Growth and Senescence of Medicago truncatula Cultured Cells Are Associated with

Characteristic Mitochondrial Morphology

Author(s): Michela Zottini, Elisabetta Barizza, Fiorenza Bastianelli, Francesco Carimi and
Fiorella Lo Schiavo
Source: New Phytologist, Vol. 172, No. 2 (2006), pp. 239-247
Published by: Wiley on behalf of the New Phytologist Trust
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Re
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Growth and senescence of Medicago truncatula cultured

cells are associated with characteristic mitochondrial
morphology
Michela Zottini'*, Elisabetta Barizzal*, Fiorenza Bastianellil, Francesco Carimi2 and Fiorella Lo
Schiavo1
'Dipartimentodi Biologia,Universitai
Degli Studidi Padova,Via U. Bassi58/B, 1-35131 Padova,Italy;2Istitutodi GeneticaVegetale,Palermo(CNR), Corso
414,
Calatafimi 1-90128 Palermo,Italy

Summary
Author
forcorrespondence: * Here mitochondrialmorphologyand dynamicswere investigatedin Medicago
MichelaZottini truncatulacell-suspensionculturesduringgrowthand senescence.
Tel:+39 049 8276247 * Cellbiologytechniqueswere used to measurecell growthand death in culture.
Fax:+39 049 8276300
Email:mzottini@bio.unipd.
it Mitochondrialmorphologywas investigatedin vivo using a membranepotential
sensorprobecoupledwith confocalmicroscopy.
Received:3 March2006
* Expression of a senescence-associatedgene (MtSAG)was evaluatedin different
Accepted:23 May2006
cell-growthphases.Mitochondriaappearedas numerous,punctuateorganellesin
cellsat the beginningof the subculturecycle,while interconnectednetworkswere
observedin activelygrowingcells.Insenescentcells,giantmitochondria were asso-
ciatedwithdyingcells.Thereleaseof cytochromec frommitochondria was detected
in differentgrowthphasesof culturedcells.
* Studieson plantcell culturesallowedus to identifyphysiologicaland molecular
markersof senescence and cell death, and to associate distinct mitochondrial
morphologywith cellsunderdifferentphysiologicalconditions.
Keywords: cell cultures, Medicago truncatula, mitochondrial morphology,
programmedcelldeath,senescence.
New Phytologist(2006) 172, 239-247

? TheAuthors(2006). Journalcompilation@ New Phytologist(2006)

doi: 10.1111/j.1469-8137.2006.01830.x

Introduction and reversibilityof processescan be analysedmore easilyin

The studyof plantcell culturesmayhelp to clarifynumerous
generalaspectsof organsenescenceand contributeto better
understanding of its relationshipwith programmedcell death
(PCD). Senescenceand PCD are associatedwith develop-
mentalprocessesin plantssuch as leaf senescenceand petal
wilting, althoughthe relationshipbetweenthe two termsis
not yetwelldefined.WhereasvanDoorn & Woltering(2004)
arguedthat senescenceis part of the programmeleadingto
cell death, Thomas et al. (2003) maintain that senescence
and PCD are,at best, only distantlyrelated.Investigations
usingculturedplantcellshavebeenperformed,as cellgrowth,
effectsof depletionor additionof nutrients,cell autonomy
*Theseauthorscontributed
equallyto thiswork.
culturedcellscomparedwith complextissues.
The growthcycle of culturedplant cells is influencedby
severalphysiological includingnutrientavailability,
parameters,
cell density,light, temperatureand hormonalconcentration.
If cells arenot subculturedat the end of the growthperiod,
they begin senescingand PCD ensues. In culturedcells of
Nicotianaplumrnbaginifolia,chromatincondensation,one of
the hallmarksof PCD, occursin cells during spontaneous
senescence;however,if the senescingcell populationis sub-
cultured,chromatincondensationis reversedandcell deathis
prevented(O'Brienetal., 1998). Senescencehas been asso-
ciatedwith PCD in two othercell-culturedspecies:carrotand
Arabidopsis thaliana,wherethe presenceof oligonucleosomal
DNA degradationhasbeenshownto occur(LoSchiavoet al.,
2000). Recentlywe reportedthat Arabidopsis culturedcells

