Plant Molecular Biology43: 621–633, 2000.Dirk Inzé (Ed.), The Plant Cell Cycle.© 2000Kluwer Academic Publishers. Printed in the Netherlands.

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The role and regulation of D-type cyclins in the plant cell cycle

Marcel Meijer and James A. H. Murray∗Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, UK (∗author forcorrespondence; e-mail: j.murray@biotech.cam.ac.uk)

Key words:D-type cyclins, differentiation, G1/S control, plant cell cycle, proliferation, retinoblastoma protein

Abstract

The G1 phase of the cell cycle represents a period of commitment to cell division, both for cells stimulated toresume division from a resting or quiescent state, and for cells involved in repeated cell cycles. During this period,various signals that affect the cells’ ability to divide must be assessed and integrated. G1 culminates in the entryof cells into S phase, when DNA replication occurs. In addition, it is likely that several types of differentiationdecision may be taken by cells in the G1 phase. In both animals and plants, it appears that D-type cyclins play animportant role in the cell cycle responses to external signals, by forming the regulatory subunit of cyclin-dependentkinase complexes. The phosphorylation targets of D-cyclin kinases in mammalian cells are the retinoblastoma(Rb) protein and close relatives. Unphosphorylated Rb can associate with E2F transcription factors, preventingtranscription of genes under E2F control until the G1/S boundary is reached. The conservation of Rb and E2Fproteins in plants suggests that this pathway is therefore conserved in all higher eukaryotes, although it is absent infungi and yeasts. Here we review the current understanding of the roles and regulations of D-type (CycD) cyclinsin plants.

Introduction

The co-ordination of cell division with cell growth anddifferentiation is necessary to create complex multicel-lular organisms, and is achieved within the frameworkof a specific developmental plan that defines the char-acteristics of the particular organism (White-Cooperand Glover, 1995; Meyerowitz, 1997). Plants alsoneed to modulate this primary pattern of growth anddevelopment in order to respond flexibly to changes intheir environment, since they are unable to physicallymove to optimal locations (De Veylder, 1998; Fran-cis, 1998). The control of cell division in plants musttherefore sense and interact with external signals.

Almost half a century ago, Howard and Pelc (1953)introduced the terminology of the eukaryotic cell divi-sion cycle, recognising the fundamental importance ofthe separation of DNA replication (S phase) from celldivision (M phase) by the two gap phases (G1 and G2)in the sequence G1-S-G2-M. This arrangement allowsfor the precise control of DNA replication and mito-

sis, and it is not surprising that an ordered series ofmolecular and cellular processes define the order andcontrol of the cycle. Cells that temporarily or perma-nently lose the capacity to divide normally stop in theG1 phase with 2C DNA content, although in plantsthere appears to be much more flexibility, with bothG1 and G2 being important cell cycle exit points. Itshould be noted however that the frequent occurrenceof endoreduplication (chromosome replication with-out subsequent mitosis) can create G1 cells with 4Cor higher DNA content (see below). Non-cycling orquiescent cells are often said to be in G0 to distinguishthem from actively cycling G1 cells, although thereis no molecular definition of this state in plants (seeLoeffler and Potten, 1996).

Progression through the eukaryotic cell cycle islargely regulated at two principal control points, onelate in G1 phase and the other at the G2/M boundary.A further important control exists at the metaphase-anaphase transition, and no doubt further subsidiarycontrols also exist. Transit through these control

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points requires activated kinase complexes, consist-ing of a cyclin-dependent serine/threonine protein ki-nase (CDK) bound to a cyclin (for review, see Pines,1995b). CDK activity is dependent on cyclin binding,which also determines substrate specificity and sub-cellular localisation of the CDK complex. The cellcycle is driven forward by the sequential activation anddestruction of CDK activities, and this indicates thatCDKs and cyclins play central roles in the regulationof cell cycle commitment and progression (for review,see Pines, 1995b).

In this paper, we focus on the molecules that regu-late transit through G1 and entry into S phase in plantcells. This period not only includes the point of com-mitment to cell division, but may also represent thetime during which differentiation decisions are made.Although there are parallels between yeast and ani-mal controls during this stage in the cell cycle, notall processes and molecules involved are equivalentbetween the two systems. We discuss recent work sug-gesting that the proteins involved in G1/S controls inplants are more closely related to mammals than tofungi, and that decisions on proliferation and differ-entiation events may therefore be made in analogousways in mammals and plants.

Cell cycle control points

The term ‘cell cycle’ tends to imply an ever-rollingcycle of events, which is true for exponentially grow-ing cell suspensions, but might not be very relevantinvivo. In all multicellular organisms, cells are part of or-gans, and have defined spatial relationships with theirneighbouring cells in creating higher-order structures.Cells must sense when they are required to divide, andwhen division must be ceased or modified, to allowdifferentiation into specialised organs to occur (Fran-cis, 1998). Cells, therefore, must be able to integrateinformation on nutrient availability and environmentalconditions, the positional and developmental contextof the cell, and intracellular information, such as DNAdamage, in order to determine whether to continue todivide or not.

