific protein phosphatases in
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PLANT CELL CYCLE 253

alfalfa by endothall caused premature, imperfect microtubular reorganizations and

activation of CDKB2;1 (4), these phosphatases might be involved in regulating
M-phase progression. The protein kinase CK2 shows discrete activity peaks at
G1/S and M in tobacco BY-2 cells, and blocking its activity during G1 abolished
the G2/M checkpoint, resulting in premature entry into prophase and showing links
between G1 processes and G2 controls (32).

Progression Through Mitosis

As discussed above, progression through mitosis involves the association of CDK
complexes with microtubular and chromatin structures, and CDK activity probably
plays an important role in microtubular dynamics and stability (135). Microinjec-
tion of active CDK into Tradescantia hair cells accelerates the early stages of
mitosis, whereas inhibition of CDK activity induces abnormal microtubular spin-
dles (6). Although the actual substrates of CDK have not yet been identified, CDK
complexes could phosphorylate microtubule-associated proteins such as MAP4
homologues, which regulate the dynamic assembly of microtubular structures, in
analogy with animals.
In animals, cytokinesis occurs by constriction of the cell cortex, but in plants it
depends on the phragmoplast, to which cell-plate formation is directed by trans-
port of Golgi-derived vesicles [reviewed by Staehelin & Hepler (124)]. A variety
of proteins involved in vesicle trafficking and microtubular-associated proteins
accumulate at the phragmoplast. In addition, a number of protein kinases such as
MMK3, p43Ntf6, and mitogen activated protein kinases (MAPKs) are detected on
the cell plate. NPK1, a MAPK kinase kinase that accumulates at the equator of
the phragmoplast, regulates the lateral expansion of the cell plate, and its targeted
action depends on the association with kinesin-like motor proteins (92).

Endoreduplication
Endoreduplication occurs in the development of mature tissues in many species
and in specific developmental situations such as endosperm development (44). En-
doreduplication can be considered a modified cell cycle, and data have emerged on
the molecular mechanisms behind the regulation of endoreduplication [reviewed
by Joubès & Chevalier (61)]. A general view is the downregulation of CYCA1,
CYCA2, CYCBs, and CDKB in endoreduplicating cells, which is consistent with
the proposed action of these components during M phase. Also, as mentioned
above, the overexpression of CYCB1;2 in endoreduplicating trichomes induces
mitotic divisions, indicating that this can be a limiting factor for entry into mitosis.
CDK action is a common driver of both mitotic cycles and endocycles. This
is supported by the observation that overexpression of KRP reduced the level
of endoreduplication as well as cell production (24, 147). From the increase of
CYCD3 and CYCA3 expression in endoreduplicating maturing tomato tissues
it was proposed that some D-type cyclins are involved in endoreduplication by
promoting the G1/S transition (61). However, this view cannot be generalized
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254 DEWITTE ¥ MURRAY

toward all D-type cyclins, as overexpression of Arabidopsis CYCD3;1 induced

cell divisions in normally endoreduplicating trichomes. Furthermore, constitutive
overexpression of CYCD3;1 prevented exit from the mitotic cycle and conferred
hyperproliferation of leaf tissues while strongly inhibiting endoreduplication (28).
In contrast, overexpression of the E2Fa/DP transcription factor, which stimulates
genes involved in S phase, promotes both the mitotic cycle and endoreduplication
(23). CDC6, which plays an essential role in the initiation of DNA replication in
animals and yeast, is regulated by E2F transcription factors and upregulated in
endoreduplicating cells (8, 101).
As mentioned above, endoreduplication is only initiated after the mitotic cell
cycle is stopped (24, 37) and it correlates with loss of mitotic regulators. A further
link is an activator of the anaphase-promoting complex, CCS52, the anaphase-
promoting complex being responsible for the ubiquitin-related degradation of mi-
totic cyclins. Expression of CCS52 was confined to dividing and endoreduplicating
cells but was absent from cells with no cell-cycle activity. Antisense expression
of Medicago CCS52 reduced endoreduplication in hypocotyls, again suggesting
that loss of mitotic cyclins is involved in the switch from mitotic cycles to endocy-
cles (9). However, the upstream mechanisms influencing this mechanism are still
unknown.

