cell cycle regulation in plant development

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Cell Cycle Regulationin Plant Development1

Dirk Inze and Lieven De Veylder

Department of Plant Systems Biology, Flanders Interuniversity Institute forBiotechnology (VIB), Ghent University, Technologiepark 927, B-9052 Gent,Belgium; email: [email protected], [email protected]

Annu. Rev. Genet. 2006. 40:77105

First published online as a Review inAdvance on September 1, 2006

TheAnnual Review of Geneticsis online athttp://genet.annualreviews.org

This articles doi:10.1146/annurev.genet.40.110405.090431

Copyright c2006 by Annual Reviews.All rights reserved

0066-4197/06/1215-0077$20.00

1NOMENCLATURE

By convention all names for plant genesare in uppercase italics, proteins inuppercase roman, and mutant genes or

alleles in lowercase italics. Much of thework described has been generated withArabidopsis thalianaas experimentalsystem. When no species reference isgiven for any gene, protein, or mutant, itshould be understood that it refers toArabidopsisresearch. In all other cases, thespecies name is mentioned.

Key Words

Arabidopsis, cell cycle, cyclin, cyclin-dependent kinase,

endoreduplication, plant development

Abstract

Cell cycle regulation is of pivotal importance for plant growth an

development. Although plant cell division shares basic mechanism

with all eukaryotes, plants have evolved novel molecules orchestrat

ing the cell cycle. Some regulatory proteins, such as cyclins and

inhibitors of cyclin-dependent kinases, are particularly numerous in

plants, possibly reflecting the remarkable ability of plants to modu

late their postembryonicdevelopment. Many plant cells also cancon

tinue DNA replication in the absence of mitosis, a process known a

endoreduplication, causing polyploidy. Here, we review the molec

ular mechanisms that regulate cell division and endoreduplicationand we discuss our understanding, albeit very limited, on how th

cell cycle is integrated with plant development.

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Meristem: tissueundergoing mitosis,giving rise to newcells and tissues

Pluripotent:property ofdifferentiated cell toreinitiate celldivision to generatenew plant organs

SAM: shoot apicalmeristem

Contents

INTRODUCTION . . . . . . . . . . . . . . . . . 78

UNIQUE FEATURES OF PLANT

CELL DIVISION . . . . . . . . . . . . . . . 78

THE BASIC CELL CYCLE

MACHINERY. . . . . . . . . . . . . . . . . . . 79

Cyclin-Dependent Kinases . . . . . . . . 79Cyclins . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Proteolysis . . . . . . . . . . . . . . . . . . . . . . . 84

CDK Phosphorylation. . . . . . . . . . . . 85

CDK Inhibitors . . . . . . . . . . . . . . . . . . 86

The RBR/E2F/DP Pathway . . . . . . 87

CELLULAR VERSUS

ORGANISMAL THEORY. . . . . . . 89

ENDOREDUPLICATION . . . . . . . . . 90

CONCLUDING REMARKS . . . . . . . 93

INTRODUCTION

The cell cycle is one of the most compre-hensively studied biological processes, partic-

ularly given its importance for growth and

development and deregulation in many hu-

man disorders. No other field has benefited

as much as the cell cycle from an extensive in-

terplay between research performed on a di-

versity of model organisms. Studies on yeast,

worms, flies, frogs, mammals, and plants have

contributed to a universal picture on how thebasic cell cycle machinery is regulated, and

research on these many divergent organisms

is also elucidating how evolution modified

the basic cell cycle machinery to cope with

the specific developmental and environmen-

tal challenges of each organism. However, this

picture is mainly based on experiments per-

formed on single cells. Indeed, the role of thecell cycle machinery during development has

received relatively little attention. To under-

stand how, in different organisms, the basic

cell cycle machinery integrates with devel-

opment remains an important scientific chal-

lenge. With this review, we hope to convince

the reader that plants offer exceptional op-

portunities to contribute significantly to such

a challenge.

UNIQUE FEATURES OF PLANTCELL DIVISION

In contrast to animals, plant development i

largely post-embryonic. New organs, such aroots, stems, leaves, and flowers, originat

from life-long iterative cell divisions followed

by cell growth and differentiation. Such cel

divisions occur at specialized zones known ameristems. Leaves and flowers are formed a

the shoot and floral meristems, respectively

whereas the root meristems continuously add

new cells to the growing root. The cells athe meristem are pluripotent so that thei

progeny can become committed to a spectrum

of developmental fates. Initially, the shoot api

cal meristem (SAM) produces leaves, but un

der the right developmental or environmenta

conditions, the SAM will be converted into a

floral meristem that produces flowers.Another interesting aspect of many dif

ferentiated plant cells is their ability to dedifferentiate and acquire pluripotentiality, a

feature that allows an even larger develop

mental plasticity (70, 172, 173). For example

quiescent root pericycle cells can be stimu

lated to undergo cell divisions and to form

lateral roots de novo (22). On the other hand

root cortical cells ofFabaceaecan initiate cel

division to produce large nitrogen-fixing nod

ules upon infection with symbiotic bacteriathe Rhizobia (65). In another well-known ex

ample, protoplasts of leaf mesophyll cells can

construct a new cell wall, divide, and regen

erate roots or shoots upon treatment with ap

propriate plant growth factors (67).

Development of plants is also unique be

cause rigid cell walls surrounding plant cell

prevent any cell migration. Consequently, local cell-division parameters, such as the num

ber of cells produced at the meristems and

the cell division plane, are important in determining the organization of plant tissues

as illustrated by the scarecrow mutant, in

which the deficiency of a specific asymmetri

division event in one of the descendants o

the root stem cells results in lack of a complete root cell layer (44). Furthermore, the

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rigid cell wall prevents cytokinesis by means

of constriction (as is the case for animal cells).

Therefore, plants have developed an elab-

orate mechanism to generate two daughtercells that involves two unique cytoskeletal ar-

rays, termed the preprophase band (PPB) and

the phragmoplast (4, 163, 164). Another in-

teresting feature of plant cells is that, unlike

mammalian cells, they do not develop tumors,

except as specialized responses to certain

pathogens (48).

THE BASIC CELL CYCLEMACHINERY

Any discussion on the role of cell division

in plant development and growth requires a

thorough understanding of the basic machin-

ery that controls the cell cycle. As in other eu-

karyotic organisms, cyclin-dependent kinases

(CDKs) govern the plant cell cycle. Differ-

ent CDK-cyclin complexes phosphorylate aplethora of substrates at the key G1-to-S and

G2-to-M transition points, triggering the on-

set of DNA replication and mitosis, respec-

tively. The catalytic CDK subunit is respon-

sible for recognizing the target motif (a serine

or threonine followed by a proline) present in

substrate proteins, whereas the exchangeable

regulatory cyclins play a role in discriminatingdistinct protein substrates.

Cyclin-Dependent Kinases

All eukaryotic organisms studied to date pos-

sess at least one CDK with the PSTAIRE hall-

mark in their cyclin-binding domain. In plants

too, a bona fide PSTAIRE CDK, designated

CDKA, plays a pivotal role at both the G1-to-S and G2-to-M transition points (Figures 1

and 2). The universal nature of PSTAIRE

CDKs is best illustrated by their ability to

complement functionally CDK-deficient mu-

tants of yeasts (58, 80). CDKA protein lev-

els remain constant throughout the cell cycle

(116, 137, 168). Overproduction of a domi-

nant negative CDKA ofArabidopsis thaliana in

tobacco(Nicotiana tabacum)plantsresultsinan

Preprophase band(PPB): cytoskeletalarray encircling thecell plate inside theplasma membrane athe site where the

future cell wall willjoin the parent wall

Phragmoplast:cytokinetic organellconsisting of parallealigned microtubuleand actin filamentsthat participate in thbuilding of a new cewall by targeting celwall material to thegrowing cell plate

CDKs:cyclin-dependentkinases

overall reduction of the cell division rate, thus

yielding smaller plants. However, the G1/G2

ratio remains unaltered, corresponding with

the observation that CDKA activity can bedetected at both checkpoints (78, 92, 138).