This integration of information operates throughcontrols at defined points in the cell division cycle toensure completion of one phase before the next one isinitiated (Hartwell and Weinert, 1989). The two maincontrol points in the cell cycle are at the G1/S transi-tion and at the G2/M transition. Progression throughthese control points is mediated by the activation of

cyclin-CDK complexes (for review, see Pines, 1995b).The first CDK to be described was encoded by thecdc2 gene ofSchizosaccharomyces pombe(Simanisand Nurse, 1986) and genetic analysis revealed thatthe cdc2gene product, p34cdc2, is required for bothcontrol points. Subsequently,cdc2homologues havebeen isolated for many organisms, including humanand several plant CDK genes (reviewed by Jacobs,1995). In contrast to yeast, in higher eukaryotes, mul-tiple CDKs regulate different stages of the cell cycle(reviewed by Pines, 1995a). In most plant cell types,the primary control point probably operates during G1phase, as for mammalian cells and yeast. Indeed, clas-sical studies on cell suspension cultures showed thatplant cells arrest in G1 or G2 when starved of nutri-ents or hormones, with the G1 arrest being the morestringent (reviewed by Bayliss, 1985).

In intact plants, many differentiated plant cells alsohave a G1 (2C) DNA content, indicating exit fromthe cell cycle in G1. However, significant propor-tions of cells stop dividing with a 4C DNA contentand can therefore be interpreted as undergoing G2 ar-rest. The status of these cells is complicated by thefrequency of endoreduplication in differentiated plantcells (Bayliss, 1985; reviewed by Traaset al., 1998).Such 4C cells may, therefore, represent cells in G1 thathave undergone an endoreduplication event rather thanG2 arrest (Bayliss, 1985). Nevertheless, it is likely thatin most cells the most important decision to divide ordifferentiate operates in late G1 phase.

The second main control point operates in G2and determines the entry into mitosis. In both yeastand mammals, this transition is largely controlled bykinase activity of thecdc2gene product. In associa-tion with mitotic cyclins (A- and B-type cyclins), thep34cdc2phosphorylates a set of substrates at the G2/M,driving cells into mitosis (reviewed by Norbury andNurse, 1992). Both A-like and B-like cyclins havebeen isolated in plants, and it is likely that the B-likecyclins are regulators of G2/M (reviewed by Renaudinet al., 1998). The ability of a B-like cyclin to acceler-ate growth inArabidopsissuggests that this G2 controlpoint may be a limiting factor in root cell division(Doerneret al., 1996).

The control of G1/S transition in mammalian cells

In this section, we briefly outline the current view onG1/S transition controls in mammalian cells. Thesecontrols in mammalian cells and budding yeast (Sac-

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charomyces cerevisiae) are discussed in more detail ina number of recent reviews (Morgan, 1997; Johnsonand Walker, 1999; Pavletich, 1999).

External signals impinge on the cell cycle at aprincipal point in G1, called the restriction point (R)in mammalian cells, and depending on the nature ofthe signals, cells either commit to another round ofcell division or exit the cell division cycle and adoptalternative differentiation pathways (Pardee, 1989).Progression through R is mediated by D-type cyclins,whose transcription is absolutely dependent on serumgrowth factors (Matsushimeet al., 1991; Ajchenbaumet al., 1993; Andoet al., 1993; reviewed by Sherr,1993, 1994). Transcript levels decline rapidly upongrowth factor removal and reappear again upon addi-tion. Cyclin-D-dependent CDKs direct phosphoryla-tion of the retinoblastoma (Rb) protein in mid-to-lateG1 phase, thereby driving cells through R and al-lowing activation of E2F controlled genes, which arerequired for S phase.

E-type cyclins accumulate transiently in late G1phase, forming kinase complexes that accelerate thephosphorylation of Rb, thereby irreversibly drivingcells across the G1/S boundary (for review, see Sherr,1996). Recent evidence suggests functional differ-ences in the phosphorylation of Rb protein by cyclinD-CDK4/CDK6 and cyclin E-CDK2 complexes (Mitt-nacht, 1998), sincein vitro different sites on Rb arephosphorylated (Kitagawaet al., 1996; Zarkowskaet al., 1997).

A defining characteristic of D-type cyclins is thepresence of a specific motif consisting of the sequenceLxCxE (single-letter code, x being any amino acid)that is responsible for the binding of D-type cyclins toRb and related proteins. Interestingly, the same motifis shared by the transforming proteins of several DNAtumour viruses, which inactivate Rb as part of theirinfection and replication mechanism.

Isolation of plant D-type cyclins

G1/S control in mammalian cells is of primary im-portance for understanding both cellular proliferationand differentiation. These issues are also important forplants, where the majority of post-embryonic divisionactivity is concentrated in the meristematic regions.Moreover, many plant cells can dedifferentiate in re-sponse to wounding, pathogen attack or exogenousapplication of plant hormones, suggesting more plas-

ticity in controlling cell cycle entry and exit than thatnormally found in mammalian cells.

It was therefore interesting to investigate whetherplant controls operating in G1 phase were more closelyrelated to those of yeast or mammals. Plant D-typecyclin (CycD cyclin) cDNAs were first isolated fromArabidopsis (Soni et al., 1995) and alfalfa (Dahlet al., 1995) by their ability to functionally comple-ment yeast strains that were defective in two of theirthree G1 (CLN) cyclins, with the third cyclin undercontrol of a galactose promoter (Xionget al., 1991).They were defined as D-type cyclins on the basis oflow sequence homology to mammalian D-type cy-clins, and the presence of the conserved LxCxE motif.Plant CycD proteins as a class have higher homologyto animal D-type cyclins than any other class of cy-clins, although residues are identical between plantand animal D-type cyclins at only about 20–25% ofpositions (Renaudinet al., 1996).

