departamento de ingeniería y ciencias agrarias Área de
TRANSCRIPT
Structuralandmetaboliccellwallmodificationsrelatedtothe
short‐termhabituationofmaizecellsuspensionsto
dichlobenil
MemoriapresentadaporlaLicenciadaMaríadeCastroRodríguezparaoptaral
gradodeDoctorporlaUniversidaddeLeón
Leon,2013
DepartamentodeIngenieríayCienciasAgrarias
ÁreadeFisiologíaVegetal
Modificacionesmetabolicasyestructuralesdelaparedcelularasociadasalahabituacionincipientedesuspensionescelulares
demaızadiclobenil
Amispadres,amisdirectoresyaDani
Dudasiempredetimismo,hastaquelosdatosnodejenlugaradudas
LouisPasteur
Agradecimientos
Antetodo,quierocomenzaragradeciendoamisdirectores,elDr.JoséLuisAcebesylaDra.PenélopeGarcíaAngulo,sugranlabor,dedicacióneinmensotrabajoeneldesarrollodeestaTesisDoctoral.Graciasporlaconfianzadepositadaenmípararealizaresteproyecto,porvuestraconstantepreocupaciónpormiformación,porelapoyotantoprofesionalcomopersonal, por vuestra extraordinaria categoría humana. Si a día de hoy algo soy comocientífico, sin duda todo os lo debo a vosotros. De manera personal a José Luis quieroagradecerlesusánimos,sensibilidad,pacienciaycariño.APenélope,elsermuchísimomásquedirectora,granamigayconsejera:miángelde laguarda.Hasidotodounhonorhaberpodidocontarconvosotros.
AgradezcoalMinisteriodeCienciaeInnovación(actualEconomíayCompetitividad)labecaconcedidaparaeldesarrollodelapresenteTesisDoctoral,asícomolafinanciacióndelas estancias breves desarrolladas. También, agradezco a la Junta de Castilla y León,Ministerio de Ciencia e Innovación y al Ministerio de Economía y Competitividad laconcesión de los proyectos LE004A10‐2, CGL2008‐02470BOS y AGL2011‐30545‐C02‐02respectivamente,quehancontribuidoalafinanciacióndeestetrabajo. Al Laboratorio de Técnicas Instrumentales de la Universidad de León y a susresponsables por permitirme la realización de experimentos en sus instalaciones. De lamismamanera,agradezcoaláreadeBioquímicayBiologíaMolecularposibilitarmeelusodesus dependencias. Especialmente quiero agradecerle al Dr. Javier Rúa toda la atenciónprestadacadavezquerequerísuayuda,siempreconunasonrisa.ACharo,quesepaquesu“escocesita”noseolvidadeella. AlDr.StephenFry,delEdinburghCellWallGroup,poracogermeensulaboratorioytutelarmedurantelarealizacióndegranpartedeestaTesisDoctoral.AmiscompañerosdelaboratoriodeEdimburgo,enespecialaDimitrayaAirianah,porquefuimuyafortunadaalencontrarlas.Nomepuedoolvidarde JaniceMiller, a laqueagradezcosuexcelenteapoyotécnico,peromásaúnsuafectoypalabrasdealiento.
AgradezcoalDr.DavidCaparróselabrirmelaspuertasdesulaboratoriodelCentredeRecerçaenAgrigenómica.Amiscompañerosdeallí:Pedro,Eric,Silvia,Jorge,Isabel,Chusy Joao, porque su ayuda profesional y personal hizo de mi estancia una experienciainolvidable. ADaniCaudepón: eresúnico,maño.AlDr. Sami Irar, sin el que el capítulodeproteómica de este trabajo hubiera sido imposible. Gracias Sami por tu incalculable yconstantecontribuciónaél,perotambiénportodoelapoyoquemebrindasymebrindaste.
Al Dr. Jesús Álvarez Fernández quiero agradecerle el descubrirme elmundo de laFisiología Vegetal. Al Dr. Antonio Encina su inestimable implicación e ilusión durante eldesarrollo de este trabajo, que hanmantenido encendida la llamade lamotivación en losmomentosmásdifíciles.Hasidotodounlujohabertenidolaoportunidaddeaprenderdeungran científico como él. A la Dra. Mari Luz Centeno profesionalmente le agradezco elhabermeintroducidojuntoconPenélopeenelmundodelaciencia(quiénmeloibaadeciramícuandomeenseñasteisaquellasvaretasdelclonIMC…).Personalmente,leagradezcosusconsejos,complicidadyvalorcomoprofesional,quelahacensertodounejemplo.AlaDra.Ana Alonso su ayuda con el análisis multivariante, y por supuesto la transferencia deconocimiento establecida entre las Semanas Santas leonesa y zamorana. A Laura, nuestratécnico,leagradezcotodoelcariñoquemehadado,quehasidomucho.AlDr.HugoMélidale agradezco toda la ayuda prestada (que es incontable) y su valiosa amistad. Un lujoempezarlatesisteniéndoleaélcomocompañero,referenteyoráculo.
Atodosmiscompañerosdelaboratorio,LauraGiner,CarmenHerrera,Romina,BorjayÁfrica(mipequeñinaparticular).Graciaschicosporvuestroapoyoincondicional,porllenarde color el laboratorio con vuestra risa. A Laura García Calvo quiero agradecerle sucomprensión, amistady compañerismo,yaque siemprees laprimerapersonadispuesta a
echarteunamanonadamás lonecesitas.Ycómono,graciasamiSasi,pordejardeserunsimple compañero nadamás empezar para convertirte en amigo. Gracias Sasi, por ser lapersonamástransparentequeheconocido,portupacienciaycariño,poresaespontaneidadyalegríaquehacequecuandonoestástodoseaincreíblementegris.
GraciasatodosvosotrosquesoisohabéissidopartedelÁreadeFisiologíaVegetal.Porque cuando vuelvo la vista atrás estos cuatro años se encuentran plagados de buenosrecuerdos,porquesientoellaboratoriocomomipropiacasayavosotroscomomisegundafamilia.Gracias,porquehesidomuyfelizentreestasparedes.
A todas mis amigas, por el apoyo y los ánimos en los periodos más difíciles deejecucióndeestaTesisDoctoral, pero también enmividapersonal.Graciaspor acogermeentrevosotras,pordarmelaoportunidaddevivirtantosytantosmomentosjuntas.ANani,porserlahermanaquenuncatuve,jamásolvidaréaquellamanoapoyadaenelcristal.
ADani,santovaróndondeloshaya,graciasportuinagotablepacienciaconmigo.Portodaslasvecesqueintentastecomprenderlaestructuradeunarabinoxilanotremendamenteentrecruzadomediantepolimerizaciónoxidativaenunesquemaenunaservilletadepapel,sonriendo como un japonés. Por acompañarme a lo largo de este viaje inyectandopositividad,porintentarcomprendermecuandoniyosoycapaz.GraciasDani,porhabermeenseñadoaverlavidaconotrosojos,porhacermidíaadíainfinitamentemásfácil,portuenormehumanidad.
Amispadres,porhabérmelodadotodo,porsermisreferentesenlavida.Porsufrirconmigocuandohesufrido,porserfelicessóloporqueyoloera,porquerermecomonadiepuede imaginar. Solo espero que todo el esfuerzo empleado en este trabajo sirva parahacerosllegarunamínimapartedemigratitudhaciavosotros.Amimadre,porenseñarmecadadíalaleccióndeamormásgrandealpermanecerincondicionalmenteamilado.GraciasMacuportuternuraytubondadsinlímites,porcuidarmeyalentarme.Ojaláalgúndíapuedallegaramerecerteporqueteestasganandoelcieloconmigo.Amipadre,porqueaunquetúyateloganaste,séqueestaráscontusonrisadelado,mordiendolapatilladetusgafasmásorgullosoquenadiedeestaTesisDoctoral, porqueparaalgoya se sabeque losdeCastrosiemprehemosdescendidodelapatadelCid.
Atodosvosotros,graciasdetodocorazón.
Losresultadosrecogidosen lapresenteTesisDoctoralhansidopresentadosen los
siguientescongresosyreunionescientíficas:
Cell wall modifications during short‐term habituation of maize cell
suspensionstodichlobenil.M.deCastro,A.Largo,H.Mélida,A.Alonso‐Simón,A.E.Encina,
J.M.Álvarez,J.L.Acebes,P.García‐Angulo.XIICellWallMeeting.Oporto,Portugal.Julio2010.
Metabolismo de polisacáridos hemicelulósicos de suspensiones celulares de
maíz habituadas a bajos niveles deDCB.M. de Castro, A. Largo,H. Mélida, A. Alonso‐
simón,A.E.Encina,J.M.Álvarez,J.L.Acebes,S.Fry,P.García‐Angulo.XIXCongresoSociedad
EspañolaFisiologíaVegetal.CastellóndelaPlana,España.Junio2011.
The biosynthesis and molecular weight distribution of hemicelluloses in
cellulose‐deficientmaize cells: an example ofmetabolic plasticity.M. de Castro, A.E.
Encina, J.L. Acebes, P. García‐Angulo, S. Fry. XIII Cell Wall Meeting.Nantes, Francia. Julio
2013.
DecipheringtheGolgiproteomeincellulose‐deficientcells.M.deCastro,S.Irar,
L.García,A.Largo,D.Caparrós,J.L.Acebes,P.García‐Angulo.XIIICellWallMeeting.Nantes,
Francia.Julio2013.
Cell wall modifications during short‐term habituation of maize cell
suspensionstodichlobenil.M.deCastroandA.Largo,A.E.Encina,J.M.Álvarez,J.L.Acebes,
P. García‐Angulo. XX Congreso Sociedad Española de Fisiología Vegetal. Lisboa (Portugal).
Julio2013.
Además, parte de los resultados de la presente Tesis Doctoral han dado lugar a la
siguientepublicación:
de CastroM, Largo‐Gosens A, Álvarez JM, García‐Angulo P, Acebes JL (2013)
Early cell‐wall modifications of maize cell cultures during habituation to dichlobenil. In
press.JPlantPhysiol(doi:10.1016/j.jplph.2013.10.010)
Abbreviations
2,4‐D:2,4‐dichloro‐phenoxy‐aceticacid
2‐D:two‐dimensional
ACC:1‐aminocyclopropane‐1‐carboxylicacid
ACS:adenosylmethioninesynthetase
ACO1:1‐aminocyclopropane‐1‐carboxylateoxidase1
AIR:alcoholinsolubleresidue
ANOVA:analysisofvariance
Ara:arabinose
CAZy:carbohydrateactiveenzymes
CBB:coomassiebrilliantblue
CBI:cellulosebiosynthesisinhibitor
CCoAOMT1:caffeoyl‐CoAO‐methyltransferase1
cDNA:complementarydeoxyribonucleicacid
CDTA:cyclohexane‐trans‐1,2‐diamine‐N,N,N´,N´‐tetraaceticsodiumsalt
CESA:cellulosesynthaseprotein
CFM:cell‐freemediumfraction
CHAPS:3‐3‐(3‐cholamidopropyl)diethyl‐ammonio‐1‐propanesulfonate
Cinn:cinnamicacid
COMT:caffeicacid3‐O‐methyltransferase
CSC:cellulosesynthasecomplex
CSLC:cellulosesynthase‐likeC
DCB:dichlobenil
DMSO:dimethylsulfoxide
DNA:deoxyribonucleicacid
Dt:doublingtime
DTT:dithiothreitol
DW:dryweight
EDTA:ethylene‐di‐amine‐tetra‐aceticacid
ESI‐MS/MS:electrosprayionizationwithtandemmassspectrometry
Fer:ferulicacid
FTIR:Fouriertransforminfrared
Fuc:fucose
Gal:galactose
GalA:galacturonicacid
GC:gaschromatography
Glc:glucose
GlcA:glucuronicacid
GPC:gel‐permeationchromatography
GT:glycosyltransferase
HEPES:2‐[4‐(2‐hydroxyethyl)‐l‐piperazinyl]‐ethanesulfonicacid
I50value:dichlobenilconcentrationcapableofinhibitingdryweightincreaseby50%respectingtothecontrol
IDA:immunodotassay
IDP:inosine5‐diphosphate
IDPase:triton‐dependentionidinedi‐phosphatase
IPG:immobilizedpHgradient
Irx:irregularxylem
Kav:partitioncoefficientforagivengel‐permeationchromatographycolumn
LiDS:lithiumdodecylsulfate
Man:mannose
MALDI‐TOF/MS:matrix‐assistedlaserdesorptionionization‐timeofflightmassspectrometry
MAP:microtubule‐associatedprotein
MeGlcA:methyl‐glucuronicacid
MM:molecularmass
MPBS:phosphate‐bufferedsalinewith4%fat‐freemilkpowder
Mr:relativemolecularmass
Mw:weight‐averagerelativemolecularmass
NIR:nearinfrared
P41:commercialpectinwith41%degreeofmethylesterification
PAGE:polyacrylamidegelelectrophoresis
PBS:phosphate‐bufferedsaline
PC:paperchromatography
PCA:principalcomponentanalysis
PCR:polymerasechainreaction
PCs:principalcomponents
p‐Cou:p‐coumaricacid
pI:isoelectricpoint
PMF:peptidemassfingerprint
RGP:reversibleglycosylationpolypeptide
Rha:rhamnose
RNA:ribonucleicacid
RT‐PCR:reversetranscriptionpolymerasechainreaction
SEPs:solubleextracellularpolymers
SDS:sodiumdodecylsulphate
Shx(n):habituatedsuspension‐culturedcellstoxµMDCBduring(n)numberofculturecycles
Snh:non‐habituatedsuspension‐culturedcells
TFA:trifluoroaceticacid
TIFF:taggedimagefileformat
TLC:thinlayerchromatography
UDP:uridine5‐diphosphate
V0:voidvolume
Vi:includedvolume
Wo:suspensioncellculturedryweightatthebeginningoftheculturecycle
Wt:suspensioncellculturedrytimeattime´t´oftheculturecycle
Xyl:xylose
Contents
INTRODUCTION.............................................................................................................................................1
THEPLANTCELLWALL.............................................................................................................................2
I.TypeIIplantcellwallcompositionandbiosynthesis..........................................................3
I.a.Cellulose...........................................................................................................................................3
I.b.Non‐cellulosiccellwallpolysaccharides............................................................................7
I.c.Proteins.........................................................................................................................................17
I.d.Phenoliccompounds...............................................................................................................20
II.ThearchitectureofprimarytypeIIcellwalls .......................................................... 22
III.Structuralplasticityoftheprimarycellwall......................................................................23
III.a.Mutants......................................................................................................................................24
III.b.Tolerancetoseveralstresses............................................................................................24
III.c.Cellulosebiosynthesisinhibitorsandhabituation...................................................25
IV.Objectives................................................................................................................................................29
MATERIALSANDMETHODS.................................................................................................................33
I.Cellcultures..............................................................................................................................................34
I.a.Invitrocultureconditions.....................................................................................................34
I.b.Habituationtodichlobenil....................................................................................................34
I.c.Culturescharacterization......................................................................................................35
II.Obtainingofdiscretecellcompartmentsandfractionsfrommaizecell
suspensions...................................................................................................................................................35
II.a.Cell‐freemediumcollectionandprotoplasmiccontentextraction....................35
II.b.ObtainingGolgi‐enrichedfractions..................................................................................35
II.c.Cellwallextractionandfractionation.............................................................................36
III.Cellwallanalyses................................................................................................................................38
III.a.Cellulosecontentdetermination.....................................................................................38
Contents
III.b.Sugaranalyses.........................................................................................................................38
III.c.Fouriertransforminfraredspectroscopy....................................................................38
III.d.Immunoanalysis.....................................................................................................................38
IV.Geneexpressionanalyses..............................................................................................................39
IV.a.IsolationoftotalRNA,RT–PCRandPCR......................................................................39
IV.b.ZmCESAgeneexpressionanalysis..................................................................................39
IV.c.IRXarabidopsishomologousgeneexpressionanalysis.........................................40
V.Invivoradiolabellingexperiments.............................................................................................40
V.a.Studiesinvolving[3H]arabinoseasmetabolicprecursor.......................................40
V.b.Studiesinvolving[14C]cinnamicacidasmetabolicprecursor..............................40
V.c.Assayofradioactivity.............................................................................................................41
V.c.1.Studiesinvolving[3H]arabinoseasmetabolicprecursor.........................41
V.c.2.Studiesinvolving[14C]cinnamicacidasmetabolicprecursor...............41
VI.Enzymaticdigestions........................................................................................................................42
VII.Chromatography................................................................................................................................42
VII.a.Paperchromatography......................................................................................................42
VII.b.Gel‐permeationchromatography..................................................................................43
VII.c.Gaschromatography...........................................................................................................43
VII.d.Thinlayerchromatography.............................................................................................43
VIII.Electrophoresis................................................................................................................................43
VIII.a.SDS‐PAGEanalysis..............................................................................................................43
VIII.b.Two‐dimensionalPAGEanalysis..................................................................................44
IX.Statisticalanalyses.............................................................................................................................45
Contents
CHAPTERI:Monitoringandcharacterisationofmodificationsthatoccurduring
thefirststepsofthedichlobenil‐habituationprocess..........................................................47
I.Introduction..............................................................................................................................................49
II.Materialsandmethods.....................................................................................................................51
II.a.Cellcultures...............................................................................................................................51
II.b.Habituationtodichlobenil...................................................................................................52
II.c.Growthmeasurements..........................................................................................................52
II.d.ZmCESAgeneexpressionanalysis:isolationoftotalRNA,
RT–PCRandPCR..............................................................................................................................52
II.e.Preparationandfractionationofcellwalls..................................................................53
II.f.Cellwallanalyses......................................................................................................................54
II.f.1.Cellulosecontentdetermination........................................................................54
II.f.2.Fouriertransforminfraredspectroscopy......................................................54
II.f.3.Sugaranalyses...........................................................................................................54
II.f.4.Analysisofpolysaccharidesbyenzymaticepitopedeletion..................55
II.f.5.Immunodotassaysrelatedheatmaps..............................................................56
II.g.Statisticalanalyses.................................................................................................................56
III.Results.....................................................................................................................................................57
III.a.Monitoringofearlycellwallmodificationsduring
dichlobenil‐habituation.................................................................................................................57
III.a.1.Fouriertransforminfraredspectroscopyandmultivariate
analysis....................................................................................................................................57
III.a.2.Cellulosecontent....................................................................................................60
III.a.3.ZmCESAgenesexpression..................................................................................61
III.b.Culturecharacteristics........................................................................................................62
III.c.Cellwallfeatures....................................................................................................................64
Contents
III.c.1.Cellwallfractionationandsugarcomposition..........................................64
III.c.2.Polysaccharideepitopescreeningafterenzymaticdigestion.............65
IV.Discussion...............................................................................................................................................72
CHAPTERII:Studyofthemetaboliccapacityofcellulose‐deficientmaizecells....77
I.Introduction..............................................................................................................................................79
II.Materialsandmethods.....................................................................................................................81
II.a.Cell‐suspensionculturesandhabituationtodichlobenil.......................................81
II.b.Radio‐labellingandsamplecollectionof3H‐hemicellulosesand
3H‐polymers......................................................................................................................................82
II.b.1.Cellfreemediumcollection................................................................................82
II.b.2.Protoplasmiccontentextraction......................................................................83
II.b.3.Extractionofcellwallfractions.........................................................................83
II.c.Assayofradioactivityintheuptakeof[3H]arabinoseandincorporation
into3H‐polymers..............................................................................................................................84
II.d.Fractionscompositionassay..............................................................................................84
III.Results.....................................................................................................................................................85
III.a.Uptakeof[3H]arabinosefromtheculturemedium.................................................85
III.b.Kineticsofincorporationof[3H]arabinoseintohemicellulosesand
polymers..............................................................................................................................................86
III.c.Kineticsofsynthesis,cellwallintegrationandsloughingof3H‐labelled
diagnosticfragments......................................................................................................................88
III.c.1.Protoplasmiccompartment...............................................................................89
III.c.2.Cellwallcompartment.........................................................................................89
III.c.3.Extracellularculturemedium...........................................................................91
III.d.Compositionofthe3H‐polymersand3H‐hemicelluloses......................................91
Contents
IV.Discussion...............................................................................................................................................95
CHAPTERIII:Analysisofthemolecularmassdistributionofpolysaccharides
andthephenolicmetabolisminmaizecellswithamoderatereductionin
theircellulosecontent.........................................................................................................................103
I.Introduction...........................................................................................................................................105
II.Materialsandmethods..................................................................................................................108
II.a.Cellculturesandhabituationtodichlobenil.............................................................108
II.b.Analysesofmolecularrelativeweightdistributions.............................................108
II.b.1.Radio‐labellingofliquidcultureswith[3H]arabinose..........................108
II.b.2.Obtaining3H‐hemicellulosesand3H‐polymersfromdiscrete
maizecellsuspensionscompartments....................................................................109
II.b.3.Gel‐permeationchromatography..................................................................110
II.c.Characterisationofphenolicmetabolism...................................................................111
II.c.1.[14C]cinnamicacidsynthesis............................................................................111
II.c.2.[14C]cinnamicacidconsumptionandincorporationinto
differentcellcompartments........................................................................................111
II.c.3.Obtainingofcinnamicacid[14C]derivativesfromdiscretemaize
cellcompartments...........................................................................................................112
II.d.Assayofradioactivity.........................................................................................................113
II.d.1Analysisofmolecularrelativeweightdistribution.................................113
II.d.2.[14C]cinnamicacidconsumptionandincorporationinto
differentcellcompartments.........................................................................................114
II.d.3.Obtainingcinnamicacid[14C]derivativesfromdiscrete
maizecellcompartments.............................................................................................114
Contents
II.e.ArabidopsisIRXhomologuegeneexpressionanalysis........................................114
III.Results..................................................................................................................................................115
III.a.Relativemolecularweight‐averageandrelativemolecularmass
distributionof3H‐polysaccharides........................................................................................115
III.b.Relativemolecularmassdistributionof3H‐labelledfragments
from3H‐polysaccharides............................................................................................................118
III.b.1.Relativemolecularmassdistributionof0.1MNaOH‐extracted
hemicellulosesdiagnosticfragments.......................................................................118
III.b.2.Relativemolecularmassdistributionof
6MNaOH‐extractedhemicellulosesdiagnosticfragments............................119
III.b.3.Relativemolecularmassdistributionofsoluble
extracellularpolymers...................................................................................................120
III.c.Analysisofphenolicmetabolism..................................................................................124
III.c.1.Uptakeof[14C]cinnamicacidfromtheculturemedium
anditsincorporationintocellcompartments......................................................124
III.c.2.[14C]cinnamicacidmetabolisminthecellwall.......................................124
III.d.ArabidopsishomologueIRXgeneexpressionanalysis.......................................130
IV.Discussion............................................................................................................................................130
CHAPTERIV:StudyoftheGolgiproteomeindichlobenil‐habituatedcells...........143
I.Introduction...........................................................................................................................................145
II.Materialsandmethods..................................................................................................................146
II.a.Cellculturesandhabituationtodichlobenil.............................................................146
II.b.ObtainingGolgi‐enrichedfractions...............................................................................147
II.c.Testforproteinsolubilisationfromtransmembranecomplex.........................148
II.d.SDS‐PAGEanalysis...............................................................................................................148
Contents
II.e.PreparationofGolgi‐enrichedfractionsandproteinextraction......................149
II.f.TwodimensionalPAGEanalysis.....................................................................................149
III.Results..................................................................................................................................................152
III.a.ObtainingGolgi‐enrichedfractions.............................................................................152
III.b.AnalysisofGolgiproteome.............................................................................................152
III.b.1.Procedure..............................................................................................................152
III.b.2.Two‐dimensionalelectrophoresis...............................................................155
III.b.3.Sequencing............................................................................................................157
IV.Discussion............................................................................................................................................158
DISCUSSION................................................................................................................................................167
CONCLUSIONS...........................................................................................................................................177
RESUMEN ..................................................................................................................................................181
REFERENCES..............................................................................................................................................219
APPENDIX ..................................................................................................................................................251
Introduction
Introduction
2
THEPLANTCELLWALL
Plant cell walls are partially‐rigid structures between 0.1 and 10 µm thick which
surround the protoplast. In spite of their apparently impenetrability and stasis, they are
dynamiccellcompartmentswithimportantstructuralandphysiologicalfunctions.Cellwalls
guide, restrict and determine cell growth and have a crucial role in the functional
specialisationofthedifferentcelltypes.Astheexternalcellbarrier,thecellwallcontrolsthe
metaboliteexchangebetweenprotoplastandexternalmedium(Baron‐Epeletal.,1988)and
takespartincell‐cellandcell‐pathogenrecognitionprocesses(Vorwerketal.,2004;Seifert
andBlaukopf,2010;Wolfetal.,2012).Moreover,cellwallsareinvolvedinthemodulationof
stress signaling responses (Roberts, 2001; Hückelhoven, 2007; Driouich et al., 2012), and
also regulate growth activity by being a source of oligosaccharines, biologically active
oligosaccharides (Fry et al., 1993, Franková et al., 2012). Furthermore, they show a
remarkablecompositionalandstructuralplasticitywhichallowscells torespondtoabiotic
and biotic stresses (Hamann et al., 2009). In addition to their important physiological
functions, cellwallshave significant economic implications: they influence the textureand
nutritionalvalueofmostplant‐basedproducts,areakeyfactorintheprocessingproperties
ofplant‐based foods forhumanandanimalconsumptionandareconsidered thesourceof
energyfromplantmaterialsinbiofuelproduction(BurtonandFincher,2012).
In higher plants, the genesis of the cell wall takes place during the cell division
process,whenthepolymerscomposingthecellwallaredepositedstartinginthetelophase.
Golgivesiclestransportingnon‐cellulosicpolysaccharidesareresponsiblefortheformation
ofthecellplate,thestructurethatwillgiverisetoatotallyfunctionalcellwall(Staehelinand
Hepler,1996;CutlerandEhrhardt,2002;Segui‐Simarroetal.,2004),whereasGolgivesicle
membranes give rise to theplasmamembraneof thedaughter cells (Aspinall, 1980).As a
consequence of the development of the cell plate, themiddle lamella appears, which is a
structure enriched in pectic polysaccharides shared by adjacent cells, and therefore,
responsible for cell adhesion (Aspinall, 1980). Immediately, cells start to synthesise the
Introduction
3
primary cell wall, located between the plasma membrane and the middle lamella. By
definition, a primarywall layer is the one inwhich cellulosicmicrofibrilswere deposited
whilethecellwasgrowing.Aprimarywalllayerwillnotacquiremorecelluloseoncegrowth
has stopped, although in some cell types other compounds, such as lignin or cutin, are
deposited; nevertheless, it remains a primary cell wall (Fry, 2011). In other cell types, a
thicker and more regularly arranged second layer of cellulose microfibrils, often
impregnatedwith lignin and suberin, is deposited on the inner face of the wall once cell
growthhasceased,andthisiscalledsecondarycellwall.
Two types of primary cellwalls can be distinguished (Carpita and Gibeaut, 1993),
whichdifferinchemicalcompositionaswellasinthetaxatowhichtheyarerelated:typeIis
characteristic of dicots and non‐commelinoids monocots whereas type II is exclusive to
commelinoidmonocots(Popper,2008).InthischapterwewillfocusonthetypeIIstructure,
which is the cellwall type corresponding to the specie analysed in thepresent study (Zea
mays).
I.TYPEIIPLANTCELLWALLCOMPOSITIONANDBIOSYNTHESIS
I.a.Cellulose
Cellulose is considered the scaffolding of the cellwall, towhich the remaining cell
wall constituentsareattached.Thecellulosecontent inboth typesofprimarywall, types I
and II, varies from 15% to 30%, whereas in secondary cell walls it may be up to 50%
(Carpita andMcCann, 2000). In the caseof invitro cultured cells, cellulose accounts for is
around20%(Blascheketal.,1981).
In primary walls, cellulose is deposited in microfibrils, traditionally defined as a
crystallinestructureconsistingof36β‐1,4‐glucanchainsarrangedinparallel(Delmer,1999;
SaxenaandBrown,2000;Lerouxeletal.,2006).This linkedconformationmeansthateach
glucose (Glc) residue is positioned at 180° with respect to the contiguous one, with
Introduction
4
cellobiose being the disaccharide repeated in cellulose (Saxena et al., 1995). This
circumstance,inadditiontotheabsenceoflateralchainsubstitutions,isresponsibleforthe
plainspatial structureof theβ‐1,4‐glucanchains,whichallows the formationof inter‐and
intra‐strandhydrogenbonds,leadingtothehighlystablecrystallinemicrofibrillarstructure
characteristicofcellulose(Somerville,2006).
BIOSYNTHESIS
Cellulose is known tobe synthesised at theplasmamembrane (Figure 1; formore
detailsseeGuerrieroetal.,2010).Subsequently,itisdirectionallydepositedonthecellwall
(Somerville, 2006; Taylor, 2008), undergoing dynamic spatial re‐arrangement after
deposition to allow for anisotropic expansion (Anderson et al., 2010). A protein complex
namedrosettesorcellulosesynthasecomplex(CSC)isresponsibleforcellulosebiosynthesis
(Brown,1996;Kimuraet al., 1999). Inplants,CSCsareorganised inhexameric structures,
with each hexamer containing six cellulose synthase (CESA) proteins. The complete
structureconsistsof36CESAunits,witheachCSCbeingcapableofsynthesisingthe36β‐1,4‐
glucanchainsofacompletemicrofibril.Thisassumptionisbasedonthesix‐foldsymmetryof
therosettestructuresaswellastheestimatedlateralsizeofmicrofibrilsfromprimarywalls.
However, since the number of active CESA proteins per rosette has never been
experimentallyconfirmed,thepossibilitythatdifferentrosettesmaycontainalowernumber
of catalytically active subunits, leading to a number lower than 36 β‐1,4‐glucan chains,
cannot be ruled out. Therefore, a more correct definition of a microfibril would be a
morphologic entity that corresponds to the minimum number of β‐1,4‐glucan chains
requiredtoformacrystallinestructure,orequally,theelementarystructureproducedbyan
individualrosette(Guerrieroetal.,2010).
CSCs are presumably synthesised within the endoplasmic reticulum and then
deliveredtotheGolgiapparatusforassemblybeforebeingfinallytransportedtotheplasma
membrane (Li et al., 2012). Since they are only thought to be functional in the plasma
membrane,itisherealonethatcellulosebiosynthesisisbelievedtooccur.Nevertheless,the
Introduction
5
possibility that cellulose synthesis or initiation may take place in another intracellular
compartment,forexampleintheGolgiapparatus,cannotbeexcluded.Theassumptionthat
cellulosesynthesistakesplaceexclusivelyintheplasmamembraneisbasedonthefactthat
crystalline microfibrillar structures of β‐1,4‐glucan have never been observed in the
intracellularcompartmentsofhigherplants.However,non‐crystallineβ‐1,4‐glucanmaybe
synthesisedintheGolgibyindependentcellulosesynthasecatalyticsubunitswhicharenot
partofarosette;thus,theassemblyofthisβ‐1,4‐glucanintomicrofibrillarstructureswould
notbeproduced(Guerrieroetal.,2010).Severalhypotheseshaveemergedtoclarifythis:a)
recentlysynthesisedchainsareproducedbyspatiallyseparatecatalyticsubunitswhichare
randomly distributed in the Golgi and therefore, the spatial proximity essential for the
spontaneous formation of interchain hydrogen bonds is not present, b) a completely
assembled rosette or the presence of accessory proteinswould be required, which is not
possibleintheGolgi,c)theinteractionofchainsintheorganellewithothercompounds,such
ascarbohydratesoraglycones,wouldpreventmicrofibrilformation(Guerrieroetal.,2010).
CESA proteins are multigene families with a number of genes that varies among
species.Mostofthemhavebeenidentifiedthroughthestudyofmutantsshowinganaltered
cell wall structure or composition (Arioli et al., 1998; Taylor et al., 1999; 2000; 2003;
Desprezetal.,2002;Darasetal.,2009).TenandtwelveCESAgeneshavebeenidentifiedin
arabidopsis andmaize, respectively (Holland et al., 2000; Appenzeller et al., 2004). In an
individual cell, each CESA complex requires three different types of CESA subunits to
functionproperly(Tayloretal.,2000).Forexample, inarabidopsis,AtCESA1,3andCESA6‐
relatedproteinsareessential forcellulosesynthesisinprimarywalls(Desprezetal.,2007;
Perssonetal.,2007),whileAtCESA4,7and8areessentialinthesecondarycellwall(Taylor
etal.,2003).Likewise,inmaize,ZmCESA1to9areinvolvedinbuildingtheprimarycellwall
whereasZmCESA10to12areinvolvedinthesecondarywall.ZmCESA6,7and8werethought
to be involved in secondarywall synthesis (Holland et al., 2010);However, other authors
have suggested that these genes form transitional sequences between the primary and
Introduction
6
secondarywallsynthesis,andareinvolvedinbuildingtheprimarywalllaterindevelopment
beforetheonsetofsecondarywallformation,duetothecloserproximityofthesegenesto
ZmCESA12 rather than toZmCESA10 and11 (Appenzeller et al., 2004). The structure of a
CESAproteinconsistsofeighttrans‐membranedomains,withtheactivesiteandtheNandC
terminalendslocatedatthecytosol(Lerouxeletal.,2006).CorticalmicrotubulescontrolCSC
movementandtrajectorythroughtheplasmamembrane,andarethereforeresponsiblefor
theorientationofcellulosemicrofibrils(Paredezetal.,2006).
In addition to CESA, other proteins are involved in cellulose biosynthesis, such as
sucrosesynthase(Amoretal.,1995),amembraneboundendo‐β‐1,4‐glucanase(KORRIGAN;
Nicoletal.,1998)andmicrotubule‐associatedproteins(MAP;Sedbrook,2004).
It has been suggested that sucrose synthase is directly involved in cellulose
biosynthesisbychannellinguridine5‐diphosphate‐(UDP)GlctotheCESAcomplex,sinceit
iscapableofsynthesisingUDP‐GlcfromsucroseandUDP(Baroja‐Fernándezetal.,2012;Li
etal.,2012). Ithasalsobeenreportedthatsucrosesynthase is locatedclosetotheplasma
membraneorcellwallswherecellulosesynthesisisveryactive(Salnikovetal.,2001;2003;
AlbrechtandMustroph,2003;Persiaetal.,2008).Inaddition,ahighexpressionofseveralof
itsisoformshasbeendetectedintissuesactiveintheproductionofcellulose(Geisler‐Leeet
al., 2006).Nevertheless, itsphysicalassociationwith the cellulose synthasemachineryhas
neverbeenexperimentallydemonstrated(Guerrieroetal.,2010).
It has been suggested that KORRIGAN in arabidopsis, and its orthologues in other
species, is involved in several possible roles, such as the release of newly synthesised
cellulose microfibrils in the cell wall, post‐synthetic trimming of imperfections along the
newly‐synthesisedmicrofibrilsorcontrolofthedegreeofβ‐1,4‐glucanchainpolymerisation
(Mølhøj et al., 2002). It would only be required at a specific stage of the cellulose
biosynthesis process, having a transitory association to cellulose synthase complex
(Guerrieroetal.,2010).
Introduction
7
MAPs are small cytosolic proteins that are also thought to be involved in cellulose
synthesissincetheyparticipateinthecorrectassemblyandpolymerisationofmicrotubules
(Sedbrook, 2004; Wasteneys and Yang, 2004) and in coupling CESA proteins to these
(Rajangametal.,2008).
Figure1.Hypotheticalmodelforthecellulosebiosynthesisinhigherplants,Kor:endo‐β‐1,4‐glucanaseKORRIGAN;SuSy:sucrosesynthase(Guerrieroetal.,2010)
I.b.Non‐cellulosiccellwallpolysaccharides
Pectins are matrix polysaccharides enriched in galacturonic acid (GalA). Their
content inthePoales is lowerthanindicotandgymnospermwalls,approximately10%by
weight(Carpita,1996).InspiteoftheirsignificantlylowercontentintypeIIcellwalls,they
perform important physiological functions such as water retention, ion transport, cell
adhesion and pore size determination, as well as acting as a defence mechanism against
pathogens, wounds and abiotic stresses (Ridley et al., 2001; Jarvis et al., 2003;
VerhertbruggenandKnox,2007;Peaucelleetal.,2012;WolfandGreiner,2012).Theycanbe
Introduction
8
classified into 4 different domains depending on their composition (Scheller et al., 2007):
homogalacturonan,rhamnogalacturonantypesIandIIandxylogalacturonan.Thesedomains
are thought to be conjoined domains (Fry, 2011) supporting the idea that inmuro, each
differentdomain isnot an independent, singlepolysaccharide (Ishii andMatsunaga,2001;
Coenenetal.,2007).
Homogalacturonan isapolymerofα‐1,4‐linkedGalAwhichcanbemethyl‐esterifiedor
acetylated(Figure2),accountingformorethan60%ofpectinsintheplantcellwall(Ridley
et al., 2001). The degree of methyl‐esterification has an important role in the cell wall,
becausehomogalacturonan can forma stable gel structurewithotherpecticmoleculesby
meansofCa2+bridgesthroughthenegativelychargedunmethylatedGalAresidues(Linerset
al., 1989).Thebackboneofhomogalacturonan is covalently linked to rhamnogalacturonan
typesIandII,andisalsothoughttobecrosslinkedtoxyloglucaninmuro(PopperandFry,
2008).
Figure2.Schematicrepresentationofhomogalacturonanstructure
Rhamnogalacturonan I is a heteropolymer of rhamnose (Rha) and GalA formed by
repeating units of the disaccharide 1,2‐α‐Rha‐1,4‐α‐GalA (Figure 3) (Carpita andMcCann,
2000).Three typesof galactanpolysaccharidesareassociatedwith rhamnogalacturonan I:
galactan, and types I and II arabinogalactan. Type I arabinogalactan is only found in
associationwith pectins and is composed of β‐1,4‐galactose (Gal) chains substitutedwith
mostlyarabinose(Ara)units.TypeIIarabinogalactaniscomposedofabackboneofβ‐1,3‐Gal
with branch points of β‐1,6‐Gal and is associated with specific proteins, called
arabinogalactanproteins(CaffallandMohnen,2009)(seesectionproteins).
Introduction
9
Figure3.SchematicrepresentationofrhamnogalacturonanIstructure
RhamnogalacturonanII isasubstitutedgalacturonanthat isaubiquitouscomponentof
plant walls (Caffall and Mohnen, 2009). It has the richest diversity of sugars and linkage
structures known, including apiose, aceric acid, methyl‐fucose, methyl‐xylose, 3‐deoxy‐D‐
manno‐2‐octulosonic acid, and3‐deoxy‐D‐lyxo‐2‐heptulosaric acid.Rhamnogalacturonan II
moleculesareknowntoself‐associate,formingdimersviaaboronbond(Albersheimetal.,
2011).Althoughhighlycomplex,thestructureofrhamnogalacturonanIIishighlyconserved,
suggestingthatitplaysanimportantroleinwallfunction(CarpitaandMcCann,2000).
Hemicelluloses are a group of neutral polysaccharides with a non‐crystalline
structure, composedof a lineal chain ofmonosaccharides,mainlyXyl (xylose), Glc orMan
(mannose),andgenerallyhaveshortbranches(fordetailsseePaulyetal.,2013).Themain
hemicellulosepolysaccharidesintypeIIcellwallsarexylansandmixed‐linkedglucan,buta
smallproportionofxyloglucanisalsofound.Throughtheestablishmentofhydrogenbonds,
xylansandmixed‐linkedglucanbindtightlytoadjacentcellulosemicrofibrilsandtherefore,
maintaintheminthecorrectspatialconformation.
Introduction
10
Xylans are composed of a backbone of 1,4 linked β‐Xyl residues, often substituted
with GlcA (glucuronic acid) andMeGlcA (methyl‐glucuronic acid; glucuronoxylan) (Figure
4A), Ara (arabinoxylan) or a combination of acidic and neutral sugars
(glucuronoarabinoxylan) (Figure 4B). Glucuronoxylan is a major component of the
secondary walls of dicots, whereas arabinoxylans and, to a lesser extent,
glucuronoarabinoxylan,aremajorcomponentsofgrasses(YorkandO’Neill,2008).
TheyarethemainhemicellulosepolysaccharidesintypeIIcellwalls(Fincher,2009),
representing 20% to 40% of thewall dryweight (Vogel, 2008). Poalean xylans show the
generalstructureofallxylans(abackboneofβ‐1,4‐XylwithAra,GlcA,orMeGlcAresidues
attached), but also someunique features.These include the attachmentofAra residues to
position3(ratherthantoposition2,characteristicofdicots)insomebackboneXylresidues
(Fry, 2011), which often show further substitutions with oligosaccharide side chains
containingXyl,Galandacetylgroups(Carpita,1996).Anotheroftheirdistinctivefeaturesis
thepresenceofhydroxycinnamates,mainlyFer (ferulicacid)andp‐Cou (p‐coumaricacid),
esterifiedon theAraresidues (SmithandHartley,1983;KatoandNevins,1985).Through
peroxidase action, these hydroxycinnamates are susceptible to oxidative coupling
(Geissmann and Neukom, 1971), forming dehydrodiferulates and hence contributing to
assemblybycross‐linkingcellwallpolysaccharides(Fry,2004;Parkeretal.,2005).
Xyloglucan consists of a lineal chain of β‐1,4‐glucan, substituted with α‐1,6‐Xyl
residues which in turn may be branched with Ara or Gal, depending on the species; in
addition, Gal residues may also have fucose (Fuc) substitutions attached (Hayashi, 1989)
(Figure 5). Although it is the main hemicellulose polysaccharide in type I cell walls,
representingaroundthe20%ofwalldryweight,itscontentissubstantiallylowerintypeII
cellwalls,contributingonly1‐5%.Poaleanxyloglucanalsodiffersqualitativelyfromthatof
dicots,showingaconsiderablylowerXyl/GlcratioandalowerAra,GalandFucsubstitution
content(Fry,2011).DespiteitsreducedpresenceintypeIIcellwalls,xyloglucanisthought
Introduction
11
toplay an important functional role, since grass tissuesoften showhigh xyloglucan‐endo‐
transglucosylaseactivities(Fryetal.,1992).
Figure4.SchematicrepresentationofA:glucuronoxylanandB:glucuronoarabinoxylanstructure(modifiedfrom
Paulyetal.,2013)
Figure5.Schematicrepresentationofxyloglucanstructure(modifiedfromPaulyetal.,2013)
Mixed‐linkageglucanisahemicelluloseofPoaleanprimarywalls,consistingofanun‐
branchedchainofβ‐Glcresidueslinkedby1,3and1,4bonds(Figure6).Playingacrucialrole
Introduction
12
inprimarywallexpansion,itslevelsarehighlyrelatedtothegrowthphase(Obeletal.,2003;
Gibeaut et al., 2005). It has been reported that it is not exclusive to the Poales, but also
appearsinliverwortsandhorsetails(PopperandFry,2003;Fryetal.,2008).
Figure6.Schematicrepresentationofmixed‐linkedglucanstructure(modifiedfromPaulyetal.,2013)
BIOSYNTHESIS
One of the crucial functions of the Golgi apparatus concerns the synthesis of non‐
cellulosiccellwallpolysaccharides.Unlikecellulose,whichispresumablysynthesisedatthe
plasma membrane, non‐cellulosic polysaccharides (hemicelluloses and pectins) are
synthesisedandassembledwithintheGolgicisternae,andsubsequentlytransportedtothe
cellsurfacebyGolgi‐derivedvesicles(Driouichetal.,1993;2012;Lerouxeletal.,2006;Day
et al., 2013). Synthesis of non‐cellulosic polysaccharides is catalysed by the coordinated
actionofglycosyltransferases(GTs),enzymesthattransferasugarresiduefromanactivated
nucleotide‐sugar onto a specific acceptor. Most plant GTs are type II membrane proteins
withasingle‐passtrans‐membranetopologyandacatalyticdomainintheGolgilumen,but
someofthemaremulti‐passtrans‐membraneproteins,withatopologicalstructuresimilar
to the GTs of other organisms (Varki et al., 2009; Rini et al., 2009). As cell wall matrix
polysaccharidesshowahighdegreeofstructuralcomplexity,aspatialorganisationoftheir
biosynthesiswithinthedifferentGolgistacksshouldoccur,notexclusivelybetweentheGTs
themselves, but also between GTs, nucleotide‐sugar transporters and nucleotide‐sugar
Introduction
13
interconvertingenzymes(Seifert,2004;Reiter,2008). Inaddition, interactionamongGolgi
proteins has been reported in recent studies (Oikawa et al., 2012). Furthermore, when
protein members of a synthesising complex vary, the latter’s biochemical and catalytic
function also differs (Oikawa et al., 2012). GTs are classified into 92 families in the CAZy
(carbohydrateactiveenzymes)database.Abriefsummaryofsomeofthegenescodifyingfor
GTs involved in pectin, xylan and xyloglucan synthesis is shown in Tables 1, 2 and 3,
respectively.
Due to the high diversity of monosaccharide and glycosidic linkages in pectic
polysaccharides, it has been suggested that a minimum of 67 GTs is involved in pectin
biosynthesis (Mohnen, 2008). In addition, control of the degree of homogalacturonan
methyl‐esterification during plant development requires specific methyltransferase and
esteraseactivities(Wolfetal.,2009).GAUT1,belongingtotheGT8family,hasbeenidentified
as a candidate for encoding the galacturonosyltransferase required to form the
homogalacturonan backbone in arabidopsis (Sterling et al., 2006). Another gene named
GAUT7, presenting a 77% similarity to GAUT1, is required for galacturonosyltransferase
activity, and it is related to the Golgi membrane anchor of GAUT1 (Atmodjo et al., 2011;
2013).QUA1 (GAUT8) also belongs to the GT8 family (Sterling et al., 2006), and since its
correspondingmutantshowsreducedgalacturonosyltransferaseactivities,itisthoughttobe
related to homogalacturonan synthesis (Orfila et al., 2005). The existence of a protein
complexcontaininggalacturonosylandmethyltransferaseactivitieshasalsobeensuggested
(Mouille et al., 2007). Similarly, QUA2 and QUA3 have been proposed to encode
methyltransferases (Miao et al., 2011) since both of them are highly co‐regulated or co‐
expressedwithQUA1 (Drouich et al., 2012) andGAUT1 andGAUT7 (Atmodjo et al., 2011;
2013) respectively. It has also been proposed that another gene, CGR3, encodes a
methyltransferase activity (Held et al., 2011).The XGD1 gene is involved in transferring
β‐1,3‐Xyl residues to thehomogalacturonanbackbone to formxylogalacturonan(Jensenet
al.,2008).Fourothergenes,RGXT1–4(GT77),havebeendemonstratedtobeinvolvedinthe
Introduction
14
synthesisofrhamnogalacturonanII,encodingα‐1,3‐xylosyltransferaseactivities(Egelundet
al.,2006;2008;Fangeletal.,2011;Liuetal.,2011).LittleisknownabouttheGTsinvolvedin
rhamnogalacturonan type I biosynthesis and only ARAD1, probably having α‐1,5‐
arabinosyltransferaseactivity,ARAD2 (bothof themmembersofGT47 family) (Harholt et
al.,2012)aswellasthreeGALS (1‐3),encodinggalactosyltransferaseactivities(Liwanaget
al.,2012),havebeencharacterised.Finally,anUDP‐arabinosemutasehasbeencharacterised
inrice(Konishietal.,2007).
Table1.Somegenesinvolvedinthepectinsynthesis(modifiedfromDrouichetal.,2012andAtmodjoetal.,2013),HGA:homogalacturonan;RGI:rhamnogalacturonantypeI;RGII:rhamnogalacturonantypeII;XGA:
xylogalacturonan;Pr:proven;Pu:putative
Recent years have witnessed great advances in our knowledge about xylan
biosynthesis.Severalarabidopsismutants(namedirxduetotheirregularxylemphenotype
they present)with lowXyl content in theirwalls have been described. Irx9 (Brown et al.,
2007;Penaetal.,2007;belongingtoGT43family),irx10[Brownetal.,2009;Wuetal.,2009
(GT47)] and irx14 [Brown et al., 2007 (GT43)]were found to be defective inmaking the
xylanbackbone(Dhuggaetal.,2012),indicatingthattheirencodedproteinsareinvolvedin
xylanchainelongation.Ontheotherhand,irx7/fra8[Brownetal.,2007(GT47)],irx8[Leeet
al.,2007;Penaetal.,2007(GT8)]andparvus[Laoetal.,2003;Leeetal.,2009(GT8)]were
observedtobedefectiveinthesynthesisoftheuniquesequenceatthereducingendofthe
xylanchain.Thesuggestedroleforthissequenceiseitherasaprimerorterminatorofthe
Gene CAZy Activity ReferenceSterlingetal.,2006Atmodjoetal.,2011
GAUT7 GT8 GolgimembraneanchorofGAUT1(Pr) Atmodjoetal.,2011Boutonetal.,2002Orfilaetal.,2005
QUA2/TSD2 – HGA:Methyltransferase(Pu) Mouilleetal.,2007QUA3 – HGA:Methyltransferase(Pu) Miaoetal.,2011CGR3 ‐ HGA:Methyltransferase(Pu) Heldetal.,2011XGD1 GT47‐C XGA:Xylosyltransferase(Pr) Jensenetal.,2008
Egelundetal.,2006;2008Fangeletal.,2011Liuetal.,2011
ARAD1 GT47‐B RGI:Arabinosyltransferase(Pu) Harholtetal.,2012ARAD2 GT47‐B RGI:Arabinosyltransferase(Pu) Harholtetal.,2012GALS1‐3 GT92 Galactosyltransferase(Pr) Liwanagetal.,2012
GAUT1 GT8 HGA:Galacturonosyltransferase(Pr)
GAUT8/QUA1 GT8 HGA:Galacturonosyltransferase(Pu)
RGXT1‐4 GT77 RG‐II:Xylosyltransferase(Pr)
Introduction
15
synthesis(YorkandO’Neill,2008).TheresultsreportedinPenaetal.(2007)indicatethatit
could be acting as a terminator of the synthesis, since a reduction in the number of
glucuronoxylan chains but an increase in their length was observed. Regarding the
substitutionofthexylanchain,GlcAandMeGlcAresiduesareaddedtothebackbonebyGUX
enzymes,withfivemembersinarabidopsis(Paulyetal.,2013).ThedifferentGUXenzymes
leadtodifferentGlcAxylansubstitutionpatterns(Bromleyetal.,2013).Othergenes,closely
relatedtoIRXbutwithamoregeneralexpressionpatternandlowerexpressionlevels,have
been named as their redundant homologue but with the suffix “LIKE”, i.e. IRX9L, IRX10L,
IRX14L(Brownetal.,2009;2011;Wuetal.,2010).
Focusing on grasses, the coordinated action of members of a protein complex
consistingof aβ‐1,4‐xylosyltransferase (GT43), anα‐1,3‐arabinosyltransferase (GT47)and
an α‐1,2‐glucuronyltransferase (GT43) has been reported in the glucuronoarabinoxylan
synthesisinwheat(Zengetal.,2010).Inthecaseofmaize,Boschetal.(2011)reportedthe
sequencesfortheIRX9,IRX10andIRX10Larabidopsisorthologues.Morerecently,threerice
genesorthologuesofarabidopsis IRX9, IRX9Land IRX14havebeen identified,encoding for
putativeβ‐1,4‐xylanbackbone‐elongatingGTs(Chiniquyetal.,2013).Inaddition,theunique
sequence acting as a primer or terminator is not found in grass glucuronoarabinoxylans,
leadingtothequestionofwhetherxylansynthesisingrassesinvolvesaprimer/terminator
freemechanism,orwhetherthereisanother,undiscoveredmoleculeinvolved(Paulyetal.,
2013). As regards xylan substitutions, a member of the GT61 family, XAX1, has been
describedasbeingresponsibleforaddingβ‐Xylunitstoformthesidechainβ‐Xyl‐α‐1,2‐Ara
inrice(Chiniquyetal.,2012).Thecorrespondingriceosxax1mutantisalsodeficientinFer
andp‐Cou,althoughthereasonforthisisunknown(Chiniquyetal.,2012).Inwheat,XAT1
andXAT2actasα‐1,3‐arabinosyltransferases(Andersetal.,2012).Finally,amemberofthe
TBL family,TBL29,hasbeenreported toberesponsible forxylanacetylation (Xionget al.,
2013). However, and although advances have been made in our understanding of xylan
synthesisingrassesinrecentyears,someaspectsofthisprocessstillremainunknown.
Introduction
16
Table2.Somegenesinvolvedinthexylansynthesis(modifiedfromPaulyetal.,2013andJensenetal.,2013)
Thexyloglucanbackbone is thought tobesynthesisedbyoneormoremembersof
thecellulosesynthase‐likeC(CSLC)genefamily(Paulyetal.,2013),inwhichtheCSLC4gene
hasbeenidentifiedasaglucansynthase(Cocuronetal.,2007).Regardingthepatterningof
Xyl substitutions, five genes of the GT34 family have been reported in arabidopsis as
xylosyltransferases (Faiket al.,2002;Vuttipongchaikijetal.,2012).TheseXyl substituents
canbefurthersubstitutedwithGalunits, throughtheactionof thecorrespondingproteins
codifiedbyMUR3andXLT2geneswhichbelongtoGT47family(Madsonetal.,2003;Jensen
etal.,2012).XUT1isasimilargenetoMUR3andXLT2andisincludedinthesamesubclade
oftheGT47family(Paulyetal.,2013)thatwasidentifiedinarabidopsisasbeingnecessary
forthepresenceofGalAacidinxyloglucan(Penaetal.,2012).
Inaddition,afucosyltransferase,FUT1,hasbeenshowntoaddterminalFucgroups
toGalorGalAresidues(Perrinetal.,1999;Faiketal.,2000;Penaetal.,2012).Xyloglucan
Brownetal.,2007Penaetal.,2007Brownetal.,2009Wuetal.,2009Brownetal.,2009Wuetal.,2009Brownetal.,2007Penaetal.,2007
IRX8 GT8 Xylansynthase Penaetal.,2007Laoetal.,2003Leeetal.,2009
Mortimeretal.,2010Leeetal.,2012
Rennieetal.,2012XAX1 GT61 Xylosyltransferase Chiniquyetal.,2012XAT1‐2 GT61 Arabinosyltransferase Andersetal.,2012GXMT ‐ Methyltransferase Urbanowiczetal.,2012GXM1‐3 ‐ Methyltransferase Leeetal.,2012
Brownetal.,2011Jensenetal.,2011
TBL29 ‐ Acetyltransferases Xiongetal.,2013IRX14/IRX14L GT43 Xylansynthase Wuetal.,2010
IRX9L GT43 Xylansynthase Wuetal.,2010
Gene CAZy Activity Reference
IRX9 GT43 Xylansynthase
PARVUS GT8 Xylansynthase
IRX10 GT47 Xylansynthase
XylansynthaseGT47IRX10L/GUT1
IRX7/FRA8 GT47 Xylansynthase
GUX1‐5 GT8 Glucurounosyltransferase
IRX15,IRX15L ‐ Methyltransferase
Introduction
17
alsocontainsacetylsubstituentsatvariouspositionsonthepolymersdependingontheplant
species (GilleandPauly,2012).TheAXY4 genehasbeen identified inarabidopsisasbeing
responsible for these substitutionsand is includedasamemberof theTBLprotein family
(Bischoffetal.,2010a;2010b).
Table3.Somegenesinvolvedinthexyloglucansynthesis(modifiedfromPaulyetal.,2013)
Mixed‐linkedglucanbiosynthesisiscarriedoutbyproteinsfromtheCSLFandCSLH
families(Burtonetal.,2006;Doblinetal.,2010).Bothgenesdifferinstructureaswellasin
the proteins that they encode (Wang et al., 2010). It has been reported that the
overexpressionofCSLF genes inbarleysignificantly increased theamountofmixed‐linked
glucaninleavesandendosperm(Burtonetal.,2011).
I.c.Proteins
Proteins are also essential constituents of cell walls, being involved in the
modificationofcellwallcomponentsandalsointeractingwithplasmamembraneproteinsat
the cell surface (Jamet et al., 2006). Two main groups of proteins can be distinguished:
structuralproteins,usually found immobilisedat thecellwall, andsolubleproteins,which
arelocatedattheapoplastandnormallyhaveenzymaticactivities(Leeetal.,2004).