Thus, CDKA is essential at both the G1-to-

S and G2-to-M transitions of the cell cycle.

The requirement of CDKA at least for entry

into mitosis has been demonstrated by null

mutants, whose primary defect appears to be

a failure to progress through the second mi-

tosis during male gametophytic development

(86, 132).No orthologs of the mammalian G1/S-

specificCDK4andCDK6genes are present in

plants. As such, CDKA is seemingly the only

CDK active at the G1 and S phases in plant

cells, whereas the entry into mitosis is proba-

bly controlled by multiple CDKs (Figure 2).

Plants possess a unique class of CDKs, the so-

called B-type CDKs that have not been de-

scribed for any other organism (16, 80, 91).The PSTAIRE hallmark present in CDKAs is

replaced by either PPTALRE or PPTTLRE,

reflecting the existence of two subgroups,

CDKB1 and CDKB2 (186). Arabidopsishar-

bors two CDKB1 (CDKB1;1 and CDKB1;2)

and CDKB2 (CDKB2;1 and CDKB2;2) fam-

ily members. The two CDKB subgroups are

found in both monocotyledonous and di-cotyledonous species, suggesting a conserved

unique role for each of the CDKB subgroups

in cell cycle regulation, but have a slightly dif-

ferent timing in cell cycle phasedependent

transcription.CDKB1transcripts accumulate

during S, G2, and M phases, whereasCDKB2

expression is specific to the G2 and M phases

(18, 31, 60, 116, 123, 125, 138, 159, 168, 185).

The accumulation of CDKB proteins followstheir transcription pattern, and their associ-

ated kinase activity reaches a maximum during

mitosis. The requirement for CDKB1 activity

to progress through mitosis has been demon-

strated with a dominant negative approach,

illustrating that a reduction in CDKB1 activ-

ity results in an increased 4C/2C ratio be-

cause of a block at the G2-to-M transition(15, 138).

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KRP

CYCD2/4

CYCD3

Sucrose

CytokininBR

CDKA Auxin

CDKA

CYCD

CDKA

CYCD

T160

InactiveCDK/cyclincomplex

ActiveCDK/cyclincomplex

CDKD

ABACold

Auxin

DPTargeted fordestruction

DPE2Fc

E2Fc

DPE2Fa/b

E2Fa/b

SCF

DP

Expression of S phase genes

RBR

RBR

RBR

CYCH

InactiveE2F/DPcomplex

Active E2F/DPcomplex

S

G1

G1-S transition

DEL

CDKF

P PP

P

P

Figure 1

Schematic representation of the regulation of the G1-to-S transition in plants. In the presence of growthfactors [such as sucrose, auxin, cytokinin, and brassinosteroids (BR)] D-type cyclins (CYCD) associatewith the A-type CDK (CDKA), forming an inactive CDKA/CYCD complex. This complex is probablyactivated through phosphorylation by the CDK-activating kinase pathway, which involves CDKF andCDKD associated with an H-type cyclin (CYCH). Full activation of the CDKA/CYCD complexrequires as well the phosphorylation of the CYCD subunit by an as-yet unknown kinase (not shown). Inresponse to antimitogenic stimuli, such as abscisic acid (ABA) and cold, KRPs can inhibit the activatedCDK/CYCD complexes. CDKA/CYCD complexes trigger the G1-to-S transition via two parallelpathways. On the one hand, CDKA/CYCD phosphorylation will initiate the destruction of theE2Fc/DP/RBR transcriptional repressor complex by the SCF E3-ubiquitin-protein ligase; on the otherhand, RBR phosphorylation will release the transcriptional activity of the E2Fa-b/DP/RBR complexes.As a result, the expression of S-phase genes is activated. The atypical E2F-like DEL transcription factor

might fine-tune the expression of a subset of E2F target genes.

Remarkably, both Chlamydomonas rein-

hardtiiandOstreococcus tauricontain only one

CDKBgene that resembles theCDKB1genes

of higher plants (11, 149). The O. tauriCDKB

contains a novel cyclin-binding motif that is

midway between the PSTAIRE CDKA andthe P[S/P]T[A/T]LRE CDKB motifs. Just a

observed for CDKBs of higher plants, the

CDKB protein levels and activity in O. taur

appear to be regulated by the cell cycle (31)

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Surprisingly, O. tauriCDKB complements a

CDK mutant of budding yeast (Saccharomyces

cerevisiae), whereas the higher plant orthologs

are unable to do so (31, 80, 137).Plants that overexpress a dominant nega-

tive allele ofCDKB1;1have abnormal stom-

ata and a decreased number of stomatal com-

plexes. This feature indicates a specific role

for CDKB1 in the series of sequential divi-

sions required to form a stomatal complex,

as substantiated by the strong expression of

CDKB1;1observed in all progenitor cells of

stomatal complexes (15). In addition, CDKB1has also been demonstrated to control the on-

set of cell cycle exit (17) (see below).

CDK activity is regulated by phosphory-

lation. Phosphorylation of Thr160 (or the

equivalent residue) of CDKs induces a con-

formational change allowing proper recog-

nition of substrates and is performed by

CDK-activating kinases (CAKs). Arabidop-

sis contains four CAK-encoding genes, di-vided into two functional classes (CDKD and

CDKF; Figures 1 and 2) (182, 183, 186,

207). CDKD is functionally related to ver-

tebrate CAKs, whereas CDKF is a plant-

specific CAK displaying unique enzyme char-

acteristics. The functional diversity between

the two CAK classes is exemplified by their

substrate specificity and cyclin dependence.Only CDKDs phosphorylate the C-terminal

domain (CTD) of the largest subunit of RNA

polymerase II. Moreover, in contrast to the

CDKDs, activation of CDKF requires no as-

sociation with H-type cyclin (CYCH) (206).

Recently, CDKF has been shown to phos-

phorylate and activate CDKDs inArabidopsis

(161) (Figures 1and2).

Through their regulation of CDK activity,CAKs have been shown to be potentially im-

portant regulators of the cell cycle in response

to endogenous hormone gradients. Whereas

normally undifferentiated callus cells can be

induced only from leaf tissue by applying both

auxin and cytokinin, calli derived from plants

overproducing a rice (Oryza sativa) CDKD

grew independently of cytokinin. This effectrelied on the activation of CDK activity and

Ostreococcus tauri:unicellular green algthat was discoveredin the MediterraneaThau lagoon

CAKs:

CDK-activatingkinases

CTD: C-terminaldomain

CYC: cyclin

RBR:retinoblastoma-relatedprotein

HEN3: HUAENHANCER3

could even be enhanced by the co-production

of CYCH (206). Likewise, inducible down-

regulation of CDKF activity resulted in a

gradual reduction in CDK activity and a pre-mature differentiation of root meristem cells

(184), whereas increased expression of the

riceCDKD;1(also known asR2) accelerated

S-phase progression and the overall growth

rate of suspension cells (56). These data in-

dicate that CAKs play an important role in

determining the growth rate and the differen-

tiation status of cells by controlling the overall

level of CDK activity.Plants also contain C-type and E-type

CDK-related genes, designated CDKC and

CDKE, with no clear role in cell cycle con-

trol. CDKCs contain PITAIRE or SPTAIRE

hallmarks, interact with CYCT, and play a

presumed role in transcription elongation

by phosphorylating the CTD of RNA poly-

merase II (6, 63, 94). A CDKC/CYCT com-

plex ofMedicago truncatula phosphorylates theretinoblastoma-related (RBR) protein as well,

suggesting that, like their mammalian coun-

terparts, plant CDKCs might control cellu-

lar differentiation through RBR inactivation

(63). In agreement with this postmitotic role

for CDKC, transcripts were found mainly in

differentiated tissues (6, 93).