Subsequently, CycD cyclin cDNAs have been iso-lated from Antirrhinum (Gaudin et al., 2000), He-lianthus (Freeman and Murray, unpublished results),tobacco (Sorrellet al., 1999; Nakagamiet al., 1999;M. Sekine, personal communication),Pisum sativum(Shimizu and Mori, 1998), tomato (A. Kvarnheden,personal communication; C. Chevalier, unpublished)andChenopodium(Renzet al., 1997; Fountainet al.,1999) by screening cDNA libraries with CycD cyclincDNA probes. These CycD cyclins form three distinctgroups designated CycD1, CycD2 and CycD3 (Fig-ure 1; Renaudinet al., 1996; Murrayet al., 1998).Using a two-hybrid screen in yeast with Cdc2aAt as abait, De Veylderet al. (1999) recently isolated a fourthD-type cyclin inArabidopsis. At this stage it is unclearwhether this CycD cyclin belongs to a separate CycDgroup or is part of the CycD2 group (Figure 1). It isimportant to note that all the groups of plant CycD cy-clins are more closely related to each other than to anyof the animal D-type cyclins, and that the numberingof the CycD groups in plants is not related to the num-bering of different cyclin D’s in animals (Renaudinet al., 1996).

When multiple genes have been identified in onegroup (e.g. CycD3) the suffix number indicates onlythe order of isolation in a given species. Thus,CycD3;1 from tobacco is not most closely related toCycD3;1 from Antirrhinum (see Figure 1). In fact,the relationship between the different CycD3 cyclinssuggests the probable existence of three subgroups,which we propose to designate CycD3a, CycD3b,and CycD3c (Figure 1), and for three species (An-

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Figure 1. CycD cyclin relationships. The relationship between plantCycD cyclins was determined by generating a multiple alignment ofthe sequences using the CLUSTALX program. This alignment wasthen optimised by manual editing, and displayed using the PILEUPprogram in the GCG package. Cyclin nomenclature is accordingto Renaudinet al. (1996). Antma,Antirrhinum majus(Gaudinet al., 2000); Arath,Arabidopsis thaliana(Soni et al., 1995; DeVeylder et al., 1999; T. Jack, personal communication); Cheru,Chenopodium rubrum(Renzet al., 1997; Fountainet al., 1999)Heltu, Helianthus tuberosus(D. Freeman and J. A. H. Murray, un-published data); Lyces,Lycopersicon esculentum(A. Kvarnheden,personal communication; C. Chevalier, unpublished data); Medsa,Medicago sativa(Dahlet al., 1995); Nicta,Nicotiana tabacum(Sor-rell et al., 1999; Nakagamiet al., 1999; M. Sekine, unpublisheddata); Pissa,Pisum sativum(Shimizu and Mori, 1998). The threedistinct groups of CycD cyclins, CycD1, CycD2/4 and CycD3, areindicated on the right. The relationship between the different CycD3cyclins suggests the probable existence of at least three subgroups.These have been designated CycD3a, CycD3b and CycD3c.

tirrhinum, tomato and tobacco) CycD3s have beenisolated from more than one subgroup. We suggestthe use of the subgroup designation CycD3a, CycD3b,CycD3c, as foreseen by Renaudinet al. (1996), isuseful to indicate sequence relationships within theCycD3 group. The relationship of a secondArabidop-sisCycD3 gene (CycD3;2, T. Jack, personal commu-nication) to the subgroups is currently unclear. Thepresence of multiple CycD3 genes raises the questionof functional redundancy of these genes, and the extentto which they may have distinct or overlapping roles,but the greater conservation of subgroup members be-

tween species than similarity with other subgroups inthe same species indicates a possible conserved func-tion. Interestingly, distinct CycD3s that have beenisolated from bothAntirrhinumand tobacco are differ-entially expressed inAntirrhinummeristems (Gaudinet al., 2000) and tobacco cell suspension cultures (Sor-rell et al., 1999). CycD3a (Antma;CycD3;1) inAntir-rhinum is expressed in organ primordia only, whereasCycD3b (Antma;CycD3;2) is probably expressed inall dividing cells (Doonan, 1998; Gaudinet al., 2000).Moreover, differences exist in the regulation of sub-group members by external signals (Gaudinet al.,2000). Furthermore, unlike theArabidopsisCycD3;1gene, which is highly cytokinin-inducible (see below;Riou-Khamlichi et al., 1999), cytokinin did not in-duce the alfalfa CycD3 gene and its overexpressionhad no obvious effect (reported in Inzéet al., 1999).Taken together, these results suggest that cyclins indifferent CycD groups and subgroups may have dis-tinct functions, although the extent to which these arenon-overlapping will have to await the identificationof insertion mutants.

