a plant cyclin b2 is degraded early in mitosis and its …488 mitosis (resnitzky et al., 1995; den...

IntroductionProgress through the eukaryotic cell cycle is regulated bysequential waves of different cyclin and cyclin-dependentkinase (Cdk) activities (Pines and Rieder, 2001). Both in plantsand in animals, several classes of Cdks exist, which act incombination with different cyclins at the major control stepsof the cell cycle (Joubes et al., 2000). In animals, S phase isinduced by Cdk2 bound to the S-phase-specific E- and A-typecyclins, and M phase is triggered by Cdk1 associated with themitotic A- and B-type cyclins (Pines, 1999). While entry intomitosis is mainly controlled by cyclin A-associated Cdk1kinase, final commitment to mitosis is mediated by the rapidactivation of cyclin B-Cdk1 (Pines and Rieder, 2001). Thisprocess is highly regulated and involves several levels ofcontrol, such as phosphorylation and dephosphorylation eventson conserved sites of the Cdk kinase, binding of inhibitors, andthe subcellular localization of the Cdk-cyclin complex (Ohiand Gould, 1999).

The first functional evidence that Cdks are pivotal for mitoticcontrol in plants came from experiments in whichmicroinjection of an active Cdk complex induced entry intomitosis (Hush et al., 1996). The plant functional homologue of

the animal Cdk1 is named CdkA and has a protein kinaseactivity that peaks at the G1-S and the G2-M transitions (Bögreet al., 1997). Treatment of cells with roscovitine, a drug thatmost specifically inhibits CdkA, blocks cell cycle progressionboth in G1 and G2 phases, further indicating multiple roles forplant CdkA during G1 and G2 phases (Binarova et al., 1998).Another group of plant Cdks is divided into two subgroups,CdkB1 and CdkB2, and exists only in plants (Joubes et al.,2000). The tightly regulated expression and activity of B-typeCdks, as well as the block in G2 phase by the expression of adominant-negative mutant form of CdkB1, suggest a specificfunction during G2-phase and mitosis (Magyar et al., 1997;Porceddu et al., 1999; Porceddu et al., 2001).

There are three A-type and two B-type cyclin groups inplants (Renaudin et al., 1998). In synchronized BY2 cellscyclin A3 is expressed during an earlier time window, from theG1-S transition until onset of mitosis compared with cyclin A1and A2, which are expressed during mid S phase until mid-mitosis (Reichheld et al., 1996). Cyclin A2-associated kinaseactivity peaks both in S phase and mitosis in synchronizedalfalfa cells (Roudier et al., 2000). In animals a single A-typecyclin associates with different Cdks during S phase and

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Mitotic progression is timely regulated by the accumulationand degradation of A- and B-type cyclins. In plants, thereare three classes of A-, and two classes of B-type cyclins,but their specific roles are not known. We have generatedtransgenic tobacco plants in which the ectopic expressionof a plant cyclin B2 gene is under the control of atetracycline-inducible promoter. We show that theinduction of cyclin B2 expression in cultured cells duringG2 phase accelerates the entry into mitosis and allows cellsto override the replication checkpoint induced byhydroxyurea in the simultaneous presence of caffeine orokadaic acid, drugs that are known to alleviate checkpointcontrol. These results indicate that in plants, a B2-typecyclin is a rate-limiting regulator for the entry into mitosisand a cyclin B2-CDK complex might be a target for

checkpoint control pathways. The cyclin B2 localizationand the timing of its degradation during mitosiscorroborate these conclusions: cyclin B2 protein is confinedto the nucleus and during mitosis it is only present duringa short time window until mid prophase, but it is effectivelydegraded from this timepoint onwards. Although cyclin B2is not present in cells arrested by the spindle checkpoint inmetaphase, cyclin B1 is accumulating in these cells. Ectopicexpression of cyclin B2 in developing plants interferes withdifferentiation events and specifically blocks rootregeneration, indicating the importance of controlmechanisms at the G2- to M-phase transition during plantdevelopmental processes.

Key words: Cyclin B, Mitosis, Checkpoint, Plant development

Summary

A plant cyclin B2 is degraded early in mitosis and itsectopic expression shortens G2-phase and alleviatesthe DNA-damage checkpoint Magdalena Weingartner 1,4, Helvia R. Pelayo 2, Pavla Binarova 3, Karin Zwerger 1, Balázs Melikant 1,Consuelo de la Torre 2, Erwin Heberle-Bors 1 and László Bögre 4,*1Institute of Microbiology and Genetics, University of Vienna, Vienna Biocenter, Dr Bohrgasse 9, A-1030 Vienna, Austria2Centro de Investigaciones Biológicas CSIC, Velázquez 144, E-28006-Madrid, Spain3Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4, Czech Republic4School of Biological Sciences, Royal Holloway, University of London, Egham TW20 0EX, UK*Author for correspondence (e-mail: [email protected])

Accepted 31 October 2002Journal of Cell Science 116, 487-498 © 2003 The Company of Biologists Ltddoi:10.1242/jcs.00250

Research Article

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mitosis (Resnitzky et al., 1995; den Elzen and Pines, 2001),while in plants two A-type cyclins, the tobacco cyclin A1 andthe alfalfa cyclin A2, were found only associated with CdkAboth in vitro (Nakagami et al., 1999), and in the yeast two-hybrid system (Roudier et al., 2000).

The expression of the B1- and B2-type plant cyclins istightly controlled and restricted to late G2 and M phases (Hirtet al., 1992; Fobert et al., 1994). A tobacco Cyclin B1-GFPfusion was found to be distributed between the cytoplasm andnucleus in interphase and to associate with the chromatinduring prophase and metaphase until it is degraded at the onsetof anaphase (Criqui et al., 2000; Criqui et al., 2001). Theseauthors also found that ectopically expressed cyclin B1 did notalter mitotic progression in cultured cells, while expression ofcyclin B1 in Arabidopsis was shown to stimulate rootformation by producing more cells as building blocks fortissue enlargement (Doerner et al., 1996). Evidence forspecialized functions of closely related plant B-type cyclinscame from the finding that ectopic expression of theArabidopsis cyclin B1;2,but not of cyclin B1;1 inhibitedendoreduplication and induced cell divisions, resulting inmulticellular trichomes (Schnittger et al., 2002). Recently, wepresented evidence for the existence of a topoisomerase II-dependent checkpoint in plant cells by showing that theectopic expression of Cyclin B2 alone is able to override thecheckpoint and induce cells to enter into mitosis without delay(Gimenez-Abian et al., 2002).