Gene CAZy Activity Reference
CSLC4 GT2 Glucansynthase Cocuronetal.,2007Faiketal.,2002
CavalierandKeegstra,2006Cavalieretal.,2008
MUR3 GT47 Galactosyltransferase Madsonetal.,2003XLT2 GT47 Galactosyltransferase Jensenetal.,2012XUT1 GT47 Galacturonosyltransferase Penaetal.,2012
Perrinetal.,1999Vanzinetal.,2002Penaetal.,2012
AXY4/AXY4L ‐ Acetyltransferase Gilleetal.,2011
XXT1‐5 GT34 Xylosyltransferase
FUT1/MUR2 GT37 Fucosyltransferase
Introduction
18
StructuralproteinsarelessabundantintypeIIthanintypeIcellwalls(1%and10%
respectively) (Vogel, 2008). They are generally glycoproteins with repeated sequences
enrichedinoneortwoaminoacids,andcanbedividedinfourmainclasses:hydroxyproline‐
richglycoproteins,proline‐richproteins,glycine‐richproteinsandarabinogalactanproteins.
Of these, this latter group isoneof thebest‐studied.Arabinogalactanproteins consist of a
coreproteinofvaryinglengthanddomaincomplexity,oneormorearabinogalactantypeII
side chains, and often a glycosylphosphatidylinositol lipid anchor. They are involved in
important biological processes such as cell division, programmed cell death, pattern
formationgrowth,pollentubeguidance,secondarycellwalldeposition,abscissionandplant
microbe interactions (Seifert andRoberts, 2007). Interestingly, anarabinogalactanprotein
which covalently links xylans to pectic polysaccharides has recently been reported in
arabidopsis (Tan et al., 2013). This finding has important implications for polymer
biosynthesis,networkformationandapoplasticpolymermetabolism.
Regarding the soluble proteins, most of them are enzymes related to important
processes such as cell wall extension, molecular transport, cell recognition and pathogen
resistance (Rose et al., 2002), and include hydrolases, transglycosylases, peroxidases,
expansinsandkinases.
Hydrolases form themain group of soluble cell wall proteins, including important
enzymes such as glycanases,which are responsible forbreaking glycosidic bonds (Gilbert,
2010).Dependingonwhethertheircleavingactiontakesplaceatthenon‐reducingterminus
or inside of the polysaccharide chain, they can be exoglycanases or endoglycanases
respectively(Fry,2000).
Transglycosylation reactions are also essential in cell growth and are catalysed by
transglycosylases, enzymes that cleave glycosidic linkages transferring either a
monosaccharide(exotransglycosylases)oranoligosaccharide(endotransglycosylases)from
thedonorpolysaccharidetothenon‐reducingendoftheacceptor(Fry,2000).
Introduction
19
Throughhydrogenperoxideororganichydroperoxides,peroxidasesareresponsible
for the oxidative coupling of phenolic residues, and are involved in arabinoxylan cross‐
linking,theformationofisodityrosinebridgesamonghydroxyproline‐richglycoproteinsand
thelignificationprocess(Fry,2004;LindsayandFry,2008).
Expansinsareinvolvedinacidgrowth(McQueen‐Masonetal.,1992),andhencehave
a crucial role in the regulation of cell wall elongation in growing cells. Basically, their
operation mechanism consists of breaking non‐covalent interactions among cellulose
microfibrils andhemicelluloses (McQueen‐Mason et al., 2007). Specifically inmaize, it has
been proposed that an expansin (EXPB1) is involved in remodelling the arabinoxylan‐
cellulosenetworkduringcellwallrelaxation(Yennawaretal.,2006).
Another important group of proteins are the kinases, known to be involved in the
signallingpathwaysofawiderangeofcellprocesses.Thoseassociatedwiththecellwallare
involvedincellelongationandthemodulationofsugarmetabolism(Kohornetal.,2006)as
well as in signalling responses to impairment of cell wall integrity (Seifert and Blaukopf,
2010),servingaspectinreceptors(KohornandKohorn,2012).
BIOSYNTHESIS
Synthesisandassemblyofcellwallproteinsishighlycontrolled:whereassomeofthem
aredevelopmentallyregulatedorexpressedinatissue‐dependentmanner,thesynthesisof
othersoccursinresponsetowounding,infectionsorenvironmentalstresses(Albersheimet
al., 2011).Cellwallproteins are synthesisedandassembled in the endoplasmic reticulum.
Proteoglycans suchas arabinogalactan,hydroxyproline‐richandglycine‐richproteins later
undergoglycosylation,usuallyintheGolgiapparatus(Figure7)(CarpitaandMcCann,2000).
Introduction
20
Figure7.Siteofbiosynthesisofsomecellwallcomponents.HGA:homogalacturonan;RGI:rhamnogalacturonantypeI;RGII:rhamnogalacturonantypeII;HRGPs:hydroxyproline‐richglycoproteins;PRPs:prolyne‐richproteins;
GRPs:glycine‐richproteinsandAGPs:arabinogalactanproteins(CarpitaandMcCann,2000)
I.d.Phenoliccompounds
Phenols are also important compounds in the cell wall of grasses, specifically the
hydroxycinnamicacids, theprincipalofwhich incellwallsareFerandp‐Cou(Wallaceand
Fry, 1994). Theymay both be esterified each otherwhen lignin is formed (Higuchi et al.,
1967),withAra andXyl residuesof arabinoxylans (Wende andFry, 1997) and xyloglucan
(Ishiietal.,1990)respectively,andprobablywithglycoproteins(Obeletal.,2003).Although
Introduction
21
aminoritycomponent inquantitativeterms,theyplaycrucialroles inthecellwall,asthey
aresusceptibletooxidativecouplingbyperoxidases(GeissmannandNeukom,1971)toform
ester‐linked dehydrodiferulates (Fry, 2004; Parker et al., 2005), contributing to cell wall
polysaccharide assembly, growth cessation and cell wall reinforcement against abiotic or
bioticstresses(Buanafina,2009).
BIOSYNTHESIS
Fer and p‐Cou are synthesised in the cytosol following the first part of the
phenylpropanoidspathway(Figure8)(Vogt,2010),andareesterifiedontheAraresiduesof
thearabinoxylanchains,aprocessthattakesplaceintheGolgiapparatus(LindsayandFry,
2008). Regarding the proteins responsible for this incorporation, members of the PFAM
familyhavebeendescribedasferuloyltransferases(Pistonetal.,2010).
Figure8.Schematiccellularlocalizationofhydroxycinammatessynthesispathwayinplantcells.Glc:glucose;Fer:ferulicacid.Notconfirmedstepsofthepathwayareindicatedindashedlines(ModifiedfromLindsayand
Fry,2008)
Introduction
22
II.THEARCHITECTUREOFPRIMARYTYPEIICELLWALLS
Primary walls are composed of three independent but interconnected structural
networks, consisting of cellulose microfibrils acting as scaffolding and tethered by
hemicelluloses embedded in a matrix of pectin polysaccharides (Figure 9) (Carpita and
McCann,2000).Othercellwallelements,suchasglycoproteins,phenolicresidues,polymers
andpolyesterssuchascutinorsuberin,arehighlyvariablebetweentissues,developmental
stagesandtaxa(Fry,2011).
Two types of primary cellwalls can be distinguished (Carpita and Gibeaut, 1993),
which differ in chemical composition aswell as in the taxa towhich they are related. As
mentioned earlier, the type I primary cell wall is characteristic of dicots and the non‐
commelinoidmonocots.Inbothcases,themainhemicellulosicpolysaccharideisxyloglucan
which is responsible for tethering the cellulosemicrofibrils, andboth groupshave similar
contents.Thexyloglucan‐celluloseframeworkisembeddedinamatrixofhomogalacturonan
and types Iand IIof rhamnogalacturonan. Inaddition, sometype Icellwallscontain large
amountofproteins,whichcaninteractwiththepecticpolysaccharides(CarpitaandMcCann,
2000).
Type II primary cell wall composition essentially differs from type I in its non‐
cellulosicpolysaccharidecontent,withloweramountsofxyloglucansandpectins,whichare
predominantly replaced by glucuronoarabinoxylans. Type II cell walls also show lower
proteincontentsbutincontrast,theamountofhydroxycinnamates(mainlyFerandp‐Cou)is
higher.Un‐branchedarabinoxylanscanhydrogen‐bondtocelluloseortoeachother(Carpita
andMcCann,2000).ThislatterpossibilitycanalsotakeplacethroughtheFerresiduesfrom
different arabinoxylanmoleculesby covalent cross‐linkingmediatedby oxidative coupling
(Fry,2000).Little isknownabouthowtype II cellwallnetworks interactwitheachother.
Nevertheless, such information would be very valuable since these interactions could be
involved in the reinforcing mechanism of the wall structure, especially when walls are
subjectedtostressconditionsaffectingoneormoreoftheircomponents.
Introduction
23
Figure9.StructuralmodelsofA:typeIandB:typeIIprimarycellwall(ModifiedfromCarpitaandMcCann,2000)
III.STRUCTURALPLASTICITYOFTHEPRIMARYCELLWALL
Part of the dynamism of cell walls is due to their structure and composition, which
endowthemwiththecapacitytoadapttonewenvironmentalconditions,aswellastobiotic
and abiotic stresses. Significant advances in the characterisation of altered cellwalls have
been achieved in recent years through the study of in vitro plant cultured cells, tissues
and/or organs. These modified walls are the result of genetic modifications as well as
adaptation to biotic and abiotic stresses, such as habituation to cellulose biosynthesis
inhibitors. The information gained from the study of these cell wall modifications is
extremely valuable, not only because it sheds light on the contribution of the cell wall to
adaptation to stress conditions, but also because it improves our knowledge about the
preciseroleofthecellwallcomponentinthesecopingmechanisms.
Introduction
24
Thestudyofcellwallplasticityalsohassignificanteconomic implications, sincean
improvement in our knowledge about cell wall synthesis and assembly would have an
impactonseveralimportantindustries,suchastextiles,wood,paperandbiofuels.
III.a.Mutants
Theuseofmutantspresentingamodifiedcellwallstructureorcompositionhasled
to thedetectionofgenes involved in cellwall constituent synthesis, inmuroprocessingor
assembly. For instance, it has recently been demonstrated that arabidopsis and
brachypodium plants expressing hemicellulose‐ and pectin‐specific fungal acetylesterases
areresistanttofungalpathogens,butnottobacterialones,indicatingthatthedegreeofcell
wall polysaccharide acetylation plays an important role in fungal defence mechanisms
(Pogorelkoetal.,2013).However, thismethodologicalapproachhas important limitations,
sincethemutationmayaffectessentialgenes,maybelethalforthemutantorcouldrender
thealteredphenotypeimperceptible,ormultigenefamiliesmaycomplicatetheanalysisdue
toredundant functions.Modifications in thecellwallof thesemutantshavebeenscreened
usinggaschromatographyandmassspectrometry(Reiteretal.,1997)orFouriertransform
infrared(FTIR)andnearinfrared(NIR)spectroscopy(Chenetal.,1998;Mouilleetal.,2003;
McCannetal.,2007;Penningetal.,2009),andthestudyofmutantsusingtheseapproaches
has led in recent years to the identificationof several of the enzymes, includingGTs (see
section non‐cellulosic polysaccharides biosynthesis), involved in polysaccharide synthesis
(Brownetal.,2007;2011;Drouichetal.,2012).
III.b.Tolerancetoseveralstresses
Cellwallmodificationsarerelatedtoplantcellstrategiestocopewithseveralkindof
stress. Asmentionedbefore, several culturedplant cell lines showing tolerance to diverse
types of stress have been obtained in recent decades with in vitro culture techniques
whetherusingmutagenicagentsornot.Inthislattertypeofstudy,itisexpectedthoughthat
Introduction
25
thosecellscapableofgrowinganddividingundersuchrestrictedgrowthconditionsbecome
tolerant,whichwouldeventuallybeaccumulatedintheculture.Binzeletal.(1988)obtained
culturedtobaccocellshabituatedtohighsalinityandwaterdeficiency.Thesecellsshoweda
diminished cell volume and weaker cell walls with lower cellulose and higher
hydroxyproline‐rich protein content (Iraki et al., 1989a; 1989b). Although no quantitative
differences were detected in the total pectin proportion, qualitative modifications in the
organisation and composition of the pectic networks were observed, showing a relaxed
homogalacturonan‐rhamnogalacturonan networkwhich partially substituted the cellulose‐
xyloglucanone.Another studyon cellwallmodifications and stress conditions showedan
increase in peroxidase activity in the culture medium concomitant with greater lignin
contentsinthecellwalloftomatocellsadaptedtosalinestress(Sanchoetal.,1996).
Throughimmobilisationofcellwalls,plantsarecapableofadaptingtoandsurviving
inhigherconcentrationsofaluminium(Vázquezetal.,1999),lead(JiangandLiu,2010)and
otherheavymetals (Carpenaet al., 2000).Furthermore, cellwalls are also involved in the
coping response to other stresses such as low temperatures (Yamada et al., 2002), hypo‐
osmotic shock (Cazalé et al., 1998) and mechanical stress (Yahraus et al., 1995). In this
regard, it has been reported that cell wall arabinose‐enriched polymers (such as pectin‐
arabinans, arabinogalactan proteins and arabinoxylans)make amajor contribution to the
generalmechanismagainstdesiccationinawidevarietyofresurrectionplants(Mooreetal.,
2013).
III.c.Cellulosebiosynthesisinhibitorsandhabituation
Several compounds affecting the biosynthesis of cell wall components have been
described (for a review see Acebes et al., 2010; García‐Angulo et al., 2012). Their use is
consideredavaluabletoolinthestudyofthegenesisandarchitectureofcellwallaswellas
of theprocesses inwhichtheyare involved.Likewise,advanceshavebeenachieved inour
knowledge of the biosynthesis, organisation and structure of primary and secondary cell
Introduction
26
walls through theuseof these inhibitors (Satiat‐JeunemaitreandDarzens,1986;Suzukiet
al., 1992; Taylor et al., 1992; Vaughn and Turley, 1999; 2001), in addition to obtaining
important information about cell plate formation and cytokinesis (Vaughn et al., 1996;
DeBolt et al., 2007) and cell wall extension and the relaxation mechanism (Hoson and
Masuda,1991;EdelmannandFry,1992;Montague,1995).Otheraspectsrelatedtocellulose
biosynthesishavealsobeenstudiedusingtheseinhibitors,suchastheconnectionbetween
cytoskeletonandcellulose(FisherandCyr,1998;DeBoltetal.,2007;BrabhamandDeBolt,
2013),CESAcomplexorganisation(MizutaandBrown,1992)andtherelationshipbetween
celluloseandcallosesynthesis(Delmer,1987;DelmerandAmor,1995).
Cellulose biosynthesis inhibitors (CBIs) are a structurally heterogeneous group of
compounds that affects cellulose synthesis, acting specifically on cellulose coupling or
depositioninhigherplants.Theycaninduceaberranttrajectories inCESAproteins,reduce
theirvelocityorevenclearthemattheplasmamembrane(Acebesetal.,2010;Brabhamand
DeBolt,2013).Severalofthem,includingdichlobenil(DCB),theinhibitorusedinthepresent
study,havebeencommercialisedasherbicidesandareclassifiedasgroupIbythe“Herbicide
ResistanceActionCommittee”.
In agriculture DCB is employed as a wide spectrum pre‐emergent herbicide.
However, it has been used in plant research as means to induce cellulose inhibition
(BrummellandHall,1985;HosonandMasuda,1991;Corio‐Costetetal.,1991;Edelmannand
Fry,1992;Shedletzkyetal.,1992;García‐Anguloetal.,2006;Mélidaetal.,2009)sinceitacts
specificallyonthisprocesswithoutaffectingcellwallpolysaccharidesynthesis(Montezinos
andDelmer,1980;Blascheketal.,1985;Franceyetal.,1989)orothermetabolicprocesses
such as deoxyribonucleic acid (DNA) and protein synthesis, oxidative phosphorylation,
cellular respiration or lipid, Glc and nucleotide metabolism (Meyer and Herth, 1978;
MontezinosandDelmer,1980;GalbraithandShields,1982;Delmer,1987).
SeveralmodesofactionhavebeenproposedforDCBinthelastdecade.First,Penget
al. (2002) suggested that DCB inhibited the synthesis of sitosterol‐β‐glycoside, thereby
Introduction
27
blocking cellulose biosynthesis. However, since the experimental conditions used in that
study were forced and it has not been proven that cellulose biosynthesis begins from
sitosterol‐β‐glycoside,thishypothesiswasdebatable.AnotherDCBactionmechanismwhich
has been proposed concerns the alteration of cellulose crystallisation, probably due to
disruption of the spatial arrangement of microtubules rather than to glucan chain
polymerisation. A study of the rsw1 cellulose‐deficient‐mutant aswell as arabidopsis cells
treatedwith1µMDCBrevealedthatwhencellulosesynthesisisreduced,theorientationof
theremainingcellulosemicrofibrilsisaltered(Ariolietal.,1998;Himmelspachetal.,2003).
Consistingwiththis,andinsupportofthishypothesisonthemodeofaction, isthefinding
that the reduction in cellulose content detected in bean cells habituated to DCB occurred
concomitantlywith theaccumulationofanon‐crystallineβ‐1,4‐glucan(Encinaetal.,2002;
García‐Anguloetal.,2006).
Although the target of DCB still remains unclear, several possibilities have been
proposed, the most plausible of which is a MAP20 described by Rajangam et al. (2008).
MAP20isinvolvedincellulosesynthesissinceitisresponsibleforcouplingCESAproteinsto
microtubules. These authors demonstrated that DCB interactedwithMAP20, blocking the
couplingofCESAproteinstomicrotubulesandthereforecausingthe inhibitionofcellulose
biosynthesis.
Duringthelasttwodecades,ithasbeendemonstratedthatitispossibletohabituate
cultured cells to lethal DCB concentrations by subculturing them in stepwise increasing
concentrationsof the inhibitor in theculturemedium(Shedletzkyetal.,1992;Wellsetal.,
1994;Nakagawa and Sakurai, 1998; Sabba et al., 1999; Encina et al., 2001; 2002; Alonso‐
Simónetal.,2004;García‐Anguloetal.,2006;2009;Mélidaetal.,2009;2010a;2010b;2011).
It could be assumed from the studies on DCB‐habituation that the habituation
mechanismresides in theabilityshownbyculturedcells togrowanddividewithreduced
cellulosecontents.Asaconsequenceofthisprocess,aseriesofchangesintheirmetabolism
and cell wall composition takes place which differs depending on the cell wall type, DCB
Introduction
28
concentrationand lengthofexposuretime(Alonso‐Simónetal.,2004;Mélidaetal.,2009).
MostofthestudiesinvolvingculturedcellshabituatedtoDCBhavebeenperformedoncells
witha type I cellwall, suchas tomato (Shedletzkyet al., 1990), tobacco (Shedletzkyet al.,
1992;Wellsetal.,1994;NakagawaandSakurai,1998;2001;Sabbaetal.,1999)andFrench
bean(Encinaetal.,2001;2002;Alonso‐Simónetal.,2004;García‐Anguloetal.,2006),where
the reduction in cellulose is, generally compensated for by an increase in pectic content,
whereasthehemicelluloseisreduced.Othermodificationshavealsobeendescribed,suchas
theapparitionoftheabove‐mentionednon‐crystallineβ‐1,4‐glucanassociatedwithcellulose
(Encinaetal.,2002;García‐Anguloetal.,2006;Alonso‐Simónetal.,2007)andanincreasein
antioxidant activities (García‐Angulo et al., 2009).Only twokindsof cultured cell showing
type II cellwalls have beenhabituated toDCB: barley (Shedletzky et al., 1992) andmaize
(Mélidaetal.,2009).InthecaseofmaizecellshabituatedtolethalDCBconcentrations,the
coping strategy for counteracting reduced cellulose levels is mainly based on a more
extensivenetworkofagreaternumberofcross‐linkedarabinoxylanswithahigherrelative
molecularmass(Mr).
Introduction
29
IV.OBJECTIVES
Themainaimofthepresentstudyistoshedlightonthemechanismsunderlyingthe
structural plasticity of type II primary cell walls, paying special attention to metabolic
strategies. To this end, culturedmaize cells habituated to growing in DCB and showing a
moderatereductionintheircellulosecontentwillbeused.Thiscentralgoalhasbeenfurther
dividedintofourobjectiveswhicharedetailedbelow:
ObjectiveI
Monitorandcharacterisemodificationsthatoccurduringthefirststepsofthe
DCB‐habituationprocess
DCB‐habituated cultured cells havedeveloped counteractingmechanisms thatmay
varydependingonthetypeofprimarywallexhibitedinquestion,DCBconcentrationandthe
lengthoftimethatcellsaregrowninitspresence(Alonso‐Simónetal.,2004).Modifications
produced in the cell walls of DCB‐habituated maize cultured cells have been analysed
previouslyusinghighDCBconcentrationsandlong‐termhabituationperiods(Mélidaetal.,
2009),but littleattentionhasbeenpaid to theearlymodificationswhichoccurduring the
habituationprocess.Therefore, the firstobjectiveof thepresentstudy isa) tomonitor the
modificationsgeneratedthroughouttheearlystepsoftheDCB‐habituationprocess,b)select
acell linewhichshowsthecellwall featuresofaninitialDCB‐habituationwithamoderate
reductionincellulosecontent,c)subsequentlycharacteriseitsgrowthpatternandcellwall
composition. To this end, a series of spectrophotometric, chromatographic and
immunocytochemicaltechniqueswillbeemployed.
Introduction
30
ObjectiveII
Obtainfurtherinformationaboutthemetaboliccapacityof
cellulose‐deficientmaizecells
Since maize cell suspensions habituated to DCB compensate for their cellulose‐
impoverished walls by acquiring a quantitatively and qualitatively modified arabinoxylan
network (Mélida et al., 2009; 2010a; 2010b; 2011), it is highly probable that their
hemicellulosemetabolism is altered.Hence, the second objective is to gain further insight
into the metabolism of maize cells with a moderate reduction in their cellulose content
throughouttheculturecycle.Tothisend,aninvivopulse‐chaseexperimentalapproachwill
be employed, feeding DCB‐habituated cell suspension cultures with [3H]Ara as the
hemicelluloseprecursoratlag,earlylogarithmicandlatelogarithmicphasesofgrowth.The
synthesisandcellfateof3H‐hemicellulosesand3H‐polymerswillbesubsequentlytrackedin
severalcellcompartmentsusinga5htime‐window.
ObjectiveIII
Analysisofthemolecularweightdistributionofpolysaccharidesandthephenolicmetabolism
inmaizecellswithamoderatereductionintheircellulosecontent
PreviousstudiesonmaizecellshabituatedtoDCBhavedescribedareinforcedwall
acquired throughmore cross‐linked arabinoxylanswith highermolecularmass and lower
extractability(Mélidaetal.,2009;2011).However, thereportedchangeswereobservedin
hemicellulosesextractedsolelyfromthecellwallandinthefinalstageoftheculturecycle.
Therefore, the third objective will be the study of molecular mass distribution of
polysaccharides in several cell compartments throughout the culture cycle in DCB‐
habituatedcellswithamildreduction in theircellulosecontent. Inaddition,ananalysisof
phenolicmetabolismwillbeperformedsinceit isknowntobehighlyrelatedtochangesin
the molecular weight of hemicelluloses in maize cells. To this end, in vivo feeding
Introduction
31
experimentswillbeperformedwiththeradiolabeledprecursors,[3H]Araand[14C]Cinnamic
acid(Cinn)forhemicellulosesandhydroxycinnamates,respectively.
ObjectiveIV
StudyoftheGolgiproteomeinDCB‐habituatedcells
Plasticity of the walls involving modified and remodeled hemicelluloses networks
undoubtedly relies on cellmetabolism, and therefore on theproteins responsible for such
responses.SincecrucialmetabolicenzymesareknowntobeGolgi‐residentproteins,Golgi‐
enrichedfractionswillbeobtainedfromnon‐habituatedandseveralDCB‐habituatedmaize
cells lines.First,acomparativeanalysisof theGolgi‐proteomeindifferentcell lineswillbe
conductedbyusingtwo‐dimensional(2‐D)polyacrylamidegelelectrophoresis(PAGE),and
subsequently,proteinsexhibitingmis‐regulationwillbesequencedbymatrix‐assistedlaser
desorption ionization‐time of flight mass spectrometry (MALDI‐TOF/MS) or electrospray
ionizationwithtandemmassspectrometry(ESI‐MS/MS).
Introduction
32
Materialsand
Methods
MaterialsandMethods
34
I.CELLCULTURES
I.a.Invitrocultureconditions
Maizecell‐suspensioncultures(ZeamaysL.,BlackMexicansweetcorn)fromimmature
embryos were grown in Murashige and Skoog media (Murashige and Skoog, 1962)
supplemented with 9 μM 2,4‐dichloro‐phenoxyacetic acid (2,4‐D), 20 g L‐1 sucrose and
adjustedtopH5.6,at25°Cunder lightandrotaryshaken,androutinelysubculturedevery
15days.
I.b.HabituationtoDCB
Inorder toobtainhabituatedcell cultures toDCB,non‐habituatedcells (Snh)were
stepwisesubculturedinamediumsuppliedwithincreasingconcentrationsofDCBdissolved
in dimethyl sulfoxide (DMSO) which does not affect cell growth at this range of
concentrations.Likewise, inthecaseofcellssuspensionshabituatedto lowDCBlevelsSnh
cellswere cultured in amediumcontaining threeDCB concentrations: 0.3μM,0.5μM (I50
value:DCBconcentrationcapableofinhibitingdryweight(DW)increaseby50%respecting
tothecontrol)and1μM.Afterseveralsubcultures,someofthecellshabituatedtogrowin
1μMDCBweretransferredtomediuminwhichtheDCBconcentrationwasincreasedupto
1.5μM.Habituatedsuspensioncultures(Sh)arereferredtoasShx(n),where´x´indicates
DCBconcentration(μM)and(n)numberofsubculturesinthatDCBconcentration.
The procedure to obtain habituated maize cells suspensions to high DCB levels
(6μM)wasslightlydifferentfromthatdescribedabovefortheobtainingofmaizecultures
habituatedtolowDCBconcentrations.Inthiscase,maizecalluscultureshabituatedtogrow
in 12 μMDCBwere the starting cellularmaterial for the habituation process,whichwere
thentransferredintoliquidmediumsupplementedwith6μMDCB(Sh6).
MaterialsandMethods
35
I.c.Culturescharacterization
Growthcurvesofmaizecellsuspensionswereobtainedbymeasuringtheincreasein
DW at different culture times and several growth kinetics parameters were estimated:
doublingtime(dt),relativegrowthrateandmaximumgrowthrate.
Cell cluster size was determined after vacuum filtration of cell suspensions using
differentpore‐sizemeshes,andtherelativeabundanceoftheclusterswasexpressedasthe
percentageofDWretainedineachfilter.
II.OBTAININGOFDISCRETECELLCOMPARTMENTSANDFRACTIONSFROMMAIZE
CELLSUSPENSIONS
II.a.Cell‐freemediumcollection(CFM)andprotoplasmiccontentextraction
Aliquots of maize cell suspensions were passed through a Poly‐Prep column. The
filtrate was then collected and labelled as CFM, whereas cells retained in the Poly‐Prep
column filter were quickly resuspended in homogenisation buffer and subsequently
transferred to the glassmortar for being homogenisedwith amotor‐driven Teflon pestle.
ResultanthomogenatewasagainpassedthroughasecondPoly‐Prepcolumnandthefiltrate
collected. Solid KClwas added to the filtrate, and after 30min at 4°C, the productswere
centrifugedseveraltimesuntilthesupernatantturnedclear.Then,theresultantsupernatant
wascollectedandlabelledastheprotoplasmicfraction.
II.b.ObtainingGolgi‐enrichedfractions
The entire procedure was carried out at 4°C. Maize cultured cells were first
pulverised in liquid and then groundwith anomni‐mixer in extractionbuffer (Zeng et al.,
2008).Thehomogenatewascentrifugedandtheresultantsupernatantwasloadedontopof
2Msucrosebufferandsubjectedtoultracentrifugation.Theupperphasewasthendiscarded
andadiscontinuoussucrosegradientwasdevelopedbyoverlaying1.3M,1.1Mand0.25M
MaterialsandMethods
36
sucrose buffers. The discontinuous sucrose gradient was again ultracentrifuged and the
0.25M/1.1MsucroseinterfacewascollectedasaGolgi‐enrichedfraction.
Golgi‐enrichedfractionswerethensubjectedtoroutineanalysiswhichincludedtotal
proteincontentbyusingBradfordreagentandaGolgi‐membranemarkeractivityassay:the
triton‐dependentionidinedi‐phosphataseactivity(IDPase;Morréetal.,1977).
II.c.Cellwallextractionandfractionation
Twomethodologicalprocedureswereconductedtoobtaincellwallsanditsfractions,
depending on the experimental approach. Likewise, when non‐radiolabelling experiments
wereperformed,amoreexhaustiveprotocol for thepreparationandobtainingofcellwall
fractions was carried out (protocol A). However, experiments in which radioactivity was
involved,asimplifiedversionwasusedforsafetyreasons,inordertominimizethehandling
ofradiolabelledproducts(protocolsB1andB2).
ProtocolA
Cellscollectedfromsuspensioncultureswerefrozenandhomogenizedwith
liquidnitrogenusingapestleandmortar,and treatedwithethanol for5days.The
suspensionobtainedwascentrifugedandthepelletwaswashedseveral timeswith
ethanol and acetone, and subsequently air dried to obtain the alcohol insoluble
residue(AIR).TheAIRwastreatedwithDMSOandincubatedwithα‐amylase from
porcinepancreasinordertode‐starchit.Thesuspensionwasfilteredandtheresidue
washedagainseveraltimeswithethanolandacetone,airdriedandthentreatedwith
phenol/acetic acid/water. This resistant material was finally washedwith ethanol
andacetoneforseveraltimesandairdriedtoobtainthecellwalls.
Driedcellwallsweresubjectedtoseveraltreatmentsinordertosequentially
solubilizedifferentpolysaccharidetypesleadingtoacellwallfractionation.Likewise,
theywerefirstlyextractedatroomtemperaturewith50mMcyclohexane‐trans‐1,2‐
MaterialsandMethods
37
diamine‐N,N,N´,N´‐tetraacetic sodium salt (CDTA) and the resulting pellet was
treatedwith0.1MKOH.Theremainingresiduewasthentreatedwith4MKOHand,
afterwardsthe4MKOH‐resistantmaterialwassubjectedtoasubsequentextraction
with6MKOH.
ProtocolB1:Studiesinvolving[3H]Araasmetabolicprecursor
Maizecellsuspensionswerehomogenisedwithamotor‐drivenTeflonpestle.
Theresultanthomogenatewas filteredthroughaPoly‐Prepcolumnandthe filtrate
was then discarded. Cell fragments retained in the filter of the Poly‐Prep column
weretreatedwith0.1MNaOHcontainingNaBH4.Thefiltratewasthencollectedand
labelledas0.1MNaOH fraction.The remainingresiduewas then treatedwith6M
NaOH containing NaBH4. The filtrate was again collected and labelled as the 6 M
NaOHfraction.The6MNaOH‐resistantmaterialwasconsideredaspolymersfirmly
boundtocelluloseresidue.
ProtocolB2:Studiesinvolving[14C]Cinnasmetabolicprecursor
Cellswereresuspendedin80%ethanolandincubatedonarotarywheel,the
sampleswerefilteredandtheresultantfiltratewasagaindiscarded.Theremaining
cellfragmentswereconsideredtheAIR.Insomecases,thatAIRwasde‐esterifiedby
saponificationwith0.5MNaOH.Solublefractionfromthistreatmentwascollectedas
the 0.5MNaOH fraction. This fractionwas acidified by addition of acetic acid and
partitionedagainstethylacetate, vacuum‐driedandre‐dissolved inacidifiedwater.
Samples were again subjected to a second ethyl acetate partition and the organic
phaseswerecollected,vacuum‐driedandre‐dissolvedinpropan‐1‐ol.
MaterialsandMethods
38
Alkali fractions from the cell wall compartmentswere acidified by the addition of
acetic acid. All the fractions obtained fromdiscrete cellwall compartmentswere dialysed
andstoredat‐20°Cuntilused.
III.CELLWALLANALYSES
III.a.Cellulosecontentdetermination
Cellulose content was quantified in crude cell walls by the Updegraff method
(Updegraff, 1969) using the hydrolytic conditions described by Saeman et al. (1963) and
quantifyingtheGlcreleasedbytheanthronemethod(Dische,1962).
III.b.Sugaranalyses
Total sugar content of each fraction was determined by the phenol‐sulfuric acid
method(Duboisetal.,1956)andwasexpressedas theGlcequivalent.Uronicacidcontent
was determined by the m‐hydroxydiphenyl method (Blumenkrantz and Asboe‐Hansen,
1973)usingGalAasstandard.Neutralsugarcontentanalysiswasperformedasdescribedby
Albersheimetal.(1967).
III.c.FTIRspectroscopy
Monitoringofcellwallchangesinmaizecell lineswereperformedbyFTIR.Tablets
wereprepared in aGraseby‐Specacpressusing cellwall samplesmixedwithKBr. Spectra
wereobtained,areanormalizedandbaseline‐corrected.Differencespectrawereobtainedby
digitalsubtractionofnon‐habituatedandhabituatedcellwallFTIRspectra.
III.d.Immunoanalysis
Anextensiveanalysisofpolysaccharidedistributionandcompositionofthecellwall
fractionswascarriedoutby immunodotassays (IDAs).Briefly,aliquots fromeach fraction
werespottedontonitrocellulosemembraneasareplicateddilutionseriesofthepreceding
MaterialsandMethods
39
one.Then,membraneswereincubatedwithanendo‐polygalacturonanase,a‐xylanaseand
an endo‐arabinanase (for details see enzymatic digestions section) following the
experimental approach described by Øbro et al. (2007) with minor modifications. After
enzymatic digestion, nitrocellulose membranes were blocked with phosphate‐buffered
saline(PBS)containing4%fat‐freemilkpowder(MPBS)priortoincubationinmonoclonal
primaryantibodies,withspecificity fordifferentpecticandhemicellulosicpolysaccharides.
AfterwashingextensivelyunderrunningtapwaterandPBS,membraneswereincubatedin
secondary antibody (anti‐rathorseradishperoxidase conjugate).Membraneswerewashed
asdescribedaboveandsubjectedtocolordevelopmentinsubstratesolution.
Immunolabelling quantification for each fraction was performed by scoring the
number of dilutions labelled. These valueswere then entered into the statistical software
(seeStatisticalanalysessectionfordetails)inordertoobtainheatmaps.
IV.GENEEXPRESSIONANALYSES
IV.a.Isolationoftotalribonucleicacid(RNA),reversetranscription‐polymerasechain
reaction(RT–PCR)andpolymerasechainreaction(PCR)
TotalRNAwasextractedwithTrizolReagent,andwasreverse‐transcribedusingthe
Superscript III first strand synthesis system for RT‐PCR. First‐strand complementaryDNA
(cDNA)wasgeneratedusinganoligo(dT)20primerandusedas a template in subsequent
PCR reactions. For each assay, several numbers of cycles were tested to ensure that
amplificationwaswithintheexponentialrange.Ubiquitinwasusedashousekeeping.
IV.b.ZmCESAgeneexpressionanalysis
The gene‐specific primers used for the analysis ofZmCESA1 (AF200525),ZmCESA2
(AF200526), ZmCESA3 (AF200527), ZmCESA5 (AF200529), ZmCESA6 (AF200530) and
ZmCESA7(AF200531)geneswerethosepreviouslydescribedbyHollandetal.(2000).Dueto
MaterialsandMethods
40
thehigh‐sequencesimilaritybetweenZmCESA1andZmCESA2,thesameprimerwasusedfor
the analysis of both genes (ZmCESA1/2). The primers used for the analysis of ZmCESA4
(AF200528)andZmCESA8(AF200532)werethosedescribedinMélidaetal.(2010a).
IV.c.IRXarabidopsishomologousgeneexpressionanalysis
Primers were used for the analysis of maize genes GRMZM2G100143,
GRMZM2G059825 and gbIBT036881.1, homologous of arabidopsis IRX10 (AT1G27440),
IRX10‐L(AT5G61840)andIRX9(AT2G37090)respectively(Boschetal.,2011).Sequencesfor
GRMZM2G100143andGRMZM2G059825werederivedfrommaizegenomebrowserandthat
forgbIBT036881.1fromPhytozome.
V.INVIVORADIOLABELLINGEXPERIMENTS
V.a.Studiesinvolving[3H]Araasmetabolicprecursor
Aliquotsofmaizecellcultureswerecollectedatseveralgrowthphasesoftheculture
cycleandindependentlyfedwithL‐[1‐3H]Ara.Duetothedisparitythatthedifferentcelllines
exhibited in the length of time of the culture phases, the days inwhich the aliquot of the
culture was sampled and [3H]Ara added were adjusted depending on the cell line. The
incorporation of [3H]Ara into discrete cell compartments was measured at different
labelling‐time points (30, 60, 120 and 300 min after [3H]Ara was fed) for 5 h in each
differentculturephase.
V.b.Studiesinvolving[14C]Cinnasmetabolicprecursor
[14C]CinnwaspreparedfromL‐[U‐14C]phenylalaninefollowingthemethoddescribed
by Lindsay and Fry. (2008) with minor modifications. Aliquots of Snh and Sh1.5 cell‐
suspensionsatdifferentphasesoftheculturecycleweretransferredintovials,andshakento
allowthemtoacclimatisetotheirnewenvironmentalconditions.Then,cellswerefedwith
MaterialsandMethods
41
[14C]Cinn, and its incorporation and consumption was measured at selected time‐points.
Whereindicated,H2O2wasaddedtosomeofthesamples120minafter[14C]Cinn,andthen
shakenfor60min.
V.c.Assayofradioactivity
V.c.1.Studiesinvolving[3H]Araasmetabolicprecursor
Origin of the radioactivity samples imply disparity in the “format” in which it is
presented (i.e dissolved, on/within paper chromatogram, polypropylene filter pad) and
therefore would determine some radioactivity assay parameters as the type of liquid
scintillationusedaswellascorrectionfactorforcountingefficiencyinordertoperformthe
quantification.
Fortheanalysisofliquidsamples,aliquotsofthemwereassayedforradioactivityby
liquidscintillationcountingmixingthesampleswithOptiPhaseHiSafeina1/10dilution.
PaperchromatogramspotscorrespondingtomonosaccharideandpolymericmaterialatRF
0werecutandseparatelyassayedbyscintillationcountingin2mlofOptiScintHiSafe.
When radioactivity of the spotswas low, a protocol for the improving of counting
sensibilitywasapplied.Likewise,spotsweresoakedovernightinwaterandthen,OptiPhase
HiSafescintillationliquidwasaddedina1/10dilution,andvialswereshakenfor24h.
Inasimilarway,thequantificationofradioactivitypresentedinpolypropylenefilterpad,the
entirefilterwasexcisedandmixedwithOptiPhaseHiSafescintillationliquid.Aftersoaking
for24hradioactivitywasthenassayedbycounting.
V.c.2.Studiesinvolving[14C]Cinnasmetabolicprecursor
Liquid samplesweremixedwith EcoscintA and radioactivitywas then assayed by
liquidscintillationcounting.
MaterialsandMethods
42
InthecaseoftheradioactivityincorporatedintotheAIR,cellfragmentswerefirstly
resuspended in distilled water, EcoscintA was subsequently added and radioactivity was
thenassayedbycounting.
Trackscorrespondingtosamplesfromthinlayerchromatography(TLC)plates,were
cutoffintopiecesandanalysedbyscintillationcountingthroughtheadditionofEcoscintA.
VI.ENZYMATICDIGESTIONS
Driselase enzymatic digestionwas performed as follows: prior to it sampleswere
dried invacuo, subjected toamild‐hydrolysis in trifluoroaceticacid (TFA)and re‐dried to
removeTFA.Thedriedmaterialwasre‐dissolvedin0.5%(w/v)Driselaseinpyridine/acetic
acid/waterandincubatedat37°Cfor96h.Thereactionwasstoppedbytheadditionof15%
formicacid.
Treatments with endo‐polygalacturonanase (M2) from Aspergillus aculeatus (E‐
PGALUSP) (EC 3.2.1.15), a recombinant ‐xylanase from Neocallimastix patriciarum (E‐
XYLNP) (EC 3.2.1.8) and an endo‐arabinanase from Aspergillus niger (E‐EARAB) (EC
3.2.1.99)werecarriedoutat37°C for24h.Theenzymesweredissolved to1U/ml in350
mMacetatebufferadjustedtopH4.7bytheadditionofpyridine.
VII.CHROMATOGRAPHY
VII.a.Paperchromatography(PC)
Internalmarkerswereaddedwithinsamplespreviousthechromatographyanalysis,
whichwasdevelopedin3MMWhatmanpaper.PCconditionsslightlydiffereddependingon
the sample components requested to be resolved. Likewise, when separation of
monosaccharidesfrompolymericmaterialatRF0wasrequired,samplesweresubjectedto
PCinbutan‐1‐ol/aceticacid/waterfor16h.Inthecaseofsamplesinwhichtheresolutionof
MaterialsandMethods
43
monosaccharides, disaccharides and polymeric RF 0material would be desirable, PC was
conductedbyusingethylacetate/pyridine/waterfor18h(ThompsonandFry,1997).
The internal markers were stained slightly by dipping the paper in 10% of the
standardconcentrationofanilinehydrogenphthalate(Fry,2000),dried,andthenheatedat
105°Cuntiltherequiredintensityofcolordevelopmentwasobtained.
VII.b.Gel‐permeationchromatography(GPC)
Polymerswere size‐fractionatedonSepharoseCL‐4B inpyridine/acetic acid/water
at 12.5 ml/h. The Sepharose CL‐4B column was calibrated with commercial dextrans of
knownweight‐average relativemolecularmass (Mw).Mwwas obtained using the Kav (1/2)
(Kav: partition coefficient for a givenGPC column)method (Kerr and Fry, 2003)with the
calibrationcurve[logMw=‐3.547Kav(1/2)+7.2048]obtainedforthiscolumn.
VII.c.Gaschromatography(GC)
Lyophilized samples were hydrolyzed with 2 M TFA at 121°C for 1 h and the
resultingsugarswerederivatizedtoalditolacetatesandanalyzedusingaSupelcoSP‐2330
column.
VII.d.TLC
TLC was carried out on plastic‐backed silica‐gel with a fluorescent indicator in
benzene/aceticacidand,duringdevelopment,plateswereexposedto312nm.
VIII.ELECTROPHORESIS
VIII.a.Sodium‐dodecyl‐sulphate(SDS)‐PAGEanalysis
ForSDS‐PAGEseparation,proteinswere treatedunder reducingconditions (boiled
samples)orpartialnon‐reducingconditions(notboiledsamples)andseparatedbystandard
MaterialsandMethods
44
SDS‐PAGEona7.5%,10%or12.5%gel.Theresultantgelswerethenstainedwithcoomassie
brilliantblue(CBB)R‐250.
VIII.b.2‐DPAGEanalysis
Previously to 2‐D electrophoresis, preparation of samples was required. Briefly,
aliquotsofeachsamplewerefreeze‐driedandthendissolvedinthelysisbuffer,incubatedin
an ultrasound bath, centrifuged and the resultant supernatantwas collected and dialysed.
Afterwards, sampleswere concentratedbyVivaspin columnsuntil the requiredamountof
proteinforthe2‐Delectrophoresiswasobtained.
Proteinsampleswereloadedontonon‐linearpH4–7immobilizedpHgradient(IPG)
strips to carry out the first dimension, performed in EttanTM IPGphor Isoelectric Focusing
System.Afterwards, IPG stripswere collectedandequilibratedwithadithiothreitol (DTT)
treatmentfollowedbyasecondtreatmentwithiodoacetamide,loadedontopofaSDS–PAGE
10%polyacrylamidegelandsubjectedtotheseconddimensionusingEttanDALTsixSystem.
Gelswerefinallyresolvedwithcorrespondingstain,CBBG‐250orsilverstain(Shevchenko
etal.,1996;Iraretal.,2006),leadingtotwoindependentbutcomplementaryanalyses.The
experimentwascarriedoutwiththreetechnicalreplicatesperbiologicalsample.Thestained
gelswere scanned and analysed (Farinha et al., 2011) and after automatic spot detection,
manualspoteditingwascarriedout.Toevaluateproteinexpressiondifferencesamonggels,
relativespotvolume(%Vol)wasused.Thosespotsshowingaquantitativevariation>Ratio
1 and positive GAP were selected as differentially accumulated. Statistically significant
proteinabundancevariationwasvalidatedbyStudent’st‐test(p,0.05)(Farinhaetal.,2011).
The selecteddifferential spotswereexcised from thegels and identifiedeitherbypeptide
mass fingerprint (PMF) using MALDI–TOF/MS or by ESI‐MS/MS. The software packages
ProteinProspectorv3.4.1fromtheUniversityofCaliforniaSanFranciscoandMASCOTwere
used to identify the proteins from the PMF data as previously reported (Carrascal et al.,
2002). The SEQUEST software was used for preliminary protein identification from the
MaterialsandMethods
45
MS/MS analysis followed by manual sequence data confirmation. Swiss‐Prot and non‐
redundantNCBIdatabaseswereused for thesearch.Searcheswereperformed for the full
range of molecular weight and isoelectric point (pI). No species restriction was applied.
Whenanidentitysearchproducednomatches,thehomologymodewasused.
IX.STATISTICALANALYSES
Principal component analysis (PCA) was performed using a maximum of five
principal components (PCs). All these analyses were carried out using the Statistica 6.0
softwarepackage.
Significant differences were tested applying a one‐factor analysis of variance
(ANOVA) (performing Tukey´s test as post‐hoc analysis), using the PASW Statistics 18
softwarepackageorbyStudent’st‐test(p,0.05).
HeatmapswereperformedbyusingRKWardstatisticalsoftware.
MaterialsandMethods
46
ChapterI
Monitoringandcharacterisationof
modificationsthatoccurduringthe
firststepsoftheDCB‐habituation
process
ChapterI
48
ABSTRACT
Studiesinvolvingthehabituationofplantcellculturestocellulosebiosynthesis
inhibitors have achieved significant progress as regards understanding the structural
plasticity of cell walls. However, since habituation studies have typically used high
concentrations of inhibitors and long‐term habituation periods, information on initial
changes associated with habituation has usually been missed. This study focuses on
monitoring and characterising the earlier cell wall changes occurring during the
habituationprocessofmaizecellsuspensionstoDCB.CellulosequantificationandFTIR
spectroscopy of cellwalls from 20 cell lines obtained during the first steps of aDCB‐
habituation process showed transitory decreases in cellulose levels which tended to
revertdependingontheinhibitorconcentrationandthelengthoftimethatcellswerein
contactwithit.Variationsinthecellulosecontentwereconcomitantwithchangesinthe
expressionof severalZmCESAgenes,mainly involvingoverexpressionofZmCESA7 and
ZmCESA8.Inordertoexploresubsequentchanges,acelllinehabituatedto1.5µMDCB
was identified as representative of incipient DCB habituation and selected for further
analysis.Theircellsgrewwithlowerrelativeandmaximumgrowthrates,determinedby
longer lagphasesanddt, and formedclusterswith largeraveragevolumeandgreater
varietyofsizes.Theircellwallsshoweda33%reductionincellulosecontent,whichwas
mainly counteracted by an increase in arabinoxylans, which presented increased
extractability.ThisresultwasconfirmedbyIDAsgraphicallyplottedbyheatmaps,since
habituatedcellwallshadamoreextensivepresenceofepitopes forarabinoxylansand
xylans, but also for homogalacturonan with a low degree of esterification and for
galactansidechainsofrhamnogalacturonanI. Inaddition,apartialshiftofxyloglucan
epitopestowardsmoreeasily‐extractablefractionswasfound.However,otherepitopes,
suchasforarabinansidechainsofrhamnogalacturonanIorforhomogalacturonanwith
a higher degree of esterification, seemed to be not affected. In conclusion, initial
modificationsoccurringinmaizecellwallsasaconsequenceofDCB‐habituationincludes
quantitative and qualitative changes of arabinoxylans and different polysaccharides.
Therebysomeofthechangesthatoccurredinthecellwallsinordertocompensatefor
the lackof cellulosediffered according to theDCB‐habituation level, and illustrate the
abilityofplantcellstoadoptappropriatecopingstrategiesdependingontheherbicide
concentrationandlengthofexposuretime.
ThisChapterpartiallycorrespondstothemanuscript:deCastroM,Largo‐GosensA,ÁlvarezJM,García‐AnguloP,AcebesJL
(2013)Earlycell‐wallmodificationsofmaizecellculturesduringhabituationtodichlobenil.InPressJPlantPhysiol
(doi:10.1016/j.jplph.2013.10.010)
ChapterI
49
I.INTRODUCTION
Plantcellwallsaredynamicstructureswhose importanceresidesprincipally inthe
key role they play in plant growth and development, enabling cells to adapt to abiotic or
biotic stresses (Wolf et al., 2012). Cell walls influence the properties ofmost plant‐based
products, including their texture and nutritional value, and condition the processing
propertiesofplant‐basedfoodsforhumanandanimalconsumption(Doblinetal.,2010).Cell
walls are mainly composed of polysaccharides, which are classified as cellulose,
hemicellulosesandpectins,andhaveloweramountsofproteins,phenolicsandotherminor
components.Cellwallcompositionvariesaccordingtotheplanttypeandspecies(Sarkaret
al., 2009), cell type and position within the plant, developmental stage and history of
responses to stresses (Doblinet al., 2010).This variability in the compositionof cellwalls
reflects a certain degree of plasticity as regards cell wall structure and composition. One
suitable method to study the mechanisms underlying the plasticity of plant cell wall
structure and composition consists in habituating cell cultures to grow in the presence of
highconcentrationsofdifferentCBIs(forareviewseeAcebesetal.,2010).
CBIs are a group of compounds that specifically affect the assembly and/or
depositionofcelluloseinthecellwall,byinducingaberranttrajectories,avelocityreduction
or even the clearance of CESA subunits at the plasma membrane (Acebes et al., 2010;
BrabhamandDeBolt,2013).Inthelasttwodecadesithasbeenshownthatitispossibleto
habituate undifferentiated cell cultures (calluses and cell suspensions) of various
dicotyledonous plants, such as arabidopsis, tobacco, tomato, bean or poplar, to lethal
concentrationsofdiverseCBIsandrelatedcompounds,suchasisoxaben,thaxtominA,DCB
orquinclorac,bygradually increasing the concentration in the culturemedium. Ingeneral
terms, in order to copewith the stressful conditions, the cell cultures habituated to these
inhibitors modify the characteristic architecture of the type I cell wall (typical of
gymnosperms,dicotsandmostmonocots),havingreduced levelsofcelluloseaccompanied
byadecreaseinthehemicellulosiccontentandasignificantincreaseinpectins(Shedletzky
ChapterI
50
et al., 1992; Díaz‐Cacho et al., 1999; Encina et al., 2001; 2002; García‐Angulo et al., 2006;
2009).Themodificationsproducedincellshabituatedtotheseinhibitorsnotonlydependon
theinhibitorconcentrationbutalsoontheperiodoftimethatcellsgrowincontactwiththe
inhibitor(Alonso‐Simónetal.,2004).
Thereareonly tworeportedstudies inwhichcell cultures fromplantswith type II
cell walls (characteristic of graminaceous plants, together with the other commelinoid
monocots)havebeenhabituatedtoaCBI.Thesewerebarleycellsuspensions(Shedletzkyet
al.,1992)andmaizecalluscultures(Mélidaetal.,2009;2010a;2010b),habituatedtoDCBin
both cases. Themodifications found in these type II cell walls differed considerably from
those described for type I cell walls, since the drastic reduction in cellulose content was
compensatedforanincreaseinthecontentofheteroxylans,whichhadlowerextractability
and higher Mr and Mw. Furthermore, in the case of maize cell cultures obtained in our
laboratory, DCB habituation has implied an enrichment of hydroxycinnamates and
dehydroferulatesesterifiedonarabinoxylans.Thecontentofotherpolymers,suchasmixed
glucan,xyloglucan,mannan,pectinsandproteins,wasunchangedorreduced(Mélidaetal.,
2009).AsthesecharacteristicsdifferedfromthosedescribedforDCB‐habituatedbarleycell
cultures,anddidnotlooklikeanyotherstructurepreviouslydescribedfortypeIIcellwalls,
we proposed that our DCB‐habituated maize cells had a unique structure, which was of
particular interest in order to gain a deeper understanding of the compositional and
structuralplasticityofthetypeIIcellwall(Mélidaetal.,2009).
ChangesassociatedwiththehabituationofcellculturestoCBIshavecommonlybeen
analysed using high concentrations of herbicide and long‐termhabituation periods. These
kinds of study take advantage of the fact that the cell culture characteristics have been
“fixed”.However,informationabouttheinitialchangesthattakeplaceduringtheprocessof
habituation is lost, although cells at these stages probably present a huge variability in
relationtotheircellwallcomposition,propertiesandabilitytohabituate.Apreviouswork
conducted in bean calluses in order tomonitor cellwall changes during DCB‐habituation,
ChapterI
51
showed that when calluses were cultured in 0.5 µM DCB for less than 7 subcultures no
appreciable changes in cellwall FTIR spectrawere detected, but after 13 subcultures the
spectraof the cellwallsunderwent several changes, including anattenuationof thepeaks
related tocellulose (Alonso‐Simónetal.,2004).So, this studyestablished thata)an initial
periodwouldbenecessaryuntil cellwallmodifications arise andb) that a set of cellwall
modifications,notonlyareductionincellulosecontent,wouldbeimplied.
Accordingly to these results, theaimof this studywas tomonitorandcharacterise
theearlychangesthatoccurduringtheDCB‐habituationofcellswithtypeIIwalls,usingasa
modelmaizecell suspensions.Firstofall,wemonitored thechangesproduced throughout
theDCB‐habituationprocess,inordertoselectacelllinethatshowedaninitialcontrastable
modificationinsomecellwallfeatures.Next,wecomparedthecellgrowthpatternandcell
wall composition of Sh1.5 and Snh cells, paying special attention to the variation in
compositionofpolysaccharides,usingasetoftechniquessuchascellwallfractionation,GC,
IDAs,enzymaticdigestionsandheatmaps.
II.MATERIALSANDMETHODS
II.a.Cellcultures
Maize cell suspensioncultures (Zeamays L.,BlackMexican sweet corn,donatedby
Dr.S.C.Fry,InstituteofMolecularPlantSciences,UniversityofEdinburgh,UK)fromcalluses
obtained from immature embryo explants were grown in Murashige and Skoog media
(MurashigeandSkoog,1962)supplementedwith9μM2,4‐Dand20gL‐1 sucrose,at25°C
underlightandrotaryshaken,androutinelysubculturedevery15days.
ChapterI
52
II.b.HabituationtoDCB
Maize cell suspensions were cultured in DCB (supplied by Fluka) concentrations
rangingfrom0.3to1.5μM.DCBwasdissolvedinDMSO,whichdidnotaffectcellgrowthat
thisrangeofconcentrations.
Initially, Snh maize cells were cultured in media containing three DCB
concentrations:0.3μM(lowerthantheI50),0.5μM(theI50value)and1μM(higherthanthe
I50value).Aftersevensubcultures,someofthecellshabituatedtogrowthin1μMDCBwere
transferredintomediacontaining1.5μMDCB.
II.c.Growthmeasurements
GrowthcurvesofSnhandSh1.5celllineswereobtainedbymeasuringtheincreasein
DWatdifferentculturetimes.Dtwasdefinedasthetimerequiredforacelltodivideoracell
populationtodoubleinsizeinthelogarithmicphaseofgrowth,andwasestimatedbyusing
theequation lnWt= lnWo+0.693t/dt.Thus,WtorWo logarithmicplottingversusculture
timeisastraightlinewheretheordinateattheorigincorrespondstotheWtorWonatural
logarithm.Relativegrowthratewasdeterminedbytheslopeofthestraightline(0.693t/dt).
Maximum growth rate was the maximum difference in DW per time unit between two
consecutivemeasurementsingrowthkinetics.
Cellclustersizewasdeterminedaftervacuumfiltrationofcellsuspensionsthatwere
sequentially passed throughdifferentpore‐sizemeshes, and the relative abundance of the
clusterswasexpressedasthepercentageofDWretainedineachfilter.
II.d.ZmCESAgeneexpressionanalysis:isolationoftotalRNA,RT–PCRandPCR
TotalRNAwasextractedwithTrizolReagent(Invitrogen),and2μgoftotalRNAwas
reverse‐transcribed using the Superscript III first strand synthesis system for RT‐PCR
(Invitrogen).First‐strandcDNAwasgeneratedusinganoligo(dT)20primer,and1μlofthe
first‐strandcDNAwasusedasatemplateinsubsequentPCRreactions. ‘No‐RT’PCRassays
ChapterI
53
were performed to confirm the absence of genomic DNA contamination. For each assay,
several numbers of cycles were tested to ensure that amplification was within the
exponentialrange.