CDKE contains a SPTAIRE motif and isidentical to HUA ENHANCER3 (HEN3)

(197). Phenotypic characterization of hen3mutants demonstrated that CDKE acts in cell

expansion in leaves and cell-fate specification

in floral organs. Like CDKCs, CDKE phos-

phorylatesCTD of RNApolymerase II, but in

contrast, is produced in dividing tissues (197).

Cyclins

Little is known to date on the interaction

of cyclins with CDKs. This lack of informa-

tion stems in part from the observation that

plants contain many more cyclins than pre-

viously described in other organisms (186).

For example, despite its small genome size,

A. thalianahas at least 32 cyclins with a puta-

tive role in cell cycle progression. The plant



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CYCD

CYCA

CDKA

CDKA/B

CYC

CYC

T160

CDK/cyclincomplexformation


CDKD

CYCH

G2

G2-M transition

CDKF

CYCB

CDKB

WEE1Stress

CDKA/B

CYC

CDKA/B


Y15T14

CYC


CDKA/B

CDC25

CCS52

T160

Y15T14

Entry into mitosis

Cyclindegradation

?

CYC


CDKA/B

T160

Exit of mitosis

APC

M-G1 transition

M

G1

P P

P P

P

P

P

DPE2Fa/b

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cyclin nomenclature is based on the functional

similarity with the mammalian counterparts.

Arabidopsisgene annotation identified 10 A-

type, 11 B-type, 10 D-type, and 1 H-type cy-clins (186, 193). In addition, at least 17 other

cyclin-related genes have been found in the

Arabidopsisgenome and are classified in types

C, P, L, and T (179, 193). Although some of

these cyclin members have been found to as-

sociate with CDKs, a function in the cell cycle

has not been demonstrated for any of them.

In a broad sense, D-type cyclins are

thought to regulate the G1-to-S transition,A-type cyclins, the S-to-M phase control,

and B-type cyclins both the G2-to-M transi-

tion and intra-M-phase control (19, 126, 140)

(Figures 1 and 2). A number of deviations

of this general functional assignment have

been reported. For Medicago sativa, CYCA2

has been shown to contribute to cell cycle

specific kinase activity, not only at the entry

to the S phase, but also during the G2-to-Mtransition (151). In contrast to animals, some

preliminary evidence points to an additional

function of D-type cyclins at the G2-to-M

transition. For example, CYCD4;1 has been

found to associate and activate the G2/M-

specific CDKB2;1 in vitro (100). In addition,

ectopic expression ofCYCD3;1 in trichomes

not only promotes S-phase entry but also in-duces mitosis (155). Likewise, induced over-

expression of the tobacco CYCD3;3 and the

snapdragon (Anthirrhinum majus) CYCD1;1in tobacco Bright Yellow-2 (BY-2) cell sus-

pensions stimulated both S phase and mitotic

BY-2: fast-growingtobacco cell culture,comparable to Helacells for humanresearch

E2F: E2

promoter-bindingfactor

entry (101, 129). These data should, how-

ever, be interpreted with caution because a

mechanism might exist by which the activa-

tion of DNA replication stimulates mitoticentry, as demonstrated by the observation that

transcription of theCDKB1;1gene is at least

in part controlled by the G1/S-specific E2F

transcription factors (17, 115). In such a sce-

nario, any positive effect on the G1-to-S tran-

sition would result in the activation of later

cell cycle phases as well. In addition, it re-

mains possible that by ectopic expression of

the cyclin subunit, CDK/CYCD complexesare formed that do not exist during normal

development. On the other hand, in favor of a

direct involvement of D-type cyclins in con-

trolling the G2-to-M transition, some D-type

cyclins show a transcriptional peak at the G2-

to-M transition(124, 125, 166). Furthermore,

CYCD3;3-associated kinases were found to

be active at both the G1/S and G2/M bound-

aries (129).D-type cyclins have a large sequence di-

vergence and were originally identified by

functional complementation of a yeast strain

deficient for G1 cyclins (34, 165). In Ara-

bidopsis, the 10 CYCDs are classified into

seven groups, designatedCYCD1 to CYCD7,

with the CYCD3 and CYCD4 groups consist-

ing of three and two members, respectively(186). Although the complexity of plant cy-

clins can be attributed partly to extensive du-

plications of theArabidopsisgenome (162), the

large number of cyclins might reflect the high

developmental plasticity of sessile plants to

Figure 2

Schematic representation of the regulation of the G2-to-M transition in plants. During the G2 phase ofthe cell cycle, cyclins of the A, B, and probably D types (CYCA, CYCB, and CYCD) associate with bothCDKs of the A and B types (CDKA and CDKB). Some B-type CDKs are under transcriptional control

of the E2F pathway, probably providing a mechanism by which the G1-to-S and G2-to-M transitionscommunicate. The CDK-activating kinase pathway, involving CDKF and CDKD associated with anH-type cyclin (CYCH), controls the activity of the distinct CDK/CYC complexes. CDK activity can benegatively regulated by phosphorylation by the WEE1 kinase, which is triggered upon loss of DNAintegrity. The CDC25-related kinase, if existing, which removes the inhibitory phosphate groups, stillneeds to be identified. Once the CDK/CYC complexes are active, they trigger the G2-to-M transitionthrough the phosphorylation of a plethora of different substrates. Exit from mitosis requires theproteolytic destruction of the cyclin subunits. This destruction is initiated by the activation of theanaphase-promoting complex (APC) through its association with the CCS52 protein.



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Physcomitrellapatens: mossenabling directloss-of-functionstudies by genetargeting

respond to both intrinsic developmental sig-

nals and extrinsic environmental cues. Pos-

sibly, the complex cell cycle machinery is the

trade-off for the tremendous plasticity and ro-bustness of plant growth, which require the

presence of flexible regulatory networks. The

different cyclins might possess a wide range of

expression patterns and confer different sub-

strate specificities.

The expression of D-type cyclin genes is

modulated by plant growth factors, such as

cytokinins, auxins, brassinosteroids, sucrose,

and gibberellins (62, 68, 81, 121, 133, 146148, 154, 165). Some D-type cyclins proba-

bly act as key switches in triggering hormonal

effects. For example, expression ofCYCD3;1appears to be rate-limiting for cell division

in calli deprived of cytokinin. Correspond-

ingly, overexpression ofCYCD3;1is sufficient

to compensate for the lack of cytokinins in the

culture medium (147). Similarly, CYCD2;1

seems to stimulate the progression throughG1 in roots and shoots, leading to a faster

growth rate of transgenic tobacco plants over-

expressing CYCD2;1(29). There is probably

an extensive functionalredundancy among D-

type cyclins, because the genome-wide inser-

tional mutagenesis surveys have yet to report

severe phenotypes for D-cyclin knockouts

(21, 177). To date, some specific D-type cy-clin knockouts displayed only a slightly de-

layed cell cycle reactivation in the root meris-

tem during seed germination, supporting the

anticipated important role for D-type cyclins

in the regulation of cell cycle entry upon

meristem activation (119). Analogously, a cycD

knockout in the mossPhyscomitrella patenshas

a surprisingly limited phenotype. While wild-

type plants react to exogenous glucose sourceswith prolonged growth of juvenile stages and

retarded differentiation, cycD knockouts ex-

hibited developmental progression indepen-

dently of the sugar supply. However, growth

rate, cell size, or plant size were not affected.