www.newphytologist.org 239

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240 Research
I
expresssenescence-associated gene 12 (SAG12),a geneencod-
ing a cysteine protease,at the end of subculturingcyclebefore
entering PCD (Carimi et al., 2004). The expressionof this
gene, initiallyidentifiedby Lohman et al. (1994), appearsto
be closelylinked to leaf senescenceand is not increasedby
many of the stresstreatmentsthat induce other senescence-
associatedgenes(Weaveret al., 1998;Noh & Amasino,1999).
In cell cultures,senescencecan occur spontaneously(cells
allowed to grow without subculturing),but it can also be
inducedby chemicals:in Arabidopsis cell-suspensioncultures,
we demonstratedthat high levels of 6-benzylaminopurine
(BA) induceearlyexpressionof SAG12,which precedescell
death and DNA fragmentation(Carimiet al., 2003, 2004).
The abovedatasuggestthatmanymoleculareventsoccurring
in plant cell culturesare similarto those occurringin plant
organs,and supportthe convictionthatculturedcellscan be
usedas a modelsystemfor studiesof cellularsenescence.
Variouscelldeath-signalling pathways arecritically
dependent
on mitochondria,which are key playersin the regulation
mechanismin both animals(Kroemer& Reed, 2000) and
plants(Zottiniet al., 2002;Yaoet al., 2004), not onlythrough
the releaseof pro-apoptoticfactors(such as cytochromec,
which constitutes a key event in cell death), but also by
alteringtheirmorphology(Franket al., 2001). In animalcells,
mitochondriacan form dynamic,interconnectednetworks,
andthe relativeratesof fusionandfissionhavebeenimplicated
in regulationof theirnumber,sizeandshape(Mozdy& Shaw,
2003). During apoptosis,mitochondrialnetworksare frag-
mentedand activationof the fissionmachineryis one of the
primarytriggersof this process(Bossy-Wetzelet al., 2003). A
markeddeclinein mitochondrialfunctionalityandan increase
in abnormalmitochondrialcristaestructureshavebeen asso-
ciatedwith cell ageing(Lenaz,1998).
Recentlyit hasbeenreportedthatthe fusion-fissionprocess
alsooccursin plants,asdescribedin onion epidermalcellsand
BY-2tobaccocells(Arimura etaL,2004b).In Arabidopsis, mito-
chondrialmorphologyappearsto be controlledbydynamin-like
proteins(Arimura etaL,2004a;LoganetaL,2004).InArabidopsis
leaves,it hasbeenreportedrecentlythatmorphological changes
in mitochondriaareone of the featuresof cell death that is
inducedby reactiveoxygenspecies(Yoshinagaet al., 2005).
Medicagotruncatula is a modellegumespecieswith several
featuresthat makeit attractivefor basicand appliedresearch
studies,in additionto the factthatsequencingof its genomeis
nearlycomplete(Cannonet al., 2005; Younget al., 2005). In
thisstudywe characterized M. truncatula cell-suspensioncultures
by defining severalparametersof cell growth and ageing,
eventuallyleadingto PCD. Specifically, we focusedourstudies
on changesin mitochondrial morphology duringdifferent growth
phasesand duringsenescence.In particular,we observeda