Characteristics of plant D-type cyclins

As mentioned above, a defining characteristic of allcyclin D proteins identified to date is the presence ofthe Rb interaction motif LxCxE or a very closely re-lated sequence near their N-terminus. In mammalianD cyclins, this motif is within a few amino acids of theinitiation methionine. All plant CycD cyclins, with theexception of tobacco CycD3;4, contain the same Lx-CxE motif near the N-terminus. In contrast, CycD3;4contains a LxCxD motif, in which aspartate replacesthe glutamate residue. In common with animal D-typecyclins, all CycD cyclins isolated to date, with the ex-ception ofArabidopsisCycD2;2 (CycD4), contain atleast one acidic residue (D or E) at positions−1 or−2relative to the LxCxE motif (Renaudinet al., 1996; DeVeylderet al., 1999).

All cyclins contain a defining homologous regionof ca. 100 amino acids known as the cyclin box, whichis involved in the interactions with the CDK partner(Lees and Harlow, 1993; Jeffreyet al., 1995; Renaudinet al., 1998). The corresponding region in CycD cy-clins has relatively low homology to mitotic cyclins,but nine residues are invariant between the cyclin boxof all cyclins of the A, B, and D classes in animals andplants, including five residues that have been shownexperimentally to be essential for catalytic activity.

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The majority of cyclins are destroyed rapidly atcertain points in the cell cycle and this ability to ac-tivate turn-over of the cyclins is central to cell cyclecontrol (Murrayet al., 1998; Renaudinet al., 1998).In the case of mitotic cyclins, a specific N-terminalsequence called the destruction box targets cyclins forspecific ubiquitin-mediated destruction during mitosis(Glotzeret al., 1991; Pines, 1995b; Genschicket al.,1998). The types of destruction motifs in plant mitoticcyclins have recently been reviewed by Renaudinet al.(1998), and analysed by Genschicket al. (1998).

G1 cyclins in both yeast and mammals have shorthalf-lives and their rapid turn-over depends on thepresence of so-called PEST sequences, regions richin these four amino acids, which are characteristicof many proteins with a rapid turn-over (Rechsteinerand Rogers, 1996; Rogerset al., 1996). Examinationof all CycD cyclins using the program PESTFIND(Rogerset al., 1996) identified PEST sequences in allCycD cyclins, apart from tobacco CycD2;1 (Sorrellet al., 1999). This suggests that PEST sequences area general feature of G1 cyclins and that most plant Dcyclins, like their animal homologues, may be short-lived proteins. However, in no case has the role ofPEST sequences in determining the half-life of plantCycD protein been experimentally verified.

Regulation of D-type cyclins in cell cultures

In mammalian cell cultures, the transcription of D-type cyclins is highly growth-factor-dependent (Mat-sushimeet al., 1991; Baldinet al., 1993; Sewinget al., 1993). Growth-factor-starved cells or quiescentcells show low levels of Cyclin D mRNA, and, uponaddition of growth factor, show rapidly increasing ex-pression levels that reach a maximum after 10 h ofserum stimulation and 8 h before DNA synthesis be-gins (Sewinget al., 1993). However, the abundance ofcyclin D1 transcript does not change significantly incycling cells, suggesting that cyclin D1-kinase activ-ity is post-transcriptionally regulated. Indeed, cyclinD protein levels are high in G1 cells and decline sig-nificantly by late S phase (Matsushimeet al., 1991;Sewinget al., 1993).

In human cells, D-type cyclins act as growth fac-tor sensors with their expression depending more onextracellular stimuli than on the position of the cellcycle (Sherr, 1993, 1996). Therefore, D-type cyclinsare proposed to provide the link between stimuli fromthe environment and the cell cycle. Support for this hy-

pothesis comes from observations that over-expressionof D-type cyclins does not only reduce the length ofthe G1 phase, but also partially overrides the needof dividing cells for mitogens (Kato and Sherr, 1993;Zwijssenet al., 1996).

Analysis of D-type cyclin expression in partiallysynchronisedArabidopsis cell suspension culturesshowed that CycD3 accumulated rapidly upon releaseof the G1/S block before accumulation of histone H4expression and the onset of S phase (Soniet al., 1995;Fuerstet al., 1996). CycD1 is expressed at very lowlevels in liquid cultured cells and CycD2 mRNA levelswere unaffected by a G1/S block and release using lowconcentrations of cycloheximide. Levels of CycD3remained relatively constant after the initial accumula-tion at the G1/S boundary (Fuerstet al., 1996), whichmirrors the behaviour of CycD1 mRNA in mammaliancells (Matsushimeet al., 1991; Sewinget al., 1993).

In tobacco BY-2 cell suspension cultures, whichare highly synchronisable (Nagataet al., 1992),CycD3;2 is induced in G1 upon re-entering the cellcycle after synchronisation and remains at a constantlevel throughout the cell cycle (Sorrellet al., 1999).Surprisingly, CycD2;1 and CycD3;1 both showed theirgreatest abundance in mitotic cells (Sorrellet al.,1999). It could be that these cyclins are required forentry into or progression through mitosis. In pro-liferating mammalian cells, retinoblastoma proteinsare further phosphorylated in G2/M before being de-phosphorylated in the later stages of mitosis (De-Caprioet al., 1992; Ludlowet al., 1993; Taya, 1997).However, it is also possible that mitotic accumulationis a BY-2 cell-specific phenomenon and not a normalfeature of the plant cell cycle. In the human HeLa tu-mour cell line an increase in human cyclin D1 is alsoobserved in G2/M phase, but not in other cell lines orin primary cultures (Motokuraet al., 1992). It has beensuggested that selection may have occurred in this cellline for altered or deregulated expression of cyclin D1as a consequence of extended proliferation (Sewinget al., 1993). Similarly, tobacco BY-2 cells have beengrowing in culture for over 30 years and show an ex-ceptional ability for rapid cell growth (Nagataet al.,1992).