These data suggest that the Physcomitrella

CYCD is not essential for cell cycle progres-

sion but seems important for coupling the de-velopment to nutrient availability (111).

Just as for D-type cyclins, only margina

phenotypes have been reported for CYCA

mutants. In tardy asynchronous meiosis (tam)the substitution of a conserved amino acid

residue probably results in an incorrect fold

ing of CYCA1;2, causing a slower cell cycl

progression during male meiosis (113, 198)

Knockouts for CYCA2;3 display a slight in

crease in their DNA ploidy level (85). In

both cases, the occurrence of only relatively

mild phenotypes can be explained by the fac

that these particular A-type cyclins are parof a family of closely related genes; as such

multiple knockouts will presumably have to

be combined before any severe phenotyp

is revealed. It is even likely that combin

ing multiple knockouts will result in embryo

lethality, as suggested by the observed de

fects on embryo formation upon antisens

expression of the tobacco CYCA3;2 cyclin

(208).In contrast to their knockdown, overex

pression of the A-type cyclin genes triggers an

acute phenotype:Arabidopsisplants that over

produce the tobacco CYCA3;2 cyclin show

ectopic cell division and delayed differentia

tion, correlated with an increase in expression

of S phasespecific genes and CYCA3;2

associated CDK activity. In addition, overproduction of CYCA3;2 impairs shoot and roo

regeneration in tissue culture (208). Thes

data indicate that for cell differentiation the

CDK activity must be down-regulated.

The potential of B-type cyclins to trig

ger the G2-to-M transition was originally

shown by microarray injection experiment

in oocytes ofXenopus laevis(77) and later by

illustrating that ectopic CYCB1;1 expressionpromotes root growth (47). CYCB1;1 wa

demonstrated in vitro to interact with and ac

tivate both A- and B-type CDKs (199).

Proteolysis

Proteolysis ensures that the cell cycle move

unidirectionally by triggering the rapid pro

teolysis of target proteins, thus providing an

irreversible mechanism that drives the cycl

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forward. In all cases, the route to destruction

runs via the ubiquitin-proteasome system,

which uses the highly conserved polypeptide

ubiquitin as a tag to mark target proteins fordegradation by the 26S proteasome. Ubiquiti-

nation requires the generation of polyubiqui-

tin chains on target proteins through the com-

bined action of ubiquitin-carrying enzymes

(or E2s) and ubiquitin-protein ligases (or E3s)

that bring targets and E2s together (135).

Two related E3 complexes are most intimately

dedicated to basic cell cycle control, namely

the anaphase-promoting complex (APC)and the Skp1/Cullin/F-box (SCF)-related

complex (192).

Plant cyclins are, as in other organisms,

subject to extensive regulation by proteoly-

sis. A- and B-type cyclins contain destruction

box (D-box) sequences that mediate protein

degradation (69, 145). B1 cyclins, a subclass

of B-type cyclins, are the substrate for the

ubiquitin-dependent protein ligase complexthat strongly resembles APC (32). The illus-

tration for the functional significance of this

cyclin destruction is that constitutive overex-

pression of a non-degradable B1 cyclin, lack-

ing the destruction box, causes severe growth

retardation and abnormal development with

a higher percentage of cells exhibiting dupli-

cated ploidy levels than the controls (199).Ectopic expression ofCYCA2;3 that lacks a

functional destruction box stabilizes protein

levels in plants and results in dwarfism (85).

On the other hand, proteolysis of B2 cyclins

at prometaphase appears to be proteasome in-

dependent (200).

Recently, CYCD3;1, but not CYCD2;1,

has been shown to be a highly unstable

protein whose proteolysis is mediated bya proteasome-dependent pathway (136). In

concert, the CYCD3;1 protein is stabilized in

mutant plants, defective in a ring-box (RBX1)

protein part of the plant SCF complexes

(108). Many D cyclins isolated to date contain

PEST sequences, regions rich in Pro, Glu,

Ser, and Thr, suggesting that they are labile

proteins degraded through a pathway similarto that of animal D-type cyclins (133, 165).

Proteasome:multisubunitcomplex involved inprotein breakdown

E2s:ubiquitin-carrying

enzymes

E3s:ubiquitin-proteinligases

APC:anaphase-promotingcomplex

SCF:Skp1/Cullin/F-boxprotein

CYCD1 instability also depends on the pro-

teasome, whereas CDKA amounts are unaf-

fected by the proteasome inhibitor MG132

(101). Other cell cycle regulators, such asCDC6 (24), CDT1a (23), E2Fc (42), and

the CDK inhibitor ICK2/KRP2 (188), are

destroyed via the ubiquitin/26S proteasome

pathway as well. It is not yet known which of

the 694 F-box proteins ofArabidopsis(189) are

involved in the specific recognition of the cell

cycle regulatory proteins.

Proteins that are degraded through the

proteasome frequently require prior phos-phorylation. TheArabidopsisCDT1a contains

multiple CDK phosphorylation sites, suggest-

ing that they might affect CDT1a regula-

tion (23). In concert, treatment ofArabidop-

sis seedlings with roscovitine, a well-known

CDK inhibitor, causes CDT1a to accu-

mulate (23). Also ICK2/KRP2 proteolysis

seems to be preceded by CDK-dependent

phosphorylation (188).The activation and substrate specificity of

the APC complex is in part determined by

two adaptor proteins, CDC20 and CDH1.

TheArabidopsisgenome encodes fiveCDC20

genes, as well as three CDH1-related pro-

teins, designated CCS52A1, CCS52A2, and

CCS52B (178). In Schizosaccharomyces pombe,

expression of the three Arabidopsis CCS52genes elicits distinct phenotypes, supporting

a nonredundant function of the CCS52 pro-

teins. Consistent with these different func-

tions,CCS52Bis expressed from G2/M to M,

whereas CCS52A1 and CCS52A2 arefromlate

M until early G1, suggesting consecutive ac-

tions of these APC activators in the plant cell

cycle. In addition, the CCS52 proteins inter-

act with different subsets of mitotic cyclins,either in free or CDK-bound forms (64).

CDK Phosphorylation

Similarly to that in yeasts and animals, the ac-

tivity of plant CDK/cyclin complexes is regu-

lated by phosphorylation/dephosphorylation

and the interaction with regulatory proteins.

Yeast CDK/cyclin complexes are subject to an



10/31

CKIs:cyclin-dependentkinase inhibitors

inhibitory phosphorylation of an N-terminal

Tyr residue in the CDK partner, whereas

in vertebrates CDKs are phosphorylated on

both an N-terminal Tyr and Thr residue. Tyrphosphorylation is catalyzed by the WEE1 ki-

nase, and both Tyr and Thr phosphorylation

is counteracted by the dual-specificity phos-

phatase CDC25.

Tyr phosphorylation of A-type CDK has

been detected unambiguously and shown

to down-regulate CDKA activity under cy-

tokinin deprivation, osmotic stress, or DNA

damage (158,210; D.I. & L.D.V., unpublisheddata). The inhibitory phosphorylation sites

are also conserved in B-type CDKs, but bio-

chemical evidence for CDKB phosphoryla-

tion is still lacking, the only known exception

being the picoeukaryote O. tauri, in which

CDKB, but not CDKA, is phosphorylated

on Tyr upon activation of the DNA integrity

checkpoint (31, 57).