constantassociationbetweenspontaneous/induced cell death
and the presenceof giant mitochondriain cells showing
condensed and stretchednuclei. In addition, cytoskeletal
organizationwas analysedin healthy and senescentcells.
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Materialsand Methods
Cellculturesand treatments
The cell line JR was generatedfrom roots of plantletsof
MedicagotruncatulaL. cv. Jemalong (genotype 2HA) and
routinelysubculturedin modifiedMurashige& Skoog(1962)
liquid medium (MSR4: 2.70 mM KH2PO4, 40 pM nicotinic
acid,33 pMthiaminehydrochloride,60 pMpyridoxalhydro-
chloride)supplementedwith 0.5 g 1-1malt extract,30 g 1-1
sucrose, 18 IpM BA and 4.5 IIM 2,4-dichlorophenoxyacetic
acid(2,4-D). Forsubculturecycles,1.2 ml packedcellvolume
was placed in 100-ml Erlenmeyerflaskscontaining20 ml
liquid medium. Cells were subculturedin freshmedium at
10-d intervalsand maintainedin a climate chamberon a
horizontalrotaryshaker(80 rpm) at 25 ? 10C undera 16 h
daylength.
The pH of the media was adjustedto 5.7 ? 0.1 with
NaOH before autoclavingat 1210C for 15 min. Growth
regulators[2,4-D, BA,6-benzylaminopurine riboside(BA-R),
1,3-diphenylurea (DPU)], when needed, were filter-sterilized
and addeddirectlyto the medium.To determinethe effectof
highconcentrations of cytokinin,4-d-oldcellswereincubated
with 27 IMDPU, 27 jIMBA or 27 piMBA-R.Cell deathwas
determined by spectrophotometricmeasurementsof the
uptake of Evan'sblue stain, as described by Shigaki &
Bhattacharyya (1999).
To determinedryweight,integercellswereseparatedfrom
theculturemediumandcelldebristhrougha vacuumfiltration
unit (Sartorius,Florence,Italy).The collectedcellsweredried
overnightat 600C. Forcell suspension-culture experiments,a
randomized complete block design was used with three
replicates(individualErlenmeyerflasks). Each experiment
was repeatedthreetimes.
Nuclearmorphology
Medicago truncatulacellswerepreparedformicroscopeanalysis
according to a previouslydescribedprocedure(Traaset al.,
1992) with minor modifications:cells were fixed by adding
0.5 ml of a solutioncontaining4% (v/v)paraformaldehyde in
Pipes, EGTA, MgSO4 (PEM) buffer (100 mMPipespH 6.9,
10 mmEGTA,10 mMMgSO4)to 0.5 ml culture.After15 min,
cells were washed three times in PEM buffer and finally
resuspendedin PEM containing0.2% (w/v) TritonX-100
(Sigma-Aldrich, Milan,Italy)and1 pg ml-1 of theDNA specific
dye4',6-diamidino-2-phenylidone (DAPI)(AlexisChemical,
Vinci, Italy).The cells were overlaid on poly-L-lysine-coated
(Sigma-Aldrich) microscope slidesand nucleiwerevisualized
using a Leica DMR epifluorescencemicroscope (Leica
MicrosystemsWetzlarGmbH, Wetzlar,Germany)with an
excitationfilterof 330-380 nm anda barrierfilterof 400 nm.
Fornuclearmorphologyexperiments,a randomizedcomplete
block design was used with three replicates (individual
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Erlenmeyer flasks).Eachexperimentwasrepeatedat leastthree loadingand transferof proteinswerecheckedby stainingthe