Plant B2-type cyclins are known to be expressed from lateG2 phase to mitosis (Hirt et al., 1992; Ferreira et al., 1994) butthe role and localization of their protein has not been studied.We show that the cyclin B2 protein is localized to the nucleusand its expression is restricted to a short time window duringearly prophase by its proteolysis. We found that the inductionof ectopic cyclin B2 expression during G2-phase drives cellsto enter mitosis earlier and allows caffeine and okadaic acid toovercome a replication checkpoint block induced byhydroxyurea. Thus, ectopic cyclin B2 expression interfereswith the control of progression from G2 to M phase, whichprobably causes the developmental abnormalities observed intransgenic plants.

Materials and MethodsPlasmid constructions and transformation techniquesThe alfalfa Medsa;CycB2;2gene was amplified by PCR, creating aKpnI site at the N terminus and NotI and XbaI restriction sites at theC terminus and cloned into the BinHygTX binary vector (Weinmannet al., 1994) as a KpnI-XbaI fragment. The triple HA epitope (Tyerset al., 1993) and GFP sequences (Sheen et al., 1995) were inserted asNotI-NotI fragments. Transgenic plants were obtained byAgrobacterium-mediated leaf disk transformation with the tobaccoline Samsung Tb-Tet(Weinmann et al., 1994). Suspension cultureswere initiated by placing stem segments of transgenic CycB2-HA andCycB2-GFP plants on 1 mg/l 2,4-D containing MS solid medium andthe resulting calli were transferred after 1 month into liquid LS-modified medium (Sano et al., 1999) supplemented with 40 mg/lhygromycin B.

Synchronization and cell cycle analysisSynchronization was carried out by diluting a 7-day-old culture 1:5and adding 10 mg/l aphidicolin (Sigma) 8 hours later for 16 hours.

After removal of the aphidicolin by washing cells five times withmedium, chlortetracycline (Sigma) was added at a concentration of0.1 mg/l to induce cycB2-expression. Drugs were added 8 hours afteraphidicolin removal in G2 phase. The following drug concentrationswere used in these experiments: 10 µM amiprophos methyl, 50 µMtaxol, 10 µM propyzamide, 100 µM roscovitine (a gift from M.Strnad, Olomouc, Czech Republic), 100 µM MG 132 (Calbiochem),5 µM lactacystin (Affinity, UK) and 1 µM epoxomicin (Affiniti, UK).To follow cell cycle progression, flow cytometric analysis wasperformed as described (Bögre et al., 1997) using a PAS2 flowcytometer (Partec, Münster, Germany). For mitotic index, cells werefixed in a 3:1 (v/v) ethanol/acetic acid mixture, then washed with 70%(v/v) ethanol. The DNA was stained with 1 µg/ml DAPI and observedby epifluorescence microscopy.

BrdU incorporation and labelingA stationary phase cell culture (7-day-old culture) was diluted 1:10with fresh medium, and bromo-deoxyuridine labeling reagent wasadded immediately at the dilution as recommended in the cellproliferation kit (RPN20) (Amersham Pharmacia Biotech). Cells weregrown for 15 hours and samples were taken every 3 hours, fixed andprocessed for immunostaining for incorporated bromo-deoxyuridine(BrdU) using monoclonal antibody to BrdU (Amersham PharmaciaBiotech) or Cycb2-HA using HA.11 monoclonal antibody (BabCO,Richmond, California). For each timepoint, 1000 cells were analyzedfor BrdU or HA-staining.

Immunostaining and microscopyFor indirect immunofluorescence, cells were fixed and stained aspreviously described (Bögre et al., 1997). The HA.11 monoclonalantibody (BabCO, Richmond, California) was used in a 1:1000dilution and with the secondary anti-mouse Cy3-conjugated antibody(Sigma) at a dilution of 1:200. DNA was stained with 1 µg/ml DAPIin PBS. Microtubule staining was performed using an anti α-tubulinmouse monoclonal antibody DM1A (Sigma) at a dilution of 1:200,and anti mouse FITC-conjugated secondary antibody (Sigma). ForGFP observation, a drop of cell suspension was transferred on a slide,carefully covered with a coverslip, and observed with an uprightfluorescence microscope (Axioplan 2; Zeiss, Jena, Germany)equipped with a GFP filter (HQ480/20X; HQ510/20M; AFAnalysentechnik, Jena, Germany). Typical exposure times were in arange of few seconds. Images were taken using a cooled charge-coupled device black-and-white digital camera (SPOT-2; DiagnosticInstruments, Burroughs, Michigan) and Metaview imaging software(Diagnostic Instruments, Burroughs, Michigan).

RNA and protein blotting, CDK activity measurementsNorthern blots were performed as described (Hirt et al., 1992).Proteins on western blots were detected by using anti-rabbit PSTAIREantibody (Bögre et al., 1997), the HA.11 monoclonal antibody(BabCO, Richmond, California) or the polyclonal rabbit anti N-terminal cyclin B1;1 antibody (kindly provided by Pascal Genschik,Strasbourg, France). Cdk activities were measured in the samples afterimmunoprecipitation with the HA.11 monoclonal antibody or afterbinding to p13suc1, as described previously (Bögre et al., 1997).

Regeneration from tobacco leaf disksLeaf disks with 1 cm diameter were excised from sterile plants andplaced on MS medium (Sigma) with 0.5/0.1 (rooting), 0.1/0.5(shooting) and 0.5/0.5 (callusing) naphtalene acetic acid (NAA)/6-benzylaminopurine (BAP)-containing media with and without 0.1mg/l Cl-tetracycline for 3 weeks, when roots on 10 inocula for eachtreatments were counted and plates were photographed.