The gene‐specific primers used for the analysis ofZmCESA1 (AF200525),ZmCESA2
(AF200526), ZmCESA3 (AF200527), ZmCESA5 (AF200529), ZmCESA6 (AF200530), and
ZmCESA7(AF200531)geneswerethosepreviouslydescribedbyHollandetal.(2000).Dueto
thehigh‐sequencesimilaritybetweenZmCESA1andZmCESA2,thesameprimerwasusedfor
the analysis of both genes (ZmCESA1/2). The primers used for the analysis of ZmCESA4
(AF200528)andZmCESA8(AF200532)werethosedescribedinMélidaetal.(2010a).
II.e.Preparationandfractionationofcellwalls
Cells collected from suspension cultures in the stationary phase of growth were
frozen and homogenisedwith liquid nitrogen using a pestle andmortar, and treatedwith
70%ethanolfor5daysatroomtemperature.Thesuspensionobtainedwascentrifugedand
thepelletwaswashedwith70%ethanol(x6)andacetone(x6),andsubsequentlyairdriedto
obtaintheAIR.TheAIRwastreatedwith90%DMSOfor8hatroomtemperature(x3)and
thenwashedwith0.01MphosphatebufferpH7.0(x2).ThewashedAIRwasthenincubated
with 2.5 Uml‐1 α‐amylase from porcine pancreas (Sigma type VI‐A) in 0.01M phosphate
bufferpH7.0for24hat37°C(x3).Thesuspensionwasfilteredthroughaglassfiberfilter,
andtheresiduewashedwith70%ethanol(x6)andacetone(x6),airdriedandthentreated
withphenol/aceticacid/water(2/1/1byvol.)for8hatroomtemperature(x2).Thisresidue
wasfinallywashedwith70%ethanol(x6)andacetone(x6)andthenairdriedtoobtainthe
cellwalls.
Inordertocarryoutacellwallfractionation,driedcellwallswereextractedatroom
temperature with 50 mM CDTA (10 mg ml‐1) and then washed with distilled water. The
resultingpelletwastreatedwith0.1MKOH(10mgml‐1)for2h(x2)andwashedagainwith
distilledwater.Theremainingresiduewasthentreatedwith4MKOH(10mgml‐1)for4h
ChapterI
54
(x2)andsubsequentlywashedwithdistilledwater.Then6MKOH(10mgml‐1)wasfinally
addedtothepelletandtreatedat37°Cfor24h.KOHextractswereneutralisedwithacetic
acidtopH5.
Extracts of each treatment were dialysed (48 h) and lyophilised, and referred to
CDTA,0.1MKOH,4MKOHand6MKOHfractions,respectively.
II.f.Cellwallanalyses
II.f.1.Cellulosecontentdetermination
Cellulose content was quantified in crude cell walls by the Updegraff method
(Updegraff, 1969) using hydrolytic conditions described by Saeman et al. (1963) and
quantifyingtheGlcreleasedbytheanthronemethod(Dische,1962).
II.f.2.FTIRspectroscopy
Tablets for FTIR spectroscopywere prepared in a Graseby‐Specac press using cell
wallsamples(2mg)mixedwithKBr(1:100,w/w).SpectrawereobtainedonaPerkin‐Elmer
instrument at a resolution of 1 cm‐1. A window between 800 and 1800 cm‐1, containing
informationaboutcharacteristicpolysaccharides,wasselectedinordertomonitorcellwall
structure modifications. All the spectra were normalized and baseline‐corrected with
Spectrum v 5.3.1 software, by Perkin‐Elmer. Data were then exported to Microsoft Excel
2003 and all spectra were area‐normalized. Difference spectra were obtained by digital
subtractionofSnhandShcellwallFTIRspectra.
II.f.3.Sugaranalyses
Total sugar content of each fraction was determined by the phenol‐sulfuric acid
method(Duboisetal.,1956)andwasexpressedas theGlcequivalent.Uronicacidcontent
was determined by the m‐hydroxydiphenyl method (Blumenkrantz and Asboe‐Hansen,
1973)usingGalAasstandard.Neutralsugarcontentanalysiswasperformedasdescribedby
ChapterI
55
Albersheimetal.(1967).Briefly,alyophilizedsampleofeachfractionwashydrolyzedwith2
M TFA at 121°C for 1 h and the resulting sugarswere derivatized to alditol acetates and
analyzedbyGCusingaSupelcoSP‐2330column.
II.f.4.Analysisofpolysaccharidesbyenzymaticepitopedeletion
Anextensiveanalysisofpolysaccharidedistributionandcompositionofthecellwall
fractionswascarriedoutby IDAs.Briefly,aliquotsof1μl fromeach fractionwerespotted
ontonitrocellulosemembrane (Schleicher& Schuell,Dassel,Germany) as a replicated1/5
dilutionseriesoftheprecedentone,being5differentdilutionsforeachfractioninall.Then,
these membranes were incubated with either an endo‐polygalacturonanase (M2) from
Aspergillus aculeatus (E‐PGALUSP) (EC 3.2.1.15), a recombinant ‐xylanase from
Neocallimastixpatriciarum(E‐XYLNP)(EC3.2.1.8)andanendo‐arabinanasefromAspergillus
niger (E‐EARAB) (EC 3.2.1.99) (all of them purchased fromMegazyme) at 37°C for 24 h,
following the experimental approach described by Øbro et al. (2007) with minor
modifications.Theenzymesweredissolvedto1U/mlin350mMacetatebufferadjustedto
pH4.7bytheadditionofpyridine.Afterenzymaticdigestion,nitrocellulosemembraneswere
blockedwithPBS (0.14MNaCl, 2.7mMKCl, 7.8mMNa2HPO4·12H2O,1.5mMKH2PO4,pH
7.2) containing 4% fat‐freemilk powder (MPBS) prior to incubation in primary antibody
(hybridoma supernatants diluted 1/10 in MPBS) for 1.5 h at room temperature. After
washing extensively under running tap water and PBS, membranes were incubated in
secondary antibody (antirat horseradish peroxidase conjugate, Sigma) diluted 1/1000 in
MPBSfor1.5hatroomtemperature.Membraneswerewashedasdescribedabovepriorto
colourdevelopmentinsubstratesolution[25mlde‐ionizedwater,5mlmethanolcontaining
10mgml‐14‐chloro‐1‐naphtol,30ml6%(v/v)H2O2].Colourdevelopmentwasstoppedby
washing themembranes. Samplesof commercial pectin (P41,Danisco), xyloglucan (kindly
donatedbyDr.THayashi)andanarabinoxylan‐enrichedfraction(AX;obtainedfrommaize
calluseshabituatedto12μMDCB,kindlydonatedbyDr.H.Mélida)wereusedasreference
ChapterI
56
compounds. Monoclonal antibodies assayed and their corresponding epitope recognized
were: JIM5 (J5; homogalacturonanwith low degree ofmethylesterification; Clausen et al.,
2003), JIM7 (J7; homogalacturonan with high degree of methylesterification; Knox et al.,
1990),LM5(L5;β‐1,4‐galactansidechainofrhamnogalacturonantypeI;Jonesetal.,1997),
LM6 (L6; α‐1,5‐arabinan side chain of rhamnogalacturonan type I and arabinogalactan
proteins; Willats et al., 1998), LM10 (L10; unsubstituted and relative low substituted
β‐1,4‐D‐xylan).NocrossactivitywithLM11(L11;unsubstitutedandrelativelowsubstituted
β‐1,4‐D‐xylan;McCartneyetal.,2005)andLM15(L15;non‐fucosylatedxyloglucan;Marcus
etal.,2008)wereobserved.
II.f.5.IDAsrelatedheatmaps
Immunolabelingsemi‐quantificationforeachfractionwasperformedbyscoringthe
numberofdilutions labeled.Thus,avalueranging from1to5wasassigneddependingon
thenumberofcoloredspotswhichappearedafterincubationwiththesecondaryantibody.
Thesevalueswerethenenteredintothestatisticalsoftware(seeStatisticalanalysessection
fordetails)inordertoobtainheatmaps.
II.g.Statisticalanalyses
PCAwasperformedusingamaximumoffivePCs.Alltheseanalyseswerecarriedout
usingtheStatistica6.0softwarepackage.
Significant differences in the cellulose content were tested applying a one‐factor
ANOVA (performing Tukey´s test as post‐hoc analysis), using the PASW Statistics 18
softwarepackage.
HeatmapswereperformedbyusingRKWardstatisticalsoftware.
ChapterI
57
III.RESULTS
III.a.MonitoringofearlycellwallmodificationsduringDCB‐habituation
III.a.1.FTIRspectroscopyandmultivariateanalysis
FTIRspectrafromcellwallscorrespondingtoSnhandhabituated(Sh)to0.3(Sh0.3),
0.5 (Sh0.5), 1 (Sh1) and 1.5 (Sh1.5) μM DCB maize cell suspensions which had been for
different number of culture cycles growing in the presence of DCB were obtained and
analyzed(Figure1).Themaindifferencesamongcelllinesweredetectedinthefingerprint
area(900‐1200cm‐1).Inthisareaofthespectra,manypolysaccharidesabsorbIRradiation,
includingcellulose.
Figure1.FourierTransformInfrared(FTIR)spectraobtainedfromcellwallsfromnon‐habituated(Snh)and
differentdichlobenil‐(DCB)habituated(Sh)maizecellsuspensions(Shx(n);xindicatesDCBconcentration(μM)and(n)numberofsubculturesinthatDCBconcentration)
Snh
80010001200140016001800
Sh0.3(4)
Sh0.3(6)
Sh0.3(8)
Sh0.3(10)
Sh0.5(2)
Sh0.5(4)
Sh0.5(6)
Sh0.5(8)
Sh0.5(10)
Sh1(1)
Sh1(4)
Sh1(6)
Sh1(8)
Sh1(10)
Sh1.5(3)
Wavenumber(cm‐1)
80010001200140016001800
ChapterI
58
In order to better appreciate the variations between Sh and Snh cell wall spectra,
difference spectra for each cell line were obtained (Figure 2). These spectra showed
noticeable variation amongSnh andmost of the Sh cell lines subjected to short‐termDCB
habituation[i.e.seeSnh‐Sh0.3(2);Snh‐Sh0.5(2)andSnh‐Sh1(1)].SpectraobtainedfromSh
cellsshoweddecreasedpeaksinwavenumbersassociatedwithcellulose(1039,1056,1060,
1105 and 1160 cm‐1) (Alonso‐Simón et al., 2004; 2011). However, these variations were
graduallyattenuatedinthedifferencespectracorrespondingtocellwallsofSh0.3andSh0.5
celllinesasthenumberofculturecyclesinpresenceofDCBwasincreased[seeSnh‐Sh0.3(4‐
10);Snh‐Sh0.5(4‐10)]. Incontrast, thedifferencesobserved in theSh1cell lineweremore
markedandtheydidnotrevertasthenumberofsubcultureswasincreased[seeSnh‐Sh1(4‐
10)].Inadditionotherwavenumbersnotassociatedtocellulose,suchas950cm‐1‐ascribed
topectins‐or1520cm‐1‐attributedtophenolicrings‐(Alonso‐Simónetal.,2011),andothers
unattributedsuchas1190and1480cm‐1werealsoaffected, indicatingthatothercellwall
componentshavebeenmodified.
PCA of FTIR spectra showed two groups (Figure 3) and the spectra were mainly
discriminatedbyPC1(approx.71%oftotalvarianceexplained).Spectraobtainedfromcell
walls corresponding to Snh and Sh suspensions habituated to lower DCB concentrations
(Sh0.3andSh0.5)whichhadbeengrowinginpresenceoftheinhibitorforagreaternumber
ofculturecycleswerelocatedonthepositivesideofPC1.Incontrast,spectracorresponding
tocellshabituatedtohigherDCBconcentrations(Sh1andSh1.5)andthosecorrespondingto
cellswhich had been growing for a lesser number of culture cycles in contactwith lower
concentrationsoftheinhibitorweregroupedonthenegativesideofPC1.
ChapterI
59
Snh‐Sh0.5(4)
Snh‐Sh0.5(6)
Snh‐Sh0.5(8)
Snh‐Sh0.5(10)
Wavenumber(cm‐1)
9001000
11001200
13001400
15001600
1700
Snh‐Sh0.3(4)
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
Snh‐Sh0.3(6)
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
Snh‐Sh0.3(8)
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
Snh‐Sh0.3(10)
800900
10001100
12001300
14001500
16001700
1800-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
Snh‐Sh1(6)
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
Snh‐Sh1(8)
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
Snh‐Sh1.5(3)
800900
10001100
12001300
14001500
16001700
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
Snh‐Sh1(10)
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
Snh‐Sh1(4)
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
Wavenumber(cm‐1)
Snh‐Sh0.5(2)800
9001000
11001200
13001400
15001600
1700
Snh‐Sh0.3(2)800
9001000
11001200
13001400
15001600
17001800
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4Snh‐Sh1(1)
800900
10001100
12001300
14001500
16001700
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
Figure2.DifferencespectraobtainedfromthedigitalsubtractionoftheFTIRspectraofcellwallsfromSnhanddifferentDCB‐habituatedShmaizecellsuspensions(legendasFigure1)
ChapterI
60
Figure3.Principalcomponentsanalysis(PCA)ofmaizecellsuspensionsFTIRspectra.AplotofthefirstandsecondPrincipalComponents(PCs)isrepresentedbasedontheFTIRspectraofcellwallsfromSnhandShmaize
cellsuspensionsalongseveralculturecycles(legendasFigure1)
III.a.2.Cellulosecontent
Cellulose contentwas influencedbyDCB concentration aswell as by the time that
maizecellsweregrowinginitspresence(Figure4).InSnhcellwalls,celluloserepresented
roughly 27% of the cellwallweight. However, a reduction in the cellulose level occurred
when maize cells were exposed to DCB for one or two culture cycles. As the number of
subcultures in presence of DCBwas increased, the cellulose content of cellwalls from Sh
suspensionshabituatedtolowerDCBconcentrations(Sh0.3andSh0.5)graduallyrevertedto
thatof the control level. In fact, after growing in contactwith the inhibitor for ten culture
cycles, their cellulosecontent reachedvalues thatdidnotdiffer significantly fromthoseof
theSnh.Incontrast,thiscompletereversiondidnotoccurintheSh1cellline,atleastnotin
thesamenumberofculturecyclesasSh0.3andSh0.5celllinesdid.
Sh1(1)
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61
Figure4.CellulosecontentofcellwallsfromSnhandShmaizecellsuspensions(legendasFigure1).Theasteriskreflectssignificantdifferencesamongnon‐habituatedandDCB‐habituatedcelllinesaccordingtoTukeytest(p<0.05).Valuesrepresentedaremean±standarderror(SE)ofninemeasurements(n=9).Referencesinbold
refertothecelllineswhichwereselectedforfurtherZmCESAgenesexpressionanalysis(Figure5)
III.a.3.ZmCESAgenesexpression
ZmCESA8 and ZmCESA7 gene expression was induced in those cell cultures which
were grown for a high number of culture cycles in the presence of DCB ‐Sh0.3(10) and
Sh0.5(10)‐respectively(Figure5).Aninterestingtrendwasobservedincellshabituatedto1
µMDCB: an induction of ZmCESA7 expressionwas detected after 8 and 10 culture cycles
‐Sh1(8) and Sh1(10)‐, and in the former line, ZmCESA8 expression was also enhanced.
However,noneofthesegeneswasinducedinDCBhabituatedcellsintheirfirstculturecycle
‐Sh1(1)‐.Lastly,theexpressionofZmCESA7wasalsoinducedinSh1.5(3).Noexpressionof
ZmCESA3 was observed in any of the cell lines analyzed, and no differences in mRNA
accumulationinZmCESA1/2,ZmCESA4,ZmCESA5andZmCESA6withrespecttotheSnhwere
detected.
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62
Figure5.RelativeZmCESAgeneexpressionanalyzedbyRT–PCRofdifferentmaizecellsuspensions(legendasFigure1).;moremRNAaccumulationthancontrol(Snh);ZmCESA3wasnotincludedbecauseno
expressionwasdetected.ZmUBI:Ubiquitinegeneexpression.Ubiquitinewasusedasthehousekeepinggeneduetoitsconstitutiveexpression
TheSh1.5celllinewasselectedforfurtheranalysissinceitwasrepresentativeofan
early DCB‐habituation based on the following criteria: a) a 33% reduction of cellulose
content, later demonstrated to be irreversible (data not shown) was observed, b) FTIR
spectrumindicatedthatcellulose,aswellasotherpolysaccharides,wereaffected,andc)PCA
resultspointedtodifferenceswithSnhcellsbutalsowiththeremainingShcells,indicating
featuresofanincipientDCBhabituation.
III.b.Culturecharacteristics
Growth of Snh and Sh1.5maize cell suspensions throughout the culture cyclewas
analyzed (Figure 6A). Sh1.5 cells showed a longer lag phase and a less pronounced
logarithmicphaseofgrowthwhencomparedwiththeSnhcells,whichdeterminedlongerdt
andlowerrelativeandmaximumgrowthrates(Figure6B).
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63
Snhcellsshowedhomogeneouscluster‐sizesince88%oftheirDWwasfoundtohave
diametersrangingbetween710and400m(Figure7).Incontrast,alargeraveragesizeand
greatervarietyofsizeswasobservedintheSh1.5cells,and73%oftheirDWwasretainedin
filterswithporesizesrangingfrom3000to710m.
Figure6.A:Growthofmaizecellsuspensionsduringtheculturecycle.Opencircle;Snh.Filledcircle;Sh1.5.Valuesrepresentedaremean±SEofninemeasurements(n=9);B:Growthparametersobtainedfromthegrowthcurves
ofSnhandSh1.5maizecellsuspensions.Dt:doublingtime
Time(days)
0 2 4 6 8 10 12 14
DW(g/l)
0
2
4
6
8
A
B
ChapterI
64
Figure7.Sizeofcellclustersofmaizecellsuspensionsatthestationaryphase.Whitebars;Snh.Blackbars;Sh1.5.
Valuesrepresentedaremean±SEofthreemeasurements(n=3)
III.c.Cellwallfeatures
III.c.1.Cellwallfractionationandsugarcomposition
The results showed thatmostpolysaccharideswereextractedbyalkali treatments,
especiallyin4MKOHand6MKOHfractions.Sh1.5cellwallswereenrichedintotalsugars,
mainlyextractedwith4MKOHfollowedby0.1MKOH(Figure8A).
GC and uronic acid analysis of cell wall fractions (Figure 8B) revealed that alkali
fractions were composed mainly of Ara and Xyl, followed by Glc, uronic acids and Gal,
whereas the CDTA fraction was enriched in uronic acids and Ara. Ara and Xyl were
responsible for the increase in neutral sugars detected in the 0.1 M KOH and 4 M KOH
fractionsinSh1.5cellwalls.AdecreaseinGlc,especiallyinfractionsextractedwith4MKOH
and6MKOH,wasobserved in Sh1.5 cells. Finally, Sh1.5 cells showeda slight increase in
uronicacids,mainlythoseextractedinalkalifractions.
Filterporesize(m)3000 2000 1000 710 400 120 22 0
DW(%)
0
20
40
60
80
100
ChapterI
65
Figure8.A:Totalsugars,neutralsugarsanduronicacidscontentsoffractionsfromcellwallsofwhitebars;Snhandblackbars;Sh1.5maizecellsuspensions.Valuesrepresentedaremean±SEofthreemeasurements(n=3);B:Monosaccharidecompositionoffractionsfromcellwallsofwhitebars;Snhandblackbars;Sh1.5maizecellsuspensions.Rha:rhamnose,Fuc:fucose,Ara:arabinose,Xyl:xylose,Man:mannose,Gal:galactose,Glc:
glucose,UA:uronicacids
III.c.2.Polysaccharideepitopescreeningafterenzymaticdigestion
Inordertodetectcellwallchangesinthedistributionofepitopesinshort‐termDCB‐
habituatedcells,aliquotsofcellwallfractionsofSnhandSh1.5cellsuspensionswereloaded
ontonitrocellulosemembranesas1/5dilutions,subjectedtoenzymaticdigestionsbyusing
4MKOH
g/mgcellwall
25
50
75
100
125
150
175
200
CDTA10
0.1MKOH
20
40
6MKOH
Rha Fuc Ara Xyl Man Gal Glc UA0
20
40
60
80
100
120
140
TotalSugars
0
50
100
150
200
250
300
350
400
NeutralSugars
g/mgcellwall
0
50
100
150
200
250
300
350
UronicAcids
CDTA
0.1MKOH
4MKOH
6MKOH
0
50
A B
ChapterI
66
an endo‐polygalacturonanase, a β‐xylanase and an endo‐arabinanase and subsequently
probedwithmonoclonalantibodiesforpecticandhemicellulosicpolysaccharides.
Heatmapsweregeneratedwiththeresultingpoolofdata(Figures9Band10).Asthe
totalnumberofdilutionsassayedwas5,avaluerangingfrom0to5wasassigned.Thisvalue
dependedonthesumofcoloredspots(correspondingtolabeledepitopesineachdilution)
foundintheheatmap(seemethods).Inordertoillustratehowheatmapwereperformed,a
representativeIDAsforLM10isshowninFigure9A.Likewise,whenLM10wasassayed,the
scoringvaluesassignedwere0foralltheSnhcellwallfractions,and0,1,1and2forSh1.5
CDTA,0.1MKOH,4MKOHand6MKOH fractions, respectively. In addition, it shouldbe
takenintoconsiderationthatinthecaseofSnhheatmapthemaximumvalueassignedwas4
duetothefactthatinanycase5colouredspotswerefound.
Twodifferent typesofgroupingsappeared in theheatmap:adendrogramwithcell
wall fractions and a dendrogram with the different antibodies probed. These groupings
dependedon thedistributionpatternof labeledepitopes:proximity ingrouping isdirectly
relatedwithsimilarityofepitopesdistributionacrossthecellwallfractions.
Infigure9Bitisshownaheatmapperformedonlywiththeresultsobtainedfromthe
non‐enzymatic digested nitrocellulosemembranes (controls). Closest cellwall fractions in
theSnhandSh1.5celllines,andconsequentlythemostsimilarintermsofthedistributionof
labeledepitopes,were6MKOHand4MKOH,followedby0.1MKOHandCDTA.Epitopes
forβ‐1,4‐xylans,recognizedbyLM10,wereexclusively foundinall thealkali‐extractedcell
wallfractionsfromSh1.5.ThepatternofLM11labelingwhichdetectedspecificallyepitopes
forxylansandarabinoxylanswassimilarinbothcelllines,withtheexceptionthattheSh1.5
6MKOHcellwallfractionshowedanincreasedintensityoflabelling.
Respecttopecticpolysaccharides,cellwallfractionsfrombothcelllineswereprobed
withantibodiesrecognizinghomogalacturonanandrhamnogalacturonanI.Resultsobtained
for JIM5 and JIM7, antibodies for homogalacturonan with low and high degree of
methylesterificationrespectively,showedthatnodifferencesinthepatternoflabelingwere
ChapterI
67
foundforJIM7amongcelllinefractions,whereasagreaterintensityoflabelingforJIM5was
foundinCDTAand0.1MKOHSh1.5celllinefractionswhencomparedtoSnh.
Detectionofepitopes forβ‐1,4‐galactansidechainsofrhamnogalacturonanI(LM5)
andnon‐fucosylatedxyloglucan(LM15)revealedagreaterdegreeoflabelinginallthealkali‐
extractedfractionsfromSh1.5cells,especiallyinthe4MKOHfraction.Moreover,thesetwo
epitopeswerealwaysrelatedtoSh1.5cells.Incontrast,inSnhalkali‐extractedfractions,an
associationwasobservedbetweenthedistributionofLM5andLM6epitopes.Nodifferences
betweencelllineswerefoundinthesefractionswhenLM6wasprobed.Nevertheless,α‐1,5‐
arabinan side chains of rhamnogalacturonan I epitopes were only detected in the CDTA
fractionofSnhcells.
In order to better appreciate the differences induced after enzymatic digestions,
individualheatmapsweregeneratedforSnhandSh1.5cells(Figure10).Intheheatmapfor
theSnhcell line(Figure10A),theclosestcellwallfractionswere0.1MKOHand4MKOH,
followedby6MKOHand finallyCDTA,whereas in theSh1.5heatmap(Figure10B),0.1M
KOHwasfirstlygroupedwith6MKOH,followedby4MKOHandlastlywithCDTA.Ascould
bededucedfromthegreaterintensityofcolour,itwasinthe6MKOHand4MKOHcellwall
fractionsthatthegreatestamountoflabelledepitopeswasdetectedinbothcelllines.
The dendrogram illustrating the enzymatic treatments, controls and antibodies
probedshowstwoprincipalbranchesforbothcelllines:1and2.Eachofthesesubsequently
dividedintoanothertwosub‐branches(Figure10).However,groupingsdiffereddepending
onthecellline,indicatingvariationsbetweencelllinesinepitopedistributioninthedifferent
polysaccharides. InSnh cells, thedistributionof epitopes for JIM7,LM15,LM10andLM11
was not substantially affected by any enzymatic treatment; however, endo‐
polygalacturonanase treatment produced modifications in JIM5, LM5 and LM6 epitope
distribution.EpitopesforJIM5disappearedfromtheCDTAandinthe0.1Mand4Malkali‐
extractedfractions.DisappearanceofepitopesforLM6wasdetectedintheCDTAand0.1M
alkali‐extracted fractions,whereas those for LM5 appeared in the 6MKOH fractionwhen
ChapterI
68
subjected to endo‐polygalacturonanase enzymatic digestion. Endo‐arabinanase treatment
also inducedchanges inLM5andLM6epitopes.Similarly to theendo‐polygalacturonanase
treatment,theintensityofLM5epitopelabellingwasgreater inthe6MKOHfractionafter
endo‐arabinanasetreatment;howeverintheLM6epitopesthisintensitywasreducedinall
thecellwallfractions.
The influence of enzymatic digestions on the pattern of labelling of epitopes
extracted in the Sh1.5 cell wall fractions showed differences when compared with Snh
(Figure10B).Agreaterproportionofepitopeswereaffected in theSh1.5 fractions than in
Snhcellsafterenzymaticdigestions.Aswasexpected,endo‐polygalacturonanasetreatment
led to thedisappearanceofepitopes for JIM5(in the0.1MKOHfraction)and JIM7(in the
CDTA fraction). In Sh1.5 cell wall fractions, both endo‐polygalacturonanase and endo‐
arabinanase treatments affected epitope distribution for xylans (LM10) as well as those
specific for rhamnogalacturonan I side chains (LM5 and LM6). Finally, LM10 and LM11
epitopes disappeared from all cell wall fractions except the CDTA fraction after xylanase
digestion.
Figure9.A:Representativeimmunodotassay(IDA).CellwallfractionsobtainedfromSnhandSh1.5wereprobedwithLM10,monoclonalantibodyspecificforxylans.AnylabellingwasobservedforSnhfractions,whereas0,1,1and2coloredspotsweredetectedrespectivelyinCDTA,0.1MKOH,4MKOHand6MKOHfractionsfromSh1.5cells.AX:arabinoxylan‐enrichedfractionusedasstandard.B:HeatmapofdataobtainedfromIDAsofSnhandSh
cellwallfractions(seenextpage).Avaluerangingfrom0(nocoloredspotdetected)to5(5coloredspotsdetected)wasassignedtoeachantibody(J5:JIM5,J7:JIM7,L5:LM5,L6;LM5,L10:LM10,L11:LM11,L15:LM15)andineachcellwallfraction(CDTA,0.1MKOH,4MKOH,6MKOH),dependingontheamountofcoloredspots
(correspondingtodilutions)thatwereshownafterrevealing
A
ChapterI
69
B
ChapterI
70
Figure10.HeatmapsofdataobtainedfromIDAsofA:SnhandB:Sh1.5(seenextpage)maizecellsuspensionsobtainedaftertheenzymaticdigestionofthecellwallfractions.Avaluerangingfrom0(nocoloredspotdetected)to5(5coloredspotsdetected)wasassignedtoeachenzymatictreatment(EPG:endo‐polygalacturonanase,Xyl:β‐xylanase,Ara:endo‐arabinanaseand–E:control,wherenoenzymewasadded),antibody(J5:JIM5,J7:JIM7,L5:LM5,L6;LM5,L10:LM10,L11:LM11,L15:LM15)ineachcellwallfraction(CDTA,0.1MKOH,4MKOH,6MKOH),
dependingontheamountofcoloredspots(correspondingtodilutions)thatwereshownafterrevealing
A
ChapterI
71
B
ChapterI
72
IV.DISCUSSION
Oneof themostconspicuouschangesdetectedbyFTIRmonitoringof thecellwalls
from the cell lines studied was a reduction in the cellulose content. In fact, the main
differencesamongspectraofSnhandDCB‐habituated(Sh)maizecellwallsappearedinthe
fingerprint region (900‐1200 cm‐1), in which a wide range of polysaccharides absorb IR
radiation, including cellulose. These differences became more marked as the DCB
concentrationintheculturemediumwasincreased.However,differencesamongthespectra
ofSnhandShexposedtolowerDCBconcentrations(Sh0.3andSh0.5)wereattenuatedwhen
the number of culture cycles that cells were grown in contact with the inhibitor was
increased.Thesedatawereconfirmedbytheresultsobtainedfromtheanalysisofcellulose
content.A23%and27%reductioninthecellulosecontentwasobservedwhenmaizecells
were exposed to 0.3 and 0.5 µMDCB, respectively, but after 10 culture cycles growing in
presence of the inhibitor, these levels reverted to values close to the Snh value.At higher
DCB concentrations (Sh1 and Sh1.5), differences in the fingerprint region among spectra
weregreaterwithrespecttotheSnhspectraandeventualrecoveryofthecellulosecontent
was not observed. The gradual reversion in cellulose content produced when cells were
growing in lowDCB concentrations and the number of culture cycles in its presencewas
increased, had previously been described in bean cell suspensions (García‐Angulo et al.,
2006)andcallus‐culturedcells (Alonso‐Simónetal.,2004),but interestingly, in thesecells
withtypeIwalls,aninitialperiodofaboutsevensubcultureswasrequiredpreviouslytothe
detectionofanychangesintheircellwallsFTIRprofiles,whereasinourmaizecells,which
havetypeIIcellwalls,thislagperiodhasnotbeenobserved.
Onepossibleexplanationforthisobservedtransientreversionincellulosecontentat
the initial stepsof theDCB‐habituationprocess couldbe that increasing concentrationsof
herbicidemaypromotesomechangeintheexpressionlevelofZmCESAgenes.Infact,ithas
been found that ZmCESA gene expression was mis‐regulated in maize cultured cells
habituated to high (12 µM) DCB concentrations (Mélida et al., 2010a). An induction of
ChapterI
73
ZmCESA7andZmCESA8,thoughttobeinvolvedinproducingtheprimarycellwallbeforethe
onsetofsecondarywallformation(Appenzelleretal.,2004;Boschetal.,2011),wasdetected
insomeofourshort‐termhabituatedcelllines.Itseemsthatmaizecellsstarttooverexpress
theseisoformswhenthenumberofculturecyclesgrowinginpresenceofDCBisincreasedor
whentheconcentrationoftheinhibitorexceedsathreshold.Similarly,maizecellssubjected
tolong‐termhabituationtohighDCBconcentrationsalsoshowedaninductionofZmCESA7
andZmCESA8genes(Mélidaetal.,2010a).Thiscouldbeindicativeofanimportantrolefor
these two CESA isoforms in habituation to all DCB levels. It could be hypothesized that,
althoughlessefficientincellulosesynthesis,CESA7andCESA8maybemoreresistanttothe
effectsofDCB.
Inadditiontocellulose,FTIRmonitoringrevealedthatotherpolysaccharidesseemto
be also affectedbyDCB‐habituation. So, in order to unmask other actors playing a role in
earlyDCB‐habituationevents,Sh1.5celllinewasselectedforfurtherstudies.
In the Sh1.5 cell line the observed 33% reduction in cellulose content was
compensatedbyanetincreasein0.1MKOHand4MKOHextractedarabinoxylans.IDAsalso
showed in this cell line an increase in the proportion of epitopes for arabinoxylan/xylan
(LM10andLM11).Interestingly,noLM10labelingwasobservedneitherinSnhcellwallsin
our work, nor in maize calluses habituated to high DCB concentrations ‐4, 6 or 12 µM‐
(Mélidaetal.,2009).Thedivergencebetweenbothstudiescouldberelatedtothedifference
of plant material (calluses or suspension cell cultures), but would be also linked to a
transient modification in chemical composition in some subsets of arabinoxylans
populationsatthefirststepsofthehabituation.
Other hemicelluloses, such as xyloglucan, seems also to be involved in the early
eventsofDCB‐habituation,sinceanincreaseinthedetectionofLM15epitopeswasobserved
in the Sh1.5 4 M KOH fraction, pointing to more easily‐extractable xyloglucanmolecules.
Reinforcing this finding, a lower presence of LM15 epitopes was found in the 6 M KOH
fractionfromSh1.5cells,treatmentwhichwouldextractstrongly‐linkedmolecules.
ChapterI
74
Furthermore, sugar composition analysis points to an increase of uronic acid‐rich
polysaccharides, especially in alkali‐extracted fractions, in the early steps of habituation.
Likewise, insuchalkali‐extracted fractions, IDAsrevealedanenhancementofLM5labeling
and,therefore,anincreaseingalactansidechainsofrhamnogalacturonanIinthehabituated
cell line.Also,anenrichmentofepitopes for JIM5(homogalacturonanwitha lowdegreeof
esterification)wasfoundinthemild‐alkali(0.1MKOH)extractedfraction,aswellasinthe
CDTA‐extractedfraction.
The fact that other related epitopes such as those for homogalacturonan with a
higherdegreeof esterification (JIM7)or for arabinanside chainsof rhamnogalacturonan I
(LM6) did not change between cell lines, indicates that only some specific subsets of
polysaccharides were affected during the early DCB‐habituation. Enzymatic treatments
affected differently depending on the cell line. Thus, these results may be indicative of
structuraldifferencesinpolysaccharidesamongcell lines,whichcouldpreventor facilitate
theenzymeaction.However,furtheranalysesarerequiredinordertoclarifyit.
ItisinterestingtonotehowinthetypeIIcellwallsofourcellcultures,characterised
byaratherlowamountofpecticpolysaccharides,anincreaseinsuchtypeofpolysaccharides
isenhancedinthefirststepsofDCB‐habituation.Thisfactiscomparablewiththeobserved
trend in DCB‐habituated plant cultured cells having type I walls, as bean, in which the
reduction in the cellulose content is mainly counteracted by an increase in pectic
polysaccharides(Encinaetal.,2001;García‐Anguloetal.,2006).Previousworkcarriedout
inDCBorisoxabenhabituatedculturedcellshavingtypeIcellwalls,arecharacterisedbya
substantialincreaseinJIM5‐reactivelow‐esterifiedpectins,whichwouldpointtomoreCa+2‐
cross‐linked pectins (Wells et al., 1994; Sabba et al., 1999; Manfield et al., 2004; García‐
Anguloetal.,2006).Aspecticpolysaccharidesare involved incell‐celladhesion(Willatset
al.,2001),thehigheramountofthesepolysaccharidesobservedinourhabituatedcellscould
explainthefactthattheseculturedcellsdevelopedlargercellclustersthantheSnhcultures,
resemblingtothatdescribedforDCB‐habituatedbeancellsuspensions(Encinaetal.,2001;
ChapterI
75
García‐Anguloetal.,2009).Nevertheless,andasasupplementaryexplanation,suchslower
andalteredgrowthpatternsdetectedinSh1.5cellscouldbealsorelatedwiththeircellulose‐
deficientwall,whichwould, therefore, cause an impaired cell division.On theotherhand,
andalthoughxyloglucanisknownnottobeanabundanthemicelluloseintypeIIcellwalls
(Fry,2000),ourresultsshownthatthishemicellulosemayplaysomeroleduringthisearly
stresssituation,asthedetectionofLM15epitopesincreased(atleastin4MKOHfraction)
duringthefirststepsofDCBhabituation.
InterestingdifferencesincellwallmodificationsappearwhenearlyDCB‐habituation
eventsarecomparedwiththosedescribedformaizecalluseshabituatedtointermediateand
high DCB levels (Mélida et al., 2009; 2010a; 2010b). In long‐termDCB habituated cells, a
75% of reduction in cellulose content was detected, and this value remained stable even
whenDCBconcentration in theculturemedium increased from6 to12µM. Incontrast, in
short term DCB‐habituation, the reduction in cellulose content was lower (23‐33%) and
tendedtorevertwhenDCBconcentrationintheculturemediumrangedfrom0.3and0.5µM.
Moreover, in long‐term DCB‐habituation the reduction in the amount of cellulose was
compensatedbyanincreaseinarabinoxylans,butthecontentofotherpolysaccharides,such
as pectins or xyloglucan, was not affected or even was reduced. However, as mentioned
above, in Sh1.5 cultures besides the not so drastic increase in the arabinoxylan amount,
otherpolysaccharidessuchasrhamnogalacturonanI,homogalacturonanwithalowdegree
ofesterification,andevenxyloglucanseemedtobeinvolved.Finally,althoughfurtherstudies
are required, our preliminary results suggest that antioxidant activities play an important
role during the first steps of the DCB habituation process (Largo‐Gosens, personal
communication),incontrasttothosedescribedincultureshabituatedlong‐termtohighDCB
concentrations(Mélidaetal.,2010a).
In sum, monitoring of short‐term DCB habituation of maize cell suspensions has
revealedthatthemainmodificationproducedduringthefirststepsofthisprocessconsisted
in the reduction of cellulose content, and it has been confirmed that changes in these
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76
cellulose levels were promoted by two main factors: the inhibitor concentration and the
numberofculturecyclesthatcellswereinitspresence.WallcompositionofSh1.5cellswas
modifiedasaconsequenceofhabituationtolowDCBlevels,showingareductionof33%in
thecellulosecontent.Furthermore,an inductionofZmCESA7andZmCESA8 tookplaceand
this effectwas revealed as a constant featureofDCBhabituation. Sh1.5 cells counteracted
the lackof cellulosewithan increase in arabinoxylans. In addition to arabinoxylans, other
polysaccharides, such as galactan side chains of rhamnogalacturonan I, homogalacturonan
withalowdegreeofesterificationandxyloglucan,seemedtohavearoleinthefirststepsof
DCB‐habituation. Thereby someof themodifications occurred in the cellwalls in order to
compensate for the lack of cellulose differed according to the DCB‐habituation level, and
demonstratestheremarkabledynamicplasticityofmaizecellcultures,whichenablesthem
tocopewithdifferentDCBhabituationconditionsbyalteringtheircellwallcomposition.
ChapterII
Studyofthemetaboliccapacityof
cellulose‐deficientmaizecells
ChapterII
78
ABSTRACT
The capacity of DCB‐habituatedmaize cells to survive with reduced cellulose
content derives from their ability to counteract the lack in cellulose by acquiring a
modifiedarabinoxylannetwork. Since it isprobable that this fact entailsalterations in
their metabolism of polysaccharides, the aim of this study was to investigate such
alterations in DCB‐habituated maize cell cultures showing a mild reduction in their
cellulose content at different stages in the culture cycle. Using a pulse‐chase radio‐
labellingexperimentalapproach,theDCB‐habituatedcellcultureswerefedwith[3H]Ara
as radio‐labelledprecursor, and themetabolismof 3H‐hemicellulosesand 3H‐polymers
wastrackedinseveralcellcompartmentsfor5h.
Thehabituatedcellsexhibiteddifficultiesintheuptakeofthe[3H]Ara,showinga
slowerandlessefficientmetabolismregardlessoftheculturestageofgrowth.Moreover,
lower proportions of strongly cell wall bound hemicelluloses ([3H]xylans and
[3H]xyloglucan) as well as 3H‐polymers ([3H]Ara‐containing and Driselase‐digestion
resistantpolymers)weregraftedontothecellwall,whileahigherpercentageofsoluble
extracellularpolymersweresloughedintotheculturemedium.Thesefindingscouldbe
relate,atleastpartially,tothecellwallcellulose‐deficiency,andthereforetoareduction
in“bindingpoints”tocelluloseand/orareducedcapacitytoincorporatearabinoxylans
intothecellwallbyextra‐protoplasmicphenoliccross‐linking.
ChapterII
79
I.INTRODUCTION
Plant cellwalls are dynamic structures essential for cell division, enlargement and
differentiation and are also involved in the modulation of stress signalling responses
(Roberts, 2001;Huckelhoven, 2007;Driouich et al., 2012).Thegeneric composition of the
primary plant cell wall consists of a cellulose framework tethered by hemicelluloses
embedded in a matrix of pectins (Demura and Ye, 2010; Pauly and Keegstra, 2010). In
grassesandcereals,thecelluloseframeworkisstrengthenedbyheteroxylans,whichpresent
the general structure of all xylans (a backbone of Xyl with Ara, GlcA, orMeGlcA residues
attached)butalsosomeuniquefeaturessuchasthepresenceofhydroxycinnamates,mainly
Ferandp‐CouesterifiedonAraresidues(SmithandHartley,1983;KatoandNevins,1985).
Hydroxycinnamates are susceptible to oxidative coupling by peroxidases (Geissmann and
Neukom,1971) to formester‐linkeddehydrodiferulateswhichcontribute towallassembly
bycross‐linkingcellwallpolysaccharides(Fry,2004;Parkeretal.,2005).
Heteroxylansfromgrassesarethethirdmostabundantcomponentofplantbiomass,
andtheyarethemainnon‐cellulosicpolysaccharideinthistypeofwall(Zhongetal.,2009;
Fincher, 2009). Non‐cellulosic polysaccharides are potential sugar sources for ethanol
fermentation and therefore, for bioethanol production (Cosgrove, 2005; Komatsu and
Yanagawa,2013). Inadditionto theireconomic importance,heteroxylansplayanessential
roleincellexpansion.Inorderforthistotakeplace,primarywallsrequiretheintegrationof
theseheteroxylansandothernewlysynthesisedpolymers,aswellasamodificationof the
pre‐existingwall boundones (Kerr andFry,2003).Thus, an improvedknowledgeof their
synthesis and assembly is crucial to understanding and manipulating plant growth and
morphogenesis,withtheconsequenteconomicimplications.
SincemRNAmightnotbe translatedand invitroassaysmaynotreproducenatural
cell conditions (with the subsequent loss of real information), in vivo metabolic studies
appear to be the best experimental approach, making it possible to monitor in vivo the
behaviour of the hemicellulosic substrates themselves (Kerr and Fry, 2003). Such in vivo
ChapterII
80
analyses have typically been carried out in suspension‐cultured cells (Edelmann and Fry,
1992;ThompsonandFry,1997;2000;EncinaandFry,2005;KerrandFry,2003;2004;Burr
andFry,2009),andalthoughthesemaydifferinsomedetailsfromplantletsorplants,they
areexcellent,easytomanipulatemodelsystems.Moreover,somesuspension‐culturedcells
(includingmaize)releasepolysaccharidesintotheculturemedium(whichcanbeconsidered
as an extension of the apoplast). Since these sloughed polysaccharides do not require
chemical treatment tobeextracted fromthecellwall, theyretain theirmolecular integrity
andhavebeendemonstrated to constitute an excellentmodel system for studying several
aspectsof cellwallbiology (KerrandFry,2003;2004;BurrandFry,2009).Feeding these
cellsuspensionsystems(cellsplusculturemedium)withradio‐labelledprecursorshasbeen
widelyshowntobeasauseful,sensitiveandprecisemethodologyfortrackingthesynthesis
and cellular traffic (from synthesis in the Golgi apparatus to integration in the cell wall
and/or sloughing into the culturemedium) of hemicelluloses in living cells: this approach
has the advantage that all the substrates are endogenous and therefore the reactions
detectedareonesthatoccurnaturally(ThompsonandFry,1997;KerrandFry,2003;2004;
LindsayandFry,2008;Mélidaetal.,2011).Specifically,when[3H]Arawassuppliedtomaize
cell‐suspensioncultures,theinvivocareersofendogenousxyloglucanandxylanmoleculesin
several cell compartments were successfully tracked, confirming the effectiveness of this
experimentalapproachfortrackingheteroxylanmetabolism(KerrandFry,2003).
Habituation of cultured cells to CBIs has proven a valuable tool to study cell wall
plasticityandbiogenesis(forareviewseeAcebesetal.,2010).WithinCBIs,DCBspecifically
inhibits thepolymerisationofGlc intoβ‐1,4‐linkedglucan(Delmer,1987;García‐Anguloet
al.,2012) therebycausinga reduction incellulosecontent.Severalplant cell cultureshave
been habituated to grow in presence of DCB (Shedletzky et al., 1992; Encina et al., 2001;
2002;García‐Anguloetal.,2006;2009;Mélidaetal.,2009),and ithasbeen foundthat the
magnitudeofthecellulosereductionprovokedbytheinhibitordependsonitsconcentration
aswell as on the length of time that cells are grown in its presence (Alonso‐Simón et al.,
ChapterII
81
2004).Thecapacityofhabituatedcellstosurviveresidesintheirabilitytomodifytheircell
wall composition in order to counteract the lack of cellulose. In this respect, it has been
proven that maize cell suspensions habituated to DCB compensate for the reduction in
cellulose levels by modifying their cell wall matrix, involving quantitative and qualitative
changesinthearabinoxylannetwork.Basedonpreviousresults(Mélidaetal.,2009;2010a;
2010band2011),itishighlyprobablethatarabinoxylanmetabolisminDCB‐habituatedcells
is both quantitatively and qualitatively altered. Therefore, the aim pursued in the present
studywastogainagreaterinsightinthemetaboliccapacityofcellulose‐deficientmaizecells
throughouttheculturecycle.Theelucidationofdifferencesinlocation,timingandintensity
ofhemicellulosebiosynthesisinthesecellulose‐deficientcellscouldhelpusto1)shedlight
on the mechanisms that account for the rearrangement of the hemicellulose network in
cellulosedeficientconditionsand2)improveourknowledgeaboutthegeneralmechanisms
underlyingthestructuralplasticityofprimarycellwalls.Tothisend,DCB‐habituatedmaize
cellcultureswerefedwith[3H]Araasradio‐labelledprecursoratlag,earlylogarithmicand
latelogarithmicgrowthphase,andthesynthesisandcellulartrafficof3H‐hemicellulosesand
3H‐polymersweretrackedinseveralcellcompartmentsfor5h.
II.MATERIALSANDMETHODS
II.a.Cell‐suspensionculturesandhabituationtoDCB
Maize cell‐suspension cultures (Zea mays L., Black Mexican sweet corn) from
immatureembryosweregrowninMurashigeandSkoogmedia(MurashigeandSkoog,1962)
supplementedwith9μM2,4‐Dand20gL‐1sucrose,at25°Cunderlightandrotaryshaken,
androutinelysubculturedevery15days.
In order to obtain habituated cell cultures, Snh cells were cultured in a medium
suppliedwith1μMDCB.DCBwasdissolved inDMSO,whichdoesnotaffectcellgrowthat
thisrangeofconcentrations.Aftersevensubcultures,someofthecellshabituatedtogrowin
ChapterII
82
1μMDCBweretransferredtomediumcontaining1.5μMDCB(Sh1.5).GrowthcurvesofSnh
and Sh1.5 maize cell lines were obtained by measuring the increase in DW at different
culturetimesanditwasobservedthatSh1.5cellspresentedlongerculturephasesthanSnh
cells (de Castro et al., 2013; Chapter I, Figure 6A). Therefore, these growth curves were
subsequentlyusedtoselectthemostappropriatedaysforsamplinginordertoensurethat
SnhandSh1.5celllineswereatthesamestageoftheculturecycle.
II.b.Radio‐labellingandsamplecollectionof3H‐hemicellulosesand3H‐polymers
Thirteen‐ml aliquots from Snh and Sh1.5maize cell cultureswere collected at lag,
logarithmicandearlystationarygrowthphases.Each13‐mlaliquotwas independently fed
with1MBqofL‐[1‐3H]Ara(148GBq/mmol;AmershamInternational,Bucks.,U.K).Thedays
onwhich the13‐ml aliquotof culturewas sampledand [3H]Arawas addedwere adjusted
dependingonthecelllineatearlyandlatelogarithmicphases.Thus,forlagphase,thefirst
dayofculturewasselectedforbothcelllines.Inthecaseoftheearly‐logarithmicphase,4th
and8thdaysofculturewerechoseninSnhandSh1.5celllines,respectively.Finally,forthe
late‐ logarithmic phase, the 8th and 12th days were selected for Snh and Sh1.5 cell lines
respectively.
The incorporation of [3H]Ara into 3H‐hemicelluloses and 3H‐polymers in both cell
lineswasmeasuredatdifferentlabelling‐timepointsfor5hineachdifferentculturephase.
Thus, 1‐ml samples were collected 30, 60, 120 and 300 min (labelling‐time points) after
[3H]Arawasfedfromtheindependent13‐mlaliquotsofculture.
II.b.1.CFMcollection
Each1‐mlsamplecollectedatthedifferentlabelling‐timepointswasfilteredthrough
an empty Poly‐Prep column (Biorad). Then, cells were rinsed with 5 ml of ice‐cold fresh
medium.ThefiltratewaspooledwiththerinsesandlabelledasCFMfraction.
ChapterII
83
II.b.2.Protoplasmiccontentextraction
ThewashedcellsretainedinthePoly‐Prepcolumnfilter,werequicklyresuspended
in 1ml of homogenisation buffer [50mM collidine acetate (pH 7.0) containing 2% (w/v)
lithiumdodecylsulfate(LiDS),10%(v/v)glycerol,5mMsodiumthiosulfateand10mMDTT
(addedfreshly)]andstoredat‐20°CinthePoly‐Prepcolumn.
Thecellsuspensionwasthawedat1.5°Candtransferredtothe10‐mlglassmortarof
aPotter–Elvehjemhomogeniserbyrinsingwith1mlofchilledhomogenisationbuffer(x3).
The cell suspensionwas then homogenisedwith amotor‐drivenTeflon pestle for 10min.
The homogenate was passed through a second empty Poly‐Prep column and the filtrate
collected.Thecellfragmentswererinsedwith1mlH2O(x4).
SolidKClwas added to the filtrate and rinses to a final concentrationof 0.67M in
order to precipitate the dodecyl sulphate as its insoluble K+ salt. After 30min at 4°C, the
productswere centrifuged at 3000 rpm for 5min. The supernatantwas removed and re‐
centrifuged. This procedure was repeated until the supernatant turned clear. Then, the
supernatantwascollectedandlabelledastheprotoplasmicfraction.
II.b.3.Extractionofcellwallfractions
CellfragmentsretainedinthefilterofthePoly‐Prepcolumnweretreatedwith1ml
of0.1MNaOHcontaining1%(w/v)NaBH4for24hatroomtemperature.Thefiltratewas
then collected and the residuewashedwith1ml of the same extractant (x3). Filtrate and
washeswerepooledand labelledas0.1MNaOHfraction.Theremainingresiduewasthen
treatedwith1mlof6MNaOHcontaining1%(w/v)NaBH4for24hat37°C.Thefiltratewas
again collected and the residuewashedwith 1ml of the same solution (x3). Filtrate and
washeswerethenpooledandlabelledas6MNaOHfraction.Boththe0.1MNaOHand6M
NaOHfractions,wereacidifiedtopH4.7bytheadditionofaceticacid.
Theresidue in thePoly‐Prepcolumnwas thenwashedwith1mlofdistilledwater
slightlyacidifiedwithaceticacid(x3).Theremainingresidue(6MNaOH‐insolublematerial)
ChapterII
84
still retained in thePoly‐Prepcolumn filterwasconsidered tobe from3H‐polymers firmly
boundtocelluloseresidue.
II.c.Assayofradioactivityintheuptakeof[3H]Araandincorporationinto3H‐polymers
An analysis of theCFMandprotoplasmic fractionswasperformed as describedby
KerrandFry(2003)withminormodifications.Briefly,5μlof10%(w/v)coldArawasadded
toanaliquotof60μlofeachsampleasaninternalmarker.Thesamplewasthensubjectedto
PC in butan‐1‐ol/acetic acid/water (12/3/5, by vol.) for 16 h. The internal marker was
stained slightly by dipping the paper in 10% of the standard concentration of aniline
hydrogenphthalate(Fry,2000),dried,andthenheatedat105°Cfor2min.Thisrevealedthe
position of the Ara without appreciably decreasing the efficiency of scintillation counting
(KerrandFry,2003).TheAraspotandtheoriginzone(RF0;containingthepolymers)were
thencut andseparately assayed for 3Hby liquid scintillationcounting in2mlofOptiScint
HiSafe(Fisher).
Fortheanalysisofcellwallfractions,analiquotof500μlofeachsamplewasassayed
forradioactivitybyliquidscintillationcountingin5mlofOptiPhaseHiSafe(Fisher).
In the case of the 3H‐polymers firmly bound to cellulose, the entire polypropylene
filterpadwas excised andmixedwith15ml ofOptiPhaseHiSafe scintillation liquid.After
soakingfor24h,radioactivitywasthenassayedbycounting.
II.d.Fractionscompositionassay
SamplesofCFM,protoplasmicandcellwallfractionsweredialysedagainstdistilled
water with 0.1% (w/v) chlorobutanol at 4°C for 72 h. Then, the dialysed samples were
centrifuged for 15 min at 3000 rpm in order to separate the soluble from the insoluble
materialinwater,andthesolubleportionwascollected.Samplesweredriedinvacuoandre‐
dissolvedin500μlof0.1MTFA,heatedat85°Cfor1htocleavearabinofuranosyllinkages,
and re‐dried to remove TFA. The dried material was re‐dissolved in 20 μl 0.5% (w/v)
ChapterII
85
Driselase (Sigma Chemical Co.; partially purified by the method of Fry 2000) in
pyridine/acetic acid/water (1/1/23 by vol. pH 4.7 containing 0.5% chlorobutanol), and
incubatedat37°Cfor96h.Thereactionwasstoppedbytheadditionof15%formicacid.The
mild acid pre‐treatment greatly increased the yield of Xyl and xylobiose generated during
subsequentdigestionwithDriselaseanddidnotdecreasetheyieldofisoprimeverose(Kerr
and Fry, 2003). An internal marker mixture containing Xyl, Ara, Glc, isoprimeverose and
xylobiose (≈ 50 g each) was then added to the digest, which was subjected to PC on
Whatman3MMinethylacetate/pyridine/water(9/3/2byvol.)for18h(ThompsonandFry,
1997).The internalmarkerswereslightlystainedwithanilinehydrogen‐phthalateandthe
identified spots were cut out and assayed for 3H (as described above). Results from the
radioactivity counting of the 3H‐labelled diagnostic fragmentswere plotted and named as
follows:polymerswhichhad[3H]Ara(radioactivitydetectedin[3H]Araspotfromthepaper
chromatogram); [3H]xylans (summation of the amounts of radioactivity from [3H]Xyl and
[3H]xylobiose spots), [3H]xyloglucan (radioactivity detected when counting the
[3H]isoprimeverose spot) and undigested 3H‐polymers (radioactivity on the origin spot of
thepaperchromatogram).
III.RESULTS
III.a.Uptakeof[3H]Arafromtheculturemedium
Snh and Sh1.5 maize cell suspensions showed differences in [3H]Ara uptake
depending on the culture phase, with consumption being more efficient at the late
logarithmic phase in both cell lines (0.114 and 0.055 KBq min‐1 for Snh and Sh1.5
respectively)(Figure1).However,Sh1.5cellswerelessefficientin[3H]ArauptakethanSnh
cells, regardless of the culture phase (Figure 1B vs 1A), showing much lower estimated
consumptionratesof[3H]Ara(roughlyhalf)thanSnhcellsinalltheculturestagesanalysed
(datanotshown).Thiswasparticularlymarkedatthelatelogarithmicphaseoftheculture
ChapterII
86
cycle,whenSnhcellscompletelydepleted the [3H]Arawithin the300minmonitored.This
depletionwasnotaccomplishedbySh1.5cells.
Figure1.Consumptionof[3H]ArainA:SnhandB:Sh1.5DCBmaizecellsuspensionsduringsolidline;lag,dottedline;earlylogarithmicanddashedline;latelogarithmicphasesofculturecycle.Resultsshownare
representativeoftwoindependentexperiments
III.b.Kineticsofincorporationof[3H]Araintohemicellulosesandpolymers
As previously shown for [3H]Ara uptake, differences in the kinetics of the
incorporationof3Hintohemicellulosesandpolymerswereobserveddependingonthecell
line (Figure 2). In Snh cells, a gradual increase in the capacity tometabolise [3H]Arawas
observed throughout the culture cycle (Figure 2A). Thus, as culture cycle progressed, the
lengthof time thatunmetabolised [3H]Araand 3H‐polymerswhichwerebeing synthesised
remained in the protoplasm began to decrease. Furthermore, the time required for 3H‐
polymers tobegraftedonto thecellwallor sloughed into themediumdecreasedover the
course of the cell culture cycle. This trendwas not observed in the Sh1.5 cell line,which
showeddelayedkineticswhencomparedwithSnhcells(Figure2B).