times.Foreachtime point and treatment,100 representative membraneswith PonceauS. Densitometric analysesof the
nucleiwerecounted.Imagesacquired
byfluorescencemicroscopy blots were performedwith a digitalimaginganalysissystem
wereprocessedusingCorelPHOTO-PAINT (CorelCorporation, (CHEMI Doc; Bio-Rad).
Dallas,TX, USA).
Cytoskeletondetection
Analysisof mitochondria
Medicagotruncatula suspensioncells(1 ml) werefixedfor 1 h
A Nikon PCM2000 laser scanning confocal microscope in freshlyprepared
3% (v/v)paraformaldehyde (Sigma-Aldrich)
(Nikon,SestoFiorentino,Italy)wasusedforanalysisof mito- solution in microtubule-stabilizingbuffer (MSB: 0.05 M
chondrialmorphology.The tetramethylrhodamine methyl HepespH 6.9, 1 mMEGTA,0.5 mMMgCl2).Afterfixation,
esterdye(TMRM)(Molecular Probes,Leiden, Netherlands), cellswerewashedthreetimesfor 15 min eachin MSB, laidon
the
a mitochondrialmembranepotential sensor,was used for poly-L-lysine-coatedmicroscopeslidesand treatedfor 10 min
visualizing mitochondria in cell culture as described by with 1% bovineserum albumin(BSA)in PBS (137 mMNaCI,
Zottini et al. (2002). Cell suspensions(300 pl) werecollected 2.7 mMKC1,4.3 mMNa2HPO4x 7H20, 1.4 mMKH2PO4
at differenttimes during their growth cycle and following pH 7.2). Cells were collectedat 7 and 14 d of a subculture
differentcytokinintreatments,andincubatedin 700 pl MSR4 cycleandat 3 d after27 pM6-BAtreatment.Eachsamplewas
medium containing 1 pMTMRM for 15 min on a rotary incubatedovernightat40Cwiththeprimarymousemonoclonal
shaker.Cells were centrifugedfor 3 min at 10 000 g, the anti-p-tubulinantibody[1 : 200 dilutioninto PBScontaining
supernatant was discarded and the pellet washedtwice with 1%(w/v)BSA;Sigma-Aldrich] andrinsedthreetimes(15 min
700 pl MSR4.Cellswerethen resuspendedin 500 pl MSR4. each) with PBS containing 1% (w/v) BSA. Cells were then
Formicroscopeanalysis,100 pilcellsuspensionwasplacedon incubatedfor 2 h at roomtemperature in the secondaryfluo-
a microscopeslideandvisualizedundera confocalmicroscope resceinisothiocyanate-conjugated antibody (1 : 50 dilution
548
(excitation nm, emission 573 nm). Control experiments into PEM-BSA;Sigma-Aldrich). applicationof the Slow
After
were performedin the presenceof the uncouplercarbonyl FadeAntifadereagent(MolecularProbes),cellswereoverlaid
cyanide p-(trifluoromethoxy)phenylhydrazone(Zottini on a microscopeslideandvisualizedwith a Nikon PCM2000
et al., 2002). ImageswereprocessedusingCorelPHOTO-PAINT.laser scanning confocal microscope (excitation 488 nm,
Mitochondrialdimensionswereestimatedwith the IMAGENT- emission510-540 nm). The imagesacquiredwith confocal
MICROIMAGE software (Casti Imaging, Venice, Italy). For microscopywere processedusing Corel PHOTO-PAINT. Each
mitochondrialmorphologyexperiments,a randomizedcom- was
experiment repeated threetimes.
plete block designwas used with threereplicates(individual
Erlenmeyerflasks).Eachexperimentwasrepeatedthreetimes. RNAanalysis
Cellswereharvested,frozenin liquidN2 andstoredat-80'C.
Detectionof cytochromec release
RNA was isolated using Trizol (Invitrogen,San Giuliano
Forproteinextraction,3 g (FW) cellswereharvested,frozen, Milanese, Italy) following the manufacturer's instructions,
powderedin liquid N2 and homogenizedin two volumesof and treatedwith DNase I (AmbionInc., Austin,TX, USA).
proteinextractionbuffer [0.3 Msucrose,0.1 MTrispH 7.5, TotalRNA fromeachsample(2.5 pg)wasreverse-transcribed
1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM usingPowerScript reversetranscriptase
(ClontechLaboratories,
dithiothreitol(DTT) containing2 mMphenylmethylsulfonyl PaloAlto,CA, USA)followingthe manufacturer's instructions.
fluoride(PMSF), 1 mMbenzamidine,5 mMe-aminocaproic RT-PCR was carriedout by following the manufacturer's
acid,5 pg ml-1leupeptin,2 pg ml-1aprotininand0.7 pg ml-1 instructions(Taq DNA Polymerase,Eppendorf,Hamburg,
pepstatin].The homogenatewas centrifugedat 1500 g for Germany).The 18S rRNAprimersand competimersof the
15 min at 40C to eliminate debris.The supernatantwas QuantumRNAUniversal18SInternalStandards Kit(Ambion)
centrifugedat 10 000 gfor 15 min at 40C, andmitochondria were used as an internal standard.The competimerswere
werecollectedin the pellet. specially modified primersthat anneal to the 18S rRNA
Proteinconcentration
wasdetermined in eachfractionstudied templatesbut cannotbe extended,resultingin the production
by the Bradfordmethod,usingthe Bio-Radproteinassay(Bio- of an attenuated315-bp internalfragment(He et al., 2002).
Rad, Segrate,Italy).Mitochondrial(30 pg)and cytoplasmic The primers used for RT-PCR analysis of MtSAGwere
(40 pg)proteinswere separatedby 15% (w/v) SDS-PAGE, 5'-GAAGGCTGCAATGGTGGTCTCA-3' (forward)and
transferredto a nitrocellulose membrane (Sartorius)and 5'-CGGCCTCAATAGCAACGCTCAC-3' (reverse).The
analysedwith antibodiesraisedagainsthumancytochromec followingcyclingconditionswereused:950C for30 s followed
(SantaCruz,CA,USA)thatallowidentification ofa 12.4-kDa by 26 cyclesat 940C for20 s, 650C for30 s, and72?Cfor60 s
band (Bossy-Wetzelet al, 1998; Zottini et al., 2002). Equal using a Hybaid PCR expressthermalcycler(VWR, Milan,