Plants possess a WEE1 kinase that is puta-tively involved in the inhibitory phosphoryla-

tion of CDKs (167, 176, 186; D.I. & L.D.V.,

unpublished results). Overexpression of the

maize (Zea mays) or Arabidopsis WEE1 genes

inS. pombecauses cell cycle arrest (167, 176),

although its in planta inhibitory effect on the

cell cycle remains to be proven.

Recently, in the primitive unicellular al-gae, O. tauri, a bona fide CDC25 has been

found (97) that presumably controls the activ-

ity of the B-type CDK (57). However, inAra-bidopsis,rice,and Chlamydomonas,whoseentire

genome sequences are available, no CDC25

gene encoding a CDK-activating phosphatase

could be identified (11, 186). Nevertheless,

biochemical and genetic evidence suggest that

higher plants also have a phosphatase thatcan activate CDK/cyclin complexes; for in-

stance, inactive CDK/cyclin complexes puri-

fied from cytokinin-starved and G2-arrested

tobacco cells can be activated in vitro by the

yeast CDC25 (210). Furthermore, overex-

pression of theS. pombe cdc25gene in trans-

genic tobacco andArabidopsisplants promotes

mitosis (10, 120, 134, 204, 209). In general,overexpression of the S. pombe cdc25mimics

the developmental effect caused by cytokinin

(174), in agreement with the pivotal role o

cytokinins for the G2-to-M transition in tobacco cells (107, 210).

Recently, an Arabidopsis gene has been

identified that encodes a CDC25-like pro

tein, capable of binding a phosphothreo

nine 14/phosphotyrosine 15 peptide and o

activating ArabidopsisCDK activity in vitro

(105). However, unlike the classical CDC25s

this CDC25 lacks the important N-termina

regulatory domain. Additionally, Arabidopsi

CDC25cannot complement yeastcdc25mu

tants and does not affect the cell cycle when

overexpressed or knocked-down in plant

(D.I. & L.D.V., unpublished data). The plan

CDC25 protein also displays similarity with

the yeast Arsenate [As(V)] reductase and, cor

respondingly, has recently been demonstrated

to possess reductase As(V) activity. Moreover

its overexpression improves the tolerance otransgenic Arabidopsis plants to mildly toxi

levels of As(V), whereas its knockout increased

As(V) sensitivity (13). As such, it is unlikely

that theArabidopsis CDC25-like gene encode

a true otholog of the classical CDC25, bu

rather has a function totally unrelated to cel

division.

Cyclins are also subject to phosphorylation. The tobacco D cyclin CYCD3;3 is phos

phorylated on Thr191, and this phosphoryla

tion is required for nuclear localization and

maximum kinase activity (129). In vivo phos

phorylation of the ArabidopsisCYCD3;1 oc

curs when cells are sucrose starved (136).

CDK Inhibitors

Cyclin-dependent kinase inhibitors (CKIsregulate cell cycle progression by binding an

inhibiting CDKs (127). Budding yeast ha

three CKIs: Far1p inhibits G1 CDK activ

ity; Sic1p controls S-phase entry by regulat

ing G1/S CDK complexes; and Pho81p in

activates a CDK/cyclin complex that play

a role in regulating gene expression un

der low-phosphate conditions. The situation

in fission yeast (Schizosaccharomyces pombe) i

86 Inz e De Veylder


11/31

considerably simpler because only one CKI,

designated Rum1, is known to control mitotic

CDK complexes. Mammals have seven CKIs,

which are subdivided into two very differentclasses. The INK4 family proteins contain an

ankyrin repeat and selectively inhibit the G1

CDKs, CDK4 and CDK6. The Cip/Kip fam-

ilyofCKIscomprisesp21 Cip1 (also called Sdi1,

Waf1, or CAP20), p27Kip1, and p57Kip2, all

of which have a conserved domain at the N

terminus. The Cip/Kip CKIs inhibit a broad

range of CDK/cyclin complexes involved in

the control of both G1-to-S and G2-to-M(131).

The first plant CKIs have been character-

ized in a yeast two-hybrid screen for CDKA-

interacting proteins (40, 89, 112, 194, 211).

Additional plant CKIs have been identified

in silico through genome data mining (30,

40). The specific interaction of ICK2/KRP2

with CDKA has also been confirmed in vivo

(188). Plant CKIs have a peculiar struc-ture: They all share a C-terminally located

31-amino-acid domain. This conserved do-

main is involved in binding CDKs and cy-

clins and is essential for the inhibitory ac-

tivity of the proteins (30, 40, 88, 89, 112,

157, 194, 212). Because of its similarity with

the N-terminally located CKI domain of the

mammalian Cip/Kip proteins, plant CKIsare most commonly known as Kip-RelatedProteins or KRPs, although the two found-

ing members are also known as Interactors

of Cdc2 Kinase or ICK (40, 112, 194). Ara-bidopsis encodes seven ICKs/KRPs, denom-

inated ICK1/KRP1, ICK2/KRP2, KRP3,

KRP4, KRP5, KRP6, and KRP7. All seven

ICKs/KRPs also interact with D cyclins

(CYCD1, CYCD2, and CYCD3) (40, 112,195, 211). Proof of in vivo binding speci-

ficity between plant ICKs/KRPs and D-type

cyclins is that the cell division inhibitory

activity caused by ICK/KRP overexpression

(see below) can be complemented by co-

overexpression of D-type cyclins (89, 157,

212). Monocotyledonous ICKs/KRPs might

differ slightly in their biochemistry from their

dicotyledonous counterparts, because maize

ICKs/KRPs:inhibitors of plantCDKs

ICKs/KRPs inhibit both A- and D-type cy-

clin/CDK complexes (30).

Length and primary structure of the

N-terminal part are highly variable withinICKs/KRPs of a single plant species (40). A

functional analysis of ICK1/KRP1 indicates

that the N-terminal domain contains a se-

quence that decreases the protein stability in

vivo. Possibly, such a sequence could drive the

temporal selective degradation of ICK/KRP

proteins at different time points in the cell cy-

cle (157, 195, 212). In accordance with a role

for proteolysis as an important mechanism tocontrol ICK/KRP activity, the ICK2/KRP2

levels have been shown to be controlled by

the proteasome (188). So far, no INK-type

inhibitors have been found in plants, in agree-

ment with the absence of the known INK sub-

strates (at least in mammals) being CDK4-

and CDK6-related CDKs.

The RBR/E2F/DP Pathway

Despitethe billion years of evolution that sep-

arate animals from plants, both types of or-

ganisms use the same Rb/E2F/dimerization

partner (DP) pathway to control the G1-

to-S transition. In the two cases, Rb pro-

teins interact with the E2F/DP complex to

repress transcription of E2F-regulated genes

(50, 144, 160). Even the canonical DNA se-quence (TTTCCCGC) recognized by the

E2F/DP transcription factors of animals and

plantsisidentical(2,102).Thisidentityargues

infavorofthehypothesisthattheRb/E2F/DP

pathway had already evolved in primitive or-

ganisms, before the branching between ani-

mal and plant taxa. However, whereas Ara-

bidopsisencodes multiple CDKs, cyclins, andKRPs, its genome contains only one Rb-

relatedgene (RBR). All known plant RBR pro-

teins have, like their animal counterpart, two

blocks of conserved sequences that form the

so-called A/B pocket domain (1, 49, 72, 205),

which is the docking place for the E2F tran-

scription factors.