A
Timeafteradditionof[3H]arabinose(min)
0 30 60 120 300
3 H(kBq/mlculture)
0
5
10
15
20
25
30
35 B
0 30 60 120 300
B
ChapterII
87
Figure2.Incorporationof[3H]AraintothepolymersofdiscretecompartmentsofA:SnhandB:Sh1.5maizecellsuspensionsduringlag(I),earlylogarithmic(II)andlatelogarithmic(III)phasesofculturecycle.Samplesweretakenduring5hatdifferentlabelingtimepoints(30,60,120and300min).Thecompartmentsassayedfortotal3H‐polymerswereprotoplasmic(),cellwall‐bound[0.1MNaOH‐extractable()and6MNaOH‐extractable()]andsolubleextracellularpolymers(SEPs)sloughedtoculturemedium().3H‐polymersthatwerefirmlyboundtotheα‐celluloseresidue()andunmetabolised[3H]Aradetectedwithintheprotoplasm()werealso
assayed.Resultsshownarerepresentativeoftwoindependentexperiments
In both cell lines, the most active phase of the culture cycle as regards [3H]Ara
incorporation intohemicellulosesandpolymerswas the late logarithmic (Figure2AIIIand
2BIII). The kinetics obtained from this phase of the culture cycle clearly illustrated the
differences mentioned above regarding to the metabolic capacity of synthesis and
incorporationof3H‐hemicellulosesand3H‐polymers.Atthisphaseoftheculturecycle,Snh
cellsrapidlymetabolisedandincorporatedthe[3H]Arainto3H‐polymersintheprotoplasm
reaching a maximum peak of synthesis 60 min after [3H]Ara feeding. Subsequently, the
synthesisof 3H‐polymers intheprotoplasmdecreased,showingconstantvaluesduringthe
remaining240minmonitored.Thissuddendecreasecouldberelatedtotheremovalofthese
I
III
0 30 60 120 300
IIII
3 H(kBq/mlculture)
0
2
4
6
8
10
12
III
Timeafteradditionof[3H]Ara(min)0 30 60 120 300
0
2
4
6
8
10
12
14
16
I
0
2
4
A B
ChapterII
88
3H‐polymers from the protoplasm and their incorporation into the cell wall as 3H‐
hemicelluloses, or their sloughing into the culture medium ([3H]soluble extracellular
polymers; [3H]SEPs) (Figure2AIII). Incontrast,Sh1.5cellsdidnot showamarkedpeakof
3H‐polymer synthesis in the protoplasm, and the incorporation of [3H]Ara into any 3H‐
hemicellulosesor[3H]SEPsdidnotreachaplateauphase,ashappenedinSnhcells(Figure
2BIII). Thismay indicate that Sh1.5 cells had a delayedmetabolism and, therefore, lower
ratesofsynthesisof3H‐polymersand3H‐hemicelluloses.
The fateof the3H‐polymerswhen leavingtheprotoplasmwasalsoaltered inSh1.5
cellswhencomparedwithSnhcells(Figure2).Snhcellspreferentiallyincorporated[3H]Ara
intostrongalkali‐extracted‐hemicelluloses(6MNaOH)and/orSEPs(Figure2A)followedby
mild alkali‐extracted hemicelluloses and 3H‐polymers firmly bound to cellulose residue.
Sh1.5 cells incorporated lower amounts of [3H]Ara into cell wall polymers (3H‐polymers
firmly bound to cellulose residue and NaOH‐extracted 3H‐hemicelluloses, mainly those
extractedwith6M),(Figure2B)buttheamountof[3H]SEPsdetectedintheculturemedium
of Sh1.5 cells at late logarithmic phase of the culture cycle was roughly equal to that
observedinSnhcells(Figure2AIIIvs2BIII).However,whendatafromtheincorporationof
the [3H]Ara into the different types of polymers and hemicelluloses were expressed as a
percentageofthetotalradioactivityincorporatedattheendofthe300minofmonitoring,a
relative increase in the incorporationof [3H]Ara into0.1MNaOH‐extractedhemicelluloses
wasdetectedinSh1.5cells.
III.c.Kineticsofsynthesis,cellwallintegrationandsloughingof3H‐labelleddiagnostic
fragments
3H‐polymersand3H‐hemicellulosesfromthedifferentcellcompartmentswerefirstly
Driselase‐digestedandthensubjectedtoPC inordertoobtainthediagnostic fragments. In
each cell line (Snh and Sh1.5), polymers which had [3H]Ara residues, [3H]xylans,
[3H]xyloglucanand 3H‐polymers resistant toDriselasedigestionshowedsimilarkinetics in
ChapterII
89
each cell compartment and culture phase analysed (Figures 3, 4, 5 and 6). Nevertheless,
variations between cell lines andwithin the same cell line, among cell compartments and
phasesofculturecyclewerefound.
III.c.1.Protoplasmiccompartment
Synthesisofthedifferentpolymersandhemicellulosesoccurredexponentiallyduring
thelagphaseinbothcelllines(Figure3).Aswiththegeneralkinetics,60minafter[3H]Ara
wasfedatearlyandlatelogarithmicphasesoftheculturecycle,Snhcellsreachedapeakin
synthesis of [3H]xylans, [3H]xyloglucan, polymers which had [3H]Ara and undigested 3H‐
polymers,whichsubsequentlydecreased(Figure3A). Incontrast,Sh1.5cellsdidnotshow
any peak in synthesis of [3H]xylans, [3H]xyloglucan, polymers which had [3H]Ara and
undigested 3H‐polymers during the early logarithmic phase of the culture cycle. At late
logarithmicphase,maximumsynthesiswasdetectedlaterinSh1.5thaninSnhcells,120min
after[3H]Arawassupplied,andtherapiddecreaseinthepeakinsynthesisdetectedinSnh
was not observed in the Sh1.5 protoplasm (Figure 3B). This indicates that [3H]xylans,
[3H]xyloglucan, polymerswhich had [3H]Ara and undigested 3H‐polymers remained in the
protoplasmofthehabituatedcellsforalongerperiodoftimeafterbeingwall‐integratedor
sloughed.
III.c.2.Cellwallcompartment
Thekineticsof3H‐labelleddiagnosticfragmentsfrompolysaccharidesextractedwith
mild (0.1MNaOH) and strong alkali treatment (6MNaOH) from Snh and Sh1.5 cells are
showninFigures4and5,respectively.SnhandSh1.5cellsintegratedasimilarproportionof
[3H]xylans, [3H]xyloglucan, polymers which had [3H]Ara and undigested 3H‐polymers
extractedwithmildalkalitreatment(Figure4),whereastheamountofthoseextractedwith
strongalkalitreatmentwasmuchlowerinSh1.5cells(Figure5).However,thekineticsof3H‐
labelled diagnostic fragments obtained by both (0.1 M and 6 M NaOH) alkali treatments
ChapterII
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showed differences depending on the cell line (Figure 4A vs Figure 4B and Figure 5A vs
Figure5B).
Figure3.Pulse‐chasekineticsofradiolabelingofpolymersintheprotoplasmafterDriselaseenzymaticdigestionofA:SnhandB:Sh1.5maizecellsuspensions.Diagnostic3H‐labelledfragmentswerethosefor:polymerswhichhaveAra();xylans();xyloglucan()andundigestedpolymers().Samplesweretakenduring5hat
differentlabelingtimepointsduringlag(I),earlylogarithmic(II)andlatelogarithmic(III)phasesofculturecycle.Resultsshownarerepresentativeoftwoindependentexperiments
Duringthelagphase,polymerswhichhad[3H]Ara,[3H]xylans,[3H]xyloglucanandthe
Driselase‐resistant3H‐polymers,eitherextractedwithmildorstrongalkalitreatment,were
graduallyintegratedintothecellwallsofSnhandSh1.5cells(Figure4AI,4BI,5AIand5BI).
At the early logarithmic phase, the cell lines started to show disparate trends in the
incorporation of different types of 3H‐polymers and 3H‐hemicelluloses (Figure 4AII, 4BII,
5AII and 5BII), and this disparitywasmoremarked at the late logarithmic culture phase
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(Figure4AIII, 4BIII, 5AIII and5BIII). This incorporation of polymers extractedwith either
mildorstrongalkalitreatmentoccurredmorerapidlywithinthefirst120minintheSnhcell
walls.However,althoughpolymerswhichhad[3H]Ara,[3H]xylans,[3H]xyloglucanaswellas
Driselase‐resistant3H‐polymerscontinuedbeingintegratedintothecellwallfrom120min
to300min,thisincreasewasnotsopronouncedasthatobservedduringthefirst120min
(Figure 4AIII and Figure 5AIII). In contrast, the incorporation of 3H‐polymers or 3H‐
hemicellulosesintohabituatedcellwallsmainlyoccurredfrom120minto300min(Figure
4BIIIandFigure5BIII),indicatingadelayedintegrationwhencomparedwithSnhcells.
III.c.3.Extracellularculturemedium
The kinetics of the appearance of polymers which had [3H]Ara, [3H]xylans,
[3H]xyloglucanandDriselase‐resistant3H‐polymers intheculturemediumofbothSnhand
Sh1.5 cell lines were similar, showing gradual sloughing at the lag and early logarithmic
phasesoftheculturecycle(Figures6AI,6AII,6BIand6II).Atthelatelogarithmicphase,an
increase in the sloughing of polymers which had [3H]Ara, [3H]xylans, [3H]xyloglucan and
Driselase‐resistant3H‐polymersintotheculturemediumwasobservedfrom60minto120
mininbothcell lines, indicatingthattheirreleaseintotheculturemediumtookplacelater
thantheirbindingtothecellwall(Figures6AIIIand6BIII).From120minto300min,Snh
cellsdidnotincreasetheamountsofpolymerswhichhad[3H]Ara,[3H]xylans,[3H]xyloglucan
andDriselase‐resistant3H‐polymerswhichweresloughedwithrespecttothevaluedetected
at 120min, showing a constant value. In contrast, within this period of time, Sh1.5 cells
continuedreleasingthemexponentiallyuntiltheendofthemonitoringperiod.
III.d.Compositionofthe3H‐polymersand3H‐hemicelluloses
Thecompositionofthe3H‐polymersand3H‐hemicellulosesdidnotdifferdepending
ontheculturestageandcellularcompartmentassayed(Figures3,4,5and6).
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92
Figure4.Pulse‐chasekineticsofradiolabelingofhemicelluloses0.1MNaOH‐extractedafterDriselaseenzymaticdigestionofA:SnhandB:Sh1.5maizecellsuspensions.Diagnostic3H‐labelledfragmentswerethosefor:
polymerswhichhaveAra();xylans();xyloglucan()andundigestedpolymers().Samplesweretakenduring5hatdifferentlabelingtimepointsduringlag(I),earlylogarithmic(II)andlatelogarithmic(III)phasesof
culturecycle.Resultsshownarerepresentativeoftwoindependentexperiments
Both types of 3H‐hemicelluloses (those extracted with mild or strong alkali
treatments) fromShcellsshowedanincreasedAra/xylanratiowhencomparedwiththose
fromSnhcells.However, this ratiowas lowerwhenconsidering to the 3H‐polymers in the
protoplasmand[3H]SEPs(Table1).
Moreover,differencesintheAra/xylanratiosdependingonthecelllinewerefound
when polysaccharides reached the cell wall as cellular fate. Likewise, 3H‐hemicelluloses
extracted from the Snh cell wall showed lower ratios than those observed for the 3H‐
polymersthatwerebeingsynthesisedintheprotoplasm,regardlessoftheculturephase.In
contrast,Sh1.5cellsshowedhigherratiosattheearlylogarithmicphaseoftheculturecycle,
whereasacleartrendwasnotobservedatlatelogarithmicphase(Table1).
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.
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Figure5.Pulse‐chasekineticsofradiolabelingofhemicelluloses6MNaOH‐extractedafterDriselase
enzymaticdigestionofA:SnhandB:Sh1.5maizecellsuspensions.
Diagnostic3H‐labelledfragmentswerethosefor:polymerswhichhaveAra();xylans();xyloglucan()andundigestedpolymers().Samplesweretakenduring5hatdifferentlabelingtimepointsduringlag(I),earlylogarithmic(II)andlate
logarithmic(III)phasesofculturecycle.Resultsshownare
representativeoftwoindependentexperiments
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Figure6.Pulse‐chasekineticsofradiolabelingofSEPsafterDriselaseenzymaticdigestionofA:SnhandB:Sh1.5maizecellsuspensions.Diagnostic3H‐labelledfragmentswerethosefor:polymerswhichhaveAra();xylans();xyloglucan()andundigestedpolymers().Samplesweretakenduring5hatdifferentlabelingtimepointsduringlag(I),earlylogarithmic(II)andlatelogarithmic(III)phasesofculturecycle.Resultsshownare
representativeoftwoindependentexperiments
Meanwhile,thexylan/xyloglucanratiowasparticularlyhighintheprotoplasmic3H‐
polymersfromSh1.5cells,anddecreasedwhencalculatedforanykindof3H‐hemicellulose
or for [3H]SEPs.However, ingeneral terms, this ratiowashigher inSh1.5whencompared
withSnhcells(Table1).
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95
Ara/Xylan Xylan/XyG Ara/Xylan Xylan/XyG Ara/Xylan Xylan/XyG Ara/Xylan Xylan/XyG
Protoplasm 3.0 2.5 2.2 10.2 4.1 4.2 2.6 14.0
0.1MNaOH 1.8 4.0 3.0 5.7 1.3 5.8 2.4 6.6
6MNaOH 1.8 3.1 2.9 2.6 1.8 3.1 3.1 3.3
SEPs 3.5 4.1 3.3 5.0 3.8 3.7 2.7 4.4
Sh1.5latelogarithmicSnhearlylogarithmic Sh1.5earlylogarithmic Snhlatelogarithmic
Table1.Ara/XylanandXylan/Xyloglucan(XyG)ratiosfromthedifferentcellcompartments[protoplasm,0.1MNaOH‐extractedpolymers(0.1MNaOH),6MNaOH‐extractedpolymers(6MNaOH)andsolubleextracellularpolymers(SEPs)]fromSnhandSh1.5maizecellsuspensions.Datashownarefromthediagnostic3H‐labelledfragmentsobtainedafterDriselasedigestionofsamplescollected300minafter[3H]Arawasfedatearly
logarithmicandlatelogarithmicphasesofculturecycle.Resultsshownarerepresentativeoftwoindependentexperiments
IV.DISCUSSION
MaizecellcultureshabituatedtodifferentconcentrationsofDCB(Mélidaetal.,2009;
2010a;2010band2011,deCastroetal.,2013;ChapterI)arecharacterisedbyareductionin
their cellulose content, which is compensated for by a coping strategy involving an
enhancementoftheirarabinoxylancontentand/ornetwork.However,themagnitudeofthis
cellulosecontentreductionandtheircopingstrategydependsontheDCBconcentrationas
wellasthelengthoftimethatcellsareexposedtoit(Alonso‐Simónetal.,2004;Mélidaetal.,
2009).SeveralmaizecellcultureshabituatedtodifferentDCBlevelshavebeenobtained,and
modifications in their walls have been characterised. Maize cells habituated to high DCB
levels show a 75% of reduction in cellulose content, counteracted by a reinforced
hemicellulosic network in theirwalls, achieved by a greater content ofmore cross‐linked
arabinoxylanswithlowerextractabilityandhigherMrandMw.Thecontentofothercellwall
polysaccharides is not affected or even reduced (Mélida et al., 2009; 2010a; 2010b and
2011).Ontheotherhand,maizecellsuspensionshabituatedtolowDCBlevels(1.5µMDCB)
‐whichwasthecellmaterialstudiedforthepresentresearch‐showa33%reductionintheir
cellulose content, mainly compensated for by a slighter increase in arabinoxylan content,
ChapterII
96
molecules which are more easily extractable. In addition, other polysaccharides such as
xyloglucanandrhamnogalacturonanIseemtobeinvolvedinthiscopingstrategy(deCastro
etal.,2013;Chapter I).The fact thatDCB‐habituatedcellsshowplasticity in themetabolic
machineryresponsibleforcellwallpolysaccharidesynthesisindicatesthattheyconstitutea
goodsystemforinvestigatingthemetabolismofheteroxylans,andtheirconnectionwiththe
biosynthesisofothercellwallpolymers.
Ithasbeendemonstratedthatfeedingmaizecellsuspensionswith[3H]Araisnotonly
asuitablebutalsoasuccessfulexperimentalmethodologyfortrackingtheinvivocareersof
endogenous [3H]xyloglucan and [3H]xylan molecules in several cell pools (Kerr and Fry,
2003).Thus,inthepresentstudy,[3H]ArawasaddedtoSnhandtolowlevelDCB‐habituated
cells(Sh1.5)duringasetoflabeling‐timepointsatdifferentstagesoftheculturecycle,and
fractionsfromseveralcellcompartments(protoplasmic,cellwallboundpolymersextracted
with0.1Mor6MNaOH)aswellasSEPswereobtained.
SnhandSh1.5cellsshoweddifferencesintheircapacityfor[3H]Arauptakeinallthe
culturestagesanalysed,withSh1.5cellsbeinglessefficientinallcases.Forinstance,atthe
late logarithmic phase of the culture cycle, Snh cells took up approximately 75% of
exogenous [3H]Ara within the first hour, which was in good agreement with data from
previous studies on maize (Kerr and Fry, 2003) and rose (Edelmann and Fry, 1992;
Thompson et al., 1997) cultured cells,whereas Sh1.5 cells only tookup37%.The [3H]Ara
uptaketrendsshownbySnhandSh1.5cellswerelinkedtohemicellulosemetabolisminboth
cell lines. DCB‐habituated cells showed a slower and less efficient metabolism when
comparedwithSnhorwithothermaize cultured cells (Kerr andFry,2003), andalthough
this finding was consistent throughout all the culture phases of growth, it was especially
obviousatthelatelogarithmicphaseoftheculturecycle.Inordertodeterminewhetherthis
altered metabolism was affecting the synthesis and cellular traffic of a specific type of
hemicellulose or polymer, samples were subjected to Driselase enzymatic digestion,
releasingdiagnostic fragmentswhichwerethenseparatedbyPC.Someof thesediagnostic
ChapterII
97
fragments are exclusive of xylans ([3H]xylobiose) and xyloglucan ([3H]isoprimeverose),
whereasothersarenotsohighlyspecificandcouldberelatedtoawiderrangeofmolecules,
such as Ara‐containing polymers ([3H]Ara) or those resistant to Driselase treatment (3H‐
material at RF 0 on the paper chromatogram). The pulse‐chase kinetics of 3H‐labeled
diagnostic fragments mimicked the general kinetics of [3H]Ara incorporation into 3H‐
polymers and 3H‐hemicelluloses. This proved, firstly, the solidity of the procedure and
secondly,thatsynthesis,graftingintothecellwallorsloughingintotheculturemediumofall
types of polymers and hemicelluloses, [3H]xylans, [3H]xyloglucans, [3H]Ara‐containing
polymersandDriselase‐undigested 3H‐polymers, fromSh1.5cellsweredelayedbecauseof
theirreducedmetaboliccapacity.Inaddition,adelayedgraftingofSh1.5arabinoxylansonto
thecellwallortheirsloughingintotheculturemedium,comparedtoxyloglucanmolecules,
could be taking place, as deduced from the reduction observed in the
[3H]xylan/[3H]xyloglucan ratio of Sh cell wall bound fractions when compared with the
protoplasm. The incorporation of [3H] into [3H]xylans and [3H]xyloglucans occurred
simultaneously. This has been observed previously in maize cultured cells, and it was
theorisedthatbothkindsofmoleculesprobablydrewfromthesamepoolof [3H]Xyl(Kerr
and Fry, 2003). Likewise, the same assumption can be made about the incorporation of
[3H]Ara into polymers which had [3H]Ara residues and the Driselase‐resistant polymers.
Thus, both kinds of 3H‐hemicelluloses ([3H]xylans and [3H]xyloglucan) and 3H‐polymers
(thosewhichcontained[3H]Araandtheundigestedones)lefttheprotoplasmcompartment,
andwerewallboundorsloughedsimultaneously.However,variationsinthetimingofthese
processesweredetectedwhenSnhandSh1.5cellswerecompared,beingdelayed inSh1.5
cells.
Anotherinterestingobservationconcernedthehemicellulosecompositionofthecell
wall fractions extracted with different alkali treatments, 0.1 M NaOH (able to cleave
hydrogen and ester bonds) and 6 M NaOH (with sufficient strength to cleave ether‐like
bonds). As mentioned before, it has been demonstrated that DCB‐habituated maize cells
ChapterII
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reinforced theirhemicellulosenetwork inorder to counteract cellulosedeficit.The results
obtained inprevious studies and in thepresentone, reveal that arabinoxylans seem tobe
responsible for this effect since theywereobserved tohaveahigher radioactivity content
than xyloglucan one. However, maize cultured cells have a lower xyloglucan than
arabinoxylan content it cannot be ruled out that xyloglucan may play an important role,
aboveall in cellulose‐deficientwalls.Thus, it couldbehypothesised thatSh1.5cellswould
preferentiallyattach[3H]xyloglucanor[3H]xylaninordertotightlyorweaklyintegratethem
into theirwalls as part of their strategy to copewith their reduced cellulose content.Our
resultsdidnotshowhabituation‐baseddifferencesinthecompositionofbothfractions(0.1
Mand6MNaOH‐extracted),indicatinginsteadthesamekindof3H‐hemicellulosesbutlinked
bydifferentbonds.However,resultsfrompulse‐chasekineticsand[3H]xylan/[3H]xyloglucan
ratiosshowedthatinbothcelllines,xyloglucanmoleculeswerepreferentiallyextractedwith
6MNaOH,andtherefore,weremorestronglylinkedinthecellwall.
The next question to emergewaswhether these 3H‐hemicelluloses or 3H‐polymers
from Sh1.5 cells differed in any respectwhen compared to those from Snh. An increased
[3H]Ara/[3H]xylan ratio was detected in Sh1.5 3H‐hemicelluloses that might reflect the
synthesisofmoresubstitutedarabinoxylans.However,taking intoconsiderationthatthere
are alternative sources for Ara residues besides arabinoxylans such as arabinogalactan
proteinsorrhamnogalacturonanIsidechains(forareviewseeFry,2011),thisresultshould
be interpreted with caution. Nevertheless, the sugar analysis of 0.1 M and 6 M alkali‐
extractedfractionsfromSh1.5cellwalls,revealedapoorcontentofuronicacids,GalorRha
residues(deCastroetal.,2013;ChapterI).Therefore,itcanbeassumedthatmostoftheAra
residuesactuallyderivedfromarabinoxylanmolecules.
When 3H‐polymers in synthesis left the protoplasm of DCB‐habituated cells,
variations in their fate were observed when compared to Snh. Results from the late
logarithmicphaseoftheculturecycleprovidedagoodexampleofthisalteredfate:asmaller
amount of strongly‐linked 3H‐hemicelluloses and 3H‐polymers firmly bound to the
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α‐cellulose residue were grafted into the Sh1.5 cell walls, and a relative increase in 3H‐
hemicellulosesextractedwith0.1Malkalitreatmentwasdetected.Thesefindingsledtothe
deduction that in Sh1.5 cells, strong‐alkali or even alkali‐resistant bonds did not play a
crucialroleinlinking3H‐hemicellulosesatanystageoftheculturecycle,atleastduringthe
period of time (300min)monitored. This is in contrast to the findings described for long
termhabituationofcellstohighDCBlevels(Mélidaetal.,2009;2011).Inthesestudiesithas
been shown that these kinds of strong linkages seem to be of relevance. For instance,
followingasimilarexperimentalapproachtotheoneusedinthepresentstudy,inMélidaet
al.(2011)habituatedcellstohighDCBlevelsandnon‐habituatedcellswerepulse‐chasefed
with [14C]Cinnand the appearanceof radio‐labelled compoundswas then tracked through
different cell pools.Their results showed that theproportionof radio‐labelled compounds
detectedinbothlabileandstablealkalifractionswashigherinthehabituatedcelllinethan
in the non‐habituated one. Another surprising finding related to the altered polymer and
hemicellulosefatesinhabituatedcellswasthehigherproportionof[3H]SEPssloughedinto
theculturemediumwhencomparedwithSnhcells(56%vs42.3%ofthetotalradioactivity
added).This resultwasevenmore interestingwhenpercentageswere comparedwith the
proportionofcellwall‐bound3H‐hemicelluloses(56%vs30%and42.3%vs55%forSh1.5
and Snh cells respectively), mainly in the 6 M NaOH fraction, in which the amount of
extracted molecules was much lower when compared with Snh cells. These results may
indicate that this population of polysaccharides, otherwise extracted with 6 M NaOH, is
sloughed into the culturemedium. Polymerswhich are sloughed into the culturemedium
could represent newly synthesised polymers that pass quickly through the wall without
being bound and/or undergoing any chemical change, or alternatively, wall‐grafted
moleculesthatarelaterloosenedandthereforereleasedintotheculturemedium(Kerrand
Fry,2003;Mélidaetal.,2011).However,iftheSEPsdetectedintheculturemediumofSh1.5
cellshadtheirorigininpreviouslywall‐graftedandlaterloosenedhemicellulosesonewould
be expect to find a decrease into the radioactivity incorporated into the cell wall bound
ChapterII
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fractionsconcurrentwithanincreaseofradioactivityintheculturemedium,probablyatthe
latestlabelingtimepointsmonitored(120‐300min).Threepossibleexplanationsemerge:a)
asSh1.5cellshaveacellulose‐impoverishedwall, it isprobablethatnewlysynthesised3H‐
hemicellulosespassthroughthewallwithoutbeingboundduetothelackoflinkingpoints,
b) thecapacityofSh1.5cells to integrate [3H]arabinoxylansbyextraprotoplasmicphenolic
cross‐linking is reduced and in this case, a lower degree of arabinoxylan feruloylation, a
reducedperoxidaseactivityoralimitationinthesupplyofH2O2couldariseasalternatives,
andc)[3H]arabinoxylansfromthehabituatedcellsaresynthesisedwithahigherdegreeof
substitution and consequently, with an increased solubility, which would reduce the
possibility of establishing linkages with cellulose (as can be deduced from the
[3H]Ara/[3H]xylan ratioswhen compared to protoplasm and culturemedium fractions). It
has previously been reported that in developing barley coleoptiles arabinoxylans were
newlysynthesisedwith80%oftheirXylbackbonesubstitutedwithAraresiduesshowinga
substituted tounsubstituted4‐linkedxylosylunit ratioof4:1 (Gibeaut et al., 2005),which
decreased to 1:1 after 3 days, since hydrolytic enzymes remove these residues in grasses
during growth (Fincher, 2009). Thus, it could be theorised that in Sh1.5 cells, these
arabinosyl‐hydrolyticactivitieswereaffectedandtherefore,[3H]arabinoxylanshadagreater
degree of substitution and higher solubility. However, since (as previously mentioned)
results for the [3H]Ara/[3H]xylan ratios should be interpreted with caution, it is more
feasible to assume that the hypotheses described above in options a and b probably
contributed to a greater or lesser extent to the altered fate observed in Sh1.5 cells.
Nevertheless,furtheranalysesarerequiredinordertoconfirmthis.
In sum, synthesis, grafting and sloughing of [3H]xylans, [3H]xyloglucans, [3H]Ara‐
containingpolymersaswellasDriselase‐digestionresistant3H‐polymershavebeentracked
inthreestagesofculturecycle(lag,earlyandlatelogarithmic)anditwasobservedthatDCB‐
habituated cells: 1) exhibited less efficiency in uptake of the radio‐labelled substrate
([3H]Ara),2)presentedaslowerandlessefficientmetabolisminalltheculturecyclepoints
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analysed, and 3) showed a delayed cellular traffic of 3H‐hemicelluloses ([3H]xylans and
[3H]xyloglucan)and3H‐polymers([3H]Ara‐containedandDriselase‐digestionresistant),but
thisoccurredsimultaneously.4)Compositionalstudiesofhemicellulosicfractionsextracted
withmildorstrongalkalitreatmentsrevealedthatneitherSnhnorSh1.5cellspreferentially
boundanykindof3H‐hemicellulose(i.e[3H]xyloglucanor[3H]xylan)tothewallintermsof
thestrengthoflinkage.5)Variationswerefoundinthefateof3H‐polymersfromhabituated
cellswhenleavingtheprotoplasm,withareducedamountofwallbound3H‐hemicelluloses
andanincreasedpresenceof[3H]SEPsintheculturemedium.TheresultsindicatethatSh1.5
cells have a diminished capacity to graft hemicelluloses onto their walls, which could be
consequence of their reduced cellulose content and/or impeded polysaccharide cross‐
linking.
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ChapterIII
Analysisofthemolecularmass distributionof
polysaccharidesandthephenolicmetabolismin
maizecellswithamoderatereductionintheir
cellulosecontent
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ABSTRACT
Asaconsequenceof thehabituation to low levelsofDCB, culturedmaizecells
presented an altered hemicellulose cell fatewith a lower proportion of stronglywall‐
boundhemicellulosesandanincreaseinsolubleextracellularpolymersreleasedintothe
culture medium. These findings could be related to a reduction in sites linking
polysaccharides to cellulose and/or a diminished capacity to incorporate
polysaccharidesintothecellwallbyextra‐protoplasmicphenoliccross‐linking.Asmaize
cells habituated to high DCB levels have been reported to have more cross‐linked
arabinoxylans with lower extractability and higher Mr, the aim of this study was to
investigatetheMrdistributionofpolysaccharidesaswellasphenolicmetabolismincells
habituatedtolowlevelsofDCBthroughouttheculturecycle.Generally,cellwallbound
hemicellulosesandsloughedpolymersfromhabituatedcellsweremorehomogeneously
sizedandhadalowerMw.Inaddition,polysaccharidesunderwentmassivecross‐linking
after being secreted into the cell wall, whichwas less pronounced in habituated cells
thaninnon‐habituatedones.Thesefindingssupportthehypothesisofareducedcross‐
linkingcapacityinhabituatedcellsforoxidativecouplingofhydroxycinnamateresidues
of arabinoxylan molecules. However, when relativised, Fer and p‐Cou contents was
higherinthiscellline,whichpartiallyrulesoutthisidea.Feasibly,cellshabituatedtolow
levelsofDCBsynthesisedmoleculeswithalowerMw,althoughcross‐linked,asapartof
theirstrategytocompensateforthelackofcellulose. Inaddition,apossiblerolearises
for xyloglucan in maize cells habituated to low levels of DCB. This variety of coping
mechanism indicates the remarkable plasticity of DCB‐habituated maize cells, which
appeartoadopttheappropriatestrategydependingontheDCBhabituationframework.
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I.INTRODUCTION
Heteroxylansarethemainnon‐cellulosicpolysaccharidesinthePoalesprimarycell
wall (for general composition and structure see general introduction section) (Fincher,
2009), playing a major structural role in tethering cellulose microfibrils; hence, they are
involvedincellexpansionandplantgrowth.Heteroxylansshowgreaterstructuraldiversity
andcomplexitythanothertypesofcellwallcomponents,sinceawiderangeofdifferentside‐
chain substitutions are found connected to the backbone (Faik, 2010). A set of genes has
been described related to heteroxylan synthesis in grasses, orthologues of IRX from
arabidopsis. Likewise, three orthologous of the arabidopsis genes IRX9, IRX9L and IRX14
encodingputativeGTsresponsibleforxylanbackboneelongationhavebeenreportedinrice
(Chiniquy et al., 2013). In wheat, the coordinated action has been described of a protein
complex including a xylosyltransferase, a glucuronyltransferase and an
arabinosyltransferase involved in glucuronoarabinoxylan synthesis (Zeng et al., 2010).
Specifically in the case ofmaize, sequences have also been reported for some arabidopsis
orthologuessuchasIRX9,IRX10andIRX10L(Boschetal.,2011).
One of the unique structural features of arabinoxylan in Poales is that it contains
hydroxycinnamate residues, such as Fer or p‐Cou, esterified on the C‐5 position of Ara
residues (Bacicetal.,1988;WendeandFry,1997).TheattachmentofFer residuesoccurs
withintheprotoplasm,beforesecretionintothecellwall(Fryetal.,2000).Fermayundergo
oxidative coupling when exposed to hydrogen peroxide plus peroxidase (Geissmann and
Neukom, 1971). Dimerisation of feruloyl polysaccharides may occur in the protoplasm
beforepolysaccharidesaresecretedintothecellwall,inthecellwalljustaftersecretion,or
later in the cell wall after binding to thewall (Fry et al., 2000;Mastrangelo et al., 2009).
Larger coupling products, such as trimers or oligomers, are also formed and have been
described to predominate over diferulates (Fry et al., 2000). Phenolic residues are also
attached to polysaccharides as ether‐like (alkali‐stable) bonds (Burr andFry, 2009). Since
they are thought to be involved in the heteroxylan cross‐linking, they are essential for
ChapterIII
106
importantbiologicalprocessessuchaswallassemblyandrestrictionofcellexpansion(Fry,
2004;Parkeretal.,2005).
One constraint industries based on the use of grass‐derived products is cell wall
digestibility,which ishighly related toheteroxylancontent, structureand interactionwith
the components of the remaining cellwall formingnetworks.Despite notable advances in
ourknowledgeaboutheteroxylansynthesisandassemblyinrecentyears,someaspectsstill
remain unclear. Elucidation of its metabolism is particularly challenging, and also has
important economic implications. In line with this, the use of cell cultures habituated to
cellulosebiosynthesisinhibitors(forareviewseeAcebesetal.,2010)suchasDCBhasbeen
demonstrated tobea valuable tool to gain insight into themechanismresponsible for the
metabolicandstructuralplasticityexhibitedbyplantcellwalls.
DCB habituation is a dynamic process based on the capacity of cultured cells to
implementcopingstrategiesinordertosurvivewithacellulose‐deficientwall.Inthecaseof
maize suspension‐cultured cells, a quantitatively and qualitatively a re‐structured and
modifiedarabinoxylannetworkisinvolvedinmanyofthesestrategies(Mélidaetal.,2009).
Inaddition, the featuresandprocesses involvedof thecopingstrategiesexhibited in these
culturedcellsdependonDCBhabituationlevel,whichishighlyrelatedtothemagnitudeof
thereductionincellulosecontent(deCastroetal.,2013;seeChapterI).Hydroxycinnamates
seems to play a crucial role in strengthening cellulose‐deficient cell walls in maize cells
habituatedtohighDCBlevels,astheyareinvolvedinpolysaccharidecross‐linking(Mélidaet
al., 2010a; 2010b; 2011). Hemicellulosemetabolism is alsomodified as a consequence of
DCBhabituation.Inapreviousstudy,maizecellsuspensioncultureshabituatedtolowlevels
of DCB (Sh1.5; for a complete characterization of the cell line see de Castro et al., 2013;
ChapterI)werefedwith[3H]Ara(ChapterII).
Cells suspension cultures present several advantages in in vivo metabolic studies
performed using an isotopic approach, such as homogeneity of cell material, a lack of
nutrients,O2orexogenousprecursorgradientsintheextracellularculturemediumandease
ChapterIII
107
ofsampling(BurrandFry,2009). Inaddition, theconsistentresultsobtained in thisstudy
once again confirmed the suitability of radio‐labelled experimental approaches based on
feedingliveplantculturedcellsuspensionsinordertotrackthe invivo fateofendogenous
polysaccharides (formore details, precedents and references see Chapter II). The feeding
experimentsdemonstratedthatthesecellshadalessefficientandslowermetabolisminall
phasesoftheculturecycle,aswellasanalteredcellularfateofpolysaccharides,withalower
amount of strongly wall bound 3H‐hemicelluloses and an increased presence of [3H]SEPs.
One possible explanation for this observation is that as Sh1.5 cells exhibited a reduced
cellulose content, which would lead to a decrease in “binding points” for hemicelluloses.
Nevertheless, another possibility also emerged, namely that Sh1.5 cells would have a
diminished capacity to incorporate polysaccharides via phenolic cross‐linking into the cell
wall. This latter alternative is particularly interesting since an increased cross‐linking of
arabinoxylans leadingtohigherMwof thesemoleculeshasbeenshowntobecrucial inthe
habituationofcellstohighlevelsofDCB(Mélidaetal.,2009;2011).
Thus,anextensiveanalysiswasperformedinthepresentstudyoftheMrdistribution
of hemicelluloses and polymers as well as the phenolic metabolism in several cell
compartmentsofSnhandmildlycellulose‐deficientmaizecellsuspensioncultures(Sh1.5)at
theearlyand late logarithmicphasesof theculturecycle.To thisend,apulse‐chaseradio‐
labelling experimental approachwas employed,which consisted of feeding both cell lines
with the corresponding radio‐labelled metabolic precursors ([3H]Ara and [14C]Cinn
respectively). In addition, a preliminary studywas carried out of IRX9, IRX10 and IRX10L
arabidopsisorthologuegeneexpressioninthesecelllines.Theresultsobtainedinthisstudy
contribute to enhancing our understanding of hemicellulose metabolism, providing new
clueswhich help clarify themechanisms involved in the structural adaptability shown by
primarycellwallswhenexposedtoenvironmentalconstraints.
ChapterIII
108
II.MATERIALSANDMETHODS
II.a.CellculturesandhabituationtoDCB
Maize cell‐suspension cultures (Zea mays L., Black Mexican sweet corn) from
immatureembryosweregrowninMurashigeandSkoogmedia(MurashigeandSkoog,1962)
supplementedwith9μM2,4‐Dand20gL‐1sucrose,at25°Cunderlightandrotaryshaken,
androutinelysubculturedevery15days.
InordertoobtaincellcultureshabituatedtoDCB,Snhwerestepwisesubculturedin
amedium suppliedwith increasing concentrations ofDCB, dissolved inDMSOwhichdoes
notaffectcellgrowthatthisrangeofconcentrations.Likewise,inthecaseofcellsuspensions
habituatedtolowDCBlevels,Snhcellsweretransferredtoamediumcontaining1μMDCB,
increasing the DCB concentration up to 1.5 μM DCB (Sh1.5) after seven subcultures. Cell
suspensions habituated to high DCB levels were derived from maize callus cultures
habituatedtogrowin12μMDCBwhichweretransferredintoliquidmediumsupplemented
with 6 μMDCB (Sh6). Growth curves of Snh and Sh1.5maize cell lineswere obtained by
measuringtheincreaseinDWatdifferentculturetimesanditwasobservedthatSh1.5cells
presentedlongerculturephasesthanSnhcells(deCastroetal.,2013;ChapterI,Figure6A).
Therefore,thesegrowthcurvesweresubsequentlyusedtoselectthemostappropriatedays
forsampling inordertoensurethatSnhandSh1.5cell lineswereat thesamestageof the
culturecycle.
II.b.AnalysisofMrdistributions
II.b.1.Radio‐labellingofliquidcultureswith[3H]Ara
Thirteen‐ml aliquots from Snh and Sh maize cells cultures were collected at
logarithmicandearlystationarygrowthphasesandeach13‐mlaliquotwas independently
fedwith1MBqofL‐[1‐3H]Ara(148GBq/mmol;AmershamInternational,Bucks.,U.K).The
days on which the 13‐ml aliquot of culture was sampled and [3H]Ara was added, were
ChapterIII
109
adjusteddependingonthecell lineatearlyandlatelogarithmicphases.Thus,fortheearly
logarithmic phase, the 4th and 8th days of culture were chosen for Snh and Sh cell lines,
respectively(seedeCastroetal.,2013;ChapterI).Inthecaseoflatelogarithmicphase,the
8thand12thwereselectedforSnhandShcelllines,respectively.Inordertoobtainanoptimal
quantityofradio‐labelled3H‐hemicellulosesand3H‐polymers,samplesforanalysisoftheMr
werecollectedatdifferentlabelling‐timepointsselectedaccordingtothemaximumvalueof
incorporated[3H]Araobservedinthekineticspreviouslydescribed(ChapterII).Thus,inthe
case of protoplasmic polymers, 1‐ml samples were collected from independent 13‐ml
aliquotsofculture60minand30minafterfeeding[3H]AraforSnhandShcells,respectively.
For the remaining 3H‐hemicelluloses and 3H‐polymers, samplingwas carried out 300min
afterfeeding[3H]Araforbothcelllines.
II.b.2.Obtaining 3H‐hemicelluloses and 3H‐polymers fromdiscretemaize cell
suspensionscompartments
Each1‐mlsamplecollectedatthedifferentlabelling‐timepointswasfilteredthrough
anemptyPoly‐Prepcolumn.Then,cellswererinsedwith5mlofice‐coldfreshmedium.The
filtratewaspooledwiththerinsesandlabelledasCFM.
ThewashedcellsretainedinthePoly‐Prepcolumnfilterwerequicklyresuspended
in 1ml of homogenisation buffer [50mM collidine acetate (pH 7.0) containing 2% (w/v)
LiDS,10%(v/v)glycerol,5mMsodiumthiosulphateand10mMDTT(addedfreshly)]and
storedat‐20°CinthePoly‐Prepcolumn.
The cell suspension was then thawed at 1.5°C and transferred to the 10‐ml glass
mortar of a Potter–Elvehjemhomogeniser by rinsingwith 1ml of chilled homogenisation
buffer (x3), and then homogenised with a motor‐driven Teflon pestle for 10 min. The
homogenatewaspassedthroughasecondemptyPoly‐Prepcolumnandthefiltratecollected
andthecellfragmentswererinsedwith1mlH2O(x4).
ChapterIII
110
SolidKClwas added to the filtrate and rinses to a final concentrationof 0.67M in
order to precipitate the dodecyl sulphate as insoluble K+ salt. After 30 min at 4°C, the
productswere centrifuged at 3000 rpm for 5min. The supernatantwas removed and re‐
centrifugedandthisprocedurewasrepeateduntilthesupernatantturnedclear,momentin
whichwascollectedandconsideredtheprotoplasmicfraction.
CellfragmentsretainedinthefilterofthePoly‐Prepcolumnweretreatedwith1ml
of0.1MNaOHcontaining1%(w/v)NaBH4for24hatroomtemperature.Thefiltratewas
then collected and the residuewashedwith1ml of the same extractant (x3). Filtrate and
washeswerepooledand labelledas0.1MNaOHfraction.Theremainingresiduewasthen
treatedwith6MNaOHcontaining1%(w/v)NaBH4for24hat37°C.Thefiltratewasagain
collectedand theresiduewashedwith1mlof thesamesolution(x3).Filtrateandwashes
werethenpooledand labelledas6MNaOHfraction.Boththe0.1MNaOHand6MNaOH
fractionswereacidifiedtopH4.7bytheadditionofaceticacid.
II.b.3.GPC
SamplesofCFM,protoplasmicandcellwallfractionsweredialysedagainstdistillated
water with 0.1% (w/v) chlorobutanol at 4°C for 72 h. Then, the dialysed samples were
centrifuged for 15 min at 3000 rpm in order to separate the soluble from the insoluble
materialinwater.
Toidentifythevoidvolume(V0)andtotallyincludedvolume(Vi),6mgdextran(5–
40MDa) and 0.8mg Glc, respectively,were added to 4ml of this 3H‐polymer solution as
markers.Thepolymerswerethensize‐fractionatedonSepharoseCL‐4B(72mlbedvolume
in a 1.5‐cm‐diameter column) in pyridine/acetic acid/water (1/1/23 by vol. pH 4.7
containing 0.5% chlorobutanol) at 12.5ml/h. Fractionswere assayed for total 3H and the
markerswere quantified by the anthrone assay (Dische, 1962). Estimated recovery of 3H‐
hemicellulosesfromtheSepharosecolumnswasroutinely>80%(KerrandFry,2003).
ChapterIII
111
TheSepharoseCL‐4BcolumnwascalibratedwithcommercialdextransofknownMw.
Each dextran preparation was run as a mixture with a trace of very high‐Mw 3H‐
hemicellulose frommaize culturemediumand [14C]Glc as internalmarkers,whichdefined
theV0andVi(Kav0and1)respectively.MwwasobtainedusingtheKav(1/2)method(Kerr
andFry,2003)withthecalibrationcurve[logMw=‐3.547Kav(1/2)+7.2048]obtainedforthis
column. The Mw estimates were nominal rather than absolute due to the conformational
differencesbetweendextranandhemicelluloses.
II.c.Characterisationofphenolicmetabolism
II.c.1.[14C]Cinnsynthesis
[14C]Cinn was prepared from L‐[U‐14C]phenylalanine (Perkin‐Elmer; 487 Ci/mol)
followingthemethoddescribedbyLindsayandFry(2008)withminormodifications.
II.c.2. [14C]Cinn consumption and incorporation into different cell
compartments
Fifteen‐ml aliquots of Snh and Sh cell suspensions (20% of settled cell volume) at
early and late logarithmic phases of the culture cycle were transferred into 100‐ml glass
flasks, looselycappedandshaken(150rpm)for1hat25°C, inordertoadaptthemtothe
newenvironmentalconditions.Then,[14C]Cinn(0.29μCi)wasaddedtoeachcellculture.At
selectedtime‐points,400µlofcellcultureswassampledandacidifiedbytheadditionof99%
formicacid,stoppingtheconsumptionandincorporationof[14C]Cinn.
Eachaliquotsampledatthedifferentlabelling‐timepointswasthencentrifugedfor4
minat10000rpmandtheresultantsupernatantwascollected.Cellswerewashedwith200
μlofdistilledwaterandsubjectedagaintocentrifugation.Supernatantandwashingswere
thenpooled,labelledandstoredasCFM.
Remainingcellswerethenincubatedfor18hwith800μlof80%ethanolonarotary
wheel, the samples were subsequently centrifuged at 10000 rpm for 4 min and the
ChapterIII
112
supernatantwas collected. Cellswere rinsedwith 200 μl of 80% ethanol and centrifuged
again. Supernatant and washings were then pooled and labelled as the protoplasmic‐like
fraction(alcoholsolublematerial).TheresidualcellfragmentswereconsideredAIR.
II.c.3.ObtainingofCinn[14C]derivativesfromdiscretemaizecellcompartments
Fourhundredμl‐aliquotsofSnhandShcell‐suspensionsatearlyandlatelogarithmic
phasesoftheculturecycleweretransferredintoflat‐bottomedvialsandshaken(75rpm)for
1hat25°Ctoallowthemtoacclimatisetotheirnewenvironmentalconditions.Then,vials
were fedwith[14C]Cinn(0.116μCi).Atselectedtime‐points,aliquotswereacidifiedbythe
additionof99%formicacid,stopping[14C]Cinnincorporation.Whereindicated,40mMH2O2
(final concentration)was added to some of the samples 120min after [14C]Cinn and then
shakenfor60min.Aliquotsofcellsuspensionscontainedinthevialswerethentransferred
intoPoly‐PrepscolumnsandthefiltratewascollectedandconsideredtheCFMfraction.
Theremainingcellswereresuspendedin1ml80%ethanolandincubatedfor18h
on a rotary wheel. Cell suspensions were filtered and the alcohol‐soluble fraction was
considered the protoplasmic‐like fraction. The AIR retained on the Poly‐Prep columnwas
then de‐esterified by saponification with 0.5 M NaOH for 18 h at 25°C. The filtrate was
collectedandtheremainingcellfragmentswererinsedwith1mlofdistilledwater.Filtrate
andwashingswerethenpooledandcollectedasthe0.5MNaOHfraction.Thisfractionwas
acidified by addition of acetic acid and partitioned against ethyl acetate (x2). The ethyl
acetatephaseswerevacuum‐driedandre‐dissolvedin0.5mlofacidifiedwater(0.01NHCl).
These later sampleswere again subjected to a secondethyl acetatepartition (x3) and the
organicphaseswerecollected,vacuum‐driedandre‐dissolvedinpropan‐1‐ol.
ChapterIII
113
II.d.Assayofradioactivity
II.d.1.AnalysisofMrdistribution
Toobtainthegeneralelutionprofilesof3H‐hemicellulosesand3H‐polymers,a200µl
aliquotofcolumnfractionswasassayedforradioactivitybyliquidscintillationcountingin2
mlofOptiPhaseHiSafe(Fisher).
In order to obtain the elution profiles corresponding to 3H‐labelled diagnostic
fragments, fractions obtained from the column were pooled in pairs and vacuum‐dried.
Then,driedsamplesweresubjectedtoamild‐hydrolysisbyre‐dissolvingthemin500μlof
0.1MTFA,heatedat85°Cfor1htocleavearabinofuranosyllinkages,andfinallyre‐driedin
vacuo to remove TFA. The driedmaterialwas re‐dissolved in 20 μl 0.5% (w/v) Driselase
(partially purified by the method reported by Fry, 2000) in pyridine/acetic acid/water
(1/1/23byvol.pH4.7containing0.5%chlorobutanol),andincubatedat37°Cfor96h.The
reaction was stopped by the addition of 15% formic acid. The mild acid pre‐treatment
greatlyincreasedtheyieldofXylandxylobiosegeneratedduringsubsequentdigestionwith
Driselaseanddidnotdecreasetheyieldofisoprimeverose(KerrandFry,2003).Aninternal
markermixture containing Xyl, Ara, Glc, isoprimeverose and xylobiose (≈50g each)was
then added to the digest, which was subjected to PC on Whatman 3MM in ethyl
acetate/pyridine/water (9/3/2 by vol.) for 18 h (Thompson and Fry, 1997). The internal
markerswereslightlystainedwithanilinehydrogen‐phthalateandtheidentifiedspotswere
cutoutandsoakedovernightin1mlwater[containing0.5%(w/v)chlorobutanol].Then,10
ml of OptiPhase HiSafe was added, and vials were shaken continuously for 24 h.
Radioactivitywassubsequentlyassayedbycounting.
ChapterIII
114
II.d.2. [14C]Cinn consumption and incorporation into different cell
compartments
To analyse CFM samples and the protoplasmic‐like fraction, 5 and 3 ml of
scintillation liquidEcoscintA (NationalDiagnostics)was added to the sample respectively,
andradioactivitywasthenassayedbycounting.
InthecaseoftheradioactivityincorporatedintotheAIR,cellfragmentswerefirstly
resuspended in 0.2 ml of distilled water; 3 ml of scintillation liquid EcoscintA was
subsequentlyadded,andtheradioactivitywasassayedbycounting.
II.d.3.ObtainingCinn[14C]derivativesfromdiscretemaizecellcompartments
Samplesofthe0.5MNaOHcellwallfractionweresubjectedtoTLC.TLCwascarried
outonplastic‐backedsilica‐gelwitha fluorescent indicator(Merck) inbenzene/aceticacid
(9/1) (v/v). During development, TLC plates were exposed to 312 nm, which maintains
hydroxycinnamatesasrapidlyinterconvertingsinglespotsofcis/trans isomers.Standarsof
Fer,p‐Couand5‐5´‐Diferulatewereusedasexternalmarkers.
Tracks corresponding to different sampleswere cut off in pieces of 0.5x1.0 cm or
1.0x1.0cmfromtheTLCandanalysedbyscintillationcountingthroughtheadditionof3ml
ofscintillationliquidEcoscintA.
II.e.ArabidopsisIRXhomologuegeneexpressionanalysis
Total RNAwas extractedwithTrizol Reagent (Invitrogen) and reverse‐transcribed
usingtheSuperscriptIII firststrandsynthesissystemforRT‐PCR(Invitrogen).First‐strand
cDNAwasgeneratedwithanoligo(dT)20primerandusedasatemplateinsubsequentPCR
reactions.Foreachassay,severalcyclesweretestedtoensurethatamplificationwaswithin
theexponentialrange.Ubiquitinwasusedashousekeepinggene.
Primers were used for the analysis of maize genes GRMZM2G100143,
GRMZM2G059825 and gbIBT036881.1, homologues of arabidopsis IRX10 (AT1G27440),
ChapterIII
115
IRX10‐L(AT5G61840)andIRX9(AT2G37090),respectively(Boschetal.,2011).Sequencesfor
GRMZM2G100143andGRMZM2G059825werederivedfromthemaizegenomebrowserand
thatforgbIBT036881.1fromthePhytozome.
III.RESULTS
III.a.MwandMrdistributionof3H‐polysaccharides
Ingeneral,Sh1.5cellspresentedmorehomogeneouslysizedpopulationsthanthose
fromSnh,mostofthemelutingatanintermediateKavandthereforehavingalowerMw.In
addition, the proportion of 3H‐polymers and 3H‐hemicelluloses thatwere eluted in theV0
waslowerinSh1.5cells(Figure1).3H‐polysaccharidesfromSh1.5cellshadalowerMwthan
thosefromSnhinallcellcompartmentsandculturephasesanalysed,withthesoleexception
ofthoseobservedwithintheprotoplasmatlatelogarithmicphase(Figure2).
Protoplasmic3H‐polymerswereconsistedofbyahighlypoly‐dispersepopulationof
molecules,which in both cell lines showed a lowerMwwhen comparedwith the cellwall
bound 3H‐hemicelluloses or [3H]SEPs. This result indicates that they underwent massive
cross‐linkingonce incorporated into thecellwallorsloughed. In linewith this,whenboth
celllineswerecompared,Snhcellsshowedamuchmorepronounceddegreeofcrosslinking
thanSh1.5cells,independentlyoftheculturephaseconsidered(Figure2).
The general elution profiles of [3H]SEPs showed that those from Sh1.5 cells were
almost identical inboth culturephases,whereas inSnhcells agreaterproportionof them
elutedintheV0attheearlylogarithmicphaseandalowerKavwasdetected(Figure1).This
observation was confirmed by the Mw data, in which the Mw of [3H]SEPs did not vary
MaizegeneID(arabidopsishomologous)
Forwardprimer Reverseprimer
GRMZM2G100143(IRX10) GATGCAGGCTCACCTTATCC CGCTGCATGTCCTCGTAGTA
GRMZM2G059825(IRX10‐L) ACGTTGGTTCAGACCTTTGG TCATTGCCGGTGTCATAGAA
gbIBT036881.1(IRX9) TGAACTCGAGAATGCTGTGG GCTCAATTACCCACCCTTGA
ChapterIII
116
dependingontheculturephaseinSh1.5cells,whereasitdecreasedintheSnhcelllineatthe
latelogarithmicphase(Figure2).Inaddition,[3H]SEPsfromSh1.5cellshadalowerMwthan
Snhonesinbothculturephases.
Figure1.Molecularrelativeweight(Mr)distributionprofileof3H‐polysaccharides:protoplasmic3H‐polymers,
cellwallbound3H‐hemicellulosesextractedwith0.1Mor6MNaOHandSEPsobtainedfromsolidline;Snhanddashedline;Sh1.5maizecellcultures.DatashownarefromtheA:earlylogarithmicandB:latelogarithmic(seenextpage)phasesofculturecycle.Samplesusedweretaken300minafterfeedingof[3H]Ara,exceptinthecaseofprotoplasmic3H‐polymers,collected30and60minafterfeedingof[3H]AraforSnhandSh1.5respectively.3H‐
polymersand3H‐hemicellulosesweresize‐fractionatedonSepharoseCL‐4B.OnecKavisdefinedasanintervalof0.01onthex‐axis.Theprofilesarescaledsuchthattheareaunderthecurveisequal(100%)ineachframe
Kav0.0 0.5 1.0
SEPs
Kav0.0 0.5 1.0
Proportionof3H(%oftotalpercKav)
0.0
0.5
1.0
1.5
2.0
2.5
6MNaOH
0.0
0.5
1.0
1.5
2.0
2.5
0.1MNaOH
0.0
0.5
1.0
1.5
2.0
2.5
Protoplasm
0.1MNaOH
6MNaOH
SEPs
Protoplasm
0.0
0.5
1.0
1.5
2.0
2.5
3.0A
ChapterIII
117
Bothtypesof3H‐hemicellulose(0.1Mor6MNaOH‐extracted)showedalowerMwin
Sh1.5 cells (Figure 2). In Snh cells, the Mw of 0.1 M NaOH‐extracted wall bound 3H‐
hemicellulosesdidnotvaryaccordingtotheculturephasewhereasitappearedtodecrease
Kav0.0 0.5 1.0
SEPs
Kav0.0 0.5 1.0
0.0
0.5
1.0
1.5
2.0
2.5
6MNaOH
0.0
0.5
1.0
1.5
2.0
2.5
Protoplasm
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.1MNaOH
0.0
0.5
1.0
1.5
2.0
2.5
Protoplasm
0.1MNaOH
6MNaOH
SEPs
Proportionof3H(%oftotalpercKav)
B
ChapterIII
118
atthelatelogarithmicinSh1.5cells(Figure2).Likewise,thegeneralelutionprofileforthis
type of 3H‐hemicellulosewas very similar in the case of Snh cells in both culture phases,
whereasSh1.5cellsshowedaslightlygreaterproportionof3H‐moleculeselutinginahigher
Kav at the late logarithmic phase (Figure 1). In the case of 6 M NaOH‐extracted 3H‐
hemicelluloses, theirMwbarely increased inSnhcells,whereas inSh1.5 cells itonceagain
decreased(Figure2).Thesefindingswererepeatedinthegeneralelutionprofiles,wherean
increaseintheproportionof3HdetectedatalowerKavwasobservedatthelatelogarithmic
phaseinSnhcells,whereasitdecreasedinSh1.5cells(Figure1).