Journal of Cell Science 116 (3)

489Role of plant Cyclin B2 in prophase

ResultsOverexpression of cyclin B2 under the control of atetracycline-regulated promoterWe studied the role of cyclin B2 during plant cell cycleprogression by ectopically expressing an alfalfa cyclin B2 gene(Medsa;cycB2;2) in transgenic tobacco plants. As theexpression of cycB2-HA from a constitutive promoter severelyinhibited shoot and root development, and did not allow theregeneration of plants from transformed leaf disks, wegenerated plants in which the expression of cycB2 can beinduced by tetracycline analogues (Weinmann et al., 1994). Tofollow the expression of the introduced gene, it was tagged witheither the epitope haemagglutinin antigen (cycB2-HA) or withthe green fluorescent protein (cycB2-GFP). Five independentlines were obtained with the cycB2-HA construct and namedCycB2-HA-1 to 5, and five independent lines harboring thecycB2-GFPconstruct were obtained and named CycB2-GFP-1to 5. Northern blot analysis revealed that in four of these linesthe cycB2-HAexpression was increased 50- to 1000-fold indetached leaves, which were incubated in 0.1 mg/l Cl-tetracycline for 24 hours (Fig. 1A). One of the lines showed alow level of constitutive expression (see lane 1 in Fig. 1A) andthis line was retarded in both shoot and especially root growth.

To facilitate the studies of cell cycle progression, wegenerated cell cultures from the CycB2-HA and CycB2-GFPtransgenic plants, which displayed tight transcriptionalregulation by Cl-tetracycline. In cultured cells derived fromline CycB2-HA-2 the cycB2-HA transcript was clearlydetectable 10 minutes after adding Cl-tetracycline, and reachedits maximal level of expression within 1 hour (Fig. 1B). Weestablished the maximal induction of CycB2-HA protein at0.01 mg/ml Cl-tetracycline (Fig. 1C). Correspondingly, in vitrokinase assays after immunoprecipitation using an anti HAantibody revealed that the CycB2-HA protein associated withand activated a histone H1 kinase (Fig. 1D), indicating that thecyclin moiety in these fusion proteins retained its normalfunction to bind and activate Cdk. The level of cycB2-HAmRNA expression was slightly lower than that of theendogenous mitotic cyclin Nt;tCycB1;1 (Fig. 2B). In view ofthe lack of a cyclin B2-specific antibody, we could notdetermine the endogenous cyclin B2 levels, but we found thatthe CycB2-HA-associated histone H1 kinase activity reachedonly around 20% of the total cellular Cdk activity purified fromthese cells by binding to p13suc1 (Fig. 1D). Thus, it is likelythat the cyclin B2 protein was not overproduced, but itsexpression was temporally unscheduled.

Ectopically expressed cyclin B2 accelerates entry intomitosisTo assess the consequences of altered cyclin B2 expression oncell cycle regulation, we synchronized the CycB2-HA-2 cellculture by aphidicolin treatment, which blocks cell cycleprogression specifically during S phase. Following removal ofthe inhibitor, we induced the expression of CycB2-HA atvarious time points during the cell cycle. We found that ectopicexpression of CycB2-HA during either S or early G2 phase didnot initiate mitosis. However, cells entered mitosis 2 hoursearlier compared with control cells when expression of CycB2-HA was ectopically induced at mid G2 phase (Fig. 2). Severallines of evidence supported the finding of this advanced entry

into mitosis by 2 hours. First, the mitotic index peaked at 10hours in cells ectopically expressing CycB2-HA, instead of 12hours in control cells (Fig. 2A). Second, monitoring cell cycleprogression by flow cytometry revealed that after cells hadpassed through mitosis, the percentage of cells with a G1 DNAincreased 2 hours earlier in CycB2-HA expressing cells (datanot shown). Third, we followed the mRNA expression of thetobacco cyclin B1and of histone H4, as stage-specific markersfor mitosis and S phase, respectively. Mirroring the results ofmitotic index above, the peak of the endogenous cyclin B1expression also appeared 2 hours earlier in tetracycline-treatedcells (Fig. 2B).

Fig. 1.Cl-tetracycline-inducible expression of cyc-B2-HA mRNAand Cyc-B2-HA-associated Cdk activity in tobacco plants and incultured cells. (A) Northern blot of mRNA samples from leaves fromlines 1, 2, 4, 5 and 6 incubated with (+) or without (–) 0.1 mg/l Cl-tetracycline for 1 day. (B) Time course of cycB2-HA mRNAinduction by 0.1 mg/l Cl-tetracycline in cultured cells generated fromline 2. In both A and B the upper panel represents CycB2-HA mRNAand the lower panel represents control (cnt6) mRNA. (C) Westernblot of total protein samples from cultured cells generated from lineCycB2-HA-2, which was induced for 12 hours by indicatedconcentrations of Cl-tetracycline. CycB2-HA and Cdc2 protein weredetected using HA-specific and PSTAIRE-specific antibodies,respectively. (D) Histone H1 kinase activity of immunopurifiedCycB2-HA-associated kinase and p13suc1-bound Cdks from theextracts shown in C.

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Because microtubule structures are highly specificlandmarks for the different stages of mitosis, we used thesefeatures to further refine and confirm the above interpretation.The pre-prophase band (PPB) appears between late G2 phaseand late prophase, the spindle structures during mitosis, andthe phragmoplast marks the cytokinetic plate during lateanaphase and telophase (Staiger and Lloyd, 1991). Wequantified the relative proportion of PPB, spindle andphragmoplast visualized by tubulin immunostaining insynchronized cells expressing cycB2-HA. Disassembly of thePPB, which occurred 2 hours earlier than in control cells, wasthe most pronounced effect of ectopic CycB2-HA expression(Fig. 2C).

Ectopic cyclin B2 expression overrides the hydroxyurea-induced S-phase checkpoint only in the presence ofcaffeine or okadaic acid Blocking DNA synthesis arrests cells before mitosis, andblocks the expression of mitotic A- and B-type cyclins in plants(Renaudin et al., 1998). First, we tested whether ectopic cyclinB2 expression could induce S-phase-arrested cells to enter intomitosis. The induction of CycB2-HA expression inasynchronously dividing cells by treatment with tetracyclinefor 16 hours slightly increased the mitotic index (Fig. 3A, lanes1 and 7) and this effect was abolished when cells were treatedat the same time with HU (Fig. 3A, lanes 4 and 10). Thus,

cyclin B2 alone is unable to override the HU-activatedcheckpoint block and we found a similar result in aphidicolintreated cells.