? The Authors(2006). Journalcompilation? New Phytologist

(2006) www.newphytologist.org New Phytologist
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 New
24
1 •: 1esac Phytologist

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Fig.1 Growth and senescence of Medicagotruncatulacellcultures.(a) Dryweightof cells.(b) Celldeathestimatedby Evansbluestainingof
cells.(c) Percentageof normaland apoptotic(condensedand stretched)nucleithroughoutallphasesof cellculture.(d-f) Nuclearmorphology
of M. truncatulacellsfixed,stainedwith DAPIand observedwith a LeicaDMRmicroscope:(d) normalnuclei(7-d-oldcells);(e) condensed
nuclei(17-d-oldcells);(f) stretchednuclei(21-d-oldcells).Bar,20 pm. (g) Analysisof MtSAGexpressionduringcell cultureby relative
quantitativeRT-PCR analysis.Upperpanel,relativeabundanceof the transcript,measuredas the ratiobetweenthe intensityof the MtSAGand
18S rRNAbands.Lowerpanel,RT-PCRproductsvisualizedby a typicalethidiumbromidegel. (h) Pairwisesequencealignmentof MtSAGand
ArabidopsisthalianaSAG12.Aminoacidresiduesthat are identicalareshownagainsta blackbackground; residuesthat aresimilarareshaded
grey.Box,cysteineproteasefamilydomain.Alignmentwas performedusing BLAST2SEQUENCE analysis (http://www.ncbi.nlm.nih.gov). Values (a-
c,g) representmean? SD (n = 9).

Italy).PCRproductswerevisualizedon 2% (w/v)agarosegels Results

containingethidiumbromide(Sambrooket al., 1989), and
densitometric analysis of the gels was performed using
QUANTITY ONEsoftware(Bio-Rad).The relativeabundanceof
the transcriptwithinthe sampleswascalculatedasthe ratioof
the intensitiesof the MtSAGamplicon relativeto the 18S
rRNAamplicon.
Productsof RT-PCRwererecovered fromagarosegelsusing
theQiaexIIgelextractionkit (Qiagen,Milan,Italy),clonedinto
the plasmidpCR2.1-TOPO (Invitrogen),and sequenced.
Cell growth and spontaneous senescence in M.
truncatula suspension cell culture
Medicago truncatula cells were subcultured with a cycle of
approx. 10 d. If the medium was not changed at this time, a
rapid decrease (decline phase) in dry cell weight was observed
(Fig. la). This decreasecorrelatedwell with a rapid increase in
cell death, as suggestedby analysisof the survivalcurve (Fig. ib).