III.b.Mrdistributionof3H‐labelleddiagnosticfragmentsfrom3H‐polysaccharides
AfterDriselasedigestion,theelutionprofileshapesofpolymerscontaining[3H]Ara,
[3H]Xyl or [3H]xylobiose were similar to the general ones, indicating the solidity of the
experimentalapproach(Figures3,4and5vsFigure1).Ingeneralterms,elutionprofilesof
polymerscontaining[3H]Ara,[3H]Xylor[3H]xylobioseshowedsimilarMrdistributions.This
findingsuggeststhatthesediagnosticfragmentswereadigestionproductofauniquetypeof
polysaccharide, putatively [3H]arabinoxylan. Reinforcing this idea was the disparity
observedbetweenthese,andfragmentsfrompolymerswhichcontained[3H]isoprimeverose,
adiagnosticfragmentfor[3H]xyloglucan.Undigested3H‐polymerswerefoundinalldigested
fractions,mostofthemelutinginaKavrangingfrom0to0.5(Figures3,4and5).
III.b.1. Mr distribution of 0.1 M NaOH‐extracted hemicelluloses diagnostic
fragments
The elution profiles of polymers which contained [3H]Ara residues and those
characteristicfor[3H]xylans([3H]Xyland[3H]xylobiose)wereverysimilarinbothcelllines
and culture stages. However, the elution profiles of [3H]isoprimeverose ([3H]xyloglucan)
differedfromthosecharacteristicof[3H]xylansand[3H]Ara‐containingpolymers(Figure3).
Mr distributions of [3H]Ara‐containing polymers and 3H‐labeled diagnostic fragments for
ChapterIII
119
xylansinbothculturephases,andthoseforundigested3H‐polymersattheearlylogarithmic
phase,wereverysimilartothegeneralelutionprofileshowninFigure1.
Mrdistributionsof[3H]Araresidues,[3H]Xyland[3H]xylobiosefromSnhcellsdidnot
varysubstantiallyaccordingtoculturephase. InthecaseofSh1.5cells, theelutionprofiles
for these diagnostic fragments aswell as [3H]isoprimeverose for [3H]xyloglucan showed a
slightlygreaterproportionofmoleculeswithalowMw(elutingKavrangingfrom0.5to1)at
thelatelogarithmicphaseofgrowth.Theseobservationsareinagreementwiththeresultsof
theestimatedMwshowninFigure2.
III.b.2. Mr distribution of 6 M NaOH‐extracted hemicelluloses diagnostic
fragments
InSnhcells, theelutionprofilesofpolymerscontaining [3H]Araresiduesand those
for [3H]xylans ([3H]Xyl and [3H]xylobiose) and [3H]xyloglucan ([3H]isoprimeverose) were
very similar in both culture stages (Figure 4). In contrast, the elution profile of
[3H]xyloglucan from Sh1.5 cells was different from that characteristic for [3H]xylans. The
undigested 3H‐polymers profile at early logarithmic phase was more similar to
[3H]xyloglucan, and at late logarithmic phase to polymers containing [3H]Ara, [3H]Xyl and
[3H]xylobiose. Inbothcell lines, theMrdistributionofpolymerswith [3H]Araresiduesand
3H‐labelled diagnostic fragments for xylans mimicked the general elution profile of 6 M
NaOH‐extractedhemicelluloses(Figure4vsFigure1).
Mostofthepolymerswhichcontained[3H]Ara,[3H]Xyland[3H]xylobioseaswellas
[3H]isoprimeveroseandundigested 3H‐polymersextracted fromboth cell lines, eluted in a
Kavrangingfrom0to0.4inbothculturephases.Inaddition,theproportionof3Hdetectedat
theV0waslowinbothcelllinesandculturephases(Figure4).Nevertheless,aremarkable
differencewas found in the elution profile of [3H]xyloglucan and undigested 3H‐polymers
fromSh1.5 cells, inwhich amarked elutionpeakwas observednear aKav of 0.1 at early
logarithmicphaseofculture.
ChapterIII
120
III.b.3.MrdistributionofSEPs
Inbothcelllines,theelutionprofilesof[3H]Ara,[3H]Xylor[3H]xylobiose‐containing
polymers were very similar (Figure 5 vs Figure 1), suggesting that [3H]SEPs are mainly
constitutedbyarabinoxylanssloughedintotheculturemedium.Agreaterproportionof3H‐
moleculeswithaslightlyhigherMwatearlylogarithmicphasewasdetectedinbothcelllines
whencomparedwiththelatelogarithmicstageoftheculturecycle(Figure5).
Protoplasmic
0.1MNaOH
6MNaOH SE
Ps0
500
1000
1500
2000
Mw(kDa)
0
500
1000
1500
2000
2500
Figure2.Relativeaveragemolecularmass(Mw)ofprotoplasmic3H‐polymers(Protoplasmic),cellwallbound3H‐hemicellulosesextractedwith0.1M(0.1MNaOH)or6M(6MNaOH)NaOHandSEPsobtainedfromA:SnhandB:Sh1.5maizecellcultures.Datashownarefromthegreybars;earlylogarithmicandblackbars;latelogarithmicphasesofculturecycle.Samplesusedweretaken300minafterfeedingof[3H]Ara,exceptinthecaseofprotoplasmic3H‐
polymers,collected30and60minafterfeedingof[3H]AraforSnhandSh1.5respectively
B
A
ChapterIII
121
Figure3.Mrdistributionprofileof3H‐labeleddiagnosticfragmentsfrom3H‐polysaccharides:cellwallbound3H‐hemicellulosesextractedwith0.1MNaOHobtainedfromsolidline;Snhanddashedline;Sh1.5maizecellcultures.DatashownarefromtheA:earlylogarithmicandB:latelogarithmic(seenextpage)phasesofculturecycle.
Samplesusedweretaken300minafterfeedingof[3H]Ara.OtherdetailsasinFigure1
Xyloglucan(Isoprimeverose)
0
1
2
3
4
5
Ara
Proportionof3H(%oftotalpercKav)
0
1
2
3
4
5
6
Xylan(Xyl)
0
1
2
3
4
5
Xylan(xylobiose)
0
1
2
3
4
5
Xyloglucan(Isoprimeverose)
Undigestedpolymers
Kav0.0 0.5 1.0
Ara
Xylan(Xyl)
Xylan(xylobiose)
Undigestedpolymers
Kav0.0 0.5 1.0
0
1
2
3
4
5
A
ChapterIII
122
Ara
Xylan(Xyl)
Xylan(xylobiose)
Xyloglucan(Isoprimeverose)
Undigestedpolymers
Kav0.0 0.5 1.0
Ara
Proportionof3H(%oftotalpercKav)
0
1
2
3
4
5
6
Xylan(Xyl)
0
1
2
3
4
5
Xyloglucan(Isoprimeverose)
0
1
2
3
4
5
Undigestedpolymers
Kav0.0 0.5 1.0
0
1
2
3
4
5
Xylan(xylobiose)
0
1
2
3
4
5
B
ChapterIII
123
In Snh cells, the elution profiles of the different diagnostic fragments at early
logarithmicphaseweresimilar,mainlyelutingataKavrangingfrom0to0.5andattheV0
(Figure5A).However,Mrdistributionoftheundigested3H‐polymersandthepolymersfrom
which[3H]xylobiosewasreleasedshowedahigherdegreeofsimilaritybetweenthemthan
withtheremainingprofiles.
At late logarithmic phase, Snh elution profiles differed from those observed at the
early logarithmic culture stage described above (Figure 5B). The elution profiles of
[3H]xylans, ([3H]Xyl and [3H]xylobiose diagnostic fragments) were similar and molecules
mainly eluted in a Kav ranging from 0.1 to 0.4. Similarly, the elution profiles of
[3H]xyloglucan ([3H]isoprimeverose) and undigested 3H‐polymerswere alike. Surprisingly,
theelutionprofileof 3H‐polymerswhich contained [3H]Ara residueswas for the first time
different to those for [3H]xylans, andshowedawidedistributionamong thedifferentKav,
withtwowell‐defined“elutionareas”.Thefirstofthese, inaKavfrom0.2to0.4,coincided
with that observed for the [3H]xylan diagnostic fragments, suggesting a presumable
sloughingofarabinoxylans.However,thesecondelutionareawasdetectedinaKavranging
from0.5 to0.8,anobservation thatwould indicate therelease into theculturemediumof
anothertypeof3H‐moleculepopulationcontaining[3H]Araresidues.Thiswasnotobserved
intheSh1.5cells, inwhichtheelutionprofileofpolymerscontaining[3H]Araresidueswas
verysimilartothosefor[3H]xylans.
In Sh1.5 cells, the digestion of [3H]SEPs with Driselase showed that all the 3H‐
polymers (thosewhich contained [3H]Ara, thosewith diagnostic fragments for [3H]xylans
and[3H]xyloglucanandtheundigested3H‐polymers)elutedinahigherKavthanthosefrom
Snh cells, and therefore had a lowerMw (Figure 5). Thiswas especially clear at the early
logarithmic phase (Figure 5A). The most remarkable difference was found in the elution
profile of [3H]xyloglucan and undigested 3H‐polymers at late logarithmic culture phase
(Figure 5B), where a marked peak was observed close to a Kav of 0.3, similar to that
ChapterIII
124
previouslydescribed for theelutionprofileof6MNaOH‐extracted [3H]xyloglucanat early
logarithmicphase.
III.c.Analysisofphenolicmetabolism
III.c.1.Uptakeof[14C]Cinnfromtheculturemediumanditsincorporationinto
cellcompartments
No differences in [14C]Cinn consumptionwere observed amongmaize cell lines or
culturephases(Figure6).Ingeneral,consumptionpercentagesof[14C]Cinnrangedbetween
40%‐55%ofthetotal[14C]Cinnaddedattimezero.Mostofthe[14C]Cinnconsumption(90%)
occurredduringthefirst15minafter[14C]Cinnwasfed.
The kinetics of [14C]Cinn incorporation into the protoplasmic‐like fraction and cell
wall compartment did not show major differences between either cell lines or culture
phases,andaswasexpected, itsincorporationintotheprotoplasmic‐likefractionpreceded
its incorporation into the cell wall. However, relative incorporation of [14C]Cinn into the
Sh1.5 protoplasmic‐like fraction and cell wall was, on average, half‐fold reduced when
comparedwithSnhcells(Figure6).
III.c.2.[14C]Cinnmetabolisminthecellwall
Ananalysisofhydroxycinnamate14C‐labelledmetabolites incorporatedintothecell
wallduringearly logarithmicphaseofgrowth isshowninFigures7and9A.The [14C]Cinn
added to the culture medium was rapidly (1‐3 min) metabolised to p‐[14C]Cou and then
incorporated into the cellwall. In both cell lines, a decreasewas observed in the relative
incorporation of p‐[14C]Cou into the cell wall the first 30 min after [14C]Cinn feeding.
Concomitantlythe[14C]Feresterifiedintothecellwall,abruptly increasinguntilreachinga
plateauphase.Furthermore,at30to60minafter[14C]Cinnfeedingadecreasewasdetected
intheamountofcellwall‐esterified[14C]Fer.Thiswasobservedinbothcelllines,althoughit
wasespeciallymarkedinSnhcells.
ChapterIII
125
Figure4.Mrdistributionprofileofcellwallbound3H‐hemicellulosesextractedwith6MNaOHobtainedfromsolidline;Snhanddashedline;Sh1.5maizecellcultures.DatashownarefromtheA:earlylogarithmicandB:latelogarithmic(seenextpage)phasesofculturecycle.Samplesusedweretaken300minafterfeedingof[3H]Ara.
OtherdetailsasinFigure1
Ara
Proportionof3H(%oftotalpercKav)
0
1
2
3
4
5
6
Xylan(Xyl)
0
1
2
3
4
5
Xylan(xylobiose)
0
1
2
3
4
5
Ara
Xylan(Xyl)
Xylan(xylobiose)
Xyloglucan(Isoprimeverose)
Undigestedpolymers
Kav0.0 0.5 1.0
Xyloglucan(Isoprimeverose)
0
1
2
3
4
5
Undigestedpolymers
Kav0.0 0.5 1.0
0
1
2
3
4
5
A
ChapterIII
126
Ara
0
1
2
3
4
5
6
Xylan(Xyl)
0
1
2
3
4
5
Xylan(xylobiose)
Proportionof3H(%oftotalpercKav)
0
1
2
3
4
5
Xyloglucan(Isoprimeverose)
0
1
2
3
4
5
Undigestedpolymers
Kav0.0 0.5 1.0
0
1
2
3
4
5
Ara
Xylan(Xyl)
Xylan(xylobiose)
Xyloglucan(Isoprimeverose)
Undigestedpolymers
Kav0.0 0.5 1.0
B
ChapterIII
127
Figure5.Mrdistributionprofileof[H3]SEPsobtainedfromtheculturemediumofsolidline;Snhanddashedline;Sh1.5maizecellcultures.DatashownarefromtheA:earlylogarithmicandB:latelogarithmic(seenextpage)phasesofculturecycle.Samplesusedweretaken300minafterfeedingof[3H]Ara.OtherdetailsasinFigure1
Ara
Proportionof3H(%oftotalpercKav)
0
1
2
3
4
5
6
Xylan(Xyl)
0
1
2
3
4
5
Xylan(xylobiose)
0
1
2
3
4
5
Ara
Xylan(Xyl)
Xyloglucan(Isoprimeverose)
Undigestedpolymers
Kav0.0 0.5 1.0
Xyloglucan(Isoprimeverose)
0
1
2
3
4
5
Undigestedpolymers
Kav0.0 0.5 1.0
0
1
2
3
4
5
Xylan(xylobiose)
A
ChapterIII
128
Ara
Proportionof3H(%oftotalpercKav)
0
1
2
3
4
5
6
Xylan(Xyl)
0
1
2
3
4
5
Xylan(xylobiose)
0
1
2
3
4
5
Xyloglucan(Isoprimeverose)
0
1
2
3
4
5
Undigestedpolymers
Kav0.0 0.5 1.0
0
1
2
3
4
5
Ara
Xylan(Xyl)
Xylan(xylobiose)
Xyloglucan(Isoprimeverose)
Undigestedpolymers
Kav0.0 0.5 1.0
B
ChapterIII
129
Figure6.Kineticsofconsumptionandincorporationofradioactivityfromexogenous[14C]Cinnamicacid
([14C]Cinn)inA,B:SnhandC,D:Sh1.5maizecellsuspensions.DatafromtheA,C:earlylogarithmicandB,D:latelogarithmicphasesofgrowthareshown.The[14C]Cinnconsumption()wascalculatedfromtheradioactivityremainingintheculturemediumafter[14C]Cinnfeeding.Theincorporationof[14C]‐labeledcompoundsintheprotoplasmic‐likefraction()andAIR()isrepresented.Thevaluesshowedaretheaverage±standard
deviation(n=3)
In all cases, [14C]Fer was the main cell wall hydroxycinnamate detected, although
when both cell lines were compared, the relative quantification of Cinn [14C]derivatives
indicatedthatSh1.5cellwallswereenrichedinp‐[14C]Couandimpoverishedin[14C]Fer in
comparison with Snh cell walls. 14C‐diferuloyl groups and 14C‐oligomers (compounds that
migrateatlowRFregions)weredetectedinbothcelllines30‐60minafter[14C]Cinnfeeding,
depending on the cell culture phase. The increase in 14C‐diferulates and 14C‐oligomers
occurredconcurrentlywithadecrease in theamountof [14C]Fer. Inaddition, theirrelative
quantificationindicatedthat[14C]Fer,14C‐dimersand14C‐oligomersweremoreabundantin
Sh1.5cellwalls.Lastly,andalsoattheearlylogarithmicphase,thesupplyofH2O2provided
120minafter[14C]Cinnfeedinginducedanincreaseinthedimerisationoroligomerisationof
[14C]ferulate,occurringtoasimilardegreeinbothcelllines.
A
[14C]Cinnincorporation
(%oftotalmCi)
0
10
20
406080
100 B
C
Time(min)0 20 40 60 80 100 120
0
10
20
406080
100D
0 20 40 60 80 100 120Time(min)
ChapterIII
130
No differences were found in the kinetics of Snh cell wall coumaroylation and
feruloylation between growth phases (Figures 7 and 9A vs 8 and 9B). However, some
differenceswereobservedinthecaseofhabituatedcells:Sh1.5cellwallsaccumulatedmore
p‐[14C]Cou, 14C‐dimers and 14C‐oligomers at the late logarithmic phase (Figures 8 and9B).
Accordingly, therelativedecrease in [14C]Ferwasmorepronounced in the late logarithmic
thanintheearlylogarithmicphase.Lastly,theadditionofH2O2didnotincreasethelevelof
[14C]ferulatedimerisationoroligomerisation.
III.d.ArabidopsishomologueIRXgeneexpressionanalysis
TherelativeexpressionofthemaizegenesGRMZM2G100143,GRMZM2G059825and
gbIBT036881.1, homologues of arabidopsis IRX10, IRX10‐L and IRX9, respectively, was
analysedbyRT‐PCR, shown inFigure10. InSh6cells, theexpressionofarabidopsis IRX10
andIRX9homologueswasinduced,whereasadecreasewasdetectedintheexpressionofthe
arabidopsis IRX10‐L homologue when compared with Snh cells. In Sh1.5 cells, a reduced
expressionofthearabidopsisIRX10homologuewasobserved.
IV.DISCUSSION
IthaspreviouslybeenreportedthatDCB‐habituatedmaizecellsconstituteasuitable
cellularmodeltoinvestigateheteroxylanmetabolism,sincetheircopingstrategiesarebased
on altered arabinoxylan networks which show a certain degree of response plasticity
depending on the habituation level (Mélida et al., 2009; de Castro et al., 2013; Chapter I).
Likewise, previous studies on maize cells habituated to high DCB levels have described
compensating strategies based on a reinforced hemicellulosic network acquired by more
cross‐linked arabinoxylanswith a higherMw and lower extractability (Mélida et al., 2009;
2011).Theseresultsareincontrasttothoseobservedinmaizecellsuspensionshabituated
to lowDCB levels (Sh1.5), inwhich the feedingof [3H]Ararevealeda lowerproportionsof
ChapterIII
131
stronglywall‐bound3H‐hemicellulosesandanincreasein[3H]SEPs,aswellasaslowerand
lessefficientmetabolismofpolysaccharidesthroughouttheentireculturecycle(ChapterII).
Figure7.Thinlayerchromatography(TLC)of[14C]‐labeledcompoundsesterifiedontheAIRaftertheadditionofexogenous[14C]CinntoA:SnhandB:Sh1.5maizecellssuspensionsattheearlylogarithmicphaseofculturecycle.Theposition(―)ofp‐Coumaricacid(p‐Cou),Ferand5,5’‐dehydrodiferulate(Di‐Fer)usedas
externalmarkersisshown.CompoundswithlowRF(Rdiferulate0.0‐0.9)areregardedasoligomers(Fryetal.,2000).TheinsertgraphicsshowwithdetailtheoligomersandFerdimersappearance.+60H2O2minshowsa
treatmentfor60minwith40mMH2O2,120minafter[14C]Cinnfeeding
t=5min
1000
2000
3000
t=30min
1000
2000
3000
+60min
H2O2
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1000
2000
3000+60min
H2O2
Distancefromtheorigin(cm)0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1000
2000
3000
t=30min
1000
2000
3000
t=60min
1000
2000
3000
t=120min
1000
2000
3000
t=1min
1000
2000
3000
4000
t=3min
1000
2000
3000
t=5min
1000
2000
3000
At=1min
1000
2000
3000
4000B
1000
2000
3000
1000
2000
3000
1000
2000
3000t=3min
t=60min
t=120min
0 1 2 3 4 5 6
100
200
300
400
500
0 1 2 3 4 5 6
100
200
300
400
500
0 1 2 3 4 5 6
100
200
300
400
500
0 1 2 3 4 5 6
100
200
300
400
500
p‐Cou Fer Fer
Oligomers
p‐Cou
Oligomers
dpm
Di‐Fer
Oligomers Di‐Fer
Oligomers
Oligomers
Oligomers
Di‐Fer
Di‐Fer
Di‐Fer
Di‐Fer
p‐Cou
p‐Cou
p‐Cou
p‐Cou
p‐Cou p‐Cou
p‐Cou
p‐Coup‐Cou
p‐Cou
p‐Cou
p‐Cou
FerFer
FerFer
Fer Fer
Fer
Fer Fer
Fer
FerFer
ChapterIII
132
Figure8.TLCof[14C]‐labeledcompoundsesterifiedontheAIRaftertheadditionofexogenous[14C]CinntoA:SnhandB:Sh1.5maizecellsuspensionsatthelatelogarithmicphaseofculturecycle.Theposition(―)ofp‐
Cou,FerandDi‐Ferusedasexternalmarkersisshown.CompoundswithlowRF(Rdiferulate0.0‐0.9)areregardedasoligomers(Fryetal.,2000).TheinsertgraphicsshowwithdetailtheoligomersandFerdimersappearance.+60H2O2minshowsatreatmentfor60minwith40mMH2O2,120minafter[14C]Cinnfeeding
t=1min
2000
4000
6000
8000
t=30min
dpm
2000
4000
6000
8000
t=60min
2000
4000
6000
8000
t=120min
2000
4000
6000
8000
+60min
H2O2
0 1 2 3 4 5 6 7 8 9 10 11 12
2000
4000
6000
8000
t=3min
2000
4000
6000
8000
t=5min
2000
4000
6000
8000
t=30min
2000
4000
6000
8000
t=5min
2000
4000
6000
8000
t=3min
2000
4000
6000
8000
0 1 2 3 4 5
200
400
600
800
1000
t=60min
2000
4000
6000
8000
t=120min
2000
4000
6000
8000
+60
H2O2
0 1 2 3 4 5 6 7 8 9 10 11 12
2000
4000
6000
8000
0 1 2 3 4 5
200
400
600
800
1000
0 1 2 3 4 5
200
400
600
800
1000
0 1 2 3 4 5
200
400
600
800
1000
t=1min
2000
4000
6000
8000
A B
Distance from the origin (cm)
p‐Cou Fer p‐Cou Fer
Oligomers
Oligomers
Oligomers
Oligomers
Oligomers
Di‐Fer
Di‐Fer Di‐Fer
Di‐FerDi‐Fer
p‐Cou
p‐Coup‐Cou
p‐Coup‐Cou
p‐Cou
p‐Cou
p‐Cou
p‐Cou
p‐Cou
p‐Cou
p‐Cou
Fer
Fer
Fer
FerFer
FerFer
Fer
Fer
Fer
Fer
Fer
ChapterIII
133
Figure10.RelativeexpressionofmaizegenesorthologuesofseveralIRXgenesfromarabidopsisanalyzedbyRT–PCRofSnh,Sh1.5andhabituatedto6µMDCB(Sh6)maizecellsuspensions.:moremRNAaccumulationthan
control(Snh);lessmRNAaccumulationthancontrol(Snh).ZmUBI:Ubiquitinegeneexpression.Ubiquitinewasusedasthehousekeepinggeneduetoitsconstitutiveexpression
0 20 40 60 80 100 120 140 160 180 200
Fer
Dimers
andoligomers
Timeafterthefeedingof[14C]Cinn(min)0 20 40 60 80 100 120 140 160 180 200
[14 C]Cinnincorporation
(%oftotaldpm
)
0
20
40
60
80
p‐Cou
0
20
40
60
80
100
Fer
0
20
40
60
80
A B
p‐Cou
Dimers
andoligomers
Figure9.Kineticsofincorporationof[14C]‐labelledcompoundsesterifiedonAIRaftertheadditionofexogenous[14C]Cinntofilledcircle;Snhandopencircle;Sh1.5maizecellsuspensionsattheA:earlylogarithmicandB:late
logarithmicphaseofculturecycle.[14C]‐labelledcompoundswereseparatedbyTLCandassayedforradioactivitybyscintillationcounting.Dataareexpressedaspercentoftotalpolymer‐esterifiedderivatives.Arrowindicatedthe
additionofH2O2(40mMfinalconcentration)
ChapterIII
134
Thisfindingwasespeciallysurprising,andtherearetwopossiblehypotheseswhich
couldexplainthisalteredcellularfate:itmaybecausedby1)areductionin“bindingpoints”
to cellulose and/or 2) a reduced capacity to incorporate polysaccharides, thought to be
mainlyarabinoxylans,intothecellwallviaextra‐protoplasmicphenoliccross‐linking.
As cross‐linking is closely related to the Mw of polysaccharides and to phenolic
metabolism, bothwere investigated in the present study, in several cell compartments of
Sh1.5 cells throughout the culture cycle. This was achieved through in vivo feeding
experimentswithradio‐labelledprecursors([3H]Araand[14C]Cinn,respectively),sinceuse
of this methodology has been successful in comparable experimental studies (Fry et al.,
2000;KerrandFry,2003;BurrandFry,2009;Mélidaetal.,2011).
The first question to arise was whether low DCB habituation levels entailed
modificationsintheMwofhemicellulosesandpolymers.Ingeneral,Sh1.5cellsshowedmore
homogeneously sized and smaller 3H‐polysaccharides populations thatweremore similar
among cell compartments, regardless of the culture phase, than those from Snh. These
results,andthesuitabilityoftheexperimentalapproach,weresupportedbytheobservation
thatthesetrendswereconsistentintheelutionprofilesofthedifferentdiagnosticfragments
(unique for [3H]xyloglucan, [3H]xylans, polymers which containing [3H]Ara and those
undigestedbyDriselase)andwereperfectlyillustratedinthecaseof[3H]SEPs.Theselatter
polymers are sloughed into the extracellular medium and no extraction treatment is
required inorder toobtain them; theirmolecular integrity ismaintained intact.Therefore,
thedataobtainedfromthestudyof[3H]SEPsareespeciallyvaluableprovidinginformation
aboutfeaturesofpolysaccharidesintheirnativestructure(KerrandFry,2003).Inaddition,
some enzymes, substrates such as H202 and inhibitors of cross‐linking (Encina and Fry,
2005),alsodiffuse into theextracellularmedium(BurrandFry,2009). 3H‐polysaccharides
fromSh1.5cellsalsohadalowerMwinallcellcompartments,independentlyoftheculture
cyclestage.AcomparisonoftheMwfromprotoplasmic3H‐polymersandcellwallbound3H‐
hemicelluloses indicated that after secretion into the cell wall, protoplasmic 3H‐polymers
ChapterIII
135
underwent massive cross‐linking in both cell lines, although this increase was much less
pronounced in Sh1.5 cells. This increase in the Mw of hemicelluloses once they were
integratedintocellwallhasalreadybeendescribedinmaizecellsuspensions(KerrandFry,
2003),suggesting theoccurrenceofcellwallgraftingprocesses.Theseprocessescomprise
the formation of non‐covalent and covalent interactions among molecules. Non‐covalent
cross‐links include the formation of hydrogen and ionic bonds, whereas covalent
interactions are mainly produced by oxidative coupling or glycosidic bonds catalysed by
transglycosylation reactions (Fry, 2000). It has been reported that xyloglucan becomes
glycosidic‐linked to acidicpectins in rose cultured cells (ThompsonandFry,2000)during
wall binding. In addition, the existence has recently been demonstrated of heteroxylan
transglycosylase in, which can use a wide range of xylans from different species as the
acceptor substrate, including wheat arabinoxylans (Johnston et al., 2013). Although this
heteroxylan transglycosylasewas isolated from the dicotCaricapapaya,which presents a
typeIcellwall,sinceitcanusearabinoxylanfromagrasslikewheat(whichhastypeIIcell
walls)assubstrate,theoccurrenceofthistypeoftransglycosylationreactioninmaizecells
cannot be ruled out. Nevertheless, it is to be expected that hydrogen and ester bonds or
oxidativecouplingcross‐linkswouldberesponsibleofcellwallgraftingprocessesinvolving
arabinoxylanmolecules.However,theincreasedetectedintheMwof3H‐hemicellulosesand
3H‐polymersinSh1.5cellwallswasmuchlesspronouncedthaninSnh.
Apossible explanation for these findings is that cells habituated to lowDCB levels
synthesised3H‐moleculeswithalowerandsimilarMwasastrategytocopewiththelackof
cellulose,consideredas“mild”whencomparedwiththatobservedincellshabituatedtohigh
DCB levels (33% vs 75% of reduction compared with Snh). The pattern observed in the
expressionofarabidopsisIRXorthologuegenesinhabituatedcells(Sh1.5andSh6)supports
this hypothesis. As mentioned above, cells habituated to high DCB levels synthesised
hemicelluloseswithahigherMwinordertocounteracttheirimpoverishmentincellulose.In
good agreementwith this, the expression of genes involved in the xylan elongation chain
ChapterIII
136
(Brownetal.,2009;Wuetal.,2009)(GRMZM2G100143orthologueofAtIRX10;Boschetal.,
2011)aswellasgbIBT036881.1,theorthologueofAtIRX9(Boschetal.,2011)involvedinthe
initiationof thexylansynthesis(Brownetal.,2007;Penaetal.,2007)wasfoundtobeup‐
regulated in Sh6 cells. Interestingly, in cells habituated to low‐DCB levels (Sh1.5),
GRMZM2G100143 (AtIRX10orthologue)was foundtoberepressedwhereas theexpression
of gbIBT036881.1 (AtIRX9 orthologue) remained unaltered. Such preliminary results may
indicatethatSh1.5cells,donotinfactpresentadefectiveinitiationofxylansynthesisbutare
impaired in the elongation of hemicellulose chains. This would lead to the production of
shorterxylanchainsinSh1.5cells,contrarytowhathappensinSh6.
In addition, a previous study on maize cells habituated to medium and high DCB
levelsrevealeddifferences in theMwofwall‐boundhemicelluloses (Mélidaetal.,2009). In
thisstudy,polysaccharidesweresubsequentlyextractedfromthecellwallusingtwoalkali‐
treatments, 0.1 M KOH and 4 M KOH, known to solubilise weakly‐ and firmly‐bound
polysaccharide populations respectively. The results showed that in medium DCB‐
habituationlevels,theMwof0.1MKOH‐extractedhemicellulosesincreasedwithrespectto
the same fraction obtained fromnon‐habituated cells, cells habituated to highDCB levels,
and even with respect to 4 M KOH‐extracted hemicelluloses. Interestingly, in maize cells
habituatedtohighDCB levels, theMwof4MKOH‐extractedhemicelluloses increasedwith
respect to non‐habituated cells, cells habituated to medium DCB levels and to those
solubilised by 0.1 M KOH treatment. It is probable that, as the level of DCB habituation
increases,aprogressiveinputofhigherMwhemicellulosesthataremoretightlyboundtakes
place in order to reinforce the cellwalls. However, it should be borne inmind that these
resultswereobtainedusingadifferentexperimentalapproachtoours.Themaizecellsused
inMélidaet al. (2009)were collectedat anadvancedstageof the culture cycle.Thus, it is
feasibletoassumethathemicellulosesinsuchwallshavebeentherefora longerperiodof
time,andhavepresumablebeensubjectedtograftingprocesswithothermolecules,which
would lead to an increase in their Mw. Furthermore, it is important to note that massive
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cross‐linkingofhemicellulosesinmaizecellsuspensionculturesoccurred11‐daysaftersub‐
culturing (Burr and Fry, 2009). In contrast, our experimental approach involved a pulse‐
chaseexperiment,inwhichthescreenedmoleculeswereformed,integratedintothewallor
sloughedoverashortperiodof time(5h).Despitethis, the ideathatmaizecellsgradually
requirelessextractableandhigherMwhemicellulosesastheDCBhabituationlevelincreases
is ingoodagreementwithobservations inprevious studies,where cellshabituated to low
andmediumDCBlevelsseemedtocompensatefortheirlackincellulosethroughanincrease
inhemicellulosesextractedwithmildalkalitreatment(0.1MKOH)(Mélidaetal.,2009;de
Castro et al., 2013; Chapter I). The finding that Sh1.5 cells had molecules, and more
specifically hemicelluloses, with a lower Mw than those from Snh may indicate that cells
habituatedtolowDCBlevelsincreasetheamountoflowMwhemicellulosesasapartoftheir
copingstrategy.Inthisrespect,ithaspreviouslybeensuggestedthatadecreaseintheMwof
xyloglucanimproves itscapacitytobindtocellulose(Limaetal.,2004),and inaccordance
withthis,ithasbeenfoundthatxyloglucanfromcellulose‐deficientbeancellshasalowerMw
thanthatofcontrolcells(Alonso‐Simónetal.,2007).Inthislattercase,itwashypothesised
that smaller xyloglucan molecules together with an increased xyloglucan‐endo‐
transglucosylase activity would produce a more rigidified cell wall (Alonso‐Simón et al.,
2007).However, it should be borne inmind that this findingwas described in bean cells,
which have a type I cell wall. Therefore, it still remains unclear whether thismechanism
couldplayanykindofroleincellswithatypeIIcellwall.
AnotherpossibleexplanationiscloselyrelatedtothecapacityofSh1.5cellstocross‐
link cell wall hemicelluloses and SEPs. It should be borne in mind that, as previously
mentioned,Sh1.5cellsshowedareducedamountofcellwallbound3H‐hemicellulosesand
anincreasedpresenceof[3H]SEPsintheculturemedium(ChapterII).Inaddition,thetiming
of cross‐linking may also be delayed in Sh1.5 cells due to their slower and less efficient
metabolism(ChapterII)thuspreventingdetectionduringthetimewindowemployedinthis
experiment(5h).Inordertoexplorethesepossibilities,ananalysiswasperformedofCinn
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metabolismaswellasacharacterisationofitsderivativesinthecellwall.First,nomarked
differences were detected in the capacity to uptake [14C]Cinn from the culture medium
dependingonthecell line,afindingthatit is inagreementwithobservationsinaprevious
study onmaize cells habituated to highDCB levels and employing a similar experimental
approach(Mélidaetal.,2011).Nevertheless,SnhandSh1.5cellsshoweddifferencesintheir
capacity to incorporate [14C]Cinn into the protoplasm and cell wall; habituated cells
presented a deficient capacity for the biosynthesis and incorporation into the cell wall of
[14C]feruloylated/coumaroylated molecules. Similar altered kinetics in Sh1.5 cells were
observedwhen[3H]Arawasfedastheradioactiveprecursor,showingthatadelayedcellular
traffic and cellwall binding of 3H‐hemicelluloses and 3H‐polymers tookplace (Chapter II).
However, and despite this reduced capacity, the relative content in ferulate dimers and
oligomerswashigher inSh1.5cells.Theseresultsare inaccordancewith thosepreviously
observedinmaizecellshabituatedtohighDCBlevels(Mélidaetal.,2010b;2011)supporting
thehypothesisthatagreatercross‐linkednetworkofhemicelluloses,mainlyarabinoxylans,
wouldbecrucial for thecellwall reinforcingstrategy inDCB‐habituatedmaizecells.Thus,
theresultssuggestthatthelowerMwofhemicellulosesandpolymersdetectedinSh1.5cells
wasnotrelatedtoalowerdegreeofcross‐linkagethroughFer.Alternatively,hemicelluloses
andpolymersfromhabituatedcellswereinfactshorter,albeitextensivelycross‐linked.
Another possibility could be that dimerisation (or oligomerisation) of Fer residues
contributed to the formation of intra‐polymeric loops to a greater extent. Consequently,
Sh1.5hemicelluloseswouldbedepositedon the cellwall asa coagulum (Fryet al., 2000).
However,anegativeassociationbetweenintra‐polymericarabinoxylancross‐linkingandcell
wall reinforcement has been suggested (Fry et al., 2000). Consequently, this latter finding
wouldrenderthispossibilityunlikelyinthescenarioofcellwallrigidification.
ChangesintheMwofmaizecellwallhemicellulosesandpolymersduringtheculture
cyclehavebeendemonstrated,whichisnotsurprisingsincecellswouldneedtomodifythem
inordertoalloworrestrictcellexpansion(KerrandFry,2003).Inaddition,ferulatecross‐
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139
linkinginmaizecellshasalsobeenshowntovarythroughouttheculturecycle,accordingto
the physiological condition of the cells (Burr and Fry, 2009). In this respect, and as a
precedent,differencesinthephenolicprofileandmetabolismofcellshabituatedtohighDCB
levels havepreviously been reported as the culture cycleprogresses (Mélida et al., 2011).
Thus, the study of how the Mw of hemicelluloses and polymers as well as the phenolic
metabolism vary during the culture cycle would be of interest in the case of maize cells
habituatedtolowDCBlevels,whichhavebeendemonstratedtopresentalteredpatternsand
kinetics of growth (de Castro et al., 2013; Chapter I), as well as a delayed metabolism
throughouttheentirecycle(ChapterII).
Generally, the elution profiles of Driselase‐digested diagnostic fragments for
polymers containing [3H]Ara and those for [3H]xylans were very similar, although Ara
residues can derive from other polymers besides arabinoxylans, such as
rhamnogalacturonantypeI,orarabinogalactanproteins.However,asugaranalysisof0.1M‐
and6Malkali‐extractedfractionsfromSh1.5cellwallsrevealedalowcontentofuronicacid,
GalandRharesidues (deCastroet al.,2013;Chapter I).Therefore, it canbeassumed that
[3H]Ara, [3H]Xyl and [3H]xylobiose are, in fact, digestion products of putative
[3H]arabinoxylanmolecules.
In both, Snh and Sh1.5 cell lines, a greater proportion of smaller molecule
populationsweredetectedatthelatelogarithmicphasecomparedwiththeearlylogarithmic
phase. In general, this trend was observed for arabinoxylans, xyloglucan and Driselase‐
undigested elution profiles regardless of the cell compartment, andwas confirmed by the
datafromtheMwcalculation.Someofthemainfactorscontrollingferulatecross‐linking(and
therefore involved in changes in theMwof hemicelluloses), such as peroxidase action and
H2O2availability,variedduringtheculturecycle(BurrandFry,2009).Thus,itisfeasiblethat
changesinthesefactorswereresponsibleforthelowerMwdetectedatthelatelogarithmic
phaseofcultureinbothcelllines.
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140
TheincreasedMwofSEPscomparedwithcellwallboundhemicellulosesfromSnhat
the early logarithmic phase may indicate that polysaccharides sloughed into the culture
medium underwent additional cross‐linking. Indeed, SEPs differ from wall bound
hemicellulosesinhavinggreaterconformationalfreedomandmobility,renderingthemmore
accessibletoenzymes,whichwouldincreasethedegreeofferulatepolymerisation(Burrand
Fry,2009).Nevertheless, the trenddescribedwasnotobservedeither in Snh cells at late‐
logarithmicphaseorinSh1.5cellsindependentlyofthecellculturephase.
Inthecellwallcompartment,theMwofpolysaccharidesinSnhcellswasverysimilar
inbothculturestagesandthesametrendofsimilaritybetweenculturephaseswasobserved
inSnhkineticsofcellwallcoumaroylationandferuloylation.Incontrast,theMwofSh1.5cell
wallhemicellulosesandpolymersdecreasedinthelatelogarithmicphase,butsurprisingly,
greater amounts of ferulate 14C‐dimers and 14C‐oligomerswere detected. In linewith this,
exogenousH2O2wasobservedtostimulateferuloyldimerisationinSnhcellsindependently
of the growth phase, suggesting H2O2 availability as the limiting factor for ferulate
dimerisationandoligomerisationinthecaseofSnhcells.Incontrast,sinceexogenousH2O2in
Sh1.5 cells at the late logarithmic growth phase did not produce any change, the limiting
factor seems to be the availability of free ferulate for an extra dimerisation. This latter
finding once again demonstrated that hemicelluloses and polymers from habituated cells
would in fact be shorter, albeit widely cross‐linked, and/or underwent an additional
trimmingprocessthatdidnotoccurinSnhcells.
Another interesting finding was the marked elution peak in the Sh1.5 xyloglucan
molecules extractedwith6MNaOHaswell as in those sloughed into the culturemedium
observedatKav0.1and0.3respectively.AveryhighMwofwallboundxyloglucanmolecules
inmaizesuspensioncellshaspreviouslybeenreported,suggestingthatahighMwcomplexof
xyloglucan, possibly linked to other polysaccharides, may play a more effective tethering
role. This finding would partially explain why graminaceous monocots can survive with
muchsmallerquantitiesof xyloglucan thandicots (KerrandFry,2003). Indeed,neitherof
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themarkedelutionpeaksofSh1.5xyloglucanwasdetectedintheV0,wheremoleculeswith
thehighestMwwouldelute.However,whenbothKavwerecompared,stronglywallbound
xyloglucan from Sh1.5 cells was detected in a lower Kav than that for the sloughed
xyloglucan (0.1 vs 0.3). This would in fact indicate that Mw increased in wall bound
xyloglucan in Sh1.5, probably through an association with other polysaccharides or
polymers. Since it has previously been suggested that xyloglucan could be involved in the
copingresponseinducedat lowDCBlevels(deCastroetal.,2013;ChapterI), theseresults
would partially support this hypothesis, although further experiments are required for
confirmation.
In sum, the study of the Mw of [3H]xylans, [3H]xyloglucans, [3H]Ara‐containing
polymers as well as Driselase‐digestion resistant 3H‐polymers as well as the phenolic
metabolism in maize cells habituated to low DCB levels revealed that: a) Sh1.5 cells
synthesisedmoleculeswithalowerMwandamorehomogeneoussizethroughouttheentire
culture cycle, b) although hemicelluloses were shorter than those observed in the non‐
habituated cells, this was not related to a diminished capacity for polysaccharide cross‐
linking,c)bothcelllinessynthesisedmoleculeswithalowerMwatthelatelogarithmicphase
oftheculturecycle,probablyassociatedwiththecessationofgrowth,andd)thehypothesis
ofasignificantroleforxyloglucaninhabituationtolowlevelsofDCBwassupportedbythe
results.
Our results indicate that Sh1.5 cells synthesise shorter, albeit extensively cross‐
linked, molecules as part of their coping strategy. These results demonstrate that DCB
habituationisagradualanddynamicprocessinwhichmaizecellsshowhighlyplasticcoping
responsesdependingon theDCBhabituation frameworkor, theequivalent,on the levelof
cellulosedeficiency.
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ChapterIV
StudyoftheGolgiproteome
inDCB‐habituatedcells
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144
ABSTRACT
Culturedmaize cells habituated to DCB have the capacity to adopt different
mechanism to counteract the reduction in wall cellulose content, including metabolic
adaptations. Since it is well known that the Golgi is an important organelle in
polysaccharide and proteinmetabolism, the aim in the present studywas to obtain a
proteomic picture of Golgi‐enriched fractions of habituated maize cell suspensions in
ordertogainabetterunderstandingoftheirmetabolicactivity.Tothisend,aprotocol
was developed for obtaining Golgi‐enriched fractions and conducting a 2‐D proteomic
study.UsingMALDI‐TOF/MSandESI‐MS/MS,atotalof13proteinsdetectedasuniqueor
mis‐regulated in habituated cells were sequenced. Some of them were probably mis‐
regulatedasaconsequenceoftheDCBaction,suchaschaperonin60andthereversible
glycosylationpolypeptideα‐1,4‐glucanproteinsynthase(UDP‐forming),whereasothers
maybeinvolvedinvariouscopingstrategies.Thislattergroupincludedenzymesrelated
to stress signalling when cell wall integrity is impaired, such as enolase 1 or 1‐
aminocyclopropane‐1‐carboxylate oxidase 1; and others such as caffeoyl‐CoA O‐
methyltransferase1, which is related toqualitativeandquantitative changes in lignin
composition, and ascorbate peroxidase, in turn related to an oxidative coping
mechanism.TheresultsofthisstudyshedlightnotonlyontheDCB‐habituationprocess
but also on the counteracting mechanisms that maize cells adopt to survive with an
impairedcellwall.
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I.INTRODUCTION
Cell walls are responsible for crucial functions in plants, such as growth,
morphogenesisandmodulationofstresssignallingresponses toabioticandbioticstresses
(Hamann et al., 2009; Driouich et al., 2012). Basically, the cell wall structure consists of
cellulose acting as a scaffold to which hemicelluloses are attached, and the entire
arrangement is surroundedby a pectinmatrix (Carpita andGibeaut, 1993). In addition to
theirphysiological importance,cellwallsalsohavesignificanteconomicimplications.Their
components are important constituents of dietary fibre, with notable implications for
industryaswellashavingapositive impactonhealth.Anotherpromisingrolearosewhen
theybegantobeconsideredasasourceofenergyfromplantmaterialsinbiofuelproduction
(BurtonandFincher,2012).
PrimarycellwallcompositioninPoalesdiffersfromthatindicotsorothermonocots,
essentially inthenon‐cellulosicpolysaccharideportion,with loweramountsofxyloglucans
and pectins which are predominantly substituted by heteroxylans (Carpita and McCann,
2000).Thoseingrasseshavethebasicstructureofallxylans,abackboneofXylresidueswith
Ara, GlcA, andMeGlcA residues attached aswell asp‐Cou and Fer rests esterified on Ara
residues(WendeandFry,1997;Faik,2010),buttheyalsopresentuniquefeatures,suchas
thepresenceofXyl‐Ara‐furanosylsidechains(Ebringerovaetal.,2005).
Cell wall polysaccharides are synthesised in the Golgi apparatus, a fundamental
organelle in the eukaryotic secretory pathway. As a component of the endomembrane
system,itishighlyinvolvedinmembranesignallingprocessesandvesiculartrafficking,and
thisorganelleiswherepost‐translationalmodification,processingofproteinsandsynthesis
of complex carbohydrates takes place, endowing the Golgi apparatus with multiple
regulatoryfunctionsincomplexmetabolicprocesses(Parsonsetal.,2012).Inhigherplants,
vesiclessecretedbytheGolgiapparatusareresponsibleforcellplateformationduringcell
division (Driouich et al., 2012). Furthermore, the Golgi apparatus plays an important role
duringplantcellgrowth:newlysynthesisedpolysaccharidesintheGolgiaretransportedinto
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vesicles until they coalesce with the plasmamembrane to generate the growing cell wall
(Cosgrove,2005).
Whenmaize cells are habituated to growwith cellulose‐impoverished cellwalls, a
modifiedandremodelledheteroxylan(predominantlyarabinoxylan)networkcompensates
for this deficiency (Mélida et al., 2009). In addition, previous studies performed using a
biochemical approach have demonstrated that metabolism in habituated cells showed an
overall alteration (Chapter II). Thus, habituated maize cell cultures would be a suitable
starting cellular material for the study of metabolic modifications, and more specifically
thoseconcerningheteroxylansynthesis.
Therefore,theaimofthepresentstudywastodeterminewhetherDCBhabituation
entails metabolic modifications from a proteomic point of view. Important metabolic
enzymes are known to be Golgi‐resident proteins, so taking advantage of our habituated
maize cultured cells as a cellularmodel, Golgi‐enriched fractionswere obtained from Snh
andhabituatedtolow(Sh1.5)andhigh(Sh6)DCBconcentrationsmaizeculturedcells.Then,
a comparative proteomic expression analysis was performed of their Golgi‐enriched
snapshots. The results shed light on the metabolism of maize cultured cells, as well as
clarifyingunknownaspectsrelatedtoDCBcopingstrategiesfromaproteomicperspective.
II.MATERIALSANDMETHODS
II.a.CellculturesandhabituationtoDCB
Maize cell‐suspension cultures (Zea mays L., Black Mexican sweet corn) from
immatureembryosweregrowninMurashigeandSkoogmedia(MurashigeandSkoog,1962)
supplementedwith9μM2,4‐Dand20gL‐1sucrose,at25°Cunderlightandrotaryshaken,
androutinelysubculturedevery15days.
In order to obtain habituated cell cultures to DCB, Snh cells were cultured in a
medium supplied with DCB increased concentrations, dissolved in DMSO which does not
ChapterIV
147
affect cell growthat this rangeof concentrations. Likewise, in the caseof cell suspensions
habituatedtolowDCBlevels,Snhcellsweretransferredinamediumcontaining1μMDCB,
andaftersevensubculturestheDCBconcentrationwasincreasedupto1.5μMDCB(Sh1.5).
Habituated cells suspensions to high DCB levels were derived frommaize callus cultures
habituatedtogrowin12μMDCBwhichweretransferredintoliquidmediumsupplemented
with6μMDCB(Sh6).
II.b.ObtainingGolgi‐enrichedfractions
CellsfromSnh,Sh1.5andSh6suspensioncultureswerecollectedatthelogarithmic
phase of growth and frozen at ‐80°C until used. Since habituated cells (Sh1.5 and Sh6)
presented longer culture phases than Snh cells, the days of sampling were adjusted
dependingonthegrowthkineticsshowedbythedifferentcell lines(deCastroetal.,2013;
Chapter I), inorder tohavesynchronizedat thestageof theculturecycleall thecell lines.
Thus, the4th, the8thandthe12thdaysof theculturecyclewereselected for theharvestof
Snh,Sh1.5andSh6cells,respectively.
The entire procedure was carried out at 4°C, and all the sucrose buffers were
dissolved in 0.1 M HEPES‐ (2‐[4‐(2‐hydroxyethyl)‐l‐piperazinyl]‐ethanesulfonic acid) KOH
pH7supplementedwith1mMDTT.Cellmaterialwasfirstpulverisedinliquidnitrogenwith
amortarandpestleandthengroundfor20minwithanomni‐mixerinextractionbuffer(0.1
MHEPES‐KOHpH7containing0.4Msucrose,0.1%(w/v)bovineserumalbumin,1mMDTT
[added freshly], 5mMMgCl2, 5mMMnCl2, 1mMphenylmethylsulphonyl fluoride and one
Rochecompleteproteaseinhibitorcocktailtablet;Zengetal.,2008)inaproportionof1.5ml
extractionbuffer/1gfreshweight.Thehomogenatewasthencentrifugedat4000rpmfor20
min(x2)inordertodiscardcelldebris,andtheresultantsupernatantwascollectedtoobtain
Golgi‐enriched fractions as described in Porchia et al. (2002) with minor modifications.
Briefly, the supernatantwas loadedon topof6mlof2M sucrosebuffer (also referred to
throughout the textascushionbuffer)andcentrifugedat100000g for90min inorder to
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148
separatethemicrosomalmembranes,whichaftercentrifugationwerelocatedontopofthe
cushion buffer. The upper phase was then discarded without disturbing the crude
microsomal interface with the cushion buffer, and afterwards, a discontinuous sucrose
gradientwasdevelopedbyoverlaying5mlof1.3Msucrosebufferontopofthemicrosomes,
followedbyanother5mlof1.1Msucroseand5mlof0.25Msucrose.Thediscontinuous
sucrosegradientwascentrifugedat100000gfor2h,andfinallythe0.25M/1.1Msucrose
interfacewascollectedasaGolgi‐enrichedfraction,andstoredat‐80°Cuntiluse.
The Golgi‐enriched fractions were then subjected to routine analysis. Golgi‐
membranemarkeractivityassayswereperformedusingTriton‐dependentIDPase(Morréet
al., 1977). Total protein content was determined by using Bradford reagent (Sigma) and
bovinealbuminserumasstandard.TheTriton‐dependentIDPaseactivityassaywascarried
outbydiluting200µlofthesamplein200µlof50mMTrisbufferpH7.2[adjustedbythe
addition of 2‐morpholino‐ethanesulfonic acid (MES)], containing: 3 mM inosine‐5‐
diphosphate (IDP), 1 mM MgCl2 and 0.01% Triton X‐100. The reaction mixture was
incubated at 37°C for 1 h (Weinecke et al., 1982) and released phosphate was then
determinedbytheAmesmethod(Ames,1966).
II.c.Testforproteinsolubilisationfromtransmembranecomplex
Golgi‐enriched fractions were incubated for 10 min on ice with two different
detergents(0.5%TritonX‐100and1%digitonine)inordertodeterminewhetheranyofthe
treatmentspartiallysolubilisedthemfromtrans‐membranecomplex(Zengetal.,2010).
II.d.SDS‐PAGEanalysis
Prior toSDS‐PAGE,proteinswere incubated for10minon icewith0.5%TritonX‐
100. For SDS‐PAGE separation, proteins were treated under reducing conditions (boiled
samples)orpartialnon‐reducingconditions(notboiledsamples)andseparatedbystandard
SDS‐PAGEona7.5%,10%or12.5%gel.Undernon‐reducingconditions,SDSwassubstituted
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149
by0.1%TritonX‐100onthestackingandseparatinggelstofacilitatethemobilityofprotein
complexes. The resultant gels were stained with CBB R‐250 (Bio‐Rad) following
manufacturer’sinstructions.
II.e.PreparationofGolgi‐enrichedfractionsandproteinextractionfor2‐DPAGE
InordertosolubiliseproteinsfromtheGolgi‐enrichedfractionmembranesaswellas
to shift the buffer (samples were dissolved in extraction buffer) to the appropriate for
carryingout2‐Delectrophoresis[lysisbuffer:20mMTris‐HClpH8.8,7Murea,2Mthiourea,
4% 3‐3‐(3‐cholamidopropyl) diethyl‐ammonio‐1‐propanesulfonate (CHAPS)], an aliquot of
eachsamplecontainingapproximately4.2µg/µlofproteinwas freeze‐dried.Theresultant
freeze‐driedsampleswerethendissolved in2mlof lysisbuffer, incubated for5min inan
ultrasoundbathandcentrifugedat8000rpmfor20min.Thesolublefraction(supernatant)
was collected and dialysed against 20 mM Tris‐HCl pH 8.8, 7 M urea and 2 M thiourea.
Dialysed samples were finally concentrated by Vivaspin columns (supplied by Sartorius)
untiltherequiredamountofproteinfor2‐Delectrophoresiswasobtained.
II.f.2‐DPAGEanalysis
Two independentbut complementary analyses of the sampleswere carriedoutby
resolvingtheresultantgelswithdifferentstains,CBBG‐250(Bio‐Rad)(Camposetal.,2010)
andsilverstain(Shevchenkoetal.,1996;Iraretal.,2006),andperforming4and3technical
replicatesofeachanalysis,respectively.FortheCBBG‐250stain,gelswereinitiallyfixedin
40%methanol and 10% acetic acid, for 3 h (or alternatively overnight). Then they were
incubated in a mixture of two different solutions: 98% of solution A, containing 2%
orthophosphoricacidand10%ammoniumsulphate,and2%ofsolutionBcomposedof5%
CBBG‐250,for3h(oralternativelyovernight).Finally,gelswerewashedinMilli‐Qwaterfor
2h(x3). In thecaseofsilvernitrate, theprocedurewascarriedoutas follows: firstly,gels
werefixedin40%ethanoland10%aceticacidfor3h(oralternativelyovernight)andthen
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150
rinsedwithMilli‐Qwater for5min.Theywere thensoaked in6.8%sodiumacetate,0.2%
sodium thiosulfate and 30%ethanol for 30min andwashedwithMilli‐Qwater for 5min
(x3). Subsequently, they were incubated for 20 min in a solution containing 0.1% silver
nitrate andwashed inMilli‐Qwater for 1min (x2). Colourwas developed for 3‐5min by
adding25µlof37%formaldehydeina3%sodiumcarbonatesolution.Afterwards,gelswere
rinsed inMilli‐Qwater for20 s, soaked in1.46%di‐sodiumethylene‐di‐amine‐tetra‐acetic
acid(EDTA) inorder tostopthestainand finally,washedagain inMilli‐Qwater for5min
(x3).
