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

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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.

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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)

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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

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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;

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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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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

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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.

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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

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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.,

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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

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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.

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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)

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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)

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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

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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

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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

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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

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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

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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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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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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,

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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

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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

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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

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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

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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

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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.

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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

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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).

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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).

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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

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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.

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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.

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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),

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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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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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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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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

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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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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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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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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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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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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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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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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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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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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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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

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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

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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   

 

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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

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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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195  

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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198  

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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204  

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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205  

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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206  

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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207  

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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208  

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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209  

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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210  

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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213  

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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215  

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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216  

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.

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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: maria.decastro@unileon.es (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).

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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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