In animals, Rb is phosphorylated by

CDK4/CYCD, CDK6/CYCD, CDK2/



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Megagametophyte:the femalegametophyte, whichdevelops from amegaspore

CYCE, and CDK2/CYCA complexes at

the G1-to-S transition. A PP1 phosphatase

dephosphorylates Rb at G2/M. Much less is

known about the kinases and phosphatasesthat govern the plants RBR phosphorylation

state. Plant RBR proteins interact with D

cyclins through a conserved LxCxE motif at

the N terminus of the latter (1, 34, 84, 129,

130, 165). The plant RBR-associated kinase

complex contains CDKA and a D cyclin (14,

101, 129) (Figure 1). Phosphorylation of

plant RBR proteins by CDKs depends on the

cell cycle phase (55, 129).

ArabidopsisRBR knock-outs are sterile, be-

cause they fail to arrest the mitotic divisions

in the mature female megagametophyte be-

fore fertilization, resulting in excessive nu-

clear divisions in the embryo sac. In addi-

tion, the central cell nucleus, which gives rise

to the endosperm after fertilization, initiates

autonomous endosperm development. These

data indicate that RBR controls the arrestof the mature unfertilized megagametophyte

(51). Analogously, RBR regulates the differ-

entiation of root stem cells (202).

Whereas in dicots the cell cycle and post-

cell cycle-related functions of RBR appear to

be performed by a single RBR gene, mono-

cotyledonous species possess two types of

RBR genes. In maize, two RBR proteinshave a complementary accumulation pattern:

RBR3 is exclusivelypresent during themitotic

phase of endosperm development, whereas

RBR1 is mainly observed post-mitotically

in endoreduplicating and differentiating cells

(152). These data suggest a division of labor,

whereby RBR3 participates in cell cycle con-

trol and RBR1 in cell differentiation.

Rb proteins control the activity of the ade-novirus E2 promoter-binding factor (E2F)

family of transcription factors that, in turn,

control the expression of many genes required

for entry into and execution of S phase and

cell cycle progression (3, 12, 46, 170). Ara-

bidopsiscontains six E2Fs (E2Fa, E2Fb, E2Fc,

E2Fd/DEL2, E2Fe/DEL1, and E2Ff/DEL3)

and two DPs (DPa and DPb). E2Fa and E2Fbact as transcriptional activators as illustrated

by their ability to induce reporter genes har

boring the E2F consensus cis-activating el

ement (102, 118, 171). Moreover, transienoverexpression ofE2FaandDPainduces qui

escent mesophyll cells to re-enter S phase

(150), whereas ectopic co-expression ofE2Faand DPa induces sustained cell proliferationin

differentiated cotyledon and hypocotyl cell

(39, 103, 150). Also co-expression of E2F

with DPa stimulates cell division, resulting

in shortening of the cell cycle (115). By con

trast, E2Fc, which lacks a strong activationdomain, operates as a negative regulator o

the E2F-responsive genes (42, 102, 118, 150

(Figure 1) and, consequently, its overexpres

sion inhibits cell division (42).

In mammals, numerous E2F target gene

are well characterized. These genes encod

proteins active during DNA replication, mi

tosis, DNAcheckpoint control, apoptosis, an

differentiation (46). By contrast, only a fewplant E2F targets have been validated experi

mentally, including MCM3, CDC6, CDT1aPCNA, RBR, and RNR (26, 27, 37, 39, 53

54, 102, 142, 152, 171). In silico analysis o

the Arabidopsisgenome for the presence o

the TTTCCCGCC canonical motif identi

fied 183 putative E2F target genes, includ

ing genes involved in DNA replication andcell cycle regulation (142). A more profoun

analysis using microarray data and bioinfor

matics tools allowed the identification of 70

putativeE2F targetgenes conserved overAra

bidopsisand rice. These genes encode protein

involved in cell cycle regulation, DNA repli

cation, and chromatin dynamics. In addition

several genes have been identified with po

tentially novel roles in the regulation of the Sphase (187).

E2Fs and DPs contain only one DNA

binding domain and, therefore, requir

dimerization to interact with the canonica

E2F motif (2, 102, 114, 141). By contrast

E2Fd/DEL2, E2Fe/DEL1, and E2Ff/DEL3

proteins are atypical E2F factors becaus

they contain two DNA-binding domainthat allow them to bind as a monome

in a DP-independent manner to the E2F

88 Inz e De Veylder


13/31

DNA-binding sequence. Additionally, these

proteins do not contain a transactivation do-

main and, as such, might play a role in a neg-

ative feedback loop to repress E2F-activatedpromoters (118, 186) (Figure 1). The atyp-

ical E2F/DEL proteins lack an Rb-binding

motif. Only very recently have two mam-

malianhomologs,designatedE2F7 andE2F8,

been discovered (28, 36, 45, 110, 117), high-

lighting that cell cycle research in plants can

yield novel insights into cell cycle research in

animals.

The biphasic expression pattern of

E2Fe/DEL1during the cell cycle, with tran-

scripts peaking at the G1-to-S and G2-to-M

transition points, while absent during S

phase (118), suggests that E2Fe/DEL1 might

control the temporal expression of E2F

target genes. E2Ff /DEL3 overexpression in

Arabidopsisreduces the size of differentiated

cells in roots and hypocotyls, whereas cells

with reduced E2Ff /DEL3 levels are larger,especially in the hypocotyls. These effects

of E2Ff/DEL3 are not associated with

changes in the nuclear ploidy levels or in

the expression of cell cycle genes. However,

expression of a subset of cell wall biogenesis

genes is misregulated in an E2Ff/DEL3-

dependent manner and, based on chromatin

immunoprecipitation experiments, theseseem to be direct E2Ff/DEL3 targets (143).

In contrast to E2Ff/DEL3, E2Fe/DEL1

plays an important role in the control of

endoreduplication (see below).

CELLULAR VERSUSORGANISMAL THEORY

The role of the cell cycle machinery in plantdevelopment has been subject to debate. Ob-

viously, cell division is essential in generating

the cells that constitute tissues and organs.

However, whether cell division is the driver

of growth and development (the cellular the-

ory) or, alternatively, cell division merely fol-

lows a developmental plan (the organismal

theory) is more difficult to answer. Basically,

to what extent do oriented cell divisions con-

tribute to the determination of form? Exper-

iments in the late 1950s and early 1960s sug-

gested that cell division has little function in

growth and morphogenesis. Seedlings fromheavily-irradiated wheat (Triticum aestivum)

grains grow to some extent without negligible

cell division and resemble untreated seedlings

in many aspects of metabolism (74), growth

(73, 75), and maturation of cells present in the

embryo (59). A 10-day-old -irradiated wheat

seedling is as large as a 3-day-old unirradiated

seedling, but contains threefold and 8.5-fold

fewer, albeit much larger, epidermal and mes-ophyll cells, respectively (73). Habers work

provided the first experimental evidence that

genetic information specifies leaf form inde-

pendently of the extents and orientations of

cell divisions.

Thanks to the ability to alter cell cycle pa-

rameters in transgenic plantsthe longstanding

question on the importance of cell division indetermining organ size and shape has been re-

addressed recently. In agreement with Habers

work (59, 7375), at least for leaves, develop-

ment seems to follow the concept laid down in

the organismal theory. Transgenic Arabidop-

sisplants constitutively overproducing any of

the ICKs/KRPs have smaller leaves that con-

sist of tenfold fewer cells with sixfold greater

average size than control cells. The reduc-tion in cell number is thus compensated for

by an increased cell size (40, 89, 196). Also in

rice,OsKRP1overexpression causes a reduc-

tion in cell number that is compensated for

by an increased cell size (D.I. & L.D.V., un-

published data). Similarly, overexpression of a

dominant negative CDKA or a nondegradable

CYCB1;1 in tobacco retards the cell cycle andcauses the formation of larger cells (78, 199).