Depending on the sensitivity of the stain used, the protein concentration required
varied, using400µg and100µg in forCBBG‐250and silver stain, respectively. Thus, the
requiredamountofproteinwasdilutedinafinalvolumeof350µlofrehydrationsolution(7
Murea,2Mthiourea,18mMTris‐HClpH8.0,4%CHAPS,0.5%IPGbufferinthesamerange
as the IPG strip, and 0.002% Bromophenol Blue) containing 1.6% DeStreak Reagent
(AmershamBiosciences),andloadedontonon‐linearpH4–7,18‐cmIPGstripstocarryout
thefirstdimension.IsoelectricfocusingwasperformedusinganEttanTMIPGphorIsoelectric
FocusingSystem(AmershamBiosciences)withthefollowingsettings:50Vfor10h,500Vin
gradient for 1.5 h, 1000 V in gradient for 1.5 h, 2000 V in gradient for 1.5 h, 4000 V in
gradientfor1.5h,8000Vingradientfor2h,and8000Vholdingfor10h.Afterwards,IPG
strips were collected and equilibrated. First, a 15 min treatment with 10 mg/ml of DTT
dissolvedinequilibrationbuffer(50mMTris‐HCl(pH8.8),6Murea,30%(v/v)glycerol,2%
SDSandatraceofBromophenolBlue),wasfollowedbyasecond15minequilibrationstepof
25mg/mlof iodoacetamidedissolved in theequilibrationbuffer,bothof them inshacking
conditions (at 180 rpm).The focused stripswere then loadedon topof a SDS–PAGE10%
polyacrylamidegel(26x20x0.1cm)andsubjectedtotheseconddimensionusinganEttan
DALTsix System (AmershamBiosciences) and the following running conditions:2.5W/gel
for30min,followedby20W/gelfor4h.Gelswerefinallyresolvedwiththecorresponding
stain (CBB G‐250 or silver stain). The experiment was carried out with three technical
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151
replicates per biological sample. The stained gels were scanned with an ImageScanner
desktop instrument (AmershamBiosciences)and imageswereacquiredusing theLabScan
scanningapplicationintransmissionmode,at(16‐bits)grayscalelevel,300dpi,andsavedas
TIFF(TaggedImageFileFormat)files.ImageanalysiswasperformedusingImageMasterTM
2D Platinum 5.0 Software (Amersham Biosciences) (Farinha et al., 2011). The optimal
parametersforspotdetectionwere:smooth=4,saliency=1.0andminimumarea=5.After
automaticspotdetection,manualspoteditingwascarriedout.Gel replicateswereused to
obtain synthetic gels with averaged positions, shapes, and optical densities. To evaluate
proteinexpressiondifferencesamonggels,relativespotvolume(%Vol)wasused.Thisisa
normalised value and represented the ratio of a given spot volume to the sum of all spot
volumes detected in the gel. Those spots showing a quantitative variation > Ratio 1 and
positive GAP were selected as differentially accumulated. Statistically significant protein
abundance variationwas validated by Student’s t‐test (p, 0.05) (Farinha et al., 2011). The
selected differential spots were excised from the gels and identified either by PMF using
MALDI–TOF/MS or ESI‐MS/MS on the Proteomics Platform (Barcelona Science Park,
Barcelona, Spain). The MALDI–TOF/MS analysis was performed using a Voyager DEPRO
(Applied Biosystems) instrument in reflectron positive‐ion mode. Spectra were mass
calibrated externally using a standard peptide mixture. For the analysis, 0.5 ml peptide
extract and 0.5mlmatrix [5mgml‐1 cyano‐4‐hydroxycinnamic acid (CHCA)]were loaded
onto theMALDIplate.When ionscorresponding toknown trypsinautolyticpeptides (m/z
842.5100, 1045.5642, 2111.1046, 2283.1807) were detected at sufficient intensities, an
automaticinternalcalibrationofthespectrawasperformed.DataweregeneratedinPKLfile
format and were submitted for database searching in the MASCOT server. The software
packages Protein Prospector v 3.4.1 from the University of California San Francisco and
MASCOT were used to identify the proteins from the PMF data (Carrascal et al., 2002).
SEQUEST software (Thermo‐Instruments, Spain) was used for preliminary protein
identification from the MS/MS analysis followed by manual sequence data confirmation.
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Swiss‐Prot and non‐redundant NCBI databases were used for the search. Searches were
performed for the full range of molecular mass (MM) and pI. No species restriction was
applied.Whenanidentitysearchproducednomatches,thehomologymodewasused.
III.RESULTS
III.a.ObtainingGolgi‐enrichedfractions
Golgi‐enrichedfractionsfromSnh,Sh1.5andSh6maizeculturedcellswereobtained
by ultracentrifugation on a discontinuous sucrose gradient (Figure 1). Different
combinationsof sucrose concentrations for thegradientwere tested inorder to select the
mostsuitableforanoptimumseparationofGolgimembranesfromSnh,Sh1.5andSh6maize
cells.Todeterminethis,gradientfacesandinterfacesweresubjectedtotheIDPaseactivity
assay,findingthatthebestresultsforseparationoftheGolgibodieswereachievedbyusing
2M,1.3M,1.1Mand0.25Mdiscontinuoussucrosegradient(datanotshown).Inaddition,
thehighestvalueofIDPaseactivitywasdetectedatthe0.25M/1.1Msucroseinterfaceinall
the cell lines (Figure 1F), and therefore it was considered the Golgi‐enriched fraction.
Moreover, whereas the total protein concentration was lower in the Sh Golgi‐enriched
fractionsthaninthosefromSnh,theIDPaseactivityshowedhighervaluesinthehabituated
celllines(Table1).
III.b.AnalysisofGolgiproteome
III.b.1.Procedure
First since some of the proteins containedwithin Golgi stacks are known to have
trans‐membrane domains, Golgi‐enriched fractions were incubated with Triton X‐100 or
digitonin in order to determine whether any of these treatments would improve their
solubility.Nodifferences,at least in termsofproteinpopulations,werefoundbetweenthe
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detergenttreatmentsandthecontrol(Figure2).However,aprevioustreatmentwithTriton
X‐100wasperformedforthefollowingassaysasapreventivemeasure.
Figure1.PerformingasucrosediscontinuousgradientfortheobtainingofGolgi‐enrichedfractions.AnextractfromSnhmaizecellsuspensionswasloadedontopofthecushionbuffer(A)andultracentrifugedinordertogetthemicrosomalfraction.Afterdiscardingtheupperphase,1.3M(B),1.1M(C)and0.25M(D)sucrosebufferswereloadedontopofthecrudemicrosomesformingadiscontinuoussucrosegradient,thensubjectedagaintoultracentrifugation.Afterwards,triton‐dependentionidinedi‐phosphataseactivity(IDPase)activitywastestedineveryinterfaceandface(E),selectingtheinterfacelocatedbetween1.1Mand0.25MsucroseasaGolgienriched
fractionsinceitshowedthehighestGolgienzymaticactivitymarkervalues(F)
Table1.ProteincontentandIDPasespecificactivityonGolgi‐enrichedfractionsfromSnh,Sh1.5andSh6maizecells
2‐D electrophoresiswas used for differential proteomic expression analyses. Since
thesearecomparativeanalyses,aminimumdegreeofhomology(≈60%)amongtheprotein
populations of the Golgi proteome from the different cell lines was required. In order to
verifythis,sampleswerepreviouslysubjectedtomono‐dimensionalPAGE,showingthatthe
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degreeofproteinpopulationhomology forbothhighand lowMM,wassatisfactoryamong
Snh,Sh6(Figure3)andSh1.5(anexacttestasinFigure3wasperformedwiththiscellline:
datanotshown)celllines.
Figure2.IncubationofGolgi‐enrichedfractionsobtainedfromSnhmaizecellswithdifferentdetergents.Assayswerecarriedoutbyincubatingthemonicefor10minwith1%digitonineor0.5%TritonX‐100.Tocontrolsamplesnodetergentwasadded.Sampleswereboiled(B)ornotboiled(NB)andsubsequentlysubjectedto(A)10%or(B)7.5%polyacrylamidegelelectrophoresis(PAGE).0.1%TritonX‐100wasaddedongelsin
substitutionofsodiumdodecylsulphate(SDS).Gelswerecoomassiebrilliantblue(CBB)R‐250stained.Molecularmass(MM)markersinkDareindicatedattherightofthegels
Other adjustments were necessary before conducting the 2‐D analysis due to the
concurrenceofthreecircumstances:a)toobtainGolgi‐enrichedfractions,Snh,Sh1.5andSh6
maize cultured cells had been homogenised in extraction buffer, which differed in
composition (see materials and methods) from that used to carry out the 2‐D
electrophoresis,b)aspreviouslymentioned,someoftheproteinswithintheGolgibodiesare
knowntohavetrans‐membranedomainsor to formtrans‐membranecomplexes,andsuch
structuralfeaturesrendereditindispensabletosolubilisetheminordertoperformthe2‐D
analysis, requiring treatmentwith a powerful detergent such as CHAPS,which is typically
usedforthistechnique,andc)therequiredtotalamountofproteinforthe2‐Danalysishad
tobeconcentratedinaspecificvolumetobeloadedontheIPGstrip;thus,concentrationof
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sampleswasrequired.Therefore,aprotocolformeetingtheserequisiteswasdeveloped,and
checkinganalyseswereroutinelyperformedaftereachkeystepoftheprotocolinorderto
detectvariationsinthetotalamountofproteinaswellastodeterminewhetheranyprotein
population was being lost as a consequence of the procedure. Neither the Golgi‐enriched
fraction from Snh or from Sh1.5 and Sh6 cells suffered any loss of a specific protein
population(Figure4).However,asexpected,alowertotalproteinvaluewasobtainedwhen
aVivaspinconcentrationstepwasincluded(Table2).Throughouttheentireprocedure,this
totalproteinlosswasgreaterinthecaseofthehabituatedcelllines(Table2).
Figure3.Golgi‐enrichedfractionsobtainedfromSnhorSh6maizecellssuspensions,incubatedonicefor10minwith0.5%(v/v)TritonX‐100.Sampleswereboiled(B)ornotboiled(NB)andsubsequentlysubjectedtoA:10%orB:7.5%PAGE.0.1%TritonX‐100wasaddedongelsinsubstitutionofSDS.GelswereCBBR‐250stained.MM
markersinkDareindicatedattherightofthegel
III.b.2.2‐Delectrophoresis
Once those critical points were successfully resolved, 2‐D electrophoresis was
performed and the resultant gels were subsequently CBB G‐250 or silver nitrate stained,
obtaining two complementary but independent studies of the Golgi‐proteome (Figure 5).
Stainedgelswerethenanalysedinordertodetectdifferentiallyaccumulatedproteinsamong
celllines.InthecaseoftheCBBG‐250study,Sh6showedthelowestvaluesinthenumberof
totalproteinspotsdetectedonthegels,exhibitingan80%reductionwhencomparedwith
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Snh.AlthoughthenumberoftotalproteinsobservedwasalsolowerinthecaseofSh1.5cells,
thisvaluewasmoresimilartoSnhshowingonlya22%reductionwhenbothcelllineswere
compared(Table3A).Aswasexpected,thegreatestnumberofmis‐regulatedproteinswas
observedintheSnhvsSh6comparativestudy,regardlessofthestainused.Thenumberof
proteinsthatwereonlypresentinonecell linewasconsiderableincomparativestudiesin
whichSh6cellswereinvolved.ThesilvernitratestudyconfirmedthesetrendsintheCBBG‐
250analysis(Table3B).
Figure4.VerificationofpresenceandintegrityofGolgi‐enrichedproteinspopulationsfromSnh,Sh1.5andSh6maizecellsuspensionsbycomparingthefirst(solubilization:S)withthelast(vivaspinconcentration:V)stepofthepreparationprocessforthetwodimensional(2‐D)electrophoresis.Sampleswereboiledandsubjectedto
12.5%SDS‐PAGE.GelwasCBBR‐250stained.MMmarkersinkDareindicatedattherightofthegels
Table2.TotalproteincontentofGolgi‐enrichedfractionsfromSnh,Sh1.5orSh6maizecellssuspensionsthroughoutthedifferentsteps(resuspension,solubilization,dialysisandvivaspinsamplesconcentration)ofthe
preparationprocessforthe2‐Delectrophoresis.Valuesshownwerestimatedinapooloffour‐technicalreplicates
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Figure5.2‐DelectrophoresisCBBG‐250(A,B,C)andsilver‐nitrate(D,E,F)stainedgelsofGolgi‐enrichedfractionsfromA,D:Snh;B,E:Sh1.5andC,F:Sh6maizecellsuspensions
III.b.3.Sequencing
Subsequentlyuniqueandmis‐regulatedproteinswereselectedandexcisedfromthe
gel,andatotalof13proteinswereidentifiedusingMALDI–TOF/MSorESI‐MS/MS(Table4).
Enzymes related to carbohydrate metabolism (enolase 1) and ethylene biosynthesis (1‐
aminocyclopropane‐1‐carboxylate oxidase 1: ACO 1 and adenosylhomocysteinase) were
affected in the habituated cells. Habituation to DCB also entailed changes in the level of
expression of caffeoyl‐CoA O‐methyltransferase 1 (CCoAOMT 1), an enzyme involved in
ligninbiosynthesis,whichwas found tobedown‐regulated. Surprisingly, in Sh1.5 andSh6
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cells,proteinsrelatedtostressresponses,suchasascorbateperoxidaseandchaperonin60,
were also found to be down‐regulated. Another interesting findingwas that an alpha‐1,4‐
glucan‐protein synthase responsible forUDP‐formingwas repressed in thehabituated cell
lines.
Table3.AgeneraloverviewofGolgi‐enrichedfractionsproteomefromSnh,Sh1.5andSh6maizeculturedcells.A:numberoftotalproteinsinCBBG‐250andsilvernitrateproteomicstudiesineachcellline.B:numberofmis‐regulatedandexclusiveproteinsdetectedinthecomparativeproteomicstudiesamongcelllines(SnhvsSh1.5orSh6,andSh1.5vsSh6)inCBBG‐250andsilvernitratestained2‐Dgels.ResultsfromtheCBBG‐250studywereobtainedfromfourtechnicalreplicates.ThosefromthesilvernitrateforSnhandSh1.5cellswereobtainedfrom
threetechnicalreplicates,whereasthevalueforSh6cellswasfromjusttworeplicates
IV.DISCUSSION
Previous studies performedusing a biochemical approach have demonstrated that
metabolismincellulose‐impoverishedcellsisaltered(ChapterII).Therefore,itisfeasibleto
B
A
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159
hypothesise thatmetabolic alterations from a proteomic perspective could also be taking
place.
As the relevantmetabolic enzymes areGolgi‐residentproteins, itwasnecessary to
obtainGolgi‐enriched fractions fromthedifferentcell lines.Typically, the isolationof such
Golgi‐enriched fractions has been carried out by ultracentrifugation on a discontinuous
sucrosegradient.Thedifferentcellularorganellesspreadoverthegradient,stabilisingatthe
interfaces (among the different sucrose concentrations) depending on its density. In the
present study, a protocol for obtaining Golgi‐enriched fractions from Snh, Sh1.5 and Sh6
maizeculturedcellswasoptimisedbasedonthosepreviouslyreportedinotherspecies,such
aswheat(Porchiaetal.,2002;Faiketal.,2002;Zengetal.,2008),arabidopsis(Brownetal.,
2007)andothers(Leelavathietal.,1970).Thisprocedureledtotwomainconclusions:a)an
improvedseparationofGolgistackswasachievedbyusing2M,1.3M,1.1Mand0.25Mas
sucrose concentrations in the gradient, and b) the Golgi‐enriched fraction was the one
locatedatthe0.25M/1.1Msucroseinterfaceinallthecelllines.
Differences were found in Golgi‐enriched fractions from Sh and Snh cells. An
interestingfindingwasthatthehighestvaluesofIDPaseactivityweredetectedinSh,mainly
Sh6,Golgi‐enrichedfractions,comparedtothoseobtainedfromSnhcells.Furthermore,the
amount of total protein estimated in the Sh cell lineswas lower than in Snh cells. IDPase
activity is a well‐known and widely used Golgi‐enzymatic marker (Gibeaut and Carpita,
1990; Turner et al., 1998; Zeng et al., 2010). It is considered a nucleoside diphosphatase
reaction showing specificitymainly for IDP, UDP and guanosine‐5‐diphosphate substrates
(Goldfischer et al., 1964). Thus, reactions in which UDP substrates are involved, such as
polysaccharidesynthesis,couldbeco‐relatedwiththismarkeractivity.Apreviousstudyhas
demonstrated a concomitant increase in IDPase activity in maize tissues, where the
carbohydratesynthesiswithintheGolgiapparatusanditssecretionwereveryactive(such
asrootcapandtheepidermis)(Dauwalderetal.,1969). Incontrast, in tissueswheresuch
secretoryandsynthesisactivitieshadceased(suchasthevacuolatedoutermostcapcellsof
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the roots) and, therefore Golgi apparatus had reverted to a compact form, the values
observed for IDPase were residual. These findings evidenced a correlation between
polysaccharide synthesis and morphological differentiation for secretion of the Golgi
apparatusandIDPaseactivity,endowingthisenzymaticmarkeractivitywithaconsiderable
functional significance. We hypothesised that a more active synthesis of polysaccharides
wouldoccurincellshabituatedtoDCB,inordertocounteracttheircellulosedeficit.Thus,it
wouldbe feasible to suggest that thehighest IDPase values exhibitedby Sh cells couldbe
relatedtoanenhancedpolysaccharidesynthesisactivitywithintheGolgiapparatusand/or
itsmorphologicalspecialisationforasecretoryfunction.
Table4.Identificationofproteinsbymatrix‐assistedlaserdesorptionionization‐timeofflightmassspectrometry(MALDI‐TOF/MS)orbyelectrosprayionizationwithtandemmassspectrometry(ESI‐MS/MS)afterspotexcisionfrom2‐Dgels.Itisindicated:mis‐regulationbehaviour;studyinwhichproteinappearsCBBG‐250(C)orsilver
nitrate(S);isoelectricpoint(pI)andMM
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Despite the differences observed in the IDPase activity values, no disparity was
detected in the different cell lines in terms of protein populations. This was especially
relevanttoensuresuccessincarryingoutthesubsequentcomparativeproteomicanalysisof
the fractions, which was performed by 2‐D electrophoresis. A crucial requisite for this
technique is that different samples must show the degree of homology required for the
comparison, essential for theaccuracyof the subsequent statistical tests for validating the
study.Therefore,thefactthatnoqualitativedifferenceswerefoundinourdifferentsamples
regarding protein populations guaranteed the minimum degree of homology required to
conductthe2‐Danalysis.
In spite of the structural complexity of the Golgi apparatus, which renders it a
complicatedsubcellularcompartmenttostudyusingmodernproteomictechniques(Parsons
et al., 2012), several studieshaveused2‐D electrophoresis to carryout an analysis of the
Golgi proteome (Taylor et al., 2000;Wu et al., 2000). The proteome of a cell reflects the
proteinsexpressedinagivenmoment.Undoubtedly,expressioncouldvarydependingona
wide range of factors, such as development stage or response to stress conditions, for
instance, DCB‐habituation itself. Therefore, the information contained in a proteome
provides crucial clues to themetabolic and physiological changes occurring in a cell at a
particular time (Faik, 2012). In this study, two independent but complementary analyses
wereconductedtoexaminetheproteomesfromthedifferentcell lines,usingtwodifferent
stains for the gels: CBBG‐250 and silver nitrate. The protein detection threshold of these
compoundsdiffers:whereasCBBG‐250stainsthemostabundantproteins,silvernitrate is
moresensitive,allowingthedetectionofproteinsinlowerquantities.Inaddition,thestains
mayevendetectdifferentproteins.ThenumberoftotalproteinsobservedintheSnhGolgi‐
enriched fractionwasmuch lower than observed in the total proteome of non‐habituated
maizeculturedcells(Mélidaetal.,2010a).Thisfindingwaspredictable,aswewereworking
withapartiallypurifiedfractionof theproteome.Shcellsshowedlowerquantitiesof total
proteinsonthegels,andthiscircumstancewasespeciallypronouncedintheSh6cells,which
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162
exhibited an 80% reductionwhen compared to Snh. Again, this resultwas not surprising
since this trend had previously been observed in the determination of the total protein
concentration assayed by the Bradfordmethod. It should be noted that the Sh6 cells had
beenhabituatedtoahighlevelofDCB,whichcouldbeexpectedtoprovokedrasticmetabolic
andphysiologicalalterations.Thiswouldalsoexplainthegreaternumberofuniqueormis‐
regulatedproteinsdetectedincomparativestudiesinvolvingSh6cells.
DifferentiallyaccumulatedanduniquespotsandwereidentifiedbyMALDI‐TOF/MS
andESI‐MS/MS.Themagnitudeofmis‐regulationseemedtohaveapositivecorrelationwith
DCBhabituationlevel,showinghighervaluesintheSh6cellGolgi‐enrichedfractions.Some
ofthesequencedproteinsareknownnottobeGolgi‐residentproteins;however,itshouldbe
borneinmindthat1)thestudywasconductedwithGolgi‐enriched(notpurified)fractions
therefore, contamination with other endomembrane system organelles was impossible to
avoidsincetheyarehighlyinterconnectedandhavesimilardensitiesorsizes(Hantonetal.,
2005;Parsonsetal.,2012),2) it iswellknownthatmanyproteinmodifications takeplace
within Golgi apparatus, making it very complicated to distinguish between Golgi‐resident
proteins and transient proteins, 3) one of the methodological restrictions of 2‐D
electrophoresis is that abundant proteins canmaskminority ones (Timperio et al., 2008).
Thismeansthatifanon‐specificGolgiproteinundergoesanykindofmis‐regulationandis
present in a greater amount thanGolgi‐resident ones, themagnitude of itsmis‐regulation
would probably be higher than that exhibited by minority Golgi‐resident proteins, which
would make it a firm candidate for sequencing. This could have been the case of the
chaperonins, known to be involved in a wide range of protein‐assisted functions and
therefore their presence is to be expected in an organelle in which such an extensive
modificationofproteinsistakingplace.
Interestingly,severalofthemis‐regulatedproteinsreportedhere,suchasenolase1,
chaperonin60andACO1,werealsodetectedasbeingmis‐regulatedinapreviousanalysisof
thetotalproteomeofmaizeDCB‐habituatedcells(Mélidaetal.,2010a).This indicatesthat
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163
they play an important metabolic role in cellulose‐deficient maize cells and confirms the
reliability of the methodological procedure designed for the present study as well as the
results obtained. Another concurrence when comparing both studies was related to
ascorbateperoxidase.Assaysofitsdetoxifyingactivityinhabituatedcellsrevealedthatthis
was slightly lowerwhen comparedwith non‐habituated cells (Mélida et al., 2010a). Thus,
thisfindingisingoodagreementwithitsdown‐regulatedexpressiondetectedinthisstudy.
Enolase1wasfoundtobeup‐regulated inthehabituatedcellsandits levelofmis‐
regulationwas correlatedwith DCB habituation level, being higher in Sh6 cells. Although
enolaseisinvolvedincarbohydratemetabolism,throughcatalysingthereversalconversion
of the hexose glucose to pyruvate, it has also been related inmaize to different stressors,
suchasanaerobicconditions(Sachsetal.,1996).Moreinterestingly,aseriesofstudieshave
indicatedthathexosesmayactasstressindicatorswhencellwallsaredamaged(Hamannet
al., 2003; Wolf et al., 2012). This concurs with our observation of a higher level of up‐
regulationinmaizecellshabituatedtohighDCBconcentrations(Sh6)whencomparedwith
low‐levelhabituation(Sh1.5)andSnh.Sh6cellwallsweremoreseriously impaired,witha
70%impoverishmentincellulosecontent(Mélidaetal.,2009),whereasthisfigurewasthe
33%forSh1.5(deCastroetal.,2013;ChapterI),whencomparedwithSnh.Sinceenolase1
was identified several times (andwas probably the same protein butwith different post‐
transcriptionalmodifications), itwouldbereasonabletoassumethat itplaysan important
roleasastressindicator.
Anothercoincidentproteinwaschaperonin60,whichwasdown‐regulatedinSh1.5
andSh6cell lines.Chaperoninsareknowntoplayanactivepart inprotein foldingandre‐
folding,assembly,translocationanddegradationinmanycellularprocesses,andbecauseof
these functions they can re‐establish cellular homeostasis when plants are under stress
conditions (Wanget al., 2004;Huet al., 2009). Ithasbeendescribedelsewhere that some
membersofchaperonin60classareinvolvedincelldeathandactivatingsystemicacquired
resistance(Ishikawaetal.,2003).Focusingonthecontextofstressconditions,suchasDCB
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164
habituation, one could expect to find them up‐regulated. Nevertheless, members of the
chaperonin60classhavealsobeenreportedtobeinvolvedinthefoldingoftubulinandactin
(Gutsche et al., 1999), and it should be noted that the DCB action mechanism affects
microtubul integrity(BisgroveandKropf,2001;Himmelspachetal.,2003;Rajangametal.,
2008).Thus,thisfindingwouldbeapossiblehypothesisfortherepressionobservedinthe
habituatedcells,althoughfurtherevidenceisneeded.
Another down‐regulated protein found in the habituated cells was CCoAOMT 1,
involved in lignin biosynthesis,more specifically in the G and S units. Two enzymesmay
catalysetheproductionofbothunits,butdifferencesintheircatalyticpreferenceshavebeen
suggestedalthoughnotproven:whereasCCoAOMTpreferentiallysynthesisesGunits,caffeic
acid3‐O‐methyltransferase (COMT) tends towardsSones (Guoetal.,2001). Inaddition, it
has been suggested that CCoAOMT is partially replaced in DCB‐habituated cells by COMT
(Mélida et al., 2010a), and its down‐regulation has been related to qualitative and
quantitativechangesinlignincompositioninalfalfa,showingadecreaseinGunitswithno
apparenteffectonSones(Guoetal.,2001).Thesequalitativedifferencesareconsistentwith
unpublished results obtained in our laboratory, which showed that the DCB‐habituation
entailed a reduction in the amount ofG lignin unitswhereas S levels remainedunaltered.
Thus, the CCoAOMT 1 down‐regulation exhibited by habituated cells in this study is in
agreementwiththesefindings,suggestingaroleforthisproteinintheDCBcopingstrategy.
An α‐1,4‐glucan protein synthase (UDP‐forming)was another of themis‐regulated
proteins,showingahigherlevelofrepressionintheSh6cellline.Thisenzymeisareversible
glycosylation polypeptide (RGP), a group of Golgi‐resident proteins (Dhugga et al., 1991)
involved in cellwallpolysaccharide synthesis (Dhuggaet al., 1997;Langeveldet al.,2002)
andithasbeensuggestedthatitformspartofalargeGolgi‐complexwhichincludesnotonly
GTsbutalsoRGPsandmultimembrane‐spanningtransporterproteins(Oikawaetal.,2012).
Consistentwiththisstatement, its involvement inxyloglucansynthesishadbeendescribed
inpeaandpotato(Dhuggaetal.,1997;Waldetal.,2003).Apreviousstudycarriedouton
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165
arabidopsiswith a doublemutant for twoRGP genes showed that its pollen development
wasextremelyabnormal,exhibitingapoorlydefinedinnercellwall layer(Drakakakietal.,
2006).ThiswouldsuggestthattheabsenceofRGPsisrelatedtoaweakenedcellwall,which
is in good agreement with the down‐regulation of the α‐1,4‐glucan protein synthase
observedinthecellulose‐deficientwallsinhabituatedcells.
The enzymes related to ethylenemetabolism, ACO 1 and adenosylhomocysteinase,
were also found to be differentially accumulated in the habituated cells. Focusing on the
former,ACO1,whichisinvolvedinethylenebiosynthesis,itwasfoundtobedown‐regulated
in the habituated cell lines. Ethylene synthesis requires the conversion of methionine to
adenosylmethioninebytheadenosylmethioninesynthetase(ACS)to1‐aminocyclopropane‐
1‐carboxylic acid (ACC), which is then used as a substrate of ACO to produce ethylene.
Pathway regulation is normally conductedbyACS, but in conditions of excessive ethylene
concentrations, ACO could also act as a regulating enzyme. A role for ACC in signalling,
independently of its involvement in ethylene biosynthesis, has also been reported
(Vandenbussche et al., 2012; Yoon and Kieber, 2013). Interestingly, this suggestion arises
from two studies in which reduced cellulose content conditions were involved (Xu et al.,
2008; Tsang et al., 2011). Furthermore, the later one hypothesised a role for ACC in the
regulationofthecellwallsensingsystemwhenitsintegrityisimpaired,basedontheresults
obtained from the inhibition of cellulose biosynthesis by isoxaben in root epidermal cells
(Tsang et al., 2011). This connection between reduced cellulose content and ACC as a
signallingmolecule renders it feasible tohypothesise that inourcellulose‐deficientcells,a
down‐regulation of ACO 1would lead to an increase in ACC content, activating signalling
pathwaysforthesubsequenttriggeringofcopingstrategies.
Overall, our results strongly suggest that some of the identified proteins were
differentiallyaccumulatedbecause theyplaya role in theDCB‐coping strategyadoptedby
habituatedcells.However,inothercases,themis‐regulationofproteinexpressionobserved
seemedtoberelatedtoDCBeffects,probablynothavinganyspecific “tactical” functionas
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166
partofaresponseagainsttheinhibitor.Nevertheless,recentstudieshavedemonstratedthe
existenceofGolgiproteininteraction.Furthermore,whenproteinmembersofthiscomplex
vary, their biochemical and catalytic functions also differ (Oikawa et al., 2012). Thus, the
possibility cannot be ruled out that some of the mis‐regulated proteins observed in this
study,althoughatfirstglancenotclearlyinvolvedinanyexpectedphysiologicalresponseto
DCB,mayplayarolethroughtheirinteractionwithothers.
Discussion
Discussion
168
Habituation toDCB involves various strategies formodifying cellwall architecture
and thus overcoming the constraint implied by having cellulose‐deficient walls. In the
specific case ofmaize cells, it has previously been reported that in cultures habituated to
otherwise lethal DCB concentrations, a quantitatively and qualitatively re‐modelled and
modified arabinoxylannetworkplays a key role inmanyof these strategies (Mélida et al.,
2009; 2010a; 2010b; 2011). However, it has also been demonstrated that the variety of
coping responses depends on the cell wall type aswell as on the DCB concentration and
lengthofexposuretimetoit(Alonso‐Simónetal.,2004;Mélidaetal.,2009).Sinceprevious
studiesonDCB‐habituatedmaizeculturedcellshavebeenachievedbytheuseofhighDCB
concentrationsandlong‐termhabituationperiods,dataabouttheearlymodificationstaking
place during the habituation process are lost. Such informationwould be highly valuable
because in the earliest stages of DCB habituation, cells would present a wide variety of
responses. In addition to cell wall alterations, DCB‐habituated cells are likely to have a
modifiedmetabolism.Therefore,thegeneralaimpursuedinthepresentthesiswastoclarify
themechanismsresponsibleforthestructuralplasticityofmaizecellwallsduringearlyDCB‐
habituation,whilstalsofocusingonmetabolicstrategies.
However, itwas first necessary to obtain a cell linewith these features. Extensive
monitoringwasperformedofmaize cell lineshabituated to lowDCB levels, using a set of
techniquessuchasFTIRspectroscopyandcellulosecontentdetermination(deCastroetal.,
2013;Chapter I,Figures1 to4).Theresultsobtained fromthispreliminarystagerevealed
theSh1.5celllinewasthemostsuitableforuseinordertoaccomplishtheobjective.
Asexpected, themainmodificationdetected inthewallsofSh1.5cellspertainedto
cellulose content,with a 33% reduction.When compared to the value reported formaize
cellshabituatedtohighDCBlevels(75%;Mélidaetal.,2009),thecellulosereductioninour
cells can be consideredmild. Likewise, our results showed an induction of ZmCESA7 and
ZmCESA8genesinseveralshort‐termhabituatedmaizecells(deCastroetal.,2013;Chapter
I, Figure5), aswas also observed in themaize cells subjected to long‐termhabituation to
Discussion
169
highDCBconcentrations(Mélidaetal.,2010a).Thiscoincidencecould indicatethatCESA7
andCESA8maybemoreresistant to theeffectsofDCB,playingarole inhabituationatall
levels.CharacterisationoftheSh1.5celllineandwallsrevealedfeaturescommontoalllevels
ofDCBhabituation inmaize cells, such asdelayed growthkinetics (deCastro et al., 2013;
ChapterI,Figure6),cellulardevelopmentinlargerclusters(deCastroetal.,2013;ChapterI,
Figure7)aswellasanetincreaseinarabinoxylans(deCastroetal.,2013;ChapterI,Figure
8).However,interestingdifferenceswerealsofound.
In our mildly cellulose‐deficient cells an increase was observed in the amount of
easily extractable arabinoxylan populations when compared to those extracted from Snh
cellswalls(deCastroetal.,2013;ChapterI,Figure8).Thiswasinclearcontrasttothelower
extractability degree reported for arabinoxylans frommaize cells habituated to high DCB
levels (Mélidaetal.,2009;2011), indicating that thedegreeof arabinoxylanextractability
fromthecellwalldiffereddependingonhabituationlevel.Furthermoreandsurprisingly,the
Mr andMw analyses of recently synthesised arabinoxylans in Sh1.5 cells revealed smaller
moleculesandamorehomogeneouslysizedpopulationthanarabinoxylansobservedinthe
Snhcellline(ChapterIII,Figures1and2).Thistrendwasconsistentthroughouttheculture
cycle,andwasapplicabletoallhemicellulosesandpolymersobtainedfromtheprotoplasm,
cell wall and extracellularmedium.When theMw of protoplasmic polymers and cell wall
bound hemicelluloses was compared, the results showed that newly synthesised
protoplasmic polymers underwent extensive cross‐linking when bound to the cell wall
(Chapter III, Figure 2). This finding has been reported previously in cultured maize cell
suspensions(KerrandFry,2003),andthemostplausibleexplanationisthatpolysaccharides
undergo a grafting process after being secreted into the cell wall. In maize cells the
dimerisation or oligomerisation by peroxidase action of Fer residues attached to the
arabinoxylanchains seems tobemainly responsible for this arabinoxylangraftingprocess
(Fry et al., 2000). However, Sh1.5 cells showed a less pronounced degree of cross‐linking
whencomparedwithSnhcells,andthisfindingisincontrasttothecrucialroleattributedto
Discussion
170
this type of molecular interaction in maize cells habituated to high DCB concentrations
(Mélidaetal.,2011).AnanalysisofCinnmetabolismandcharacterisationofitsderivativesin
thecellwall(ChapterIII,Figures6to9)showedthatSh1.5cellsexhibitedapoorcapacityto
synthesise polysaccharides bearing Fer or p‐Cou residues or graft them to the wall.
Furthermore, arabinoxylans and polysaccharides in general had an altered cellular fate in
Sh1.5 cells, showing a reduced amount of strongly wall bound hemicelluloses and an
increased presence of SEPs in the culturemedium (Chapter II, Figures 2, 5 and 6). Taken
togethertheseresultssupportthehypothesisofadiminishedcapacityofSh1.5tocarryout
cross‐linking of polysaccharides. However, when Fer dimer and oligomer content was
relativised,itwasobservedtobehigherinSh1.5cells.Thisfindingindicatedthatthelower
MwdetectedinarabinoxylansfromSh1.5cellswasnotrelatedtoadeficientcapacitytocarry
outphenolicoxidativecouplingofarabinoxylans.Thus,itisfeasibletoassumethatalthough
they have a lower Mw, arabinoxylans from Sh1.5 cells are actually oxidative cross‐linked.
Therefore, thecouplingof arabinoxylansbyperoxidaseactionseems tobeanothershared
feature of DCB‐habituation in maize cells to all concentrations, despite the Mw of the
molecule.
Regarding the increased presence of SEPs detected in the extracellularmedium of
Sh1.5 cells, it should be noted that this polymer population may have its origin in a)
polymers that are sloughed into the culturemedium immediately after synthesis without
beinggraftedontothecellwall,orb)inpolymersthatwereactuallyintegratedintothewall
butlaterloosenedandwerereleasedintotheculturemedium(KerrandFry,2003;Mélidaet
al.,2011).However,thislatterpossibilitycanberuledoutsincewallboundhemicelluloses
were not observed to decrease simultaneously with an increase of SEPs in the culture
medium. Hence, the most probable explanation is that due to the reduction in cellulose
content, and therefore in polysaccharide‐cellulose linking points, Sh1.5 cells sloughed an
increasedamountofpolysaccharidesintotheculturemediumbecauseoftheimpossibilityof
graftingthemontothecellwall.
Discussion
171
Since the reduction in the cellulose content detected in cells habituated to low
concentrations of DCB was considerably lower than in those habituated to high
concentrations(33%vs75%),thepossibilityarisesthatSh1.5cellssynthesisedanincreased
amountofhemicelluloseswithalowerMwaspartoftheircopingstrategy.Inthisrespect,it
has previously been reported that a lower Mw would facilitate binding to cellulose in
hemicelluloses,andmorespecifically inxyloglucan(Limaetal.,2004).Furthermore, ithas
been suggested that lower Mw xyloglucan molecules in conjunction with an increased
xyloglucan‐endo‐transglucosylase activity would contribute to rigidifying the cellulose‐
deficientwallsofDCB‐habituatedbeancells(Alonso‐Simónetal.,2007).Previousdatafrom
theanalysisoftheMwofhemicellulosesinmaizecellshabituatedtomediumandhighDCB
levels(Mélidaetal.,2009),suggestthatasthelevelofDCBhabituationrises(andtherefore
cellulose content decreases) the Mw of wall bound hemicelluloses also increases
progressively. The results obtained in this study from an analysis of the expression of
arabidopsis IRXorthologue genes are consistentwith this hypothesis. Likewise, the genes
involvedininitiationandelongationofxylanchainswerefoundtobeinducedinmaizecells
habituated to high DCB levels. In contrast, initiation of xylan synthesis was unaffected in
Sh1.5cells,whereaselongationwasimpaired,whichwouldpartiallyexplainwhySh1.5cells
hadshorterhemicellulosechains(ChapterIII,Figure10).
ThenextquestiontoarisewaswhetheranyfeatureofarabinoxylansfromSh1.5cells
differed when compared with those from Snh. The increased Ara/xylan ratio detected
(Chapter II, Table 1) would suggest the synthesis of more substituted arabinoxylans.
Certainly, other sources for Ara residues besides arabinoxylans, such as arabinogalactan
proteins or rhamnogalacturonan side chains (for a review see Fry, 2011), can exist.
Nevertheless, a sugar analysis of 0.1M and 6M alkali‐extracted fractions from Sh1.5 cell
walls, revealedapoor contentofuronic acids,Gal orRha residues (deCastroet al., 2013;
Chapter I, Figure 8). Therefore, it can be assumed that most of the Ara residues actually
derivedfromarabinoxylanmolecules.
Discussion
172
Another interesting finding was that other polysaccharides, such as xyloglucan,
rhamnogalacturonanIandhomogalacturonanwithalowdegreeofesterification,seemtobe
involvedintheearlyeventsofDCBhabituation(deCastroetal.,2013;ChapterI,Figure9B).
ThereductionincellulosecontentinDCB‐habituatedbeancellswascompensatedforbyan
increase in pectic polysaccharides (Encina et al., 2001; García‐Angulo et al., 2006).
Interestingly, although habituatedmaize cells have type II walls in which xyloglucan and
pectin polysaccharides are notmajor components, they nevertheless shared featureswith
habituated bean cells which have type I walls where these polysaccharides are more
abundant. In addition, pectin or xyloglucan content inmaize cells habituated to highDCB
levels generally remained unmodified, but in some caseswas even reduced (Mélida et al.,
2009).
The continued albeit reduced presence of xyloglucan in the cell walls of
graminaceousmonocotsplants,suggestsitplaysanimportantrole.Thus,ahigh‐Mwcomplex
ofxyloglucan thought tobe linked tootherpolysaccharideswouldexplain,at least inpart,
whygraminaceousmonocotscansurvivewithconsiderablysmalleramountsofthistypeof
hemicellulosethandicots(KerrandFry,2003).Itwasthereforeremarkabletofindahigher
Mw population of strongly wall bound xyloglucan in Sh1.5 cells (de Castro et al., 2013;
Chapter I, Figures 9B and 10; Chapter III, Figure 5B). Hence, a role for xyloglucan in the
coping strategies of mildly cellulose‐deficient cells cannot be ruled out. Since the
compositional analysis of cellwall fractions revealed that neither Snh or Sh1.5 cellswere
preferentially tightly or weakly‐grafting xyloglucan or arabinoxylans onto the cell wall
(ChapterII,Figures4and5),itcouldbehypothesisedthatthereinforcingstrategyisbased
onchangesintheirMwratherthaninthestrengthoftheirbondtothecellwall.
Theobservationthatepitopesofothercellwallpolymers,suchashomogalacturonan
withahigherdegreeofesterificationorthearabinansidechainsofrhamnogalacturonanI,
didnotappeartobemodifiedinSh1.5cellsindicatesthatratherthananoverallreshuffleof
Discussion
173
thecellwalltakingplace,onlysomespecificsubsetsofpolysaccharideswereaffectedduring
earlyDCBhabituation.
Therefore, these results demonstrate the capacity ofmaize cell cultures tomodify
their cell wall architecture in order to counteract different DCB habituation frameworks.
However, reduced cellulose content coping strategies were not limited to cell wall
architecturealone.DCB‐habituatedmaizecellsalsoshowedgeneralmetabolicchanges,not
onlypertainingtothesynthesisandcellularfateofhemicellulosesandpolymers,whichwere
delayed, less efficient and altered (Chapter II and III), but also to other important cellular
processes(Mélidaetal.,2010a).Withregardtometabolism,theGolgiapparatusisknownto
beaoneofthemostimportantorganellesintheeukaryotes;itiswherepolysaccharidesin
plants are synthesised and proteins are processed and undergo post‐translational
modifications (Parsons et al., 2012). Thus, information about the Golgi proteomic picture
fromcellulose‐deficient cellswouldprovidevaluable informationabouthowproteinsmay
contributetotheplasticityofresponsesshownbysuchcelllines.Tothisend,aprotocolfor
obtainingofGolgi‐enrichedfractionsfrommaizecellshabituatedtohighandlowDCBlevels
andnon‐habituated,wasoptimisedusingdiscontinuoussucrosegradientultracentrifugation
(Chapter IV,Figure1).Ageneral characterisationof these fractionsrevealed thatalthough
they contained a lower amount of total proteins, habituated cells exhibited higher IDPase
activity (aGolgi‐enzymaticmarker) values (Chapter IV,Table 1). TheGolgi apparatuswas
observed to undergo morphological differentiation when synthesis and secretion of
polysaccharides took place, but reverted to its compactmorphologywhen both processes
ceased (Dauwalder et al., 1969). Interestingly, in this latter study, higher IDPase activity
valuesweredetectedintissueswherepolysaccharidesynthesisandsecretionwasoccurring.
In contrast residual IDPase activity values were observed once the processes had halted.
These findings suggest that IDPase activity is co‐related to polysaccharide synthesis and
Golgi‐morphological differentiation for secretion. Therefore, our results may indicate an
enhancedsynthesisofpolysaccharidesincellulose‐deficientcellsinordertocounteractthis
Discussion
174
lack. Golgi‐enriched fractions were then subjected to 2‐D electrophoresis and the mis‐
regulatedproteinsdetectedwerestudied(ChapterIV,Table4).
Coincidences were found between some mis‐regulated proteins (enolase 1,
chaperonine60andACO1) fromGolgi‐enrichedfractionsandapreviousstudyof thetotal
proteomeofhabituatedmaizecells(Mélidaetal.,2010a).Theseconcurrencessuggestthey
play an important role in cellulose‐deficient cells. In addition, ascorbate peroxidase was
foundtobedown‐regulatedinaccordancewithitsloweractivitydetectedinDCB‐habituated
cells (Mélida et al., 2010a). Mis‐regulation of some of the sequenced proteins would be
related to some of the effects provoked by DCB. For instance, chaperonin 60 has been
describedtobeinvolvedinactinandtubulinefolding(Gutscheetal.,1999),andsinceDCB
disrupts microtubule structure (Bisgrove and Kropf, 2001; Himmelspach et al., 2003;
Rajangam et al., 2008), this would be a possible explanation for its down‐regulation in
habituatedcells.
Another examplewould be the repression observed in habituated cells of theRGP
α‐1,4‐glucanproteinsynthase(UDP‐forming).AlthoughsomeRGPsareinvolvedincellwall
polysaccharidesynthesis(Dhuggaetal.,1997;Langeveldetal.,2002),theirabsencecouldbe
relatedtoaweakenedcellwall,asinthecaseofanarabidopsisdoublemutantfortwoRGP
genes reported in Drakakaki et al. (2006). In contrast, other differentially accumulated
proteinsinhabituatedcellsseemtoparticipateinDCBcopingstrategies,whilstothersactas
indicators when cell wall integrity is damaged. For example, enolase 1, which was up‐
regulatedinhabituatedcells,hasbeenreportedtoactasastressindicatorinmaize(Sachset
al.,1996),andalsoasitssubstrate(hexoses)whencellwallintegrityisimpaired(Hamannet
al., 2003; Wolf et al., 2012). In addition, another enzyme found to be down‐regulated in
habituatedcellswasACO1,whichparticipatesinethylenebiosynthesis.Butmorethanthe
protein, it is its substrate, ACC, which is of considerable physiological importance when
cellulosebiosynthesis is inhibited,as it is involvedincellwallsensingwhenits integrity is
damaged(Tsangetal.,2011).Therefore,apossibleexplanation for thedown‐regulationof
Discussion
175
ACO1 inourhabituatedcellswouldbe that itprovokesan increase inACCcontentwhich
wouldinducetheactivationofthecorrespondingsignallingpathwaystoinitiatethecoping
strategy.
CCoAOMT1involvedintheGandSunitsoflignin,wasfoundtobedown‐regulated
intheDCB‐habituatedcells.Thisenzymewaspreviouslythoughttobesubstitutedtosome
extentinDCBhabituatedcellsbyCOMT(involvedinSunitsynthesis)(Mélidaetal.,2010a).
Furthermore,qualitativeandquantitativechangesinlignincompositioninalfalfahavebeen
related to CCoAOMT repression (Guo et al., 2001). Interestingly, similar qualitative
differences such as those reported in the previously mentioned study have also been
demonstrated in unpublished results from our laboratory. This study showed that the
proportionofGligninunitswasreducedasaconsequenceoftheDCB‐habituation,withno
apparenteffectonSunits.
It ispossible thatnotall thesequencedproteinswereGolgi‐resident.Nevertheless,
the study was carried out on Golgi‐enriched fractions, which could be expected to be
contaminated with other, extremely difficult to avoid organelles (Hanton et al., 2005;
Parsons et al., 2012). In addition, since one of the most important functions of the Golgi
apparatus is theprocessingofproteinmodifications, it isdifficult todiscriminatebetween
Golgi‐resident and others merely in transit. Although the methodology used, 2‐D
electrophoresis,hasbeendemonstratedtobesuccessfulintheanalysisofsimilarsamplesin
otherspecies(Tayloretal.,2000;Wuetal.,2000),italsopresentsanimportantlimitation,
namelythatabundantproteinscanmaskminorityones(Timperioetal.,2008).Nevertheless,
an interaction has recently been reported in the Golgi protein forming complex and,
dependingontheproteincomplexcomposition,itscatalyticfunctionalsodiffers(Oikawaet
al.,2012).Therefore,althoughnotexpectedtohaveaclearphysiologicroleintheresponse
to DCB, the possibility that some of the mis‐regulated proteins may be involved through
interactionwithothersshouldbetakenintoconsideration.
Discussion
176
Since habituated cells showed modified arabinoxylan networks, it is feasible to
assume some degree of mis‐regulation in their synthesising‐enzymes. Cell wall
polysaccharides synthesis and maturation has recently been discovered to initiate in
differentGolgicisternae,and localisationvariesdependingonthecell type(Driouichetal.,
2012;Wordenetal.,2012).Thus,itistobeexpectedthattheGTsresponsibleforaparticular
stepofthesynthesisand/ormaturationwouldalsobelocatedintheircorrespondingGolgi‐
cisternae. Therefore, it would be of interest in future experiments to perform a further
fractionationofthemembranescontainedintheGolgi‐enrichedfractionsinordertoobtain
glucuronoarabinoxylansynthase‐enrichedfractionsandassaytheirenzymaticactivities.
Theresultsobtained in thepresent studyconfirmthatmaizecell cultureshave the
capacitytoadoptdifferentcopingstrategiesdependingontheDCBhabituationframework,
using mechanisms based not only on modification of cell wall composition but also on
important variations inmetabolic and gene expression. These strategies showed common
features throughout the differentDCBhabituation levels, such as amodified arabinoxylan
network, changes in the level of expression of genes involved in cellulose or xylan
biosynthesis,andthemis‐regulationofmetabolicproteins;however,alterationswhichwere
exclusively associated with particular DCB‐habituation levels were also found, thus
demonstrating the remarkable dynamic plasticity of maize cells in coping with different
degreesofcellulosecontentreduction.
Conclusions
Conclusions
178
V.CONCLUSIONS
1. Amaizecellsuspensionlinehabituatedto1.5µMdichlobenil‐DCB‐(Sh1.5)showed
featuresof earlyDCB‐habituation. Sh1.5 cellspresentedalterations in theirgrowth
pattern,withdelayedgrowthkinetics,longerdoublingtimesandlowergrowthrates
thannon‐habituatedcells(Snh).Inaddition,Sh1.5cellsdevelopedlargercellclusters.
Their cell wall composition was also modified, with a mild reduction (33%) in
cellulosecontentthatwaspartiallycompensatedforbyanincreaseinarabinoxylans.
However, other polysaccharides such as xyloglucan or rhamnogalacturonan I also
seemedtobeinvolved,althoughtoalesserextent.
2. Arabinoxylans from Sh1.5 cells were more homogeneously sized with higher
extractabilityandlowerrelativemolecularmassandweight‐averagethanthosefrom
Snh cells. This finding was consistent throughout the culture cycle in both the
protoplasmandcellwall,aswellasintheextracellularmedium.Thecellularfateof
polysaccharidesalsomodified,withreducedproportionsofstronglywallboundones
andanincreasedpresenceofthepolysaccharidessloughedintotheculturemedium.
However,ananalysisofphenolicmetabolismrevealedthatthislatterfindingwasnot
relatedtoadiminishedcapacityforcross‐linking.
3. Expressionof thegenes involved in cellulosebiosynthesiswasalsoaffectedduring
short‐termhabituationtoDCB.ZmCESA7andZmCESA8isoformswereinducedwhen
thenumberofculturecyclesgrownin thepresenceofDCBwas increasedorwhen
theinhibitorconcentrationexceededagiventhreshold,leadingtothehypothesisthat
althoughlessefficientinthecellulosesynthesis,CESA7andCESA8proteinswouldbe
more resistant to the effects of DCB. Furthermore, the lower expression of
arabidopsis IRX orthologues, involved in initiating the synthesis and elongation of
Conclusions
179
xylan chains, suggested that only elongation was impaired in Sh1.5 cells, which
wouldpartiallyexplainthelowermolecularrelativeweightsdetected.
4. Low‐levelDCBhabituation also entailedmodifications in cultured cellmetabolism,
which was slower and less efficient throughout the entirety of the culture cycle.
Nevertheless,althoughdelayedwithrespecttoSnhcells,synthesis,wall‐graftingand
sloughing of the different types of polysaccharides and polymers occurred
synchrony.
5. Importantmetabolicenzymesrelatedtostressconditions,ethylenebiosynthesisand
carbohydrate and ligninmetabolismwere also differentially accumulated in Golgi‐
enrichedfractionsfromDCB‐habituatedcells.Themis‐regulationobservedcouldbe
relatedtothedirecteffectsofDCBonthecells,asinthecaseofchaperonin60and
the reversible glycosylation polypeptide α‐1,4‐glucan protein synthase (UDP‐
forming),ormayhavereflectedarole in theDCB‐copingstrategiesadoptedby the
habituated cells such as enolase 1, 1‐aminocyclopropane‐1‐carboxylate oxidase 1,
ascorbateperoxidaseandcaffeoyl‐CoAO‐methyltransferase1.
Mostoftheseresultsshowthatsomeofthechangesthatoccurredinthecellwallsin
order to compensate for the lackof cellulose1)differedaccording to theDCB‐habituation
level,2)impliedmodificationsofcellwallarchitectureandseveralmetabolicfeatures,and3)
illustratethecapacityofplantcellstoadoptappropriatecopingstrategiesdependingonthe
herbicideconcentrationandlengthofexposuretime.
Conclusions
180
Resumen
Resumen
182
INTRODUCCIÓN
Laparedcelulardelasplantas
Losprotoplastosdelascélulasdelasplantasterrestresestánrodeadosporunacapa
semirrígida:laparedcelular.Apesardequeeltérminoparadesignarlainduzcaapensaren
unaestructuraimpenetrableyestática,setratadeuncompartimentocelularmuydinámico
conimportantesfuncionestantoestructuralescomofisiológicas.Lasparedescelularesguían,
restringenydeterminanelcrecimientocelularytienenunpapelcrucialenlaespecialización
funcionaldelosdiferentestiposcelulares.Alconstituirlabarreraexternaentrelacélulayel
medio se encuentran implicadas en la señalización de respuestas a situaciones de estrés
(Roberts, 2001; Hückelhoven, 2007; Driouich y col., 2012) y procesos de reconocimiento
célula‐célulaasícomocélula‐patógeno(Vorwerkycol.,2004;SeifertyBlaukopf,2010;Wolf
ycol.,2012).Además,presentanunaelevadaplasticidadestructuralydecomposición,que
permite a las células adaptarse a condiciones de estrés, tanto abióticos como bióticos
(Hamanny col.,2009).Apartedesus funciones fisiológicas, lasparedescelulares también
tienenunimportantevaloreconómico,yaqueinfluyenenlatexturayelvalornutricionalde
la mayor parte de los productos que se obtienen de plantas, son un factor clave en su
procesamiento,yseconsideranfuenteyreservoriodeenergíaenplantasparalaproducción
debiocombustibles(BurtonyFincher,2012).
Laparedcelularprimariaesexclusivadecélulasqueaúnmantienenlacapacidadde
dividirse y/o elongarse. Finalmente, en algunas células especializadas aparece una
estructura multicapa, que es la pared secundaria, de mayor espesor y ordenación más
regular de las microfibrillas de celulosa y que suele presentar depósitos de lignina y
suberina.
La síntesis de los diferentes componentes de la pared celular ocurre en distintos
compartimentos celulares. Las microfibrillas de celulosa se sintetizan en la membrana
plasmática por acción del complejo enzimático celulosa sintasa (CSC), y se depositan
Resumen
183
directamente en la pared. Los polisacáridos no celulósicos (hemicelulosas y pectinas) se
sintetizan en el aparato de Golgi, y las diferentes proteínas, tanto estructurales como
enzimáticas, se sintetizan en el retículo endoplásmico rugoso, aunque con frecuencia las
proteínasestructuralessufrenglicosilacionesyotrasmodificacionesenelGolgi.Losdistintos
componentesnocelulósicosdelaparedcelularseempaquetanenvesículassecretorasyse
transportan a la superficie celular, donde se integran con las microfibrillas de celulosa
sintetizadasdenovo(CarpitayMcCann,2000).
Arquitecturadelaparedcelularprimaria
La pared celular primaria se compone de tres redes estructuralmente
independientespero interconectadas (CarpitayMcCann,2000).Laprimeradeestas redes
estáconformadaporunarmazóndecelulosa‐hemicelulosas,lasegundaredestáconstituida
porpolisacáridospécticos,ylaterceraredconsisteenproteínasestructuralesycompuestos
fenólicos.
En las plantas superiores se distinguen dos tipos de paredes celulares primarias
(Carpita y Gibeaut, 1993) que difieren en la composición química y en la asociación a
diferentestaxones:laparedcelularprimariatipoI,queescaracterísticadedicotiledóneasy
algunasmonocotiledóneas(nocommelinoides),ylaparedcelulartipoIIpresenteenalgunas
monocotiledóneas(commelinoides)(Popper,2008).
ComposicióndelaparedcelulartipoII
Celulosa
Elcontenidodecelulosaenlasparedescelularesprimarias,tantotipoIcomotipoII,
varíaentreel15%yel30%,siendoesteporcentajemayorenlaparedcelularsecundaria
(CarpitayMcCann,2000).Enelcasodecélulascultivadasinvitro,laproporcióndecelulosa
sesitúaentornoaun20%(Blaschekycol.,1981).
La celulosa en las paredes celulares primarias se dispone formandomicrofibrillas,
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184
quesonestructuras insolubles compuestaspor36cadenasdeβ‐1,4‐glucanodispuestasen
paralelo(Delmer,1999;SaxenayBrown,2000;Lerouxelycol.,2006).Lascadenasdeβ‐1,4‐
glucano adquieren una estructura espacial plana que permite la interacción de unas con
otrasmediantelaformacióndepuentesdehidrogenointraeintercatenarios,quedanlugara
unaestructuracristalinamuyestable(Somerville,2006).
Lasíntesisdelacelulosaocurreenlamembranaplasmática,siendodepositadaenla
paredcelular(Guerrieroycol.,2010).Lamaquinariaencargadadesusíntesisesuncomplejo
proteico CSC o rosetas (Brown, 1996; Kimura y col., 1999). En plantas, los CSCs se
encuentran organizados en hexámeros constituido por proteínas denominadas celulosa
sintasa(CESA).
Las proteínas CESA son codificadas por familias multigénicas, con un número
variable de genes dependiendode la especie. En arabidopsis sehan identificado10 genes
CESA,mientrasqueenmaízhansido12(Hollandycol.,2000;Appenzellerycol.,2004).En
maízlosgenesZmCESA1al9seencuentranimplicadosenlasíntesisdecelulosaenparedes
primariasyZmCESA10,11y12enlacorrespondientealassecundarias.Sinembargodebido
a su homología con el genZmCESA12más que con ZmCESA10 y11, se ha propuesto que
están implicados en la síntesis de celulosa en paredes celulares primarias durante los
estadiostardíosdeldesarrollo(Appenzellerycol.,2004).