Similar to the response of mammalian CycD cy-clins to serum growth factors, plant CycD cyclins areregulated in response to exogenous signals known toaffect growth of plant cells. Auxin and cytokinin areimportant plant growth regulators (PGRs or hormones)that are required for most plant cell cultures. In ad-dition to these plant hormones, sucrose is of central

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importance to plant metabolism as the major transportproduct of photosynthesis, and a possible signallingmolecule in the regulation of a large number of genes(Koch, 1996; Jyung and Sheen, 1997). Sucrose isa favourable candidate for modulating cell divisionrates because its availability to dividing cells in shootand root meristems will be a reflection on the over-all photosynthetic capacity of the plant and thereforeon the environmental conditions to which the plant isexposed (Koch, 1996).

UsingArabidopsissuspension cell cultures, it wasshown that sucrose can induce both CycD2 and CycD4in starved suspension cells (Soniet al., 1995; DeVeylderet al., 1999). A more comprehensive analysisof all possible combinations of sugars and PGRs incell cultures that were deprived of cytokinin, auxin andsucrose for 48 h showed that CycD3 was specificallyinduced by cytokinin and CycD2 by sucrose. Recently,more detailed analysis has shown that cytokinin induc-tion of CycD3 is dependent on the presence of sucrose,and sucrose alone can also induce CycD3 (Riou-Khamlichi et al., submitted). When cycloheximidewas used at a concentration that inhibits both pro-tein synthesis and cell cycle progression, CycD2 andCycD3 levels were still stimulated by sucrose. Fromthese experiments it can be concluded that neithercell cycle progression norde novoprotein synthesisis required for increases in CycD2 and CycD3 mRNAlevels and this is consistent with their proposed rolesas sensors of nutrient status in cell cycle control (Riou-Khamlichi et al., submitted). The response of CycD2and CycD3 to different stimuli suggests that each ofthem is involved in a separate signal transduction path-way, a conclusion supported by the different responsesof the two genes to the presence of inhibitors of proteinphosphatases (Riou-Khamlichiet al., submitted).

Regulation of D-type cyclinsin vivo

Data on CycD cyclin expression levels in intact tissuesare more limited.In situ hybridisation studies on thetwo CycD3 homologues inAntirrhinum shoot apicaland floral meristems confirmed that CycD3 mRNAdoes not accumulate in a cell-cycle-dependent man-ner, unlike cyclin B genes that have been studied(Doonan, 1998; Gaudinet al., 2000). InAntirrhinum,CycD1 is expressed throughout the meristem and atlow levels in other tissues. Both CycD3 genes ofAn-tirrhinum are expressed within regions that exhibit celldivision activity, but CycD3a is only expressed in the

peripheral region of the meristem, in particular in or-gan primordia (Gaudinet al., 2000). CycD3b appearsto be generally expressed in dividing cells. In floralmeristems, CycD3b expression is modulated in cellssurrounding the base of organ primordia and, later indevelopment, in the ventral petals and is repressed inthe dorsal stamen. These results add weight to the con-cept of differential function within apical meristems(Doonan, 1998), and it suggests that changes in cellcycle control could be early events in the switch to adifferentiated state (discussed later).

In Arabidopsis, CycD3;1 is shown by RNA gel blotanalysis to be highly expressed in mature roots andto show somewhat lower expression levels in above-ground and callus tissue (Soniet al., 1995; reviewedby Murrayet al., 1998).In situ hybridisation studieson the recently isolated CycD4 inArabidopsisshowedthat CycD4 is expressed during vascular tissue de-velopment, embryogenesis and formation of lateralroot primordia (De Veylderet al., 1999). The threetranscripts of CycD1 show differential abundance indifferent tissues with the longest transcript particularlyprevalent in flowers, and to a lesser extent in roots andcallus material, whereas in leaves the intermediate andshorter transcripts prevail (Soniet al., 1995).

Fusion of the CycD3;1 promoter to theβ-glucuronidase (GUS) marker gene in transgenicAra-bidopsisresulted in GUS activity in the shoot apicalmeristem, and in stele cells associated with lateralroot primordia. In older roots, more general expres-sion was observed in the stele, but GUS activity wasnever observed in the root apex (C. Riou-Khamlichiand Murray, unpublished observations). This expres-sion pattern distinguishes CycD3;1 from ‘general’ cellcycle genes that are expressed in all dividing cells,since tissue specificity is observed.

The role of cytokinin in regulating the CycD3;1 cy-clin in Arabidopsishas been reported recently (Riou-Khamlichi et al., 1999). In both cell cultures andin whole plants, application of cytokinin inducesCycD3. Moreover, leaf tissue from transgenic plantsthat constitutively express CycD3 were able to gen-erate callus in the absence of exogenous cytokinin,which could be maintained on auxin alone. It has beensuggested that cytokinin activatesArabidopsiscell di-vision through induction of CycD3 at the G1/S phasetransition in whole plants, as well as in tissue culture(Riou-Khamlichiet al., 1999).