On the other hand, constitutive overexpres-

sion of positive regulators of the cell cycle,

such asE2Fa,CYCA3;2, orCYCD3;1, results

in more cells (39, 43, 208). Here, the increase

in cell number is compensated for by a de-

crease in cell size. All these observations sup-

port the organismal theory of plant develop-

ment in which the size and shape of an organor organism is set to some extent by a certain



14/31

endogenous mechanism independently of cell

division processes (96). The phenomenon by

which cell numbers is inversely correlated to

cell size is called compensated cell enlarge-ment (181). Also in animals, cell division

and cell expansion can compensate each other

to achieve an optimal species-specific organ

size (35, 139). InDrosophila melanogaster, this

phenomenon is also known as the total mass

checkpoint (139).

It is not known what determines the

blueprint for leaf shape and size, but hor-

monal gradients probably play an importantrole. However, not all experimental data fit

the organismal theory. For example, overex-

pression of a dominant negative CDKA un-

der the control of a seed-storage promoter

deregulates embryo development (79). Fur-

thermore, overexpression ofCYCB1;1under

the control of theCDKA;1promoter acceler-

ates growth of roots, as would be expected un-

der the cellular theory (47). Previously, we hadproposed that these two theoriesthe cellu-

lar and the organismalare too polarized (9).

Rather, cell division, cell differentiation, and

morphogenesis can be considered as integral

parts of a higher-level ontogenetic program

defined by both short-range and long-range

signaling between growth zones (9). The fu-

ture challenge is to elucidate how the devel-opmental signaling components interact with

the cell cycle machinery in animals and plants.

ENDOREDUPLICATION

The normal cell cycle mode is characterized

by a round of DNA replication (S phase) fol-

lowed by mitosis and cytokinesis (M phase).

Two gap phases (G1 and G2) usually separatetheSandMphases.However,manyplantcells

have a different cell cycle mode with cells un-

dergoing iterative DNA replications without

any subsequent cytokinesis. This endoredu-

plication is frequently observed in some, but

not all, plants (20, 33, 38, 66, 128, 175, 180).

The level of ploidy varies between species

and tissues. In Arabidopsis, nuclei with up to

32C are detected (66), but some maize en-

dosperm cells attain a DNA content as high

as 96C or 192C (104, 109). The physiologi

cal role of endoreduplication is subject to de

bate, and several hypotheses have been proposed. Endoreduplication probably plays an

important role in the differentiation proces

of postmitotic cells because the onset of th

endocycle often characterizes the switch be

tween cell proliferation and differentiation

as observed during hypocotyl elongation, tri

chome growth, andfruit andleaf developmen

(7, 17, 95, 99, 106). In addition, plant specie

that endoreduplicate are often characterizedby a rapid life cycle and improved yield sta

bility, implying that endoreduplication migh

support fast development (5). Endoredupli

cation was long believed to be essential to

support cell growth and to maintain an opti

mal balance between cell volume and nuclear

DNA (61, 82, 83, 122, 180). However, thi

concept is currently being challenged (175)For example, cell size and overall ploidy leve

are not correlated in root cells of differen

Arabidopsisecotypes (8). Similarly, recent ex

periments in which the level of endoredupli

cation is modified do not support this hy

pothesis. For example, high overexpression o

ICK/KRPgenes causes a remarkable overal

decrease in the ploidy level but, at the same

time, greatly increases cell size (40, 89, 211)Also, cell size of the starchy endosperm did

not change dramatically when the endoredu

plication process in the maize endosperm wa

specifically inhibited by overexpression of a

dominant negative allele of the CDKA gen

(109). An alternative hypothesis is that muta

tions, which plants accumulate during thei

sessile life, are buffered by endoreduplication. Indeed, many plants are exposed dur

ing their life cycle to less favorable condi

tions, and the various copies, such as 8, 16

32, 64, etc., of the genome would safeguard

that the genome retains functional copies

Another hypothesis is that endoreduplication

is important to maintain an optimal balanc

between organellar and nuclear DNA. Ad

ditionally, endoreduplication might enhancthe metabolic capacity of plant cells. Thi

90 Inze De Veylder


15/31

hypothesis is based on the high polyploidy of

the endosperm cells of cereal seeds. An in-

crease in the gene copy numbers of metabolic

genes might allow the endosperm cells to syn-thesize very large amounts of storage prod-

ucts, such as starch and storage proteins (90,

99, 106, 180). In favor of this hypothesis,

genome amplification of maize endosperm

cells coincides with cell enlargement and the

onset of starch and protein accumulation.

However, recent experiments argue against

a role of endoreduplication in enhancing

metabolic activity, because by overexpressionof the dominant negativeCDKAin the maize

endosperm, starch and protein accumulation

were only slightly reduced as a consequence

of the decrease in the endoreduplication level

(109).

How are cells triggered to undergo en-

doreduplication? InArabidopsis, the exit of the

mitotic cycle correlates with the onset of en-

doreduplication, suggesting that this processcan be perceived as a continuum of the mi-

totic cell cycle in which mitosis, but not DNA

replication, is inhibited (87). In the maize en-

dosperm, the tomato (Lycopersicon esculentum)

fruit, and theArabidopsisleaf, the onset of en-

doreduplication coincides with a decrease in

M phasespecific CDK activity (7, 17, 71, 95,

106). In S. pombe andin D. melanogasterinhibi-tion of the M phaseassociated CDK activity

issufficienttodrivecellsintotheendoredupli-

cation cycle (52, 76, 153). As such, it is reason-

able to believe that in plants too an active pro-

cess controls the decrease in M-phase CDK

activity. Over the past few years, considerable

progress has been made in understanding the

molecular basis of endoreduplication in plants

(Figure 3). The mitotic cycle and endocyclehave DNA replication in common, and both

cell cycle modes probably make use of the

same machinery. In agreement, constitutive

overexpression of genes that stimulate DNA

replication has been shown to promote both

the mitotic cell cycle and the endocycle (23,

39, 103, 155). Thus, the difference between

the mitotic cycle and the endocycle must belooked for in the mechanism regulating the

MIF:mitosis-inducingfactor

G2-to-M transition. The decision of a cell to

either undergo endoreduplication or continue

mitosis might depend on a cellular factor, des-

ignatedMitosis-Inducing Factor (MIF) (39), a

hypothesis based on extensive ectopic cell di-visions in some tissues (such as root cap) and

extensive endoreduplication in other tissues

(such as root cortex)caused by the overexpres-

sion of bothE2Faand DPa. The ectopic cell

division phenotype has mostly been observed

in the proximity of stomata that are known to

be able to divide. Possibly, the cells in which

E2Fa-DPaoverexpression triggers additionalcell division might possess a MIF that is inac-

tive or absent in the endoreduplicating cells.

Sorting cell types in whichE2Fa-DPahas dif-

ferent effects and comparing their transcrip-

tome should help in unraveling the nature of

MIF. A likely candidate to be part of MIF is the

CDKB1;1 kinase whose role in the mitosis-

to-endocycle switch has been hinted by its

temporal expressionpattern duringleaf devel-opment. CDKB1;1 is highly expressed in di-

viding cells and is down-regulated at the onset

of endoreduplication. In concert, transgenic

plants overproducing a dominant negative

CDKB1;1 undergo increased endoreduplica-

tion. When the dominant negative CDKB1;1

is co-expressed with the heterodimeric tran-

scription factor E2Fa-DPa, the endoredupli-cation phenotype is enhanced, whereas the

extra mitotic cell divisions normally induced

by ectopicE2Fa-DPa expression are repressed

(17). Thus, CDKB1;1 activity seemingly con-

trols the balance between mitotic cell divi-

sion and endoreduplication. Candidate cy-

clin genes that act together with CDKB1;1

are CYCA3;2, CYCA2;3, and CYCD3;1, as

they inhibit the endoreduplication processupon their overproduction (43, 85, 208). Also

the cyclin CYCB1;2 is a possible candidate,

as illustrated by its ectopic expression that

changes the developmental fate of trichome

cells from endocycling into mitotically divid-

ing cells (156).