Drugs such as caffeine or okadaic acid are known to cancelthe DNA-replication checkpoint in yeast and animal cells, butwere found to be ineffective in plant cells (Amino and Nagata,1996; Pelayo et al., 2001). A reason for this could be that inplants mitotic cyclins are not expressed in S-phase-arrestedcells. Thus, to gain further evidence that CycB2 is a potentinducer for mitosis, we tested whether its ectopic expressioncould help caffeine or okadaic acid to override the S-phaseblock. In an asynchronously growing control culture themitotic index was determined to be 7% and the addition ofcaffeine or okadaic acid for 1 hour did not modify thepercentage of mitotic cells. The addition of hydroxyurea (HU)for 16 hours, which blocks cells in S-phase, decreased themitotic index (MI) and this low MI was not increased by asubsequent 1 hour treatment with caffeine or okadaic acid (Fig.3A, left panel). Thus, caffeine or okadaic acid were unable tooverride the HU-induced S-phase block, which is in agreementwith the results found before in onion root cells (Amino andNagata, 1996; Pelayo et al., 2001). However, when caffeine orokadaic acid were added to the HU-blocked cells expressingCycB2-HA, the mitotic indices were raised to a level of around50 and 25%, respectively, when compared with control cellswithout HU. Caffeine was slightly more effective than okadaicacid in stimulating mitosis in HU blocked cells, together withectopic CycB2-HA expression (Fig. 3A, right panel). Thus,CycB2-HA expression allowed caffeine or okadaic acid tooverride replication checkpoints in S phase.


Fig. 2.Entry into mitosis is advanced by Cl-tetracycline-inducedCycB2-HA expression. (A) Mitotic indices after release fromaphidicolin-induced S phase block in the presence (+) or absence (–)of 0.1 mg/l Cl-tetracycline. (B) Northern blot analysis of tobaccocyclin B1,1, histone H4, cyclin B2-HA and cnt6 mRNA levels insynchronized cells as in A. (C) Proportion of mitotic microtubulestructures [pre-prophase band (PPB), spindle and phragmoplast] insynchronized cells in the same experiment.

Fig. 3.The tetracycline-induced expression of cyclin B2 allowscaffeine and okadaic acid to override the HU-induced replicationcheckpoints. (A) Mitotic indices of the asynchronously growingtransgenic cell line without (lanes 1-6) or with (lanes 7-12)tetracycline-induced cyclin B2 expression. Cells were either treatedwith no drug (1 and 7), with caffeine (3 and 8) or okadaic acid (4 and9) for 1 hour, or first with hydroxyurea for 16 hours (4 and 10), andthen incubated for 1 hour with caffeine (5 and 12) or okadaic acid (6and 12). (B) Cyclin B2-associated histone H1 kinase activities afterimmunopurification using the HA-epitope tag (H1 CycB2-HA),immunodetection of CycB2-HA protein using an anti HA-antibody,and CDKs using a PSTAIRE antibody, from cells treated in the sameway as in A.


To address whether caffeine and okadaic acid stimulatedcells to enter into mitosis by elevating the CycB2-HA-associated Cdk activity in HU-blocked cells, weimmunopurified the CycB2-HA-associated kinase complexwith an antibody directed against the HA-tag and measured itshistone H1-kinase activity. In the absence of tetracycline, noCycB2-HA protein or CycB2-HA-associated Cdk activity werefound, while the Cdk amounts, as detected by the PSTAIREantibody, were constant in these extracts (Fig. 3B, left panel).In the presence of tetracycline, CycB2-HA protein expressionwas increased by equal amounts after all treatments, showingthat the tetracycline-induction of CycB2-HA expression wassimilar in all the treatments. We found that the CycB2-HA-associated Cdk activity in cells treated with tetracycline wasincreased after treatment with caffeine and okadaic acid. Theaddition of HU abolished the CycB2-HA-associated Cdkactivity. In agreement with its ability to induce mitosis in HU-blocked cells, caffeine and okadaic acid elevated the activityof CycB2-associated Cdk in HU-treated cells (Fig. 3B, rightpanel). Okadaic acid was again less effective in raising the Cdkactivity than was caffeine. Thus, we conclude that the activityof CycB2-associated Cdk is inhibited in the presence of HU,and this inhibition is cancelled by treatment with caffeine andokadaic acid, which supports our findings that caffeine orokadaic acid together with CycB2 expression override thereplication-checkpoint in plant cells.

Cyclin B2 degradation is initiated in between pre-prophase and prophase and persists until mid-G1 phaseTo learn more about how cyclin B2 promotes the G2-Mtransition, we determined its localization in cultured cells byindirect immunofluorescence using an anti HA-antibody (Fig.4). In interphase cells CycB2-HA was localized to the nucleus.In late G2-phase cells, which display a pre-prophase band,CycB2-HA was still present within the nucleus, but it wasabsent in pro-metaphase cells with condensed chromatin andvisible spindle microtubules (Fig. 4A, PM). This suggests thatCycB2-HA is degraded as cells progress from pre-prophase topro-metaphase. No CycB2-HA staining was observed in cellsin meta-, ana- or in telophase, and it was missing in around40% of interphase cells (Fig. 4A).

To confirm the localization of the cyclin B2 protein in livecells, we used a cell line expressing a cyclin B2-GFP fusion.Similar to the CycB2-HA protein visualized byimmunostaining with the HA antibody, CycB2-GFP was alsofound within the nucleus in interphase cells and it was neverfound in mitotic cells (Fig. 4B, left panel). We were unable tofollow the degradation of CycB2-GFP by time-lapsemicroscopy, because UV irradiation of cells during late G2-phase blocked cell cycle progression, presumably by activatinga prophase checkpoint, similar to that described in animal cells(Rieder and Cole, 1998).

In an asynchronously growing culture around 60% ofinterphase cells displayed CycB2-HA or CycB2-GFP stainingwithin the nucleus after treatment with tetracycline. To definemore precisely at what stage of interphase cyclin B2 wasstable, we blocked cell cycle progression at the G1-S transitionby treatment with aphidicolin. Both CycB2-HA and CycB2-GFP signals were detectable in around 90% of aphidicolin-blocked cells. After release from the block, a high proportion

of cells (around 90%) remained positive for nuclear CycB2-GFP or CycB2-HA signals, which decreased when cellsprogressed through mitosis (data not shown). Thus, CycB2-HAand CycB2-GFP were stable during S and G2 phases, andbecame unstable as cells entered mitosis and G1 phase. To mapthe time window within G1 during which CycB2-HA wasstable, we observed stationary phase cells arrested with a G1DNA content. In these cells we never found CycB2 proteinexpression, indicating that it is unstable or not translated (Fig.4C). Adding back fresh medium reinitiated the cell cycle andcells reached S phase within 0 to 15 hours, which was shownby monitoring incorporation of BrdU. CycB2-HA proteinreappeared at around 6-9 hours in a large proportion of cells(Fig. 4C). Thus, there is a transition point within G1 whencyclin B2 becomes stabilized, similar to B-type cyclins of yeastand animal cells (Amon et al., 1993; Brandeis and Hunt, 1996).