The only otherin planta study on CycD cyclingene expression levels is on CycD3;Ms in alfalfa(Dahl et al., 1995), which appears to be induced

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before the onset of DNA synthesis. CycD3;Ms expres-sion levels are high in the root and is limited to thepericycle, endodermis and outer cortex (Dahlet al.,1995).

In summary, there is still a lot to learn about theexpression of CycD cyclins. So far, there is clear ev-idence for tissue- and meristem-specific expression,and cell cycle behaviour that may be similar to that inmammalian cells. It has been established that CycD3in Arabidopsis is stimulated by cytokinin, but ad-ditional signals, such as sucrose, nitrate, cell cycleprogress and cellular position in specific tissues alsoplay a role. Nevertheless, all results clearly impli-cate plant CycD cyclins as important integrators ofproliferating signals in G1 phase.

Interactions of D-type cyclins with CDKs and Rbproteins

Progression through the eukaryotic cell cycle is me-diated by the phosphorylation of key substrates bycyclin-dependent kinases (CDKs). Potential substratesof CDKs include cytoskeletal proteins (e.g. lamins),chromatin-associated proteins (e.g. histone H1), andregulatory proteins, such as Rb (reviewed by Nigg,1993). The substrate specificity of different CDKs isthought to be regulated by the targeting of CDKs todistinct cellular compartments by binding of CDKs todifferent types of cyclins (Pines, 1995b).

The interaction of CycD cyclins in plants withCDKs has been shown with a yeast two-hybrid ap-proach. InArabidopsis, a CDK (Cdc2aAt; De Veylderet al., 1997) was used as a bait to identify putativesubstrates of CDKs. Using this approach, cyclin D1;1was identified as a partner of Cdc2aAt and Cdc2bAt(De Veylderet al., 1997). Likewise, recent results us-ing immunoprecipitation with specific antisera showthat inArabidopsisboth CycD2 and CycD3 associatewith Cdc2aAt. Kinase activity could be assayed usinghistone H1 as a substrate with both CycD2 and CycD3immunoprecipitates (S. Healey and J.A.H. Murray,manuscript in preparation).

As mentioned earlier, all D-type cyclins contain N-terminal LxCxE motifs, which are capable of bindingthe pocket domain of Rb (Dowdyet al., 1993; Katoet al., 1993). The Rb family of proteins play an im-portant role in mammalian cell cycle by controllingtransit through G1 and inhibition of inappropriate cellproliferation (Weinberg, 1995). Rb exerts its negativeregulatory control by inactivating the E2F family of

transcription factors (Sherr, 1996), through recruit-ment of histone deacetylases (HDAC) to the promot-ers of E2F-regulated genes (reviewed by Brehm andKouzarides, 1999). The Msi1 protein identified intomato interacts with Rb, and is a homologue of ahuman component of an HDAC complex (Achet al.,1997b; Figure 2). E2F activity is normally essential forS phase, and Rb inhibits its activation by binding E2Fvia a region of Rb containing two conserved sequenceblocks, which form the so-called ‘A/B pocket domain’(reviewed by Dyson, 1998). This pocket domain isalso required for the growth restraining ability of Rb,and cyclin D interaction (Katoet al., 1993).

Binding and inactivation of E2F by Rb is reg-ulated by Rb phosphorylation. During most of G1,Rb is hypo-phosphorylated, but phosphorylation ofRb by the cyclin-dependent kinase complexes cyclinD/CDK4, cyclin E/CDK2 and cyclin A/CDK2 inacti-vates Rb (Sherr, 1996). This activates E2F and leadsto S-phase onset. Mutational analysis has shown therequirement for the intact LxCxE motif in D-type cy-clins as well as integrity of the pocket domain of Rb(Dowdy et al., 1993; Ewenet al., 1993). The recentisolation of E2F cDNA clones from wheat, tobaccoandArabidopsisreinforces the idea that regulation ofG1/S transition is more closely related to that foundin animals than that in yeast (Ramirez-Parraet al.,1999; Sekineet al., 1999; de Jageret al., manuscriptin preparation).

The importance of cyclin D-Rb interactions wasshown by several lines of evidence. First, the cyclin Dcomponent of the cyclin D/CDK4 complex links Rb toenvironmental clues, since cyclin D is an unstable pro-tein whose transcription is totally dependent on serumgrowth factors in the growth media (Sewinget al.,1993; Sherr, 1993). Second, at least one residue onRb that is required for Rb inactivation by phosphory-lation can only be phosphorylated by cyclin D/CDK4(Kitagawaet al., 1996), so S-phase entry is ultimatelydependent on cyclin D/CDK activity. Finally, cyclinD-kinases are unnecessary for progression throughthe restriction point in cells that lack Rb, suggestingthat Rb may be the only essential substrate of cyclinD/CDK4 (Sherr, 1996).