Recently, the ICKs/KRPs have been

demonstrated to contribute to the control ofthe mitosis-to-endocycle transition. Whereas



16/31

CDKB

CYCA/BCYCA/B

WEE1

DEL1

CCS52

KRP

CDKA

CYC

Destruction

Destruction

?

?

P

?

DK

CYCA/B

KRPWEE1

DEL1

CCS52

P

CDKA

CYC

Destruction

Destruction

?

?

?

E2Fa b

ba

High CDKAactivity

Low CDKAactivity

Mitotic cell cycle Endoreduplication cycle

DP

E2Fa/b

Figure 3

Schematic overview of the mitosis-to-endocycle transition. (a) Mitotic cell cycle. (b) Endoreduplication

cycle. Colored and gray-shaded symbols correspond to active and inactive molecules, respectively.Mitotically dividing cells are characterized by high CDKA/CYC and CDKB/CYC activities. B-typeCDKs, of which some family members are under transcriptional control of the E2F pathway, prevent theinhibition of the CDKA/CYC complexes through phosphorylation of the CDK inhibitory ICK/KRPproteins, triggering their destruction. Exit from the mitotic cell cycle is also negatively regulated by theatypical E2F-like E2Fe/DEL1 transcription factor, in a manner yet to be determined, and isaccompanied with the activation of the CCS52 protein that probably targets B-type CDK-associatedcyclins for destruction. The decrease in B-type CDK activity will result in a stabilization of theICK/KRP proteins, which, in turn, will down-regulate the activity of the CDKA/CYC complexes. TheWEE1 kinase might also be partly responsible for the decrease in CDKA/CYC activity. The decrease inCDKA/CYC activity results in inhibition of mitosis, but still allows DNA replication to occur withendoreduplication as a consequence.

high ICK2/KRP2 levels repress both cell di-

vision and endoreduplication, weak overex-

pressioninhibitsthemitoticcellcyclespecific

CDKA complexes only, resulting in a pre-

mature onset of the endoreduplication pro-

gram (188). A similar dose-dependent ef-

fect on the cell cycle has been observed for

ICK1/KRP1 (201). The difference in sensi-tivity of CDK complexes toward ICK2/KRP2

inhibitionmightbecausedbythenatureofthe

cyclins in complex with CDKA at either theG1-to-S or the G2 phases (188).

The abundance of the ICK2/KRP2 pro-

tein is developmentally regulated and con-

trolled through CDK phosphorylation and

proteasome-dependent degradation. Its ac-cumulation in plants overexpressing a dom-

inant negative allele of CDKB1;1 strongly

suggests that this particular CDK target

ICK2/KRP2 for degradation by phosphory

lation. In this model, the CDKB1;1 kinase

inhibits ICK2/KRP2 activity, resulting in

higher CDKA activity at the G2-to-M transi

tion and consequently less endoreduplication

(188) (Figure 3).At this stage, the possibility that th

CDKB1;1 kinase suppresses the endocycl

alsovia other mechanisms cannotbe excludedIn fission yeast, the mitotic CDK complex ha

been demonstrated to prevent the relicens

ing of replicated DNA by its association with

the replication complex during chromosom

duplication, preventing reinitiation of DNAsynthesis (203). Similarly, CDKB1;1 migh

92 Inze De Veylder


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negatively regulate pre-replication complex

assembly and thereby influence the progres-

sion of endocycles as well as the mitosis-to-

endocycle progression. In concert with thishypothesis, and despite its anticipated role

during mitosis, CDKB1;1 transcripts and as-

sociated protein activity can already be de-

tected during S-phase progression (18, 124,

138, 168). Actually, the kinetics ofCDKB1;1

transcription during S phase are identical to

those of genes that encode the pre-replication

complex itself (such as MCMs and CDC6).

The reason could be that CDKB1;1 pro-moter activity is controlled by E2F activity,

as shown by its up-regulation in E2Fa/DPa-

overexpressing plants and mutational analysis

of the E2F-binding site in theCDKB1;1pro-

moter (17, 115) (Figures 2and3).

Another well-documented molecular

switch for the mitotic cycle to the endocycle

is the CCS52A protein. InM. sativa, expres-

sion ofCCS52 correlates with the onset ofendoreduplication, and down-regulation of

CCS52A expression significantly reduces the

ploidy level (25, 178, 190). Mitotic cyclins

are likely candidates of CCS52A-mediated

proteolysis (64, 98). We hypothesize that the

cyclin partner of CDKB1;1 is the target for

CCS52A-mediated proteolysis (Figure 3).

Mitotic CDK activity could also be negativelyregulated by WEE1, because in the maize en-

dosperm theWEE1transcript levels increase

when cells undergo endoreduplication (176).

Through a still unspecified molecular

mechanism, E2Fe/DP-E2F-like protein 1

(DEL1) also regulates the onset to en-

doreduplication (191). Loss of function ofE2Fe/DEL1 results in increased ploidy levels,

whereas ectopic expression ofE2Fe/DEL1re-

duces endoreduplication. The observed DNA

ploidy changes are correlated with altered ex-

pression of a subset of E2F target genes that

encode proteins necessary for DNA replica-

tion. BecauseE2Fe/DEL1transcripts are de-

tected exclusively in mitotically dividing cells,the E2Fe/DEL1 operates as an important in-

hibitor of the endocycle onset by preserving

the mitotic state of proliferating cells and bysuppressing transcription of genes that are re-

quired for cells to enter the DNA endoredu-

plication cycle (191) (Figure 3).

CONCLUDING REMARKS

Considerable progress has been made in re-

cent years in our knowledge of the basicmechanisms that regulate cell division and en-

doreduplication in higher plants. In contrast,

very little is known on how the cell cycle ma-

chinery communicates with both intrinsic de-

velopment signals and external cues. Under-

standing this process might help to explain

growth rates and architecture of plants. Fur-

thermore, it might open new perspectives in

the breeding or engineering of plants with im-proved yield.

SUMMARY POINTS

1. The cell cycle machinery of plants is regulated by components that are conserved in

other eukaryotes as well as by molecules that are plant specific.

2. Plants contain many more cyclins than animals, possibly reflecting their role in ren-

dering plant development very plastic.

3. Plants are particularly well suited to study how the cell cycle machinery is regulated

by intrinsic developmental signals and environmental cues.

4. An increase in cell number in leaves is compensated for by a decrease in cell size and

vice versa.



18/31

5. Endoreduplication results from a controlled down-regulation of cyclin-dependentkinase activity at the G2-to-M transition. Various mechanisms can account for this

down-regulation.

ACKNOWLEDGMENTS

The authors thank Aurine Verkest for critical reading of the manuscript, Martine De Cock fo

help preparing it, and Karel Spruyt for artwork. This work was supported by a grant from th

Interuniversity Poles of Attraction Program-Belgian Science Policy (P5/13) and the Research

Foundation-Flanders (grant no. G008306). L.D.V. is a Postdoctoral Fellow of the Research

Foundation-Flanders.

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