We analyzed the timing of the initiation of CycB2-HAdegradation by using drugs that inhibited cell cycle progressionat specific time points. Inhibition of Cdk activity by addingroscovitine to synchronized cells during mid G2 phase (6 hoursafter the release from aphidicolin treatment) blocks cells at lateG2 phase and G2-M interface, characterized by a fullydeveloped prophase spindle and condensed chromatinsurrounded by a persistent nuclear envelope (Binarova et al.,

Fig. 4.Cyclin B2 is located in the nucleus and degrades in pro-metaphase. (A) Triple-labeling of CycB2-HA-expressing cells withantibodies against the HA-epitope (red) and α-tubulin (green);chromatin is stained with DAPI (blue). A representative cell is shownin interphase (Int), preprophase (PPB), pro-metaphase (PM),metaphase (Met), anaphase (Ana) and telophase (Tel). (B) CycB2-GFP fluorescence and the corresponding DIC images in culturedcells. Arrows in the top images indicate metaphases, and in thebottom images a cell after cytokinesis. (C) CycB2-HA is absent instationary-phase cells and becomes detectable before S phase.Stationary phase cells from line CycB2-HA-2 were diluted with freshmedium and immediately incubated with BrdU labeling reagent.Aliquots were taken at 3 hour intervals and the initiation of S phasewas determined by immunostaining for BrdU incorporation using anantibody against BrdU. The percentage of cells with CycB2-HAprotein was determined by immunostaining using an antibody againstthe HA-epitope. Scale bars: 10 µm.

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1998). Inducing such a G2-arrest kept CycB2-HA intact withinthe nucleus, while in control cells CycB2-HA becamedegraded after prophase (Fig. 5A). Thus, the degradation ofcyclin B2 requires either an active Cdk or nuclear envelopebreakdown.

Cyclin B2 destruction during mitosis is not affected bythe spindle checkpointActivation of the spindle checkpoint by treatment of cells withmicrotubule disrupting drugs leads to a metaphase arrest andstabilization of cyclin B1 (Criqui et al., 2000). To studywhether the spindle checkpoint might influence cyclin B2

degradation in plants, we treated the synchronized CycB2-HA-2 cells in late G2-phase with the microtubule stabilizing drugtaxol, as well as with the microtubule disrupting drugamiprophos methyl (APM), and followed CycB2-HAlocalization with immunostaining. Both APM and taxolarrested a large proportion of cells in metaphase, without anydetectable CycB2-HA-derived signal (Fig. 5B,C).

To confirm that CycB2 is unstable in metaphase-arrestedcells, and that the signal is not masked and thereforeundetectable by immunofluorescence, we used the CycB2-GFPcell line and followed the CycB2-GFP fluorescence in livingcells during activation of the spindle checkpoint bydepolymerizing microtubules with propyzamide (Fig. 6A).Again, we found that the CycB2-GFP-derived fluorescencewas clearly present in propyzamide-treated interphase cells,but it was missing in metaphase-arrested cells (Fig. 6A, rightpanel, arrow). When the same treatment was applied to a cellline expressing a cyclin B1-GFP fusion, we found that the GFPsignal was strongly present in association with chromosomesin metaphase-arrested cells (Fig. 6A, left panel) as it wasreported previously (Criqui et al., 2000; Criqui et al., 2001).

To further ascertain that the CycB2-HA protein is indeeddecreased when the spindle checkpoint has been activated andthat cyclin B1 is stabilized in these cells, we analyzed proteinextracts of synchronized cells by immunoblotting using the antiHA-antibody for detection of CycB2-HA protein and the anticyclin B1;1 antibody for detection of endogenous cyclin B1(Criqui et al., 2000). In control cells, both the expression ofCycB2-HA and cyclin B1 increased during early mitosis,reaching the maximum at 9 hours after aphidicolin removal,from which point both cyclin levels declined rapidly (Fig. 7).Treatment with propyzamide at 7 hours during G2 phase didnot affect the levels of CycB2-HA protein, and it decreased at15 hours to a similar level as in the control cells (Fig. 7A).Contrary to this, the endogenous cyclin B1 protein levelremained high in propyzamide-treated cells (Fig. 7B). Thisstrongly suggests, that cyclin B1, but not cyclin B2 is stabilizedby the spindle-checkpoint in plants.

Treatment with the proteosome inhibitor MG 132 in thepresence of ectopic expression of Cyclin B2 abrogatesthe metaphase alignment of chromosomesDegradation of A- and B-type cyclins is blocked byproteosome inhibitors such as MG 132, epoxomicin orlactacystin, both in plant and in animal cells and they arrestcells with over-condensed metaphase chromosomes (Genschiket al., 1998). Surprisingly, we found that the CycB2-HAderived signal was barely detectable in MG 132-treated cells(Fig. 5C).

To confirm that proteosome inhibitors differently affect B-type cyclin levels in vivo, we treated CycB1-GFP- and CycB2-GFP-expressing cells with MG 132, as well as with two otherinhibitors: epoxomicin and lactacystin. Similar to our findingsby immunolocalization in MG 132-treated cells, CycB2-GFPwas barely detectable, while CycB1-GFP was strongly presentand localized to the condensed chromosomes in cells whichwere arrested in metaphase after treatment with epoxomicin(Fig. 6B), MG 132 or lactacystin (data not shown).

Immunodetection of the CycB2-HA and the endogenouscyclin B1 protein levels further substantiated the above results.