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Nuclearmorphology,observedat differenttimesaftercul-
tureinitiation,wasinvestigatedusingDAPI stainingcoupled
with fluorescencemicroscopy(Houot et al., 2001). Healthy
cellnucleishoweddiffusestaining(Fig.Id). Twomorphotypes
of nucleicouldbedistinguished in oldcells(17 and21 d):nuclei
with highly condensedchromatinin micronuclei(Fig. le);
and stretchednuclei (Fig. If). In the decline phase of the
culture,the numberof condensed/stretched nucleiincreased,
reaching 20% at 14 d and up to 80% at 21 d (Fig.Ic).
A searchin theTIGR GeneIndicesM. truncatula database
identifieda transcript(TC89773), subsequentlyreferredto as
MtSAG,which codes for a proteindisplayingan amino acid
residueidentityof 50%with Arabidopsis SAG12 (Fig. 1h). In
M. truncatula cell-suspension cultures, a low basallevel of
MtSAGexpressionwas detectedby RT-PCR analysisduring
the first7 d of the subculturecycle.RT-PCR analysisof the
expressionof MtSAGshowedthatthe transcriptaccumulated
at day 8, definingthe beginningof the reversiblesenescent
phase(Fig. Ig). The relativeabundanceof the MtSAGtran-
scriptin 11-d-oldcellswas 80%higherthan in 7-d-old cells,
and this levelof expressionwas maintainedduringthe senes-
cent phase.The increaseof the MtSAGtranscriptpreceded
thatof cell deathand nuclearchromatincondensation.In the
standardcultureconditionsmentionedabove,the mediumis
renewedafter10 d. However,the mediumchangeshouldbe
performedbeforereaching40-50% cell deathand 60% con-
densed/stretchednuclei, a physiologicalcondition in which
the reversiblesenescencephaseendsandthe entirecellculture
entersPCD.
Mitochondrialmorphologyand dynamicsduringcell-
culturegrowthand spontaneoussenescence
Mitochondrialmorphologyand dynamicsduringthe growth
cycleweredeterminedin culturedcellsusingTMRM staining
coupled with confocalmicroscopy.At differenttimes after
cultureinitiation,cells were harvestedfrom the flasksand
stainedwith TMRM to visualizemitochondria.After4 d of
subculture,M. truncatula cellsinitiatedthe exponentialgrowth
phase, and mitochondria appearedas numerous,punctuate
spheres distributed uniformly throughout the cytoplasm
In
(Fig. 2a). dividing cells, punctiformmitochondriawere
observedat the newly formed cell plate (Fig. 2b). At 7 d,
when cells are in the exponentialphase,mitochondriawere
present as interconnectednetworks (Fig.2c,i; vermiform
mitochondria).At day 10, at the end of the exponentialphase
when the cells stop dividing,the numberof mitochondrial
network structures decreased considerably (Fig. 2d). At
this time, if cells arekept in spent medium they proceedin
the senescencepathwayand enter a cell-deathprogram.At
14 d, mitochondriaappearedas small,punctiformorganelles
(Fig.2e,l) andthe totalnumberofmitochondriamaintaining
the membranepotentialdecreasedprogressively(Fig.2e-g).
At day 21, giant spherical mitochondriawere observed
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Reseaftwl- ?:24
(Fig.2g,m), sometimesassociatedwith a clumped nuclear
distribution(Fig.2h).
Throughoutall phasesof cell cultureand senescence,we
observedalterationsin both the morphologyanddynamicsof
mitochondria.In Fig.3, a representativetime-lapseseriesof
confocalimagesof 4-d-old cellsis shown,demonstratingthat
the mitochondrialdistributionpatternmutatesvery rapidly
(VideoClip S1 in Supplementary Material).Thesealterations
areprobablyaccountableforthe activefusion-fissionprocess
in which the organellesareinvolved.By contrast,in steady-
statecells,mainlypunctiformmitochondriaareobservedthat
show very raremovements,completelyabsentin senescent
cells (datanot shown).
Releaseof cytochromec in M. truncatulacell culture
An earlyeventtriggeredin severalformsof PCD is the release
of cytochromec into the cytoplasmafterpermeabilization of
the mitochondrialoutermembrane(Sunet al., 1999; Carimi
et al., 2003). In orderto define the differentphysiological
phases of M. truncatulacell culture in more detail, we
evaluatedthereleaseof cytochromecbyWesternblot analysis.
As shown in Fig.4, the releaseof cytochromec into the
cytosolicfractionwas seen clearlyat the end of the log phase
(atday8), in the initialreversiblephaseof senescence.Itslevel
remainsstablein the cytoplasmfor few days, and increased
consistentlyin the verylastphaseof cell culture.However,it
could alsobe detectedas earlyas 5-7 d.
PCDin M. truncatulacellculture
Cytokinin-induced
Recentlywe reportedthat high dosageof the cytokininBA
wasableto inducePCD by accelerating the senescenceprocess
in bothArabidopsis andcarrotcell lines (Carimiet al, 2004).
Treatmentof 4-d-old M. truncatulacell cultureswith 27 ipM
BA similarlyinduceda threefoldincrementof cell deathafter
3 d. The ribosylatedform of BA or DPU, an inhibitorof
cytokininoxidase(bothat 27 pM),induceda similarincrease
in cell death(Fig.5a).We observeda concomitantincreasein
the percentageof condensedplus stretchednuclei(Fig. 5b).
We next analysedmitochondrialmorphologyby confocal
microscopyto determineif cytokinin-inducedcell deathwas
alsoassociatedwith structuralalterationsat the mitochondrial
level.Aftertreatmentfor 3 d (27 PMDPU, BA, BA-R),the
typicaltubularmitochondrialstructureobservedin healthy
cells disintegratedinto round organelles(Fig.5c). Giant,
sphericalmitochondriawere also observedin treatedcells,
showingthe morphologytypicallyobservedin culturecells
when the spontaneoussenescencephase proceedsinto the
PCD phase.In addition,cytokininsalso stoppedmitochon-
drialmovementafter48 h of treatment(datanot shown).
When cellsweretreatedwith 27 pMBA-Rfor48 h, MtSAG
transcriptlevelsincreasedby 65% (Fig.5d) concomitantwith
an increasedreleaseofcytochromecin thecytoplasm(Fig.5e).
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I Phytologist

Cell subculture cycle

Spontaneous senescence

1 "