The recent isolation of Rb homologues in plants(Grafi et al., 1996; Xie et al., 1996; Ach et al.,1997a) and the interaction of these homologues withplant CycD cyclins through the conserved LxCxE mo-tif (Ach et al., 1997a; Huntleyet al., 1998) indicatesthat the pathway of G1/S control involving cyclin D-mediated Rb phosphorylation and E2F activation is

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Figure 2. Model for control of G1/S transition in plants, indicating the stimulatory and inhibitory signals that are incorporated into control ofa START-like point in plant cells, and modulate the activity of CDKs. CycD2 and CycD3 respond to sucrose, whereas only CycD3 is inducedby cytokinin. CDK inhibitors (CKI) prevent kinase activity of cyclin-CDK complexes. ICK1, a CDK inhibitor fromArabidopsis, was isolatedand it was shown that ICK1 interacts with both CycD3 and Cdc2a (Wanget al., 1998) and is induced by abscisic acid (ABA). This suggeststhat ABA might provide an inhibitory mechanism for the cell cycle, operating through CycD activity. Plants also contain homologues of humanRb-binding proteins (RbAp48; Achet al., 1997b), itself a homologue of yeast MSI1. RbAp48 is a component of a human histone deacetylasecomplex, and its plant homologue MSI1 may play a similar role (Achet al., 1997b). The presence of an Rb-binding motif in a gemini virusreplication protein indicates that viral proteins in plants may drive cells into S phase by directly promoting release of E2F from Rb (Xieet al.,1995; Achet al., 1997a).

conserved among higher eukaryotes. This indicatesthat G1/S control in plants is more closely related tomammalian G1/S regulation than to yeast, which doesnot involve proteins with homology to cyclin D, Rbor E2F (Pines, 1995b; Nasmyth, 1996; Huntleyet al.,1998).

Recently, Nakagamiet al. (1999) have also shownthat a tobacco Rb-related protein (NtRb1) can bephosphorylatedin vitro by a kinase assembled from to-bacco Cdc2a and cyclin Nicta;CycD3;3. An antibodyagainst the cyclin can immunoprecipitate a complexfrom tobacco BY-2 cells that can also phosphorylateNtRb1, suggesting that NtRb1 phosphorylation is in-

deed mediated by a CycD kinasein vivo (Nakagamiet al., 1999).

So far, little is known about the regulation ofcyclin-CDK complexes in plants. Studies in yeast andmammalian systems have shown that activation ofCDK involves not only binding of a cyclin, but alsoinvolves a CDK-activating kinase (CAK) and CDC25protein phosphatase (Lees, 1995). Another level ofregulation of cyclin/CDK complex activity is providedby CDK inhibitors, which stoichiometrically inhibitCDK activity (reviewed by Pines, 1995; Harper andElledge, 1996). The discovery of a CDK inhibitor inArabidopsis(ICK1; Wanget al., 1997) indicates thatsimilar regulatory pathways exist in plants.ICK1 was

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subsequently shown to be induced by abscisic acid,and uponICK1 induction a decrease in histone H1 ki-nase activity was observed (Wanget al., 1998).ICK1clones were also identified in yeast two-hybrid screenswith CycD3 as bait, and it was subsequently confirmedthat ICK1 protein could interact with both Cdc2a andCycD3 byin vitro binding assays (Wanget al., 1998).These results indicate a role for CDK inhibitors inregulation of CycD kinases.

Role of D-type cyclins in cell differentiation anddevelopment

An emerging field is the role of D-type cyclins incellular differentiation in human tumours. Cyclin D1was originally identified as a proto-oncogene activatedby translocation to a thyroid promoter in parathyroidadenomas (Motokuraet al., 1991). Subsequently, itwas show that cyclin D1-deficient mice had a reducedbody size and a reduced number of cells in their reti-nas, a tissue that has a very high demand for cyclinD1 (Sicinki et al., 1995). Recent developments inmammalian systems showed that increased expressionlevels of cyclin D2 is associated with testicular cancerdevelopment (Bartkovaet al., 1999). In more gen-eral terms, it appears that the cyclin D-CDK4 andD-CDK6 complexes interact with the proto-oncogenec-myc. Loss of c-myc causes profound growth de-fects correlated with a 12-fold reduction in cyclinD1-CDK4 activity (Mateyaket al., 1999). It is there-fore imperative that we develop our understanding ofthe interaction between proliferation and differentia-tion in plant cells and in particular the reversibilityof this switch, its relationship to meristem function,and regulation of cell cycle decision by organ identitygenes.

Some data already point to the likely importanceof D-type cyclins in cellular differentiation in plants.The discovery of the differential expression of twoCycD3 genes inAntirrhinum, with one of them onlyexpressed in incipient and developing primordia andthe other down-regulated in boundary layers of cellsthat lie between proliferating zones (Doonan, 1998;Gaudinet al., 2000) hints at the possibility that CycDcyclins are involved in meristem function and the con-trol of proliferation and differentiation. So, althoughit may seem intuitive that cell cycle regulation shouldfollow on from developmental controls, it is possiblethat changes in cell cycle regulation could drive down-stream differentiation events. This hypothesis finds a

parallel in the discovery of the change from controlledcell division to tumourous growth upon disruptionof cyclin D2 expression levels in the human testis(Bartkovaet al., 1999).