Además de las CESA, existen otras proteínas implicadas en la síntesis de celulosa,
comolasacarosasintasa(Amorycol.,1995),laproteínaKORRIGAN(Nicolycol.,1998)olas
proteínasasociadasamicrotúbulos(MAP)(Sedbrook,2004).
Polisacáridosnocelulósicos:pectinasyhemicelulosas
Laspectinassonpolisacáridosmatricialesricosenácidogalacturónico.Lasparedes
tipo II presentan menor contenido en pectinas que las paredes tipo I. Aun siendo un
componenteminoritario, laspectinas jueganun importantepapel en la retencióndeagua,
transporte de iones, adhesión celular y determinación de tamaño de poro de la pared.
Resumen
185
Además,estánimplicadasenmecanismosdedefensafrenteapatógenos,heridasyestreses
abióticos (Ridley y col., 2001; Jarvis y col., 2003; Verhertbrugger yKnox, 2007). Entre los
polisacáridos pécticos se encuentran el homogalacturonano, el ramnogalacturonano I y el
ramnogalacturonanoII.
Elhomogalacturonanoesunhomopolímeroderestosdeácidogalacturónicoquepuede
presentar diferentes grados de metilesterificación. En principio, se deposita en la pared
celular con un alto grado de metilesterificación, y una vez allí sufre la pérdida de restos
metiloporlaaccióndepectinmetilesterasas.Estapérdidademetilacionesesnecesariapara
que se establezcan puentes de calcio entre cadenas antiparalelas de homogalacturonano
(Liners y col., 1989). La cadena principal del homogalacturonano se encuentra
covalentementeunidaaambostiposderamnogalacturonano,yademássecreequetambién
puedeinteraccionarinmuroconelxiloglucano(PopperyFry,2008).
Elramnogalacturonano tipo I secomponedeunacadena formadapor larepeticióndel
disacáridoα‐1,4‐ácido‐galacturónico‐α‐1,2‐ramnosa(CarpitayMcCann,2000).Losrestosde
ramnosapuedentenerunidascadenaslateralesdegalactano,arabinanooarabinogalactano.
El ramnogalacturonano tipo II es un galacturonano con una gran diversidad de
sustituciones. Las moléculas de ramnogalacturonano tipo II se pueden asociar entre sí
formandodímerosmedianteelenlaceatravésderestosborato(Albersheimycol.,2011).
Las hemicelulosas son un grupo de polisacáridos neutros constituidos por una
cadena lineal demonosacáridos, principalmente xilosa, glucosa omanosa, en general con
ramificaciones cortas y estructura no cristalina. La pared celular tipo II contiene como
principales polisacáridos hemicelulósicos xilanos, y glucano mixto. Además, aparece una
pequeñaproporcióndexiloglucano(CarpitayMcCann,2000).
Losxilanosyelglucanomixtounenmicrofibrillasdecelulosaadyacentesmedianteel
establecimiento de puentes de hidrogeno, y las mantienen en su disposición espacial
Resumen
186
correcta, asumiendo las funciones que el xiloglucano desempeña en las paredes celulares
tipoI.
Los xilanos son los principales polisacáridos hemicelulósicos en paredes celulares
tipo II (Fincher, 2009) constituyendo alrededor del 20‐40% del peso de la pared (Vogel,
2008). Suestructura sebasaenuna cadenaprincipaldeβ‐1,4‐xilosas, ypuedenpresentar
sustituciones de arabinosa (arabinoxilanos) y en menor medida de ácido glucurónico
(glucuronoarabinoxilanos).EnparedescelularestipoIImuyfrecuentementelasunidadesde
arabinosa del arabinoxilano/glucurono‐arabinoxilano pueden encontrarse sustituidas por
ácidoshidroxicinámicos(principalmenteácidoferúlicoyenmenormedidaácidocumárico)
unidosmedianteenlaceéster(WendeyFry,1997).Estosrestosdeácidoshidroxicinámicos,
mediante acoplamiento oxidativomediado por peroxidasas (Geissmann yNeukom, 1971),
sonsusceptiblesdeunirsedemaneraqueentrecruzanlascadenasdearabinoxilanossobre
lasqueseencuentranesterificados(Fry,2004).
Elxiloglucanoesunβ‐1,4‐glucanoconnumerosasramificacionesdeα‐1,6‐xilosaque
presentan a su vez sustituciones de arabinosa, galactosa y/o fucosa (Hayashi, 1989). En
paredescelularestipoIIesunpolisacáridominoritario,enelquelafucosanoestápresente
(Carpita,1996).
Labiosíntesisasícomoelensamblajedelospolisacáridosnocelulósicostienelugar
en el aparato de Golgi. Posteriormente, son transportados en vesículas derivadas de este
orgánulo hasta lamembrana plasmática, donde se produce la fusión de las vesículas y el
consecuente vertido de su contenido hacia la pared celular (Driouich y col., 1993; 2012;
Lerouxel y col., 2006;Day y col., 2013). Su síntesis corre a cargodeuna seriede enzimas
denominadasglicosiltransferasas(GTs),clasificadasen92familiasenlabasededatosCAZy.
Proteínas
En la pared primaria se pueden encontrar tanto proteínas estructurales como
solubles. En cuanto a las proteínas estructurales, las paredes tipo II presentan también
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contenidosreducidos(1%)encomparaciónconlastipoI(10%)(Vogel,2008).Estetipode
proteínasseencuentraninmovilizadasenlapared,yfrecuentementesonglicoproteínascon
secuenciasrepetidasdeunoodosaminoácidos.
Respectoa lasproteínas solubles, lamayorpartedeellas sonenzimas relacionadas
conlaextensióndelaparedcelular,eltransportemolecular,elreconocimientocelularo la
resistencia a patógenos (Rose y col., 2002). Dentro de ellas encontramos hidrolasas,
transglicosilasas,peroxidasas,expansinasykinasas.
Fenoles
Los ácidos ferúlico y p‐cumárico son los principales fenilpropanoides o
hidroxicinamatosdelaparedcelular(WallaceyFry,1994)y,aunquesoncuantitativamente
minoritarios,sonimportantes.Lascadenasdearabinoxilanopuedenentrecruzarsegraciasa
la polimerización oxidativa de estos restos de ácido ferúlico, generando dímeros
(dehidroferulatos)oinclusotrímerosuoligómeros,unidosporenlacefenil‐fenilofenil‐éter.
El proceso de esterificación y entrecruzamiento se produce de manera mayoritaria en el
Golgiperotambiénpuederealizarseinmuro.Estasunionesconducenaunreforzamientode
la estructura de la pared celular, promueven la cohesión celular, restringen la expansión
celular, contribuyen al ensamblaje de la pared y participan en el reforzamiento de esta
estructuraenrespuestaafactoresabióticosybióticos(Buanafina,2009).
Plasticidadestructuraldelaparedcelular
Lascélulaspuedenmodificarlacomposiciónyestructuradesusparedescelularesen
condicionesdeestrés,tantoabióticoscomobióticos.Elgradodeflexibilidaddelasparedes
dependedelaespeciedequesetrate,deltipodetejidoeinclusodelgradodediferenciación
deltipocelulardado.Elestudiodelaplasticidadestructuraldelaparedcelularadquiereuna
gran importancia, no sólo desde un punto de vista de investigación básica con el fin de
conocer cómo se regula la síntesis y el ensamblaje de sus componentes, sino también en
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investigación aplicada, con el fin de obtener productos de interés en las industrias textil,
papelera,maderera,odebiocombustibles.
En los últimos años el empleo de técnicas de cultivo in vitro de células, tejidos y
órganosvegetaleshapermitidoavanzarconsiderablementeenlacaracterizacióndeparedes
celularesalteradaspormodificacionesgenéticas(mutaciones)yporadaptacionesaestreses
abióticos, comosalinos (Binzely col., 1988),metalespesados (Carpenay col., 2000),bajas
temperaturas (Yamada y col., 2002) o desecación (Moore y col., 2013), así como por la
habituaciónainhibidoresdelabiosíntesisdelaparedcelular(Satiat‐JeunemaitreyDarzens,
1986;Suzukiycol.,1992;VaughnyTurley,1999;Encinaycol.,2001;García‐Anguloycol.,
2006;2009;Mélidaycol.,2009).
Habituaciónaherbicidasinhibidoresdelabiosíntesisdecelulosa
Lahabituacióndecultivoscelularesainhibidoresdelabiosíntesisdelaparedcelular
reflejalacapacidaddelascélulasdesobrevivirconunaparedcelularmodificada,siendopor
tantounsistemamuyútilparaavanzarenelconocimientodelaplasticidadestructuraldela
pared celular. Varios compuestos han sido descritos como inhibidores de algunos de los
componentesdelaparedcelularprimaria(Acebesycol.,2010;García‐Anguloycol.,2012),
entre los que se encuentra el diclobenil (2,6‐diclorobenzonitrilo o DCB), que inhibe
específicamente la síntesis de celulosa enplantas superiores (Vaughn, 2002), sin afectar a
otrosprocesosfisiológicoscomolasíntesisdeADNoproteínas(GalbraithyShields,1982),la
respiración(MontezinosyDelmer,1980)oelmantenimientodelosnivelesdeUDP‐Glucosa,
fosfolípidosonucleósidosmonootrifosfato(Delmer,1987). Aunque ladianadelDCBaún
permanece desconocida, parece tratarse de una proteína MAP, en concreto la MAP20
(Rajangamy col., 2008).EstosautorespostularonqueelDCB interaccionaría conMAP20,
bloqueandoelensamblajedelasproteínasCESAalosmicrotúbulosyportantolasíntesisde
celulosa.
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En las dos últimas décadas se ha demostrado que es posible habituar cultivos de
células indiferenciadas (callos y suspensiones celulares) a concentraciones letales de DCB
aumentando paulatinamente su concentración en el medio de cultivo (Shedletzky y col.,
1992;Wellsycol.,1994;NakagawaySakurai,1998;Sabbaycol.,1999;Encinaycol.,2001;
2002; Alonso‐Simón y col., 2004; García‐Angulo y col., 2006; 2009; Mélida y col., 2009;
2010a;2010b;2011).
La habituación a DCB reside en la capacidad que las células tienen de dividirse y
crecerconuncontenidoreducidoencelulosaenlaparedcelular.Enesteprocesoseproduce
unaseriedecambiosestables,quedifierenencuantoaltipodeparedcelularquepresentan
lascélulas.Enelcasodecultivosdemaízhabituadosaconcentraciones letalesdeDCB, los
cambios que acompañan a la habituación residen, sobre todo, en la compensación de la
pérdidade celulosa con incrementos en el contenidode ácidos fenólicos y arabinoxilanos.
Ademásestosarabinoxilanosdifierendelosquepresentanlascélulasnohabituadas,yaque
tienen mayor masa molecular, se unenmás fuertemente a la pared y se encuentran más
entrelazadospordehidroferulatos(Mélidaycol.,2009;2010a;2010by2011).Loscambios
asociados a la habituación a altas concentraciones de DCB son muy drásticos y se han
observadotraslargosperiodosenpresenciadelinhibidor.Sinembargo,esposiblequeestas
modificaciones tengan lugar de manera paulatina y que por lo tanto estemos perdiendo
valiosa información acerca del mecanismo de habituación durante los primeros estadios.
Además, ha sido previamente demostrado en cultivos celulares de alubia que los cambios
producidos por la habituación a DCB difieren (tanto cualitativamente como
cuantitativamente) dependiendo de su concentración en el medio como del periodo de
tiempoquelascélulaspermanecenexpuestasaél(Alonso‐Simónycol.,2004;Mélidaycol.,
2009). Por otro lado, el hecho de que altas concentraciones del inhibidor provoquen
importantesmodificacionescelularespodríalimitarlarealizacióndedeterminadostiposde
análisis, como los estudios metabólicos in vivo, entre otros. Por tanto, el empleo de
suspensiones celulares demaíz habituadas a bajas concentraciones deDCB es una valiosa
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herramientaparaestudiarenprofundidadlaplasticidadestructuraldelasparedescelulares
tipoII,particularmentedesdeunpuntodevistametabólico.
OBJETIVOS
El principal objetivo de esta tesis es esclarecer los mecanismos subyacentes a la
plasticidad estructural de las paredes celulares primarias tipo II, haciendo énfasis en las
estrategias metabólicas. Para llevarlo a cabo, se utilizarán cultivos celulares de maíz
habituadosabajosnivelesdeDCB.Esteobjetivoprincipalsedivideasuvezencuatrosub‐
objetivosparciales,enumeradosacontinuación:
ObjetivoI
Monitorizaciónycaracterizacióndelasmodificacionesquesucedendurantelosprimerospasos
delprocesodehabituaciónaDCB
LascélulashabituadasaDCBsoncapacesdedesarrollarestrategiasparasobrevivir
en presencia del inhibidor, que varían dependiendo del tipo de pared celular, la
concentracióndeDCBoelperiododetiempoenelcuallascélulasseencuentrancreciendo
encontactoconel(Alonso‐Simónycol.,2004).Lasmodificacionesproducidasenlasparedes
decélulasdemaízhabituadasaDCBhansidopreviamenteestudiadasmedianteelempleode
concentraciones letalesdel inhibidor y tiemposprolongadosdehabituación (Mélida y col.,
2009). Sin embargo, se desconocen los cambios que acontecen durante los primeros
momentos del proceso de habituación. Por este motivo, el primer objetivo del presente
estudioesa)lamonitorizacióndelasmodificacionesquetienenlugardurantelahabituación
tempranaaDCB,b)laseleccióndeunalíneacelularquemuestretantocaracterísticasdeesta
habituación incipiente, comouna reducciónmoderadaenel contenidoencelulosa y, c) la
consiguientecaracterizacióndesuspatronesdecrecimientoycomposicióndesusparedes
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191
celulares. Para acometerlo, se emplearan técnicas tanto espectrofotométricas como
cromatográficasydeinmunoanálisis.
ObjetivoII
AnálisisdelmetabolismohemicelulósicodecélulasconnivelesincipientesdehabituaciónaDCB
LassuspensionescelularesdemaízhabituadasaDCBsoncapacesdecontrarrestarel
empobrecimientoen celulosapresente en susparedesmediante la adquisicióndeuna red
modificada de arabinoxilanos tanto a nivel cuantitativo como cualitativo (Mélida y col.,
2009;2010a;2010b;2011).Araízdeestosantecedentes,surgeelsegundoobjetivodeeste
trabajo, que será el estudio delmetabolismo de polisacáridos en células demaíz con una
reducciónmoderadaenelcontenidoencelulosa.Esteestudioserealizaráalolargodelciclo
de cultivo a través experimentos de radio‐marcaje in vivo, suministrando a las células
[3H]arabinosa como precursor metabólico en fase de acomodación, exponencial y
estacionaria. Posteriormente, se rastreará tanto la síntesis como el destino celular de
hemicelulosasypolímerosmarcadosradioactivamente,envarioscompartimentoscelulares
durante5h.
ObjetivoIII
Estudiodeladistribucióndemasasmolecularesdepolisacáridosymetabolismofenólicoen
célulasdemaízconunareducciónmoderadaenelcontenidoencelulosa
EnestudiospreviosrealizadosencélulasdemaízhabituadasaDCB,sehandescrito
paredes celulares reforzadas a través del desarrollo de una red de arabinoxylanos con
mayoresmasasmolecularesymásentrecruzados,queademáspresentabanunmenorgrado
deextractabilidaddelapared(Mélidaycol.,2009;2011).Sinembargo,estosestudiosfueron
realizadosenarabinoxilanosaisladosúnicamentedelaparedcelularyenunafasefinaldel
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ciclo de cultivo. Por esta razón, el tercer objetivo del presente trabajo será el análisis, en
células demaíz con una reducción leve en el contenido en celulosa, de la distribución de
masasmolecularesdepolisacáridosenvarioscompartimentoscelularesa lo largodelciclo
de cultivo. Para llevarlo a cabo, empleando como metodología experimentos de radio‐
marcaje in vivo, se administrará a las suspensiones celulares [3H]arabinosa y ácido
[14C]cinámico como precursores metabólicos para hemicelulosas e hidroxicinamatos,
respectivamente.
ObjetivoIV
AnálisisdelproteomadeGolgiencélulasdemaízhabituadasaDCB
Laplasticidadestructuralmostradapor lasparedesde célulasdemaíz cuandoson
habituadasaDCB innegablemente incurreen sumetabolismoypor ende, en lasproteínas
responsables de las respuestas exhibidas. Teniendo en cuenta que importantes enzimas
metabólicas son proteínas residentes en el aparato de Golgi, se obtendrán fracciones
enriquecidasendichoorgánulo tantodecélulasdemaíznohabituadascomohabituadasa
diferentes niveles de DCB. En primer lugar, se realizará un análisis comparativo del
proteomadeGolgide lasdiferentes líneascelularesmedianteelectroforesisbidimensional.
Posteriormente,aquellasproteínasdeinterésqueseacumulendiferencialmenteentrelíneas
sesecuenciaránmedianteMALDI‐TOF/MS(matrix‐assistedlaserdesorptionionization‐time
of flight mass spectrometry) o ESI‐MS/MS (electrospray ionization with tandem mass
spectrometry).
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MATERIALESYMÉTODOS
Cultivoscelulares
Las suspensiones celulares se obtuvieron a partir de callos de maíz (Zeamays L.,
BlackMexican)procedentesdeembrionesinmaduros,enmediolíquidoMurashigeySkoog
que contenía 2,4‐D 9 μM, sacarosa 20 g L‐1 (pH5,6). Las suspensiones semantuvieron en
agitaciónorbital,a25°C,fotoperiodode16hyfueronsubcultivadascada15días.
HabituaciónaDCB
ElprocesodehabituaciónaDCBserealizócultivandosuspensionescelularesdemaíz
no habituadas (Snh) en presencia del inhibidor. En el caso de suspensiones habituadas a
bajos niveles de DCB, células Snh se subcultivaron y fueron mantenidas en tres
concentracionesinicialesdeDCB:0,3μM(Sh0,3),0,5μM(Sh0,5)y1μM(Sh1).Despuésde
variossubcultivos,partedelascélulasprocedentesdelassuspensionesmantenidasenDCB
1μMsetransfirieronaunmedioqueconteníaDCB1,5μM(Sh1,5).
LascélulasdemaízhabituadasaaltasconcentracionesdeDCBseobtuvieronapartir
decallosdemaízhabituadosaDCB12μM,quesetransfirieronamediolíquidoconteniendo
DCB6μM(Sh6).
Caracterizacióndeloscultivoscelulares
Se realizó una cinética de crecimiento de las suspensiones celulares de maíz
midiendoelincrementoenpesosecoparacadaintervalodetiempoalolargodetodoelciclo
decultivo,yseestimaronvariosparámetrosdecrecimientocomoeltiempodeduplicación,
latasarelativaylatasamáximadecrecimiento.
Paraladeterminacióndeltamañodeagregado,lassuspensionescelularesdemaízse
filtraronatravésdefiltrosdenylondediferentetamañodeporo, expresandoelresultado
finalcomoporcentajedepesosecoretenidoencadafiltrorespectoaltotal.
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Aislamiento de fracciones de diversos compartimentos celulares de suspensiones
celularesdemaíz
Obtencióndelmedioextracelularyextraccióndelcontenidoprotoplasmático
Alícuotasde suspensiones celulares se filtrarona travésde columnasPoly‐Prep.El
filtrado resultante libre de células, se recogió y consideró la fracción correspondiente al
medioextracelular(CFM).Enlosestudiosllevadosacabocon[3H]arabinosacomoprecursor
metabólico, las células que quedaron retenidas en el filtro de la columna Poly‐Prep se
resuspendieron en un tampón de extracción del contenido protoplasmático y fueron
homogeneizadas.Acontinuación,elhomogeneizadoresultantesehizopasaratravésdeuna
segunda columnaPoly‐Preppara recoger el filtrado correspondiente, que fue centrifugado
varias veces hasta que el sobrenadante se tornó transparente, momento en el cual se
considerócomolafracciónprotoplásmica.
En el caso de los estudios realizados con ácido [14C]cinámico como precursor
metabólico, se tomaron alícuotas de suspensiones celulares que se filtraron a través de
columnas Poly‐Prep o, alternativamente, se sometieron a centrifugación. El filtrado o
sobrenadantesedescartó,ylascélulasseincubaronconetanolal80%.Posteriormente,las
células se filtraron o centrifugaron y el filtrado o sobrenadante se recogió y se consideró
comocontenidosimilaralprotoplásmico.
ObtencióndefraccionesenriquecidasenGolgi
Célulasprocedentesdesuspensionescelularesdemaízsepulverizaronennitrógeno
líquidoysehomogeneizaronentampóndeextracción(Zengycol.,2008).Elhomogeneizado
secentrifugóyserecogióelsobrenadante.Estesobrenadantesecargóencimadetampónde
sacarosa 2 M y fue sometido a ultracentrifugación para la obtención de la fracción
microsomal. Después de esta primera ultracentrifugación, los microsomas quedaron
localizadosenunabandadefinidainmediatamenteporencimadelafasecorrespondienteal
tampóndesacarosa2M.Lafracciónsuperioraestabandafueretiradaysobrelafracción
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microsomal se añadieron superpuestos una serie de tampones de sacarosa de
concentraciones1,3M,1,1My0,25M, conobjetode crearungradientediscontinuo.Este
gradiente discontinuo se sometió de nuevo a ultracentrifugación, y la interfase generada
entrelostamponesdeconcentraciones0,25My1,1Mserecogiócomofracciónenriquecida
enGolgi.
Extracciónyfraccionamientodeparedescelulares
Dependiendo del enfoque experimental, se siguieron dos metodologías diferentes
para laobtenciónde lasparedescelularesysuposterior fraccionamiento.Deestamanera,
cuando los experimentos realizados no incluían el uso de compuestos radio‐marcados, se
llevóacabounprotocolomásexhaustivo(protocoloA).Encambio,cuandolametodología
incluíaelusoymanejodecompuestosradioactivos,seempleóunaversiónsimplificadapor
razonesdeseguridad(protocolosB1yB2).
ProtocoloA
Paralaobtencióndeparedescelulares,lassuspensionescelularesdemaízse
sometieron a una homogeneización con nitrógeno líquido hasta obtener un polvo
fino, y el homogeneizado se trató con etanol al 70% durante 5 días. El residuo
resultante se trató con etanol al 70% y acetona y se dejó secar a temperatura
ambienteparaobtenerelresiduoinsolubleenalcohol(AIR).PosteriormenteelAIR
fue tratado con dimetilsulfóxido y el residuo obtenido se incubó con α‐amilasa de
páncreasporcinodisueltaen tampón fosfato.Tras retirar la soluciónenzimática,el
residuoselavóconetanolal70%yacetonaysedejósecaratemperaturaambiente.
Posteriormente, se trató con fenol/acético/agua y finalmente se lavó con
etanolal70%yacetonay sedejó secara temperaturaambiente. Se consideróque
esteresiduoeranlasparedescelulares.
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Las paredes celulares fueron sometidas a una serie de tratamientos para
extraer de manera secuencial distintos tipos de polímeros. Primeramente, fueron
tratadas con ácido t‐1,2‐diaminociclohexano‐N,N,N´,N´‐tetra‐acético (CDTA) y el
residuo insoluble obtenido se incubó con KOH 0,1 M. Al residuo resistente a esta
extracción se le añadió KOH 4 M y de nuevo, el residuo obtenido de la fracción
anterior fuesometidoaunúltimotratamientoconKOH6M.Lossobrenadantesde
cada uno de los tratamientos constituyeron las fracciones CDTA; KOH 0,1M; KOH
4MyKOH6M.
ProtocoloB1:estudiosqueimplicaronelusode[3H]arabinosacomoprecursor
metabólico
En este caso, células procedentes de las suspensiones celulares demaíz se
homogeneizaron y el homogeneizado resultante se filtró a través de una columna
Poly‐Prep. A los fragmentos celulares retenidos en el filtro se les trató con una
solucióndeNaOH0,1MqueconteníaNaBH4.Elresiduoinsolublefuesometidoaun
segundotratamientoconNaOH6MconteniendoNaBH4,yalmaterialresistentesele
considerócomopolímerosfuertementeunidosalacelulosa.
ProtocoloB2:estudiosqueimplicaronelusodeácido[14C]cinámicocomo
precursormetabólico
Las células de suspensiones de maíz se re‐suspendieron e incubaron en
etanol al 80%. Posteriormente, se filtraron o, alternativamente, centrifugaron y el
correspondientefiltradoosobrenadantesedescartó,considerandoalosfragmentos
celulares residuales el AIR. El AIR se saponificó mediante tratamiento con NaOH
0,5MylafracciónsolubleresultantedelmismoseconsiderólafracciónNaOH0,5M.
Dichafracciónfueacidificadaconácidoacéticoyseprocedióarealizarunaprimera
partición en acetato de etilo. Consecutivamente, lasmuestras se secaronmediante
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197
vacío, y se re‐disolvieron en agua acidificada, para volver a ser sometidas a una
segundaparticiónenacetatodeetilo.Las fasesorgánicasserecogieron,secaronen
vacíoyre‐disolvieronenpropanol.
Lasfraccionessolublesobtenidasdelosdiferentestratamientosfueronneutralizadas
a pH 5 con ácido acético, dializadas y etiquetadas con el nombre del tratamiento
correspondienteutilizadoparasuextracción.
Análisisdelasparedescelulares
Valoracióndelcontenidoencelulosa
Lacantidaddecelulosasecuantificóespectrofotométricamentemedianteelmétodo
de Updegraff (Updegraff, 1969) con las condiciones hidrolíticas descritas por Saeman
(Saeman y col., 1963). La glucosa liberada se valoró por elmétodo de la antrona (Dische,
1962).
Valoracióndelcontenidoenazúcares
La determinación del contenido de azúcares totales de cada fracción se realizó
espectrofotométricamentemedianteelmétododelfenol‐sulfúrico(Duboisycol.,1956)yla
deácidosurónicosmedianteelmétododelm‐hidroxibifenil(BlumenkrantzyAsboe‐Hansen,
1963) utilizando como estándares glucosa y ácido galacturónico respectivamente. El
contenidoenazúcaresneutrosserealizómediantecromatografíadegasessegúnelmétodo
descritoporAlbersheimycol.(1967).
EspectroscopíadeinfrarrojoportransformadadeFourier(FTIR)
Con objeto de monitorizar los cambios ocurridos en las paredes celulares de las
diferentes líneas demaíz, se utilizó FTIR. Para ello, las paredes celulares procedentes de
suspensiones, semezclaron con KBr y se homogeneizaron en unmortero de ágata, hasta
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conseguir un polvo fino. Posteriormente se comprimieron en una prensa Graseby‐Specac,
obteniéndoselaspastillasnecesariasparaelanálisisyqueserealizómedianteelempleode
unespectroscopioPerkin‐Elmer.Unavezobtenidos losespectrosFTIRsecorrigiósu línea
baseysenormalizaronsusáreas.
Inmuno‐ensayos
La composiciónde lasdiferentes fraccionesobtenidas en el fraccionamientode las
paredescelularesseanalizómedianteensayosdeinmunodot.Paraello,setomaronalícuotas
de 1 μl de distintas diluciones de las fracciones, y se colocaron en orden decreciente en
membranas de nitrocelulosa. Posteriormente, lasmembranas se incubaron con una endo‐
poligalacturonanasa, una ‐xilanasa y una endo‐arabinanasa (para más detalle ver el
apartadodedigestionesenzimáticas)aplicandolametodologíadescritaenØbroycol.(2007)
con ligeras modificaciones. Después de los tratamientos enzimáticos, las membranas de
nitrocelulosa fueron bloqueadas con tampón fosfato salino (PBS) con leche en polvo, y se
incubaron con anticuerpos primarios específicos para diferentes tipos de polisacáridos
pécticosyhemicelulósicos.SelavaronlasmembranasconaguayPBSparaeliminarelexceso
deanticuerpoprimarioyseincubaronconunanticuerposecundarioanti‐rataconjugadocon
peroxidasaderábano.DespuésdelavarlasmembranasconPBSyaguamilli‐Qparaeliminar
elexcesodeanticuerposecundario,seañadiólasoluciónsustratoparaprocederalrevelado
delasmembranas.
La cuantificación del inmuno‐marcaje mostrado en cada fracción se realizó
otorgando valores correspondientes al número de diluciones que exhibíanmarcaje. Estos
valores fueron después utilizados para generar los heatmaps (para más detalle ver el
apartadocorrespondientealanálisisestadístico).
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Análisisdeexpresióngénica:aislamientodeARNtotal,RT‐PCRyPCR
ElARNtotalseextrajoempleandoelreactivocomercialTrizol,yfueposteriormente
retro‐transcritomedianteel sistema“Superscript III firststrandsynthesissystem”.ElADN
complementario segeneróusandouncebadoroligo(dT)20,que fueutilizadocomocadena
moldeen las consiguientes reaccionesdePCR.Para cadaanálisisde expresión sehicieron
pruebas variando el número de ciclos de las reacciones de PCR con objeto de determinar
aquellas condiciones óptimas en las que la amplificación se encontrase dentro del rango
exponencial.Comogencontrolseempleóeldelaubiquitina.
AnálisisdeexpresióndegenesZmCESA
Para llevar a cabo el análisis de los genes ZmCESA1 (AF200525), ZmCESA2
(AF200526),ZmCESA3(AF200527),ZmCESA5(AF200529),ZmCESA6(AF200530)yZmCESA7
(AF200531)seusaronloscebadoresespecíficosparaellosdescritosenHollandycol.(2000).
Debido a que los genes ZmCESA1 y ZmCESA2 poseen una alta homología de secuencia, se
utilizóelmismocebadorparaestudiarlaexpresióndeambosgenes.EnelcasodeZmCESA4
(AF200528)yZmCESA8(AF200532),seemplearon loscebadoresdescritosenMélidaycol.
(2010a).
AnálisisdeexpresióndegeneshomólogosIRXdearabidopsis
Sediseñaronyemplearoncebadoresespecíficosparaelanálisisenmaízdelosgenes
GRMZM2G100143, GRMZM2G059825 y gbIBT036881.1 homólogos de IRX10 (AT1G27440),
IRX10‐L (AT5G61840) y IRX9 (AT2G37090) respectivamente en arabidopsis (Bosch y col.,
2011). Las secuencias de dichos cebadores derivaron de la base de datos “maize genome
browser” para el caso de GRMZM2G100143 y GRMZM2G059825 y “Phytozome” para
gbIBT036881.1.
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Experimentosderadiomarcajeinvivo
Estudiosqueimplicaroncon[3H]arabinosacomoprecursormetabólico
Se recogieronalícuotasde suspensiones celularesdemaíz endiferentes etapasdel
ciclodecultivoyselessuministróL‐[1‐3H]arabinosa.Laincorporaciónde[3H]arabinosaen
los diferentes compartimentos celulares se midió a distintos tiempos en cada etapa de
cultivo.
Estudiosconácido[14C]cinámicocomoprecursormetabólico
El ácido [14C]cinámico se sintetizó partiendo de L‐[U‐14C]fenilalanina, siguiendo
básicamenteelmétododescritoenLindsayyFry.(2008).Alasalícuotasdesuspensionesde
maízselessuministróácido[14C]cinámicoendiferentespuntosdelciclodecultivoy,tantola
incorporacióndelprecursorcomosuconsumo,semidieronadiferentestiempos.
Cuandofuerequerido,aalgunasdelasmuestrasselesañadióH2O2120mindespués
de la administración de ácido [14C]cinámico, y se mantuvieron durante 60 min más en
condicionesdeagitación.
Laradioactividadpresenteenlasmuestrasseanalizóenuncontadordecentelleo.
Digestionesenzimáticas
Previamente al tratamiento enzimático con Driselasa las muestras se secaron en
vacíoysesometieronaunahidrólisissuaveenácidotrifluoroacético(TFA).Finalmentese
volvieronasecaryelmaterialsecoseredisolvióenunasolucióndeDriselasaal0,5%(p/v)
en piridina/ácido acético/agua y se incubó a 37°C durante 96 h. La reacción se paró
mediantelaadicióndeácidofórmicoal15%.
Los tratamientos con endo‐poligalacturonanasa (M2) extraída de Aspergillus
aculeatus(E‐PGALUSP)(EC3.2.1.15),‐xilanasarecombinantedeNeocallimastixpatriciarum
(E‐XYLNP)(EC3.2.1.8)yendo‐arabinanasadeAspergillusniger (E‐EARAB)(EC3.2.1.99)se
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llevaronacaboa37°Cdurante24h.Paraello,lasenzimassedisolvieronentampónacetato
350mMpH4.7aunaconcentraciónde1U/ml.
Cromatografía
Cromatografíaenpapel
ParallevaracabolacromatografíaenpapelseutilizópapelWhatman3MM,yeltipo
de solvente y la duración variaron dependiendo de los componentes a separar. De esta
manera, para la separación de monosacáridos, se realizó en butanol/ácido acético/agua
durante 16 h. En aquellos casos en que fue necesaria la separación de monosacáridos,
disacáridos y material polimérico a RF 0, la cromatografía en papel se llevó a cabo
empleando acetato de etilo/piridina/agua durante 18 h (Thompson y Fry, 1997).
Posteriormente, seprocedió a la tinciónde los cromatogramas con ftalatode anilina (Fry,
2000)yserevelarona105°C.
CromatografíadeFiltraciónenGel
LospolímerossefraccionaronportamañosusandounacolumnadeSefarosaCL‐4B
porlaquesehizopasarunflujodepiridina/ácidoacético/aguade12,5ml/h.Lacolumnafue
previamentecalibradacondextranosdemasasmolecularesrelativasmedias(Mw)conocidas.
Mediante el método del Kav (1/2) (Kerr y Fry, 2003) se obtuvo la ecuación de calibración
[log Mw = ‐3,547 Kav(1/2) + 7,2048] que se empleó posteriormente para los cálculos
necesarios.
CromatografíadeGases
Las muestras fueron previamente liofilizadas e hidrolizadas en TFA 2 M a 121°C
durante 1 h. Los azúcares resultantes se derivatizaron a alditol acetatos y se analizaron
medianteunacolumnaSupelcoSP‐2330(Albersheimycol.,1967).
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CromatografíaenCapaFina
Se realizó en placas de plástico con silica gel como fase inerte, conteniendo
indicadores de fluorescencia. Como solvente se empleó benceno/ácido acético y para su
reveladolasplacasseexpusierona321nm.
Electroforesis
Electroforesisenpoliacrilamida(SDS‐PAGE)
Los análisis se llevaron a cabo tanto en condiciones semi‐nativas como en
desnaturalizantes,nohirviendoohirviendo,respectivamente,lasmuestras.Posteriormente
sesepararonengelesdeacrilamidadel7,5%,10%o12,5%.Comotincionesseemplearon
coomassiebrilliantblue(CBB)R‐250onitratodeplata.
Electroforesisbidimensional
Antesde larealizaciónde laelectroforesisbidimensional, fueronnecesariosciertos
pasospreviosparalapreparaciónde lasmuestras.Paraello,se liofilizaronalícuotasdelas
mismas que posteriormente se disolvieron en el tampón específico para la electroforesis
bidimensional, el tampón de lisis. Una vez disueltas, se incubaron en un baño de
ultrasonidos, se centrifugaron y se recogió el sobrenadante, que fue dializado. A
continuación,lasmuestrasseconcentraronmedianteelsistemadecolumnasVivaspin,hasta
queseobtuvolaconcentracióndeproteínanecesaria.
Subsiguientemente,conobjetoderealizarlaseparaciónenlaprimeradimensiónse
utilizaron gradientes inmovilizados con un rango de pH 4‐7. La separación en la segunda
dimensiónserealizóengelesSDS‐PAGEal10%.Losgelesresultantessetiñeronempleando
CBBG‐250(Camposycol.,2010)omediantetincióndeplata(Shevchenkoycol.,1996;Irary
col., 2006)en funciónde la cantidaddeproteínasa resolver, siendoesteúltimodemayor
sensibilidad.Unavezteñidos,losgelesfueronescaneadosylaadquisicióndelasimágenesse
realizó a 16‐bits/canal, 300 dpi (en escala de grises) y en formato TIFF. Las imágenes
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generadas se utilizaron para realizar el análisis proteómico (Farinha y col., 2011), fueron
integradasylosspotsidentificados.Aestosspotsdemaneraautomáticalesfueronasignados
unaintensidad,volumen,áreayelparámetrosaliency,queintegraatodosellos.Enestecaso
el parámetro normalizado utilizado fue el%Volumen o volumen normalizado. Cuando los
datosmostraronvariacionessuperioresaunvalordel0,5delvolumennormalizadoy1del
ratio se realizó el análisis estadístico, con la consecuente validación de los resultados
medianteelTest‐tconunvalorpdel0,05(Farinhaycol.,2011).Losspotsdeinterésfueron
cortados y se procedió a su identificaciónmedianteMALDI‐TOF y porESI‐MS/MS. Para la
identificación de proteínas se utilizaron las bases de datos de NCBI y Swissprot. Los
programasdebúsquedasutilizadosfueronSEQUESTyMASCOT.
Análisisestadísticos
El análisis de componentes principales (PCA) se realizó con un máximo de cinco
componentesprincipalesenelsoftwareStatistica6.0.
ParaelanálisisdediferenciassignificativasserealizóuntestANOVAdeunavíacon
unapruebadeTukey’s comoanálisispost‐hocenel softwarePASWStatistics18obienel
Test‐tconunvalorpdel0,05.
LosheatmapsserealizaronempleandoelsoftwareRKWard.
RESULTADOSYDISCUSIÓN
LahabituaciónaDCBsebasaeneldesarrollodeestrategiasque implicancambios
tanto en el metabolismo como en la arquitectura de la pared celular, para superar la
limitaciónquesuponeelhechodetenerparedesdeficitariasencelulosa.Enelcasoespecífico
de las células de maíz habituadas a crecer en concentraciones letales de DCB, se ha
observadoqueunaremodelacióncuantitativaycualitativadelareddearabinoxilanosjuega
unpapel clave enuna granparte de estas estrategias (Mélida y col., 2009; 2010a; 2010b;
2011).Sinembargo,tambiénsehademostradoquelavariedadderespuestasdeadaptación
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depende del tipo de pared celular, así como de la concentración de DCB y el tiempo de
exposición al inhibidor (Alonso‐Simón y col., 2004; Mélida y col., 2009). Dado que los
estudios previos en células de maíz habituadas a DCB se han llevado a cabo con altas
concentracionesdelinhibidoryperíodosdehabituaciónalargoplazo,lainformaciónacerca
delasprimerasmodificacionesquetienenlugarduranteelprocesodehabituaciónsepierde.
Tal información puede resultar muy valiosa ya que durante los primeros estadios de la
habituaciónlascélulaspodríanpresentarunagranvariedadderespuestas.Porlotanto,enla
presentetesisseharealizadounextensoanálisisdelasmodificacionesquetienenlugaren
losprimerospasosdelprocesodehabituaciónaDCB.
Monitorización de los cambios acontecidos en los primeros pasos del proceso de
habituaciónaDCB
En primer lugar, se procedió a identificar una línea celular demaíz quemostrara
características de una temprana o incipiente habituación a DCB (de Castro et al., 2013;
Capítulo I). Por esta razón, suspensiones celulares de maíz Snh fueron sub‐cultivadas
durantevariosciclosdecultivoendiferentesconcentracionesdeDCB(partiendode0,3µM
hasta 1,5 µM), y las modificaciones en la pared celular se monitorizaron mediante
espectroscopíaFTIR (Figuras I.1 a3) y valoracionesdel contenidoen celulosa (Figura I.4)
Los resultados revelaron que la principal modificación de la pared celular durante la
habituaciónaDCBestárelacionadaconelcontenidoencelulosa.Lascélulasquecrecíanen
las concentraciones más bajas de DCB presentaron inicialmente una reducción en el
contenido en celulosa que posteriormente revirtió hasta valores próximos a los de Snh, a
medida que se incrementaban los ciclos de cultivo enpresencia del inhibidor (Figura I.4).
Dadoquesehabíaobservadopreviamentequelascélulashabituadasaaltasconcentraciones
deDCBpresentabanfluctuacionesenelniveldeexpresióndealgunosgenesZmCESA(Mélida
y col., 2010a) se procedió a evaluar su expresión también en células habituadas a bajas
concentraciones de DCB (Figura I.5). Al igual que en los procesos de habituación a altos
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niveles (Mélida y col., 2010a), los resultados mostraron una inducción de ZmCESA7 y
ZmCESA8 en células demaíz habituadas a bajos niveles. Dicha coincidencia podría indicar
queCESA7yCESA8actúandemaneramáseficienteenpresenciadeDCB,porloquepodrían
tenerunpapelenlahabituaciónatodoslosniveles.
Los resultados de lamonitorización dieron lugar a la selección de una línea Sh1,5
comolamásadecuadaparalosposterioresanálisis,basándonosenlossiguientescriterios:
a) presentó una reducción no reversible de un 33%del contenido en celulosa respecto al
control (Figura I.4),dichareducciónpuedeconsiderarse levesi secomparaconel75%de
reducciónquepresentanlaslíneashabituadasalargostiemposyelevadasconcentraciones
deDCB (Méliday col., 2009);b) los espectrosFTIRmostraronque, ademásde la celulosa,
también otros polisacáridos estaban afectados en esta línea (Figuras I.1 y 2); c) los
resultados de PCA apuntan a diferencias no solo con las células control sino también con
respecto al resto de las líneas habituadas monitorizadas (Figura I.3), lo que indicaría
característicasdeunahabituaciónincipiente.
CaracterizacióndelalíneacelulardemaízhabituadaabajosnivelesdeDCB
LaampliacaracterizacióndelalíneaSh1,5ydesusparedescelularesrevelófactores
comunesenlahabituacióndemaízaDCBatodoslosniveles.Algunasdelascaracterísticas
comunes de las células habituadas a DCB es que muestran cinéticas de crecimiento
retrasadas y parámetros de crecimiento alterados respecto a las de las células control
(FiguraI.6),desarrollocelularenformadegrandesagregados(FiguraI.7)yunincremento
netoenarabinoxilanos(FiguraI.8).Sinembargo,tambiénsehanencontradodiferenciasen
los tipos de modificaciones de la pared celular. En primer lugar, durante los primeros
estadios de la habituación, el incremento en arabinoxilanos se produjo en fracciones de
polisacáridos fácilmente extraíbles (Figura I.8), contrastando con los arabinoxilanos más
fuertementeunidosquepresentabanlaslíneashabituadasaaltosnivelesdeDCB(Méliday
col., 2009).Además, otrospolisacáridos comoel xiloglucano, el ramnogalacturonano I y el
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homogalacturonanoconbajogradodemetilesterificaciónparecenestarinvolucradosenlos
primeros pasos de la habituación (Figura I.9B). Los tratamientos enzimáticos específicos
para pectinas, hemicelulosas y cadenas laterales de algunos polisacáridos, afectaron de
maneradiferencialalaslíneascelularescontrolyestaslíneashabituadas(FiguraI.10).Esto
podría indicar diferencias estructurales en los polisacáridos de ambas líneas, lo cual
permitiríauna acción enzimáticamásomenos eficaz.Noobstante, sonnecesarios análisis
másprofundosparaverificarestahipótesis.
Estudios similares realizados en cultivos celulares de alubia habituados a DCB
mostraron que estas células conpared celular tipo I compensaban su pérdida en celulosa
modificandolaredpécticayhemicelulósica(Encinaycol.,2001;García‐Anguloycol.,2006).
LascélulasdemaízpresentanparedesdetipoIIenlasqueelxiloglucanoylaspectinasno
son componentesmayoritarios. Sin embargo, es de destacar que cuando son habituadas a
DCB, comparten características con células de alubia. En sus paredes celulares tipo I,
xiloglucano y pectinas sí son componentes principales y tienen un papel relevante en la
habituación a DCB (Encina y col., 2001; 2002; Alonso‐Simón y col., 2004; 2011; García‐
Anguloycol.,2006;2009).Porotrolado,enlascélulasdemaízhabituadasaaltosnivelesde
DCB el contenido en pectinas o xiloglucano no se vio modificado o incluso fue reducido
(Mélidaycol.,2009).
Estosresultadosponenenevidencialaelevadacapacidaddeloscultivoscelularesde
maízparamodificarlaarquitecturadelaparedcelular,conelfindeadaptarsealosdistintos
nivelesdehabituaciónaDCB.
Análisis del metabolismo de polisacáridos de células de maíz habituadas a bajos
nivelesdeDCB
Dado que es esperable que tales respuestas de plasticidad estructural estén
relacionadas con el metabolismo, se eligieron células habituadas a DCB como sistema
experimentalparaindagarenelmetabolismodelospolisacáridosdelaparedcelularyenlas
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interaccionesqueentreellostenganlugar.Deestemodo,célulasdemaízSnhySh1,5fueron
cultivadas en presencia de [3H]arabinosa como precursormetabólico en diferentes etapas
delciclodecultivo,conelobjetivodeaislardistintoscompartimentoscelularesyfracciones
deparedparasuposterioranálisis(CapítuloII).Además,conelfindecomprobarsiestaba
siendo particularmente afectado un tipo específico de polisacárido, las muestras se
sometieronadigestiónenzimáticaconDriselasa.
Tráficocelularycaracterizacióndehemicelulosasyotrospolímeros
Se realizó un seguimiento del metabolismo, así como del tráfico celular de
hemicelulosas([3H]arabinoxilanosy [3H]xiloglucano)yotrospolímeros([3H]polímerosque
contienenarabinosaypolímerossindigerirporDriselasa).Enprimerlugar,seobservóque
ambas líneas celulares diferían en la toma del precursor radio‐marcado.Mientras que las
células Snh mostraron una tendencia similar a la observada previamente en cultivos
celularesdemaíz(KerryFry,2003)yrosa(EdelmannyFry,1992;Thompsonycol.,1997),
lascélulasSh1,5mostraronunacapacidadmenorenlatomade[3H]arabinosa(FiguraII.1).
Además, en todas las fases de cultivo analizadas (Figura II.2) las células Sh1,5 fueron
metabólicamente más lentas y menos eficientes que las células Snh y que otros cultivos
celularesdemaíz(KerryFry,2003).Dichareducciónenlacapacidadmetabólicaafectópor
igual a la síntesis, la incorporación a pared celular o la liberación al medio de cultivo de
arabinoxilanos,xiloglucano,polímerosquecontienenarabinosaypolímerosnodigeridospor
Driselasa(FigurasII.3a6).
La extractabilidad de los arabinoxilanos de la pared celular también se vio
modificada en función del nivel de habituación. Los cultivos celulares habituados a altos
niveles de DCB presentaron arabinoxilanosmás difícilmente extraíbles que las líneas Snh
(Mélidaycol.,2009;2011)ySh1,5.
Lasiguientecuestiónqueseplanteófuesilas3H‐hemicelulosasolos3H‐polímerosde
lascélulasShdifierendealgunamanerade loscorrespondientesa lascélulasSnh.Las 3H‐
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hemicelulosas de las células Sh presentaron un incremento en la relación
[3H]arabinosa/[3H]xilanolocualpodríaestarindicandolapresenciadearabinoxilanosmás
sustituidos en estos cultivos (Tabla II.1). No obstante, este resultado se debe tomar con
cautela ya que otros polisacáridos también pueden aportar restos de arabinosa, como los
arabinogalactanos de las arabinogalactano proteínas y determinadas cadenas laterales del
ramnogalacturonano (para más detalle ver Fry, 2011). Sin embargo, el análisis de las
fraccionesobtenidasde laparedcelulardecélulasSh1,5revelóbajoscontenidosdeácidos
urónicos así como de restos de galactosa y ramnosa (Figura I.8). Por lo tanto, se puede
asumir que la mayor parte de los restos de arabinosa derivan de moléculas de
arabinoxilanos.
Destinocelulardehemicelulosasyotrospolímeros
Eldestinocelulardelos3H‐polímeroscuandoabandonanelprotoplasmatambiénfue
distintoenSh1,5(FiguraII.2).Alcontrariode loobservadoenaltosnivelesdehabituación
(Méliday col., 2009;2011), las células Sh1,5presentaronuna reducciónen la cantidadde
hemicelulosas fuertemente unidas a la pared (las extraídas con NaOH 6 M). Además, las
célulasSh1,5mostraronunincrementorelativoenhemicelulosasextraídascontratamientos
suaves(NaOH0,1M)(FiguraII.4),locualcoincideconresultadosanteriores(FiguraI.8).Por
último,seobservóunincrementodepolisacáridosextracelulares(SEPs)liberadosalmedio
de cultivo (Figura II.6). Estos SEPs pueden incluir polímeros que han sido sintetizados
recientementeyexcretadosalmediosinunirsealapared,ypolímerosquefueronintegrados
alaparedymástardehansidoliberadosalmediodecultivo(KerryFry,2003;Mélidaycol.,
2011).Sinembargoestaúltimaposibilidadpuededescartarse,yaquenoseobservóen las
células Sh1,5 una disminución en la incorporación de radiactividad en las fracciones
obtenidasdelapared,sincrónicamenteconunaumentodelamismaenelmediodecultivo.
Este hecho puede tener dos posibles explicaciones: a) dado que las células Sh1,5
contienenmenoscelulosaensusparedes,seproduciríaundescensoenlosposiblespuntos
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deuniónparaotrospolisacáridos,y/ob)laslíneasSh1,5podríantenerunamenorcapacidad
para incorporar polisacáridos a través de enlaces fenólicos. Esta alternativa es
particularmente interesanteyaqueen lahabituaciónde célulasdemaíz aniveles altosde
DCB, se ha demostrado que la presencia de una red más desarrollada de arabinoxilanos
unidos mediante acoplamiento fenólico oxidativo es decisiva en las estrategias de
reforzamiento de la pared (Mélida y col., 2009; 2011). La importancia del incremento del
acoplamientooxidativoresideenqueproducemoléculasdearabinoxilanoconmayoresMw,
cruciales para reforzar unas paredes celulares que muestran un 75% de reducción en
celulosa.
AnálisisdeladistribucióndemasasmolecularesyMwdehemicelulosasyotros
polímeros
Las Mw de hemicelulosas y polímeros recién sintetizados en células Sh1,5 fueron
menoresquelosdeSnh(CapítuloIII),locualapoyalahipótesisdequeestaslíneaspudieran
presentanmenorcapacidadparaentrecruzarpolisacáridos(FigurasIII.1y2).Estatendencia
fue consistente en todo el ciclo de cultivo y en todos los compartimentos celulares
(protoplasmayparedcelular),asícomoenelmedioextracelularparalosdiferentestiposde
polisacáridos y polímeros (Figuras III.1 a 5). Además, cuando se compararon las Mw de
polímerosprotoplásmicosconlosdehemicelulosasunidasalapared,secomprobóquelos
polímerosprotoplásmicosreciénsintetizadossufríanun intensoentrecruzamientounavez
incorporados a la pared (Figura III.2). Este comportamiento ya se había observado con
anterioridad en suspensiones demaíz (Kerr y Fry, 2003), aunque fuemenosmarcado en
Sh1,5queenSnh.Laexplicaciónmásplausibleesquelospolisacáridossonsometidosaun
procesodeunióndespuésdesersecretadosenlaparedcelular.Talesmecanismosincluyen
interaccionesentremoléculastantodetiponocovalentecomodetipocovalente,talescomo
formación de puentes iónicos, de hidrógeno, glicosídicos o acoplamiento oxidativo (Fry,
2000).Aunqueconvieneconsiderartodotipodeinteracciones,esesperablequelospuentes
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dehidrógenooelacoplamientooxidativoseanespecialmenteimportantesenelprocesode
entrecruzamientoentrearabinoxilanos.
Estudiodelmetabolismofenólico
Enmaíz, se sabe que la formación de dímeros u oligómeros de ácido ferúlico que
entrelazanarabinoxilanosporaccióndeperoxidasaseselprincipalmecanismoporel cual
aumentalaMwdeestashemicelulosas(Fryycol.,2000).Porotraparte,ycomosemencionó
anteriormente,enlascélulasdemaízhabituadasaaltasconcentracionesdeDCBestetipode
entrecruzamiento fenólico tieneunpapel crucial en las estrategias de reforzamientode la
pared(Mélidaycol.,2011).Porestemotivo,serealizóunanálisisdelmetabolismodelácido
cinámico, así como una caracterización de sus derivados en la pared celular, en células
habituadasabajosnivelesdeDCB(FigurasIII.6a9).LascélulasSh1,5mostraronunaescasa
capacidadparaincorporarenlaparedcelularpolisacáridosconrestosdeácidoferúlicoop‐
cumárico(Figuras III.6a9), locualapoyaría losresultadosobtenidospreviamente(Figura
II.2). Los resultados que se han expuesto hasta ahora reforzarían la hipótesis de una
disminuciónen lacapacidadde lascélulasSh1,5para llevaracaboelentrecruzamientode
lospolisacáridos.Sinembargo,entérminosrelativos,elcontenidoendímerosyoligómeros
deácidoferúlicofuemayorencélulasSh1,5.EstehechopodríaindicarquelasMwmenores
observadasenestaslíneasnoestánrelacionadasconunmenoracoplamientofenólicodelos
arabinoxilanos.
Dadoquelareducciónenelcontenidodecelulosaenlascélulashabituadasabajos
nivelesesconsiderablementemenorqueen lashabituadasaconcentracionesaltasdeDCB
(33%vs 75%), surge laposibilidaddeque las células Sh1,5 estén sintetizandounamayor
cantidaddemoléculas conmenorMw comopartede su estrategiadehabituación. En este
sentido, se ha descrito con anterioridad que las hemicelulosas, específicamente el
xiloglucano,conmenoresMwpuedenunirsemásfácilmentealacelulosa(Limaycol.,2004).
Además,Alonso‐Simóny col. (2007)propusieronqueun xiloglucano conmenorMw, junto
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con un incremento de la actividad xiloglucano‐endo‐transglucosilasa, podría contribuir a
aumentar la rigidez de la pared en células de alubia habituadas a DCB. Estudios previos,
realizadosennivelesdehabituaciónaDCBmediosyaltos,parecenindicarqueamedidaque
disminuyeelcontenidoencelulosaenlasparedesaumentaprogresivamenteeltamaño(Mw)
de las hemicelulosas unidas a pared (Mélida y col., 2009). En concordancia con esto se
comprobóquelascélulasdemaízhabituadasaaltosnivelesdeDCBpresentabanunmayor
niveldeexpresióndelosgenesIRXortólogosdearabidopsisimplicadostantoenlainiciación
comoenlaelongacióndelascadenasdexilano(FiguraIII.10).SinembargolascélulasSh1,5
nomostraroncambiosenlaexpresióndelosgenesimplicadosenlainiciacióndelasíntesis
de xilanos, pero sí presentaron una menor expresión para los genes involucrados en la
elongación.EstehechopodríaexplicarporquélascélulasSh1,5contienenhemicelulosasde
cadenasmáscortas.
Como consecuencia, es factible suponer que a pesar de tener menores Mw, las
hemicelulosas y polímeros de células Sh1,5 están más entrelazados. Por lo tanto, la
contribucióndeeste tipodeenlacespareceserotracaracterísticacomúnenelprocesode
habituaciónaDCBencélulasdemaíz.
EstudiodelproteomadeGolgiencélulasdemaízhabituadasaDCB
Como hemos visto, las células habituadas a crecer en presencia de DCB presentan
alteracionesmetabólicas que afectan entre otras cosas a la síntesis de polisacáridos de la
pared. En lo referente almetabolismo de polisacáridos, el aparato de Golgi es uno de los
orgánulosmásimportanteseneucariotas.Enplantaseselorgánuloencargadonosolodela
síntesis de polisacáridos sino también del procesamiento de algunas proteínas (Parsons y
col.,2012).Por lotanto,unestudiodelproteomadeGolgipodríaproporcionarunavaliosa
información sobre cómo las proteínas contribuyen a la plasticidad de las respuestas que
muestranlascélulashabituadasaDCB(CapítuloIV).
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212
ObtencióndefraccionesenriquecidasenGolgi
Para abordar este objetivo se desarrolló un protocolo de obtención de fracciones
enriquecidasenGolgidecélulasdemaíz,nohabituadasyhabituadasanivelesbajosyaltos
deDCB, empleandoun gradientediscontinuode sacarosa (Figura IV.1). La caracterización
general mostró que las fracciones obtenidas de células habituadas presentaban menor
cantidaddeproteínatotalperomayoractividadIDPasa(actividadmarcadoradeGolgi;Tabla
IV.1). Cuando la síntesis y secreción de polisacáridos tiene lugar, el aparato de Golgi
experimentaciertasdiferenciacionesmorfológicas,revirtiendoasuestadocompactocuando
estasactividadescesan(Dauwalderycol.,1969).Enesteestudioseobservóunaumentoen
losvaloresdeactividadIDPasaentejidosdondelasíntesisysecrecióndepolisacáridoseran
muy activas, valores que revirtieron a basales cuando ambos procesos dejaron de
producirse. Por lo tanto, estos resultados sugieren que la actividad IDPasa está
correlacionada con la síntesis de polisacáridos, más concretamente con los cambios
morfológicos que experimenta el aparato de Golgi durante la secreción de este tipo de
moléculas. Consecuentemente, nuestros resultados podrían apuntar a una síntesis
potenciada de polisacáridos no celulósicos en las líneas habituadas, precisamente para
contrarrestarlapérdidaencelulosa.