Vermiform mitochondria Punctiform mitochondria Giant mitochondria

(6.46 1 2.96
im')2) (2.62 2.79 m2) (28.17 1 8.31 im')2)
? methylesterdye:(a)4-d-oldcellsshowing
Fig.2 Confocalmicroscopeanalysisof Medicagotruncatulacellsstainedwithtetramethylrhodamine
numerouspunctiformmitochondria distributed uniformlyinthe cytoplasm;(b) recentlydividedcells,arrowspointto mitochondria accumulated
at the newlyformedcell plate;(c) 7-d-oldcellsshowingmitochondrial interconnectednetworks;(d) 10-d-oldcellswithdecreasednumberof
mitochondrialnetworkstructures; with a reducednumberof smallpunctiformmitochondria;
(e,f) 14- and 17-d-oldcells,respectively, (g)
oversizedmitochondriaobservedin 21-d-oldcells.Bar(a-g), 20 pm. (h) Exampleof a mitochondrial clusteraroundthe nucleusobservedin 21-
d-oldcells.Higher-magnificationimagespermittedvisualization morphologyof 7- (i), 17- (1)and 21- (m) d-old
of alterationsin mitochondrial
areais also reported.Valuesrepresentthe mean? SD (n = 9). Bar(h-m), 5 pm.
cells.Mitochondrial

methylester
time-lapseseriesof confocalimagesof 4-d-oldMedicagotruncatulacellsstainedwithtetramethylrhodamine
Fig.3 Representative
dye. Imagesweretakenat 5-s intervals.Bar,20
im.

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r, 4

days 5 7 8 11 17 21
Fig.4 Analysisof cytochromec released
frommitochondria intothe cytoplasmof Mitochondria - 12.4 kDa
cellscollectedat differentdaysafter ..
4•:••
cultureinitiation.Mitochondrial(30 pg)
and cytoplasmic(40 pg) proteinswere Cytoplasm )
loadedonto gelsforWesternblotanalysis.
.12.4kDa

(a) (c)
50

S30 IL

10

Control DPU BA BA-R

d) S0.8 (e)
(b)
120-
CNT BAR
. 0.4 --
80 - Mitochondria
stretched
-C condensed Cytoplasm
-"
-
40- l normal
,m
CNT BAR

18S
Control DPU BA BA-R MltS.IG
Fig.5 Effectsof cytokininson Medicagotruncatulacells.Cellsat 4 d old weretreatedfor3 d with27 pm1,3-diphenylurea (DPU),
6-benzylaminopurine (BA),or 6-benzylaminopurineriboside(BA-R)(a-c, respectively).(a) Celldeathestimatedby Evansbluestaining;
(b) percentageof normal,condensedandstretchednuclei;(c) mitochondrial morphologyobservedin cellstreatedwithdifferentcytokininsfor
48 h. Bar,20 pm.(d)RT-PCR analysisof MtSAGexpressionpattern;(e) detectionof cytochromec inmitochondriaandcytoplasmof cellstreated
with27 pmBA-Rfor48 h. Values(a,b,d)representmean? SD (n = 9).

Fig.6 Organization of microtubules in

Medicagotruncatulacells.Cellswere At.
analysedby indirectimmunofluorescence 'Al

microscopyusingantiP-tubulinantibodies
(see MaterialsandMethods).Cellsat (a)7 d;
(b) 14 d subculture;
(c) 4-d-oldcellstreated
for 3 d with27 pmBA-R.Bar,20 pm.

Mitochondriaand cytoskeletonorganization Cellsat 7 d with mitochondriaobservedas interconnected

networks,containa fine microtubulenetworkas determined
The mitochondrial association
with the cytoskeleton
prompted by immunofluorescence experiments usingan antibodyagainst
us to examineits organizationin differentphasesof the sub- P-tubulin(Fig.6a). At 14 d, when the cellsaresenescentand
culturecycle, correspondingto differencesin mitochondrial mitochondriaarepresentas small,punctiformorganelles,cells
morphology. showeddisorganization of the microtubulenetwork(Fig.6b).