Fig. 5. CycB2-HA localization in cells treated with roscovitine,APM, taxol and MG-132. (A) Cells were treated in G2 phase with100 µM roscovitine and samples were taken after five hoursincubation; arrows indicate a prophase-arrested cell. Immunostainingof CycB2-HA expressing cells with the anti HA-antibody is shown inthe left-hand panels and DAPI-staining of the chromatin in the right-hand panels. (B) Cells were treated in G2 phase with 10 µMamiprophos methyl (APM) (upper panels) or 50 µM taxol (lowerpanels) and samples were taken after 5 hours incubation; arrowsindicate metaphase-arrested cells. (C) Cells were treated in G2-phasewith 100 µM MG 132 and samples were taken after 5 hours. Thearrows indicate a cell arrested in mitosis. Scale bars: 10 µm.


We treated cells before mitosis, at 7 hours after aphidicolinerelease with epoxomicin, lactacystin or MG 132, and preparedextracts from cells at 15 hours for immunoblotting. We foundthat CycB2-HA protein levels were unaffected by theproteosome inhibitors, and were as low in the treated cells asin the control cells (Fig. 7A), while the cyclin B1 protein levelsincreased (Fig. 7B).

In contrast to wild-type cells, we found that CycB2-HA-expressing cells arrested in the presence of MG 132 at a stagewhen chromosomes were not aligned along the metaphaseplate (Fig. 5C). In untreated cells, which ectopically expressedCycB2-HA, we observed a slight increase in the percentage ofmetaphase cells compared with control cells, but we neverfound abnormalities in chromosome alignment. This indicatesthat increased levels of cyclin B2 during prophase mightinterfere with chromosome alignment at the metaphase plate.No such abnormalities were observed in control cells orCycB1-overexpressing cells treated with the proteosomeinhibitors.

Ectopic expression of cyclin B2 interferes with rootdevelopmentWe initially observed, that constitutive expression of CycB2severely retards plant growth with most pronounced effectson root development. To investigate how ectopic expressionof CycB2 could interfere with shoot and root regeneration,we used a transgenic line with Cl-tetracycline induciblecycB2-HA expression. Detached leaves from transgenicplants were placed on Cl-tetracycline-containing medium, incombination with different ratios of cytokinin and auxin. It iswell established that high ratios of cytokinin to auxin favorshoot regeneration, while the reverse would privilege rootgrowth (Sugiyama, 1999). Our results show that ectopicexpression of CycB2 specifically blocked root developmentin a medium containing high auxin to cytokinin ratios,conditions in which root development is normally encouraged(Fig. 8A). However, the initiation of shoot development inthese transgenic plants was not detectably modified on highratios of cytokinin to auxin containing medium (data notshown).

To determine whether the inhibition of root development can

Fig. 6.Cyclin B1-GFP but not cyclin B2-GFP is stabilized in living cells arrested in mitosis by activation of the spindle checkpoint or byinhibition of the proteosome. (A) Visualization of GFP fluorescence in living cells expressing cyclin B1-GFP (left panels) or cyclin B2-GFP(right panels) after treatment with propyzamide. Arrows indicate cells arrested in mitosis. (B) Visualization of GFP fluorescence of Cyclin B1-GFP (left panels) or cyclin B2-GFP (right panels) in living cells after treatment with epoxomicin. Arrows indicate cells arrested in mitosis.Upper panels, Fluorescent images; lower panels, DIC images. Scale bars: 10 µm.

Fig. 7.Protein levels of ectopically expressed CycB2-HA andendogenous cyclin B1 in synchronized CycB2-HA-2 cells and aftertreatment with propyzamide, MG 132, epoxomicin or lactacystin.(A) Immunodetection of CycB2-HA protein with an anti HA-antibody in protein samples prepared from non induced control cellsor in samples prepared at 2 hour intervals from 7 to 15 hours afteraphidicolin release and at 15 hours in cells that were treated in lateG2-phase with propyzamide (P), epoxomicin (E), lactacystin (L) orMG132 (Mg). (B) Immunodetection of endogenous CycB1 proteinwith an anti N-terminal CycB1;1 peptide antibody in the samesynchronized samples as described in A, and in samples treated withpropyzamide (P) epoxomicin (E) and lactacystin (L).

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be traced to an altered cell cycle progression, we measured thedistribution of cells with a G1 and G2 DNA content. Most cellsin leaves without hormone treatment have G1 DNA content,irrespective of tetracycline-induced cycB2-HA expression(data not shown). Control experiments, in which leaves wereincubated on auxin-containing medium but without inductionof cycB2-HA expression, showed that after 3 days the cellcycle was initiated in a proportion of cells (as judged fromBrdU incorporation for S-phase and observing mitotic figuresin leaf pieces) and 30% of the cells displayed a G2 DNAcontent. By contrast, in leaves that were incubated with auxinwhile the expression of cycB2-HA was induced, only about15% of the cells displayed G2 DNA content after 3 days (Fig.8B), while similar percentages of S-phase and mitosis, ascompared with control samples, were observed. The reducedproportion of cells with G2 DNA content indicates that, as inthe previous experiments with cultured cells, the G2-phase isaccelerated upon ectopic expression of cycB2 in the presenceof auxin.

DiscussionIn plants, cell division can be influenced by growth ordevelopmental signals not only in G1 phase, but also duringG2 phase, but the molecular mechanisms for the control of G2progression is poorly understood. We show that ectopicexpression of a B2-type cyclin interferes with the timing ofentry into mitosis and affects plant development.

The plant cyclin B2 appears to play a similar role to cyclinA in animal cells during entry into mitosis. In animals,microinjection of exogenous cyclin A into G2 cells rapidlyinduces chromosome condensation, while inhibition of cyclinA-Cdk-complex by p21cip1/waf1 prevents G2 cells fromentering prophase and induces early prophase cells to returnback to interphase (Furuno et al., 1999). Irradiation-inducedDNA damage identifies a late G2 control point in animal cells,from where the chromosomes return to an uncondensedinterphase stage (Rieder and Cole, 1998). An analogous G2-checkpoint was observed in onion cells, where chromosomecondensation is reversed by inhibiting protein synthesis beforemid-prophase (Gracia-Herdugo et al., 1974). This reversiblecondensation phase coincides with the timing of cyclin A-associated Cdk1 activation in animal cells (Clute and Pines,1999), and our findings suggest that it might be a cyclin B2-associated Cdk kinase in plant cells. Although animal cyclinA is expressed during a broad time window and has a role bothin S-phase and during entry into mitosis (Rosenblatt et al.,1992), plant cyclin B2 expression is confined to late G2-phaseand early mitosis. The existence of several subgroups of plantA- and B-type cyclins, which are specifically expressed duringS, G2 or mitotic phases, suggests a high degree ofspecialization in cyclin function in plants (Renaudin et al.,1998).