Tras varios pasos de preparación de las muestras (Figuras IV.2 a 4 y Tabla 2), la
fracción enriquecida en Golgi de cada una de las líneas fue sometida a electroforesis 2‐D
(Figura IV.5), ya que este tipo de metodología ha demostrado ser adecuada en estudios
similaresrealizadosconotrasespecies (Taylorycol.,2000;Wuycol.,2000).Detodas las
proteínasdesreguladas(TablaIV.3)aquellasdemayorinterésfueronextraídasdelgelpara
suposterioridentificación.
Identificacióndeproteínasdesreguladas
LosresultadosdelasecuenciacióndedichasproteínassemuestranenlaTablaIV.4.
Algunasdelasproteínassecuenciadas(enolasa1,chaperonina60y1‐aminociclopropano‐1‐
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carboxílicooxidasa1‐ACO1‐) fueroncoincidentesconotrasencontradasenunestudiode
proteoma total llevado a cabo anteriormente por nuestro grupo en células habituadas
(Mélida y col., 2010a). Estos resultados parecen sugerir que estas proteínas juegan un
importante papel en la habituación a DCB. Además, se detectó una ascorbato peroxidasa
menosdiferencialmenteacumuladaen las líneashabituadas, lo cual coincidecon lamenor
actividadquepresentaestaenzimaendichaslíneas(Mélidaycol.,2010a).
Los resultadosapuntan aque algunasde lasproteínas secuenciadas se encuentran
desreguladasporefectodirectodelDCB,ynotantoporlahabituaciónalmismo.Porejemplo,
se ha descrito que la chaperonina 60 está implicada en el ensamblaje de la actina y la
tubulina(Gutscheycol.,1999).DadoqueelDCBactúaimpidiendolapolimerizacióndelos
microtúbulos (Bisgrove y Kropf, 2001; Himmelspach y col., 2003; Rajangam y col., 2008),
estehechopodríaexplicarporquéencélulashabituadaslachaperonina60aparecemenos
diferencialmente acumulada. Otro caso puede ser la represión observada en células
habituadas para la α‐1,4‐glucano sintasa, clasificada dentro del grupo de polipéptidos de
glicosilación reversible (RGP). Aunque algunos RGPs están involucrados en la síntesis de
polisacáridosdepared(Dhuggaycol.,1997;Langeveldycol.,2002),suausenciapuedeestar
relacionada conunaparedcelularmásdebilitada, tal y comohandemostradoparadobles
mutantesdegenesRGPsDrakakakiycol.(2006).
Porotrolado,otrasproteínasdiferencialmenteacumuladasenlascélulashabituadas
parecen formar parte de la estrategia de adaptación a DCB. Algunas de ellas podrían
participarcomoindicadoresrelacionadoscon la integridadde laparedcelularcuandoésta
se ve dañada. La enolasa 1, que aparece más acumulada en las células habituadas, se ha
descrito como una señal de estrés en maíz (Sachs y col., 1996), así como su sustrato
(hexosas)cuandolaintegridaddelaparedcelularsevedañada(Hamannycol.,2003;Wolfy
col.,2012).LaACO1,enzimaimplicadaenlasíntesisdeetileno,tambiénseacumulómenos
enlaslíneashabituadas.Enestecaso,essusustrato,amino‐ciclopropano‐carboxilico(ACC),
másquelaenzima,elquepresentaunarelevanciafisiológicacuandolasíntesisdecelulosa
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214
se ve afectada (Tsang y col., 2011). Si los niveles de ACO 1 son menores en las células
habituadas,susustratoACCsufriráunaacumulaciónenlascélulas,locualpodríaactivarla
correspondienterutadeseñalizaciónparacomenzarconlasestrategiasdehabituación.
Laenzimacafeoil‐CoAO‐metiltransferasa1(CCoAOMT1),queparticipaenlasíntesis
deunidadesGySdelalignina,apareciómenosacumuladaenlascélulashabituadasaDCB.
Ya se había descrito anteriormente que esta enzima era sustituida durante el proceso de
habituaciónporlaácidocafeico3‐O‐metiltransferasa(COMT),queparticipaenlasíntesisde
unidadesS(Mélidaycol.,2010a).Además,sehavistoenalfalfaqueloscambioscualitativos
y cuantitativos en la composición de la lignina están relacionados con la represión de la
enzima CCoAOMT (Guo y col., 2001). Curiosamente, resultados preliminares de nuestro
laboratorio indican que nuestras líneas celulares habituadas a DCB presentan diferencias
cualitativasenlalignina,yaqueéstapresentaunmenorcontenidoenunidadesGsintener
afectadoelcontenidodelasunidadesS.
NotodaslasproteínasquefueronsecuenciadassonresidentesdeGolgi.Sinembargo,
hayqueconsiderarqueelestudiosehallevadoacaboconfraccionesenriquecidasenGolgi,
por lo que es esperable que puedan tener contaminaciones con otros orgánulos celulares
(Hanton y col., 2005; Parsons y col., 2012). Además, dado que una de las funciones más
importantesdelaparatodeGolgiesrealizarmodificacionespost‐traduccionalesenmuchas
proteínas,resultadifícildiscriminarentreproteínasentránsitooresidentes.
Aunque la metodología empleada ha demostrado ser efectiva para el análisis de
muestras similares en otras especies (Taylor y col., 2000; Wu y col., 2000), tiene la
restriccióndequelasproteínasmásabundantesenmascaranalasminoritarias(Timperioy
col.,2008).Porotro lado,recientementesehavistoque la interacciónentreproteínasque
forman parte de los complejos proteicos del Golgi puede diferir, y que este hecho afecta
también a la función catalítica de losmismos (Oikawa y col., 2012). Por lo tanto, hay que
considerarlaposibilidaddequealgunasdelasproteínasdesreguladas,aunquenotenganun
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papelfisiológicoclaroenlarespuestadehabituaciónaDCB,podríanparticiparatravésdesu
interacciónconotras.
Puesto que las células habituadas tienen modificada la red de arabinoxilanos, es
plausibleasumirquetambiénpresentenalgúntipodedesregulaciónenlasenzimasquelos
sintetizan.Sehadescubiertorecientementequela iniciacióndelasíntesisdepolisacáridos
delaparedcelularysumaduraciónseproduceendiferentescisternasdeGolgi,yquedicha
localizaciónvaríadependiendodeltipodecélula(Driouichycol.,2012;Wordenycol.,2012).
Por lo tanto, sería esperable que las enzimas GTs encargadas de dicha síntesis y/o
maduración estuvieran también ubicadas en distintas cisternas del aparato de Golgi. En
futurosestudiosseríainteresanterealizarunanálisismásfinodelasmembranascontenidas
enlafracciónenriquecidaenGolgiycomprobarenellasdistintasactividadesGTs.
CONCLUSIONES
1.LalíneacelularhabituadaaDCB1,5µM(Sh1,5)mostrólascaracterísticasdeuna
habituación incipiente al inhibidor. Estas células presentaron patrones de crecimiento
alterados, entre los que se observaron cinéticas de crecimiento más lentas, tiempos de
duplicaciónmáslargosytasasdecrecimientomásbajasquelascélulasnohabituadas(Snh).
Además,crecierondesarrollandoagregadoscelularesdemayorestamaños.Lacomposición
desusparedescelularestambiénresultómodificadacomoconsecuenciadelahabituacióna
DCB: se observóuna reduccióndel 33%en el contenido en celulosaque fueparcialmente
compensadoporun incremento en arabinoxilanos.Además, aunque enmenormedida, los
contenidos de otros polisacáridos como el xiloglucano y el ramnogalacturonano tipo I
parecenencontrarsemodificados.
2. Los arabinoxilanos de las células Sh1,5 constituyeron poblaciones más
homogéneas en términos demasasmoleculares, con tamañosmediosmás bajos y mayor
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gradodeextractabilidadquelosdelascélulasSnh.Estascaracterísticasfueronconsistentes
en todas las fasesdel ciclodecultivoasí comoen losdiferentescompartimentoscelulares
analizados:protoplasma,paredcelularyenelmedioextracelular.Eldestinocelularde los
polisacáridos se encontró también modificado en las células habituadas a DCB, que
mostraron proporciones reducidas de hemicelulosas fuertemente unidas a la pared y un
incremento de los polímeros liberados al medio de cultivo. El análisis del metabolismo
fenólicodescartóqueesteúltimohechofuesedebidoaunamenorcapacidaddelascélulas
Sh1,5paraentrelazardichospolisacáridos.
3.Laexpresióndealgunosdelosgenesimplicadosenlabiosíntesisdecelulosayde
xilanosresultóalteradadurantelahabituaciónabajosnivelesdeDCB.Deestamanera,según
aumentabaelnúmerodeciclosdecultivoenelquelascélulascrecíanenpresenciadeDCB,o
cuandolaconcentracióndelmismosuperóciertoumbral,lasisoformasZmCESA7yZmCESA8
resultaroninducidas.Estehallazgodalugaralahipótesisdequeaunquemenoseficientesen
lasíntesisdecelulosa, lasproteínasCESA7yCESA8seríanmásresistentesa losefectosde
DCB. Los resultados del análisis de expresión de los genes ortólogos IRX de arabidopsis,
implicados tanto en la iniciación de la síntesis como en la elongación de las cadenas de
xilanos, sugieren que solo ésta última se encontraría afectada en las células Sh1,5, lo cual
contribuiría parcialmente a explicar las menores masas moleculares medias de estas
moléculasobservadasenestalíneacelular.
4.ComoconsecuenciadelahabituaciónabajosnivelesdeDCB,elmetabolismodelas
célulasdemaíz también resultóalterado, siendomenoseficienteymás lentoa lo largode
todoelciclodecultivo.Noobstante,aunquelasíntesis, integraciónenlaparedasícomola
liberaciónde losdistintos tiposdepolisacáridosalmediodecultivoseencontróretrasada
conrespectoalosdelascélulasSnh,ocurriódemanerasincronizada.
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217
5.Importantesenzimasmetabólicasrelacionadasconcondicionesdeestrés,síntesis
de etileno o metabolismo de carbohidratos y lignina se encontraron diferencialmente
acumuladasenlasfraccionesenriquecidasenGolgidelascélulashabituadasaDCB.Algunas
de las proteínas desreguladas parecen estarlo debido a efectos directos del DCB, como la
chaperonina60ylaα‐1,4‐glucanosintasa.Sinembargootrasparecenestarimplicadasenlas
estrategiasdehabituaciónaDCB, como laenolasa1, la1‐aminociclopropano‐1‐carboxilato
oxidasa1,laascorbatoperoxidasaylacafeoil‐CoAO‐metiltransferasa1.
Losresultadosdelpresentetrabajoconfirmanqueloscultivoscelularesdemaízson
capacesde adoptardistintas estrategias en funcióndel nivel dehabituación aDCB, lo que
implica no sólo cambios en la composición de la pared celular, sino también importantes
alteracionesmetabólicas.Estasestrategiasmostraronrasgoscomunesatodoslosnivelesde
habituación, como la presencia de una red de arabinoxilano modificada, cambios en la
expresióndegenesrelacionadosconlabiosíntesisdecelulosaoxilanos,o ladesregulación
en el metabolismo de algunas proteínas. Sin embargo, también se han observado
alteracionesexclusivasasociadasalosdistintosprocesosdeadaptaciónaDCB.Todosestos
resultadosponendemanifiestolanotableplasticidadestructuraldelascélulasdemaízpara
hacerfrenteadiferentesgradosdereducciónensucontenidodecelulosa.
Resumen
218
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Please cite this article in press as: de Castro M, et al. Early cell-wall modifications of maize cell cultures during habituation to dichlobenil. J PlantPhysiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.10.010
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Journal of Plant Physiology xxx (2013) xxx– xxx
Contents lists available at ScienceDirect
Journal of Plant Physiology
j o ur nal homepage: www.elsev ier .com/ locate / jp lph
Physiology1
Early cell-wall modifications of maize cell cultures during habituationto dichlobenil
2
3
María de Castro ∗, Asier Largo Gosens, Jesús Miguel Alvarez, Penélope García-Angulo,Q1
José Luis Acebes4
5
Área de Fisiología Vegetal, Facultad de CC Biológicas y Ambientales, Universidad de León, E-24071 León, Spain6
7
a r t i c l e i n f o8
9
Article history:10
Received 3 May 201311
Received in revised form 21 October 201312
Accepted 22 October 201313
Available online xxx
14
Keywords:15
Arabinoxylan16
Cellulose17
FTIR18
Immunodot assay19
ZmCesA genes20
a b s t r a c t
Studies involving the habituation of plant cell cultures to cellulose biosynthesis inhibitors have achievedsignificant progress as regards understanding the structural plasticity of cell walls. However, since habit-uation studies have typically used high concentrations of inhibitors and long-term habituation periods,information on initial changes associated with habituation has usually been lost. This study focuses onmonitoring and characterizing the short-term habituation process of maize (Zea mays) cell suspensions todichlobenil (DCB). Cellulose quantification and FTIR spectroscopy of cell walls from 20 cell lines obtainedduring an incipient DCB-habituation process showed a reduction in cellulose levels which tended to revertdepending on the inhibitor concentration and the length of time that cells were in contact with it. Varia-tions in the cellulose content were concomitant with changes in the expression of several ZmCesA genes,mainly involving overexpression of ZmCesA7 and ZmCesA8. In order to explore these changes in moredepth, a cell line habituated to 1.5 �M DCB was identified as representative of incipient DCB habituationand selected for further analysis. The cells of this habituated cell line grew more slowly and formed largerclusters. Their cell walls were modified, showing a 33% reduction in cellulose content, that was mainlycounteracted by an increase in arabinoxylans, which presented increased extractability. This result wasconfirmed by immunodot assays graphically plotted by heatmaps, since habituated cell walls had a moreextensive presence of epitopes for arabinoxylans and xylans, but also for homogalacturonan with a lowdegree of esterification and for galactan side chains of rhamnogalacturonan I. Furthermore, a partial shiftof xyloglucan epitopes toward more easily extractable fractions was found. However, other epitopes,such as these specific for arabinan side chains of rhamnogalacturonan I or homogalacturonan with a highdegree of esterification, seemed to be not affected.
In conclusion, the early modifications occurring in maize cell walls as a consequence of DCB-habituationinvolved quantitative and qualitative changes of arabinoxylans, but also other polysaccharides. Therebysome of the changes that took place in the cell walls in order to compensate for the lack of cellulose differedaccording to the DCB-habituation level, and illustrate the ability of plant cells to adopt appropriate copingstrategies depending on the herbicide concentration and length of exposure time.
© 2013 Published by Elsevier GmbH.
Introduction21
Plant cell walls are dynamic structures whose importance22
resides principally in the key role they play in plant growth and23
development, enabling cells to adapt to abiotic or biotic stresses24
(Wolf et al., 2012). Cell walls influence the properties of most25
plant-based products, including their texture and nutritional value,26
and condition the processing properties of plant-based foods for27
human and animal consumption (Doblin et al., 2010). Cell walls28
are mainly composed of polysaccharides, which are classified as29
∗ Corresponding author. Tel.: +34 987295304.E-mail address: [email protected] (M. de Castro).
cellulose, hemicelluloses and pectins, and have lower amounts of 30
proteins, phenolics and other minor components. Cell wall compo- 31
sition varies according to the plant type and species (Sarkar et al., 32
2009), cell type and position within the plant, developmental stage 33
and history of responses to stresses (Doblin et al., 2010). This vari- 34
ability in the composition of cell walls reflects a certain degree of 35
plasticity as regards cell wall structure and composition. One suit- 36
able method to study the mechanisms underlying the plasticity 37
of plant cell wall structure and composition consists in habituat- 38
ing cell cultures to grow in the presence of high concentrations of 39
different cellulose biosynthesis inhibitors (CBIs) (for a review see 40
Acebes et al., 2010). CBIs are a structurally heterogeneous group of 41
compounds that affects cellulose synthesis, acting specifically on 42
cellulose coupling or deposition in higher plants. They can induce 43
0176-1617/$ – see front matter © 2013 Published by Elsevier GmbH.http://dx.doi.org/10.1016/j.jplph.2013.10.010
Please cite this article in press as: de Castro M, et al. Early cell-wall modifications of maize cell cultures during habituation to dichlobenil. J PlantPhysiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.10.010
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aberrant trajectories in CESA proteins, reduce their velocity or even44
clear them at the plasma membrane (Acebes et al., 2010; Brabham45
and DeBolt, 2013). In the last two decades it has been shown that46
it is possible to habituate undifferentiated cell cultures (calluses47
and cell suspensions) of various dicotyledonous plants, such as ara-48
bidopsis, tobacco, tomato, bean or poplar, to lethal concentrations49
of diverse cellulose biosynthesis inhibitors and related compounds,50
such as isoxaben, thaxtomin A, dichlobenil (DCB) or quinclorac, by51
gradually increasing the concentration in the culture medium. In52
general terms, in order to cope with the stressful conditions, the cell53
cultures habituated to these inhibitors modify the characteristic54
architecture of the type I cell wall (typical of gymnosperms, dicots55
and most monocots), having reduced levels of cellulose accompa-56
nied by a decrease in the hemicellulose content and a significant57
increase in pectins (Shedletzky et al., 1992; Díaz-Cacho et al., 1999;58
Encina et al., 2001, 2002; García-Angulo et al., 2006, 2009). The59
modifications produced in cells habituated to these inhibitors not60
only depend on the inhibitor concentration but also on the period61
of time that cells grow in contact with the inhibitor (Alonso-Simón62
et al., 2004).63
There are only two reported studies in which cell cultures from64
plants with type II cell walls (characteristic of graminaceous plants,65
together with the other commelinoid monocots) have been habit-66
uated to a CBI. These were barley cell suspensions (Shedletzky67
et al., 1992) and maize callus cultures (Mélida et al., 2009, 2010a,b),68
habituated to DCB in both cases. The modifications found in these69
type II cell walls differed considerably from those described for70
type I cell walls, since the drastic reduction in cellulose content71
was compensated for an increase in the content of heteroxylans,72
which had lower extractability and higher relative molecular mass.73
Furthermore, in the case of maize cell cultures obtained in our74
laboratory, DCB habituation has implied an enrichment of hydrox-75
ycinnamates and dehydroferulates esterified on arabinoxylans. The76
content of other polymers, such as mixed glucan, xyloglucan, man-77
nan, pectins and proteins, was unchanged or reduced (Mélida et al.,78
2009). As these characteristics differed from those described for79
DCB-habituated barley cell cultures, and did not look like any other80
structure previously described for type II cell walls, we proposed81
that our DCB-habituated maize cells had a unique structure, which82
was particularly interesting in order to gain a deeper understand-83
ing of the structural plasticity of the type II cell wall (Mélida et al.,84
2009).85
Changes associated with the habituation of cell cultures to CBIs86
have commonly been analyzed using high concentrations of herbi-87
cide and long-term habituation periods. These kinds of study take88
advantage of the fact that the cell culture characteristics have been89
“fixed”. However, information about the initial changes that take90
place during the process of habituation is lost, although cells at91
these stages probably present a huge variability in relation to their92
cell wall composition, properties and ability to habituate. A previ-93
ous study conducted in order to monitor cell wall changes during94
DCB-habituation in bean calluses showed that when them were cul-95
tured in 0.5 �M DCB for less than 7 culture cycles no appreciable96
changes in cell wall FTIR spectra were detected, but when the num-97
ber of culture cycles growing in DCB presence increased up to 13,98
the spectra of the cell walls underwent several changes, including99
an attenuation of the peaks related to cellulose (Alonso-Simón et al.,100
2004). So, this study established that (a) an initial period would be101
necessary until cell wall modifications arise and (b) that a set of cell102
wall modifications, not only a reduction in cellulose content, would103
be taking place.104
Accordingly to these results, the aim of this study was to monitor105
and characterize the early changes happening during the DCB-106
habituation of cells with type II walls using maize cell suspensions107
as a cellular model. First of all, we monitored the changes produced108
throughout the DCB-habituation process, in order to select a cell109
line that showed an initial contrastable modification in some cell 110
wall features. Next, we compared the cell growth pattern and cell 111
wall composition of 1.5 �M DCB-habituated and non-habituated 112
cells, paying special attention to the variation in composition of 113
polysaccharides, using a set of techniques such as cell wall frac- 114
tionation, gas-chromatography, immunodot assays and heatmaps. 115
Materials and methods 116
Cell cultures 117
Maize cell suspension cultures (Zea mays L., Black Mexican sweet 118
corn, donated by Dr. S. C. Fry, Institute of Molecular Plant Sciences, 119
University of Edinburgh, UK) from calluses obtained from imma- 120
ture embryo explants were grown in Murashige and Skoog media 121
(Murashige and Skoog, 1962) supplemented with 9 �M 2,4-d and 122
20 g L−1 sucrose, at 25 ◦C under light and rotary shaken, and rou- 123
tinely subcultured every 15 days. 124
Habituation to DCB 125
Maize cell suspensions were cultured in DCB (supplied by Fluka) 126
concentrations ranging from 0.3 to 1.5 �M. DCB was dissolved in 127
dimethyl sulfoxide (DMSO), which did not affect cell growth at this 128
range of concentrations. 129
Initially, non-habituated maize cells were cultured in media 130
containing three DCB concentrations: 0.3 �M (lower than the 131
I50-concentration of DCB capable of inhibiting dry weight (DW) 132
increase by 50% with respect to the control-value), 0.5 �M (the I50 133
value) and 1 �M (higher than the I50 value). After seven subcul- 134
tures, some of the cells habituated to growth in 1 �M DCB were 135
transferred into media containing 1.5 �M DCB. Habituated suspen- 136
sion cultures are referred to as Shx (n), where ‘x’ indicates DCB 137
concentration (�M) and (n) number of subcultures in that DCB 138
concentration. 139
Growth measurements 140
Growth curves of non-habituated and 1.5 �M DCB-habituated 141
cell lines were obtained by measuring the increase in DW at dif- 142
ferent culture times. Doubling time (dt) was defined as the time 143
required for a cell to divide or a cell population to double in size 144
in the logarithmic phase of growth, and was estimated by using 145
the equation lnWt = lnWo + 0.693t/dt (in which Wt: suspension cell 146
culture DW at time ‘t’ of the culture cycle; and Wo: suspension cell 147
culture DW at the beginning of the culture cycle). Thus, Wt or Wo 148
logarithmic plotting versus culture time is a straight line where the 149
ordinate at the origin corresponds to the Wt or Wo natural log- 150
arithm. Relative growth rate (�) was determined by the slope of 151
the straight line (0.693t/dt). Maximum growth rate was the max- 152
imum difference in DW per time unit between two consecutive 153
measurements in growth kinetics. 154
Cell cluster size was determined after vacuum filtration of 155
cell suspensions that were sequentially passed through different 156
pore-size meshes, and the relative abundance of the clusters was 157
expressed as the percentage of DW retained in each filter. 158
ZmCesA gene expression analysis: isolation of Total RNA, RT–PCR 159
and PCR 160
Total RNA was extracted with Trizol Reagent (Invitrogen), and 161
2 �g of total RNA was reverse-transcribed using the Superscript III 162
first strand synthesis system for RT-PCR (Invitrogen). First-strand 163
cDNA was generated using an oligo (dT) 20 primer, and 1 �l of 164
the first-strand cDNA was used as a template in subsequent PCR 165
Please cite this article in press as: de Castro M, et al. Early cell-wall modifications of maize cell cultures during habituation to dichlobenil. J PlantPhysiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.10.010
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reactions. ‘No-RT’ PCR assays were performed to confirm the166
absence of genomic DNA contamination.167
For each assay, several numbers of cycles were tested to ensure168
that amplification was within the exponential range.169
The gene-specific primers used for the analysis of ZmCesA1170
(AF200525), ZmCesA2 (AF200526), ZmCesA3 (AF200527), ZmCesA5171
(AF200529), ZmCesA6 (AF200530), and ZmCesA7 (AF200531) genes172
were those previously described by Holland et al. (2000). Due to the173
high-sequence similarity between ZmCesA1 and ZmCesA2, the same174
primer was used for the analysis of both genes (ZmCesA1/2). The175
primers used for the analysis of ZmCesA4 (AF200528) and ZmCesA8176
(AF200532) were those described in Mélida et al. (2010a).177
Preparation and fractionation of cell walls178
Cells collected from suspension cultures in the stationary phase179
of growth were frozen and homogenized with liquid nitrogen using180
a pestle and mortar, and treated with 70% ethanol for 5 days at181
room temperature. The suspension obtained was centrifuged and182
the pellet was washed with 70% ethanol (x6) and acetone (x6), and183
subsequently air dried to obtain the alcohol insoluble residue (AIR).184
The AIR was treated with 90% DMSO for 8 h at room temperature185
(x3) and then washed with 0.01 M phosphate buffer pH 7.0 (x2). The186
washed AIR was then incubated with 2.5 U ml−1 �-amylase from187
porcine pancreas (Sigma type VI-A) in 0.01 M phosphate buffer pH188
7.0 for 24 h at 37 ◦C (x3). The suspension was filtered through a189
glass fiber filter, and the residue washed with 70% ethanol (x6)190
and acetone (x6), air dried and then treated with phenol–acetic191
acid–water (2:1:1 by vol.) for 8 h at room temperature (x2). This192
residue was finally washed with 70% ethanol (x6) and acetone (x6)193
and then air dried to obtain the cell walls.194
In order to carry out a cell wall fractionation, dried195
cell walls were extracted at room temperature with 50 mM196
cyclohexane-trans-1,2-diamine-N,N,N′,N′-tetraacetic sodium salt197
(CDTA; 10 mg ml−1) and then washed with distilled water. The198
resulting pellet was treated with 0.1 M KOH (10 mg ml−1) for 2 h199
(x2) and washed again with distilled water. The remaining residue200
was then treated with 4 M KOH (10 mg ml−1) for 4 h (x2) and sub-201
sequently washed with distilled water. Then 6 M KOH (10 mg ml−1)202
was finally added to the pellet and treated at 37 ◦C for 24 h. KOH203
extracts were neutralized with acetic acid to pH 5.0.204
Extracts of each treatment were dialyzed (48 h) and lyophilized,205
and referred to CDTA, 0.1 M KOH, 4 M KOH and 6 M KOH fractions,206
respectively.207
Cell wall analyses208
Cellulose content was quantified in crude cell walls by theQ2209
Updegraff method (Updegraff, 1962) using hydrolytic conditions210
described by Saeman et al. (1963) and quantifying the glucose211
released by the anthrone method (Dische, 1962).212
Tablets for Fourier transform infrared (FTIR) spectroscopy were213
prepared in a Graseby-Specac press using cell wall samples (2 mg)214
mixed with KBr (1:100, w/w). Spectra were obtained on a Perkin-215
Elmer instrument at a resolution of 1 cm−1 (Online Resource 1).216
A window between 800 and 1800 cm−1, containing information217
about characteristic polysaccharides, was selected in order to218
monitor cell wall structure modifications. All the spectra were nor-219
malized and baseline-corrected with Spectrum v 5.3.1 software, by220
Perkin-Elmer. Data were then exported to Microsoft Excel 2003 and221
all spectra were area-normalized. Difference spectra were obtained222
by digital subtraction of non-habituated and habituated cell wall223
FTIR spectra.224
Total sugar content of each fraction was determined by the phe-225
nol–sulfuric acid method (Dubois et al., 1956) and was expressed as226
the glucose equivalent. Uronic acid content was determined by the227
m-hydroxydiphenyl method (Blumenkrantz and Asboe-Hansen, 228
1973) using galacturonic acid as standard. Neutral sugar content 229
analysis was performed as described by Albersheim et al. (1967). 230
Briefly, a lyophilized sample of each fraction was hydrolyzed with 231
2 M trifluoroacetic acid at 121 ◦C for 1 h and the resulting sugars 232
were derivatized to alditol acetates and analyzed by gas chromatog- 233
raphy (GC) using a Supelco SP-2330 column. 234
Analysis of polysaccharides by immunodot assays 235
An extensive analysis of polysaccharide distribution and com- 236
position of the cell wall fractions was carried out. Briefly, aliquots 237
of 1 �l from each fraction were spotted onto nitrocellulose mem- 238
brane (Schleicher & Schuell, Dassel, Germany) as a replicated 1/5 239
dilution series of the preceding one, with 5 different dilutions for 240
each fraction. Then, nitrocellulose membranes were blocked with 241
phosphate-buffered saline (PBS, 0.14 M NaCl, 2.7 mM KCl, 7.8 mM 242
Na2HPO4·12H2O, 1.5 mM KH2PO4, pH 7.2) containing 4% fat-free 243
milk powder (MPBS) prior to incubation in primary antibody 244
(hybridoma supernatants diluted 1/10 in MPBS) for 1.5 h at room 245
temperature. After washing extensively under running tap water 246
and PBS, membranes were incubated in secondary antibody (anti- 247
rat horseradish peroxidase conjugate, Sigma) diluted to 1/1000 in 248
MPBS for 1.5 h at room temperature. Membranes were washed as 249
described above prior to color development in substrate solution 250
[25 ml de-ionized water, 5 ml methanol containing 10 mg ml−1 4- 251
chloro-1-naphtol, 30 ml 6% (v/v) H2O2]. Color development was 252
stopped by washing the membranes. Samples of commercial 253
pectin (10 mg/ml; P41, Danisco), xyloglucan (10 mg/ml; kindly 254
donated by Dr. T. Hayashi) and an arabinoxylan-enriched frac- 255
tion (10 mg/ml; obtained from maize calluses habituated to 12 �M 256
DCB, kindly donated by Dr. H. Mélida) were used as reference 257
compounds. 258
Monoclonal antibodies were assayed and their corresponding 259
recognized epitopes were: JIM5 (homogalacturonan with a low 260
degree of methyl esterification; Clausen et al., 2003), JIM7 (homo- 261
galacturonan with a high degree of methyl esterification; Knox 262
et al., 1990), LM5 (�-1,4-galactan side chain of rhamnogalactur- 263
onan type I; Jones et al., 1997), LM6 (�-1,5-arabinan side chain of 264
rhamnogalacturonan type I and arabinogalactan proteins; Willats 265
et al., 1998), LM10 (unsubstituted and relatively low substituted 266
(1,4)-�-d-xylan; McCartney et al., 2005), LM11 (xylans and ara- 267
binoxylans; McCartney et al., 2005), and LM15 (non-fucosylated 268
xyloglucan; Marcus et al., 2008). 269
Immunodot assay related heatmaps 270
Immunolabeling semi-quantification for each fraction was per- 271
formed by scoring the number of dilutions labeled. Thus, a value 272
ranging from 1 to 5 was assigned depending on the number of 273
colored spots which appeared after incubation with the secondary 274
antibody. These values were then entered into the statistical soft- 275
ware (see Statistical analysis section for details) in order to obtain 276
heatmaps. 277
Statistical analysis 278
Principal component analysis (PCA) was performed using a max- 279
imum of five principal components. All these analyses were carried 280
out using the Statistica 6.0 software package. 281
Significant differences in the cellulose content were tested 282
applying a one-factor ANOVA (performing Tukey’s test as post hoc 283
analysis), using the PASW Statistics 18 software package. 284
Heatmaps were performed by using RKWard statistical soft- 285
ware. 286
Please cite this article in press as: de Castro M, et al. Early cell-wall modifications of maize cell cultures during habituation to dichlobenil. J PlantPhysiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.10.010
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Fig. 1. Difference spectrum obtained from the digital subtraction of the FTIR spectraof cell walls from non-habituated (Snh) and habituated to 1.5 �M DCB (Sh1.5) maizecell suspensions, growing in that DCB concentration for three subcultures.
Results287
Monitoring of early cell wall modifications during288
DCB-habituation289
FTIR spectroscopy and multivariate analysis290
FTIR spectra from cell walls corresponding to non-habituated291
(Snh) and habituated (Sh) to 0.3 (Sh0.3), 0.5 (Sh0.5), 1 (Sh1) and292
1.5 (Sh1.5) �M DCB maize cell suspensions which had been for dif-293
ferent number of culture cycles growing in the presence of DCB294
were obtained and analyzed (Supplementary Data 1). The main295
differences among cell lines were detected in the fingerprint area296
(900–1200 cm−1). In this area of the spectra, many polysaccharides297
absorb IR, including cellulose. In order to better appreciate the vari-298
ations between Sh and Snh cell wall spectra, difference spectra for299
each cell line were obtained (Supplementary Data 2; Fig. 1). These300
spectra showed noticeable variation among Snh and most of the301
Sh cell lines subjected to short-term DCB habituation [i.e. see Snh-302
Sh0.3(2); Snh-Sh0.5(2) and Snh-Sh1(1)]. Spectra obtained from Sh303
cells showed decreased peaks in wavenumbers associated with304
cellulose (1039, 1056, 1060, 1105 and 1160 cm−1) (Alonso-Simón305
et al., 2004, 2011). However, these variations were gradually atten-306
uated in the difference spectra corresponding to cell walls of Sh0.3307
and Sh0.5 cell lines as the number of culture cycles in presence308
of DCB was increased [see Snh-Sh0.3(4–10); Snh-Sh0.5(4–10)]. In309
contrast, the differences observed in the Sh1 cell line were more310
marked and they did not revert as the number of subcultures was311
increased [see Snh-Sh1(4–10)]. In addition, other wavenumbers312
not associated to cellulose such as 950 cm−1 – ascribed to pectins313
– or 1520 cm−1 -attributed to phenolic rings (Alonso-Simón et al.,314
2011), and others unattributed such as 1190 and 1480 cm−1, were315
also affected indicating that other cell wall components have been316
modified.317
Principal component analysis (PCA) of FTIR spectra showed two318
groups (Fig. 2) and the spectra were mainly discriminated by PC1319
(approx. 71% of total variance explained). Spectra obtained from cell320
walls corresponding to Snh and suspensions habituated to lower321
DCB concentrations (Sh0.3 and Sh0.5) which had been growing in322
presence of the inhibitor for a greater number of culture cycles323
were located on the positive side of PC1. In contrast, spectra cor-324
responding to cells habituated to higher DCB concentrations (Sh1325
and Sh1.5) and those corresponding to cells which had been grow-326
ing for a lower number of culture cycles in contact with lower327
Fig. 2. Principal components analysis (PCA) of maize cell suspensions FTIR spectra.A plot of the first and second principal components (PCs) is represented based on theFTIR spectra of cell walls from non-habituated (Snh) and DCB-habituated (Sh) maizecell suspensions along several culture cycles. (Shx (n); x indicates DCB concentration(�M) and (n) number of subcultures in that DCB concentration).
concentrations of the inhibitor were grouped on the negative side 328
of PC1. 329
Cellulose content 330
Cellulose content was influenced by DCB concentration as well 331
as by the time that maize cells were growing in its presence (Fig. 3). 332
In Snh cell walls, cellulose represented roughly 27% of the cell wall 333
weight. However, a reduction in the cellulose level occurred when 334
maize cells were exposed to DCB for one or two culture cycles. As 335
the number of subcultures in presence of DCB was increased, the 336
cellulose content of cell walls from suspensions habituated to lower 337
DCB concentrations (Sh0.3 and Sh0.5) gradually reverted to that of 338
the control level. In fact, after growing in contact with the inhibitor 339
for ten culture cycles, their cellulose content reached values that 340
did not differ significantly from those of the Snh. In contrast, this 341
complete reversion did not occur in the Sh1 cell line, at least in the 342
same number of culture cycles as Sh0.3 and Sh0.5 cell lines did. 343
Fig. 3. Cellulose content of cell walls from non-habituated maize cell suspen-sions (Snh) and different DCB-habituated (Sh) maize cell suspensions (legend asFig. 1). The asterisk reflects significant differences among non-habituated and DCB-habituated cell lines according to Tukey test (p < 0.05). Values represented aremean ± SE of nine measurements (n = 9). References in bold refer to the cell lineswhich were selected for further ZmCesA genes expression analysis (Fig. 4).
Please cite this article in press as: de Castro M, et al. Early cell-wall modifications of maize cell cultures during habituation to dichlobenil. J PlantPhysiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.10.010
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Fig. 4. Relative ZmCesA gene expression analyzed by RT-PCR of different maize cellsuspensions (legend as Fig. 1). ↑; more mRNA accumulation than control (Snh);ZmCesA3 was not included because no expression was detected. ZmUbi: ubiqui-tine gene expression. Ubiquitine was used as the housekeeping gene due to itsconstitutive expression.
ZmCesA genes expression344
ZmCesA8 and ZmCesA7 gene expression was induced in those cell345
cultures which were grown for a high number of culture cycles in346
the presence of DCB -Sh0.3(10) and Sh0.5(10), respectively (Fig. 4).347
An interesting trend was observed in cells habituated to 1 �M348
DCB: an induction of ZmCesA7 expression was detected after 8 and349
10 culture cycles – Sh1(8) and Sh1(10), and in the former line,350
ZmCesA8 expression was also enhanced. However, none of these351
genes were induced in DCB habituated cells in their first culture352
cycle – Sh1(1). Lastly, the expression of ZmCesA7 was also induced353
in Sh1.5(3). No expression of ZmCesA3 was observed in any of the354
cell lines analyzed, and no differences in mRNA accumulation in355
ZmCesA1/2, ZmCesA4, ZmCesA5 and ZmCesA6 with respect to the Snh356
were detected.357
The Sh1.5 cell line was selected for further analysis since it was 358
representative of an early DCB-habituation based on the following 359
criteria: (a) a 33% reduction of cellulose content, later demonstrated 360
to be irreversible (data not shown) was observed (b) FTIR spec- 361
trum indicated that cellulose, as well as other polysaccharides, were 362
affected (c) PCA results pointed to differences with Snh cells but also 363
with the remaining Sh cells, indicating features of an incipient DCB 364
habituation. 365
Culture features 366
Growth of Snh and Sh1.5 maize cell suspensions throughout the 367
culture cycle was analyzed (Fig. 5a). Sh1.5 cells showed a longer lag 368
phase and a less pronounced logarithmic phase of growth when 369
compared with the Snh cells, which determined longer doubling 370
times and lower relative and maximum growth rates (Fig. 5b). 371
Snh cells showed homogeneous cluster-size since 88% of their 372
DW was found to have diameters ranging between 710 and 400 �m. 373
In contrast, a larger average size and greater variety of sizes was 374
observed in the Sh1.5 cells, and 73% of their DW was retained in 375
filters with pore sizes ranging from 3000 to 710 �m (Fig. 6). 376
Cell wall characterization 377
Cell wall fractionation and sugar analysis 378
Most polysaccharides were extracted by alkali treatments, espe- 379
cially in 4 M KOH and 6 M KOH fractions. Sh1.5 cell walls were 380
enriched in total sugars, mainly extracted with 4 M KOH followed 381
by 0.1 M KOH (Fig. 7a). 382
Gas chromatography and uronic acid analysis of cell wall frac- 383
tions (Fig. 7b) revealed that alkali fractions were composed mainly 384
of arabinose and xylose, followed by glucose, uronic acids and 385
galactose, whereas the CDTA fraction was enriched in uronic acids 386
and arabinose. Arabinose and xylose were responsible for the 387
increase in neutral sugars detected in the 0.1 M KOH and 4 M KOH 388
fractions in Sh1.5 cell walls. A decrease in glucose, especially in 389
fractions extracted with 4 M KOH and 6 M KOH, was observed in 390
Sh1.5 cells. Finally, an slight increase in uronic acids, mainly in 391
alkali-extracted fractions, was shown by Sh1.5 cells. 392
Fig. 5. (a) Growth of maize cell suspensions during the culture cycle. Open circle; non-habituated maize cell suspensions (Snh). Filled circle; habituated to 1.5 �M DCB maizecell suspensions (Sh1.5). Values represented are mean ± SD of nine measurements (n = 9); (b) growth parameters obtained from the growth curves of non-habituated (Snh)and habituated to 1.5 �M DCB (Sh1.5) maize cell suspensions.
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Fig. 6. Size of cell clusters of maize cell suspensions at the stationary phase. Whitebars; non-habituated maize cell suspensions (Snh). Black bars; habituated to 1.5 �MDCB maize cell suspensions (Sh1.5). Values represented are mean ± SE of three mea-surements (n = 3).
Polysaccharide epitope screening393
In order to detect cell wall changes in the distribution of epitopes394
in short-term DCB-habituated cells, aliquots of cell wall fractions395
of Snh and Sh1.5 cell suspensions were loaded onto nitrocellulose 396
membranes as 1/5 dilutions and subjected to immunodot assays 397
(Fig. 8a). Monoclonal antibodies for different pectic and hemicel- 398
lulosic epitopes were probed. Regarding to the specific antibodies 399
probed for hemicelluloses epitopes for �-1,4-xylans, recognized by 400
LM10, were exclusively found in all the alkali-extracted cell wall 401
fractions from Sh1.5. The pattern of LM11 labeling -which detected 402
specifically epitopes for xylans and arabinoxylans- was similar in 403
both cell lines, with the exception that the Sh1.5 6 M KOH cell 404
wall fraction showed an increased intensity of labeling. Lastly, the 405
degree of labeling for non-fucosylated xyloglucan epitopes (LM15) 406
was revealed to be stronger in the Sh1.5 4 M KOH cell wall fraction, 407
whereas the opposite trend was observed in the 6 M KOH fraction. 408
Respect to pectic polysaccharides, cell wall fractions from both 409
cell lines were probed with specific antibodies for homogalactur- 410
onan and rhamnogalacturonan I. Results obtained for JIM5 and 411
JIM7, antibodies for homogalacturonan with low and high degree 412
of methylesterification respectively, showed that no differences in 413
the pattern of labeling were found among cell wall-fractions from 414
the Snh and Sh1.5 cell lines for JIM7, whereas a greater intensity of 415
labeling for JIM5 was found in CDTA and 0.1 M KOH Sh1.5 cell line 416
wall-fractions when compared to Snh. 417
LM5 and LM6 were probed in order to detect �-1,4-galactan 418
and �-1,5-arabinan side chains of rhamnogalacturonan I. A more 419
intense label for LM5 was found in all the alkali extracted-fractions 420
from Sh1.5 cells. In contrast, no differences between cell lines were 421
Fig. 7. (a) Total sugars, neutral sugars and uronic acids contents of fractions from cell walls of non-habituated (White bars) and habituated to 1.5 �M DCB (Black bars) maizecell suspensions. Values represented are mean ± SE of three measurements (n = 3); (b) monosaccharide composition of fractions from cell walls of non-habituated (Whitebars) and habituated to 1.5 �M DCB (Black bars) maize cell suspensions. Rha: rhamnose, Fuc: fucose, Ara: arabinose, Xyl: xylose, Man: mannose, Gal: galactose, Glc: glucose,UA: uronic acids.
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Fig. 8. (a) Representative immunodot assay (IDA). Cell wall fractions obtained from non-habituated cells (Snh) and habituated to 1.5 �M DCB (Sh1.5) were probed withLM10, monoclonal antibody specific for xylans. Any labeling was observed for Snh fractions, whereas 0, 1, 1 and 2 colored spots were detected respectively in CDTA, 0.1 MKOH, 4 M KOH and 6 M KOH fractions from Sh1.5 cells. AX: arabinoxylan-enriched fraction used as standard. (b) Heatmap of data obtained from IDAs of non-habituated (Snh)and habituated to 1.5 �M DCB (Sh) cell wall fractions. A value ranging from 0 (no colored spot detected) to 5 (5 colored spots detected) was assigned to each antibody (J5:JIM5, J7: JIM7, L5: LM5, L6; LM5, L10:LM10, L11:LM11, L15:LM15) and in each cell wall fraction (CDTA, 0.1 M KOH, 4 M KOH, 6 M KOH), depending on the amount of coloredspots (corresponding to dilutions) that were shown after revealing.
found in these fractions when LM6 was probed. Nevertheless, �-422
1,5-arabinan side chains of rhamnogalacturonan I epitopes were423
solely detected in the CDTA fraction of Snh cells.424
A heatmap was generated with the resulting pool of data425
(Fig. 8b). As the total number of dilutions assayed was 5, a value426
ranging from 0 to 5 was assigned. This value depended on the427
sum of colored spots (corresponding to labeled epitopes in each428
dilution) found in the heatmap (see methods). In order to illus-429
trate this process, a representative immunodot assay for LM10 is430
shown in Fig. 8a. Likewise, when LM10 was assayed, the scoring431
values assigned were 0 for all the Snh cell wall fractions and 0, 1, 1432
and 2 for Sh1.5 CDTA, 0.1 M KOH, 4 M KOH and 6 M KOH fractions,433
respectively.434
Two different types of groupings appeared in the heatmap: a435
dendrogram with cell wall fractions and a dendrogram with the436
different antibodies probed. These groupings depended on the dis-437
tribution pattern of labeled epitopes in the fractions.438
The closest cell wall fractions in the Snh and Sh1.5 cell lines, and439
consequently the most similar in terms of the distribution of labeled440
epitopes, were 6 M KOH and 4 M KOH, followed by 0.1 M KOH and441
CDTA. In the dendrogram generated from the antibodies probed,442
two main branches were formed, each of which then divided again443
into another two principal sub-branches. Branch 1.1 grouped anti-444
bodies which recognized xylans (LM10) and arabinoxylans (LM11).445
For these, a darker intensity of color was observed in the heatmap446
for Sh1.5 cells when compared with that for Snh cells, indicating a447
more widespread presence of these polysaccharides in the alkali- 448
extracted cell wall fractions of the habituated cell line. Although 449
not so marked, a similar trend appeared in the labeling pattern of 450
antibodies which recognized �-1,4-galactan (LM5) side chains of 451
rhamnogalacturonan I, non-fucosylated xyloglucan (LM15) (branch 452
2.1 vs. 2.2) and homogalacturonan with a low degree of esteri- 453
fication (JIM5) (branch 1.2), pointing again to a more extensive 454
occurrence of epitopes for these polysaccharides throughout the 455
cell wall fractions of the Sh1.5 cells. 456
Discussion 457
One of the most conspicuous changes detected by FTIR monitor- 458
ing of the cell walls from the cell lines studied was a reduction in 459
the cellulose content. In fact, the main differences among spectra 460
of non-habituated (Snh) and DCB-habituated (Sh) (Fig. 1 and Sup- 461
plementary Data 1, 2) maize cell walls appeared in the fingerprint 462
region (900–1200 cm−1), in which a wide range of polysaccharides 463
absorb IR radiation, including cellulose. These differences became 464
more marked as the DCB concentration in the culture medium was 465
increased. However, differences among the spectra of Snh and Sh 466
exposed to lower DCB concentrations (Sh0.3 and Sh0.5) were atten- 467
uated when the number of culture cycles that cells were grown in 468
contact the inhibitor was increased. These data were confirmed by 469
the results obtained from the analysis of cellulose content (Fig. 3). 470
A 23% and 27% reduction in the cellulose content was observed 471
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when maize cells were exposed to 0.3 and 0.5 �M DCB, respectively,472
but after 10 culture cycles growing in presence of the inhibitor,473
these levels reverted to values close to the Snh value. At higher474
DCB concentrations (Sh1 and Sh1.5), differences in the fingerprint475
region among spectra (Fig. 1 and see Supplementary Data 1, 2)476
were greater with respect to the Snh spectra and eventual recov-477
ery of the cellulose content was not observed (Fig. 3). The gradual478
reversion in cellulose content produced when cells were growing479
in low DCB concentrations and the number of culture cycles in its480
presence was increased, had previously been described in bean481
cell suspensions (García-Angulo et al., 2006) and callus-cultured482
cells (Alonso-Simón et al., 2004), but interestingly, in these cells483
with type I walls, an initial period of about seven subcultures was484
required previously to the detection of any changes in their cell485
walls FTIR profiles, whereas in our maize cells, which have type II486
cell walls, this lag period has not been observed.487
One possible explanation for this observed transient reversion in488
cellulose content at the initial steps of the DCB-habituation process489
could be that increasing concentrations of herbicide may promote490
some change in the expression level of ZmCesA genes. In fact, it491
has been found that ZmCesA gene expression was mis-regulated in492
maize cultured cells habituated to high (12 �M) DCB concentra-493
tions (Mélida et al., 2010a). An induction of ZmCesA7 and ZmCesA8,494
thought to be involved in producing the primary cell wall before495
the onset of secondary wall formation (Appenzeller et al., 2004;496
Bosch et al., 2011), was detected in some of our short-term habitu-497
ated cell lines (Fig. 4). It seems that maize cells start to overexpress498
these isoforms when the number of culture cycles growing in pres-499
ence of DCB is increased or when the concentration of the inhibitor500
exceeds a threshold. Similarly maize cells subjected to long-term501
habituation to high DCB concentrations also showed an induction502
of ZmCesA7 and ZmCesA8 genes (Mélida et al., 2010a). This could be503
indicative of an important role for these two CesA isoforms in habit-504
uation to all DCB levels. It could be hypothesized that, although less505
efficient in cellulose synthesis, ZmCesA7 and ZmCesA8 may be more506
resistant to the effects of DCB.507
In addition to cellulose, FTIR monitoring revealed that other508
polysaccharides seem to be also affected by DCB-habituation. So,509
in order to unmask other actors playing a role in early DCB-510
habituation events, Sh1.5 cell line was selected for further studies.511
In the Sh1.5 cell line the observed 33% reduction in cellulose512
content was compensated by a net increase in 0.1 M KOH and 4 M513
KOH extracted arabinoxylans (Figs. 7a and b). Immunodot assays514
also showed in this cell line an increase in the proportion of epi-515
topes for arabinoxylan/xylan (LM10 and LM11). Interestingly, no516
LM10 labeling was observed neither in Snh cell walls in our work,517
nor in maize calluses habituated to high DCB concentrations – 4,518
6 or 12 �M (Mélida et al., 2009). The divergence between both519
studies could be related to the difference of plant material (cal-520
luses or suspension cell cultures), but would be also linked to a521
transient modification in chemical composition in some subsets of522
arabinoxylans populations at the first steps of the habituation.523
Other hemicelluloses, such as xyloglucan, seems also to be524
involved in the early events of DCB-habituation, since an increase in525
the detection of LM15 epitopes was observed in the Sh1.5 4 M KOH526
fraction, pointing to more easily extractable xyloglucan molecules.527
Reinforcing this finding, a lower presence of LM15 epitopes was528
found in the in 6 M KOH fraction from Sh1.5 cells, treatment which529
would extract strongly linked molecules.530
Furthermore, sugar composition analysis points to an increase531
of uronic acid-rich polysaccharides, especially in alkali-extracted532
fractions, in the early steps of habituation. Likewise, in such alkali-533
extracted fractions, immunodot assays revealed an enhancement534
of LM5 labeling and, therefore, an increase in galactan side chains535
of rhamnogalacturonan I in the habituated cell line. Also, an enrich-536
ment of epitopes for JIM5 (homogalacturonan with a low degree of537
esterification) was found in the mild-alkali (0.1 M KOH) extracted 538
fraction, as well as in the CDTA-extracted fraction. 539
The fact that other related epitopes such as those for homogalac- 540
turonan with a higher degree of esterification (JIM7) or for arabinan 541
(LM6) did not change between cell lines, indicates that only some 542
specific subsets of polysaccharides were affected during the early 543
DCB-habituation. 544
It is interesting to note how in the type II cell walls of our cell 545
cultures, characterized by a rather low amount of pectic polysac- 546
charides, an increase in such type of polysaccharides is enhanced 547
in the first steps of DCB-habituation. This fact is comparable with 548
the observed trend in DCB-habituated plant cultured cells hav- 549
ing type I walls, as bean, in which the reduction in the cellulose 550
content is mainly counteracted by an increase in pectic polysac- 551
charides (Encina et al., 2001; García-Angulo et al., 2006). Previous 552
work carried out in DCB or isoxaben habituated cultured cells hav- 553
ing type I cell walls, are characterized by a substantial increase in 554
JIM5-reactive low-esterified pectins, which would point to more 555
Ca+2-cross-linked pectins (Wells et al., 1994, Sabba et al., 1999, Q3556
Manfield et al., 2004; García-Angulo et al., 2006). As pectic polysac- 557
charides are involved in cell-cell adhesion (Willats et al., 2001), the 558
higher amount of these polysaccharides observed in our habitu- 559
ated cells could explain the fact that these cultured cells developed 560
larger cell clusters than the Snh cultures (Fig. 6), resembling to 561
that described for DCB-habituated bean cell suspensions (Encina 562
et al., 2001; García-Angulo et al., 2009). Nevertheless, and as a 563
supplementary explanation, such slower and altered growth pat- 564
terns detected in Sh1.5 cells could be also related with their 565
cellulose-deficient wall which would therefore, cause an impaired 566
cell division. On the other hand, and although xyloglucan is known 567
not to be an abundant hemicellulose in type II cell walls (Fry, 2000), 568
our results shown that these hemicelluloses may play some role 569
during this early stress situation, as the detection of LM15 epitopes 570
increased (at least in 4 M KOH fraction) during the first steps of DCB 571
habituation. 572
Interesting differences in cell wall modifications appear when 573
early DCB-habituation events are compared with those described 574
for maize calluses habituated to intermediate and high DCB lev- 575
els (Mélida et al., 2009, 2010a,b). In long-term DCB habituated 576
cells, a 75% of reduction in cellulose content was detected, and 577
this value remained stable even when DCB concentration in the 578
culture medium increased from 6 to 12 �M. In contrast, in short 579
term DCB-habituation, the reduction in cellulose content was lower 580
(23–33%) and tended to revert when DCB concentration in the 581
culture medium ranged from 0.3 and 0.5 �M. Moreover, in long- 582
term DCB-habituation the reduction in the amount of cellulose 583
was compensated by an increase in arabinoxylans, but the content 584
of other polysaccharides, such as pectins or xyloglucan, was not 585
affected or even was reduced. However, as mentioned above, in 586
Sh1.5 cultures besides the not so drastic increase in the arabinoxy- 587
lan amount, other polysaccharides such as rhamnogalacturonan I, 588
homogalacturonan with a low degree of esterification, and even 589
xyloglucan seemed to be involved. Finally, although further stud- 590
ies are required, our preliminary results suggest that antioxidant 591
activities play an important role during the first steps of the DCB 592
habituation process (Largo, personal communication), in contrast 593
to those described in cultures habituated long-term to high DCB 594
concentrations (Mélida et al., 2010a). 595
Concluding remarks 596
In sum, monitoring of short-term DCB habituation of maize cell 597
suspensions has revealed that the main modification produced dur- 598
ing the first steps of this process consisted in the reduction of 599
cellulose content, and it has been confirmed that changes in these 600
Please cite this article in press as: de Castro M, et al. Early cell-wall modifications of maize cell cultures during habituation to dichlobenil. J PlantPhysiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.10.010
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cellulose levels were promoted by two main factors: the inhibitor601
concentration and the number of culture cycles that cells were in602
its presence. Wall composition of Sh1.5 cells was modified as a603
consequence of habituation to low DCB levels, showing a reduc-604
tion of 33% in the cellulose content. Furthermore, an induction of605
ZmCesA7 and ZmCesA8 took place and this effect was revealed as a606
constant feature of DCB habituation. Sh1.5 cells counteracted the607
lack of cellulose with an increase in arabinoxylans. In addition to608
arabinoxylans, other polysaccharides, such as galactan side chains609
of rhamnogalacturonan I, homogalacturonan with a low degree of610
esterification and xyloglucan, seemed to have a role in the first steps611
of DCB-habituation. Thereby some of the modifications occurred in612
the cell walls in order to compensate for the lack of cellulose dif-613
fered according to the DCB-habituation level, and demonstrates the614
remarkable dynamic plasticity of maize cell cultures, which enables615
them to cope with different DCB habituation conditions by altering616
their cell wall composition.617
Acknowledgements618
The authors thank Dr. Encina, Dr. Alonso-Simón and Dr. Mél-619
ida for their helpful scientific discussion and Denise Phelps for620
the English revision of the manuscript. This study was supported621
by grants from the Spanish Ministry of Science and Innovation622
(CGL2008-02470 and AGL2011-30545-C02-02). María de Castro623
received funding through a PhD grant from the Spanish Ministry624
of Science and Innovation FPI program, and Asier Largo from the625
University of León, Spain.626
Appendix A. Supplementary data627
Supplementary material related to this article can be628
found, in the online version, at http://dx.doi.org/10.1016/629
j.jplph.2013.10.010.630
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