HORMONES AND THE CELL CYCLE

Studies spanning the past 50 years have shown the effects of plant hormones on
cell proliferation. However, because most hormones also provoke morphogenetic
effects, the cell-cycle consequences may be direct or part of the morphogenetic
response.
Cytokinins and auxins are indispensable for maintaining undifferentiated cells
in proliferation during in vitro culture and are the hormones most directly linked to
cell proliferation. A detailed review by Jacqmard et al. (59) suggests that cytokinins
have effects on the G1/S and G2/M transitions, as well as on progression through S
phase. Cytokinins and brassinosteroids induce the expression of CYCD3;1 (5, 22,
107), and the overexpression of CYCD3;1 conferred cytokinin-autotrophic initia-
tion and growth of Arabidopsis leaf calli, showing that in this system high levels
of CYCD3;1 are sufficient to replace exogenous cytokinin (107). A mutation in
the RPN12 subunit of the 26S proteasome conferred cytokinin insensitivity and
a partially constitutive cytokinin response such as anthocyanin accumulation and
inhibition of root growth and lateral root development. Interestingly, this muta-
tion also conferred overexpression of CYCD3;1 and NIA1 (119). Therefore, it
seems that pathways involving brassinosteroid and cytokinin signaling converge
on CYCD3;1 expression, which promotes entry into the mitotic cell cycle.
Tobacco BY2 cells do not require exogenous cytokinin, and zeatin-type cy-
tokinins accumulate at the end of S phase and early M phase (102). Inhibition of
isoprenoid biosynthesis at the G2/M transition combined with rescue of the inhi-
bition of mitosis by exogenous zeatin showed that this transition requires these
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PLANT CELL CYCLE 255

zeatin-type cytokinins (72), which might be linked to the nuclear localization re-
ported for this type of cytokinin (27). G1 progression was not as dependent on
cytokinins in these highly proliferating cultures (73). Similarly, in a cytokinin-
dependent N. plumbaginifolia cell suspension culture cells arrested in G2 owing to
the lack of cytokinins. Cytokinin induced dephosphorylation and hence activation
of CDKA in these N. plumbaginifolia cells in G2, as well as in tobacco pith cells
grown in the presence of auxin alone (146). Cytokinin is therefore linked with
CDK activation at the G2/M boundary either by direct activation of a phosphatase
or by downregulation of the WEE1 kinase.
Cell division is inhibited by abscisic acid. Abscisic acid induces the expression
of the CDK inhibitor KRP1/ICK1, causing a decrease in the kinase activity asso-
ciated with CDKA (138). Abscisic acid prevents tobacco BY2 cells from entering
S phase but does not affect the progression through the other cell-cycle phases,
consistent with the role of KRP1/ICK1 in regulating CDKA/CYCD3 activity (138).
Jasmonic acid application during G1 prevented DNA synthesis, whereas jasmonic
acid application during early S phase had only a minor effect on DNA synthesis
but inhibited M phase progression (128). Ethylene treatment of cell suspensions
provoked cell death at the G2/M boundary (50).

CONCLUSION
As in other eukaryotes, the cell cycle is driven by CDK-controlled phosphoryla-
tion. In plants the core cell-cycle players have been identified, and it is clear that
there is a larger number of genes than in animals, as well as completely new types
of regulators including novel CDKs and E2F-like proteins. This complexity poses
a challenge in the unravelling of the rules of this complex game. In particular,
the mechanisms by which signal transduction cascades and developmental regu-
lation impinges on the cell cycle are still largely unknown. However, considerable
progress has been made, and the application of genomics tools is likely to lead to
rapid further advances.

ACKNOWLEDGMENTS
Research in J. Murray’s laboratory is funded by BBSRC and the EU. The authors
are very grateful for the contribution by Graham Armstrong to the manuscript.

The Annual Review of Plant Biology is online at http://plant.annualreviews.org

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