The PPB is a microtubule structure formed in late G2-phase,and it disassembles as cells enter mitosis in late prophase(Utrilla et al., 1993). Further evidence that ectopic cyclin B2expression affects the G2 to M phase transition is given by thelower percentage of PPBs in these cells. This indicates thateither the time window during which the PPB is presentbecomes shorter or a cyclin B2-associated Cdk activity directlyinduces the disassembly of the PPB. It has been shown thatmicroinjections of an active Cdk complex into plant cells cancause the disassembly of PPB and nuclear envelope breakdown(Hush et al., 1996).

Ectopic Cyclin B2 expression could not initiate mitosis in Sor early G2 phase. In yeast and animals, entry into mitosis isregulated by the phosphorylation of the Thr14 and Tyr15inhibitory sites of CDK (Walworth, 2001). The existence ofphosphorylation-mediated Cdk-regulatory pathways duringplant mitosis is indicated by the presence of a wee1-kinasehomolog in maize and Arabidopsis, which inactivates Cdksthrough its phosphorylation at the conserved Tyr15 residue (Sunet al., 1999). Surprisingly, no apparent homologs of the cdc25phosphatase have been found in the fully sequenced genomeof Arabidopsis, but dephosphorylation of Tyr15 is tightlyregulated in response to cytokinin or during stress treatment(Reichheld et al., 1999). Expression of S. pombe cdc25intobacco can induce cells to resume cell divisions that have beenarrested in G2 phase by cytokinin starvation (John, 1996;Zhang et al., 1996). Constitutive expression of cdc25 intobacco plants leads to a number of developmentalabnormalities, such as precocious flowering, aberrant leaves


Fig. 8. Root regeneration is inhibited by ectopic expression ofCycB2-HA, which correlates with shortening of the G2 phase in leafcells. (A) Leaf disks from line CycB2-HA-2 were incubated on root-inducing medium containing 0.5 mg/l auxin (NAA) and 0.1 mg/lcytokinin (BAP) in the presence (+) or absence (–) of 0.1 mg/l Cl-tetracycline and pictures of regenerates were taken after 3 weeks.(B) Flow cytometry measurements of G1 and G2 DNA contents inisolated nuclei from leaves treated as in A and incubated for 3 days.


and flowers, and a smaller size of dividing cells in lateral rootscompared with control plants (Bell et al., 1993).

There is a conserved DNA-synthesis checkpoint-signalingpathway in yeast and animal cells that regulates CDK activitythrough the phosphorylation of the Thr14 and Tyr15 inhibitorysites. Caffeine is a drug that inhibits checkpoint function inanimal cells (Schlegel and Pardee, 1986; Andreassen andMargolis, 1992; Moser et al., 2000), such as the replicationcheckpoint during S phase (Schlegel and Pardee, 1986) and theDNA damage checkpoint in G2 phase (Blasina et al., 1999). Inplants, caffeine is able to override the G2 DNA damagecheckpoint (Hartley-Asp et al., 1980; González-Fernández etal., 1985) but it is unable to cancel replication checkpoints inS phase (Amino and Nagata, 1996; Pelayo et al., 2001).Okadaic acid, a protein phosphatase 2A inhibitor, inducespremature mitosis in animal cells in a similar way to caffeine(Ghosh et al., 1996). Correspondingly, endothal, anotherPP2A-specific phosphatase inhibitor, was found to prematurelyactivate a plant mitotic CDK resulting in microtubule andchromosome condensation abnormalities (Ayaydin et al.,2000). Caffeine was found to releases the checkpoint-inducedblock in G2 phase by inhibiting the ATM kinase, which worksupstream of the two checkpoint kinases: Chk1 and Chk2(Blasina et al., 1999; Brondello et al., 1999). In a current modelthe Chk1 kinase directly inhibits the Cdc25 phosphatase andthus stops activation of CDK by keeping it in a phosphorylatedstate at the Thr14 and Tyr15 inhibitory sites. The animal targetof caffeine, the ATM kinase, has been conserved in plants(Garcia et al., 2000), but little is understood about its functionduring checkpoint pathways in plants.

We show that, in hydroxyurea-treated cells, cyclin B2expression stimulated the entry into mitosis only when thereplication checkpoint pathway was compromised by caffeineor okadaic acid. Moreover, under these conditions, the CycB2-associated Cdk activity was also induced. This suggests that atleast one of the targets of the replication checkpoint in plantsis the CycB2-associated Cdk. In animal cells, the nuclear-localized cyclin A stimulates entry into mitosis (Furuno et al.,1999), and cyclin B1 is able to abolish the replicationcheckpoint in response to caffeine when experimentally alteredto localize to the nucleus (Tam et al., 1995; Toyoshima et al.,1998). The plant cyclin B2 is nuclear, and during a period ofS phase, we found the localization of CycB2 to be speckledwithin the nucleus (M. W., unpublished).

In animal cells, A-type cyclins are degraded in pro-metaphase, while B-type cyclins undergo proteolysis inmetaphase. Degradation of both cyclins requires the anaphase-promoting complex (APC). Although the APC is activatedalready during prometaphase, destruction of cyclin B in animalcells appears to be selectively inhibited by the spindle assemblycheckpoint, resulting in stabilization of cyclin B until allchromosomes are aligned along the metaphase plate (Geley etal., 2001). Both cyclin types contain the conserved destructionbox motif recognized by the APC, but in A-type cyclins, thismotif is not sufficient to trigger proteolysis during prophase.Rather, prophase destruction of cyclin A is mediated by anextended destruction box including multiple overlappingelements (Kaspar et al., 2001). Plant cyclin A and Bdegradation during mitosis is also dependent on theirconserved destruction box motif and likely to be mediated bythe APC (Genschik et al., 1998). Similar to animal cyclins,