This relationship between proliferation and dif-ferentiation is likely to be subtle and complex. Thestructure of the shoot apical meristem (SAM) of di-cotyledonous plants illustrates the issues involved.The central zone of the SAM consists of a group ofcells whose size and morphology do not change dur-ing most of the post-embryonic development of theplant (Laufset al., 1998; Doerner, 1999; Lenhard andLaux, 1999). This population of cells, the central zone,represents the source of all above-ground tissues. Themeristem cells divide anticlinally (division plane per-pendicular to the surface), so the progeny cells arepushed into the surrounding peripheral zone. In thiszone, leaf or floral primordia are specified and developin a spiral pattern

Cells in the peripheral zone must therefore undergodecisions that will result in the formation of determi-nate structures, such as leaves and floral organs, whichconsist of differentiated, non-dividing cells. Thus, thefate of cells in the peripheral zone is different fromcells in the central zone, and may be thought of as hav-ing undergone a differentiation process. However, fewif any morphological differences are found betweencells in the two zones, and the rate of cell division isactually faster in cells in the peripheral zones (Lyndon,1998). Also, these peripheral cells produce progenycells which will develop into a number of differentcell types (Francis, 1998). Nevertheless, all cells inthe peripheral zone and their progeny will eventuallydifferentiate, unlike cells in the central zone.

It is likely, therefore, that several types of differen-tiation decisions are made by plant cells that involvealterations in the control of the cell cycle, suggestingintimate interconnections between proliferation anddifferentiation. One example would be the loss ofstem cell characteristics, corresponding to exit fromthe central zone of the SAM. Cells undergoing thistransition into the peripheral zone differ from cells inthe central zone in that they lose the ability to giverise to stem cells, and the progeny of these periph-eral zone cells will therefore ultimately differentiateand cease division. The transition from central zoneto peripheral zone characteristics may involve aspectsof cell cycle control as the proliferation rate increases(Lyndon, 1994).

A subset of peripheral zone cells become in-volved in the initiation of new organ primordia, ei-

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ther leaves or flowers depending on the characteris-tics of the apical meristem, and the expression ofCycD3a (Antma;CycD3;1) inAntirrhinum is a mole-cular marker for these cells (Doonan, 1998). In addi-tion, in Arabidopsisthe expression of the homeoboxgeneSTM is absent from cells participating in theformation of primordia (Longet al., 1996).

Further events occur when cells embark on cell-specific differentiation pathways and become morpho-logically or physiologically distinct. This is likelyto correspond to a reduction in cell division rates.One could speculate that Rb-related proteins may beinvolved in mediating this reduction, or may be asso-ciated with differentiation events. In the final stagesof determination, a cell ceases all division and exitsfrom the cell cycle either in the G1 or the G2 phase.Regardless of cell cycle exit point, such cells mightbe predicted to lack CycD expression and have highlevels of Rb-related proteins. Further types of differ-entiation events that relate to cell cycle controls arelikely in cells that undergo further events such as en-doreduplication (Traaset al., 1998), or programmedcell death in processes such as xylogenesis (for review,see Pennel and Lamb, 1997).

Conclusions

Mammalian D-type cyclins regulate progression ofcells through the G1 phase of the cell division cyclein response to extracellular signals through the in-teraction with Rb. The plant CycD cyclins that havebeen analysed so far show conserved regions and ex-pression patterns that have parallels to those found inmammalian systems. This suggests that CycD cyclinsmay also function as integrators of external signalsinto the cell cycle via interactions with plant Rbs.The recent discovery thatArabidopsisCycD cyclinscan bind both plant and human Rb proteins via theconserved LxCxE motif supports this hypothesis (Achet al., 1997a; Gutierrez, 1998; Huntleyet al., 1998).

The requirement for plant growth regulators suchas auxins and cytokinins for the growth of cell cultures(Murashige and Skoog, 1962) and for re-entry into thedivision cycle of quiescent cells has been known formany years (Bayliss, 1985). Studies on tobacco pithexplants showed that auxin increased the amount ofCDK protein. However, this protein did not have ki-nase activity unless cytokinin was also present (Johnet al., 1993). It was proposed that CDKs are inducedby auxin (Miao et al., 1993; Murrayet al, 1998;

reviewed by Mironovet al., 1999), whereas CycD3represents the cytokinin-limited component requiredfor the G1/S transition of the cell cycle (Murrayet al.,1998). Other cytokinin targets are likely to be impor-tant in other species or cell cycle controls (Johnet al.,1993). Recent studies on cell suspension cultures ofArabidopsisshowed the induction of expression levelsof CycD3 by cytokinin (Riou-Khamlichiet al., 1999)and CycD2 and CycD4 by sucrose (De Veylderet al.,1999; Riou-Khamlichiet al., submitted).

Here we present a model on the control of G1/Stransition in plants, incorporating the recent discov-eries of inducibility of CycD cyclins by sucrose andcytokinin (Figure 2). Various signals, both stimula-tory and inhibitory, are incorporated into control of aSTART-like point in plant cells (Murrayet al., 1994;Murray, 1998) and modulate the activity of CDKs.

The strong parallels in controlling G1/S transitionin plants and mammals suggests that common themeswill be found. Future work will be exciting as the in-volvement of CycD cyclins in plant-specific aspectsof both proliferation and differentiation is uncovered.We expect that studies on the cell cycle regulators willincorporate studies on meristematic controls, organidentity and developmental processes. This combinedeffort will result in the uncovering of the features thatare unique to plant growth and development.

Acknowledgements

We thank Thomas P. Jack, Masami Sekine and An-ders Kvarnheden for sharing sequences in advance ofpublication.

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