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246 Research
A larger extent of microtubule degradation was observed in
3-d cytokinin (27 pMBA-R)-treated cells (Fig. 6c).
Discussion
Here we show that different growth phases of cells in culture
are associatedwith alterationsin mitochondrial dynamics and
morphology. In M. truncatula (in our opinion the best plant
experimental system for visualizing mitochondrial morph-
ology in vivo), we have observed differences in mitochondrial
morphology ranging from interconnected networks to
punctiform mitochondria.
In M. truncatula cell cultures, three phases can be dis-
tinguished: initial, in which cells condition their medium; log,
in which cell division takes place; and final, in which cells stop
dividing, elongate and senesce (Fig. la). Following the expres-
sion of MtSAG (an orthologue of ArabidopsisSAG12), we
observed that it was alreadydetectable at the beginning of the
cell-subculture cycle and increased, as in Arabidopsis,at the
end of the log phase (Fig. ig). The difference in expression
pattern of the two SAG species could be caused by the pre-
sence of a somewhat higher cell-death background level in
M. truncatula(20%, cf. 8-10% in Arabidopsis),but still com-
patible with the ongoing subculture cycle. A Western blot
analysis was then performed to evaluate the release of cyto-
chrome cfrom mitochondria into the cytoplasm. This analysis
revealed, in the initial phase of the culture, a modest presence
of this marker protein, which increased at the end of the log
phase and reached a stable level in later phases (Fig. 4). The
cytochrome c pattern (Fig. 4) was similar to that observed for
MtSAG (Fig. Ig). This result suggests that initial low levels of
cytochrome c detected in the cytoplasm could depend on the
cell-death background level present in M. truncatulacultures.
Its successiveincreasecoincides with the initial reversiblephase
of senescence. This could suggest a cell-autonomous cell-
death process, at least during the initial phases of cell culture.
During the senescence phase, the level of cell death in the
cell population increasesslowly but constantly. If the medium
is renewed, another subculture cycle reinitiates. In Arabidopsis
cell cultures, the reversibilityperiod is over when DNA lad-
dering becomes detectable and the level of cell death reaches
approx. 40% (Carimi et al., 2005). In the final phase of
M. truncatulacell culture, the increase in cell death (Fig. lb)
is concomitant with the increased number of nuclei showing
chromatin condensation (Fig. l c); unless the medium is
renewed before reaching 40-50% of cell death and 60% of
condensed/stretched nuclei, the entire population of cells
undergoes a massive, irreversiblePCD.
Healthy, growing cells arecharacterizedby a typical reticular
arrangement of mitochondria; when cells go into senescence
the network disintegrates and punctiform mitochondria are
observed (Fig. 2). The number of punctiform mitochondria
appearsreduced with respect to that detected at the beginning
of the log phase, probably because of a decrease in their
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membrane potential. Giant mitochondria are observed when
high levels of cell death are reached in the cell culture and, at
this stage, cytochrome cis detected principallyin the cytoplasm,
the entire cell population has entered into PCD and cannot
be rescued. Giant mitochondria have been reported to appear
during hypoxic stressin tobacco cells (Van Gestel & Verbelen,
2002).
During the process of senescence and cell death induced by
cytokinins, the reticular arrangement of mitochondria dis-
integrates rapidly,MtSAG transcriptlevels increase, and giant
mitochondria are detected together with an increasing release
of cytochrome c in the cytoplasm (Fig. 5). This scenario is
similarto that observedwhen spontaneous senescenceproceeds
into PCD. Hence mitochondrial changes in morphology and
release of cytochrome c support a role of these organelles in
the death process.
Mitochondria have been shown to associate with both
microtubules and actin (Logan, 2003; Sheahan et al., 2004),
and for this reason we analysed microtubule organization in
cultured cells under different physiological conditions. In
M. truncatula,during cell growth a normal microtubule array
is observed that appears disorganized when senescence takes
place, becoming almost disrupted in cytokinin-treated cells
induced to enter PCD (Fig. 6). These results suggest a rela-
tionship between morphological alterations of mitochondria
and cytoskeletal organization.
In conclusion, the analysis of mitochondrial changes in
morphology, performed at different cell-growth phases, pro-
mpted us to identify giant mitochondria as a markerfor senes-
cent cells entering PCD. In addition, two significant results
from the characterizationof senescence in M. truncatulacell
cultures allowed us to confirm MtSAGas a senescence marker,
and to hypothesize that the release of cytochrome c in the
cytoplasm could be an autonomous signal for cell death.
Acknowledgements
We are grateful to Professor Mario Terzi for helpful scientific
discussion and critical reading of the manuscript. This work
was supported by the national FIRB program:Post Genomica
di Leguminose Foraggere.
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SupplementaryMaterial
The following supplementary material is available for this
articleonline:
Video Clip S1. Movement of TMRM-stained mitochondria
cells.Frameswererecordedat
in 4-d-oldMedicagotruncatula
5-s intervals.
This materialis availableas part of the online articlefrom
http://www.blackwell-synergy.com
New Phytologist
(2006) 172: 239-247