plant cyclin A is undetectable in metaphase (Roudier et al.,2000), while plant cyclin B1 is present in metaphase and itsdegradation is inhibited upon activation of the spindleassembly checkpoint (Criqui et al., 2000). The plant cyc B2;2genes are grouped together with B-type cyclins, based onsimilarities within the cyclin box, but the arrangement of theN-terminal destruction box motifs of the plant cyclin B2sequence better resembles that of animal A-type cyclins whichhave multiple destruction box elements. Our studies indicatethat the plant cyclin B2 protein uses a similar degradationmechanism as animal A-type cyclins and that it is degradedduring late prophase, again similar to animal cyclin A.Treatment of cells with the proteosome inhibitors MG 132,epoxomicin or lactacystin arrested cells in metaphase, asexpected, but surprisingly, cyclin B2 protein was undetectableby indirect immunofluorescence, suggesting that it is degradedin spite of the presence of the inhibitors in these cells. Toexclude the possibility that the cyclin B2 was undetectable dueto technical difficulties, such as masking the epitope, werepeated these experiments by treating cells expressing CycB1-GFP or CycB2-GFP with the same proteosome inhibitors andfollowed GFP fluorescence in living cells. Furthermore, wedirectly compared the protein level of CycB2-HA with that ofendogenous cyclin B1 in proteosome-inhibitor-treated cells byimmunoblotting. Both experiments led to the same conclusion:the degradation of cyclin B2 is hardly affected by treatmentswith proteosome inhibitors, while cyclin B1 destruction isreadily blocked and cells are arrested in metaphase. Theseresults indicate that cyclin B2 is either not targeted by theproteosome at pro-metaphase and proteolysed by some othermechanism or its destruction is more effective than that of othersubstrates, including cyclin B1. Recently, it was shown thatcyclin A and cyclin F are degraded by proteolysis independentof proteosome in animal cells (Welm et al., 2002; Fung et al.,2002). Collectively, the data showing that cyclin B2 is onlypresent until pro-metaphase promote our conclusion that plantcyclin B2 functions during early mitosis.

The expression of cyclin B2 to a level not significantlyhigher than that of endogenous cyclin somewhat delayedmetaphase, without affecting subsequent chromosomealignment along the metaphase plate. Compromisingproteolysis in these cells by treatment with proteosomeinhibitors resulted in an accumulation of cells withchromosomes that were not correctly aligned at the metaphaseplate. By contrast, MG 132-treatment of wild-type cells arrestscells in metaphase with normal spindle and chromosomealignment (Genschik et al., 1998). This shows that Cyclin B2ectopic expression, together with impaired proteolysissynergistically affects chromosome alignment duringmetaphase. A similar effect has been reported in animal cellsupon overexpression of cyclin A during late G2-phase andmitosis (den Elzen and Pines, 2001). This again suggests thatthe plant cyclin B2 has analogous functions to animal cyclin Ain regulating mitosis.

Proper chromosome alignment at the metaphase plate iscontrolled by a complex anchor of many proteins associatedwith chromosomes mainly at their kinetochore/centomericregion. Kinetochore localization of phosphoepitopesrecognized by the MPM2 antibody suggests that the functionof these proteins is regulated by mitosis-specificphosphorylation in plants (Binarova et al., 1993). In

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Drosophila, Cyclin A inhibits chromosome disjunction byacting synergistically with other proteins involved in thisprocess (Sigrist et al., 1995; Parry and O’Farrell, 2001).Among these are the Drosophilasecurin Pim, which stabilizessister chromosome cohesion until it is degraded at themetaphase to anaphase transition (Leismann et al., 2000), andthe Cdk inhibitor Rux, which interacts genetically andphysically with Cyclin A and inhibits its in vitro kinase activity(Foley et al., 1999).

The increased number of metaphase cells observed uponectopic cyclin B2 expression suggests that in plants B2-typecyclins might have a similar role to cyclin A in animal cells,delaying the progression through metaphase.

We found that cyclin B2 was non-detectable in stationaryphase cells, which are arrested in G1, and begins to accumulatebefore cells enter S phase. This confirms the results obtainedin yeast and animal cells (Amon et al., 1994; Brandeis andHunt, 1996; Huang et al., 2001) and shows that APC activityis switched off during late G1 to S phase in plants. In yeast andanimal cells, APC activity is required during G1 phase toprevent expression of proteins that may interfere with cellularpolarization events or lead to premature DNA replication ordisturb spindle assembly. Before mitosis, the APC must beturned off to allow mitotic substrates to accumulate.Inactivation of the APC has been linked to mid-G1, which iscorresponding to the start in yeast, and is suggested to dependon raising G1 cyclin activity (Amon et al., 1994). Recently, itwas shown that during a normal cell cycle the APC isinactivated in a graded manner, and that its completeinactivation occurs in S phase and requires S phase cyclins(Huang et al., 2001).

We found that ectopic expression of cyclin B2 in leafsegments interferes with root regeneration induced by auxin.This re-differentiation process is initiated in a singledifferentiated cell within the vascular bundle and was mappedby hormone shift-experiments to a relatively short timewindow, about 24 hours, during which auxin induces the cellto re-enter cell division and re-polarize (Attfield and Evans,1991). The majority of leaf cells enter the cell cycle from theG1 phase, and a 24 hour period might coincide with the time,when a proportion of cells are in G2-phase. Shortening of G2-phase may disrupt some events, such as cell polarization, whichis required for the determination of the root fate. Timing of cellcycle phases was shown to be an important factor indetermining polarized growth in yeast (Lew and Reed, 1993).In plants, the timing of the presence of the PPB, which isparticularly altered upon ectopic CycB2-HA expression, couldbe an important factor for the orientation of cell division. Inline with this is a recent report which implicates Cdk1 as animportant factor in regulating the orientation of asymmetriccell divisions in Drosophila (Tio et al., 2001). Our resultssuggest that enhancing the expression of one of the key cellcycle regulators, a cyclin B2 gene, is sufficient to preventformation of a root meristem under conditions when it isnormally formed.

M.W. was supported by a PhD fellowship from the AustrianAcademy of Sciences. This work was supported by an AustrianFörderung der Wissenschaftlichen Forschung grant to E.H.-B.; byEuropean Union Framework V project ECCO Grant QLRT-1999-00454 to E.H.-B., L.B. and P.B.; by a Biotechnology and Biological

Science Research Council grant (111/P133340) to L.B.; by grantLN00A081 from the Czech Ministry of Education; by a collaborativeWellcome Trust grant to P.B. and L.B.; and by a Spanish Ministeriode Ciencia y Tecnologia grant (BMC2001-2195) to C.T. Moreover,we thank Gireg Weingartner for his patience and Giovanna Vinti,among others, for taking care of him.

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